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Human induced pluripotent stem cells (iPSCs) hold great promise for biomedical applications. However, establishment of new iPSC lines still presents many challenges. Here we describe a simple yet highly efficient two-step protocol for the isolation and purification of human iPSC lines. The first step adapts iPSCs to single cell culture and passaging, promoting survival and self-renewal; the second step enables the isolation and purification of bona fide iPSCs from a mixed population using column-based positive selection of cells expressing pluripotency markers such as TRA-1-60. Both steps utilize commercially available reagents. Using this protocol, iPSCs can be purified from cell preparations containing differentiated or unreprogrammed cells, or even be isolated directly from reprogramming vessels. The protocol could be adopted for high throughput isolation and expansion of iPSC lines and facilitate the widespread use of iPSCs in future applications.

Hepatocyte-like cells (HLCs) generated from human induced pluripotent stem cells (iPSCs) could provide an unlimited source of liver cells for regenerative medicine, disease modeling, drug screening, and toxicology studies. Here we describe a stepwise improved protocol that enables highly efficient, homogeneous, and reproducible differentiation of human iPSCs into functional hepatocytes through controlling all three stages of hepatocyte differentiation, starting from a single cell (non-colony) culture of iPSCs, through homogeneous definitive endoderm induction and highly efficient hepatic specification, and finally arriving at matured HLCs. The final population of cells exhibits morphology closely resembling that of primary human hepatocytes, and expresses specific hepatic markers as evidenced by immunocytochemical staining. More importantly, these HLCs demonstrate key functional characteristics of mature hepatocytes, including major serum protein (e.g., albumin, fibronectin, and alpha-1 antitrypsin) secretion, urea synthesis, glycogen storage, and inducible cytochrome P450 activity.

Directed differentiation of human induced pluripotent stem cells (iPSCs) into hepatocytes could provide an unlimited source of liver cells, and therefore holds great promise for regenerative medicine, disease modeling, drug screening and toxicology studies. Various methods have been established during the past decade to differentiate human iPSCs into hepatocyte-like cells (HLCs) using growth factors and/or small molecules. However, direct comparison of the differentiation efficiency and the quality of the final HLCs between different methods has rarely been reported. In the current study, two hepatocyte differentiation methods were devised, termed Method 1 and 2, through modifying existing well-known hepatocyte differentiation strategies, and the resultant cells were compared phenotypically and functionally at different stages of hepatocyte differentiation. Compared to Method 1, higher differentiation efficiency and reproducibility were observed in Method 2, which generated highly homogeneous functional HLCs at the end of the differentiation process. The cells exhibited morphology closely resembling primary human hepatocytes and expressed high levels of hepatic protein markers. More importantly, these HLCs demonstrated several essential characteristics of mature hepatocytes, including major serum protein (albumin, fibronectin and α-1 antitrypsin) secretion, urea release, glycogen storage and inducible cytochrome P450 activity. Further transcriptomic comparison of the HLCs derived from the two methods identified 1,481 differentially expressed genes (DEGs); 290 Gene Ontology terms in the biological process category were enriched by these genes, which were further categorized into 34 functional classes. Pathway analysis of the DEGs identified several signaling pathways closely involved in hepatocyte differentiation of pluripotent stem cells, including 'signaling pathways regulating pluripotency of stem cells', 'Wnt signaling pathway', 'TGF-beta signaling pathway' and 'PI3K-Akt signaling pathway'. These results may provide a molecular basis for the differences observed between the two differentiation methods and suggest ways to further improve hepatocyte differentiation in order to obtain more mature HLCs for biomedical applications.

The application of silver nanoparticles (AgNPs) in consumer products has been increasing rapidly over the past decades. Therefore, in vitro models capable of accurately predicting the toxicity of AgNPs are much needed. Hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (iPSCs) represent an attractive alternative in vitro hepatotoxicity model. Yet, the use of iPSC-derived HLCs (iPSC-HLCs) for the study of nanoparticle toxicity has not been reported so far. In the present study, transcriptomic changes induced by varying concentrations (5-25 μg/ml) of AgNPs were characterized in iPSC-HLCs after 24-h exposure. AgNPs caused concentration-dependent gene expression changes in iPSC-HLCs. At all the concentrations, members of the metallothionein (MT) and the heat shock protein (HSP) families were the dominating upregulated genes, suggesting that exposure to AgNPs induced oxidative stresses in iPSC-HLCs and as a result elicited cellular protective responses in the cells. Functional analysis showed that the differentially expressed genes (DEGs) were majorly involved in the biological processes of metabolism, response to stress, and cell organization and biogenesis. Ingenuity Pathway Analysis revealed that cancer was at the top of diseases and disorders associated with the DEGs at all concentrations. These results were in accordance with those reported previously on hepatoma cell lines and primary hepatocytes. Considering the advantages iPSC-HLCs have over other liver cell models in terms of unlimited supply, consistency in quality, sustainability of function in long-term culture, and, more importantly, affordability of donor specificity, the results of the current study suggest that iPSC-HLCs may serve as a better in vitro model for liver nanotoxicology.

Purpose Dihydroxyacetone (DHA) is the only ingredient approved by the U.S. FDA as a color additive in sunless tanning (self-tanning) products. Consumer sunless tanning products available for retail purchase contain 1% to 15% DHA. Although originally thought to only interact with the stratum corneum, more recent research has shown that DHA penetrates beyond the stratum corneum to living keratinocytes indicating a possible route of exposure in the epidermis. Materials and methods Normal Human Epidermal Keratinocytes (NHEK) were used to determine any potential in vitro toxicological effects of DHA in the epidermis. NHEK cells exposed to DHA concentrations up to 0.90% (100 mM) in dosing media were evaluated for viability, genotoxicity (Comet Assay), and gene expression changes by microarray analysis. Results Cell viability significantly decreased ∼50% after 3-hour exposure to 50 and 100 mM DHA. DNA damage was only found to be significantly increased in cells exposed to cytotoxic DHA concentrations. A subtoxic dose of DHA induced significant gene expression changes. Particularly, expression of cyclin B1, CDK1, and six other genes associated with the G2/M cell cycle checkpoint were significantly decreased which correlates well with a G2/M block reported in existing literature. Advanced Glycation End Product (AGE) formation significantly increased after 24 hours of DHA exposure at and above 10 mM. In summary, these data show that DHA is cytotoxic above 25 mM in primary keratinocytes. Genotoxicity was detected only at cytotoxic concentrations, likely indicative of non-biologically relevant DNA damage, while subtoxic doses induce gene expression changes and glycation. Conclusion DHA treatment had a significant and negative effect on primary keratinocytes consistent with in vitro cultured cell outcomes; however, more information is needed to draw conclusions about the biological effect of DHA in human skin.

Induced pluripotent stem cells (iPSCs) offer the potential to generate tissue cells with donor diversity therefore promising to have widespread applications in regenerative medicine, disease modeling, drug discovery, and toxicity testing. Several somatic cell types have been utilized, with varying efficiencies, as source cells for the reprogramming of iPSCs. Recently, it has been reported that endothelial progenitor cells (EPCs) derived from umbilical cord blood (CB) or adult peripheral blood (PB) afford a practical and efficient cellular substrate for iPSC generation, and possess several advantages over other cell types. In this chapter, we describe a protocol that covers all steps of reprogramming iPSCs from blood-derived EPCs, including (1) isolation of mononuclear cells (MNCs) from blood samples, (2) derivation of EPCs from MNCs, and (3) generation of iPSCs from EPCs. The final step of reprogramming EPCs into iPSCs is achieved through ectopic expression of four transcription factors, OCT4, KLF4, SOX2, and c-MYC, using self-replicative RNA (srRNA) technology.

Hepatocytes play a central role in toxicological studies, yet the paucity and variability in supply and instability under in vitro conditions limit the use of primary human hepatocytes in such applications. In the last decade, there has been burgeoning interest in the differentiation of human induced pluripotent stem cells (iPSCs) to hepatocyte-like cells (HLCs) as a defined and renewable source of hepatic cells. Many protocols have been developed thus far, which generated cells with encouraging results in terms of marker expression and functional assays. However, most of these methods rely on the use of growth factors therefore suffering the disadvantages of high cost and low reproducibility. Recently, increasing studies have shown the feasibility and advantages of using small molecules (SMs) for the generation of HLCs. In this chapter, we present a historical overview and current status on the differentiation of iPSCs into HLCs, and focus on recent development of SM-based differentiation protocols. We also discuss applications of HLCs in hepatotoxicity studies and offer some perspectives on future development of the field.

Induced pluripotent stem cells (iPSCs) offer the potential to generate tissue cells with donor diversity therefore promising to have widespread applications in regenerative medicine, disease modeling, drug discovery, and toxicity testing. Several somatic cell types have been utilized, with varying efficiencies, as source cells for the reprogramming of iPSCs. Recently, it has been reported that endothelial progenitor cells (EPCs) derived from umbilical cord blood (CB) or adult peripheral blood (PB) afford a practical and efficient cellular substrate for iPSC generation, and possess several advantages over other cell types. In this chapter, we describe a protocol that covers all steps of reprogramming iPSCs from blood-derived EPCs, including (1) isolation of mononuclear cells (MNCs) from blood samples, (2) derivation of EPCs from MNCs, and (3) generation of iPSCs from EPCs. The final step of reprogramming EPCs into iPSCs is achieved through ectopic expression of four transcription factors, OCT4, KLF4, SOX2, and c-MYC, using self-replicative RNA (srRNA) technology.

Silver nanoparticles (AgNPs) have been increasingly used in a variety of consumer products over the last decades. However, their potential adverse effects have not been fully understood. In a previous study, we characterized transcriptomic changes in human induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) in response to AgNP exposure. Here, we report findings of a follow-up proteomic study that evaluated alternations at the protein level in the same cell after being exposed to 10 μg/ml AgNPs for 24 h. In total, 6287 proteins were identified across two groups of samples (n = 3). Among these proteins, 665 were found to be differentially regulated (fold change ≥1.25, p < 0.01) between the AgNP-treated group and the untreated control group, including 264 upregulated and 401 downregulated. Bioinformatics analysis of the proteomics data, in side-by-side comparison to the transcriptomics data, confirms and substantiates previous findings on AgNP-induced alterations in metabolism, oxidative stress, inflammation, and potential association with cancer. A mechanism of action was proposed based on these results. Collectively, the findings of the current proteomic study are consistent with those of the previous transcriptomic study and further demonstrate the usefulness of iPSC-derived HLCs as an in vitro model for liver nanotoxicology.

Background Hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (iPSCs) hold great promise in toxicological applications as well as in regenerative medicine. Previous efforts on hepatocyte differentiation have mostly relied on the use of growth factors (GFs) to recapitulate developmental signals under in vitro conditions. Recently, the use of small molecules (SMs) has emerged as an attractive tool to induce cell fate transition due to its superiority in terms of both quality and cost. However, HLCs derived using SMs have not been well characterized, especially on the transcriptome level. Methods HLCs were differentiated from human iPSCs using a protocol that only involves SMs and characterized by transcriptomic analysis using whole genome microarrays. Results HLCs derived using the SM protocol (HLC_SM) displayed specific hepatic marker expression and demonstrated key hepatic functions. Transcriptomic analysis of the SM-driven differentiation defined a hepatocyte differentiation track and characterized the expression of some key marker genes in major stages of hepatocyte differentiation. In addition, HLC_SM were scored with CellNet, a bioinformatics tool quantifying how closely engineered cell populations resemble their target cell type, and compared to primary human hepatocytes (PHHs), adult liver tissue, fetal liver tissue, HLCs differentiated using GFs (HLC_GF), and commercially available HLCs. Similar to HLC_GF, HLC_SM displayed a mixed phenotype of fetal and adult hepatocytes and had relatively low expression of metabolic enzymes, transporters, and nuclear receptors compared to PHHs. Finally, the differentially expressed genes in HLC_SM compared to HLC_GF and to PHHs were analyzed to identify pathways and upstream transcription regulators which could potentially be manipulated to improve the differentiation of HLCs. Conclusions Overall, the present study demonstrated the usefulness of the SM-based hepatocyte differentiation method, offered new insights into the molecular basis of hepatogenesis and associated gene regulation, and suggested ways for further improvements in hepatocyte differentiation in order to obtain more mature HLCs that could be used in toxicological studies.

The application of silver nanoparticles (AgNPs) in consumer products has been increasing rapidly over the past decades. Therefore, in vitro models capable of accurately predicting the toxicity of AgNPs are much needed. Hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (iPSCs) represent an attractive alternative in vitro hepatotoxicity model. Yet, the use of iPSC-derived HLCs (iPSC-HLCs) for the study of nanoparticle toxicity has not been reported so far. In the present study, transcriptomic changes induced by varying concentrations (5-25 μg/ml) of AgNPs were characterized in iPSC-HLCs after 24-h exposure. AgNPs caused concentration-dependent gene expression changes in iPSC-HLCs. At all the concentrations, members of the metallothionein (MT) and the heat shock protein (HSP) families were the dominating upregulated genes, suggesting that exposure to AgNPs induced oxidative stresses in iPSC-HLCs and as a result elicited cellular protective responses in the cells. Functional analysis showed that the differentially expressed genes (DEGs) were majorly involved in the biological processes of metabolism, response to stress, and cell organization and biogenesis. Ingenuity Pathway Analysis revealed that cancer was at the top of diseases and disorders associated with the DEGs at all concentrations. These results were in accordance with those reported previously on hepatoma cell lines and primary hepatocytes. Considering the advantages iPSC-HLCs have over other liver cell models in terms of unlimited supply, consistency in quality, sustainability of function in long-term culture, and, more importantly, affordability of donor specificity, the results of the current study suggest that iPSC-HLCs may serve as a better in vitro model for liver nanotoxicology.

Human induced pluripotent stem cells (iPSCs) provide a potentially unlimited source of differentiated cells from individuals with specific genetic backgrounds. Using self-replicative RNA reprogramming technology, we generated nine iPSC lines from endothelial progenitor cells (EPCs) derived from blood samples of three different ethnicities: Black or African American, Latino or Hispanic, and Non-Hispanic White. The resulting iPSC lines showed normal karyotype in large part, expressed pluripotency marker genes, and spontaneously differentiated in vitro into the three germ layers. These iPSC lines offer the potential to generate tissues with ethnic diversity, and thus afford a valuable tool for ethnic-related toxicological applications.

In this study, the effects of surface charge, dose, and cosmetic vehicle on the penetration of silver nanoparticles (AgNPs) into pig and human skin were compared. AgNPs (20 nm) with varying surface-charges (polyethylene glycol (PEG; neutral), citrate (CIT; negative), and branched polyethylenimine (bPEI; positive) were dosed onto skin in in vitro diffusion cells using an aqueous solution and an oil-in-water emulsion formulation. Samples were analyzed by inductively coupled plasma mass spectroscopy (ICP-MS) and transmission electron microscope (TEM) to assess AgNP skin penetration. The results showed that neutral and positive AgNPs penetrate human skin when applied in a high dose aqueous solution and less with the emulsion vehicle. A mass balance percutaneous penetration study in human skin found the majority of AgNPs were washed from the skin or remained mostly in the stratum corneum (3.4% of the applied dose for AgbPEI and 1.7% for AgPEG). Very little silver was found in the epidermis (1.2% AgbPEI and 0.3% AgPEG) and dermis (0.1% AgbPEI and none detected for AgPEG). These results indicate low dermal penetration of AgNPs that is not greatly affected by surface coating charge. The results will facilitate dermal exposure assessments by better understanding how nanoparticle properties affect skin absorption of nanoparticles found in personal care products.

Hepatocytes derived from human induced pluripotent stem cells (iPSCs) hold great promise as an in vitro liver model by virtue of their unlimited long-term supply, stability and consistency in functionality, and affordability of donor diversity. However, the suitability of iPSC-derived hepatocytes (iPSC-Heps) for toxicology studies has not been fully validated. In the current study, we characterized global gene expression profiles of iPSC-Heps in comparison to those of primary human hepatocytes (PHHs) and several human hepatoma cell lines (HepaRG, HuH-7, HepG2, and HepG2/C3A). Furthermore, genes associated with hepatotoxicity, drug-metabolizing enzymes, transporters, and nuclear receptors were extracted for more detailed comparisons. Our results showed that iPSC-Heps correlate more closely to PHHs than hepatoma cell lines, suggesting that iPSC-Heps had a relatively mature hepatic phenotype that more closely resembles that of adult hepatocytes. HepaRG was the sole exception but nonetheless suffers from lack of donor diversity and poor prediction of hepatotoxicity. The effects of sex differences and DMSO treatment on gene expression of the cellular models were also investigated. Overall, the results presented in the current study suggest that iPSC-Heps represent a reproducible source of human hepatocytes and a promising in vitro model for hepatotoxicity evaluation. Further studies are needed to develop a robust protocol for hepatocyte differentiation towards a more mature adult phenotype.

Human-induced pluripotent stem cells (iPSCs) hold considerable promise for future biomedical applications. However, the generation, isolation, and establishment of an iPSC line still presents many challenges. In this study, we describe a simple yet highly efficient two-step method for the isolation, purification, and passaging of human iPSC lines that utilizes commercially available reagents. The first step adapts iPSCs to single cell culture and passage, promoting survival and self-renewal; the second step enables the isolation and purification of bona fide iPSCs from a mixed population using column-based positive selection of cells expressing pluripotency markers such as TRA-1-60. Using this method, we were able to purify iPSCs from cell preparations containing differentiated or unreprogrammed cells, and even to isolate iPSC lines directly from derivation plates. The iPSC lines generated by this method maintained their pluripotency and genomic stability, as demonstrated by trilineage differentiation and karyotype analysis. The method presented here could be adopted for high-throughput isolation and expansion of iPSC lines and facilitate the widespread use of iPSCs in future applications.

Thalidomide is a potent developmental toxicant that induces a range of birth defects, notably severe limb malformations. To unravel the molecular mechanisms underpinning the teratogenic effects of thalidomide, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on the differentiation of mouse embryonic stem cells (mESCs), and published the major findings in a research article entitled “Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells” [1]. The data presented herein contains complementary information related to the aforementioned research article.

The increased use of silver nanoparticles (AgNPs) in foods and cosmetics has raised public safety concerns. However, only limited knowledge exists on the effect of AgNPs on the cellular transcriptome. This study evaluated global gene expression profiles of human liver HepG2 cells exposed to 20 and 50 nm AgNPs for 4 and 24 h at 2.5 µg ml–1. Exposure to 20 nm AgNPs resulted in 811 altered genes after 4 h, but much less after 24 h. Exposure to 50 nm AgNPs showed minimal altered genes at both exposure times. The HepG2 cells responded to the toxic insult of AgNPs by transiently upregulating stress response genes such as metallothioneins and heat shock proteins. Functional analysis of the altered genes showed more than 20 major biological processes were affected, of which metabolism, development, cell differentiation and cell death were the most dominant categories. Several cellular pathways were also impacted by AgNP exposure, including the p53 signaling pathway and the NRF2-mediated oxidative stress response pathway, which may lead to increased oxidative stress and DNA damage in the cell and potentially result in genotoxicity and carcinogenicity. Together, these results indicate that HepG2 cells underwent a multitude of cellular processes in response to the toxic insult of AgNP exposure, and suggest that toxicogenomic characterization of human HepG2 cells could serve as an alternative model for assessing toxicities of NPs. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

Highlights • Transcriptomics was used to study the effect of silver ion on developing thymus. • Maternal exposure to silver acetate was conducted during gestation and lactation. • Silver acetate up to 40.0 mg/kg did not adversely affect thymic development., Male and female rats (26-day-old) were exposed to 0.0, 0.4, 4 or 40 mg/kg body weight silver acetate (AgAc) in drinking water for 10 weeks prior to and during mating. Sperm-positive females remained within their dose groups and were exposed to silver acetate during gestation and lactation. At postnatal day 26, the effect of silver ions on the developing F1 generation rat thymus was evaluated at the transcriptional level using whole-genome microarrays. Gene expression profiling analyses identified a dozen differentially expressed genes (DEGs) in each dose group using a loose criterion of fold change (FC) >1.5 and unadjusted p

Background The widespread application of silver nanoparticles (AgNPs) and silver-containing products has raised public safety concerns about their adverse effects on human health and the environment. To date, in vitro toxic effects of AgNPs and ionic silver (Ag+) on many somatic cell types are well established. However, no studies have been conducted hitherto to evaluate their effect on cellular transcriptome in embryonic stem cells (ESCs). Results The present study characterized transcriptomic changes induced by 5.0 µg/ml AgNPs during spontaneous differentiation of mouse ESCs, and compared them to those induced by Ag+ under identical conditions. After 24 h exposure, 101 differentially expressed genes (DEGs) were identified in AgNP-treated cells, whereas 400 genes responded to Ag+. Despite the large differences in the numbers of DEGs, functional annotation and pathway analysis of the regulated genes revealed overall similarities between AgNPs and Ag+. In both cases, most of the functions and pathways impacted fell into two major categories, embryonic development and metabolism. Nevertheless, a number of canonical pathways related to cancer were found for Ag+ but not for AgNPs. Conversely, it was noted that several members of the heat shock protein and the metallothionein families were upregulated by AgNPs but not Ag+, suggesting specific oxidative stress effect of AgNPs in ESCs. The effects of AgNPs on oxidative stress and downstream apoptosis were subsequently confirmed by flow cytometry analysis. Conclusions Taken together, the results presented in the current study demonstrate that both AgNPs and Ag+ caused transcriptomic changes that could potentially exert an adverse effect on development. Although transcriptomic responses to AgNPs and Ag+ were substantially similar, AgNPs exerted specific effects on ESCs due to their nanosized particulate form. Electronic supplementary material The online version of this article (doi:10.1186/s12951-017-0265-6) contains supplementary material, which is available to authorized users.

Additional file 2: Figure S2. Morphological changes of the differentiating EBs after exposure for 24Â h to varying concentrations of AgNPs (A) or Ag+ (B). The concentrations of AgNPs or Ag+ used for the exposure (in Âľg/ml) are indicated by the numbers at the top left corner of each image.

Anti-malarial 8-aminoquinolines drugs cause acute hemolytic anemia in individuals with glucose-6-phosphate dehydrogenase deficiency (G6PDD). Efforts to develop non-hemolytic 8-aminoquinolines have been severely limited caused by the lack of a predictive in vivo animal model of hemolytic potential that would allow screening of candidate compounds. This report describes a G6PDD mouse model with a phenotype closely resembling the G6PDD phenotype found in the African A-type G6PDD human. These G6PDD mice, given different doses of primaquine, which used as a reference hemolytic drug, display a full array of hemolytic anemia parameters, consistently and reproducibly. The hemolytic and therapeutic indexes were generated for evaluation of hemotoxicity of drugs. This model demonstrated a complete hemolytic toxicity response to another known hemolytic antimalarial drug, pamaquine, but no response to non-hemolytic drugs, chloroquine and mefloquine. These results suggest that this model is suitable for evaluation of selected 8-AQ type candidate antimalarial drugs for their hemolytic potential.

Biomarkers play a pivotal role in the early detection and diagnosis of cancer. Accurate quantitation of certain biomarkers is crucial to reach correct treatment decisions. In practice, immunohistochemistry (IHC) remains the most important diagnostic technique to evaluate protein biomarker expression in tissue biopsies. However, IHC has largely been qualitative. Low specificity of the mammalian IgG antibodies used to capture the analytes and instability of fluorescence from the organic dyes used as the detecting agents are among the major factors that have impeded the development of quantitative IHC. Avian IgY antibodies have many attractive biochemical, immunological and production advantages over IgGs and are, therefore, better substitutes in diagnostic applications. Using IgY in immunoassays can potentially eliminate false positives and often results in low background and interference. Quantum dots (QDs) have recently emerged as a novel class of fluorophores, promising for many biomedical imaging applications. Fluorescence from QDs is significantly brighter and more photostable than organic dyes. In addition, QDs offer the capacity of multiplexed detection of several biomarkers simultaneously. Combining the high sensitivity and specificity of IgY antibodies and the high brightness and photostability of QDs in IHC has been demonstrated to improve biomarker detection and quantitation.

Background Sulfur mustard (SM) inhalation causes apoptosis and death of airway epithelial cells as well as inflammation in the airway. Efficient clearance of the cell debris by alveolar macrophages is necessitated to reduce the inflammation. Macrolide antibiotics have been reported to have anti-inflammatory properties by modulating the production of proinflammatory cytokines and mediators, and by improving macrophage functions. The present study investigated the effects of four commonly used macrolide antibiotics, namely azithromycin, clarithromycin, erythromycin, and roxithromycin, on chemotactic and phagocytotic function and on inflammatory cytokines/mediators production in vitro in SM-exposed monocyte THP-1 cells. Results Chemotaxis and phagocytosis of the monocytes reduced upon exposure to 10 μM SM (8.1% and 17.5%, respectively) were restored by treatment with 10 μM of any of the four macrolides. Overexpression of inflammatory cytokines following SM exposure was decreased by 50–70% with macrolide treatment. Similarly, exaggerated iNOS expression and nitric oxide (NO) production induced by SM exposure was largely inhibited by treatment with macrolides. Conclusion The data demonstrate that macrolide antibiotics were effective in improving the degenerated chemotactic and phagocytotic functions of monocytes following SM exposure, and in reducing SM-induced overproduction of proinflammatory cytokines and mediators. Thus, treatment with macrolide antibiotics may lead to improved clearance of apoptotic material in the airway and ultimately result in reduced airway inflammation and injury caused by SM inhalation, suggesting that macrolide antibiotics may serve as potential vesicant respiratory therapeutics.

Background: Human epidermal growth factor receptor 2 (HER2) is an important biomarker whose status plays a pivotal role in therapeutic decision-making for breast cancer patients and in determining their clinical outcomes. Ensuring the accuracy and reproducibility of HER2 assays by immunohistochemistry (IHC) and by fluorescence in situ hybridization (FISH) requires a reliable standard for monitoring assay sensitivity and specificity, and for assessing methodologic variation. A prior NIST workshop addressed this need by reaching a consensus to create cell lines as reference materials for HER2 testing.Methods: Breast carcinoma cell lines SK-BR-3 and MCF-7 were characterized quantitatively by IHC with chicken anti-HER2 IgY antibody and by FISH with biotinylated bacterial artificial chromosome DNA probes; both assays used quantum dots as detectors. Formalin-fixed and paraffin-embedded (FFPE) cell blocks were prepared and tested for suitability as candidate reference materials by IHC and FISH with commercially available reagents. IHC and FISH results were also compared with those obtained by laser-scanning cytometry and real-time PCR, respectively.Results: MCF-7 cells had typical numbers of gene copies and very low production of HER2 protein, whereas SK-BR-3 cells contained approximately 10-fold more copies of the gene and exhibited approximately 15-fold higher amounts of HER2 protein than MCF-7 cells. FFPE SK-BR-3 cells showed results similar to those for fresh SK-BR-3 cells.Conclusions: SK-BR-3 and MCF-7 are suitable as candidate reference materials in QC of HER2 testing. Coupled with the associated assay platforms, they provide valuable controls for quantitative measurement of HER2 amplification and production in breast cancer samples, irrespective of the antibody/probe or detector used.

Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72h after exposure to 0.25mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment.

Ubiquinol cytochrome c oxido-reductase (EC. 1.10.2.2, bc1) is an integral membrane protein complex essential to cellular respiration. Structures of the 11-subunit mitochondrial bc1 complex were determined with and without the fungicide famoxadone. Specific inhibition by famoxadone is achieved through a coordinated optimization of aromatic-aromatic interactions where conformational rearrangements in famoxadone and in residues lining the inhibitor-binding pocket produce a network of aromatic-aromatic interactions that mimic the crystal lattice of benzene. The profound aromatic-aromatic interactions as supported by prior mutagenesis provide a structural basis for specific protein-ligand interaction in a hydrophobic environment. Dramatic conformational changes, both in cyt. b and ISP subunits in the inhibitor-protein complex, confer experimental evidence for a functional role of cytochrome b in the induced conformational arrest of ISP and allow the identification of a possible intrasubunit signal transduction pathway that controls the movement of ISP. These results support an inhibitory mechanism that is consistent with the requirement for ISP movement in the electron transfer of this complex.

Mustard gas (sulfur mustard [SM], bis-[2-chloroethyl] sulfide) is a vesicating chemical warfare agent and a potential chemical terrorism agent. Exposure of SM causes debilitating skin blisters (vesication) and injury to the eyes and the respiratory tract; of these, the respiratory injury, if severe, may even be fatal. Therefore, developing an effective therapeutic strategy to protect against SM-induced respiratory injury is an urgent priority of not only the US military but also the civilian antiterrorism agencies, for example, the Homeland Security. Toward developing a respiratory medical countermeasure for SM, four different classes of therapeutic compounds have been evaluated in the past: anti-inflammatory compounds, antioxidants, protease inhibitors and antiapoptotic compounds. This review examines all of these different options; however, it suggests that preventing cell death by inhibiting apoptosis seems to be a compelling strategy but possibly dependent on adjunct therapies using the other drugs, that is, anti-inflammatory, antioxidant, and protease inhibitor compounds.

Organophosphorus (OP) compounds represent an important group of chemical warfare nerve agents that remains a significant and constant military and civilian threat. OP compounds are considered acting primarily via cholinergic pathways by binding irreversibly to acetylcholinesterase, an important regulator of the neurotransmitter acetylcholine. Many studies over the past years have suggested that other mechanisms of OP toxicity exist, which need to be unraveled by a comprehensive and systematic approach such as genome-wide gene expression analysis. Here we performed a microarray study in which cultured human neural cells were exposed to 0.1 or 10 μM of VX for 1 h. Global gene expression changes were analyzed 6, 24, and 72 h post exposure. Functional annotation and pathway analysis of the differentially expressed genes has revealed many genes, networks and canonical pathways that are related to nervous system development and function, or to neurodegenerative diseases such as Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease. In particular, the neuregulin pathway impacted by VX exposure has important implications in many nervous system diseases including schizophrenia. These results provide useful information valuable in developing suitable antidotes for more effective prevention and treatment of, as well as in developing biomarkers for, VX-induced chronic neurotoxicity.

Macrolide antibiotics have been shown to protect airway epithelial cells and macrophages from sulfur mustard (SM)–induced cytotoxicity. In the current study, the efficacy of roxithromycin in ameliorating SM-induced respiratory injury was further evaluated in a rat model. Anesthetized rats ( N = 8/group) were intratracheally exposed to SM by vapor inhalation. For the drug treatment groups, rats were orally given 10, 20, or 40 mg/kg roxithromycin one hr prior to exposure and every twenty-four hr thereafter. After one, three, or seven days of treatment, sections of the lung were examined and scored for histopathological parameters. Treatment with roxithromycin ameliorated many of the symptoms caused by SM in some animals. In particular, treatment at 40 mg/kg for three days showed significant improvements ( p < .05) over the untreated group. When the evaluation was focused on trachea, treatment with roxithromycin for three days showed a trend of dose-dependent protection; moreover, the groups treated with 20 or 40 mg/kg of roxithromycin were statistically different ( p < .001 and p < .05, respectively) from the untreated group. These results suggest that roxithromycin protects against some damages associated with SM injury in the lung, particularly in the upper respiratory tract.

The sections in this article are Introduction Cancer A Primer on Cancer Biology The Importance of Early Cancer Detection and Diagnosis The Role of Biomarkers in Cancer Early Detection and Diagnosis Quantum Dots: Physics and Chemistry Photophysical Properties of QDs Quantum Dot Chemistry Synthesis Surface Passivation Water Solubilization Bioconjugation Cancer Imaging with QDs In Vitro Screening and Detection of Cancer Biomarkers Using Microarrays In Vitro Cellular Labeling of Cancer Biomarkers Labeling of Fixed Cells and Tissues Live Cell Imaging in Cancer Cells In Vivo Cancer Imaging In Vivo Tracking of Cancer Cells Tumor Vasculature Imaging Sentinel Lymph Node Mapping and Fluorescence Lymphangiography In Vivo Whole-Body Tumor Imaging in Animals Multimodality Tumor Imaging Dual-Functionality QDs for Cancer Imaging and Therapy Quantum Dot Cytotoxicity and Potential Safety Concerns Concluding Remarks and Future Perspectives Acknowledgments Keywords: quantum dot; nanocrystal; cancer; tumor; imaging; fluorescence; biomarker; early detection

Background The rapid growth of the nanotechnology industry and the wide application of various nanomaterials have raised concerns over their impact on the environment and human health. Yet little is known about the mechanism of cellular uptake and cytotoxicity of nanoparticles. An array of nanomaterials has recently been introduced into cancer research promising for remarkable improvements in diagnosis and treatment of the disease. Among them, quantum dots (QDs) distinguish themselves in offering many intrinsic photophysical properties that are desirable for targeted imaging and drug delivery. Results We explored the kinetics and mechanism of cellular uptake of QDs with different surface coatings in two human mammary cells. Using fluorescence microscopy and laser scanning cytometry (LSC), we found that both MCF-7 and MCF-10A cells internalized large amount of QD655-COOH, but the percentage of endocytosing cells is slightly higher in MCF-7 cell line than in MCF-10A cell line. Live cell fluorescent imaging showed that QD cellular uptake increases with time over 40 h of incubation. Staining cells with dyes specific to various intracellular organelles indicated that QDs were localized in lysosomes. Transmission electron microscopy (TEM) images suggested a potential pathway for QD cellular uptake mechanism involving three major stages: endocytosis, sequestration in early endosomes, and translocation to later endosomes or lysosomes. No cytotoxicity was observed in cells incubated with 0.8 nM of QDs for a period of 72 h. Conclusions The findings presented here provide information on the mechanism of QD endocytosis that could be exploited to reduce non-specific targeting, thereby improving specific targeting of QDs in cancer diagnosis and treatment applications. These findings are also important in understanding the cytotoxicity of nanomaterials and in emphasizing the importance of strict environmental control of nanoparticles.

Background Nanocarrier-based antibody targeting is a promising modality in therapeutic and diagnostic oncology. Single-walled carbon nanotubes (SWNTs) exhibit two unique optical properties that can be exploited for these applications, strong Raman signal for cancer cell detection and near-infrared (NIR) absorbance for selective photothermal ablation of tumors. In the present study, we constructed a HER2 IgY-SWNT complex and demonstrated its dual functionality for both detection and selective destruction of cancer cells in an in vitro model consisting of HER2-expressing SK-BR-3 cells and HER2-negative MCF-7 cells. Methods The complex was constructed by covalently conjugating carboxylated SWNTs with anti-HER2 chicken IgY antibody, which is more specific and sensitive than mammalian IgGs. Raman signals were recorded on Raman spectrometers with a laser excitation at 785 nm. NIR irradiation was performed using a diode laser system, and cells with or without nanotube treatment were irradiated by 808 nm laser at 5 W/cm2 for 2 min. Cell viability was examined by the calcein AM/ethidium homodimer-1 (EthD-1) staining. Results Using a Raman optical microscope, we found the Raman signal collected at single-cell level from the complex-treated SK-BR-3 cells was significantly greater than that from various control cells. NIR irradiation selectively destroyed the complex-targeted breast cancer cells without harming receptor-free cells. The cell death was effectuated without the need of internalization of SWNTs by the cancer cells, a finding that has not been reported previously. Conclusion We have demonstrated that the HER2 IgY-SWNT complex specifically targeted HER2-expressing SK-BR-3 cells but not receptor-negative MCF-7 cells. The complex can be potentially used for both detection and selective photothermal ablation of receptor-positive breast cancer cells without the need of internalization by the cells. Thus, the unique intrinsic properties of SWNTs combined with high specificity and sensitivity of IgY antibodies can lead to new strategies for cancer detection and therapy.

Sulfur mustard (SM) inhalation causes apoptosis and death of airway epithelial cells as well as inflammation in the airway. Efficient clearance of the cell debris by alveolar macrophages (AMs) is necessitated to reduce the inflammation. Macrolide antibiotics have been reported to have anti-inflammatory properties by modulating the production of proinflammatory cytokines and mediators, and by improving macrophage functions. The present study investigated the effects of four FDA-approved macrolide antibiotics, namely azithromycin, clarithromycin, erythromycin, and roxithromycin, on macrophage chemotactic and phagocytotic function and on inflammatory cytokines/mediators production in vitro using SM-exposed monocyte THP-1 cells. Using flow cytometry we found that chemotaxis and phagocytosis of the monocytes reduced upon exposure to 10 micrometer SM (8.1% and 17.5%, respectively) were restored by treatment with 10 micrometer of any of the four macrolides. Cytokine measurements using real-time RT-PCR and ELISA revealed that overexpression of proinflammatory cytokines following SM exposure was decreased by 50%- 70% with macrolide treatment. Similarly, immunocytochemical detection of inducible nitric oxide synthase (iNOS) showed that exaggerated expression of iNOS induced by SM exposure was totally inhibited by treatment with macrolides. Together, these data demonstrate that macrolide antibiotics were effective in improving the degenerated chemotactic and phagocytotic functions of macrophages following SM exposure, and in reducing SM-induced overproduction of proinflammatory cytokines and mediators. These effects may lead to improved clearance of apoptotic material in the airway and ultimately result in reduced airway inflammation and injury caused by SM inhalation. Our results suggest that macrolide antibiotics may serve as potential vesicant respiratory therapeutics.

Sulfur mustard, a vesicant chemical warfare agent, causes airway injury due to massive release of destructive enzymes and mediators of inflammation. Nitric oxide plays an important yet controversial role in inflammation. An impressive number of reports suggest that excessive amount of nitric oxide may promote inflammation-induced cell injury and death. Overproduction of nitric oxide is catalysed by up-regulated expression of the inducible isoform of nitric oxide synthase (iNOS). In this study, we used quantum dot-mediated immunocytochemistry to analyse iNOS expression and flow cytometry to analyse the intracellular nitric oxide production in sulfur mustard-exposed normal human small airway epithelial cells and bronchial/tracheal epithelial cells and studied the effect of four US Food and Drug Administration-approved macrolide antibiotics, namely, azithromycin, clarithromycin, erythromycin and roxithromycin. Exposure to 100 µM sulfur mustard significantly up-regulated iNOS expression and resulted in overproduction of nitric oxide in these cells. Addition of macrolide antibiotics to 100 µM in the medium reduced both iNOS expression and nitric oxide production to near normal level. Thus, the current study provides in vitro evidence of the immunomodulatory effects of macrolide antibiotics in sulfur mustard-exposed airway epithelial cells. These results suggest that macrolide antibiotics may serve as potential vesicant respiratory therapeutics through mechanisms independent of their antibacterial activity.

In an effort to improve affinity biomarker validation in fixed patient tissue specimens, we have developed a novel quantum dot-based bioimaging system that utilizes chicken IgY antibody for high sensitivity and specificity relative quantitation of cancer proteins. Monospecific, polyclonal IgYs were generated against human HER2 and telomerase, and analytically validated for specificity by western blot and immunohistochemistry on tumor and normal cells and for relative affinity by layered peptide array (LPA). IgYs bound desired targets in cell lines and fixed tissues and showed greater affinity than commercial mammalian antibodies for both HER2 and telomerase proteins. In tissue microarray experiments, HER2 quantitation with IgY antibody and quantum dot imaging correlated well with chromogenic in situ hybridization (CISH), whereas telomerase quantitation suggested a trend toward correlation with prostate cancer Gleason Grade and differentiation. Although patient numbers were small, these findings demonstrate the feasibility of relative quantitation of cancer biomarkers with IgY and quantum dot fluorophores, and show promise for rigorous clinical validation in large patient cohorts.