DNA Methylation in the Anti-Mullerian Hormone Gene and the Risk of Disease Activity in Multiple Sclerosis.

OBJECTIVE: Multiple sclerosis (MS) has a complex pathobiology, with genetic and environmental factors being crucial players. Understanding the mechanisms underlying heterogeneity in disease activity is crucial for tailored treatment. We explored the impact of DNA methylation, a key mechanism in the genetics-environment interplay, on disease activity in MS. METHODS: Peripheral immune methylome profiling using Illumina Infinium MethylationEPIC BeadChips was conducted on 249 untreated relapsing-remitting MS patients, sampled at the start of disease-modifying treatment (DMT). A differential methylation analysis compared patients with evidence of disease activity (EDA) to those with no evidence of disease activity (NEDA) over 2 years from DMT start. Utilizing causal inference testing (CIT) and Mendelian randomization (MR), we sought to elucidate the relationships between DNA methylation, gene expression, genetic variation, and disease activity. RESULTS: Four differentially methylated regions (DMRs) were identified between EDA and NEDA. Examining the influence of single nucleotide polymorphisms (SNPs), 923 variants were found to account for the observed differences in the 4 DMRs. Importantly, 3 out of the 923 SNPs, affecting DNA methylation in a DMR linked to the anti-Mullerian hormone (AMH) gene, were associated with disease activity risk in an independent cohort of 1,408 MS patients. CIT and MR demonstrated that DNA methylation in AMH acts as a mediator for the genetic risk of disease activity. INTERPRETATION: This study uncovered a novel molecular pathway implicating the interaction between DNA methylation and genetic variation in the risk of disease activity in MS, emphasizing the role of sex hormones, particularly the AMH, in MS pathobiology. ANN NEUROL 2024.

Clinical relevance of the left brachiocephalic vein anatomy for vascular access in dialysis patients.

INTRODUCTION: Most hemodialysis patients start renal replacement therapy with a central venous catheter (CVC). The left internal jugular vein (LIJV) is the second-choice vein for CVC positioning, after the right IJV. However, to reach the right atrium, the CVC must pass through the left brachiocephalic vein (LBV), which also drains blood from the left arm through the subclavian vein. The purpose of this study is to describe how the anatomy of the central venous system and in particular that of the LBV affects vascular access in hemodialysis patients. MATERIALS AND METHODS: Three-dimensional (3D) virtual model reconstructions of the central thoracic veins of three hemodialysis patients were obtained from contrast-enhanced computed tomography scans acquired in the venous phase. The images were exported as DICOM files and loaded on open-source software for visualizing and analyzing the medical imaging (3D Slicer, Windows version 4.8.1). RESULTS: As expected, the 3D reconstructions showed that the LBV has a tortuous path with three main angulations that could be associated with external compression and stenosis. These could determine the difficulties and increased risks of venous injury during CVC placement, and an increased risk of medium to long-term catheter-associated vein thrombosis and stenosis. CONCLUSIONS: The anatomical features of the LBV indicate that the path of a CVC from the LIJV to the right atrium is tortuous and can easily be complicated by vein injury, negatively affecting the creation of future arterio-venous vascular accesses in the left arm.

Assessing seasonal dynamics of Guillain-Barré syndrome with search engine query data.

BACKGROUND AND OBJECTIVE: In previous studies, data deriving from Google Trends showed promising correlation with disease incidence trends assessed with public health control systems. The aim of this work is to use search engine query data to investigate seasonal dynamics in Guillain-Barré syndrome (GBS) in the USA. METHODS: Average Google monthly search volumes for GBS from 2008 to 2017 were analysed for the USA overall and on regional base with generalized estimating equation models. Association with monthly historical temperature variations was tested. RESULTS: Monthly search volume for GBS displayed the greatest positive anomaly for October, clustering with September and November. Region-wide analysis confirmed this pattern and showed secondary spring (Feb/Apr) subpeaks in Pacific and Midwest. Association of GBS search volume with month-to-month temperature variations showed J-shaped relationship, with the highest peak occurring in months with greatest temperature falls, and subpeak in months with sharpest temperature rises. CONCLUSIONS: This study represents the first approach in investigating digital epidemiology of GBS and establishing possible links with traditional epidemiology. Cold season GBS peak has been observed by some traditional studies; hypothetical pathogenic relationship with infectious antecedents is supported from finding GBS peaks clustering with greatest temperature change. Further studies are needed to compare these findings to traditional public health approaches.

Genetic Contribution to Medium-Term Disease Activity in Multiple Sclerosis.

Multiple sclerosis (MS) is a complex disorder characterized by high heterogeneity in terms of phenotypic expression, prognosis and treatment response. In the present study, we aimed to explore the genetic contribution to MS disease activity at different levels: genes, pathways and tissue-specific networks. Two cohorts of relapsing-remitting MS patients who started a first-line treatment (n = 1294) were enrolled to evaluate the genetic association with disease activity after 4 years of follow-up. The analyses were performed at whole-genome SNP and gene level, followed by the construction of gene-gene interaction networks specific for brain and lymphocytes. The resulting gene modules were evaluated to highlight key players from a topological and functional perspective. We identified 23 variants and 223 genes with suggestive association to 4-years disease activity, highlighting genes like PON2 involved in oxidative stress and in mitochondria functions and other genes, like ILRUN, involved in the modulation of the immune system. Network analyses led to the identification of a brain module composed of 228 genes and a lymphocytes module composed of 287 genes. The network analysis allowed us to prioritize genes relevant for their topological properties; among them, there are MPHOSPH9 (connector hub in both brain and lymphocyte module) and OPA1 (in brain module), two genes already implicated in MS. Modules showed the enrichment of both shared and tissue-specific pathways, mainly implicated in inflammation. In conclusion, our results suggest that the processes underlying disease activity act on shared mechanisms across brain and lymphocyte tissues.

Risk HLA Variants Affect the T-Cell Repertoire in Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: The major histocompatibility complex (MHC) locus has a predominant role in the genetic predisposition to multiple sclerosis (MS), with 32 associations found to be involved. We aimed to investigate the impact of MHC MS-risk alleles on T-cell repertoire in patients with MS. METHODS: We studied 161 untreated patients with relapsing-remitting MS for whom Class I and II human leukocyte antigen (HLA) alleles were inferred from whole-genome genotyping data, and T-cell receptor (TCR) CDR3 sequences were obtained through next-generation sequencing. T-cell repertoire features including diversity, public clones, and architecture were evaluated. RESULTS: We identified 5 MS-risk loci associated with TCR diversity: HLA-DRB1*15:01 (7.65 × 10-3), rs9271366 (1.96 × 10-3), rs766848979 A (1.89 × 10-2), rs9277626 (2.95 × 10-2), and rs11751659 (1.92 × 10-2), with evidence of expanded clonotypes in carriers of risk alleles. Moreover, HLA-DRB1*15:01 (4.99 × 10-3), rs9271366 (6.54 × 10-3), rs1049079 C (4.37 × 10-2), AA DQΒ1 position -5 L (1.05 × 10-3), and AA DQΒ1 position 221 Q (9.39 × 10-4) showed an association with the CDR3 aminoacidic sequence architecture, suggesting an impact on the antigen recognition breadth as well. Evaluating the sharing of clones across MS-risk allele carrier individuals revealed the presence of highly shared clonotypes predicted to target viral antigens, including Epstein-Barr virus. DISCUSSION: Our study supports the association between MHC-risk alleles and macrofeatures of the T-cell repertoire in the context of MS. Further studies are needed to understand the underlying molecular mechanisms.

Defective Bile Acid Signaling Promotes Vascular Dysfunction, Supporting a Role for G-Protein Bile Acid Receptor 1/Farnesoid X Receptor Agonism and Statins in the Treatment of Nonalcoholic Fatty Liver Disease.

BACKGROUND: Patients with nonalcoholic fatty liver disease are at increased risk to develop atherosclerotic cardiovascular diseases. FXR and GPBAR1 are 2 bile acid-activated receptors exploited in the treatment of nonalcoholic fatty liver disease: whether dual GPBAR1/FXR agonists synergize with statins in the treatment of the liver and cardiovascular components of nonalcoholic fatty liver disease is unknown. METHODS AND RESULTS: Investigations of human aortic samples obtained from patients who underwent surgery for aortic aneurysms and Gpbar1-/-, Fxr-/-, and dual Gpbar1-/-Fxr-/- mice demonstrated that GPBAR1 and FXR are expressed in the aortic wall and regulate endothelial cell/macrophage interactions. The expression of GPBAR1 in the human endothelium correlated with the expression of inflammatory biomarkers. Mice lacking Fxr and Gpbar1-/-/Fxr-/- display hypotension and aortic inflammation, along with altered intestinal permeability that deteriorates with age, and severe dysbiosis, along with dysregulated bile acid synthesis. Vasomotor activities of aortic rings were altered by Gpbar1 and Fxr gene ablation. In apolipoprotein E-/- and wild-type mice, BAR502, a dual GPBAR1/FXR agonist, alone or in combination with atorvastatin, reduced cholesterol and low-density lipoprotein plasma levels, mitigated the development of liver steatosis and aortic plaque formation, and shifted the polarization of circulating leukocytes toward an anti-inflammatory phenotype. BAR502/atorvastatin reversed intestinal dysbiosis and dysregulated bile acid synthesis, promoting a shift of bile acid pool composition toward FXR antagonists and GPBAR1 agonists. CONCLUSIONS: FXR and GPBAR1 maintain intestinal, liver, and cardiovascular homeostasis, and their therapeutic targeting with a dual GPBAR1/FXR ligand and atorvastatin holds potential in the treatment of liver and cardiovascular components of nonalcoholic fatty liver disease.

Activation of GPBAR1 attenuates vascular inflammation and atherosclerosis in a mouse model of NAFLD-related cardiovascular disease.

While patients with nonalcoholic fatty liver disease (NAFLD) are at increased risk to develop clinically meaningful cardiovascular diseases (CVD), there are no approved drug designed to target the liver and CVD component of NAFLD. GPBAR1, also known as TGR5, is a G protein coupled receptor for secondary bile acids. In this study we have investigated the effect of GPBAR1 activation by BAR501, a selective GPBAR1 agonist, in Apolipoprotein E deficient (ApoE-/-) mice fed a high fat diet and fructose (Western diet), a validated model of NAFLD-associated atherosclerosis. Using aortic samples from patients who underwent surgery for abdominal aneurism, and ex vivo experiments with endothelial cells and human macrophages, we were able to co-localize the expression of GPBAR1 in CD14+ and PECAM1+ cells. Similar findings were observed in the aortic plaques from ApoE-/- mice. Treating ApoE-/- mice with BAR501, 30 mg/kg for 14 weeks, attenuated the body weight gain while ameliorated the insulin sensitivity by increasing the plasma concentrations of GLP-1 and FGF15. Activation of GPBAR1 reduced the aorta thickness and severity of atherosclerotic lesions and decreased the amount of plaques macrophages. Treating ApoE-/- mice reshaped the aortic transcriptome promoting the expression of anti-inflammatory genes, including IL-10, as also confirmed by tSNE analysis of spleen-derived macrophages. Feeding ApoE-/- mice with BAR501 redirected the bile acid synthesis and the composition of the intestinal microbiota. In conclusion, GPBAR1 agonism attenuates systemic inflammation and improve metabolic profile in a genetic/dietetic model of atherosclerosis. BAR501 might be of utility in the treatment for NAFLD-related CVD.

Early use of fingolimod is associated with better clinical outcomes in relapsing-remitting multiple sclerosis patients.

BACKGROUND: Multiple sclerosis (MS) is a chronic autoimmune disease with huge heterogeneity in terms of clinical course, disease severity and treatment response. The need for a tailored treatment approach has emerged over the last few years. The present observational study aims to assess fingolimod (FTY) effectiveness in RRMS patients, stratifying them according to the disease-modifying treatments used before FTY, to identify subjects who could benefit more from this treatment. METHODS: We prospectively included 554 RRMS patients who started FTY at San Raffaele Hospital between 2012 and 2018. We classified them into three categories according to previous treatments: naïve patients, subjects previously treated with first-line drugs, and patients previously treated with second-line drugs. We compared disease activity during a 2-years follow-up using No-Evidence-of-Disease-Activity (NEDA-3) and Time-To-First-Relapse (TTFR) outcomes, applying logistic and Cox proportional hazard regression respectively. RESULTS: The proportion of patients who maintained NEDA-3 status was higher in the naïve group despite a higher level of baseline disease activity (naïve versus first-line p = 0.025, naïve versus second-line p < 0.001). In the multivariable analyses, patients switching to FTY from first- and second-line treatments showed a higher risk of disease reactivation (p = 0.041, OR = 1.86 and p = 0.002, OR = 2.92, respectively) and a shorter TTFR (p = 0.017, HR = 4.35 and p = 0.001, HR = 8.19, respectively). CONCLUSION: Naïve patients showed a better response to FTY compared to patients switching to FTY from other drugs. Our findings support the early use of FTY in patients with active MS.

BDNF Val66Met Polymorphism Is Associated With Motor Recovery After Rehabilitation in Progressive Multiple Sclerosis Patients.

Background: Rehabilitation is fundamental for progressive multiple sclerosis (MS), but predictive biomarkers of motor recovery are lacking, making patient selection difficult. Motor recovery depends on synaptic plasticity, in which the Brain-Derived Neurotrophic Factor (BDNF) is a key player, through its binding to the Neurotrophic-Tyrosine Kinase-2 (NTRK2) receptor. Therefore, genetic polymorphisms in the BDNF pathway may impact motor recovery. The most well-known polymorphism in BDNF gene (rs6265) causes valine to methionine substitution (Val66Met) and it influences memory and motor learning in healthy individuals and neurodegenerative diseases. To date, no studies have explored whether polymorphisms in BDNF or NTRK2 genes may impact motor recovery in MS. Objectives: To assess whether genetic variants in BDNF and NTRK2 genes affect motor recovery after rehabilitation in progressive MS. Methods: The association between motor recovery after intensive neurorehabilitation and polymorphisms in BDNF (rs6265) and NTKR2 receptor (rs2289656 and rs1212171) was assessed using Six-Minutes-Walking-Test (6MWT), 10-Metres-Test (10MT) and Nine-Hole-Peg-Test (9HPT) in 100 progressive MS patients. Results: We observed greater improvement at 6MWT after rehabilitation in carriers of the BDNF Val66Met substitution, compared to BDNF Val homozygotes (p = 0.024). No significant association was found for 10MT and 9HPT. NTRK2 polymorphisms did not affect the results of motor function tests. Conclusion: BDNF Val66Met was associated with walking function improvement after rehabilitation in progressive MS patients. This result is in line with previous evidence showing a protective effect of Val66Met substitution on brain atrophy in MS. Larger studies are needed to explore its potential as a predictive biomarker of rehabilitation outcome.

Involvement of NINJ2 Protein in Inflammation and Blood-Brain Barrier Transmigration of Monocytes in Multiple Sclerosis.

Multiple sclerosis (MS) is an inflammatory neurodegenerative disorder of the central nervous system (CNS). The migration of immune cells into the CNS is essential for its development, and plasma membrane molecules play an important role in triggering and maintaining the inflammation. We previously identified ninjurin2, a plasma membrane protein encoded by NINJ2 gene, as involved in the occurrence of relapse under Interferon-ß treatment in MS patients. The aim of the present study was to investigate the involvement of NINJ2 in inflammatory conditions and in the migration of monocytes through the blood-brain barrier (BBB). We observed that NINJ2 is downregulated in monocytes and in THP-1 cells after stimulation with the pro-inflammatory cytokine LPS, while in hCMEC/D3 cells, which represent a surrogate of the BBB, LPS stimulation increases its expression. We set up a transmigration assay using an hCMEC/D3 transwell-based model, finding a higher transmigration rate of monocytes from MS subjects compared to healthy controls (HCs) in the case of an activated hCMEC/D3 monolayer. Moreover, a positive correlation between NINJ2 expression in monocytes and monocyte migration rate was observed. Overall, our results suggest that ninjurin2 could be involved in the transmigration of immune cells into the CNS in pro-inflammatory conditions. Further experiments are needed to elucidate the exact molecular mechanisms.

[Standard procedures in dialysis during the Covid-19 epidemic].

Scope: The aim of this document has been to define standard procedures for dealing with dialysis patients once the first cases of novel Coronavirus 2019-nCoV (Covid-19) were confirmed among the Italian population. Applicability: These procedures, that refer exclusively to the hospital's dialysis rooms, are currently implemented at the ASST Santi Paolo e Carlo in Milan and two smaller centers in the Milan area. Description: We describe the preemptive measures adopted by the staff at our dialysis unit since 24/02/2020, in order to slow down the transmission of Covid-19. They have allowed us to adopt a uniform approach towards all patients, streamlining the way we identify and deal with suspected, likely and confirmed Coronavirus infections. To start with, all patients coming to the hospital for their dialytic session have been treated as potentially infectious and everybody has been following closely the standard protocols regarding personal protective equipment (PPE).

Diagnosing autoimmune encephalitis in a real-world single-centre setting.

BACKGROUND: Early recognition and treatment of autoimmune encephalitis (AE) are crucial for patients, but diagnosis is often difficult and time-consuming. For this purpose, a syndrome-based diagnostic approach was published by Graus et al. (Lancet Neurol 15:391-404, 2016), but very little is known in the literature about its application in clinical practice. AIM: Our aims are to test the feasibility of such approach in a real-world single-centre setting and to analyse the most relevant factors in criteria fulfilment. METHODS: We retrospectively applied these criteria to our cohort of patients discharged from our hospital with diagnosis of autoimmune encephalitis (n = 33, 58% antibody-positive). RESULTS: All the subjects fulfilled criteria for possible AE (pAE), with EEG and MRI playing a central role in diagnosis, while CSF was useful mainly to rule out other conditions. Three patients respected criteria for probable anti-NMDA-R encephalitis (pNMDA). Definite anti-NMDAR encephalitis was diagnosed in 4 patients with detection of the autoantibody but, surprisingly, none of these subjects had fulfilled criteria for pNMDA. 18 patients were diagnosed with definite limbic AE (15 patients were antibody-positive, three antibody-negative). Need for MRI bilateral involvement in antibody-negative limbic AE limited diagnosis. One patient fulfilled criteria for probable antibody-negative AE, while ten patients remained classified as pAE. CONCLUSION: From our retrospective analysis, some suggestions for a better definition of the criteria may emerge. Larger studies on prospective cohorts may be more helpful to explore possible important issues.

Refractory anti-NMDAR encephalitis successfully treated with bortezomib and associated movements disorders controlled with tramadol: a case report with literature review.

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a potentially fatal autoimmune disease, characterized by autoantibody-mediated neurotransmission impairment in multiple brain locations. The course of this condition often comprises altered mental status, autonomic dysfunctions, refractory seizures and hyperkinetic movement disorders. Available disease-modifying therapies include corticosteroids, i.v. immunoglobulins, plasma exchange, rituximab and cyclophosphamide. In a subgroup of patients not responding to B-cell depletion, bortezomib, a proteasome inhibitor, has shown promising evidence of efficacy. The time course of recovery from acute phase may be very slow (weeks/months), and only few data are available in literature about the concurrent management of encephalitis-associated movement disorders. We report a case of severe anti-NMDAR encephalitis in a 29-year-old woman, not responsive to first- and second-line treatments, with persistent involuntary motor manifestations. Starting three months after symptom onset, four cycles of bortezomib have been administered; subsequently we observed a progressive improvement of neurological status. Meanwhile, motor manifestations were controlled after the administration of tramadol, a non-competitive NMDA receptor antagonist.

Dialysis access: issues related to conversion from peritoneal dialysis to hemodialysis and vice versa.

ABSTRACTHemodialysis (HD) and peritoneal dialysis (PD) represent two complementary modalities of renal replacement therapy (RRT) for end-stage renal disease patients. Conversion between the two modalities is frequent and more likely to happen from PD to HD. Every year, 10% of PD patients convert to HD, suggesting the need for recommendations on how to proceed with the creation of a vascular access in these patients. Criteria for selecting patients who would likely fail PD, and therefore take advantage of a backup access, are undefined. Creating backup fistulas at the time of PD treatment start to allow emergency access for HD has proved to be inefficient, but it may be considered in patients with progressive difficulty in achieving adequate depuration and/or peritoneal ultrafiltration. A big challenge is represented by patients switching from PD to HD for unexpected infectious complications. Those patients need to start HD with a central venous catheter (CVC), but an alternative approach might be using an early cannulation graft, provided that infection has been cleared by the circulation. An early cannulation graft might also be used to considerably shorten the time spent using a CVC. In patients who need a conversion from HD to PD, urgent-start PD is now an accepted and well-established approach.

Optimization of dialysis catheter function.

Central venous catheters (CVCs) are essential in the management of hemodialysis patients, but they also carry unintended negative consequences and in particular thrombosis and infection, adversely affecting patient morbidity and mortality. This review will focus on the etiology, prevention, and management of CVC-related dysfunction, which is mainly associated with inadequate blood flow. CVC dysfunction is a major cause of inadequate depuration. Thrombus, intraluminal and extrinsic, as well as fibrous connective tissue sheath (traditionally indicated as fibrin sheath) formation play a central role in establishing CVC dysfunction. Thrombolysis with urokinase or recombinant tissue plasminogen activator (rTPA) can be undertaken in the dialysis unit, restoring adequate blood flow in most patients, preserving the existing catheter, and avoiding an interventional procedure. If thrombolytics fail, mainly because of the presence of fibrous connective tissue sheath, catheter exchange with fibrin sheath disruption may be successful and preserve the venous access site. Prevention of CVC dysfunction is important for containing costly pharmacologic and interventional treatments, which also affect patients' quality of life. Prevention is based on the use of anticoagulant and/or thrombolytic CVC locks, which are only partially effective. Chronic oral anticoagulation with warfarin has also been proposed, but its use for this indication is controversial and its overall risk-benefit profile has not been clearly established.

Tunneled central venous catheter exchange: techniques to improve prevention of air embolism.

Malfunctioning tunneled hemodialysis central venous catheters (CVCs), because of thrombotic or infectious complications, are frequently exchanged. During the CVC exchanging procedure, there are several possible technical complications, as in first insertion, including air embolism. Prevention remains the key to the management of air embolism. Herein, we emphasize the technical tricks capable of reducing the risk of air embolism in long-term CVC exchange. In particular, adoption of a 5 to 10 degrees Trendelenburg position, direct puncture of the previous CVC venous lumen for guide-wire insertion, as opposed to guide-wire introduction after cutting the CVC, a light manual compression of the internal jugular vein venotomy site after catheter removal. The Valsalva maneuvre in collaborating patients, valved introducers, and correction of hypovolemia are also useful precautions. Principles of air embolism diagnosis and treatment are also outlined in the article.