High-density transcranial direct current stimulation to improve upper limb motor function following stroke: study protocol for a double-blind randomized clinical trial targeting prefrontal and/or cerebellar cognitive contributions to voluntary motion.

BACKGROUND: Focal brain lesions following a stroke of the middle cerebral artery induce large-scale network disarray with a potential to impact multiple cognitive and behavioral domains. Over the last 20 years, non-invasive brain neuromodulation via electrical (tCS) stimulation has shown promise to modulate motor deficits and contribute to recovery. However, weak, inconsistent, or at times heterogeneous outcomes using these techniques have also highlighted the need for novel strategies and the assessment of their efficacy in ad hoc controlled clinical trials. METHODS: We here present a double-blind, sham-controlled, single-center, randomized pilot clinical trial involving participants having suffered a unilateral middle cerebral artery (MCA) stroke resulting in motor paralysis of the contralateral upper limb. Patients will undergo a 10-day regime (5 days a week for 2 consecutive weeks) of a newly designed high-definition transcranial direct current stimulation (HD-tDCS) protocol. Clinical evaluations (e.g., Fugl Meyer, NIHSS), computer-based cognitive assessments (visuo-motor adaptation and AX-CPT attention tasks), and electroencephalography (resting-state and task-evoked EEG) will be carried out at 3 time points: (I) Baseline, (II) Post-tDCS, and (III) Follow-up. The study consists of a four-arm trial comparing the impact on motor recovery of three active anodal tDCS conditions: ipsilesional DLPFC tDCS, contralesional cerebellar tDCS or combined DLPFC + contralesional cerebellar tDCS, and a sham tDCS intervention. The Fugl-Meyer Assessment for the upper extremity (FMA-UE) is selected as the primary outcome measure to quantify motor recovery. In every stimulation session, participants will receive 20 min of high-density tDCS stimulation (HD-tDCS) (up to 0.63 mA/[Formula: see text]) with [Formula: see text] electrodes. Electrode scalp positioning relative to the cortical surface (anodes and cathodes) and intensities are based on a biophysical optimization model of current distribution ensuring a 0.25 V/m impact at each of the chosen targets. DISCUSSION: Our trial will gauge the therapeutic potential of accumulative sessions of HD-tDCS to improve upper limb motor and cognitive dysfunctions presented by middle cerebral artery stroke patients. In parallel, we aim at characterizing changes in electroencephalographic (EEG) activity as biomarkers of clinical effects and at identifying potential interactions between tDCS impact and motor performance outcomes. Our work will enrich our mechanistic understanding on prefrontal and cerebellar contributions to motor function and its rehabilitation following brain damage. TRIAL REGISTRATION: ClinicalTrials.gov NCT05329818. April 15, 2022.

Nonalcoholic Steatohepatitis Modifies Serum Iron-Related Variables in Patients with Morbid Obesity.

Nonalcoholic steatohepatitis (NASH) is frequently associated with severe obesity. The liver is the principal storage repository for iron, and the excessive accumulation of this metal may promote hepatic inflammation. Laparoscopic sleeve gastrectomy (LSG) results in weight loss and improvement in comorbidities such as NASH. The aim of this study was to assess the specific NASH-related changes in iron metabolism and to investigate whether these changes are reversed by LSG. We included 150 patients with morbid obesity who provided 12-h fasting blood samples immediately before LSG together with an intraoperative wedge-liver biopsy. Thirty-eight patients with NASH underwent a second blood extraction 12 months postsurgery. Serum samples were collected from a control group comprising 50 healthy volunteers. We found significantly higher serum iron and transferrin concentrations in patients with NASH along with the highest degrees of steatosis, fibrosis, hepatocellular ballooning, and lobular inflammation. However, we did not find any significant accumulation of iron in the hepatic biopsies. Presurgery serum iron concentrations were lower in the patient group than in the control group and increased 1 year postsurgery. Serum ferritin levels showed changes in the opposite direction. We did not observe any significant change in serum transferrin concentrations. These changes were reversed by LSG. We conclude that alterations in serum iron-related variables are related to the severity of NASH in patients with morbid obesity, and these alterations are reversed by LSG. We also found that severe forms of NASH can be found in the absence of increased iron stores.

Long-term protection of HPV test in women at risk of cervical cancer.

OBJECTIVE: To evaluate the 9-year incidence of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) and cumulative adherence to perform a next test in a cohort of women aged 40+ years with no cervical screening cytology within a window of 5 years (underscreened women), after baseline cervical cytology and HPV tests. METHODS: In Catalonia, Spain, co-testing with cytology and HPV test has been recommended in the Public Health system since 2006 for underscreened women. In 2007, 1,594 women with underscreened criteria were identified and followed through medical records form Pathological Department. 9-year cumulative incidence of histologically confirmed CIN2+ and cumulative adherence to perform a next test were estimated using Kaplan-Meier statistics. RESULTS: Follow-up was available for 1,009 women (63.3%) resulting in 23 women with. CIN2+ (2.3%). Of them, 4 women (17%) had both tests negative at baseline (3CIN2 and 1CIN3) with cumulative incidence of CIN2+ of 0.4% (95% CI: 0.1-1.4) at 5-years and 1.3% (95% CI: 0.4-3.7) at 9-years. During the first year, the prevalence among women with both tests positive was 27.0% (95% CI: 13.0-50.6) for CIN2+. Lost to follow-up was higher among women with both tests negative compared to those with both positive tests (38.7% vs 4.2%, p-value <0.001). 40.5% of the women HPV-/cyto- had a re-screening test during the 4 years following the baseline, increasing until 53.5% during the 6 years of follow-up. CONCLUSIONS: HPV detection shows a high longitudinal predictive value at 9-year to identify women at risk to develop CIN2+. The data validate a safe extension of the 3-year screening intervals (current screening interval) to 5-year intervals in underscreened women that had negative HPV result at baseline. It is necessary to establish mechanisms to ensure screening participation and adequate follow-up for these women.

Chemokine (C-C motif) ligand 2 and coronary artery disease: Tissue expression of functional and atypical receptors.

Chemokines, particularly chemokine (C-C- motif) ligand 2 (CCL2), control leukocyte migration into the wall of the artery and regulate the traffic of inflammatory cells. CCL2 is bound to functional receptors (CCR2), but also to atypical chemokine receptors (ACKRs), which do not induce cell migration but can modify chemokine gradients. Whether atherosclerosis alters CCL2 function by influencing the expression of these receptors remains unknown. In a necropsy study, we used immunohistochemistry to explore where and to what extent CCL2 and related receptors are present in diseased arteries that caused the death of men with coronary artery disease compared with unaffected arteries. CCL2 was marginally detected in normal arteries but was more frequently found in the intima. The expression of CCL2 and related receptors was significantly increased in diseased arteries with relative differences among the artery layers. The highest relative increases were those of CCL2 and ACKR1. CCL2 expression was associated with a significant predictive value of atherosclerosis. Findings suggest the need for further insight into receptor specificity or activity and the interplay among chemokines. CCL2-associated conventional and atypical receptors are overexpressed in atherosclerotic arteries, and these may suggest new potential therapeutic targets to locally modify the overall anti-inflammatory response.

Laparoscopic sleeve gastrectomy reverses non-alcoholic fatty liver disease modulating oxidative stress and inflammation.

BACKGROUND & AIMS: Hepatic alterations, such as in non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are frequently associated with obesity. To investigate the molecular mechanisms of these alterations and to identify molecules that could be used as potential therapeutic targets, we investigated the modulation of hepatic indices of oxidative stress and inflammation in obese patients undergoing laparoscopic sleeve gastrectomy (LSG). METHODS: Patients (n = 436) attending our obesity clinic underwent LSG for weight loss. We obtained a diagnostic intraoperative liver biopsy, and a sub-cohort (n = 120) agreed to a 1-year follow-up that included donation of blood samples and additional liver biopsies. Selected key molecules in blood and liver tissue were used to investigate the hepatic alterations in obesity, and their response to LSG. RESULTS: One year post-surgery, the prevalence of diabetes, dyslipidemia and hypertension decreased significantly. LSG improved liver histology features in all patients. Improvement was greater in severe cases of NAFLD including those with steatohepatitis, bridging fibrosis or cirrhosis. Significant pre-surgery differences in plasma, and liver markers of oxidative stress and inflammation (including chemokine C-C motif ligand 2, paraoxonase-1, galectin-3, and sonic hedgehog) were observed between patients with, and those without, NASH; post-surgery indicated consistent improvements in these parameters. CONCLUSION: Our study shows that the histology and liver function of patients with morbid obesity are significantly improved after LSG via mechanisms that involve the reduction of oxidative stress and inflammatory processes. These data encourage the use of LSG as a therapeutic option to improve, or resolve, NAFLD.

Plasma Energy-Balance Metabolites Discriminate Asymptomatic Patients with Peripheral Artery Disease.

Peripheral artery disease (PAD) is a common disease affecting 20-25% of population over 60 years old. Early diagnosis is difficult because symptoms only become evident in advanced stages of the disease. Inflammation, impaired metabolism, and mitochondrial dysfunction predispose to PAD, which is normally associated with other highly prevalent and related conditions, such as diabetes, dyslipidemia, and hypertension. We have measured energy-balance-associated metabolite concentrations in the plasma of PAD patients segregated by the severity of the disease and in plasma of healthy volunteers using a quantitative and targeted metabolomic approach. We found relevant associations between several metabolites (3-hydroxybutirate, aconitate, (iso)citrate, glutamate, and serine) with markers of oxidative stress and inflammation. Metabolomic profiling also revealed that (iso)citrate and glutamate are metabolites with high ability to discriminate between healthy participants and PAD patients without symptoms. Collectively, our data suggest that metabolomics provide significant information on the pathogenesis of PAD and useful biomarkers for the diagnosis and assessment of progression.

Assessment of extracellular matrix-related biomarkers in patients with lower extremity artery disease.

BACKGROUND: The prevalence of lower extremity artery disease (LEAD) is high (20%-25%) in the population older than 65 years, but patients are seldom identified until the disease is advanced. Circulating markers of disease activity might provide patients with a key opportunity for timely treatment. We tested the hypothesis that measuring blood-specific fragments generated during degradation of the extracellular matrix (ECM) could provide further insight into the pathophysiologic mechanism of arterial remodeling. METHODS: The protein profile of diseased arteries from patients undergoing infrainguinal limb revascularization was assessed by a liquid chromatography and tandem mass spectrometry, nontargeted proteomic approach. The information retrieved was the basis for measurement of neoepitope fragments of ECM proteins in the blood of 195 consecutive patients with LEAD by specific enzyme-linked immunosorbent assays. RESULTS: Histologic and proteomic analyses confirmed the structural disorganization of affected arteries. Fourteen of 81 proteins were identified as differentially expressed in diseased arteries with respect to healthy tissues. Most of them were related to ECM components, and the difference in expression was used in multivariate analyses to establish that severe arterial lesions in LEAD patients have a specific proteome. Analysis of neoepitope fragments in blood revealed that fragments of versican and collagen type IV, alone or in combination, segregated patients with mild to moderate symptoms (intermittent claudication, Fontaine I-II) from those with severe LEAD (critical limb ischemia, Fontaine III-IV). CONCLUSIONS: We propose noninvasive candidate biomarkers with the ability to be clinically useful across the LEAD spectrum.

Understanding the role of circulating chemokine (C-C motif) ligand 2 in patients with chronic ischemia threatening the lower extremities.

The role of chemokine (C-C motif) ligand 2 (CCL2) in peripheral artery disease is unclear. We measured the difference between serum and plasma levels of CCL2 in patients with chronic ischemia threatening the lower extremities following the observation that atypical chemokine receptors in blood and tissue cells may prevent CCL2 from entering the circulation and consequently modulate its function of attracting monocytes to the site of lesion. To identify the influence of CCL2, we compared the patients' values to those in bio-banked samples from a control population. Further, we explored the association with the Asp42Gly polymorphism (rs12075) in Duffy antigen chemokine receptor; one of these atypical chemokine receptors. When possible, we evaluated in surgically excised normal and affected arteries the calcium burden as well as the expression of CCL2 and related receptors reflecting the inflammatory status. Our findings indicate that circulating CCL2 was significantly associated with the severity and presence of the disease (OR 0.966, 95% CI 0.944 to 0.988, p = 0.003). Circulating CCL2 was dependent on the rs12075 genotype (AA>AG>GG), which, probably, indicates a higher expression of chemokine receptor in the arteries of AA subjects. The associations with genetic variants and the over-expression of atypical chemokine receptors in diseased arteries may have potential implications and our data indicate that CCL2 may represent a previously unrecognized factor that needs to be considered in the screening of patients with risk factors for peripheral artery disease.

Protecting the underscreened women in developed countries: the value of HPV test.

BACKGROUND: Poor attendance to cervical cancer (CC) screening is a major risk factor for CC. Efforts to capture underscreened women are considerable and once women agree to participate, the provision of longitudinal validity of the screening test is of paramount relevance. We evaluate the addition of high risk HPV test (HPV) to cervical cytology as a primary screening test among underscreened women in the longitudinal prediction of intraepithelial lesions grade 2 or worse (CIN2+). METHODS: Women were included in the study if they were older than 39 years and with no evidence of cervical cytology in the previous five years within the Public Primary Health Care System in Catalonia (Spain). 1,832 underscreened women from eight public primary health areas were identified during 2007-2008 and followed-up for over three years to estimate longitudinal detection of CIN2+. Accuracy of each screening test and the combination of both to detect CIN2+ was estimated. The risk of developing CIN2+ lesions according to histology data by cytology and HPV test results at baseline was estimated using the Kaplan-Meier method. RESULTS: At baseline, 6.7% of participants were HPV positive, 2.2% had an abnormal cytology and 1.3% had both tests positive. At the end of follow-up, 18 out of 767 (2.3%) underscreened women had a CIN2+, two of which were invasive CC. The three-year longitudinal sensitivity and specificity estimates to detect CIN2+ were 90.5% and 93.0% for HPV test and 38.2% and 97.8% for cytology. The negative predictive value was >99.0% for each test. No additional gains in validity parameters of HPV test were observed when adding cytology as co-test. The referral to colposcopy was higher for HPV but generated 53% higher detection of CIN2+ compared to cytology. CONCLUSIONS: Underscreened women had high burden of cervical disease. Primary HPV screening followed by cytology triage could be the optimal strategy to identify CIN2+ leading to longer and safe screen intervals.

Prediction of cervical intraepithelial neoplasia grade 2+ (CIN2+) using HPV DNA testing after a diagnosis of atypical squamous cell of undetermined significance (ASC-US) in Catalonia, Spain.

BACKGROUND: A protocol for cervical cancer screening among sexually active women 25 to 65 years of age was introduced in 2006 in Catalonia, Spain to increase coverage and to recommend a 3-year-interval between screening cytology. In addition, Human Papillomavirus (HPV) was offered as a triage test for women with a diagnosis of atypical squamous cells of undetermined significance (ASC-US). HPV testing was recommended within 3 months of ASC-US diagnosis. According to protocol, HPV negative women were referred to regular screening including a cytological exam every 3 years while HPV positive women were referred to colposcopy and closer follow-up. We evaluated the implementation of the protocol and the prediction of HPV testing as a triage tool for cervical intraepithelial lesions grade two or worse (CIN2+) in women with a cytological diagnosis of ASC-US. METHODS: During 2007-08 a total of 611 women from five reference laboratories in Catalonia with a novel diagnosis of ASC-US were referred for high risk HPV (hrHPV) triage using high risk Hybrid Capture version 2. Using routine record linkage data, women were followed for 3 years to evaluate hrHPV testing efficacy for predicting CIN2+ cases. Logistic regression analysis was used to estimate the odds ratio for CIN2 +. RESULTS: Among the 611 women diagnosed with ASC-US, 493 (80.7%) had at least one follow-up visit during the study period. hrHPV was detected in 48.3% of the women at study entry (mean age 35.2 years). hrHPV positivity decreased with increasing age from 72.6% among women younger than 25 years to 31.6% in women older than 54 years (p < 0.01). At the end of the 3 years follow-up period, 37 women with a diagnosis of CIN2+ (18 CIN2, 16 CIN3, 2 cancers, and 1 with high squamous intraepithelial lesions--HSIL) were identified and all but one had a hrHPV positive test at study entry. Sensitivity to detect CIN2+ of hrHPV was 97.2% (95%confidence interval (CI) = 85.5-99.9) and specificity was 68.3% (95%CI = 63.1-73.2). The odds ratio for CIN2+ was 45.3 (95% CI: 6.2-333.0), when among ASC-US hrHPV positive women were compared to ASC-US hrHPV negative women. CONCLUSIONS: Triage of ASC-US with hrHPV testing showed a high sensitivity for the detection of CIN2+ and a high negative predictive value after 3 years of follow-up. The results of this study are in line with the current guidelines for triage of women with ASC-US in the target age range of 25-65. Non adherence to guidelines will lead to unnecessary medical interventions. Further investigation is needed to improve specificity of ASC-US triage.