Association between matrix metalloproteinase (MMP)-2 polymorphisms and MMP-2 levels in hypertensive disorders of pregnancy

We examined whether two functional polymorphisms (g.-1306 C> T and g.-735 C>T) in matrix metalloproteinase (MMP)-2 gene are associated with preeclampsia (PE) or gestational hypertension (GH), and whether they modify MMP-2 or tissue inhibitor of metalloproteinase (TIMP)-2 plasma concentrations in these hypertensive disorders of pregnancy. We studied 130 healthy pregnant (HP), 130 pregnant with GH, and 133 pregnant with PE. Genomic DNA was extracted from whole blood and genotypes for g.-1306 C>T and g.-735 C>T polymorphisms were determined by Real Time-PCR, using Taqman allele discrimination assays. Haplotypes were inferred using the PHASE program. Plasma MMP-2 and TIMP-2 concentrations were measured by ELISA. The main findings were that pregnant with PE have higher plasma MMP-2 and TIMP-2 concentrations than HP (P<0.05), although the MMP-2/TIMP-2 ratios were similar (P>0.05). Moreover, pregnant with GH have elevated plasma MMP-2 levels and MMP-2/TIMP-2 ratios compared to HP (P<0.05). While MMP-2 genotypes and haplotypes are not linked with hypertensive disorders of pregnancy, MMP-2 genotypes and haplotypes are associated with significant alterations in plasma MMP-2 and TIMP-2 concentrations in preeclampsia (P<0.05). Our findings may help to understand the relevance of MMP-2 and its genetic polymorphisms to the pathophysiology of hypertensive disorders of pregnancy. It is possible that patients with PE and the MMP-2 haplotype combining the C and T alleles for the g.-1306 C>T and g.-735 C>T polymorphisms may benefit from the use of MMPs inhibitors such as doxycycline. However, this possibility remains to be determined. (C) 2012 Elsevier Inc. All rights reserved.

Analisi genetico-molecolare dei linfomi a cellule T periferiche

Molecular profiling of Peripheral T-cell lymphomas not otherwise specified Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of tumors that the WHO classification basically subdivides into specified and not otherwise specified (NOS). In Western countries, they represent around 12% of all non-Hodgkin's lymphomas. In particular, PTCL/NOS is the commonest subtype, corresponding to about 60-70% of all T-cell lymphomas. However, it remains a complex entity showing great variety regarding either morphology, immunophenotype or clinical behavior. Specially, the molecular pathology of these tumors is still poorly known. In fact, many alteration were found, but no single genes were demonstrated to have a pathogenetic role. Recently, gene expression profiling (GEP) allowed the identification of PTCL/NOS-associated molecular signatures, leading to better understanding of their histogenesis, pathogenesis and prognostication. Interestingly, proliferation pathways are commonly altered in PTCLs, being highly proliferative cases characterized by poorer prognosis. In this study, we aimed to investigate the possible role in PTCL/NOS pathogenesis of selected molecules, known to be relevant for proliferation control. In particular, we analyzed the cell cycle regulators PTEN and CDKN1B/p27, the NF-kB pathway, and the tyrosin kinase PDGFR. First, we found that PTEN and p27 seem to be regulated in PTCL/NOS as in normal T-lymphocytes, as to what expression and cellular localization are concerned, and do not present structural abnormalities in the vast majority of PTCL/NOS. Secondly, NF-kB pathway appeared to be variably activated in PTCL/NOS. In particular, according to NF-kB gene expression levels, the tumors could be divided into two clusters (C1 and C2). Specially, C1 corresponded to cases presenting with a global down-regulation of the entire pathway, while C2 showed over-expression of genes involved in TNF signaling. Notably, by immunohistochemistry, we showed that either the canonical or the alternative NK-kB pathway were activated in around 40% of cases. Finally, we found PGDFRA to be consistently over-expressed (at mRNA and protein level) and activated in almost all PTCLs/NOS. Noteworthy, when investigating possible causes for PDGFRA deregulation, we had evidences that PDGFR over-expression is due to the absence of miR-152, which appeared to be responsible for PDGFRA silencing in normal T-cells. Furthermore, we could demonstrate that its aberrant activation is sustained by an autocrine loop. Importantly, this is the first case, to the best of our knowledge, of hematological tumor in which tyrosin kinase aberrant activity is determined by deregulated miRNA expression and autocrine loop activation. Taken together, our results provide novel insight in PTCL/NOS pathogenesis by opening new intriguing scenarios for innovative therapeutic interventions.

Multicentre validation study of nucleic acids extraction from FFPE tissues

In most pathology laboratories worldwide, formalin-fixed paraffin embedded (FFPE) samples are the only tissue specimens available for routine diagnostics. Although commercial kits for diagnostic molecular pathology testing are becoming available, most of the current diagnostic tests are laboratory-based assays. Thus, there is a need for standardized procedures in molecular pathology, starting from the extraction of nucleic acids. To evaluate the current methods for extracting nucleic acids from FFPE tissues, 13 European laboratories, participating to the European FP6 program IMPACTS (www.impactsnetwork.eu), isolated nucleic acids from four diagnostic FFPE tissues using their routine methods, followed by quality assessment. The DNA-extraction protocols ranged from homemade protocols to commercial kits. Except for one homemade protocol, the majority gave comparable results in terms of the quality of the extracted DNA measured by the ability to amplify differently sized control gene fragments by PCR. For array-applications or tests that require an accurately determined DNA-input, we recommend using silica based adsorption columns for DNA recovery. For RNA extractions, the best results were obtained using chromatography column based commercial kits, which resulted in the highest quantity and best assayable RNA. Quality testing using RT-PCR gave successful amplification of 200 bp-250 bp PCR products from most tested tissues. Modifications of the proteinase-K digestion time led to better results, even when commercial kits were applied. The results of the study emphasize the need for quality control of the nucleic acid extracts with standardised methods to prevent false negative results and to allow data comparison among different diagnostic laboratories.

Elevated systemic monocyte chemoattractrant protein-1 in hepatic steatosis without significant hepatic inflammation

Non-alcoholic fatty liver disease (NAFLD) is strongly associated with obesity and the metabolic syndrome. It encompasses a clinico-pathologic spectrum of conditions ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). The latter develops upon pro-inflammatory cell infiltration and is widely considered as the first relevant pathophysiological step in NAFLD-progression. The chemokine monocyte chemoattractant protein 1 (MCP-1) plays an important role in the progression of hepatic inflammation and fibrosis, and both increased hepatic expression and circulating serum levels have been described in NASH. Here, we aimed to investigate MCP-1 expression in simple hepatic steatosis. Upon feeding a high-fat diet mice developed hepatic steatosis in the absence of significant hepatic inflammation, but elevated hepatic MCP-1 expression compared to control mice fed a standard chow. Interestingly, high-fat diet fed mice had significantly higher MCP-1 serum levels, and MCP-1 mRNA expression was significantly increased in visceral adipose tissue. Furthermore, MCP-1 serum levels were also elevated in patients with ultrasound-diagnosed NAFLD and correlated with the body-mass index and fasting glucose. In conclusion, our data indicate both the liver and adipose tissue as cellular sources of elevated circulating MCP-1 levels already in the early phase of hepatic steatosis. Since MCP-1 derived from visceral adipose tissue reaches the liver via portal circulation at high concentrations it may significantly contribute to the progression of simple steatosis to NASH.

Soluble CD163 is not increased in visceral fat and steatotic liver and is even suppressed by free fatty acids in vitro

Visceral fat differs from subcutaneous fat by higher local inflammation and increased release of IL-6 and free fatty acids (FFA) which contribute to hepatic steatosis. IL-6 has been shown to upregulate the monocyte/macrophage specific receptor CD163 whose soluble form, sCD163, is increased in inflammatory diseases. Here, it was analyzed whether CD163 and sCD163 are differentially expressed in the human fat depots and fatty liver. CD163 mRNA and protein were similarly expressed in paired samples of human visceral and subcutaneous fat, and comparable levels in portal venous and systemic venous blood of liver-healthy controls indicate that release of sCD163 from visceral adipose tissue was not increased. CD163 was also similarly expressed in steatotic liver when compared to non-steatotic tissues and sCD163 was almost equal in the respective sera. Concentrations of sCD163 were not affected when passing the liver excluding substantial hepatic removal/release of this protein. A high concentration of IL-6 upregulated CD163 protein while physiological doses had no effect. However, sCD163 was not increased by any of the IL-6 doses tested. FFA even modestly decreased CD163 and sCD163. The anti-inflammatory mediators fenofibrate, pioglitazone, and eicosapentaenoic acid (EPA) did not influence sCD163 levels while CD163 was reduced by EPA. These data suggest that in humans neither visceral fat nor fatty liver are major sources of sCD163.

Impaired 11 beta-hydroxysteroid dehydrogenase contributes to renal sodium avidity in cirrhosis: hypothesis or fact?

Exaggerated renal sodium retention with concomitant potassium loss is a hallmark of cirrhosis and contributes to the accumulation of fluid as ascites, pleural effusion, or edema. This apparent mineralocorticoid effect is only partially explained by increased aldosterone concentrations. I present evidence supporting the hypothesis that cortisol confers mineralocorticoid action in cirrhosis. The underlying molecular pathology for this mineralocorticoid receptor (MR) activation by cortisol is a reduced activity of the 11 beta-hydroxysteroid dehydrogenase type 2, an enzyme protecting the MR from promiscuous activation by cortisol in healthy mammalians.

Eotaxin/CCL11 expression by infiltrating macrophages in rat heart transplants during ongoing acute rejection

Eotaxin/CCL11 chemokine is expressed in different organs, including the heart, but its precise cellular origin in the heart is unknown. Eotaxin is associated with Th2-like responses and exerts its chemotactic effect through the chemokine receptor-3 (CCR3), which is also expressed on mast cells (MC). The aim of our study was to find the cellular origin of eotaxin in the heart, and to assess whether expression is changing during ongoing acute heart transplant rejection, indicating a correlation with mast cell infiltration which we observed in a previous study. In a model of ongoing acute heart transplant rejection in the rat, we found eotaxin mRNA expression within infiltrating macrophages, but not in mast cells, by in situ-hybridization. A five-fold increase in eotaxin protein in rat heart transplants during ongoing acute rejection was measured on day 28 after transplantation, compared to native and isogeneic control hearts. Eotaxin concentrations in donor hearts on day 28 after transplantation were significantly higher compared to recipient hearts, corroborating an origin of eotaxin from cells within the heart, and not from the blood. The quantitative comparison of eotaxin mRNA expression between native hearts, isografts, and allografts, respectively, revealed no statistically significant difference after transplantation, probably due to an overall increase in the housekeeping gene's 18S rRNA during rejection. Quantitative RT-PCR showed an increase in mRNA expression of CCR3, the receptor for eotaxin, during ongoing acute rejection of rat heart allografts. Although a correlation between increasing eotaxin expression by macrophages and mast cell infiltration is suggestive, functional studies will elucidate the role of eotaxin in the process of ongoing acute heart transplant rejection.

Reduced serum chemerin in patients with more severe liver cirrhosis

Chemerin is a well-established modulator of immune cell function and its serum levels are induced in inflammatory diseases. Liver cirrhosis is associated with inflammation which is aggravated by portal hypertension. The objective of this study was to evaluate whether chemerin is induced in patients with more severe liver cirrhosis and portal hypertension. Chemerin has been measured by ELISA in the portal venous serum (PVS), systemic venous serum (SVS) and hepatic venous serum (HVS) of 45 patients with liver cirrhosis. Chemerin is higher in HVS compared to PVS in accordance with our recently published finding. SVS, HVS and PVS chemerin decline in patients with more advanced liver injury defined by the CHILD-PUGH score. Hepatic chemerin has been determined in a small cohort and is similarly expressed in normal and cirrhotic liver. MELD score and serum markers of liver and kidney function do not correlate with chemerin. There is a positive correlation of chemerin in all compartments with Quick prothrombin time and of SVS chemerin with systolic blood pressure. PVS chemerin is induced in patients with modest/massive ascites but this does not translate into higher HVS and SVS levels. Chemerin is not associated with variceal size. Reduction of portal pressure by transjugular intrahepatic portosystemic shunt does not affect chemerin levels. These data show that low chemerin in patients with more severe liver cirrhosis is associated with reduced Quick prothrombin time.

Expression of HOXB2, a retinoic acid signaling target in pancreatic cancer and pancreatic intraepithelial neoplasia

Purpose: Despite significant progress in understanding the molecular pathology of pancreatic cancer and its precursor lesion: pancreatic intraepithelial neoplasia (PanIN), there remain no molecules with proven clinical utility as prognostic or therapeutic markers. Here, we used oligonucleotide microarrays to interrogate mRNA expression of pancreatic cancer tissue and normal pancreas to identify novel molecular pathways dysregulated in the development and progression of pancreatic cancer. Experimental Design: RNA was hybridized to Affymetrix Genechip HG-U133 oligonucleotide microarrays. A relational database integrating data from publicly available resources was created to identify candidate genes potentially relevant to pancreatic cancer. The protein expression of one candidate, homeobox B2 (HOXB2), in PanIN and pancreatic cancer was assessed using immunohistochemistry. Results: We identified aberrant expression of several components of the retinoic acid (RA) signaling pathway (RARa, MUC4, Id-1, MMP9, uPAR, HB-EGF, HOXB6, and HOXB2), many of which are known to be aberrantly expressed in pancreatic cancer and Pan IN. HOXB2, a downstream target of RA, was up-regulated 6.7-fold in pancreatic cancer compared with normal pancreas. Immunohistochemistry revealed ectopic expression of HOXB2 in 15% of early Pan IN lesions and 48 of 128 (38%) pancreatic cancer specimens. Expression of HOXB2 was associated with nonresectable tumors and was an independent predictor of poor survival in resected tumors. Conclusions: We identified aberrant expression of RA signaling components in pancreatic cancer, including HOXB2, which was expressed in a proportion of PanIN lesions. Ectopic expression of HOXB2 was associated with a poor prognosis for all patients with pancreatic cancer and was an independent predictor of survival in patients who underwent resection.

Nucleic acid amplification testing for Neisseria gonorrhoeae: An ongoing challenge

Nucleic acid amplification tests (NAATs) for the detection of Neisseria gonorrhoeae became available in the early 1990s. Although offering several advantages over traditional detection methods, N. gonorrhoeae NAATs do have some limitations. These include cost, risk of carryover contamination, inhibition, and inability to provide antibiotic resistance data. In addition, there are sequence-related limitations that are unique to N. gonorrhoeae NAATs. In particular, false-positive results are a major consideration. These primarily stem from the frequent horizontal genetic exchange occurring within the Neisseria genus, leading to commensal Neisseria species acquiring N. gonorrhoeae genes. Furthermore, some N. gonorrhoeae subtypes may lack specific sequences targeted by a particular NAAT. Therefore, NAAT false-negative results because of sequence variation may occur in some gonococcal populations. Overall, the N. gonorrhoeae species continues to present a considerable challenge for molecular diagnostics. The need to evaluate N. gonorrhoeae NAATs before their use in any new patient population and to educate physicians on the limitations of these tests is emphasized in this review.

Simplified preparation of human arterial sections for PCR analysis of Chlamydia pneumoniae and human DNA

AIMS: To investigate multiple techniques for the preparation of solid tissue for polymerase chain reaction (PCR) analysis, and to identify the most simple techniques for routine use in the laboratory. METHODS: Techniques for the preparation of arterial tissue samples including homogenisation, ultrafiltration, and treatments involving proteinase K, Gene Clean, lectin, and Fe3+ specific chelators were evaluated using the PCR to amplify both Chlamydia pneumoniae and human DNA. RESULTS: Treatment with either Gene-Clean or lectin and the Fe3+ specific chelator deferoxamine mesylate removed PCR inhibitors from tissue homogenates. Homogenisation followed by GeneClean treatment resulted in the amplification of C pneumoniae DNA from within a section of atherosclerotic carotid artery, implying that C pneumoniae elementary bodies had been disrupted. In eight further clinical samples from patients not known to have C pneumoniae infection, human DNA was amplified and no cross contamination was observed between samples. These samples contained no evidence of C pneumoniae by PCR. CONCLUSIONS: A simple preparation of solid tissue for PCR analysis, involving homogenisation followed by GeneClean treatment has been developed, and is effective for the amplification of both C pneumoniae and human DNA.

Can neurodegenerative disease be defined by four 'primary determinants':anatomy, cells, molecules, and morphology?

Traditional methods of describing and classifying neurodegenerative disease are based on the clinico-pathological concept supported by molecular pathological studies and defined by 'consensus criteria'. Disease heterogeneity, overlap between disorders, and the presence of multiple co-pathologies, however, have questioned the validity and status of many traditional disorders. If cases of neurodegenerative disease are not easily classifiable into distinct entities, but more continuously distributed, then a new descriptive framework may be required. This review proposes that there are four key neuropathological features of neurodegenerative disease (the 'primary determinants') that could be used to provide such a framework, viz., the anatomical pathways affected by the disease ('anatomy'), the cell populations affected ('cells'), the molecular pathology of 'signature' pathological lesions ('molecules'), and the morphological types of neurodegeneration ('morphology'). This review first discusses the limitations of existing classificatory systems and second provides evidence that the four primary determinants could be used as axes to define all cases of neurodegenerative disease. To illustrate the methodology, the primary determinants were applied to the study of a group of closely related tauopathy cases and to heterogeneity within frontotemporal lobar degeneration with TDP-43 proteinopathy (FTLD-TDP).

Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial.

AIMS: Mutation detection accuracy has been described extensively; however, it is surprising that pre-PCR processing of formalin-fixed paraffin-embedded (FFPE) samples has not been systematically assessed in clinical context. We designed a RING trial to (i) investigate pre-PCR variability, (ii) correlate pre-PCR variation with EGFR/BRAF mutation testing accuracy and (iii) investigate causes for observed variation. METHODS: 13 molecular pathology laboratories were recruited. 104 blinded FFPE curls including engineered FFPE curls, cell-negative FFPE curls and control FFPE tissue samples were distributed to participants for pre-PCR processing and mutation detection. Follow-up analysis was performed to assess sample purity, DNA integrity and DNA quantitation. RESULTS: Rate of mutation detection failure was 11.9%. Of these failures, 80% were attributed to pre-PCR error. Significant differences in DNA yields across all samples were seen using analysis of variance (p

A new generation of companion diagnostics: cobas BRAF, KRAS and EGFR mutation detection tests

The cobas® (Roche) portfolio of companion diagnostics in oncology currently has three assays CE-marked for in vitro diagnostics. Two of these (EGFR and BRAF) are also US FDA-approved. These assays detect clinically relevant mutations that are correlated with response (BRAF, EGFR) or lack of response (KRAS) to targeted therapies such as selective mutant BRAF inhibitors in malignant melanoma, tyrosine kinases inhibitor in non-small cell lung cancer and anti-EGFR monoclonal antibodies in colorectal cancer, respectively. All these assays are run on a single platform using DNA extracted from a single 5 µm section of a formalin-fixed paraffin-embedded tissue block. The assays provide an ‘end-to-end’ solution from extraction of DNA to automated analysis and report on the cobas z 480. The cobas tests have shown robust and reproducible performance, with high sensitivity and specificity and low limit of detection, making them suitable as companion diagnostics for clinical use.

Validation of immunocytochemistry as a morphomolecular technique

Immunocytochemistry (ICC) is a long-established means for clinical laboratories to investigate material for which it is difficult to obtain tissue samples. Unlike immunohistochemistry (IHC), the cells do not retain surrounding tissue environment/architecture. This can be of benefit in that fixation is often immediate and rapid, protecting the cells. Although fixation is frequently observed as the main preanalytic variable of test quality, all cytology preanalytic factors should be identified and controlled. In addition, the validation of ICC should take the same rigorous approach that other molecular pathology techniques follow. A three-step validation protocol is offered here. The end result is a comprehensive, morphomolecular approach to ICC, with an emphasis on therapeutic ICC.