Defects of CTLA-4 Are Associated with Regulatory T Cells in Myasthenia Gravis Implicated by Intravenous Immunoglobulin Therapy.

Myasthenia gravis (MG) is a CD4+ T cell-dependent autoimmune disease resulting from aberrant immune response mediated by circulating autoantibodies at the neuromuscular junction. Intravenous immunoglobulin (IVIg) is an expensive and commonly used immunotherapeutic approach to treat patients with MG. The mechanisms of actions involved in IVIg treatment, however, remain to be investigated. In an effort to examine the roles of various subsets of CD4+ T cells in the periphery blood of MG and uncover the mechanisms that contribute to the therapeutical effects of IVIg, we first demonstrated that a subset of CD4+ T cells, CTLA-4-expressing regulatory T (Treg) cells, were underrepresented and functionally defective in MG patients. The dynamic profiling during the IVIg therapy course further revealed an inverse relationship between the frequency of CTLA-4+ Treg and the quantitative MG (QMG) score that represents disease severity. Our mechanistic studies indicated that IVIg expands CTLA-4-Treg cells via modulating antigen-presenting dendritic cells (DCs). To determine the molecular defects of CTLA-4 in abnormities of Treg in MG patients, we demonstrated hypermethylation at -658 and -793 CpGs of CTLA-4 promoter in MG Tregs. Interestingly, IVIg therapy significantly reduced the methylation level at these two sites in MG patients. Overall, our study may suggest a role of CTLA-4 in functionally defected Treg cells in MG and its actions involved in IVIg therapy.

3-N-Butylphthalide mitigates high glucose-induced injury to Schwann cells: association with nitrosation and apoptosis.

A high glucose state readily causes peripheral axon atrophy, demyelination, loss of nerve fiber function, and delayed regeneration. However, few studies have examined whether nitration is also critical for diabetic peripheral neuropathy. Therefore, this study investigated the effects of high glucose on proliferation, apoptosis, and 3-nitrotyrosine levels of Schwann cells treated with butylphthalide. In addition, we explored potential protective mechanisms of butylphthalide on peripheral nerves. Schwann cells were cultured in vitro with high glucose then stimulated with the peroxynitrite anion inhibitors uric acid and 3-n-butylphthalide for 48 hours. Cell Counting Kit-8 and flow cytometry were used to investigate the effects of uric acid and 3-n-butylphthalide on proliferation and apoptosis of Schwann cells exposed to a high glucose environment. Effects of uric acid and 3-n-butylphthalide on levels of 3-nitrotyrosine in Schwann cells were detected by enzyme-linked immunosorbent assay. The results indicated that Schwann cells cultured in high glucose showed decreased proliferation, but increased apoptosis and intracellular 3-nitrotyrosine levels. However, intervention with uric acid or 3-n-butylphthalide could increase proliferation of Schwann cells cultured in high glucose, and inhibited apoptosis and intracellular 3-nitrotyrosine levels. According to our data, 3-n-butylphthalide may inhibit cell nitrification and apoptosis, and promote cell proliferation, thereby reducing damage to Schwann cells caused by high glucose.

MiR-106a Associated with Diabetic Peripheral Neuropathy Through the Regulation of 12/15-LOX-meidiated Oxidative/Nitrative Stress.

BACKGROUND: Diabetic peripheral neuropathy (DPN) is a common complication of diabetes with a complex pathogenesis. Study has reported that oxidative stress and nitrative stress are all involved in the DPN. However, the mechanism between them is still unclear. In the present study, we investigated whether miR-106a regulated 12/15-Lipoxygenase (12/15-LOX) of oxidative/nitrative stress (OS/NS) in DPN. METHODS: Dorsal root ganglion (DRG) was isolated from 10 healthy mice and 10 DPN mice. DPN mouse model was established by the injection of streptozotocin (STZ), and high glucose was used for inducing the in vitro model of DPN. RESULTS: In DPN mice, the levels of fasting blood-glucose (FBG) level, Methane Dicarboxylic Aldehyde (MDA) and the Inducible Nitric Oxide Synthase (iNOS) were increased, while the levels of motor nerve conduction velocity (MNCV), sensory nerve conduction velocity (SNCV) and superoxide dismutase (SOD) were decreased. MiR-106a level in DRG cells of DPN was decreased. The 12/15-LOX expression and cell apoptosis were increased. DRG cells subjected to high glucose resulted in the same effects as above. MiR-106a was observed to target 12/15-LOX and regulated its expression. MiR-106a overexpression reversed the effect that was induced by high glucose, however, overexpression of 12/15-LOX reversed the effect that was induced by miR-106a overexpression. CONCLUSION: MiR-106 may be associated with 12/15-LOX mediated OS/NS in DPN and may be considered as a potential therapeutic target.

[The Correlation between Blood Uric Acid and Goldstein Grading in Hepatolenticular Degeneration Patients with Different Chinese Medical Syndrome Types].

OBJECTIVE: To observe blood uric acid levels and Goldstein grading, as well as their correlation in Wilson's disease (WD) patients with different Chinese medical syndrome types. METHODS: Totally 906 WD patients in line with inclusive criteria were assigned to 6 groups, i.e., the heart spirit confused by phlegm group (HSCP, 26 cases), the phlegm-fire disturbing heart group (PFDH, 90 cases), the retention of damp-heat group (RDH, 113 cases), deficiency of qi and blood group (DQB, 168 cases), the deficiency of Gan-yin and Shen-yin group (DGYSY, 327 cases), the deficiency of Gan and Shen group (DGS, 182 cases) due to different Chinese medical syndrome types. Recruited were another 160 healthy subjects having similar ages and diet structures, who came for medical examinations, as the healthy control group. Venous blood was collected from the medial cubital vein of each-patient on an empty stomach in early mornings to detect blood uric acid levels. Results Blood uric acid levels were lower in each syndrome type group than in the healthy control group (146.08 +/- 67.24 micromol/L in the HSCP group; 157.08 +/- 69.77 micromol/L in the PFDH group; 162.58 +/- 97.72 micromol/L in the RDH group; 156.20 +/- 62.63 micromol/L in the DQB group; 161.83 +/- 111.23 micromol/L in the DGYSY group; 194.41 +/- 90.01 micromol/L in the DGS group; 242.39 +/- 87.55 micromol/L in the healthy control group, P < 0.01). Blood uric acid levels were higher in the DGYSY group than in the other 5 syndrome groups (P < 0.01). Correlation analyses between Goldstein grading and blood uric acid showed that, along with increased Goldstein grade (that was aggravating disease conditions), WD patients' blood uric acid levels decreased (P < 0.01). CONCLUSIONS: WD patient's blood uric acid levels decreased more. Blood uric acid levels and Goldstein grading were different in various Chinese medical syndrome types. Blood uric acid levels had certain value in assessing the severity of WD.

CHOP/ORP150 ratio in endoplasmic reticulum stress: a new mechanism for diabetic peripheral neuropathy.

BACKGROUND/AIMS: Peripheral neuropathy is a frequent and severe diabetic complication characterized by progressive loss of peripheral nerve axons and manifested by pain and eventually complete loss of sensation. Effective therapy for diabetic peripheral neuropathy (DPN) is still lacking due to our limited understanding of the mechanisms for nerve injury. METHODS: Here we tested the roles of endoplasmic reticulum (ER) stress and the ER stress-activated pro-apoptotic protein CHOP and anti-apoptotic protein ORP150 in DPN in a rat model of high-fat/streptozotocin diabetes and in cultured Schwann cells (SCs). RESULTS: No significant DPN was seen in the early stage of diabetes (4 weeks following verification of diabetes). However, after prolonged diabetes (16 weeks following verification of diabetes), DPN was severely developed as reflected by slowed motor and sensory nerve conduction velocity, blunted thermal nociception, and decreased intraepidermal nerve fiber profiles in the hindpaw. Meanwhile, while it was not noticed in sciatic nerves of early diabetes, ER stress in prolonged diabetic rats was indicated by robust increases in H2O2 production and expression of the ER chaperon glucose-regulated protein 78 (GRP78). ORP150 expression was substantially upregulated, accompanied by mild increase in CHOP expression, resulting in a low CHOP/ORP150 ratio, in early diabetes. In contrast, with prolonged diabetes, CHOP expression exceeded ORP150 expression, resulting in an increased CHOP/ORP150 ratio. In vivo knockdown of ORP150 induced DPN in early diabetes and exacerbated the DPN after prolonged diabetes, whereas knockdown of CHOP ameliorated DPN in rats with prolonged diabetic. On the other hand, in vitro knockdown of ORP150 promoted high glucose-induced SC apoptosis, whereas knockdown of CHOP protected SCs from apoptosis. CONCLUSION: Taken together, we have provided evidence for the critical role of ER stress in the development of DN and also uncovered CHOP/ORP150 ratio as an important mechanism for determining neuronal apoptosis during ER stress. In the early stage of diabetes, CHOP/ORP150 ratio was relatively low favoring neuronal cell survival, whereas after prolonged diabetes, CHOP/ORP150 ratio increased resulting in apoptotic cell death leading to accelerated DPN.

The effects of educational background on Montreal Cognitive Assessment screening for vascular cognitive impairment, no dementia, caused by ischemic stroke.

It is possible that a patient's educational background has an effect on their Montreal Cognitive Assessment (MoCA) score, which is used to evaluate patients for vascular cognitive impairment, no dementia (VCIND) after ischemic stroke. Cognitive impairment was evaluated in patients with no cognitive impairment (NCI) or VCIND using the MoCA. The receiver operating characteristic curve and maximal Youden index were used to determine the optimal cut-off values to distinguish between NCI and VCIND. The sensitivity and specificity of MoCA were calculated for patients with primary, secondary and tertiary educational levels. Patients with NCI (n=111) and VCIND (n=95) were tested. In patients with a primary education, a significant difference was found between the two groups in each of the MoCA factors, except for naming. Likewise, a significant difference was found in all factors, except for naming, attention and calculation, for patients with a secondary education. For the patients with a tertiary education, a significant difference was found only in visuospatial/executive abilities, abstraction and memory (p<0.05). The optimal cut-off value for MoCA in order to identify VCIND was 22-23. MoCA showed an overall sensitivity of 65.26% and specificity of 78.73%. The sensitivity in the primary, secondary and tertiary educated groups was 97.06%, 56.10% and 40%, respectively, with the specificity being 47.22%, 87.80% and 100%, respectively. We suggest that the MoCA score needs to be amended according to the patient's educational levels in order to improve the effectiveness of the screening.

[The value of ABCD3-I score in prediction of cerebral infarction after transient ischaemic attack].

OBJECTIVE: To assess the ability of ABCD3-I score in evaluating the early risk of cerebral infarction after transient ischemic attack (TIA). METHODS: A total of 107 TIA patients were evaluated according to ABCD2, ABCD3 and ABCD3-I criteria. The occurrences of cerebral infarction within 2 days and 7 days were observed. RESULTS: The AUC(ROC) of ABCD2, ABCD3 and ABCD3-I were 0.61, 0.66 and 0.71 in predicting the risk of cerebral infarction within 2 days, and were 0.62, 0.68 and 0.74 in predicting within 7 days, respectively. Among 107 patients with TIA, 13 evolved into cerebral infarction within 2 days, accounting for 12.1%, and 24 within 7 days, accounting for 22.4%.According to ABCD3-I criteria, 17 patients were of low risk scored 0-3; 54 patients were of medium risk scored 4-7; and 36 patients were of high risk scored 8-13. The different incidence of cerebral infarction after TIA was related to ABCD3-I score: the higher the score was, the higher incidence was. Except for age factor, every score item of ABCD3-I display obvious influence to the occurrence of cerebral infarction within 2 days and 7 days after TIA (P < 0.05). CONCLUSION: ABCD3-I criteria could more effectively predict the occurrence of early risk of cerebral infarction after TIA, which could be used in regular clinical practice for assistance in TIA risk stratification and treatment.

Protective effect of gliclazide on diabetic peripheral neuropathy through Drp-1 mediated-oxidative stress and apoptosis.

OBJECTIVE: To investigate the protective effect of gliclazide and the role of dynamin-related protein l (Drp-1)-mediated oxidative stress and apoptosis in diabetic peripheral neuropathy (DPN). METHODS: Diabetic rats developed through intra-peritoneal injection of streptozotocin were randomly assigned to treatment group receiving gliclazide or non-treatment group without gliclazide treatment. Rats in control group received intra-peritoneal injection of vehicle and no gliclazide treatment. Eight weeks later, the nerve conduction velocity (NCV) of sciatic nerve was measured and the morphological alterations, the malondialdehyde (MDA) level and superoxide-dismutase (SOD) activity, the expressions of Drp-1, caspase-3, Bax and Bcl-2 in sciatic nerve were evaluated. RESULTS: When compared to rats in control group, rats in non-treatment group showed significantly decrease of NCV, obvious demyelinative alteration of sciatic nerve, increased expressions of Drp-1, caspase-3, Bax, Bcl-2 and MDA, and decreased SOD activity. Compared to rats in non-treatment group, rats in treatment group showed significantly increase of NCV, less demyelination of sciatic nerve, decreased expressions of Drp-1, caspase-3, Bax and MDA, and increased activity of SOD. The expression of Bcl-2 was not significantly different between treatment and non-treatment groups. CONCLUSION: Gliclazide showed protective effect on DPN through modulating Drp-1-mediated oxidative stress and apoptosis.

Changes of Treg-associated molecules on CD4+CD25 +Treg cells in myasthenia gravis and effects of immunosuppressants.

OBJECTIVE: Myasthenia gravis (MG) is a CD4(+) T cell-dependent autoimmune disease, and close attention has been paid to the role of CD4(+)CD25(+)Treg cells (Tregs). Previous results regarding Tregs in MG patients have been conflicting. The discrepancy was partly ascribed to selecting different Treg-associated molecules in defining Tregs. Therefore, we considered it necessary to find a reliable index for assessing the immunologic state in MG patients and explore the effect of IS on them. METHODS: We adopted flow cytometric techniques to measure the numbers and frequencies of Tregs in peripheral blood taken from 57 patients and 91 age-matched healthy donors, and we also analyzed FOXP3 mean fluorescence intensity on Tregs. RESULTS: The number and frequency of Tregs in peripheral blood of MG patients significantly decreased, together with down-regulation of FOXP3 expression. There was dynamic change of Treg cell level and the inverse relationship with clinical symptom, suggesting that the immunologic disorder in MG patients was related to peripheral Tregs population. Meanwhile, CD4(+)CD25(+)FOXP3(+)Helios(+)T cells might be activated Tregs, rather than nTregs. Moreover, the number and frequency of CD4(+)CD25(+)FOXP3(+)Helios(+)T cells significantly decreased in MG patients, indicating that the reduction of the activated Tregs population might be a critical contributor to the pathogenesis of MG. CONCLUSIONS: The significant reduction of the peripheral Tregs population in MG patients might be responsible for the immunologic disorders in MG patients. IS such as GC took its effect possible by increasing the population size, and the underlying mechanism should be further investigated.

Expression and subcellular localization of P9-ZFD protein in patients with myasthenia gravis.

OBJECTIVE: To express and purify the protein coded by the TRAF-type zinc finger domain of myasthenia gravis (MG)-related gene P9 (P9-ZFD) and to prepare P9-ZFD antiserum for detecting expression and subcellular distribution of P9-ZFD protein in the skeletal muscles of patient with MG. METHODS: The cDNA encoding P9-ZFD was amplified by RT-PCR. The cloned P9-ZFD cDNA was ligated into pET24a, and the P9-ZFD recombinant protein was induced via E. coli. BL21 (DE3) and purified by histidine affinity chromatography. P9-ZFD antiserum was prepared and its titer and specificity were determined by ELISA and Western blot. Expression and subcellular distribution of P9-ZFD protein in the skeletal muscles of MG and control were studied. RESULTS: The molecular weight of purified P9-ZFD protein was about 30 kD. Its purity was more than 95%. Antiserum specific for P9-ZFD was excellent. P9-ZFD protein is fully confined to the cytoplasm membrane of skeletal muscle cell of MG, obvious immunostaining was absent in the A, I, and Z bands of cytoplasm and no immunoreactivity was observed in the skeletal muscle cell of control. CONCLUSION: P9-ZFD protein is expressed as a cytoplasm membrane-bound protein and has obvious distribution difference in the skeletal muscle cells of patient with MG and normal control.

Anticopper efficacy of captopril and sodium dimercaptosulphonate in patients with Wilson's disease.

The aim of this study was to explore and compare initial treatment effects of captopril (Tensiomin) and sodium dimercaptosulphonate (DMPS) on a relatively large series of Wilson's disease inpatients. Two important markers of anticopper efficacy: serum sulphydryl and 24 h urinary copper levels in the patients were evaluated before and after treatment. The patients were randomly subdivided into 4 groups to allow statistical analysis (ANOVA) of the values recorded. The protocol was an open-label study of all the patients treated for 8 weeks (i.e., all the patients except those in the no-drug group), and a further six-month follow-up (post hospitalization) of the 14 patients administered captopril. Several copper-related variables were studied to evaluate the effect of the drugs on copper, and several biochemical and clinical variables were studied to evaluate potential toxic effects. Captopril was found to have a significant anticopper effect and did not markedly raise serum sulphydryl levels within this limited patient sample; the anticopper efficacy of captopril was, however, found to be markedly lower than that of DMPS; DMPS was found to raise the patients' serum sulphydryl and urinary copper levels. Evaluation of data from individual patients revealed evidence of a toxic side effect in only 1 patient, treated with DMPS, who exhibited transiently raised serum alanine aminotransferases, while no serious adverse events, upstanding syncope, irritating cough and leukopenia induced by captopril were noted. The results obtained in this four-group sample suggest that captopril might be a mild anticopper agent for Wilson's disease, possibly relieving the hepatic portal hypertension, but that DMPS has a greater field of anticopper efficiency than captopril. The authors also discuss recent experience of the overall treatment in China.

Copper-chelating therapeutic effect in Wilson disease with different clinical phenotypes and polymorphisms of ATP7B gene.

AIM:To investigate the copper-chelating therapeutic effect in Wilson disease (WD) with different clinical phenotypes and polymorphisms of ATP7B gene.METHODS:One hundred and twenty-two WD patients with different clinicalphenotypes were given DMPS intravenously and Gandou copper-chelating tablet orally for one month. The therapeutic effect was judged by modified Goldstein mothod. Exon 18 of ATP7B gene extracted from the DNA of patients and 20 healthy volunteers was amplified with PCR mutation and polymorphism were screened with SSCP technique.RESULTS:Four kinds of abnormal migration bands in PCR-SSCP were observed in 37 WD patients, mutation frequencies of three different disease phenotypes, and curative effect between mutation group and non-mutation group showed no statistically significant difference (P > 0.05), but the total effectiveness rates in patients with Wilson type or pseudosclerosis type were significantly higher than those of patients with hepatic type (X(2) = 6.17, P < 0.05).CONCLUSION:Most WD patients are compound heterozygotes, the patients with different clinical phenotypes have different response to copper-chelating therapy. Specific mutation, at least in part, plays a role in influencing the disease phenotypes and therapeutic effect.

Comparison of long lasting therapeutic effects between succimer and penicillamine on hepatolenticular degeneration.

AIM:To compare the long-term effect of succimer (Suc) with that of penicillamine (Pen) in treating hepatolenticular degeneration (HLD).METHODS:One hundred and twenty patients with HLD were divided into 2 groups. Group A (n =60) received Suc 750mg,po.bid.Group B (n =60) received Pen 250mg, po. qid. The period of maintenance treatment varied from 6 months to 3 years, averaging 1.5 years. Symptoms and therapeutic effects were evaluated by modified Goldstein scale.RESULTS:The total effectiveness of group A in two different periods of treatment were 80% and 85% respectively, higher than those of group B (58% and 59% respectively)(P < 0.05). Suc also had obvious curative effects for the patients who failed in the use of Pen. There were fewer side effect in group A than in group B (P < 0.05). Suc and Pen could increase urinary copper excretion effectively and continually.CONCLUSION:Suc is more effective and safer than Pen. Clinically, it can replace Pen as first-choice drug for long-term maintenance therapy of HLD.