P53 oncosuppressor influences selection of genomic imbalances in response to ionizing radiations in human osteosarcoma cell line SAOS-2

PURPOSE: To investigate the impact of TP53 (tumor protein 53, p53) on genomic stability of osteosarcoma (OS). MATERIALS AND METHODS: In first instance, we expressed in OS cell line SAOS-2 (lacking p53) a wild type (wt) p53 construct, whose protein undergoes nuclear import and activation in response to ionizing radiations (IR). Thereafter, we investigated genomic imbalances (amplifications and deletions at genes or DNA regions most frequently altered in human cancers) associated with radio-resistance relative to p53 expression by mean of an array-based comparative genomic hybridization (aCGH) strategy. Finally we investigated a putative marker of radio-induced oxidative stress, a 4,977 bp deletion at mitochondrial (mt) DNA usually referred to as 'common' deletion, by mean of a polimerase chain reaction (PCR) strategy. RESULTS: In radio-resistant subclones generated from wt p53-transfected SAOS-2 cells DNA deletions were remarkably reduced and the accumulation of 'common' deletion at mtDNA (that may let the persistence of oxidative damage by precluding detoxification from reactive oxygen species [ROS]) completely abrogated. CONCLUSIONS: The results of our study confirm that wt p53 has a role in protection of OS cell DNA integrity. Multiple mechanisms involved in p53 safeguard of genomic integrity and prevention of deletion outcome are discussed.     

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines

Background

EGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and in combination with EGF.

Methods

Cell growth was determined using a variation on the MTT assay. Cell-cycle analysis was conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression and scanning electron microscopy (SEM) evidenced the ultrastructural morphology. Gene expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40 K array A.

Results

Caco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this level of cell cycle distribution after treatment, suggesting a predominantly differentiated state. Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important reduction of the microvilli (which also lose their erect position in Caco-2), possibly invalidating microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the 2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment. In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes appeared to be less stimulated than for single drug cases.

Conclusion

This is the first study to have systematically investigated the effect of cetuximab or gefitinib, alone and in combination with EGF, on human colon cancer cell lines. The EGFR inhibitors have a weaker effect in the presence of EGF that binds EGFR. Cetuximab treatment showed an expression pattern that inversely correlates with EGF treatment. We found interesting cyto-morphological features closely relating to gene expression profile. Both drugs have an effect on differentiation towards cellular death.     

Long-term effects of carvedilol in idiopathic dilated cardiomyopathy with persistent left ventricular dysfunction despite chronic metoprolol

OBJECTIVES

The purpose of this study was to analyze whether long-term treatment with the nonselective beta-adrenergic blocking agent carvedilol may have beneficial effects in patients with dilated cardiomyopathy (DCM), who are poor responders in terms of left ventricular (LV) function and exercise tolerance to chronic treatment with the selective beta-blocker metoprolol.

BACKGROUND

Although metoprolol has been proven to be beneficial in the majority of patients with heart failure, a subset of the remaining patients shows long-term survival without satisfactory clinical improvement.

METHODS

Thirty consecutive DCM patients with persistent LV dysfunction (ejection fraction ≤40%) and reduced exercise tolerance (peak oxygen consumption <25 ml/kg/min) despite chronic (>1 year) tailored treatment with metoprolol and angiotensin-converting enzyme inhibitors were enrolled in a 12-month, open-label, parallel trial and were randomized either to continue on metoprolol (n = 16, mean dosage 142 ± 44 mg/day) or to cross over to maximum tolerated dosage of carvedilol (n = 14, mean dosage 74 ± 23 mg/day).

RESULTS

At 12 months, patients on carvedilol, compared with those continuing on metoprolol, showed a decrease in LV dimensions (end-diastolic volume −8 ± 7 vs. +7 ± 6 ml/m², p = 0.053; end-systolic volume −7 ± 5 vs. +6 ± 4 ml/m², p = 0.047), an improvement in LV ejection fraction (+7 ± 3% vs. −1 ± 2%, p = 0.045), a reduction in ventricular ectopic beats (−12 ± 9 vs. +62 ± 50 n/h, p = 0.05) and couplets (−0.5 ± 0.4 vs. +1.5 ± 0.6 n/h, p = 0.048), no significant benefit on symptoms and quality of life and a negative effect on peak oxygen consumption (−0.6 ± 0.6 vs. +1.3 ± 0.5 ml/kg/min, p = 0.03).

CONCLUSIONS

In DCM patients who were poor responders to chronic metoprolol, carvedilol treatment was associated with favorable effects on LV systolic function and remodeling as well as on ventricular arrhythmias, whereas it had a negative effect on peak oxygen consumption.     

Return to Work and Quality of Life: A Psychosocial Survey After Kidney Transplant

Background

The main goals of kidney transplantation are to recreate a condition of psychophysical well-being and to improve the quality of life of the patient, including going back to work after transplant. Returning to work after a kidney transplant is an important health care indicator. The aim of the study was to assess the psychophysical well-being and work condition in kidney transplant recipients and to identify possible predictors of return to work.

Comparison of different methods of functional evaluation in patients with chronic heart failure

BACKGROUND: Stratification of the severity of heart failure has major prognostic and therapeutic implications. AIMS: To prospectively compare different methods of assessment of functional capacity in patients with chronic heart failure (CHF). METHODS AND RESULTS: We studied 143 patients (78 male and 65 female) with CHF aged less than 70 years (mean 57.3 years). Functional assessment was made clinically according to NYHA classification and according to the Goldman Activity Scale Classification (GASC). Cardiovascular performance was measured by peak O(2) consumption (pVO(2)) and anaerobic threshold (AT) at cardiopulmonary exercise test and by the distance walked during a 6-min walk test (6-MWT). Clinical scales resulted significantly related. Peak VO(2) and AT showed a mild relation with distance covered at 6-MWT (r=0.56 and r=0.46, respectively). Concordance between NYHA classification and levels of performance at cardiopulmonary exercise test or at 6-MWT was less than 50%. CONCLUSION: Our results suggest that none of the usually employed methods give a definitive assessment of functional capacity of cardiovascular system and a high degree of discordance exists among the results of different tests in the same patient. Although NYHA classification maintains its value in clinical evaluation of patients with CHF, the 6-min walk test is recommended in patients with mild-to-moderate CHF (II-III NYHA classes) as a simple and useful screening test to select patients for further diagnostic evaluation.     

Meningitis Caused by Toscana Virus Is Associated with Strong Antiviral Response in the CNS and Altered Frequency of Blood Antigen-Presenting Cells

Toscana virus (TOSV) is a Phlebotomus-transmitted RNA virus and a frequent cause of human meningitis and meningoencephalitis in Southern Europe during the summer season. While evidence for TOSV-related central nervous system (CNS) cases is increasing, little is known about the host defenses against TOSV. We evaluated innate immune response to TOSV by analyzing frequency and activation of blood antigen-presenting cells (APCs) and cytokine levels in plasma and cerebrospinal fluid (CSF) from patients with TOSV neuroinvasive infection and controls. An altered frequency of different blood APC subsets was observed in TOSV-infected patients, with signs of monocytic deactivation. Nevertheless, a proper or even increased responsiveness of toll-like receptor 3 and 7/8 was observed in blood APCs of these patients as compared to healthy controls. Systemic levels of cytokines remained low in TOSV-infected patients, while levels of anti-inflammatory and antiviral mediators were significantly higher in CSF from TOSV-infected patients as compared to patients with other infectious and noninfectious neurological diseases. Thus, the early host response to TOSV appears effective for viral clearance, by proper response to TLR3 and TLR7/8 agonists in peripheral blood and by a strong and selective antiviral and anti-inflammatory response in the CNS.     

Renal transplantation parenchymal complications: what Doppler ultrasound can and cannot do

Kidney transplantation is the treatment of choice in end-stage renal disease, given the better quality of life of transplanted patients when compared with patients on maintenance dialysis. In spite of surgical improvements and new immunosuppressive regimens, parts of transplanted grafts still develop chronic dysfunction. Ultrasonography, both in B-mode and with Doppler ultrasound, is an important diagnostic tool in case of clinical conditions which might impair kidney function. Even though ultrasonography is considered fundamental in the diagnosis of vascular and surgical complications of the transplanted kidney, its role is not fully understood in case of parenchymal complications of the graft. The specificity of Doppler is low both in case of acute complications, such as acute tubular necrosis, drugs toxicity and acute rejection, and in case of chronic conditions, such as chronic allograft nephropathy. Single determinations of resistance indices present low diagnostic accuracy, which is higher in case of successive measurements performed during the follow-up of the graft. Modern techniques such as tissue pulsatility index, maximal fractional area and contrast-enhanced ultrasound increase ultrasonography diagnostic power in case of parenchymal complications of the transplanted kidney.

Electronic supplementary material

The online version of this article (doi:10.1007/s40477-014-0118-1) contains supplementary material, which is available to authorized users.     

A meta-analysis on age-associated changes in blood DNA methylation: results from an original analysis pipeline for Infinium 450k data

Aging is characterized by a profound remodeling of the epigenetic architecture in terms of DNA methylation patterns. To date the most effective tool to study genome wide DNA methylation changes is Infinium HumanMethylation450 BeadChip (Infinium 450k). Despite the wealth of tools for Infinium 450k analysis, the identification of the most biologically relevant DNA methylation changes is still challenging. Here we propose an analytical pipeline to select differentially methylated regions (DMRs), tailored on microarray architecture, which is highly effective in highlighting biologically relevant results. The pipeline groups microarray probes on the basis of their localization respect to CpG islands and genic sequences and, depending on probes density, identifies DMRs through a single-probe or a region-centric approach that considers the concomitant variation of multiple adjacent CpG probes. We successfully applied this analytical pipeline on 3 independent Infinium 450k datasets that investigated age-associated changes in blood DNA methylation. We provide a consensus list of genes that systematically vary in DNA methylation levels from 0 to 100 years and that have a potentially relevant role in the aging process.     

Remodelling of biological parameters during human ageing: evidence for complex regulation in longevity and in type 2 diabetes

Factor structure analyses have revealed the presence of specific biological system markers in healthy humans and diseases. However, this type of approach in very old persons and in type 2 diabetes (T2DM) is lacking. A total sample of 2,137 Italians consisted of two groups: 1,604 healthy and 533 with T2DM. Age (years) was categorized as adults (≤65), old (66–85), oldest old (>85–98) and centenarians (≥99). Specific biomarkers of routine haematological and biochemical testing were tested across each age group. Exploratory factorial analysis (EFA) by principal component method with Varimax rotation was used to identify factors including related variables. Structural equation modelling (SEM) was applied to confirm factor solutions for each age group. EFA and SEM identified specific factor structures according to age in both groups. An age-associated reduction of factor structure was observed from adults to oldest old in the healthy group (explained variance 60.4% vs 50.3%) and from adults to old in the T2DM group (explained variance 57.4% vs 44.2%). Centenarians showed three-factor structure similar to those of adults (explained variance 58.4%). The inflammatory component became the major factor in old group and was the first one in T2DM. SEM analysis in healthy subjects suggested that the glucose levels had an important role in the oldest old. Factorial structure change during healthy ageing was associated with a decrease in complexity but showed an increase in variability and inflammation. Structural relationship changes observed in healthy subjects appeared earlier in diabetic patients and later in centenarians.     

Multi-detector row CT scanner angiography in the evaluation of living kidney donors

Renal vascular anomalies are frequent and are not usually problematic, especially when they have been identified and localised with preoperative imaging; computed tomography angiography is a fast and minimally invasive procedure that may afford accurate visualisation of arterial and venous anatomy. We report on our experience with the utilisation of multi-detector row angiography in the preoperative evaluation of living kidney donors. Nineteen living kidney donors underwent multidetector row scan angiography with 3D post-processing. The subjects were 12 male and 7 female donors with a mean age of 60 years. Renal vascular anomalies were identified in 52.6% of donors. A total of 10 supernumerary arteries were identified. Surgical correlation was available for 19 kidneys (17 left and 2 right). The donated kidneys were selected on the basis of CT scan and renal function. CT scan identified all 29 arteries including 10 double right or left arteries (100% specificity and sensitivity). Dual multi-phase multi-detector row CT angiography is a minimally invasive and highly accurate method for preoperative evaluation of renal donors. It affords comprehensive depiction of the arterial and venous anatomy of the kidney, which is particularly critical for planning and performing the donor nephrectomy, especially via a laparoscopic approach.