Simon S. Wong

Mechanisms of Lung Fibrosis Resolution

Fibrogenesis involves a dynamic interplay between factors that promote the biosynthesis and deposition of extracellular matrix along with pathways that degrade the extracellular matrix and eliminate the primary effector cells. Opposing the often held perception that fibrotic tissue is permanent, animal studies and clinical data now demonstrate the highly plastic nature of organ fibrosis that can, under certain circumstances, regress. This review describes the current understanding of the mechanisms whereby the lung is known to resolve fibrosis focusing on degradation of the extracellular matrix, removal of myofibroblasts, and the role of inflammatory cells. Although there are significant gaps in understanding lung fibrosis resolution, accelerated improvements in biotechnology and bioinformatics are expected to improve the understanding of these mechanisms and have high potential to lead to novel and effective restorative therapies in the treatment not only of pulmonary fibrosis, but also of a wide-ranging spectrum of chronic disorders.

In vivo comparison of epithelial responses for S-8 versus JP-8 jet fuels below permissible exposure limit

This study was designed to characterize and compare the pulmonary effects in distal lung from a low-level exposure to jet propellant-8 fuel (JP-8) and a new synthetic-8 fuel (S-8). It is hypothesized that both fuels have different airway epithelial deposition and responses. Consequently, male C57BL/6 mice were nose-only exposed to S-8 and JP-8 at average concentrations of 53mg/m(3) for 1h/day for 7 days. A pulmonary function test performed 24h after the final exposure indicated that there was a significant increase in expiratory lung resistance in the S-8 mice, whereas JP-8 mice had significant increases in both inspiratory and expiratory lung resistance compared to control values. Neither significant S-8 nor JP-8 respiratory permeability changes were observed compared to controls, suggesting no loss of epithelial barrier integrity. Morphological examination and morphometric analysis of airway tissue demonstrated that both fuels showed different patterns of targeted epithelial cells: bronchioles in S-8 and alveoli/terminal bronchioles in JP-8. Collectively, our data suggest that both fuels may have partially different deposition patterns, which may possibly contribute to specific different adverse effects in lung ventilatory function.

Dose-dependent transcriptome changes by metal ores on a human acute lymphoblastic leukemia cell line

The increased morbidity of childhood leukemia in Fallon, Nevada and Sierra Vista, Arizona has prompted great health concern. The main characteristic that these two towns share is the environmental pollution attributed to metal ore from abandoned mining operations. Consequently, we have investigated the transcriptome effects of metal ores from these endemic areas using a human T-cell acute lymphoblastic leukemia cell line (T-ALL). Metal ore from Fallon significantly increased cell growth after 24, 48 and 72 h of incubation at 1.5 mg/mL concentration, as measured by trypan-blue. Sierra Vista ore significantly increased cell growth with 0.15 and 1.5 mg/mL following 72 h of incubation. From human cDNA microarray, results indicate that in total, eight genes, mostly metallothionein (MT) genes, were up-regulated and 10 genes were down-regulated following treatment of the T-ALL cells with 0.15 and 1.5 mg/mL of metal ores at 72 h, in comparison with untreated cells. Twenty-eight metals of both ores were quantified and their presence may be associated with the cell growth rate and dose-dependent activation of transcriptomes in immature T-cells.

Prenatal exposure of mice to tungstate is associated with decreased transcriptome-expression of the putative tumor suppressor gene, DMBT1: Implications for childhood leukemia

Background. Two concurrent, childhood leukemia clusters have been identified in the southwestern United States at Fallon, Nevada, and Sierra Vista, Arizona. Additionally, Fallon, Nevada has also experienced concurrent contamination by atmospheric tungsten particles. The etiology of leukemia is not known. Hypothesized risk factors for leukemia are environmental exposure, genetic predisposition, and viral infection. Additionally, strong evidence supports a prenatal origin. Our objective is to generate testable hypotheses towards elucidating the probable, multi-factorial etiology of leukemia by identifying the exposures unique to Fallon, Nevada, and held in common with Sierra Vista, Arizona, then exposing C57BL/6 mice, while in utero, to these chemicals to ascertain their leukemogenic potential. Utilizing advances in medical geology to analyze tree rings, surface dust, lichens and atmospheric particulate matter, we have identified tungsten and arsenic as potentially relevant to leukemogenesis. Methods. We utilized microarray (Affymetrix 430A 2.0 mouse) and real-time RTPCR of Dmbt1 transcriptome-expression in spleen tissue collected from four-week-old C57BL/6 mouse pups (N = 6–8/group/gender) exposed, while in utero, to tungstate, arsenite, tungstate/arsenite and longitudinal controls at 20% of the normalized exposure a human mother would receive during gestation at mean environmental concentrations. Results. Prenatal exposure to tungstate is associated with a 37 + 1.2-fold (p = 0.012) decrease in DMBT1 transcriptome-expression in mice expressing DMBT1 at high levels. Additionally, prenatal exposure to tungstate/arsenite significantly altered a cytokine–cytokine receptor interaction pathway associated with lymphocyte activation and a network associated with hematological/immunological disease. Conclusion. Because DMBT1 protein products are known to aggregate viruses and possibly regulate immune response, additional research is warranted to determine the potential that prenatal exposure to tungstate or tungstate/ arsenite has to increase susceptibility to viruses and to induce leukemogenesis.

PULMONARY EVALUATION OF PERMISSIBLE EXPOSURE LIMIT OF SYNTROLEUM S-8 SYNTHETIC JET FUEL IN MICE

No current studies have systematically examined pulmonary health effects associated with Syntroleum S-8 synthetic jet fuel (S-8). In order to gain an understanding about the threshold concentration in which lung injury is observed, C57BL/6 male mice were nose-only exposed to S-8 for 1 h/day for 7 days at average concentrations of 0 (control), 93, 352, and 616 mg/m(3). Evaluation of pulmonary function, airway epithelial barrier integrity, and pathohistology was performed 24 h after the final exposures. Significant decreases were detected in expiratory lung resistance and total lung compliance of the 352 mg/m(3) group, for which no clear concentration-dependent alterations could be determined. No significant changes in respiratory permeability were exhibited, indicating that there was no loss of epithelial barrier integrity following S-8 exposure. However, morphological examination and morphometric analysis of distal lung tissue, by using transmission electron microscopy, revealed cellular damage in alveolar type II epithelial cells, with significant increases in volume density of lamellar bodies/vacuoles at 352 and 616 S-8 mg/m(3). Moreover, terminal bronchiolar Clara injury, as evidenced by apical membrane blebs, was observed at relatively low concentrations, suggesting if this synthetic jet fuel is utilized, the current permissible exposure limit of 350 mg/m(3) for hydrocarbon fuels should cautiously be applied.

Repeated Aerosol-vapor JP-8 Jet Fuel Exposure Affects Neurobehavior and Neurotransmitter Levels in a Rat Model

Four groups of Fischer Brown Norway hybrid rats were exposed for 5, 10, 15, or 20 d to aerosolized-vapor jet propulsion fuel 8 (JP-8) compared to freely moving (5 and 10-d exposures) or sham-confined controls (15 and 20-d exposures). Behavioral testing utilized the U.S. Environmental Protection Agency Functional Observational Battery. Exploratory ethological factor analysis identified three salient factors (central nervous system [CNS] excitability, autonomic 1, and autonomic 2) for use in profiling JP-8 exposure in future studies. The factors were used as dependent variables in general linear modeling. Exposed animals were found to engage in more rearing and hyperaroused behavior compared to controls, replicating prior JP-8 exposure findings. Exposed animals also showed increasing but rapidly decelerating stool output (autonomic 1), and a significant increasing linear trend for urine output (autonomic 2). No significant trends were noted for either of the control groups for the autonomic factors. Rats from each of the groups for each of the time frames were randomly selected for tissue assay from seven brain regions for neurotransmitter levels. Hippocampal DOPAC was significantly elevated after 4-wk JP-8 exposure compared to both control groups, suggesting increased dopamine release and metabolism. Findings indicate that behavioral changes do not appear to manifest until wk 3 and 4 of exposure, suggesting the need for longitudinal studies to determine if these behaviors occur due to cumulative exposure, or due to behavioral sensitization related to repeated exposure to aerosolized-vapor JP-8.

Thy-1 interaction with Fas in lipid rafts regulates fibroblast apoptosis and lung injury resolution.

Thy-1-negative lung fibroblasts are resistant to apoptosis. The mechanisms governing this process and its relevance to fibrotic remodeling remain poorly understood. By using either sorted or transfected lung fibroblasts, we found that Thy-1 expression is associated with downregulation of anti-apoptotic molecules Bcl-2 and Bcl-xL, as well as increased levels of cleaved caspase-9. Addition of rhFasL and staurosporine, well-known apoptosis inducers, caused significantly increased cleaved caspase-3, -8, and PARP in Thy-1-transfected cells. Furthermore, rhFasL induced Fas translocation into lipid rafts and its colocalization with Thy-1. These in vitro results indicate that Thy-1, in a manner dependent upon its glycophosphatidylinositol anchor and lipid raft localization, regulates apoptosis in lung fibroblasts via Fas-, Bcl-, and caspase-dependent pathways. In vivo, Thy-1 deficient (Thy1−/−) mice displayed persistence of myofibroblasts in the resolution phase of bleomycin-induced fibrosis, associated with accumulation of collagen and failure of lung fibrosis resolution. Apoptosis of myofibroblasts is decreased in Thy1−/− mice in the resolution phase. Collectively, these findings provide new evidence regarding the role and mechanisms of Thy-1 in initiating myofibroblast apoptosis that heralds the termination of the reparative response to bleomycin-induced lung injury. Understanding the mechanisms regulating fibroblast survival/apoptosis should lead to novel therapeutic interventions for lung fibrosis.

In vitro time- and dose-effect response of JP-8 and S-8 jet fuel on alveolar type II epithelial cells of rats.

This study was designed to characterize and compare the effects of jet propellant-8 (JP-8) fuel and synthetic-8 (S-8) on cell viability and nitric oxide synthesis in cultured alveolar type II epithelial cells of rats. Exposure times varied from 0.25, 0.5, 1, and 6 hours at the following concentrations of jet fuel: 0.0, 0.1, 0.4, and 2.0 µg/mL. Data indicate that JP-8 presents a gradual decline in cell viability and steady elevation in nitric oxide release as exposure concentrations increase. At a 2.0 µg/mL concentration of JP-8, nearly all of the cells are not viable. Moreover, S-8 exposure to rat type II lung cells demonstrated an abrupt fall in percentage cell viability and increases in nitric oxide measurement, particularly after the 2.0 µg/mL was reached at 1 and 6 hours. At 0.0, 0.2, and 0.4 µg/mL concentrations of S-8, percentage viability was sustained at steady concentrations. The results suggest different epithelial toxicity and mechanistic effects of S-8 and JP-8, providing further insight concerning the impairment imposed at specific levels of lung function and pathology induced by the different fuels.

In Vitro Pro-inflammatory Regulatory role of Substance P in Alveolar Macrophages and Type II Pneumocytes after JP-8 Exposure

The effects of JP-8 on pro-inflammatory cytokine interleukin (IL)-1α,β and nitric oxide (NO) secretion as well as the role of substance P (SP) in these processes were examined in cultured alveolar macrophages (AM), type II epithelial cells (AIIE), and AM/AIIE co-cultures. Exposure of AM to JP-8 for 24 hr exhibited release of IL-1α,β, whereas exposure to AIIE showed a concentration-dependent NO overproduction. Data indicate that there are cell-dependent inflammatory mechanisms responsible for the actual level of JP-8 exposure in alveoli. However, treatment with substance P significantly attenuated JP-8 induced the IL-1α,β secretion. This finding was confirmed by using [Sar9 Met (O2)11] SP (10− 10 M), an agonist of substance P, suggesting that substance P may have signal pathway(s) to AM in the inflammatory response mediated by IL-1. Moreover, AM/AIIE co-culture obviously reduced NO overproduction observed in AIIE alone, suggesting that there may be cell interactions or communications between AM and AIIE in response to the JP-8 exposure.

Tissue-specific patterns of neurokinin-1 receptor (NK-1R) gene expression in mice exposed to sidestream cigarette smoke

Neurokinin-1 receptor (NK-1R), a high-affinity plasma membrane-bound receptor for neurokinin substance P, plays important roles in the onset of the pathophysiological responses. To test whether the transcript levels of gene encoding NK-1R in organs are affected by sidestream cigarette smoke (SSCS) exposure, the C57BL/6 mice were randomly assigned to five groups (six/group) in a study of the dose-effect relationship. The mice were exposed to 0 (filtered room air), 2, 4, 8 and 16 mg total particulate matter (TPM) of SSCS/exposure/day, respectively, for seven days through a nose-only exposure chamber (IN-TOX, Albuquerque, NM, USA). The levels of NK-1R mRNA in the lung, heart, liver, kidney and spleen tissues were detected by reverse transcription-polymerase chain reaction (RT-PCR) techniques and normalized against GAPDH expression. NK-1R mRNA in heart tissue showed SSCS-induced dose-dependent downregulation, with minimum expression at a dose of 8.0 mg TPM. Whereas, the levels of NK-1R mRNA in the liver were upregulated to 2.86 and 5.13- fold after exposure to 2.0 and 4.0 mg TPM of SSCS respectively, then returned to 4.19 and 3.93-fold at the exposure doses of 8.0 and 16.0 mg TPM, respectively, when compared to that of the control. In the kidney, SSCS exposure at a dose of 2.0 TPM, but not higher than that level, induced significant elevation of NK-1R mRNA expression. These findings suggest that there are the paracrine and/or autocrine signalling mechanisms through receptor -ligand interactions. No alteration of NK-1R gene expression was observed in the lungs and spleen tissues in this study. The tissue-specific patterns by which SSCS affect NK-1R gene expression in these organs may partially explain dissimilarity of NK-1R activation and the associated toxicity caused by environmental tobacco smoke.