Molecular heterogeneity and prognostic implications of synchronous advanced colorectal neoplasia.

BACKGROUND: It is uncertain whether synchronous colorectal cancers (S-CRCs) preferentially develop through widespread DNA methylation and whether they have a prognosis worse than solitary CRC. As tumours with microsatellite instability (MSI) may confound the effect of S-CRC methylation on outcome, we addressed this issue in a series of CRC characterised by BRAF and MS status. METHODS: Demographics, clinicopathological records and disease-specific survival (DSS) were assessed in 881 consecutively resected CRC undergoing complete colonoscopy. All tumours were typed for BRAF(c.1799T>A) mutation and MS status, followed by search of germ-line mutation in patients with MSI CRC. RESULTS: Synchronous colorectal cancers (50/881, 5.7%) were associated with stage IV microsatellite-stable (MSS) CRC (19/205, 9.3%, P=0.001) and with HNPCC (9/32, 28%, P<0.001). BRAF mutation (60/881, 6.8%) was associated with sporadic MSI CRC (37/62, 60%, P<0.001) but not with S-CRC (3/50, 6.0%, P=0.96). Synchronous colorectal cancer (HR 1.82; 95% CI 1.15-2.87; P=0.01), synchronous advanced adenoma (HR 1.81; 95% CI 1.27-2.58; P=0.001), and BRAF(c.1799T>A) mutation (HR 2.16; 95% CI 1.25-3.73; P=0.01) were stage-independent predictors of death from MSS CRC. Disease-specific survival of MSI CRC patients was not affected by S-CRC (HR 0.74; 95% CI 0.09-5.75; P=0.77). CONCLUSION: Microsatellite-stable CRCs have a worse prognosis if S-CRC or synchronous advanced adenoma are diagnosed. The occurrence and the enhanced aggressiveness of synchronous MSS advanced neoplasia are not associated with BRAF mutation.

Van-Gogh-like 2 antagonises the canonical WNT pathway and is methylated in colorectal cancers.

BACKGROUND: Aberrant activation of the canonical WNT signaling is a feature of colorectal cancer (CRC). Van-Gogh-like 2 (VANGL2) belongs to the non-canonical WNT pathway whose activation inhibits canonical WNT signaling. In this study, we investigated the role of VANGL2 and its epigenetic regulation in CRC. METHODS: Van-Gogh-like 2 expression and promoter methylation after 5-aza-2'-deoxycytidine (5-aza) treatment were evaluated in CRC cells. DNA samples from 418 sporadic CRCs were tested for VANGL2 promoter methylation and microsatellite instability (MSI). Proliferation, colony formation and activation of the WNT pathway were tested in cells after VANGL2 overexpression. RESULTS: Van-Gogh-like 2 mRNA was significantly higher in 5-aza-treated RKO, LOVO and SW48, whereas no differences were found in SW480. Van-Gogh-like 2 was fully methylated in RKO, SW48, HCT116, DLD1 and Caco2; partially methylated in LOVO, LS174T and SW837; and unmethylated in SW480, SW620 and HT29. Higher expression of VANGL2 mRNA was found in the unmethylated cell lines. In CRC specimens (8.93% MSI), methylated VANGL2 was associated with MSI, higher grade, proximal colon location and BRAF mutation. Van-Gogh-like 2 overexpression in SW480 significantly decreased proliferation, colony formation and ß-catenin levels. CONCLUSION: Van-Gogh-like 2 is frequently methylated in MSI-CRCs with BRAF mutation and may act as a tumour suppressor gene, counteracting WNT/ß-catenin signaling.

JC virus infects the enteric glia of patients with chronic idiopathic intestinal pseudo-obstruction.

BACKGROUND AND AIMS: Chronic idiopathic intestinal pseudo-obstruction (CIIP) is characterised by severe impairment of intestinal propulsive motility that mimics bowel obstruction. JC virus (JCV) is a polyomavirus that can infect brain glial cells causing a fatal disease, but may also be found throughout the normal gastrointestinal tract. The hypothesis that JCV infects the myenteric plexuses of patients with CIIP was tested. METHODS: 10 patients with CIIP and 61 normal specimens (30 ascending colon and 31 ileum) from patients with uncomplicated colon cancer were studied. DNA was extracted from the myenteric plexuses, and JCV T antigen (TAg) DNA and the viral regulatory region were detected by PCR and sequencing. Immunohistochemistry was performed to detect JCV viral protein expression, neuronal and glial markers. Fluorescence in situ hybridisation was performed for cellular localisation of the JCV infection. RESULTS: Clinical studies demonstrated neurogenic impairment, and pathological analyses showed neuropathy in each patient with CIIP. JCV TAg DNA was found in the myenteric plexuses of 8/10 (80%) of the patients with CIIP and 3/31 (9.7%) of the control patients (p<0.001). All samples were JCV Mad-1 strains. Seven of the 10 CIIP specimens expressed both JCV TAg and the JCV viral protein VP1, while none of the controls expressed either. JCV infection co-localised with glial fibrillary acidic protein expression, a marker of enteric glial cells. CONCLUSION: JCV infection occurs in the myenteric plexuses of patients with CIIP. The JCV localisation in enteroglial cells suggests a possible pathological role for this virus in enteric neuropathy.

13C-methionine breath tests for mitochondrial liver function assessment.

13C-methionine breath test has been proposed as a non-invasive tool for the assessment of human hepatic mithocondrial function. Two methionine breath labeled with 13C in differents point of his molecular structure have been used for breath test analisys. Aim of this study was to compare two differently 13C-labeled methionines in the evaluation of mitochondrial oxidation in basal conditions and after an acute oxidative stress. 15 healthy male subjects (mean age 30.5 +/- 3.1) received [methyl-13C]-methionine dissolved in water. Breath samples were taken at baseline and and 10, 20, 30, 45, 60, 75, 90, 105 and 120 minutes after the ingestion of the labeled substrate. Forthy-eight hours later, subjects underwent the same test 30 minutes after ethanol ingestion (0,3 g/kg of body weight). Seven-day later, subjects underwent breath test using (L-methionine-1-13COOH) as substrate, in basal condition and after ethanol ingestion. At basal condition, the cumulative percentage of 13CO2 recovered in breath during the test period (%cum-dose) was higher using L-methionine-1-13COOH than [methyl-13C]-methionine (10.25 +/- 1.0 vs 4.07 +/- 0.8; p < 0.01). After ethanol ingestion, % cum dose was significantly decreased at 60 and 120 minutes with both methionines (120 min: 10.25 +/- 1.0 vs 5.03% +/- 1.8; < 0.01 and 4.07 +/- 0.8 vs 2.16% +/- 0.9; p < 0.01, respectively). However, %cum-dose during L-methionine-1-13C-breath test was significantly lower than that observed during methyl-13C-methionine breath test (120 minutes: 5.03% +/- 1.8 vs 2.16% +/- 0.9; p < 0.01). In conclusion, breath test based on L-methionine-1-13COOH seems to show a greater reliability when compared to [methyl-13C]-methionine to assess mitochondrial function because a larger amount of labeled carbon that reaches the Krebs' cicle.

Clinical features, diagnostic and therapeutic approaches to haematogenous vertebral osteomyelitis.

This article review the clinical features and the diagnostic approach to haematogenous vertebral osteomyelitis in order to optimise treatment strategies and follow-up assessment. Haematogenous spread is considered to be the most important route: the lumbar spine is the most common site of involvement for pyogenic infection and the thoracic spine for tuberculosis infection. The risk factors for developing haematogenous vertebral osteomyelitis are different among old people, adults and children: the literature reports that the incidence seems to be increasing in older patients. The source of infection in the elderly has been related to the use of intravenous access devices and the asymptomatic urinary infections. In young patients the increase has been correlated with the growing number of intravenous drug abusers, with endocarditis and with immigrants from areas where tuberculosis is still endemic. The onset of symptoms is typically insidious with neck or back pain often underestimated by the patient. Fever is present in 10-45% of patients. Spinal infections may cause severe neurological compromise in few cases, but mild neurological deficit, limited to one or two nerve roots, was detected in 28-35% of patients. The diagnosis of haematogenous vertebral osteomyelitis may be very difficult, as the symptoms can be sometimes not specific, vague or almost absent. The usual delay in diagnosis has been reported to be two to four months, despite the use of imaging techniques: in the early diagnosis of vertebral ostemyelitis is important the role of bone scintigraphy. The general principles for the management of spine infections are non operative, consisting of external immobilization and intravenous antibiotics, followed by oral antibiotics. Indications for surgery should be given in case of absence of clinical improvement after 2-3 weeks of intravenous antibiotics, persistent back pain and systemic effects of chronic infection and with presence or progression of neurological deficit in elderly or in cervical infection. Chronic ostemyelitis may require surgery in case of a development of biomechanical instability and/or a vertebral collapse with progressive deformity.

Helicobacter pylori infection and ischaemic heart disease: an overview of the general literature.

In the last years, a considerable number of studies have been performed on the correlation between Helicobacter pylori infection and ischaemic heart disease. The reason is the supposed role of some chronic infections in the genesis and development of vessel wall injury and atheromatous plaque, as already reported for Chlamydia pneumoniae and herpes viruses. While this association may be theoretically conceivable, it still remains debated from a practical point of view. Epidemiological and animal studies as well as some eradicating trials gave conflicting results, while studies investigating the specific molecular mimicry mechanisms induced by H. pylori strongly support the association. Moreover, none of the studies performed so far did take into account the effect of the genetic susceptibility to develop ischaemic heart disease or to respond to H. pylori infection. In particular, while the exposure to some known risk factor for atherosclerosis should lead to develop ischaemic heart disease, no condition or exposure, either individual or in combination, completely explains the occurrence and the progression of the disease, as many patients develop ischaemic heart disease in the absence of any risk factor. Based on these concepts, can we state that H. pylori infection may cause the same effect in patients with ischaemic heart disease as in healthy subjects? Further studies are needed in order to clarify this issue.

13C-breath tests in the study of microsomal liver function.

Conventional liver tests can be used to estimate a mixture of injury and function but none of these may be regarded as a reliable marker either to quantify functional hepatic reserve or to reflect life-threatening complications of acute and chronic liver diseases. To overcome this limit, many dynamic tests have been developed in order to evaluate the "hepatic functional mass". Among these tests we can include breath tests with 13C-labeled substrates undergoing different metabolic pathways. As concerning the evaluation of microsomal function, two main categories of breath tests have been developed based on the limiting step in the different substrates metabolism. The first group include aminopyrine, caffeine and diazepam, all substrates with a metabolism independent from hepatic blood flow and dependent almost exclusively from the enzymatic activity of different cytochromes P450. The other group is composed of substrates with flow dependent metabolism like methacetin, phenacetin, erythromycin. The aim of this review is to describe the clinical applications of microsomal liver breath tests in different hepatic diseases.

13C-breath tests in the study of mitochondrial liver function.

Breath tests for "dynamic" liver function evaluation have been proposed several years ago. A variety of carbon-labelled breath tests for the assessment of mitochondrial, microsomal and cytosolic liver function have been described with the aim to increase data on liver disease staging, prognosis, and response to therapy. In the last years a great interest is developed about the use of breath test for liver mitochondrial function evaluation since it results impaired in a wide range of liver diseases either of genetic or acquired origin. In these cases mitochondrial oxidative metabolism of some substrates, as far as recovery of the hepatic energy state after a metabolic insult, results impaired because of the disfunction of the electron transport chain and/or ATP synthesis. Ketoisocaproic acid and methionine are the best studied carbon-labelled substrates for the investigation of mitochondrial functional damages related to structural alterations that occur in many liver diseases. Although these tests are simple, cost-effective and safe, to date there is still not general approval for their usefulness in clinical settings since they should fulfill several requirements to overcome the drawbacks of traditional quantitative tests. On the other hand, this field is relatively young and further studies are needed in order to assess the suitable substrate for the evaluation of the complex mitochondrial metabolism both in healthy subjects and in patients with liver disease.

Accuracy of remote sensing of water temperature by Raman spectroscopy.

We present an experimental study of the Raman spectrum of pure and synthetic seawater with respect to its salinity and temperature dependence. Experiments made in the laboratory with both cw and pulsed excitation yield information on the limits and applicability of the technique in actual experiments in the field. We have also performed an experimental analysis to determine the presence of stimulated Raman scattering and its influence on the temperature dependence of the spectrum.

Stabilization of a femtosecond dye laser synchronously pumped by a frequency-doubled mode-locked YAG laser.

Stable synchronous pumping of a Rh6G-DODCI femtosecond dye laser by a frequency-doubled continuously modelocked Nd:YAG laser is achieved. A group-velocity-compensated linear cavity terminates in an antiresonant ring containing a saturable absorber. Critical variations of pulse characteristics with cavity-length detuning are avoided by an active stabilization of the cavity length. As the error signal, we use the variation of the dye-laser's central wavelength with the cavity mismatch.