Deep Lung Tissue Research Articles

Assessment of litter prevalence of Mycoplasma hyopneumoniae in preweaned piglets utilizing an antemortem tracheobronchial mucus collection technique and a real-time polymerase chain reaction assay.

The swine industry currently lacks validated antemortem methods of detecting baseline herd prevalence of Mycoplasma hyopneumoniae. The focus of our study was to evaluate alternative antemortem detection techniques and to determine baseline litter prevalence in preweaned pig populations utilizing the selected technique and a real-time polymerase chain reaction (qPCR) assay. Preliminary data was analyzed on weaned piglets with evidence of respiratory disease (n = 32). Five sample types (antemortem nasal swab, tracheobronchial mucus, postmortem deep airway swab, bronchoalveolar lavage, and lung tissue) were collected from each pig. Individual samples were tested for M. hyopneumoniae using qPCR. Compared to nasal swabs, tracheobronchial mucus demonstrated higher test sensitivity (P < 0.0001). Tracheobronchial mucus was collected from apparently healthy preweaned piglets (n = 1,759; sow farms 1-3) and preweaned piglets exhibiting signs of respiratory disease (n = 32; sow farm 4), ranging in age from 12 to 25 days. Samples from sow farms 1-3 were pooled into 2 groups of 5 per litter (n = 360 pools from 180 litters), and qPCR was utilized to analyze each pool. A qPCR-positive result, threshold cycle <37, from either pool designated the litter positive for M. hyopneumoniae. Two out of 180 litters revealed a positive result (1.1%). Individual qPCR assays were run on the samples collected from sow farm 4. Five out of 30 samples revealed a positive result (16.7%). Tracheobronchial mucus collection in combination with qPCR is a sensitive antemortem sampling technique that can be used to estimate the prevalence of M. hyopneumoniae in preweaned pigs, thus providing insight into the infection dynamics across the entire farrow-to-finish process.

Bioimaging and pulmonary applications of self-assembled Flt1 peptide–hyaluronic acid conjugate nanoparticles

Despite wide exploitation of corticosteroid drugs for the treatment of asthma, the poor therapeutic effect on a neutrophilic subtype of asthma prohibits the full recovery of asthma patients. In this work, dexamethasone (Dexa) was loaded in Flt1 peptide–hyaluronic acid (HA) conjugate nanoparticles to overcome the limitation of corticosteroid resistance for the treatment of neutrophilic pulmonary inflammation. Flt1 peptide–HA conjugates are self-assembled to nanoparticles because of hydrophobic Flt1 peptide conjugated to HA by benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) chemistry. In vitro bioimaging showed efficient internalization of Flt1 peptide–HA conjugate nanoparticles into lung epithelial cells by HA-receptor mediated endocytosis. Also, ex vivo imaging for the biodistribution in ICR mice revealed long-term retention of Flt1 peptide–HA conjugate nanoparticles in deep lung tissues possibly due to mucoadhesive property of HA. On the basis of bioimaging results for pulmonary drug delivery applications, we prepared Dexa-loaded Flt1 peptide–HA conjugate nanoparticles. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) confirmed the formation of nanoparticles, which reduced cytokine levels of lipopolysaccharide (LPS)-stimulated cells more efficiently than free Dexa. Furthermore, according to the bronchoalveolar lavage (BAL) cellularity and histological analysis, Dexa loaded Flt1 peptide–HA conjugate nanoparticles showed remarkable therapeutic effects in both eosinophilic and neutrophilic asthma model mice.

Abstract 3583: Subchronic exposure of carbon nanotubes to human small airway epithelial cells induces neoplastic transformation and toxicogenomic responses.

Abstract Carbon nanotubes (CNT) have been shown to penetrate and accumulate in the deep lung tissues after in vivo pulmonary exposure. This raises a major concern of long-term adverse health effects, particularly carcinogenesis due to their structural similarity to asbestos, a known human carcinogen. However, there is neither clear knowledge nor a practical method to assess this carcinogenic potential. In this study, we developed a chronic exposure model using human lung epithelial cells to address this knowledge gap. We conducted subchronic exposures of dispersed single-walled CNT (D-SWCNT), multi-walled CNT (D-MWCNT) and crocidolite asbestos (ASB) to human small airway epithelial cells (SAEC) in culture. Ultrafine carbon black (D-UFCB) and dispersant-only exposed cells (DISP) served as negative controls. SAEC were exposed to 0.02 μg/cm2 of the particles for 25 weeks and evaluated for cancer cell phenotypes. Next, mRNA samples from the exposed cells were subjected to whole genome microarray and rtPCR analyses for toxicogenomic evaluation. Differentially expressed genes were uploaded to Ingenuity Pathway Analysis to identify gene targets and molecular mechanisms of neoplastic transformation. Our results showed that both D-SWCNT and D-MWCNT-treated cells exhibited typical malignant transformation properties, such as increased proliferation, migration, invasion, anchorage-independent cell growth and angiogenesis compared to controls. Both D-SWCNT and D-MWCNT cells expressed significant changes in genes associated with cell death, movement, proliferation and cancer. Top ranked pathway along with Western blot analyses identified several altered signaling pathways and transcription factors associated with oncogenesis. These results indicate that long-term low-dose exposure of human lung epithelial cells to D-SWCNT and D-MWCNT induced neoplastic transformation of the cells which suggests potential carcinogenicity of the nanoparticles. The described cell model system could potentially be used as an in vitro predictive screening test for potential carcinogenicity of other nanomaterials. Phenotypic anchoring of toxicogenomic response to neoplastic cell transformation following in vitro subchronic nanomaterial exposure can potentially serve to identify novel mechanisms of action and facilitate human cancer risk assessment. Keywords: Carbon nanotubes, microarray, carcinogenesis, lung, cell, transformation. Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the National Institute for Occupational Safety and Health. Citation Format: Liying Wang, Todd Stueckle, Anurag Mishra, Raymond Derk, Terence Meighan, Vincent Castranova, Yon Rojanasakul. Subchronic exposure of carbon nanotubes to human small airway epithelial cells induces neoplastic transformation and toxicogenomic responses. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3583. doi:10.1158/1538-7445.AM2013-3583

Synthesis and Characterization of Lipid–Polymer Hybrid Nanoparticles with pH-Triggered Poly(ethylene glycol) Shedding

Novel lipid-polymer hybrid nanoparticles are designed with a poly(ethylene glycol) (PEG) coating that is shed in response to a low pH trigger. This allows the nanoparticles to be stable during systemic circulation and at neutral pH, but destabilize and fuse with lipid membranes in acidic environments. The hybrid nanoparticles consist of a poly(lactic-co-glycolic acid) core with a lipid and lipid-PEG monolayer shell. To make the hybrid nanoparticles pH sensitive, a lipid-(succinate)-mPEG conjugate is synthesized to provide a hydrolyzable PEG stealth layer that is shed off the particle surface at low pH. The pH-sensitivity of the nanoparticles is tunable using the molar concentration of the lipid-(succinate)-mPEG incorporated in the lipid shell of the particles. Possible uses of these pH-sensitive nanoparticles include aggregating in acidic tumor microenvironments, escaping acidified endosomes, or aggregating in deep lung tissue for improved inhalation administration.

An Intratracheal Instillation Bioassay System for Detection of Lung Toxicity Due to Fine Particles in F344 Rats

It is an urgent priority to establish in vivo bioassays for detection of hazards related to fine particles, which can be inhaled into deep lung tissue by humans. In order to establish an appropriate bioassay for detection of lung damage after particle inhalation, several experiments were performed in rats using quartz as a typical lung toxic particle. The results of pilot experiments suggest that Days 1 and 28 after intratracheal instillation of 2 mg of fine test particles in vehicle are most appropriate for detection of acute and subacute inflammatory changes, respectively. Furthermore, the BrdU incorporation on Day 1 and the iNOS level on Day 28 proved to be suitable end-point markers for this purpose. An examination of the toxicity of a series of particles was performed with the developed bioassay. Although some materials, including nanoparticles, demonstrated toxicity that was too strong for sensitive assessment, a ranking order could be clarified. The bioassay thus appears suitable for rapid hazard identification with a possible ranking of the toxicity of various particles at single concentrations.

Aerosol Infection of BALB/c Mice with Brucella melitensis and Brucella abortus and Protective Efficacy against Aerosol Challenge

Brucellosis is a zoonotic disease with a worldwide distribution that can be transmitted via intentional or accidental aerosol exposure. In order to engineer superior vaccine strains against Brucella species for use in animals as well as in humans, the possibility of challenge infection via aerosol needs to be considered to properly evaluate vaccine efficacy. In this study, we assessed the use of an aerosol chamber to infect deep lung tissue of mice to elicit systemic infections with either Brucella abortus or B. melitensis at various doses. The results reveal that B. abortus causes a chronic infection of lung tissue in BALB/c mice and peripheral organs at low doses. In contrast, B. melitensis infection diminishes more rapidly, and higher infectious doses are required to obtain infection rates in animals similar to those of B. abortus. Whether this difference translates to severity of human infection remains to be elucidated. Despite these differences, unmarked deletion mutants BADeltaasp24 and BMDeltaasp24 consistently confer superior protection to mice against homologous and heterologous aerosol challenge infection and should be considered viable candidates as vaccine strains against brucellosis.

Bioassay by intratracheal instillation for detection of lung toxicity due to fine particles in F344 male rats

We have established and documented an in vivo bioassay for detection of hazards with intratracheally instilled fine particles, which can be used for risk assessment of toxicity of materials inhaled into deep lung tissue of humans (Yokohira et al. Establishment of a bioassay system for detection of lung toxicity due to fine particle instillation: sequential histopathological changes with acute and subacute lung damage due to intratracheal instillation of quartz in F344 male rats. J Toxicol Pathol 2005;18:13–8). For validation we here examined toxicity of fine particles from quartz, hydrotalcite, potassium octatitanate, palladium oxide and carbon black with this bioassay. A total of 108, 10-week-old F344/DuCrj male rats were randomly divided into 8 groups. Groups 1 to 5 underwent intratracheal instillation of the 5 test particles (4 mg/rat) suspended in 0.2 ml vehicle (saline or 10% propylene glycol and 1% sodium carboxymethyl cellulose in saline: PG-CMC) with a specially designed aerolizer, and subgroups of 7 rats were killed on Days 1 and 28 thereafter. Groups 6 and 7 similarly were exposed to saline and PG-CMC, respectively, as vehicle controls, while group 8 was maintained untreated. Using histopathological changes and immunohistochemically assessed bromodeoxyuridine (BrdU) labeling indices, inducible nitric oxide synthase (iNOS) and matrix metalloproteinase-3 (MMP-3) levels as end points, the quartz treated group exhibited high toxicity, while the values for the other particle-treated groups pointed to only slight effects. Although additional efforts are needed to establish advantages and disadvantages with our bioassay, models featuring intratracheal instillation clearly can be useful for detection of acute or subacute lung toxicity due to inhaled fine particles by using histopathological scoring and markers like BrdU and iNOS for screening purposes in short-term studies.

Characterization of nanostructure phenomena in airborne particulate aggregates and their potential for respiratory health effects.

Airborne aggregates of nanoparticulates were collected on carbon/form-coated, 100-mesh Ni TEM grids in a thermal precipitator and observed in an analytical TEM utilizing a BF-SAED-DF-EDS characterization protocol to identify the nanocrystalline or nanoparticulate components, especially their degree of crystallinity, size, structural/morphologic features, and chemistries. Reference aggregates of TiO2 rutile and anatase as well as Si3N4 nanoparticles were used to establish these characterization protocols, which were applied to several hundred individual particulates: homogeneous aggregates of carbonaceous/diesel particulate matter, complex mixtures of carbonaceous matter, including carbon nanocrystals, and inorganic nanocrystals; and heterogeneous, nanocrystal/nanoparticulate aggregates. Most airborne particulates were aggregates ranging in aerodynamic diameters from a few nanometers to a few microns; containing as few as 2 nanocrystals to several thousand nanocrystals or nanoparticulates such as carbonaceous spherules arranged in complex branched homogeneous aggregates composing diesel exhaust, with spherule diameters ranging from 10 to 30 nm. The potential for ultrafine airborne aggregates to fragment into hundreds or thousands of nanoparticulate components in human airways and act as toxic agents in deep lung tissue is demonstrated.

Changes in biomarkers from space radiation may reflect dose not risk

This presentation evaluates differences between radiation biomarkers of dose and risk and demonstrates the consequential problems associated with using biomarkers to do risk calculations following radiation exposures to the complex radiation environment found in deep space. Dose is a physical quantity, while risk is a biological quantity. Dose does not predict risk. This manuscript discusses species sensitivity factors, tissue weighting factors, and radiation quality factors derived from relative biological effectiveness (RBE). These factors are used to modify dose to make it a better predictor of risk. At low doses, where it is not possible to measure changes in risk, biomarkers have been used incorrectly as an intermediate step in predicting risk. Examples of biomarkers that do not predict risk are reviewed. Species sensitivity factors were evaluated using the Syrian hamster and the Wistar rat. Although the frequency of chromosome damage is very similar in these two species, the Wistar rat is very sensitive to radiation-induced lung cancer while the Syrian hamster is very resistant. To illustrate problems involved in using tissue weighting factors, rat trachea and deep lung tissues were compared. The similar level of chromosome damage observed in these two tissues would predict that the risk for cancer induction would be the same. However, even though large numbers of deep lung tumors result from inhaled radon, under the same exposure conditions there has never been a tracheal tumor observed. Finally, the Relative Biological Effectiveness (RBE) used to generate “quality factors” that convert exposure and dose from different types of radiation to a single measure of risk, is discussed. Important risk comparisons are done at very low doses, where the response to the reference radiation has been shown to either increase or decrease as a function of dose. Thus, the RBE and the subsequent risk predicted is more dependent on the background response of the endpoint and the shape of the dose response to the reference radiation than it is on the radiation type of interest.A large study using micronuclei as biomarkers following exposure to different energies of mono-energetic neutrons, x-rays and gamma rays delivered at very low doses (0.0 to 0.10 Gy) is reported. As additional biomarkers of risk involved in critical steps in the carcinogenic process are developed, it may become possible to base risk estimates on biological change rather than the radiation energy deposition or dose.

Identification of vascular sphincters at the junction between alveolar capillaries and pulmonary venules of the mouse lung

A peculiar feature of lung circulation in the lung is the pronounced variations in blood volume observed in alveolar capillaries that occur because of the changes in the conformation of the alveolar wall that are associated with the respiratory movements. This phenomenon has led to the postulate that mechanisms of postcapillary control of blood flow are to be present in the lung vessels. In the present study we searched for microanatomical evidence of vascular sphincters in the deep lung tissue of mice, namely in alveolar capillaries and pulmonary veins. We have used scanning electron microscopy (SEM) to examine two types of samples of normal lung tissue of CD-1 mice: 1) vascular corrosion casts made by vascular perfusion with Mercox resin, and 2) routinely made gold/platinum-coated replicas of sectioned lung tissue. Careful scrutiny of the vessels of the deep lung tissue led to the identification of sphincters in alveolar capillaries. These sphincters were located at the junction between capillary and pulmonary veins. They corresponded to areas of the vascular wall showing circular swellings where a radial organization was observed, since they were made up of alternating grooves and bulges. Transmission electron microscopy showed that smooth muscle cells participated in the formation of the sphincters. Our data reveal a new location for vascular sphincters in pulmonary vessels and, because these novel sphincters are located at the capillary-vein junction, they offer a structural setting for the existence of postcapillary control of blood flow in the pulmonary circulation of mice.

Morphological Evidence for Migration of Particle-Laden Macrophages through the Interalveolar Pores of Kohn in the Murine Lung

We have investigated the topography of particle-laden macrophages in the pulmonary tissue of CD-1 mice after intratracheal instillation of a suspension of 250 micrograms of calcium tungstate. The mice were sacrificed 1, 3, 7 and 14 days after the particle deposition. Lung fragments were studied by scanning electron microscopy (SEM) coupled with X-ray microanalysis that allowed in situ elemental identification of tungsten in the lungs. Tungsten-positive macrophages were distinctly located in the lungs of mice sacrificed at 1-3 days when compared with samples from mice killed 7-14 days after the calcium tungstate instillation. At 1-3 days, the tungsten-carrying macrophages were accumulated near the terminal bronchioles whereas they were seen predominantly in the alveolar ducts and sacs in the 7- to 14-day groups of mice. This suggests that during pulmonary inflammation there is a redistribution of the particle-containing macrophages throughout the deep lung tissue. In high-magnification SEM views, we observed that the tungsten-positive macrophages presented numerous surface microvilli. Tungsten-laden phagocytes were detected in interalveolar fenestrae, at the so-called Kohn pores. This finding documents that the Kohn pores may be used by inflammatory cells as a pathway for the migration of phagocytes in between adjacent alveolar sacs.

Dehydration and recovery of free alveolar macrophages.

Rats fed dry chow diet were deprived of wafer for 24 or 48 hours. Free alveolar cells were recovered from the excised lungs by repeated instillation and recovery of saline. The number of cells per gram lung weight harvested from dehydrated rats was significantly below control values and was closely related to the duration of wafer deprivation and loss of body weight. This decrease in cell recovery was not primarily attributable to reduction in food intake which accompanies water deprivation, since rats allowed wafer but restricted to the same food intake did not show as significant a decrease as occurred with dehydration. Free alveolar cells are considered lung macrophages which are responsible for clearance of particulates and microorganisms reaching the deep lung tissue. These experiments indicate that dehydration may affect this important mechanism.