Complement C3 polymorphism is associated with the susceptibility of myasthenia gravis in Chinese adult patients.

Complement component 3 (C3) had been proved to be involved in the pathogenesis and exacerbation of both myasthenia gravis (MG) patients and experimental autoimmune myasthenia gravis (EAMG) models. We evaluated the underlying association between five SNPs (rs344555, rs7951, rs3745568, rs366510 and rs163913) in C3 gene and Chinese adult MG patients. Our study consisted of 409 adult MG patients and 487 healthy controls. Subgroups were classified by gender, onset age, thymoma, anti-AChR antibody, onset muscle involvement (ocular/generalized) and severity (Oosterhuis score at the maximal severity during the initial two years after the onset of MG). We found significant differences in allele frequencies between MG and the control group, between various MG subgroups and the control group in rs344555 and rs3745568. There were significant differences in genotype frequencies between MG group and the control group, between MG subgroups and the control group under the codominant and additive inheritance models in rs344555 and rs3745568. No association was found between the frequencies of these SNPs and the severity of MG. We also used a comprehensive classification which was close to the clinical scenario to minimize the interaction among clinical features. In rs344555, the T allele frequency in thymoma (-) AChR-Ab (+) subgroup was significantly higher than that in the control group. Our results indicated that rs344555 was associated with the susceptibility of Chinese adult MG patients; rs3745568 was probably associated with the susceptibility of Chinese adult MG patients. No association was found between the frequencies of these SNPs and the severity of MG.

Comparison of efficacy and tolerability of azathioprine, mycophenolate mofetil, and lower dosages of rituximab among patients with neuromyelitis optica spectrum disorder.

OBJECTIVE: To observe and compare the efficacy and tolerability of azathioprine (AZA), mycophenolate mofetil (MMF) and lower dosages of rituximab (RTX) among patients with neuromyelitis optica spectrum disorder. METHODS: In this prospective cohort, AQP4-IgG-seropositive patients with neuromyelitis optica spectrum disorder (NMOSD) were enrolled and randomly divided into three groups, using AZA, MMF or lower dosages of RTX (defined as 100mg RTX intravenous injection, once per week for 4 consecutive weeks) respectively. Annualized relapse rate (ARR), EDSS scores, CD19+ B-cell counts in peripheral blood, serum AQP-4-IgG titre and drug adverse reactions were compared between three groups. RESULTS: In the AZA group (n=22), MMF group (n=30) and RTX group (n=20), 54.5%, 60.0% and 65.0% of patients reached a relapse-free state and EDSS score improved in 90.9%, 83.3% and 90.0% of patients respectively. In addition, there was significant reduction in ARR in all the three groups. Reduced dosage of RTX exerted a significant effect in reducing CD19+ B-cell counts (P<0.01). Compared with the AZA group, the MMF group and the RTX group decreased the AQP-4-IgG titre evidently and caused fewer adverse events. Neither the Kaplan-Meier survival curves nor the Cox proportional hazard model indicated a significant difference in relapse among the three groups (P>0.05). CONCLUSIONS: AZA, MMF and reduced dosages of rituximab are all effective in reducing ARR and improving the clinical symptom of patients with NMOSD. Lower dosages of RTX are more effective than the others in decreasing the CD19 B-cell counts. MMF and reduced RTX decrease AQP-4-IgG titre more and cause fewer adverse events than AZA. However, more multicentre studies are still needed to find more effective therapeutic regimen.

Lower dosages of rituximab used successfully in the treatment of anti-NMDA receptor encephalitis without tumour.

OBJECTIVE: The aim of this study was to evaluate the use and efficacy of lower dosages of rituximab for treating anti N-methyl-d-aspartate receptor (NMDAR) encephalitis without tumour. METHODS: We performed a prospective study of 10 patients with anti-NMDAR encephalitis who did not respond to 10 to 14days first-line immunotherapy and received rituximab administered intravenously (IV) at a dosage of 100mg once per week for 4 consecutive weeks. Reinfusion of rituximab was given when CD19+ B-cell counts of total lymphocytes in peripheral blood >1%. The annualized relapse rate (ARR), modified Rankin scale (mRS) and CD19+ B-cell counts were measured every 4 to 10weeks after initial rituximab treatment in order to assess the clinical outcome and efficacy of rituximab. RESULTS: Lower dosages of rituximab led to a significant reduction of mRS and CD19+ B-cells when compared with before the rituximab infusion (P<0.05) and allowed 9 (90%) patients to maintain a stabilised neurological status. One patient experienced a relapse at 19weeks after initial rituximab infusion. Although ARR reduction of all 10 patients did not achieve statistical significance (P>0.05), in the 4 patients who had relapses before rituximab treatment there was an apparent reduction in ARR over 56weeks. At the last follow up, 9 patients (90%) had a good outcome (mRS≤2) including 3 patients (30%) who recovered completely (mRS=0). Transient infusion adverse events occurred in 2 patients. We observed no serious delayed adverse events during the 56weeks follow-up. CONCLUSIONS: In patients with anti-NMDAR encephalitis who did not respond to first-line immunotherapy, early application of lower dosages of rituximab could efficiently reduce CD19+ B-cell counts of peripheral blood and improve the prognosis of anti-NMDAR encephalitis.

Protection against cerebral infarction by Withaferin A involves inhibition of neuronal apoptosis, activation of PI3K/Akt signaling pathway, and reduced intimal hyperplasia via inhibition of VSMC migration and matrix metalloproteinases.

PURPOSE: Stroke is a major public health concern with high rates of morbidity and mortality worldwide. Cerebral ischemia and infarction are commonly associated with stroke. Currently used medications, though effective, are also associated with adverse effects. Development of effective neuroprotective agents with fewer side effects would be of clinical value. We evaluated the effects of Withaferin A (WA), a steroidal lactone derived from the plant Withania somnifera, on experimentally induced cerebral infarction. MATERIALS AND METHODS: The ability of WA to inhibit neuroapoptosis and modulate vascular smooth muscle cell (VSMC) migration and PI3K/Akt signaling was assessed. Separate groups of Sprague Dawley rats were subjected to cerebral occlusion and reperfused for 24h. RESULTS: WA treatment (25, 50 or 100mg/kg bodyweight) significantly reduced the infarct area in a carotid ligation model; WA reduced intimal hyperplasia and proliferating cell nuclear antigen (PCNA)-positive cell counts. Western blotting analysis revealed significantly suppressed PI3K/Akt signaling following cerebral ischemia/reperfusion injury. WA supplementation was found to downregulate apoptotic pathway proteins. WA suppressed PTEN and enhanced p-Akt and GSK-3ß levels and elevated mTORc1, cyclinD1 and NF-κB p65 expression, suggesting activation of the PI3K/Akt pathway. In vitro studies with PDGF-stimulated A7r5 cells revealed that WA exposure severely downregulated matrix metalloproteinases (MMP)-2 and -9 and inhibited migration of A7r5 cells. Additionally, WA reduced the proliferation of A7r5 cells significantly. CONCLUSIONS: WA exerted neuroprotective effects by activating the PI3K/Akt pathway, modulating the expression of MMPs, and inhibiting the migration of VSMCs.

Protective immunity of rAd5/NR2B vaccine against concomitant aversiveness of spontaneous neuropathic pain following spinal nerve ligation injury.

OBJECTIVE: Peripheral nerve injury elicits an aversive state of spontaneous neuropathic pain, and up to now, the modulation of this concomitant aversive state remains a major therapeutic challenge. NMDA receptor subunits NR2B in the rACC are critically involved in the processing of this aversive state and then a strategy targeted at the NR2B subunit might be promising for modulation of the aversive state. Thus, in the present study, using negative reinforcement animal model to reveal spontaneous pain, we investigated the effect of oral immunization with recombinant adenovirus serotype 5-mediated NR2B gene transfer (rAd5/NR2B) on the modulation of the tonic pain. MATERIAL AND METHODS: Following oral administration of the rAd5/NR2B vaccine, NR2B-specific antibodies were induced in serum. And the humoral response was involved in the decreased expression of NR2B protein in the rACC. RESULTS: The present study demonstrated that CPP achieved by spinal administration of clonidine in spinal nerve ligation (SNL) rats revealed the presence of aversive state of spontaneous neuropathic pain. Notably, the humoral autoimmune response blocked the CPP by spinal clonidine, suggesting the relief of the concomitant aversive of spontaneous neuropathic pain in the SNL rats. CONCLUSION: These data proved the feasibility of oral immunization with rAd5/NR2B for modulation of concomitant aversive of spontaneous neuropathic pain due to peripheral nerve injury.

Silencing the NR2B gene in rat ACC neurons by lentivirus-delivered shRNA alleviates pain-related aversion.

The N-methyl-D-aspartate (NMDA) receptor NR2B subunit on neurons in the anterior cingulate cortex (ACC) is implicated in the affective response to noxious stimuli. Selectively silencing this NR2B subunit in ACC neurons could therefore alleviate pain-related aversion. However, to date, there is no optimal approach to selectively silence the NR2B gene in ACC neurons. In the present study, we constructed lentiviral vectors and delivered shRNA (NR2B-RNAi-LV) to effectively silence the NR2B gene in ACC neurons. The use of lentivirus resulted in 95% transfection efficiency and 83% silencing of the NR2B gene in ACC neurons. Electrophysiological experiments showed that the total INMDA was similarly reduced by 48% in lentivirus-transfected ACC neurons. The biochemical and functional data demonstrated that lentiviral shRNA delivery produced a high transfection and silencing efficiency in the ACC neurons. SNI rats weighting 220-250 g were randomly divided into three groups: normal saline group (NS), lenti-siRNA/NC (LV-NC) group, and lenti-siRNA/NR2B (LV-NR2B) group, and conditioned place avoidance was conducted. The results indicated that NR2B-RNAi-LV decreased greatly the conditioning scores of F-CPA while NC-GFP-LV has no effects. NR2B mRNA expression in the NR2B-RNAi-LV group was significantly lower than that in the control group and NC-GFP-LV group. This novel approach of silencing the NR2B gene in ACC neuron could potentially be used to alleviate pain-related aversion.

Lingo-1 inhibited by RNA interference promotes functional recovery of experimental autoimmune encephalomyelitis.

Lingo-1 is a negative regulator of myelination. Repairment of demyelinating diseases, such as multiple sclerosis (MS)/experimental autoimmune encephalomyelitis (EAE), requires activation of the myelination program. In this study, we observed the effect of RNA interference on Lingo-1 expression, and the impact of Lingo-1 suppression on functional recovery and myelination/remyelination in EAE mice. Lentiviral vectors encoding Lingo-1 short hairpin RNA (LV/Lingo-1-shRNA) were constructed to inhibit Lingo-1 expression. LV/Lingo-1-shRNA of different titers were transferred into myelin oligodendrocyte glycoprotein-induced EAE mice by intracerebroventricular (ICV) injection. Meanwhile, lentiviral vectors carrying nonsense gene sequence (LVCON053) were used as negative control. The Lingo-1 expression was detected and locomotor function was evaluated at different time points (on days 1,3,7,14,21, and 30 after ICV injection). Myelination was investigated by luxol fast blue (LFB) staining.LV/Lingo-1-shRNA administration via ICV injection could efficiently down-regulate the Lingo-1 mRNA and protein expression in EAE mice on days 7,14,21, and 30 (P < 0.01), especially in the 5 × 10(8) TU/mL and 5 × 10(9) TU/mL LV/Lingo-1-shRNA groups. The locomotor function score in the LV/Lingo-1-shRNA treated groups were significantly lower than the untreated or LVCON053 group from day 7 on. The 5 × 10(8) TU/mL LV/Lingo-1-shRNA group achieved the best functional improvement (0.87 ± 0.11 vs. 3.05 ± 0.13, P < 0.001). Enhanced myelination/remyelination was observed in the 5 × 10(7) , 5 × 10(8) , 5 × 10(9) TU/mL LV/Lingo-1-shRNA groups by LFB staining (P < 0.05, P < 0.01, and P < 0.05).The data showed that administering LV/Lingo-1-shRNA by ICV injection could efficiently knockdown Lingo-1 expression in vivo, improve functional recovery and enhance myelination/remyelination. Antagonism of Lingo-1 by RNA interference is, therefore, a promising approach for the treatment of demyelinating diseases, such as MS/EAE.

Effects of fibrillar Aß(1-40) on the viability of primary cultures of cholinergic neurons and the expression of insulin signaling-related proteins.

To investigate the effects of fibrillar Aß(1-40) on the morphology and viability of cholinergic neurons and the involvement of the insulin-signaling pathway, we established primary cultures of rat basal forebrain cholinergic neurons and observed their responses to treatment with fibrillar Aß(1-40) at different concentrations for different durations. Cell morphology was examined under microscope after immunofluorescence staining for neurofilament protein, cell vitality accessed by the Methyl thiazolyl tetrazolium assay, and expressions of a panel of insulin signaling-related proteins was detected by Western blot analysis. We show here that, at low concentrations of 0.1-1.0 micromol/L, fibrillar Aß(1-40) had little effects on the cells; however, at higher concentrations of 2-10 µmicromol/L, it caused pathological changes, decreased the cell viability, and reduced the expression of insulin receptor, insulin receptor substrate-I, Protein Kinase B, and B cell lymphoma/leukemia-2 in a dose- and time-dependent manner. These results demonstrate that fibrillar Aß(1-40) not only decreases the viability of cholinergic neuron but also down regulates the expression of important proteins in the insulin signal transduction pathway. We speculate that fibrillar Aß(1-40) may contribute to the pathogenesis of Alzheimer's through disrupting the insulin signaling pathway, therefore decreasing neuronal activity and eventually leading to the apoptosis and cell loss.

[Diffusion tensor imaging of elderly leukoaraiosis and its correlation with cognitive function].

OBJECTIVE: To apply diffusion tensor imaging (DTI) for investigating the correlation between leukoaraiosis (LA) lesion's fraction anisotropy (FA) as well as average diffusion coefficient (DCavg) and LA severity, so as to explore DTI changes in microstructure of white marrow with normal ordinary MRI and its correlation with cognitive function. METHODS: Sixty LA patients and 30 healthy elderly people accepted DTI examination to detect the value of DCavg and FA of LA lesion and normal white marrow. The Mini-Mental State Examination (MMSE) was used for assessing cognitive function. RESULTS: LA severity (0 grade to 3 grade) was positively associated with DCavg, i.e. the more severe was LA, the higher DCavg was (0.66 +/- 0.05 to 1.09 +/- 0.06, P < 0.05); and it was negatively associated with FA, i.e. the more severe was LA, the lower FA was (0.42 +/- 0.04 to 0.26 +/- 0.03, P < 0.05). Neuropsychology tests (Mini-Mental State Examination, MMSE) had a significant relationship with DCavg and FA of normal appearing white matter (NAWM) in LA patients (P < 0.05), especially in anterior horn (Pearson Correlation Coefficient 0.422, P < 0.05) and in centrum semiovale (Pearson Correlation Coefficient -0.495, P < 0.01). CONCLUSIONS: In DTI examination, DCavg and FA of LA displays characteristic changes. Therefore, DTI can detect the macrostructaral changes of white marrow with normal MRI and these changes are related to cognitive function.

[Experimental study of implantation of neurotropin-3 modified olfactory ensheathing cells in experimental allergic encephalomyelitis].

OBJECTIVE: To investigate the therapeutic effect of neurotrophin-3 (NT-3) modified olfactory ensheathing cell (OEC) upon experimental allergic encephalomyelitis (EAE). METHODS: OEC-NT-3 gene engineering cell, constructed by neurotrophin-3 transinfecting GEC inducted by retrovirus, was transplanted into lateral ventricle. The migration and distribution were observed and compared with control group and OEC transplantation group. Then myelin repairing and axon regeneration were evaluated from conical somatosensory evoked potential (CSEP), function score and ultrastructural morphology. RESULTS: (1) OEC-NT-3 could survive, migrate within axons and spread diffusely away from the focus at Day 28 post-transplantation; (2) as compared with other two groups, more nerve fibers, better myelin repair and more distinct myelin structure were observed in the transgene group; (3) as compared with other two groups, the latent time was obviously shortened and the amplitude higher in the transplantation group (P < 0.05); (4) the transcription level of NT-3mRNA in the transgene group was significantly higher than the GEC group and the contrast group (212.32 +/- 16.14) x 10(-2) vs. (1.98 +/- 0.19) x 10(-2), (1.23 +/- 0.13) x 10(-2) (P < 0.01). CONCLUSION: OEC-NT-3 cell expresses NT-3 stably and effectively in EAE. It may contribute to the repairing of myelin and the regeneration of axon.

Paradoxical expression of anti-apoptotic and MRP genes on cancer stem-like cell isolated from TJ905 glioblastoma multiforme cell line.

Existence of cancer stem cell is regarded as a main reason for metastasis and/or recurrences. In this study, the cancer stem-like cell derived from TJ905 glioblastoma multiforme cell line was isolated successfully. However, it was observed that generating rate of the cancer stem-like cells was lower than that of TJ905 cells, that expression of the anti-apoptotic and multidrug resistance-associated protein (MRP) genes were paradoxical to the literatures, which showed the uncertainty of cancer stem cells, and that some stem cell was not the solo factor to maintain tumor growth and resist apoptosis and anti-tumor drugs.