Clinicopathological phenotype and outcomes of NCAM-1+ membranous lupus nephritis.

INTRODUCTION: No studies explored the long-term outcomes of neural cell adhesion molecule 1 (NCAM1) associated membranous lupus nephritis (MLN) patients. METHOD: We performed immunohistochemical studies on kidney biopsy specimens against NCAM1 in consecutive MLN patients. The clinical and histopathological characteristics and outcomes of cases of NCAM1 associated MLN patients are described and compared with NCAM1 negative patients. In addition, we detected serum circulating anti-NCAM1 antibodies through western blotting and indirect immunofluorescence assays. RESULTS: Among 361 MLN cases, 18 (5.0%) were glomerular NCAM1-positive. NCAM1 positive MLN patients were older [35 years (IQR 27-43) versus 28 (22-37); P = 0.050) and had lower systemic lupus erythematosus disease activity index [11 (IQR 8-12) versus 14 (10-18); P = 0.007], serum creatinine [60 µmol/L (IQR 50-70) versus 70 (54-114); P = 0.029], activity index [3 (IQR 2-6) versus 6 (3-9); P = 0.045] at kidney biopsy compared with NCAM1 negative patients. The percentage of positive anti-Sjogren's syndrome related antigen A antibodies in NCAM1 positive patients was significantly greater (83.3% versus 58.2%; P = 0.035) than in the NCAM1 negative patients. However, no evidence of neuropsychiatric disorders was found in these 18 patients. There were no significant differences in the treatment response and the risk of end stage renal diseases between NCAM1 positive and negative groups (P = 0.668 and P = 0.318, respectively). But the risk of death was much higher in the NCAM1 positive group than the NCAM1 negative group (27.8% vs. 8.1%, P = 0.007). Moreover, the risk of death was also much higher in the NCAM1 positive group than the matched NCAM1 negative group (Log-rank P = 0.013). Additionally, circulating anti-NCAM1 antibodies can be detected in 1/5 (20%) patients who had serum available. CONCLUSION: The prevalence of NCAM1 positivity was 5.0% in our cohort of MLN and the high mortality in these subgroup patients are needed to validate in future studies.

Enhanced electrophysiological activity and neurotoxicity screening of environmental chemicals using 3D neurons from human neural precursor cells purified with PSA-NCAM.

The assessment of neurotoxicity for environmental chemicals is of utmost importance in ensuring public health and environmental safety. Multielectrode array (MEA) technology has emerged as a powerful tool for assessing disturbances in the electrophysiological activity. Although human embryonic stem cell (hESC)-derived neurons have been used in MEA for neurotoxicity screening, obtaining a substantial and sufficiently active population of neurons from hESCs remains challenging. In this study, we successfully differentiated neurons from a large population of human neuronal precursor cells (hNPC) purified using a polysialylated neural cell adhesion molecule (PSA-NCAM), referred to as hNPCPSA-NCAM+. The functional characterization demonstrated that hNPCPSA-NCAM+-derived neurons improve functionality by enhancing electrophysiological activity compared to total hNPC-derived neurons. Furthermore, three-dimensional (3D) neurons derived from hNPCPSA-NCAM+ exhibited reduced maturation time and enhanced electrophysiological activity on MEA. We employed subdivided population analysis of active mean firing rate (MFR) based on electrophysiological intensity to characterize the electrophysiological properties of hNPCPSA-NCAM+-3D neurons. Based on electrophysiological activity including MFR and burst parameters, we evaluated the sensitivity of hNPCPSA-NCAM+-3D neurons on MEA to screen both inhibitory and excitatory neuroactive environmental chemicals. Intriguingly, electrophysiologically active hNPCPSA-NCAM+-3D neurons demonstrated good sensitivity to evaluate neuroactive chemicals, particularly in discriminating excitatory chemicals. Our findings highlight the effectiveness of MEA approaches using hNPCPSA-NCAM+-3D neurons in the assessment of neurotoxicity associated with environmental chemicals. Furthermore, we emphasize the importance of selecting appropriate signal intensity thresholds to enhance neurotoxicity prediction and screening of environmental chemicals.

Characterization of a novel T cell-engaging bispecific antibody for elimination of L1CAM-positive tumors.

Neural cell adhesion molecule L1 (L1CAM) is a cell-surface glycoprotein involved in cancer occurrence and migration. Up to today, L1CAM-targeted therapy appeared limited efficacy in clinical trials although quite a few attempts by monoclonal antibody (mAb) or chimeric antigen receptor T-cell therapy (CAR-T) have been reported. Therefore, the development of new effective therapies targeting L1CAM is highly desirable. It has been demonstrated that T cell-engaging bispecific antibody (TCE) plays an effective role in cancer immunotherapy by redirecting the cytotoxic activity of CD3+ T cells to tumor cells, resulting in tumor cell death. In this study, we designed and characterized a novel bispecific antibody (CE7-TCE) based on the IgG-(L)-ScFv format, which targets L1CAM and CD3 simultaneously. In vitro, CE7-TCE induced specific killing of L1CAM-positive tumor cells through T cells. In vivo, CE7-TCE inhibited tumor growth in human peripheral blood mononuclear cell/tumor cell co-grafting models. To overcome the adaptive immune resistance (AIR) that impairs the efficacy of TCEs, we conducted a combination therapy of CE7-TCE with Pembrolizumab (anti-PD1 mAb), which enhanced the anti-tumor activity of CE7-TCE. Our results confirmed the feasibility of using L1CAM as a TCE target for the treatment of solid tumors and revealed the therapeutic potential of CE7-TCE combined with immune checkpoint inhibitors.

Elevation of astrocyte-derived extracellular vesicles over the first month post-stroke in humans.

We sought to identify alterations in the quantity of plasma brain-derived extracellular vesicles (EV) over the first month post-stroke to shed light on related injury and repair mechanisms. We assessed plasma levels of presumed neuron-derived EVs (NDEs), astrocyte-derived EVs (ADEs), and oligodendrocyte-derived EVs (ODEs) in 58 patients 5, 15, and 30 days post-ischemic stroke and 46 controls matched for cardiovascular risk factors using sandwich immunoassays. Subsets of brain-derived EVs were identified by co-expression of the general EV marker CD9 and markers for neurons (L1CAM, CD171), astrocytes (EAAT1), and oligodendrocytes (MOG) respectively. Clinical MRIs assessed lesion volume and presence of hemorrhagic transformation. ADE levels were elevated 5, 15, and 30 days post-stroke compared to controls (p = 0.002, p = 0.002, and p = 0.005 respectively) with no significant change for NDE or ODE. ADEs were increased 15 days post-stroke in patients with hemorrhagic transformation (p = 0.04) compared to patients with no hemorrhage. We conclude that ADE levels are preferentially increased over the first month post-stroke in humans, possibly to provide trophic support to injured neurons following ischemia. ADEs hold potential as biomarkers of blood-brain barrier breakdown and hemorrhagic transformation, but this requires further study at earlier time points post-stroke.

NCAM mimetic peptide P2 synergizes with bone marrow mesenchymal stem cells in promoting functional recovery after stroke.

The neural cell adhesion molecule (NCAM) promotes neural development and regeneration. Whether NCAM mimetic peptides could synergize with bone marrow mesenchymal stem cells (BMSCs) in stroke treatment deserves investigation. We found that the NCAM mimetic peptide P2 promoted BMSC proliferation, migration, and neurotrophic factor expression, protected neurons from oxygen-glucose deprivation through ERK and PI3K/AKT activation and anti-apoptotic mechanisms in vitro. Following middle cerebral artery occlusion (MCAO) in rats, P2 alone or in combination with BMSCs inhibited neuronal apoptosis and induced the phosphorylation of ERK and AKT. P2 combined with BMSCs enhanced neurotrophic factor expression and BMSC proliferation in the ischemic boundary zone. Moreover, combined P2 and BMSC therapy induced translocation of nuclear factor erythroid 2-related factor, upregulated heme oxygenase-1 expression, reduced infarct volume, and increased functional recovery as compared to monotreatments. Treatment with LY294002 (PI3K inhibitor) and PD98059 (ERK inhibitor) decreased the neuroprotective effects of combined P2 and BMSC therapy in MCAO rats. Collectively, P2 is neuroprotective while P2 and BMSCs work synergistically to improve functional outcomes after ischemic stroke, which may be attributed to mechanisms involving enhanced BMSC proliferation and neurotrophic factor release, anti-apoptosis, and PI3K/AKT and ERK pathways activation.

Development of a predictive model to distinguish prostate cancer from benign prostatic hyperplasia by integrating serum glycoproteomics and clinical variables.

BACKGROUND: Prostate Cancer (PCa) represents the second leading cause of cancer-related death in men. Prostate-specific antigen (PSA) serum testing, currently used for PCa screening, lacks the necessary sensitivity and specificity. New non-invasive diagnostic tools able to discriminate tumoral from benign conditions and aggressive (AG-PCa) from indolent forms of PCa (NAG-PCa) are required to avoid unnecessary biopsies. METHODS: In this work, 32 formerly N-glycosylated peptides were quantified by PRM (parallel reaction monitoring) in 163 serum samples (79 from PCa patients and 84 from individuals affected by benign prostatic hyperplasia (BPH)) in two technical replicates. These potential biomarker candidates were prioritized through a multi-stage biomarker discovery pipeline articulated in: discovery, LC-PRM assay development and verification phases. Because of the well-established involvement of glycoproteins in cancer development and progression, the proteomic analysis was focused on glycoproteins enriched by TiO2 (titanium dioxide) strategy. RESULTS: Machine learning algorithms have been applied to the combined matrix comprising proteomic and clinical variables, resulting in a predictive model based on six proteomic variables (RNASE1, LAMP2, LUM, MASP1, NCAM1, GPLD1) and five clinical variables (prostate dimension, proPSA, free-PSA, total-PSA, free/total-PSA) able to distinguish PCa from BPH with an area under the Receiver Operating Characteristic (ROC) curve of 0.93. This model outperformed PSA alone which, on the same sample set, was able to discriminate PCa from BPH with an AUC of 0.79. To improve the clinical managing of PCa patients, an explorative small-scale analysis (79 samples) aimed at distinguishing AG-PCa from NAG-PCa was conducted. A predictor of PCa aggressiveness based on the combination of 7 proteomic variables (FCN3, LGALS3BP, AZU1, C6, LAMB1, CHL1, POSTN) and proPSA was developed (AUC of 0.69). CONCLUSIONS: To address the impelling need of more sensitive and specific serum diagnostic tests, a predictive model combining proteomic and clinical variables was developed. A preliminary evaluation to build a new tool able to discriminate aggressive presentations of PCa from tumors with benign behavior was exploited. This predictor displayed moderate performances, but no conclusions can be drawn due to the limited number of the sample cohort. Data are available via ProteomeXchange with identifier PXD035935.

The impact of life-long strength versus endurance training on muscle fiber morphology and phenotype composition in older men.

Aging is typically associated with decreased muscle strength and rate of force development (RFD), partly explained by motor unit remodeling due to denervation, and subsequent loss of fast-twitch type II myofibers. Exercise is commonly advocated to counteract this detrimental loss. However, it is unclear how life-long strength versus endurance training may differentially affect markers of denervation and reinnervation of skeletal myofibers and, in turn, affect the proportion and morphology of fast-twitch type II musculature. Thus, we compared fiber type distribution, fiber type grouping, and the prevalence of atrophic myofibers (≤1,494 µm2) in strength-trained (OS) versus endurance-trained (OE) master athletes and compared the results to recreationally active older adults (all >70 yr, OC) and young habitually active references (<30 yr, YC). Immunofluorescent stainings were performed on biopsy samples from vastus lateralis, along with leg press maximal strength and RFD measurements. OS demonstrated similar type II fiber distribution (OS: 52.0 ± 16.4%; YC: 51.1 ± 14.4%), fiber type grouping, maximal strength (OS: 170.0 ± 18.9 kg, YC: 151.0 ± 24.4 kg), and RFD (OS: 3,993 ± 894 N·s-1, YC: 3,470 ± 1,394 N·s-1) as young, and absence of atrophic myofibers (OS: 0.2 ± 0.7%; YC: 0.1 ± 0.4%). In contrast, OE and OC exhibited more atrophic fibers (OE: 1.2 ± 1.0%; OC: 1.1 ± 1.4%), more grouped fibers, and smaller proportion of type II fibers (OE: 39.3 ± 11.9%; OC: 35.0 ± 12.4%) than OS and YC (all P < 0.05). In conclusion, strength-trained master athletes were characterized by similar muscle morphology as young, which was not the case for recreationally active or endurance-trained old. These results indicate that strength training may preserve type II fibers with advancing age in older men, likely as a result of chronic use of high contractile force generation.NEW & NOTEWORTHY Aging is associated with loss of fast-twitch type II myofibers, motor unit remodeling, and grouping of myofibers. This study reveals, for the first time, that strength training preserves neural innervation of type II fibers, resulting in similar myofiber type distribution and grouping in life-long strength-trained master athletes as young moderately active adults. In contrast, life-long endurance-trained master athletes and recreationally active old adults demonstrated higher proportion of type I fibers accompanied by more marked grouping of type I myofibers, and more atrophic fibers compared with strength-trained master athletes and young individuals. Thus, strength training should be utilized as a training modality for preservation of fast-twitch musculature, maximal muscle strength, and rapid force capacity (RFD) with advancing age.

Interaction of L1CAM with LC3 Is Required for L1-Dependent Neurite Outgrowth and Neuronal Survival.

The neural cell adhesion molecule L1 (also called L1CAM or CD171) functions not only in cell migration, but also in cell survival, differentiation, myelination, neurite outgrowth, and signaling during nervous system development and in adults. The proteolytic cleavage of L1 in its extracellular domain generates soluble fragments which are shed into the extracellular space and transmembrane fragments that are internalized into the cell and transported to various organelles to regulate cellular functions. To identify novel intracellular interaction partners of L1, we searched for protein-protein interaction motifs and found two potential microtubule-associated protein 1 light-chain 3 (LC3)-interacting region (LIR) motifs within L1, one in its extracellular domain and one in its intracellular domain. By ELISA, immunoprecipitation, and proximity ligation assay using L1 mutant mice lacking the 70 kDa L1 fragment (L1-70), we showed that L1-70 interacts with LC3 via the extracellular LIR motif in the fourth fibronectin type III domain, but not by the motif in the intracellular domain. The disruption of the L1-LC3 interaction reduces L1-mediated neurite outgrowth and neuronal survival.

Alterations of Plasmatic Biomarkers of Neurodegeneration in Mucopolysaccharidosis Type II Patients Under Enzyme Replacement Therapy.

Mucopolysaccharidosis type II (MPS II) is a disorder caused by a deficient activity of iduronate-2-sulfatase, a lysosomal enzyme responsible for degrading glycosaminoglycans (GAGs). The abnormal storage of GAGs within lysosomes disrupts cellular homeostasis and leads to a severe symptomatology. Patients present neuropsychiatric impairment characterized by mental retardation and impaired cognition. The aim of this study was to quantify four neurodegeneration biomarkers in plasma: brain-derived neurotrophic factor (BDNF), platelet-derived growth factor (PDGF-AA), neural cell adhesion molecule (NCAM) and cathepsin-D, as well as to identify possible correlations with urinary GAGs in seven patients undergoing treatment with ERT (Elaprase® 0.5 mg/kg of body weight). Patients with both severe and attenuated forms of MPS II showed signs of neurodegeneration in neuroimaging exams. Patients have a decrease in BDNF and PDGF-AA concentrations, and an increase in NCAM level compared to controls. No alterations in cathepsin-D concentration were seen. GAGs levels were higher in patients than in controls, but no significant correlations between GAGs and biomarkers were observed. These results evidence that patients have neurodegeneration and that monitoring these biomarkers might be useful for assessing this process. To this date, this is the first work to analyze these plasmatic markers of neurodegeneration in patients.

A neural cell adhesion molecule from oyster Crassostrea gigas: Molecular identification and immune functional characterization.

Neural cell adhesion molecules (NCAMs) are large cell-surface glycoproteins playing important roles in cell-cell and cell-extracellular matrix interactions in nervous system. Recent study identified a homologue of NCAM (CgNCAM) from the Pacific oyster Crassostrea gigas. Its ORF was of 2634 bp which encodes a protein (877 amino acids) consisting of five immunoglobulin domains and two fibronectin type III domains. CgNCAM transcripts were broadly distributed in oyster tissues especially in mantle, labial palp and haemolymph. CgNCAM showed up-regulated expression in haemocytes of oysters after Vibrio splendidus and Staphylococcus aureus stimulation. The recombinant CgNCAM protein (rCgNCAM) was able to bind manose, lipopolysaccharide and glucan, as well as different microbes including Gram-negative bacteria and fungi. rCgNCAM displayed bacterial agglutination and hemagglutination activity. CgNCAM improved the phagocytosis of haemocytes towards V. splendidus by regulating the expression of CgIntegrin, CgRho J and CgMAPKK. Moreover, CgNCAM was involved in the extracellular trap establishment of haemocytes after V. splendidus stimulation. The results collectively indicated that CgNCAM acted as a recognition receptor executing multiple immune functions to recognize and eliminate invading microorganisms in innate immunity of oysters.

Neural cell adhesion molecule 1 is a cellular target engaged plasma biomarker in demyelinating Charcot-Marie-Tooth disease.

BACKGROUND AND PURPOSE: Elevated plasma concentrations of neural cell adhesion molecule 1 (NCAM1) and p75 neurotrophin receptor (p75) in patients with peripheral neuropathy have been reported. This study aimed to determine the specificity of plasma concentration elevation of either NCAM1 or p75 in a subtype of Charcot-Marie-Tooth disease (CMT) and its correlation with pathologic nerve status and disease severity. METHODS: Blood samples were collected from 138 patients with inherited peripheral neuropathy and 51 healthy controls. Disease severity was measured using Charcot-Marie-Tooth Neuropathy Score version 2 (CMTNSv2), and plasma concentrations of NCAM1 and p75 were analyzed by enzyme-linked immunosorbent assay. Eight sural nerves from CMT patients were examined to determine the relation of histopathology and plasma NCAM1 levels. RESULTS: Plasma concentration of NCAM1, but not p75, was specifically increased in demyelinating subtypes of CMT (median = 7100 pg/mL, p < 0.001), including CMT1A, but not in axonal subtype (5964 pg/mL, p > 0.05), compared to the control (3859 pg/mL). CMT1A patients with mild or moderate severity (CMTNSv2 < 20) showed higher levels of plasma NCAM1 than healthy controls. Immunofluorescent NCAM1 staining for the sural nerves of CMT patients showed that NCAM1-positive onion bulb cells and possible demyelinating Schwann cells might be associated with the specific increase of plasma NCAM1 in demyelinating CMT. CONCLUSIONS: The plasma NCAM1 levels in demyelinating CMT might be a surrogate biomarker reflecting pathological Schwann cell status and disease progression.

Dysregulation of Synaptic Plasticity Markers in Schizophrenia.

Schizophrenia is a mental disorder characterized by cognitive impairment resulting in compromised quality of life. Since the regulation of synaptic plasticity has functional implications in various aspects of cognition such as learning, memory, and neural circuit maturation, the dysregulation of synaptic plasticity is considered as a pathobiological feature of schizophrenia. The findings from our recently concluded studies indicate that there is an alteration in levels of synaptic plasticity markers such as neural cell adhesion molecule-1 (NCAM-1), Neurotropin-3 (NT-3) and Matrix-mettaloproteinase-9 (MMP-9) in schizophrenia patients. The objective of the present article is to review the role of markers of synaptic plasticity in schizophrenia. PubMed database (http;//www.ncbi.nlm.nih.gov/pubmed) was used to perform an extensive literature search using the keywords schizophrenia and synaptic plasticity. We conclude that markers of synaptic plasticity are altered in schizophrenia and may lead to complications of schizophrenia including cognitive dysfunction.

Molecular and cellular changes in the post-traumatic spinal cord remodeling after autoinfusion of a genetically-enriched leucoconcentrate in a mini-pig model.

Post-traumatic spinal cord remodeling includes both degenerating and regenerating processes, which affect the potency of the functional recovery after spinal cord injury (SCI). Gene therapy for spinal cord injury is proposed as a promising therapeutic strategy to induce positive changes in remodeling of the affected neural tissue. In our previous studies for delivering the therapeutic genes at the site of spinal cord injury, we developed a new approach using an autologous leucoconcentrate transduced ex vivo with chimeric adenoviruses (Ad5/35) carrying recombinant cDNA. In the present study, the efficacy of the intravenous infusion of an autologous genetically-enriched leucoconcentrate simultaneously producing recombinant vascular endothelial growth factor (VEGF), glial cell line-derived neurotrophic factor (GDNF), and neural cell adhesion molecule (NCAM) was evaluated with regard to the molecular and cellular changes in remodeling of the spinal cord tissue at the site of damage in a model of mini-pigs with moderate spinal cord injury. Experimental animals were randomly divided into two groups of 4 pigs each: the therapeutic (infused with the leucoconcentrate simultaneously transduced with a combination of the three chimeric adenoviral vectors Ad5/35-VEGF165, Ad5/35-GDNF, and Ad5/35-NCAM1) and control groups (infused with intact leucoconcentrate). The morphometric and immunofluorescence analysis of the spinal cord regeneration in the rostral and caudal segments according to the epicenter of the injury in the treated animals compared to the control mini-pigs showed: (1) higher sparing of the grey matter and increased survivability of the spinal cord cells (lower number of Caspase-3-positive cells and decreased expression of Hsp27); (2) recovery of synaptophysin expression; (3) prevention of astrogliosis (lower area of glial fibrillary acidic protein-positive astrocytes and ionized calcium binding adaptor molecule 1-positive microglial cells); (4) higher growth rates of regenerating ßIII-tubulin-positive axons accompanied by a higher number of oligodendrocyte transcription factor 2-positive oligodendroglial cells in the lateral corticospinal tract region. These results revealed the efficacy of intravenous infusion of the autologous genetically-enriched leucoconcentrate producing recombinant VEGF, GDNF, and NCAM in the acute phase of spinal cord injury on the positive changes in the post-traumatic remodeling nervous tissue at the site of direct injury. Our data provide a solid platform for a new ex vivo gene therapy for spinal cord injury and will facilitate further translation of regenerative therapies in clinical neurology.

Use of metabolic glycoengineering and pharmacological inhibitors to assess lipid and protein sialylation on cells.

Metabolic glycoengineering (MGE) has been developed to visualize carbohydrates on live cells. The method allows the fluorescent labeling of sialic acid (Sia) sugar residues on neuronal plasma membranes. For instance, the efficiency of glycosylation along neurite membranes has been characterized as cell health measure in neurotoxicology. Using human dopaminergic neurons as model system, we asked here, whether it was possible to separately label diverse classes of biomolecules and to visualize them selectively on cells. Several approaches suggest that a large proportion of Sia rather incorporated in non-protein components of cell membranes than into glycoproteins. We made use here of deoxymannojirimycin (dMM), a non-toxic inhibitor of protein glycosylation, and of N-butyl-deoxynojirimycin (NBdNM) a well-tolerated inhibitor of lipid glycosylation, to develop a method of differential labeling of sialylated membrane lipids (lipid-Sia) or sialylated N-glycosylated proteins (protein-Sia) on live neurons. The time resolution at which Sia modification of lipids/proteins was observable was in the range of few hours. The approach was then extended to several other cell types. Using this technique of target-specific MGE, we found that in dopaminergic or sensory neurons >60% of Sia is lipid bound, and thus polysialic acid-neural cell adhesion molecule (PSA-NCAM) cannot be considered the major sialylated membrane component. Different from neurons, most Sia was bound to protein in HepG2 hepatoma cells or in neural crest cells. Thus, our method allows visualization of cell-specific sialylation processes for separate classes of membrane constituents.

The Graphical Studies of the Major Molecular Interactions for Neural Cell Adhesion Molecule (NCAM) Polysialylation by Incorporating Wenxiang Diagram into NMR Spectroscopy.

Polysialylation is a process of polysialic acid (polySia) addition to neural cell adhesion molecule (NCAM), which is associated with tumor cell migration and progression in many metastatic cancers and neurocognition. Polysialylation can be catalyzed by two highly homologous mammalian polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST). It has been proposed that two polybasic domains, polybasic region (PBR) and polysialyltransferase domain (PSTD) in polySTs, are possible binding sites for the intermolecular interactions of polyST-NCAM and polyST-polySia, respectively, as well as the intramolecular interaction of PSTD-PBR. In this study, Chou's wenxiang diagrams of the PSTD and PBR are used to determine the key amino acids of these intermolecular and intramolecular interactions, and thus it may be helpful for the identification of the crucial amino acids in the polyST and for the understanding of the molecular mechanism of NCAM polysialylation by incorporating the wenxiang diagram and molecular modeling into NMR spectroscopy.

Autologous Genetically Enriched Leucoconcentrate in the Preventive and Acute Phases of Stroke Treatment in a Mini-Pig Model.

The natural limitations of regeneration in the CNS are major problems for the treatment of neurological disorders, including ischaemic brain strokes. Among the approaches being actively developed to inhibit post-ischaemic negative consequences is the delivery of therapeutic genes encoding neuroprotective molecules to the brain. Unfortunately, there are currently no proven and available medicines that contain recombinant human genes for the treatment of ischaemic cerebral stroke. Of particular interest is the development of treatments for patients at risk of ischaemic stroke. In the present study, we propose a proof of concept for the use of an autologous, genetically enriched leucoconcentrate temporally secreting recombinant vascular endothelial growth factor (VEGF), glial-cell-line-derived neurotrophic factor (GDNF) and the neural cell adhesion molecule (NCAM) for the treatment of stroke. In a mini-pig ischaemic stroke model, genetically enriched leucoconcentrate was infused 4 h after surgery (gene therapy in acute phase) or 2 days before stroke modelling (preventive gene therapy). On day 21, after the stroke modelling, the post-ischaemic brain recovery was examined by morphologic and immunofluorescence analysis. The benefits of treating a stroke with genetically enriched leucoconcentrate both for preventive purposes and in the acute phase were confirmed by an improved performance in behavioural tests, higher preservation of brain tissue and positive post-ischaemic brain remodelling in the peri-infarct area. These results suggest that the employment of autologous leucocytes enabling the temporary production of the recombinant therapeutic molecules to correct the pathological process in the CNS may be one of the breakthrough approaches in gene therapy.

Conditioned-medium of stem cells from human exfoliated deciduous teeth prevent apoptosis of neural progenitors.

Purpose: This study aimed to evaluate the neuroprotective ability of the conditioned medium of stem cells from human exfoliated deciduous teeth (CM-SHED) to prevent glutamate-induced apoptosis of neural progenitors. Materials and methods: Neural progenitors were isolated from two-day-old rat brains, and the conditioned medium was obtained from a mesenchymal stem cell SHED. Four groups were examined: neural progenitor cells cultured in neurobasal medium with (N + ) and without (N-) glutamate and glycine, and neural progenitor cells cultured in CM-SHED with (K + ) and without (K-) glutamate and glycine. Results: The expression of GABA A1 receptor (GABAAR1) messenger RNA (mRNA) in neural progenitor measured by real-time quantitative PCR. GABA contents were measured by enzyme-linked immunosorbent assay, whereas the apoptosis markers caspase-3 and 7-aminoactinomycin D were analysed with a Muse® cell analyzer. The viability of neural progenitor cells in the K + group (78.05 %) was higher than the control group N- (73.22 %) and lower in the N + group (68.90 %) than in the control group. The K + group showed the highest GABA content, which significantly differed from that in the other groups, whereas the lowest content was observed in the N + group. The expression level of GABAAR1 mRNA in the K + group was the highest compared to that in the other groups. CM-SHED potently protected the neural progenitors from apoptosis. Conclusions: CM-SHED may effectively prevent glutamate-induced apoptosis of neural progenitors.

Relationship Between Neural Cell Adhesion Molecule-1 and Cognitive Functioning in Schizophrenia Spectrum Disorder.

Abnormal synaptic plasticity leads to cognitive impairment in schizophrenia. Markers of synaptic plasticity are known to be altered in schizophrenia, but there are limited data available about neural cell adhesion molecule-1 (NCAM-1) levels and its association with cognitive functions in schizophrenia. The objective of the study was to analyze NCAM-1 levels and its association with various cognitive domains in schizophrenia. One hundred and seventy-six schizophrenia cases and 176 controls were recruited for the study. Serum NCAM-1 levels were analysed in both the groups. Cognitive examination was performed using Addenbrooke cognitive examination-III (ACE-III) and disease severity was assessed using Positive and negative symptoms scale (PANSS). Serum NCAM-1 levels were elevated in schizophrenia cases (p = 0.006) compared to controls. NCAM-1 was positively associated with attention (r = 0.196, p = 0.009), language (r = 0.192, p = 0.011), visuospatial abilities (r = 0.207, p = 0.006) and total ACE-III score (r = 0.189, p = 0.012). We conclude that elevated levels of NCAM-1 are associated with better cognitive functioning in schizophrenia.

New antigens involved in membranous nephropathy beyond phospholipase A2 receptor.

When the physiopathology of membranous nephropathy was first described, almost 30% of cases were recognized to be secondary to well-known diseases such as autoimmune diseases, tumors or infections. The remaining 70% cases were called primary membranous nephropathy as the exact mechanism or pathogenic factor involved was unknown. The discovery of the M type phospholipase A2 receptor and thrombospondin type 1 domain containing 7A as causative antigens in these "so called" primary membranous nephropathies provided new insights into the effective causes of a large proportion of these cases. Novel techniques such as laser microdissection and tandem mass spectrometry as well as immunochemistry with antibodies directed against novel proteins allowed the confirmation of new involved antigens. Finally, using confocal microscopy to localize these new antigens and immunoglobulin G and Western blot analysis of serum samples, these new antigens were detected on the glomerular membrane, and the related antibodies were detected in serum samples. The same antigens have been recognized in some cases of secondary membranous disease due to autoimmune diseases, tumors and infections. This has allowed examination of the relationship between antigens in primary membranous nephropathy and their presence in some secondary nephropathies. The aim of this study is to describe the characteristics of the new antigens discovered and their association with other diseases.

Interactions between the Polysialylated Neural Cell Adhesion Molecule and the Transient Receptor Potential Canonical Channels 1, 4, and 5 Induce Entry of Ca2+ into Neurons.

The neural cell adhesion molecule (NCAM) plays important functional roles in the developing and mature nervous systems. Here, we show that the transient receptor potential canonical (TRPC) ion channels TRPC1, -4, and -5 not only interact with the intracellular domains of the transmembrane isoforms NCAM140 and NCAM180, but also with the glycan polysialic acid (PSA) covalently attached to the NCAM protein backbone. NCAM antibody treatment leads to the opening of TRPC1, -4, and -5 hetero- or homomers at the plasma membrane and to the influx of Ca2+ into cultured cortical neurons and CHO cells expressing NCAM, PSA, and TRPC1 and -4 or TRPC1 and -5. NCAM-stimulated Ca2+ entry was blocked by the TRPC inhibitor Pico145 or the bacterial PSA homolog colominic acid. NCAM-stimulated Ca2+ influx was detectable neither in NCAM-deficient cortical neurons nor in TRPC1/4- or TRPC1/5-expressing CHO cells that express NCAM, but not PSA. NCAM-induced neurite outgrowth was reduced by TRPC inhibitors and a function-blocking TRPC1 antibody. A characteristic signaling feature was that extracellular signal-regulated kinase 1/2 phosphorylation was also reduced by TRPC inhibitors. Our findings indicate that the interaction of NCAM with TRPC1, -4, and -5 contributes to the NCAM-stimulated and PSA-dependent Ca2+ entry into neurons thereby influencing essential neural functions.