Semi-automated water sampling module for repeated sampling and concentration of Bacillus cereus group spores.

Monitoring the presence of pathogenic Bacillus spores is important for industrial applications, as well as necessary for ensuring human health. Bacillus thuringiensis is used as a biopesticide against several insect pests. Bacillus cereus spores are a significant cause of food poisoning, and Bacillus anthracis is a recognized biosecurity threat. Laboratory-based methods, such as polymerase chain reaction, enzyme-linked immunosorbent assay, or matrix-assisted laser desorption ionization spectroscopy provide sensitive detection of bacteria and spores, but the application of those methods for quasi-continuous environmental monitoring presents a significant challenge requiring frequent human intervention. To address this challenge, we developed a workstation for quasi-autonomous monitoring of water reservoirs for the presence of bacteria and spores, and designed and validated the functionality of a microprocessor-controlled module capable of repetitive collection and pre-concentration of spores in liquid samples tested with fiberglass (FG), polyether sulfone and polyvinylidene fluoride filters. The best results were obtained with FG filters delivering a 20× concentration of B. thuringiensis and B. cereus spores from saline suspensions. The successful 20× pre-concentration of Bacillus spores demonstrated with FG filters could be repeated up to 3 times when bleach decontamination is applied between filtrations. Taken together, our results demonstrate an attractive instrument suitable for semi-automated, quasi-continuous sampling and pre-processing of water samples for biosensing of bacterial spores originating from a complex environment.

Rapid, Sensitive, and Selective Quantification of Bacillus cereus Spores Using xMAP Technology.

Bacillus cereus is a spore-forming ubiquitous bacterium notable as a food poisoning agent. Detection of B. cereus spores using selective media is laborious and non-specific. Herein, the quantitative detection of B. cereus spores was investigated with commercial antibodies and published aptamer sequences. Several detection reagents were screened for affinity to Bacillus collagen-like protein A (BclA), an abundant exosporium glycoprotein. Sensitivity and selectivity toward B. cereus spores were tested using immunoassays and multi-analyte profiling (xMAP). A recombinant antibody developed in llama against BclA protein showed B. cereus spore selectivity and sensitivity between 102 and 105 spores/mL using xMAP. DNA aptamer sequences demonstrated sensitivity from 103 to 107 spores/mL and no cross-reaction to B. megaterium and B. subtilis. Selectivity for B. cereus spores was also demonstrated in a mixture of several diverse microorganisms and within a food sample with no compromise of sensitivity. As proof of concept for multiplexed measurement of human pathogens, B. cereus and three other microorganisms, E. coli, P. aeruginosa, and S. cerevisiae, were simultaneously detected using xMAP. These data support the development of a rapid, sensitive, and selective system for quantitation of B. cereus spores and multiplexed monitoring of human pathogens in complex matrices.

Selective Detection of Legionella pneumophila Serogroup 1 and 5 with a Digital Photocorrosion Biosensor Using Antimicrobial Peptide-Antibody Sandwich Strategy.

Rapid detection of Legionella pneumophila (L. pneumophila) is important for monitoring the presence of these bacteria in water sources and preventing the transmission of the Legionnaires' disease. We report improved biosensing of L. pneumophila with a digital photocorrosion (DIP) biosensor functionalized with an innovative structure of cysteine-modified warnericin antimicrobial peptides for capturing bacteria that are subsequently decorated with anti-L. pneumophila polyclonal antibodies (pAbs). The application of peptides for the operation of a biosensing device was enabled by the higher bacterial-capture efficiency of peptides compared to other traditional ligands, such as those based on antibodies or aptamers. At the same time, the significantly stronger affinity of pAbs decorating the L. pneumophila serogroup-1 (SG-1) compared to serogroup-5 (SG-5) allowed for the selective detection of L. pneumophila SG-1 at 50 CFU/mL. The results suggest that the attractive sensitivity of the investigated sandwich method is related to the flow of an extra electric charge between the pAb and a charge-sensing DIP biosensor. The method has the potential to offer highly specific and sensitive detection of L. pneumophila as well as other pathogenic bacteria and viruses.

Strategies for capturing Bacillus thuringiensis spores on surfaces of (001) GaAs-based biosensors.

Bacillus thuringiensis (Bt) is used as a bioinsecticide since it effectively kills insect larvae. Bt is also genetically similar to Bacillus cereus (Bc), a well recognized foodborne human pathogen; they are both members of the Bacillus cereus group (BC group). Although approved Bt bioinsecticide products have been confirmed to be non-pathogenic to humans, close monitoring of Bt during dissemination is important for cost considerations and to limit impact on biodiversity towards nontarget organisms. As such, developing rapid, sensitive, and specific tools for quantitative detection of Bt spores during and following spray operations is highly desirable. The goals of this study were to investigate commercially available detection reagents for sensitivity and selectivity in detecting Bt spores, and then functionalize a surface of (001) GaAs used in photonic biosensing. To achieve these goals, we (1) screened commercial antibodies for their capacity to bind recombinant proteins from Bt spores, (2) screened antibodies and aptamers for their sensitivity and selectivity against Bt spores, and (3) tested the efficiency of selected antibodies and aptamers in capturing Bt spores on the surface of functionalized GaAs biochips. Seven genes encoding Bt spore proteins were cloned and expressed in Escherichia coli. The binding of each purified spore antigen was tested by commercially available polyclonal and monoclonal antibodies claimed to exclusively target spores. Of the seven targets, Bacillus collagen-like protein A, was the most abundant protein on Bt spores and demonstrated the strongest binding affinity to all test antibodies. The commercial antibodies (Abs) were also tested for specificity to BC Group versus non-BC Group spores. Three of six commercial antibodies showed selectivity to Bt spores, with recombinant Abs providing the most robust lower range of detection (102 to 6 × 103 spores/mL). The sensitivity and selectivity of three published DNA aptamer sequences demonstrated a wide range of detection sensitivity for Bt spores. Two of the three test aptamers also showed reasonable selectivity towards Bt spores while the third demonstrated reactivity to non-BC Group B. megaterium and B. subtilis. Of the reagents tested, a thiolated aptamer and llama recombinant Ab showed highest Bt spore capture efficiency as measured by spore coverage of the GaAs surface. These results confirm that the selected aptamer and llama rAb can be considered strong candidates for the development of GaAs-based biosensing devices.

Short Ligand, Cysteine-Modified Warnericin RK Antimicrobial Peptides Favor Highly Sensitive Detection of Legionella pneumophila.

Culture-based methods for the detection of Legionella pneumophila are prohibitively slow and frequently inadequate. The problem has been addressed with biosensing technology that employs a variety of ligands for the specific capture of bacteria. However, the limited success of the application of mammalian antibodies, aptamers, and nucleic acid-based probes for sensitive biosensing has generated growing interest in exploring alternative biosensing architectures, such as those based on antimicrobial peptides (AMP) that are known for their attractive therapeutic applications. We report on the successful employment of cysteine-modified warnericin RK AMP for the operation of a highly sensitive biosensor of L. pneumophila based on digital photocorrosion of GaAs/AlGaAs nanoheterostructures. The replacement of the relatively cumbersome procedure commonly applied for the attachment of antibodies to COOH-terminated mercaptohexadecanoic acid self-assembled monolayers has allowed for a significant reduction in the distance at which bacteria are immobilized above the biosensor surface. An important consequence of this approach is the attractive limit of detection of L. pneumophila estimated at 2 × 102 CFU/mL. The target bacteria were captured four times more efficiently than P. fluorescens, B. subtilis, and E. coli, which is highly promising for environmental monitoring.

Hepatotoxicity of Cadmium Telluride Quantum Dots Induced by Mitochondrial Dysfunction.

The aim of this study was to investigate the detailed mechanisms of hepatotoxicity induced by cadmium telluride quantum dots (CdTe-QDs) in BALB/c mice after intravenous injection. The study investigated oxidative stress, apoptosis, and effects on mitochondria as potential mechanistic events to elucidate the observed hepatotoxicity. Oxidative stress in the liver, induced by CdTe-QD exposure, was demonstrated by depletion of total glutathione, an increase in superoxide dismutase activity, and changes in the gene expression of several oxidative stress-related biomarkers. Furthermore, CdTe-QD treatment led to apoptosis in the liver via both intrinsic and extrinsic apoptotic pathways. Effects on mitochondria were evidenced by the enlargement and increase in the number of mitochondria in hepatocytes of treated mice. CdTe-QDs also caused changes in the levels and gene expression of electron transport chain enzymes, depletion of ATP, and an increase in the level of the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a regulator of mitochondrial biogenesis. The findings from this study suggest that CdTe-QDs-induced hepatotoxicity might have originated from mitochondrial effects which resulted in oxidative stress and apoptosis in the liver cells. This study provides insight into the biological effects of CdT-QDs at the tissue level and the detailed mechanisms of their toxicity in animals. The study also provides important data for bridging the gap between in vitro and in vivo testing and risk assessment of these NPs.

Biodistribution and Systemic Effects in Mice Following Intravenous Administration of Cadmium Telluride Quantum Dot Nanoparticles.

Quantum dots (QDs) are engineered nanoparticles (NPs) of semiconductor structure that possess unique optical and electronic properties and are widely used in biomedical applications; however, their risks are not entirely understood. This study investigated the tissue distribution and toxic effects of cadmium telluride quantum dots (CdTe-QDs) in male BALB/c mice for up to 1 week after single-dose intravenous injections. CdTe-QDs were detected in the blood, lung, heart, liver, spleen, kidney, testis and brain. Most CdTe-QDs accumulated in the liver, followed by the spleen and kidney. At high doses, exposure to CdTe-QDs resulted in mild dehydration, lethargy, ruffled fur, hunched posture, and body weight loss. Histological analysis of the tissues, upon highest dose exposures, revealed hepatic hemorrhage and necrotic areas in the spleen. The sera of mice treated with high doses of CdTe-QDs showed significant increases in alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin levels, as well as a reduction in albumin. CdTe-QD exposure also led to a reduced number of platelets and elevated total white blood cell counts, including monocytes and neutrophils, serum amyloid A, and several pro-inflammatory cytokines. These results demonstrated that the liver is the main target of CdTe-QDs and that exposure to CdTe-QDs leads to hepatic and splenic injury, as well as systemic effects, in mice. By contrast, cadmium chloride (CdCl2), at an equivalent concentration of cadmium, appeared to have a different pharmacokinetic pattern from that of CdTe-QDs, having minimal effects on the aforementioned parameters, suggesting that cadmium alone cannot fully explain the toxicity of CdTe-QDs.

Acellular filtrate of a microbial-based cleaning product potentiates house dust mite allergic lung inflammation.

Microbial-based cleaning products (MBCPs) contain bacteria and chemical constituents. They are used in consumer applications such as odor reduction, unclogging drains, and surface cleaning. To determine the capacity of a model MBCP to contribute to acute allergic lung inflammation, a two-week repeated exposure regimen was used. Mice were exposed by endotracheal instillation to saline alone, MBCP alone, house dust mites (HDM) alone, or sequentially (i.e., MBCP followed by HDM, HDM followed by MBCP, or HDM + MBCP followed by HDM). Both whole MBCP and acellular MBCP filtrate were investigated, and showed minimal differences in the endpoints examined. HDM exposure caused pulmonary perivascular inflammation, bronchiolar mucous cell metaplasia, elevated bronchoalveolar lavage fluid (BALF) eosinophils, and HDM-specific IgG1. For MBCP, notable changes were associated with sequential exposures. MBCP/HDM caused elevated TH2 cytokines in BALF, and elevated neutrophils, eosinophils and IL-5 in peripheral blood. Co-administration of MBCP and HDM followed by HDM resulted in elevated blood and BALF eosinophils and HDM-specific IgE and IgG1. These results demonstrated that acellular MBCP filtrate, and not bacteria within MBCPs, potentiated the acute allergic inflammation to HDM. This methodology could be extended to investigate chronic allergic inflammation and inflammatory potential of other MBCPs and biotechnology products with complex compositions.

Incorporating Aptamers in the Multiple Analyte Profiling Assays (xMAP): Detection of C-Reactive Protein.

Aptamers are short oligonucleotide sequences used in detection systems because of their high affinity binding to a variety of macromolecules. With the introduction of aptamers over 25 years ago came the exploration of their use in many different applications as a substitute for antibodies. Aptamers have several advantages; they are easy to synthesize, can bind to analytes for which it is difficult to obtain antibodies, and in some cases bind better than antibodies. As such, aptamer applications have significantly expanded as an adjunct to a variety of different immunoassay designs. The Multiple-Analyte Profiling (xMAP) technology developed by Luminex Corporation commonly uses antibodies for the detection of analytes in small sample volumes through the use of fluorescently coded microbeads. This technology permits the simultaneous detection of multiple analytes in each sample tested and hence could be applied in many research fields. Although little work has been performed adapting this technology for use with apatmers, optimizing aptamer-based xMAP assays would dramatically increase the versatility of analyte detection. We report herein on the development of an xMAP bead-based aptamer/antibody sandwich assay for a biomarker of inflammation (C-reactive protein or CRP). Protocols for the coupling of aptamers to xMAP beads, validation of coupling, and for an aptamer/antibody sandwich-type assay for CRP are detailed. The optimized conditions, protocols and findings described in this research could serve as a starting point for the development of new aptamer-based xMAP assays.

Composition and pathogenic potential of a microbial bioremediation product used for crude oil degradation.

A microbial bioremediation product (MBP) used for large-scale oil degradation was investigated for microbial constituents and possible pathogenicity. Aerobic growth on various media yielded >108 colonies mL-1. Full-length 16S rDNA sequencing and fatty acid profiling from morphologically distinct colonies revealed ≥13 distinct genera. Full-length 16S rDNA library sequencing, by either Sanger or long-read PacBio technology, suggested that up to 21% of the MBP was composed of Arcobacter. Other high abundance microbial constituents (>6%) included the genera Proteus, Enterococcus, Dysgonomonas and several genera in the order Bacteroidales. The MBP was most susceptible to ciprofloxacin, doxycycline, gentamicin, and meropenam. MBP exposure of human HT29 and A549 cells caused significant cytotoxicity, and bacterial growth and adherence. An acellular MBP filtrate was also cytotoxic to HT29, but not A549. Both MBP and filtrate exposures elevated the neutrophil chemoattractant IL-8. In endotracheal murine exposures, bacterial pulmonary clearance was complete after one-week. Elevation of pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α, and chemokines KC and MCP-1 occurred between 2h and 48h post-exposure, followed by restoration to baseline levels at 96h. Cytokine/chemokine signalling was accompanied by elevated blood neutrophils and monocytes at 4h and 48h, respectively. Peripheral acute phase response markers were maximal at 24h. All indicators examined returned to baseline values by 168h. In contrast to HT29, but similar to A549 observations, MBP filtrate did not induce significant murine effects with the indicators examined. The results demonstrated the potentially complex nature of MBPs and transient immunological effects during exposure. Products containing microbes should be scrutinized for pathogenic components and subjected to characterisation and quality validation prior to commercial release.

Immunomodulation by gastrointestinal carbon black nanoparticle exposure in ovalbumin T cell receptor transgenic mice.

Humans could become exposed to carbon black nanoparticles (CBNPs) in consumer products or an occupational setting. In rodent models, acute respiratory, subcutaneous, and direct immune cell exposure to CBNPs has been shown to enhance allergic sensitization to co-administered ovalbumin (OVA) protein from chicken egg. However, little is known about the effects of ingested CBNPs on immunological responses and oral tolerance to food antigens. We hypothesized that ingestion of CBNPs would enhance the development of food allergy to OVA. Allergy prone DO11.10 mice were orally exposed to CBNPs every second day for 2 weeks (total dose 10.8 (LOW) or 108 µg (HI)), with and without (±) co-administered OVA. Systemic immune parameters were measured at necropsy. Exposure to OVA resulted in significant increases in serum anti-OVA IgG1, anti-OVA IgM, and anti-OVA IgA antibodies relative to vehicle control. Immunophenotyping revealed a reduction in the number of OVA-specific CD4+ T helper cells upon OVA ± CBNPHI treatment in the spleen. Yet, secretion of the allergy-associated Th2 cytokines IL-4, IL-9, and IL-13 was greater in OVA323-339 peptide-pulsed splenocytes from OVA + CBNPHI-treated mice compared with control. Transcriptome analysis at necropsy of splenocytes from OVA + CBNPHI dose mice compared with OVA mice revealed increases in the allergy associated genes Il4 and Stat6 and decreases in Csf3r and Retnlg. Although oral exposure to high-dose CBNPs did not impact OVA-specific antibody production relative to OVA, we did observe increased expression of genes and cytokines associated with allergy in peripheral splenocytes. This work suggests that CBNP gastrointestinal exposure may potentiate allergy pathways.

Toxicological evaluation of representative silver nanoparticles in macrophages and epithelial cells.

Silver nanoparticles (Ag-NPs) are highly relevant for human and environmental exposure due to their widespread use in consumer and medical products and various applications. Thus, there is a need for evaluating potential toxicity of these NPs. The objective of this study was to investigate the toxic effects of the OECD (Organization for Economic Co-operation and Development) representative Ag-NPs, NM300K, in mouse macrophage J774A.1 and human colonic epithelial HT29 cells, using multiple endpoint assays. Exposure of test cells to different concentrations (1-250 µg/mL; total silver content) of NM300K for 24h showed a dose-dependent decrease in cell viability. At high doses, NM300K altered cell shape and induced the formation of vacuolar structures, as examined by confocal and electron microscopy. Moreover, NM300K induced inflammation as evidenced by the elevated levels of pro-inflammatory cytokines. Finally, high doses of NM300K led to increased production of reactive oxygen species and induction of oxidative stress, leading to oxidative DNA damage and apoptosis in test cells. At equivalent silver concentrations, NM300K were less cytotoxic than AgNO3. However, the similar patterns in the effects of NM300K and AgNO3 throughout the assessed toxicological endpoints suggest that Ag(+) released from these NPs by dissolution could be a primary contributor to toxicity. This study is among the first to characterize the potential toxicity of OECD representative AgNPs in vitro, and provides additional insight into the biological mechanisms associated with Ag-NP toxicity.

Virulence Attributes and Host Response Assays for Determining Pathogenic Potential of Pseudomonas Strains Used in Biotechnology.

Pseudomonas species are opportunistically pathogenic to humans, yet closely related species are used in biotechnology applications. In order to screen for the pathogenic potential of strains considered for biotechnology applications, several Pseudomonas strains (P.aeruginosa (Pa), P.fluorescens (Pf), P.putida (Pp), P.stutzeri (Ps)) were compared using functional virulence and toxicity assays. Most Pa strains and Ps grew at temperatures between 28°C and 42°C. However, Pf and Pp strains were the most antibiotic resistant, with ciprofloxacin and colistin being the most effective of those tested. No strain was haemolytic on sheep blood agar. Almost all Pa, but not other test strains, produced a pyocyanin-like chromophore, and caused cytotoxicity towards cultured human HT29 cells. Murine endotracheal exposures indicated that the laboratory reference strain, PAO1, was most persistent in the lungs. Only Pa strains induced pro-inflammatory and inflammatory responses, as measured by elevated cytokines and pulmonary Gr-1 -positive cells. Serum amyloid A was elevated at ≥ 48 h post-exposure by only some Pa strains. No relationship was observed between strains and levels of peripheral leukocytes. The species designation or isolation source may not accurately reflect pathogenic potential, since the clinical strain Pa10752 was relatively nonvirulent, but the industrial strain Pa31480 showed comparable virulence to PAO1. Functional assays involving microbial growth, cytotoxicity and murine immunological responses may be most useful for identifying problematic Pseudomonas strains being considered for biotechnology applications.

Review of current methods for characterizing virulence and pathogenicity potential of industrial Saccharomyces cerevisiae strains towards humans.

Most industrial Saccharomyces cerevisiae strains used in food or biotechnology processes are benign. However, reports of S. cerevisiae infections have emerged and novel strains continue to be developed. In order to develop recommendations for the human health risk assessment of S. cerevisiae strains, we conducted a literature review of current methods used to characterize their pathogenic potential and evaluated their relevance towards risk assessment. These studies revealed that expression of virulence traits in S. cerevisiae is complex and depends on many factors. Given the opportunistic nature of this organism, an approach using multiple lines of evidence is likely necessary for the reasonable prediction of the pathogenic potential of a particular strain. Risk assessment of S. cerevisiae strains would benefit from more research towards the comparison of virulent and non-virulent strains in order to better understand those genotypic and phenotypic traits most likely to be associated with pathogenicity.

Phagocytosis-coupled flow cytometry for detection and size discrimination of anionic polystyrene particles.

Flow cytometry was evaluated for its capacity to detect and distinguish a wide size range (20-2000 nm) of fluorescent polystyrene particles (PSPs). Side scatter and fluorescence parameters could predict dispersed PSP sizes down to 200 nm, but the forward scatter parameter was not discriminatory. Confocal microscopy of flow-sorted fractions confirmed that dispersed PSPs appeared as a single sharp peak on fluorescence histograms, whereas agglomerated PSPs were detected as smaller adjacent peaks. Particles as small as 200 nm could also be detected by flow cytometry after they were first phagocytized by J774A.1 murine macrophages. Confocal microscopy demonstrated that these PSPs were internalized within the cytoplasm. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and calcein-AM (acetoxymethyl ester) assays showed that they were not cytotoxic. Internalized PSP size correlated to both cellular side scatter (R(2)=0.9821) and fluorescence intensity (R(2)=0.9993). Furthermore, PSPs of various sizes could be distinguished when J774A.1 cells were loaded with a single size of PSP and mixed with cells containing other sizes. However, spectra of cells loaded with a mixture of PSP sizes resembled those containing only the largest PSP. These data demonstrate the capacity and limitations of phagocytosis-coupled flow cytometry to distinguish between dispersed and agglomerated states and detect a wide size range of particles.

Mitochondrial Toxicity of Cadmium Telluride Quantum Dot Nanoparticles in Mammalian Hepatocytes.

There are an increasing number of studies indicating that mitochondria are relevant targets in nanomaterial-induced toxicity. However, the underlying mechanisms by which nanoparticles (NPs) interact with these organelles and affect their functions are unknown. The aim of this study was to investigate the effects of cadmium telluride quantum dot (CdTe-QD) NPs on mitochondria in human hepatocellular carcinoma HepG2 cells. CdTe-QD treatment resulted in the enlargement of mitochondria as examined with transmission electron microscopy and confocal microscopy. CdTe-QDs appeared to associate with the isolated mitochondria as detected by their inherent fluorescence. Further analyses revealed that CdTe-QD caused disruption of mitochondrial membrane potential, increased intracellular calcium levels, impaired cellular respiration, and decreased adenosine triphosphate synthesis. The effects of CdTe-QDs on mitochondrial oxidative phosphorylation were evidenced by changes in levels and activities of the enzymes of the electron transport chain. Elevation of peroxisome proliferator-activated receptor-γ coactivator levels after CdTe-QD treatment suggested the effects of CdTe-QDs on mitochondrial biogenesis. Our results also showed that the effects of CdTe-QDs were similar or greater to those of cadmium chloride at equivalent concentrations of cadmium, suggesting that the toxic effects of CdTe-QDs were not solely due to cadmium released from the NPs. Overall, the study demonstrated that CdTe-QDs induced multifarious toxicity by causing changes in mitochondrial morphology and structure, as well as impairing their function and stimulating their biogenesis.

Development of a bead-based aptamer/antibody detection system for C-reactive protein.

A multiplexing bead-based platform provides an approach for the development of assays targeting specific analytes for biomonitoring and biosensing applications. Multi-Analyte Profiling (xMAP) assays typically employ a sandwich-type format using antibodies for the capture and detection of analytes of interest, and the system permits the simultaneous quantitation of multiple targets. In this study, an aptamer/antibody assay for the detection of C-reactive protein (CRP) was developed. CRP is an acute phase marker of inflammation whose elevated basal levels are correlated with an increased risk for a number of pathologies. For this assay, an RNA aptamer that binds CRP was conjugated to beads to act as the capture agent. Biotinylated anti-CRP antibody coupled to fluorescently labeled streptavidin was used for quantification of CRP. The detection limit of the CRP assay was 0.4 mg/L in diluted serum. The assay was then used to detect spiked CRP samples in the range of 0.4 to 10mg/L in diluted serum with acceptable recoveries (extrapolated values of 70-130%), including that of a certified reference material (129% recovery). The successful incorporation of the CRP aptamer into this platform demonstrates that the exploration of other aptamer-target systems could increase the number of analytes measurable using xMAP-type assays.

Critical experimental parameters related to the cytotoxicity of zinc oxide nanoparticles.

The increasing use of zinc oxide nanoparticles (ZnO-NPs) has raised concerns about their potential hazards to human and environmental health. In this study, the characterization and cytotoxicity of two ZnO-NPs products (Z-COTE and Z-COTE HP1) were investigated. The zinc content of Z-COTE and Z-COTE HP1 was 82.5 ± 7.3 and 80.1 ± 3.5 %, respectively. Both ZnO-NP samples contained sub-cytotoxic levels of iron and copper, and silicon was detected from the surface coating of Z-COTE HP1. All samples were highly agglomerated, and the primary particles appeared as variable polyhedral structures. There was no significant difference in size distribution or average diameter of Z-COTE (53 ± 23 nm) and Z-COTE HP1 (54 ± 26 nm). A dose-dependent cytotoxicity was observed 24 h after exposure to ZnO-NPs, and monocytes were more sensitive than lung epithelial cells or lymphoblasts in both human and mouse cells. There was a significant difference in cytotoxicity between nano- and fine-forms, but only at the threshold cytotoxic dose with cellular metabolism assays. Compared to uncoated ZnO-NPs, the surface coating with triethoxycaprylylsilane marginally attenuated cellular oxidative stress and protected cellular metabolic activity. These results demonstrate the importance of model cell type, dose selection, and cytotoxicity assessment methodology to accurately evaluate the potential toxicity of various nanoparticles in vitro.

In vitro enhancement of mouse T helper 2 cell sensitization to ovalbumin allergen by carbon black nanoparticles.

Agglomerated carbon black nanoparticles (CBNPs) administered via respiratory or subcutaneous routes have been shown to promote allergic sensitization to coadministered ovalbumin (OVA) protein in rodents. In the present study, we aimed to model and elucidate the mechanism of this adjuvanticity using an in vitro assay based on T cell sensitization to ovalbumin323₋339 peptide (OVA(p)). CBNP base particles of 22 and 39 nm were characterized and termed CBNP22 and CBNP39 powders. Splenic leukocytes derived from transgenic DO11.10 mice were exposed to suspensions of media alone, concanavalin A mitogen, CBNP agglomerates smaller than 220 nm, OVA(p) alone, OVA(p) + anti-CD28 costimulant, OVA(p) + cyclosporin A immunosuppressant, or OVA(p) + CBNPs. Samples were analyzed at 72 h post-exposure. Proliferation rate, a marker of cellular mitosis, was assessed. Polymerase chain reaction arrays were used to assess genes involved in allergic response pathways. The mitogen control, costimulatory control, and immunosuppressive control chemicals modified the T helper cell proliferation rate. CBNP22 mildly reduced proliferation at 12 µg/ml, but CBNP39 did not. Gene expression analysis of cells treated with OVA(p) showed that coincubation with 12 µg/ml CBNP22 enhanced gene expression of interleukin-4 (IL-4), IL-10, and IL-13, all allergy-associated Th2 cytokines. Coincubation of OVA(p) with 12 µg/ml CBNP39 significantly enhanced IL-13 gene expression concurrent with downregulation of the Th1-associated transcription factor Stat4. IL-4 and IL-13 protein secretion reflected the mRNA trends. The changes were consistently higher in cells exposed to CBNP22 than CBNP39, suggesting that smaller particle size, higher surface area, and higher purity were associated with the direct adjuvant effect on Th2 cells in this genetically susceptible model of OVA allergy.