Quantitative assessment shows loss of antigenic epitopes as a function of pre-analytic variables

Pre-analytic variables, specifically cold ischemic time, have been implicated as key variables in the measurement of proteins by immunohistochemistry. To determine the significance and magnitude of antigenic loss due to pre-analytic variables, we have compared protein antigenicity in core needle biopsies, with essentially no cold ischemic time, with that in routinely processed tumor resection specimens. Two cohorts of matched core needle biopsies and tumor resections were collected with 20 matched pairs and 14 matched pairs, respectively. Both series were analyzed by quantitative immunofluorescence using the AQUA? method. Epitopes phospho-ERK, total ERK, phospho-AKT, total AKT, phospho-S6K1, total S6K1, estrogen receptor (ER), Ki67, cytokeratin and GAPDH were assessed. Detection levels for all phospho-epitopes were significantly decreased in tumor resections compared with biopsies while no significant change was seen in the corresponding total proteins. Of the other four proteins examined, ER and cytokeratin showed significant loss of antigenicity. This data suggest that measurement of phospho-protein antigenicity in formalin-fixed tissue by immunological methods is dramatically affected by pre-analytic variables. This study suggests that core needle biopsies are more accurate for assessment of tissue biomarkers.     

Analysis of emergence of quinolone-resistant gonococci in Greece by combined use of Neisseria gonorrhoeae multiantigen sequence typing and multilocus sequence typing

The prevalence of quinolone-resistant Neisseria gonorrhoeae (QRNG) in Greece remained low from 1997 to 2003 but increased dramatically from 11% to 56% between 2004 and 2007. N. gonorrhoeae multiantigen sequence typing (NG-MAST) and multilocus sequence typing (MLST) were used to investigate trends in quinolone resistance from 1997 to 2007 and explore the origins of the recent increase in QRNG. We characterized 295 QRNG isolates from the study period and 233 quinolone-susceptible (QS) gonococci from 2004 and 2005, when the rapid increase in QRNG occurred. From 1997 to 1999, an outbreak of QRNG was due to the dissemination of isolates of serovar Arst that belonged to two closely related genotypes. Few QRNG isolates, of diverse genotypes, were present between 2001 and 2003, whereas the sharp increase in QRNG from 2004 onwards was due to the appearance of serovar Bropyst isolates of several major NG-MAST sequence type (STs) that previously had not been identified in Greece. These isolates were shown by MLST to be variants of a single multiply antibiotic-resistant QRNG strain (ST1901) that appeared in Greece and rapidly diversified into 31 NG-MAST STs. There were no isolates of MLST ST1901 or any of the 31 NG-MAST STs among QS isolates from 2004 and 2005 or among 8 representatives of multiresistant but quinolone-susceptible serovar Bropyst isolates circulating in Greece during the 1990 s, supporting the view that the recent increase in QRNG was due to importation of a QRNG strain(s) of MLST ST1901 into Greece. Recently, multiresistant QRNG isolates of ST1901 with reduced susceptibility to the newer cephalosporins have appeared in Greece.     

Parecoxib has non-significant long-term effects on bone healing in rats when administered for a short period after fracture

Introduction  Selective and non-selective cyclo-oxygenase (COX) inhibitors impair bone healing by inhibiting prostaglandin synthesis. The purpose of this study was to evaluate the long-term effect of parecoxib, a selective COX-2 inhibitor, on bone healing in rats, when it is applied in a pattern similar to clinical treatment patterns, that is, in a high dose and for a short period after bone fracture. Method  Closed non-displaced mid-diaphyseal fractures in the middle of the left femoral shaft were generated in each animal. In the study group, parecoxib sodium (1.06 mg/kg) was administered intra-peritoneally every day for 7 days. In the control group, normal saline was administered intra-peritoneally every day for 7 days. In both groups fracture healing (bone union and callus formation) was evaluated with X-rays 28 and 42 days after surgery. Results  Bone healing was lower in the study group (60 vs. 80% in the control group 28 days after fracture and 80 vs. 90% 42 days after fracture) but this difference was not statistically significant (P > 0.05). Conclusion  Parecoxib does not have a significant long-term effect on bone healing in rats, when it is administered in a high dose and for a short period after bone fracture. Declaration  The experiments comply with the current laws of the EU, and the protocol was approved by the Local Ethics Committee on Animal Research.     

KIT-negative gastrointestinal stromal tumors with a long term follow-up： A new subgroup does exist

AIMTo investigate the incidence of KIT immunoho-stochemical staining in(GI)stromal tumors(GISTs),and to analyze the clinical manifestations of the tumors and prognostic indicators.METHODSWe retrospectively analyzed 50 cases of Previously diagnosed GISTs.Tissue samples were assessed with KIT(CD117 antigen),CD34,SMA,desmin,S-100,NSE,PCNA,Ki-67,and BCL-2 for immunohistochemical study and pathological characteristics were analyzed for prognostic factors.RESULTSFifteen tumors(30%)were negative in KIT staining.A significant association was observed between gender(male patients14/15)and KIT-negative staining P = 0.003).The patients's mean age was 56.6 years.Tumors developed in stomach(n = 8),small intestine (n = 5),large intestine(n = 1)and oesophagus(n = 1).The mean tumor size was 5.72 cm.The mitotic count ranged from 0-29/50 HPF(mean3.4)and 73% of tumors showed no necrosis.The majority of the tumors(67%)had dual or epithelioid differentiation.Tumors were classified as very low or low risk(n = 7),intermediate risk(n = 5),and high risk(n = 3)groups.Twelve(80%)patients were alive without evidence of residual tumor for an average period of 40.25 mo(12-82 mo);three patients developed metastatic disease to the liver and eventually died within 2-12 mo(median survival8.6 mo).CONCLUSIONA small subgroup of GISTs fulfils the clinical and morphological criteria of these tumors,and lacks KIT expression.These tumors predominantly developed in the stomach,being dual or epithelioid in morphology,which are classified as low risk tumors and presented a better survival status than KIT-positive tumors.The ability to diagnose GISTs still depends on immunohistochemical staining but the research should extend in gene mutations.     

Hypochromic erythrocytes (%): a reliable marker for recognizing iron-restricted erythropoiesis and predicting response to erythropoietin in anemic patients with myeloma and lymphoma

The aim of the study was to evaluate the role of hypochromic erythrocytes (HYPO%) compared to “traditional” and novel markers of iron status and erythropoiesis in recognizing iron-restricted erythropoiesis (IRE) and predicting response to erythropoietin (rHuEPO) in anemic patients with myeloma and lymphoma. Forty-one newly diagnosed patients who received epoetin-beta at a subcutaneous weekly dose of 30,000 IU for 6 weeks were studied. Response to rHuEPO was observed in 27 patients (65.8%). Twelve non-responders received, additionally, 200 mg of IV iron sucrose, weekly, for 4 weeks. Evaluation of markers was performed at baseline and on weeks 1, 2 and 6 for all patients and also on weeks 7–10 for non-responders to rHuEPO. Baseline HYPO%, at a cut-off value of <5%, and an increment in reticulocyte absolute number (RETICS-AB) ≥ 50,000/μl and reticulocyte hematocrit (RETICS-Hct) ≥ 50%, between baseline and week 2, were independent predictive factors for response to rHuEPO. We found that these markers had superior predictive value for response to rHuEPO than four widely used predictive models. Furthermore, a baseline HYPO% count of above 5% proved superior over serum ferritin < 100 ng/ml and transferrin saturation < 20% in recognizing IRE. In conclusion, baseline HYPO% either alone or in combination with RETICS-AB or RETICS-Hct after 2 weeks of rHuEPO treatment could be reliably used in predicting response to rHuEPO. Additionally, HYPO% has proved a reliable marker for recognizing IRE before rHuEPO treatment and, thus, could be used for identifying patients who will benefit from IV iron supplementation.     

The impact of graph construction scheme and community detection algorithm on the repeatability of community and hub identification in structural brain networks

A critical question in network neuroscience is how nodes cluster together to form communities, to form the mesoscale organisation of the brain. Various algorithms have been proposed for identifying such communities, each identifying different communities within the same network. Here, (using test–retest data from the Human Connectome Project), the repeatability of thirty‐three community detection algorithms, each paired with seven different graph construction schemes were assessed. Repeatability of community partition depended heavily on both the community detection algorithm and graph construction scheme. Hard community detection algorithms (in which each node is assigned to only one community) outperformed soft ones (in which each node can belong to more than one community). The highest repeatability was observed for the fast multi‐scale community detection algorithm paired with a graph construction scheme that combines nine white matter metrics. This pair also gave the highest similarity between representative group community affiliation and individual community affiliation. Connector hubs had higher repeatability than provincial hubs. Our results provide a workflow for repeatable identification of structural brain networks communities, based on the optimal pairing of community detection algorithm and graph construction scheme.     

Lipid lowering drugs and gallstones: a therapeutic option?

Cholelithiasis is a common disease worldwide. The majority of gallstones can occur when the bile is supersaturated with cholesterol. Dyslipidaemia, obesity, insulin resistance are associated with an increased risk for cholesterol gallstone formation as well as with vascular risk. Statins and ezetimibe are used to treat dyslipidaemia and appear to have some effect on bile composition and cholesterol gallstone formation. Statin (e.g. pravastatin, simvastatin, fluvastatin and lovastatin) monotherapy or combined with ursodeoxycholic acid (UDCA) have shown reductions in bile cholesterol saturation, preventing gallstone formation and even dissolving pre-existing stones. However, this effect was not consistently reported in all studies. Statin use has also been associated with a reduced risk for cholecystectomy in 2 large epidemiological studies. Ezetimibe was shown to have a beneficial action against cholelithiasis in animal studies but data in humans - although promising - are very limited. The effect of these drugs on gallstone disease warrants further investigation in large human trials. We also consider the links between cholelithiasis, vascular risk and the use of lipid lowering drugs.     

Liver enzymes: potential cardiovascular risk markers?

Several cross-sectional studies have reported a relationship between elevated serum activity of liver enzymes [e.g. alanine aminotransferase (ALT) and gamma-glutamyltransferase (γGT)] and metabolic syndrome (MetS) and/or diabetes mellitus (DM). Raised serum activity of liver enzymes independently predicted the future development of MetS and DM as well as cardiovascular (CV) events and/or total/CV mortality in prospective studies. However, this association was not consistently demonstrated and it appears to be independent of alcohol intake. Even though these associations can be partly attributed to non-alcoholic fatty liver disease (NAFLD) and insulin resistance, there may be additional underlying mechanisms that contribute to the increased CV risk (e.g. inflammation and oxidative stress). The association of γGT with atherosclerotic plaque is of particular importance.     

Conserved vertebrate mir-451 provides a platform for Dicer-independent,Ago2-mediated microRNA biogenesis

Canonical animal microRNAs (miRNAs) are generated by sequential cleavage of precursor substrates by the Drosha and Dicer RNase III enzymes. Several variant pathways exploit other RNA metabolic activities to generate functional miRNAs. However, all of these pathways culminate in Dicer cleavage, suggesting that this is a unifying feature of miRNA biogenesis. Here, we show that maturation of miR-451, a functional miRNA that is perfectly conserved among vertebrates, is independent of Dicer. Instead, structure-function and knockdown studies indicate that Drosha generates a short pre-mir-451 hairpin that is directly cleaved by Ago2 and followed by resection of its 3′ terminus. We provide stringent evidence for this model by showing that Dicer knockout cells can generate mature miR-451 but not other miRNAs, whereas Ago2 knockout cells reconstituted with wild-type Ago2, but not Slicer-deficient Ago2, can process miR-451. Finally, we show that the mir-451 backbone is amenable to reprogramming, permitting vector-driven expression of diverse functional miRNAs in the absence of Dicer. Beyond the demonstration of an alternative strategy to direct gene silencing, these observations open the way for transgenic rescue of Dicer conditional knockouts.     

The Pro12Ala polymorphism in PPARgamma2 gene affects lipid parameters in Greek primary school children: A case of gene-to-gender interaction

BACKGROUND: In the present study we sought to evaluate the impact of the PPAR-gamma2 Pro12Ala polymorphism on blood lipid levels of primary school children. METHODS: 81 male and 92 female schoolchildren were genotyped. Biochemical, anthropometric, and lifestyle variables were assessed. RESULTS: 14.1% females and 14.8% males were heterozygotes, while the rest of the subjects were homozygotes for the Pro allele. A significant interaction between the PPARgamma-2 gene and gender on blood lipid levels was detected. In particular, Pro/Pro females exhibited higher values of total cholesterol (194 +/- 32 vs 180 +/- 28 mg/dL, P = 0.06) and triglycerides (94 +/- 31 vs 77 +/- 11 mg/dL, P = 0.045) compared to Pro/Ala individuals. The gene-to-gender interaction term was highly significant (P < 0.001). On the other hand, Pro/Pro males showed higher values of HDL cholesterol (47 +/- 8 vs 43 +/- 9 mg/dL, P = 0.001), lower total cholesterol/HDL ratio (4.04 +/- 0.59 vs 4.45 +/- 0.61, P = 0.031), lower values of apoB (59.8 +/- 11.3 vs 66.8 +/- 6.6 mg/dL, P = 0.007) and lower values of apoB/apoA1 ratio (0.41 +/- 0.09 vs 0.48 +/- 0.08, P = 0.019) compared with Pro/Ala. Even after adjusting for body mass index (BMI), total energy intake, total fat intake and saturated fat intake, differences in total cholesterol/high-density lipoprotein (HDL) cholesterol and the apoB/apoA1 ratios remained significant. Regarding females, no differences were observed among genotypes concerning total cholesterol/HDL levels (P for gene-to-gender interaction = 0.001) and the apoB/apoA1 levels (P for gene-to-gender interaction = 0.029). CONCLUSION: We show for the first time a gene-to-gender interaction on total cholesterol/HDL and apoB/apoA1 ratios, in male schoolchildren genotyped for PPAR-gamma2 Pro12Ala.