Sex-specific lung functional changes in adult mice exposed only to second-hand smoke in utero.

BACKGROUND: An increasing number of epidemiological and experimental studies have associated exposure to second-hand smoke (SHS) during pregnancy with adverse outcomes in newborns. As we have previously shown in mice, in utero exposure to SHS at critical stages of fetal development, results in altered lung responses and increased disease susceptibility upon re-exposure to irritants (SHS or ovalbumin) in adulthood. In this study, we asked whether the in utero SHS exposure alone is sufficient to alter lung structure and function in adult mice. METHODS: Pregnant BALB/c mice were exposed from days 6 to 19 of pregnancy to 10 mg/m3 of SHS or HEPA-filtered air. Male and female offspring (n = 13-15/group) were sacrificed at 15 weeks of age. We measured lung function with non-invasive and invasive methods, performed lung morphometric analysis on trichrome-stained lung tissue samples, and assessed lung gene expression via RNA sequencing and protein assays. RESULTS: In utero SHS exposure significantly increased mean linear intercept and decreased the surface area per unit volume of the lungs in both males and females, indicating perturbation in alveolar developmental processes. Tidal volume, minute volume and inspiratory capacity were significantly decreased compared with the controls only in male mice exposed in utero to SHS, suggesting that males are more sensitive than females to an SHS insult during lung development. This also suggests that in our model, lung structure changes may be necessary but are not sufficient to impair lung function. SERPINA1A, the mouse ortholog of human α1-antitrypsin, deficiency of which is a known genetic risk factor for emphysema, was down-regulated at the protein level in the in utero SHS-exposed mice. Additionally, DNMT3A protein expression was dysregulated, indicating that DNA methylation occurred in the lungs. CONCLUSIONS: Our results indicate that in utero SHS exposure alone alters both lung function and structure well into adulthood (15 weeks) in male mice. Furthermore, lung function alterations in this model are sex-specific, with males being more susceptible to in utero SHS effects. Overall, our data suggest that in utero SHS exposure alone can predispose to adult lung diseases.

In utero exposure to second-hand smoke aggravates the response to ovalbumin in adult mice.

Second-hand smoke (SHS) exposure in utero exacerbates adult responses to environmental irritants. We tested the hypothesis that effects of in utero SHS exposure on modulating physiological and transcriptome responses in BALB/c mouse lungs after ovalbumin (OVA) challenge extend well into adulthood, and that the responses show a sex bias. We exposed BALB/c mice in utero to SHS or filtered air (AIR), then sensitized and challenged all offspring with OVA from 19 to 23 weeks of age. At the end of the adult OVA challenge, we evaluated pulmonary function, examined histopathology, analyzed bronchoalveolar lavage fluid (BALF), and assessed gene expression changes in the lung samples. All groups exhibited lung inflammation and inflammatory cell infiltration. Pulmonary function testing (airway hyperresponsiveness [AHR], breathing frequency [f]) and BALF (cell differentials, Th1/Th2 cytokines) assessments showed significantly more pronounced lung responses in the SHS-OVA groups than in AIR-OVA groups (AHR, f; eosinophils, neutrophils; IFN-γ, IL-1b, IL-4, IL-5, IL-10, IL-13, KC/CXCL1, TNF-α), with the majority of responses being more pronounced in males than in females. SHS exposure in utero also significantly altered lung gene expression profiles, primarily of genes associated with inflammatory responses and respiratory diseases, including lung cancer and lung fibrosis. Altered expression profiles of chemokines (Cxcl2, Cxcl5, Ccl8, Ccl24), cytokines (Il1b, Il6, Il13) and acute phase response genes (Saa1, Saa3) were confirmed by qRT-PCR. In conclusion, in utero exposure to SHS exacerbates adult lung responses to OVA challenge and promotes a pro-asthmatic milieu in adult lungs; further, males are generally more affected by SHS-OVA than are females.

Mmp12 Is Upregulated by in utero Second-Hand Smoke Exposures and Is a Key Factor Contributing to Aggravated Lung Responses in Adult Emphysema, Asthma, and Lung Cancer Mouse Models.

Matrix metalloproteinase-12 (Mmp12) is upregulated by cigarette smoke (CS) and plays a critical role in extracellular matrix remodeling, a key mechanism involved in physiological repair processes, and in the pathogenesis of emphysema, asthma, and lung cancer. While cigarette smoking is associated with the development of chronic obstructive pulmonary diseases (COPD) and lung cancer, in utero exposures to CS and second-hand smoke (SHS) are associated with asthma development in the offspring. SHS is an indoor air pollutant that causes known adverse health effects; however, the mechanisms by which in utero SHS exposures predispose to adult lung diseases, including COPD, asthma, and lung cancer, are poorly understood. In this study, we tested the hypothesis that in utero SHS exposure aggravates adult-induced emphysema, asthma, and lung cancer. Methods: Pregnant BALB/c mice were exposed from gestational days 6-19 to either 3 or 10mg/m3 of SHS or filtered air. At 10, 11, 16, or 17weeks of age, female offspring were treated with either saline for controls, elastase to induce emphysema, house-dust mite (HDM) to initiate asthma, or urethane to promote lung cancer. At sacrifice, specific disease-related lung responses including lung function, inflammation, gene, and protein expression were assessed. Results: In the elastase-induced emphysema model, in utero SHS-exposed mice had significantly enlarged airspaces and up-regulated expression of Mmp12 (10.3-fold compared to air-elastase controls). In the HDM-induced asthma model, in utero exposures to SHS produced eosinophilic lung inflammation and potentiated Mmp12 gene expression (5.7-fold compared to air-HDM controls). In the lung cancer model, in utero exposures to SHS significantly increased the number of intrapulmonary metastases at 58weeks of age and up-regulated Mmp12 (9.3-fold compared to air-urethane controls). In all lung disease models, Mmp12 upregulation was supported at the protein level. Conclusion: Our findings revealed that in utero SHS exposures exacerbate lung responses to adult-induced emphysema, asthma, and lung cancer. Our data show that MMP12 is up-regulated at the gene and protein levels in three distinct adult lung disease models following in utero SHS exposures, suggesting that MMP12 is central to in utero SHS-aggravated lung responses.

The effect of urea on refractometric total protein measurement in dogs and cats with azotemia.

BACKGROUND: While protein is the predominant solute measured in plasma or serum by a refractometer, nonprotein substances also contribute to the angle of refraction. There is debate in the current literature regarding which nonprotein substances cause factitiously high refractometric total protein measurements, as compared to the biuret assay. OBJECTIVES: The purpose of the study was to determine if the blood of azotemic animals, specifically with increased blood urea concentration, will have significantly higher refractometric total protein concentrations compared to the total protein concentrations measured by biuret assay. METHODS: A prospective case series was conducted by collecting data from azotemic (n = 26) and nonazotemic (n = 34) dogs and cats. In addition, an in vitro study was performed where urea was added to an enhanced electrolyte solution at increasing concentrations, and total protein was assessed by both the refractometer and spectrophotometer. Statistical analysis was performed to determine the effect of urea. RESULTS: The refractometric total protein measurement showed a positive bias when compared to the biuret protein measurement in both groups, but the bias was higher in the azotemic group vs the nonazotemic group. The mean difference in total protein measurements of the nonazotemic group (0.59 g/dL) was significantly less (P < .01) than the mean difference of the azotemic group (0.95 g/dL). The in vitro experiment revealed a positive bias with a proportional error. CONCLUSIONS: This study demonstrated that increasing concentrations of urea significantly increased the total protein concentration measured by the refractometer as compared to the biuret assay, both in vivo and in vitro.

Nannizziopsis guarroi infection in 2 Inland Bearded Dragons (Pogona vitticeps): clinical, cytologic, histologic, and ultrastructural aspects.

Chrysosporium-related infections have been increasingly reported in reptiles over the last 2 decades. In this report, we describe clinical, cytologic, histopathologic, and ultrastructural aspects of Chrysosporium-related infection in 2 Inland Bearded Dragons (Pogona vitticeps). Case 1 was presented for an enlarging raised lesion over the left eye and multiple additional masses over the dorsum. Case 2 was submitted to necropsy by the referring veterinarian for suspected yellow fungus disease. Impression smears of the nodules in case 1 revealed granulomatous to pyogranulomatous inflammation and many septate, variably long, 4-10 µm wide, often undulated hyphae, and very rare conidia. Postmortem impression smears of the superficial lesions of case 2 contained large numbers of solitary conidia and arthroconidia and low numbers of hyphae with similar morphology to case 1. Histopathology of the 2 cases revealed severe, multifocal, chronic, ulcerative, nodular pyogranulomatous dermatitis, with myriad intralesional septate hyphae, and arthroconidia. Fungal culture and molecular sequencing in both cases indicated infection with Nannizziopsis guarroi.