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Introduction: Idiopathic epilepsy (IE) and meningoencephalomyelitis of unknown origin (MUO) are common causes of brain diseases leading to seizures in dogs. In this study, the concentrations of 196 lipid metabolites and nitrogen oxide (NO) production in the cerebrospinal fluid (CSF) and plasma of dogs with MUO or IE were measured using a LC-MS/MS and a NOx analyzer, respectively. Methods: Nine clinically healthy dogs and 11 and 12 dogs with IE and MUO, respectively, were included in the study. Results: Lipid analysis revealed variations in the levels of four and six lipid metabolites in CSF and plasma, respectively, between the groups. The levels of 6-keto-prostaglandin (PG) F1α (PGF1α), 20-carboxy arachidonic acid (20-carboxy-AA), 9-hydroxyoctadecadienoic acid, and lyso-platelet-activating factor were high in the CSF of dogs with MUO. In addition, the plasma levels of 11,12-dihydroxyeicosatrienoic acid, 20-carboxy-AA, and oleoylethanolamide were high in dogs with IE, and those of PGF1α were high in dogs with MUO. NO production levels were high in CSF but not in plasma in dogs with MUO or IE. Discussion: It remains unknown whether these changes represent the cause or effect of diseases of the central nervous system; however, lipid metabolites and NO production in CSF and plasma may be used as diagnostic biomarkers and could be exploited for treating idiopathic or inflammatory epilepsy in dogs.
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Idiopathic normal pressure hydrocephalus in elderly people is considered a form of glymphopathy caused by malfunction of the waste clearance pathway, called the glymphatic system. Tau is a representative waste material similar to amyloid-ß. During neurodegeneration, lipocalin-type prostaglandin D synthase (L-PGDS), a major cerebrospinal fluid (CSF) protein, is reported to act as a chaperone that prevents the neurotoxic aggregation of amyloid-ß. L-PGDS is also a CSF biomarker in idiopathic normal pressure hydrocephalus and significantly correlates with tau concentration, age, and age-related brain white matter changes detected by magnetic resonance imaging. To investigate this glymphopathy, we aimed to analyze white matter changes and contributing factors in vivo and their interactions ex vivo. Cerebrospinal tap tests were performed in 60 patients referred for symptomatic ventriculomegaly. Patients were evaluated using an idiopathic normal pressure hydrocephalus grading scale, mini-mental state examination, frontal assessment battery, and timed up-and-go test. The typical morphological features of high convexity tightness and ventriculomegaly were measured using the callosal angle and Evans index, and parenchymal white matter properties were evaluated with diffusion tensor imaging followed by tract-based spatial statistics. Levels of CSF biomarkers, including tau, amyloid-ß, and L-PGDS, were determined by ELISA, and their interaction, and localization were determined using immunoprecipitation and immunohistochemical analyses. Tract-based spatial statistics for fractional anisotropy revealed clusters that positively correlated with mini-mental state examination, frontal assessment battery, and callosal angle, and clusters that negatively correlated with age, disease duration, idiopathic normal pressure hydrocephalus grading scale, Evans index, and L-PGDS. Other parameters also indicated clusters that correlated with symptoms, microstructural white matter changes, and L-PGDS. Tau co-precipitated with L-PGDS, and colocalization was confirmed in postmortem specimens of neurodegenerative disease obtained from the human Brain Bank. Our study supports the diagnostic value of L-PGDS as a surrogate marker for white matter integrity in idiopathic normal pressure hydrocephalus. These results increase our understanding of the molecular players in the glymphatic system. Moreover, this study indicates the potential utility of enhancing endogenous protective factors to maintain brain homeostasis.
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BACKGROUND: Nasal congestion in allergic rhinitis (AR) is caused by vascular hyperpermeability and vascular relaxation of the nasal mucosa. We previously detected high levels of a lipoxygenation metabolite of dihomogammalinolenic acid, 15-hydroxy-8Z,11Z,13E-eicosatrienoic acid (15-HETrE) in the nasal lavage fluid of AR model mice. Here, we investigated the effects of 15-HETrE on vascular functions associated with nasal congestion. METHODS: We measured 15-HETrE levels in the nasal lavage fluid of ovalbumin-induced AR model mice and nasal discharge of patients with AR. We also assessed nasal congestion and vascular relaxation in mice. Vascular contractility was investigated using isolated mouse aortas. RESULTS: Five ovalbumin challenges increased 15-HETrE levels in AR model mice. 15-HETrE was also detected in patients who exhibiting AR-related symptoms. Intranasal administration of 15-HETrE elicited dyspnea-related behavior and decreased the nasal cavity volume in mice. Miles assay and whole-mount immunostaining revealed that 15-HETrE administration caused vascular hyperpermeability and relaxation of the nasal mucosa. Intravital imaging demonstrated that 15-HETrE relaxed the ear vessels that were precontracted via thromboxane receptor stimulation. Moreover, 15-HETrE dilated the isolated mouse aortas, and this effect was attenuated by K+ channel inhibitors and prostaglandin D2 (DP) and prostacyclin (IP) receptor antagonists. Additionally, vasodilatory effects of 15-HETrE were accompanied by an increase in intracellular cAMP levels. CONCLUSIONS: Our results indicate that 15-HETrE, whose levels are elevated in the nasal cavity upon AR, can be a novel lipid mediator that exacerbates nasal congestion. Moreover, it can stimulate DP and IP receptors and downstream K+ channels to dilate the nasal mucosal vasculature.
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Modelos Animais de Doenças , Rinite Alérgica , Animais , Camundongos , Rinite Alérgica/metabolismo , Humanos , Masculino , Mucosa Nasal/metabolismo , Mucosa Nasal/efeitos dos fármacos , Mucosa Nasal/irrigação sanguínea , Ácidos Hidroxieicosatetraenoicos/metabolismo , Feminino , Obstrução Nasal/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Ovalbumina , Vasodilatação/efeitos dos fármacos , Líquido da Lavagem NasalRESUMO
Background: Allergic conjunctivitis (AC) is a common ophthalmologic disorder that causes symptoms that often reduces a patient's quality of life (QOL). We investigated the effects of the eicosapentaenoic acid metabolite (±)5(6)-dihydroxy-8Z,11Z,14Z,17Z-eicosatetraenoic acid ((±)5(6)-DiHETE) on AC using a mouse model. Methods: BALB/c mice were sensitized with two injections of short ragweed pollen in alum, challenged fifth with pollen in eyedrops. The clinical signs and tear volume were evaluated at 15 min after the final challenge. Histamine-induced ocular inflammation model was prepared by instilling histamine onto the surface of the eye. Fifteen minutes after histamine application, tear volume was measured using the Schirmer tear test. Miles assay was performed to investigate vascular permeability. To cause scratching behavior 10 µg of serotonin was injected in the cheek. Results: Repeated topical application of pollen induced conjunctivitis, accompanied by eyelid edema and tearing in mice. Pollen application typically degranulates mast cells and recruits eosinophils to the conjunctiva. Intraperitoneal administration of 300 µg/kg of (±)5(6)-DiHETE significantly inhibited pollen-induced symptoms. The administration of (±)5(6)-DiHETE also attenuated mast cell degranulation and eosinophil infiltration into the conjunctiva. To assess the effects of (±)5(6)-DiHETE on the downstream pathway of mast cell activation in AC, we used a histamine-induced ocular inflammation model. Topical application of 4 µg/eye histamine caused eyelid edema and tearing and increased vascular permeability, as indicated by Evans blue dye extravasation. Intraperitoneal administration of 300 µg/kg or topical administration of 1 µg/eye (±)5(6)-DiHETE inhibited histamine-induced manifestations. Finally, we assessed the effects of (±)5(6)-DiHETE on itching. An intradermal injection of 10 µg serotonin in the cheek caused scratching behavior in mice. Intraperitoneal administration of 300 µg/kg (±)5(6)-DiHETE significantly inhibited serotonin-induced scratching. Conclusion: Thus, (±)5(6)-DiHETE treatment broadly suppressed AC pathology and could be a novel treatment option for AC.
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Normal angiogenesis is essential for retinal development and maintenance of visual function in the eye, and its abnormality can cause retinopathy and other eye diseases. Prostaglandin D2 is an anti-angiogenic lipid mediator produced by lipocalin-type PGD synthase (L-PGDS) or hematopoietic PGD synthase (H-PGDS). However, the exact role of these PGD synthases remains unclear. Therefore, we compared the roles of these synthases in murine retinal angiogenesis under physiological and pathological conditions. On postnatal day (P) 8, the WT murine retina was covered with an elongated vessel. L-PGDS deficiency, but not H-PGDS, reduced the physiological vessel elongation with sprouts increase. L-PGDS expression was observed in endothelial cells and neural cells. In vitro, L-PGDS inhibition increased the hypoxia-induced vascular endothelial growth factor expression in isolated endothelial cells, inhibited by a prostaglandin D2 metabolite, 15-deoxy-Δ12,14 -PGJ2 (15d-PGJ2) treatment. Pericyte depletion, using antiplatelet-derived growth factor receptor-ß antibody, caused retinal hemorrhage with vessel elongation impairment and macrophage infiltration in the WT P8 retina. H-PGDS deficiency promoted hemorrhage but inhibited the impairment of vessel elongation, while L-PGDS did not. In the pericyte-depleted WT retina, H-PGDS was expressed in the infiltrated macrophages. Deficiency of the D prostanoid receptor also inhibited the vessel elongation impairment. These results suggest the endogenous role of L-PGDS signaling in physiological angiogenesis and that of H-PGDS/D prostanoid 1 signaling in pathological angiogenesis.
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Introduction: Conjunctivitis is a major ocular disease classified into allergic or infectious. The pathological features of conjunctivitis are not fully understood despite its high morbidity rate; thus, its differentiation can be difficult. Materials and methods: We used ovalbumin-induced allergic conjunctivitis and lipopolysaccharide-induced infectious conjunctivitis models of guinea pigs. Both models showed conjunctival swelling. Histological studies revealed that numerous eosinophils infiltrated the conjunctiva in the allergic model, whereas neutrophils infiltrated the conjunctiva in the infectious model. We collected conjunctival lavage fluid (COLF) and comprehensively analyzed lipid production using liquid chromatography-tandem mass spectrometry. Results: COLF showed increase of 20 and 12 lipid species levels in the allergic and infectious models, respectively. Specifically, the levels of a major allergic mediator, prostaglandin D2 and its three metabolites and several cytochrome P450-catalyzed lipids increased in the allergic model. In the infectious model, the levels of prostaglandin E2 and 8-iso-prostaglandin E2 increased, indicating tissue inflammation. Moreover, the level of 12-oxo-eicosatetraenoic acid, a lipoxygenase metabolite, increased in the infectious model. Conclusion: These differences in lipid production in the COLF reflected the pathological features of allergic and infectious conjunctivitis.
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Atopic dermatitis (AD) is the most common inflammatory skin disease in children. The serum level of thymus and activation-regulated chemokine (TARC) is a useful AD index to reflect disease severity; however, it requires blood collection from young children. In comparison, urine samples are easier to collect in a pediatric clinical setting. Here, we analyzed the lipids excreted in urine to identify a diagnostic biomarker for AD. We generated a murine dermatitis model by repeated topical application of 2,4-dinitrofluorobenzene (DNFB) or tape-stripping the dorsal skin. Lipid metabolites excreted in the urine were comprehensively analyzed using liquid chromatography-tandem mass spectrometry. To corroborate our findings, we also analyzed urine samples from patients with AD. DNFB application induced AD-like skin lesions, including epidermal thickening, infiltration of eosinophils and T cells, and an increase in Th2 cytokine levels. Assessment of lipids excreted in urine showed a dominance of prostaglandins (PGs), namely, a PGF2α metabolite (13,14-dihydro-15-keto-tetranor-PGF1α ), a PGE2 metabolite (13,14-dihydro-15-keto-tetranor-PGE2 ), and a PGD2 metabolite (13,14-dihydro-15-keto PGJ2 ). mRNA and protein expression of PGF2α , PGE2 , and PGD2 synthase was upregulated in DNFB-treated skin. The tape-stripping model also caused dermatitis but without Th2 inflammation; urine PGF2α and PGD2 metabolite levels remained unaffected. Finally, we confirmed that the urinary levels of the aforementioned PG metabolites, as well as PGI2 metabolite, 6,15-diketo-13,14-dihydro-PGF1α and arachidonic acid metabolite, 17-hydroxyeicosatetraenoic acid (17-HETE) increased in patients with AD. Our data highlights the unique urinary lipid profile in patients with AD, which may provide insight into novel urinary biomarkers for AD diagnosis.
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Dermatite Atópica/diagnóstico , Dermatite Atópica/urina , Prostaglandinas/urina , Índice de Gravidade de Doença , Administração Cutânea , Animais , Biomarcadores/urina , Criança , Pré-Escolar , Cromatografia Líquida/métodos , Dermatite Atópica/induzido quimicamente , Dinitrofluorbenzeno/administração & dosagem , Dinitrofluorbenzeno/efeitos adversos , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pele/efeitos dos fármacos , Pele/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Tetranor-PGDM is a metabolite of PGD2. Urinary tetranor-PGDM levels were reported to be increased in some diseases, including food allergy, Duchenne muscular dystrophy, and aspirin-intolerant asthma. In this study, we developed a monoclonal antibody (MAb) and a competitive enzyme immunoassay (EIA) for measuring tetranor-PGDM. Spleen cells isolated from mice immunized with tetranor-PGDM were utilized to generate Ab-producing hybridomas. We chose hybridomas and purified MAb against tetranor-PGDM to develop competitive EIA. The assay evaluated the optimal ionic strength, pH, precision, and reliability. Specificity was determined by cross-reactivity to tetranor-PGEM, tetranor-PGFM, and tetranor-PGAM. Recovery was determined by spiking experiments on artificial urine. Optimal ionic strength was 150 mM NaCl, and optimal pH was pH 7.5. Metabolites other than tetranor-PGDM did not show any significant cross-reactivity in the EIA. The assay exhibited a half-maximal inhibition concentration (IC50) of 1.79 ng/mL, limit of detection (LOD) of 0.0498 ng/mL, and range of quantitation (ROQ) value of 0.252 to 20.2 ng/mL. The intra- and inter-assay variation for tetranor-PGDM was 3.9-6.0% and 5.7-10.4%, respectively. The linearity-dilution effect showed excellent linearity under dilution when artificial urine samples were applied to solid-phase extraction (SPE). After SPE, recovery of tetranor-PGDM in artificial urine averaged from 82.3% to 113.5% and was within acceptable limits (80%-120%). We successfully generated one monoclonal antibody and developed a sensitive competitive EIA. The established EIA would be useful for routine detection and monitoring of tetranor-PGDM in research or diagnostic body fluids.
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Anticorpos Monoclonais/imunologia , Técnicas Imunoenzimáticas/métodos , Prostaglandina D2/análogos & derivados , Animais , Anticorpos Monoclonais/isolamento & purificação , Feminino , Camundongos , Modelos Animais , Prostaglandina D2/imunologia , Prostaglandina D2/metabolismo , Prostaglandina D2/urina , Reprodutibilidade dos TestesRESUMO
5,6-dihydroxy-8Z,11Z,14Z,17Z-eicosatetraenoic acid (5,6-DiHETE) is an eicosapentaenoic acid-derived lipid metabolite, which we previously detected in inflamed mouse colon. In this study, we investigated the pathophysiological roles of 5,6-DiHETE in murine colitis and its underlying mechanisms of action, focusing on the effects on transient receptor potential vanilloid (TRPV) channel activity. Oral administration of dextran sodium sulfate (DSS, 2%, for 4 days) caused colon inflammation, which peaked on day 7 and gradually declined by day 18. 5,6-DiHETE concentration in colon tissue was significantly increased during the healing phase of colitis (days 9 to 18). In vitro study showed that pretreatment with 5,6-DiHETE (0.1-1 µM, 30 minutes) significantly inhibited endothelial barrier disruption induced by a TRPV4 agonist (GSK1016790A, 50 nM). Intracellular Ca2+ imaging also showed that pretreatment with 5,6-DiHETE (1 µM, 10 minutes) reduced GSK1016790A-induced intracellular Ca2+ increase in HEK293T cells overexpressing TRPV4. In vivo, intraperitoneal administration of 5,6-DiHETE (50 µg kg-1 day-1 ) during the healing phase accelerated the recovery from DSS-induced colitis. Pathological studies showed that the administration of 5,6-DiHETE inhibited edema formation and leukocyte infiltration in inflamed colon tissue. In conclusion, we identified 5,6-DiHETE as a novel endogenous TRPV4 antagonist, and we also demonstrated that its administration promotes the healing of colitis by inhibiting inflammatory responses.
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Ácidos Araquidônicos/farmacologia , Colite/induzido quimicamente , Regulação da Expressão Gênica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , Animais , Colite/tratamento farmacológico , Sulfato de Dextrana/toxicidade , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Canais de Cátion TRPV/genéticaRESUMO
BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is characterized by eosinophilic rhinosinusitis, nasal polyposis, aspirin sensitivity, and asthma. Aims/Objectives: This study aims to identify a mechanism to target for the future treatment of AERD via the elucidation of the effect of systemic steroids on the expression of hematopoietic prostaglandin D2 synthase (HPGDS) and chemotaxic prostaglandin D2 (DP2) receptor relative to eosinophil activation in the nasal polyps of patients with AERD. MATERIALS AND METHODS: Among 37 patients undergoing endoscopic sinus surgery, 28 received systemic steroids preoperatively. Nasal polyps were harvested from all 37 patients. After routine processing of paraffin sections, immunohistochemistry was performed using specific antibodies for HPGDS, eosinophil peroxidase (EPX), and DP2. RESULTS: Expression of HPGDS, DP2, and EPX by eosinophils was higher and more frequent in patients with non-preoperative steroid therapy. Likewise, HPGDS and DP2 were highly expressed in activated eosinophils in the nasal polyps, but not in normal eosinophils. CONCLUSION AND SIGNIFICANCE: This study provides clear evidence that systemic steroid therapy inhibits eosinophil activation and decreases HPGDS and DP2 expression in patients with AERD, indicating a reduction in prostaglandin D2 production and hence control hyperplasia of nasal polyps.
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Corticosteroides/uso terapêutico , Asma Induzida por Aspirina/tratamento farmacológico , Eosinófilos/efeitos dos fármacos , Oxirredutases Intramoleculares/metabolismo , Pólipos Nasais/tratamento farmacológico , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Adulto , Idoso , Asma Induzida por Aspirina/metabolismo , Inibição de Migração Celular , Inibidores de Ciclo-Oxigenase/efeitos adversos , Regulação para Baixo/efeitos dos fármacos , Peroxidase de Eosinófilo/metabolismo , Eosinófilos/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pólipos Nasais/metabolismoRESUMO
Sleep is affected by the environment. In rodents, changes in the amount of rapid eye movement sleep (REMS) can precede those of other sleep/wake stages. The molecular mechanism underlying the dynamic regulation of REMS remains poorly understood. Here, we focused on the sublaterodorsal nucleus (SLD), located in the pontine tegmental area, which plays a crucial role in the regulation of REMS. We searched for genes selectively expressed in the SLD and identified copine-7 (Cpne7), whose involvement in sleep was totally unknown. We generated Cpne7-Cre knock-in mice, which enabled both the knockout (KO) of Cpne7 and the genetic labeling of Cpne7-expressing cells. While Cpne7-KO mice exhibited normal sleep under basal conditions, the amount of REMS in Cpne7-KO mice was larger compared to wildtype mice following cage change or water immersion and restraint stress, both of which are conditions that acutely reduce REMS. Thus, it was suggested that copine-7 is involved in negatively regulating REMS under certain conditions. In addition, chemogenetically activating Cpne7-expressing neurons in the SLD reduced the amount of REMS, suggesting that these neurons negatively regulate REMS. These results identify copine-7 and Cpne7-expressing neurons in the SLD as candidate molecular or neuronal components of the regulatory system that controls REMS.
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Sono REM , Água , Animais , Proteínas de Transporte , Imersão , Camundongos , SonoRESUMO
The molecular mechanism regulating sleep largely remains to be elucidated. In humans, families that carry mutations in TFAP2B, which encodes the transcription factor AP-2ß, self-reported sleep abnormalities such as short-sleep and parasomnia. Notably, AP-2 transcription factors play essential roles in sleep regulation in the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster Thus, AP-2 transcription factors might have a conserved role in sleep regulation across the animal phyla. However, direct evidence supporting the involvement of TFAP2B in mammalian sleep was lacking. In this study, by using the CRISPR/Cas9 technology, we generated two Tfap2b mutant mouse strains, Tfap2bK144 and Tfap2bK145 , each harboring a single-nucleotide mutation within the introns of Tfap2b mimicking the mutations in two human kindreds that self-reported sleep abnormalities. The effects of these mutations were compared with those of a Tfap2b knockout allele (Tfap2b-). The protein expression level of TFAP2B in the embryonic brain was reduced to about half in Tfap2b+/- mice and was further reduced in Tfap2b-/- mice. By contrast, the protein expression level was normal in Tfap2bK145/+ mice but was reduced in Tfap2bK145/K145 mice to a similar extent as Tfap2b-/- mice. Tfap2bK144/+ and Tfap2bK144/K144 showed normal protein expression levels. Tfap2b+/- female mice showed increased wakefulness time and decreased nonrapid eye movement sleep (NREMS) time. By contrast, Tfap2bK145/+ female mice showed an apparently normal amount of sleep but instead exhibited fragmented NREMS, whereas Tfap2bK144/+ male mice showed reduced NREMS time specifically in the dark phase. Finally, in the adult brain, Tfap2b-LacZ expression was detected in the superior colliculus, locus coeruleus, cerebellum, and the nucleus of solitary tract. These findings provide direct evidence that TFAP2B influences NREMS amounts in mice and also show that different mutations in Tfap2b can lead to diverse effects on sleep architecture.
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Fases do Sono , Fator de Transcrição AP-2/genética , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Feminino , Íntrons , Masculino , Camundongos , Mutação Puntual , Fator de Transcrição AP-2/metabolismoRESUMO
Marmoset wasting syndrome (MWS) is clinically characterized by progressive weight loss. Although morbidity and mortality of MWS are relatively high in captive marmosets, its causes remain unknown. Lipid mediators are bioactive metabolites which are produced from polyunsaturated fatty acids, such as arachidonic acid (AA) and eicosapentaenoic acid. These lipid metabolites regulate a wide range of inflammatory responses and they are excreted into the urine. As urinary lipid profiles reflect systemic inflammatory conditions, we comprehensively measured the levels of 141 types of lipid metabolites in the urines obtained from healthy common marmoset (Callithrix jacchus) (N = 7) or marmosets with MWS (N = 7). We found that 41 types of metabolites were detected in all urine samples of both groups. Among them, AA-derived metabolites accounted for 63% (26/41 types) of all detected metabolites. Notably, the levels of AA-derived prostaglandin (PG) E2, PGF2α, thromboxane (TX) B2 and F2-isoprostanes significantly increased in the urine samples of marmosets with MWS. In this study, we found some urinary lipid metabolites which may be involved in the development of MWS. Although the cause of MWS remains unclear, our findings may provide some insight into understanding the mechanisms of development of MWS.
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Callithrix/metabolismo , Callithrix/urina , Lipídeos/urina , Metaboloma , Doenças dos Macacos/urina , Síndrome de Emaciação/urina , Síndrome de Emaciação/veterinária , Animais , Peso Corporal , Ácidos Graxos Insaturados/urina , Redes e Vias Metabólicas , Oxirredução , Síndrome de Emaciação/metabolismoAssuntos
Hipersensibilidade Alimentar/imunologia , Receptores de Prostaglandina/imunologia , Animais , Modelos Animais de Doenças , Feminino , Hidantoínas/farmacologia , Intestinos/imunologia , Mastócitos/imunologia , Camundongos Endogâmicos BALB C , Receptores de Prostaglandina/agonistas , Receptores de Prostaglandina/antagonistas & inibidoresRESUMO
Although prostaglandins (PGs) are known to be involved in the progression of arthritis, the role of PGD2 remains unclear. In this study, we evaluated the role of PGD2 in joint inflammation using genetically modified mice. Injection of complete Freund's adjuvant (CFA) increased the production of PGD2 and induced paw swelling and cartilage erosion in wild-type (WT) mice. These phenomena were accompanied with an increase in the mRNA levels of TNF-α, IL-6, IL-1ß, and matrix-degrading metalloproteinase-9. Knockdown of hematopoietic PGD synthase (H-PGDS) abolished the PGD2 production and exacerbated all of the arthritic manifestations in the inflamed paw. Immunostaining revealed that infiltrating macrophages strongly expressed H-PGDS in the CFA-injected paw. Morphologic studies revealed vascular hyperpermeability and angiogenesis in the inflamed WT paw. H-PGDS deficiency was accelerated, whereas daily administration of a PGD2 receptor D prostanoid (DP) agonist attenuated the CFA-induced hyperpermeability and angiogenesis. We further confirmed that DP deficiency exacerbated, whereas the administration of the DP agonist improved, the CFA-induced arthritic manifestations. The findings demonstrate that H-PGDS-derived PGD2 ameliorates joint inflammation by attenuating vascular permeability and subsequent angiogenesis and indicates the therapeutic potential of a DP agonist for arthritis.-Tsubosaka, Y., Maehara, T., Imai, D., Nakamura, T., Kobayashi, K., Nagata, N., Fujii, W., Murata, T. Hematopoietic prostaglandin D synthase-derived prostaglandin D2 ameliorates adjuvant-induced joint inflammation in mice.
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Artrite Experimental/prevenção & controle , Inflamação/prevenção & controle , Oxirredutases Intramoleculares/fisiologia , Artropatias/prevenção & controle , Neovascularização Patológica/prevenção & controle , Prostaglandina D2/farmacologia , Adjuvantes Imunológicos/toxicidade , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Permeabilidade Capilar , Colágeno/toxicidade , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Artropatias/induzido quimicamente , Artropatias/metabolismo , Artropatias/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/induzido quimicamente , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologiaRESUMO
Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is responsible for the production of PGD2 in adipocytes and is selectively induced by a high-fat diet (HFD) in adipose tissue. In this study, we investigated the effects of HFD on obesity and insulin resistance in two distinct types of adipose-specific L-PGDS gene knockout (KO) mice: fatty acid binding protein 4 (fabp4, aP2)-Cre/L-PGDS flox/flox and adiponectin (AdipoQ)-Cre/L-PGDS flox/flox mice. The L-PGDS gene was deleted in adipocytes in the premature stage of the former strain and after maturation of the latter strain. The L-PGDS expression and PGD2 production levels decreased in white adipose tissue (WAT) under HFD conditions only in the aP2-Cre/L-PGDS flox/flox mice, but were unchanged in the AdipoQ-Cre/L-PGDS flox/flox mice. When fed an HFD, aP2-Cre/L-PGDS flox/flox mice significantly reduced body weight gain, adipocyte size, and serum cholesterol and triglyceride levels. In WAT of the HFD-fed aP2-Cre/L-PGDS flox/flox mice, the expression levels of the adipogenic, lipogenic, and M1 macrophage marker genes were decreased, whereas those of the lipolytic and M2 macrophage marker genes were enhanced or unchanged. Insulin sensitivity was improved in the HFD-fed aP2-Cre/L-PGDS flox/flox mice. These results indicate that PGD2 produced by L-PGDS in premature adipocytes is involved in the regulation of body weight gain and insulin resistance under nutrient-dense conditions.
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Adipócitos/metabolismo , Resistência à Insulina , Oxirredutases Intramoleculares/metabolismo , Lipocalinas/metabolismo , Obesidade/metabolismo , Prostaglandina D2/biossíntese , Adipócitos/patologia , Animais , Gorduras na Dieta/efeitos adversos , Gorduras na Dieta/farmacologia , Oxirredutases Intramoleculares/genética , Lipocalinas/genética , Camundongos , Camundongos Transgênicos , Obesidade/induzido quimicamente , Obesidade/genética , Obesidade/patologia , Prostaglandina D2/genéticaRESUMO
Acute lung injury (ALI) is caused by various stimuli such as acid aspiration and infection, resulting in severe clinical outcomes with high mortality. Prostaglandin D2 (PGD2 ) is a lipid mediator produced in the lungs of patients with ALI. There are two prostaglandin D synthases (PGDS), namely, lipocalin-type PGDS (L-PGDS) and hematopoietic PGDS (H-PGDS). We previously reported the anti-inflammatory role of H-PGDS-derived PGD2 in an endotoxin-induced murine ALI model. Therefore, in this study, we investigated the role of L-PGDS-derived PGD2 in ALI in comparison to H-PGDS-derived PGD2 . Intratracheal administration of HCl caused lung inflammation accompanied by tissue edema and neutrophil accumulation in mouse lungs. The deficiency of both L-PGDS and H-PGDS exacerbated HCl-induced lung dysfunction to a similar extent. Furthermore, a detailed investigation revealed that L-PGDS-derived PGD2 inhibited lung edema, while H-PGDS-derived PGD2 inhibited neutrophil infiltration. Immunostaining showed that inflamed endothelial/epithelial cells express L-PGDS, while macrophages and neutrophils express H-PGDS. Hematopoietic reconstitution with WT bone marrow did not rescue the exacerbated lung edema in L-PGDS deficient mice, indicating the importance of nonhematopoietic endothelial/epithelial cell-expressing L-PGDS for protection against ALI. A modified Miles assay showed that L-PGDS deficiency accelerated vascular hyper-permeability in the inflamed lung, which was suppressed by the stimulation of D prostanoid (DP) receptor, a PGD2 receptor. In vitro, DP agonism enhanced the barrier function of endothelial cells but not epithelial cells. Taken together, our results suggest that in the HCl-induced murine ALI model PGD2 was produced locally by inflamed endothelial and epithelial L-PGDS and this enhanced the endothelial barrier through the DP receptor. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Lesão Pulmonar Aguda/patologia , Células Endoteliais/metabolismo , Pneumonia/patologia , Prostaglandina D2/metabolismo , Animais , Permeabilidade Capilar/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Neutrófilos/patologiaRESUMO
In diabetic retinopathy (DR), pericyte dropout from capillary walls is believed to cause the breakdown of the blood-retina barrier (BRB), which subsequently leads to vision-threatening retinal edema. While various proinflammatory cytokines and chemokines are upregulated in eyes with DR, their distinct contributions to disease progression remain elusive. Here, we evaluated roles of stromal cell-derived factor-1α (SDF-1α) and its receptor CXCR4 in the BRB breakdown initiated by pericyte deficiency. After inhibition of pericyte recruitment to developing retinal vessels in neonatal mice, endothelial cells (ECs) upregulated the expression of SDF-1α. Administration of CXCR4 antagonists, or EC-specific disruption of the CXCR4 gene, similarly restored the BRB integrity, even in the absence of pericyte coverage. Furthermore, CXCR4 inhibition significantly decreased both the expression levels of proinflammatory genes (P < 0.05) and the infiltration of macrophages (P < 0.05) into pericyte-deficient retinas. Taken together, EC-derived SDF-1α induced by pericyte deficiency exacerbated inflammation through CXCR4 in an autocrine or paracrine manner and thereby induced macrophage infiltration and BRB breakdown. These findings suggest that the SDF-1α/CXCR4 signaling pathway may be a potential therapeutic target in DR.
Assuntos
Barreira Hematorretiniana/metabolismo , Quimiocina CXCL12/metabolismo , Retinopatia Diabética/metabolismo , Pericitos/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais , Animais , Quimiocina CXCL12/genética , Quimiocinas , Citocinas/metabolismo , Retinopatia Diabética/terapia , Progressão da Doença , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Receptores CXCR4/genética , Retina/diagnóstico por imagem , Retina/crescimento & desenvolvimento , Retina/patologia , Vasos Retinianos/crescimento & desenvolvimentoRESUMO
Prostaglandin D2 (PGD2) is a lipid mediator involved in sleep regulation and inflammation. PGD2 interacts with 2 types of G protein-coupled receptors, DP1 and DP2/CRTH2 (chemoattractant receptor homologous molecule expressed on T helper type 2 cells)/GPR44 to show a variety of biological effects. DP1 activation leads to Gs-mediated elevation of the intracellular cAMP level, whereas activation of DP2 decreases this level via the Gi pathway; and it also induces G protein-independent, arrestin-mediated cellular responses. Activation of DP2 by PGD2 causes the progression of inflammation via the recruitment of lymphocytes by enhancing the production of Th2-cytokines. Here we developed monoclonal antibodies (MAbs) against the extracellular domain of mouse DP2 by immunization of DP2-null mutant mice with DP2-overexpressing BAF3, murine interleukin-3 dependent pro-B cells, to reduce the generation of antibodies against the host cells by immunization of mice. Moreover, we immunized DP2-KO mice to prevent immunological tolerance to mDP2 protein. After cell ELISA, immunocytochemical, and Western blot analyses, we successfully obtained a novel monoclonal antibody, MAb-1D8, that specifically recognized native mouse DP2, but neither human DP2 nor denatured mouse DP2, by binding to a particular 3D receptor conformation formed by the N-terminus and extracellular loop 1, 2, and 3 of DP2. This antibody inhibited the binding of 0.5 nM [3H]PGD2 to mouse DP2 (IC50 = 46.3 ± 18.6 nM), showed antagonistic activity toward 15(R)-15-methyl PGD2-induced inhibition of 300 nM forskolin-activated cAMP production (IC50 = 16.9 ± 2.6 nM), and gave positive results for immunohistochemical staining of DP2-expressing CD4+ Th2 lymphocytes that had accumulated in the kidney of unilateral ureteral obstruction model mice. This monoclonal antibody will be very useful for in vitro and in vivo studies on DP2-mediated diseases.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Receptores Imunológicos/imunologia , Receptores de Prostaglandina/imunologia , Animais , Anticorpos Monoclonais/química , Especificidade de Anticorpos , Linfócitos T CD4-Positivos/metabolismo , Células CHO , Células COS , Cricetulus , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Mapeamento de Epitopos , Células HEK293 , Humanos , Hibridomas/metabolismo , Imunização , Imuno-Histoquímica , Rim/metabolismo , Rim/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Precursoras de Linfócitos B/imunologia , Prostaglandina D2/análogos & derivados , Prostaglandina D2/antagonistas & inibidores , Receptores Imunológicos/genética , Receptores de Prostaglandina/genética , Obstrução Ureteral/imunologia , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , beta-Arrestinas/metabolismoRESUMO
Rapid eye movement (REM) sleep loss is associated with increased consumption of weight-promoting foods. The prefrontal cortex (PFC) is thought to mediate reward anticipation. However, the precise role of the PFC in mediating reward responses to highly palatable foods (HPF) after REM sleep deprivation is unclear. We selectively reduced REM sleep in mice over a 25-48 hr period and chemogenetically inhibited the medial PFC (mPFC) by using an altered glutamate-gated and ivermectin-gated chloride channel that facilitated neuronal inhibition through hyperpolarizing infected neurons. HPF consumption was measured while the mPFC was inactivated and REM sleep loss was induced. We found that REM sleep loss increased HPF consumption compared to control animals. However, mPFC inactivation reversed the effect of REM sleep loss on sucrose consumption without affecting fat consumption. Our findings provide, for the first time, a causal link between REM sleep, mPFC function and HPF consumption.