Joseph D. Brain

Pulmonary distribution of particles given by intratracheal instillation or by aerosol inhalation

Abstract In animal studies concerned with the deposition of particulate matter in the lung, two methods for delivery of particles are commonly used, aerosol inhalation and intratracheal instillation of particle suspensions. We have attempted to evaluate the distribution patterns of each of these methods. Particles labeled with 99m Tc were administered to both rats and hamsters. The animals were subsequently killed. The lungs were excised, weighed, inflated, dried, and divided into 54 pieces which were counted individually in a Nuclear-Chicago Model 4230 Automatic Gamma Scintillation System. Groups receiving intratracheal instillations demonstrated nonuniform distribution patterns with preferential deposition in the dependent portions of the lung. The aerosol groups evidence more even distribution with preferential deposition in the apical lobes.

Elastase-mediated phosphatidylserine receptor cleavage impairs apoptotic cell clearance in cystic fibrosis and bronchiectasis

Cystic fibrosis is characterized by an early and sustained influx of inflammatory cells into the airways and by release of proteases. Resolution of inflammation is normally associated with the orderly removal of dying apoptotic inflammatory cells through cell recognition receptors, such as the phosphatidylserine receptor, CD36, and alpha v integrins. Accordingly, removal of apoptotic inflammatory cells may be impaired in persistent inflammatory responses such as that seen in cystic fibrosis airways. Examination of sputa from cystic fibrosis and non-cystic fibrosis bronchiectasis patients demonstrated an abundance of apoptotic cells, in excess of that seen in patients with chronic bronchitis. In vitro, cystic fibrosis and bronchiectasis airway fluid directly inhibited apoptotic cell removal by alveolar macrophages in a neutrophil elastase-dependent manner, suggesting that elastase may impair apoptotic cell clearance in vivo. Flow cytometry demonstrated that neutrophil elastase cleaved the phosphatidylserine receptor, but not CD36 or CD32 (Fc gamma RII). Cleavage of the phosphatidylserine receptor by neutrophil elastase specifically disrupted phagocytosis of apoptotic cells, implying a potential mechanism for delayed apoptotic cell clearance in vivo. Therefore, defective airway clearance of apoptotic cells in cystic fibrosis and bronchiectasis may be due to elastase-mediated cleavage of phosphatidylserine receptor on phagocytes and may contribute to ongoing airway inflammation.

Substrate stiffening promotes endothelial monolayer disruption through enhanced physical forces

A hallmark of many, sometimes life-threatening, inflammatory diseases and disorders is vascular leakage. The extent and severity of vascular leakage is broadly mediated by the integrity of the endothelial cell (EC) monolayer, which is in turn governed by three major interactions: cell-cell and cell-substrate contacts, soluble mediators, and biomechanical forces. A potentially critical but essentially uninvestigated component mediating these interactions is the stiffness of the substrate to which the endothelial monolayer is adherent. Accordingly, we investigated the extent to which substrate stiffening influences endothelial monolayer disruption and the role of cell-cell and cell-substrate contacts, soluble mediators, and physical forces in that process. Traction force microscopy showed that forces between cell and cell and between cell and substrate were greater on stiffer substrates. On stiffer substrates, these forces were substantially enhanced by a hyperpermeability stimulus (thrombin, 1 U/ml), and gaps formed between cells. On softer substrates, by contrast, these forces were increased far less by thrombin, and gaps did not form between cells. This stiffness-dependent force enhancement was associated with increased Rho kinase activity, whereas inhibition of Rho kinase attenuated baseline forces and lessened thrombin-induced inter-EC gap formation. Our findings demonstrate a central role of physical forces in EC gap formation and highlight a novel physiological mechanism. Integrity of the endothelial monolayer is governed by its physical microenvironment, which in normal circumstances is compliant but during pathology becomes stiffer.

An in vivo hamster bioassay to assess the toxicity of particulates for the lungs

Abstract We have developed a short-term bioassay to predict the toxicity of particulates for the lungs using hamsters exposed by intratracheal instillation. After exposure the animals were killed, their lungs were lavaged, and the pulmonary damage was characterized by cellular and biochemical assays of lavage fluid: (a) changes in in situ phagocytic ability of macrophages; (b) damage to the air-blood barrier shown by increases in albumin and red blood cells; (c) inflammation shown by increases in polymorphonuclear neutrophils (PMNs) and macrophages; and (d) cellular damage, measured by the levels of lactate dehydrogenase (LDH), β- N -acetylglucosaminidase, peroxidase, and elastase in the extracellular supernatant fraction of the lavage fluid. The system was calibrated using toxic α-quartz and two nontoxic dusts, aluminum oxide and iron oxide. Increases in albumin and red blood cells one day after exposure were greater following quartz than aluminum oxide and iron oxide; in contrast, a large part of the LDH increase was a nonspecific response to increased dust within the lungs. Most of the indicators, including red blood cell numbers, glucosaminidase, and peroxidase, either approached or were at control levels 4 days after exposure to iron oxide or α-quartz. In α-quartz-exposed animals, macrophage and PMN numbers were more elevated at 4 days that at 1 day and remained elevated for at least 14 days. In contrast, in iron oxide-exposed hamsters, macrophage numbers did not differ from control levels and PMN numbers approached control levels with time. The ability to cause a prolonged infiltration of macrophages and PMNs may be an important determinant of the toxicity of mineral dusts.

The particle has landed--characterizing the fate of inhaled pharmaceuticals

Although there is a modest body of literature on the absorption of inhaled pharmaceuticals by normal lungs and some limited information from diseased lungs, there is still a surprising lack of mechanistic knowledge about the details of the processes involved. Where are molecules absorbed, what mechanisms are involved, how well are different lung regions penetrated, what are the determinants of metabolism and dissolution, and how best can one retard the clearance of molecules deposited in the lung or induce intracellular uptake by lung cells? Some general principles are evident: (1) small hydrophobic molecules are absorbed very fast (within tens of seconds) usually with little metabolism; (2) small hydrophilic molecules are absorbed fast (within tens of minutes), again with minimal metabolism; (3) very low water solubility of the drug can retard absorption; (4) peptides are rapidly absorbed but are significantly metabolized unless chemically protected against peptidases; (5) larger proteins are more slowly absorbed with variable bioavailabilities; and 6) insulin seems to be best absorbed distally in the lungs while certain antibodies appear to be preferentially absorbed in the upper airways. For local lung disease applications, and some systemic applications as well, many small molecules are absorbed much too fast for convenient and effective therapies. For systemic delivery of peptides and proteins, absorption may sometimes be too fast. Bioavailabilities are often too low for cost-effective and reliable treatments. A better understanding of the determinants of pulmonary drug dissolution, absorption, metabolism, and how to target specific regions and/or cells in the lung will enable safer and more effective inhaled medicines in the future.

Gadolinium induces macrophage apoptosis

Gadolinium (Gd) suppresses reticuloendothelial functions in vivo by unknown mechanisms. In vitro exposure of rat alveolar macrophages to GdCl3·6H2O caused cell death, as measured by trypan blue permeability, in both dose‐ and time‐dependent fashions. Even a 10‐min exposure to Gd caused significant cell death by 24 h. The morphology of Gd‐treated cells, pyknosis and karyorrhexis prior to loss of membrane integrity, suggested apoptosis. Upon flow cytometric examination, Gd‐treated propidium iodide‐excluding cells demonstrated light scatter changes characteristic of apoptotic cells (decreased forward and increased right angle scatter). Gel electrophoresis of DNA from Gd‐treated macrophages clearly showed the ladder pattern unique to apoptotic cells. Electron‐dense structures containing Gd were observed via electron spectroscopic imaging within phagosomes and also within nuclei (associated with condensed chromatin). Gadolinium, endocytosed by macrophages and distributed to nuclei, causes apoptosis of macrophages in vitro.

Pulmonary intravascular macrophages: their contribution to the mononuclear phagocyte system in 13 species

The organ uptake of intravenously injected particles was examined in 13 species. All animals were injected intravenously with 198Au colloid and magnetic iron oxide particles. Vascular clearance kinetics of 198Au colloid was similar in all species. Pulmonary uptake of 198Au colloid ranged from 17 to 60% in sheep, calves, pigs, and cats but was <1.1% in monkeys, hyraxes, rabbits, guinea pigs, rats, mice, and chickens. For iron oxide particles, pulmonary uptake ranged from 80 to 99% in sheep, calves, pigs, goats, and cats and 15 to 18% in hamsters, hyraxes, and monkeys and was <10% in rabbits, chicken, mice, rats, and guinea pigs. In all species, the bulk of the remainder of particle uptake was in the liver. Pulmonary intravascular macrophages are the cellular site of lung uptake in calves, cats, pigs, goats, and sheep, whereas monocytes and neutrophils predominate in other species. Kupffer cells were the site of uptake in the liver. Our data show marked species differences in the fate of circulating particles; ruminants, pigs, and cats have extensive pulmonary localization due to phagocytosis by pulmonary intravascular macrophages.The organ uptake of intravenously injected particles was examined in 13 species. All animals were injected intravenously with 198Au colloid and magnetic iron oxide particles. Vascular clearance kinetics of198Au colloid was similar in all species. Pulmonary uptake of 198Au colloid ranged from 17 to 60% in sheep, calves, pigs, and cats but was <1.1% in monkeys, hyraxes, rabbits, guinea pigs, rats, mice, and chickens. For iron oxide particles, pulmonary uptake ranged from 80 to 99% in sheep, calves, pigs, goats, and cats and 15 to 18% in hamsters, hyraxes, and monkeys and was <10% in rabbits, chicken, mice, rats, and guinea pigs. In all species, the bulk of the remainder of particle uptake was in the liver. Pulmonary intravascular macrophages are the cellular site of lung uptake in calves, cats, pigs, goats, and sheep, whereas monocytes and neutrophils predominate in other species. Kupffer cells were the site of uptake in the liver. Our data show marked species differences in the fate of circulating particles; ruminants, pigs, and cats have extensive pulmonary localization due to phagocytosis by pulmonary intravascular macrophages.

Variation in genes involved in the RANKL/RANK/OPG bone remodeling pathway are associated with bone mineral density at different skeletal sites in men

In order to assess the contribution of polymorphisms in the RANKL (TNFSF11), RANK (TNFRSF11A) and OPG (TNFRSF11B) genes to variations in bone mineral density (BMD), a population-based cohort with 1,120 extreme low hip BMD cases or extreme high hip BMD controls was genotyped on five SNPs. We further explored the associations between these genetic variations and forearm BMDs by genotyping 266 offspring and 309 available parents from 160 nuclear families. A family-based association test was used. Significantly positive associations were found for A163G polymorphisms in the promoter regions of the OPG gene, a missense substitution in exon 7 (Ala192Val) of the RANK gene and rs9594782 SNP in the 5′ UTR of the RANKL gene with BMD in men only. Men with TC/CC genotypes of the rs9594782 SNP had a 2.1 times higher risk of extremely low hip BMD (P=0.004), and lower whole body BMD (P<0.001). Subjects with the TC genotype of the Ala192Val polymorphism had a 40% reduced risk of having extremely low hip BMD (P<0.01), and higher whole body BMD (P<0.01). Subjects with the GG genotype of the A163G polymorphism had a 70% reduced risk of having extremely low hip BMD (P<0.05), and higher whole body BMD (P<0.01). Significant gene–gene interactions were also observed among the OPG, RANK and RANKL genes. Our findings suggest that genetic variation in genes involved in the RANKL/RANK/OPG bone remodeling pathway are strongly associated with BMD at different skeletal sites in adult men, but not in women.

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 apparentfunction 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. Thompson, K., Molina, R. M., Donaghey, T., Schwob, J. E., Brain, J. D., Wessling‐Resnick, M. Olfactory uptake of manganese requires DMT1 and is enhanced by anemia. FASEB J. 21, 223–230 (2007)

Oxidative metabolism in the alveolar macrophage: analysis by flow cytometry.

We evaluated the reagents dichlorofluorescin (DCFH) and hydroethidine (HE) for use in flow cytometric analysis of the respiratory burst of alveolar macrophages and monocytes. DCFH and HE are non‐fluorescent precursors which can be oxidized intracellularly to the fluorescent compounds dichlorofluorescein and ethidium.