Restructuring the Gut Microbiota by Intermittent Fasting Lowers Blood Pressure.

[Figure: see text].

Lipid Receptor GPR31 (G-Protein-Coupled Receptor 31) Regulates Platelet Reactivity and Thrombosis Without Affecting Hemostasis.

OBJECTIVE: 12-LOX (12-lipoxygenase) produces a number of bioactive lipids including 12(S)-HETE that are involved in inflammation and platelet reactivity. The GPR31 (G-protein-coupled receptor 31) is the proposed receptor of 12(S)-HETE; however, it is not known whether the 12(S)-HETE-GPR31 signaling axis serves to enhance or inhibit platelet activity. Approach and Results: Using pepducin technology and biochemical approaches, we provide evidence that 12(S)-HETE-GPR31 signals through Gi to enhance PAR (protease-activated receptor)-4-mediated platelet activation and arterial thrombosis using both human platelets and mouse carotid artery injury models. 12(S)-HETE suppressed AC (adenylyl cyclase) activity through GPR31 and resulted in Rap1 (Ras-related protein 1) and p38 activation and low but detectable calcium flux but did not induce platelet aggregation. A GPR31 third intracellular (i3) loop-derived pepducin, GPR310 (G-protein-coupled receptor 310), significantly inhibited platelet aggregation in response to thrombin, collagen, and PAR4 agonist, AYPGKF, in human and mouse platelets but relative sparing of PAR1 agonist SFLLRN in human platelets. GPR310 treatment gave a highly significant 80% protection (P=0.0018) against ferric chloride-induced carotid artery injury in mice by extending occlusion time, without any effect on tail bleeding. PAR4-mediated dense granule secretion and calcium flux were both attenuated by GPR310. Consistent with these results, GPR310 inhibited 12(S)-HETE-mediated and PAR4-mediated Rap1-GTP and RASA3 translocation to the plasma membrane and attenuated PAR4-Akt and ERK activation. GPR310 caused a right shift in thrombin-mediated human platelet aggregation, comparable to the effects of inhibition of the Gi-coupled P2Y12 receptor. Co-immunoprecipitation studies revealed that GPR31 and PAR4 form a heterodimeric complex in recombinant systems. CONCLUSIONS: The 12-LOX product 12(S)-HETE stimulates GPR31-Gi-signaling pathways, which enhance thrombin-PAR4 platelet activation and arterial thrombosis in human platelets and mouse models. Suppression of this bioactive lipid pathway, as exemplified by a GPR31 pepducin antagonist, may provide beneficial protective effects against platelet aggregation and arterial thrombosis with minimal effect on hemostasis.

Differential expression and analysis of extrachromosomal circular DNAs as serum biomarkers in lung adenocarcinoma.

BACKGROUND: Extrachromosomal circular DNAs (eccDNAs) increase the number of proto-oncogenes by enhancing oncogene expression to promote tumorigenesis. However, there are limited reports on differential eccDNA expression and analysis in lung cancer, especially in lung adenocarcinoma (LAD). METHODS: Three LAD and three corresponding NT tissues samples were used for eccDNA next-generation sequencing analysis, and an additional 20 were used for quantitative PCR (qPCR) evaluations. We further performed qPCR amplification using serum samples from LAD patients and healthy medical examiners. RESULTS: eccDNAs from LAD samples were mainly 200-1000 bp in length. Gene annotation analysis revealed that most eccDNAs were derived from chromosomes 1 and 2. The top-ten increased and top-ten decreased eccDNAs in LAD tissues were CircD-ARPC1B, CircD-ARPC1A, CircD-FAM49B, CircD-SDK1, CircD-KCNG1, CircD-POLR2F, CircD-SS18L1, CircD-SLC16A3, CircD-CSNK1D, CircD-KCTD1, and CircD-TMIGD2, CircD-PDIA5, CircD-VAV2, CircD-GATAD2A, CircD-CAB39L, CircD-KHDC1, CircD-FOXN3, CircD-SULT2B1, CircD-DPP9, and CircD-CSNK1D. qPCR demonstrated that the expression of CircD-DZRN3 was higher in LAD tissues than in normal lung tissues, whereas CircD-LGR6 and CircD-UMODL1 expression levels were lower in LAD than in normal lung tissues. Furthermore, the serum CircD-PDZRN3 level increased, while CircD-LGR6 decreased in LAD. Receiver operating characteristic (ROC) analysis showed that area under curve (AUC) of serum CircD-PDZRN3 (0.991), CircD-LGR6 (0.916) was higher than that of serum carcinoembryonic antigen (CEA) (0.825), CY211 (cytokeratin 19 fragment) (0.842), SCCA(squamous cell carcinoma antigen) (0.857) for the diagnosis of LAD. CONCLUSIONS: Our study first showed that several eccDNAs were aberrantly expressed in LAD, among which CircD-PDZRN3 and CircD-LGR6 clearly distinguished LAD patients from healthy controls, indicating their potential as biomarkers.

Deficiency of TGR5 exacerbates immune-mediated cholestatic hepatic injury by stabilizing the ß-catenin destruction complex.

Intrahepatic cholestasis induced by drug toxicity may cause cholestatic hepatic injury (CHI) leading to liver fibrosis and cirrhosis. The G protein-coupled bile acid receptor 1 (TGR5) is a membrane receptor with well-known roles in the regulation of glucose metabolism and energy homeostasis. However, the role and mechanism of TGR5 in the context of inflammation during CHI remains unclear. Wild-type (WT) and TGR5 knockout (TGR5-/-) mice with CHI induced by bile duct ligation (BDL) were involved in vivo, and WT and TGR5-/- bone marrow-derived macrophages (BMDMs) were used in vitro. TGR5 deficiency significantly exacerbated BDL-induced liver injury, inflammatory responses and hepatic fibrosis compared with WT mice in vivo. TGR5-/- macrophages were more susceptible to lipopolysaccharide (LPS) stimulation than WT macrophages. TGR5 activation by its ligand suppressed LPS-induced pro-inflammatory responses in WT but not TGR5-/- BMDMs. Notably, expression of ß-catenin was effectively inhibited by TGR5 deficiency. Furthermore, TGR5 directly interacted with Gsk3ß to repress the interaction between Gsk3ß and ß-catenin, thus disrupting the ß-catenin destruction complex. The pro-inflammatory nature of TGR5-knockout was almost abolished by lentivirus-mediated ß-catenin overexpression in BMDMs. BMDM migration in vitro was accelerated under TGR5-deficient conditions or supernatant from LPS-stimulated TGR5-/- BMDMs. From a therapeutic perspective, TGR5-/- BMDM administration aggravated BDL-induced CHI, which was effectively rescued by ß-catenin overexpression. Our findings reveal that TGR5 plays a crucial role as a novel regulator of immune-mediated CHI by destabilizing the ß-catenin destruction complex, with therapeutic implications for the management of human CHI.

A paper-based ELISA for rapid sensitive determination of anaphylaxis-related MRGPRX2 in human peripheral blood.

Mas-related G-protein-coupled receptor X2 (MRGPRX2) has recently been reported to be associated with anaphylaxis. Detection of MRGPRX2 levels in human peripheral blood might serve as a powerful tool for predicting the predisposition of patients to anaphylactic reactions. For rapid measurement of MRGPRX2, we established a paper-based double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antibody and horseradish peroxidase (HRP)-labelled rabbit polyclonal antibody as capture antibody and detection antibody, respectively. We avoided chemical functionalization of the cellulose paper by introducing bovine serum albumin (BSA) to provide COOH and NH2 groups for covalent immobilization of the capture antibody. Through amide condensation, a two-layer immobilization strategy was applied with BSA-BSA and BSA-capture antibody networks as the first and second layers, respectively. This strategy improved the quantity, activity and stability of the immobilized antibody. We then established a paper-based ELISA to detect MRGPRX2 in human peripheral blood. Our method is less laborious, easier to implement, and more cost-effective than conventional ELISA, while offering similar sensitivity, specificity, and accuracy. Therefore, it could serve as an innovative clinical point-of-care diagnostic tool, especially in areas that lack advanced clinical equipment.

Minimal Age-Related Alterations in Behavioral and Hematological Parameters in Trace Amine-Associated Receptor 1 (TAAR1) Knockout Mice.

Since the discovery in 2001, the G protein-coupled trace amine-associated receptor 1 (TAAR1) has become an important focus of research targeted on evaluation of its role in the central nervous system (CNS). Meanwhile, impact of TAAR1 in the peripheral organs is less investigated. Expression of TAAR1 was demonstrated in different peripheral tissues: pancreatic ß-cells, stomach, intestines, white blood cells (WBC), and thyroid. However, the role of TAAR1 in regulation of hematological parameters has not been investigated yet. In this study, we performed analysis of anxiety-related behaviors, a complete blood count (CBC), erythrocyte fragility, as well as FT3/FT4 thyroid hormones levels in adult and middle-aged TAAR1 knockout mice. Complete blood count analysis was performed on a Siemens Advia 2120i hematology analyzer and included more than 35 measured and calculated parameters. Erythrocyte fragility test evaluated spherocytosis pathologies of red blood cells (RBC). No significant alterations in essentially all these parameters were found in mice without TAAR1. However, comparative aging analysis has revealed a decreased neutrophils level in the middle-aged TAAR1 knockout mouse group. Minimal alterations in these parameters observed in TAAR1 knockout mice suggest that future TAAR1-based therapies should exert little hematological effect and thus will likely have a good safety profile.

Imbalanced serum levels of resolvin E1 (RvE1) and leukotriene B4 (LTB4) in patients with allergic rhinitis.

Timely and successful resolution of acute inflammation plays a crucial role in preventing the development of chronic airway inflammation in allergic rhinitis (AR). This study intends to assess the serum levels of pro-inflammatory leukotriene B4 (LTB4), anti-inflammatory mediators, including resolvin E1 (RvE1), RvD1, IL-10, and TGF-ß, besides mRNA expression level of G-protein coupled receptor 120 (GPR120) and peroxisome proliferator-activated receptor-γ (PPAR-γ) receptors in peripheral blood leukocytes of AR patients. Thirty-seven AR patients and thirty age- and gender-matched healthy subjects were enrolled in this study. The serum levels of LTB4, RvE1, RvD1, IL-10, and TGF-ß were measured using enzyme-linked immunosorbent assay (ELISA) technique, and the mRNA expression level of GPR120 and PPAR-γ was assessed by the real-time PCR method. The serum levels of RvE1 and LTB4 were significantly higher in patients with AR than in healthy subjects (P < 0.01 and P < 0.0001, respectively). However, a significantly lower ratio of RvE1 and RvD1 to LTB4 was found in patients with AR relative to healthy subjects (P < 0.05 and P < 0.0001, respectively). Likewise, the serum levels of both IL-10 and TGF-ß cytokines were significantly reduced in patients with AR compared to healthy subjects (P < 0.01 and P < 0.0001, respectively). Furthermore, the mRNA expression of PPAR-γ was significantly lower in patients with AR than in healthy subjects (P < 0.05). Our findings indicate that imbalanced pro-resolving lipid mediator RvE1 and pro-inflammatory LTB4 might contribute to the defective airway inflammation-resolution and subsequent progression toward chronic inflammation in AR patients.

Relationship between circulating levels of angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis and coronary heart disease.

As a counter-regulatory arm of the renin angiotensin system (RAS), the angiotensin-converting enzyme 2-angiotensin-(1-7)-MAS axis (ACE2-Ang-(1-7)-MAS axis) plays a protective role in cardiovascular diseases. However, the link between circulating levels of ACE2-Ang-(1-7)-Mas axis and coronary atherosclerosis in humans is not determined. The object of present study was to investigate the association of circulating levels of ACE2, Ang-(1-7) and Ang-(1-9) with coronary heart disease (CHD) defined by coronary angiography (CAG). 275 patients who were referred to CAG for the evaluation of suspected CHD were enrolled and divided into two groups: CHD group (diameter narrowing ≥ 50%, n = 218) and non-CHD group (diameter narrowing < 50%, n = 57). Circulating ACE2, Ang-(1-7) and Ang-(1-9) levels were detected by enzyme-linked immunosorbent assay (ELISA). In females, circulating ACE2 levels were higher in the CHD group than in the non-CHD group (5617.16 ± 5206.67 vs. 3124.06 ± 3005.36 pg/ml, P = 0.009), and subgroup analysis showed the significant differences in ACE2 levels between the two groups only exist in patients with multi-vessel lesions (P = 0.009). In multivariate logistic regression, compared with the people in the lowest ACE2 quartile, those in the highest quartile had an OR of 4.33 (95% CI 1.20-15.61) for the CHD (P for trend = 0.025), the OR was 5.94 (95% CI 1.08-32.51) for the third ACE2 quartile and 9.58 (95% CI 1.61-56.95) for the highest ACE2 quartile after adjusting for potential confounders (P for trend = 0.022). However, circulating Ang-(1-7) and Ang-(1-9) levels had no significant differences between the two groups. In males, there were no significant differences in the levels of ACE2-Ang-(1-7)-MAS axis between two groups. Together, circulating ACE2 levels, but not Ang-(1-7) and Ang-(1-9) levels, significantly increased in female CHD group when compared with non-CHD group, increased ACE2 was independently associated with CHD in female and in patients with multi-vessel lesions even after adjusting for the confounding factors, indicating that ACE2 may participate as a compensatory mechanism in CHD.

GPER-1 and sex-hormone levels in patients with otosclerosis.

OBJECTIVE: Otosclerosis is a widespread disease but the etiopathogenesis is still not fully understood. Hormonal factors especially estrogens are accused in recent years. The study aimed to evaluate the levels of G-protein associated membrane estrogen receptor-1 (GPER-1) and sex-hormones in patients with otosclerosis. SUBJECT AND METHODS: The study included 60 people (30 otosclerosis patients, 30 control group). Serum sex-hormone (estradiol, progesterone, prolactin and total testosterone) and GPER-1 levels were measured in otosclerosis patients and compared with the normal population. For the otosclerosis group, air conduction and bone conduction thresholds and air-bone gaps were viewed from audiograms and the relationships between hearing and GPER-1 or sex-hormone levels were also investigated. RESULTS: Sex-hormone levels were not different between the groups. GPER-1 level was significantly lower in the otosclerosis group [3.1353 (0.76-8.21) ng/mL] than the control group [5.4773 (0.96-20.31) ng/mL] (p =0.017). Differential diagnosis with ROC analysis for the GPER-1 level was also significant (p=0.017). GPER-1 level was significantly lower for the females than the males in the otosclerosis group (p=0.043). Serum estradiol, progesterone, and prolactin levels were significantly higher (p=0.02, p =0.029 and p=0.019 respectively) and the GPER-1 level was significantly lower (p= 0.04) in the female patients compared to the female controls. There was no statistically significant relationship between GPER-1 or sex-hormone levels and hearing parameters. CONCLUSION: GPER-1 level was lower in the otosclerosis patients compared to healthy volunteers and also lower in females than males in the patient group. Female sex-hormone levels were higher and GPER-1 level was lower in the female patient group than the female control group. Neither GPER-1 nor sex-hormone levels were not predictive of hearing levels. These findings indicate that sex-hormones especially estrogen and GPER-1 might have a potential role in the etiopathogenesis of otosclerosis. This is the first study in the literature that investigates the GPER-1 values in otosclerosis.

Involvement of obesity-associated upregulation of chemerin/chemokine-like receptor 1 in oxidative stress and apoptosis in ovaries and granulosa cells.

In this study, we evaluated the expression and function of chemerin and CMKLR1 in the ovaries and granulosa cells of high-fat diet-induced obese (OB) mice. In vivo, chemerin/CMKLR1 system was upregulated in the serum, ovaries, and granulosa cells of OB mice compared with those in control mice. Apoptotic ovarian follicles, oxidative stress, and apoptosis biomarkers were also increased in the ovaries of OB mice. In vitro, mouse granulosa cells (mGCs) were cultured and treated with different concentrations of chemerin to investigate the effects of chemerin on viability, reactive oxygen species (ROS), and apoptosis and on the phosphorylation of AKT, AMP-activated protein kinase α (AMPKα), and nuclear factor-κB p65. Chemerin suppressed mGC viability with or without gonadotrophin and induced ROS accumulation and apoptosis in mGCs. Moreover, AMPKα and p65 were activated by chemerin, whereas AKT was suppressed. These changes in phosphorylation were blocked with CMKLR1 knockdown. Our findings showed that chemerin contributed to ROS accumulation and apoptotic cell death through three signaling pathways, suggesting that upregulation of chemerin and CMKLR1 may explain the imbalance of oxidative stress and apoptosis in the ovaries of OB mice.

Specific induction of the unique GPR15 expression in heterogeneous blood lymphocytes by tobacco smoking.

Purpose: In the peripheral blood, it has been shown that smoking is, to date, the only specific condition leading to an increase in GPR15+ T cells. We, therefore, aimed to characterize GPR15-expressing blood T cells in more detail. Materials and Methods: The whole transcriptome by RNAseq as a proxy for protein expression was analyzed in GPR15+ and GPR15- T cells. A deep immuno-phenotyping was conducted for the identification of T cell subtypes. Results: The expression of GPR15 seemed to be unique, not concomitantly accompanied with the expression of another protein. According to different T cell subtypes, there is no single cell type prominently represented in GPR15+ T cells. The individually different proportions of GPR15+ cells among each GPR15-expressing T cell subtypes in blood were strongly associated with chronic smoking. Indeed, the frequency of GPR15+ T cell subtypes can be effectively used as a highly convincing biomarker for tobacco smoking. Conclusions: While the chronic smoking-induced enrichment of GPR15+ T cells in blood might indicate a systemic inflammation, by the widespread presence in different T cell subtypes, GPR15 could feature a general impact on maintaining the systemic homeostasis to putatively prevent harm from smoking.

Type 2 diabetes with hypertensive patients results in changes to features of adipocytokines: Leptin, Irisin, LGR4, and Sfrp5.

The incidence of hypertension and diabetes is increasing, it is reported that adipocytokines might be involved in the pathogenesis of diabetes and hypertension. We aimed to investigate the features of adipocytokines, included of Leptin, Irisin, LGR4, and Sfrp5 in type 2 diabetes mellitus (T2DM) patients with hypertension, simultaneously analyzed the connection of the alteration of adipocytokines with blood pressure and glucose. 424 patients with T2DM and 90 healthy subjects were included in the study. The patients with T2DM were divided into 4 groups based on the blood pressure. The levels of adipocytokines (Leptin, Irisin, LGR4, and Sfrp5) were determined by enzyme-linked immunosorbent assay (ELISA). Significantly higher levels of Leptin and lower levels of Irisin, LGR4 and Sfrp5 were seen in patients with diabetes compared with non-diabetes (P < 0.05), the mean values of Leptin level was ascending and Irisin, LGR4, and Sfrp5 levels were declining with promoting of blood pressure in hypertension as compared to the non-hypertension with diabetic patients. Multiple stepwise linear regression analysis showed that the concentrations of Leptin, Irisin, Sfrp5, and LGR4 were found to be closely associated with the control of blood pressure and glucose. Conclusion: Four adipocytokines might play different roles and closely relate to the occurrence and development of diabetes and hypertension.

Niacin Ameliorates Neuro-Inflammation in Parkinson's Disease via GPR109A.

In this study, we used macrophage RAW264.7 cells to elucidate the molecular mechanism underlying the anti-inflammatory actions of niacin. Anti-inflammatory actions of niacin and a possible role of its receptor GPR109A have been studied previously. However, the precise molecular mechanism of niacin's action in reducing inflammation through GPR109A is unknown. Here we observed that niacin reduced the translocation of phosphorylated nuclear kappa B (p-NF-κB) induced by lipopolysaccharide (LPS) in the nucleus of RAW264.7 cells. The reduction in the nuclear translocation in turn decreased the expression of pro-inflammatory cytokines IL-1ß, IL-6 in RAW264.7 cells. We observed a decrease in the nuclear translocation of p-NF-κB and the expression of inflammatory cytokines after knockdown of GPR109A in RAW264.7 cells. Our results suggest that these molecular actions of niacin are mediated via its receptor GPR109A (also known as HCAR2) by controlling the translocation of p-NF-κB to the nucleus. Overall, our findings suggest that niacin treatment may have potential in reducing inflammation by targeting GPR109A.

An evaluation of G-protein coupled membrane estrogen receptor-1 level in stuttering.

OBJECTIVE: Stuttering is a widespread but little understood disease. There has been a recent increase in neuropathophysiological, genetic, and biochemical studies related to the etiopathogenesis. As developmental stuttering continues in adult males, hormonal factors are thought to have an effect. In this study, an evaluation was made for the first time of serum GPER-1 level in patients with a stutter. STUDY DESIGN: Prospective case control. MATERIALS AND METHODS: The study included 30 patients with a stutter, aged < 18 years, and 35 age-matched children as the control group. The Stuttering Severity Instrument-3 form was administered to the patients. Evaluations were made of serum GPER-1, TSH, estradiol, prolactin, and progesterone and testosterone levels. RESULTS: GPER-1 level was determined as 0.51 (0.42-0.67) ng/mL in the patients and as 0.19 (0.13-0.25) ng/mL in the control group, and the difference was statistically significant (p < 0.001). A statistically significant difference was determined between genders with GPER-1 level of 0.56 (0.44-0.68) ng/mL in the male stuttering patient group and 0.44 (0.35-0.49) ng/mL in the female patient group (p = 0.026). Differential diagnosis with ROC analysis for the serum GPER-1 levels was statistically significant [Area under the ROC curve (AUC): 0.998, confidence interval, CI 0.992-1.000, p < 0.001]. CONCLUSION: The GPER-1 levels of the stuttering patients were found to be higher than those of the control group and GPER-1 levels of male patients were higher than those of females. As GPER-1 has high sensitivity and sensitivity, it could be considered important in the diagnosis and treatment of stuttering.

Human relaxins (RLNH1, RLNH2), their receptor (RXFP1) and fetoplacental growth.

Relaxin, a systemic and placental hormone, has potential roles in fetoplacental growth. Human placenta expresses two RLN genes, RLNH1 and RLNH2 Maternal obesity is common and is associated with abnormal fetal growth. Our aims were to relate systemic and cord blood RLNH2, placental RLNs and their receptor (RXFP1) with fetoplacental growth in context of maternal body mass index, and associations with insulin-like growth factor 2 (IGF2) and vascular endothelial growth factor A (VEGFA) in the same placentas. Systemic, cord blood and placental samples were collected prior to term labor, divided by prepregnancy body mass index: underweight/normal (N = 25) and overweight/obese (N = 44). Blood RLNH2 was measured by ELISA; placental RLNH2, RLNH1, RXFP1, IGF2 and VEGFA were measured by quantitative immunohistochemistry and mRNAs were measured by quantitative reverse transcription PCR. Birthweight increased with systemic RLNH2 only in underweight/normal women (P = 0.036). Syncytiotrophoblast RLNH2 was increased in overweight/obese patients (P = 0.017) and was associated with placental weight in all subjects (P = 0.038). RLNH1 had no associations with birthweight or placental weight, but was associated with increased trophoblast and endothelial IGF2 and VEGFA, due to female fetal sex. Thus, while systemic RLNH2 may be involved in birthweight regulation in underweight/normal women, placental RLNH2 in all subjects may be involved in placental weight. A strong association of trophoblast IGF2 with birthweight and placental weight in overweight/obese women suggests its importance. However, an association of only RLNH1 with placental IGF2 and VEGFA was dependent upon female fetal sex. These results suggest that both systemic and placental RLNs may be associated with fetoplacental growth.

Differential Roles of the NADPH-Oxidase 1 and 2 in Platelet Activation and Thrombosis.

OBJECTIVE: Reactive oxygen species (ROS) are known to regulate platelet activation; however, the mechanisms of ROS production during platelet activation remain unclear. Platelets express different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs). Here, we investigated the role of NOX1 and NOX2 in ROS generation and platelet activation using NOX1 and NOX2 knockout mice. APPROACH AND RESULTS: NOX1(-/Y) platelets showed selective defects in G-protein-coupled receptor-mediated platelet activation induced by thrombin and thromboxane A2 analog U46619, but were not affected in platelet activation induced by collagen-related peptide, a glycoprotein VI agonist. In contrast, NOX2(-/-) platelets showed potent inhibition of collagen-related peptide-induced platelet activation, and also showed partial inhibition of thrombin-induced platelet activation. Consistently, production of ROS was inhibited in NOX1(-/Y) platelets stimulated with thrombin, but not collagen-related peptide, whereas NOX2(-/-) platelets showed reduced ROS generation induced by collagen-related peptide or thrombin. Reduced ROS generation in NOX1/2-deficient platelets is associated with impaired activation of Syk and phospholipase Cγ2, but minimally affected mitogen-activated protein kinase pathways. Interestingly, laser-induced arterial thrombosis was impaired but the bleeding time was not affected in NOX2(-/-) mice. Wild-type thrombocytopenic mice injected with NOX2(-/-) platelets also showed defective arterial thrombosis, suggesting an important role for platelet NOX2 in thrombosis in vivo but not hemostasis. CONCLUSIONS: NOX1 and NOX2 play differential roles in different platelet activation pathways and in thrombosis. ROS generated by these enzymes promotes platelet activation via the Syk/phospholipase Cγ2/calcium signaling pathway.

Urotensinergic system genes in experimental subarachnoid hemorrhage.

OBJECTIVE: Cerebral vasospasm, one of the main complications of subarachnoid hemorrhage (SAH), is characterized by arterial constriction and mainly occurs from day 4 until the second week after the event. Urotensin-II (U-II) has been described as the most potent vasoconstrictor peptide in mammals. An analysis is made of the serum U-II concentrations and mRNA expression levels of U-II, urotensin related peptide (URP) and urotensin receptor (UT) genes in an experimental murine model of SAH. DESIGN: An experimental study was carried out. SETTING: Experimental operating room of the Biomedicine Institute of Seville (IBiS), Virgen del Rocío University Hospital (Seville, Spain). PARTICIPANTS: 96 Wistar rats: 74 SAH and 22 sham intervention animals. INTERVENTIONS: Day 1: blood sampling, followed by the percutaneous injection of 100µl saline (sham) or blood (SAH) into the subarachnoid space. Day 5: blood sampling, followed by sacrifice of the animals. MAIN VARIABLES OF INTEREST: Weight, early mortality, serum U-II levels, mRNA values for U-II, URP and UT. RESULTS: Serum U-II levels increased in the SAH group from day 1 (0.62pg/mL [IQR 0.36-1.08]) to day 5 (0.74pg/mL [IQR 0.39-1.43]) (p<0.05), though not in the sham group (0.56pg/mL [IQR 0.06-0.83] day 1; 0.37pg/mL [IQR 0.23-0.62] day 5; p=0.959). Between-group differences were found on day 5 (p<0.05). The ROC analysis showed that the day 5 serum U-II levels (AUC=0.691), URP mRNA (AUC=0.706) and UT mRNA (AUC=0.713) could discriminate between sham and SAH rats. The normal serum U-II concentration range in rats was 0.56pg/mL (IQR 0.06-0.83). CONCLUSION: The urotensinergic system is upregulated on day 5 in an experimental model of SAH.

Bile Acids Increase Independently From Hypocaloric Restriction After Bariatric Surgery.

OBJECTIVES: To measure changes in the composition of serum bile acids (BA) and the expression of Takeda G-protein-coupled receptor 5 (TGR5) acutely after bariatric surgery or caloric restriction. SUMMARY BACKGROUND DATA: Metabolic improvement after bariatric surgery occurs before substantial weight loss. BA are important metabolic regulators acting through the farnesoid X receptor and TGR5 receptor. The acute effects of surgery on BA and the TGR5 receptor in subcutaneous white adipose tissue (WAT) are unknown. METHODS: A total of 27 obese patients with type 2 diabetes mellitus were randomized to Roux-en-Y gastric bypass (RYGB) or to hypocaloric diet (HC diet) restriction (NCT 1882036). A cohort of obese patients with and without type 2 diabetes mellitus undergoing vertical sleeve gastrectomy was also recruited (n = 12) as a comparison. RESULTS: After vertical sleeve gastrectomy, the level of BA increased [total: 1.17 ±â€Š1.56 µmol/L to 4.42 ±â€Š3.92 µmol/L (P = 0.005); conjugated BA levels increased from 0.99 ±â€Š1.42 µmol/L to 3.59 ±â€Š3.70 µmol/L (P = 0.01) and unconjugated BA levels increased from 0.18 ±â€Š0.24 µmol/L to 0.83 ±â€Š0.70 µmol/L (P = 0.009)]. With RYGB, there was a trend toward increased BA [total: 1.37 ±â€Š0.97 µmol/L to 3.26 ±â€Š3.01 µmol/L (P = 0.07); conjugated: 1.06 ±â€Š0.81 µmol/L to 2.99 ±â€Š3.02 µmol/L (P = 0.06)]. After HC diet, the level of unconjugated BA decreased [0.92 ±â€Š0.55 µmol/L to 0.32 ± 0.43 µmol/L (P = 0.05)]. The level of WAT TGR5 gene expression decreased after surgery, but not in HC diet. Protein levels did not change. CONCLUSIONS: The levels of serum BA increase after bariatric surgery independently from caloric restriction, whereas the level of WAT TGR5 protein is unaffected.

Protein profiles of CCL5, HPGDS, and NPSR1 in plasma reveal association with childhood asthma.

Asthma is a common chronic childhood disease with many different phenotypes that need to be identified. We analyzed a broad range of plasma proteins in children with well-characterized asthma phenotypes to identify potential markers of childhood asthma. Using an affinity proteomics approach, plasma levels of 362 proteins covered by antibodies from the Human Protein Atlas were investigated in a total of 154 children with persistent or intermittent asthma and controls. After screening, chemokine ligand 5 (CCL5) hematopoietic prostaglandin D synthase (HPGDS) and neuropeptide S receptor 1 (NPSR1) were selected for further investigation. Significantly lower levels of both CCL5 and HPGDS were found in children with persistent asthma, while NPSR1 was found at higher levels in children with mild intermittent asthma compared to healthy controls. In addition, the protein levels were investigated in another respiratory disease, sarcoidosis, showing significantly higher NPSR1 levels in sera from sarcoidosis patients compared to healthy controls. Immunohistochemical staining of healthy tissues revealed high cytoplasmic expression of HPGDS in mast cells, present in stroma of both airway epithelia, lung as well as in other organs. High expression of NPSR1 was observed in neuroendocrine tissues, while no expression was observed in airway epithelia or lung. In conclusion, we have utilized a broad-scaled affinity proteomics approach to identify three proteins with altered plasma levels in asthmatic children, representing one of the first evaluations of HPGDS and NPSR1 protein levels in plasma.

Expression of CD11c and EMR2 on neutrophils: potential diagnostic biomarkers for sepsis and systemic inflammation.

There is a need for cellular biomarkers to differentiate patients with sepsis from those with the non-infectious systemic inflammatory response syndrome (SIRS). In this double-blind study we determined whether the expression of known (CD11a/b/c, CD62L) and putative adhesion molecules [CD64, CD97 and epidermal growth factor (EGF)-like molecule containing mucin-like hormone receptor (EMR2)] on blood neutrophils could serve as useful biomarkers of infection and of non-infectious SIRS in critically ill patients. We studied 103 patients with SIRS, 83 of whom had sepsis, and 50 healthy normal subjects, using flow cytometry to characterize neutrophils phenotypically in whole blood samples. Patients with SIRS had an increased prevalence of neutrophils expressing CD11c, CD64 and EMR2 in comparison with healthy subjects (P < 0.001), but normal expression of CD11a, CD11b, CD62L and CD97. An increase in the percentage of neutrophils bearing CD11c was associated with sepsis, EMR2 with SIRS and CD64 with sepsis and SIRS. Neutrophils expressing CD11c had the highest sensitivity (81%) and specificity (80%) for the detection of sepsis, and there was an association between the percentage of neutrophils expressing EMR2 and the extent of organ failure (P < 0.05). Contrary to other reports, we did not observe an abnormal expression of CD11b or CD62L on neutrophils from patients with SIRS, and suggest that this discrepancy is due to differences in cell processing protocols. We propose that blood neutrophils expressing CD11c and EMR2 be considered as potential biomarkers for sepsis and SIRS, respectively.