Jacob N. Finkelstein

Translocation of inhaled ultrafine manganese oxide particles to the central nervous system

Background Studies in monkeys with intranasally instilled gold ultrafine particles (UFPs; < 100 nm) and in rats with inhaled carbon UFPs suggested that solid UFPs deposited in the nose travel along the olfactory nerve to the olfactory bulb. Methods To determine if olfactory translocation occurs for other solid metal UFPs and assess potential health effects, we exposed groups of rats to manganese (Mn) oxide UFPs (30 nm; ~ 500 μg/m3) with either both nostrils patent or the right nostril occluded. We analyzed Mn in lung, liver, olfactory bulb, and other brain regions, and we performed gene and protein analyses. Results After 12 days of exposure with both nostrils patent, Mn concentrations in the olfactory bulb increased 3.5-fold, whereas lung Mn concentrations doubled; there were also increases in striatum, frontal cortex, and cerebellum. Lung lavage analysis showed no indications of lung inflammation, whereas increases in olfactory bulb tumor necrosis factor-α mRNA (~ 8-fold) and protein (~ 30-fold) were found after 11 days of exposure and, to a lesser degree, in other brain regions with increased Mn levels. Macrophage inflammatory protein-2, glial fibrillary acidic protein, and neuronal cell adhesion molecule mRNA were also increased in olfactory bulb. With the right nostril occluded for a 2-day exposure, Mn accumulated only in the left olfactory bulb. Solubilization of the Mn oxide UFPs was < 1.5% per day. Conclusions We conclude that the olfactory neuronal pathway is efficient for translocating inhaled Mn oxide as solid UFPs to the central nervous system and that this can result in inflammatory changes. We suggest that despite differences between human and rodent olfactory systems, this pathway is relevant in humans.

A perpetual cascade of cytokines postirradiation leads to pulmonary fibrosis

PURPOSE Radiation-induced pulmonary reactions have classically been viewed as distinct phases--acute pneumonitis and, later, fibrosis--occurring at different times after irradiation and attributed to different target cell populations. We prefer to view these events as a continuum, with no clear distinction between the temporal sequence of the different pulmonary reactions; the progression is the result of an early activation of an inflammatory reaction, leading to the expression and maintenance of a cytokine cascade. In the current study, we have examined the temporal and spatial expression of cytokine and extracellular matrix messenger ribonucleic acid (mRNA) abundance in fibrosis-sensitive mice after thoracic irradiation. METHODS AND MATERIALS Radiation fibrosis-prone (C57BL/6) mice received thoracic irradiation of 5 and 12.5 Gy. At Day 1, and 1, 2, 8, 16, and 24 weeks after treatment, animals were killed and lung tissue processed for light microscopy and isolation of RNA. Expression of cytokine and extracellular matrix mRNA abundance was evaluated by slot-blot analysis and cellular localization by in situ hybridization and immunochemistry. RESULTS One of the cytokines responsible for the inflammatory phase (IL-1 alpha) is elevated at 2 weeks, returns to normal baseline values, then increases at 8 weeks, remaining elevated until 26 weeks when lung fibrosis appears. Transforming growth factor-beta (TGF beta), a proliferative cytokine, is elevated at 2 weeks, persists until 8 weeks, and then returns to baseline values. In parallel with the cytokine cascade, the fibrogenic markers for CI/CIII/IV (collagen genes) correlate by showing a similar early and then later elevation of activity. For instance, the collagen gene expression of CI/CIII is a biphasic response with an initial increase at 1-2 weeks that remits at 8 weeks, remains inactive from 8 to 16 weeks, and then becomes elevated at 6 months when collagen deposition is recognized histopathologically. CONCLUSION These studies clearly demonstrate the early and persistent elevation of cytokine production following pulmonary irradiation. The temporal relationship between the elevation of specific cytokines and the histological and biochemical evidence of fibrosis serves to illustrate the continuum of response, which, we believe, underlies pulmonary radiation reactions and supports the concept of a perpetual cascade of cytokines produced immediately after irradiation, prompting collagen genes to turn on, and persisting until the expression of late effects becomes apparent pathologically and clinically.

The influence of protein adsorption on nanoparticle association with cultured endothelial cells.

As materials are produced at smaller scales, the properties that make them especially useful for biological applications such as drug delivery, imaging or sensing applications also render them potentially harmful. There has been a reasonable amount of work addressing the interactions of biological fluids at material surfaces that demonstrates the high affinity of protein for particle surfaces and some looking at the role of particle surface chemistry in cellular associations, but mechanisms have been too little addressed outside the context of intended, specific interactions. Here, using cultured endothelium as a model for vascular transport, we demonstrate that the capacity of nanoparticle surfaces to adsorb protein is indicative of their tendency to associate with cells. Quantification of adsorbed protein shows that high binding nanoparticles are maximally coated in seconds to minutes, indicating that proteins on particle surfaces can mediate cell association over much longer time scales. We also remove many of the most abundant proteins from culture media which alters the profile of adsorbed proteins on nanoparticles but does not affect the level of cell association. We therefore conclude that cellular association is not dependent on the identity of adsorbed proteins and therefore unlikely to require specific binding to any particular cellular receptors.

Early alterations in extracellular matrix and transforming growth factor β gene expression in mouse lung indicative of late radiation fibrosis

PURPOSE Fibrosis, characterized by the accumulation of collagen, is a late result of thoracic irradiation. The expression of late radiation injury can be found immediately after irradiation by measuring messenger RNA (mRNA) abundance. METHODS AND MATERIALS To determine if extracellular matrix mRNA and transforming growth factor beta abundance was affected acutely after irradiation, we measured mRNA levels of collagen I (CI), collagen III (CIII), collagen IV (CIV), fibronectin (FN), and transforming growth factor beta (TGF beta 1,2&3) in mouse lungs on day 1 and day 14 after graded doses of radiation. C57BL/6 female mice were irradiated with a single dose to the thorax of 5 or 12.5 Gy. Total lung RNA was prepared and immobilized by Northern and slot blotting and hybridized with radiolabelled cDNA probes for CI, CIII, CIV, FN, TGF beta 1,2&3 and a control probe encoding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Autoradiographic data were quantified by video densitometry and results normalized to GAPDH. RESULTS Changes in the expression of CI, CIII, CIV, FN and TGF beta 1,2&3 were observed as early as 1 day after exposure. Through 14 days, changes in mRNA up to 5-fold were seen for any one dose. Dose related changes as high as 10-fold were also evident. The CI:CIII ratio increased gradually for the 5 Gy dose at 14 days postirradiation while the CI:CII ratio for the 12.5 Gy dose decreased by approximately 4-fold as compared to the control. CONCLUSION These studies suggest that alterations in expression of extracellular matrix and TGF beta mRNA occur very early after radiation injury even at low doses and may play a role in the development of chronic fibrosis.

Concept of Assessing Nanoparticle Hazards Considering Nanoparticle Dosemetric and Chemical/Biological Response Metrics

Engineered nanoparticles (NP) are being developed and incorporated in a number of commercial products, raising the potential of human exposure during manufacture, use, and disposal. Although data concerning the potential toxicity of some NP have been reported, validated simple assays are lacking for predicting their in vivo toxicity. The aim of this study was to evaluate new response metrics based on chemical and biological activity of NP for screening assays that can be used to predict NP toxicity in vivo. Two cell-free and two cell-based assays were evaluated for their power in predicting in vivo toxicity of eight distinct particle types with widely differing physicochemical characteristics. The cell-free systems comprised fluorescence- and electron spin resonance-based assays of oxidant activity. The cell-based systems also used electron spin resonance (ESR) as well as luciferase reporter activity to rank the different particle types in comparison to benchmark particles of low and high activity. In vivo experiments evaluated acute pulmonary inflammatory responses in rats. Endpoints in all assays were related to oxidative stress and responses were expressed per unit NP surface area to compare the results of different assays. Results indicated that NP are capable of producing reactive species, which in biological systems lead to oxidative stress. Copper NP had the greatest activity in all assays, while TiO2 and gold NP generally were the least reactive. Differences in the ranking of NP activity among the assays were found when comparisons were based on measured responses. However, expressing the chemical (cell-free) and biological (cells; in vivo) activity per unit particle surface area showed that all in vitro assays correlated significantly with in vivo results, with the cellular assays correlating the best. Data from this study indicate that it is possible to predict acute in vivo inflammatory potential of NP with cell-free and cellular assays by using NP surface area-based dose and response metrics, but that a cellular component is required to achieve a higher degree of predictive power.

Surface property changes from interactions of albumin with natural lung surfactant and extracted lung lipids

These experiments characterize the effects of albumin on the dynamic surface activity of natural lung surfactant (LS), and an extracted mixed lipid fraction (CLL), at physiologic temperature, humidity, and film cycling rate on an oscillating bubble apparatus. Measurements of albumin effects on the surface pressure-time (pi-t) adsorption isotherms of CLL and LS are also reported. Results show that albumin in concentrations greater than or equal to 20 mg/ml increased the minimum dynamic surface tension of LS suspensions (0.4 mg phospholipid/ml) from less than 1 dyne/cm to 21 dynes/cm at 37 degrees C. Albumin in low concentrations (2 mg/ml) had a similar detrimental effect on the dynamic surface activity of extracted surfactant lipids, CLL. In addition, albumin also inhibited the isolated adsorption facility of LS and CLL; instead of adsorbing rapidly to their maximum spreading pressures of 45 dynes/cm, both surfactant mixtures (at 0.063 and 0.125 mg phospholipid/ml) adsorbed more slowly or reached lower final surface pressures in the presence of plasma protein. A striking finding was that albumin inhibition of surface activity was moderated or abolished at high lipid concentrations. For example, minimum dynamic surface tensions less than 1 dyne/cm were reached on the oscillating bubble for natural LS at concentrations greater than 0.75 mg/ml and CLL at concentrations greater than 1.5 mg/ml, even in the presence of very large amounts of albumin (100 mg/ml). Similarly, LS and CLL adsorption facility was protected from albumin inhibition at sufficiently high phospholipid concentrations. Albumin inhibition of natural LS adsorption was also moderated by the presence of 1.4 mM Ca2+ ions. These results show that albumin in plasma transudates has the potential to seriously impair alveolar surfactant activity in vivo. However, the detrimental effect will be mitigated if a critical threshold of phospholipid is present.

Immune-mediated inflammation directly impairs pulmonary function, contributing to the pathogenesis of Pneumocystis carinii pneumonia

The clinical severity of Pneumocystis carinii pneumonia (PCP) correlates closely with the appearance of pulmonary markers of inflammation. Therefore, a model system was developed whereby physiological studies could be performed on live mice to determine the extent to which pulmonary inflammation contributes to respiratory impairment during PCP. P. carinii-infected severe combined immunodeficient mice displayed little evidence of pulmonary inflammation and exhibited normal oxygenation and dynamic lung compliance. When comparably infected littermates were immunologically reconstituted, however, an intense immune-mediated inflammatory response was observed that resulted in significant decreases in both lung compliance and oxygenation. As the pneumonia resolved pulmonary function returned toward normal. To begin to define the cell populations contributing to inflammation-associated respiratory impairment during PCP, similar studies were performed in CD4(+) T cell-depleted mice. Mice depleted of both CD4(+) and CD8(+) cells developed infection, but they demonstrated neither abnormal lung compliance nor increased respiratory rate and displayed no markers of lung injury. In contrast, mice depleted of only CD4(+) T cells exhibited severe pulmonary inflammation and injury, decreased oxygenation and lung compliance, and increased respirations. Respiratory compromise was associated with the presence of activated CD8(+) cells and neutrophils in broncho-alveolar lavage fluid. These observations provide direct experimental evidence that the hosts response to P. carinii directly impairs pulmonary function and contributes to the pathogenesis of PCP. Furthermore, CD8(+) T cells likely contribute to the respiratory compromise observed during PCP.

Newborn tracheal aspirate cytology: Classification during respiratory distress syndrome and bronchopulmonary dysplasia†

Cytopathologic examinations of tracheobronchial aspirates from 108 infants sampled during mechanical ventilation demonstrated a well-defined progression of cytologic changes in bronchial cells that could be divided into three classes. Seventy percent of infants with respiratory distress syndrome who developed bronchopulmonary dysplasia had pulmonary effluent cytology designated Class III; no infants with RDS but without BPD had these cytologic findings. Additionally, a temporal progression of events involving polymorphonuclear leukocyte and macrophage populations occurred in the absence of infection; these events were associated with duration of assisted ventilation and oxygen exposure. The technique described provides a useful way to monitor the progression of lung injury and repair and offers a cytologic method to predict and diagnose the development of bronchopulmonary dysplasia.

Validation of an LDH assay for assessing nanoparticle toxicity.

Studies showed that certain cytotoxicity assays were not suitable for assessing nanoparticle (NP) toxicity. We evaluated a lactate dehydrogenase (LDH) assay for assessing copper (Cu-40, 40nm), silver (Ag-35, 35nm; Ag-40, 40nm), and titanium dioxide (TiO(2)-25, 25nm) NPs by examining their potential to inactivate LDH and interference with β-nicotinamide adenine dinucleotide (NADH), a substrate for the assay. We also performed a dissolution assay for some of the NPs. We found that the copper NPs, because of their high dissolution rate, could interfere with the LDH assay by inactivating LDH. Ag-35 could also inactivate LDH probably because of the carbon matrix used to cage the particles during synthesis. TiO(2)-25 NPs were found to adsorb LDH molecules. In conclusion, NP interference with the LDH assay depends on the type of NPs and the suitability of the assay for assessing NP toxicity should be examined case by case.

CLARA CELL SECRETORY PROTEIN DEFICIENCY INCREASES OXIDANT STRESS RESPONSE IN CONDUCTING AIRWAYS

Little is known about the molecular basis for differential pulmonary oxidant sensitivity observed between genetically disparate members of the same species. We have generated mice that are deficient in Clara cell secretory protein (CCSP -/-) and that exhibit an oxidant-sensitive phenotype. We characterized the kinetics and distribution of altered stress-response [interleukin-6 (IL-6) and metallothionein (MT)] and epithelial cell-specific [cytochrome P-450 2F2 (CYP2F2)] gene expression to further understand the cellular and molecular basis for altered oxidant sensitivity in 129 strain CCSP -/- mice. Increases in IL-6 and MT mRNA abundance were detected by 2 h of exposure to 1 part/million ozone and preceded reductions in Clara cell CYP2F2 mRNA expression. Despite being qualitatively similar, increases in IL-6 and MT mRNA expression were enhanced in CCSP -/- mice with respect to coexposed 129 strain wild-type mice. Increased MT mRNA expression, indicative of the stress response, localized to the airway epithelium, surrounding mesenchyme, and endothelium of blood vessels. These results demonstrate a protective role for Clara cells and their secretions and indicate potential genetic mechanisms that may influence susceptibility to oxidant stress.