Case report: A compound heterozygous mutations in ARSA associated with adult-onset metachromatic leukodystrophy.

Metachromatic Leukodystrophy (MLD) is a rare autosomal recessive disease, which is caused by mutations in the arylsulfatase A (ARSA) gene. The ARSA gene is located on chromosome 22q13, containing eight exons. According to the age of onset, MLD can be divided into late infantile type, juvenile type, and adult type. Adult MLD has an insidious onset after the age of 16 years. Additionally, intellectual as well as behavioral changes, such as memory deficits or emotional instability, are commonly the first presenting symptoms. There is a study that reported an adult-onset MLD manifested cognitive impairment progressively due to compound heterozygous mutations of NM_000487: c.[185_186dupCA], p.(Asp63GlnfsTer18), and NM_000487: c.[154G>T], p.(Gly172Cys), rs74315271 in the ARSA gene, finding that the c.[154G>T], p.(Gly172Cys) is a novel missense mutation. Brain magnetic resonance imaging (MRI) revealed symmetrical demyelination of white matter. The activity of ARSA enzymatic in leukocytes decreased. Nerve conduction studies displayed that evidence of polyneuropathy was superimposed upon diffuse, uniform demyelinating, and sensorimotor polyneuropathy. Family genes revealed that each family member carried one of two heterozygous mutant genes. She has been discharged and is currently being followed up. This study found a compound heterozygous mutation in the ARSA gene associated with MLD and identified a novel missense mutation NM_000487: c.[154G>T], p.(Gly172Cys), rs74315271. This will provide a critical clue for prenatal diagnosis of the offspring in this family, and expand the mutation spectrum of MLD-related ARSA.

Isolation of Mouse Cerebral Microvasculature for Molecular and Single-Cell Analysis.

Brain microvasculature forms a specialized structure, the blood-brain barrier (BBB), to maintain homeostasis and integrity of the central nervous system (CNS). The BBB dysfunction is emerging as a critical contributor to multiple neurological disorders, including stroke, traumatic brain injury, autoimmune multiple sclerosis, and neurodegenerative diseases. The brain microvasculature exhibits highly cellular and regional heterogeneity to accommodate dynamic changes of microenvironment during homeostasis and diseases. Thus, investigating the underlying mechanisms that contribute to molecular or cellular changes of the BBB is a significant challenge. Here, we describe an optimized protocol to purify microvessels from the mouse cerebral cortex using mechanical homogenization and density-gradient centrifugation, while maintaining the structural integrity and functional activity of the BBB. We show that the isolated microvessel fragments consist of BBB cell populations, including endothelial cells, astrocyte end-feet, pericytes, as well as tight junction proteins that seal endothelial cells. Furthermore, we describe the procedures to generate single-cell suspensions from isolated microvessel fragments. We demonstrate that cells in the single-cell suspensions are highly viable and suitable for single-cell RNA-sequencing analysis. This protocol does not require transgenic mice and cell sorting equipment to isolate fluorescence-labeled endothelial cells. The optimized procedures can be applied to different disease models to generate viable cells for single-cell analysis to uncover transcriptional or epigenetic landscapes of BBB component cells.

Dimethyl fumarate attenuates reactive microglia and long-term memory deficits following systemic immune challenge.

BACKGROUND: Systemic inflammation is associated with increased cognitive decline and risk for Alzheimer's disease. Microglia (MG) activated during systemic inflammation can cause exaggerated neuroinflammatory responses and trigger progressive neurodegeneration. Dimethyl fumarate (DMF) is a FDA-approved therapy for multiple sclerosis. The immunomodulatory and anti-oxidant properties of DMF prompted us to investigate whether DMF has translational potential for the treatment of cognitive impairment associated with systemic inflammation. METHODS: Primary murine MG cultures were stimulated with lipopolysaccharide (LPS) in the absence or presence of DMF. MG cultured from nuclear factor (erythroid-derived 2)-like 2-deficient (Nrf2 -/- ) mice were used to examine mechanisms of DMF actions. Conditioned media generated from LPS-primed MG were used to treat hippocampal neuron cultures. Adult C57BL/6 and Nrf2 -/- mice were subjected to peripheral LPS challenge. Acute neuroinflammation, long-term memory function, and reactive astrogliosis were examined to assess therapeutic effects of DMF. RESULTS: DMF suppressed inflammatory activation of MG induced by LPS. DMF suppressed NF-κB activity through Nrf2-depedent and Nrf2-independent mechanisms in MG. DMF treatment reduced MG-mediated toxicity towards neurons. DMF suppressed brain-derived inflammatory cytokines in mice following peripheral LPS challenge. The suppressive effect of DMF on neuroinflammation was blunted in Nrf2 -/- mice. Importantly, DMF treatment alleviated long-term memory deficits and sustained reactive astrogliosis induced by peripheral LPS challenge. DMF might mitigate neurotoxic astrocytes associated with neuroinflammation. CONCLUSIONS: DMF treatment might protect neurons against toxic microenvironments produced by reactive MG and astrocytes associated with systemic inflammation.

A Combination of Three Repurposed Drugs Administered at Reperfusion as a Promising Therapy for Postischemic Brain Injury.

Cerebral ischemia leads to multifaceted injury to the brain. A polytherapeutic drug that can be administered immediately after reperfusion may increase protection to the brain by simultaneously targeting multiple deleterious cascades. This study evaluated efficacy of the combination of three clinically approved drugs: lamotrigine, minocycline, and lovastatin, using two mouse models: global and focal cerebral ischemia induced by transient occlusion of the common carotid arteries or the middle cerebral artery, respectively. In vitro, the combination drug, but not single drug, protected neurons against oxygen-glucose deprivation (OGD)-induced cell death. The combination drug simultaneously targeted cell apoptosis and DNA damage induced by ischemia. Besides acting on neurons, the combination drug suppressed inflammatory processes in microglia and brain endothelial cells induced by ischemia. In a transient global ischemia model, the combination drug, but not single drug, suppressed microglial activation and inflammatory cytokine production, and reduced neuronal damage. In a transient focal ischemia model, the combination drug, but not single drug, attenuated brain infarction, suppressed infiltration of peripheral neutrophils, and reduced neurological deficits following ischemic stroke. In summary, the combination drug confers a broad-spectrum protection against ischemia/reperfusion (I/R) injury and could be a promising approach for early neuroprotection after out-of-hospital cardiac arrest or ischemic stroke.

3H-1,2-Dithiole-3-thione as a novel therapeutic agent for the treatment of ischemic stroke through Nrf2 defense pathway.

Cerebral ischemic stroke accounts for more than 80% of all stroke cases. During cerebral ischemia, reactive oxygen species produced in brain tissue induce oxidative stress and inflammatory responses. D3T, the simplest compound of the cyclic, sulfur-containing dithiolethiones, is found in cruciferous vegetables and has been reported to induce antioxidant genes and glutathione biosynthesis through activation of Nrf2. In addition to antioxidant activity, D3T was also reported to possess anti-inflammatory effects. In this study, we evaluated the therapeutic potential of D3T for the treatment of ischemic stroke and investigated the mechanisms underlying the protective effects of D3T in ischemic stroke. Mice subjected to transient middle cerebral artery occlusion/reperfusion (tMCAO/R) were administered with vehicle or D3T to evaluate the effect of D3T in cerebral brain injury. We observed D3T reduced infarct size, decreased brain edema, lessened blood-brain barrier disruption, and ameliorated neurological deficits. Further investigation revealed D3T suppressed microglia (MG) activation and inhibited peripheral inflammatory immune cell infiltration of CNS in the ischemic brain. The protective effect of D3T in ischemic stroke is mediated through Nrf2 induction as D3T-attenuated brain injury was abolished in Nrf2 deficient mice subjected to tMCAO/R. In addition, in vitro results indicate the induction of Nrf2 by D3T is required for its suppressive effect on MG activation and cytokine production. In summary, we demonstrate for the first time that D3T confers protection against ischemic stroke, which is mediated through suppression of MG activation and inhibition of CNS peripheral cell infiltration, and that the protective effect of D3T in ischemic stroke is dependent on the activation of Nrf2.

Analysis for Discordance of Positive and Negative Blood Typing by Gel Card / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the method of Gel card identifying ABO blood group, determine the inconsistent cause and the distribution of disease affecting factors, and put forward a method of its solutions.</p><p><b>METHODS</b>To collect 240 positive and negative typing-discordant blood speciments from patients examined by Gel card and send these speciments to blood type reference laboratory for examining with the classic tube method and serological test, such as salivary blood-group substance, in order to performe genotyping method when serologic test can not be determined.</p><p><b>RESULTS</b>Among 240 positive and negative typing-discordant blood speciments from patients examined by Gel card, 107 blood speciments were positive and negative consistent examined by false agglutination test (44.58%), 133 blood specinents were discordent examined by false agglutination (55.42%), out of them, 35 cases (14.58%) with inconsistent cold agglutination test, 22 cases (9.17%) with weakened AB antigenicity, 16 cases (6.67%) with ABO subtyping, 12 cases (5.00%) with positive direct antiglobulin test, 11 cases (4.58%) with reduced or without antibodies, 11 cases (4.58%) with false aggregation caused by drugs or protein, 11 cases (4.58%) with salivary blood-type substances, 8 cases (3.33%) with non-ABO alloantibody, and 7 cases (2.92%) with allogeneic bone marrow transplantation. The distribution of disease were following: blood disease (16.83%), tumor (11.88%), and cardiopulmonary diseases (11.39%); chi-square test results indicated that the distribution significantly different.</p><p><b>CONCLUSION</b>The analysis of ABO blood grouping shows a variety factors influencing positive and negative blood typing, and the Gel Card identification can produc more false positive blood types. Therefore, more attention should be paid on the high incidence diseases, such as blood disease, tumor, and cardiopulmonary disease.</p>

The Status and Associated Factors of Successful Aging among Older Adults Residing in Longevity Areas in China.

OBJECTIVE: This study aims to assess the status of successful aging (SA) in longevity areas in China and explore multiple factors associated with SA among the young-old and oldest-old. METHODS: A total of 2296 elderly people aged 65 and older were interviewed in the longevity areas sub-sample of the Chinese Longitudinal Healthy Longevity Survey (CLHLS) in 2012. Baseline assessments included a researcher-administered questionnaire, physical examination, and laboratory testing. A logistic regression model was used to identify factors associated with SA. RESULTS: The prevalence of SA was 38.81% in the CLHLS in 2012. There were significant differences between ages groups, with SA compromising 56.85% among ⋝65 years group and 20.31% among ⋝100 years group (χ2trend=126.73, P<0.01). The prevalence of SA among females was 33.59%, which was significantly lower than that among males (45.58%) (χ2gender=33.65, P<0.05). In the regression analysis, having anemia (OR=0.744, 95% CI: 0.609-0.910), poor lifestyle (OR=0.697, 95% CI: 0.568-0.854), poor sleep quality (OR=0.558, 95% CI: 0.456-0.682), and central obesity (OR=0.684, 95% CI: 0.556-0.841) were the main factors associated with SA. The promoting SA rate decreased as age increased, and the group of 65-79 years had higher odds than the other age group. CONCLUSION: Preventing central obesity, improving sleep quality and promoting healthy lifestyle may contribute to achieve SA among the elderly.

3H-1,2-dithiole-3-thione as a novel therapeutic agent for the treatment of experimental autoimmune encephalomyelitis.

3H-1,2-dithiole-3-thione (D3T), the simplest member of the sulfur-containing dithiolethiones, is found in cruciferous vegetables, and has been previously reported to be a potent inducer of antioxidant genes and glutathione biosynthesis by activation of the transcription factor Nrf2. D3T is a cancer chemopreventive agent and possesses anti-inflammatory properties. Although D3T has been shown to protect against neoplasia, the effect of D3T in the autoimmune inflammatory disease multiple sclerosis/experimental autoimmune encephalomyelitis (EAE) is unknown. The present study is the first report of the therapeutic effect of D3T in EAE. Our results show D3T, administered post immunization, not only delays disease onset but also dramatically reduces disease severity in EAE. Strikingly, D3T, administered post disease onset of EAE, effectively prevents disease progression and exacerbation. Mechanistic studies revealed that D3T suppresses dendritic cell activation and cytokine production, inhibits pathogenic Th1 and Th17 differentiation, represses microglia activation and inflammatory cytokine expression, and promotes microglia phase II enzyme induction. In summary, these results indicate that D3T affects both innate and adaptive immune cells, and the protective effect of D3T in EAE might be attributed to its effects on modulating dendritic cell and microglia activation and pathogenic Th1/Th17 cell differentiation.

Interferon-ß Modulates Inflammatory Response in Cerebral Ischemia.

BACKGROUND: Stroke is a leading cause of death in the world. In >80% of strokes, the initial acute phase of ischemic injury is due to the occlusion of a blood vessel resulting in severe focal hypoperfusion, excitotoxicity, and oxidative damage. Interferon-ß (IFNß), a cytokine with immunomodulatory properties, was approved by the US Food and Drug Administration for the treatment of relapsing-remitting multiple sclerosis for more than a decade. Its anti-inflammatory properties and well-characterized safety profile suggest that IFNß has therapeutic potential for the treatment of ischemic stroke. METHODS AND RESULTS: We investigated the therapeutic effect of IFNß in the mouse model of transient middle cerebral artery occlusion/reperfusion. We found that IFNß not only reduced infarct size in ischemic brains but also lessened neurological deficits in ischemic stroke animals. Further, multiple molecular mechanisms by which IFNß modulates ischemic brain inflammation were identified. IFNß reduced central nervous system infiltration of monocytes/macrophages, neutrophils, CD4(+) T cells, and γδ T cells; inhibited the production of inflammatory mediators; suppressed the expression of adhesion molecules on brain endothelial cells; and repressed microglia activation in the ischemic brain. CONCLUSIONS: Our results demonstrate that IFNß exerts a protective effect against ischemic stroke through its anti-inflammatory properties and suggest that IFNß is a potential therapeutic agent, targeting the reperfusion damage subsequent to the treatment with tissue plasminogen activator.

The correlation analysis of the physical and mental status of organ transplanted patients' family members during the patients' rehabilitation period and the long term survival of the transplanted organ / 中国实用护理杂志

ObjectiveTo know about the correlation analysis of the physical and mental status of organ transplanted patients' family members during the patients' rehabilitation period and the long term survival of the transplanted organs.MethodsA total of 600 organ transplanted patients' family members were investigated by Zung self-rating anxiety scale (SAS) and simple coping style questionnaire.The results underwent subsequent analysis.ResultsAnxious state of organ transplanted patients' family members was more severe than that of domestic norm (P<0.01).There were some differences in terms of anxious state of family members with different genders,education backgrounds,income and the resource of medical expenditure (P<0.05).Family members most took positive coping styles,whereas fewer adopted negative coping styles (P<0.01).Positive coping styles were negatively correlated with the anxiety of family members (P<0.01) and positively correlated with long term survival of transplanted organ(P<0.05).However,negative coping styles of family members were significantly positively correlated with their anxiety (P<0.05) and were negatively correlated with long term survival of transplanted organ.ConclusionsThe anxiety generally exists in organ transplanted patients' family members.There are differences in terms of anxious state among family members of different genders,different education backgrounds,income or with the resource of medical expenditure.The more they adopt positive coping styles,the lower anxiety level they show and the longer the transplanted organ survive.Conversely,the more they adopt negative coping styles,the higher anxious level they show and the shorter the transplanted organ survive.

Synchronization measures of the scalp electroencephalogram can discriminate healthy from Alzheimer's subjects.

Three synchronization measures are applied to scalp electroencephalogram (EEG) data collected from 20 patients diagnosed to have either: (1) no dementia, (2) mild cognitive impairment (MCI), or (3) Alzheimer's disease (AD). We apply the three synchronization measures--the phase synchronization, and two measures of nonlinear interdependency--to the data collected from awake patients resting with eyes closed. We show that the synchronization in potential between electrodes near the left and right occipital lobes provides a statistically significant discriminant between the healthy and AD subjects, and the MCI and AD subjects. None of the three measures appears able to distinguish between the healthy and MCI subjects, although MCI subjects show synchronization values intermediate between healthy subjects (with high synchronization values) and AD subjects (with low synchronization values) on average.

Estrogen receptors alpha and beta in choroid plexus epithelial cells in Alzheimer's disease.

Estrogen replacement therapy (ERT) may reduce the risks of Alzheimer's disease (AD). One of the potential actions of estrogen is through its effect on beta-amyloid elimination into the cerebrospinal fluid (CSF) compartment from the brain parenchyma. CSF secretion is controlled largely by the choroid plexus (CP) epithelial cells. In this study, we evaluated the status of estrogen receptor (ER) alpha and beta subtypes in CP epithelial cells. Tissue from 49 AD and 12 non-AD patients were studied using immunohistochemistry with anti-ER antibodies. ER alpha and ER beta were present in CP epithelial cells in both cytosolic and nuclear compartments. Male and female AD patients had lower ER alpha and beta densities in CP compared to non-AD patients. We further stratified the female AD patients into four groups according to their hormonal status. Among the female AD patients, those without hysterectomy and with ERT had the highest ER alpha density. In contrast, those with hysterectomy and without ERT had the lowest ER alpha density. The effects of hysterectomy and ERT were additive. For ER beta, ERT but not the status of hysterectomy was associated with higher receptor density. The lower ER density in CP epithelial cells of AD patients supports a potential role of estrogen in the regulation of CSF secretion of beta-amyloid protein, which may affect the accumulation of beta-amyloid in the brain parenchyma. Among AD patients, the association of ER alpha density with status of hysterectomy and ERT supports estrogen effects through receptor-mediated mechanisms.