Todd M. Gambling

Air Pollution and Brain Damage

Exposure to complex mixtures of air pollutants produces infl ammation in the upper and lower respiratory tract. Because the nasal cavity is a common portal of entry, respiratory and olfactory epithelia are vulnerable targets for toxicological damage. This study has evaluated, by light and electron microscopy and immunohistochemica l expression of nuclear factor-kappa beta (NF-κB) and inducible nitric oxide synthase (iNOS), the olfactory and respiratory nasal mucosae, olfactory bulb, and cortical and subcortical structures from 32 healthy mongrel canine residents in Southwest Metropolitan Mexico City (SWMMC), a highly polluted urban region. Findings were compared to those in 8 dogs from Tlaxcala, a less polluted, control city. In SWMMC dogs, expression of nuclear neuronal NF-κB and iNOS in cortical endothelial cells occurred at ages 2 and 4 weeks; subsequent damage included alterations of the blood—brain barrier (BBB), degenerating cortical neurons, apoptotic glial white matter cells, deposition of apolipoprotein E (apoE)-positive lipid droplets in smooth muscle cells and pericytes, nonneuritic plaques , and neurofi brillary tangles. Persistent pulmonary infl ammation and deteriorating olfactory and respiratory barriers may play a role in the neuropathology observed in the brains of these highly exposed canines. Neurodegenerative disorders such as Alzheimers may begin early in life with air pollutants playing a crucial role.

Palmitoylation of claudins is required for efficient tight-junction localization

Palmitoylation of integral membrane proteins can affect intracellular trafficking, protein-protein interactions and protein stability. The goal of the present study was to determine whether claudins, transmembrane-barrier-forming proteins of the tight junction, are palmitoylated and whether this modification has functional implications for the tight-junction barrier. Claudin-14, like other members of the claudin family, contains membrane-proximal cysteines following both the second and the fourth transmembrane domains, which we speculated could be modified by S-acylation with palmitic acid. We observed that [3H]-palmitic acid was incorporated into claudin-14 expressed by transfection in both cultured epithelial cells and fibroblasts. Mutation of cysteines to serines following either the second or the fourth transmembrane segments decreased the incorporation of [3H]-palmitic acid, and mutation of all four cysteines eliminated palmitoylation. We previously reported that expression of claudin-14 in epithelial monolayers results in a fivefold increase in electrical resistance. By contrast, expression of the mutant claudin-14 resulted in smaller increases in resistance. The mutants localized less well to tight junctions and were also found in lysosomes, suggesting an alteration in trafficking or stability. However, we observed no change in protein half-life and only a small shift in fractionation out of caveolin-enriched detergent-resistant membranes. Although less well localized to the tight junction, palmitoylation-deficient claudin-14 was still concentrated at sites of cell-cell contact and was competent to assemble into freeze-fracture strands when expressed in fibroblasts. These results demonstrate that palmitoylation of claudin-14 is required for efficient localization into tight junctions but not stability or strand assembly. Decreased ability of the mutants to alter resistance is probably the result of their less efficient localization into the barrier.

Canines as Sentinel Species for Assessing Chronic Exposures to Air Pollutants: Part 1. Respiratory Pathology

A complex mixture of air pollutants is present in the ambient air in urban areas. People, animals, and vegetation are chronically and sequentially exposed to outdoor pollutants. The objective of this first of 2 studies is to evaluate by light and electron microscopy the lungs of Mexico City dogs and compare the results to those of 3 less polluted cities in MEXICO: One hundred fifty-two clinically healthy stray mongrel dogs (91 males/61 females), including 43 dogs from 3 less polluted cities, and 109 from southwest and northeast metropolitian Mexico City (SWMMC, NEMMC) were studied. Lungs of dogs living in Mexico City and Cuernavaca exhibited patchy chronic mononuclear cell infiltrates along with macrophages loaded with particulate matter (PM) surrounding the bronchiolar walls and extending into adjacent vascular structures; bronchiolar epithelial and smooth muscle hyperplasia, peribronchiolar fibrosis, microthrombi, and capillary and venule polymorphonuclear leukocytes (PMN) margination. Ultrafine PM was seen in alveolar type I and II cells, endothelial cells, interstitial macrophages (Mtheta), and intravascular Mtheta-like cells. Bronchoalveolar lavage showed significant numbers of alveolar macrophages undergoing proliferation. Exposure to complex mixtures of pollutants-predominantly particulate matter and ozone-is causing lung structural changes induced by the sustained inflammatory process and resulting in airway and vascular remodeling and altered repair. Cytokines released from both, circulating inflammatory and resident lung cells in response to endothelial and epithelial injury may be playing a role in the pathology described here. Deep concern exists for the potential of an increasing rise in lung diseases in child populations exposed to Mexico Citys environment.

Postnatal Development of Tracheal Surface Epithelium and Submucosal Glands in the Ferret

We explored the usefulness of the postnatal ferret as a model for early developmental events in the large airways, using light and scanning electron microscopy. In the first 28 postnatal days, ferret tracheal surface epithelium and glands undergo dramatic growth and development. Tracheal surface area increases 8-fold. At birth, ciliated cells are sparse (9.4 +/- 1.2% of total epithelial cells). A significant increase in ciliated cells is observed at weekly intervals and by day 28 the ciliated cell is the predominant cell type (54.2 +/- 2.8% of total epithelial cells). Secretory cells decrease from 66.4 +/- 1.0% at birth to 22.2 +/- 2.8% of total epithelial cells. Histochemical staining of the granules of the epithelial secretory cells changes from predominantly non-acidic (staining with PAS but not Alcian blue) to predominantly acidic (staining also with Alcian blue). During the same time interval, tracheal glands develop from intraepithelial cellular aggregates devoid of secretory granules at birth into complex, submucosal tubuloacinar structures composed predominantly of cells containing non-acidic secretory granules at 28 days. Therefore, infant ferrets offer an opportunity to examine the structural and functional components of the mucociliary clearance mechanism at developmental stages which occur prenatally in many laboratory animals and in humans.

Bubble hair: Case caused by an overheating hair dryer and reproducibility in normal hair with heat

BACKGROUND Bubble hair is an acquired hair shaft deformity characterized by bubble-like areas in the hair shaft seen with light microscopy and corresponding cavitary defects with scanning electron microscopy. OBJECTIVE Our purpose was to report the fourth case of bubble hair, to demonstrate a cause, and to determine whether the cavities contain gas or liquid. METHODS Light and scanning electron microscopy were performed. The patients hair dryer was examined. We applied heat to normal hair of the patient and that of 16 human volunteers. Approximate temperatures for bubble formation were measured. The bubble contents were investigated by applying aqueous and nonaqueous liquids to affected hairs. RESULTS No persons hair failed to develop bubbles when subjected to sufficient heat. The cause of bubble hair in the patient was an overheating hair dryer. The cavitations contained a gas. CONCLUSION Bubble hair is a reproducible hair shaft defect caused by heat. The use of overheating hair dryers, or any other hair care equipment that overheats, should be avoided.

Ultrastructure of airway epithelial cell membranes among patients with cystic fibrosis

Alterations in transepithelial ion fluxes are a primary pathophysiologic feature in cystic fibrosis (CF). Chronic respiratory infections and host responses are secondary aggravating pulmonary complications of this disease. In the present study, the application of the freeze-fracture technique to samples of nasal and tracheal epithelium from patients with CF has provided a perspective of large areas of cell membrane for the evaluation of possible structural correlates to the pathophysiology of this disease. A variety of aberrant configurations in stranding pattern and disorganization of the epithelial tight junctional complexes in CF airway epithelium are described. Additionally, examination of ciliary membranes revealed the presence of compound cilia and dysmorphology of ciliary necklace configuration. These features are thought to represent acquired structural lesions possibly derived from chronic infection and/or host responses which may further exacerbate abnormal ion transport properties and decrements of ciliary function that appear to be associated with the airway epithelium of individuals with CF.

Physicochemical basis for dilated intercellular spaces in non-erosive acid-damaged rabbit esophageal epithelium

Dilated intercellular spaces (DIS) within esophageal epithelium (EE) is a histopathologic feature of non-erosive reflux disease and early lesion in acid-damaged rabbit EE associated with increased paracellular permeability. Its cause remains unknown, but the lesions morphology suggests a significant fluid shift into the intercellular spaces (ICS). Since water follows osmotic forces and consequently ion movements, we explored the role of active (ion) transport and ion gradients in its pathogenesis. This was done by quantifying the effect of inhibited active transport and altered ion gradients on electrical resistance (R(T)) and ICS diameter in acid-exposed Ussing-chambered rabbit EE. Compared with normal Ringer, pH 7.5, 30 minutes of luminal HCl (100 mmol/L), pH 1.1, increased permeability (R(T): +5 +/- 4% vs-52 +/- 4%) and ICS diameter (0.25 +/- 0.01 microm vs 0.42 +/- 0.02 microm), but had no effect on cell morphology or diameter. Ouabain pretreatment significantly reduced active transport but had no effect on the acid-induced changes. However, negating the chloride gradient created by luminal HCl either by adding choline chloride, 100 mmol/L, serosally or by replacing luminal HCl, pH 1.1, with luminal H(2)SO(4), pH 1.1, prevented the development of DIS while maintaining the increase in permeability. DIS was also prevented in the presence of a 100 mmol/L (choline) chloride gradient by luminal exposure at neutral pH. DIS in HCl-damaged EE is caused by an H(+)-induced increase in epithelial permeability; this enables Cl(-) to diffuse along its gradient into the ICS, creating an osmotic force for water movement into and (hydrostatic) dilation of the ICS.

In Vitro and In Vivo Evaluation of a Water-in-Oil Microemulsion System for Enhanced Peptide Intestinal Delivery

Peptide and protein drugs have become the new generation of therapeutics, yet most of them are only available as injections, and reports on oral local intestinal delivery of peptides and proteins are quite limited. The aim of this work was to develop and evaluate a water-in-oil (w/o) microemulsion system in vitro and in vivo for local intestinal delivery of water-soluble peptides after oral administration. A fluorescent labeled peptide, 5-(and-6)-carboxytetramethylrhodamine labeled HIV transactivator protein TAT (TAMRA-TAT), was used as a model peptide. Water-in-oil microemulsions consisting of Miglyol 812, Capmul MCM, Tween 80, and water were developed and characterized in terms of appearance, viscosity, conductivity, morphology, and particle size analysis. TAMRA-TAT was loaded and its enzymatic stability was assessed in modified simulated intestinal fluid (MSIF) in vitro. In in vivo studies, TAMRA-TAT intestinal distribution was evaluated using fluorescence microscopy after TAMRA-TAT microemulsion, TAMRA-TAT solution, and placebo microemulsion were orally gavaged to mice. The half-life of TAMRA-TAT in microemulsion was enhanced nearly three-fold compared to that in the water solution when challenged by MSIF. The treatment with TAMRA-TAT microemulsion after oral administration resulted in greater fluorescence intensity in all intestine sections (duodenum, jejunum, ileum, and colon) compared to TAMRA-TAT solution or placebo microemulsion. The in vitro and in vivo studies together suggested TAMRA-TAT was better protected in the w/o microemulsion in an enzyme-containing environment, suggesting that the w/o microemulsions developed in this study may serve as a potential delivery vehicle for local intestinal delivery of peptides or proteins after oral administration.

Loss of ASP but not ROPN1 reduces mammalian ciliary motility

Protein kinase A (PKA) signaling is targeted by interactions with A‐kinase anchoring proteins (AKAPs) via a dimerization/docking domain on the regulatory (R) subunit of PKA. Four other mammalian proteins [AKAP‐associated sperm protein (ASP), ropporin (ROPN1), sperm protein 17 (SP17) and calcium binding tyrosine‐(Y)‐phosphorylation regulated protein (CABYR)] share this highly conserved RII dimerization/docking (R2D2) domain. ASP and ROPN1 are 41% identical in sequence, interact with a variety of AKAPs in a manner similar to PKA, and are expressed in ciliated and flagellated human cells. To test the hypothesis that these proteins regulate motility, we developed mutant mouse lines lacking ASP or ROPN1. Both mutant lines produced normal numbers of cilia with intact ciliary ultrastructure. Lack of ROPN1 had no effect on ciliary motility. However, the beat frequency of cilia from mice lacking ASP is significantly slower than wild type, indicating that ASP signaling may regulate ciliary motility. This is the first demonstration of in vivo function for ASP. Similar localization of ASP in mice and humans indicates that these findings may translate to human physiology, and that these mice will be an excellent model for future studies related to the pathogenesis of human disease.

Features of Developing Ferret Tracheal Epithelium: Ultrastructural Observations of in Vivo and in Vitro Differentiation of Ciliated Cells

Ultrastructural features of the developing, surface epithelium of ferrets from birth to 28 days of age were characterized. Progressive ciliogenesis in vivo was observed, beginning with cells covering the membranous portion of the trachea. Emerging cilia appeared in ultrathin sections and by scanning electron microscopy at sites correlating with accumulation of integral membrane particles seen in freeze-fracture preparations. Two patterns of ciliogenesis were observed: (1) the random emergence of cilia over the apical cell surface, and (2) initial emergence of cilia at the peripheral boundary of the luminal border of individual cells. Novel, ringlike structures were observed on the surfaces of nonciliated cells at all ages studied. Active ciliogenesis as well as the appearance of ring structures also were documented in the superficial epithelium from 1- to 5-day-old animals maintained in vitro for up to 4 days.