Assessment of Fraction of Exhaled Nitric Oxide and Soluble Receptor for Advanced Glycation End Products Biomarkers for Jordanian Asthmatic Children.

Purpose: Fraction of exhaled nitric oxide (FeNO) and soluble advanced glycation end-product receptor (sRAGE) are proposed as biomarkers of asthma, therefore we sought to assess their use in asthmatic children of Jordan. Patients and Methods: We conducted a case-control study at The University of Jordan Hospital. A total of 141 asthmatic children followed by respiratory pediatricians and 118 healthy children aged 4-18 years were recruited. FeNO was measured by NObreath device and serum sRAGE by ELISA that detect endogenously soluble isoform (esRAGE) and total soluble RAGE (sRAGE). Results: sRAGE in asthmatic was half of the control (p <0.001). In addition, ratio of esRAGE/sRAGE was two-fold higher in asthmatic (p = <0.001). Neither FeNO nor esRAGE levels were significantly different between groups. FeNO and asthma control test (ACT) score were negatively correlated corrected for age and body mass index (BMI), (r = -0.180, p= 0.034). For the uncontrolled asthma group, esRAGE/sRAGE negatively correlated with ACT score (r = -.329, p = 0.038). Receiver operating curve (ROC) analysis revealed significant predictive value (PV) for sRAGE and esRAGE/sRAGE in asthma detection with area under the curve (AUC) of (0.751 ± 0.031) and (0.711±.033), consequently. However, no biomarker had a significant PV for lack of control. Conclusion: The current study supports utilizing sRAGE as a marker for asthma and present a potential therapeutic target. However, our results indicate that both FeNO and sRAGE have a limited role in the management of asthmatic children or assessment of asthma control.

Methylglyoxal-Modified Albumin Effects on Endothelial Arginase Enzyme and Vascular Function.

Advanced glycation end products (AGEs) contribute significantly to vascular dysfunction (VD) in diabetes. Decreased nitric oxide (NO) is a hallmark in VD. In endothelial cells, NO is produced by endothelial NO synthase (eNOS) from L-arginine. Arginase competes with NOS for L-arginine to produce urea and ornithine, limiting NO production. Arginase upregulation was reported in hyperglycemia; however, AGEs' role in arginase regulation is unknown. Here, we investigated the effects of methylglyoxal-modified albumin (MGA) on arginase activity and protein expression in mouse aortic endothelial cells (MAEC) and on vascular function in mice aortas. Exposure of MAEC to MGA increased arginase activity, which was abrogated by MEK/ERK1/2 inhibitor, p38 MAPK inhibitor, and ABH (arginase inhibitor). Immunodetection of arginase revealed MGA-induced protein expression for arginase I. In aortic rings, MGA pretreatment impaired acetylcholine (ACh)-induced vasorelaxation, which was reversed by ABH. Intracellular NO detection by DAF-2DA revealed blunted ACh-induced NO production with MGA treatment that was reversed by ABH. In conclusion, AGEs increase arginase activity probably through the ERK1/2/p38 MAPK pathway due to increased arginase I expression. Furthermore, AGEs impair vascular function that can be reversed by arginase inhibition. Therefore, AGEs may be pivotal in arginase deleterious effects in diabetic VD, providing a novel therapeutic target.

COVID-19-associated mental health impact on menstruation physiology: A survey study among medical students in Jordan.

BACKGROUND: The coronavirus disease 2019 pandemic has been an extraordinarily stressful situation in recent years. Stress is a physiological reaction to negative stimuli that is regulated by different neuroendocrine pathways. The female reproductive function is maintained by the menstrual cycle, which is negatively affected by hyperstimulation of stress signals. OBJECTIVES: This study evaluates the effect of the coronavirus disease 2019 outbreak on menstrual function and mental health, exploring the relationship between them. DESIGN: The current study uses a cross-sectional, survey-based design. METHODS: During this cross-sectional study, an online self-completion questionnaire was conducted among a sample of 385 Jordanian female medical students during the pandemic. The survey compared menstrual characteristics, depression, anxiety, and stress 10 months after the coronavirus disease 2019 pandemic with 10 months prior. Paired t-test, McNemar's test, Pearson's correlation, and multiple linear regression model were employed to analyze data using SPSS software. RESULTS: The mean age of female medical student respondents was 19.89 years. Data showed that the menstrual cycle length significantly increased during the coronavirus disease 2019 pandemic compared with 10 months prior (32.23 days versus 30.02 days, p = 0.019). The average number of heavy bleeding days also increased during the coronavirus disease 2019 pandemic (2.82 days versus 2.42 days, p = 0.002). The proportion of females with heavy bleeding amount was more than doubled during the pandemic of coronavirus disease 2019 compared with before (27.3% versus 10.4%, p = 0.000). Unpleasant menstrual signs such as nausea and/or vomiting, breast pain, and urinary urgency were significantly increased during the pandemic (p = 0.000, p = 0.008, and p = 0.024, respectively). During coronavirus disease 2019, a positive association between total Depression, Anxiety, and Stress Scale-21 Questionnaire score and heavy bleeding was identified (p < 0.05). The findings also indicated that mental disorders and the incidence of amenorrhea, nausea and/or vomiting, and urinary urgency were positively correlated during the coronavirus disease 2019 pandemic. The multiple regression analysis revealed associations between several menstrual characteristics such as amenorrhea and severity of bleeding with coronavirus disease 2019-related depression, anxiety, and stress. CONCLUSION: This study revealed that the stress related to the pandemic of coronavirus disease 2019 could affect the female menstrual cycle and hence the quality of women's life. Therefore, this study could serve as a baseline for planning and introducing stress mitigation interventions in crisis situations to improve the physiological and mental well-being of females and improve their quality of life.

Assessment of Proteolysis by Pyrylium and Other Fluorogenic Reagents.

AIMS: We aim to evaluate the potential application of amine reactive fluorogenic reagents for estimating enzymatic proteolysis. BACKGROUND: Proteolytic enzymes play important roles in regulating many physiological processes in living organisms. OBJECTIVES: Assessment of protein degradation by using reagents for protein assay techniques. METHODS: We have assayed samples at the start and after 30-60 minutes incubation with trypsin by Chromeo P503 (Py 1 pyrylium compound) and CBQCA (3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde) as amine reactive reagents and NanoOrange as non-amine reactive dye. RESULTS: All BSA prepared samples with trypsin have shown significantly higher fluorescence intensity (FI) versus controls (which reflects proteolysis) when assayed by Chromeo P503 (Py 1 pyrylium compound) and CBQCA (3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde) as amine reactive reagents. However, same samples assayed with NanoOrange as non-amine reactive reagent did not show any significant variation between samples containing trypsin and controls. CONCLUSION: These results are confirming reliability of highly sensitive protein assays utilizing amine reactive fluorogenic reagents for general estimation of proteolysis.

Vitamin D deficiency in children with bronchial asthma in southern Jordan: a cross-sectional study.

OBJECTIVES: To assess serum 25-hydroxycholecalciferol (25-OH vitamin D) levels in Jordanian children with bronchial asthma, and to examine correlations between 25-OH vitamin D levels and asthma severity and control. METHODS: A cross-sectional study was conducted at the Paediatric Chest Clinic, Al-Karak Governmental Hospital, Southern Jordan, between May 2015 and February 2016. Serum 25-hydroxyvitamin D level was determined in children aged 1-14 years diagnosed with bronchial asthma (6-14 years) or recurrent wheezing episodes (<6 years). Asthma severity was determined based on the Global Initiative for Asthma assessment, the Asthma Control Test, and the Childhood Asthma Control Test. Demographic and clinical characteristics were compared between patients with low and normal 25-OH vitamin D levels, and correlations between asthma severity and 25-OH vitamin D level were assessed. RESULTS: Out of 98 included children, 25-OH vitamin D levels were deficient and insufficient in 41 (41.8%) and 34 (34.7%) children, respectively. Only 23 (23.5%) had sufficient 25-OH vitamin D levels. A significant correlation was found between severity of asthma symptoms and 25-OH vitamin D deficiency. CONCLUSION: 25-OH vitamin D deficiency is highly prevalent in Jordanian children with bronchial asthma and correlates significantly with asthma severity.

Sensitization to inhaled allergens in asthmatic children in southern Jordan: a cross-sectional study.

BACKGROUND: Sensitization to inhaled allergens in children with bronchial asthma significantly affects asthma pathogenesis, severity and persistence into late childhood and adulthood. The present study determined the prevalence of sensitization to inhaled allergens in children with bronchial asthma and wheezing episodes in order to investigate the effect of positive sensitization on the severity and control of asthma symptoms and to screen for other associated allergic conditions. METHODS: A cross-sectional study was conducted, including children between 6 months and 14 years of age attending the chest clinic of Al-Karak, south of Jordan, between November 2013 and February 2016. Skin prick tests (SPTs) using 11 standardized allergen extracts were conducted in 277 children. The severity of asthma was determined based on the Global Initiative for Asthma (GINA) assessment and the Childhood Asthma Control Test (C-ACT) in addition to the history of use of systemic steroids and hospital admissions within the past 12 months. RESULTS: Sixty-seven percent of children with bronchial asthma reported sensitization to one or more of the inhaled allergens. The most common allergens were olive pollens (18%), cat fur (13.5%), and Dermatophagoides pteronyssinus (11.9%). There was a significant increase in allergen sensitization with age (p < 0.001). The most common concomitant allergic condition among children was allergic rhinitis (71.5%); however, allergic conjunctivitis was the only allergic condition that correlated with the skin test reactivity (p = 0.01). A family history of bronchial asthma was confirmed in 40.4% of children. Children with positive SPTs had lower ACT scores and reported more frequent use of systemic steroids and admissions to hospital within the past 12 months; however, this effect was not statistically significant (p > 0.05). CONCLUSIONS: Sensitization to inhaled allergens is highly prevalent in children with asthma and wheezing episodes in southern Jordan and may be correlated with the severity of the disease. Therefore, appropriate measures to recognize and avoid these allergens are highly recommended. Most children in our study suffered from concomitant allergic rhinitis, indicating that an appropriate diagnosis and treatment of allergic rhinitis could significantly improve asthma control and thus the quality of life of these children. TRIAL REGISTRATION: This study is not a clinical trial.

Nuclear expression of renin-angiotensin system components in NRK-52E renal epithelial cells.

INTRODUCTION: Isolated nuclei of sheep proximal tubules express angiotensin (Ang) receptors as well as angiotensinogen (AGT) and renin. The present study characterized the NRK-52E tubular epithelial cell line for the intracellular expression of renin-angiotensin system (RAS) components. METHODS: RAS components were visualized by immunofluorescent staining in intact cells and protein expression in isolated nuclei. RESULTS: An antibody to the angiotensin I (Ang I) sequence of AGT (AI-AGT) revealed only cytosolic staining, while an antibody to an internal sequence of AGT (Int-AGT) revealed primarily nuclear staining. Immunoblots of nuclear and cytosolic fractions confirmed the differential cell staining of AGT. Immunostaining for renin was present on nuclei of intact cells. Nuclear renin activity averaged 0.77±0.05 nmol/mg protein/h that was reduced by aliskiren (0.13±0.01 nmol/mg/h, n=3, p<0.01); trypsin activation increased activity three-fold. Peptide staining localized angiotensin II (Ang II) and Ang-(1-7) to the nucleus and peptide content averaged 59±2 and 57±22 fmol/mg (n=4), respectively. Peptide metabolism in isolated nuclei revealed the processing of Ang I to Ang-(1-7) by thimet oligopeptidase. CONCLUSION: We conclude that the NRK-52E cells express an intracellular RAS localized to the nucleus and may be an appropriate cell model to elucidate the functional relevance of this system.

Angiotensin-(1-7) abolishes AGE-induced cellular hypertrophy and myofibroblast transformation via inhibition of ERK1/2.

UNLABELLED: Angiotensin-(1-7) (Ang-(1-7))/AT7-Mas receptor axis is an alternative pathway within the renin-angiotensin system (RAS) that generally opposes the actions of Ang II/AT1 receptor pathway. Advanced glycated end product (AGEs) including glucose- and methylglyoxal-modified albumin (MGA) may contribute to the development and progression of diabetic nephropathy in part through activation of the Ang II/AT1 receptor system; however, the influence of AGE on the Ang-(1-7) arm of the RAS within the kidney is unclear. The present study assessed the impact of AGE on the Ang-(1-7) axis in NRK-52E renal epithelial cells. MGA exposure for 48 h significantly reduced the intracellular levels of Ang-(1-7) approximately 50%; however, Ang I or Ang II expression was not altered. The reduced cellular content of Ang-(1-7) was associated with increased metabolism of the peptide to the inactive metabolite Ang-(1-4) [MGA: 175±9 vs. CONTROL: 115±11 fmol/min/mg protein, p<0.05, n=3] but no change in the processing of Ang I to Ang-(1-7). Treatment with Ang-(1-7) reversed MGA-induced cellular hypertrophy and myofibroblast transition evidenced by reduced immunostaining and protein expression of α-smooth muscle actin (α-SMA) [0.4±0.1 vs. 1.0±0.1, respectively, n=3, p<0.05]. Ang-(1-7) abolished AGE-induced activation of the MAP kinase ERK1/2 to a similar extent as the TGF-ß receptor kinase inhibitor SB58059; however, Ang-(1-7) did not attenuate the MGA-stimulated release of TGF-ß. The AT7-Mas receptor antagonist D-Ala(7)-Ang-(1-7) abolished the inhibitory actions of Ang-(1-7). In contrast, AT1 receptor antagonist losartan did not attenuate the MGA-induced effects. We conclude that Ang-(1-7) may provide an additional therapeutic approach to the conventional RAS blockade regimen to attenuate AGE-dependent renal injury.

The ins and outs of angiotensin processing within the kidney.

The kidney is a key target organ for bioactive components of the renin-angiotensin system (RAS); however, various renal cells such as the tubular epithelium contain an intrinsic RAS. The renal RAS can be functionally divided into ANG II-AT1 receptor and ANG-(1-7)-AT7/Mas receptor arms that functionally oppose one another. The current review considers both extracellular and intracellular pathways that potentially govern the formation and metabolism of angiotensin peptides within the renal proximal tubules.

Update on the Angiotensin converting enzyme 2-Angiotensin (1-7)-MAS receptor axis: fetal programing, sex differences, and intracellular pathways.

The renin-angiotensin-system (RAS) constitutes an important hormonal system in the physiological regulation of blood pressure. Indeed, dysregulation of the RAS may lead to the development of cardiovascular pathologies including kidney injury. Moreover, the blockade of this system by the inhibition of angiotensin converting enzyme (ACE) or antagonism of the angiotensin type 1 receptor (AT1R) constitutes an effective therapeutic regimen. It is now apparent with the identification of multiple components of the RAS that the system is comprised of different angiotensin peptides with diverse biological actions mediated by distinct receptor subtypes. The classic RAS can be defined as the ACE-Ang II-AT1R axis that promotes vasoconstriction, sodium retention, and other mechanisms to maintain blood pressure, as well as increased oxidative stress, fibrosis, cellular growth, and inflammation in pathological conditions. In contrast, the non-classical RAS composed of the ACE2-Ang-(1-7)-Mas receptor axis generally opposes the actions of a stimulated Ang II-AT1R axis through an increase in nitric oxide and prostaglandins and mediates vasodilation, natriuresis, diuresis, and oxidative stress. Thus, a reduced tone of the Ang-(1-7) system may contribute to these pathologies as well. Moreover, the non-classical RAS components may contribute to the effects of therapeutic blockade of the classical system to reduce blood pressure and attenuate various indices of renal injury. The review considers recent studies on the ACE2-Ang-(1-7)-Mas receptor axis regarding the precursor for Ang-(1-7), the intracellular expression and sex differences of this system, as well as an emerging role of the Ang1-(1-7) pathway in fetal programing events and cardiovascular dysfunction.

Novel roles of nuclear angiotensin receptors and signaling mechanisms.

The renin-angiotensin system (RAS) constitutes an important hormonal system in the physiological regulation of blood pressure. The dysregulation of the RAS is considered a major influence in the development and progression of cardiovascular disease and other pathologies. Indeed, experimental and clinical evidence indicates that blockade of this system with angiotensin-converting enzyme (ACE) inhibitors or angiotensin type 1 receptor (AT1R) antagonists is an effective therapy to attenuate hypertension and diabetic renal injury, and to improve heart failure. Originally defined as a circulating system, multiple tissues express a complete RAS, and compelling evidence now favors an intracellular system involved in cell signaling and function. Within the kidney, intracellular expression of the three predominant ANG receptor subtypes is evident in the nuclear compartment. The ANG type 1 receptor (AT1R) is coupled to the generation of reactive oxygen species (ROS) through the activation of phosphoinositol-3 kinase (PI3K) and PKC. In contrast, both ANG type 2 (AT2R) and ANG-(1-7) (AT7R) receptors stimulate nitric oxide (NO) formation, which may involve nuclear endothelial NO synthase (eNOS). Moreover, blockade of either ACE2-the enzyme that converts ANG II to ANG-(1-7)-or the AT7 receptor exacerbates the ANG II-ROS response on renal nuclei. Finally, in a model of fetal programmed hypertension, the nuclear ROS response to ANG II is enhanced, while both AT2 and AT7 stimulation of NO is attenuated, suggesting that an imbalance in the intracellular RAS may contribute to the development of programming events. We conclude that a functional intracellular or nuclear RAS may have important implications in the therapeutic approaches to cardiovascular disease.