Human stem cell models to study placode development, function and pathology.

Placodes are embryonic structures originating from the rostral ectoderm that give rise to highly diverse organs and tissues, comprising the anterior pituitary gland, paired sense organs and cranial sensory ganglia. Their development, including the underlying gene regulatory networks and signalling pathways, have been for the most part characterised in animal models. In this Review, we describe how placode development can be recapitulated by the differentiation of human pluripotent stem cells towards placode progenitors and their derivatives, highlighting the value of this highly scalable platform as an optimal in vitro tool to study the development of human placodes, and identify human-specific mechanisms in their development, function and pathology.

Detection of Germline Variants in 450 Breast/Ovarian Cancer Families with a Multi-Gene Panel Including Coding and Regulatory Regions.

With the progress of sequencing technologies, an ever-increasing number of variants of unknown functional and clinical significance (VUS) have been identified in both coding and non-coding regions of the main Breast Cancer (BC) predisposition genes. The aim of this study is to identify a mutational profile of coding and intron-exon junction regions of 12 moderate penetrance genes (ATM, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53) in a cohort of 450 Italian patients with Hereditary Breast/Ovarian Cancer Syndrome, wild type for germline mutation in BRCA1/2 genes. The analysis was extended to 5'UTR and 3'UTR of all the genes listed above and to the BRCA1 and BRCA2 known regulatory regions in a subset of 120 patients. The screening was performed through NGS target resequencing on the Illumina platform MiSeq. 8.7% of the patients analyzed is carriers of class 5/4 coding variants in the ATM (3.6%), BRIP1 (1.6%), CHEK2 (1.8%), PALB2 (0.7%), RAD51C (0.4%), RAD51D (0.4%), and TP53 (0.2%) genes, while variants of uncertain pathological significance (VUSs)/class 3 were identified in 9.1% of the samples. In intron-exon junctions and in regulatory regions, variants were detected respectively in 5.1% and in 32.5% of the cases analyzed. The average age of disease onset of 44.4 in non-coding variant carriers is absolutely similar to the average age of disease onset in coding variant carriers for each proband's group with the same cancer type. Furthermore, there is not a statistically significant difference in the proportion of cases with a tumor onset under age of 40 between the two groups, but the presence of multiple non-coding variants in the same patient may affect the aggressiveness of the tumor and it is worth underlining that 25% of patients with an aggressive tumor are carriers of a PTEN 3'UTR-variant. This data provides initial information on how important it might be to extend mutational screening to the regulatory regions in clinical practice.

Coronary artery disease and restenosis after peripheral endovascular intervention are predictors of poor outcome in peripheral arterial disease.

Background: Few data are available on long-term atherothrombotic events after percutaneous transluminal angioplasty (PTA) for peripheral arterial disease (PAD). Restenosis after PTA may be a marker of a more aggressive atherothrombosis.Aim: To ascertain whether restenosis detected by duplex sonography (DUS) after PTA for iliac and femoro-popliteal disease is associated with a higher risk of cardiovascular events.Methods: We conducted a prospective cohort study of patients undergoing iliac or femoro-popliteal PTA for PAD. Patients were seen at one month, six months, one year and every year thereafter after PTA. At each visit, DUS was performed and accordingly restenosis was stratified into two categories (absent/present). The outcome was the composite of major adverse cardiovascular events (MACE).Results: Two hundred and fifty patients (aged 69 ± 11 years, male 59.2%) were enrolled. During a mean follow-up of 1207 ± 904 days, 102 (40.8%) patients developed restenosis. Restenosis was more frequent in patients with diabetes and critical limb ischaemia. MACEs (n = 76) were more frequent in the patients that developed restenosis vs. those that did not (40.2 vs. 23.6%, p = .005). Predictors of MACEs were diabetes (HR 2.02, 95%CI: 1.19-3.41, p = .009), presence of coronary heart disease at enrolment (HR 2.84, 95%CI: 1.78-4.53, p = .001) and restenosis (HR 1.87, 95%CI: 1.16-3.00, p = .010).Conclusion: Restenosis at DUS, diabetes, and coronary heart disease in patients who underwent iliac or femoro-popliteal PTA for PAD are associated with increased risk of arterial thrombotic event. Intervention trials are required to show the benefit of different therapeutic approaches in such patients at high risk of clinical deterioration.

Anticoagulants (heparin, low molecular weight heparin and oral anticoagulants) for intermittent claudication.

BACKGROUND: Anticoagulant treatment for intermittent claudication might improve functional capacity and prevent acute cardiovascular complications caused by peripheral obstructive arterial disease. This is an update of the review first published in 2001. OBJECTIVES: To assess the effects of anticoagulant drugs (heparin, low molecular weight heparin (LMWH) and oral anticoagulants) in patients with intermittent claudication (Fontaine stage II) in terms of improving walking capacity (pain-free walking distance or absolute walking distance), mortality, cardiovascular events, ankle/brachial pressure index, progression to surgery, amputation-free survival and side effects of these drugs. SEARCH METHODS: For this update the Cochrane Peripheral Vascular Diseases Group Trials Search Co-ordinator searched the Specialised Register (last searched May 2013) and CENTRAL (2013, Issue 4). SELECTION CRITERIA: All randomised trials of anticoagulants used to treat patients with intermittent claudication. DATA COLLECTION AND ANALYSIS: Seven studies were included. Only three studies (two evaluating oral anticoagulants, one evaluating heparin) met the high quality methodological inclusion criteria and were included in the primary analysis. Four other studies were included in the sensitivity analysis. The authors extracted the data independently. MAIN RESULTS: No new studies were included for this update. Seven studies with a combined total of 802 participants were included in this review. No significant difference was observed between heparin treatment and control groups for pain-free walking distance or maximum walking distance at the end of treatment. There were no data to indicate that LMWHs benefit walking distance. Revascularisation or amputation-free survival rates were reported in one study only with a five year follow-up. No study reported a significant effect on overall mortality or cardiovascular events and the pooled odds ratios were not significant for these outcomes either. Major and minor bleeding events were significantly more frequent in the group treated with oral anticoagulants compared to control, with a non-significant increase in fatal bleeding events. No major bleeding events were reported in the study evaluating heparin, while a non-significant increase in minor bleeding events was reported. AUTHORS' CONCLUSIONS: The benefit of heparin, LMWHs and oral anticoagulants for treatment of intermittent claudication has not been established while an increased risk of major bleeding events has been observed, especially with oral anticoagulants. There is no clear evidence to support the use of anticoagulants for intermittent claudication at this stage.

G1691A factor V and G20210A FII mutations, acute ischemic stroke of unknown cause, and patent foramen ovale.

BACKGROUND: Genetic polymorphisms of haemostatic factors such as G1691A factor V (FV Leiden) and G20210A prothrombin (FII) may be involved in the onset of patent foramen ovale (PFO)-related cerebral ischaemia. We assessed the possible association between such inherited thrombophilic alterations and right-to-left shunt in patients with stroke. METHODS: We investigated the presence of G20210A FII and FV Leiden mutations in 340 Caucasian patients consecutively evaluated by our Angiology Unit for stroke of unknown cause. PFO was assessed in all patients with Transcranial Doppler with intravenous injection of agitated saline. Stroke patients were divided into two groups: patients with PFO (n=136), and patients without PFO (n=204). As control group, we studied 272 subjects with early venous insufficiency. RESULTS: The prevalence of FII G20210A mutation was significantly higher in patients with PFO vs. controls (OR: 2.90; 95% CI: 1.19-7.07) and in patients without PFO vs. controls (OR: 2.88; 95% CI: 1.25-6.60) but was similar in patients with and without PFO (OR: 1.11; 95% CI: 0.51-2.44). The frequency of FV Leiden mutation was similar in the three groups. Across the population the presence of the FII G20210A mutation (OR: 2.97;95% CI: 1.32-6.69), a history of DVT (OR: 1.04; 95% CI: 1.02-1.06), and oestrogen-containing contraceptive therapy (OR: 1.14; 95% CI: 1.09-1.18) were all associated with stroke of unknown cause after adjustment for other risk factors, This was not the case with PFO. CONCLUSIONS: Our data do not support the assumption that these inherited thrombophilic alterations are associated with PFO in patients with cryptogenic stroke. FII G20210A mutation may be associated with cryptogenic stroke irrespective of the presence of PFO.

Evolution of untreated calf deep-vein thrombosis in high risk symptomatic outpatients: the blind, prospective CALTHRO study.

The natural history of calf deep-vein thrombosis (DVT) is still uncertain and it is debated whether it warrants to be diagnosed and treated. We aimed to investigate the complication rate of untreated isolated calf DVT (ICDVT). Symptomatic outpatients were prospectively managed with serial compression ultrasonography (SCUS). Those without proximal DVT and with likely pre-test clinical probability (PCP) or altered D-dimer received immediate subsequent complete examination of calf deep veins (CCUS) by a different operator. The result of CCUS was kept blind both to the managing doctor and the patient and disclosed after three months. Primary outcome was the rate of venous thromboembolism at three months. We examined 431 subjects (196 males; median age 68.0 years) in whom five outcomes were recorded (1.2%; 95% confidence intervals [CI]: 0.4-2.7). If CCUS results had been available, outcomes would have been recorded in 3/424 patients (0.7%; 95% CI: 0.2-2.1) with two events in subjects negative at both serial and complete CUS. ICDVT was diagnosed in 65 subjects (15.3%; 95% CI: 12-19); of whom 59 remained uneventful (one was lost to follow-up). A significant higher rate of outcomes was recorded in subjects with than without ICDVT (5/64; 7.8%; 95% CI: 3-17 vs. 3/351; 0.8%; 95% CI: 0-2; p=0.003). However, after excluding two events picked at serial CUS in subjects with ICDVT, the difference became barely significant (3/64; 4.7%; 95% CI: 1-13; p=0.049). Thrombotic evolution of untreated ICDVT in high-risk subjects may be relevant. Larger studies are needed to address this issue.

Thrombophilic risk factors and peripheral arterial disease severity.

Few data are available on thrombophilic risk factors and progression of atherosclerotic peripheral arterial disease (PAD). Thrombophilic alterations can be an aggravating factor when arterial stenoses are present. In a cross-sectional study, we evaluated the presence of the thrombophilic factors fibrinogen, homocysteine, factor (F)VIII, lupus anticoagulant (LAC), FII G20210A, and FV R506Q mutations in 181 patients with PAD at Fontaine's stage II (claudication), in 110 patients with critical limb ischaemia (CLI), and in 210 controls. Fibrinogen was higher in patients with CLI vs. those with claudication and controls (427.9 +/- 10.5 vs. 373.1 +/- 5.2 vs. 348.9 +/- 7.0 p=0.001, respectively). Homocysteine and FVIII were higher in patients with PAD than in controls, but were similar in patients with CLI and claudication. The prevalence of LAC increased in patients with CLI vs. those with claudication and controls (21.4% vs. 7.8% vs. 5.2% p<0.001, respectively). The prevalence of FII 20210A allele was higher in patients with CLI vs. those with claudication and controls. Using a logistic model, FII G20210A mutation (odds ratio [OR] 19.8, confidence interval [CI] 4.5-87.1, p=0.001), LAC (OR 2.7, CI1.1-6.5, p=0.032), and fibrinogen (OR 1.01, CI 1.00-1.01, p=0.001) were associated with CLI, whereas homocysteine, FVIII, and FV R506Q mutation were not. CLI risk increased according to the number of thrombophilic alterations. In conclusion, altered levels of some important thrombophilic risk factors are independently associated with PAD severity. These data suggest that the presence of two or more thrombophilic risk factors raise the likelihood of PAD being more severe, justifying the need for larger longitudinal studies.

Analysis of agreement between Duplex ultrasound scanning and arteriography in patients with lower limb artery disease.

BACKGROUND: Angiography is the gold standard for therapeutic decision-making in lower limb artery disease. However, both the potentiality and safety of Duplex ultrasound scanning suggest that it may become the main diagnostic tool. The present study aimed to investigate the agreement between Duplex scanning and angiography in the diagnosis of stenosis in lower limb artery disease. METHODS: Forty-nine patients with lower limb artery disease (24 claudication, 12 critical ischemia, 13 skin lesions) underwent angiography and Duplex scanning. The lower limb arterial axis was divided into 15 segments and graded on the basis of the degree of stenosis (0-49%, 50-69%, 70-99% and occlusion). Agreement between angiography and Duplex scanning was assessed by Cohen's kappa statistics (kappa). The sensitivity and specificity of Duplex scanning in detecting significant stenosis at angiography (>/= 70%) were also calculated. RESULTS: Good diagnostic agreement (kappa = 0.70; 95% CI 0.588-0.825) was achieved in the whole arterial axis. Agreement was good for the aorto-iliac district (kappa = 0.63) with a sensitivity of 63% and a specificity of 96%, and for the femoro-popliteal district (kappa = 0.70) with a sensitivity of 74% and a specificity of 83%. In infrapopliteal arteries, kappa showed a poor agreement, but Duplex scanning detected 28 patent tibial arteries in limbs that were not opacified on arteriography. CONCLUSIONS: Duplex scanning shows good agreement with angiography in lower limb artery disease on the whole, but poor agreement in infrapopliteal districts, with a low sensitivity and high specificity in detecting significant stenoses or occlusions.

Endothelin-1 response to mental stress in early ischemic lesions of the extremities due to systemic sclerosis.

We studied circulating levels of endothelin-1, catecholamines and nitric oxide after a mental arithmetic test in 14 patients with early ischemic lesions of the extremities due to systemic sclerosis and slightly impaired peripheral vascular flow. The test induced an increase (P<0.01) in blood pressure, heart rate, endothelin-1 and catecholamine levels, whereas it did not change the low basal levels of nitric oxide. In healthy subjects (n=20) the test significantly (P<0.01) decreased endothelin-1 without affecting nitric oxide. The low basal levels of nitric oxide and the high plasma concentration of endothelin-1 after psychological stress cannot be explained by an impaired release from the limited ischemic lesions alone. This suggests a diffuse microvascular derangement that aggravates the course of peripheral microvascular ischemic lesions.

Opioid peptide response to spinal cord stimulation in chronic critical limb ischemia.

Twelve patients with chronic critical limb ischemia in whom a spinal cord stimulation (SCS) system had been implanted for at least one year had increased microvascular flow and achieved healing of trophic acral lesions. After switching off the system, the clinical improvement persisted for 10 days and the neurohormonal pattern showed high plasma values of beta-endorphin and Met-enkephalin, normal dynorphin B, endothelin-1 and catecholamines, and low nitric oxide. Met-enkephalin levels were further increased (P < 0.01) immediately after switching on the electrical stimulation again. The persistence of high plasma opioid levels after switching off the spinal cord stimulation explains the absence of subjective complaints and suggests an involvement of opioids in the regulation and improvement of the microcirculation.