The epithelial to mesenchymal transition (EMT) represents a crucial event during cancer progression and dissemination. EMT is the conversion of carcinoma cells from an epithelial to a mesenchymal phenotype that associates with a higher cell motility as well as enhanced chemoresistance and cancer stemness. Notably, EMT has been increasingly recognized as an early event of metastasis. Numerous gene expression studies (GES) have been conducted to obtain transcriptome signatures and marker genes to understand the regulatory mechanisms underlying EMT. Yet, no meta-analysis considering the multitude of GES of EMT has been performed to comprehensively elaborate the core genes in this process. Here we report the meta-analysis of 18 independent and published GES of EMT which focused on different cell types and treatment modalities. Computational analysis revealed clustering of GES according to the type of treatment rather than to cell type. GES of EMT induced via transforming growth factor-β and tumor necrosis factor-α treatment yielded uniformly defined clusters while GES of models with alternative EMT induction clustered in a more complex fashion. In addition, we identified those up- and downregulated genes which were shared between the multitude of GES. This core gene list includes well known EMT markers as well as novel genes so far not described in this process. Furthermore, several genes of the EMT-core gene list significantly correlated with impaired pathological complete response in breast cancer patients. In conclusion, this meta-analysis provides a comprehensive survey of available EMT expression signatures and shows fundamental insights into the mechanisms that are governing carcinoma progression. The epithelial to mesenchymal transition (EMT) has been originally described as an essential process of metazoan embryogenesis EMT has been classified into three subtypes Profiling of the transcriptome using microarrays has been widely used to elucidate the expression patterns during EMT under different conditions which revealed novel biomarkers and molecular mechanisms from single studies. A meta-analysis usually describes the combination of a large number of studies from different samples and tissues or the comparison of own data with published data Changes in a biological system require a concerted alteration of gene expression sets. Bioinformatic enrichment analysis tools investigate gene expression sets for such changes. These tools examine the overrepresentation of gene sets in comparison to the whole genome, map an input list of genes to biological categories in online databases and statistically assess the overrepresentation of genes for each biological category or annotation such as Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and gene ontology (GO) terms We gathered data from 18 published and independent GES of EMT and extracted gene lists of significantly up- and downregulated genes for cluster analysis. This approach revealed gene clusters according to treatment modalities rather than to cell type. We subsequently extracted an EMT-core list consisting of 130 genes with official gene symbols and names which was further investigated by enrichment analysis with several single enrichment tools. Notably, selected genes from the EMT-core list significantly correlated with impaired pathological complete response (pCR) in breast cancer patients. This analysis proposes that the EMT-core gene list is relevant for the recognition of the molecular mechanisms of EMT. In addition, the cluster analysis shows novel insights into the relationships of EMT processes across different cell types and induction modes.et al. published two related GES, of which one consisted of two datasets, resulting in three datasets of one independent study et al. reported 5 datasets published within one GES with similar expression patterns and different modes of EMT induction t-test was used to compute p-values. Significantly up- and downregulated genes were selected to meet a fold change greater than 2 or lower than 0.5 and a p-value below 0.05.To assess the similarities between published GES and define a core gene list of human EMT, we analyzed 18 independent GES of EMT. These 18 independent and published GES consisted of 24 datasets in total . SeveralWe generated a matrix containing gene symbols across the analyzed GES  that are all uniquely reported. Significantly up- and downregulated genes of each GES were transferred into the matrix according to their type of regulation. Upregulated genes were labeled with 1, downregulated genes with −1 and not differentially regulated genes with 0 . This daBased on the cluster analysis of the GES, we aimed to define a meaningful EMT-core gene list which describes the majority of the involved genes across the analyzed GES. The cluster analysis of the genes shared between at least 10 datasets contained 365 genes . HoweverTo further analyze the EMT-core list consisting of 130 genes, a rigorous single enrichment analysis combined with stringent selection criteria was performed. First, an enriched KEGG pathway or GO term had to contain at least 5 genes from the input list and a p-value below 0.05 to be considered significant. An enumeration of significantly enriched terms and pathways is shown in The EMT-core gene list contains several genes with yet unidentified roles in cancer progression and/or EMT. We aimed to investigate the clinical relevance of this selection of genes. Therefore, we correlated their expression with overall survival of patients suffering from squamous cell lung carcinomas (SCC) Over the past decade a considerable number of GES that deal with EMT have been accumulating in the literature. These cover a variety of cell types which display EMT and include different modes of EMT induction. So far, these resources have only been partially used to compare single findings with those in the literature Cluster analysis of genes shared between at least 10 GES datasets revealed clusters of GES with the same or a similar treatment type. The GES in which EMT was induced by TNF-α either alone or in combination with TGF-β, by TGF-β alone or by different transcription factors consistently grouped together. These clusters persisted when genes shared between at least 14 datasets were used for cluster analysis. A clear clustering of different types of EMT induction, however, would have only been possible if an adequate number of GES on each of these EMT initiation methods existed. Since several treatment modalities are only represented once in the literature, such GES cluster to their most related treatment type.et al; GSE23655; et al; GSE24261; et al. , TNF-α together with TGF-β (GSE12548) or TGF-β alone (GSE15205_TGFb) in order to induce EMT The cluster of GES with TNF-α as EMT inducer contained the study by Takahashi et al.  such as array IDs, expressed sequence tags and locus IDs. We used consistently enriched pathway analysis to further investigate these gene lists. Notably, our EMT-core list displayed more enriched KEGG pathways and GO terms than the gene lists of Choi et al. and Taube et al.  and AE (available: http://www.ebi.ac.uk/arrayexpress/) by using “EMT” as keyword for published GES until February 2012. The downloaded GES were annotated to retrieve official gene symbols, EntrezID and gene names using BioConductor 2.9  http://www.affymetrix.com/analysis/index.affx; accessed: 2012 June 25). BioConductor was used within the R environment t-test were calculated. Significantly up- and downregulated genes were selected and separated from each other when showing a fold change greater than 2 or below 0.5 and a p-value below 0.05. Upregulated genes were ordered from highest to lowest fold change. Vice versa, downregulated genes were arranged from lowest to highest fold change. Duplicates were removed afterwards. Gene symbols have been used for further analysis and will be referred to as genes.Processed microarray data were downloaded from the websites of GEO  by chance with the binomial distribution function provided by R (probability = 11.78%). The possibilities to draw each cutoff option by chance were compared to preliminary cluster analyses of each cutoff option in order to determine a suitable cutoff. The clustering was performed in BioConductor 2.9 embedded in R 2.14.1 (64 bit) with the packages gdata et al. with the data from FatiGO The gene lists were analyzed using five different bioinformatic enrichment tools. A comprehensive overview of the used tools and their characteristics is shown in et al.et al.http://bioinformatics.mdanderson.org/pubdata.html; accessed 2012 Sep 07). Patients were divided into high and low expressing groups for selected genes within the EMT-core list. The p-values were computed using two-sided Student's t-test. Survival analysis for the data by Raponi et al. was performed with the chi-squared test of equality using the survival package in R Microarray and clinical data for patients with squamous cell lung carcinomas (n = 130) reported by Raponi Figure S1Cluster analysis of genes shared between at least 14 GES datasets shows persistent and distinct clusters.(PDF)Click here for additional data file.Figure S2The 130 genes EMT-core list and the 365 genes list exhibit comparable enrichment ratios of GO molecular functions.(PDF)Click here for additional data file.Figure S3Flow chart depicting the generation of the EMT-core gene list.(PDF)Click here for additional data file.Table S1Matrix containing significantly up- and downregulated genes across the analyzed GES datasets.(XLS)Click here for additional data file.Table S2List of 365 genes significantly regulated in at least 10 GES datasets.(DOC)Click here for additional data file.Table S3EMT-core gene list of 130 up- or downregulated genes shared between at least 10 GES datasets.(DOC)Click here for additional data file.Table S4Enrichment tools used in this study and their properties.(DOC)Click here for additional data file.


It is now widely recognized that radiotherapy of thoracic and chest wall tumors increases the long-term risk of cardiovascular damage although the underlying mechanisms are not fully elucidated. There is increasing evidence that microvascular damage is involved. Endoglin, an accessory receptor for TGF-β1, is highly expressed in damaged endothelial cells and may play a crucial role in cell proliferation and revascularization of damaged heart tissue. We have therefore specifically examined the role of endoglin in microvascular damage and repair in the irradiated heart.+/+ or Eng+/− mice and damage was evaluated at 4, 20 and 40 weeks, relative to age-matched controls. Gated single photon emission computed tomography (gSPECT) was used to measure cardiac geometry and function, and related to histo-morphology, microvascular damage (detected using immuno- and enzyme-histochemistry) and gene expression .A single dose of 16 Gy was delivered to the heart of adult Eng+/− mice compared to Eng+/+ littermates. Fibrosis related genes, TGF-β1, ALK 5 and PDGF, were only upregulated in Eng+/+ mice during the early phase of radiation-induced cardiac damage (4 weeks). In addition, only the Eng+/+ mice showed significant upregulation of collagen deposition in the early fibrotic phase (20 weeks) after irradiation. Despite these differences in gene expression, there was no reduction in inflammatory invasion (CD45+cells) of irradiated Eng+/− hearts. Microvascular damage  was also similar in both strains.Genes categorized according to known inflammatory and immunological related disease were less prominently regulated in irradiated Eng+/− mice displayed impaired early inflammatory and fibrotic responses to high dose irradiation compared to Eng+/+ littermates. This did not result in significant differences in microvascular damage or cardiac function between the strains.Eng Nearly 5 million long-term cancer survivors were registered in 2007 in the United States alone and at least half of these patients underwent radiotherapy as part of their cancer treatment. Although radiotherapy is an effective cancer treatment, it can contribute to late toxicity in surrounded normal tissue. Much work has been done to reduce the risk of late normal tissue toxicity induced by radiotherapy over the last decade, including modified fractionation schedules and conformal image-guided-radiotherapy (IGRT). Moreover, knowledge about the molecular mechanisms underlying the development of normal tissue toxicity after radiotherapy is increasing and this should eventually help in designing methods to prevent or treat normal tissue toxicity. Nevertheless, little is known of the underlying molecular mechanism of radiation-induced cardiac toxicity in thoracic cancer patients.+/− mice and compared this to damage in wild type littermates.We and others +/− C57BL/6 mice were originally obtained from H. Arthur  and subsequently bred in the Netherlands Cancer Institute. Male Eng+/+ mice and Eng+/− littermates aged 8–12 weeks were randomly allocated (after genotyping by PCR) to receive 16 Gy or 0 Gy to the heart. Mice were housed in a temperature-controlled room with 12 hour light-dark cycle. Standard mouse chow and water were provided ad libitum. Irradiation was performed with 250 kV X-rays, operating at 12 mA and filtered with 0.6 mm Copper. The dose rate was 0.94 Gy/min with a field size of 10.6x 15 mm (including up to 30% lung volume) and the rest of the mouse was shielded with lead. Unanesthetized mice were immobilized in a prone position in acrylic perspex jigs. Separate cohorts of animals were included for analyses at 4, 20 and 40 weeks after irradiation, with age matched controls (sham irradiated with 0 Gy). Each cohort typically comprised 10 to 15 mice . This study was in agreement with the Dutch law on animal experiments and welfare, whereby the Animal Experiments Committee (AEC) of the Netherlands Cancer Institute has evaluated the set-up of the experiments and has given a positive recommendation (Permit number: 08008–1990), and in line with the international Guide for the Care and Use of Laboratory Animals (Eighth edition). No severe suffering was anticipated in this study. If mice appeared distressed, or lost >15% body weight, they were humanely sacrificed before the planned follow-up time. At termination of the experiment, mice were humanely sacrificed under lethal sodium pentobarbital anesthesia .EngTotal RNA was isolated from frozen sections (30 sections of 30 µm per mouse and 4–7 mice per group) of the mid part of the heart using Trizol® Reagent , according to the manufacturer’s protocol. The quantity of total RNA was measured using a spectrophotometer  followed by a quality check measured by a Agilent 2100 Bioanalyzer with the RNA Integrity Number (RIN) . Samples with a RIN above 7 were used for DNAse treatment and amplified (350 ng per sample) using Illumina Totalprep RNA Amplification kit . Before hybridization, individual RNA was pooled for each treatment group. Hybridization of aRNA to Illumina Expression Bead Chips Mouse Whole Genome (WG-6 vs. 2.0) and subsequent washing, blocking and detecting were performed according to the manufacturer’s protocol . Samples were scanned on the IlluminaR BeadArray™ 500GX Reader using IlluminaR BeadScan image data acquisition software (version 2.3.0.13). MouseWG-6 vs. 2.0 BeadChip contains the full set of MouseRef-8 BeadChip probes with additional 11,603 probes from RIKEN FANTOM2, NCBI REfSeq as well from the MEEBO database.Before analyzing, the database was normalized using robust spline normalization method within the microarray facility of the Netherlands Cancer Institute. Log2 ratio between expression of genes from control mice and expression of genes from irradiated mice were calculated using Excel version 2003, as well as the sum of the expression of genes from both control and irradiated mice. According to the sum of both expressions, genes with sums below 6 were discarded. The threshold for standard deviation (SD) was set to 3 and mean ± nSD was calculated to identify genes that are above expression 6 and above threshold 3 of SD. These genes were further analyzed in Ingenuity Pathway Analysis (IPA) version September 2011 core analysis. IPA calculates a significant score for each associated network. This score indicates the likelihood that the assembly of a set of focus genes in a network could be explained by random chance alone. Networks with scores of 2 or higher have at least a 99% confidence of not being generated by random chance alone. For individual gene expression profiling, RNAs of each treatment group were individually transversely transcribed into cDNA. Expression of genes of interest was detected by qPCR with SYBR Green . Changes in gene expression were analyzed with the comparative ΔCt method and corrected for the expression of the housekeeping gene GAPDH. Primers used to detect changes in gene expression are listed in the At termination of the experiment, the heart was perfused via the aortic arch (retro-grade), under lethal sodium pentobarbital anesthesia , with PBS (frozen sections) or PBS followed by 1% paraformaldehyde (paraffin sections). The heart was then quickly excised before freezing on dry ice or immersion in 1% paraformaldehyde.Cross-sections were cut at the level of the mid-horizontal plane of the heart from fixed paraffin-embedded tissues (4 µm) or frozen tissues (7 µm).Transverse sections were stained with hematoxylin and eosin (H&E) to measure the epicardial and myocardial thickness. To determine the extent of inflammation, sections were immuno-labeled with anti-CD45 antibody . Perls’-staining was performed as indicator of previous hemorrhage. Based on a Sirius red staining, interstitial collagen was determined in the subendocardium and myocardium of the left ventricle (LV). Within one follow-up time all sections were processed identically, at the same time with precisely the same incubation times for the primary and secondary antibody and diamminobenzidine (DAB) solution . Therefore, all differences between the treatments are ultimately due to DAB identification of the relevant protein.Photographs of the LV wall, excluding the septum, were taken using a 5x objective (Leica DFC320) and 12 measurements per heart were performed for the epicardial and myocardial thickness. The number of CD45+ cells per section was counted separately in the epicard and myocard to determine the extent of inflammation. Perls’ stained sections were examined for evidence of iron-containing macrophages and this was recorded as positive or negative for each section. Interstitial collagen was quantified in five randomly selected areas of the subendocardium and myocardium of the LV (40x objective) and results were expressed as percentage tissue positive for Sirius red relative to myocardial area. Morphometric parameters were analyzed using a computerized morphometry system .An anti-CD31 antibody  was used to visualize cardiac vasculature of the central part of the heart. To determine functional changes in the microvasculature, a histochemical staining with Naphtol AS-MX/DMF and fast Blue BB salt was performed to detect endothelial cell alkaline phosphatase. Sections were also reacted with antibodies against von Willebrand Factor (vWF)  as a marker of thrombotic changes. Within one time group all sections were processed identically, at the same time with precisely the same incubation times for the primary and secondary antibody and DAB solution. A double staining was performed to visualize the vasculature (anti-CD31) and the pericytes coverage . Primary antibodies were visualized with Alexa Fluor (AF) 633  and AF568 .2 were automatically excluded from the measurements. Photographs of whole sections stained for ALP and vWF were taken with an Aperio scanner  using 40x objective. Analyses of the percentage myocardium, excluding endocardium, positive for each marker were done with a computerized morphometry system (Leica Qwin V3). Photographs of the fluorescent stainings were performed on a Leica SP5 system microscope ; they were collected individually in the blue and red channels and merged thereafter. An average of 5 photographs were taken around the left ventricle and the pericyte coverage of microvessels was determined by counting NG2+/CD31+ vessels using a Image J computer analysis program.For quantification of microvessels, five random fields (40x objective) from transverse sections of the subendocardium were photographed with a CCD 2 – Color Microscope system, including a Zeiss AxioCam color camera , and a computerized morphometry system (Leica Qwin V3) was used to quantify the microvascular density (MVD). Vessels beneath a size of 1.5 or above 200 µm2O:Dormicum; 120 µl/mouse). Serum Albumin (HSA)  was labeled with 1–1.5 ml 99mTc-pertechnetate. The radiotracer (150 µl) was injected intravenously (i.v.), with a total activity of about 50 MBq per mouse. Three-lead electrodes  were attached to both hind paws and right front paw of the mouse, placed on the animal bed in the prone position and connected to the integrated electrocardiography (ECG) monitor to measure heart rate (HR). Once a stable HR was established, a short X-ray topogram was made to set the field of view (FOV) and so focus on the thorax to reduce scan time. After the FOV was set, gated SPECT acquisition was started using a quadruple-head gamma camera high precision gantry, equipped with 4 pyramid collimators and 9 pinhole apertures (diameter 1.2 mm). The axial FOV was 16 mm. A 20% window centered on the 140 keV photoelectric peak of 99mTc was used to acquire 20 projections with uniform angular sampling over a 360° radius into a 128×128 matrix Human X-ray topogram and SPECT acquisition were initiated directly after tracer administration. ECG-gated data were recorded in 8 time-bins per cardiac cycle. HiSPECT NG software  was used to perform iterative reconstruction into 3D-datasets. Quantitative analysis of the reconstructed datasets was performed on a clinical e.soft (syngo-based) workstation , using algorithms to automatically reconstruct a count based 3D model of the dimensions of the left ventricular (LV) end diastolic and systolic volumes . The ejection fraction (EF) was calculated based on the difference between EDV and ESV divided by EDV.Gated single photon emission computed tomography (gSPECT) acquisitions were made with the dedicated small-animal NanoSPECT/CT . Animals were anesthetized with Hypnorm  and Dormicum  via intraperitoneal (i.p.) injection  of unirradiated Eng+/− mice at termination of the experiment .There were no significant differences between groups in mean body weight or health, based on genotype or treatment or time-point , except +/− mice versus Eng+/+ mice, microarray and pathway analyses were performed using the software program IPA  and to generate hypotheses.In order to identify genes and pathways potentially involved in the cardiac response to irradiation in Eng+/+ mice at 4 weeks after 16 Gy was classed as “behavior/ nervous system development and function”. Immune response regulating genes interferon regulatory factor 7 (IRF7) and chemokine (C-X-C motif) ligand 10 (CXCL10) were identified as central molecules and were significantly upregulated within this top network  and chemokine (C-X-C motif) ligand 12 (CXCL12), and both were significantly upregulated . By 40 weeks after irradiation, the mRNA levels of endoglin were decreased to <50% of control values in both strains, although this did not reach statistical significance. TGF-β1 was significant increased by 4 weeks after irradiation in Eng+/+ mice and in both strains at 20 weeks and 40 weeks (not significant) after irradiation. Irradiation did not alter the mRNA expression of fibrogenesis activator ALK5 in Eng+/− mice but there was a non-significant increase in ALK5 in Eng+/+ mice at 4 weeks followed by significant decreased at 40 weeks after irradiation. PDGF showed a trend to increase in Eng+/+ mice at early times (4 weeks), with increases in both strains at later times . Irradiation led to a transient increase in CD45+ cells in the myocardium of both strains at 4 weeks and in the epicardium at 20 and 40 weeks. However, there were no differences in the inflammatory response noted between strains at the tissue level  A-B. Iror strain  C.+/− mice and in both strains 40 weeks after irradiation (Microvascular density (MVD) decreased significantly at 4 weeks after irradiation in Engadiation  A. This adiation  B–C. Micadiation .Since Eng+/− mice are known to be susceptible to microvascular defects as they age 99mTc-HSA) showed modest decreases in EDV and ESV at 20 weeks after irradiation of Eng+/− mice only. SV decreased in both strains with no changes in EF . Genes involved in both inflammatory response and immunological related disease were less prominently regulated by radiation in Eng+/− mice than in Eng+/+ mice. Similar results were previously obtained by Scharpfenecker et al. +/+ mice and Eng+/− mice. In these studies the reduced endoglin levels in Eng+/− mice were associated with both reduced expression of inflammatory cytokines  and reduced inflammatory infiltration (CD45+ cells) in irradiated kidney In this study gene expression analysis of irradiated hearts demonstrated a decreased inflammatory response in Eng+/+ mice during the early phase of radiation induced cardiac damage (4 weeks). Moreover, at 20 weeks after irradiation, immunohistochemical analysis showed significant collagen deposition in Eng+/+ mice only and not in Eng+/− mice. Again, this is consistent with previous studies, in which endoglin haplo-insufficiency reduced fibrosis in irradiated kidneys and in a model of ischemic heart damage Radiation-induced cardiac inflammation often precedes a late fibrotic response. In our study, fibrosis related genes TGF-β1, and to a lesser extent ALK5 and PDGF, were only upregulated in EngWith increased follow-up, our results indicated a shift away from the inflammatory response, although profibrotic genes TGF-β1 and PDGF were upregulated in both strains at 20 and 40 weeks follow up. This suggests that endoglin haplo-insufficiency only limits inflammation and fibrosis in the early phase of radiation-induced cardiac damage.+/− mice but functional damage in remaining vessels, ALP, vWF expression and pericyte coverage, was not greater in Eng+/− than Eng+/+ mice.We had previously demonstrated that cardiac irradiation of wildtype C57BL/6 mice induced endothelial cell damage +/− hearts may have contributed to a normalization of cardiac function at 40 weeks after irradiation.Changes in cardiac function were modest and non-progressive but significant decreases in ESV and EDV were seen at 20 weeks in endoglin haplo-insufficient mice and decreased SV in both strains. Upregulation in genes involved in cardiac contractility (top network 16 Gy 40 weeks) in irradiated EngIn summary, high dose radiation induced endothelial cell damage in cardiac microvasculature, which progressed in time. This microvascular tissue damage was independent of endoglin expression levels. However, our data do demonstrate that endoglin haplo-insufficiency limited the early inflammatory response and fibrosis in our radiation-induced mouse model of cardiac damage. Another model of acute pressure overload heart failure Table S1Primers used for quantitative real-time PCR.(DOC)Click here for additional data file.Table S2Body and organ weights of mice at sacrifice. * indicates significant differences between irradiated and age matched control groups .(DOC)Click here for additional data file.


In vitro, the effect of silencing TXNIP (siTXNIP) in osteoblasts, including its effect on human islet cells, was examined. Our major findings were: (i) The high mRNA levels of TXNIP in bone from CS patients were significantly associated with high levels of glucose and insulin, increased insulin resistance, and decreased insulin sensitivity in these patients. (ii) Silencing TXNIP in osteoblasts enhanced their OC response to insulin and glucose and down-regulated interleukin (IL)-8 levels in these cells. (iii) Conditional media from siTXNIP-treated osteoblasts promoted insulin content and anti-inflammatory responses in human islet cells. We recently demonstrated that the thioredoxin/TXNIP axis may mediate some detrimental effects of glucocorticoid excess on bone tissue in CS. Here we show that alterations in this axis also may affect glucose metabolism in these patients.Recent studies have described bone as an endocrine organ regulating glucose metabolism, with insulin signaling regulating osteocalcin secretion and osteocalcin regulating β cell function. We have previously demonstrated increased bone expression of TXNIP in patients with endogenous Cushing's syndrome (CS), and we hypothesized that TXNIP could contribute to the dysregulated glucose metabolism in CS. We studied 33 CS patients and 29 matched controls, with bone biopsies from nine patients, before and after surgical treatment.  Glucose intolerance and overt diabetes mellitus are classical parts of endogenous Cushing's syndrome (CS), and insulin resistance is a well known feature of cortisol excess vice versa, at least in experimental models Recent studies have shown a reciprocal relationship between bone and energy metabolism, whereby imbalance in bone remodeling seems to be of importance for glucose metabolism and insulin resistance and Thioredoxin 1 (TRX) is involved in the regulation of intracellular redox status and is induced and secreted in response to oxidative stress and inflammation. High circulating levels of TRX have been found in patients with diabetes mellitus, hypertension, hypercholesterolemia, atherosclerosis in vitro. Furthermore, we found that TXNIP expression in osteoblasts was a major regulator of OC production. Based on the pronounced insulin resistance observed in CS and low circulating OC levels, we hypothesized that long-term exposure to glucocorticoids could impact insulin resistance through TXNIP mediated mechanisms on OC in bone. This was investigated in vivo by looking at associations between TRX/TXNIP, OC and indices of insulin resistance and in vitro by modulating TXNIP in osteoblasts and investigating effects of these osteoblasts on pancreatic islets.We have previously performed a microarray study on mRNA isolated from bone biopsies in CS patients to identify genes that could be involved in glucocorticoid induced osteoporosis. TXNIP was one of the most regulated genes and further investigation demonstrated that it is regulated by glucocorticoids in bone cells Thirty-three patients diagnosed with CS were recruited based on clinical evaluation and biochemical workup as previously detailed Undercarboxylated OC was measured by enzyme immunoassays (EIA) provided by Takara  and insulin by radioimmunoassay (RIA) . TRX and cortisol was measured as previously reported Extraction of total RNA was performed using Trizol  as previously described Microarray analysis was performed as previously described Reverse transcription was performed using a High Capacity cDNA Archive Kit . Quantification of mRNA was performed using standard curve method of the ABI Prism 7500 (Applied Biosystems). For real-time RT-PCR, sequence specific exon-exon spanning oligonucleotide primers were designed using Primer Express software version 2.0 (Applied Biosystems) and can be supplied on request.Extraction of protein from the bone biopsies was performed using the Trizol method  after RNA and DNA was removed. The protein pellet was dissolved in 1% SDS and stored in −70 C. Eighty micrograms of total protein of each biopsy were incubated at 97°C for 2 minutes before separated on Any kD SDS-polyacrylamide gels  and transferred onto polyvinylidene difluoride membranes . Membranes were incubated with mouse anti-human TXNIP  antibody over night at 4°C and 1 hour at room temperature with anti-mouse IgG  horseradish peroxidase (HRP) -conjugated secondary antibody. HRP signals were developed using the ECL Plus Western blotting detection system  and visualized by Fujifilm Luminiscent Image Analyzer 4000 mini. To ensure equal loading of protein, the blots were stained with Ponceau S solution .5′-GAAUACAUGUUCCCGAAUUUU-3′, TXNIP2: 5′-GCAAACAGACUUCGGAGUAUU3′, TXNIP3:5′-CGAACCAGCUCUGAGAUGAUU-3′, TXNIP4: 5′GUCAGAGGCAAUCAUA UUAUU-3′. hFOB cells  were grown and transfected in twelve-well plates (5×104 cells/well) for 24 hours at 33.5°C (40% confluent) in antibiotic-free medium containing 10% FCS. For transfection, 50 nM siRNA duplexes and 6 µl HiPerFect transfection reagent  were prepared in OptiMem with glutamax-1 (Gibco-Invitrogen) at a final volume of 300 µl in 12-well plates . After 4 hours, 300 µl medium with 0.6% Ultrozer  was added to the cells for the remaining period of 24 hours at 33.5°C, and the temperature was shifted to 39.5°C for 48 hours to induce differentiation. Finally, cells were treated with glucose (5.6 and 50 mM), insulin (2.5 and 100 nM) and vehicle in quadruplicate cultures for different times, as indicated. In different experiments media from the silenced TXNIP osteoblasts were used to stimulate islet cells. The media was prepared as the vehicle above for the time point of 24 and 48 hours. A combination of the supernatant of these two time points was used for treatment experiments on the islets. The glucose level in the supernatants was measured in different batches of supernatant with Accu-chek compact strips .A pool of four siRNA duplexes specific for human TXNIP (siTXNIP) and scramble control pool (siSCR) were designed by Dharmacon , and experiments preformed as previously reported Following obtained consent from the organ donor registry or relatives, pancreata from deceased donors were transported from the donor hospital to the laboratory for islet isolation in Uppsala, Sweden. Islets were isolated according to the automated method, refined by the Nordic Network for Islet Transplantation p<0.05 was considered significant. All in vitro studies were performed three times with representative experiments presented.Unless stated otherwise, all values are expressed as mean±SEM. In vivo data were analyzed by non-parametric statistics  while in vitro data were analyzed by parametric statistics . A p-value of vs. controls (n = 29) there were no significant differences in age , gender . As expected, patients with CS were characterized by markedly elevated cortisol levels .Comparing patients (n = 33) We have previously demonstrated markedly increased TXNIP mRNA levels in iliac crest biopsies in CS patients with active disease, significantly correlated to high plasma levels of TRX, with decreased mRNA levels of TXNIP post-treatment Insulin signaling in osteoblasts has recently been shown to influence glucose metabolism by regulating circulating ucOC We have previously shown that silencing TXNIP in the osteoblast cell line hFOB for 2 days reduced mRNA expression (>50%) and diminished protein expression compared to scrambled treated controls, and that silencing TXNIP mRNA in osteoblasts has a strong effect on the regulation of OC in these cells (∼50-fold upregulation) Our findings so far suggest that TXNIP may modulate osteoblast expression of factors that may have impact on human pancreatic islet function (i.e. OC and chemokines). To further elucidate these issues we stimulated human islets with conditioned media from hFOB treated with siTXNIP or siSCR control. As depicted in We recently demonstrated that the TRX/TXNIP axis may mediate some detrimental effects of glucocorticoid excess on bone tissue in CS. Here we show that alterations in this axis also may affect glucose metabolism in these patients. Thus, in the present study we show (i) an association between high TXNIP mRNA expression in bone and high levels of glucose and insulin, increased insulin resistance, and decreased insulin sensitivity in CS patients; (ii) that decreased TXNIP levels in osteoblasts enhanced their OC response to insulin and glucose and down-regulated IL-8 levels in these cells; and that (iii) conditional media from siTXNIP-treated osteoblasts promoted insulin content and anti-inflammatory responses in human islets cells.in vivo situation in CS patients, displayed increased levels of TXNIP. The positive association between TXNIP from bone and insulin levels is in contrast to the reciprocal relationship in healthy individuals reported by Parikh et al TXNIP has been shown to modulate overall gene transcription and enhance β-cell death resulting in impaired insulin secretion in vivo data in different patient populations characterized by perturbed glucose metabolism are limited, at least in relation to glucocorticoid excess. In mice lacking the INSR in osteoblasts, a decrease in both total OC and ucOC was observed in serum, leading to decreased bone formation and development of peripheral adiposity and insulin resistance in vivo in CS as well. Based on the low OC, and in particular ucOC, in these patients, the potential beneficial effects of OC on β-cell function and insulin secretion could be reduced. Indeed, within healthy controls, high circulating OC was associated with markers of lower insulin resistance and increased insulin sensitivity. Although not as augmented, these associations were also present in CS, supporting that decreased OC could mediate some adverse effects of enhanced TXNIP expression in bone on insulin signaling and glucose metabolism in these patients. However, a local elevation of TXNIP in other tissues could also have direct negative effects on glucose metabolism in these patients. Indeed, hyperglycemia has been shown to increase TXNIP in adipocytes, and enhance inflammation in adipose tissue Recently, OC has been suggested as a metabolic hormone regulating glucose homeostasis in experimental models In vivo, this could indicate that a beneficial increase in OC in response to elevated blood glucose and/or insulin levels may be reduced in the presence of high TXNIP expression. Although glucocorticoids directly may modulate OC expression in osteoblasts by repressing activity of the OC gene promoter in vivo. Our finding that silencing TXNIP in osteoblasts was associated with a twofold increased INSR expression further supports that TXNIP can influence insulin signaling in these cells. Interestingly, the phenotype of transgenic mice lacking the INSR in osteoblasts, with decreased circulating OC, decreased bone formation and insulin resistance We recently reported that silencing TXNIP expression in human osteoblasts markedly increases OC expression and secretion in these cells in vivo data in CS patients may suggest that TXNIP could mediate some adverse effects of glucocorticoids on glucose metabolism through its influence on OC in osteoblasts. We have previously reported that patients with CS are characterized by elevated circulating levels of IL-8, inversely correlated with bone mass and serum OC levels TXNIP has been shown to modulate inflammation and oxidative stress in β-cells, including IL-1β which is known to be a key mediator of islet dysfunction and destruction in diabetes Although relatively few patients were examined, our study indicates that a dysregulated TRX/TXNIP axis could mediate some of the detrimental effects of glucocorticoid excess on glucose homeostasis in CS patients, and suggests that skeletal tissue may be a key regulator of energy metabolism in these patients.


Spinal muscular atrophy (SMA) is a leading genetic cause of infant mortality, resulting primarily from the degeneration and loss of lower motor neurons. Studies using mouse models of SMA have revealed widespread heterogeneity in the susceptibility of individual motor neurons to neurodegeneration, but the underlying reasons remain unclear. Data from related motor neuron diseases, such as amyotrophic lateral sclerosis (ALS), suggest that morphological properties of motor neurons may regulate susceptibility: in ALS larger motor units innervating fast-twitch muscles degenerate first. We therefore set out to determine whether intrinsic morphological characteristics of motor neurons influenced their relative vulnerability to SMA. Motor neuron vulnerability was mapped across 10 muscle groups in SMA mice. Neither the position of the muscle in the body, nor the fibre type of the muscle innervated, influenced susceptibility. Morphological properties of vulnerable and disease-resistant motor neurons were then determined from single motor units reconstructed in Thy.1-YFP-H mice. None of the parameters we investigated in healthy young adult mice – including motor unit size, motor unit arbor length, branching patterns, motor endplate size, developmental pruning and numbers of terminal Schwann cells at neuromuscular junctions - correlated with vulnerability. We conclude that morphological characteristics of motor neurons are not a major determinant of disease-susceptibility in SMA, in stark contrast to related forms of motor neuron disease such as ALS. This suggests that subtle molecular differences between motor neurons, or extrinsic factors arising from other cell types, are more likely to determine relative susceptibility in SMA. Spinal muscular atrophy (SMA) is the most common childhood form of motor neuron disease affecting 1∶6,000–1∶10,000 live births SMN1) with retention of a near identical gene (SMN2) only capable of producing low levels of full-length SMN protein SMN2 determines disease severity, with higher copy numbers resulting in increased levels of full length SMN protein and less severe disease phenotypes SMA is caused by reduced levels of the ubiquitously expressed Survival Motor Neuron (SMN) protein Previous studies have shown that one of the earliest pathological events affecting motor neurons in SMA is a breakdown of motor nerve terminals at the neuromuscular junction (NMJ), rendering skeletal muscle fibres denervated G93A mouse, where large diameter motor axons were preferentially decreased in number in the ventral roots We set out to establish whether core intrinsic morphological features of motor neurons, such as motor unit size or branching patterns, predisposed them to degeneration in SMA. We chose to investigate motor neuron morphology as a possible regulator of vulnerability in SMA as a result of recent evidence obtained during studies of the adult onset form of motor neuron disease, amyotrophic lateral sclerosis (ALS). A growing body of evidence suggests that SMA and ALS share biochemical pathways Here, we report that, in sharp contrast to ALS, morphological characteristics of motor neurons, such as motor unit size, do not determine their relative vulnerability to degeneration in SMA.All animal experiments were approved by a University of Edinburgh internal ethics committee and were performed under license by the UK Home Office (project license number 60/3891).Smn+/−;SMN2 mice (Jackson labs strain no. 005024) on a congenic FVB background were maintained as heterozygote breeding pairs under standard SPF conditions in animal care facilities in Edinburgh. All animal procedures and breeding were performed in accordance with Home Office and institutional guidelines. Litters produced from SMA colonies were retrospectively genotyped using standard PCR protocols (JAX® Mice Resources).Thy.1-YFP-H mice Smn−/−;SMN2Neonatal Thy.1-YFP-H Muscles were permeabilised in 2% Triton X in 0.1 M PBS for 30 minutes and then blocked in 4% bovine serum albumin and 1% Triton X in 0.1 M PBS for 30 minutes. For endplate occupancy and synapse elimination counts, muscles were then incubated overnight at 4°C in primary antibodies against neurofilament (anti-mouse 2H3)  and anti-mouse SV2  diluted in blocking solution. For Schwann cell counts, muscles were incubated overnight at 4°C in primary antibody anti-rabbit S100 . After washing for 2 h with four changes of 0.1 M PBS, muscles were incubated with 5 mg/ml TRITC-conjugated α-bungarotoxin  for 10 minutes at room temperature. Muscles were then washed in 0.1 M PBS for 5 minutes. Muscles for NMJ counts and synapse elimination studies were then incubated with 1∶100 dilution of Donkey anti-Mouse Dylight488 (H+L) (Stratech Scientific) in 0.1 M PBS for 4 h at room temperature. Muscles for Schwann cell counts were incubated with a 1∶60 dilution of Swine anti-rabbit FITC (Dako) in 0.1 M PBS for 4 hours at room temperature. Muscles were then washed for 30 minutes in three changes of 0.1 M PBS. Muscles for Schwann cell counts were then further incubated with TO-PRO3 for 10 minutes and washed with 0.1 M PBS. Muscles were then mounted in Mowiol  on glass slides and coverslipped for subsequent imaging.Muscle preparations were viewed using a standard epifluorescence microscope equipped with a chilled CCD camera , and a laser scanning confocal microscope . TRITC labelled preparations were imaged using 543 nm, excitation and 590 nm emission optics; YFP-labelled preparations were imaged using 488 nm excitation and 520 nm emission optics. For confocal microscopy, 488 nm, 543 nm and 633 nm laser lines were used for excitation and confocal Z-series were merged using Zen software.Reconstructions of whole motor units were obtained by montaging fluorescent micrographs from TRITC-conjugated α-bungarotoxin and YFP-H labelled muscle preparations in Adobe Photoshop software. Individual motor neurons were traced by hand and arbor lengths and endplate areas were measured using ImageJ software.For synaptic vulnerability studies, a minimum of 80 endplates per muscle per mouse, selected at random, were assessed in each muscle preparation. Muscles with poor staining and/or damage were excluded from further analysis. For occupancy counts, the occupancy of individual NMJs was evaluated by categorising endplates as either fully occupied (neurofilament and SV2 entirely overlie the endplate), partially occupied (neurofilament and SV2 cover less than 50% of the endplate), or vacant (no neurofilament or SV2 overlying the endplate).Motor unit reconstructions were used to quantify morphological parameters of single motor neurons. Motor unit size was determined by identifying the number of motor end plates per axon from traced reconstructions. The total intramuscular arbor length of motor units was determined by tracing the axons in the reconstructed images by hand from the point of entering the muscle all the way to the nerve terminals. The arbor length of the axons was determined as the sum of the length of the primary axon trunk and all the distal branches.The number of branch points in individual motor units was evaluated by marking the branch points by hand in the motor unit reconstructions using Adobe Photoshop. Where more than one motor unit was labelled with YFP, differences in cytosolic levels of YFP expression were used to distinguish between individual motor units. The branching pattern of the axons was further investigated by recreating schematic branching diagrams based on the whole motor unit reconstructions. The branch order for each terminal branch was determined by counting the number of branch points between the nerve terminus and the site where the axon entered the muscle.End plate area was determined from the Maximum Intensity Z-stack images. The outline of the motor endplates was manually traced in the Image J software to enable the software to calculate the area.To evaluate the number of axonal inputs in neonatal muscles, the number of axons converging on a single endplate was counted. A minimum of 80 endplates per muscle were counted from each mouse.The number of terminal Schwann cells per motor end plate was quantified by analysing Maximum Intensity confocal projection images in Adobe Photoshop. To be counted as a tSC, the cell had to be positive for S100 with a TO-PRO-3 labelled nucleus.All data were collected into Microsoft Excel spreadsheets and analysed using GraphPad Prism software. Figures were produced on Adobe Photoshop. All bar charts shown are mean ± SEM. Statistical significance was considered to be p<0.05 for all analyses. Individual statistical tests used are detailed in figure legends.Smn−/−;SMN2; In order to determine the extent of heterogeneity in motor neuron pathology occurring between distinct pools of motor neurons in a severe mouse model of SMA  are a specialised form of glial cell that overlies the nerve terminal of the motor neuron at the neuromuscular junction. It is well documented that tSCs are intricately involved in the development, maintenance, and stability of neuromuscular junctions tSCs were immunohistochemically labelled in whole-mount P5 muscles from wild-type P5 mice using primary antibodies raised against S100 protein and the nuclear marker TO-PRO-3 . There wThe current study demonstrates that, in sharp contrast to ALS, morphological characteristics of motor neurons do not determine their relative susceptibility to degeneration in SMA. Neither the position of the innervated muscle in the body, nor the muscle fibre type being innervated, modulated the susceptibility of a motor neuron. Similarly, we could find no correlation between factors such as motor unit size, total length of intramuscular axonal arbors, motor unit branching patterns, numbers of synaptic glia and intrinsic remodelling capabilities, in healthy young adult mice, and the relative susceptibility of some motor neurons in SMA.The clear difference between our findings in a mouse model of SMA and previous studies of ALS patients and mouse models SMN2 gene is lower in motor neurons compared to other cells in the spinal cord As morphological characteristics of motor neurons do not appear to be a major determinant of their relative susceptibility to degeneration in SMA, it remains unclear what factors are regulating their vulnerability. One possibility is that intrinsic differences persist between pools of vulnerable and disease-resistant motor neurons at a molecular level. The key determining factor of a motor neuron’s relative susceptibility to degeneration in SMA could therefore be dependent upon baseline levels of SMN protein. It has recently been established that the splicing efficiency of exon 7 in the Although intrinsic differences in the molecular composition of vulnerable and disease-resistant motor neurons may contribute to their relative susceptibility to degeneration, our current findings also raise the possibility that interactions between motor neurons and other cell types may also be important. For example, intrinsic defects in skeletal muscle have been identified in SMA Our study highlights the importance of animal models for examining factors regulating disease pathogenesis in SMA. The decision to examine motor unit morphology in healthy young adult mice expressing YFP  does leave open the remote possibility that subtle intrinsic morphological properties of motor neurons specific to SMA mouse models may contribute to relative vulnerability in disease. However, the finding that motor neurons in pre-symptomatic SMA mice are grossly indistinguishable from those in wild-type littermates


The proposed target cell isolation technique can provide a practical means to collect significant quantities of viable intact cells required to translate stem cell biology to regenerative medicine in a simple label-free manner.Passive and label-free isolation of viable target cells based on intrinsic biophysical cellular properties would allow for cost savings in applications where molecular biomarkers are known as well as potentially enable the separation of cells with little-to-no known molecular biomarkers. We have demonstrated the purification of adrenal cortical progenitor cells from digestions of murine adrenal glands utilizing hydrodynamic inertial lift forces that single cells and multicellular clusters differentially experience as they flow through a microchannel. Fluorescence staining, along with gene expression measurements, confirmed that populations of cells collected in different outlets were distinct from one another. Furthermore, primary murine cells processed through the device remained highly viable and could be cultured for 10 days  There exists extensive interest in the enrichment and purification of specific subpopulations of cells from heterogeneous biological samples for immunology research, tissue engineering, and medicine in vitro. Here, to test the ability to collect cells from real tissue samples, we purified primary tissue digests containing adrenal cortical progenitor cells using a label-free microfluidic approach.Recent advances in microfluidic technologies have resulted in the development of numerous approaches, allowing simple, fast, and label-free target cell separation in vitro, and these adrenal cells can be transplanted into the patient and potentially restore adrenal functionality or reverse adrenal insufficiency The adrenal cortex is located at the perimeter of the adrenal gland, a major hormone-secreting organ responsible for synthesis of steroid hormones dim) expressed the steroidogenic factor 1 (Sf1) gene  but lacked expression of zonal-specific adrenal cortical genes, such as Cyp11b1 (zona fasciculata) and Cyp11b2 (zona glomerulosa). This result suggests the successful enrichment and identification of a potential biomarker for a progenitor cell population.Previously, adrenal cortical progenitor cells were isolated based on increased intracellular cholesterol content within fully differentiated cells compared with progenitor cells, with differences potentially originating from different steroid hormone synthesis capabilities However, the discrimination of progenitor cells from heterogeneous tissue digests was based on the intensity level of fluorescent stains, which can vary widely due to staining procedures and other challenges. For instance, dyes are often unsuitable for cells that will eventually be re-implanted in humans. Moreover, the function and proliferation rate of target cells isolated using immunoaffinity-based techniques could be adversely affected due to the strong binding between the labeling probes and targeting antigens Here, we hypothesized that differences in intracellular cholesterol contents and phenotypic characteristics found in fully differentiated vs. progenitor cells would result in a variation in biophysical properties of such cells. It is known that cell adhesion molecules and junctional proteins are differentially expressed on cells with varying degrees of differentiation and lineage commitment Murine adrenal cortical progenitor cells were purified from a heterogeneous tissue digest using DACS in a simple and label-free manner. Heterogeneity in biophysical properties of digested cells in the injected solution allows the inertial focusing mechanism to locate subpopulations differentially at distinctive distances from the channel walls . Based on these differences in lateral equilibrium position, smaller single cells were directed toward the outermost outlets  and diluted in 5 mL Knockout (KO) media prior to separation. Our previous study has demonstrated that the current flow condition approaches the maximum flow rate and throughput of a single DACS, given that a further increase in fluid inertia would result in uniform focusing positions regardless of cell size variation W = 40 µm, H = 90 µm and L = 4.5 cm), a gradually expanding region, and 5 branched outlets with fluidic resistors. The angle between the channel wall and the flow direction in a gradual expansion was increased 2° for every 100 µm. The dimension of a gradual expansion was carefully designed through numerous empirical tests to create a smooth and low transition in shear that processed cells would experience. The low-shear transition design was essential to confine focused cells to their predetermined streamlines while enhancing eqX differences among cell types, as well as maintaining minimum distances between cells flowing toward designated outlets, without sacrificing the purity of collected samples. Additionally, long serpentine channels with dominating fluidic resistance were incorporated into outlet designs to ensure undistorted and uniform flow through individual outlets during the entire course of the purification process regardless of small fluctuations in fluidic resistances. Regardless, those resistors were designed to have negligible shear effects on flowing cells . Detailed dimensions and empirical supporting evidence can be found elsewhere To isolate adrenal cortical progenitor cells from the digested tissue samples, we used the DACS as previously reported Dulbecco's modified Eagle's medium and Ham's F12 medium (DMEM/F12), KnockOut™ DMEM, Hank's balanced salt solution (HBSS), fetal bovine serum, horse serum, KnockOut™ serum replacement, GlutaMAX™, and antibiotics were purchased from Invitrogen . Collagenase I, deoxyribonuclease I, and bovine serum albumin were purchased from Sigma-Aldrich . Collagen sponge Helistat was purchased from Integra LifeSciences . Adrenal Media was prepared with DMEM/F12 medium, 15% horse serum, 2.5% fetal bovine serum, and antibiotics. Knockout (KO) media was prepared with KnockOut™ DMEM, 15% KnockOut™ serum replacement, GlutaMAX™, and antibiotics.Female C57/BL6 mice, 8 weeks in age (20–22 g), were purchased from Charles River Laboratory . All animals were maintained in an animal barrier as a non-breeding colony in a temperature- and light-controlled room and were allowed free access to food and water. In each experiment, C57 mice served as donors. The use of the animals was approved by the UCLA Animal Research Committee (IRB 2003-178).Adrenal glands were removed from mice after euthanasia. After removing the surrounding fat, the harvested adrenal glands were incubated in the digestion mixture at 37°C for 1 hour with gentle shaking. The digestion mixture consisted of 10 mL of HBSS containing 2 mg/mL collagenase I, 0.05 mg/mL DNase I, and 5 mg/mL bovine serum albumin. After dispersing the cells through a pipette, the cells were washed with Adrenal Media and then filtered through a 70-µm strainer . Cells collected from two adrenal glands were suspended with KO media (5 mL).5 mL of cell suspensions containing 400,000 cells/mL were loaded into a glass syringe (Hamilton GASTIGHT®), and the solutions were injected into DACS while being continuously agitated using a custom-built cell shaker. The overall flow rate was maintained at 60 µL/min using a syringe pump (Harvard Apparatus PHD2000), as we had previously found that the maximum separation was achievable at this flow condition using the current device T method (CT−CA)−(CG−CB). Here, CT and CG are the threshold cycle number for adrenal cortical gene and GAPDH in the sample , respectively, whereas CA and CB are the cycle number for the same adrenal cortical gene and GAPDH in the pooled neonatal adrenal glands, respectively. RNA from multiple adrenal glands of neonatal mice was extracted and pooled in order to prepare a large batch of adrenal RNA. It served as a constant normalization standard for multiple qRT-PCR analyses. Statistically significant differences between the two sets of mRNA levels were determined using Student's t-test.RNA extraction from sorted and unsorted cells was performed with RNeasy Mini kit , following the manufacturer's protocol. The expression of specific mRNAs was obtained with quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), Qiagen qRT-PCR kit. Primers and probes for amplifying adrenal gland genes of interest  were purchased from Operon Technologies , while the housekeeping gene (GAPDH) primers were purchased from Applied Biosystems . qRT-PCR data for each sample were obtained using ABI PRISM 7900 Sequence Detection System. The reverse transcription was performed at 50°C for 30 min, followed by the initial activation at 95°C for 15 min. Then, samples were processed for 45 cycles of 94°C for 15 sec and 60°C for 60 sec. The threshold cycle number for each sample indicates the cycle number at which the fluorescence signal exceeded the preset threshold value, which was the value ten times greater than the standard deviation of the baseline. Gene expression results were normalized to that of the mRNA level in pooled neonatal adrenal glands using the comparative CTo assess whether the current enrichment method adversely affected processed cells, we performed colorimetric live/dead assay using Calcein AM and Ethidium homodimer-2 (Invitrogen) in addition to long-term culture with KO media following previously established protocols We have demonstrated that target cells in the form of dispersed single cells can be passively purified from multicellular clusters from complex heterogeneous tissue digests using DACS. Putative adrenal cortical progenitor cells with little-to-no cholesterol content can be isolated as single cells at the outermost outlets, whereas more differentiated adrenal cells with higher cholesterol content can be collected as multicellular clusters at the inner outlets. Our preliminary results show that DACS has practical applications in tissue engineering and regenerative medicine, which often require the processing of fragile primary cells or stem cells with significant yields/throughputs. Furthermore, the ability to isolate multicellular clusters from heterogeneous cell solutions can provide a useful tool to study native organized structures, which is often required for cancer prognostics


In the development of psychotic symptoms, environmental and genetic factors may both play a role. The reported association between childhood trauma and psychotic symptoms could therefore be moderated by single nucleotide polymorphisms (SNPs) associated with the stress response, such as FK506-binding protein 5 (FKBP5) and brain-derived neurotrophic factor (BDNF). Recent studies investigating childhood trauma by SNP interactions have inconsistently found the hippocampus to be a potential target underlying these interactions. Therefore, more detailed modelling of these effects, using appropriate covariates, is required. We examined whether BDNF/FKBP5 and childhood trauma interactions affected two proxies of hippocampal integrity: (i) hippocampal volume and (ii) cognitive performance on a block design (BD) and delayed auditory verbal task (AVLT). We also investigated whether the putative interaction was different for patients with a psychotic disorder (n = 89) compared to their non-psychotic siblings (n = 95), in order to elicit possible group-specific protective/vulnerability effects. SNPs were rs9296158, rs4713916, rs992105, rs3800373 (FKBP5) and rs6265 (BDNF). In the combined sample, no BDNF/FKBP5 by childhood trauma interactions were apparent for either outcome, and BDNF/FKBP5 by childhood trauma interactions were not different for patients and siblings. The omission of drug use and alcohol consumption sometimes yielded false positives, greatly affected explained error and influenced p-values. The consistent absence of any significant BDNF/FKBP5 by childhood trauma interactions on assessments of hippocampal integrity suggests that the effect of these interactions on psychotic symptoms is not mediated by hippocampal integrity. The importance of appropriate statistical designs and inclusion of relevant covariates should be carefully considered. A history of childhood trauma (CT) is prevalent in individuals suffering from psychotic symptoms Exposure to CT/abuse has been reported to affect hypothalamus-pituitary-adrenal axis (HPA) function later in life: changes in cortisol FK506-binding protein 5 (FKBP5) gene Studies that have investigated the effect of either CT To date, two studies have investigated the effect of BDNF/FKBP5 by CT interactions on psychotic symptoms in adulthood The present study investigated whether synergistic effects of CT and BDNF/FKBP5 genotype influenced two proxies of hippocampal integrity in a large sample of individuals with psychotic disorder and siblings. These proxies were: (i) assessments of hippocampal volume and (ii) delayed performance on an auditory verbal learning task and performance on a spatial memory task, cognitive tasks consistently demonstrated to be dependent on hippocampal function We first investigated the role of BDNF and FKBP5 in the association between CT and hippocampal volume/cognitive performance in adulthood, regardless of illness status, further investigating recent mixed results on the role of the BDNF gene in the association between childhood adversity and hippocampal volume All data described in this manuscript pertain to a longitudinal magnetic resonance imaging (MRI) study in Maastricht, the Netherlands 2. The number of excitations was one. Two sequences were used because of a scanner update during data collection. The MPRAGE and MDEFT are similar, but to prevent any systematic bias, the total proportion of MPRAGE scans (approximately one third) was balanced between the groups MRI scans were obtained on a 3T Siemens scanner . Acquisition parameters: Modified Driven Equilibrium Fourier Transform (MDEFT) sequence with 176 slices, isotropic voxel size of 1 mm, echo time = 2.4 ms, repetition time = 7.92 ms, inversion time = 910 ms, flip angle = 15°, total acquisition time = 12 m51 s. Magnetization Prepared Rapid Acquisition Gradient-Echo (MPRAGE) sequence with 192 slices, isotropic voxel size of 1 mm, echo time = 2.6 ms, repetition time = 2250 ms, inversion time =  900 ms, flip angle =  9°, total acquisition time = 7 m23 s. Matrix size was 256×256 and field of view was 256×256 mmhttp://surfer.nmr.mgh.harvard.edu/), published in detail previously Preprocessing was performed and structural volumetric measures were obtained using reconstruction and volumetric segmentation procedures from the freely available Freesurfer stable release version v5.0.0 (CT was assessed with the Dutch version of the Childhood Trauma Questionnaire Short Form (CTQ). The short CTQ consists of 25 items rated on a 5-point Likert scale (1 = never true to 5 =  very often true) inquiring about traumatic experiences in childhood. Five types of childhood maltreatment were assessed: emotional , physical  and sexual abuse , and emotional  and physical neglect . Five questions covered each type of trauma Two neuropsychological tasks that rely on hippocampal functioning were included: the auditory verbal learning task (AVLT) and block design (BD) task, part of the Dutch version of the Wechsler Adult Intelligence Scale (WAIS IV). Delayed performance on the AVLT and BD performance assess recall capacity and spatial memory respectively, processes dependent on hippocampal integrity http://www.ncbi.nlm.nih.gov/gene/627) and selected FKBP5 SNPs have been consistently associated with functional outcomes 2 = 2.34, p = .18), rs4713916 , rs992105 , rs3800373  and rs6265 . The distribution of genotypes among groups was not significantly different: rs9296158 , rs4713916 , rs992105 , rs3800373  and rs6265 (BDNF) . The linkage disequilibrium (LD) between FKBP5 SNPs has been described previously The FKBP5 SNP selection was based on previous work Antipsychotic medication use was determined by multiplying the number of days of antipsychotic medication use with the corresponding haloperidol equivalents and summing scores for all periods of antipsychotics use (including the exposure period between baseline assessment for the study and moment of MRI scan), using the published converting formulas for antipsychotic medication dose equivalents described by Andreasen and colleagues' For hippocampal volume, the number of voxels in the left and right hippocampus was used as continuous dependent variable, an indication of the structure's size. For cognitive performance, we used delayed AVLT and (exponentiated) BD performance as a continuous measure. The effect of group , genotype  and CT  on hippocampal volume/cognitive performance was first explored separately. Association analyses between hippocampal volume and antipsychotic medication use were also performed. Next, we investigated the role of BDNF/FKBP5 genotype in the association between CT and hippocampal volume/cognitive performance in adulthood in the whole sample , guided by recent mixed results on the role of the BDNF gene in the association between childhood adversity and hippocampal volume Demographic analyses were performed using linear regression and chi-square tests (REGRESS and TAB command in STATA 11.0 respectively). All remaining regression analyses were mixed models and were performed using the XTMIXED command in Stata 11.0  with family ID as random effect Volumetric analyses were corrected for intracranial volume, age, gender Demographics are shown in Given that groups differed on some sociodemographic variables (e.g. drug use) , model fA strong group difference was found in left hippocampal volume (patients<siblings) , which rHigher levels of CT were negatively associated with BD performance, even when substance use was excluded from the model . CT was Best-estimates of lifetime cumulative antipsychotic medication use were not associated with left or right hippocampal volume in patients . No association between lifetime cumulative antipsychotic medication and delayed AVLT  or BD performance  was observed.Convincing CT by group interactions in the model of left and right hippocampal volume were not observed , even when substance use was included . Variations within the selected FKBP5 SNPs, rs9296158, rs4713916, rs992105, rs3800373 and BDNF did not significantly interact with CT to influence hippocampal volume , regardless of substance use. Variations within rs9296158, rs4713916, rs992105, rs3800373 and BDNF did not interact with CT in the model of AVLT/BD performance  were not affected by a BDNF/FKBP5, CT and group status interaction, at least, when adequately controlling for the undesired influence of drug use and alcohol consumption.BDNF and FKBP5 genotype did not moderate the association between CT and hippocampal volume assessed later in life, regardless of group status. This is the first investigation looking at FKBP5 genotype by CT interactions in the model of hippocampal volume. Our BDNF findings parallel those of a recent study No main effects of BDNF genotype on hippocampal volume were observed, in agreement with a recent inconclusive meta-analysis In concordance with our volumetric results, CT by BDNF/FKBP5 genotype interactions, with or without the inclusion of group status, were not observed in the model of delayed AVLT and BD performance. The presented results are the first to indicate that FKBP5 genotype does not affect the association between CT (or CT by group status interactions) and hippocampal-dependent cognition. Although BDNF did not influence the association between CT interactions and cognition, BDNF genotype has been shown to impact on the association between sexual abuse and cognition Although studies investigating BDNF/FKBP5 by CT interactions in the model of cognition are scarce, and associations between BDNF genotype on cognition are modestly strong at best Previously cited studies demonstrate a potential role for BDNF and FKBP5 minority alleles in the association between CT and changes in affective domains later in life. The absence of consistent association with cognition, including those presented in the current manuscript, could suggest that BDNF/FKBP5 genoype by CT interactions impact on an affective, rather than cognitive, pathway towards psychopathology later in life The consistent absence of any FKBP5/BDNF genotype × CT interactions could have been the result of a power problem (e.g. CT by group by BDNF interaction in the model of delayed AVLT). Although similar in sample size to other published work In order to provide an accurate reflection of hippocampal integrity, two proxies were investigated. The degree of consistency between those results may have benefitted the accuracy of the conclusions. Furthermore, including influential sample characteristics  and the use of appropriate covariates (times G/E) Data presented in this manuscript is available to collaborators upon request.Table S1BDNF/FKBP5 genotype distribution for siblings and patients.(DOC)Click here for additional data file.Table S2Model fit tests comparing a statistical model including substance use to a model without substance use.(DOC)Click here for additional data file.


However, a precise understanding of how nanomaterial design parameters such as size, shape and composition affect the mammalian immune system is a prerequisite for the realization of nanomedicine's translational promise. Herein, we make use of the recently developed Particle Replication in Non-wetting Template (PRINT) fabrication process to precisely fabricate particles across and the nano- and micro-scale with defined shapes and compositions to address the role of particle design parameters on the murine innate immune response in both in vitro and in vivo settings. We find that particles composed of either the biodegradable polymer poly(lactic-co-glycolic acid) (PLGA) or the biocompatible polymer polyethylene glycol (PEG) do not cause release of pro-inflammatory cytokines nor inflammasome activation in bone marrow-derived macrophages. When instilled into the lungs of mice, particle composition and size can augment the number and type of innate immune cells recruited to the lungs without triggering inflammatory responses as assayed by cytokine release and histopathology. Smaller particles (80×320 nm) are more readily taken up in vivo by monocytes and macrophages than larger particles (6 µm diameter), yet particles of all tested sizes remained in the lungs for up to 7 days without clearance or triggering of host immunity. These results suggest rational design of nanoparticle physical parameters can be used for sustained and localized delivery of therapeutics to the lungs.Nanomedicine has the potential to transform clinical care in the 21 The application of nanoparticles in medicine for disease treatment is a potentially transformative area of research. The possibility of potent instruction and modulation of host physiology through nanomaterials has been abundantly demonstrated. These efforts include modulation of cell-specific gene expression through delivery of antisense oligonucleotides, dose-sparing and targeted delivery of pharmacologics, as well as enhanced multi-functional imaging diagnostics through nanoformulations in situ require a comprehensive understanding of the immune response, or lack thereof, engendered to introduced nanocarriers Researchers in the emerging field of immune-engineering are capitalizing on these exciting advances to open up the possibility to direct and instruct immunological outcomes to a variety of pathological conditions In order for the gamut of potential downstream therapeutic applications of nanomedicine to be realized, we must first understand how the physical properties of nanomaterials augment host immune responses. These principles will then enable the appropriate design of nano- and micro-scale interventions for specific purposes. For example, the use of nanoparticles to deliver potent biological molecules, such as oligonucleotides or small molecules augmenting intracellular signaling pathways, may squander the regulatory opportunity to reach the clinic if the nanocarriers for such entities initiate off-target events that activate host immune responses. Conversely, vaccines can be made more potent if nanocarriers are designed to activate the appropriate innate immune response to tailor adaptive immune responses to delivered antigens. As an example, the current state of the art is to use Alum as a non-specific immunomodulatory adjuvant in vaccine formulations. This could explain, in part, why some of the most pressing pathogens do not yet have useful vaccines because Alum is known to engender a mixed Th1/Th2-biased humoral immune response that does not reflect the immune activation which occurs during an actual microbial infection A great issue in advancing nanotechnology from a laboratory pursuit into a component of clinical care is a robust understanding of how physical particle properties augment biological outcomes. There is a wealth of literature promoting the use of nanotechnology in modern medicine, but much of this literature relies on particle fabrication methods, such as oil-in-water emulsion, that generate heterogeneous populations of particles that can vary widely between batches and across labs Most currently employed fabrication methods do not allow precise control over particle physical parameters, thus it is difficult to draw conclusions as to how size, shape and composition affect the innate immune response to particles in the nano and micron range. To address this lack of knowledge, we employed our recently developed top-down nanofabrication technique termed Particle Replication in Non-Wetting Templates (PRINT) in vitro assays with murine derived macrophages and in vivo delivery of particles to the lungs of mice to test the inflammatory potential of these particles. The lung is a highly desired site for therapeutic delivery of nanomedicine and we chose it for both clinical relevance and its sensitivity as an immunological organ To lend both clinical and field relevance to our findings, particles were designed with either the F.D.A approved polymer Poly-lactic co-gloycolic acid (PLGA) or derivatives of the commonly published hydrogel polymer polyethylene glycol (PEG). While PLGA is an attractive polymer given its long history of clinical use, our study was aimed in part to clarify discrepancy in the literature as to whether particles made of PLGA trigger inflammation. As our main purpose was to define the ‘baseline’ status of whether particles of defined size, shape and composition triggered inflammation, we did not augment particles in this study to include additional biologically active molecules, such as oligonucleotides, small molecules or adjuvants as previously published by our group and others n 700) (PEG700DA), 2-aminoetheyl methacrylate hydrochloride (AEM), Diphenyl -phoshine oxide (TPO), and poly lactic co-glycolic acid  were purchased from Sigma-Aldrich. Tetraethylene glycol monoacrylate (HP4A) was synthesized in-house as previously described Poly(ethylene glycol) diacrylate , 10 wt% PEG700DA (crosslinker), 1 wt% TPO (photo initiator) and 1.5 wt% Dylight 650 maleimide. This composition was then dissolved at 3.5 wt% in methanol and drawn as a thin film using a # 3 Mayer rod  onto a roll of corona treated PET using an in-house custom-made roll-to-roll lab line (Liquidia Technologies) running at 12 ft/min. The solvent was evaporated from this delivery sheet by exposing the film to heat guns. The delivery sheet was laminated  to the patterned side of the mold, followed by delamination at the nip. Particles were cured by passing the filled mold through a UV-LED . A PVOH harvesting sheet was hot laminated to the filled mold . Upon cooling to room temperature, particles were removed from the mold by splitting the PVOH harvesting sheet from the mold. Particles were then harvested by dissolving the PVOH in a bead of water (1 mL of water per 5 ft of harvesting sheet). The particle suspension was passed through a 2 µm filter (Agilent) to remove any large particulates. To remove the excess PVOH, particles were centrifuged (Eppendorf Centrifuge 5417R) at 14000 rpm for 15 min, the supernatant was removed and the particles were re-suspended in sterile water. This purification process was repeated 4 times prior to lyophilization as detailed above. The 80×320 nm particles were acetylated prior to experimental use to match negative charge of micron sized hydrogel particles.The process of fabricating 80×320 nm hydrogel particles was conceptually similar to PLGA fabrication, but with important differences. The pre-particle solution (PPS) contained a composition of 67.5 wt% HP700-DA, 78% HP4A, 1% TPO (photoinitiator), and 1% Dylight 650. The solution was spread onto a fluorocur mold and a poly (PET) sheet was laminated on top of the mold and polymer mixture and run through a heated, pressurized laminator to fill the molds. The mold was then cured with a UV LED lamp for 30 seconds. Particles were transferred out of the mold onto a Luvitec harvesting layer by laminating the mold and Luvitec sheet together and running them through a heated laminator nip. The mold and harvesting sheet was separated, leaving free particles on the harvest layer. Particles were collected from the harvest sheet by bead harvesting with water and pelleted by centrifugation. The particles were re-suspended in tert-butanol and lyophilized overnight.The 1.5 and 6 µm donut shaped hydrogel particles were fabricated using a dropcast method. The pre-particle solution (PPS) was composed of 20% PEGThermogravimetric analysis (TGA) was used to determine stock particle concentrations (TA Instruments Q5000 TGA). Briefly, 20 µL of the stock nanoparticle solution was pipetted into a tared aluminum sample pan. The sample was heated at 30°C/min to 130°C and held at this temperature for 10 minutes. The sample was then cooled at 30°C/min down to 30°C and held for 2 minutes. A Hitachi S-4700 scanning electron microscope (SEM)was used to visualize particles. Prior to imaging, the SEM samples were coated with 1.5 nm of gold-palladium alloy using a Cressington 108 auto sputter coater. Particle size and zeta potential were measured by dynamic light scattering (DLS) on a Zetasizer Nano ZS .All studies were conducted in accordance with National Institutes of Health guidelines for the care and use of laboratory animals and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of North Carolina at Chapel Hill. All animals were maintained in pathogen-free facilities at the University of North Carolina at Chapel Hill.MH-S murine alveolar macrophages were plated in complete DMEM at 20,000 cells per well in 8-well chamber slides (LabTek) 48 hours prior to treatment with particles. Particles were resuspended in DMEM at 20 µg/ml and 300 µl of particle solution were added to each well. Particles were incubated with cells at 37°C for 4 hours. Cells were then washed twice with PBS and cells fixed with 4% Paraformaldehyde (PFA) solution and later stained with Alexa Fluor 488 Phalloidin (Invitrogen) and DAPI . Fluorescent imaging of stained cells was performed on a Zeiss 710 laser scanning confocal imaging system (Zeiss).Bone marrow macrophages were isolated from the femurs of C57Bl/6 and BALB/c mice using standard procedures. Bone marrow-derived macrophages were cultured for six days in DMEM supplemented with 10% fetal bovine serum, L-Glutamine, pen/strep and 20% L929-conditioned medium prior to use in particle experiments. Adherent cells were isolated and plated in complete Dulbecco's Modifed Eagle Medium (Gibco) with 10% fetal calf serum, 1% penicillin/streptomycin and 1% L-glutamine at 200,000 cells per well in a 96-well dish for 24 hours prior to treatment with particles for up to 24 hours. Some cells were primed with LPS (50 ng/ml) for 24 prior to particle treatment to provide signal 1 for inflammasome activation. Monosodium urate  treated at 300 µg/ml or ATP  was used as a positive control for inflammasome activation. Particles were resuspended in PBS prior to dosing at various concentrations. After 24 hours of particle treatment in triplicate, supernatants were harvested and analyzed by murine IL-1β, TNF-α and IL-6 ELISA (BD Biosciences). Limit of detection for ELISAs was 31.3 pg/ml (IL-1β) and 15.6 pg/ml (TNF-α and IL-6). Lactate dehydrogenase release was used to measure particle-induced cytotoxicity (Roche Applied Sciences).To assess whether PRINT particles induce airway inflammation, 10–12 week old female C57Bl/6 mice were anesthetized with isofluorane inhalation and particles were instilled via intratracheal (i.t.) administration. 50 µg of particles were dosed in 50 µl of PBS. Intratracheal administration of PBS (50 µl) or LPS (20 µg in 50 µl PBS) served as negative and positive controls for airway inflammation, respectively, as previously described Serum was collected from animals by cardiac puncture and centrifuged at 15,000 RPM for 10 minutes. The serum supernatant was collected and used for ELISA analysis of inflammatory markers. Bronchoalveolar lavage fluid (BALF) was also collected to evaluate local leukocyte and cytokine levels in the lungs. For this purpose, lungs were lavaged three times with 1 ml Hanks Balanced Salt Solution . After centrifugation at 1500 RPM for 5 minutes, cell-free supernatants were collected and used to assess cytokine levels of IL-1β, TNF-α and IL-6 via ELISA (BD Biosciences). RBC were lysed via brief hypotonic saline treatment and the cell pellet was resuspended in PBS. Total BALF cellularity was assessed with a hemacytometer. The cellular composition was determined by cytospin of BALF aliquots onto slides and staining with Diff-Quik (Dade Behring) for differential cell counts. Leukocytes were identified based on the morphology of ≥200 cells per sample. Following BALF harvest, the lungs were fixed by inflation (20-cm pressure) and immersed in 10% buffered formalin.Inflammation was evaluated in 5 µm sections of the left lung lobe after hematoxylin and eosin (H&E) staining. Serial paraffin-embedded sections were set and cut to reveal maximum longitudinal visualization of the intrapulmonary main axial airway and inflammation was scored by one of the authors (I.C.A.) who was blinded to genotype and treatment. As previously described, histology images were evaluated on each of the following inflammatory parameters and scored between 0 (absent) to 3 (severe): mononuclear cell infiltration, polymorphonuclear cell infiltration, airway epithelial cell hyperplasia/injury, extravasation, perivascular cuffing, and estimated percentage of the lung involved with inflammation BALF aliquots from PEG treated mice were fixed in 2% paraformaldehyde and stained with DAPI (nuclei) and Phalloidin 488 (actin) and then viewed via epifluorescence microscopy for particle uptake (Dylight 650). Five distinct fields of view (FOV) were captured for each slide. The percentage of cell uptake was determined by dividing the number of cells showing particle internalization by the total number of cells in each field of view.GraphPad Prism 5 software was used to identify statistical significance. Single data point comparisons were evaluated by Student's two-tailed t-test, whereas multiple comparisons were evaluated for statistical significance using Analysis of Variance (ANOVA) followed by Tukey-Kramer HSD post-test. All cytokine and cell count data are presented as mean +/− standard deviation (SD) or standard error of the mean (SEM), respectively, with a p-value less than 0.05 considered statistically significant.We employed Particle Replication in Non-Wetting Templates (PRINT) in an effort to address whether particles of defined size, shape and composition trigger an inflammatory response in mice. This fabrication platform enables production of homogenous and monodisperse particles with user-defined physical parameters. As a large amount of literature shows crucial biological differences depending on size and shape, the PRINT technique enables reproducible probing of basic cell biology with nearly complete control of design parameters For the purposes of our studies, we fabricated particles across the nano and micron range to reflect biologically relevant sizes. These include 80×320 nm particles , 1 µm and 1.5 µm particles (commensurate with bacteria and platelets), and 6 µm particles (akin to a red blood cell in size) poly(lactic-co-glycolic acid) PLGA, we also incorporated studies using hydrogel particles fabricated with derivatives of biocompatible poly (ethylene glycol) (PEG). Chemical modification of PEG is more feasible than with PLGA and thus it is often used to add increased functionality to nanocarriers, such as decoration of cell-targeting ligands, imaging agents, and pharmacologic cargo incorporation. Characteristics of fabricated PEG particles were similar to that of the PLGA particles, with low PDI and negative surface charge  and respiratory infections by microbial pathogens such as tuberculosis and influenza Bolstered by our in vitro findings and suggest that 80×320 nm PLGA particles can be delivered to the lungs without causing innate immune activation and inflammation.While BALF cellularity is widely used as a marker of lung inflammation, lung histopathology enables a deeper understanding of inflammatory effects on the lung parenchyma. We examined representative sections of histopathology slides of the main bronchi of the left lobe to further delineate leukocyte infiltration around lung vasculature, parenchyma and the large and small airways . Whereasin vivo findings, we fabricated a series of particles using PEG polymers and their derivatives (hydrogels) that incorporated fluorescent dyes which enabled us to track them in vivo over time after lung instillation. The hydrogel particles ranged in size from 80×320 nm to 1.5 µm and 6 µm as characterized in In vitro experiments indicated they did not elicit inflammatory cytokines or cell death from bone marrow-derived macrophages  immunologic stability in the lungs of mice.Given the diverse therapeutic potential of nano- and microscale particles, this study sought to define whether particles composed of either PLGA or PEG-derivatives induced inflammatory responses in an in situ. Some groups suggest PLGA particles are inflammatory in vitro and in vivo, whereas others have not found this to be the case in vivo delivery does not trigger an inflammatory reaction, contrary to a previous report The wide array of polymers and particle fabrication techniques used in nanomedicine studies makes it difficult to reach definitive conclusions regarding particle effects on innate immune functions. We initially used particles fabricated from PLGA as this is a commonly used polymer with attractive clinical potential given its F.D.A approval. There is some discrepancy in the literature as to whether PLGA particles are inflammatory In addition, our studies using PEG particles enabled us to broaden our understanding of innate immune activation by particles comprised of a polymer composition that enables wide-ranging chemical modifications for enhanced functionality, such as cell targeting, pH-specific cargo release and siRNA incorporation as previously reported by our group and others The issue of innate immune activation by particles is of central relevance to the translational application of nanotechnology. While particulate vaccines against some pathogens and cancers will likely be designed to trigger localized inflammation as part of the general innate immune activation required for robust adaptive immune responses, most other biomedical applications for nano- and microparticles will benefit by avoiding such responses. Additionally, a strong immune response might lead to the undesirable outcome of rapid particle clearance as well as hypersensitivity responses. Drug delivery, diagnostic imaging and physiological bio-mimicry are examples of nanoengineering applications that may be impeded by innate immune activation. Importantly, many advances in immune modulation made available through rationally designed nano- and microscale particles such as tolerance induction in the setting of autoimmunity or organ transplantation, direct targeting of immune cell subsets and immune-skewing of pathological microenvironments such as tumors or sites of chronic inflammation, require that particles be designed initially from an inert immunological state in vivo studies reveal that particle size augments uptake into innate immune cells of the lungs, with larger particles taken up less than smaller particles. This finding suggests a duality in design considerations depending on therapeutic application. For example, drug delivery to the lungs to ameliorate asthma would likely be best served by larger particles that can release their cargo to extracellular spaces. Conversely, if trying to deliver a respiratory vaccine, smaller particles that are more readily taken up by antigen presenting cells and traffic to lymph nodes would be more appropriate. Our finding that particles of all tested sizes remain in the lungs up to 7 days post instillation also suggest the ability to provide sustained localized delivery of therapeutically attractive molecules via particulate formulations. This is far different than the rapid clearance seen for smaller particles (<50 nm diameter) and reflects the importance of particle design parameters when considering therapeutic interventions Our The lung serves as an attractive route for therapeutic delivery due to its ease of access and its large absorptive surface area. There are several important particle characteristics that need to be considered for effective pulmonary delivery such as size, shape, surface charge, toxicity, and potential inflammatory effects. Inhaled particles with mass median aerodynamic diameters (MMAD) larger than 5 µm tend to be deposited in the upper conducting airway while particles with MMAD between 1–5 µm deposit in the lower respiratory airways Having identified particles across the nano- and microscale that do not trigger inflammatory responses in mice while remaining in the lungs, we plan to next use these particles as delivery devices for a range of biologically relevant molecules, including siRNAs, anti-inflammatory agents, and immune-skewing compounds. It should also be noted that mucoadhesive components may be incorporated within the PRINT particle system to enhance adsorption in the mucosal region which may enable differential deposition and enhanced temporal localization to the lungs. These studies will test the hypothesis that targeted modulation of lung immunology via nanoengineering may enable a new class of therapeutics for lung disorders that avoid systemic side-effects while also reducing administration doses. While we have tested a 50 µg dose of inert particles in this manuscript, PRINT enables a high weight percent loading of bioactive molecules and thus local and sustained pulmonary delivery may show therapeutic efficacy at low particle doses Importantly, we will move studies into human cells to provide much needed data regarding the immunological response to nanomaterials in our own species. Using the design control inherent to PRINT technology, we will also be able to systematically address the role of particle size and shape during delivery of bioactive molecules. Such results may be crucial to advancing next-generation respiratory vaccines and treatments for asthma, allergies and chronic disorders of lung function.