Thrombomodulin reduces α-synuclein generation and ameliorates neuropathology in a mouse model of Parkinson's disease.

The neurotoxic α-synuclein (α-syn) oligomers play an important role in the occurrence and development of Parkinson's disease (PD), but the factors affecting α-syn generation and neurotoxicity remain unclear. We here first found that thrombomodulin (TM) significantly decreased in the plasma of PD patients and brains of A53T α-syn mice, and the increased TM in primary neurons reduced α-syn generation by inhibiting transcription factor p-c-jun production through Erk1/2 signaling pathway. Moreover, TM decreased α-syn neurotoxicity by reducing the levels of oxidative stress and inhibiting PAR1-p53-Bax signaling pathway. In contrast, TM downregulation increased the expression and neurotoxicity of α-syn in primary neurons. When TM plasmids were specifically delivered to neurons in the brains of A53T α-syn mice by adeno-associated virus (AAV), TM significantly reduced α-syn expression and deposition, and ameliorated the neuronal apoptosis, oxidative stress, gliosis and motor deficits in the mouse models, whereas TM knockdown exacerbated these neuropathology and motor dysfunction. Our present findings demonstrate that TM plays a neuroprotective role in PD pathology and symptoms, and it could be a novel therapeutic target in efforts to combat PD. Schematic representation of signaling pathways of TM involved in the expression and neurotoxicity of α-syn. A TM decreased RAGE, and resulting in the lowered production of p-Erk1/2 and p-c-Jun, and finally reduce α-syn generation. α-syn oligomers which formed from monomers increase the expression of p-p38, p53, C-caspase9, C-caspase3 and Bax, decrease the level of Bcl-2, cause mitochondrial damage and lead to oxidative stress, thus inducing neuronal apoptosis. TM can reduce intracellular oxidative stress and inhibit p53-Bax signaling by activating APC and PAR-1. B The binding of α-syn oligomers to TLR4 may induce the expression of IL-1ß, which is subsequently secreted into the extracellular space. This secreted IL-1ß then binds to its receptor, prompting p65 to translocate from the cytoplasm into the nucleus. This translocation downregulates the expression of KLF2, ultimately leading to the suppression of TM expression. By Figdraw.

Next-generation sequencing combined with serological tests based pathogen analysis for a neurocysticercosis patient with a 20-year history:a case report.

BACKGROUND: Neurocysticercosis (NCC) is the most common helminthic infection of the central nervous system (CNS) caused by the larval stage of Taenia solium. Accurate and early diagnosis of NCC remains challenging due to its heterogeneous clinical manifestations, neuroimaging deficits, variable sensitivity, and specificity of serological tests. Next-generation sequencing (NGS)-based pathogen analysis in patient's cerebrospinal fluid (CSF) with NCC infection has recently been reported indicating its diagnostic efficacy. In this case study, we report the diagnosis of a NCC patient with a symptomatic history of over 20 years using NGS analysis and further confirmation of the pathology by immunological tests. CASE PRESENTATION: This study reports the clinical imaging and immunological features of a patient with a recurrent headache for more than 20 years, which worsened gradually with the symptom of fever for more than 7 years and paroxysmal amaurosis for more than 1 year. By utilizing NGS technique, the pathogen was detected in patient's CSF, and the presence of Taenia solium-DNA was confirmed by a positive immunological reaction to cysticercus IgG antibody in CSF and serum samples. The symptoms of the patient were alleviated, and the CSF condition was improved substantially after the anti-helminthic treatment. CONCLUSIONS: This study suggests that combining CSF NGS with cysticercus IgG testing may be a highly promising approach for diagnosing the challenging cases of NCC. Further studies are needed to evaluate the parasitic DNA load in patients' CSF for the diagnosis of disease severity, stage, and monitoring of therapeutic responses.

Prevalence and Risk Factors of Restless Legs Syndrome in Hemodialysis Patients.

OBJECTIVE: The current study aimed to investigate the prevalence and risk factors of restless legs syndrome (RLS) in patients undergoing hemodialysis, as well as the mortality and risks of cardiovascular and cerebrovascular events. METHODS: A total of 354 hemodialysis patients from four hospitals were enrolled. RLS was diagnosed using the International RLS Study Group (IRLSSG) criteria. The patients were evaluated face-to-face using the IRLSSG rating scale, Epworth Sleepiness Scale (ESS), Hamilton Anxiety Scale, Hamilton Depression Scale, and Pittsburgh Sleep Quality Index (PSQI). The patients were followed up for 9 months. Death was considered an endpoint event. The cardiovascular and cerebrovascular events were investigated. RESULTS: The prevalence of RLS in hemodialysis patients was 40.7% and was associated with factors such as duration of hemodialysis, hypersensitive C-reactive protein, hyperparathyroidism, glycosylated serum protein, and erythropoietin treatment. The scores of the PSQI, ESS, and Hamilton Depression Scale in the RLS group were significantly higher than those in the non-RLS group (p < 0.05). During follow-ups, the incidence rate of cardiovascular diseases was 18.8% in the RLS group and 8.6% in the non-RLS group (p < 0.005). The IRLSSG rating scores were significantly higher in RLS patients with kidney transplantation failure compared with those without transplantation (p < 0.05). CONCLUSION: The prevalence of RLS was high in hemodialysis patients. The risk factors of RLS included duration of hemodialysis, hypersensitive C-reactive protein, hyperparathyroidism, glycosylated serum protein, and erythropoietin treatment. RLS affected sleep quality and emotion and increased the risk of cardiovascular diseases in hemodialysis patients. RLS was more severe in patients with kidney transplantation failure compared with those without transplantation.

A case report: 1-year follow-up of cerebral sparganosis mansoni with a stroke-like onset.

BACKGROUND: Sparganosis mansoni is a parasitic disease caused by infection with the larvae of Spirometra spp. tapeworms. Its clinical manifestations and severity depend on the migration and the location of the parasites. The proportion of cerebral sparganosis in all Spirometra mansoni infections is 13.5% in Thailand and 12.4% in China. In the clinical setting, cerebral sparganosis is often misdiagnosed due to atypical characteristics, irregular intracranial location, and atypical epidemiology. CASE PRESENTATION: The patient in the case study suffered from an acute paroxysmal attack of lateral numbness, accompanied with focal epilepsy. He was admitted to the neurology department as a stroke patient but was later diagnosed with cerebral sparganosis mansoni following lab and radiology investigations. He was fully recovered and free of Spirometra mansoni one year after initial consultation following several courses of oral praziquantel. The current report focuses on the diagnosis, treatment and follow up of this patient. CONCLUSIONS: A case of cerebral sparganosis mansoni with a stroke-like onsetsuggests that in the clinical diagnosis, neurologists should pay attention to brain lesions and look out for the possibility of neuroparasitic infections when dealing with patients with stroke-like onset accompanied by epilepsy. Detections of relevant antibodies in blood and cerebrospinal fluid may be necessary. The combination of the epidemiological history, clinical manifestations, detection of parasite antibody, head radiology, pathological biopsy, and identification of parasites will help us in diagnosis and differential diagnosis.

Restless legs syndrome secondary to pontine infarction: Clinical analysis of five cases.

OBJECTIVE: Pontine infarction is a common type of stroke in the cerebral deep structures, resulting from occlusion of small penetrating arteries, may manifest as hemi-paralysis, hemi-sensory deficit, ataxia, vertigo, and bulbar dysfunction, but patients presenting with restless legs syndrome (RLS) are extremely rare. Herein, we reported five cases with RLS as a major manifestation of pontine infarction. METHODS: Five cases of pontine infarction related RLS were collected from July 2013 to February 2016. The diagnosis of RLS was made according to criteria established by the International RLS Study Group (IRLSSG) in 2003. Neurological functions were assessed according to the National Institutes of Health Stroke Scale (NIHSS) and modified Rankin Scale (mRS). Severity of RLS was based on the International RLS Rating Scale (IRLS-RS). Sleep quality was assessed by Epworth Rating Scale (ERS), and individual emotional and psychological states were assessed by Hamilton Depression Scale (HDS) and Hamilton Anxiety Scale (HAS). RESULTS: The laboratory data at the onset including hemoglobin, serum concentration of homocysteine, blood urea nitrogen (BUN), creatinine, electrolytes, and thyroid hormones were normal. The electroencephalogram (EEG), lower-extremity somatosensory evoked potential (SEP), and nerve conduction velocity (NCV) in four limbs were normal. The average period of follow-up was 34.60 ± 12.76 months. The MRI examination showed acute or subacute pontine infarction lesions, 3 cases in the rostral inner side, 1 case in the rostral lateral and inner side, and 1 case in rostral lateral side. The neurological deficits included weakness in 4 cases, contralateral sensory deficit in 1 case, and ataxia in 2 cases. All 5 patients presented with symptom of RLS at or soon after the onset of infarction and 4 patients experienced uncomfortable sensations in the paralyzed limbs contralateral to the ischemic lesion. Their neurological deficits improved significantly 2 weeks later, but the symptoms of RLS did not resolve. Among them, 3/5 patients were treated with dopaminergic drugs. At the end of the follow-up, RLS symptom eventually resolved in 3 patients but persisted in two. The IRLS-RS, NIHSS and mRS scores were significantly lower at the onset than those at the last follow-up (P = 0.035, 0.024 and 0.049, respectively). However, there was no significant difference in the ERS, HDS and HAS scores (P = 0.477, 0.226 and 0.778, respectively). CONCLUSION: RLS can be an onset manifestation of pontine infarction, clinicians should be aware of this potential symptom. RLS usually occurs in the paralyzed limbs contralateral to the infarction lesion. The pathogenesis still needs further investigation.

Human cytomegalovirus-IgM seropositivity is not associated with atherogenic alterations of lipid profiles and inflammatory status in ischemic stroke patients: a preliminary study.

BACKGROUND: Past exposure to human cytomegalovirus has been suggested to participate in the pathogenetic events associated with atherosclerotic lesion establishment and progression. However, whether ongoing human cytomegalovirus infection is related to plaque instability, and subsequent acute cerebral ischemia, is relatively unknown. The purpose of this study was to evaluate the potential relationships between active human cytomegalovirus infection and ischemic stroke, especially in regard to metabolism and inflammation. METHODS: Ninety-nine acute ischemic stroke patients, associated with large artery atherosclerosis, were divided into two groups based on the presence or absence of human cytomegalovirus immunoglobulin M (IgM) (human cytomegalovirus-IgM-positive/human cytomegalovirus-IgM-negative = 33:66). Baseline clinical characteristics, inflammatory factors, and biochemical assessments were compared in both groups. Then, all patients and human cytomegalovirus-IgM-positive patients were divided into quartiles according to their high-sensitivity C-reactive protein levels, respectively, and risk factors were compared. Finally, correlations between inflammatory factors (high-sensitivity C-reactive protein and white blood cell count) and other atherosclerosis risk factors in both human cytomegalovirus-IgM-positive and -negative subjects were evaluated. RESULTS: An association between human cytomegalovirus-IgM seropositivity and atherogenic modification of metabolism and inflammatory status were not found in this study. Both age and white blood cell count increased across quartiles of high-sensitivity C-reactive protein in all subjects (P = 0.001), while age and low-density lipoprotein cholesterol increased across quartiles of high-sensitivity C-reactive protein in the human cytomegalovirus-IgM-positive group (P = 0.02 and 0.007, respectively). Multivariate linear regression analysis showed that high-sensitivity C-reactive protein was associated with age in human cytomegalovirus-IgM-positive group (P = 0.002), while no other factor was associated with white blood cell count in these subjects. CONCLUSION: Our study provided no evidence for the direct implication of active systemic human cytomegalovirus infection, represented by human cytomegalovirus-IgM positivity, in the pathogenesis of acute ischemic strokes, particularly those involving plaque instability and metabolic disorders.

[Fluvastatin's effect on atherogenesis in apolipoprotein-E knockout mice infected by cytomegalovirus].

OBJECTIVE: The goal of this study was to investigate whether murine cytomegalovirus (MCMV) is able to exacerbate the atherosclerotic process in apolipoprotein E knockout (apoE -/-) mice, and the effect of fluvastatin on the atherogenesis. METHODS: The apoE-/- mice kept on a west diet were given low dosage of MCMV. At 14,18 and 24 weeks post infection, AS lesion were measured on aorta. The fluvastatin was administered, and AS lesion were measured accordingly above. RESULTS: We observed that in the chronic phase of the infection, AS lesion area was significantly increased. MCMV gB mRNA was not amplified by real-time PCR from the arterial wall. The IgG antibody level of MCMV in blood plasma and the content of virus DNA in salivary gland were not correlated with AS lesions. After the administration of fluvastatin, there was no significant difference of AS lesions between MCMV infected group and mock-infected group. CONCLUSION: MCMV may aggravate the AS lesion in apoE -/- mice in the chronic phase of infection, and promote more severe type of AS lesions. But it might not be the direct effects of mechanism of MCMV on the local lesion of AS. Fluvastatin could meliorate the progression of AS after MCMV infection, but this was not accomplished by decreasing MCMV duplication.

[Effect of cytomegalovirus infection on expression of matrix metalloproteinase-2 in human endothelial cells].

OBJECTIVE: To investigate the effect of cytomegalovirus infection on expression of matrix metalloproteinase-2 in human endothelial cells. METHODS: Human umbilical vein endothelial cells were cultured in vitro. Cells between 3-6 passages were infected with cytomegalovirus for different time. Expression of matrix metalloproteinase-2 mRNA was analyzed by reverse transcription-polymerase chain reaction, matrix metalloproteinase-2 activity was detected by gel zymography. RESULTS: Expression of matrix metalloproteinase-2 mRNA and its activity 6 hours after infection was almost equal to control, and was greatly enhanced 12 and 24 hours after infection. CONCLUSION: Cytomegalovirus infection up-regulates expression of matrix metalloproteinase-2 in human endothelial cells. It might be one of the mechanisms that cytomegalovirus is involved in atherosclerosis.

[Construction and expression of the fusion protein DT389-hIL-13 and its cytotoxicity to glioma cell lines].

OBJECTIVE: To construct a fusion protein toxin DT389-hIL-13 which comprises the N-terminal 389 amino acids of diphtheria toxin (DT389) and human interlukin 13 (hIL-13), and to explore its cytotoxicity on U251 glioma cells. METHODS: The cDNA of hIL-13 gene was amplified by PCR and linked with the 3'-terminus of the gene encoding the N-terminal 389 amino acids, which correspond to the enzyme domain and transmembrane domain of diphtheria toxin. The tandem constructed gene was then inserted into an E. coli expression vector pET30a. The resulted expression vector was transformed into E. coli BL21 and induced by IPTG. The expressed protein was analyzed by SDS-PAGE and Western blot analysis. U(251) glioma cells were cultured DT389-hIl-13 was added into the culture. The cytotoxicity was determined using colorimetric MTS proliferation assay. RESULTS: The expression plasmid pET30a/DT389-hIL13 was constructed with correct sequence. The recombinant protein was successfully expressed in E. coli in manner of inclusion body and with a relative molecular weight of about 55 000, which reacted well with both anti-diphtheria toxin and anti-hIL-13 polyclonal antibody in Western blot assay. The purified recombinant chimeric toxin was found to effectively inhibit the prolifieration of glioblastoma multiforme cells bearing high affinity hIL-13 receptors, and resulted in dose-dependent relationship with 50% inhibition concentration (IC(50)) of 5 x 10(-)11mol/L. CONCLUSION: Prokaryotic expression system can be recruited to produce recombinant chimeric toxin DT389-hIL-13. The results may lay a foundation for preparing specific the agent targets for tumors overexpressing IL-13 receptor.

[Expression, purification and specific monoclonal antibodies preparation of diphtheria toxin A fragment].

Diphtheria toxin A fragment (DTA) is an essential catalytic domain of diphtheria toxin (DT)-based immunotoxin. DTA protein and its antibodies play an important role in the studies on toxicology, purification and identification of DT-based immunotoxins. In this paper, DTA was expressed and purified from E. coli. After Q-Sepharose FF chromatography and (Ni+)-Sepharose affinity chromatography, 6 x His-DTA fusion protein with 90% purity was achieved. Using the purified DTA as antigen to immunize BalB/c mice, 2 hybridoma cell lines (designated as 3B6 and 3B9, respectively) secreting monoclonal antibodies (McAbs) against DTA were established. Investigations showed that both McAbs were characterized as IgG1 with titers of 1: 10(6). The binding of the McAbs to DTA was competitively inhibited by horse sera against DT. The fact that anti-DTA McAbs could be used in western blot analysis and affinity chromatography purification of DT-based immunotoxins implied that they will be useful agents in the studies on DT-based immunotoxins.