Neurology ethics at the end of life.

Ethical challenges in medical decision making are commonly encountered by clinicians caring for patients afflicted by neurological injury or disease at the end of life (EOL). In many of these cases, there are conflicting opinions as to what is right and wrong originating from multiple sources. There is a particularly high prevalence of impaired patient judgment and decision-making capacity in this population that may result in a misrepresentation of their premorbid values and goals. Conflict may originate from a discordance between what is legal or from stakeholders who view and value life and existence differently from the patient, at times due to religious or cultural influences. Promotion of life, rather than preservation of existence, is the goal of many patients and the foundation on which palliative care is built. Those who provide EOL care, while being respectful of potential cultural, religious, and legal stakeholder perspectives, must at the same time recognize that these perspectives may conflict with the optimal ethical course to follow. In this chapter, we will attempt to review some of the more notable ethical challenges that may arise in the neurologically afflicted at the EOL. We will identify what we believe to be the most compelling ethical arguments both in support of and opposition to specific EOL issues. At the same time, we will consider how ethical analysis may be influenced by these legal, cultural, and religious considerations that commonly arise.

Reversible Encephalopathy Due to Venous Hypertension From Arteriovenous Hemodialysis Graft.

Venous congestive encephalopathy is a rare complication in patients with arteriovenous hemodialysis grafts. It commonly manifests as encephalopathy of fluctuating severity, often with seizures. Because these patients typically have multiple significant chronic health problems, venous hypertension's contribution to the patient's cognitive decline can easily be overlooked. This nonspecific presentation can make diagnosis challenging, therefore delaying treatment. We describe a case of progressive, fluctuating encephalopathy with seizures due to cerebral venous congestion caused by arterial shunting from an upper limb arteriovenous (AV) fistula to the proximal venous system, that was initially unrecognized, yet ultimately reversed by elimination of the source of venous hypertension.

Monomethyl fumarate treatment impairs maturation of human myeloid dendritic cells and their ability to activate T cells.

BACKGROUND: Dimethyl fumarate (DMF) and its active metabolite monomethyl fumarate (MMF) effectively lead to reduction in disease relapses and active magnetic resonance imaging (MRI) lesions. DMF and MMF are known to be effective in modulating T- and B-cell responses; however, their effect on the phenotype and function of human myeloid dendritic cells (mDCs) is not fully understood. OBJECTIVE: To investigate the role of MMF on human mDCs maturation and function. METHODS: mDCs from healthy controls were isolated and cultured in vitro with MMF. The effect of MMF on mDC gene expression was determined by polymerase chain reaction (PCR) array after in vitro MMF treatment. The ability of mDCs to activate T cells was assessed by in vitro co-culture system. mDCs from DMF-treated multiple sclerosis (MS) patients were analyzed by flow cytometry and PCR. RESULTS: MMF treatment induced a less mature phenotype of mDCs with reduced expression of major histocompatibility complex class II (MHC-II), co-stimulatory molecules CD86, CD40, CD83, and expression of nuclear factor κB (NF-κB) subunits RELA and RELB. mDCs from DMF-treated MS patients also showed the same immature phenotype. T cells co-cultured with MMF-treated mDCs showed reduced proliferation with decreased production of interferon gamma (IFN-γ), interleukin-17 (IL-17), and granulocyte-macrophage colony-stimulating factor (GM-CSF) compared to untreated cells. CONCLUSION: We report that MMF can modulate immune response by affecting human mDC function.

Differences in Intercellular Communication During Clinical Relapse and Gadolinium-Enhanced MRI in Patients With Relapsing Remitting Multiple Sclerosis: A Study of the Composition of Extracellular Vesicles in Cerebrospinal Fluid.

This study was designed based on the hypothesis that changes in both the levels and surface marker expression of extracellular vesicles (EVs) isolated from the cerebrospinal fluid (CSF) may be associated with the clinical form, disease activity, and severity of multiple sclerosis (MS). The analyzes were performed on subjects affected by MS or other neurological disorders. EVs, which were isolated by ultracentrifugation of CSF samples, were characterized by flow cytometry. A panel of fluorescent antibodies was used to identify the EV origin: CD4, CCR3, CCR5, CD19, and CD200, as well as isolectin IB4. The Mann-Whitney U-test and Kruskal-Wallis test were used for statistical analyzes. EVs isolated from the CSF were more abundant in patients with progressive MS and in those with a clinically isolated syndrome than in all the other groups examined. Furthermore, an important change in the number of EVs and in their surface marker expression occurred during active phases of MS [i.e., clinical relapses and the presence of enhancing lesions on magnetic resonance imaging (MRI)]. In particular, the number of CSF-EVs increased in patients affected by MS during clinical relapse; this finding was associated with a decrease in the number of CD19+/CD200+ (naïve B cells) EVs. These markers are expressed by immature and naïve B lymphocytes, and to the best of our knowledge, this double staining has never been associated with MS, but their reduction has been observed in patients with another type of Th1 cell-mediated autoimmune disease. In contrast, the presence of lesions in the brain and spine on gadolinium-enhanced MRI was associated with an increase in the numbers of CCR3+/CCR5+ (subset of CD8 memory T cells), CD4+/CCR3+ (Th2 cells), and CD4+/CCR5+ (Th1 cells) CSF-EVs. Two points are worth emphasizing: (i) the data obtained in this study confirm that CSF-EVs represent a potentially promising tool to identify biomarkers specific for different phases of MS; and (ii) Considering the role of EVs in intercellular communication, our results provide some insights that improve our understanding of the relationships among some of the cell types that are mainly involved in MS pathogenesis (e.g., lymphocytes, glia, and neurons).

Identification of MS-specific serum miRNAs in an international multicenter study.

OBJECTIVE: To identify circulating microRNAs (miRNAs) linked to disease, disease stage, and disability in MS across cohorts. METHODS: Samples were obtained from the Comprehensive Longitudinal Investigation of Multiple Sclerosis (CLIMB, Boston, MA), EPIC (San Francisco, CA), AMIR (Beirut, Lebanon) as part of the SUMMIT consortium, and Stockholm Prospective Assessment of Multiple Sclerosis (Stockholm, Sweden) cohorts. Serum miRNA expression was measured using locked nucleic acid-based quantitative PCR. Four groups were compared: (1) MS vs healthy control (HC), (2) relapsing-remitting (RR) vs HC, (3) secondary progressive (SP) vs HC, and (4) RR vs SP. A Wilcoxon rank-sum test was used for the comparisons. The association between each miRNA and the Expanded Disability Status Scale (EDSS) score was assessed using the Spearman correlation coefficient. For each comparison, the p values were corrected for multiple comparisons using the approach of Benjamini and Hochberg to control the false discovery rate. RESULTS: In the CLIMB cohort, 5 miRNAs (hsa-miR-484, hsa-miR-140-5p, hsa-miR-320a, hsa-miR-486-5p, and hsa-miR-320c) showed a significant difference between patients with MS and healthy individuals; among these, miR-484 remained significant after accounting for multiple comparisons (p = 0.01). When comparing RRMS with HCs, hsa-miR-484 showed a significant difference (p = 0.004) between the groups after accounting for multiple group comparisons. When SP and HC were compared, 6 miRNAs (hsa-miR-484, hsa-miR-140-5p, hsa-miR-142-5p, hsa-miR-320a, hsa-miR-320b, and hsa-miR-320c) remained significantly different after accounting for multiple comparisons. Disability correlation analysis with miRNA provided 4 miRNAs (hsa-miR-320a, hsa-miR-337-3p, hsa-miR-199a-5p, and hsa-miR-142-5p) that correlated with the EDSS during the internal reproducibility phase. Among these, hsa-miR-337-3p was the most statistically significant miRNA that negatively correlated with the EDSS in three of the MS cohorts tested. CONCLUSIONS: These findings further confirm the use of circulating serum miRNAs as biomarkers to diagnose and monitor disease status in MS. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that levels of circulating miRNAs identify patients with MS.

Correlating serum micrornas and clinical parameters in amyotrophic lateral sclerosis.

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a debilitating neurologic disorder with poor survival rates and no clear biomarkers for disease diagnosis and prognosis. METHODS: We compared serum microRNA (miRNA) expression from patients with ALS with healthy controls and patients with multiple sclerosis and Alzheimer disease. We also correlated miRNA expression in cross-sectional and longitudinal cohorts of ALS patients with clinical parameters. RESULTS: We identified 7 miRNAs (miR-192-5p, miR-192-3p, miR-1, miR-133a-3p, miR-133b, miR-144-5p, miR-19a-3p) that were upregulated and 6 miRNAs (miR-320c, miR-320a, let-7d-3p, miR-425-5p, miR-320b, miR-139-5p) that were downregulated in patients with ALS compared with healthy controls, patients with Alzheimer disease, and patients with multiple sclerosis. Changes in 4 miRNAs (miR-136-3p, miR-30b-5p, miR-331-3p, miR-496) correlated positively and change in 1 miRNA (miR-2110) correlated negatively with changes in clinical parameters in longitudinal analysis. DISCUSSION: Our findings identified serum miRNAs that can serve as biomarkers for ALS diagnosis and progression. Muscle Nerve 58: 261-269, 2018.

Association between multiple sclerosis, cancer risk, and immunosuppressant treatment: a cohort study.

BACKGROUND: The association between multiple sclerosis (MS) and cancer has long been investigated with conflicting results. Several reports suggest an increased cancer risk among MS patients treated with immunosuppressant (IS) drugs. METHODS: We performed a cohort study including MS patients recruited at the Neurological Department of the University of Palermo. Mean follow-up period was ten years for the whole cohort. We calculated cancer incidence among patients treated with IS. Incidence rates were compared in the cohort by calculating the relative risk according to length and dose of exposure to IS. Cancer incidence among MS patients was compared to cancer incidence in the general population of Sicily in similar age groups. RESULTS: On an overall cohort of 531 MS patients (346 women and 185 men) exposed to IS, we estimated a crude incidence rate for cancer of 2.26% (2.02% in women, 2.7% in men). Cancer risk was higher compared to rates observed among an equal number of patients not exposed to IS, and to the risk in the general population in Sicily at similar age groups (adjusted HR: 11.05; CI 1.67-73.3; p = 0.013). CONCLUSION: The present study showed a higher cancer risk in MS patients associated only to previous IS exposure. Studies on long-term outcomes are essential to evaluate the possibility that treatment options that need to be considered for a long time-period may modify risk for life threatening diseases.

Dynamic regulation of serum aryl hydrocarbon receptor agonists in MS.

OBJECTIVE: Several factors influence the clinical course of autoimmune inflammatory diseases such as MS and inflammatory bowel disease. Only recently, the complex interaction between the gut microbiome, dietary factors, and metabolism has started to be appreciated with regard to its potential to modulate acute and chronic inflammation. One of the molecular sensors that mediates the effects of these environmental signals on the immune response is the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor with key functions in immune cells. METHODS: In this study, we analyzed the levels of AHR agonists in serum samples from patients with MS and healthy controls in a case-control study. RESULTS: We detected a global decrease of circulating AHR agonists in relapsing-remitting MS patients as compared to controls. However, during acute CNS inflammation in clinically isolated syndrome or active MS, we measured increased AHR agonistic activity. Moreover, AHR ligand levels in patients with benign MS with relatively mild clinical impairment despite longstanding disease were unaltered as compared to healthy controls. CONCLUSIONS: Collectively, these data suggest that AHR agonists in serum are dynamically modulated during the course of MS. These findings may guide the development of biomarkers to monitor disease activity as well as the design of novel therapeutic interventions for MS.

Comprehensive evaluation of serum microRNAs as biomarkers in multiple sclerosis.

OBJECTIVE: To identify circulating microRNAs (miRNAs) linked to disease stage and disability in multiple sclerosis (MS). METHODS: Sera from 296 participants including patients with MS, other neurologic diseases (Alzheimer disease and amyotrophic lateral sclerosis), and inflammatory diseases (rheumatoid arthritis and asthma) and healthy controls (HCs) were tested. miRNA profiles were determined using LNA (locked nucleic acid)-based quantitative PCR. Patients with MS were categorized according to disease stage and disability. In the discovery phase, 652 miRNAs were measured in sera from 26 patients with MS and 20 HCs. Following this, significant miRNAs (p < 0.05) from the discovery set were validated using quantitative PCR in 58 patients with MS, 30 HCs, and in 74 samples from other disease controls (Alzheimer disease, amyotrophic lateral sclerosis, asthma, and rheumatoid arthritis). RESULTS: We validated 7 miRNAs that differentiate patients with MS from HCs (p < 0.05 in both the discovery and validation phase); miR-320a upregulation was the most significantly changing serum miRNA in patients with MS. We also identified 2 miRNAs linked to disease progression, with miR-27a-3p being the most significant. Ten miRNAs correlated with the Expanded Disability Status Scale of which miR.199a.5p had the strongest correlation with disability. Of the 15 unique miRNAs we identified in the different group comparisons, 12 have previously been reported to be associated with MS but not in serum. CONCLUSIONS: Our findings identify circulating serum miRNAs as potential biomarkers to diagnose and monitor disease status in MS. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that circulating serum miRNAs can be used as biomarker for MS.

Alterations of the human gut microbiome in multiple sclerosis.

The gut microbiome plays an important role in immune function and has been implicated in several autoimmune disorders. Here we use 16S rRNA sequencing to investigate the gut microbiome in subjects with multiple sclerosis (MS, n=60) and healthy controls (n=43). Microbiome alterations in MS include increases in Methanobrevibacter and Akkermansia and decreases in Butyricimonas, and correlate with variations in the expression of genes involved in dendritic cell maturation, interferon signalling and NF-kB signalling pathways in circulating T cells and monocytes. Patients on disease-modifying treatment show increased abundances of Prevotella and Sutterella, and decreased Sarcina, compared with untreated patients. MS patients of a second cohort show elevated breath methane compared with controls, consistent with our observation of increased gut Methanobrevibacter in MS in the first cohort. Further study is required to assess whether the observed alterations in the gut microbiome play a role in, or are a consequence of, MS pathogenesis.

Identification of a novel mechanism of action of fingolimod (FTY720) on human effector T cell function through TCF-1 upregulation.

BACKGROUND: Fingolimod (FTY720), the first oral treatment for multiple sclerosis (MS), blocks immune cell trafficking and prevents disease relapses by downregulation of sphingosine-1-phosphate receptor. We determined the effect of FTY720 on human T cell activation and effector function. METHODS: T cells from MS patients and healthy controls were isolated to measure gene expression profiles in the presence or absence of FTY720 using nanostring and quantitative real-time polymerase chain reaction (qPCR). Cytokine protein expression was measured using luminex assay and flow cytometry analysis. Lentivirus vector carrying short hairpin RNA (shRNA) was used to knock down the expression of specific genes in CD4+ T cells. Chromatin immunoprecipitation was performed to assess T cell factor 1 (TCF-1) binding to promoter regions. Luciferase assays were performed to test the direct regulation of interferon gamma (IFN-γ) and granzyme B (GZMB) by TCF-1. Western blot analysis was used to assess the phosphorylation status of Akt and GSK3ß. RESULTS: We showed that FTY720 treatment not only affects T cell trafficking but also T cell activation. Patients treated with FTY720 showed a significant reduction in circulating CD4 T cells. Activation of T cells in presence of FTY720 showed a less inflammatory phenotype with reduced production of IFN-γ and GZMB. This decreased effector phenotype of FTY720-treated T cells was dependent on the upregulation of TCF-1. FTY720-induced TCF-1 downregulated the pathogenic cytokines IFN-γ and GZMB by binding to their promoter/enhancer regions and mediating epigenetic modifications. Furthermore, we observed that TCF-1 expression was lower in T cells from multiple sclerosis patients than in those from healthy individuals, and FTY720 treatment increased TCF-1 expression in multiple sclerosis patients. CONCLUSIONS: These results reveal a previously unknown mechanism of the effect of FTY720 on human CD4+ T cell modulation in multiple sclerosis and demonstrate the role of TCF-1 in human T cell activation and effector function.

Identification of biomarkers in cerebrospinal fluid and serum of multiple sclerosis patients by immunoproteomics approach.

Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the central nervous system. At present, the molecular mechanisms causing the initiation, development and progression of MS are poorly understood, and no reliable proteinaceous disease markers are available. In this study, we used an immunoproteomics approach to identify autoreactive antibodies in the cerebrospinal fluid of MS patients to use as candidate markers with potential diagnostic value. We identified an autoreactive anti-transferrin antibody that may have a potential link with the development and progression of MS. We found this antibody at high levels also in the serum of MS patients and created an immunoenzymatic assay to detect it. Because of the complexity and heterogeneity of multiple sclerosis, it is difficult to find a single marker for all of the processes involved in the origin and progression of the disease, so the development of a panel of biomarkers is desirable, and anti-transferrin antibody could be one of these.

Tumor diagnosis preceding Alzheimer's disease onset: is there a link between cancer and Alzheimer's disease?

Studies reporting an inverse association between Alzheimer's disease (AD) and cancer are scant. Available data are mostly based on ancillary findings of mortality data or obtained from studies evaluating frequency of neoplasms in AD patients independently if they occurred before or after AD. Moreover, some studies estimated frequencies of neoplasms in demented individuals, who were not necessarily AD patients. We estimated frequency of tumors preceding the onset of AD in AD patients and compared it to that of age- and gender-matched AD-free individuals. Occurrence of tumors preceding AD onset was assessed through a semi-structured questionnaire. Tumors were categorized as benign, malignant, or of uncertain classification and as endocrine-related or not. Odds ratios (OR), used as measure of the association between the two diseases, were adjusted for tumor categories and known risk factors for AD and tumors. We included 126 AD patients and 252 matched controls. Tumor frequency before AD onset was 18.2% among cases and 24.2% among controls. There was a suggestive trend of an overall inverse association between the two diseases (adjusted OR 0.6; 95% CI 0.4-1.1; p = 0.11). Risk for neoplasms was significantly reduced only for women (adjusted OR, 0.5; 95% CI 0.3-0.9; p = 0.03) and for endocrine related tumors (adjusted OR, 0.5; 95% CI 0.2-1; p = 0.04). Our study confirms the inverse association reported in previous epidemiological studies. Though our findings might be explained by processes playing an opposite role in tumors development and neurodegeneration, they are also suggestive for a possible role of estrogen.

Reversible posterior leukoencephalopathy syndrome in a patient with thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is an autoimmune disorder characterised by fever, microangiopathic haemolytic anaemia, renal insufficiency, and thrombocytopenia. Neurological involvement, a prominent component of TTP, is characterised by a variety of brain lesions which include reversible cerebral oedema or magnetic resonance imaging (MRI) features of reversible posterior leukoencephalopathy syndrome (RPLS). TTP is frequently associated with deficiency of the von Willebrand factor-cleaving protease, ADAMTS13.Here, we report a case of TTP with severe acute encephalopathy. Posterior leukoencephalopathy and brainstem oedema with triventricular hydrocephalus were observed on MRI. The low activity of ADAMTS13 was not observed and ADAMTS-13 antibodies were absent. Neurological symptoms and patient's condition were completely resolved by plasma exchange therapy in addition to high dose of methylprednisolone.

Leprosy: report of a case with severe peripheral neuropathy.

Leprosy (Hansen's disease) is a chronic granulomatous infectious disease, caused by Mycobacterium leprae, with cutaneous and neurological manifestations. Leprosy is very rare in Europe but some cases are reported, especially among people coming from endemic areas. Here, we report a case of Hansen's disease and emphasize the importance of a prompt diagnosis and treatment also in non-endemic areas.

Frequency and determinants for hemorrhagic transformation of cerebral infarction.

BACKGROUND: Frequency and associated risk factors for hemorrhagic transformation (HT), a worrying complication of ischemic stroke (IS), are not clearly defined. Our aim was to estimate the overall frequency and risk factors for HT in a hospital-based population. METHODS: A retrospective review of medical records of patients discharged from our department during the period 2004-2006 with a diagnosis of anterior IS. Demographic, clinical and hematological information was collected. Uni- and multivariate logistic regression analyses were used to estimate risk for spontaneous HT. RESULTS: We included 240 patients (125 males, 52%), mean age at admission was 72.5 years. HT was observed in 29 patients (12%). At univariate analysis, consciousness impairment at admission (OR 5.6, 95% CI 1.3-28.2), the presence of early CT signs (OR 2.4, 95% CI 1.1-5.3), infarcts of medium-large size (OR 11.3, 95% CI 4.1-30.8), cardioembolic stroke (OR 2.3, 95% CI 1.1-5.2) and low total cholesterol levels (OR 3.3, 95% CI 1.3-8.2) were significantly associated with HT. At multivariate analysis, only infarct size (OR 10.2, 95% CI 3.2-32.1) was still significantly associated with HT. CONCLUSIONS: Frequency of HT in our study was 12%. Consistently with previous results, HT was associated with the size of ischemic area. As patients included in our study did not receive thrombolytic therapy, our results are applicable to those patients whom clinicians, working in a hospital setting, usually deal with.