Whole‐genome methylation analysis of benign and malignant colorectal tumours

Changes in DNA methylation, whether hypo‐ or hypermethylation, have been shown to be associated with the progression of colorectal cancer. Methylation changes substantially in the progression from normal mucosa to adenoma and to carcinoma. This phenomenon has not been studied extensively and studies have been restricted to individual CpG islands, rather than taking a whole‐genome approach. We aimed to study genome‐wide methylation changes in colorectal cancer. We obtained 10 fresh‐frozen normal tissue–cancer sample pairs, and five fresh‐frozen adenoma samples. These were run on the lllumina HumanMethylation27 whole‐genome methylation analysis system. Differential methylation between normal tissue, adenoma and carcinoma was analysed using Bayesian regression modelling, gene set enrichment analysis (GSEA) and hierarchical clustering (HC). The highest‐rated individual gene for differential methylation in carcinomas versus normal tissue and adenomas versus normal tissue was GRASP (p_adjusted = 1.59 × 10^–5, BF = 12.62, p_adjusted = 1.68 × 10^–6, BF = 14.53). The highest‐rated gene when comparing carcinomas versus adenomas was ATM (p_adjusted = 2.0 × 10^–4, BF = 10.17). Hierarchical clustering demonstrated poor clustering by the CIMP criteria for methylation. GSEA demonstrated methylation changes in the Netrin–DCC and SLIT–ROBO pathways. Widespread changes in DNA methylation are seen in the transition from adenoma to carcinoma. The finding that GRASP, which encodes the general receptor for phosphoinositide 1‐associated scaffold protein, was differentially methylated in colorectal cancer is interesting. This may be a potential biomarker for colorectal cancer. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

NF‐κB‐inducing kinase is a key regulator of inflammation‐induced and tumour‐associated angiogenesis

Angiogenesis is essential during development and in pathological conditions such as chronic inflammation and cancer progression. Inhibition of angiogenesis by targeting vascular endothelial growth factor (VEGF) blocks disease progression, but most patients eventually develop resistance which may result from compensatory signalling pathways. In endothelial cells (ECs), expression of the pro‐angiogenic chemokine CXCL12 is regulated by non‐canonical nuclear factor (NF)‐κB signalling. Here, we report that NF‐κB‐inducing kinase (NIK) and subsequent non‐canonical NF‐κB signalling regulate both inflammation‐induced and tumour‐associated angiogenesis. NIK is highly expressed in endothelial cells (ECs) in tumour tissues and inflamed rheumatoid arthritis synovial tissue. Furthermore, non‐canonical NF‐κB signalling in human microvascular ECs significantly enhanced vascular tube formation, which was completely blocked by siRNA targeting NIK. Interestingly, Nik^?/? mice exhibited normal angiogenesis during development and unaltered TNFα‐ or VEGF‐induced angiogenic responses, whereas angiogenesis induced by non‐canonical NF‐κB stimuli was significantly reduced. In addition, angiogenesis in experimental arthritis and a murine tumour model was severely impaired in these mice. These studies provide evidence for a role of non‐canonical NF‐κB signalling in pathological angiogenesis, and identify NIK as a potential therapeutic target in chronic inflammatory diseases and tumour neoangiogenesis. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Exploring the validity of estimating EQ-5D and SF-6D utility values from the health assessment questionnaire in patients with inflammatory arthritis

Background  

Utility scores are used to estimate Quality Adjusted Life Years (QALYs), applied in determining the cost-effectiveness of health care interventions. In studies where no preference based measures are collected, indirect methods have been developed to estimate utilities from clinical instruments. The aim of this study was to evaluate a published method of estimating the EuroQol-5D (EQ-5D) and Short Form-6D (SF-6D) (preference based) utility scores from the Health Assessment Questionnaire (HAQ) in patients with inflammatory arthritis.     

Contribution of interleukin-1 to activation of coagulation and fibrinolysis, neutrophil degranulation, and the release of secretory- type phospholipase A2 in sepsis: studies in nonhuman primates after interleukin-1 alpha administration and during lethal bacteremia

Although studies with interleukin-1 receptor antagonist (IL-1ra) in animal models have shown that IL-1 contributes to mortality in sepsis, the mechanisms whereby IL-1 mediates lethal effects are not well established. A possible mechanism is that IL-1 enhances the activation and release of other inflammatory mediator systems such as coagulation, fibrinolysis, neutrophils, and secretory-type phospholipase A2 (sPLA2). We investigated this possibility by assessing the effect of intravenously injected recombinant human IL-1 alpha (rhIL-1 alpha) on these plasma parameters in baboons. In addition, we examined the course of these inflammatory parameters in baboons after a challenge with a lethal dose of Escherichia coli and while receiving a 24-hour constant infusion of IL-1ra or placebo. Intravenous administration of IL-1 alpha (10 micrograms/kg) induced the formation of thrombin, as evidenced by the appearance of thrombin-antithrombin III (TAT) complexes into the circulation (peak levels, 188 +/- 92 ng/mL at 2 hours), as well as the activation of fibrinolysis, assessed by circulating plasmin-alpha 2- antiplasmin complexes (PAP complexes; peak levels, 0.4% +/- 0.03% of fully activated plasma at 1 hour), the release of tissue-type plasminogen activator (t-PA; peak levels, 6 +/- 2 ng/mL at 2 hours), and its inhibitor, plasminogen activator inhibitor (PAI; peak levels, 724 +/- 246 ng/mL at 4 hours). Il-1 alpha administration also induced the release of sPLA2 (maximal levels, 336 +/- 185 ng/mL at 8 hours), but not degranulation of neutrophils. In the septic baboons, a significant reduction of the formation of thrombin (peak TAT levels decreased from 582 +/- 78 ng/mL to 219 +/- 106 ng/mL; P < .005), the release of t-PA (peak levels decreased from 37 +/- 11 ng/mL to 17 +/- 2 ng/mL; P < .001), and its inhibitor, PAI (peak levels decreased from 2,639 +/- 974 ng/mL to 1,110 +/- 153 ng/mL; P <.001), was observed in the group receiving IL-1ra compared to that receiving placebo. The release of neutrophilic elastase was also significantly attenuated in IL-1a-treated animals (peak levels, 1,024 +/- 393 and 655 +/- 104 ng/mL in control and treatment groups, respectively; P < .05). The difference between sPLA2 levels in both groups, although higher in the controls (maximal levels, 3,140 +/- 1,435 ng/mL in control v 2,217 +/- 1,375 ng/mL in IL-1ra-treated group), was not significant. Thus, IL-1 contributes to activation of various other mediator systems in severe sepsis in nonhuman primates. We propose that these effects may explain the lethal actions of IL-1 in this sepsis model and suggest a similar role for IL-1 in severe human sepsis.     

Rapid Rh D genotyping by polymerase chain reaction-based amplification of DNA

Rh (rhesus) D is the dominant antigen of the Rh blood group system. Recent advances in characterization of the nucleotide sequence of the cDNA(s) encoding the Rh D polypeptide allow the determination of the Rh D genotype at the DNA level. This can be of help in cases in which red blood cells are not available for phenotyping, eg, when in concerns a fetus. We have tested three independent DNA typing methods based on the polymerase chain reaction (PCR) for their suitability to determine the Rh D genotype. DNA derived from peripheral blood mononuclear cells from 234 Rh-phenotyped healthy donors (178 Rh D positive and 56 Rh D negative) was used in the PCR. The Rh D genotypes, as determined with a method based on the allele-specific amplification of the 3' noncoding region of the Rh D gene described by Bennett et al (N Engl J Med 329:607, 1993), were not concordant with the serologically established phenotypes in all cases. We have encountered 5 discrepant results, ie, 3 false-positive and 2 false-negative (a father and child). Rh D genotyping with the second method was performed by PCR amplification of exon 7 of the D gene with allele-specific primers. In all donors phenotyped as D positive tested so far (n = 178), the results of molecular genotyping with this method were concordant with the serologic results, whereas a false-positive result was obtained in one of the D-negative donors (also false-positive in the first method). Complete agreement was found between genotypes determined in the third method, based on a 600-bp deletion in intron 4 of the Rh D gene described by Arce et al (Blood 82:651, 1993), and serologically determined phenotypes. The Rh blood group system is complex, and unknown polymorphisms at the DNA level are expected to exist. Therefore, although genotypes determined by the method of Arce et al were in agreement with serotypes, it cannot yet be regarded as the golden standard. More experience with this or other methods is still needed.     

Enhanced proteolysis of plasma von Willebrand factor in thrombotic thrombocytopenic purpura and the hemolytic uremic syndrome

To examine whether enhanced in vivo proteolysis of von Willebrand factor (vWF) would account for the reported loss of larger multimers in acute thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS), we studied eight patients with acute TTP/HUS whose blood samples were collected into an anticoagulant containing a cocktail of protease inhibitors to impede in vitro proteolysis. In all, enhanced proteolytic degradation of vWF was expressed as a relative decrease in the intact 225-Kd subunit of vWF and a relative increase in the 176-Kd fragment. However, instead of the loss of larger forms of normal multimers reported by other investigators, the plasma of all but one of our patients (whether they had TTP or HUS) contained a set of larger than normal (supranormal) multimers. Hence, although proteolytic fragmentation of vWF was enhanced during acute TTP/HUS, this phenomenon was not associated with the loss of larger multimers. In the five patients who survived the acute disease and underwent plasma exchange (three with HUS and two with chronic relapsing TTP), subunits and fragments returned to normal values, and supranormal multimers were no longer detected in plasma. In conclusion, even though vWF proteolysis is enhanced in acute TTP/HUS, it does not lead to loss of larger multimers.     

Th activation of maternal and cord blood

Th activation of red cells is characterized by agglutination with the peanut lectin from Arachis hypogaea and is diminished by treatment with proteolytic enzymes. The first cases of Th activation were associated with bacterial infections. More recently, a high incidence of Th activation in congenital hypoplastic anemia has been reported, along with the finding that 13.5 percent of cord bloods are Th activated. The incidence of Th reactivity in newborn infants was confirmed by studying 200 paired samples of maternal and cord blood. Twenty-two (11%) of the cord samples and 13 (6.5%) of the maternal samples were Th activated. In 6 paired samples (6/22), both the mother and child had Th activation, a finding that demonstrates a high degree of concordance. Additionally, 3 (6%) of 50 pregnant women were Th positive. These findings indicate that Th activation is another of the red cell antigen alterations related to pregnancy.     

The new era of ventricular pacing during structural interventions

• Ventricular pacing is mandatory in many structural interventions, with associated risks like tamponade and capture loss.
• The Tempo pacing wire was designed to deal with these issues incorporating an elastomeric balloon on the tip and an active fixation system.
• In this initial multicenter experience, Tempo pacing wire demonstrated an outstanding performance in both safety and efficacy aspects of ventricular pacing during and after structural interventions.

     

Imbalance in redox status is associated with tumor aggressiveness and poor outcome in lung adenocarcinoma patients

Purpose

The expression levels of human antioxidant genes (HAGs) and oxidative markers were investigated in light of lung adenocarcinoma aggressiveness and patient outcome.

Methods

We assayed in vitro the tumoral invasiveness and multidrug resistance in human lung adenocarcinoma (AdC) cell lines (EKVX and A549). Data were associated with several redox parameters and differential expression levels of HAG network. The clinicopathological significance of these findings was investigated using microarray analysis of tumor tissue and by immunohistochemistry in archival collection of biopsies.

Results

An overall increased activity (expression) of selected HAG components in the most aggressive cell line (EKVX cells) was observed by bootstrap and gene set enrichment analysis (GSEA). In vitro validation of oxidative markers revealed that EKVX cells had high levels of oxidative stress markers. In AdC cohorts, GSEA of microarray datasets showed significantly high levels of HAG components in lung AdC samples in comparison with normal tissue, in advanced stage compared with early stage and in patients with poor outcome. Cox multivariate regression analysis in a cohort of early pathologic (p)-stage of AdC cases showed that patients with moderate levels of 4-hydroxynonenal, a specific and stable end product of lipid peroxidation, had a significantly less survival rate (hazard ratio of 8.87) (P < 0.05).

Conclusions

High levels of oxidative markers are related to tumor aggressiveness and can predict poor outcome of early-stage lung adenocarcinoma patients.