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1.
Commun Biol ; 7(1): 129, 2024 01 25.
Article in English | MEDLINE | ID: mdl-38272969

ABSTRACT

Neudesin, originally identified as a neurotrophic factor, has primarily been studied for its neural functions despite its widespread expression. Using 8-week-old neudesin knockout mice, we elucidated the role of neudesin in the spleen. The absence of neudesin caused mild splenomegaly, shortened lifespan of circulating erythrocytes, and abnormal recovery from phenylhydrazine-induced acute anemia. Blood cross-transfusion and splenectomy experiments revealed that the shortened lifespan of erythrocytes was attributable to splenic impairment. Further analysis revealed increased erythrophagocytosis and decreased iron stores in the splenic red pulp, which was linked to the upregulation of Fcγ receptors and iron-recycling genes in neudesin-deficient macrophages. In vitro analysis confirmed that neudesin suppressed erythrophagocytosis and expression of Fcγ receptors through ERK1/2 activation in heme-stimulated macrophages. Finally, we observed that 24-week-old neudesin knockout mice exhibited severe symptoms of anemia. Collectively, our results suggest that neudesin regulates the function of red pulp macrophages and contributes to erythrocyte and iron homeostasis.


Subject(s)
Anemia , Iron , Animals , Mice , Iron/metabolism , Macrophages/metabolism , Mice, Knockout , Phagocytosis/physiology , Receptors, IgG/metabolism , Spleen/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Nerve Tissue Proteins/metabolism
2.
Poult Sci ; 100(2): 452-460, 2021 Feb.
Article in English | MEDLINE | ID: mdl-33518096

ABSTRACT

Ovomucoid is a major egg white protein which is considered as the most dominant allergen in chicken eggs. Owing to the difficulty of separating ovomucoid from egg whites, researchers have adopted genetic deletion for development of hypoallergenic eggs. Previously, we used CRISPR/Cas9 to establish chickens with ovomucoid gene (OVM) mutations, but it remained unknown whether such hens could produce eggs at maturity. Here, we have reported on eggs laid by OVM-targeted hens. Except for watery egg whites, the eggs had no evident abnormalities. Real-time PCR revealed alternative splicing of OVM mRNA in hens, but their expression was limited. Immunoblotting detected neither mature ovomucoid nor ovomucoid-truncated splicing variants in egg whites. Sixteen chicks hatched from 28 fertilized eggs laid by OVM-targeted hens, and fourteen of the sixteen chicks demonstrated healthy growth. Taken together, our results demonstrated that OVM knockout could almost completely eliminate ovomucoid from eggs, without abolishing fertility. Thus, the eggs developed in this study have potential as a hypoallergenic food source for most patients with egg allergies.


Subject(s)
Chickens/genetics , Eggs/standards , Mutation , Ovomucin/genetics , Allergens/genetics , Animals , Chickens/growth & development , Chickens/physiology , Egg White/adverse effects , Egg White/chemistry , Egg White/standards , Female , Gene Deletion , Male , Oviposition/genetics , Ovomucin/adverse effects , Ovum
3.
Genes (Basel) ; 12(1)2020 12 30.
Article in English | MEDLINE | ID: mdl-33396657

ABSTRACT

Increased commercial demand for monoclonal antibodies (mAbs) has resulted in the urgent need to establish efficient production systems. We previously developed a transgenic chicken bioreactor system that effectively produced human cytokines in egg whites using genome-edited transgenic chickens. Here, we describe the application of this system to mAb production. The genes encoding the heavy and light chains of humanized anti-HER2 mAb, linked by a 2A peptide sequence, were integrated into the chicken ovalbumin gene locus using a CRISPR/Cas9 protocol. The knock-in hens produced a fully assembled humanized mAb in their eggs. The mAb expression level in the egg white was 1.4-1.9 mg/mL, as determined by ELISA. Furthermore, the antigen binding affinity of the anti-HER2 mAb obtained was estimated to be equal to that of the therapeutic anti-HER2 mAb (trastuzumab). In addition, antigen-specific binding by the egg white mAb was demonstrated by immunofluorescence against HER2-positive and -negative cells. These results indicate that the chicken bioreactor system can efficiently produce mAbs with antigen binding capacity and can serve as an alternative production system for commercial mAbs.


Subject(s)
Antibodies, Monoclonal/biosynthesis , CRISPR-Cas Systems , Chickens/genetics , Egg White/chemistry , Receptor, ErbB-2/antagonists & inhibitors , Animals , Animals, Genetically Modified , Antibodies, Monoclonal/isolation & purification , Bioreactors , Female , Gene Editing/methods , Humans , Plasmids/chemistry , Plasmids/metabolism , Receptor, ErbB-2/genetics , Receptor, ErbB-2/immunology , Trastuzumab/biosynthesis , Trastuzumab/isolation & purification , Zygote/chemistry , Zygote/metabolism
4.
Int Immunopharmacol ; 67: 408-416, 2019 Feb.
Article in English | MEDLINE | ID: mdl-30586664

ABSTRACT

Dendritic cells (DCs) play a primary role in antigen presentation to CD4+ and CD8+ T cells and induce acquired immune response against cancer cells. Therefore, determining positive modulators of DC activation to improve therapeutic approaches for cancer immunotherapy is greatly needed. In this study, we investigated the effect of maitake α-glucan YM-2A, isolated from Grifola frondosa, on the maturation and function of DCs and its adjuvant effect on a tumor-associated antigen (TAA)-loaded DC vaccine against murine tumor. We showed that YM-2A induced morphological changes and increased cell-surface maturation markers and cytokine production in DCs. In a mixed lymphocyte reactions assay, YM-2A-treated DCs increased proliferation and production of IFN-γ by allogeneic CD4+ and CD8+ T cells. These results indicate that YM-2A phenotypically and functionally activates DCs. Furthermore, YM-2A-treated TAA-loaded DC vaccine significantly reduced tumor growth and improved survival in two murine tumor models, CT-26 tumor-bearing BALB/c mice and B16 melanoma-bearing C57BL/6 mice. YM-2A-treated TAA-loaded DC vaccine increased splenic IFN-γ producing CD4+ and CD8+ T cells in CT-26 tumor-bearing BALB/c mice. Antibody neutralization studies indicated that YM-2A-induced DC maturation is mediated, in part, by the Dectin-1-dependent pathway. Overall, YM-2A-treatment with a TAA-loaded DC vaccine could be an excellent candidate for immunotherapy against cancer.


Subject(s)
Cancer Vaccines/therapeutic use , Dendritic Cells/drug effects , Glucans/pharmacology , Grifola/chemistry , Neoplasms, Experimental/therapy , Animals , CD4-Positive T-Lymphocytes , CD8-Positive T-Lymphocytes , Dendritic Cells/immunology , Dendritic Cells/physiology , Female , Glucans/chemistry , Interferon-gamma/genetics , Interferon-gamma/metabolism , Lectins, C-Type/metabolism , Mice
5.
J Pharmacol Sci ; 131(1): 64-7, 2016 May.
Article in English | MEDLINE | ID: mdl-26883455

ABSTRACT

Pregabalin (PGB) is a valuable therapeutic drug against chronic pain. Here we attempted to perform the combinatorial drug therapy with P-glycoprotein (P-gp) inhibitors to lower therapeutic dosage of PGB in the intermittent cold stress-induced fibromyalgia-like pain model. Single intracerebroventricular (i.c.v.) PGB injection exerted long-lasting anti-hyperalgesic effects for 72 h, while the effect of PGB given intraperitoneally (i.p.) disappeared within 3 h. Importantly, the pretreatment with P-gp inhibitors markedly prolonged the PGB (i.p.) effects, which lasted for 72 h. These results suggest that the combinatorial treatment with P-gp inhibitor enables the prolongation of dose-interval for PGB.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , Analgesics/therapeutic use , Cyclosporins/therapeutic use , Fibromyalgia/drug therapy , Pregabalin/therapeutic use , Animals , Chronic Pain/drug therapy , Cold Temperature , Drug Therapy, Combination , Hyperalgesia/drug therapy , Male , Mice, Inbred C57BL , Neuralgia/drug therapy , Sciatic Nerve/surgery
6.
J Pharmacol Sci ; 128(4): 208-11, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26318673

ABSTRACT

Neuropathic pain is often insensitive to morphine. Our previous study has demonstrated that neuron-restrictive silencer factor represses mu opioid receptor (MOP) gene expression in the dorsal root ganglion (DRG) via histone hypoacetylation-mediated mechanisms after peripheral nerve injury, thereby causing loss of peripheral morphine analgesia. Here, we showed that histone deacetylase (HDAC) inhibitors, such as trichostatin A and valproic acid, restored peripheral and systemic morphine analgesia in neuropathic pain. Also, these agents blocked nerve injury-induced MOP down-regulation in the DRG. These results suggest that HDAC inhibitors could serve as adjuvant analgesics to morphine for the management of neuropathic pain.


Subject(s)
Analgesics , Down-Regulation/drug effects , Drug Resistance/drug effects , Gene Expression/drug effects , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylase Inhibitors/therapeutic use , Morphine/pharmacology , Morphine/therapeutic use , Neuralgia/drug therapy , Neuralgia/etiology , Peripheral Nerve Injuries/complications , Receptors, Opioid, mu/genetics , Valproic Acid/pharmacology , Valproic Acid/therapeutic use , Acetylation , Analgesia , Animals , Ganglia, Spinal/metabolism , Histone Deacetylases/metabolism , Histone Deacetylases/physiology , Histones/metabolism , Hydroxamic Acids , Male , Mice, Inbred C57BL , Receptors, Opioid, mu/metabolism
7.
J Pharmacol Exp Ther ; 353(3): 471-9, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25805256

ABSTRACT

Treatment of fibromyalgia is an unmet medical need. To develop novel therapies for the treatment of fibromyalgia, we explored pain therapeutic actions of existing pharmaceuticals, which inhibit the somatic symptoms frequently observed in fibromyalgia patients. This study first examined the therapeutic actions of pilocarpine, which inhibits dry-eye and dry-mouth symptoms, using an experimental fibromyalgia-like chronic pain model produced by intermittent cold stress (ICS) in mice. A single intraperitoneal and intracerebroventricular, but not intrathecal, pilocarpine administration attenuated ICS-induced thermal hyperalgesia and mechanical allodynia, and this action was abolished by muscarinic antagonist pirenzepine (i.c.v.). Treatment with 1-10 µg/kg donepezil (i.p.), which can easily penetrate into the brain, also showed similar therapeutic effects. Importantly, we found that both pilocarpine and donepezil produced antihyperalgesic effects via supraspinal action. Furthermore, repeated donepezil treatments completely cured the ICS-induced hyperalgesia and allodynia even after the cessation of drug treatments. Acute and chronic treatments of these cholinomimetics had no effects on the nociceptive threshold in control animals. By contrast, the lack of morphine (i.c.v.) analgesia initially observed in the ICS model remained in ICS model mice treated with long-term donepezil. Collectively, these findings suggest that stimulation of the muscarinic cholinergic system effectively inhibits some mechanisms underlying chronic pain in the ICS model, but does not inhibit the lack of descending pain-inhibitory mechanisms, which are driven by central morphine.


Subject(s)
Chronic Pain/drug therapy , Chronic Pain/etiology , Fibromyalgia/drug therapy , Indans/therapeutic use , Nootropic Agents/therapeutic use , Piperidines/therapeutic use , Stress, Psychological/complications , Analgesics, Opioid/therapeutic use , Animals , Donepezil , Hyperalgesia/drug therapy , Hyperalgesia/etiology , Indans/administration & dosage , Injections, Intraventricular , Male , Mice , Mice, Inbred C57BL , Morphine/therapeutic use , Muscarinic Agonists/therapeutic use , Nootropic Agents/administration & dosage , Pain Measurement/drug effects , Pilocarpine/therapeutic use , Piperidines/administration & dosage
8.
Br J Pharmacol ; 170(5): 991-8, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24032674

ABSTRACT

BACKGROUND AND PURPOSE: Hypoesthesia is a clinical feature of neuropathic pain. The feature is partly explained by the evidence of epigenetic repression of Nav 1.8 sodium channel in the dorsal root ganglion (DRG). EXPERIMENTAL APPROACH: We investigated the possibility of trichostatin A (TSA), valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) to reverse the unique C-fibre sensitivity observed following partial ligation of sciatic nerve in mice. KEY RESULTS: Nerve injury-induced down-regulation of DRG Nav 1.8 sodium channel and C-fibre-related hypoesthesia were reversed by TSA, VPA and SAHA treatments, which inhibit histone deacetylase (HDAC), and increase histone acetylation at the regulatory sequence of Nav 1.8. CONCLUSIONS AND IMPLICATIONS: Taken together, these studies provide the evidence that hypoesthesia and underlying down-regulation of Nav 1.8, negative symptoms observed in nerve injury-induced neuropathic pain models are regulated by an epigenetic chromatin remodelling through HDAC-related machineries.


Subject(s)
Analgesics/pharmacology , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Hypesthesia/drug therapy , Nerve Fibers, Unmyelinated/drug effects , Pain Threshold/drug effects , Sciatic Nerve/drug effects , Sciatic Neuropathy/drug therapy , Acetylation , Animals , Chromatin Assembly and Disassembly/drug effects , Disease Models, Animal , Epigenesis, Genetic/drug effects , Ganglia, Spinal/drug effects , Ganglia, Spinal/enzymology , Ganglia, Spinal/physiopathology , Histones/metabolism , Hydroxamic Acids/pharmacology , Hypesthesia/enzymology , Hypesthesia/genetics , Hypesthesia/physiopathology , Ligation , Male , Mice , Mice, Inbred C57BL , NAV1.8 Voltage-Gated Sodium Channel/drug effects , NAV1.8 Voltage-Gated Sodium Channel/genetics , NAV1.8 Voltage-Gated Sodium Channel/metabolism , Nerve Fibers, Unmyelinated/enzymology , Pain Measurement , Sciatic Nerve/physiopathology , Sciatic Nerve/surgery , Sciatic Neuropathy/enzymology , Sciatic Neuropathy/genetics , Sciatic Neuropathy/physiopathology , Time Factors , Valproic Acid/pharmacology , Vorinostat
9.
Curr Pharm Des ; 19(42): 7355-61, 2013.
Article in English | MEDLINE | ID: mdl-23448475

ABSTRACT

Herein, we investigated the role of periaqueductal gray (PAG)-resident microglia in the development of morphine tolerance and its underlying mechanisms. We showed that clodronate and minocycline known as microglia inhibitors reversed morphine tolerance, providing proof that microglia activation has key role in the development of morphine tolerance. The microglia-mediated anti-opioid mechanism occurs via sequential BDNF release and NMDA expression. Experimental evidence is provided here as conditional bdnf knockout mice (bdnf⁻/⁻) failed to develop tolerance following Cre-recombinase adenovirus treatment. Increased BDNF expression followed microglia activation in acute minocycline treatment reversible manner. Following BDNF release, NR2A subunit of NMDA receptor was upregulated in anti-BDNF reversible manner showing the contribution of BDNF signaling in the control of NMDA receptor expression following chronic morphine treatment. Our data provide compelling evidence that microglia activation and BDNF release are key regulators in opioid tolerance mechanism via glutaminergic synapse plasticity.


Subject(s)
Analgesics, Opioid/pharmacology , Brain-Derived Neurotrophic Factor/metabolism , Microglia/drug effects , Morphine/pharmacology , Receptors, N-Methyl-D-Aspartate/metabolism , Animals , Blotting, Western , Male , Mice , Mice, Inbred C57BL , Phagocytosis/drug effects
10.
Mol Pain ; 7: 69, 2011 Sep 21.
Article in English | MEDLINE | ID: mdl-21933442

ABSTRACT

BACKGROUND: Fibromyalgia (FM) is characterized by chronic widespread pain, which is often refractory to conventional painkillers. Numerous clinical studies have demonstrated that antidepressants are effective in treating FM pain. We previously established a mouse model of FM-like pain, induced by intermittent cold stress (ICS). RESULTS: In this study, we find that ICS exposure causes a transient increase in plasma corticosterone concentration, but not in anxiety or depression-like behaviors. A single intrathecal injection of an antidepressant, such as milnacipran, amitriptyline, mianserin or paroxetine, had an acute analgesic effect on ICS-induced thermal hyperalgesia at post-stress day 1 in a dose-dependent manner. In addition, repeated daily antidepressant treatments during post-stress days 1-5 gradually reversed the reduction in thermal pain threshold, and this recovery was maintained for at least 7 days after the final treatment. In addition, relief from mechanical allodynia, induced by ICS exposure, was also observed at day 9 after the cessation of antidepressant treatment. In contrast, the intravenous administration of these antidepressants at conventional doses failed to provide relief. CONCLUSIONS: These results suggest that the repetitive intrathecal administration of antidepressants permanently cures ICS-induced FM pain in mice.


Subject(s)
Antidepressive Agents/therapeutic use , Cold Temperature , Fibromyalgia/complications , Fibromyalgia/drug therapy , Pain/complications , Pain/drug therapy , Stress, Physiological , Analgesics/administration & dosage , Analgesics/pharmacology , Analgesics/therapeutic use , Animals , Antidepressive Agents/administration & dosage , Antidepressive Agents/pharmacology , Anxiety/blood , Anxiety/complications , Behavior, Animal/drug effects , Body Weight/drug effects , Corticosterone/blood , Depression/blood , Depression/complications , Fibromyalgia/blood , Hyperalgesia/blood , Hyperalgesia/complications , Hyperalgesia/drug therapy , Injections, Spinal , Male , Mice , Mice, Inbred C57BL , Pain/blood , Stress, Physiological/drug effects
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