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Background: Critical limb-threatening ischemia (CLTI) constitutes the most severe manifestation of peripheral artery disease, usually induced by atherosclerosis. CLTI patients suffer from high risk of amputation of the lower extremities and elevated mortality rates, while they have low options for surgical revascularization due to associated comorbidities. Alternatively, cell-based therapeutic strategies represent an effective and safe approach to promote revascularization. However, the variability seen in several factors such as cell combinations or doses applied, have limited their success in clinical trials, being necessary to reach a consensus regarding the optimal "cellular-cocktail" prior further application into the clinic. To achieve so, it is essential to understand the mechanisms by which these cells exert their regenerative properties. Herein, we have evaluated, for the first time, the regenerative and vasculogenic potential of a combination of endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) isolated from adipose-tissue (AT), compared with ECFCs from umbilical cord blood (CB-ECFCs) and AT-MSCs, in a murine model of CLTI., Methods: Balb-c nude mice (n:32) were distributed in four different groups (n:8/group): control shams, and ischemic mice (after femoral ligation) that received 50 µl of physiological serum alone or a cellular combination of AT-MSCs with either CB-ECFCs or AT-ECFCs. Follow-up of blood flow reperfusion and ischemic symptoms was carried out for 21 days, when mice were sacrificed to evaluate vascular density formation. Moreover, the long-term molecular changes in response to CLTI and both cell combinations were analyzed in a proteomic quantitative approach., Results: AT-MSCs with either AT- or CB-ECFCs, promoted a significant recovery of blood flow in CLTI mice 21 days post-ischemia. Besides, they modulated the inflammatory and necrotic related processes, although the CB group presented the slowest ischemic progression along the assay. Moreover, many proteins involved in the repairing mechanisms promoted by cell treatments were identified., Conclusions: The combination of AT-MSCs with AT-ECFCs or with CB-ECFCs promoted similar revascularization in CLTI mice, by restoring blood flow levels, together with the modulation of the inflammatory and necrotic processes, and reduction of muscle damage. The protein changes identified are representative of the molecular mechanisms involved in ECFCs and MSCs-induced revascularization (immune response, vascular repair, muscle regeneration, etc.)., (© 2024. The Author(s).)

Neurogenesis is the process of forming new neurons from neural stem cells (NSCs). In adults, this process takes place in specific areas of the brain, known as neurogenic niches. These regions have unique anatomical features that have been studied in animal models and in the human brain; however, there are differences between these models that need to be addressed. The most studied areas are the subventricular zone, the lateral and latero-dorsal walls of the lateral ventricles, and the dentate gyrus of the hippocampus (Hp), which are known as the canonical areas. Other, less-studied niches, such as the hypothalamus, the cerebellum, and the amygdala, are known as non-canonical areas. Anatomy occupies a relevant place in adult neurogenesis, in which the tissue architecture and cellular location are necessities for the interaction and release of diverse molecules that allow this phenomenon. The cell arrangement within the niche and the location of the niche itself are of particular relevance to the state in which the NSCs are found. Consequently, the majority of previous discoveries have been related to pathology. While many studies are based on animal models, discoveries related to neurogenesis in humans have also been made; however, in this case, opinions vary, leading to extensive controversy in recent years. In this review, we address the anatomical characteristics of the different brain regions to better understand their relationships within neurogenesis.

Background: Neurogenesis is stimulated in the subventricular zone (SVZ) of mice with cortical brain injuries. In most of these injuries, newly generated neuroblasts attempt to migrate toward the injury, accumulating within the corpus callosum not reaching the perilesional area., Methods: We use a murine model of mechanical cortical brain injury, in which we perform unilateral cortical injuries in the primary motor cortex of adult male mice. We study neurogenesis in the SVZ and perilesional area at 7 and 14 dpi as well as the expression and concentration of the signaling molecule transforming growth factor alpha (TGF-α) and its receptor the epidermal growth factor (EGFR). We use the EGFR inhibitor Afatinib to promote neurogenesis in brain injuries., Results: We show that microglial cells that emerge within the injured area and the SVZ in response to the injury express high levels of TGF-α leading to elevated concentrations of TGF-α in the cerebrospinal fluid. Thus, the number of neuroblasts in the SVZ increases in response to the injury, a large number of these neuroblasts remain immature and proliferate expressing the epidermal growth factor receptor (EGFR) and the proliferation marker Ki67. Restraining TGF-α release with a classical protein kinase C inhibitor reduces the number of these proliferative EGFR + immature neuroblasts in the SVZ. In accordance, the inhibition of the TGF-α receptor, EGFR promotes migration of neuroblasts toward the injury leading to an elevated number of neuroblasts within the perilesional area., Conclusions: Our results indicate that in response to an injury, microglial cells activated within the injury and the SVZ release TGF-α, activating the EGFR present in the neuroblasts membrane inducing their proliferation, delaying maturation and negatively regulating migration. The inactivation of this signaling pathway stimulates neuroblast migration toward the injury and enhances the quantity of neuroblasts within the injured area. These results suggest that these proteins may be used as target molecules to regenerate brain injuries., (© 2023. The Author(s).)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection significantly affects the cardiovascular system, causing vascular damage and thromboembolic events in critical patients. Endothelial dysfunction represents one of the first steps in response to COVID-19 that might lead to cardiovascular complications and long-term sequelae. However, despite the enormous efforts in the last two years, the molecular mechanisms involved in such processes remain poorly understood. Herein, we analyzed the protein changes taking place in endothelial colony forming cells (ECFCs) after the incubation with the serum from individuals infected with COVID-19, whether asymptomatic or critical patients, by application of a label free-quantitative proteomics approach. Specifically, ECFCs from healthy individuals were incubated ex-vivo with the serum of either COVID-19 negative donors (PCR-/IgG-, n:8), COVID-19 asymptomatic donors at different infective stages (PCR+/ IgG-, n:8and PCR-/IgG+, n:8), or hospitalized critical COVID-19 patients (n:8), followed by proteomics analysis. In total, 590 proteins were differentially expressed in ECFCs in response to all infected serums. Predictive analysis highlighted several proteins like CAPN5, SURF4, LAMP2 or MT-ND1, as highly discriminating features between the groups compared. Protein changes correlated with viral infection, RNA metabolism or autophagy, among others. Remarkably, the angiogenic potential of ECFCs in response to the infected serums was impaired, and many of the protein alterations in response to the serum of critical patients were associated with cardiovascular-related pathologies., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Submicron particles have been produced from an ethanolic extract of Myrtus communnis leaves using supercritical carbon dioxide technology, hereinafter referred to as Supercritical Antisolvent Extraction (SAE). The influence of pressure (9-20 MPa), temperature (308 and 328 K) and injection rate (3 and 8 mL/min) on the particles' precipitation has been investigated, and it has been confirmed that increases in pressure and temperature led to smaller particle sizes. The obtained particles had a quasi-spherical shape with sizes ranging from 0.42 to 1.32 μm. Moreover, the bioactivity of the generated particles was assessed and large contents of phenolic compounds with a high antioxidant activity were measured. The particles were also subjected to in vitro studies against oxidative stress. The myrtle particles demonstrated cytoprotective properties when applied at low concentrations (1 μM) to macrophage cell lines.

The addition of natural substances with pharmacoactive properties to polymeric biomedical devices would provide beneficial regarding the assimilation of these endoprostheses when implanted into a patient's body. The added drug would facilitate endothelization by regulating the inflammatory processes that such interventions entail, preventing contamination hazards and favoring the angiogenesis or formation of blood vessels in the tissue. The present work used mango leaf extract (MLE) obtained through pressurized ethanol for this purpose. Polylactic acid (PLA) in the form of filaments or 3D-printed disks was impregnated by means of supercritical technology with MLE for the culture essays. The release kinetics has been studied and the polymer matrices have been examined by scanning electron microscopy (SEM). The impregnated devices were subjected to in vitro culture of colony-forming endothelial cells. The influence of the different impregnation conditions used for the production of the MLE impregnated polymeric devices on the development of the cell culture was determined by fluorescence microscopy. The best results were obtained from the calcein cultures on 35 °C MLE impregnated into 3D-printed polymer disks.

Background: Endothelial colony forming cells (ECFCs), alone or in combination with mesenchymal stem cells, have been selected as potential therapeutic candidates for critical limb-threatening ischemia (CLTI), mainly for those patients considered as "no-option," due to their capability to enhance revascularization and perfusion recovery of ischemic tissues. Nevertheless, prior to translating cell therapy to the clinic, biodistribution assays are required by regulatory guidelines to ensure biosafety as well as to discard undesired systemic translocations. Different approaches, from imaging technologies to qPCR-based methods, are currently applied., Methods: In the current study, we have optimized a cell-tracking assay based on DiR fluorescent cell labeling and near-infrared detection for in vivo and ex vivo assays. Briefly, an improved protocol for DiR staining was set up, by incubation of ECFCs with 6.67 µM DiR and intensive washing steps prior cell administration. The minimal signal detected for the residual DiR, remaining after these washes, was considered as a baseline signal to estimate cell amounts correlated to the DiR intensity values registered in vivo. Besides, several assays were also performed to determine any potential effect of DiR over ECFCs functionality. Furthermore, the optimized protocol was applied in combination with qPCR amplification of specific human Alu sequences to assess the final distribution of ECFCs after intramuscular or intravenous administration to a murine model of CLTI., Results: The optimized DiR labeling protocol indicated that ECFCs administered intramuscularly remained mainly within the hind limb muscle while cells injected intravenously were found in the spleen, liver and lungs., Conclusion: Overall, the combination of DiR labeling and qPCR analysis in biodistribution assays constitutes a highly sensitive approach to systemically track cells in vivo. Thereby, human ECFCs administered intramuscularly to CLTI mice remained locally within the ischemic tissues, while intravenously injected cells were found in several organs. Our data corroborate the need to perform biodistribution assays in order to define specific parameters such as the optimal delivery route for ECFCs before their application into the clinic., (© 2022. The Author(s).)

Cardiovascular diseases remain the leading cause of death worldwide, mainly triggered by the formation of atherosclerotic plaques that reduce blood flow. Angiogenic cell therapy based on endothelial colony forming cells (ECFCs) constitutes a promising alternative to promote vascular revascularization; however, under the oxidative environment that prevails in ischemic areas, these cells become impaired. Thus, it is necessary to investigate strategies to enhance their regenerative properties. Antioxidant substances, such as polyphenols, have been shown to be useful for this purpose. In the current study we evaluated the potential of mango leaves, olive leaves and red grape pomace extracts, rich in polyphenols, to promote ECFC reparative effects. For this, aqueous and ethanolic extracts of the aforementioned raw materials were obtained by pressurized liquid extraction (PLE). After evaluating the polyphenol content and the antioxidant activity, in vitro assays were carried out, and we found that ethanolic extracts at low concentrations improved angiogenic capacities of ECFCs and reduced proliferation, apoptosis, and the inflammatory response of these cells. Overall, mango leaves ethanolic extract provided the most promising results, but all three extracts ameliorated the functionality of ECFCs.

Atherosclerosis remains the underlying process responsible for cardiovascular diseases and the high mortality rates associated. This chronic inflammatory disease progresses with the formation of occlusive atherosclerotic plaques over the inner walls of vascular vessels, with oxidative stress being an important element of this pathology. Oxidation of low-density lipoproteins (ox-LDL) induces endothelial dysfunction, foam cell activation, and inflammatory response, resulting in the formation of fatty streaks in the atherosclerotic wall. With this in mind, different approaches aim to reduce oxidative damage as a strategy to tackle the progression of atherosclerosis. Special attention has been paid in recent years to the transcription factor Nrf2 and its downstream-regulated protein heme oxygenase-1 (HO-1), both known to provide protection against atherosclerotic injury. In the current review, we summarize the involvement of oxidative stress in atherosclerosis, focusing on the role that these antioxidant molecules exert, as well as the potential therapeutic strategies applied to enhance their antioxidant and antiatherogenic properties.

Colorectal cancer (CRC) is one of the main causes of cancer death in the world. Post-translational modifications (PTMs) have been extensively studied in malignancies due to its relevance in tumor pathogenesis and therapy. This review is focused on the dysregulation of glycosyltransferase expression in CRC and its impact in cell function and in several biological pathways associated with CRC pathogenesis, prognosis and therapeutic approaches. Glycan structures act as interface molecules between cells and their environment and in several cases facilitate molecule function. CRC tissue shows alterations in glycan structures decorating molecules, such as annexin-1, mucins, heat shock protein 90 (Hsp90), β1 integrin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), insulin-like growth factor-binding protein 3 (IGFBP3), transforming growth factor beta (TGF-β) receptors, Fas (CD95), PD-L1, decorin, sorbin and SH3 domain-containing protein 1 (SORBS1), CD147 and glycosphingolipids. All of these are described as key molecules in oncogenesis and metastasis. Therefore, glycosylation in CRC can affect cell migration, cell-cell adhesion, actin polymerization, mitosis, cell membrane repair, apoptosis, cell differentiation, stemness regulation, intestinal mucosal barrier integrity, immune system regulation, T cell polarization and gut microbiota composition; all such functions are associated with the prognosis and evolution of the disease. According to these findings, multiple strategies have been evaluated to alter oligosaccharide processing and to modify glycoconjugate structures in order to control CRC progression and prevent metastasis. Additionally, immunotherapy approaches have contemplated the use of neo-antigens, generated by altered glycosylation, as targets for tumor-specific T cells or engineered CAR (Chimeric antigen receptors) T cells.

Cannabis is the third most commonly used psychoactive substance of abuse, yet it also receives considerable attention as a potential therapeutic drug. Therefore, it is essential to fully understand the actions of cannabis in the human brain. The olfactory neuroepithelium (ON) is a peripheral nervous tissue that represents an interesting surrogate model to study the effects of drugs in the brain, since it is closely related to the central nervous system, and sensory olfactory neurons are continually regenerated from populations of stem/progenitor cells that undergo neurogenesis throughout life. In this study, we used ON cells from chronic cannabis users and healthy control subjects to assess alterations in relevant cellular processes, and to identify changes in functional proteomic pathways due to cannabis consumption. The ON cells from cannabis users exhibited alterations in the expression of proteins that were related to the cytoskeleton, cell proliferation and cell death, as well as, changes in proteins implicated in cancer, gastrointestinal and neurodevelopmental pathologies. Subsequent studies showed cannabis provoked an increase in cell size and morphological alterations evident through β-Tubulin III staining, as well as, enhanced beta-actin expression and a decrease in the ability of ON cells to undergo cell attachment, suggesting abnormalities of the cytoskeleton and cell adhesion system. Furthermore, these cells proliferated more and underwent less cell death. Our results indicate that cannabis may alter key processes of the developing brain, some of which are similar to those reported in mental disorders like DiGeorge syndrome, schizophrenia and bipolar disorder.

Glioblastoma (GB), the most aggressive malignant glioma, is made up of a large percentage of glioma-associated microglia/macrophages (GAM), suggesting that immune cells play an important role in the pathophysiology of GB. Under physiological conditions, microglia, the phagocytes of the central nervous system (CNS), are involved in various processes such as neurogenesis or axonal growth, and the progression of different conditions such as Alzheimer's disease. Through immunohistochemical studies, markers that enhance GB invasiveness have been shown to be expressed in the peritumoral area of ​​the brain, such as Transforming Growth Factor α (TGF-α), Stromal Sell-Derived Factor 1 (SDF1/CXCL12), Sphingosine-1-Phosphate (S1P) and Neurotrophic Factor Derived from the Glial cell line (GDNF), contributing to the increase in tumor mass. Similarly, it has also been described 17 biomarkers that are present in hypoxic periarteriolar HSC niches in bone marrow and in hypoxic periarteriolar GSC niches in glioblastoma. Interestingly, microglia plays an important role in the microenvironment that supports GB progression, being one of the most important focal points in the study of therapeutic targets for the development of new drugs. In this review, we describe the altered signaling pathways in microglia in the context of GB. We also show how microglia interact with glioblastoma cells and the epigenetic mechanisms involved. Regarding the interactions between microglia and neurogenic niches, some authors indicate that glioblastoma stem cells (GSC) are similar to neural stem cells (NSC), common stem cells in the subventricular zone (SVZ), suggesting that this could be the origin of GB. Understanding the similarities between SVZ and the tumor microenvironment could be important to clarify some mechanisms involved in GB malignancy and to support the discovering of new therapeutic targets for the development of more effective glioblastoma treatments., Competing Interests: Authors LG and EN were employed by company Care4You. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Geribaldi-Doldán, Fernández-Ponce, Quiroz, Sánchez-Gomar, Escorcia, Velásquez and Quiroz.)

Background: Bone Marrow Mononuclear Cells (BM-MNC) constitute a promising alternative for the treatment of Chronic Limb-Threatening ischemia (CLTI), a disease characterized by extensive blockade of peripheral arteries, clinically presenting as excruciating pain at rest and ischemic ulcers which may lead to gangrene and amputation. BM-MNC implantation has shown to be efficient in promoting angiogenesis and ameliorating ischemic symptoms in CLTI patients. However, the variability seen between clinical trials makes necessary a further understanding of the mechanisms of action of BM-MNC, and moreover, to improve trial characteristics such as endpoints, inclusion/exclusion criteria or drug product compositions, in order to implement their use as stem-cell therapy. Materials: Herein, the effect of REX-001, a human-BM derived cell suspension enriched for mononuclear cells, granulocytes and CD34+ cells, has been assessed in a murine model of CLTI. In addition, a REX-001 placebo solution containing BM-derived red blood cells (BM-RBCs) was also tested. Thus, 24 h after double ligation of the femoral artery, REX-001 and placebo were administrated intramuscularly to Balb-c nude mice (n:51) and follow-up of ischemic symptoms (blood flow perfusion, motility, ulceration and necrosis) was carried out for 21 days. The number of vessels and vascular diameter sizes were measured within the ischemic tissues to evaluate neovascularization and arteriogenesis. Finally, several cell-tracking assays were performed to evaluate potential biodistribution of these cells. Results: REX-001 induced a significant recovery of blood flow by increasing vascular density within the ischemic limbs, with no cell translocation to other organs. Moreover, cell tracking assays confirmed a decrease in the number of infused cells after 2 weeks post-injection despite on-going revascularization, suggesting a paracrine mechanism of action. Conclusion: Overall, our data supported the role of REX-001 product to improve revascularization and ischemic reperfusion in CLTI., Competing Interests: MD-R, MR-T, and MJ-P collaborated with Rexgenero Biosciences SL in the project, supported by Rexgenero, where the current results were obtained from. JM-R and IS-G are employees of Rexgenero Biosciences SL. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Rojas-Torres, Jiménez-Palomares, Martín-Ramírez, Beltrán-Camacho, Sánchez-Gomar, Eslava-Alcon, Rosal-Vela, Gavaldá and Durán-Ruiz.)

Aquaporin-4, present in ependymal cells, in glia limiting and abundantly in pericapillary astrocyte foot processes, and aquaporin-1, expressed in choroid plexus epithelial cells, play an important role in cerebrospinal fluid production and may be involved in the pathophysiology of age-dependent hydrocephalus. The finding that brain aquaporins expression is regulated by low oxygen tension led us to investigate how hypoxia and elevated levels of cerebral aquaporins may result in an increase in cerebrospinal fluid production that could be associated with a hydrocephalic condition. Here we have explored, in young and aged mice exposed to hypoxia, whether aquaporin-4 and aquaporin-1 participate in the development of age-related hydrocephalus. Choroid plexus, striatum, cortex and ependymal tissue were analyzed separately both for mRNA and protein levels of aquaporins. Furthermore, parameters such as total ventricular volume, intraventricular pressure, cerebrospinal fluid outflow rate, ventricular compliance and cognitive function were studied in wild type, aquaporin-1 and aquaporin-4 knock-out animals subjected to hypoxia or normoxia. Our data demonstrate that hypoxia is involved in the development of age-related hydrocephalus by a process that depends on aquaporin-4 channels as a main route for cerebrospinal fluid movement. Significant increases in aquaporin-4 expression that occur over the course of animal aging, together with a reduced cerebrospinal fluid outflow rate and ventricular compliance, contribute to produce more severe hydrocephalus related to hypoxic events in aged mice, with a notable impairment in cognitive function. These results indicate that physiological events and/or pathological conditions presenting with cerebral hypoxia/ischemia contribute to the development of chronic adult hydrocephalus., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Detection of IgG anti-Aquaporin-4 (AQP4) in serum of patients with Neuromyelitis optica syndrome disorders (NMOSD) has improved diagnosis of these processes and differentiation from Multiple sclerosis (MS). Recent findings also claim that a subgroup of patients with NMOSD, serum negative for IgG-anti-AQP4, present antibodies anti-AQP1 instead. Explore the presence of IgG-anti-AQP1 using a previously developed cell-based assay (CBA) highly sensitive to IgG-anti-AQP4. Serum of 205 patients diagnosed as NMOSD (8), multiple sclerosis (94), optic neuritis (39), idiopathic myelitis (29), other idiopathic demyelinating disorders of the central nervous system (9), other neurological diseases (18) and healthy controls (8), were used in a CBA over fixed HEK cells transfected with hAQP1-EGFP or hM23-AQP4-EGFP, treated with Triton X-100 and untreated. ELISA was also performed. Analysis of serum with our CBA indicated absence of anti-AQP1 antibodies, whereas in cells pretreated with detergent, noisy signal made reliable detection impossible. ELISA showed positive results in few serums. The low number of NMOSD serums included in our study reduces its power to conclude the specificity of AQP1 antibodies as new biomarkers of NMOSD. Our study does not sustain detection of anti-AQP1 in serum of NMOSD patients but further experiments are expected.

AQP3 has been correlated with higher transport of glycerol, increment of ATP content, and larger proliferation capacity. Recently, we described the gold(III) complex Auphen as a very selective and potent inhibitor of AQP3's glycerol permeability (Pgly ). Here we evaluated Auphen effect on the proliferation of various mammalian cell lines differing in AQP3 expression level: no expression (PC12), moderate (NIH/3T3) or high (A431) endogenous expression, cells stably expressing AQP3 (PC12-AQP3), and human HEK293T cells transiently transfected (HEK-AQP3) for AQP3 expression. Proliferation was evaluated in the absence or presence of Auphen (5 μM) by counting number of viable cells and analyzing 5-bromo-2'-deoxyuridine (BrdU) incorporation. Auphen reduced ≈50% the proliferation in A431 and PC12-AQP3, ≈15% in HEK-AQP3 and had no effect in PC12-wt and NIH/3T3. Strong arrest in the S-G2/M phases of the cell cycle, supported by analysis of cyclins (A, B1, D1, E) levels, was observed in AQP3-expressing cells treated with Auphen. Flow-cytometry of propidium iodide incorporation and measurements of mitochondrial dehydrogenases activity confirmed absence of cytotoxic effect of the drug. Functional studies evidenced ≈50% inhibition of A431 Pgly by Auphen, showing that the compound's antiproliferative effect correlates with its ability to inhibit AQP3 Pgly . Role of Cys-40 on AQP3 permeability blockage by Auphen was confirmed by analyzing the mutated protein (AQP3-Ser-40). Accordingly, cells transfected with mutated AQP3 gained resistance to the antiproliferative effect of Auphen. These results highlight an Auphen inhibitory effect on proliferation of cells expressing AQP3 and suggest a targeted therapeutic effect on carcinomas with large AQP3 expression., (© 2014 Wiley Periodicals, Inc.)

Background: Cell-based assays for neuromyelitis optica (NMO) diagnosis are the most sensitive and specific methods to detect anti-aquaporin 4 (AQP4) antibodies in serum, but some improvements in their quantitative and specificity capacities would be desirable. Thus the aim of the present work was to develop a sensitive quantitative method for detection of anti-AQP4 antibodies that allows clear diagnosis of NMO and distinction of false labeling produced by natalizumab treatment., Methods: Sera from 167 individuals, patients diagnosed with NMO (16), multiple sclerosis (85), optic neuritis (24), idiopathic myelitis (21), or other neurological disorders (13) and healthy controls (8), were used as the primary antibody in an immunofluorescence assay on HEK cells transfected with the M23 isoform of human AQP4 fused with enhanced green fluorescent protein. Cells used were freshly transfected or stored frozen and then thawed just before adding the serum., Results: Microscopic observation and fluorescence quantification produced similar results in fresh and frozen samples. Serum samples from patients diagnosed with NMO were 100% positive for anti-AQP4 antibodies, while all the other sera were negative. Using serum from patients treated with natalizumab, a small and unspecific fluorescent signal was produced from all HEK cells, regardless of AQP4 expression., Conclusions: Our cell-based double-label fluorescence immunoassay protocol significantly increases the signal specificity and reduces false diagnosis of NMO patients, especially in those receiving natalizumab treatment. Frozen pretreated cells allow faster detection of anti-AQP4 antibodies.