Fluorescence Immunocytochemistry Research Articles

Glucocorticoid Receptor Isoforms in Breast Cancer Raise Implications for Personalised Supportive Therapies.

Glucocorticoid receptor (GR) activation may promote metastasis in oestrogen receptor-negative and triple-negative breast cancer (TNBC). However, the role of the GRβ isoform, which has opposing effects to the main isoform, has not been studied in clinical samples. We aimed to analyse the intracellular localisation of total GR and GRβ in vitro using plasmid constructs and fluorescent immunocytochemistry. Additionally, our goal was to perform immunostaining for total GR and GRβ on two cohorts: (i) on 194 clinical breast cancer samples to compare the expression in different molecular subtypes, and (ii) on 161 TNBC samples to analyse the association of GR with survival. We supplemented our analysis with RNA data from 1097 TNBC cases. We found that in the absence of the ligand, GR resided in the cytoplasm of breast cancer cells, while upon ligand activation, it translocated to the nucleus. A negative correlation was found between cytoplasmic GRtotal and Ki67 in luminal A tumours, while the opposite trend was observed in TNBC samples. Tumours with strong lymphoid infiltration showed higher cytoplasmic GRtotal staining compared to those with weaker infiltration. Patients with high nuclear GRtotal staining had shorter progression-free survival in univariate analysis. High cytoplasmic GRβ was a marker for better overall survival in multivariate analysis (10-year overall survival HR [95% CI]: 0.46 [0.22-0.95], p = 0.036). As a conclusions, this study is the first to investigate GRβ expression in breast tumours. Different expression and cellular localisation of GRtotal and GRβ were observed in the context of molecular subtypes, underscoring the complex role of GR in breast cancer. An inverse association between cytoplasmic GRtotal and the Ki67 proliferation index was observed in luminal A and TNBC. Regarding the impact of GR on outcomes in TNBC patients, while cytoplasmic GRβ was associated with a better prognosis, patients with nuclear GRtotal staining may be at a higher risk of disease progression, as it negatively affects survival. Caution should be exercised when using glucocorticoids in patients with nuclear GR staining, as it may negatively impact survival.

Pterostilbene ameliorates oxidative stress and neuronal apoptosis after intracerebral hemorrhage via the sirtuin 1-mediated Nrf2 pathway in vivo and in vitro

IntroductionOxidative stress and neuroapoptosis are significant pathological processes that occur in response to intracerebral hemorrhage (ICH), however, the optimal therapeutic strategy to treat these responses remains unknown. Pterostilbene (PTE) influences neural cell survival in in the pathology of a number of neurological diseases, but the mechanisms underlying this influence at present are not clear. The objective of the present study was to examine the potential impact of PTE on mitigating oxidative stress and neuronal apoptosis following ICH, while also elucidating the potential underlying pathways. Material & methodFor in vivo experimentation, male C57BL/6 mice were used to establish ICH models. Wet-to-dry weight ratios were utilized to assess the degree of cerebral edema in the context of PTE intervention. Behavioral experiments were conducted to evaluate neurological dysfunction and cognitive impairment, and hematoxylin and eosin staining was employed to observe histopathological changes in the brain. Furthermore, oxidative stress levels in hippocampal tissues were measured, and cell apoptosis was examined using TUNEL staining and western blotting techniques. In vitro experiments were conducted to evaluate the extent of oxidative stress and neural apoptosis after sirtuin 1 (SIRT1) siRNA treatment. Immunofluorescence cytochemistry was used to analyze the immunofluorescence colocalization of SIRT1 and NeuN. ResultMice that experienced ICH exhibited worsening neurological deterioration, increased oxidative stress and neuronal cell apoptosis. However, the addition of PTE was found to lessen these effects. Furthermore, PTE was found to activate the SIRT1-mediated Nrf2 pathway in mice with ICH. When SIRT1 was inhibited, levels of oxidative stress and neuronal apoptosis increased, even in the presence of PTE. ConclusionThe present study provided evidence to indicate that PTE can suppress oxidative damage and neuronal apoptosis following ICH by activating the SIRT1/Nrf2 pathway.

Bafilomycin 1A Affects p62/SQSTM1 Autophagy Marker Protein Level and Autophagosome Puncta Formation Oppositely under Various Inflammatory Conditions in Cultured Rat Microglial Cells.

Regulation of autophagy through the 62 kDa ubiquitin-binding protein/autophagosome cargo protein sequestosome 1 (p62/SQSTM1), whose level is generally inversely proportional to autophagy, is crucial in microglial functions. Since autophagy is involved in inflammatory mechanisms, we investigated the actions of pro-inflammatory lipopolysaccharide (LPS) and anti-inflammatory rosuvastatin (RST) in secondary microglial cultures with or without bafilomycin A1 (BAF) pretreatment, an antibiotic that potently inhibits autophagosome fusion with lysosomes. The levels of the microglia marker protein Iba1 and the autophagosome marker protein p62/SQSTM1 were quantified by Western blots, while the number of p62/SQSTM1 immunoreactive puncta was quantitatively analyzed using fluorescent immunocytochemistry. BAF pretreatment hampered microglial survival and decreased Iba1 protein level under all culturing conditions. Cytoplasmic p62/SQSTM1 level was increased in cultures treated with LPS+RST but reversed markedly when BAF+LPS+RST were applied together. Furthermore, the number of p62/SQSTM1 immunoreactive autophagosome puncta was significantly reduced when RST was used but increased significantly in BAF+RST-treated cultures, indicating a modulation of autophagic flux through reduction in p62/SQSTM1 degradation. These findings collectively indicate that the cytoplasmic level of p62/SQSTM1 protein and autophagocytotic flux are differentially regulated, regardless of pro- or anti-inflammatory state, and provide context for understanding the role of autophagy in microglial function in various inflammatory settings.

Endothelial cells in chronic thromboembolic pulmonary hypertension exhibit cytoskeletal alteration

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Health Research Fund (Fondo de Investigación Sanitaria [FIS]) from the Instituto de Salud Carlos III Introduction Endothelial dysfunction (ED) is pivotal in Chronic Thromboembolic Pulmonary Hypertension (CTEPH) due to structural and functional alterations in endothelial. Previous studies have shown the critical function of cytoskeletal elements, particularly actin microfilaments and microtubules, along with associated cell adhesion complexes, in the endothelial integrity. Nevertheless, precise alterations in the cytoskeletal elements of endothelial cells (ECs) in CTEPH patients remain unclear. Purpose This study aims to investigate the cytoskeleton changes in CTEPH-ECs compared to healthy ECs, characterizing morphological and structural differences. Objectives involve assessing microtubules and microfilaments expression, and their influence on ECs migratory capacity. Finally, our study aims to gain novel insights into the spatial organization of cytoskeleton components in CTEPH patients. Methods CTEPH-ECs were isolated from freshly resected pulmonary endarterectomy specimens of CTEPH patients, while human pulmonary artery ECs (HPAE) were used as healthy controls. Fluorescence immunocytochemistry assessed phenotypic and compositional characteristics of the cytoskeleton. Morphometric analysis described morphology and filament organization of cytoskeletal proteins using a custom-built pipeline. X-ray microscopy examined the phenotype and cytoskeletal components distribution. The migration assay evaluated how these structural differences affected ECs migratory capacity and the cytoskeletal elements expression. Results CTEPH-ECs exhibited larger surface area (1352 vs 662µm2, p<0.01) and a more stellate morphology (0.365 vs 0.289, p<0.01) than HPAE-ECs. Immunostaining indicated altered cytoskeletal composition in CTEPH-ECs with significantly higher tubulin expression, expressed in microtubules, (3327 vs 2238AU, p<0.01) and F-actin microfilaments (2068 vs 1300AU, p<0.01) compared to HPAE. Similarly, CTEPH-ECs showed reduced G-actin expression (3002 vs 7461AU, p<0.01). Morphometric analysis demonstrated that microfilaments were thinner (0.623 vs 0.831AU, p<0.01), more straight (0.135 vs. 0.172, p<0.01) and more aligned (0.132 vs. 0.156, p<0.01) in CTEPH-ECs. Microtubule thickness decreased compared to HPAE-ECs (0.341 vs 1.371AU, p<0.01). CTEPH-ECs showed impaired wound healing (60 vs 89% closure, p<0.05). CTEPH group showed an increase in microtubules and microfilaments expression during wound healing, but with irregular patterns, unlike the constant levels in HPAE. TXM-technology revealed spatial organization differences, especially expansive microtubules distribution in CTEPH-ECs (4.2*10^7 vs. 8.7*10^6 nm³, p<0.05). Conclusion These preliminary findings highlight significant structural and compositional differences in CTEPH-ECs, along with altered wound healing capacity, suggesting dynamic cytoskeletal and cellular changes in CTEPH. Understanding these alterations is crucial for advancing CTEPH knowledge and identifying potential therapeutic targets.

GLP-1(7–36) protected against oxidative damage and neuronal apoptosis in the hippocampal CA region after traumatic brain injury by regulating ERK5/CREB

BackgroundGlucagon-like peptide-1 (GLP-1) (7–36) amide, an endogenous active form of GLP-1, has been shown to modulate oxidative stress and neuronal cell survival in various neurological diseases.ObjectiveThis study investigated the potential effects of GLP-1(7–36) on oxidative stress and apoptosis in neuronal cells following traumatic brain injury (TBI) and explored the underlying mechanisms.MethodsTraumatic brain injury (TBI) models were established in male SD rats for in vivo experiments. The extent of cerebral oedema was assessed using wet-to-dry weight ratios following GLP-1(7–36) intervention. Neurological dysfunction and cognitive impairment were evaluated through behavioural experiments. Histopathological changes in the brain were observed using haematoxylin and eosin staining. Oxidative stress levels in hippocampal tissues were measured. TUNEL staining and Western blotting were employed to examine cell apoptosis. In vitro experiments evaluated the extent of oxidative stress and neural apoptosis following ERK5 phosphorylation activation. Immunofluorescence colocalization of p-ERK5 and NeuN was analysed using immunofluorescence cytochemistry.ResultsRats with TBI exhibited neurological deterioration, increased oxidative stress, and enhanced apoptosis, which were ameliorated by GLP-1(7–36) treatment. Notably, GLP-1(7–36) induced ERK5 phosphorylation in TBI rats. However, upon ERK5 inhibition, oxidative stress and neuronal apoptosis levels were elevated, even in the presence of GLP-1(7–36).ConclusionIn summary, this study suggested that GLP-1(7–36) suppressed oxidative damage and neuronal apoptosis after TBI by activating ERK5/CREB.

Taming Microglia in Alzheimer's Disease: Exploring Potential Implications of Choline Alphoscerate via α7 nAChR Modulation.

Alzheimer's disease (AD), marked by cognitive impairment, predominantly affects the brain regions regulated by cholinergic innervation, such as the cerebral cortex and hippocampus. Cholinergic dysfunction, a key contributor to age-related cognitive decline, has spurred investigations into potential therapeutic interventions. We have previously shown that choline alphoscerate (α-GPC), a cholinergic neurotransmission-enhancing agent, protects from Aβ-mediated neurotoxicity. Herein, we investigated the effects of α-GPC on the microglial phenotype in response to Aβ via modulation of the nicotinic alpha-7 acetylcholine receptor (α7 nAChR). BV2 microglial cells were pre-treated for 1 h with α-GPC and were treated for 24, 48, and 72 h with Aβ1-42 and/or α-BTX, a selective α7nAchR antagonist. Fluorescent immunocytochemistry and Western blot analysis showed that α-GPC was able to antagonize Aβ-induced inflammatory effects. Of note, α-GPC exerted its anti-inflammatory effect by directly activating the α7nAChR receptor, as suggested by the induction of an increase in [Ca2+]i and Ach-like currents. Considering that cholinergic transmission appears crucial in regulating the inflammatory profiles of glial cells, its modulation emerges as a potential pharmaco-therapeutic target to improve outcomes in inflammatory neurodegenerative disorders, such as AD.

Immunocytochemistry of Primary Cultured Cerebral Cortical Neurons.

Immunocytochemistry combined with confocal or superresolution microscopy allows us to observe molecular localization and intracellular structures. However, it is challenging to analyze individual neurons in brain tissue, where neurons are densely packed. In contrast, we can easily observe structures such as the axonal growth cone and dendritic spines in dissociated individual neurons. Thus, the immunocytochemistry of primary cultured neurons is often used because it reflects the in vivo condition at least in part. Here, we describe a method for indirect fluorescence immunocytochemistry of primary cultured neurons from the embryonic cerebral cortex. This involves multiple steps including fixation, permeabilization, and antibody reaction, and in particular, we introduce an optimized protocol for permeabilization to enable the precise localization of target molecules.

Gestational VPA exposure reduces the density of juxtapositions between TH+ axons and calretinin or calbindin expressing cells in the ventrobasal forebrain of neonatal mice.

Gestational exposure to valproic acid (VPA) is a valid rodent model of human autism spectrum disorder (ASD). VPA treatment is known to bring about specific behavioral deficits of sociability, matching similar alterations in human autism. Previous quantitative morphometric studies from our laboratory showed a marked reduction and defasciculation of the mesotelencephalic dopaminergic pathway of VPA treated mice, along with a decrease in tissue dopamine in the nucleus accumbens (NAc), but not in the caudatoputamen (CPu). In the present study, the correlative distribution of tyrosine hydroxylase positive (TH+) putative axon terminals, presynaptic to the target neurons containing calretinin (CR) or calbindin (CB), was assessed using double fluorescent immunocytochemistry and confocal laser microscopy in two dopamine recipient forebrain regions, NAc and olfactory tubercle (OT) of neonatal mice (mothers injected with VPA on ED13.5, pups investigated on PD7). Representative image stacks were volumetrically analyzed for spatial proximity and abundance of presynaptic (TH+) and postsynaptic (CR+, CB+) structures with the help of an Imaris (Bitplane) software. In VPA mice, TH/CR juxtapositions were reduced in the NAc, whereas the TH/CB juxtapositions were impoverished in OT. Volume ratios of CR+ and CB+ elements remained unchanged in NAc, whereas that of CB+ was markedly reduced in OT; here the abundance of TH+ axons was also diminished. CR and CB were found to partially colocalize with TH in the VTA and SN. In VPA exposed mice, the abundance of CR+ (but not CB+) perikarya increased both in VTA and SN, however, this upregulation was not mirrored by an increase of the number of CR+/TH+ double labeled cells. The observed reduction of total CB (but not of CB+ perikarya) in the OT of VPA exposed animals signifies a diminished probability of synaptic contacts with afferent TH+ axons, presumably by reducing the available synaptic surface. Altered dopaminergic input to ventrobasal forebrain targets during late embryonic development will likely perturb the development and consolidation of neural and synaptic architecture, resulting in lasting changes of the neuronal patterning (detected here as reduced synaptic input to dopaminoceptive interneurons) in ventrobasal forebrain regions specifically involved in motivation and reward.

Clearance of intracellular lipids by lipophagy impaired in APOE ε4 human astrocytes

AbstractBackgroundApolipoprotein E (APOE) ɛ4 is the strongest known genetic risk factor for Alzheimer’s disease. APOE ɛ4 has been found to impair lipid metabolism in human glia causing accumulation of intracellular lipid droplets. We hypothesized that this phenotype in human APOE ɛ4/ɛ4 astrocytes was due to defective clearance via lipophagy.MethodGlobal transcriptomic analyses were performed on astrocytes derived from isogenic and population model human induced pluripotent stem cells (iPSCs). Fluorescent reporters, immunocytochemistry, and western blotting were used to quantify lipid droplets and autophagic machinery in iPSC‐based models.ResultGlobal transcriptomic analyses reveal that APOE ε4 drives autophagy dysregulation in astrocytes from human iPSCs. High‐content imaging of fluorescent autophagy reporters and immunocytochemistry revealed accumulation of lipid droplets and autophagy machinery in APOE ɛ4/ɛ4 astrocytes which was ameliorated by induction of autophagy.ConclusionHuman iPSC‐derived APOE ɛ4/ɛ4 astrocytes exhibit impaired lipophagy.

Impact of COVID-19 on sperm quality and the prostaglandin and polyamine systems in the seminal fluid.

The implications of SARS-CoV-2 infection on male fertility remain largely unknown. Besides their well-known pro- and anti-inflammatory actions, prostaglandins and polyamines are present in semen, where they play key roles in sperm quality. To analyze semen parameters, oxidative profile and the seminal fluid prostaglandin and polyamine systems in samples collected from individuals without coronavirus disease 2019 diagnosis and men who recovered from coronavirus disease 2019. This study compared semen collected from men without positive coronavirus disease 2019 diagnosis with samples obtained from individuals 1-6 months and 7-30 months post SARS-CoV-2 infection. Semen parameters, thiobarbituric acid reactive substances, cyclooxygenase 2 expression by fluorescence immunocytochemistry and immunoblotting, prostaglandin levels by enzyme immunoassay, ornithine decarboxylase activity by a radioactive assay, and polyamine and acetylated polyamine levels by thin-layer chromatography were assessed. In both groups of semen samples from coronavirus disease 2019 recovered men, sperm vitality, total and progressive sperm motility, and putrescine levels were significantly decreased when compared with samples from the uninfected group. In contrast, lipid peroxidation, leukocyte-associated cyclooxygenase 2 expression, and prostaglandin D2 levels were higher in semen from coronavirus disease 2019 recovered men than in samples from uninfected individuals. While sperm concentration and morphology, ornithine decarboxylase activity, and N-acetylputrescine levels were statistically diminished in semen obtained up to 6 months after coronavirus disease 2019 recovery, these parameters remained unchanged when samples were collected 7-30 months after coronavirus disease 2019 recovery. Coronavirus disease 2019 vaccination did not show negative effects on any of the parameters evaluated. Our work provides insights into the detrimental impact of coronavirus disease 2019 on several sperm parameters, in some cases, even more than a year after SARS-CoV-2 infection, which would be accompanied by alterations in the seminal fluid prostaglandin and polyamine profiles. Therefore, future treatments targeting the prostaglandin and polyamine pathways in coronavirus disease 2019 recovered men could lead to a successful reinstatement of semen parameters.

L-Arginine Enhances Oral Keratinocyte Proliferation under High-Glucose Conditions via Upregulation of CYP1A1, SKP2, and SRSF5.

High glucose inhibits oral keratinocyte proliferation. Diabetes can lead to delayed oral wound healing and periodontal disease. L-Arginine, one of the most versatile amino acids, plays an important role in wound healing, organ maturation, and development. In this study, L-Arginine was found to enhance oral keratinocyte proliferation under high-glucose conditions. RNA sequencing analysis discovered a significant number of genes differentially upregulated following L-Arginine treatment under high-glucose conditions. Cytochrome P450 family 1 subfamily A member 1 (CYP1A1) was the most significantly upregulated gene at 24 and 48 h after L-Arginine treatment. Gene Ontology enrichment analysis found that cell proliferation- and mitosis-related biological processes, such as mitotic nuclear division, mRNA processing, and positive regulation of cell cycle processes, were significantly upregulated. Pathway enrichment analysis found that S-phase kinase-associated protein 2 (SKP2) and serine- and arginine-rich splicing factor 5 (SRSF5) were the top upregulated genes in cell cycle and spliceosome pathways, respectively. Indirect immunofluorescent cytochemistry confirmed increased protein levels of CYP1A1, SKP2, and SRSF5 after L-Arginine treatment. Knockdown of CYP1A1, SKP2, and SRSF5 abolished the enhanced proliferative effect of L-Arginine on oral keratinocytes under high-glucose conditions. In conclusion, L-Arginine enhances oral keratinocyte proliferation under high-glucose conditions via upregulation of CYP1A1, SKP2, and SRSF5, suggesting that supplemental L-Arginine in oral care products may be beneficial for oral tissue repair and regeneration.

Mollugin activates GLP-1R to improve cognitive dysfunction in type 2 diabetic mice

AimsThe incidence of diabetic cognitive dysfunction is increasing year by year, and it has gradually become a research hot spot. Studies have shown that glucagon-like peptide-1 receptor (GLP-1R) agonists can improve cognitive dysfunction in diabetic patients. This study focuses on whether small molecule GLP-1R agonists from traditional Chinese medicine (TCM) can improve the diabetic cognitive dysfunction. Materials and methodsThe small molecules from TCM were screened by cell membrane chromatography (CMC) with GLP-1R-HEK293 cell membrane column. MTT assay, flow cytometry, immunofluorescence cytochemistry and other methods were used to determine the effects of mollugin on the apoptosis rate and reactive oxygen species (ROS) level of high glucose (HG)/hydrogen peroxide (H2O2) induced PC12 cells. Real-Time PCR was used to detect mRNA expression in mouse cerebral cortex. Water maze test was further used to confirm the effect of mollugin on cognitive dysfunction in T2DM mice. Key findingsMollugin bound to GLP-1R, promoted Ca2+ influx, increased insulin secretion and cAMP content in β-TC-6 cells. Mollugin enhanced the cell viability, ameliorated apoptosis, reduced intracellular ROS levels in HG/H2O2-injured PC12 cells. Mollugin reduced the T2DM mice's escape latency, improved neuronal cell damage, decreased the expression of Pik3ca, Akt1 and Mapk1 mRNA in the cerebral cortex tissue. SignificanceThe results suggest that mollugin could improve cognitive dysfunction in T2DM mice through activating GLP-1R/cAMP/PKA signal pathway.

Regulation of the ATP-binding cassette transporters ABCB1, ABCG2 and ABCC5 by nuclear receptors in porcine blood-brain barrier endothelial cells.

Blood-brain barrier (BBB) ABCB1, ABCG2 and ABCC5 transporters influence central therapeutic drug distribution. Transporter expression is regulated by the NR3C1, NR1I3 and NR1I2 nuclear receptors, but their precise roles in brain are poorly understood. We investigated the effects of selective ligand-based activation of NR3C1, NR1I3, NR1I2 and NR2B1 in porcine brain endothelial cells (PBECs). Primary cultures of PBECs were exposed to NR3C1, NR1I3 and NR1I2 ligands and ABCB1, ABCG2 and ABCC5 transporter activities determined by measuring intracellular accumulation of fluorescent probes. Western blotting was used to determine the effects of receptor ligands on expression of ABCB1, ABCG2, ABCC5, NR1I2, NR1I3, NR3C1 and NR2B1. Fluorescent immunocytochemistry was employed to assess the effects of receptor ligands on the cellular localisation of NR1I2 and NR1I3. The NR1I2 agonist rifampicin significantly up-regulated ABCG2 activity, which is counteracted by co-treatment with NR1I2 antagonist l-sulforaphane. The NR1I3 agonist 6-(4-chlorophenyl)-imidazo[2,1-b]thiazole-5-carbaldehyde and inverse agonist meclizine significantly down-regulated ABCB1, ABCG2 and ABCC5 activity. NR3C1 agonist dexamethasone significantly increased ABCB1, ABCG2 and ABCC5 activity and ABCG2 and ABCC5 protein expression, which was counteracted by co-treatment with the NR3C1 antagonist mifepristone. This first study demonstrates that NR1I3 and NR3C1 regulate ABCC5 activity and protein expression in BBB endothelial cells. In PBECs, expression of key ATP-binding cassette (ABC) transporters and nuclear receptors is differentially regulated by NR1I3, NR1I2, NR3C1 and NR2B1. This will help to better understand the response of the BBB to physiological and pharmacological activation of nuclear receptors.

Stem cell therapy based on human periodontal ligament stem cells preactivated with TNF-α as for human head and neck squamous cell carcinoma

ObjectiveMesenchymal stem cells (MSCs) express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) following activation by various molecules and have antitumor effects against several cancer types. In particular, preactivation of MSCs with tumor necrosis factor-α (TNF-α) enhances TRAIL expression. We focused on the application of human periodontal ligament-derived stem cells (hPDLSCs) to stem cell-based cancer therapy, and evaluated their effects on head and neck squamous cell carcinoma (HNSCC) cells. MethodsTRAIL expression in hPDLSCs preactivated with TNF-α (A-hPDLSCs) was evaluated using real-time PCR, western blotting, and fluorescent immunocytochemistry. Subsequently, non-treated hPDLSCs (N-hPDLSCs) or A-hPDLSCs were cocultured with three types of HNSCC cells: HSC2, HSC3, and HSC4, and HNSCC cell apoptosis was assessed using flow cytometry. The migration ability of N-hPDLSCs and A-hPDLSCs toward HNSCC cells was also evaluated using a 3D gel invasion assay. In addition, conditioned medium (CM) derived from A-hPDLSCs was added to HNSCC cells, and cell proliferation ability was measured. ResultsA-hPDLSCs preactivated with 10 ng/mL TNF-α significantly upregulated the expression of TRAIL, increased apoptosis in HNSCC cells, and exhibited increased migration toward HNSCC cells compared to N-hPDLSCs. However, CM derived from A-hPDLSCs neither increased apoptosis nor suppressed HNSCC cell proliferation. ConclusionshPDLSCs are proposed to be beneficial for stem cell therapies for HNSCC. Upon TNF-α preactivation, hPDLSCs may migrate toward HNSCC cells and directly induce apoptosis.

Inhibition of IL-4/STAT6/IRF4 signaling reduces the epithelial-mesenchymal transition in eosinophilic chronic rhinosinusitis with nasal polyps.

Previous studies have shown that epithelial-to-mesenchymal transition (EMT) in nasal epithelial cells is critical for tissue remodeling of chronic rhinosinusitis with nasal polyps (CRSwNP). However, the precise mechanism underlying the EMT remains poorly understood. This study aimed to investigate the role of interleukin-4 (IL-4)/signal transducer and activator of transcription 6 (STAT6)/interferon regulatory factor 4 (IRF4) signaling pathway on EMT in eosinophilic CRSwNP. We performed quantitative real-time polymerase chain reaction, immunohistochemistry, immunofluorescent staining, and Western blotting to evaluate the expression of STAT6, IRF4, and EMT markers in sinonasal mucosal samples. Effects of IL-4-induced EMT were determined using primary human nasal epithelial cells (hNECs) from patients with eosinophilic CRSwNP. Wound scratch assay, cell morphology, Western blotting, and immunofluorescence cytochemistry were performed to evaluate EMT, and EMT-related markers. Next, human THP-1 monocytic cells were stimulated by phorbolate-12-myristate-13-acetate to differentiate into M0 and were subsequently polarized into M1 with lipopolysaccharide and interferon-γ, M2 with IL-4. The markers of the macrophage phenotype were assessed by Western blotting. The co-culture system was built to explore the interaction between macrophages (THP-1 cells) and hNECs. After co-culture with M2 macrophages, EMT-related markers of primary hNECs were evaluated by immunofluorescence cytochemistry and Western blotting. Enzymelinked immunosorbent assays were used to detect transforming growth factor beta 1 (TGF-β1) in THP-1-derived supernatants. STAT6 and IRF4 mRNA and protein expression were significantly upregulated in both eosinophilic and noneosinophilic nasal polyps compared with control tissues. The expression of STAT6 and IRF4 in eosinophilic nasal polyps was higher than those in noneosinophilic nasal polyps. STAT6 and IRF4 were not only expressed in epithelial cells but also in macrophages. The number of STAT6+CD68+ cells and IRF4+CD68+ cells in eosinophilic nasal polyps was higher than those in noneosinophilic nasal polyps and control tissues. EMT was enhanced in eosinophilic CRSwNP compared to the healthy controls and noneosinophilic CRSwNP. IL-4-stimulated human nasal epithelial cells exhibited EMT characteristics. The hNECs co-cultured with M2 macrophages demonstrated high levels of EMT-related markers. The TGF-β1 level was significantly induced by IL-4 and elevated (M2) rather than control macrophages. The inhibition of STAT6 by AS1517499 reduced the expression of IRF4 in epithelial cells and macrophages and counteracted IL-4-induced EMT in epithelial cells. In eosinophilic nasal polyps, IL-4 induces STAT6 signaling to upregulate IRF4 expression in epithelial cells and macrophages. IL-4 promotes EMT of hNECs through the STAT6/IRF4 signaling pathway. IL-4-induced M2 macrophages enhanced EMT of hNECs. Inhibition of STAT6 can downregulate the expression of IRF4 and suppress the EMT process, thus providing a new strategy for the treatment of nasal polyps.

Bell's palsy was associated with TRPV2 downregulation of Schwann cell by cold stress

ObjectiveEpidemiological and clinical studies have shown that sharp changes in the ambient temperature are associated with the occurrence and development of Bell's palsy. However, the specific pathogenesis of peripheral facial paralysis remains nebulous. This study investigated the effect of cold stress on transient receptor potential cation channel subfamily V member 2 (TRPV2) secretion by Schwann cells and its role in Bell's palsy. Materials and methodsSchwann cell morphology was observed using transmission electron microscopy (TEM). Cell proliferation, apoptosis and cell cycle were analysed using CCK8 and flow cytometry. ELISA, Reverse transcription-quantitative PCR, western blotting and immunocytochemical fluorescence staining were used to detect the effects of cold stress on TRPV2, neural cell adhesion molecule (NCAM) and nerve growth factor (NGF) expression in Schwann cells. ResultsCold stress resulted in a widening of the intercellular space, and the particles on the membrane showed different degrees of loss. Cold stress may cause Schwann cells to enter a cold dormant state. ELISA, RT-qPCR, western blotting and immunocytochemical fluorescences staining indicated that cold stress inhibited the expression of TRPV2, NCAM, and NGF. ConclusionsDrastic temperature difference between cold and heat can downregulate TRPV2 and the secretome of Schwann cells. The imbalance of Schwann cell homeostasis under such stress may contribute to nerve signalling dysfunction leading to the development of facial paralysis.

Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs.

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication of soft tissue constructs. However, granular gel formulations have been restricted to a limited number of biomaterials that allow for the cost-effective generation of large amounts of hydrogel microparticles. Therefore, granular gel support media have generally lacked the cell-adhesive and cell-instructive functions found in the native extracellular matrix (ECM). To address this, a methodology has been developed for the generation of self-healing annealable particle-extracellular matrix (SHAPE) composites. SHAPE composites consist of a granular phase (microgels) and a continuous phase (viscous ECM solution) that, together, allow for both programmable high-fidelity printing and an adjustable biofunctional extracellular environment. This work describes how the developed methodology can be utilized for the precise biofabrication of human neural constructs. First, alginate microparticles, which serve as the granular component in the SHAPE composites, are fabricated and combined with a collagen-based continuous component. Then, human neural stem cells are printed inside the support material, followed by the annealing of the support. The printed constructs can be maintained for weeks to allow the differentiation of the printed cells into neurons. Simultaneously, the collagen continuous phase allows for axonal outgrowth and the interconnection of regions. Finally, this works provides information on how to perform live-cell fluorescence imaging and immunocytochemistry to characterize the 3D-printed human neural constructs.

GPR160 is not a receptor of anorexigenic cocaine- and amphetamine-regulated transcript peptide

Cocaine- and amphetamine-regulated transcript peptide (CARTp) is an anorexigenic neuropeptide whose receptor is undisclosed. Previously, we reported the specific binding of CART(61–102) to pheochromocytoma PC12 cells, where CART(61–102) affinity and the number of binding sites per cell corresponded to ligand-receptor binding. Recently, Yosten et al. designated orphan GPR160 as the CARTp receptor, because the GPR160 antibody abolished neuropathic pain and anorexigenic effects induced by CART(55–102) and exogenous CART(55–102) coimmunoprecipitated with GPR160 in KATOIII cells. As no direct evidence that CARTp is a ligand for GPR160 has been described, we decided to verify this hypothesis by testing CARTp affinity to the GPR160 receptor. We investigated the GPR160 expression in PC12 cells since it is cell line known to specifically bind CARTp. Moreover, we examined the specific CARTp binding in THP1 cells, with high endogenous GPR160 expression and GPR160-transfected cell lines U2OS and U-251 MG. In PC12 cells, the GPR160 antibody did not compete for specific binding with 125I-CART(61–102) or with 125I-CART(55–102), and GPR160 mRNA expression and GPR160 immunoreactivity were not detected. Moreover, THP1 cells did not show any 125I-CART(61–102) or 125I-CART(55–102) specific binding despite GPR160 detection by fluorescent immunocytochemistry (ICC). Finally, no 125I-CART(61–102) or 125I-CART(55–102) specific binding in the GPR160-transfected cell lines U2OS and U-251 MG, selected due to their negligible endogenous expression of GPR160, was detected, despite the detection of GPR160 by fluorescent ICC. Our binding studies clearly demonstrated that GPR160 cannot be a receptor for CARTp. Further studies are needed to identify true CARTp receptors.

Changes in the Localization of Polyamine Spermidine in the Rat Retina with Age.

Polyamines (PAs) in the nervous system has a key role in regeneration and aging. Therefore, we investigated age-related changes in the expression of PA spermidine (SPD) in the rat retina. Fluorescent immunocytochemistry was used to evaluate the accumulation of SPD in retinae from rats of postnatal days 3, 21, and 120. Glial cells were identified using glutamine synthetase (GS), whereas DAPI, a marker of cell nuclei, was used to differentiate between retinal layers. SPD localization in the retina was strikingly different between neonates and adults. In the neonatal retina (postnatal day 3-P3), SPD is strongly expressed in practically all cell types, including radial glia and neurons. SPD staining showed strong co-localization with the glial marker GS in Müller Cells (MCs) in the outer neuroblast layer. In the weaning period (postnatal day 21-P21), the SPD label was strongly expressed in all MCs, but not in neurons. In early adulthood (postnatal day 120-P120), SPD was localized in MCs only and was co-localized with the glial marker GS. A decline in the expression of PAs in neurons was observed with age while glial cells accumulated SPD after the differentiation stage (P21) and during aging in MC cellular endfoot compartments.

Evaluating the clinical utility of circulating tumor cells (CTC) profiling to predict selection of preferred therapeutic regimens in newly diagnosed or pretreated refractory renal cell carcinomas (RCC).

717 Background: There is an unmet need for biomarkers enabling therapeutic selection and prediction of clinical outcomes in kidney cancer . We evaluated the feasibility and utility of profiling of circulating tumor cells (CTCs) via multiplexed fluorescence immunocytochemistry (ICC) to identify liquid biopsy biomarkers linked to treatment response (or resistance) in advanced RCC patients. In addition, transcriptome analysis for 20802 genes from exosomal RNA is planned to evaluate novel prognostic and predictive signatures. Methods: Patients with either untreated or pretreated advanced RCC were eligible prior to starting a new systemic therapy regimen. IRB approved written informed consent was obtained. Serial blood samples were collected at baseline, and at 3, 6, 12 and 24 months. Primary endpoint of the study was to detect the proportion of patients with RCC in whom CTCs can be detected and profiled. Secondary endpoints are to correlate the profiling and changes in CTC with response and clinical outcomes. The study will meet its primary endpoint if the assay provides adequate detection and profiling for at least 12 patients. With an overall sample size of 50 the width of a 95% confidence interval for the rate of providing a therapeutic intervention is guaranteed to be less than 26%. Results: 44 evaluable patients have been enrolled; 11 females, 33 males. Median age was 64 years (range 40-85 years). 38 white patients, 2 black patients and 4 of other ethnicity have been enrolled.16 patients were untreated, 22 were pretreated and 6 were undergoing adjuvant therapy post nephrectomy. 37 patients had clear cell histology, 4 non clear cell and 3 unclassified histology. 42 of the 44 (95.5%) patients had detectable CTCs in the baseline sample. 21 on treatment samples have been collected to date, with detectable CTCs in all except one sample. 53 of 65 samples (81.5%) demonstrated detection of at least one biomarker by ICC. VEGFA expression was the most commonly detected biomarker on ICC (detected in 28 of 63 samples). Conclusions: The primary endpoint was met, and feasibility of the test was demonstrated with 95% of the baseline samples showing CTC detection and 81.5% showing biomarker expression. This blood-based, non-invasive liquid biopsy demonstrated high sensitivity for detection of cancer cells, and presents a potential opportunity for biomarker profiling to predict therapeutic efficacy of conventional RCC therapeutic agents. Correlation of longitudinal CTC with clinical outcomes will be reported. Transcriptome analysis is under evaluation for novel prognostic and predictive signatures and will be reported.