- Email: [email protected]
Buccal-Lung Comparison of Quantitative Expression of Carcinogen and Oxidant Metabolism Genes in Human Subjects
blast cell lines of laboratory animals, whereas the predominant cell type transformed in the sheep host is an epithelial cell of the lung, the alveolar type II (ATII) cell. To provide a system for investigating the effects of the JSRV envelope protein on its target cell, we report here on the isolation and extended culture (ie, 7 to 14 days) of primary sheep ATII cells, and their infection by JSRV. The maintenance of the cells in culture led to a homogeneous population of proliferating ATII cells with numerous lamellar bodies, well-organized microvilli, apical/basolateral polarity, and prominent tight junctions. Moreover, the cells exhibited high levels of agonist-induced surfactant lipid secretion. Primary ATII cells also could be infected using the JSRV molecular clone JSRVJS7, demonstrating that the cultured cells expressed the specific receptor for JSRV, described as hyaluronidase-2. Furthermore, the viral genome was shown to be transcribed and translated as demonstrated by reverse transcriptase activity in the supernatant of virus-adsorbed cells. The examination of the effects of the JSRV envelope protein expressed in primary ATII cells and its role in ovine pulmonary adenocarcinoma is the current focus of our work.
Selective Suppression of Dendritic Cell Functions by Cigarette Smoke Extract* Robert Vassallo, MD, FCCP; and Lieping Chen, MD, PhD
(CHEST 2004; 125:107S) Abbreviation: CSE ⫽ cigarette smoke extract
cells are potent antigen-presenting cells and D endritic critical regulators of T-cell responses. We postulated
that an important mechanism by which smoking predisposes a person to cancer is by suppressing dendritic cell function, thus inhibiting the ability of dendritic cells to stimulate appropriate T-cell responses. To test this hypothesis, human monocyte-derived dendritic cells were incubated with freshly prepared cigarette smoke extract (CSE) [0 to 2%; mean (⫾ SD) nicotine content in 1% CSE, 2.3 ⫾ 0.8 g/mL] for the final 18 to 24 h of culture. Dendritic cells were then washed to remove all residual CSE and were incubated with allogenic purified T cells in a mixed lymphocyte reaction for 72 h. T-cell proliferation was measured by the incorporation of tritiated thymidine *From the Mayo Clinic, Rochester, MN. This research was supported by an American Lung Association research grant, a Parker B. Francis fellowship (RV), and National Institutes of Health grants CA79915 and CA85721 (LC). Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: [email protected]). Correspondence to: Robert Vassallo, MD, FCCP, Assistant Professor of Medicine, Division of Pulmonary and Critical Care Medicine, Mayo Clinic, Stabile Bldg 8 –54, Rochester, MN 55905; e-mail: [email protected] www.chestjournal.org
and cytokine levels into the mixed lymphocyte reaction measured by enzyme-linked immunosorbent assay. The transient incubation of immature dendritic cells with CSE at doses that did not affect viability (as measured by the XTT assay, trypan blue exclusion, and annexin V/propidium iodide staining) resulted in significant suppression of dendritic cell-induced T-cell proliferation. This decrease in T-cell proliferation was accompanied by reduced interferon-␥ production, but not in interleukin-4 or interleukin-10 secretion. Dendritic cell surface expression of major histocompatibility complex class II, major histocompatibility complex class I, CD-40, CD-80, CD-86, CD-83, and CD-1a was not altered by transient exposure (ie, for 24 h) to CSE at the same concentrations that diminished dendritic cell-induced T-cell proliferation. These data indicate that transient exposure to CSE causes the selective suppression of dendritic cell functions in the induction of T-cell proliferation and the production of interferon-␥.
Buccal-Lung Comparison of Quantitative Expression of Carcinogen and Oxidant Metabolism Genes in Human Subjects* R.J. Jain, PhD; S. Varma, MS; G.J. Hurteau, BS; and Simon D. Spivack, MD, MPH, FCCP
(CHEST 2004; 125:107S–108S) human lung is inaccessible for direct examination. T heBuccal cells lining the inner cheek of tobacco smokers
are carcinogen- and oxidant-exposed, easily accessible, and potentially valuable surrogates for lung tissue. Lung cancer susceptibility phenotyping might include carcinogenmetabolism and oxidant-metabolism gene expression assayed in buccal cells.
Materials and Methods For buccal cell analysis, we adapted known techniques of RNA preservation and extraction. We have employed our novel RNAspecific reverse transcription polymerase chain reaction strategy that is unconfounded by genomic DNA pseudogene sequence encoding target and reference “housekeeper” sequences. All primers were blasted against bacterial sequences to assure human *From the Wadsworth Center, New York State Department of Health, State University of New York School of Public Health, and Albany Medical College, Pulmonary and Critical Care Medicine, Albany, NY. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: [email protected]). Correspondence to: Simon Spivack, MD, MPH, FCCP, Human Toxicology & Molecular Epidemiology, Wadsworth Center, Biggs E622, New York State Department of Health, SUNY School of Public Health, Empire State Plaza, PO Box 509, Albany, NY 12201-0509; e-mail: [email protected] CHEST / 125 / 5 / MAY, 2004 SUPPLEMENT
107S
cell specificity of the amplification. Precise real-time quantitation of the expression of these genes employs ratios of target crossover point to reference housekeeper CRO (LightCycler; Roche; Basel, Switzerland). Genes that were analyzed included the aromatic hydrocarbon receptor gene (Ahr), cytochrome P450 genes CYP1A1 and CYP1B1, glutathione-S-transferase genes GSTM1, GSTM3, GSTP1, and GSTT1, nicotinamide adenine dinucleotide phosphate-quinone oxidoreductase (NQO1), copper-zinc superoxide dismutase (CuZn-SOD), catalase (CAT), and glutathione peroxidase (GPx). For any given individual among our initial group of human subjects, we have been able to reliably quantitate gene expression in the buccal cytologic specimens. We have compared buccal gene expression to laser capture microdissected lung gene expression, both by identical techniques of real-time quantitative reverse transcription polymerase chain reaction.
Results We have found close concordance of phase I, phase II, and antioxidant genes in constitutive expression (not expressed, CYP1A1, GSTM1, and GSTM3; all expressed, GSTP1, GSTT1; NQO1, SOD, CAT, and GPx) between the two tissues. There is often buccal-lung discordance for the Ahr gene. However, for all 11 transcripts studied in our first 11 individuals to date, 88 to 96% of the genespecific buccal-lung expression comparisons have shown qualitatively concordant expression (2 ⫽ 62.4; p ⬍ 0.001). The quantitative covariation between the two tissues is under study.
Conclusion We have in preliminary fashion shown that (1) gene expression is quantifiable in cytologically collected buccal cells, and (2) there appears to be good concordance between those carcinogen and oxidant metabolism genes expressed in the buccal mucosa and those expressed in the laser microdissected human lung epithelium. We have enlarged the group undergoing buccal-lung comparison, and have commenced stratification of the expression data for covariation with tobacco exposure, to define each individual’s unique gene expression signature in this cancer-relevant pathway.
1992, Aguayo et al described six patients with slowly I nprogressive cough and breathlessness, obstructive and/ 1
or restrictive respiratory impairment, and diffuse reticulonodular infiltrates seen on thoracic tomographic scanning, in whom a pulmonary biopsy revealed widespread intraluminal and extraluminal proliferation of pulmonary neuroendocrine cells (PNCs) in all patients, accompanied by peripheral carcinoid tumors in three patients. There was submucosal fibrosis of affected airways, but none of the conditions usually associated with the reactive proliferation of PNCs was evident. This and subsequent studies have further defined the clinical and pathologic parameters of this rare condition, now conventionally known as diffuse idiopathic PNC hyperplasia or DIPNECH. DIPNECH raises interesting questions about the relationship between the proliferation of PNCs, hitherto considered a reactive process, and the genesis of bona fide pulmonary neuroendocrine tumors (PNTs), such that it now appears as a preneoplastic condition in the latest World Health Organization histologic classification of tumors of the lung and pleura. However, the carcinoid tumors that accompany DIPNECH and are presumed to develop from it appear to be invariably peripheral and always of low grade. The disease runs a chronic, indolent course, and the emergence from DIPNECH of PNTs in the central airways or of higher grades of malignancy has not been described. It is probable, therefore, that DIPNECH is a precursor exclusively of typical carcinoid tumors in a peripheral location. In this regard, it may be related to the more localized and less florid proliferation of PNCs that are commonly seen in the vicinity of such tumors. A role for the condition as a precursor to the predominantly central, high-grade PNTs (ie, the large cell and small cell neuroendocrine carcinomas) seems unlikely, however, any precursor to these more common and dangerous tumors remains unknown.
Reference 1 Aguayo SM, Miller YE, Waldron JA, et al. Brief report: idiopathic diffuse hyperplasia of pulmonary neuroendocrine cells and airways disease. N Engl J Med 1992; 327:1285–1288
Diffuse Idiopathic Pulmonary Neuroendocrine Cell Hyperplasia as a Precursor to Pulmonary Neuroendocrine Tumors* John R. Gosney, BSc, MD
(CHEST 2004; 125:108S) Abbreviations: DIPNECH ⫽ diffuse idiopathic PNC hyperplasia; PNC ⫽ pulmonary neuroendocrine cell; PNT ⫽ pulmonary neuroendocrine tumor 108S
*From the Royal Liverpool University Hospital, Liverpool, UK. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: [email protected]). Correspondence to: J.R. Gosney, BSc, MD, Department of Pathology, 5th floor, Duncan Bldg, Royal Liverpool University Hospital, Daulby St, Liverpool L69 3GA, UK; e-mail: j.gosney@ rlbuh-tr.nwest.nhs.uk
Thomas L. Petty 46th Annual Aspen Lung Conference; Lung Cancer: Early Events, Early Interventions
Selective Suppression of Dendritic Cell Functions by Cigarette Smoke Extract* Robert Vassallo, MD, FCCP; and Lieping Chen, MD, PhD
(CHEST 2004; 125:107S) Abbreviation: CSE ⫽ cigarette smoke extract
cells are potent antigen-presenting cells and D endritic critical regulators of T-cell responses. We postulated
that an important mechanism by which smoking predisposes a person to cancer is by suppressing dendritic cell function, thus inhibiting the ability of dendritic cells to stimulate appropriate T-cell responses. To test this hypothesis, human monocyte-derived dendritic cells were incubated with freshly prepared cigarette smoke extract (CSE) [0 to 2%; mean (⫾ SD) nicotine content in 1% CSE, 2.3 ⫾ 0.8 g/mL] for the final 18 to 24 h of culture. Dendritic cells were then washed to remove all residual CSE and were incubated with allogenic purified T cells in a mixed lymphocyte reaction for 72 h. T-cell proliferation was measured by the incorporation of tritiated thymidine *From the Mayo Clinic, Rochester, MN. This research was supported by an American Lung Association research grant, a Parker B. Francis fellowship (RV), and National Institutes of Health grants CA79915 and CA85721 (LC). Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: [email protected]). Correspondence to: Robert Vassallo, MD, FCCP, Assistant Professor of Medicine, Division of Pulmonary and Critical Care Medicine, Mayo Clinic, Stabile Bldg 8 –54, Rochester, MN 55905; e-mail: [email protected] www.chestjournal.org
and cytokine levels into the mixed lymphocyte reaction measured by enzyme-linked immunosorbent assay. The transient incubation of immature dendritic cells with CSE at doses that did not affect viability (as measured by the XTT assay, trypan blue exclusion, and annexin V/propidium iodide staining) resulted in significant suppression of dendritic cell-induced T-cell proliferation. This decrease in T-cell proliferation was accompanied by reduced interferon-␥ production, but not in interleukin-4 or interleukin-10 secretion. Dendritic cell surface expression of major histocompatibility complex class II, major histocompatibility complex class I, CD-40, CD-80, CD-86, CD-83, and CD-1a was not altered by transient exposure (ie, for 24 h) to CSE at the same concentrations that diminished dendritic cell-induced T-cell proliferation. These data indicate that transient exposure to CSE causes the selective suppression of dendritic cell functions in the induction of T-cell proliferation and the production of interferon-␥.
Buccal-Lung Comparison of Quantitative Expression of Carcinogen and Oxidant Metabolism Genes in Human Subjects* R.J. Jain, PhD; S. Varma, MS; G.J. Hurteau, BS; and Simon D. Spivack, MD, MPH, FCCP
(CHEST 2004; 125:107S–108S) human lung is inaccessible for direct examination. T heBuccal cells lining the inner cheek of tobacco smokers
are carcinogen- and oxidant-exposed, easily accessible, and potentially valuable surrogates for lung tissue. Lung cancer susceptibility phenotyping might include carcinogenmetabolism and oxidant-metabolism gene expression assayed in buccal cells.
Materials and Methods For buccal cell analysis, we adapted known techniques of RNA preservation and extraction. We have employed our novel RNAspecific reverse transcription polymerase chain reaction strategy that is unconfounded by genomic DNA pseudogene sequence encoding target and reference “housekeeper” sequences. All primers were blasted against bacterial sequences to assure human *From the Wadsworth Center, New York State Department of Health, State University of New York School of Public Health, and Albany Medical College, Pulmonary and Critical Care Medicine, Albany, NY. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: [email protected]). Correspondence to: Simon Spivack, MD, MPH, FCCP, Human Toxicology & Molecular Epidemiology, Wadsworth Center, Biggs E622, New York State Department of Health, SUNY School of Public Health, Empire State Plaza, PO Box 509, Albany, NY 12201-0509; e-mail: [email protected] CHEST / 125 / 5 / MAY, 2004 SUPPLEMENT
107S
cell specificity of the amplification. Precise real-time quantitation of the expression of these genes employs ratios of target crossover point to reference housekeeper CRO (LightCycler; Roche; Basel, Switzerland). Genes that were analyzed included the aromatic hydrocarbon receptor gene (Ahr), cytochrome P450 genes CYP1A1 and CYP1B1, glutathione-S-transferase genes GSTM1, GSTM3, GSTP1, and GSTT1, nicotinamide adenine dinucleotide phosphate-quinone oxidoreductase (NQO1), copper-zinc superoxide dismutase (CuZn-SOD), catalase (CAT), and glutathione peroxidase (GPx). For any given individual among our initial group of human subjects, we have been able to reliably quantitate gene expression in the buccal cytologic specimens. We have compared buccal gene expression to laser capture microdissected lung gene expression, both by identical techniques of real-time quantitative reverse transcription polymerase chain reaction.
Results We have found close concordance of phase I, phase II, and antioxidant genes in constitutive expression (not expressed, CYP1A1, GSTM1, and GSTM3; all expressed, GSTP1, GSTT1; NQO1, SOD, CAT, and GPx) between the two tissues. There is often buccal-lung discordance for the Ahr gene. However, for all 11 transcripts studied in our first 11 individuals to date, 88 to 96% of the genespecific buccal-lung expression comparisons have shown qualitatively concordant expression (2 ⫽ 62.4; p ⬍ 0.001). The quantitative covariation between the two tissues is under study.
Conclusion We have in preliminary fashion shown that (1) gene expression is quantifiable in cytologically collected buccal cells, and (2) there appears to be good concordance between those carcinogen and oxidant metabolism genes expressed in the buccal mucosa and those expressed in the laser microdissected human lung epithelium. We have enlarged the group undergoing buccal-lung comparison, and have commenced stratification of the expression data for covariation with tobacco exposure, to define each individual’s unique gene expression signature in this cancer-relevant pathway.
1992, Aguayo et al described six patients with slowly I nprogressive cough and breathlessness, obstructive and/ 1
or restrictive respiratory impairment, and diffuse reticulonodular infiltrates seen on thoracic tomographic scanning, in whom a pulmonary biopsy revealed widespread intraluminal and extraluminal proliferation of pulmonary neuroendocrine cells (PNCs) in all patients, accompanied by peripheral carcinoid tumors in three patients. There was submucosal fibrosis of affected airways, but none of the conditions usually associated with the reactive proliferation of PNCs was evident. This and subsequent studies have further defined the clinical and pathologic parameters of this rare condition, now conventionally known as diffuse idiopathic PNC hyperplasia or DIPNECH. DIPNECH raises interesting questions about the relationship between the proliferation of PNCs, hitherto considered a reactive process, and the genesis of bona fide pulmonary neuroendocrine tumors (PNTs), such that it now appears as a preneoplastic condition in the latest World Health Organization histologic classification of tumors of the lung and pleura. However, the carcinoid tumors that accompany DIPNECH and are presumed to develop from it appear to be invariably peripheral and always of low grade. The disease runs a chronic, indolent course, and the emergence from DIPNECH of PNTs in the central airways or of higher grades of malignancy has not been described. It is probable, therefore, that DIPNECH is a precursor exclusively of typical carcinoid tumors in a peripheral location. In this regard, it may be related to the more localized and less florid proliferation of PNCs that are commonly seen in the vicinity of such tumors. A role for the condition as a precursor to the predominantly central, high-grade PNTs (ie, the large cell and small cell neuroendocrine carcinomas) seems unlikely, however, any precursor to these more common and dangerous tumors remains unknown.
Reference 1 Aguayo SM, Miller YE, Waldron JA, et al. Brief report: idiopathic diffuse hyperplasia of pulmonary neuroendocrine cells and airways disease. N Engl J Med 1992; 327:1285–1288
Diffuse Idiopathic Pulmonary Neuroendocrine Cell Hyperplasia as a Precursor to Pulmonary Neuroendocrine Tumors* John R. Gosney, BSc, MD
(CHEST 2004; 125:108S) Abbreviations: DIPNECH ⫽ diffuse idiopathic PNC hyperplasia; PNC ⫽ pulmonary neuroendocrine cell; PNT ⫽ pulmonary neuroendocrine tumor 108S
*From the Royal Liverpool University Hospital, Liverpool, UK. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: [email protected]). Correspondence to: J.R. Gosney, BSc, MD, Department of Pathology, 5th floor, Duncan Bldg, Royal Liverpool University Hospital, Daulby St, Liverpool L69 3GA, UK; e-mail: j.gosney@ rlbuh-tr.nwest.nhs.uk
Thomas L. Petty 46th Annual Aspen Lung Conference; Lung Cancer: Early Events, Early Interventions