Exposure to Phthalates Affects Calcium Handling and Intercellular Connectivity of Human Stem Cell-Derived Cardiomyocytes

Background

The pervasive nature of plastics has raised concerns about the impact of continuous exposure to plastic additives on human health. Of particular concern is the use of phthalates in the production of flexible polyvinyl chloride (PVC) products. Di-2-ethylhexyl-phthalate (DEHP) is a commonly used phthalate ester plasticizer that imparts flexibility and elasticity to PVC products. Recent epidemiological studies have reported correlations between urinary phthalate concentrations and cardiovascular disease, including an increased risk of high blood pressure and coronary risk. Yet, there is little direct evidence linking phthalate exposure to adverse effects in human cells, including cardiomyocytes.

Methods and Results

The effect of DEHP on calcium handling was examined using monolayers of gCAMP3 human embryonic stem cell-derived cardiomyocytes, which contain an endogenous calcium sensor. Cardiomyocytes were exposed to DEHP (5 – 50 μg/mL), and calcium transients were recorded using a Zeiss confocal imaging system. DEHP exposure (24 – 72 hr) had a negative chronotropic and inotropic effect on cardiomyocytes, increased the minimum threshold voltage required for external pacing, and modified connexin-43 expression. Application of Wy-14,643 (100 μM), an agonist for the peroxisome proliferator-activated receptor alpha, did not replicate DEHP’s effects on calcium transient morphology or spontaneous beating rate.

Conclusions

Phthalates can affect the normal physiology of human cardiomyocytes, including DEHP elicited perturbations in cardiac calcium handling and intercellular connectivity. Our findings call for additional studies to clarify the extent by which phthalate exposure can alter cardiac function, particularly in vulnerable patient populations who are at risk for high phthalate exposure.     

Leishmanicidal Effect of Synthetic trans-Resveratrol Analogs

Background

Stilbene-based compounds show antitumoral, antioxidant, antihistaminic, anti-inflammatory and antimicrobial activities. Here, we evaluated the effect of the trans-resveratrol analogs, pterostilbene, piceatannol, polydatin and oxyresveratrol, against Leishmania amazonensis.

Methodology/Principal Findings

Our results demonstrated a low murine macrophage cytotoxicity of all four analogs. Moreover, pterostilbene, piceatannol, polydatin and oxyresveratrol showed an anti-L. amazonensis activity with IC₅₀ values of 18 μM, 65 μM, 95 μM and 65 μM for promastigotes, respectively. For intracellular amastigotes, the IC₅₀ values of the analogs were 33.2 μM, 45 μM, 29 μM and 30.5 μM, respectively. Among the analogs assayed only piceatannol altered the cell cycle of the parasite, increasing 5-fold the cells in the Sub-G0 phase and decreasing 1.7-fold the cells in the G0-G1 phase. Piceatannol also changed the parasite mitochondrial membrane potential (ΔΨm) and increased the number of annexin-V positive promastigotes, which suggests incidental death.

Conclusion/Significance

Among the analogs tested, piceatannol, which is a metabolite of resveratrol, was the more promising candidate for future studies regarding treatment of leishmaniasis.     

Is There a Threshold Concentration of Cat Allergen Exposure on Respiratory Symptoms in Adults?

Background and Objective

Cat allergen concentrations higher than 8 μg/g in settled house dust, have been suggested to provoke exacerbation of allergic respiratory symptoms. However, whether the 8μg/g of indoor cat allergen concentration is indeed the minimal exposure required for triggering the asthma related respiratory symptoms or the development of sensitization has not yet been confirmed. We studied the associations between domestic cat allergen concentrations and allergic symptoms in the European Community Respiratory Health Survey II, with the aim of confirming this suggested threshold.

Methods

Cat allergen concentrations were measured in the mattress dust of 3003 participants from 22 study centres. Levels of specific immunoglobulin E to cat allergens were measured in serum samples using an immunoassay. Information on allergic symptoms, medication use, home environment and smoking was obtained from a face-to-face interview.

Results

Domestic cat allergen concentrations were not associated with allergic/ asthmatic symptoms in the entire study population, nor in the subset sensitized to cat allergen. We also found no association among individuals exposed to concentrations higher than 8 μg/g. However, exposure to medium cat allergen concentrations (0.24-0.63 μg/g) was positively associated with reported asthmatic respiratory symptoms in subjects who have experienced allergic symptoms when near animals.

Conclusions

The proposed 8 μg/g threshold of cat allergen concentrations for the exacerbation of allergic/ respiratory symptoms was not confirmed in a general European adult population. Potential biases attributable to avoidance behaviours and an imprecise exposure assessment cannot be excluded.     

Fecal Calprotectin Concentrations in Healthy Children Aged 1-18 Months

Objective

Fecal calprotectin (FC) is an established biomarker of gut inflammation. The aim of this study was to evaluate FC concentrations in healthy children between 1 and 18 months of age.

Methods

Healthy children aged 1-18 months were enrolled in this study at the Department of Children''s Health Care in Shanghai, China. Children’s stool samples were collected and analyzed, and FC concentration was determined using a commercially available enzyme-linked immunosorbent assay (ELISA). The children''s weights and lengths were measured. Parents were asked to complete a brief questionnaire regarding several clinical and sociodemographic factors.

Results

The FC concentrations were unevenly distributed; the median FC concentration was 174.3 μg/g (range: 6.0-1097.7 μg/g) or 2.241 log10 μg/g (range: 0.775-3.041 log10 μg/g) for all 288 children. The children were divided into several age groups: 1-3 months, 3-6 months, 6-9 months, 9-12 months and 12-18 months. The median FC concentrations for these age groups were 375.2 μg/g (2.574 log10 μg/g), 217.9 μg/g (2.338 log10 μg/g), 127.7 μg/g (2.106 log10 μg/g), 96.1 μg/g (1.983 log10 μg/g) and 104.2 μg/g (2.016 log10 μg/g), respectively. A significant correlation between age and FC concentration was found (r=-0.490, p<0.001). A simple correlation analysis of weight-for-length Z-scores or weight-for-age Z-scores vs. FC concentrations showed that these variables were negatively correlated (Spearman’s rho=-0.287, p<0.001; Spearman’s rho=-0.243, p<0.001, respectively).

Conclusions

The FC levels of children aged 1-18 months exhibit a downward trend with increasing age and are greater than the normal levels observed in healthy adults. In healthy children aged <6 months, FC levels are high. In children aged 6-18 months, FC concentrations are relatively low but are still higher than those of children aged >4 years.     

Ocular Dominance Is Associated with the Ganglion Cell-Inner Plexiform Layer Thickness Profile in the Macula

Purpose

To investigate the characteristics of macular ganglion cell-inner plexiform layer (GCIPL) thickness profiles associated with ocular dominance.

Setting

Private practice, Seoul, Republic of Korea.

Design

Comparative case-control study.

Methods

Both eyes of 199 participants with no ophthalmic abnormalities were included. Participants were imaged by spectral-domain optical coherence tomography, and underwent dominant eye testing using a hole-in-a-card test (sighting dominance) at the same visit. Macular GCIPL, as well as circumpapillary retinal nerve fiber layer (RNFL) thickness were compared for individual patients, according to ocular dominance.

Results

Ocular dominance occurred predominantly in the right eye (right vs. left: 72.36 vs. 27.60%; P < 0.001). In the comparison of macular GCIPL thickness, the average (81.27±5.01 μm vs. 80.66±6.31 μm in dominant vs. non-dominant eyes), inferonasal (81.39±5.47μm vs. 80.33±6.82μm, and inferior sectors (77.95±6.05μm vs. 76.97±8.15μm) were significantly different between dominant and non-dominant eyes (P = 0.040, 0.005, and 0.032, respectively). Significant predictors of average GCIPL thickness were spherical equivalent (β = 1.37, P<0.001), astigmatic power (β = 1.44, P = 0.009), disc area (β = 3.90, P < 0.001), average RNFL thickness (β = 0.22, P<0.001), average cup-to-disc ratio (β = 5.74, P = 0.002), difference between the inferior and superior quadrant RNFL thicknesses (β = 0.08, P = 0.024), and ocular dominance (β = 2.10, P = 0.020). On multivariate regression analysis, ocular dominance was correlated with average GCIPL thickness after adjusting for potential confounders (β = 1.63, P = 0.048).

Conclusions

Dominant eyes accompanied significantly thicker average macular GCIPL. This information suggests that macular GCIPL thickness may provide an indicator of the relative dominance of an eye.     

Fecal Calprotectin in Healthy Children Aged 1-4 Years

Objective

Calprotectin has been well emulated recently in adults as well as in children. The aim of this study was to assess fecal calprotectin concentrations in healthy children aged from 1 to 4 years.

Methods

Volunteers were enlisted from 3 nurseries. A brief questionnaire was used to ensure these children meet the inclusion criteria, and some clinical and sociodemographic factors were collected. Anthro software (version 3.1) was used to calculated Length-for-age Z-scores (LAZ), weight-for-age Z-scores (WAZ), and weight-for-length Z-scores (WLZ) respectively. Fecal calprotectin was detected by a commercially available ELISA.

Results

In total 274 children were recruited, with age ranging from 1 to 4 years old. The median FC concentration was 83.19 μg/g [range 4.58 to 702.50 μg/g, interquartile range (IQR) 14.69–419.45 μg/g] or 1.92 log10 μg/g (range 0.66 log10 to 2.85 log10 μg/g, IQR 1.17 log10-2.62 log10 μg/g). All of the children were divided into three groups, 1–2 years (12–24 months), 2–3 years (24–36 months), 3–4 years (36–48 months), with median FC concentrations 96.14 μg/g (1.98 log10 μg/g), 81.48 μg/g (1.91 log10 μg/g), 65.36 μg/g (1.82 log10 μg/g), respectively. There was similar FC level between boys and girls. FC concentrations showed a downward trend by the growing age groups. A statistic difference was found in FC concentrations among groups 1–2 years, 2–3 years and 3–4 years (P = 0.016). In inter-groups comparison, a significant difference was found between children aged 1–2 years and children aged 3–4 years (P = 0.007). A negative correlation trend was found between age and FC concentration (Spearman''s rho = -0.167, P = 0.005) in all the participants. A simple correlation was performed among WLZ, WAZ, birth weight, or birth length with FC, and there was no correlation being observed.

Conclusion

Children aged from 1 to 4 years old have lower FC concentrations compared with healthy infants (<1years), and higher FC concentrations when comparing with children older than 4 years and adults.     

Blood Lead Levels in Children Aged 0–6 Years Old in Hunan Province,China from 2009–2013

Objectives

The aim of this study is to describe blood lead levels (BLLs) and the prevalence of elevated blood lead levels (EBLLs) in children aged 0–6 years old and to analyze the BLL trend in children from 2009 to 2013 in China.

Methods

A total of 124,376 children aged 0–6 years old were recruited for this study from January 1^st 2009 to December 31^st 2013. Their blood lead levels were analyzed using atomic absorption spectrometry.

Results

The median BLL was 64.3 μg/L (IQR: 49.6–81.0), and the range was 4.3–799.0 μg/L. Blood lead levels were significantly higher in boys (66.0 μg/L) than in girls (61.9 μg/L) (P<0.001). The overall prevalence of BLLs≥100 μg/L was 10.54% in children aged 0–6 years in Hunan Province. Between 2009 and 2013, the prevalence of EBLLs (≥100 μg/L) decreased from 18.31% to 4.26% in children aged 0–6 years and increased with age. The prevalence of EBLLs has dramatically decreased in two stages (2009–2010 and 2012–2013), with a slight fluctuation in 2010 and 2011.

Conclusions

Both BLLs and the prevalence of EBLLs in children aged 0–6 years old declined substantially from 2009 to 2013 in Hunan Province; however, both remain at unacceptably high levels compared to developed countries. Comprehensive strategies are required to further reduce blood lead levels in children.     

The Effects of Acute Exercise and Exercise Training on Plasma Homocysteine: A Meta-Analysis

Background

Although studies have demonstrated that physical exercise alters homocysteine levels in the blood, meta-analyses of the effects of acute exercise and exercise training on homocysteine blood concentration have not been performed, especially regarding the duration and intensity of exercise, which could affect homocysteine levels differently.

Objective

The aim of this meta-analysis was to ascertain the effects of acute exercise and exercise training on homocysteine levels in the blood.

Method

A review was conducted according to the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses using the online databases PubMed, SPORTDiscus, and SciELO to identify relevant studies published through June 2015. Review Manager was used to calculate the effect size of acute exercise and exercise training using the change in Hcy plasmaserum concentration from baseline to post-acute exercise and trained vs. sedentary control groups, respectively. Weighted mean differences were calculated using random effect models.

Results

Given the abundance of studies, acute exercise trials were divided into two subgroups according to exercise volume and intensity, whereas the effects of exercise training were analyzed together. Overall, 22 studies with a total of 520 participants indicated increased plasma homocysteine concentration after acute exercise (1.18 μmol/L, 95% CI: 0.71 to 1.65, p < .01). Results of a subgroup analysis indicated that either long-term exercise of low-to-moderate intensity (1.39 μmol/L, 95% CI: 0.9 to 1.89, p < .01) or short-term exercise of high intensity (0.83 μmol/L, 95% CI: 0.19 to 1.40, p < .01) elevated homocysteine levels in the blood. Increased homocysteine induced by exercise was significantly associated with volume of exercise, but not intensity. By contrast, resistance training reduced plasma homocysteine concentration (-1.53 μmol/L, 95% CI: -2.77 to -0.28, p = .02), though aerobic training did not. The cumulative results of the seven studies with a total of 230 participants in exercise training analysis did not demonstrate a significant impact on homocysteine levels in the blood (-0.56 μmol/L, 95% CI: -1.61 to 0.50, p = .23).

Conclusions

Current evidence demonstrates that acute exercise increases homocysteine levels in the blood independent of exercise duration and intensity. Resistance, but not aerobic training decreases plasma homocysteine levels.     

Hierarchy Low CD4+/CD8+ T-Cell Counts and IFN-γ Responses in HIV-1+ Individuals Correlate with Active TB and/or M.tb Co-Infection

Objective

Detailed studies of correlation between HIV-M.tb co-infection and hierarchy declines of CD8+/CD4+ T-cell counts and IFN-γ responses have not been done. We conducted case-control studies to address this issue.

Methods

164 HIV-1-infected individuals comprised of HIV-1⁺ATB, HIV-1⁺LTB and HIV-1⁺TB^- groups were evaluated. Immune phenotyping and complete blood count (CBC) were employed to measure CD4+ and CD8+ T-cell counts; T.SPOT.TB and intracellular cytokine staining (ICS) were utilized to detect ESAT6, CFP10 or PPD-specific IFN-γ responses.

Results

There were significant differences in median CD4+ T-cell counts between HIV-1⁺ATB (164/μL), HIV-1⁺LTB (447/μL) and HIV-1⁺TB^- (329/μL) groups. Hierarchy low CD4+ T-cell counts (<200/μL, 200-500/μL, >500/μL) were correlated significantly with active TB but not M.tb co-infection. Interestingly, hierarchy low CD8+ T-cell counts were not only associated significantly with active TB but also with M.tb co-infection (P<0.001). Immunologically, HIV-1⁺ATB group showed significantly lower numbers of ESAT-6-/CFP-10-specific IFN-γ+ T cells than HIV-1⁺LTB group. Consistently, PPD-specific IFN-γ+CD4+/CD8+ T effector cells in HIV-1⁺ATB group were significantly lower than those in HIV-1⁺LTB group (P<0.001).

Conclusions

Hierarchy low CD8+ T-cell counts and effector function in HIV-1-infected individuals are correlated with both M.tb co-infection and active TB. Hierarchy low CD4+ T-cell counts and Th1 effector function in HIV-1+ individuals are associated with increased frequencies of active TB, but not M.tb co-infection.     

Urinary Bladder Dysfunction in Transgenic Sickle Cell Disease Mice

Background

Urological complications associated with sickle cell disease (SCD), include nocturia, enuresis, urinary infections and urinary incontinence. However, scientific evidence to ascertain the underlying cause of the lower urinary tract symptoms in SCD is lacking.

Objective

Thus, the aim of this study was to evaluate urinary function, in vivo and ex vivo, in the Berkeley SCD murine model (SS).

Methods

Urine output was measured in metabolic cage for both wild type and SS mice (25-30 g). Bladder strips and urethra rings were dissected free and mounted in organ baths. In isolated detrusor smooth muscle (DSM), relaxant response to mirabegron and isoproterenol (1nM-10μM) and contractile response to (carbachol (CCh; 1 nM-100μM), KCl (1 mM-300mM), CaCl₂ (1μM-100mM), α,β-methylene ATP (1, 3 and 10 μM) and electrical field stimulation (EFS; 1-32 Hz) were measured. Phenylephrine (Phe; 10nM-100μM) was used to evaluate the contraction mechanism in the urethra rings. Cystometry and histomorphometry were also performed in the urinary bladder.

Results

SS mice present a reduced urine output and incapacity to produce typical bladder contractions and bladder emptying (ex vivo), compared to control animals. In DSM, relaxation in response to a selective β3-adrenergic agonist (mirabegron) and to a non-selective β-adrenergic (isoproterenol) agonist were lower in SS mice. Additionally, carbachol, α, β-methylene ATP, KCl, extracellular Ca²⁺ and electrical-field stimulation promoted smaller bladder contractions in SS group. Urethra contraction induced by phenylephrine was markedly reduced in SS mice. Histological analyses of SS mice bladder revealed severe structural abnormalities, such as reductions in detrusor thickness and bladder volume, and cell infiltration.

Conclusions

Taken together, our data demonstrate, for the first time, that SS mice display features of urinary bladder dysfunction, leading to impairment in urinary continence, which may have an important role in the pathogenesis of the enuresis and infections observed the SCD patients.     

Ketamine Causes Mitochondrial Dysfunction in Human Induced Pluripotent Stem Cell-Derived Neurons

Purpose

Ketamine toxicity has been demonstrated in nonhuman mammalian neurons. To study the toxic effect of ketamine on human neurons, an experimental model of cultured neurons from human induced pluripotent stem cells (iPSCs) was examined, and the mechanism of its toxicity was investigated.

Methods

Human iPSC-derived dopaminergic neurons were treated with 0, 20, 100 or 500 μM ketamine for 6 and 24 h. Ketamine toxicity was evaluated by quantification of caspase 3/7 activity, reactive oxygen species (ROS) production, mitochondrial membrane potential, ATP concentration, neurotransmitter reuptake activity and NADH/NAD⁺ ratio. Mitochondrial morphological change was analyzed by transmission electron microscopy and confocal microscopy.

Results

Twenty-four-hour exposure of iPSC-derived neurons to 500 μM ketamine resulted in a 40% increase in caspase 3/7 activity (P < 0.01), 14% increase in ROS production (P < 0.01), and 81% reduction in mitochondrial membrane potential (P < 0.01), compared with untreated cells. Lower concentration of ketamine (100 μM) decreased the ATP level (22%, P < 0.01) and increased the NADH/NAD⁺ ratio (46%, P < 0.05) without caspase activation. Transmission electron microscopy showed enhanced mitochondrial fission and autophagocytosis at the 100 μM ketamine concentration, which suggests that mitochondrial dysfunction preceded ROS generation and caspase activation.

Conclusions

We established an in vitro model for assessing the neurotoxicity of ketamine in iPSC-derived neurons. The present data indicate that the initial mitochondrial dysfunction and autophagy may be related to its inhibitory effect on the mitochondrial electron transport system, which underlies ketamine-induced neural toxicity. Higher ketamine concentration can induce ROS generation and apoptosis in human neurons.     

Low Goiter Rate Associated with Small Average Thyroid Volume in Schoolchildren after the Elimination of Iodine Deficiency Disorders

Background

After the implementation of the universal salt iodization (USI) program in 1996, seven cross-sectional school-based surveys have been conducted to monitor iodine deficiency disorders (IDD) among children in eastern China.

Objectives

This study aimed to examine the correlation of total goiter rate (TGR) with average thyroid volume (Tvol) and urinary iodine concentration (UIC) in Jiangsu province after IDD elimination.

Design

Probability-proportional-to-size sampling was applied to select 1,200 children aged 8–10 years old in 30 clusters for each survey in 1995, 1997, 1999, 2001, 2002, 2005, 2009 and 2011. We measured Tvol using ultrasonography in 8,314 children and measured UIC (4,767 subjects) and salt iodine (10,184 samples) using methods recommended by the World Health Organization. Tvol was used to calculate TGR based on the reference criteria specified for sex and body surface area (BSA).

Results

TGR decreased from 55.2% in 1997 to 1.0% in 2009, and geometric means of Tvol decreased from 3.63 mL to 1.33 mL, along with the UIC increasing from 83 μg/L in 1995 to 407 μg/L in 1999, then decreasing to 243 μg/L in 2005, and then increasing to 345 μg/L in 2011. In the low goiter population (TGR < 3.9%), TGR was positively associated with average Tvol (r = 0.99); UIC showed a non-linear association with average Tvol, and UIC > 300 μg/L was associated with a smaller average Tvol in children.

Conclusions

After IDD elimination in Jiangsu province in 2001, lower TGR was associated with smaller average Tvol. Average Tvol was more sensitive than TGR in detecting the fluctuation of UIC. A UIC of 300 μg/L may be defined as a critical value for population level iodine status monitoring.     

Arsenic in Drinking Water and Mortality for Cancer and Chronic Diseases in Central Italy, 1990-2010

Background

In several volcanic areas of Italy, arsenic levels exceed European regulatory limits (10 μg/L in drinking water). There is still uncertainty about health risks from arsenic at low-medium doses (<100 μg/L).

Objectives

A large population-based study using an administrative cohort of residents in the Viterbo province (Central Italy), chronically exposed to low-medium arsenic levels via drinking water, was investigated to evaluate the effects of a lifetime exposure to arsenic on mortality from cancers and chronic diseases.

Methods

The study population consisted of 165,609 residents of 17 municipalities, followed from 1990 until 2010. Average individual arsenic exposure at the first residence (As_I) was estimated through a space-time modeling approach using residential history and arsenic concentrations from water supply. A time-dependent Cumulative Arsenic dose Indicator (CAI) was calculated, accounting for daily water intake and exposure duration. Mortality Hazard Ratios (HR) were estimated by gender for different diseases using Cox proportional models, adjusting for individual and area-level confounders. A flexible non-parametric approach was used to investigate dose-response relationships.

Results

Mean As_I exposure was 19.3 μg/L, and average exposure duration was 39.5 years. Associations of As_I and CAI indicators with several diseases were found, with greatest risks found for lung cancer in both sexes (HR = 2.61 males; HR = 2.09 females), myocardial infarction, peripheral arterial disease and COPD in males (HR = 2.94; HR = 2.44; HR = 2.54 respectively) and diabetes in females (HR = 2.56). For lung cancer and cardiovascular diseases dose-response relationship is modelled by piecewise linear functions revealing effects even for doses lower than 10 μg/L, and no threshold dose value was identified as safe for health.

Conclusions

Results provide new evidence for risk assessment of low-medium concentrations of arsenic and contribute to the ongoing debate about the threshold-dose of effect, suggesting that even concentrations below 10 μg/L carry a mortality risk. Policy actions are urgently needed in areas exposed to arsenic like in the Viterbo province, to comply with current EU regulations.     

Nevirapine,Sodium Concentration and HIV-1 RNA in Breast Milk and Plasma among HIV-Infected Women Receiving Short-Course Antiretroviral Prophylaxis

Introduction

Risk factors for breast milk transmission of HIV-1 from mother to child include high plasma and breast milk viral load, low maternal CD4 count and breast pathology such as mastitis.

Objective

To determine the impact of nevirapine and subclinical mastitis on HIV-1 RNA in maternal plasma and breast milk after intrapartum single-dose nevirapine combined with either 1-week tail of Combivir (zidovudine/lamivudine) or single-dose Truvada (tenofovir/emtricitabine).

Methods

Maternal plasma and bilateral breast milk samples were collected between April 2008 and April 2011 at 1, 4 and 6 weeks postpartum from HIV-infected Tanzanian women. Moreover, plasma samples were collected at delivery from mother and infant.

Results

HIV-1 RNA was quantified in 1,212 breast milk samples from 273 women. At delivery, 96% of the women and 99% of the infants had detectable nevirapine in plasma with a median (interquartile range, IQR) of 1.5 μg/mL (0.75–2.20 μg/mL) and 1.04 μg/mL (0.39–1.71 μg/mL), respectively (P < 0.001). At 1 week postpartum, 93% and 98% of the women had detectable nevirapine in plasma and breast milk, with a median (IQR) of 0.13 μg/mL (0.13–0.39 μg/mL) and 0.22 μg/mL (0.13–0.34 μg/mL), respectively. Maternal plasma and breast milk HIV-1 RNA correlated at all visits (R = 0.48, R = 0.7, R = 0.59; all P = 0.01). Subclinical mastitis was detected in 67% of the women at some time during 6 weeks, and in 38% of the breast milk samples. Breast milk samples with subclinical mastitis had significantly higher HIV-1 RNA at 1, 4 and 6 weeks (all P < 0.05).

Conclusion

After short-course antiretroviral prophylaxis, nevirapine was detectable in most infant cord blood samples and the concentration in maternal plasma and breast milk was high through week 1 accompanied by suppressed HIV-1 RNA in plasma and breast milk.     

TLR4/NF-κB Signaling Contributes to Chronic Unpredictable Mild Stress-Induced Atherosclerosis in ApoE-/- Mice

Objective

Chronic stress is an important risk factor for atherosclerotic diseases. Our previous studies have shown that chronic unpredictable mild stress (CUMS) accelerates atherosclerosis and up-regulates TLR4/NF-κB expression in apoE^-/- mice. However, TLR4/NF-κB signaling whether directly contributes to the development of atherosclerosis in CUMS mice is unclear. We hypothesized that the interference of TLR4/NF-κB can ameliorate CUMS-induced inflammation and atherosclerosis in apoE^-/- mice.

Methods

ApoE^-/- mice were exposed to 12 weeks CUMS. Ad-siRNA TLR4 was given by tail vein injection (10 μl/mouse, every 5 days), and PDTC (an inhibitor of NF-κB) was given by intraperitoneal injection (60 mg/kg, once a day). Plasma corticosterone concentrations were determined by solid-phase ¹²⁵I radioimmunoassay. Atherosclerosis lesions in aortic sinuses were evaluated and quantified by IMAGEPRO PLUS. Western blotting was used to detect the expression of TLR4, NF-κB, and IL-1β in aortas of the mice. Plasma lipid profiles, IL-1β, TNF-α, and MCP-1 were measured by ELISA.

Results

Our results indicated that CUMS apoE^-/- mice treatment with siRNA TLR4 significantly decreased atherosclerosis and down-regulated TLR4, NF-κB, and inflammatory cytokines. PDTC also remarkably reduced atherosclerosis and the levels of IL-1β, TNF-α and MCP-1 in plasma. However, Treatment with siRNA TLR4 or PDTC had no effect on plasma corticosterone levels, and lipid profiles.

Conclusions

TLR4/NF-κB pathway may participate in CUMS-induced atherosclerosis through activation of proinflammatory cytokines in apoE^-/- mice. Our data may provide a new potential therapeutic target for prevention of CUMS -induced atherosclerosis.     

Lacrimal Cytokines Assessment in Subjects Exposed to Different Levels of Ambient Air Pollution in a Large Metropolitan Area

Background

Air pollution is one of the most environmental health concerns in the world and has serious impact on human health, particularly in the mucous membranes of the respiratory tract and eyes. However, ocular hazardous effects to air pollutants are scarcely found in the literature.

Design

Panel study to evaluate the effect of different levels of ambient air pollution on lacrimal film cytokine levels of outdoor workers from a large metropolitan area.

Methods

Thirty healthy male workers, among them nineteen professionals who work on streets (taxi drivers and traffic controllers, high pollutants exposure, Group 1) and eleven workers of a Forest Institute (Group 2, lower pollutants exposure compared to group 1) were evaluated twice, 15 days apart. Exposure to ambient PM_2.5 (particulate matter equal or smaller than 2.5 μm) was 24 hour individually collected and the collection of tears was performed to measure interleukins (IL) 2, 4, 5 and 10 and interferon gamma (IFN-γ) levels. Data from both groups were compared using Student’s t test or Mann- Whitney test for cytokines. Individual PM_2.5 levels were categorized in tertiles (lower, middle and upper) and compared using one-way ANOVA. Relationship between PM_2.5 and cytokine levels was evaluated using generalized estimating equations (GEE).

Results

PM_2.5 levels in the three categories differed significantly (lower: ≤22 μg/m³; middle: 23–37.5 μg/m³; upper: >37.5 μg/m³; p<0.001). The subjects from the two groups were distributed unevenly in the lower category (Group 1 = 8%; Group 2 = 92%), the middle category (Group 1 = 89%; Group 2 = 11%) and the upper category (Group 1 = 100%). A significant relationship was found between IL-5 and IL-10 and PM_2.5 levels of the group 1, with an average decrease of 1.65 pg/mL of IL-5 level and of 0.78 pg/mL of IL-10 level in tear samples for each increment of 50 μg/m3 of PM_2.5 (p = 0.01 and p = 0.003, respectively).

Conclusion

High levels of PM_2.5 exposure is associated with decrease of IL-5 and IL-10 levels suggesting a possible modulatory action of ambient air pollution on ocular surface immune response.     

The p70S6K Specific Inhibitor PF-4708671 Impedes Non-Small Cell Lung Cancer Growth

Background

As a serine/threonine protein kinase, p70S6K plays an important role in tumor cells. Evidence has revealed overexpression of p70S6K and phosphorylated p70S6K (p-p70S6K) in various tumor tissues, with these proteins identified as independent prognostic markers in non-small cell lung cancer (NSCLC). In this study, we explored the role of the p70S6K specific inhibitor PF-4708671 in NSCLC.

Methods

Three NSCLC cell lines (A549, SK-MES-1, and NCI-H460) were treated with PF-4708671 at five different concentrations, including 0.1μM, 0.3μM, 1μM, 3μM and 10μM, and protein levels were determined by Western-blot. Then, PF-4708671’s effects were assessed both in vitro (cell proliferation, apoptosis, cell cycle distribution, and invasion) and in vivo.

Results

The expression levels of p-p70S6K and the downstream effector S6 were significantly reduced by PF-4708671. Diametrically opposite, the downstream protein levels of BAD, Caspase3 and ERK had increased after treatment with PF-4708671. In addition, PF-4708671 drastically inhibited cell proliferation and invasion ability in A549, SK-MES-1 and NCI-H460 cells in vitro, causing cell cycle arrest in G0-G1 phase. Limited effects of PF-4708671 were observed on apoptosis in the three NSCLC cell lines assessed. Importantly, PF-4708671 could inhibit tumorigenesis in nude mice in vivo.

Conclusion

These findings demonstrated that the p70S6K specific inhibitor PF-4708671 has inhibitory effects on NSCLC tumorigenesis in vitro and in vivo. Therefore, P70S6K should be considered a new potential therapeutic target, and PF-470867 may be used as targeted drug for cancer treatment.     

Attention Deficit/Hyperactivity Disorder and Urinary Nonylphenol Levels: A Case-Control Study in Taiwanese Children

Objective

Nonylphenol (NP) belongs to the family of endocrine disruptors, and it is widely used in industrial applications and is ubiquitous in daily foods. Animal studies have suggested that NP exposure might promote motor hyperactivity, likely by causing deficits in dopaminergic neurons. However, research assessing NP exposure and epidemiology studies on human populations are limited. The aim of this study was to explore the association between child NP exposure and ADHD while considering particular covariants, such as lead levels and dopamine-related gene variations.

Methods

A case-control study was conducted on patients with clinically diagnosed ADHD; the Swanson, Nolan and Pelham, Fourth Revision (SNAP-IV) questionnaire was used to identify normal controls aged 4–15 years. Participants were examined for urinary NP concentrations, blood lead levels, and select single-nucleotide polymorphisms of two dopamine-related genes (D4 dopamine receptor, DRD4, and dopamine transporter, DAT1). Socio-demographic variables, maternal lifestyle factors during pregnancy and family medical history were obtained using a questionnaire.

Results

A total of 97 children with doctor-diagnosed ADHD and 110 normal controls were enrolled. The blood lead levels in both groups were similar (1.57±0.73 vs. 1.73±0.77 μg/dL, p = 0.15). No significant difference in urinary NP concentration was found between the children with ADHD and the control subjects (4.52±3.22 μg/g cr. vs. 4.64±2.95 μg/g cr., p = 0.43). ADHD was significantly more prevalent among males in this study (male to female ratio: 5:1 for the ADHD group and 1.3:1 for the control group, p<0.01). The analysis was repeated after excluding the females, but this had no effect on the association between NP and ADHD. The regression model, including or excluding females, indicated no increased odds of having ADHD in the context of NP exposure after adjusting for covariants.

Conclusion

This study indicated that NP exposure might not promote ADHD in children, even though children in Taiwan had relatively high levels of NP compared to those reported previously and those in developed nations.     

Nasal Lipopolysaccharide Challenge and Cytokine Measurement Reflects Innate Mucosal Immune Responsiveness

Background

Practical methods of monitoring innate immune mucosal responsiveness are lacking. Lipopolysaccharide (LPS) is a component of the cell wall of Gram negative bacteria and a potent activator of Toll-like receptor (TLR)-4. To measure LPS responsiveness of the nasal mucosa, we administered LPS as a nasal spray and quantified chemokine and cytokine levels in mucosal lining fluid (MLF).

Methods

We performed a 5-way cross-over, single blind, placebo-controlled study in 15 healthy non-atopic subjects (n = 14 per protocol). Doses of ultrapure LPS (1, 10, 30 or 100μg/100μl) or placebo were administered by a single nasal spray to each nostril. Using the recently developed method of nasosorption with synthetic adsorptive matrices (SAM), a series of samples were taken. A panel of seven cytokines/chemokines were measured by multiplex immunoassay in MLF. mRNA for intercellular cell adhesion molecule-1 (ICAM-1) was quantified from nasal epithelial curettage samples taken before and after challenge.

Results

Topical nasal LPS was well tolerated, causing no symptoms and no visible changes to the nasal mucosa. LPS induced dose-related increases in MLF levels of IL-1β, IL-6, CXCL8 (IL-8) and CCL3 (MIP-1α) (AUC at 0.5 to 10h, compared to placebo, p<0.05 at 30 and 100μg LPS). At 100μg LPS, IL-10, IFN-α and TNF-α were also increased (p<0.05). Dose-related changes in mucosal ICAM-1 mRNA were also seen after challenge, and neutrophils appeared to peak in MLF at 8h. However, 2 subjects with high baseline cytokine levels showed prominent cytokine and chemokine responses to relatively low LPS doses (10μg and 30μg LPS).

Conclusions

Topical nasal LPS causes dose-dependent increases in cytokines, chemokines, mRNA and cells. However, responsiveness can show unpredictable variations, possibly because baseline innate tone is affected by environmental factors. We believe that this new technique will have wide application in the study of the innate immune responses of the respiratory mucosa.

Key Messages

Ultrapure LPS was used as innate immune stimulus in a human nasal challenge model, with serial sampling of nasal mucosal lining fluid (MLF) by nasosorption using a synthetic absorptive matrix (SAM), and nasal curettage of mucosal cells. A dose response could be demonstrated in terms of levels of IL-1β, IL-6, CXCL8 and CCL3 in MLF, as well as ICAM-1 mRNA in nasal curettage specimens, and levels of neutrophils in nasal lavage. Depending on higher baseline levels of inflammation, there were occasional magnified innate inflammatory responses to LPS.

Trial Registration

Clinical Trials.gov NCT02284074