Enhanced Chemiluminescence Method Research Articles

Pathological (Dis)Similarities in Neuronal Exosome-Derived Synaptic and Organellar Marker Levels Between Alzheimer's Disease and Frontotemporal Dementia.

Alzheimer's disease (AD) and frontotemporal dementia (FTD) are pathologically distinct neurodegenerative disorders with certain overlap in cognitive and behavioral symptoms. Both AD and FTD are characterized by synaptic loss and accumulation of misfolded proteins, albeit, in different regions of the brain. To investigate the synaptic and organellar markers in AD and FTD through assessment of the levels of synaptic protein, neurogranin (Ng) and organellar proteins, mitofusin-2 (MFN-2), lysosomal associated membrane protein-2 (LAMP-2), and golgin A4 from neuronal exosomes. Exosomes isolated from the plasma of healthy controls (HC), AD and FTD subjects were characterized using transmission electron microscopy. Neurodegenerative status was assessed by measurement of neurofilament light chain (NfL) using Simoa. The pooled exosomal extracts from each group were analyzed for Ng, MFN-2, LAMP-2, and golgin A4 by western blot analysis using enhanced chemiluminescence method of detection. The densitometric analysis of immunoreactive bands demonstrated a 65% reduction of Ng in AD and 53% in FTD. Mitochondrial protein MFN-2 showed a significant reduction by 32% in AD and 46% in FTD. Lysosomal LAMP-2 and Golgi complex associated golgin A4 were considerably increased in both AD and FTD. Changes in Ng may reflect the ongoing synaptic degeneration that are linked to cognitive disturbances in AD and FTD. Importantly, the rate of synaptic degeneration was more pronounced in AD. Changes to a similar extent in both the dementia groups in organellar proteins indicates shared mechanisms of protein accumulation/degradation common to both AD and FTD.

Assessment of Serum Vitamin D Status and Its Associated Health Problem Among Children With Protein Energy Malnutrition: A Cross-Sectional Research Study in Central Rural India.

Background Protein energy malnutrition (PEM) is a condition that results from inadequate intake of both macronutrients and micronutrients, leading to a lack of energy. The condition can manifest quickly or gradually, ranging from mild to severe. It predominantly affects children in low-income countries who do not receive enough calories and proteins. In developed nations, it is more prevalent among older individuals. PEM is more common in children as they consume less protein. In rare cases in developed countries, it may result from fad diets or a lack of knowledge about children's nutritional requirements, especially in cases of milk allergy. Vitamin D plays a critical role in bone growth and development by facilitating the absorption of calcium and phosphorus from food and supplements. Additionally, vitamin D has been linked to a reduced risk of infections, immune system disorders, diabetes, high blood pressure, and heart disease. Aims and objectives The primary objective of this study is to evaluate serum vitamin D levels and their relationship with health complications in children affected by PEM. The specific aim is to estimate the serum vitamin D levels in children with PEM who exhibit symptoms of being underweight, stunting (limited linear growth), wasting (abrupt weight loss), or edematous malnutrition (kwashiorkor). Additionally, this study seeks to analyze the correlation between serum vitamin D levels and the associated health problems in children with PEM. Materials and methods This cross-sectional study employed an analytical research approach. A total of 45 children with PEM participated in the study. Data were collected through a venipuncture method, and serum vitamin D level was assessed using an enhanced chemiluminescence method. The children's pain was assessed using a visual analogue scale, and developmental delay was evaluated using an assessment chart. Data were analyzed using SPSS Version 22 (IBM Corp., Armonk, NY). Results The study's results indicate that a considerable proportion of children, specifically 46.6%, were deficient in vitamin D, whereas 42.2% exhibited insufficiency and only 11.2% had sufficient levels of the vitamin. Pain assessment using the visual analogue scale classification revealed that 15.6% of children reported no pain, 60% reported mild pain, and 24.4% reported moderate pain. The mean and standard deviation of vitamin D levels correlated with developmental delay were 4.22±0.212 and 5.34±0.438, respectively. Similarly, the mean and standard deviation of vitamin D levels correlated with pain were 4.22±0.212 and 2.98±0.489, respectively. The Pearson correlation coefficient for vitamin D levels and pain was 0.010, with a significant level of 0.989, significantly lower than the tabulated value at a 5% significance level. Conclusion Based on the study's findings, it was concluded that children who suffer from PEM are at a risk of developing vitamin D deficiency, which can result in adverse health outcomes, including developmental delay and pain.

A high‐salt diet induces synaptic loss and memory impairment via gut microbiota and butyrate in mice

A high‐salt diet induces synaptic loss and memory impairment via gut microbiota and butyrate in mice

Features of the population immunity to SARS-CoV-2 development in staff members of medical organizations during the spread of COVID-19

Background. The ongoing COVID-19 pandemic generates the urgent need of monitoring the herd immunity among healthcare workers as a high-risk group for infection.
 Aim. Determining the level and structure of population immunity to SARS-CoV-2 in the medical workers during the second peak of COVID-19 from August to November 2020.
 Material and methods. The study involved 15,158 medical workers from 91 medical organizations of the Republic of Tatarstan. The age of the examined stuff members varied from 18 to 74 years, the average age was 45.480.1016 years, most of them were women (13 627, 89.870.258%). Testing for the presence of total antibodies (immunoglobulin classes G, A and M) to SARS- CoV-2 was performed once by the enhanced chemiluminescence method. Statistical processing was carried out by methods of variation statistics and correlation analysis according to the Pearson method using MS Excel and WinPepi.
 Results. Seroprevalence to SARS-CoV-2 in medical workers was 35.230.388%. An increase in seropositive individuals level from 27.440.265% in August to 57.910.499% in November and an increase in the average geometric mean titer of total antibodies (immunoglobulin classes A, M, G) from 84.43 in August to 101.09 in November 2020 were detected. The level of individuals with antibodies increased with age from 18 to 59 years and decreased in group of 6070 years. The largest proportion of seropositive to SARS-CoV-2 individuals was found among junior (42.011.384%) and middle grade (37.50.527%) medical staff of hospitals for patients with COVID-19, ambulance and emergency stations. Among the seropositive to the SARS-CoV-2 workers the proportion of asymptomatic forms of infection was 84.970.498%. According to the level of seroprevalence, municipal districts of the Republic of Tatarstan were divided into three groups: moderate 8.939.0; average 42.949.6, high level 53.164.4.
 Сonclusion. An increase in seroprevalence to SARS-CoV-2 was revealed among medical workers of the Republic of Tatarstan from August to November 2020 during the second peak of COVID-19.

SARS-Coronavirus-2 seroprevalence in asymptomatic healthy blood donors: Indicator of community spread

BackgroundThe Corona virus disease 2019 (COVID-19) pandemic caused by SARS -Corona virus-2 (SARS-CoV-2) has been a major concern the world over. Serological surveillance is an important tool to assess the spread of infection in the community. This study attempted to assess the prevalence of antibodies to SARS-CoV-2 among blood donors in Delhi, India during the pre-vaccination period. MethodsSeroprevalence of SARS-CoV2-2 IgG antibodies were determined in blood donors reporting to the Department of Transfusion medicine at a tertiary care hepatobiliary center, in India from September to October 2020. The SARS-CoV-2 IgG antibodies against spike subunit 1 protein were measured using the enhanced chemiluminescence method. ResultsA total of 1066 blood donors were screened. The overall seropositivity for SARS-CoV-2 IgG antibodies was 27.57 % (294/1066). The highest seropositivity was seen in the age group 26−35 years, 46.6 % (137/492), followed by 18−25 years, 28.2 % (83/260), 36−45 years, 19.4 % (57/244), and more than 45 years, 5.8 % (17/70). The seropositivity in the donors who had donated blood previously was 26.1 % (189/723). There was no statistically significant difference amongst seroprevalence in the blood groups, AB blood group (32.6 %, 95 % CI 23.02−43.3), group B (27.2 %, 95 % CI 22.8−32.09 %), group A (27.1 %, 95 % CI 21.8−32.9 %), and group O (27.02 %, 95 % CI 22.3−32.1 %) (p 0.539). ConclusionsThere was significantly higher seropositivity for SARS-CoV-2 antibodies in the voluntary healthy blood donors indicating community spread and large number of asymptomatic cases in Delhi. Higher seroprevalence in younger adults indicated increased exposure to the virus and lack of COVID appropriate behaviour.

Occurrence of Hepatitis E virus infection in patients screened for Hepatitis A virus

: Infectious hepatitis can be caused by Hepatitis A and Hepatitis E viruses. Hepatitis E infection is a water-borne disease transmitted by faeco-oral route. It is a cause of major health problems in developing countries. Outbreaks can happen due to improper hygiene and poor sanitation. Hepatitis E infection shows similar mode of transmission and clinical features of Hepatitis A infection. : This study was conducted to investigate the occurrence of Hepatitis E virus infection in patients screened for hepatitis A virus.: This prospective surveillance study was conducted between March 2020 to March 2021 on 57 serum samples which is came for the screening of anti-Hepatitis A virus IgM by enhanced Chemiluminescence method. These samples were collected and tested for anti- Hepatitis E virus IgM by Enzyme linked immunosorbent assay.: Among 57 samples, 11 samples (19.3%) were positive for Hepatitis A Virus IgM, 13 samples (22.8%) were positive for Hepatitis E virus IgM, and 7 samples (12.3%) were positive for both.: Hepatitis E virus infections were more than Hepatitis A virus. Hepatitis E and Hepatitis A virus co- infection was also observed. Screening of both viruses is suggested for infectious hepatitis.

The relationship of plasma renin, angiotensin, and aldosterone levels to blood pressure variability and target organ damage in children with essential hypertension

BackgroundTo investigate the relationships of plasma renin, angiotensin, and aldosterone levels to blood pressure variability and target organ damage in children with essential hypertension.MethodsA case-control study was conducted on 132 children diagnosed with essential hypertension (103 males and 29 females with the mean age of 11.8 ± 2.4 years). The plasma RAAS levels were measured using the enhanced chemiluminescence method, the ambulatory blood pressure was monitored for 24 h, and then the average real variability (ARV) was calculated. Data on indicators were used for assessing cardiac and renal damages. The correlations of plasma renin, angiotensin, and aldosterone (RAAS) levels to blood pressure variability (BPV) and target organ damage (TOD) were studied. A comparison between the groups was conducted using SPSS 20.ResultsAmong the 132 children, 55 cases had target organ damage. The 24-h ARV and the daytime ARV of the systolic blood pressure of the high angiotensin II (AT II) group was significantly higher than that of the normal AT II group (t = 2.175, P = 0.031; t = 2.672, P = 0.009). Plasma AT II and aldosterone levels were significantly associated with the left ventricular mass index (r = 0.329, P = 0.0001; r = 0.175, P = 0.045). Linear regression analysis showed that AT II [β ± s.e. = 0.025 ± 0.006, 95% CI (0.013–0.038), P = 0.0001] and aldosterone [β ± s.e. = 0.021 ± 0.007, 95% CI (0.008–0.034), P = 0.002] were risk factors for LVH.ConclusionsThe AT II level in children with essential hypertension affected the variability of the 24-h and the daytime SBP. Plasma AT II and aldosterone levels were associated with cardiac damage. Results from this study indicated that AT II and aldosterone are risk factors for LVH in childhood hypertension and are of great significance for improving the clinical prognosis of pediatric patients with hypertension.

Quantification of Free Radical Scavenging Properties and SOD-Like Activity of Cerium Dioxide Nanoparticles in Biochemical Models

The enhanced chemiluminescence method in combination with mathematical modeling has been used to quantify the antioxidant properties and to study the superoxide dismutase-like (SOD-like) activity of a citrate-stabilized colloid solution of ultrasmall CeO2 nanoparticles (3 nm) in biochemical models. The trolox (a water-soluble analogue of vitamin E) equivalent antioxidant capacity of a 1 µmol/L CeO2 sol was 0.049 ± 0.004 µmol/L; thus, the ability of CeO2 to act as a free radical scavenger is about 20 times lower than that of trolox. The antioxidant activity was estimated by the mathematical modeling method. The free radical scavenging rate constants were 2000 nM–1 min–1 for trolox and k1 = 300 nM–1 min–1 and k2 = 4 nM–1 min–1 for cerium dioxide. The SOD-like activity of 1 mmol/L СеО2 in SOD activity units was 2.00 ± 0.03 nmol/L. Thus, the activity of CeO2 as a SOD mimetic is about six orders of magnitude lower than the activity of the native enzyme.

Generation of hydrogen peroxide in protein solutions under the influence of thermal and optical electromagnetic radiation

Using the method of induced chemiluminescence in solutions of bovine serum albumin (BSA) and bovine gamma globulin (BGG) under the influence of thermal and optical electromagnetic radiation, the formation of long-lived reactive protein species (LRPS) with a half-life of about 4-5 hours was registered. The oxygen effect was established, namely, the dependence of the chemiluminescence intensity of protein solutions on the concentration of dissolved oxygen. Using the enhanced chemiluminescence method in the luminol-p-iodophenol-peroxidase system, the ability of LRPS, induced by heat and laser radiation, to generate hydrogen peroxide in solution was studied. The dependence of the formation of H2O2 under the influence of LRPS at different times after exposure was established. Using the fluorescent probe coumarin-3-carboxylic acid, the formation of hydroxyl radicals in protein solutions after exposure to laser radiation and heat was detected. The obtained experimental data suggest that the generation of hydrogen peroxide by LRPS after exposure to thermal and optical electromagnetic radiation may be one of the mechanisms for activating protective cellular mechanisms that help overcome diseases, including those associated with oxidative stress.

Early predictors of cardiac dysfunction in Egyptian children with chronic kidney disease.

Background:Cardiovascular morbidity (CVM) is the main etiology of mortality in children and adolescents with chronic kidney disease (CKD). CKD associated cardiovascular mortality is more common in children with diastolic cardiac dysfunction which was considered as an early indicator for death, while increased left ventricular mass (LVM) is a strong independent risk factor for these patients. Vitamin D deficiency was previously studied as one of the risk factors for CVM.Aim:The aim of the work was to investigate the relationship between biomarkers of mineral bone disorder including serum 25(OH) Vitamin D3 (25-OH D3), phosphorus and calcium × phosphorus (Ca×Po4) product with diastolic cardiac function and LVM in children and adolescents with CKD.Subjects and Methods:This was a cross-sectional observational study. Participants were classified into two groups: Group I including 86 pediatric patients with CKD (stages 4 or 5) and Group II including 40 healthy controls. Group I was subdivided into IA included children with diastolic dysfunction and IB included cases without diastolic dysfunction. 25-OH D3 level was measured by enhanced chemiluminescence method and intact parathyroid hormone (iPTH) by electrochemiluminescence method. Parameters for diastolic function and LVM were assessed by Doppler echocardiography, tissue Doppler imaging, and M-mode echocardiography.Results:25-OH D3 level was significantly lower in Group I when compared to Group II. Diastolic dysfunction was present in 48.8% of the studied patients and was significantly associated with increased serum phosphorus and calcium-phosphorus product but not with decreased level of 25-OH D3. There was a significant positive correlation between LVM and iPTH.Conclusions:Hyperphosphatemia and high Ca×Po4 product were considered of prognostic value as they predict early diastolic dysfunction and increased LVM in children with CKD.

Mitochondria as a Source of Superoxide Anion Radical in Human Platelets

The radical-producing activity of human platelets has been studied using the enhanced chemiluminescence method. It is shown that chemiluminescence of isolated platelets is observed only in the presence of lucigenin, a selective probe for superoxide anion; the luminescence is amplified many times upon the addition of NADH and NADPH, the substrates of oxidative chains. The chemiluminescence is not affected by diphenyliodonium, an inhibitor of NADPH oxidase, but it is inhibited in a dose-dependent manner by the oxidative phosphorylation uncouplers dinitrophenol and rotenone. Thus, a superoxide anion radical is the main free radical generated by platelets, and mitochondria are one of the superoxide anion radical sources in platelets.

Antioxidant potential, tannin and polyphenol contents of seed and pericarp of three Coffea species.

To investigate the antioxidant activity, total phenolic and total tannin content of the pericarp and the seed of Coffea benghalensis (C.benghalensis) and Coffea liberica compared to Coffea arabica (C.arabica). The antioxidant potential, total tannin and polyphenol contents of the immature and mature seed and pericarp of C.benghalensis and Coffea liberica were quantified and compared to C.arabica. Enhanced chemiluminescence (ECL), 2,2-diphenyl-1-picrylhydrazyl (DPPH), oxygen radical absorbance capacity, Folin-Ciocalteau method and total tannin content assays were used. Trolox equivalent (TE/g plant material) values obtained by ECL and DPPH methods showed loose correlation (r(2)=0.587) while those measured by oxygen radical absorbance capacity assay were higher without correlation in each plant. A closer correlation was detected between the ECL method and the percentage antioxidant activity of the DPPH technique (r(2)=0.610 7) in each species, however the immature pericarp of C.benghalensis showed much higher DPPH scavenging potential than was seen in the ECL assay. The immature pericarp of C.benghalensis expressed the highest tannin and polyphenol content, and a high polyphenol level was also detected in the immature seed of C.arabica. The immature pericarp of Bengal and Liberian coffees showed the largest amount of phenolic contents. The obtained data highlight the potential role of C.benghalensis as a new source of natural antioxidants and polyphenols compared to C.arabica.

Analytical performance of high-sensitivity cardiac troponin I detection by VITROS ECIQ with enhanced chemiluminescence method

Objective To evaluate the performances of high-sensitivity cardiac troponin I (cTnI) on VITRO ECIQ with enhanced chemiluminescence method. Methods This verification study validated the limited of detection, function sensitivity, imprecision, linearity of the high-sensitivity cardic troponin used VITROS ECIQ according to the document EP-17A, EP-6A, EP-15A published by Clinical and Laboratory Standards Institute (CLSI) and determined 99th percentiles. All 652 patients with chest pain on immediate admission in TEDA International Cardiovascular Hospital during January to November 2013 were enrolled in this study. Including 323 cases of acute ST segment elevation myocardial infarction and non ST segment elevation myocardial infarction patients as the case group, exclude 329 cases of other diagnosis, 303 cases of apparent normal people as control group. The receiver operating characteristic curve was used to evaluated the sensitivity and the specificity of cTnI. Non-normal distribution of quantitative data were used nonparametric test Mann-Whitney U, With P<0.05 for the difference was statistically significant. Results The LoB was 0.006 5 ng/ml and the LoD was 0.015 5 ng/ml; the FS was 0.016 76 ng/ml; repeatability CV was 1.73 %-2.33 %, reproducibility CV was 4.93%-9.96%.The imprecision were lower than that declared by assay producer. The linearity was 0.015 5-78.4 ng/ml(R2=0.999 9); the 99th percentile reference value was 0.017 ng/ml. The area under the curve(AUC)of cTnI was 0.986, 95%CI 0.973-0.994, with the cut-off value as 0.017 ng/ml, the diagnostic sensitivity and specificity in CIN were 90.09 % and 99.34%. Compared between STEMI and NSTEMI groups after admission, the levels of cTnI had no significantly difference, Z were -0.485, P>0.05; compared between STEMI and control groups after admission, the levels of cTnI had significantly difference, Z were -19.567, P<0.001; compared between NSTEMI and control groups after admission, the levels of cTnI had significantly difference, Z were -14.598, P<0.001. Conclusions High-sensitivity cardiac troponin I detection by VITROS ECIQ with enhanced chemiluminescence method has good performances of sensitivity, linearity, specificity, which meet the clinical needs.(Chin J Lab Med, 2015, 38: 461-465) Key words: Troponin I; Myocardial infarction; Luminescence; Immunoassay; Sensitivity and specificity

A comprehensive serological and supplemental evaluation of hepatitis B "seroyield" blood donors: A cross-sectional study from a tertiary healthcare center in India.

Background:The present study addressed the interesting findings of supplemental evaluation of hepatitis B “seroyield” donors.Materials and Methods:Each blood donor sample was tested for anti-human immunodeficiency virus type I (HIV-I)/HIV type II (HIV-II), HBsAg, and anti-hepatitis C virus (HCV) antibody by enhanced chemiluminescence method and subjected to individual donor-nucleic acid testing (NAT) for HIV-I, hepatitis B virus (HBV), and HCV. NAT test was performed using the eSAS system, Procleix Ultrio Assay, Novartis Diagnostics, CA, US. Confirmation of HBsAg was done using HBsAg Confirmatory Kit (Ortho Clinical Diagnostics, Johnson & Johnson, USA) and viral load assessment was done using Cobas TaqMan real time-polymerase chain reaction (RT-PCR) assay (Roche Molecular Systems, Branchburg, NJ, USA). To provide information on the stage of infection, specimens were tested for anti-HBc total (IgG + IgM), anti-HBc IgM and HBeAg. HBeAg-negative samples were tested for anti-HBe antibody.Results:A total of 60 hepatitis B seroyield donors which showed mean initial sample/cutoff of 1.6 with enhanced chemiluminescence assay were investigated further for confirmation of disease status. All 60 cases were confirmed positive with neutralization assay (VITROS HBsAg Confirmatory Kit) while no target was detected on viral load assessment with RT-PCR. Sixteen donors were HBeAg positive (4 IgM anti-HBc positive and 12 IgM anti-HBc negative) and 44 were IgM anti-HBc negative, anti-HBc total positive, and anti-HBe positive.Conclusion:About 7.7% of HBsAg positive and NAT nonreactive donors (nondetectable HBV DNA) could be potentially infectious (HBeAg positive), whereas rest of the donors were consistent with chronic HBV infection.

Hepatitis B Virus Core Promoter Mutations in Patients With Chronic Hepatitis B and Hepatocellular Carcinoma in Bucharest, Romania

Background:Accurate and personalized molecular virological diagnosis of hepatitis B virus (HBV) infection is crucial for individualized selection of patients for antiviral therapy in Romania.Objectives:We aimed to investigate HBV mutations in Romanian patients with chronic HBV infection, also to match HBV genotypes with HBV mutations identified and clinical outcomes.Patients and Methods:This was a cross-sectional study. A total of 484 Romanian patients with chronic HBV infection and hepatocellular carcinoma (HCC) were investigated. This was performed in Fundeni Clinical Institute, Bucharest, Romania during January 2005 to August 2010. HBsAg positive patients with chronic HBV infection admitted to Fundeni Clinical Institute were randomly enrolled in the study. Analysis was performed in the Centre for Immunogenetics and Virology, Fundeni Clinical Institute, Bucharest, Romania. Indirect diagnosis was performed with enhanced chemiluminescence method using Architect i2000SR and HBV-DNA was quantified with COBAS TaqMan HBV PCR. Direct sequencing of the PCR-products was performed with the PCR-product sequencing kit. HBV genotyping was performed with INNO-LiPA DR Amplification and INNO-LiPA HBV precore-core.Results:We detected two HBV genotypes; A (8.1%) and D (60.5%), and a mixture of genotypes A and D (31.4%) (P < 0.001). Basal core promoter (BCP) A1762T/G1764A and precore (PC) G1896A mutations were detected in these Romanian patients with chronic HBV infection. HBV chronic carriers had mainly genotype D (54.4%) and HBV WT (64.0%). BCP A1762T, G1764A and PC G1896A were significantly associated with HCC-tissue HBV sequencing (75.3%) (P < 0.001). PC G1896A alone was detected in HCC-serum HBV sequencing group (66.7%).Conclusions:Genotype D was the main genotype detected in Romanian patients with chronic HBV infection. Genotype D presented both BCP and PC mutations more frequently.

Determination of sulpiride in pharmaceutical preparations and biological fluids using a Cr (III) enhanced chemiluminescence method

A highly sensitive and simple method for identifying sulpiride in pharmaceutical formulations and biological fluids is presented. The method is based on increased chemiluminescence (CL) intensity of a luminol-H2O2 system in response to the addition of Cr (III) under alkaline conditions. The CL intensity of the luminol-H2O2-Cr (III) system was greatly enhanced by the addition of sulpiride and the CL intensity was proportional to the concentration of sulpiride in a sample solution. Various parameters affecting the CL intensity were systematically investigated and optimized for determination of the sulpiride in a sample. Under the optimum conditions, the CL intensity was proportional to the concentration of sulpiride in the range of 0.068-4.0 µg/mL, with a good correlation coefficient of 0.997. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 8.50 × 10(-6) µg/mL and 2.83 × 10(-5) µg/mL, respectively. The method presented here produced good reproducibility with a relative standard deviation (RSD) of 2.70% (n = 7). The effects of common excipients and metal ions were studied for their interference effect. The method was validated statistically through recovery studies and successfully applied for the determination of sulpiride in pure form, pharmaceutical preparations and spiked human plasma samples. The percentage recoveries were found to range from 99.10 to 100.05% for pure form, 98.12 to 100.18% for pharmaceutical preparations and 97.9 to 101.4% for spiked human plasma.

EPLERENONE ATTENUATES MYOCARDIAL FIBROSIS IN THE ANGIOTENSIN II-INDUCED HYPERTENSIVE MOUSE: INVOLVEMENT OF OSTEOPOTIN INDUCED BY NADPH OXIDASE

EPLERENONE ATTENUATES MYOCARDIAL FIBROSIS IN THE ANGIOTENSIN II-INDUCED HYPERTENSIVE MOUSE: INVOLVEMENT OF OSTEOPOTIN INDUCED BY NADPH OXIDASE

Mutant HFE H63D Protein Is Associated with Prolonged Endoplasmic Reticulum Stress and Increased Neuronal Vulnerability

A specific polymorphism in the hemochromatosis (HFE) gene, H63D, is over-represented in neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer disease. Mutations of HFE are best known as being associated with cellular iron overload, but the mechanism by which HFE H63D might increase the risk of neuron degeneration is unclear. Here, using an inducible expression cell model developed from a human neuronal cell line SH-SY5Y, we reported that the presence of the HFE H63D protein activated the unfolded protein response (UPR). This response was followed by a persistent endoplasmic reticulum (ER) stress, as the signals of UPR sensors attenuated and followed by up-regulation of caspase-3 cleavage and activity. Our in vitro findings were recapitulated in a transgenic mouse model carrying Hfe H67D, the mouse equivalent of the human H63D mutation. In this model, UPR activation was detected in the lumbar spinal cord at 6 months then declined at 12 months in association with increased caspase-3 cleavage. Moreover, upon the prolonged ER stress, the number of cells expressing HFE H63D in early apoptosis was increased moderately. Cell proliferation was decreased without increased cell death. Additionally, despite increased iron level in cells carrying HFE H63D, it appeared that ER stress was not responsive to the change of cellular iron status. Overall, our studies indicate that the HFE H63D mutant protein is associated with prolonged ER stress and chronically increased neuronal vulnerability.

Regulatory Effects of Ribosomal S6 Kinase 1 (RSK1) in IFNλ Signaling

Although the mechanisms of generation of signals that control transcriptional activation of Type III IFN (IFNλ)-regulated genes have been identified, very little is known about the mechanisms by which the IFNλ receptor generates signals for mRNA translation of IFNλ-activated genes. We provide evidence that IFNλ activates the p90 ribosomal protein S6 kinase 1 (RSK1) and its downstream effector, initiation factor eIF4B. Prior to its engagement by the IFNλ receptor, the non-active form of RSK1 is present in a complex with the translational repressor 4E-BP1 in IFNλ-sensitive cells. IFNλ-inducible phosphorylation/activation of RSK1 results in its dissociation from 4E-BP1 at the same time that 4E-BP1 dissociates from eIF4E to allow formation of eIF4F and initiation of cap-dependent translation. Our studies demonstrate that such IFNλ-dependent engagement of RSK1 is essential for up-regulation of p21(WAF1/CIP1) expression, suggesting a mechanism for generation of growth-inhibitory responses. Altogether, our data provide evidence for a critical role for the activated RSK1 in IFNλ signaling.

Cytochrome P450 2B1 Mediates Complement-dependent Sublytic Injury in a Model of Membranous Nephropathy

Membranous nephropathy is a disease that affects the filtering units of the kidney, the glomeruli, and results in proteinuria accompanied by loss of kidney function. Passive Heymann nephritis is an experimental model that mimics membranous nephropathy in humans, wherein the glomerular epithelial cell (GEC) injury induced by complement C5b-9 leads to proteinuria. We examined the role of cytochrome P450 2B1 (CYP2B1) in this complement-mediated sublytic injury. Overexpression of CYP2B1 in GECs significantly increased the formation of reactive oxygen species, cytotoxicity, and collapse of the actin cytoskeleton following treatment with anti-tubular brush-border antiserum (anti-Fx1A). In contrast, silencing of CYP2B1 markedly attenuated anti-Fx1A-induced reactive oxygen species generation and cytotoxicity with preservation of the actin cytoskeleton. Gelsolin, which maintains an organized actin cytoskeleton, was significantly decreased by complement C5b-9-mediated injury but was preserved in CYP2B1-silenced cells. In rats injected with anti-Fx1A, the cytochrome P450 inhibitor cimetidine blocked an increase in catalytic iron and ROS generation, reduced the formation of malondialdehyde adducts, maintained a normal distribution of nephrin in the glomeruli, and provided significant protection at the onset of proteinuria. Thus, GEC CYP2B1 contributes to complement C5b-9-mediated injury and plays an important role in the pathogenesis of passive Heymann nephritis.