A case of NMDAR Encephalitis with muscular pain as the main presentation.

BACKGROUND: Persistent somatoform pain disorder (PSPD) is often the initial diagnosis in patients seeking treatment in psychiatric departments, making it challenging to consider organic nervous system diseases. However, autoimmune encephalitis can present with atypical initial symptoms, leading to misdiagnosis or missed diagnosis. Lumbar puncture, with antibody support, plays a crucial role in diagnosing autoimmune encephalitis. CASE PRESENTATION: This report describes a 40-year-old male adult patient who was initially diagnosed with persistent somatoform pain disorder in 2022. The patient reported a reduction in pain while resting on his back. There were no fever or relevant medical history. Despite 8 months of symptomatic treatment, the symptoms did not improve. Moreover, the patient developed confusion, gibberish speech, non-cooperation during questioning, and increased frequency and amplitude of upper limb convulsions. Lumbar puncture revealed elevated protein levels and protein-cell dissociation. The autoimmune encephalitis antibody NMDAR (+) was detected, leading to a diagnosis of autoimmune encephalitis (NMDAR). CONCLUSION: Autoimmune encephalitis (NMDAR), starting with persistent somatoform pain (PSPD), often presents with atypical symptoms and can be easily misdiagnosed. Therefore, it is important to consider the possibility of organic nervous system disease in time, and to test serum or cerebrospinal fluid antibodies to rule out organic nervous system disease after symptomatic treatment of mental disorders is ineffective. This approach facilitates the early diagnosis of autoimmune encephalitis and other underlying organic neurological disorders.

Lower glomerular filtration rate after mild stroke induces cognitive impairment by causing endothelial dysfunction.

The incidence of post stroke cognitive impairment (PSCI) is high in patients with mild stroke (MIS), and the risk factors and mechanism are uncertain. Increased cystatin C (CysC) levels after stroke may reflect lower glomerular filtration rate (GFR) and renal impairment. Previous studies have suggested endothelial dysfunction (ED) is closely related to renal impairment and cognitive impairment, respectively. We aimed to observe whether lower GFR estimated by CysC after MIS leaded to a high incidence of PSCI, and the role of ED in this process. 256 patients were enrolled in this prospective observational study. Renal function was assessed using GFR estimated by serum CysC. Endothelial function was evaluated by reactive hyperemia index (RHI) which calculated automatically by peripheral arterial tonometry (PAT). The cognitive function at baseline and 3 months was evaluated by MoCA score, and MoCA score ≤ 26 indicates the presence of PSCI. Spearman correlation analysis and linear regression were conducted to explore the factors affecting ED. Univariate and multivariate analysis was used to identify the independent risk factors of PSCI. The receiver operating characteristic (ROC) curve was applied to explore the optimal cutoff value of the independent risk factors levels for predicting PSCI. A total of 141 patients (55.1%) suffered from ED. Multiple linear regression analysis showed that there was a strong linear correlation between eGFRcys and RHI (p < 0.001). At the three-month follow-up, a total of 150 (58.6%) patients had been diagnosed with PSCI. Multivariate logistic regression analysis showed that RHI was an independent factor affecting the occurrence of PSCI (p < 0.05). ROC curve showed that the area under the curve was 0.724, and the optimal cut-off value of RHI was 1.655, with the sensitivity and specificity for PSCI were 72.7% and 73.6%, respectively. The lower eGFRcys level after MIS was significantly associated with ED, and ED may mediate the higher incidence of PSCI at 3 months after MIS.

Case Report: Paroxysmal weakness of unilateral limb as an initial symptom in anti-LGI1 encephalitis: a report of five cases.

Anti-leucine-rich glioma-inactivated 1 (LGI1) encephalitis is the second most common kind of autoimmune encephalitis following anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis. Anti-LGI1 encephalitis is characterized by cognitive impairment or rapid progressive dementia, psychiatric disorders, epileptic seizures, faciobrachial dystonic seizures (FBDS), and refractory hyponatremia. Recently, we found an atypical manifestation of anti-LGI1 encephalitis, in which paroxysmal limb weakness was the initial symptom. In this report, we describe five cases of anti-LGI1 encephalitis with paroxysmal limb weakness. Patients had similar presentations, where a sudden weakness involving a unilateral limb was observed, which lasted several seconds and occurred dozens of times each day, with the anti-LGI1 antibody being positive in both serum and cerebrospinal fluid (CSF). FBDS occurred after a mean of 12 days following paroxysmal limb weakness in three of five patients (Cases 1, 4, and 5). All patients were given high-dose steroid therapy, which had a good effect on their condition. Based on this report, we suggest that paroxysmal unilateral weakness may be a kind of epilepsy and be connected to FBDS. As an unusual neurological presentation, paroxysmal weakness can be included in the clinical manifestations of anti-LGI1 encephalitis, helping to raise awareness of the recognition of anti-LGI1 encephalitis in patients with this symptom and leading to early diagnosis and early treatment, which would contribute to improved clinical outcomes.

Association between estimated pulse wave velocity and silent lacunar infarct in a Korean population.

Aims: Previous studies have proposed the estimated pulse wave velocity (ePWV) as a simple and cost-effective measure of arterial stiffness. Since arterial stiffness plays a role in the progression of silent lacunar infarct (SLI), our present work aims to evaluate the association between ePWV and the presence of SLI. Methods: The present work was based on a cross-section study. Our study included 1,011 neurologically healthy Korean participants. The SLI was evaluated using brain magnetic resonance images (MRI). The ePWV was derived from a published equation using age and mean blood pressure (MBP). Logistic regression analyses were performed to investigate the association between ePWV and SLI. The linear relationship and robustness were evaluated using smooth curve fitting and subgroup analyses, respectively. Results: The prevalence of SLI was 11.87%. After fully adjusting for covariates, per 1 m/s increase of ePWV casted 31% additional risk for SLI (P = 0.009). When dividing the ePWV into quartiles, the top quartile had 4.01 times risk compared with the bottom quartile. The increasing trend across the quartiles was statistically significant (P for trend < 0.001). Consistently, smooth curve fitting revealed that the risk of SLI elevated linearly with the increase of ePWV. Finally, subgroup analysis suggested that the association was robust in several sub-populations divided by age, sex, smoking, hypertension, diabetes mellitus (DM), coronary artery occlusive disease (CAOD), hyperlipidemia, and statin medication (all P for interaction > 0.05). Conclusion: The current study revealed an independent and positive association between ePWV and the presence of SLI in a neurologically healthy Korean population.

Human urinary kallidinogenase decreases the incidence of post-stroke cognitive impairment in acute ischemic stroke patients.

BACKGROUND: Post-stroke cognitive impairment (PSCI) is a common symptom of stroke and affects the quality of life and prognosis of stroke survivors. In our study, we evaluated the efficacy of Human urinary kallidinogenase (HUK) on cognitive function in acute ischemic stroke (AIS) patients, and discussed the role of cystatin C (CysC) in improving PSCI. METHODS: We enrolled a retrospective cohort with prospective follow-up. From August 2020 to May 2021, 130 patients completed the final follow-up. Among them, 61 patients received HUK combined with basic treatment, which we defined as the HUK group, and 69 patients received basic treatment, which we defined as the control group. We compared the changes of CysC, urea nitrogen and creatinine levels after one week of treatment between the two groups. Cognitive function was assessed by Montreal Cognitive Assessment (MoCA) at 3-month after AIS. RESULTS: No significant differences in demographic data and Laboratory tests between two groups before treatment. A total of 67 patients (51.5%) were diagnosed as PSCI at 3-month follow-up, among which, 25 patients were in the HUK group and 42 patients were in the control group. Compared with the control group (60.9%), the incidence of PSCI was significantly lower in the HUK group (41.0%). In addition, the serum CysC level after a week of treatment significantly decreased from baseline in HUK group (p = 0.037), in comparison, the serum CysC level in the control group was basically unchanged (p = 0.951). There was a significant negative correlation between MoCA score and the level of CysC after treatment (p = 0.003, r = -0.373). CONCLUSIONS: HUK can reduce the risk of PSCI at 3-month in AIS patients. The decrease of serum CysC level may be one of the mechanisms by which HUK reduces the incidence of PSCI.

Association between serum cystatin C level and post-stroke cognitive impairment in patients with acute mild ischemic stroke.

BACKGROUND: Mild ischemic stroke (MIS) has been proved to be closely related to post-stroke cognitive impairment (PSCI). However, there are relatively few studies on the risk factors of MIS. We aimed to evaluate the relationship between serum cystatin C (CysC) level and cognitive function in patients with acute MIS. METHODS: Four hundred consecutive patients with acute MIS were screened and 281 patients were eligible for this study. The serum CysC levels were detected within 24 h after admission. Cognitive function was assessed by Montreal Cognitive Assessment (MoCA) at 3 months after acute MIS. Logistic regression was used to identify the predictors of PSCI, and the receiver operating characteristic (ROC) curve was applied to explore the optimal cut-off value. RESULTS: One hundred sixty-four (58.4%) patients were diagnosed with PSCI at 3 months follow-up. The serum CysC levels in patients with PSCI were significantly higher than patients without PSCI (p < .001). The binary logistic regression analysis showed that higher serum CysC level was an independent predictor for PSCI at 3 months (odds ratio [OR], 5.745; 95% confidence interval, [CI], 1.089-30.311; p = 0.039). The ROC curve showed that area under the curve (AUC) was 0.723, and at a 0.945 mg/l CysC cut-off point, the sensitivity and specificity for PSCI at 3 months were 79.9% and 58.1%, respectively. CONCLUSION: Our findings suggested that the serum CysC levels were increased after acute MIS, and higher serum CysC levels at baseline might be an independent risk factor for PSCI in patients with acute MIS, but further research are warranted.

Pseudogene RPL32P3 regulates the blood-tumor barrier permeability via the YBX2/HNF4G axis.

The existence of the blood-tumor barrier (BTB) severely hinders the transport of anti-tumor drugs to brain tumor tissues. Selectively opening BTB is of great significance to improve the chemotherapy effect of glioma. Pseudogenes have been recognized as important regulators in various biologic processes. In this study, we identified that ribosomal protein L32 pseudogene 3 (RPL32P3) was highly expressed in glioma-exposed endothelial cells (GECs). Knockdown of RPL32P3 decreased the expression of tight junction-related proteins (TJPs) and increased BTB permeability. Subsequent analysis of the underlying mechanism indicated that RPL32P3 recruited lysine methyltransferase 2 A (KMT2A) to the Y-box binding protein 2 (YBX2) promoter region and mediated H3K4me3 to promote YBX2 transcription. Highly expressed YBX2 bound and stabilized hepatocyte nuclear factor 4 gamma (HNF4G) mRNA. Highly expressed HNF4G directly bound to the promoters of TJPs ZO-1, occludin and claudin-5 to promote their transcriptional activities and regulated BTB permeability. The simultaneous knockdown of RPL32P3, YBX2, and HNF4G combined with doxorubicin (DOX) increased the apoptosis of glioma cells. In conclusion, the current study indicated that RPL32P3 knockdown increased BTB permeability through the YBX2/HNF4G pathway. These findings may provide new targets for the comprehensive treatment of glioma.

Post-stroke Anxiety Analysis via Machine Learning Methods.

Post-stroke anxiety (PSA) has caused wide public concern in recent years, and the study on risk factors analysis and prediction is still an open issue. With the deepening of the research, machine learning has been widely applied to various scenarios and make great achievements increasingly, which brings new approaches to this field. In this paper, 395 patients with acute ischemic stroke are collected and evaluated by anxiety scales (i.e., HADS-A, HAMA, and SAS), hence the patients are divided into anxiety group and non-anxiety group. Afterward, the results of demographic data and general laboratory examination between the two groups are compared to identify the risk factors with statistical differences accordingly. Then the factors with statistical differences are incorporated into a multivariate logistic regression to obtain risk factors and protective factors of PSA. Statistical analysis shows great differences in gender, age, serious stroke, hypertension, diabetes mellitus, drinking, and HDL-C level between PSA group and non-anxiety group with HADS-A and HAMA evaluation. Meanwhile, as evaluated by SAS scale, gender, serious stroke, hypertension, diabetes mellitus, drinking, and HDL-C level differ in the PSA group and the non-anxiety group. Multivariate logistic regression analysis of HADS-A, HAMA, and SAS scales suggest that hypertension, diabetes mellitus, drinking, high NIHSS score, and low serum HDL-C level are related to PSA. In other words, gender, age, disability, hypertension, diabetes mellitus, HDL-C, and drinking are closely related to anxiety during the acute stage of ischemic stroke. Hypertension, diabetes mellitus, drinking, and disability increased the risk of PSA, and higher serum HDL-C level decreased the risk of PSA. Several machine learning methods are employed to predict PSA according to HADS-A, HAMA, and SAS scores, respectively. The experimental results indicate that random forest outperforms the competitive methods in PSA prediction, which contributes to early intervention for clinical treatment.

Score for Predicting Active Cancer in Patients with Ischemic Stroke: A Retrospective Study.

BACKGROUND: We aimed to examine the differences of clinical characteristics between patients with ischemic stroke with active cancer and those without cancer to develop a clinical score for predicting the presence of occult cancer in patients with ischemic stroke. METHODS: This retrospective study enrolled consecutive adult patients with acute ischemic stroke who were admitted to our department between December 2017 and January 2019. The demographic, clinical, laboratory, and neuroimaging characteristics were compared between patients with ischemic stroke with active cancer and those without cancer. Multivariate analysis was performed to identify independent factors associated with active cancer. Subsequently, a predictive score was developed using the areas under the receiver operating characteristic curves based on these independent factors. Finally, Bayesian decision theory was applied to calculate the posterior probability of active cancer for finding the best scoring system. RESULTS: Fifty-three (6.63%) of 799 patients with ischemic stroke had active cancer. The absence of a history of hyperlipidemia (odds ratio (OR) = 0.17, 95% confidence interval (CI): 0.06-0.48, P < 0.01), elevated serum fibrinogen (OR = 1.72, 95% CI: 1.33-2.22, P < 0.01) and D-dimer levels (OR = 1.43, 95% CI: 1.24-1.64, P <0.01), and stroke of undetermined etiology (OR = 22.87, 95% CI: 9.91-52.78, P < 0.01) were independently associated with active cancer. A clinical score based on the absence of hyperlipidemia, serum fibrinogen level of ≥4.00 g/L, and D-dimer level of ≥2.00 µg/mL predicted active cancer with an area under the curve of 0.83 (95% CI: 0.77-0.89, P < 0.01). The probability of active cancer was 59% at a supposed prevalence of 6.63%, if all three independent factors were present in a patient with ischemic stroke. CONCLUSIONS: We devised a clinical score to predict active cancer in patients with ischemic stroke based on the absence of a history of hyperlipidemia and elevated serum D-dimer and fibrinogen levels. The use of this score may allow for early intervention. Further research is needed to confirm the implementation of this score in clinical settings.

Usefulness of atherogenic index of plasma for estimating reduced eGFR risk: insights from the national health and nutrition examination survey.

AIMS: Previous studies have identified Atherogenic index of plasma (AIP) as a simple measure of atherosclerosis. Because atherosclerosis plays a role in the development of renal damage, our study aims to evaluate the effect of AIP on the risk of reduced eGFR and assess its usefulness to refine the risk stratification of reduced estimated glomerular filtration rate (eGFR). METHODS: Our study included 15,836 participants from the National Health and Nutritional Survey (NHANES) 2009-2016. Association was investigated by logistic regression. AIP was calculated as log (triglycerides/high-density lipoprotein cholesterol). Reduced eGFR was determined as eGFR < 60 ml/min per 1.73 m*2. RESULTS: The prevalence of reduced eGFR was 8.01%. In the full model, each SD increase of AIP leaded to 27.4% additional risk for reduced eGFR. After dividing AIP into quartiles, the fourth quartile had a 1.649 times risk than the first quartile. Moreover, smooth curve fitting suggested that the risk of reduced eGFR elevated linearly with the increase of AIP. Subgroup analysis demonstrated that the association between AIP and reduced eGFR was robust in sex, body mass index, hypertension, and diabetes subpopulation, but the association was significantly stronger in black race and people aged less than 50 years old. Additionally, AUC displayed an advancement when introducing AIP into established risk factors (0.875 cs. 0.897, P < 0.001), category-free net reclassification index (0.249, 95% CI: 0.192-0.306, P < 0.001) and integrated discrimination index (0.007, 95% CI: 0.004-0.009, P < 0.001) also suggested the improvement from AIP. CONCLUSION: The present work suggested a linear association between AIP and reduced eGFR. Furthermore, the results showed that the association was stronger in black race and people aged less than 50 years old. Most importantly, our work implicated the usefulness of AIP to refine the risk stratification of reduced eGFR.

Bilateral middle cerebellar peduncle lesions: Neuroimaging features and differential diagnoses.

OBJECTIVES: Lesions limited to the bilateral middle cerebellar peduncles (MCPs) are uncommon. This retrospective study investigated diseases with a proclivity for the bilateral MCPs and explored the associations between their neuroimaging features and clinical findings for the differential diagnosis of such lesions. METHODS: We enrolled 26 patients who were admitted to our department between January 2016 and March 2019 with bilateral MCP abnormalities on magnetic resonance imaging (MRI). The demographic, clinical, and neuroimaging characteristics, and the biomarkers and diagnoses were evaluated. RESULTS: Although all patients exhibited symmetrical bilateral MCP hypointensities on T1-weighted imaging and hyperintensities on T2-weighted and fluid-attenuated inversion recovery imaging, they were diagnosed with different conditions. Diagnoses included acute cerebral infarction (ACI) (n = 9, 34.62%), Wallerian degeneration (WD) (n = 8, 30.77%), multiple system atrophy (MSA) (n = 6, 23.08%), neuromyelitis optica (NMO) (n = 1, 3.85%), heroin-induced leukoencephalopathy (n = 1, 3.85%), and primary central nervous system lymphoma (PCNSL) (n = 1, 3.85%). Patients with ACI exhibited bilateral MCP-restricted diffusion hyperintensities on diffusion-weighted imaging and corresponding stenosis or occlusion of the vertebrobasilar system. The initial MRI of patients with WD depicted pontine infarctions, while symmetrical MCP lesions were observed on follow-up MRI. Symmetrical MCP lesions, cruciform hyperintensity, and marked atrophy in the posterior fossa were characteristic manifestations of MSA. Longitudinally extensive myelitis affecting more than three vertebral segments on cervical MRI and positive serum AQP4-IgG may be indicative of NMO. Heroin-induced leukoencephalopathy was characterized by extra-symmetrical lesions in the posterior limbs of the internal capsules, while the anterior limbs were spared. PCNSL was indicated by a significant and characteristic "fist" sign on contrast-enhanced MRI. CONCLUSIONS: Bilateral MCP lesions were most frequently observed in cerebrovascular diseases, followed by neurodegenerative diseases, inflammatory diseases, toxic encephalopathies, and lymphomas. Our findings demonstrate that bilateral MCP signal abnormalities are more common in patients with ACI and WD, with fewer degenerative processes than previously believed. The high frequency of WD may be attributed to the specific awareness of this pathology. WD can also present with stage-related restricted diffusion and should not be mistaken for a new infarction. The symmetrical bilateral MCP hypointensities on T1-weighted imaging and hyperintensities on T2-weighted imaging often raise concern regarding a demyelinating process. Our findings emphasize that neurologists should consider the aforementioned conditions and correlate the specific neuroimaging characteristics and medical history before arriving at the final diagnosis.

C-Reactive Protein Can Be an Early Predictor of Poststroke Apathy in Acute Ischemic Stroke Patients.

BACKGROUND: Apathy is a multidimensional syndrome referring to a primary lack of motivation that occurs frequently in survivors of stroke. Higher C-reactive protein (CRP) level was associated with higher apathy scores among Alzheimer disease cases. However, data on the relationship between CRP levels and apathy in patients with stroke are lacking. So, we hypothesized an association between CRP and poststroke apathy (PSA). METHODS: Two hundred ninety-two consecutive patients with stroke were recruited within 7 days after stroke. Apathy symptoms were assessed at baseline and at 1, 3, and 6 months after stoke using the Apathy Evaluation Scale-Clinical (AES-C). Demographic and clinical information were obtained using the National Institutes of Health Stroke Scale (NIHSS) scores, Barthel Index (BI) scores, Mini-Mental State Examination (MMSE) scores, Hamilton Depression Scale (HAMD) scores, and Hamilton Anxiety Scale (HAMA) scores. CRP was measured at baseline. The presence and the location of infarcts were evaluated using magnetic resonance imaging. RESULTS: Apathy at baseline was significantly associated with body mass index (BMI), NIHSS, BI, MMSE, HAMD, and CRP (P < .05) upon admission. PSA at 6 months was significantly associated with elevated CRP concentrations, high AES-C score, and low BI score (P < .05) upon admission. The AES-C scores peaked 3 months after stroke, but then abated over 6 months. CONCLUSIONS: CRP, BMI, MMSE, depression, and disability are closely related to apathy during the acute stage of ischemic stroke. Lower BI scores, higher CRP concentrations, and apathy in acute stroke phase increased the risk of PSA at 6 months.

S14G-humanin alleviates insulin resistance and increases autophagy in neurons of APP/PS1 transgenic mouse.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by Aß plaque deposition in the brain, which is related to the disorder of autophagosome maturation, transport, and formation of autolysosome. Notably, abnormal insulin signaling is connected with cognitive dysfunction in AD. In this study, using APP/PS1 transgenic mice as AD model, we investigated the mechanism by which S14G-humanin (HNG) improved autophagy and insulin signaling in AD brain. Immunohistochemistry was used to determine the levels of mTOR and Aß deposition, and Western blot analysis was used to determine IRS-1, IRS-1 pSEr636, ULK1, p62, LC3 I/LC3 II protein levels. Our results demonstrated that HNG could improve the learning ability and memory in APP/PS1 transgenic mice, possibly through decreasing IRS-1 Ser636 phosphorylation and mTOR protein expression in the hippocampus, thus improving insulin resistance in the brain. In addition, HNG increased ULK1 expression, decreased p62 and LC3 I/LC3 II protein levels, thus enhancing autophagy and decreasing Aß deposition in the brain. Taken together, our results suggest that through the regulation of IRS-1/mTOR insulin signaling in the hippocampus, HNG increases the activity of autophagy and decreases Aß deposition in the brain, and improves learning ability and memory of AD mice.

Glaucocalyxin B Alleviates Lipopolysaccharide-Induced Parkinson's Disease by Inhibiting TLR/NF-κB and Activating Nrf2/HO-1 Pathway.

BACKGROUND/AIMS: Parkinson's disease (PD) is a common neurodegenerative disease in the old population, characterized by dopaminergic neuron loss, inflammation and oxidative stress injury in the substantia nigra. Glaucocalyxin B (GLB), an ent-kauranoid diterpenoid isolated from Rabdosia japonica, has anti-inflammation and anti-tumor effects. However, its effects on PD remain unclear. METHODS: PD was introduced in rats via injection of lipopolysaccharide (LPS) into cerebral corpus striatum, and GLB was given intracerebroventricularly to these rats. Their walking, climbing and sensory states were detected by Stepping, Whisker and Cylinder Tests. The expression of tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), CD11b and ionized calcium binding adaptor molecule (IBA)-1 were detected by immunohischemical staining. The levels of a series of inflammatory factors, oxidative stress-related factors and apoptosis-related factors were measured by real-time PCR, immunoblotting and ELISA. In addition, Toll-like receptor (TLR)/nuclear factor kappa B (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase (HO)-1 pathways were investigated to illustrate the underlying mechanism. In vitro, microglial cells exposed to LPS were treated with GLB. RESULTS: The injection of LPS caused walking, climbing and sensory disturbances in rats, induced inflammation, oxidative stress response and apoptosis, and activated TLR/NF-κB and Nrf2/ HO-1 pathways in the cerebral tissue. GLB administration attenuated LPS-induced alterations. The TLR/NF-κB pathway was deactivated and Nrf2/HO-1 was activated after application of GLB. In vitro, cytotoxic effects induced by the conditioned medium derived from microglial cells exposed to LPS in PC12 cells were attenuated by GLB. CONCLUSION: GLB suppresses LPS-induced PD symptoms by modification of TLR/NF-κB and Nrf2/HO-1 pathways in vivo and in vitro.

DT(270-326) , a Truncated Diphtheria Toxin, Increases Blood-Tumor Barrier Permeability by Upregulating the Expression of Caveolin-1.

AIM: The nontoxic mutant of diphtheria toxin (DT) has been demonstrated to act as a receptor-specific carrier protein to delivery drug into brain. Recent research showed that the truncated "receptorless" DT was still capable of being internalized into cells. This study investigated the effects and potential mechanisms of DT(270-326) , a truncated "receptorless" DT, on the permeability of the blood-tumor barrier (BTB). METHODS: BTB and GECs were subjected to DT(270-326) treatment. HRP flux assays, immunofluorescent, co-immunoprecipitation, Western blot, CCK-8, and Flow cytometry analysis were used to evaluate the effects of DT(270-326) administration. RESULTS: Our results revealed that 5 µM of DT(270-326) significantly increased the permeability of BTBin vitro, which reached its peak at 6 h. The permeability was reduced by pretreatment with filipinIII. DT(270-326) co-localized and interacted with caveolin-1 via its caveolin-binding motif. The mRNA and protein expression levels of caveolin-1 were identical with the changes of BTB permeability. The upregulated expression of caveolin-1 was associated with Src kinase-dependent tyrosine phosphorylation of caveolin-1, which subsequently induced phosphorylation and inactivation of the transcription factor Egr-1. The combination of DT(270-326) with doxorubicin significantly enhanced the loss of cell viability and apoptosis of U87 glioma cells in contrast to doxorubicin alone. CONCLUSIONS: DT(270-326) might provide a novel strategy to increase the delivery of macromolecular therapeutic agents across the BTB.

The long noncoding RNA TUG1 regulates blood-tumor barrier permeability by targeting miR-144.

Blood-tumor barrier (BTB) limits the delivery of chemotherapeutic agent to brain tumor tissues. Long non-coding RNAs (lncRNAs) have been shown to play critical regulatory roles in various biologic processes of tumors. However, the role of lncRNAs in BTB permeability is unclear. LncRNA TUG1 (taurine upregulated gene 1) was highly expressed in glioma vascular endothelial cells from glioma tissues. It also upregulated in glioma co-cultured endothelial cells (GEC) from BTB model in vitro. Knockdown of TUG1 increased BTB permeability, and meanwhile down-regulated the expression of the tight junction proteins ZO-1, occludin, and claudin-5. Both bioinformatics and luciferase reporter assays demonstrated that TUG1 influenced BTB permeability via binding to miR-144. Furthermore, Knockdown of TUG1 also down-regulated Heat shock transcription factor 2 (HSF2), a transcription factor of the heat shock transcription factor family, which was defined as a direct and functional downstream target of miR-144. HSF2 up-regulated the promoter activities and interacted with the promoters of ZO-1, occludin, and claudin-5 in GECs. In conclusion, our results indicate that knockdown of TUG1 increased BTB permeability via binding to miR-144 and then reducing EC tight junction protein expression by targeting HSF2. Thus, TUG1 may represent a useful future therapeutic target for enhancing BTB permeability.

Overexpression of Roundabout4 predicts poor prognosis of primary glioma patients via correlating with microvessel density.

Roundabout4 (Robo4), a new member of Robo proteins family, is specifically expressed in endothelial cells. Recent studies have indicated that Robo4 could regulate tumor angiogenesis and vascular permeability. However, the role and function of Robo4 are not well understood. This study was performed to investigate the expression of Robo4 in primary glioma patients, and thus to determine the association of Robo4 expression with microvessel density and survival of glioma patients. In this study, real-time PCR and immunohistochemistry were performed to examine the mRNA level and protein expression of Robo4 in both 43 cases of glioma samples and 10 cases of normal brain tissue samples. The results demonstrated that Robo4 was significantly up-regulated in glioma tissues compared with normal brain tissues. In addition, double immunofluorescent staining revealed that Robo4 expression co-localized with CD34 expression in the vessel of glioma tissues. The expression of Robo4 positively correlated with patients' age (P = 0.0139) and glioma grade (P < 0.0001). A linear correlation was observed between the relative mRNA expression of Robo4 values and corresponding microvessel density values (r = 0.9735, P < 0.0001). Kaplan-Meier analysis and log-rank test result showed that the overall survival of patients with Robo4 high expression was significantly shorter than that of patients with Robo4 low expression (P < 0.001). The results of present study verify that overexpression of Robo4 is related to poor prognosis of primary gliomas patients through correlating with microvessel density.

Endophilin-1 regulates blood-brain barrier permeability via EGFR-JNK signaling pathway.

Endophilin-1 (Endo1), a multifunctional protein, is essential for synaptic vesicle endocytosis. However, the role and mechanism of endophilin-1 in blood-brain barrier (BBB) function are still unclear. This study was performed to determine whether endophilin-1 regulated BBB permeability via the EGFR-JNK signaling pathway. In the present study, we found that endophilin-1 over-expression in human cerebral microvascular endothelial cell (hCMEC/D3) increased BBB permeability and meanwhile reduced the expression levels of epidermal growth factor receptor (EGFR), phosphorylated c-Jun N-terminal kinase (p-JNK). While endophilin-1 knockdown led to the contrary results. After JNK inhibitor SP600125 was administered to the endophilin-1 silenced hCMEC/D3 cells, the transendothelial electrical resistance (TEER) value was decreased and the permeability coefficient values to 4kDa and 40kDa FITC-dextran were increased. Results observed by Transmission electron microscopy (TEM) showed that tight junctions (TJs) were opened. Moreover, immunofluorescence and Western blot assays revealed the discontinuous distribution of TJ-associated proteins ZO-1, occludin on cell-cell boundaries and a significant decrease in protein expressing levels. Therefore, these results indicated that endophilin-1 positively regulated BBB permeability via the EGFR-JNK signaling pathway in hCMEC/D3 cells, which would provide an experimental basis for further research on endophilin-1 mediated the opening of BBB.

MiR-449a exerts tumor-suppressive functions in human glioblastoma by targeting Myc-associated zinc-finger protein.

Glioblastoma (GBM) is one of the most common and aggressive primary brain tumors in adults. Deregulated expression of microRNAs (miRNAs) has been associated with GBM progression through alterations in either oncogenic or tumor suppressor targets. Here, we elucidated the function and the possible molecular mechanisms of miR-449a in human GBM cell lines and tumor specimens-derived glioblastoma stem cells (GSCs). Quantitative real-time PCR demonstrated that miR-449a was down-regulated in human GBM cell lines and GSCs. Functionally, miR-449a acted as a tumor suppressor by reducing cell proliferation, migration and invasion as well as inducing apoptosis in human GBM cell lines and GSCs. Myc-associated zinc-finger protein (MAZ) was identified as a direct target of miR-449a, mediating these tumor-suppressive effects, demonstrated by Western blot assay and luciferase assays. Moreover, over-expression of miR-449a inhibited the expression of Podoplanin (PDPN) by down-regulating MAZ which could positively control the promoter activities via binding to the promoter of PDPN, demonstrated by luciferase assays and chromatin immunoprecipitation assays. Further, the PI3K/AKT pathway was blocked when MAZ was down-regulated by miR-449a. This process was coincided with the up-regulation of apoptotic proteins and the down-regulation of anti-apoptotic proteins, MMP2 and MMP9. Furthermore, nude mice carrying over-expressed miR-449a combined with knockdown MAZ tumors produced the smallest tumors and the highest survival. These results elucidated a novel molecular mechanism of GBM progression, and may thus suggest a promising application for GBM treatment.

Roundabout 4 regulates blood-tumor barrier permeability through the modulation of ZO-1, Occludin, and Claudin-5 expression.

The blood-tumor barrier (BTB) restricts the delivery of chemotherapeutic drug molecules to tumor tissues. We found that the endothelial cell (EC) receptor molecule Roundabout 4 (Robo4) is endogenously expressed in human brain microvascular ECs and that it is upregulated in a BTB model of glioma cocultured ECs. Knockdown of Robo4 in this BTB model increased permeability; short hairpin RNA targeting Robo4 (shRobo4) led to decreased transendothelial electric resistance values, increased BTB permeability, and downregulated expression of the EC tight junction proteins ZO-1, occludin, and claudin-5. Roundabout 4 influenced BTB permeability via binding with its ligand, Slit2. Short hairpin RNA targeting Robo4 also increased matrix metalloproteinase-9 (MMP-9) activity and expression in glioma cocultured ECs; pretreatment with the MMP inhibitor GM6001 partially blocked the effects of shRobo4 on the transendothelial electric resistance values and ZO-1 and occludin expression. Short hairpin RNA targeting Robo4 also upregulated the phosphorylation of Src and Erk1/2; the Src inhibitor PP2 and the Erk1/2 inhibitor PD98059 blocked shRobo4-mediated alteration in ZO-1 and occludin expression. Together, our results indicate that knockdown of Robo4 increased BTB permeability by reducing EC tight junction protein expression, and that the Src-Erk1/2-MMP-9 signal pathways are involved in this process. Thus, Robo4 may represent a useful future therapeutic target for enhancing BTB permeability.