Soluble ANPEP Released From Human Astrocytes as a Positive Regulator of Microglial Activation and Neuroinflammation: Brain Renin-Angiotensin System in Astrocyte-Microglia Crosstalk.

Astrocytes are major supportive glia and immune modulators in the brain; they are highly secretory in nature and interact with other cell types via their secreted proteomes. To understand how astrocytes communicate during neuroinflammation, we profiled the secretome of human astrocytes following stimulation with proinflammatory factors. A total of 149 proteins were significantly upregulated in stimulated astrocytes, and a bioinformatics analysis of the astrocyte secretome revealed that the brain renin-angiotensin system (RAS) is an important mechanism of astrocyte communication. We observed that the levels of soluble form of aminopeptidase N (sANPEP), an RAS component that converts angiotensin (Ang) III to Ang IV in a neuroinflammatory milieu, significantly increased in the astrocyte secretome. To elucidate the role of sANPEP and Ang IV in neuroinflammation, we first evaluated the expression of Ang IV receptors in human glial cells because Ang IV mediates biological effects through its receptors. The expression of angiotensin type 1 receptor was considerably upregulated in activated human microglial cells but not in human astrocytes. Moreover, interleukin-1ß release from human microglial cells was synergistically increased by cotreatment with sANPEP and its substrate, Ang III, suggesting the proinflammatory action of Ang IV generated by sANPEP. In a mouse neuroinflammation model, brain microglial activation and proinflammatory cytokine expression levels were increased by intracerebroventricular injection of sANPEP and attenuated by an enzymatic inhibitor and neutralizing antibody against sANPEP. Collectively, our results indicate that astrocytic sANPEP-induced increase in Ang IV exacerbates neuroinflammation by interacting with microglial proinflammatory receptor angiotensin type 1 receptor, highlighting an important role of indirect crosstalk between astrocytes and microglia through the brain RAS in neuroinflammation.

Subsequent correlated changes in complement component 3 and amyloid beta oligomers in the blood of patients with Alzheimer's disease.

INTRODUCTION: Alzheimer's disease (AD) involves the complement cascade, with complement component 3 (C3) playing a key role. However, the relationship between C3 and amyloid beta (Aß) in blood is limited. METHODS: Plasma C3 and Aß oligomerization tendency (AßOt) were measured in 35 AD patients and 62 healthy controls. Correlations with cerebrospinal fluid (CSF) biomarkers, cognitive impairment, and amyloid positron emission tomography (PET) were analyzed. Differences between biomarkers were compared in groups classified by concordances of biomarkers. RESULTS: Plasma C3 and AßOt were elevated in AD patients and in CSF or amyloid PET-positive groups. Weak positive correlation was found between C3 and AßOt, while both had strong negative correlations with CSF Aß42 and cognitive performance. Abnormalities were observed for AßOt and CSF Aß42 followed by C3 changes. DISCUSSION: Increased plasma C3 in AD are associated with amyloid pathology, possibly reflecting a defense response for Aß clearance. Further studies on Aß-binding proteins will enhance understanding of Aß mechanisms in blood.

Distinct cerebral cortical perfusion patterns in idiopathic normal-pressure hydrocephalus.

The aims of the study are to evaluate idiopathic normal-pressure hydrocephalus (INPH)-related cerebral blood flow (CBF) abnormalities and to investigate their relation to cortical thickness in INPH patients. We investigated cortical CBF utilizing surface-based early-phase 18 F-florbetaben (E-FBB) PET analysis in two groups: INPH patients and healthy controls. All 39 INPH patients and 20 healthy controls were imaged with MRI, including three-dimensional volumetric images, for automated surface-based cortical thickness analysis across the entire brain. A subgroup with 37 participants (22 INPH patients and 15 healthy controls) that also underwent 18 F-fluorodeoxyglucose (FDG) PET imaging was further analyzed. Compared with age- and gender-matched healthy controls, INPH patients showed statistically significant hyperperfusion in the high convexity of the frontal and parietal cortical regions. Importantly, within the INPH group, increased perfusion correlated with cortical thickening in these regions. Additionally, significant hypoperfusion mainly in the ventrolateral frontal cortex, supramarginal gyrus, and temporal cortical regions was observed in the INPH group relative to the control group. However, this hypoperfusion was not associated with cortical thinning. A subgroup analysis of participants that also underwent FDG PET imaging showed that increased (or decreased) cerebral perfusion was associated with increased (or decreased) glucose metabolism in INPH. A distinctive regional relationship between cerebral cortical perfusion and cortical thickness was shown in INPH patients. Our findings suggest distinct pathophysiologic mechanisms of hyperperfusion and hypoperfusion in INPH patients.

Glycan-Controlled Human PD-1 Variants Displaying Broad-Spectrum High Binding to PD-1 Ligands Potentiate T Cell.

Although therapeutic immunoglobulin G (IgG) antibodies that regulate the activity of immune checkpoints bring innovation to the field of immuno-oncology, they are still limited in their efficiency to infiltrate the tumor microenvironment due to their large molecular size (150 kDa) and the necessity of additional engineering works to ablate effector functions for antibodies targeting immune cells. To address these issues, the human PD-1 (hPD-1) ectodomain, a small protein moiety of 14-17 kDa, has been considered as a therapeutic agent. Here, we used bacterial display-based high-throughput directed evolution to successfully isolate glycan-controlled (aglycosylated or only single-N-linked glycosylated) human PD-1 variants exhibiting over 1000-fold increased hPD-L1 binding affinity compared to that of wild-type hPD-1. The resulting hPD-1 variants, aglycosylated JYQ12 and JYQ12-2 with a single-N-linked glycan chain, showed exceptionally high binding affinity to hPD-L1 and very high affinity to both hPD-L2 and mPD-L1. Moreover, the JYQ12-2 efficiently potentiated the proliferation of human T cells. hPD-1 variants with significantly improved binding affinities for hPD-1 ligands could be used as effective therapeutics or diagnostics that can be differentiated from large-sized IgG antibody-based molecules.

Role of M2-like macrophages in the progression of ovarian cancer.

In this study, we noninvasively assessed whether M2-like macrophages accelerate the progression of ovarian cancer by performing molecular imaging of ovarian cancer cells expressing enhanced firefly luciferase (Effluc) in living mice. First, murine ovarian cancer ID8 cells expressing Effluc (ID8/Effluc cells) were established by retroviral infection. Subsequently, macrophages were isolated from the peritoneal exudate of mice injected with thioglycollate medium and differentiated into M2-like macrophages by adding interleukin 4. To characterize these M2-like macrophages, F4/80 and cluster of differentiation 206 expression levels were determined. Then, the M2-like macrophages were co-cultured with the ID8/Effluc cells and bioluminescence imaging (BLI) of signals from the ID8/Effluc cells was completed. Additionally, migration and wound healing were assessed to evaluate the effects of conditioned medium (CM) from M2-like macrophages on ID8/Effluc cell motility. In the in vivo study, mice were first given either liposome-phosphate-buffered saline or liposome-clodronate (lipo-clodronate). After 24 h, ID8/Effluc cells were intraperitoneally injected into the mice and BLI was completed at the designed time points. Next, histological analysis was conducted to characterize the infiltrated tumor. Flow cytometric analysis revealed high levels of CD206 expression in the differentiated M2-like macrophages. Meanwhile, ID8/Effluc cells co-cultured with these M2-like macrophages proliferated rapidly in an M2-like macrophage, number-dependent manner. The migration of the ID8/Effluc cells was also increased by the application of CM from M2-like macrophages. In vivo BLI revealed that the growth rate of intraperitoneally injected ovarian cancer cells was inhibited following macrophage depletion by treatment with lipo-clodronate. M2-like macrophages accelerated the progression of ovarian cancer, suggesting they are a new therapeutic target for ovarian cancer and that ovarian cancer could be managed by altering the nature of communication between ovarian cancer and macrophages.

Saline Load Test and Quilting Sutures to Treat Intractable Lateral Malleolar Bursitis.

The purpose of this study was to evaluate the clinical outcomes of patients with intractable lateral malleolar bursitis who were treated using the intraoperative saline load test to find communication between the bursal sac and the ankle joint and the quilting sutures after bursectomy to reduce the dead space. We reviewed a total of 28 patients who had been treated with quilting sutures after bursectomy between April 2014 and June 2017. When there was capsular opening detected with the saline load test, it was closed with sutures or augmented with periosteum. On the final follow-up office visit, the lateral malleolus was examined for the recurrence of bursitis. Patient medical records were reviewed for postoperative wound dehiscence, skin necrosis, infections, and nerve symptoms. The saline load test was positive in 11 (42%) cases. The mean foot function index improved from 25.94 ± 20.46 to 11.73 ± 5.27 (p = .003). Fourteen (54%) patients were very satisfied with the results, 9 (35%) were satisfied, 2 (8%) rated their satisfaction as fair, and 1 (4%) was dissatisfied. No cases required skin graft or flap surgery. Intractable lateral malleolar bursitis was successfully treated using the saline load test and quilting sutures after bursectomy. Closure of the capsular opening prevented fluid drainage around the wound. Quilting sutures after bursectomy reduced dead space underneath the wound to prevent fluid accumulation and promoted healing of the skin on the underlying soft tissue.

Human Allogeneic Bone Marrow-Derived Mesenchymal Stem Cell Therapy for Cerebellar Ataxia: A Case Report.

To date, there is no curable treatment option for non-hereditary degenerative cerebellar ataxia. Here we report the case of a patient with sporadic adult-onset ataxia (SAOA) who underwent allogeneic bone marrow-derived mesenchymal stem cell (MSC) therapy via the intrathecal route. A 60-year-old male patient visited our clinic complaining of progressive gait disturbance that commenced two years ago. Upon neurologic examination, the patient exhibited limb dysmetria and gait ataxia. Brain magnetic resonance imaging (MRI) revealed cerebellar atrophy whereas the autonomic function test was normal. The patient was diagnosed with SAOA. The medications that were initially prescribed had no significant effects on the course of this disease and the symptoms deteriorated progressively. At the age of 64, the patient was treated with allogeneic bone marrow-derived MSC therapy. The subsequent K-SARA (Korean version of the Scale for the Assessment and Rating of Ataxia) scores demonstrated a distinct improvement up until 10 months post-administration. No adverse events were reported. The improved post-treatment K-SARA scores may suggest that the MSC therapy can have a neuroprotective effect and that stem cell therapy may serve as a potential therapeutic option for degenerative cerebellar ataxia.

White blood cell labeling with Technetium-99m (99mTc) using red blood cell extracellular vesicles-mimetics.

BACKGROUND: Extracellular vesicles, have gained increasing attention for their application in drug delivery. Here, we developed a novel method for radiolabeling WBCs with 99mTc using RBC-derived extracellular vesicles -mimetics (EVMs), and monitored in vivo inflammation tracking of 99mTc-WBC using gamma camera in acute inflammation mouse model. METHODS: Engineered EVMs from RBCs were produced by a one-step extrusion method. RBC-EVMs were analyzed by NTA and TEM. Cells were labeled with 99mTc by using 99mTc-RBC-EVMs. Inflammation mice model was prepared and confirmed by 18F-FDG PET/CT. 99mTc-WBCs were injected in mice, and their biodistribution was analyzed by gamma camera. FINDING: The radiochemical purity of 99mTc-RBC-EVMs was 100%. The 99mTc-labeling did't affect the size and morphology. The 99mTc in the cytoplasm of RBC-EVMs was successfully confirmed by high angle annular dark field STEM (scanning transmission electron microscope). Cells were successfully labeled with 99mTc using 99mTc-RBC-EVMs, and the counts per minute was increased in dose- and time-dependent manners. The 18F-FDG PET/CT images confirmed establishment of acute inflammation (left mouse foot). 99mTc-WBCs showed higher uptake in the inflamed foot than non-inflamed foot. INTERPRETATION: This novel method for radiolabeling WBCs using RBC-EVMs. 99mTc labeling may be a feasible method to monitor the in vivo biodistribution of cells.

Lower Glucose Level Associated With Increased Risk for Post-Dural Puncture Headache.

OBJECTIVE AND BACKGROUND: Post-dural puncture headache is the most common significant adverse event following lumbar puncture. In this study, we investigated the possible systemic factors associated with risk for post-dural puncture headache (PDPH). METHODS: We performed a retrospective cohort study in 969 patients who underwent diagnostic lumbar puncture following a standardized protocol. We compared the clinical and laboratory profiles of the post-dural puncture headache group and non-headache group. We also identified independent factors associated with the incidence of post-dural puncture headache. RESULTS: A total of 48 patients (5%) reported headache; 12 of these patients (25%) received a therapeutic epidural blood patch and the remaining 36 patients improved with conservative treatment. After adjusting for other variables that could be related to PDPH, we found that the development of post lumbar puncture headache was independently associated with age (OR: 0.97, 95% CI: 0.95-0.99, P = .001) and serum glucose levels (OR: 0.98, 95% CI: 0.97-0.99, P = .008).When the patients were classified by age, serum glucose levels were persistently lower in patients with PDPH vs those patients without PDPH in all age groups, with more clearly significant differences observed in the elderly (age <30 years, 103.4 mg/dL vs 106.3 mg/dL, P = .716; >60 years, 111.8 mg/dL vs 137.3 mg/dL, P = .023). CONCLUSIONS: Low glucose levels were inversely associated with risk for post-dural puncture headache. Patients with low serum glucose should be carefully monitored for headache after lumbar puncture.

Behavioural and trait changes in parkinsonian patients with impulse control disorder after switching from dopamine agonist to levodopa therapy: results of REIN-PD trial.

OBJECTIVE: In this multicentre open-label trial, we compared behavioural and neuropsychiatric symptoms in Parkinson's disease (PD) patients with impulse control disorders (ICD) treated with dopamine agonists before and 12 weeks after substituting dopamine agonists with an equivalent dose of levodopa/carbidopa slow-release formulation. METHODS: Baseline characteristics of 50 PD patients with ICD were compared with those of 60 medicated and 40 drug-naive PD control groups. Neuropsychiatric trait changes in the PD-ICD group were investigated 12 weeks after the intervention. ICD behaviours were assessed via modified Minnesota Impulsive Disorders Interview (mMIDI), whereas parkinsonian severity and neuropsychiatric characters were systematically assessed with the Unified PD Rating Scale (UPDRS) and a predefined neuropsychological assessment battery. RESULTS: At baseline, ICD patients showed higher scores in the Neuropsychiatric Inventory and anxiety, anger and obsessive-compulsive traits compared with both PD control groups. In contrast, the three PD groups showed indifference in the impulsivity scales. At 12 weeks post intervention, ICD behaviours significantly improved (p<0.001, Δ modified MIDI score=‒5.27 ± 5.75) along with the UPDRS II daily activity scores (p=0.02, Δ=‒2.07 ± 4.53). Behavioural disinhibition tended to improve (p=0.06), although no significant changes were observed in the Neuropsychiatric Inventory and personality trait scores. Dopamine agonist withdrawal syndrome developed in 5.3% of the PD-ICD group. CONCLUSIONS: This study provides class IV evidence suggesting that switching from dopamine agonists to levodopa/carbidopa slow-release formulations alleviated ICD behaviours in PD patients leading to improvement in daily activities whereas neuropsychiatric traits associated with ICD persisted after the 12-week therapy. TRIAL REGISTRATION NUMBER: NCT01683253.

Characteristics of obstructive sleep apnea in myasthenia gravis patients: a single center study.

BACKGROUND: Recent studies have shown a high prevalence of obstructive sleep apnea in myasthenia gravis compared to the normal population. The aim of this study was to elucidate clinical and polysomnographic differences between clinically stable Korean MG patients with and without OSA. METHODS: A total of 18 consecutively stable MG patients were included in this prospective study. We compared MG patients with OSA (n = 7) and without OSA (n = 11) with respect to the baseline characteristics and overnight polysomnography (PSG) parameters. Demographic parameters, prescribed medication status, thymectomy status, myasthenia gravis foundation of America score, and antibody status were obtained from their medical records. We performed the Korean version of Pittsburg sleep quality index to assess the subjective quality of sleep. Statistical analyses were performed using SPSS version 18.0 with Wilcoxon rank sum test, chi-square test, Fisher's exact test, and Spearman correlation test. RESULTS: Among the clinical parameters, MG patients with OSA showed a higher proportion of male sex (p = 0.016) and increased body mass index (p = 0.033). The PSG showed an 11-fold higher supine apnea-hyponea index (AHI) in MG patients with OSA. AHI was further analyzed with supine and non-supine position. MG patients with OSA had a higher supine AHI (19.5 ± 15.8) compared to those without OSA (1.9 ± 1.2, P = 0.008). Most of MG patients with OSA (85.7%) showed more than two times higher supine AHI than non-supine AHI. CONCLUSIONS: This study showed that the occurrence of OSA in patients with MG is associated with male sex and obesity, which is in accordance with the normal population. Moreover, PSG data showed a high prevalence of supine dominant OSA in MG patients with OSA.

Neuropsychiatric Traits Associated with Refractory Impulse Control Disorder in Parkinson's Disease.

INTRODUCTION: Impulse control disorder (ICD) in Parkinson's disease (PD) is a critical nonmotor symptom with personality or neuropsychiatric traits contributing to ICD. OBJECTIVE: This study aimed to identify predictive traits for persistent or paradoxical aggravation of ICD after dopamine agonist substitution therapy for ICD in PD. METHODS: We conducted a case-control study using a database of a multicenter intervention trial for ICD in PD. The poor-outcome group was defined by showing paradoxical increases in ICD behaviors after the substitution of dopamine agonists with levodopa. We analyzed the pre-intervention personality traits associated with the poor outcome and also evaluated the risk traits for refractory ICD using a receiver-operating characteristic (ROC) curve analysis. RESULTS: The poor-outcome group showed higher levels of anger expression (p =0.007) and obsessive-compulsive traits (p =0.009) compared with the good-outcome group at the pre-intervention state. In the ROC curve analysis, the Obsessive-Compulsive Inventory showed the highest area under the curve with 80.0% sensitivity and 74.3% specificity in discriminating against the poor-outcome group. CONCLUSIONS: Our results suggest that assessment of obsessive compulsiveness may be useful for predicting the refractoriness of ICD behaviors in planning an interventional treatment for ICD in PD.

Dual reconstruction of lateral collateral ligament is safe and effective in treating posterolateral rotatory instability of the elbow.

PURPOSE: Although reconstruction of the lateral ulnar collateral ligament (LUCL) has been considered the procedure of choice for posterolateral rotatory instability (PLRI), recent studies have reported that the entire lateral collateral ligament complex (LCLC), rather than its posterior part only, contributes to preventing PLRI. Thus, it was hypothesized that dual reconstruction of the radial collateral ligament (RCL) and LUCL for the treatment of elbow PLRI could provide favourable clinical results regardless of the mechanism of injury. METHODS: This retrospective study reviewed the clinical results of 21 patients who underwent dual reconstruction of the RCL and LUCL between 2011 and 2016. Functional outcomes were assessed using the numeric rating scale (NRS) score, Mayo Elbow Performance Score (MEPS), quick Disabilities of the Arm, Shoulder, and Hand (quick DASH) score, and manual varus instability. To identify any difference in outcomes according to the aetiologies for LCLC insufficiency, our patients were divided into LCLC insufficiency associated with elbow dislocation and that with lateral epicondylitis. RESULTS: At a median follow-up of 27 months (range 13-65 months), all patients showed resolved instability and achieved a functional arc of motion. In addition, lateral pivot shift tests were negative in all patients. The median MEPS significantly improved after surgery from 70 (range 60-75) to 85 (range 75-100) (p < 0.001), while the median quick DASH score improved from 38.6 (range 26.6-54.5) to 11.4 (range 0-34.1) (p < 0.001). Clinical outcomes according to the aetiology of LCLC insufficiency were not significantly different except for the NRS score. CONCLUSION: The results suggest that the dual reconstruction technique leads to a clinical outcome similar to that of conventional LUCL reconstruction in LCLC insufficiency regardless of aetiology. In addition, the dual reconstruction technique was technically easier than the conventional LUCL reconstruction technique and may be a potential alternative when a bone tunnel created at the proximal ulna by the original technique has failed. LEVEL OF EVIDENCE: IV.

Migration of mesenchymal stem cells to tumor xenograft models and in vitro drug delivery by doxorubicin.

Mesenchymal stem cells (MSCs) show therapeutic effects in various types of diseases. MSCs have been shown to migrate towards inflamed or cancerous tissues, and visualized after sacrificing the animal. MSCs are able to deliver drugs to target cells, and are an ideal candidate for cancer therapy. The purpose of this study was to track the migration of MSCs in tumor-bearing mice; MSCs were also used as drug delivery vehicles. Human breast cancer cells (MDA-MB-231) and anaplastic thyroid cancer cells (CAL62) were transduced with lentiviral particles, to express the Renilla luciferase and mCherry (mCherry-Rluc) reporter genes. Human bone marrow-derived MSCs were transduced with lentiviral particles, to express the firefly luciferase and enhanced green fluorescence protein (Fluc2-eGFP) reporter genes (MSC/Fluc). Luciferase activity of the transduced cells was measured by bioluminescence imaging (BLI). Further in vitro migration assays were performed to confirm cancer cells conditioned medium dependent MSC and doxorubicin (DOX) treated MSC migration. MSCs were loaded with DOX, and their therapeutic effects against the cancer cells were studied in vitro. In vivo MSC/Fluc migration in mice having thyroid or breast cancer xenografts was evaluated after systemic injection. Rluc activity of CAL62/Rluc (R2=0.911), MDA-MB-231/Rluc (R2=0.934) cells and Fluc activity of MSC/Fluc (R2=0.91) cells increased with increasing cell numbers, as seen by BLI. eGFP expression of MSC/Fluc was confirmed by confocal microscopy. Similar migration potential was observed between MSC/Fluc and naïve MSCs in migration assay. DOX treated MSCs migration was not decreased compared than MSCs. Migration of the systemically injected MSC/Fluc cells into tumor xenografts (thyroid and breast cancer) was visualized in animal models (p<0.05) and confirmed by ex vivo (p<0.05) BLI. Additionally, MSCs delivered DOX to CAL62/Rluc and MDA-MB-231/Rluc cells, thereby decreasing their Rluc activities. In this study, we confirmed the migration of MSCs to tumor sites in cancer xenograft models using both in vivo and ex vivo BLI imaging. DOX-pretreated MSCs showed enhanced cytotoxic effects. Therefore, this noninvasive reporter gene (Fluc2)-based BLI may be useful for visualizing in vivo tracking of MSCs, which can be used as a drug delivery vehicle for cancer therapy.

Amyloid Deposits and Idiopathic Normal-Pressure Hydrocephalus: An 18F-Florbetaben Study.

BACKGROUND: The first aim of our study was to determine whether cortical 18F-florbetaben retention was different between healthy controls and idiopathic normal-pressure hydrocephalus (INPH) patients. Our second aim was to investigate whether there were any relationships between 18F-florbetaben retention and either hippocampal volume or clinical symptoms in INPH patients. METHODS: Seventeen patients diagnosed with INPH and 8 healthy controls underwent studies with magnetic resonance imaging and 18F-florbetaben positron emission tomography imaging. RESULTS: Automated region-of-interest analysis showed significant increases in 18F-florbetaben uptake in several brain regions in INPH patients compared to control subjects, with especially remarkable increases in the frontal (bilateral), parietal (bilateral), and occipital (bilateral) cortices. In the INPH group, right hippocampal volume was found to be negatively correlated with right frontal 18F-florbetaben retention. Korean-Mini Mental State Examination scores negatively correlated with right occipital 18F-florbetaben retention. Higher 18F-florbetaben retention correlated significantly with a higher Clinical Dementia Rating Scale score in the right occipital cortex. CONCLUSIONS: Our results indicate that INPH might be a disease exhibiting a characteristic pattern of cortical 18F-florbetaben retention. 18F-florbetaben retention in the frontal cortex may be related to hippocampal neuronal degeneration. Our findings may also help us understand the potential pathophysiology of cognitive impairments associated with INPH.

Regulated Mesenchymal Stem Cells Mediated Colon Cancer Therapy Assessed by Reporter Gene Based Optical Imaging.

Colorectal cancer is the most common cancer in both men and women and the second most common cause of cancer-related deaths. Suicide gene-based therapy with suicide gene-transduced mesenchymal stem cells (MSCs) is a promising therapeutic strategy. A tetracycline-controlled Tet-On inducible system used to regulate gene expression may be a useful tool for gene-based therapies. The aim of this study was to develop therapeutic MSCs with a suicide gene that is induced by an artificial stimulus, to validate therapeutic gene expression, and to monitor the MSC therapy for colon cancer using optical molecular imaging. For our study, we designed the Tet-On system using a retroviral vector and developed a response plasmid RetroX-TRE (tetracycline response element) expressing a mutant form of herpes simplex virus thymidine kinase (HSV1-sr39TK) with dual reporters (eGFP-Fluc2). Bone marrow-derived MSCs were transduced using a RetroX-Tet3G (Clontech, CA, USA) regulatory plasmid and RetroX-TRE-HSV1-sr39TK-eGFP-IRES-Fluc2, for a system with a Tet-On (MSC-Tet-TK/Fluc2 or MSC-Tet-TK) or without a Tet-On (MSC-TK/Fluc2 or MSC-TK) function. Suicide gene engineered MSCs were co-cultured with colon cancer cells (CT26/Rluc) in the presence of the prodrug ganciclovir (GCV) after stimulation with or without doxycycline (DOX). Treatment efficiency was monitored by assessing Rluc (CT26/Rluc) and Fluc (MSC-Tet-TK and MSC-TK) activity using optical imaging. The bystander effect of therapeutic MSCs was confirmed in CT26/Rluc cells after GCV treatment. Rluc activity in CT26/Rluc cells decreased significantly with GCV treatment of DOX(+) cells (p < 0.05 and 0.01) whereas no significant changes were observed in DOX(-) cells. In addition, Fluc activity in also decreased significantly with DOX(+) MSC-Tet-TK cells, but no signal was observed in DOX(-) cells. In addition, an MSC-TK bystander effect was also confirmed. We assessed therapy with this system in a colon cancer xenograft model (CT26/Rluc). We successfully transduced cells and developed a Tet-On system with the suicide gene HSV1-sr39TK. Our results confirmed the therapeutic efficiency of a suicide gene with the Tet-On system for colon cancer. In addition, our results provide an innovative therapeutic approach using the Tet-On system to eradicate tumors by administration of MSC-Tet-TK cells with DOX and GCV.

Pathogenic Upregulation of Glial Lipocalin-2 in the Parkinsonian Dopaminergic System.

UNLABELLED: Lipocalin-2 (LCN2) is a member of the highly heterogeneous secretory protein family of lipocalins and increases in its levels can contribute to neurodegeneration in the adult brain. However, there are no reports on the role of LCN2 in Parkinson's disease (PD). Here, we report for the first time that LCN2 expression is increased in the substantia nigra (SN) of patients with PD. In mouse brains, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment for a neurotoxin model of PD significantly upregulated LCN2 expression, mainly in reactive astrocytes in both the SN and striatum. The increased LCN2 levels contributed to neurotoxicity and neuroinflammation, resulting in disruption of the nigrostriatal dopaminergic (DA) projection and abnormal locomotor behaviors, which were ameliorated in LCN2-deficient mice. Similar to the effects of MPTP treatment, LCN2-induced neurotoxicity was also observed in the 6-hydroxydopamine (6-OHDA)-treated animal model of PD. Moreover, treatment with the iron donor ferric citrate (FC) and the iron chelator deferoxamine mesylate (DFO) increased and decreased, respectively, the LCN2-induced neurotoxicity in vivo In addition to the in vivo results, 1-methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity in cocultures of mesencephalic neurons and astrocytes was reduced by LCN2 gene deficiency in the astrocytes and conditioned media derived from MPP(+)-treated SH-SY5Y neuronal enhanced glial expression of LCN2 in vitro Therefore, our results demonstrate that astrocytic LCN2 upregulation in the lesioned DA system may play a role as a potential pathogenic factor in PD and suggest that inhibition of LCN2 expression or activity may be useful in protecting the nigrostriatal DA system in the adult brain. SIGNIFICANCE STATEMENT: Lipocalin-2 (LCN2), a member of the highly heterogeneous secretory protein family of lipocalins, may contribute to neuroinflammation and neurotoxicity in the brain. However, LCN2 expression and its role in Parkinson's disease (PD) are largely unknown. Here, we report that LCN2 is upregulated in the substantia nigra of patients with PD and neurotoxin-treated animal models of PD. Our results suggest that LCN2 upregulation might be a potential pathogenic mechanism of PD, which would result in disruption of the nigrostriatal dopaminergic system through neurotoxic iron accumulation and neuroinflammation. Therefore, inhibition of LCN2 expression or activity may be useful in protecting the nigrostriatal dopaminergic projection in PD.

Astrocyte-derived lipocalin-2 mediates hippocampal damage and cognitive deficits in experimental models of vascular dementia.

Lipocalin-2 (LCN2) has diverse functions in multiple pathophysiological conditions; however, its pathogenic role in vascular dementia (VaD) is unknown. Here, we investigated the role of LCN2 in VaD using rodent models of global cerebral ischemia and hypoperfusion with cognitive impairment and neuroinflammation. Mice subjected to transient bilateral common carotid artery occlusion (tBCCAo) for 50 min showed neuronal death and gliosis in the hippocampus at 7 days post-tBCCAo. LCN2 expression was observed predominantly in the hippocampal astrocytes, whereas its receptor was mainly detected in neurons, microglia, and astrocytes. Furthermore, Lcn2-deficient mice, compared with wild-type animals, showed significantly weaker CA1 neuronal loss, cognitive decline, white matter damage, blood-brain barrier permeability, glial activation, and proinflammatory cytokine production in the hippocampus after tBCCAo. Lcn2 deficiency also attenuated hippocampal neuronal death and cognitive decline at 30 days after unilateral common carotid artery occlusion (UCCAo). Furthermore, intracerebroventricular (i.c.v) injection of recombinant LCN2 protein elicited CA1-neuronal death and a cognitive deficit. Our studies using cultured glia and hippocampal neurons supported the decisive role of LCN2 in hippocampal neurotoxicity and microglial activation, and the role of the HIF-1α-LCN2-VEGFA axis of astrocytes in vascular injury. Additionally, plasma levels of LCN2 were significantly higher in patients with VaD than in the healthy control subjects. These results indicate that hippocampal damage and cognitive impairment are mediated by LCN2 secreted from reactive astrocytes in VaD.

Serum methylmalonic acid correlates with neuropathic pain in idiopathic Parkinson's disease.

Recent studies have shown a relatively higher prevalence of peripheral neuropathy in idiopathic Parkinson's disease (IPD). The hypothesis is that prolonged levodopa exposure causes vitamin B12 deficiency, which leads to peripheral neuropathy. The aim of our study was to find the relationship between vitamin B12 and its precursor methylmalonic acid (MMA) in IPD patients with neuropathic pain. We performed a cross-sectional study by enrolling consecutive 43 patients who were clinically tested positive for F-18 FP-CIT PET and 15 patients were diagnosed with peripheral neuropathy according to the Toronto clinical scoring system (TCSS). The severity of neuropathic pain was evaluated using total neuropathy scale, revised (TNSr), and Korean Neuropathic Pain Questionnaire (KNPQ). The correlations between age, IPD duration, levodopa equivalent dose (LED), UPDRS III, vitamin B12, MMA, and homocysteine levels were assessed. The prevalence rate of peripheral neuropathy in IPD patients was 35%. Among the serums assessed, MMA levels showed a positive correlation to TNSr and KNPQ in the IPD patients with peripheral neuropathy (TNSr r = 0.882, p < 0.001, KNPQ r = 0.710, p = 0.004), while Vitamin B12 and homocysteine showed no statistically significant correlation. Our study showed a prevalence of peripheral neuropathy in 35% of Korean IPD patients. The serum MMA positively correlated with the severity of neuropathic pain and this can be used as a useful marker in assessment of peripheral neuropathy in Parkinson's disease.

Frontal Assessment Battery and Cerebrospinal Fluid Tap Test in Idiopathic Normal-Pressure Hydrocephalus.

BACKGROUND AND PURPOSE: Our aim in this study was to assess whether the frontal assessment battery (FAB) could contribute to the differential diagnosis of cerebrospinal fluid tap test (CSFTT) responders and nonresponders with the hypothesis that CSFTT nonresponders had greater frontal lobe dysfunction. We also explored whether a relationship exists between FAB scores and gait disturbance in idiopathic normal-pressure hydrocephalus (INPH) patients. METHODS: INPH subjects were selected in a consecutive order from a prospectively enrolled INPH registry. Fifty-one INPH patients constituted the final sample for analysis. RESULTS: Logistic regression analysis using the FAB score as independent variable showed a significant influence of the FAB on the differential diagnosis of CSFTT responders and nonresponders (p = 0.025; OR 1.186; 95% CI 1.022-1.377). The FAB scores were negatively correlated with the Timed Up and Go test score (r = -0.382; p = 0.007), 10-meter walking test score (r = -0.351; p = 0.014), Gait Status Scale score (r = -0.382; p = 0.007), and INPH Grading Scale gait score (r = -0.370; p = 0.009). CONCLUSIONS: Our findings may indicate a possibility for considering FAB scores in patients with ventriculomegaly as potential cognitive markers for the prediction of CSFTT response. Association between gait function and FAB scores suggests the involvement of similar circuits producing gait symptom and frontal lobe functions in INPH.