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Eosinophil recruitment and enhanced production of NO are characteristic features of asthma. However, neither the ability of eosinophils to generate NO-derived oxidants nor their role in nitration of targets during asthma is established. Using gas chromatography-mass spectrometry we demonstrate a 10-fold increase in 3-nitrotyrosine (NO2Y) content, a global marker of protein modification by reactive nitrogen species, in proteins recovered from bronchoalveolar lavage of severe asthmatic patients (480 ± 198 μmol/mol tyrosine; n = 11) compared with nonasthmatic subjects (52.5 ± 40.7 μmol/mol tyrosine; n = 12). Parallel gas chromatography-mass spectrometry analyses of bronchoalveolar lavage proteins for 3-bromotyrosine (BrY) and 3-chlorotyrosine (ClY), selective markers of eosinophil peroxidase (EPO)- and myeloperoxidase-catalyzed oxidation, respectively, demonstrated a dramatic preferential formation of BrY in asthmatic (1093 ± 457 μmol BrY/mol tyrosine; 161 ± 88 μmol ClY/mol tyrosine; n = 11 each) compared with nonasthmatic subjects (13 ± 14.5 μmol BrY/mol tyrosine; 65 ± 69 μmol ClY/mol tyrosine; n = 12 each). Bronchial tissue from individuals who died of asthma demonstrated the most intense anti-NO2Y immunostaining in epitopes that colocalized with eosinophils. Although eosinophils from normal subjects failed to generate detectable levels of NO, NO2−, NO3−, or NO2Y, tyrosine nitration was promoted by eosinophils activated either in the presence of physiological levels of NO2− or an exogenous NO source. At low, but not high (e.g., >2 μM/min), rates of NO flux, EPO inhibitors and catalase markedly attenuated aromatic nitration. These results identify eosinophils as a major source of oxidants during asthma. They also demonstrate that eosinophils use distinct mechanisms for generating NO-derived oxidants and identify EPO as an enzymatic source of nitrating intermediates in eosinophils.

ClinicalTrials.gov; No.: NCT02232750; URL: www.clinicaltrials.gov.

No abstract

BackgroundThe purpose of this study was to test the hypothesis that mast cells that are present in fibrotic regions of cancer can suppress the growth of tumor cells through an indirect mechanism involving peri-tumoral fibroblasts.MethodsWe first immunostained a wide variety of human cancers for the presence of degranulated mast cells. In a subsequent series of controlled in vitro experiments, we then co-cultured UACC-812 human breast cancer cells with normal fibroblasts in the presence or absence of different combinations and doses of mast cell tryptase, mast cell heparin, a lysate of the human mast cell line HMC-1, and fibroblast growth factor-7 (FGF-7), a powerful, heparin-binding growth factor for breast epithelial cells.ResultsDegranulating mast cells were localized predominantly in the fibrous tissue of every case of breast cancer, head and neck cancer, lung cancer, ovarian cancer, non-Hodgkin's lymphoma, and Hodgkin's disease that we examined. Mast cell tryptase and HMC-1 lysate had no significant effect on the clonogenic growth of cancer cells co-cultured with fibroblasts. By contrast, mast cell heparin at multiple doses significantly reduced the size and number of colonies of tumor cells co-cultured with fibroblasts, especially in the presence of FGF-7. Neither heparin nor FGF-7, individually or in combination, produced any significant effect on the clonogenic growth of breast cancer cells cultured without fibroblasts.ConclusionDegranulating mast cells are restricted to peri-tumoral fibrous tissue, and mast cell heparin is a powerful inhibitor of clonogenic growth of tumor cells co-cultured with fibroblasts. These results may help to explain the well-known ability of heparin to inhibit the growth of primary and metastatic tumors.

S U M M A R YWe describe an improved immunohistochemical procedure for detecting regions of hypoxia in normal organs and tumors in mice. The method employs a primary fluorescein-conjugated mouse monoclonal antibody directed against pimonidazole protein adducts that are created in hypoxic tissues and a secondary mouse anti-fluorescein antibody that is conjugated to horseradish peroxidase. Using these reagents, we clearly visualized the regions of relative hypoxia in implanted tumors in mice as well as in normal organs such as liver and kidney. Significantly, the resulting tissue sections were remarkably free of the background staining that is characteristically observed when rodent antibodies are used to detect antigens in rodent tissues.

IntroductionAccumulating evidence suggests that fibroblasts play a pivotal role in promoting the growth of breast cancer cells. The objective of the present study was to characterize and validate an in vitro model of the interaction between small numbers of human breast cancer cells and human fibroblasts.MethodsWe measured the clonogenic growth of small numbers of human breast cancer cells co-cultured in direct contact with serum-activated, normal human fibroblasts. Using DNA microarrays, we also characterized the gene expression profile of the serum-activated fibroblasts. In order to validate the in vivo relevance of our experiments, we then analyzed clinical samples of metastatic breast cancer for the presence of myofibroblasts expressing α-smooth muscle actin.ResultsClonogenic growth of human breast cancer cells obtained directly from in situ and invasive tumors was dramatically and consistently enhanced when the tumor cells were co-cultured in direct contact with serum-activated fibroblasts. This effect was abolished when the cells were co-cultured in transwells separated by permeable inserts. The fibroblasts in our experimental model exhibited a gene expression signature characteristic of 'serum response' (i.e. myofibroblasts). Immunostaining of human samples of metastatic breast cancer tissue confirmed that myofibroblasts are in direct contact with breast cancer cells.ConclusionSerum-activated fibroblasts promote the clonogenic growth of human breast cancer cells in vitro through a mechanism that involves direct physical contact between the cells. This model shares many important molecular and phenotypic similarities with the fibroblasts that are naturally found in breast cancers.

Human breast cancer is extensively infiltrated by mast cells that contain powerful anticoagulants such as heparin, tryptase and chymase. To determine if human breast cancer is associated with mast cell activation, we measured the levels of mast cell tryptase (an indicator of mast cell activation) in the blood of 20 women with varying stages of breast cancer. The mean level of tryptase in women with breast cancer (10.3 ؎ 4.2 g/L) was significantly higher than in 50 normal healthy women without breast cancer (3.0 ؎ 2.5 g/L, p < 0.05 by two-tailed t-test). To explore the role of mast cells in breast cancer in more detail, we then carried out experiments that were aimed at determining if an inhibitor of mast cell function, sodium cromolyn, could increase blood clotting and hypoxia within subcutaneous implants of the 4T1 mammary adenocarcinoma cell line in mice. We treated tumor-bearing mice with 5 consecutive daily doses of sodium cromolyn (10 mg/kg, i.p.). An average of 30% of the periphery of the tumors from the 5 drug-treated mice contained large lakes of clotted blood that were not evident in any of the tumors from the control (untreated) mice. By computerized image analysis of tumors immunostained for a hypoxia marker (pimonidazole), the tumors from the treated mice had significantly more hypoxia (35 ؎12 % hypoxic regions, n ‫؍‬ 5) than the tumors from untreated (control) mice (16 ؎ 7%, n ‫؍‬ 5). We conclude that sodium cromolyn enhanced peri-tumoral blood clotting and intratumoral hypoxia. These results suggest that mast cells may play an important role in regulating blood clotting and hypoxia within breast cancer.