Environmental Lead Exposure among Preschool Children in Shanghai,China: Blood Lead Levels and Risk Factors

Objective

To determine blood lead levels and to identify related risk factors among children in Shanghai; to explore the lead change trend of children after industrial transformation and to provide data for policy development to control environmental lead pollution in Shanghai.

Methods

A stratified-clustered-random sampling method was used. A tungsten atomizer absorption spectrophotometer was employed to determine blood lead levels.

Results

The arithmetic mean, geometric mean and median of blood lead levels of 0- to 6-year-old children from Shanghai were 22.49 µg/L, 19.65 µg/L and 19.5 µg/L, including 0.26% (6/2291) with concentrations ≥100 µg/L and 2.7% (61/2291) with concentrations ≥50 µg/L. Boys'' levels (23.57 µg/L) were greater than those of girls (21.2 µg/L). The blood lead levels increased with age. This survey showed that the Chongming district was the highest and Yangpu district was the lowest, this result is completely opposite with the earlier survey in Shanghai. Risk factors for lead contamination included housing environment, parents'' education levels, social status, hobbies, and children''s nutritional status.

Conclusions

The blood lead levels of children in Shanghai were lower than the earlier data of Shanghai and those of published studies in China, but higher than the blood lead levels of developed countries. The blood lead levels of urban districts are higher than the central districts with the industrial transformation. Society and the government should take an active interest in childhood lead poisoning of urban areas.     

Intravitreal Docosahexaenoic Acid in a Rabbit Model: Preclinical Safety Assessment

Purpose

The purpose of the present study was to evaluate the retinal toxicity of a single dose of intravitreal docosahexaenoic acid (DHA) in rabbit eyes over a short-term period.

Methods

Sixteen New Zealand albino rabbits were selected for this pre-clinical study. Six concentrations of DHA (Brudy Laboratories, Barcelona, Spain) were prepared: 10 mg/50 µl, 5 mg/50 µl, 2’5 mg/50 µl, 50 µg/50 µl, 25 µg/50 µl, and 5 µg/50 µl. Each concentration was injected intravitreally in the right eye of two rabbits. As a control, the vehicle solution was injected in one eye of four animals. Retinal safety was studied by slit-lamp examination, and electroretinography. All the rabbits were euthanized one week after the intravitreal injection of DHA and the eyeballs were processed to morphologic and morphometric histological examination by light microscopy. At the same time aqueous and vitreous humor samples were taken to quantify the concentration of omega-3 acids by gas chromatography. Statistical analysis was performed by SPSS 21.0.

Results

Slit-lamp examination revealed an important inflammatory reaction on the anterior chamber of the rabbits injected with the higher concentrations of DHA (10 mg/50 µl, 5 mg/50 µl, 2''5 mg/50 µ) Lower concentrations showed no inflammation. Electroretinography and histological studies showed no significant difference between control and DHA-injected groups except for the group injected with 50 µg/50 µl.

Conclusions

Our results indicate that administration of intravitreal DHA is safe in the albino rabbit model up to the maximum tolerated dose of 25 µg/50 µl. Further studies should be performed in order to evaluate the effect of intravitreal injection of DHA as a treatment, alone or in combination, of different retinal diseases.     

An Investigation of Outpatient Children's Blood Lead Level in Wuhan China

Objective

Blood lead levels (BLLs) and possible influencing factors in children in Wuhan China were investigated in order to understand current lead pollution exposure and provide a scientific basis for prevention and policy making.

Materials and Methods

BLL data were collected from 15,536 out-patients in Wuhan Children Hospital in 2012 full year. All of them were under 18 years of age (Mean ± SD: 4.32±3.2, 64.4% boys). The BLLs were measured by an atomic absorption spectrometry (BH2100).

Results

The geometric mean of BLLs for all the subjects was 44.75 µg/L (95%CI: 44.46 µg/L – 45.05 µg/L), much lower than that reported in previous studies. The prevalence of the elevated BLLs (≥ 100 µg/L) in the children tested was 2% in 2012 and the prevalence of BLLs (≥ 50 µg/L) was 44%. Age and sex could be possible influencing factors for BLLs in the children (p<0.001). In addition, the BLLs in different seasons were different (p<0.001).

Conclusions

These results demonstrate that BLLs have significantly decreased in children in Wuhan during recent years. However, we should continuously pay attention to lead pollution and emphasize that prevention is much more important than treatment for controlling children''s BLLs.     

A Novel Immunological Assay for Hepcidin Quantification in Human Serum

Background

Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Increased hepcidin concentrations lead to iron sequestration in macrophages, contributing to the pathogenesis of anaemia of chronic disease whereas decreased hepcidin is observed in iron deficiency and primary iron overload diseases such as hereditary hemochromatosis. Hepcidin quantification in human blood or urine may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a valuable tool for clinicians for the differential diagnosis of anaemia. This study describes a specific and non-operator demanding immunoassay for hepcidin quantification in human sera.

Methods and Findings

An ELISA assay was developed for measuring hepcidin serum concentration using a recombinant hepcidin25-His peptide and a polyclonal antibody against this peptide, which was able to identify native hepcidin. The ELISA assay had a detection range of 10–1500 µg/L and a detection limit of 5.4 µg/L. The intra- and interassay coefficients of variance ranged from 8–15% and 5–16%, respectively. Mean linearity and recovery were 101% and 107%, respectively. Mean hepcidin levels were significantly lower in 7 patients with juvenile hemochromatosis (12.8 µg/L) and 10 patients with iron deficiency anemia (15.7 µg/L) and higher in 7 patients with Hodgkin lymphoma (116.7 µg/L) compared to 32 age-matched healthy controls (42.7 µg/L).

Conclusions

We describe a new simple ELISA assay for measuring hepcidin in human serum with sufficient accuracy and reproducibility.     

Comparative acute toxicity of gallium(III), antimony(III), indium(III), cadmium(II), and copper(II) on freshwater swamp shrimp (Macrobrachium nipponense)

Background

Acute toxicity testing were carried out the freshwater swamp shrimp, Macrobrachium nipponense, as the model animal for the semiconductor applied metals (gallium, antimony, indium, cadmium, and copper) to evaluate if the species is an suitable experimental animal of pollution in aquatic ecosystem.

Results

The static renewal test method of acute lethal concentrations determination was used, and water temperature was maintained at 24.0 ± 0.5°C. Data of individual metal obtained from acute toxicity tests were determined using probit analysis method. The median lethal concentration (96-h LC₅₀) of gallium, antimony, indium, cadmium, and copper for M. nipponense were estimated as 2.7742, 1.9626, 6.8938, 0.0539, and 0.0313 mg/L, respectively.

Conclusions

Comparing the toxicity tolerance of M. nipponense with other species which exposed to these metals, it is obviously that the M. nipponense is more sensitive than that of various other aquatic animals.     

Effect of Antioxidant Supplementation on Exercise-Induced Cardiac Troponin Release in Cyclists: A Randomized Trial

Background

Cardiac troponin is the biochemical gold standard to diagnose acute myocardial infarction. Interestingly however, elevated cardiac troponin concentrations are also frequently observed during and after endurance-type exercise. Oxidative stress associated with prolonged exercise has been proposed to contribute to cardiac troponin release. Therefore, the aim of this study was to assess the effect of 4 week astaxanthin supplementation (a potent cartenoid antioxidant) on antioxidant capacity and exercise-induced cardiac troponin release in cyclists.

Methods

Thirty-two well-trained male cyclists (age 25±5, weight 73±7 kg, maximum O₂ uptake 60±5 mL·kg⁻¹·min⁻¹, W_max 5.4±0.5 W·kg⁻¹; mean ± SD) were repeatedly subjected to a laboratory based standardized exercise protocol before and after 4 weeks of astaxanthin (20 mg/day), or placebo supplementation in a double-blind randomized manner. Blood samples were obtained at baseline, at 60 min of cycling and immediately post-exercise (≈ 120 min).

Results

The pre-supplementation cycling trial induced a significant rise of median cardiac troponin T concentrations from 3.2 (IQR 3.0–4.2) to 4.7 ng/L (IQR 3.7–6.7), immediately post-exercise (p<0.001). Four weeks of astaxanthin supplementation significantly increased mean basal plasma astaxanthin concentrations from non-detectable values to 175±86 µg·kg⁻¹. However, daily astaxanthin supplementation had no effect on exercise-induced cardiac troponin T release (p = 0.24), as measured by the incremental area under the curve. Furthermore, the elevation in basal plasma astaxanthin concentrations was not reflected in changes in antioxidant capacity markers (trolox equivalent antioxidant capacity, uric acid, and malondialdehyde). Markers of inflammation (high-sensitivity C-reactive protein) and exercise-induced skeletal muscle damage (creatine kinase) were equally unaffected by astaxanthin supplementation.

Conclusion

Despite substantial increases in plasma astaxanthin concentrations, astaxanthin supplementation did not improve antioxidant capacity in well-trained cyclists. Accordingly, exercise-induced cardiac troponin T concentrations were not affected by astaxanthin supplementation.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01241877","term_id":"NCT01241877"}}NCT01241877     

Electronegative Low-Density Lipoprotein Increases C-Reactive Protein Expression in Vascular Endothelial Cells through the LOX-1 Receptor

Objectives

Increased plasma C-reactive protein (CRP) levels are associated with the occurrence and severity of acute coronary syndrome. We investigated whether CRP can be generated in vascular endothelial cells (ECs) after exposure to the most electronegative subfraction of low-density lipoprotein (LDL), L5, which is atherogenic to ECs. Because L5 and CRP are both ligands for the lectin-like oxidized LDL receptor-1 (LOX-1), we also examined the role of LOX-1.

Methods and Results

Plasma LDL samples isolated from asymptomatic hypercholesterolemic (LDL cholesterol [LDL-C] levels, 154.6±20 mg/dL; n = 7) patients and normocholesterolemic (LDL-C levels, 86.1±21 mg/dL; P<0.001; n = 7) control individuals were chromatographically resolved into 5 subfractions, L1-L5. The L5 percentage (L5%) and the plasma L5 concentration ([L5]  =  L5% × LDL-C) in the patient and control groups were 8.1±2% vs. 2.3±1% (P<0.001) and 12.6±4 mg/dL vs. 1.9±1 mg/dL (P<0.001), respectively. In hypercholesterolemic patients treated with atorvastatin for 6 months (10 mg/day), [L5] decreased from 12.6±4 mg/dL to 4.5±1.1 mg/dL (P = 0.011; n = 5), whereas both [L5] and L5% returned to baseline levels in 2 noncompliant patients 3 months after discontinuation. In cultured human aortic ECs (HAECs), L5 upregulated CRP expression in a dose- and time-dependent manner up to 2.5-fold (P<0.01), whereas the least electronegative subfraction, L1, had no effect. DiI-labeled L1, internalized through the LDL receptor, became visible inside HAECs within 30 seconds. In contrast, DiI-labeled L5, internalized through LOX-1, became apparent after 5 minutes. L5-induced CRP expression manifested at 30 minutes and was attenuated by neutralizing LOX-1. After 30 minutes, L5 but not L1 induced reactive oxygen species (ROS) production. Both L5-induced ROS and CRP production were attenuated by ROS inhibitor N-acetyl cysteine.

Conclusions

Our results suggest that CRP, L5, and LOX-1 form a cyclic mechanism in atherogenesis and that reducing plasma L5 levels with atorvastatin disrupts the vascular toxicity of L5.     

Effects of Serum Lipid Smoothness on the Progression and Vulnerability of Atherosclerotic Plaques in Rabbits

Objective

We aimed to explore the effects of lipid smoothness on the progression and vulnerability of atherosclerotic plaques.

Approach

24 rabbits were divided into three groups randomly. Group 1 was given standard chow diet; group 2 was fed with cholesterol-rich diet; for group 3, subjects were planned to take cholesterol-rich diet at the first phase for 12 weeks and during the second phase, low-fat and cholesterol-rich diet was then applied alternately every three weeks till the end of the experiment. Lipid profiles, inflammatory factors, endothelium functions, pathological and histological changes were examined. Expressions of matrix metalloproteinase-9 and lectin-like oxidized LDL receptor-1 were measured by immunohistochemical staining.

Results

According to data collected during the whole experiment, lipid smoothness index of group 3 was the lowest. Compared with group 2, statistics of the group 3 indicated that: the development of plaques progressed faster; the plaque area and plaque thickness (53.53[22.6]% vs 33.90[24.91]% , 800.38[98.25]µm vs 675.00[109.67]µm) were higher while the fibrous cap thickness (103.50[45.66]µm vs 295.83[97.90]µm) was lower; hs-CRP (0.53[0.07]mg/dL vs 0.45[0.06]mg/dL), interleukin-18 (186.01[8.41]ng/L vs 158.08[2.37]ng/L), OX-LDL (177.15[5.93]µg/L vs 139.57[2.35] µg/L) and endothelin-1 (164.66[9.54]ng/L vs 131.52[4.39]ng/L) were higher while nitric-oxide (22.41[1.69]µmol/L vs 27.23[1.36]µmol/L) was lower; expressions of matrix metalloproteinase-9 (IOD: 37375.87[5634.52] vs 20956.57[4616.93]) and lectin-like oxidized LDL receptor-1 (IOD: 45213.04[16653.81] vs 21921.68[6142.32]) were higher.

Conclusions

Lipids fluctuation could accelerate the progression and vulnerability of atherosclerotic plaques through worsening arterial endothelium dysfunction and inflammatory reactions.     

Burnt Sugarcane Harvesting – Cardiovascular Effects on a Group of Healthy Workers,Brazil

Background

Brazil is the world''s largest producer of sugarcane. Harvest is predominantly manual, exposing workers to health risks: intense physical exertion, heat, pollutants from sugarcane burning.

Design

Panel study to evaluate the effects of burnt sugarcane harvesting on blood markers and on cardiovascular system.

Methods

Twenty-eight healthy male workers, living in the countryside of Brazil were submitted to blood markers, blood pressure, heart rate variability, cardiopulmonary exercise testing, sympathetic nerve activity evaluation and forearm blood flow measures (venous occlusion plethysmography) during burnt sugarcane harvesting and four months later while they performed other activities in sugar cane culture.

Results

Mean participant age was 31±6.3 years, and had worked for 9.8±8.4 years on sugarcane work. Work during the harvest period was associated with higher serum levels of Creatine Kinase – 136.5 U/L (IQR: 108.5–216.0) vs. 104.5 U/L (IQR: 77.5–170.5), (p = 0.001); plasma Malondialdehyde–7.5±1.4 µM/dl vs. 6.9±1.0 µM/dl, (p = 0.058); Glutathione Peroxidase – 55.1±11.8 Ug/Hb vs. 39.5±9.5 Ug/Hb, (p<0.001); Glutathione Transferase– 3.4±1.3 Ug/Hb vs. 3.0±1.3 Ug/Hb, (p = 0.001); and 24-hour systolic blood pressure – 120.1±10.3 mmHg vs. 117.0±10.0 mmHg, (p = 0.034). In cardiopulmonary exercise testing, rest-to-peak diastolic blood pressure increased by 11.12 mmHg and 5.13 mmHg in the harvest and non-harvest period, respectively. A 10 miliseconds reduction in rMSSD and a 10 burst/min increase in sympathetic nerve activity were associated to 2.2 and 1.8 mmHg rises in systolic arterial pressure, respectively.

Conclusion

Work in burnt sugarcane harvesting was associated with changes in blood markers and higher blood pressure, which may be related to autonomic imbalance.     

Artemisinin-Derived Dimers Have Greatly Improved Anti-Cytomegalovirus Activity Compared to Artemisinin Monomers

Background

Artesunate, an artemisinin-derived monomer, was reported to inhibit Cytomegalovirus (CMV) replication. We aimed to compare the in-vitro anti-CMV activity of several artemisinin-derived monomers and newly synthesized artemisinin dimers.

Methods

Four artemisinin monomers and two novel artemisinin-derived dimers were tested for anti-CMV activity in human fibroblasts infected with luciferase-tagged highly–passaged laboratory adapted strain (Towne), and a clinical CMV isolate. Compounds were evaluated for CMV inhibition and cytotoxicity.

Results

Artemisinin dimers effectively inhibited CMV replication in human foreskin fibroblasts and human embryonic lung fibroblasts (EC₅₀ for dimer sulfone carbamate and dimer primary alcohol 0.06±0.00 µM and 0.15±0.02 µM respectively, in human foreskin fibroblasts) with no cytotxicity at concentrations required for complete CMV inhibition. All four artemisinin monomers (artemisinin, artesunate, artemether and artefanilide) shared a similar degree of CMV inhibition amongst themselves (in µM concentrations) which was significantly less than the inhibition achieved with artemisinin dimers (P<0.0001). Similar to monomers, inhibition of CMV with artemisinin dimers appeared early in the virus life cycle as reflected by decreased expression of the immediate early (IE1) protein.

Conclusions

Artemisinin dimers are potent and non-cytotoxic inhibitors of CMV replication. These compounds should be studied as potential therapeutic agents for the treatment of CMV infection in humans.     

Serum Uric Acid Concentrations in Meat Eaters,Fish Eaters,Vegetarians and Vegans: A Cross-Sectional Analysis in the EPIC-Oxford Cohort

Introduction

Circulating concentrations of uric acid may be affected by dietary components such as meat, fish and dairy products, but only a few studies have compared uric acid concentrations among individuals who exclude some or all of these foods from their diet. The aim of this study was to investigate differences in serum uric acid concentrations between meat eaters, fish eaters, vegetarians and vegans.

Subjects and Methods

A sample of 670 men and 1,023 women (424 meat eaters, 425 fish eaters, 422 vegetarians and 422 vegans, matched on age and sex) from the European Prospective Investigation into Cancer and Nutrition Oxford cohort were included in this cross-sectional analysis. Diet was assessed using a semi-quantitative food frequency questionnaire and serum concentrations of uric acid were measured. Mean concentrations of uric acid by diet group were calculated after adjusting for age, body mass index, calcium and alcohol intake.

Results

In both men and women, serum uric acid concentrations differed significantly by diet group (p<0.0001 and p = 0.01, respectively). The differences between diet groups were most pronounced in men; vegans had the highest concentration (340, 95% confidence interval 329–351 µmol/l), followed by meat eaters (315, 306–324 µmol/l), fish eaters (309, 300–318 µmol/l) and vegetarians (303, 294–312 µmol/l). In women, serum uric acid concentrations were slightly higher in vegans (241, 234–247 µmol/l) than in meat eaters (237, 231–242 µmol/l) and lower in vegetarians (230, 224–236 µmol/l) and fish eaters (227, 221–233 µmol/l).

Conclusion

Individuals consuming a vegan diet had the highest serum concentrations of uric acid compared to meat eaters, fish eaters and vegetarians, especially in men. Vegetarians and individuals who eat fish but not meat had the lowest concentrations of serum uric acid.     

Clinical and Biochemical Correlates of Serum L-Ergothioneine Concentrations in Community-Dwelling Middle-Aged and Older Adults

Background

Despite the increasing interest towards the biological role of L-ergothioneine, little is known about the serum concentrations of this unusual aminothiol in older adults. We addressed this issue in a representative sample of community-dwelling middle-aged and older adults.

Methods

Body mass index, estimated glomerular filtration rate, serum concentrations of L-ergothioneine, taurine, homocysteine, cysteine, glutathione, cysteinylglycine, and glutamylcysteine were evaluated in 439 subjects (age 55–85 years) randomly selected from the Hunter Community Study.

Results

Median L-ergothioneine concentration in the entire cohort was 1.01 IQR 0.78–1.33 µmol/L. Concentrations were not affected by gender (P = 0.41) or by presence of chronic medical conditions (P = 0.15). By considering only healthy subjects, we defined a reference interval for L-ergothioneine serum concentrations from 0.36 (90% CI 0.31–0.44) to 3.08 (90% CI 2.45–3.76) µmol/L. Using stepwise multiple linear regression analysis L-ergothioneine was negatively correlated with age (rpartial = −0.15; P = 0.0018) and with glutamylcysteine concentrations (rpartial = −0.13; P = 0.0063).

Conclusions

A thorough analysis of serum L-ergothioneine concentrations was performed in a large group of community-dwelling middle-aged and older adults. Reference intervals were established. Age and glutamylcysteine were independently negatively associated with L-ergothioneine serum concentration.     

Hyperhomocysteinemia in Patients with Polypoidal Choroidal Vasculopathy: A Case Control Study

Purpose

To determine whether elevated plasma homocysteine and serum high sensitivity C-reactive protein (hsCRP) levels, two established risk factors of vascular diseases, are associated with polypoidal choroidal vasculopathy (PCV).

Design

Retrospective case-control study.

Methods

One hundred and nineteen consecutive patients with PCV and 119 matched controls were enrolled in a tertiary hospital from September 2008 to June 2013. Plasma homocysteine and serum hsCRP levels were measured. Associations among plasma homocysteine, serum hsCRP levels and PCV were further evaluated using multivariable logistic regression analysis.

Results

The median plasma homocysteine level was significantly higher in patients with PCV than in the controls (12.20 µmol/L vs. 9.80 µmol/L, p<0.001). The median serum hsCRP level was slightly higher in the PCV group (0.16 mg/dl vs. 0.11 mg/dl in control group, p = 0.07). After multivariable logistic regression analysis, each 1 µmol/L increase of plasma homocysteine was associated with a 1.5-fold increase in likelihood of having PCV (OR, 1.54; 95% confidence interval (CI), 1.33–1.79, p<0.001).

Conclusions

Hyperhomocysteinemia was associated with PCV and might play a role in the pathogenesis of PCV.     

Vancomycin Dosing in Neutropenic Patients

Background

To compare vancomycin pharmacokinetic parameters in patients with and without neutropenia.

Methods

Patients ≥18 years admitted on general wards were included. Routinely vancomycin trough and peak plasma concentrations were measured with a fluorescence polarization immunoassay. Pharmacokinetic parameters of individual patients were determined with maximum a posterior Bayesian estimation (MW Pharm 3.60). Neutropenia was defined as neutrophils <0.5×10⁹ cells/L.

Principal Findings

A total of 171 patients were included. Patients with neutropenia (n = 56) had higher clearance of vancomycin (CLva), 67 (±26) mL/min, compared to patients without neutropenia (n = 115), CLva 50 (±22) mL/min (p<0.001). No significant difference was found in serum creatinine and vancomycin volume of distribution. Neutropenia was positively associated with CLva, independently of relevant co-variables (B: 12.122, 95%CI: 1.095 to 23.149, p = 0.031). On average patients with neutropenia needed 33% higher doses of vancomycin to attain adequate exposure, i.e. AUC₂₄≥400 mg×h/L. Furthermore, 15 initially neutropenic patients in our study group received vancomycin for a second administration period. Ten patients received the second administration period during another neutropenic period and 5 patients during a non-neutropenic phase. All 5 patients with vancomycin during both neutropenic and non-neutropenic phase had higher CLva (91 (±26) mL/min) during the neutropenic period and lower CLva (45 (±10) mL/min) during the non-neutropenic phase (p = 0.009).

Conclusion

This study shows that most patients with neutropenia have augmented CLva. In a small group of patients that received vancomycin during two episodes, the augmented CLva seems to be reversible in the non-neutropenic period. Our data indicate that it is important to increase the daily dose with one third in patients with neutropenia (from 15 mg/kg twice daily to 13 mg/kg three times daily). Frequent performance of therapeutic drug monitoring in patients with neutropenia may prevent both therapy failure due to low AUCs and overcomes toxicity due to high vancomycin trough concentrations during recovery from neutropenia.     

A Possible Genetic Link between MTHFR Genotype and Smoking Behavior

Background

Hyperhomocysteinemia is an independent risk factor for stroke and other vascular events. The variant methylenetetrahydrofolate reductase (MTHFR) C677T is associated with elevated homocysteine levels, cardiovascular disease and stroke, which supports a causal relationship between hyperhomocysteinemia and vascular disease. However, MTHFR variants have also been reported to be associated with smoking behavior, which could be an important confounder.

Methodology/Principal Findings

We analyzed the MTHFR variants C677T and A1298C in two independent samples of 525 and 535 individuals, respectively. 21% of the non-smokers, but only 12% of the smokers were homozygous carriers of both MTHFR wildtype alleles, i.e. 677CC and 1298AA (Chi² = 15.8; p<0.001; binary regression). Plasma homocysteine levels were higher in smokers (13.9±4.1 µmol/L) than in non-smokers (12.6±4.0 µmol/L; F = 11.4; p = 0.001; ANOVA). Smoking MTHFR 677TT individuals had the highest plasma homocysteine levels (16.2±5.2 µmol/L), non-smoking 677CC individuals had the lowest (12.2±13.6 µmol/L).

Conclusions/Significance

In our study samples, MTHFR variants and smoking behaviour were associated with homocysteine plasma levels. In addition, the MTHFR variants were associated with smoking behaviour. Such an association may be a relevant confounder between MTHFR variants, homocysteine plasma levels and vascular diseases.     

Low-Dose Levodopa Protects Nerve Cells from Oxidative Stress and Up-Regulates Expression of pCREB and CD39

Objective

This study aimed to investigate the influence of low-dose levodopa (L-DOPA) on neuronal cell death under oxidative stress.

Methods

PC12 cells were treated with L-DOPA at different concentrations. We detected the L-DOPA induced reactive oxygen species (ROS). Meanwhile, MTT and LDH assay were performed to determine the proliferation and growth of PC12 cells with or without ROS scavenger. In addition, after pretreatment with L-DOPA at different concentrations alone or in combination with CD39 inhibitor, PC12 cells were incubated with hydrogen peroxide (H₂O₂) and the cell viability was evaluated by MTT and LDH assay. In addition, the expression of pCREB and CD39 was detected by immunofluorescence staining and Western blot assay in both cells and rat’s brain after L-DOPA treatment.

Results

After treatment with L-DOPA for 3 days, the cell proliferation and growth were promoted when the L-DOPA concentration was <30 µM, while cell proliferation was comparable to that in control group when the L-DOPA concentration was >30 µM. Low dose L-DOPA could protect the PC12 cells from H₂O₂ induced oxidative stress, which was compromised by CD39 inhibitor. In addition, the expression of CD39 and pCREB increased in both PC12 cells and rats’ brain after L-DOPA treatment.

Conclusions

L-DOPA at different concentrations has distinct influence on proliferation and growth of PC12 cells, and low dose (<30 µM) L-DOPA protects PC12 cells against oxidative stress which might be related to the up-regulation of CD39 and pCREB expression.     

Improved Methods of Carnivore Faecal Sample Preservation,DNA Extraction and Quantification for Accurate Genotyping of Wild Tigers

Background

Non-invasively collected samples allow a variety of genetic studies on endangered and elusive species. However due to low amplification success and high genotyping error rates fewer samples can be identified up to the individual level. Number of PCRs needed to obtain reliable genotypes also noticeably increase.

Methods

We developed a quantitative PCR assay to measure and grade amplifiable nuclear DNA in feline faecal extracts. We determined DNA degradation in experimentally aged faecal samples and tested a suite of pre-PCR protocols to considerably improve DNA retrieval.

Results

Average DNA concentrations of Grade I, II and III extracts were 982pg/µl, 9.5pg/µl and 0.4pg/µl respectively. Nearly 10% of extracts had no amplifiable DNA. Microsatellite PCR success and allelic dropout rates were 92% and 1.5% in Grade I, 79% and 5% in Grade II, and 54% and 16% in Grade III respectively. Our results on experimentally aged faecal samples showed that ageing has a significant effect on quantity and quality of amplifiable DNA (p<0.001). Maximum DNA degradation occurs within 3 days of exposure to direct sunlight. DNA concentrations of Day 1 samples stored by ethanol and silica methods for a month varied significantly from fresh Day 1 extracts (p<0.1 and p<0.001). This difference was not significant when samples were preserved by two-step method (p>0.05). DNA concentrations of fresh tiger and leopard faecal extracts without addition of carrier RNA were 816.5pg/µl (±115.5) and 690.1pg/µl (±207.1), while concentrations with addition of carrier RNA were 49414.5pg/µl (±9370.6) and 20982.7pg/µl (±6835.8) respectively.

Conclusions

Our results indicate that carnivore faecal samples should be collected as freshly as possible, are better preserved by two-step method and should be extracted with addition of carrier RNA. We recommend quantification of template DNA as this facilitates several downstream protocols.     

Concentrations of the Stress Hormone Copeptin Increase upon Hypoglycaemia in Patients with Type 1 Diabetes Dependent of Hypoglycaemia Awareness

Objective

Copeptin, a marker for stress mirroring vasopressin concentrations, has been shown to increase upon insulin-induced hypoglycaemia in patients after transsphenoidal surgery of pituitary adenomas. Patients with type 1 diabetes mellitus are prone to hypoglycaemia, but no data about copeptin levels upon hypoglycaemia are available. Furthermore, the perception of hypoglycaemia can vary from total unawareness to disabling episodes. The aim of this study was to investigate whether copeptin increases upon hypoglycaemia in patients with type 1 diabetes mellitus and is associated with the degree of hypoglycaemia awareness.

Materials and Methods

In this prospective observational study, 17 patients with type 1 diabetes underwent a standardized insulin infusion test. Blood sampling for glucose and copeptin was performed at baseline and after 60 minutes (min). To assess hypoglycaemia associated symptoms the Mood and Symptom Questionnaire (MSQ) was conducted at baseline and after 60 min.

Results

During insulin infusion, blood glucose decreased from 5.1 (SD±0.2) to 3.0 (±0.5) mmol/L at 60 min (p<0.001). Copeptin concentrations increased from 3.2 (±1.7) to 3.8 (±1.9) pmol/L (p = 0.03). Mood and Symptoms Questionnaire scores increased from 14 (±3.0) to 18 (±5.8), (p = 0.006). Patients with good hypoglycaemia awareness had an increase in copeptin from 3.0 (±1.8) to 4.2 (±2.4) pmol/L (p = 0.03) in contrast to patients more unaware of hypoglycaemia who only showed an increase in copeptin from 3.3 (±1.6) to 3.6 (±1.4) pmol/L (p = 0.4). There was a trend to a larger copeptin increase in patients aware of hypoglycemia compared to patients unaware of hypoglycemia (p = 0.074).

Conclusion

Copeptin increases in patients with type 1 diabetes upon insulin induced hypoglycaemia. Interestingly, the copeptin increase seems associated with the degree of hypoglycaemia awareness. This hypothesis warrants further verification.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00515801","term_id":"NCT00515801"}}NCT00515801     

A Pro-Inflammatory Role of C5L2 in C5a-Primed Neutrophils for ANCA-Induced Activation

Background

The complement system is crucial for the development of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). In particular, C5a and its receptor on neutrophils, CD88, play a central role. The functional role of the second receptor of C5a, C5L2, remains unclear. In the current study, we investigated the role of C5L2 in C5a-primed neutrophils for ANCA-induced activation.

Methods

The effect of blocking C5L2 by anti-human C5L2 blocking antibody were tested on respiratory burst and degranulation of C5a-primed neutrophils activated with ANCA, as well as on membrane-bound proteinase 3 (mPR3) and concentration of myeloperoxidase (MPO) in supernatant of C5a-primed neutrophils. An antagonist for CD88 was also employed.

Results

Blocking C5L2 resulted in a significantly decreased MPO concentration in the supernatant of C5a-primed neutrophils. mPR3 expression increased from 209.0±43.0 in untreated cells to 444.3±60.8 after C5a treatment (P<0.001), and decreased to 375.8±65.44, 342.2±54.3 and 313.7±43.6 by pre-incubating blocking C5L2 antibody at 2.5 µg/ml, 5 µg/ml or 10 µg/ml (compared with C5a-priming group, P<0.001, P<0.001, and P<0.001), respectively. In C5a-primed neutrophils, subsequently activating with MPO-ANCA-positive IgG, the MFI value was 425.8±160.6, which decreased to 292.8±141.2, 289.7±130.0 and 280.3±136.4 upon pre-incubation with mouse anti-human C5L2 blocking antibody at 2.5 µg/ml, 5 µg/ml or 10 µg/ml (compared with C5a-primed neutrophils, for MPO-ANCA-positive IgG-induced activation, P<0.05, P<0.05, and P<0.05), respectively. Blocking C5L2 also resulted in significantly decreased C5a-primed neutrophils for PR3-ANCA-positive IgG-induced activation. Moreover, the lactoferrin concentration in the supernant significantly decreased in pre-incubation with anti-human C5L2 blocking antibody, compared with C5a-primed neutrophils induced by PR3- or MPO-ANCA-positive IgG.

Conclusions

C5L2 may be implicated in the pro-inflammatory role in C5a-primed neutrophils for ANCA-induced activation.     

Different Effects of Propofol and Isoflurane on Cochlear Blood Flow and Hearing Function in Guinea Pigs

Objectives

To investigate the effects of isoflurane and propofol on mean arterial pressure (MAP), cochlear blood flow (CoBF), distortion-product otoacoustic emission (DPOAE), and the ultrastructure of outer hair cells (OHCs) in guinea pig cochleae.

Methods

Forty-eight male guinea pigs were randomly assigned to one of six treatment groups. Groups 1 to 3 were infused (i.v.) with a loading dose of propofol (5 mg/kg) for 5 min and three maintenance doses (10, 20, or 40 mg kg⁻¹·h⁻¹, respectively) for 115 min. Groups 4 to 6 were inhaled with isoflurane at concentrations of 1.15 vol%, 2.30 vol% or 3.45 vol% respectively for 120 min. CoBF and MAP were recorded prior to and at 5 min intervals during drug administration. DPOAE was measured before, immediately after, and 1 h after administration. Following the final DPOAE test, cochleae were examined using scanning electron microscopy.

Results

Propofol treatment reduced MAP in a dose-dependent manner. CoBF and DPOAE showed increases at propofol maintenance doses of 10 and 20 mg kg⁻¹·h⁻¹. Inhalation of isoflurane at concentrations of 2.30 vol% and 3.45 vol% reduced MAP and CoBF. DPOAE amplitude increased following inhalation of 1.15 vol% isoflurane, but decreased following inhalations of 2.30 vol% and 3.45 vol%. Cochlear structure was changed following inhalation of either 2.30 vol% or 3.45 vol% isoflurane.

Conclusions

Propofol could decrease MAP and increase both CoBF and DPOAE without affecting OHC structure. Inhalation of isoflurane at concentrations >2.30 vol% decreased CoBF and DPOAE, and produced injury to OHCs.