Nanoparticle Wettability Influences Nanoparticle-Phospholipid Interactions.

We explored the influence of nanoparticle (NP) surface charge and hydrophobicity on NP-biomolecule interactions by measuring the composition of adsorbed phospholipids on four NPs, namely, positively charged CeO2 and ZnO and negatively charged BaSO4 and silica-coated CeO2, after exposure to bronchoalveolar lavage fluid (BALf) obtained from rats, and to a mixture of neutral dipalmitoyl phosphatidylcholine (DPPC) and negatively charged dipalmitoyl phosphatidic acid (DPPA). The resulting NP-lipid interactions were examined by cryogenic transmission electron microscopy (cryo-TEM) and atomic force microscopy (AFM). Our data show that the amount of adsorbed lipids on NPs after incubation in BALf and the DPPC/DPPA mixture was higher in CeO2 than in the other NPs, qualitatively consistent with their relative hydrophobicity. The relative concentrations of specific adsorbed phospholipids on NP surfaces were different from their relative concentrations in the BALf. Sphingomyelin was not detected in the extracted lipids from the NPs despite its >20% concentration in the BALf. AFM showed that the more hydrophobic CeO2 NPs tended to be located inside lipid vesicles, whereas less hydrophobic BaSO4 NPs appeared to be outside. In addition, cryo-TEM analysis showed that CeO2 NPs were associated with the formation of multilamellar lipid bilayers, whereas BaSO4 NPs with unilamellar lipid bilayers. These data suggest that the NP surface hydrophobicity predominantly controls the amounts and types of lipids adsorbed, as well as the nature of their interaction with phospholipids.

Repeated pulmonary exposures to zinc ions enhance inflammatory responses to subsequent metal exposures.

AIM OF STUDY: Metal contaminants contribute to adverse human health effects via acute and chronic exposures. Acute metal exposures followed by prolonged secondary metal exposures may elicit exaggerated inflammatory responses in certain individuals. The aim of this study is to determine whether repeated pulmonary exposures to zinc chloride (ZnCl2) alter subsequent responses to zinc or cerium exposures. MATERIALS AND METHODS: Rats were intratracheally (IT) instilled with physiologic saline (n = 24) or 0.05 mg/kg ZnCl2 (n = 16) twice weekly for 4 weeks. Four days after last dosing, the saline group was divided into three subgroups, each IT-instilled with either saline, ZnCl2 or CeCl3 (both at 0.1 mg/kg). The ZnCl2 pre-instilled rats were divided into two subgroups, each instilled with 0.1 mg/kg ZnCl2 or CeCl3. Biomarkers of lung injury/inflammation were assessed in bronchoalveolar lavage (BAL) fluid collected 24 hours later. Oxidative stress was evaluated as total and reduced glutathione in BAL. RESULTS: Increases in inflammatory cells, LDH, albumin, leptin, MCP-1, IP-10, fractalkine, TNFα and RANTES were observed in rats instilled with multiple PBS and then with 0.1 mg/kg ZnCl2 and CeCl3. However, rats pre-exposed repeatedly to 0.05 mg/kg ZnCl2 and then challenged with 0.1 mg/kg ZnCl2 or CeCl3 showed even more eosinophils, lymphocytes, and increased concentrations of hemoglobin and MIP-1α. Significant reduction in GSH/GSSG ratios in BAL in response to all ZnCl2 or CeCl3 exposures indicated oxidative stress. CONCLUSION: Previous exposure to zinc ions increases responsiveness to subsequent exposures to zinc and cerium ions. These findings suggest enhanced sensitization possibly due to a reduction in antioxidant defenses.

Protein corona: implications for nanoparticle interactions with pulmonary cells.

BACKGROUND: We previously showed that cerium oxide (CeO2), barium sulfate (BaSO4) and zinc oxide (ZnO) nanoparticles (NPs) exhibited different lung toxicity and pulmonary clearance in rats. We hypothesize that these NPs acquire coronas with different protein compositions that may influence their clearance from the lungs. METHODS: CeO2, silica-coated CeO2, BaSO4, and ZnO NPs were incubated in rat lung lining fluid in vitro. Then, gel electrophoresis followed by quantitative mass spectrometry was used to characterize the adsorbed proteins stripped from these NPs. We also measured uptake of instilled NPs by alveolar macrophages (AMs) in rat lungs using electron microscopy. Finally, we tested whether coating of gold NPs with albumin would alter their lung clearance in rats. RESULTS: We found that the amounts of nine proteins in the coronas formed on the four NPs varied significantly. The amounts of albumin, transferrin and α-1 antitrypsin were greater in the coronas of BaSO4 and ZnO than that of the two CeO2 NPs. The uptake of BaSO4 in AMs was less than CeO2 and silica-coated CeO2 NPs. No identifiable ZnO NPs were observed in AMs. Gold NPs coated with albumin or citrate instilled into the lungs of rats acquired the similar protein coronas and were cleared from the lungs to the same extent. CONCLUSIONS: We show that different NPs variably adsorb proteins from the lung lining fluid. The amount of albumin in the NP corona varies as does NP uptake by AMs. However, albumin coating does not affect the translocation of gold NPs across the air-blood barrier. A more extensive database of corona composition of a diverse NP library will develop a platform to help predict the effects and biokinetics of inhaled NPs.

Repeated Mouse Lung Exposures to Stachybotrys chartarum Shift Immune Response from Type 1 to Type 2.

After a single or multiple intratracheal instillations of Stachybotrys chartarum (S. chartarum or black mold) spores in BALB/c mice, we characterized cytokine production, metabolites, and inflammatory patterns by analyzing mouse bronchoalveolar lavage (BAL), lung tissue, and plasma. We found marked differences in BAL cell counts, especially large increases in lymphocytes and eosinophils in multiple-dosed mice. Formation of eosinophil-rich granulomas and airway goblet cell metaplasia were prevalent in the lungs of multiple-dosed mice but not in single- or saline-dosed groups. We detected changes in the cytokine expression profiles in both the BAL and plasma. Multiple pulmonary exposures to S. chartarum induced significant metabolic changes in the lungs but not in the plasma. These changes suggest a shift from type 1 inflammation after an acute exposure to type 2 inflammation after multiple exposures to S. chartarum. Eotaxin, vascular endothelial growth factor (VEGF), MIP-1α, MIP-1ß, TNF-α, and the IL-8 analogs macrophage inflammatory protein-2 (MIP-2) and keratinocyte chemoattractant (KC), had more dramatic changes in multiple- than in single-dosed mice, and parallel the cytokines that characterize humans with histories of mold exposures versus unexposed control subjects. This repeated exposure model allows us to more realistically characterize responses to mold, such as cytokine, metabolic, and cellular changes.

Pulmonary distribution of nanoceria: comparison of intratracheal, microspray instillation and dry powder insufflation.

Particles can be delivered to the respiratory tract of animals using various techniques. Inhalation mimics environmental exposure but requires large amounts of aerosolized NPs over a prolonged dosing time, varies in deposited dose among individual animals, and results in nasopharyngeal and fur particle deposition. Although less physiological, intratracheal (IT) instillation allows quick and precise dosing. Insufflation delivers particles in their dry form as an aerosol. We compared the distribution of neutron-activated 141CeO2 nanoparticles (5 mg/kg) in rats after (1) IT instillation, (2) left intrabronchial instillation, (3) microspraying of nanoceria suspension and (4) insufflation of nanoceria dry powder. Blood, tracheobronchial lymph nodes, liver, gastrointestinal tract, feces and urine were collected at 5 min and 24 h post-dosing. Excised lungs from each rat were dried at room temperature while inflated at a constant 30 cm water pressure. Dried lungs were then sliced into 50 pieces. The radioactivity of each lung piece and other organs was measured. The evenness index (EI) of each lung piece was calculated [EI = (µCi/mgpiece)/(µCi/mglung)]. The degree of EI value departure from 1.0 is a measure of deposition heterogeneity. We showed that the pulmonary distribution of nanoceria differs among modes of administration. Dosing by IT or microspraying resulted in similar spatial distribution. Insufflation resulted in significant deposition in the trachea and in more heterogeneous lung distribution. Our left intrabronchial instillation technique yielded a concentrated deposition into the left lung. We conclude that animal dosing techniques and devices result in varying patterns of particle deposition that will impact biokinetic and toxicity studies.

Silica coating influences the corona and biokinetics of cerium oxide nanoparticles.

BACKGROUND: The physicochemical properties of nanoparticles (NPs) influence their biological outcomes. METHODS: We assessed the effects of an amorphous silica coating on the pharmacokinetics and pulmonary effects of CeO2 NPs following intratracheal (IT) instillation, gavage and intravenous injection in rats. Uncoated and silica-coated CeO2 NPs were generated by flame spray pyrolysis and later neutron-activated. These radioactive NPs were IT-instilled, gavaged, or intravenously (IV) injected in rats. Animals were analyzed over 28 days post-IT, 7 days post-gavage and 2 days post-injection. RESULTS: Our data indicate that silica coating caused more but transient lung inflammation compared to uncoated CeO2. The transient inflammation of silica-coated CeO2 was accompanied by its enhanced clearance. Then, from 7 to 28 days, clearance was similar although significantly more (141)Ce from silica-coated (35%) was cleared than from uncoated (19%) (141)CeO2 in 28 days. The protein coronas of the two NPs were significantly different when they were incubated with alveolar lining fluid. Despite more rapid clearance from the lungs, the extrapulmonary (141)Ce from silica-coated (141)CeO2 was still minimal (<1%) although lower than from uncoated (141)CeO2 NPs. Post-gavage, nearly 100% of both NPs were excreted in the feces consistent with very low gut absorption. Both IV-injected (141)CeO2 NP types were primarily retained in the liver and spleen. The silica coating significantly altered the plasma protein corona composition and enhanced retention of (141)Ce in other organs except the liver. CONCLUSION: We conclude that silica coating of nanoceria alters the biodistribution of cerium likely due to modifications in protein corona formation after IT and IV administration.

Biokinetics and effects of barium sulfate nanoparticles.

BACKGROUND: Nanoparticulate barium sulfate has potential novel applications and wide use in the polymer and paint industries. A short-term inhalation study on barium sulfate nanoparticles (BaSO4 NPs) was previously published [Part Fibre Toxicol 11:16, 2014]. We performed comprehensive biokinetic studies of ¹³¹BaSO4 NPs administered via different routes and of acute and subchronic pulmonary responses to instilled or inhaled BaSO4 in rats. METHODS: We compared the tissue distribution of ¹³¹Ba over 28 days after intratracheal (IT) instillation, and over 7 days after gavage and intravenous (IV) injection of ¹³¹BaSO4. Rats were exposed to 50 mg/m³ BaSO4 aerosol for 4 or 13 weeks (6 h/day, 5 consecutive days/week), and then gross and histopathologic, blood and bronchoalveolar lavage (BAL) fluid analyses were performed. BAL fluid from instilled rats was also analyzed. RESULTS: Inhaled BaSO4 NPs showed no toxicity after 4-week exposure, but a slight neutrophil increase in BAL after 13-week exposure was observed. Lung burden of inhaled BaSO4 NPs after 4-week exposure (0.84 ± 0.18 mg/lung) decreased by 95% over 34 days. Instilled BaSO4 NPs caused dose-dependent inflammatory responses in the lungs. Instilled BaSO4 NPs (0.28 mg/lung) was cleared with a half-life of ≈ 9.6 days. Translocated ¹³¹Ba from the lungs was predominantly found in the bone (29%). Only 0.15% of gavaged dose was detected in all organs at 7 days. IV-injected ¹³¹BaSO4 NPs were predominantly localized in the liver, spleen, lungs and bone at 2 hours, but redistributed from the liver to bone over time. Fecal excretion was the dominant elimination pathway for all three routes of exposure. CONCLUSIONS: Pulmonary exposure to instilled BaSO4 NPs caused dose-dependent lung injury and inflammation. Four-week and 13-week inhalation exposures to a high concentration (50 mg/m³) of BaSO4 NPs elicited minimal pulmonary response and no systemic effects. Instilled and inhaled BaSO4 NPs were cleared quickly yet resulted in higher tissue retention than when ingested. Particle dissolution is a likely mechanism. Injected BaSO4 NPs localized in the reticuloendothelial organs and redistributed to the bone over time. BaSO4 NP exhibited lower toxicity and biopersistence in the lungs compared to other poorly soluble NPs such as CeO2 and TiO2.

Bioavailability, distribution and clearance of tracheally-instilled and gavaged uncoated or silica-coated zinc oxide nanoparticles.

BACKGROUND: Nanoparticle pharmacokinetics and biological effects are influenced by several factors. We assessed the effects of amorphous SiO2 coating on the pharmacokinetics of zinc oxide nanoparticles (ZnO NPs) following intratracheal (IT) instillation and gavage in rats. METHODS: Uncoated and SiO2-coated ZnO NPs were neutron-activated and IT-instilled at 1 mg/kg or gavaged at 5 mg/kg. Rats were followed over 28 days post-IT, and over 7 days post-gavage. Tissue samples were analyzed for 65Zn radioactivity. Pulmonary responses to instilled NPs were also evaluated at 24 hours. RESULTS: SiO2-coated ZnO elicited significantly higher inflammatory responses than uncoated NPs. Pulmonary clearance of both 65ZnO NPs was biphasic with a rapid initial t1/2 (0.2 - 0.3 hours), and a slower terminal t1/2 of 1.2 days (SiO2-coated ZnO) and 1.7 days (ZnO). Both NPs were almost completely cleared by day 7 (>98%). With IT-instilled 65ZnO NPs, significantly more 65Zn was found in skeletal muscle, liver, skin, kidneys, cecum and blood on day 2 in uncoated than SiO2-coated NPs. By 28 days, extrapulmonary levels of 65Zn from both NPs significantly decreased. However, 65Zn levels in skeletal muscle, skin and blood remained higher from uncoated NPs. Interestingly, 65Zn levels in bone marrow and thoracic lymph nodes were higher from coated 65ZnO NPs. More 65Zn was excreted in the urine from rats instilled with SiO2-coated 65ZnO NPs. After 7 days post-gavage, only 7.4% (uncoated) and 6.7% (coated) of 65Zn dose were measured in all tissues combined. As with instilled NPs, after gavage significantly more 65Zn was measured in skeletal muscle from uncoated NPs and less in thoracic lymph nodes. More 65Zn was excreted in the urine and feces with coated than uncoated 65ZnO NPs. However, over 95% of the total dose of both NPs was eliminated in the feces by day 7. CONCLUSIONS: Although SiO2-coated ZnO NPs were more inflammogenic, the overall lung clearance rate was not affected. However, SiO2 coating altered the tissue distribution of 65Zn in some extrapulmonary tissues. For both IT instillation and gavage administration, SiO2 coating enhanced transport of 65Zn to thoracic lymph nodes and decreased transport to the skeletal muscle.

Influence of DMT1 and iron status on inflammatory responses in the lung.

Divalent metal transporter 1 (DMT1) is the major iron transporter responsible for duodenal dietary iron absorption and is required for erythropoiesis. Recent studies suggest that loss of DMT1 activity could be involved in metal-related lung injury, but little is known about the effects of iron status and DMT1 function on pulmonary inflammation. To better define the role of DMT1 and iron status in pulmonary inflammatory responses, we performed bronchoalveolar lavage (BAL) following intratracheal instillation of lipopolysaccharide (LPS) to the Belgrade rat, an animal model deficient in DMT1 function. In the basal state, the BAL fluid of Belgrade rats had more macrophages and higher lactate dehydrogenase, myeloperoxidase, albumin, and hemoglobin levels compared with heterozygote control rats. Following LPS instillation, the macrophage fraction relative to total BAL cell content and levels of albumin and IgM were increased in Belgrade rats compared with controls. In contrast, heterozygote Belgrade rats made anemic by diet-induced iron deficiency exhibited attenuated inflammatory responses to LPS. These combined results show that pulmonary inflammation can be modified by both DMT1 and iron status. Loss of DMT1 alters pulmonary responses necessary for lung homeostasis in the basal state and enhances LPS-induced inflammation and therefore would contribute to progression of lung injury.

Effects of Ingested Nanomaterials on Tissue Distribution of Co-ingested Zinc and Iron in Normal and Zinc-Deficient Mice.

Cellulose nanofibers (CNF) reduced serum triglyceride levels in rats when co-administered with heavy cream by gavage. Do CNF and other nanomaterials (NMs) alter the tissue distribution and retention of co-administered metal ions? We evaluated whether 5 different NMs affected tissue distribution of co-ingested 65Zn++ and 59Fe+++ in zinc-replete versus zinc-deficient mice. Male C57BL/6J mice were fed either zinc-replete or zinc-deficient diets for 3 weeks, followed by gavage with NM suspensions in water containing both 65ZnCl2 and 59FeCl3. Urine and feces were measured for 48 h post-gavage. Mice were euthanized and samples of 22 tissues were collected and analyzed for 65Zn and 59Fe in a gamma counter. Our data show that zinc deficiency alters the tissue distribution of 65Zn but not of 59Fe, indicating that zinc and iron homeostasis are regulated by distinct mechanisms. Among the tested NMs, soluble starch-coated chitosan nanoparticles, cellulose nanocrystals, and TiO2 reduced Zn and Fe tissue retention in zinc-deficient but not in zinc-replete animals.

Development and characterization of a Versatile Engineered Nanomaterial Generation System (VENGES) suitable for toxicological studies.

A novel system for generation of engineered nanomaterials (ENMs) suitable for in situ toxicological characterization within biological matrices was developed. This Versatile Engineered Nanomaterial Generation System (VENGES) is based on industry-relevant, flame spray pyrolysis aerosol reactors that can scaleably produce ENMs with controlled primary and aggregate particle size, crystallinity, and morphology. ENMs are produced continuously in the gas phase, allowing their continuous transfer to inhalation chambers, without altering their state of agglomeration. Freshly generated ENMs are also collected on Teflon filters for subsequent physicochemical and morphological characterization and for in vitro toxicological studies. The ability of the VENGES system to generate families of ENMs of pure and selected mixtures of iron oxide, silica, and nanosilver with controlled physicochemical properties was demonstrated using a range of state-of-the-art-techniques. Specific surface area was measured by nitrogen adsorption using the Brunauer-Emmett-Teller method, and crystallinity was characterized by X-ray diffraction. Particle morphology and size were evaluated by scanning and transmission electron microscopy. The suitability of the VENGES system for toxicological studies was also shown in both in vivo and in vitro studies involving Sprague-Dawley rats and human alveolar-like monocyte derived macrophages, respectively. We demonstrated linkage between physicochemical ENM properties and potential toxicity.

Effects of ingested nanocellulose on intestinal microbiota and homeostasis in Wistar Han rats.

Micron scale cellulose materials are "generally regarded as safe" (GRAS) as binders and thickeners in food products. However, nanocellulose materials, which have unique properties that can improve food quality and safety, have not received US-Food and Drug Administration (FDA) approval as food ingredients. In vitro and in vivo toxicological studies of ingested nanocellulose revealed minimal cytotoxicity, and no subacute in vivo toxicity. However, ingested materials may modulate gut microbial populations, or alter aspects of intestinal function not elucidated by toxicity testing, which could have important health implications. Here, we report the results of studies conducted in a rat gavage model to assess the effects of ingested cellulose nanofibrils (CNF) on the fecal microbiome and metabolome, intestinal epithelial expression of cell junction genes, and ileal cytokine production. Feces, plasma, and ilea were collected from Wistar Han rats before and after five weeks of biweekly gavages with water or cream, with or without 1% CNF. CNF altered microbial diversity, and diminished specific species that produce short chain fatty acids, and that are associated with increased serum insulin and IgA production. CNF had few effects on the fecal metabolome, with significant changes in only ten metabolites of 366 measured. Exposure to CNF also altered expression of epithelial cell junction genes, and increased production of cytokines that modulate proliferation of CD8 T cells. These perturbations likely represent initiation of an adaptive immune response, however, no associated pathology was seen within the duration of the study. Additional studies are needed to better understand the health implications of these changes in long term.

Toxicological effects of ingested nanocellulose in in vitro intestinal epithelium and in vivo rat models.

Cellulose is widely used as a thickener and filler in foods and drugs. It has been designated "generally regarded as safe" (GRAS). Nanocellulose (NC) has many additional potential applications designed to improve food quality and safety, but has not yet been designated as GRAS. Here we present results of toxicological studies of ingested NC in physiologically relevant in vitro and in vivo systems. In vitro studies employed a gastrointestinal tract simulator to digest two widely-used forms of NC, nanocellulose fibrils (CNF) and cellulose nanocrystals (CNC), at 0.75 and 1.5% w/w, in a fasting diet as well as in a standardized food model based on the average American diet. A triculture model of small intestinal epithelium was used to assess effects of a 24-hour incubation with the digested products (digesta) on cell layer integrity, cytotoxicity and oxidative stress. Other than a 10% increase over controls in reactive oxygen species (ROS) production with 1.5% w/w CNC, no significant changes in cytotoxicity, ROS or monolayer integrity were observed. In vivo toxicity was evaluated in rats gavaged twice weekly for five weeks with 1% w/w suspensions of CNF in either water or cream. Blood, serum, lung, liver, kidney, and small intestine were collected for analysis. No significant differences in hematology, serum markers or histology were observed between controls and rats given CNF suspensions. These findings suggest that ingested NC has little acute toxicity, and is likely non-hazardous when ingested in small quantities. Additional chronic feeding studies are required to assess long term effects, and potential detrimental effects on the gut microbiome and absorbance of essential micronutrients. These studies are underway, and their outcome will be reported in the near future.

Fate of Barium Sulfate Nanoparticles Deposited in the Lungs of Rats.

We have shown that barium [from BaSO4 nanoparticles (NPs)] was cleared from the lungs faster than other poorly soluble NPs and translocated mostly to bone. We now studied barium biokinetics in rats during Study 1: two-year inhalation exposure to 50 mg/m3 BaSO4 NP aerosols, and Study 2: single intratracheal (IT) instillation of increasing doses of BaSO4 NPs or BaCl2. Study 1 showed that lung barium content measured by inductively coupled plasma mass spectrometry increased during 360 days of BaSO4 NP aerosol exposures. An equilibrium was established from that time until 2 years. Barium concentrations in BaSO4-exposed animals were in the order (lungs > lymph nodes > hard bone > bone marrow > liver). In Study 2, there was an increase in lung barium post-IT instillation of BaSO4 NPs while barium from BaCl2 was mostly cleared by day 28. Transmission electron microscopy showed intact BaSO4 NPs in alveolar macrophages and type II epithelial cells, and in tracheobronchial lymph nodes. Using stimulated Raman scattering microscopy, specific BaSO4 Raman spectra were detected in BaSO4 NP-instilled lungs and not in other organs. Thus, we posit that barium from BaSO4 NPs translocates from the lungs mainly after dissolution. Barium ions are then incorporated mostly into the bone and other organs.

Olfactory uptake of manganese requires DMT1 and is enhanced by anemia.

Manganese, an essential nutrient, can also elicit toxicity in the central nervous system (CNS). The route of exposure strongly influences the potential neurotoxicity of manganese-containing compounds. Recent studies suggest that inhaled manganese can enter the rat brain through the olfactory system, but little is known about the molecular factors involved. Divalent metal transporter-1 (DMT1) is the major transporter responsible for intestinal iron absorption and its expression is regulated by body iron status. To examine the potential role of this transporter in uptake of inhaled manganese, we studied the Belgrade rat, since these animals display significant defects in both iron and manganese metabolism due to a glycine-to-arginine substitution (G185R) in their DMT1 gene product. Absorption of intranasally instilled 54Mn was significantly reduced in Belgrade rats and was enhanced in iron-deficient rats compared to iron-sufficient controls. Immunohistochemical experiments revealed that DMT1 was localized to both the lumen microvilli and end feet of the sustentacular cells of the olfactory epithelium. Importantly, we found that DMT1 protein levels were increased in anemic rats. The apparent function of DMT1 in olfactory manganese absorption suggests that the neurotoxicity of the metal can be modified by iron status due to the iron-responsive regulation of the transporter.

Diagnostic performance of assays for the detection of anti-Porcine reproductive and respiratory syndrome virus antibodies in serum and muscle transudate ("meat juice") based on samples collected under experimental conditions.

Three assays were evaluated for their ability to detect antibodies against Porcine reproductive and respiratory syndrome virus (PRRSV) in porcine muscle transudate ("meat juice") samples. Samples were derived from 91 pigs inoculated with PRRSV isolate VR-2332 and 46 age-matched controls. Serum and muscle (Musculus longissimus dorsi) samples were collected from randomly selected animals euthanized at approximately 14-day intervals from 28 to 202 days postinoculation. Serum samples were assayed at a dilution of 140, and muscle transudate samples were assayed at 5 dilutions (12, 15, 110, 120, 140) using a commercial PRRSV antibody enzyme-linked immunosorbent assay (ELISA). In addition, muscle transudate samples were tested using an indirect fluorescent antibody test (IFAT) at 5 dilutions (12, 15, 110, 120, 140). Attempts to assay muscle transudate samples for neutralizing antibodies using a modified fluorescent focus neutralization assay were unsuccessful. Receiver operator characteristic (ROC) curve analyses were used to estimate cutoff thresholds and the associated diagnostic sensitivities and specificities for ELISA and IFAT at each dilution. For ELISA, muscle transudate samples at the ROC-optimized cutoffs were >95% sensitive and 100% specific at each dilution. At a cutoff dilution of > or =15, the IFAT diagnostic sensitivity and specificity of muscle transudate was estimated at 63.3% and 100%, respectively. These findings validated the use of muscle transudate samples in PRRSV surveillance programs based on ELISA antibody testing.

Effects of a vinegar-based multi-micronutrient supplement in rats: a multi-pronged assessment of dietary impact.

We determined the effects of continuous access to drinking of water with a vinegar-based multi-micronutrient (VMm) supplement containing rice and fruit vinegars, vitamins, organic acids and sugars during gestation, lactation, and early adulthood in rats. Pregnant rats were provided with reverse-osmosis water or VMm water from the start of pregnancy through the time of weaning. Weaned pups consumed the same drinking water for 3-12 additional weeks. We examined fecal metabolite and microbial profiles, and other physiological parameters. Body weights were less in rats that drank VMm water. Thirty fecal metabolites involved in amino acid and dipeptide metabolism were significantly altered in VMm-supplemented rats. Analysis of microbial 16S rRNA showed enrichment of bacteria in the family S24-7 in VMm-supplemented rats, and one in Ruminococcaceae in controls. Our data show that a VMm-containing beverage can alter growth, and gut metabolism and microbial community. Future work to correlate these parameters is warranted.

Reducing Intestinal Digestion and Absorption of Fat Using a Nature-Derived Biopolymer: Interference of Triglyceride Hydrolysis by Nanocellulose.

Engineered nanomaterials are increasingly added to foods to improve quality, safety, or nutrition. Here we report the ability of ingested nanocellulose (NC) materials to reduce digestion and absorption of ingested fat. In the small intestinal phase of an acellular simulated gastrointestinal tract, the hydrolysis of free fatty acids (FFA) from triglycerides (TG) in a high-fat food model was reduced by 48.4% when NC was added at 0.75% w/w to the food, as quantified by pH stat titration, and by 40.1% as assessed by fluorometric FFA assay. Furthermore, translocation of TG and FFA across an in vitro cellular model of the intestinal epithelium was significantly reduced by the presence of 0.75% w/w NC in the food (TG by 52% and FFA by 32%). Finally, in in vivo experiments, the postprandial rise in serum TG 1 h after gavage with the high fat food model was reduced by 36% when 1.0% w/w NC was administered with the food. Scanning electron microscopy and molecular dynamics studies suggest two primary mechanisms for this effect: (1) coalescence of fat droplets on fibrillar NC (CNF) fibers, resulting in a reduction of available surface area for lipase binding and (2) sequestration of bile salts, causing impaired interfacial displacement of proteins at the lipid droplet surface and impaired solubilization of lipid digestion products. Together these findings suggest a potential use for NC, as a food additive or supplement, to reduce absorption of ingested fat and thereby assist in weight loss and the management of obesity.

Surface modification of zinc oxide nanoparticles with amorphous silica alters their fate in the circulation.

Nanoparticle (NP) pharmacokinetics and biological effects are influenced by many factors, especially surface physicochemical properties. We assessed the effects of an amorphous silica coating on the fate of zinc after intravenous (IV) injection of neutron activated uncoated (65)ZnO or silica-coated (65)ZnO NPs in male Wistar Han rats. Groups of IV-injected rats were sequentially euthanized, and 18 tissues were collected and analyzed for (65)Zn radioactivity. The protein coronas on each ZnO NP after incubation in rat plasma were analyzed by SDS-PAGE gel electrophoresis and mass spectrometry of selected gel bands. Plasma clearance for both NPs was biphasic with rapid initial and slower terminal clearance rates. Half-lives of plasma clearance of silica-coated (65)ZnO were shorter (initial - <1 min; terminal - 2.5 min) than uncoated (65)ZnO (initial - 1.9 min; terminal - 38 min). Interestingly, the silica-coated (65)ZnO group had higher (65)Zn associated with red blood cells and higher initial uptake in the liver. The (65)Zn concentrations in all the other tissues were significantly lower in the silica-coated than uncoated groups. We also found that the protein corona formed on silica-coated ZnO NPs had higher amounts of plasma proteins, particularly albumin, transferrin, A1 inhibitor 3, α-2-hs-glycoprotein, apoprotein E and α-1 antitrypsin. Surface modification with amorphous silica alters the protein corona, agglomerate size, and zeta potential of ZnO NPs, which in turn influences ZnO biokinetic behavior in the circulation. This emphasizes the critical role of the protein corona in the biokinetics, toxicology and nanomedical applications of NPs.