15-Hydroxyeicosatrienoic acid induces nasal congestion by changing vascular functions in mice.

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.

Roles of lipocalin-type and hematopoietic prostaglandin D synthases in mouse retinal angiogenesis.

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.

5,6-dihydroxy-8Z,11Z,14Z,17Z-eicosatetraenoic acid accelerates the healing of colitis by inhibiting transient receptor potential vanilloid 4-mediated signaling.

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.

Urinary lipid profile of atopic dermatitis in murine model and human patients.

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.

Hematopoietic prostaglandin D synthase-derived prostaglandin D2 ameliorates adjuvant-induced joint inflammation in mice.

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.

L-PGDS-derived PGD2 attenuates acute lung injury by enhancing endothelial barrier formation.

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.

Lipid metabolites and nitric oxide production in the cerebrospinal fluid and plasma of dogs with meningoencephalitis of unknown origin and idiopathic epilepsy: a pilot study.

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.

Lipocalin-type prostaglandin D synthase: a glymphopathy marker in idiopathic hydrocephalus.

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.

PGD2-CRTH2 pathway promotes tubulointerstitial fibrosis.

Urinary excretion of lipocalin-type PGD(2) synthase (L-PGDS), which converts PG H(2) to PGD(2), increases in early diabetic nephropathy. In addition, L-PGDS expression in the tubular epithelium increases in adriamycin-induced nephropathy, suggesting that locally produced L-PGDS may promote the development of CKD. In this study, we found that L-PGDS-derived PGD(2) contributes to the progression of renal fibrosis via CRTH2-mediated activation of Th2 lymphocytes. In a mouse model, the tubular epithelium synthesized L-PGDS de novo after unilateral ureteral obstruction (UUO). L-PGDS-knockout mice and CRTH2-knockout mice both exhibited less renal fibrosis, reduced infiltration of Th2 lymphocytes into the cortex, and decreased production of the Th2 cytokines IL-4 and IL-13. Furthermore, oral administration of a CRTH2 antagonist, beginning 3 days after UUO, suppressed the progression of renal fibrosis. Ablation of IL-4 and IL-13 also ameliorated renal fibrosis in the UUO kidney. Taken together, these data suggest that blocking the activation of CRTH2 by PGD(2) might be a strategy to slow the progression of renal fibrosis in CKD.

Production profile of lipid mediators in conjunctival lavage fluid in allergic and infectious conjunctivitis in guinea pigs.

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.

Alleviation of allergic conjunctivitis by (±)5(6)-dihydroxy-8Z,11Z,14Z,17Z-eicosatetraenoic acid in mice.

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.

[Endogenous sleep-promoting substance].

Prostaglandin (PG) D2 is the endogenous somnogen that accumulates in the brain during prolonged wakefulness. PGD2 is produced by lipocalin-type PGD synthase localized in the leptomeninges, choroid plexus and oligodendrocytes, and circulates in the cerebrospinal fluid as a sleep hormone. PGD2 stimulates DP1 receptors and increases the local extracellular concentration of adenosine in the basal forebrain as a paracrine sleep-promoting molecule. Adenosine activates sleep-active neurons in the ventrolateral preoptic area (VLPO) through adenosine A2A receptors. Sleep-promoting neurons in the VLPO send inhibitory signals to suppress the histaminergic neurons in the tuberomammillary nucleus, which contribute to arousal through histamine H1 receptors. The neural network between VLPO and TMN is considered to play a key role in the regulation of sleep.

Suppression of adipocyte differentiation by aldo-keto reductase 1B3 acting as prostaglandin F2alpha synthase.

Prostaglandin (PG) F(2alpha) suppresses adipocyte differentiation by inhibiting the function of peroxisome proliferator-activated receptor gamma. However, PGF(2alpha) synthase (PGFS) in adipocytes remains to be identified. Here, we studied the expression of members of the aldo-keto reductase (AKR) 1B family acting as PGFS during adipogenesis of mouse 3T3-L1 cells. AKR1B3 mRNA was expressed in preadipocytes, and its level increased about 4-fold at day 1 after initiation of adipocyte differentiation, and then quickly decreased the following day to a level lower than that in the preadipocytes. In contrast, the mRNA levels of Akr1b8 and 1b10 were clearly lower than that level of Akr1b3 in preadipocytes and remained unchanged during adipogenesis. The transient increase in Akr1b3 during adipogenesis was also observed by Western blot analysis. The mRNA for the FP receptor, which is selective for PGF(2alpha), was also expressed in preadipocytes. Its level increased about 2-fold within 1 h after the initiation of adipocyte differentiation and was maintained at almost the same level throughout adipocyte differentiation. The small interfering RNA for Akr1b3, but not for Akr1b8 or 1b10, suppressed PGF(2alpha) production and enhanced the expression of adipogenic genes such as peroxisome proliferator-activated receptor gamma, fatty acid-binding protein 4 (aP2), and stearoyl-CoA desaturase. Moreover, an FP receptor agonist, Fluprostenol, suppressed the expression of those adipogenic genes in 3T3-L1 cells; whereas an FP receptor antagonist, AL-8810, efficiently inhibited the suppression of adipogenesis caused by the endogenous PGF(2alpha). These results indicate that AKR1B3 acts as the PGFS in adipocytes and that AKR1B3-produced PGF(2alpha) suppressed adipocyte differentiation by acting through FP receptors.

Increased expression of lipocalin-type-prostaglandin D synthase in ulcerative colitis and exacerbating role in murine colitis.

The pathogenesis of ulcerative colitis (UC) is unclear, but enhancement of disease activity by usage of nonsteroidal anti-inflammatory drugs suggests involvement of prostanoid in its pathophysiology. However, biological effect of prostaglandin (PG) D(2) on intestinal inflammation remains unknown. We investigated the expression of enzymes for PGD(2) synthesis, prostaglandin D synthase (PGDS), and its relation to the activity of colitis in UC patients. The role of lipocalin-type PGDS (L-PGDS) using a murine colitis model was also assessed. Tissue samples were obtained by colonic biopsies from patients with UC. Expression levels of mRNAs for L-PGDS and hematopoietic-type PGDS were investigated by quantitative RT-PCR. COX-2 and L-PGDS expression was investigated by immunohistochemistry. Localization of L-PGDS expression was also determined by in situ hybridization. In experimental study, mice were treated with dextran sodium sulfate in the drinking water to induce colitis. The degree of colonic inflammation was compared with L-PGDS(-/-) mice and control mice. The level of L-PGDS mRNA expression was increased in UC patients in parallel with disease activity. Colocalization of L-PGDS and cyclooxygenase (COX) 2 was observed in lamina proprial infiltrating cells and muscularis mucosa in UC patients. The level of hematopoietic PGDS mRNA expression did not differ from control mucosa. Dextran sodium sulfate treatment to L-PGDS(-/-) mice showed lower disease activity than control mice. We reported for the first time the presence of L-PGDS in the COX-2-expressing cells in the mucosa of active UC patients and that only L-PGDS increased with disease activity. An animal model study suggests that PGD(2) derived from L-PGDS-expressing cells plays proinflammatory roles in colitis.

Crystallization and preliminary X-ray diffraction analysis of mouse prostaglandin F2α synthase, AKR1B3.

Aldo-keto reductase 1B3 (AKR1B3) catalyzes the NADPH-dependent reduction of prostaglandin H(2) (PGH(2)), which is a common intermediate of various prostanoids, to form PGF(2α). AKR1B3 also reduces PGH(2) to PGD(2) in the absence of NADPH. AKR1B3 produced in Escherichia coli was crystallized in complex with NADPH by the sitting-drop vapour-diffusion method. The crystal was tetragonal, belonging to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 107.62, c = 120.76 Å. X-ray diffraction data were collected to 2.4 Å resolution at 100 K using a synchrotron-radiation source.

Development of Monoclonal Antibody-Based EIA for Tetranor-PGDM which Reflects PGD2 Production in the Body.

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.

Copine-7 is required for REM sleep regulation following cage change or water immersion and restraint stress in mice.

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.

Steroids Inhibit Eosinophil Accumulation and Downregulate Hematopoietic Chemotaxic Prostaglandin D2 Receptor in Aspirin-Exacerbated Respiratory Disease.

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.

Inhibition of prostaglandin D synthase suppresses muscular necrosis.

Duchenne muscular dystrophy is a fatal muscle wasting disease that is characterized by a deficiency in the protein dystrophin. Previously, we reported that the expression of hematopoietic prostaglandin D synthase (HPGDS) appeared in necrotic muscle fibers from patients with either Duchenne muscular dystrophy or polymyositis. HPGDS is responsible for the production of the inflammatory mediator, prostaglandin D(2). In this paper, we validated the hypothesis that HPGDS has a role in the etiology of muscular necrosis. We investigated the expression of HPGDS/ prostaglandin D(2) signaling using two different mouse models of muscle necrosis, that is, bupivacaine-induced muscle necrosis and the mdx mouse, which has a genetic muscular dystrophy. We treated each mouse model with the HPGDS-specific inhibitor, HQL-79, and measured both necrotic muscle volume and selected cytokine mRNA levels. We confirmed that HPGDS expression was induced in necrotic muscle fibers in both bupivacaine-injected muscle and mdx mice. After administration of HQL-79, necrotic muscle volume was significantly decreased in both mouse models. Additionally, mRNA levels of both CD11b and transforming growth factor beta1 were significantly lower in HQL-79-treated mdx mice than in vehicle-treated animals. We also demonstrated that HQL-79 suppressed prostaglandin D(2) production and improved muscle strength in the mdx mouse. Our results show that HPGDS augments inflammation, which is followed by muscle injury. Furthermore, the inhibition of HPGDS ameliorates muscle necrosis even in cases of genetic muscular dystrophy.