Carotid stenosis and cryptogenic stroke.

OBJECTIVES: Cryptogenic stroke represents a type of ischemic stroke with an unknown origin, presenting a significant challenge in both stroke management and prevention. According to the Trial of Org 10,172 in Acute Stroke Treatment criteria, a stroke is categorized as being caused by large artery atherosclerosis only when there is >50% luminal narrowing of the ipsilateral internal carotid artery. However, nonstenosing carotid artery plaques can be an underlying cause of ischemic stroke. Indeed, emerging evidence documents that some features of plaque vulnerability may act as an independent risk factor, regardless of the degree of stenosis, in precipitating cerebrovascular events. This review, drawing from an array of imaging-based studies, explores the predictive values of carotid imaging modalities in the detection of nonstenosing carotid plaque (<50%), that could be the cause of a cerebrovascular event when some features of vulnerability are present. METHODS: Google Scholar, Scopus, and PubMed were searched for articles on cryptogenic stroke and those reporting the association between cryptogenic stroke and imaging features of carotid plaque vulnerability. RESULTS: Despite extensive diagnostic evaluations, the etiology of a considerable proportion of strokes remains undetermined, contributing to the recurrence rate and persistent morbidity in affected individuals. Advances in imaging modalities, such as magnetic resonance imaging, computed tomography scans, and ultrasound examination, facilitate more accurate detection of nonstenosing carotid artery plaque and allow better stratification of stroke risk, leading to a more tailored treatment strategy. CONCLUSIONS: Early detection of nonstenosing carotid plaque with features of vulnerability through carotid imaging techniques impacts the clinical management of cryptogenic stroke, resulting in refined stroke subtype classification and improved patient management. Additional research is required to validate these findings and recommend the integration of these state-of-the-art imaging methodologies into standard diagnostic protocols to improve stroke management and prevention.

Retinoic Acid Receptor ß Loss in Hepatocytes Increases Steatosis and Elevates the Integrated Stress Response in Alcohol-Associated Liver Disease.

In alcohol-associated liver disease (ALD), hepatic reductions in vitamin A and perturbations in vitamin A metabolism are common. However, the roles that the vitamin A receptors, termed retinoic acid receptors (RARs), may have in preventing the pathophysiology of ALD remains unclear. Our prior data indicate that a RARß agonist limits the pathology of alcohol-related liver disease. Thus, we generated liver-specific AlbCre-RARß knockout (BKO) mice and compared them to wild type (WT) mice in an early ALD model. Both strains showed similar blood ethanol concentrations and ETOH-metabolizing enzymes. However, the livers of pair-fed-BKO and ETOH-BKO mice developed higher levels of steatosis and triglycerides than pair-fed-WT and ETOH-WT mice. The increased hepatic steatosis observed in the pair-fed-BKO and ETOH-BKO mice was associated with higher lipid synthesis/trafficking transcripts and lower beta-oxidation transcripts. ETOH-BKO mice also exhibited a higher integrated stress response (ISR) signature, including higher transcript and protein levels of ATF4 and its target, 4-EBP1. In human hepatocytes (HepG2) that lack RARß (RARß-KO), ETOH treatments resulted in greater reactive oxygen species compared to their parental cells. Notably, even without ETOH, ATF4 and 4-EBP1 protein levels were higher in the RARß-KO cells than in their parental cells. These 4-EBP1 increases were greatly attenuated in cultured ATF4-deficient and RARß/ATF4-deficient HepG2, suggesting that RARß is a crucial negative regulator of 4-EBP1 through ATF4 in cultured hepatocytes. Here, we identify RARß as a negative regulator of lipid metabolism and cellular stress in ALD.

A retinoic acid receptor ß2 agonist attenuates transcriptome and metabolome changes underlying nonalcohol-associated fatty liver disease.

Nonalcohol-associated fatty liver disease (NAFLD) is characterized by excessive hepatic accumulation of fat that can progress to steatohepatitis, and currently, therapeutic options are limited. Using a high-fat diet (HFD) mouse model of NAFLD, we determined the effects of the synthetic retinoid, AC261066, a selective retinoic acid receptor ß2 (RARß2) agonist, on the global liver transcriptomes and metabolomes of mice with dietary-induced obesity (DIO) using genome-wide RNA-seq and untargeted metabolomics. We found that AC261066 limits mRNA increases in several presumptive NAFLD driver genes, including Pklr, Fasn, Thrsp, and Chchd6. Importantly, AC261066 limits the increases in the transcript and protein levels of KHK, a key enzyme for fructose metabolism, and causes multiple changes in liver metabolites involved in fructose metabolism. In addition, in cultured murine hepatocytes, where exposure to fructose and palmitate results in a profound increase in lipid accumulation, AC261066 limits this lipid accumulation. Importantly, we demonstrate that in a human hepatocyte cell line, RARß is required for the inhibitory effects of AC261066 on palmitate-induced lipid accumulation. Finally, our data indicate that AC261066 inhibits molecular events underpinning fibrosis and exhibits anti-inflammatory effects. In conclusion, changes in the transcriptome and metabolome indicate that AC261066 affects molecular changes underlying multiple aspects of NAFLD, including steatosis and fibrosis. Therefore, we suggest that AC261066 may have potential as an effective therapy for NAFLD.

Incidence and spatial distribution of adult-onset primary malignant and other central nervous system tumors in Southern Sardinia, Italy.

PURPOSE: To study for the first time the incidence of adult-onset CNS tumors in Southern Sardinia, Italy. METHODS: Clinical records of patients > 18 years old who were diagnosed with primary CNS tumors during 2016-2019 in the study area were reviewed. Meningiomas, cranial/paraspinal nerve tumors, lymphomas, and pituitary tumors were excluded. Cases were classified according to the 2016 WHO classification of CNS tumors and to the morphology codes from the International Classification of Diseases-Oncology, third edition. Age-adjusted incidence rates were calculated by the direct method to the 2011-2020 European standard population. Kulldorff's spatial scan statistic was used to identify geographic clusters of patients who shared increased/decreased tendency to develop CNS tumors. RESULTS: CNS tumors were diagnosed in 234 incident patients, but histological diagnosis was available in 222/234 patients (95%) aged 64.3 ± 13.5 years at diagnosis. Crude incidence rate was 7.1 per 100,000 persons-year (95% CI, 6.2-8.1), 6.2 per 100,000 persons-year (95% CI, 5.4-7.0) when age-adjusted. CNS tumors were more frequent in men and after age 40. Glioblastoma accounted for 76% of the total (adjusted rate, 4.7 per 100,000 persons-year; 95% CI, 4.0-5.4). Spatial analysis revealed geographic variations of glioblastoma incidence within the study area. CONCLUSION: Although the distribution of tumor diagnoses in Sardinia reflects expected age and gender-related patterns in western populations, our findings would indicate a slightly higher incidence of glial tumors, glioblastoma in particular, in Sardinia than in other European countries. The identification of spatial clusters of high/low risk will serve as a resource for etiological research.

Fabry-Stroke Italian Registry (FSIR): a nationwide, prospective, observational study about incidence and characteristics of Fabry-related stroke in young-adults. Presentation of the study protocol.

BACKGROUND: TIA and stroke, both ischemic and hemorrhagic, may complicate Fabry disease at young-adult age and be the first manifestation that comes to the clinician's attention. No definite indications have yet been elaborated to guide neurologists in Fabry disease diagnostics. In current practice, it is usually sought in case of cryptogenic strokes (while Fabry-related strokes can also occur by classical pathogenic mechanisms) or through screening programs in young cerebrovascular populations. Data on recurrence and secondary prevention of Fabry's stroke are scanty. METHODS: The study had a prospective observational design involving 33 Italian neurological Stroke Units. Considering the incidence of TIA/stroke in the European population aged < 60 years and the frequency of Fabry disease in this category (as foreseen by a pilot study held at the Careggi University-Hospital, Florence), we planned to screen for Fabry disease a total of 1740 < 60-year-old individuals hospitalized for TIA, ischemic, or hemorrhagic stroke. We investigated TIA and stroke pathogenesis through internationally validated scales and we gathered information on possible early signs of Fabry disease among all cerebrovascular patients. Every patient was tested for Fabry disease through dried blood spot analysis. Patients who received Fabry disease diagnosis underwent a 12-month follow-up to monitor stroke recurrence and multi-system progression after the cerebrovascular event. DISCUSSION: The potential implications of this study are as follows: (i) to add information about the yield of systematic screening for Fabry disease in a prospective large cohort of acute cerebrovascular patients; (ii) to deepen knowledge of clinical, pathophysiological, and prognostic characteristics of Fabry-related stroke.

Fenretinide Reduces Intestinal Mucin-2-Positive Goblet Cells in Chronic Alcohol Abuse.

INTRODUCTION: Alcohol-induced thickening of the gut mucosal layer and increased expression of goblet cell gel-forming mucins, such as mucin-2 (MUC2) are associated with disruptions to the gut barrier in alcoholic liver disease (ALD). Interest in drugs that can target gut mucins in ALD has grown; however to date, no studies have examined the properties of drugs on expression of gut mucins in models of ALD. We previously demonstrated that at 10 mg/kg/day, the drug fenretinide (N-[4-hydroxyphenyl] retinamide [Fen]), a synthetic retinoid, mitigates alcohol-associated damage to the gut barrier and liver injury in a murine model of ALD. METHODS: In this study, we specifically sought to examine the effects of Fen on gut goblet cells, and expression of mucins, including MUC2 using a 25-day Lieber-DeCarli model of chronic alcohol intake. RESULTS: Our results show that chronic alcohol intake increased gut-mucosal thickening, goblet cell numbers, and mRNA and protein expression of MUC2 in both the ileum and colon. Alcohol intake was associated with marked decreases in ileal and colonic Notch signaling, levels of Notch ligands Dll1 and Dll4, and increases in the expression of Notch-associated genes indispensable for goblet cell specification, including Math1 and Spdef. Interestingly, ileal and colonic expression of KLF4, which is involved in terminal differentiation of goblet cells, was reduced in mice chronically fed alcohol. Coadministration of alcohol with Fen at 10 mg/kg/day significantly reduced alcohol-associated increases in ileal and colonic mucosal thickening, ileal Muc2, colonic Muc2, Muc5ac and Muc6 mRNAs, and goblet cell numbers. We also found that Fen strongly prevented alcohol-mediated suppression of the Notch ligand Dll1, Notch signaling, and alcohol-induced increases in expression of Notch-associated goblet cell specification genes in both the ileum and colon. In the absence of alcohol, Fen treatments alone at 10 mg/kg/day had no effects on any of the goblet cell-related endpoints. CONCLUSION: These data show for the first time that the drug Fen possesses mucosal layer-modulating properties in response to chronic alcohol abuse. These data warrant further preclinical examination of Fen given the need for anti-ALD drugs and emerging evidence of a role for intestinal goblet cell mucins in the progression of ALD.

Gut microbiota and metabolome distinctive features in Parkinson disease: Focus on levodopa and levodopa-carbidopa intrajejunal gel.

BACKGROUND AND PURPOSE: Recent data suggest that imbalances in the composition of the gut microbiota (GM) could exacerbate the progression of Parkinson disease (PD). The effects of levodopa (LD) have been poorly assessed, and those of LD-carbidopa intestinal gel (LCIG) have not been evaluated so far. The aim of this study was to identify the effect of LD and LCIG, in particular, on the GM and metabolome. METHODS: Fecal DNA samples from 107 patients with a clinical diagnosis of PD were analyzed by next-generation sequencing of the V3 and V4 regions of the 16S rRNA gene. PD patients were classified in different groups: patients on LCIG (LCIG group, n = 38) and on LD (LD group, n = 46). We also included a group of patients (n = 23) without antiparkinsonian medicaments (Naïve group). Fecal metabolic extracts were evaluated by gas chromatography mass spectrometry. RESULTS: The multivariate analysis showed a significantly higher abundance in the LCIG group of Enterobacteriaceae, Escherichia, and Serratia compared to the LD group. Compared to the Naïve group, the univariate analysis showed a reduction of Blautia and Lachnospirae in the LD group. Moreover, an increase of Proteobacteria, Enterobacteriaceae, and a reduction of Firmicutes, Lachnospiraceae, and Blautia was found in the LCIG group. No significant difference was found in the multivariate analysis of these comparisons. The LD group and LCIG group were associated with a metabolic profile linked to gut inflammation. CONCLUSIONS: Our results suggest that LD, and mostly LCIG, might significantly influence the microbiota composition and host/bacteria metabolism, acting as stressors in precipitating a specific inflammatory intestinal microenvironment, potentially related to the PD state and progression.

Mutations in long-lived epithelial stem cells and their clonal progeny in pre-malignant lesions and in oral squamous cell carcinoma.

Oral squamous cell carcinomas (OSCCs) are the most common cancers of the oral cavity, but the molecular mechanisms driving OSCC carcinogenesis remain unclear. Our group previously established a murine OSCC model based on a 10-week carcinogen [4-nitroquinoline 1-oxide (4-NQO)] treatment. Here we used K14CreERTAM;Rosa26LacZ mice to perform lineage tracing to delineate the mutational profiles in clonal cell populations resulting from single, long-lived epithelial stem cells, here called LacZ+ stem cell clones (LSCCs). Using laser-capture microdissection, we examined mutational changes in LSCCs immediately after the 10-week 4-NQO treatment and >17 weeks after 4-NQO treatment. We found a 1.8-fold ±0.4 (P = 0.009) increase in single-nucleotide variants and insertions/deletions (indels) in tumor compared with pre-neoplastic LSCCs. The percentages of indels and of loss of heterozygosity events were 1.3-fold±0.3 (P = 0.02) and 2.2-fold±0.7 (P = 0.08) higher in pre-neoplastic compared with tumor LSCCs. Mutations in cell adhesion- and development-associated genes occurred in 83% of the tumor LSCCs. Frequently mutated genes in tumor LSCCs were involved in planar cell polarity (Celsr1, Fat4) or development (Notch1). Chromosomal amplifications in 50% of the tumor LSCCs occurred in epidermal growth factor receptor, phosphoinositide 3-kinase and cell adhesion pathways. All pre-neoplastic and tumor LSCCs were characterized by key smoking-associated changes also observed in human OSCC, C>A and G>T. DeconstructSigs analysis identified smoking and head and neck cancer as the most frequent mutational signatures in pre-neoplastic and tumor LSCCs. Thus, this model recapitulates a smoking-associated mutational profile also observed in humans and illustrates the role of LSCCs in early carcinogenesis and OSCCs.

Genetic Creutzfeldt-Jakob disease in Sardinia: a case series linked to the PRNP R208H mutation due to a single founder effect.

In genetic prion diseases (gPrD), five genetic variants (E200K, V210I, V180I, P102L, and D178N) are responsible for about 85% of cases. The R208H is one of the several additional rare mutations and to date, only 16 cases carrying this mutation have been reported worldwide. To describe the phenotypic features of 5 affected patients belonging to apparently unrelated Sardinian (Italian) families with R208H gPrD, and provide evidence for a possible founder effect are the aims of this study. The R208H PRNP mutation has a much higher relative frequency in Sardinia than elsewhere in Italy (72% vs. 4.4% of gCJD cases). Our cohort shared similar phenotypic features to the previously described patients with R208H-129M haplotype with most patients showing the classical Creutzfeldt-Jakob disease (CJD) phenotype. The analysis of 10 controls and 5 patients by NGS sequencing identified 4 haplotypes, 3 associated with the wild type variant, and one (H1) shared by all patients carrying the 208His variant. This is the first report of a regional cluster for R208H mutation in gPrD and the first report of the presence of a common ancestor for this Sardinian R208H cluster, confirming the probable consequences of genetic isolation process even for rare diseases.

Infection with hepatitis C virus depends on TACSTD2, a regulator of claudin-1 and occludin highly downregulated in hepatocellular carcinoma.

Entry of hepatitis C virus (HCV) into hepatocytes is a complex process that involves numerous cellular factors, including the scavenger receptor class B type 1 (SR-B1), the tetraspanin CD81, and the tight junction (TJ) proteins claudin-1 (CLDN1) and occludin (OCLN). Despite expression of all known HCV-entry factors, in vitro models based on hepatoma cell lines do not fully reproduce the in vivo susceptibility of liver cells to primary HCV isolates, implying the existence of additional host factors which are critical for HCV entry and/or replication. Likewise, HCV replication is severely impaired within hepatocellular carcinoma (HCC) tissue in vivo, but the mechanisms responsible for this restriction are presently unknown. Here, we identify tumor-associated calcium signal transducer 2 (TACSTD2), one of the most downregulated genes in primary HCC tissue, as a host factor that interacts with CLDN1 and OCLN and regulates their cellular localization. TACSTD2 gene silencing disrupts the typical linear distribution of CLDN1 and OCLN along the cellular membrane in both hepatoma cells and primary human hepatocytes, recapitulating the pattern observed in vivo in primary HCC tissue. Mechanistic studies suggest that TACSTD2 is involved in the phosphorylation of CLDN1 and OCLN, which is required for their proper cellular localization. Silencing of TACSTD2 dramatically inhibits HCV infection with a pan-genotype effect that occurs at the level of viral entry. Our study identifies TACSTD2 as a novel regulator of two major HCV-entry factors, CLDN1 and OCLN, which is strongly downregulated in malignant hepatocytes. These results provide new insights into the complex process of HCV entry into hepatocytes and may assist in the development of more efficient cellular systems for HCV propagation in vitro.

Odor Identification Performance in Idiopathic Parkinson's Disease Is Associated With Gender and the Genetic Variability of the Olfactory Binding Protein.

Deficits in olfaction are among the most frequent non-motor symptoms in Parkinson's disease (PD) and can be detected early compared with motor symptoms. The reason for the early onset, as well as the mechanism involved remains unknown. We aimed to characterize the olfactory performance of patients with PD and age-matched healthy control (HC) participants in association with gender and a specific polymorphism in the odorant-binding protein IIa (OBPIIa) gene, which plays a crucial role in the perception of odors. The olfactory performance was assessed using the odor identification part of the Sniffin' Sticks test in 249 participants (patients with PD: n = 131 and HC participants: n = 118). All participants were genotyped for the rs2590498 polymorphism of the OBPIIa gene, whose major allele A is associated with a higher retronasal perception than the minor allele G. A higher number of men with PD than women with PD exhibited hyposmia. Importantly, OBPIIa gene polymorphism showed an effect on PD-related olfactory deficits only in women. Women with PD carrying two sensitive alleles (AA) showed a better olfactory performance than women with PD with at least one insensitive allele (G); the olfactory scores of the AA genotype women with PD were not different from those of HC participants. In conclusion, our results confirmed a sex effect on the reduced olfactory performance of patients with PD and identified the OBPIIa locus, which may provide a mechanism to determine the risk factor for olfactory deficits in women with PD at the molecular level.

Management of epilepsy in brain tumors.

Epilepsy in brain tumors (BTE) may require medical attention for a variety of unique concerns: epileptic seizures, possible serious adverse effects of antineoplastic and antiepileptic drugs (AEDs), physical disability, and/or neurocognitive disturbances correlated to tumor site. Guidelines for the management of tumor-related epilepsies are lacking. Treatment is not standardized, and overall management might differ according to different specialists. The aim of this document was to provide directives on the procedures to be adopted for a correct diagnostic-therapeutic path of the patient with BTE, evaluating indications, risks, and benefits. A board comprising neurologists, epileptologists, neurophysiologists, neuroradiologists, neurosurgeons, neuro-oncologists, neuropsychologists, and patients' representatives was formed. The board converted diagnostic and therapeutic problems into seventeen questions. A literature search was performed in September-October 2017, and a total of 7827 unique records were retrieved, of which 148 constituted the core literature. There is no evidence that histological type or localization of the brain tumor affects the response to an AED. The board recommended to avoid enzyme-inducing antiepileptic drugs because of their interference with antitumoral drugs and consider as first-choice newer generation drugs (among them, levetiracetam, lamotrigine, and topiramate). Valproic acid should also be considered. Both short-term and long-term prophylaxes are not recommended in primary and metastatic brain tumors. Management of seizures in patients with BTE should be multidisciplinary. The panel evidenced conflicting or lacking data regarding the role of EEG, the choice of therapeutic strategy, and timing to withdraw AEDs and recommended high-quality long-term studies to standardize BTE care.

6-n-propylthiouracil taste disruption and TAS2R38 nontasting form in Parkinson's disease.

BACKGROUND: The few studies that evaluated taste function in Parkinson's disease (PD) showed inconsistent results. The inherited ability to taste the bitter compound of 6-n-propylthiouracil has been considered to be a paradigm of general taste perception. 6-n-propylthiouracil taste perception is mediated by the TAS2R38 receptor, and reduced 6-n-propylthiouracil sensitivity has been associated with several diseases not typically related to taste function. OBJECTIVES: We evaluated the 6-n-propylthiouracil taste perception and the TAS2R38 gene as genetic risk factors for the development of idiopathic PD in PD patients and healthy controls (HC). METHODS: The 6-n-propylthiouracil taste perception was assessed by testing the responsiveness, and the ability to recognize, 6-n-propylthiouracil and sodium chloride. The participants were classified for 6-n-propylthiouracil taster status and genotyped for the TAS2R38 gene. RESULTS: A significant increase in the frequency of participants classified as 6-n-propylthiouracil nontasters and a reduced ability to recognize bitter taste quality of 6-n-propylthiouracil were found in PD patients when compared with healthy controls. The results also showed that only 5% of PD patients had the homozygous genotype for the dominant tasting variant of TAS2R38, whereas most of them carried the recessive nontaster form and a high number had a rare variant. CONCLUSIONS: Our results show that 6-n-propylthiouracil taster status and TAS2R38 locus are associated with PD. The 6-n-propylthiouracil test may therefore represent a novel, simple way to identify increased vulnerability to PD. Moreover, the presence of the nontasting form of TAS2R38 in PD may further substantiate that disease-associated taste disruption may represent a risk factor associated with the disease. © 2018 International Parkinson and Movement Disorder Society.

Parkinsonism and dementia are negative prognostic factors for the outcome of subdural hematoma.

To determine, among a population with subdural hematoma (SH), whether patients affected by neurodegenerative disorders (parkinsonism and dementia) have a worse clinical outcome. We reviewed the data of patients diagnosed with fall-related SH discharged from the Departments of Neurology/Stroke unit, Neurosurgery, Intensive Care Unit at Brotzu General Hospital (Cagliari, Italy) between January 2010 and December 2013. Patients with severe traumatisms, evidence of spontaneous intracerebral bleeding or aged less than 50 were excluded. 332 patients were selected: 69 with a neurodegenerative parkinsonism or dementia (N-group), 217 with history of chronic non-neurological medical conditions with significant disability, previous falls and/or balance problems (NND-group) and 46 with a history of "minor" chronic non-neurological disorder. (NN-group). The clinical status at admission and discharge was assessed by modified Rankin Scale (mRS). The time-span between trauma and hospital admission was also calculated. At hospital admission we found a significantly longer delay in SH's diagnosis (χ (2) test p < 0.001) and a worse mRS score (Kruskal Wallis p < 0.001) in the N-group compared to both NN and NND-groups. During hospital stay we observed the lack of significant variation in mRS score in N-group (Wilcoxon test p = 0.86), in contrast with NN and NND-groups who significantly improved (Wilcoxon test p < 0.001). Our results demonstrate that the consequences of SH are more severe in the N-group compared to NN and NND-groups. The longer interval between trauma and hospital admittance plays a critical role in worsening the outcome of patients with parkinsonism and dementia compared to subjects without neurodegenerative disorders.

Influence of treatments in multiple sclerosis disability: a cohort study.

BACKGROUND AND OBJECTIVE: A critical aspect of multiple sclerosis (MS) treatments is understanding the effect of disease-modifying drugs (DMDs) on the long-term risk of disability and whether the effect is related to disability at start of treatment. METHODS: We performed an observational study on 3060 MS patients. The effect of therapy on progression to Expanded Disability Status Scale (EDSS) 3.0 and 6.0 from onset was analysed in treated vs untreated (UTP) patients using Cox regression analysis adjusted for propensity score and immortal time bias. RESULTS: Compared to UTP, the risks of EDSS 3.0 were 94% and 73% lower in immunomodulant (IMTP-) and immunosuppressant (ISTP-) treated patients, respectively, while the risk of EDSS 6.0 was 86% lower in IMTP. The risk of EDSS 6.0 was, respectively, 91% and 75% lower in 1275 IMTP before and 114 after EDSS 3.0 than in 539 UTP; the risk was higher in IMTP starting therapy after EDSS 3.0 than before (HR = 4.42). CONCLUSIONS: DMDs delayed long-term disability in MS patients treated either in the early or, to a lesser extent, in the later phase of the disease. Thus, the window of therapeutic opportunity is relatively extended, assuming that early is better than late treatment, but late is better than never.

Neuroactive steroid levels in plasma and cerebrospinal fluid of male multiple sclerosis patients.

Neuroactive steroid family includes molecules synthesized in peripheral glands (i.e., hormonal steroids) and directly in the nervous system (i.e., neurosteroids) which are key regulators of the nervous function. As already reported in clinical and experimental studies, neurodegenerative diseases affect the levels of neuroactive steroids. However, a careful analysis comparing the levels of these molecules in cerebrospinal fluid (CSF) and in plasma of multiple sclerosis (MS) patients is still missing. To this aim, the levels of neuroactive steroids were evaluated by liquid chromatography-tandem mass spectrometry in CSF and plasma of male adults affected by Relapsing-Remitting MS and compared with those collected in control patients. An increase in pregnenolone and isopregnanolone levels associated with a decrease in progesterone metabolites, dihydroprogesterone, and tetrahydroprogesterone was observed in CSF of MS patients. Moreover, an increase of 5α-androstane-3α,17ß-diol and of 17ß-estradiol levels associated with a decrease of dihydrotestosterone also occurred. In plasma, an increase in pregnenolone, progesterone, and dihydrotestosterone and a decrease in dihydroprogesterone and tetrahydroprogesterone levels were reported. This study shows for the first time that the levels of several neuroactive steroids, and particularly those of progesterone and testosterone metabolites, are deeply affected in CSF of relapsing-remitting MS male patients. We here demonstrated that, the cerebrospinal fluid and plasma levels of several neuroactive steroids are modified in relapsing remitting multiple sclerosis male patients. Interestingly, we reported for the first time that, the levels of progesterone and testosterone metabolites are deeply affected in cerebrospinal fluid. These findings may have an important relevance in therapeutic and/or diagnostic field of multiple sclerosis.

Viral expression and molecular profiling in liver tissue versus microdissected hepatocytes in hepatitis B virus-associated hepatocellular carcinoma.

BACKGROUND: The molecular mechanisms whereby hepatitis B virus (HBV) induces hepatocellular carcinoma (HCC) remain elusive. We used genomic and molecular techniques to investigate host-virus interactions by studying multiple areas of the same liver from patients with HCC. METHODS: We compared the gene signature of whole liver tissue (WLT) versus laser capture-microdissected (LCM) hepatocytes along with the intrahepatic expression of HBV. Gene expression profiling was performed on up to 17 WLT specimens obtained at various distances from the tumor center from individual livers of 11 patients with HCC and on selected LCM samples. HBV markers in liver and serum were determined by real-time polymerase chain reaction (PCR) and confocal immunofluorescence. RESULTS: Analysis of 5 areas of the liver showed a sharp change in gene expression between the immediate perilesional area and tumor periphery that correlated with a significant decrease in the intrahepatic expression of HB surface antigen (HBsAg). The tumor was characterized by a large preponderance of down-regulated genes, mostly involved in the metabolism of lipids and fatty acids, glucose, amino acids and drugs, with down-regulation of pathways involved in the activation of PXR/RXR and PPARα/RXRα nuclear receptors, comprising PGC-1α and FOXO1, two key regulators critically involved not only in the metabolic functions of the liver but also in the life cycle of HBV, acting as essential transcription factors for viral gene expression. These findings were confirmed by gene expression of microdissected hepatocytes. Moreover, LCM of malignant hepatocytes also revealed up-regulation of unique genes associated with cancer and signaling pathways, including two novel HCC-associated cancer testis antigen genes, NUF2 and TTK. CONCLUSIONS: Integrated gene expression profiling of whole liver tissue with that of microdissected hepatocytes demonstrated that HBV-associated HCC is characterized by a metabolism switch-off and by a significant reduction in HBsAg. LCM proved to be a critical tool to validate gene signatures associated with HCC and to identify genes that may play a role in hepatocarcinogenesis, opening new perspectives for the discovery of novel diagnostic markers and therapeutic targets.

Post-natalizumab clinical and radiological findings in a cohort of multiple sclerosis patients: 12-month follow-up.

There is an urgent need to identify the best strategies to prevent the loss of natalizumab (N) beneficial effects after its suspension. The objective is to evaluate the clinical and radiological disease activity and to test the efficacy of immunomodulatory/immunosuppressive drugs (IT) after N suspension. Clinical and radiological data from 54 patients 2 years before treatment (pre-N), during treatment (on-N) and after interruption, during 1-year follow-up (post-N) were retrospectively collected. Annualized relapse rate (ARR), expanded disability status scale (EDSS), presence of new T2 lesions and Gd+ (gadolinium enhancing) T1 lesions were evaluated. Pre-N ARR at 1 year was 1.74 while post-N ARR was 0.94 (p = 0.0053). We observed that post-N disease activity never raised over pre-N levels, neither post-N ARR nor post-N EDSS. In patients retreated with N after suspension, post-N ARR was significantly lower than pre-N ARR (p = 0.017), but not in patients treated with other IT or in patients not treated with any disease modifying drugs (DMD). The mean time of freedom from new T2 lesions and new Gd+ lesions was lower in post-N period compared to on-N (T2 lesions p = 0.0000, Gd+ lesions p = 0.0000). In conclusion, a "rebound" pattern was not identified in our cohort, though the disease activity rapidly returned after N, regardless of the treatment used.

Mechanism and Effect of HNF4α Decrease in a Rat Model of Cirrhosis and Liver Failure.

BACKGROUND & AIMS: HNF4α, a master regulator of liver development and the mature hepatocyte phenotype, is down-regulated in chronic and inflammatory liver disease. We used contemporary transcriptomics and epigenomics to study the cause and effects of this down-regulation and characterized a multicellular etiology. METHODS: Progressive changes in the rat carbon tetrachloride model were studied by deep RNA sequencing and genome-wide chromatin immunoprecipitation sequencing analysis of transcription factor (TF) binding and chromatin modification. Studies compared decompensated cirrhosis with liver failure after 26 weeks of treatment with earlier compensated cirrhosis and with additional rat models of chronic fibrosis. Finally, to resolve cell-specific responses and intercellular signaling, we compared transcriptomes of liver, nonparenchymal, and inflammatory cells. RESULTS: HNF4α was significantly lower in 26-week cirrhosis, part of a general reduction of TFs that regulate metabolism. Nevertheless, increased binding of HNF4α contributed to strong activation of major phenotypic genes, whereas reduced binding to other genes had a moderate phenotypic effect. Decreased Hnf4a expression was the combined effect of STAT3 and nuclear factor kappa B (NFκB) activation, which similarly reduced expression of other metabolic TFs. STAT/NFκB also induced de novo expression of Osmr by hepatocytes to complement induced expression of Osm by nonparenchymal cells. CONCLUSIONS: Liver decompensation by inflammatory STAT3 and NFκB signaling was not a direct consequence of progressive cirrhosis. Despite significant reduction of Hnf4a expression, residual levels of this abundant TF still stimulated strong new gene expression. Reduction of HNF4α was part of a broad hepatocyte transcriptional response to inflammation.