Effects of carbon nanotubes on barrier epithelial cells via effects on lipid bilayers

Indiana University-Purdue University Indianapolis (IUPUI)Carbon nanotubes (CNTs) are one of the most common nanoparticles (NP) found in workplace air. Therefore, there is a strong chance that these NP will enter the human body. They have similar physical properties to asbestos, a known toxic material, yet there is limited evidence showing that CNTs may be hazardous to human barrier epithelia. In previous studies done in our laboratory, the effects of CNTs on the barrier function in the human airway epithelial cell line (Calu-3) were measured. Measurements were done using electrophysiology, a technique which measures both transepithelial electrical resistance (TEER), a measure of monolayer integrity, and short circuit current (SCC) which is a measure of vectorial ion transport across the cell monolayer. The research findings showed that select physiologically relevant concentrations of long single-wall (SW) and multi-wall (MW) CNTs significantly decreased the stimulated SCC of the Calu-3 cells compared to untreated cultures. Calu-3 cells showed decreases in TEER when incubated for 48 hours (h) with concentrations of MWCNT ranging from 4µg/cm2 to 0.4ng/cm2 and SWCNT ranging from 4µg/cm2 to 0.04ng/cm2. The impaired cellular function, despite sustained cell viability, led us to investigate the mechanism by which the CNTs were affecting the cell membrane. We investigated the interaction of short MWCNTs with model lipid membranes using an ion channel amplifier, Planar Bilayer Workstation. Membranes were synthesized using neutral diphytanoylphosphatidylcholine (DPhPC) and negatively charged diphytanoylphosphatidylserine (DPhPS) lipids. Gramicidin A (GA), an ion channel reporter protein, was used to measure changes in ion channel conductance due to CNT exposures. Synthetic membranes exposed to CNTs allowed bursts of currents to cross the membrane when they were added to the membrane buffer system. When added to the membrane in the presence of GA, they distorted channel formation and reduced membrane stability

The needs of informal grandparent caregivers and how they are met at the Kinship Family Center

Seventeen grandparent caregivers, who have primary responsibility for one or more grandchildren and do not have a parent of the grandchildren living in the household, were interviewed to evaluate the effectiveness of the services offered by Kinship Family Center (KFC), a non-profit support agency for relative caregivers and the children in their care. Kinship Family Center is a program of the Central City Lutheran Mission and is funded by the San Bernardino County Department of Children\u27s Services. The researchers found that KFC was an effective asset for informal grandparent caregivers. For grandparents, it was a source of support and comfort in their role of second time around parents. For children, it served as a means to help identify with other children in similar situations

Bioinformatic Analysis of Proteomic Changes That Occur in an Airway Epithelial Cell Line in Response to Exposure to Physiologically Relevant Concentrations of Carbon Nanotubes

poster abstractCarbon nanomaterials are widely produced and used in industry, medicine and scientific research. To examine the impact of acute exposure to nanoparticles on human health, the human airway epithelial cell line, Calu-3, was used to evaluate potential alterations in cellular function of airway epithelia after 24 hours exposure to different concentrations of two common carbon nanoparticles, single- and multi-wall carbon nanotubes (SWCNT, MWCNT). After exposure to the nanoparticles, label-free quantitative mass spectrometry (LFQMS) was used to study the differential protein expression in Calu-3 cells. Ingenuity Pathway Analysis (IPA) was used to conduct a bioinformatic analysis of proteins identified in LFQMS. Changes in protein abundance generated in response to 100 ng/ml exposure of both MWCNT and SWCNT suggest that cell functions of cell death and survival, cell-to-cell signaling and interaction, cellular assembly and organization, cellular growth and proliferation, infectious disease, molecular transport and protein synthesis are predicted to be effected. The majority of the protein changes represent a decrease in amount suggesting a shut down of metabolism to protect cells. The STRING database was used to analyze the protein networks in different functions. Interestingly some proteins like cadherin 1 (CDH1), signal transducer and activator of transcription 1 (STAT1), junction plakoglobin (JUP), apoptosis-associated speck-like protein containing a CARD (PYCARD), appear in several functions and tend to be in the center of the networks, which suggest they may play important roles in the cell function and activity

Effects of Carbon Nanotubes in Barrier Epithelial Cells via Effects on Lipid Bilayers

poster abstractCarbon nanotubes (CNTs) are one of many nanoparticles (NP) which are being developed as part of the burgeoning nanotechnology. The tubes have similar physical properties to known toxic materials, such as asbestos; yet there is a lack of evidence showing that they may be hazardous to humans, specifically to our barrier epithelial cells. We measured the effects of CNTs on human airway epithelial cells (Calu-3 cell line) using electrophysiology. This is a technique which measures transepithelial electrical resistance (TEER), a measure of monolayer integrity; and short circuit current (SCC) a measure of net ion transport across the cell. Exposed cells showed significant decreases in TEER when incubated for 48 hours with physiologically relevant concentrations of 4μg/cm2 - 0.4ng/cm2 of multi-wall (MW) and 4μg/cm2 - 0.04ng/cm2 single-wall (SW) CNT. TEER is a measure of barrier function which is important in cells that maintain separate compartments in the body. The impaired barrier function, despite sustained cell viability, led us to investigate the mechanism by which the CNT were interacting with the cell when applied topically. Model lipid membranes connected to an ion channel amplifier, Planar Bilayer Workstation (BLM), were used. Membranes were formed using the neutral diphytanoylphosphatidylcholine (DPhPC) and negatively charged diphytanoyl phosphatidylserine (DPhPS) lipids. CNTs caused random, transient currents ranging from 0pA to 6479pA to traverse the membrane. In the presence of Gramicidin A, an ion channel reporter protein, the tubes induced increased gramicidin channel formation in the membrane to saturation level and then membrane lysis. This CNT- lipid interaction indicated that short MWCNTs permits unregulated ion movement across the lipid membrane. Disruption in the selective permeability of the plasmalemma may impact the tissue’s barrier function

Effect of carbon nanotubes on transepithelial resistance in barrier epithelial cells

poster abstractThe burgeoning of the nanotechnology industry has revolutionized engineering, medicine and the fashion industry amongst many other technologies. The arrays of products that are synthesized from nanomaterials include high definition TV and computer screens, artificial organs, as well as antibacterial food containers. With these novel applications there is a paralleled concern of the negative implications of nano-material contamination in our environment and food chains. We are interested in carbon nanotubes because they are the most abundantly occurring nanoparticles that are found in the workplace. We have recently conducted studies in barrier epithelial cells to show that long single-wall and multi-wall carbon nanotubes (CNTs) caused a decrease in the transepithelial electrical resistance, a measure of the barrier function ,of renal principal cells at very low concentrations (0.4 ng/cm2- 4 g/cm2). These results suggested that nanoparticles may also cause an effect on other barrier epithelial cells such as those lining the human digestive and respiratory tracks. After 48 hours of CNT exposure, to airway and colon cell lines, Calu-3 and t84 respectively, the calculated resistances were approximately half of the control monolayers’, indicating that the barrier function of the tissue had been compromised, while the cellular monolayer remained intact. We sought to determine the mechanism of action of the nanoparticles, by investigating the interaction of CNTs with model lipid membranes using a bilayer clamp amplifier. Measurements showed that the presence of nanoparticles caused transient disruptions in lipid membranes made of phosphatidylcholine lipids. Nanotubes also caused transient interruptions in the current allowed by the ion channel reporter (gramicidin A). These results began to elucidate the mode of action of the particles and indicated that it is important to develop a complete understanding of how nanoparticles interact with cells if we are to safeguard against changes that these materials will cause in vivo

Epinephrine stimulation of anion secretion in the Calu-3 serous cell model

Calu-3 is a well-differentiated human bronchial cell line with the characteristics of the serous cells of airway submucosal glands. The submucosal glands play a major role in mucociliary clearance because they secrete electrolytes that facilitate airway hydration. Given the significance of both long- and short-term β-adrenergic receptor agonists in the treatment of respiratory diseases, it is important to determine the role of these receptors and their ligands in normal physiological function. The present studies were designed to characterize the effect of epinephrine, the naturally occurring β-adrenergic receptor agonist, on electrolyte transport of the airway serous cells. Interestingly, epinephrine stimulated two anion secretory channels, the cystic fibrosis transmembrane conductance regulator and a Ca2+-activated Cl− channel, with the characteristics of transmembrane protein 16A, thereby potentially altering mucociliary clearance via multiple channels. Consistent with the dual channel activation, epinephrine treatment resulted in increases in both intracellular cAMP and Ca2+. Furthermore, the present results extend previous reports indicating that the two anion channels are functionally linked

The study of women, infant feeding and type 2 diabetes after GDM pregnancy and growth of their offspring (SWIFT Offspring study): prospective design, methodology and baseline characteristics

Abstract Background Breastfeeding is associated with reduced risk of becoming overweight or obese later in life. Breastfed babies grow more slowly during infancy than formula-fed babies. Among offspring exposed in utero to maternal glucose intolerance, prospective data on growth during infancy have been unavailable. Thus, scientific evidence is insufficient to conclude that breastfeeding reduces the risk of obesity among the offspring of diabetic mothers (ODM). To address this gap, we devised the Study of Women, Infant Feeding and Type 2 Diabetes after GDM Pregnancy and Growth of their Offspring, also known as the SWIFT Offspring Study. This prospective, longitudinal study recruited mother-infant pairs from the SWIFT Study, a prospective study of women with recent gestational diabetes mellitus (GDM). The goal of the SWIFT Offspring Study is to determine whether breastfeeding intensity and duration, compared with formula feeding, are related to slower growth of GDM offspring during the first year life. This article details the study design, participant eligibility, data collection, and methodologies. We also describe the baseline characteristics of the GDM mother-infant pairs. Methods The study enrolled 466 mother-infant pairs among GDM deliveries in northern California from 2009–2011. Participants attended three in-person study exams at 6–9 weeks, 6 months and 12 months after delivery for infant anthropometry (head circumference, body weight, length, abdominal circumference and skinfold thicknesses), as well as maternal anthropometry (body weight, waist circumference and percent body fat). Mothers also completed questionnaires on health and lifestyle behaviors, including infant diet, sleep and temperament. Breastfeeding intensity and duration were assessed via several sources (diaries, telephone interviews, monthly mailings and in-person exams) from birth through the first year of life. Pregnancy course, clinical perinatal and newborn outcomes were obtained from health plan electronic medical records. Infant saliva samples were collected and stored for genetics studies. Discussion This large, racially and ethnically diverse cohort of GDM offspring will enable evaluation of the relationship of infant feeding to growth during infancy independent of perinatal characteristics, sociodemographics and other risk factors. The longitudinal design provides the first quantitative measures of breastfeeding intensity and duration among GDM offspring during early life

Rare copy number variation in posttraumatic stress disorder

Posttraumatic stress disorder (PTSD) is a heritable (h2 = 24-71%) psychiatric illness. Copy number variation (CNV) is a form of rare genetic variation that has been implicated in the etiology of psychiatric disorders, but no large-scale investigation of CNV in PTSD has been performed. We present an association study of CNV burden and PTSD symptoms in a sample of 114,383 participants (13,036 cases and 101,347 controls) of European ancestry. CNVs were called using two calling algorithms and intersected to a consensus set. Quality control was performed to remove strong outlier samples. CNVs were examined for association with PTSD within each cohort using linear or logistic regression analysis adjusted for population structure and CNV quality metrics, then inverse variance weighted meta-analyzed across cohorts. We examined the genome-wide total span of CNVs, enrichment of CNVs within specified gene-sets, and CNVs overlapping individual genes and implicated neurodevelopmental regions. The total distance covered by deletions crossing over known neurodevelopmental CNV regions was significant (beta = 0.029, SE = 0.005, P = 6.3 × 10-8). The genome-wide neurodevelopmental CNV burden identified explains 0.034% of the variation in PTSD symptoms. The 15q11.2 BP1-BP2 microdeletion region was significantly associated with PTSD (beta = 0.0206, SE = 0.0056, P = 0.0002). No individual significant genes interrupted by CNV were identified. 22 gene pathways related to the function of the nervous system and brain were significant in pathway analysis (FDR q < 0.05), but these associations were not significant once NDD regions were removed. A larger sample size, better detection methods, and annotated resources of CNV are needed to explore this relationship further

Effects of Carbon Nanotubes in Barrier Epithelial Cells via Effects on Lipid Bilayers

Carbon nanotubes (CNTs) are one of the most common nanoparticles (NP) found in workplace air. Therefore, there is a strong chance that these NP will enter the human body. They have similar physical properties to asbestos, a known toxic material, yet there is limited evidence showing that CNTs may be hazardous to human barrier epithelia. In previous studies done in our laboratory, the effects of CNTs on the barrier function in the human airway epithelial cell line (Calu-3) were measured. Measurements were done using electrophysiology, a technique which measures both transepithelial electrical resistance (TEER), a measure of monolayer integrity, and short circuit current (SCC) which is a measure of vectorial ion transport across the cell monolayer. The research findings showed that select physiologically relevant concentrations of long single-wall (SW) and multi-wall (MW) CNTs significantly decreased the stimulated SCC of the Calu-3 cells compared to untreated cultures. Calu-3 cells showed decreases in TEER when incubated for 48 hours (h) with concentrations of MWCNT ranging from 4µg/cm2 to 0.4ng/cm2 and SWCNT ranging from 4µg/cm2 to 0.04ng/cm2. The impaired cellular function, despite sustained cell viability, led us to investigate the mechanism by which the CNTs were affecting the cell membrane. We investigated the interaction of short MWCNTs with model lipid membranes using an ion channel amplifier, Planar Bilayer Workstation. Membranes were synthesized using neutral diphytanoylphosphatidylcholine (DPhPC) and negatively charged diphytanoylphosphatidylserine (DPhPS) lipids. Gramicidin A (GA), an ion channel reporter protein, was used to measure changes in ion channel conductance due to CNT exposures. Synthetic membranes exposed to CNTs allowed bursts of currents to cross the membrane when they were added to the membrane buffer system. When added to the membrane in the presence of GA, they distorted channel formation and reduced membrane stability

Complement modulation in the retinal pigment epithelium rescues photoreceptor degeneration in a mouse model of Stargardt disease

Recessive Stargardt macular degeneration (STGD1) is caused by mutations in the gene for the ABCA4 transporter in photoreceptor outer segments. STGD1 patients and Abca4-/- (STGD1) mice exhibit buildup of bisretinoid-containing lipofuscin pigments in the retinal pigment epithelium (RPE), increased oxidative stress, augmented complement activation and slow degeneration of photoreceptors. A reduction in complement negative regulatory proteins (CRPs), possibly owing to bisretinoid accumulation, may be responsible for the increased complement activation seen on the RPE of STGD1 mice. CRPs prevent attack on host cells by the complement system, and complement receptor 1-like protein y (CRRY) is an important CRP in mice. Here we attempted to rescue the phenotype in STGD1 mice by increasing expression of CRRY in the RPE using a gene therapy approach. We injected recombinant adeno-associated virus containing the CRRY coding sequence (AAV-CRRY) into the subretinal space of 4-wk-old Abca4-/- mice. This resulted in sustained, several-fold increased expression of CRRY in the RPE, which significantly reduced the complement factors C3/C3b in the RPE. Unexpectedly, AAV-CRRY-treated STGD1 mice also showed reduced accumulation of bisretinoids compared with sham-injected STGD1 control mice. Furthermore, we observed slower photoreceptor degeneration and increased visual chromophore in 1-y-old AAV-CRRY-treated STGD1 mice. Rescue of the STGD1 phenotype by AAV-CRRY gene therapy suggests that complement attack on the RPE is an important etiologic factor in STGD1. Modulation of the complement system by locally increasing CRP expression using targeted gene therapy represents a potential treatment strategy for STGD1 and other retinopathies associated with complement dysregulation