Lung Tissue Culture Research Articles

Sarcoidosis Presenting With Cystic Lung Lesions and Autoimmune Hemolytic Anemia

Introduction Hemolytic anemia is an unusual finding in sarcoidosis. We report an intriguing case of hemolytic anemia in cystic sarcoidosis. Case Presentation A 34-year-old African American male with bronchial asthma presented with wheezing, exertional dyspnea, cough, fever, night sweats, weight loss, and fatigue. Physical examination revealed pallor, palpable peripheral adenopathy, and expiratory rhonchi. Initial laboratory findings were remarkable for leukopenia, anemia, reticulocytosis, elevated lactate dehydrogenase, and abnormal liver function test. Serum haptoglobin was low. Sickling test was negative. Direct Coombs test was positive, and cold agglutinins were demonstrated. Chest radiograph revealed cystic lesions in both upper lung fields, and chest computed tomographic scan confirmed these lesions. Also seen were mediastinal adenopathy and hepatosplenomegaly. Tuberculin skin test was negative in 48 hours. Three sputum smears for acid-fast bacilli were negative. Serum angiotensin converting enzyme level was elevated. He underwent diagnostic bronchoscopy with carinal and transbronchial lung biopsies revealing non-necrotizing granulomatous inflammation consistent with sarcoidosis. Acid-fast bacilli and silver stains were negative. Respiratory and lung tissue cultures were negative for fungi and Mycobacteria. Diagnosis of sarcoidosis with autoimmune hemolytic anemia was established and systemic corticosteroid was instituted with resolution of hemolysis and modest roentgenographic regression of the cystic lung lesions. Conclusions Some cases of cystic/bullous sarcoidosis had been reported and the occurrence of autoimmune disease with sarcoidosis well known. To the best of our knowledge, this is the first reported case of sarcoidosis presenting with cystic lung lesions and autoimmune hemolytic anemia in a single patient.

Prevalidation of in vitro continuous flow exposure systems as alternatives to in vivo inhalation safety evaluation experimentations: Outcome from MAAPHRI-PCRD5 research program

Diesel engine emission aerosol-induced toxicity patterns were compared using both in vitro (organotypic cultures of lung tissue) and in vivo experimentations mimicking the inhalation situation with continuous aerosol flow exposure designs. Using liquid media resuspended diesel particles, we show that toxic response pattern is influenced by the presence of tensioactive agent in the medium which alter particle-borne pollutant bioavailability. Using continuous aerosol exposure in vitro, we show that with high sulfur fuel (300 ppm) in the absence of oxidation catalysis, particulate matter was the main toxic component triggering DNA damage and systemic inflammation, while a very limited oxidant stress was evidenced. In contrast, with ultra-low sulfur fuel in the presence of strong diesel oxidation catalysis, the specific role of particulate matter is no longer evidenced and the gas phase then becomes the major component triggering strong oxidant stress, increased NO 2 being the most probable trigger. In vivo, plasma tumor necrosis factor alpha (TNFalpha), lung superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) activity levels varied in agreement with in vitro observations. Diesel emission treatment with oxycat provokes a marked systemic oxidant stress. Again NO 2 proved to account for a major part of these impacts. In conclusion, similar anti-oxidant responses were observed in in vitro and in vivo experiments after diesel emission aerosol continuous flow exposures. The lung slice organotypic culture model-exposed complex aerosol appears to be a very valuable alternative to in vivo inhalation toxicology experimentations in rodents.

Pneumococcal Necrotizing Pneumonia in Utah: Does Serotype Matter?

Streptococcus pneumoniae is the most common cause of bacterial pneumonia in children. Despite the use of the 7-valent pneumococcal conjugate vaccine, the incidence of pneumococcal necrotizing pneumonia (PNP) has been increasing. Our objectives were to describe temporal trends in PNP and to evaluate pneumococcal serotypes associated with PNP in Utah. We performed a retrospective review of all children <18 years of age who were cared for at a tertiary care children's hospital and who had blood, lung tissue, broncheoalveolar lavage, or pleural fluid cultures that grew S. pneumoniae, as well as radiographic evidence of pneumonia, from January 1997 through March 2006. All S. pneumoniae isolates were typed. A total of 124 children with pneumococcal pneumonia were identified, and 33 (27%) of these children had radiographic evidence of PNP. During the period 1997-2000, 5 (13%) of 39 cases of culture-confirmed pneumococcal pneumonia were associated with PNP. In contrast, during the period 2001-2006, 28 (33%) of 85 pneumococcal pneumonia cases were complicated by PNP (odds ratio, 3.34; 95% confidence interval, 1.11-12.03). Non-7-valent pneumococcal conjugate vaccine serotypes comprised 49% of the isolates during 1997-2000 and 88% of isolates during 2001-2006 (odds ratio, 7.89; 95% confidence interval, 2.91-21.90). Pneumonia due to serotype 3 was most often associated with PNP. Eleven (79%) of 14 cases of serotype 3-associated pneumonia were associated with PNP. When compared with all other serotypes, serotype 3 was strongly associated with necrosis (odds ratio, 14.67; 95% confidence interval, 3.39-86.25). PNP is a serious and increasingly common complication of S. pneumoniae pneumonia in Utah. Infection with serotype 3 is associated with an increased risk of developing PNP. The increase in the incidence of infection due to nonvaccine serotypes reported worldwide and the changing epidemiology of invasive pneumococcal disease should be considered when developing vaccine strategies.

Down-regulation of Caveolin-1, an Inhibitor of Transforming Growth Factor-β Signaling, in Acute Allergen-induced Airway Remodeling

Asthma can progress to subepithelial airway fibrosis, mediated in large part by transforming growth factor-beta (TGF-beta). The scaffolding protein caveolin-1 (cav1) can inhibit the activity of TGF-beta, perhaps by forming membrane invaginations that enfold TGF-beta receptors. The study goals were 1) to evaluate how allergen challenge affects lung expression of cav1 and the density of caveolae in vivo 2) to determine whether reduced cav1 expression is mediated by interleukin (IL)-4 and 3) to measure the effects of decreased expression of cav1 on TGF-beta signaling. C57BL/6J, IL-4-deficient mice, and cav1-deficient mice, sensitized by intraperitoneal injections of phosphate-buffered saline or ovalbumin (OVA) at days 0 and 12, received intranasal phosphate-buffered saline or OVA challenges at days 24, 26, and 28. Additionally, another group of C57BL/6J mice received IL-4 by intratracheal instillation for 7 days. We confirmed that the OVA-allergen challenge increased eosinophilia and T-helper type 2-related cytokine levels (IL-4, IL-5, and IL-13) in bronchoalveolar lavage. Allergen challenge reduced lung cav1 mRNA abundance by 40%, cav1 protein by 30%, and the number of lung fibroblast caveolae by 50%. Administration of IL-4 in vivo also substantially decreased cav1 expression. In contrast, the allergen challenge did not decrease cav1 expression in IL-4-deficient mice. The reduced expression of cav1 was associated with activation of TGF-beta signaling that was further enhanced in OVA-sensitized and challenged cav1-deficient mice. This study demonstrates a previously unknown modulation of TGF-beta signaling by IL-4, via cav1, suggesting novel therapeutic targets for controlling the effects of TGF-beta and thereby ameliorating pathological airway remodeling.

Nocardiose pulmonaire compliquée d’ostéomyélite

We report a case of a 49-year-old female who developed a bronchopneumonia associated with atelectasis of the upper right lobe and back pain of bone origin. Bronchoscopy revealed an endobronchial mass at the origin of the right upper lobe bronchus. Scintigraphy showed three paravertebral spots of the seventh and eighth thoracic vertebrae, without any radiological modification. Culture of lung tissue obtained by transparietal punction under CT scan control became positive to Nocardia belonging to the pneumoniae complex. Positive diagnosis of pulmonary nocardiosis associated with two rare localisations was set, one was an endobronchial mass, the other was osteomyelitis of the posterior chest wall. The patient was treated with Trimethoprim–Sulfamethoxazole and recovered completely.

Mouse Strains Differ in Their Susceptibility to Poststroke Infections

Objective: Severe infections, in particular pneumonia, have a major impact on the clinical management and outcome of stroke patients. In a mouse model we have recently demonstrated that stroke induces immunodepression which can result in life-threatening infections. Here, we investigated whether the susceptibility to infections after stroke is strain dependent. Methods and Results: Mice from 129SV, C57/B6, and Balb/C strains were subjected to experimental stroke by filament occlusion of the middle cerebral artery (MCAO) for 60 min. Infarct volumes were measured 3 days after MCAO. Microbiological assessment was based on cultures of bronchoalveolar lavage (BAL), lung tissue and blood of animals obtained 3 days after stroke. Three days after stroke 129SV mice did not only develop bacterial chest infection, but also had a strongly increased susceptibility to bacteremia. In contrast, C57BL/6 and Balb/C mice acquired bacterial lung infections only. In addition, bacterial load in BAL was significantly higher in 129SV mice than in the other mice strains. These differences in susceptibility to infection did not correlate with infarct volumes. Conclusions: Stroke-associated pneumonia developed in three commonly used mouse strains while severity of infections differed between strains. Since infections affect outcome, monitoring of infections is highly relevant for the interpretation of results in experimental stroke research.

Upregulation of substance P receptor expression by calcitonin gene-related peptide, a possible cooperative action of two neuropeptides involved in airway inflammation

Airway hyperresponsiveness (AHR) is present in almost all patients with symptomatic asthma, yet its mechanism is not well understood. Airway inflammation is thought to be an important underlying mechanism involved in causing AHR. Recent studies indicate release of neuropeptides from C-fiber endings plays a pivotal role in airway inflammation. Substance P (SP) is a critical neurotransmitter of sensory C-fiber and a well-known effector of inflammatory response. However, roles of other neuropeptides and interaction among these neuropeptides in airway inflammation and AHR were largely unknown. Calcitonin gene-related peptide (CGRP), another intrapulmonary neuropeptide that functions as a potent vasodilator and neutrophils activator, is released from the same C-fiber ending as SP is released. By using an ozone-stressing animal model, previously we had demonstrated that CGRP might be involved in the development of AHR in rabbits. To extend the functional roles of SP, and to explore the possible interactive roles of SP and CGRP in airway inflammation, we examined expressions of SP, SP receptor (neurokinin 1, or NK-1R) and CGRP in vivo and ex vivo. We exposed guinea pigs at intervals to inhalation of ozone to induce airway inflammation. Animals were sacrificed at different time points; SP, SP receptor and CGRP expression were determined during the onset and progression of airway inflammation by radioimmunoassay, immunohistochemistry and in situ hybridization. Our data showed that after exposure to ozone, the concentration of SP in lung homogenate and the number of SP-immunoreactive cell bodies in lung slides increased within 24 h, peaked on day 2, and then decreased slowly. Interestingly, CGRP expressions exhibited a similar temporal and spatial pattern, and there was a strong correlation between SP expression and CGRP expression, indicating a possible cooperative action of these two neuropeptides. We also noted an increased expression of SP receptor NK-1R in the development of airway inflammation. In order to test the hypothesis that CGRP as a coexisting neurotransmitter with SP can regulate the expression of NK-1R in the lung, and contribute to the SP-mediated inflammatory response, we used in vitro lung tissue culture to determine the effect of CGRP on NK-1R expression. We found that NK-1R expression was induced by CGRP incubation at both mRNA and protein levels, and the induction was attenuated by additions of the inhibitors of Protein Kinase A (PKA) pathway, Calmodulin-dependent Kinase pathway, and Tyrosine Protein Kinase pathway. In conclusion, our data provide compelling evidence that SP and CGRP are involved in the development of airway inflammation. The interaction between SP and CGRP is likely to contribute to the pathogenesis of AHR and other lung inflammatory diseases.

Oxidative response of rat lung tissue after crown ethers exposure and the effects of alpha-tocopherol treatment

Background Ionophoric properties of crown ethers enable them ideally to transport across membranes and interfere with different living systems. We studied cytotoxicity effects of 18-crown-6 and 15-crown-5 and the role of oxidative stress in rat lung tissue culture. Methods We assayed the effects of these crown ethers in a range of doses (0.1 to 2 mmol/l) on lipids and proteins oxidative damages end products; malondialdehyde (MDA) and dityrosine and on the activity of antioxidant enzymes; superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT), in rat lung tissue culture by spectrometry and HPLC. Results Both compounds significantly increased the levels of MDA, dityrosine and enzyme activities at doses > 0.5 mmol/l after 48-h incubation in the lung tissue, representing promotion of ROS generation with respect to control. These effects were more considerable for 18-crown-6 than 15-crown-5. Treatment of lung tissue with 30 μm of α-tocopherol in addition to 2 mmol/l of crown ethers showed significant decrease on the levels of enzyme activities, MDA and dityrosine. Conclusion We showed the oxidative radicals forming ability of crown ethers as documented in their toxicity effects through lipid and proteins oxidation damages.

Feasibility of assessment of regulatory lipids in breath condensate as potential presymptomatic harbingers of pulmonary pathobiology

Regulatory lipids from the airway surface readily form aerosols that can be recovered non-invasively by cooling expired breath to form breath condensate (BC). Regulatory lipids have been detected previously utilizing enzyme-linked-immunosorbent serologic assay (ELISA). Here we test the feasibility of assessment of regulatory lipids in BC by mass spectrometry so presently unknown lipid regulatory components can be detected without addition of specific antibodies as in the ELISA procedure. Baseline regulatory lipids were detected in > pg/mL BC in control animals or human lung tissue culture cells. In nearly every case animals exposed to toxins or infectious bacteria showed increases in the BC regulatory components. Lipids were recovered from BC by solid phase extraction. Phosphatidylcholine (PC) based lipids were detected as the progenitor (parent) ions of isomers that fragmented in producing product positive ions at m/ z 184 (of phosphocholine) in tandem MS using capillary HPLC and electrospray ionization. BC eicosanoids such as prostaglandins, thromboxane, and isoprostanes require capillary gas chromatography for separation and detection that necessitates methoximation, pentafluorobenzyl (PFB) ester formation, and trimethyl silylation of hydroxyls prior to gas chromatography/ion trap tandem mass spectrometry of negative ions after chemical ionization (NICI). Tetradeuterated internal standards were utilized for quantitation with the GC/NICI/MS. Changes in concentrations of lipids and eicosanoids were observed in piglets, and rats exposed to aerosolized 100 μg/kg lipopolysaccharide (LPS), or 50 μg/kg and 150 μg/kg aerosolized Staphylococcal enterotoxin B (SEB) in BC as well as in human THP-1 cell culture cell supernatants and bronchoalveolar lavage (BAL) samples in rats. Responses of the molecular species of phosphatidylcholines (PCs), platelet activating factors (PAFs) and specific eicosanoids correlated to the toxin and bacterial infections suggesting that patterns of differential responses could be detected with further experimentation. Initial targets included prostaglandins (PGE 2, PGF 2α), thromboxane (TXB 2), and prostacyclin (as 6-Keto PGF 1α) that show differential responses to inflammation, the leukotriene (LTB 4) and PGD 2 for allergic responses, isoprostanes (8- iso-PGF 2α) for free radical oxidative stress responses, and HETEs for differential lipoxygenase activities. PAFs and lysoPAFs have been shown to increase with inflammation and in the feasibility experiments reported here. Preliminary studies show pulmonary responses of piglets to intrathecal exposure of toxicants (LPS and SEB) or infections with Actinobacillus pleuropneumoniae induce increased levels of lipids and two eicosanoids with the suggestion that differential patterns might be detected with expanded testing. Preliminary experience indicates numerous other eicosanoids were available for assay in BC. This suggests an important potential application of BC to observe a wide array of factors to establish comprehensive profiles for physiological and pathophysiological states. Ultimately this technique could be used as a non-invasive possibly presymptomatic assessment of pulmonary pathobiology.

Preventive antibacterial treatment improves the general medical and neurological outcome in a mouse model of stroke.

Epidemiological studies have demonstrated a high incidence of infections after severe stroke and their prominent role in morbidity and mortality in stroke patients. In a mouse model, it has been shown recently that stroke is coupled with severe and long-lasting immunosuppression, which is responsible for the development of spontaneous systemic infections. Here, we investigated in the same model the effects of preventive antibiotic treatment on survival and functional outcome of experimental stroke. Mice were subjected to experimental stroke by occlusion of the middle cerebral artery (MCAO) for 60 minutes. A group of mice received moxifloxacin (6x100 mg/kg body weight every 2 hours over 12 hours) either immediately or 12 hours after MCAO. Control animals received the vector only. Behavior, neurological deficit, fever, survival, and body weight were monitored over 14 days. In a subgroup, infarct volume was measured 4 days after MCAO. Microbiological assessment was based on cultures of lung tissue, blood, and feces of animals 3 days after stroke. For a dose-response study, moxifloxacin was given immediately after MCAO in different doses and at different time points. Microbiological analyses of blood and lung tissue demonstrated high bacterial burden, mainly Escherichia coli, 3 days after stroke. Accordingly, we observed clinical and histological signs of septicemia and pneumonia. Moxifloxacin prevented the development of infections and fever, significantly reduced mortality, and improved neurological outcome. Preventive antibiotic treatment may be an important new therapeutical approach to improve outcome in patients with severe stroke.

Therapeutic effect of intravenous delivery of lipoplexes containing the interferon-beta gene and poly I: poly C in a murine lung metastasis model.

We have evaluated and compared the efficacy of systemic administration of lipoplex formulations containing plasmids encoding IFN-beta or IFN-gamma, and a synthetic double-strand RNA poly I:poly C (pI:pC), a type I IFN inducer, in a lung metastasis model in which colon carcinoma CT-26 cells were inoculated intravenously into immunocompatible mice. Injection of lipoplexes containing plasmid DNA, regardless of IFN gene insertion, stimulated a transient increase in the serum concentration of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and IFN-gamma, while injection of lipoplexes containing pI:pC led to a low level of TNF-alpha and undetectable IFN-gamma production. Furthermore, injection of these lipoplexes containing plasmids resulted in the production of a mixture of type I and type II IFNs, partly derived from the inserted IFN genes, in lung tissue cultures. In tumor-prophylactic experiments, intravenous injection of lipoplexes containing plasmid, regardless of IFN gene insertion, showed a significant reduction in lung metastatic nodules probably due to proinflammatory cytokines such as TNF-alpha and IFN-gamma nonspecifically induced by the CpG motifs in the plasmid and the type I IFNs produced. On the other hand, the antimetastatic effect of pI:pC-lipoplex seemed to be due mainly to IFN-beta induced by pI:pC. In established lung metastasis experiments, a single intravenous administration of lipoplexes containing IFN-beta gene or pI:pC, but not other lipoplexes, showed a significant therapeutic effect on the tumor metastasis: reduction in tumor nodules and prolongation of survival time of tumor-burden mice. The therapeutic effects were specifically impaired by anti-IFN-beta antibody treatment, indicating that IFN-beta produced by the lipoplexes played an important role in the suppression of established metastatic lung tumors. Thus, the local IFN-beta in the lung delivered by intravenous administration of lipoplex containing IFN-beta gene or pI:pC may be a convenient and useful method of inhibiting established metastatic lung tumors.

Isoflavones protect against diesel engine exhaust injury in organotypic culture of lung tissue

The wide range of potential health beneficial effects of isoflavones, including a chemoprentive action, have prompted us to study the potential benefits of genistein and daidzein in an experimental model of environmental pollution impact on lung tissue. A diesel engine placed was used to generate reproducible emissions including both gaseous and particulate matters that are commonly found in urban atmospheres. Isoflavones were added to culture medium of rat lung slices 2 h prior to their exposure to pollutants for 3 h. Intracellular ATP and GSH levels, TNFα production, nucleosome assay and TUNEL labeling were monitored. Isoflavones showed almost total in vitro protection against inflammatory and pro-apoptotic responses in lung slices. Isoflavones 0.3 and 1 μmol/l protected against exhaust induced GSH depletion. Isoflavones 0.3 μmol/l appeared to exert the most beneficial effects. In conclusion, this study points out the potential interest of soy isoflavones consumption in polluted areas. Further studies should be undertaken to verify that similar effects could be obtained after in vivo administration of isoflavones.

Experimental Chlamydia pneumoniae infection in NIH/S mice: effect of reinoculation with chlamydial or cell preparation on culture, PCR and histological findings of lung tissue

The cellular components present in chlamydial preparations may contribute to the course of the experimental infection. NIH/S mice were inoculated and reinoculated intranasally with Chlamydia pneumoniae or a cellular preparation. The mock inoculation induced only mild histological changes in the lungs, which possibly induced partial protection against subsequent C. pneumoniae infection and, when given as reinoculation, possibly reactivated the culture-negative infection as culture-positive. In addition, serum antibodies against mouse heat shock protein 60 (Hsp60) were found in a few mice. In conclusion, the main immunopathogenic factors in a C. pneumoniae mouse model are chlamydial components. However, a cellular preparation may participate in an inflammatory reaction. Autoimmunity against Hsp60 may also play a role in the pathogenesis of C. pneumoniae infection.

Lung pathology in premature infants with Ureaplasma urealyticum infection.

Respiratory tract colonization with Ureaplasma urealyticum in preterm infants has been associated with a higher incidence of pneumonia, severe respiratory failure, bronchopulmonary dysplasia (BPD), and death. In this report, we characterize the lung pathology and expression of tumor necrosis factor-alpha (TNF-alpha) associated with U. urealyticum pneumonia in very low-birth weight infants (VLBW; < or =1500 g). Lung pathology of archived autopsy specimens was retrospectively reviewed in three groups of VLBW infants: 5 gestational controls who died from nonpulmonary causes, 13 infants with pneumonia who were culture and/or PCR negative for U. urealyticum, and 5 infants with pulmonary disease and positive for U. urealyticum by tracheal aspirate and/or lung tissue culture or polymerase chain reaction (PCR). Presence and extent of alveolar macrophages and neutrophils, as well as interstitial lymphocytic infiltration and fibrosis were evaluated. PCR was performed on formalin-fixed, paraffin-embedded lung sections. Additional sections were immunostained for TNF-alpha. The peripheral total white blood cell counts and absolute neutrophil counts were three-fold higher in infants with U. urealyticum pneumonia than cell counts in infants infected with other organisms. There was a trend toward a predominance of neutrophils in alveoli of non- Ureaplasma pneumonia infants, but a trend toward a predominance of alveolar macrophages in U. urealyticum-infected infants. The most striking finding was the presence of increased interstitial fibrosis in all Ureaplasma-infected infants. TNF-alpha immunoreactive cell density was very low in the gestational controls, but increased in both pneumonia groups. We conclude that persistent lung U. urealyticum infection may contribute to chronic inflammation and early fibrosis in the preterm lung.

Lung Pathology in Premature Infants withUreaplasma urealyticumInfection

Respiratory tract colonization with Ureaplasma urealyticum in preterm infants has been associated with a higher incidence of pneumonia, severe respiratory failure, bronchopulmonary dysplasia (BPD), and death. In this report, we characterize the lung pathology and expression of tumor necrosis factor-α (TNF-α) associated with U. urealyticum pneumonia in very low–birth weight infants (VLBW; ≤1500 g). Lung pathology of archived autopsy specimens was retrospectively reviewed in three groups of VLBW infants: 5 gestational controls who died from nonpulmonary causes, 13 infants with pneumonia who were culture and/or PCR negative for U. urealyticum, and 5 infants with pulmonary disease and positive for U. urealyticum by tracheal aspirate and/or lung tissue culture or polymerase chain reaction (PCR). Presence and extent of alveolar macrophages and neutrophils, as well as interstitial lymphocytic infiltration and fibrosis were evaluated. PCR was performed on formalin-fixed, paraffin-embedded lung sections. Additional sections were immunostained for TNF-α. The peripheral total white blood cell counts and absolute neutrophil counts were three-fold higher in infants with U. urealyticum pneumonia than cell counts in infants infected with other organisms. There was a trend toward a predominance of neutrophils in alveoli of non- Ureaplasma pneumonia infants, but a trend toward a predominance of alveolar macrophages in U. urealyticum–infected infants. The most striking finding was the presence of increased interstitial fibrosis in all Ureaplasma-inf ected infants. TNF-α immunoreactive cell density was very low in the gestational controls, but increased in both pneumonia groups. We conclude that persistent lung U. urealyticum infection may contribute to chronic inflammation and early fibrosis in the preterm lung.

Telomerase in alveolar epithelial development and repair.

Telomerase expression and activity were examined in the developing lung and in the adult lung during repair after injury. Both whole lung tissue and primary cultures of type 2 alveolar epithelial cells (AEC2) isolated from fetal and adult rodents were analyzed for 1) telomerase expression by immunohistochemistry and 2) telomerase activity with a telomerase repeat amplification protocol. We found that telomerase was expressed in a temporally regulated manner in fetal lung through the late stages of gestation, with peak expression just before birth. Expression persisted for a brief period in neonates, then decreased to nearly undetectable levels by postnatal day 9. Telomerase expression and activity were reinduced in normally quiescent adult lung by in vivo treatment with hyperoxia. In populations of AEC2 isolated from both developing and repairing lungs, telomerase expression and activity showed a strong correlation with the proliferation marker proliferating cell nuclear antigen. It has been suggested that telomerase expression and activity are hallmarks of stem or progenitor cells. Our observations suggest that a telomerase-positive subpopulation is present within the general AEC2 population. Telomerase may act as a marker for the proliferative status of this subpopulation.

Oncogenic ras results in increased cell kill due to defective thermoprotection in lung cancer cells

Background. The survival response of normal cells to heat stress is an upregulation of heat shock proteins and ras protein activation. We hypothesized that in lung cancer cells the presence of oncogenic ras interferes with thermoprotective mechanisms resulting in cell death. Methods. An equal number of lung tissue culture cells (normal and cancerous) were subjected to either heat stress and then recovery (43°C for 180 minutes, 37°C for 180 minutes) or recovery alone (37°C for 360 minutes). End points were surviving number of cells, cell-death time course, heat shock protein (HSP70, HSC70, HSP27) expression before and after heat stress, and time course for HSP70 expression during heat stress and recovery. Heated cells were compared with unheated control cells, then this difference was compared between cell types. Results. Heat stress in normal cells caused an 8% decrease in cell number versus a 78% ± 5% decrease in cancer cells ( p < 0.05). In normal cells, heat stress caused a 4.4-fold increase in HSP70, no change in HSC70, and a 1.7-fold increase in HSP27. In contrast, cancer cells initially contained significantly less HSP70 ( p < 0.05), and there was a 27-fold increase in HSP70 and a 2-fold increase in HSC70 with no HSP27 detected (comparison significant, p < 0.05). HSP70 time course in normal cells showed that HSP70 increased 100-fold, reaching a vertex at 2 hours and remaining elevated for 24 hours; in cancer cells, HSP70 maximum expression (100-fold) peaked at 5 hours, then decreased to slightly elevated at 24 hours. Conclusions. Cancer cells with oncogenic ras have defective thermoprotective mechanism(s) causing increased in vitro cell death, which provides an opportunity for thermal treatment of lung cancer.

Blinded Invasive Diagnostic Procedures in Ventilator-Associated Pneumonia

Blinded Invasive Diagnostic Procedures in Ventilator-Associated Pneumonia

Identification of a novel NOTCH-4/INT-3 RNA species encoding an activated gene product in certain human tumor cell lines.

Ectopic expression of the intracellular domain of NOTCH-4/INT-3 leads to tumorigenesis in the mouse mammary gland. This results from a gain-of-function mutation. To evaluate gain-of-function NOTCH-4/INT-3 activity in human cancers we have surveyed human breast, lung, and colon carcinoma tissue culture cell lines for evidence of increased NOTCH-4/INT-3 RNA expression. High levels of a 1.8 Kb NOTCH-4/INT-3 RNA species are detected in normal human testis but not in other tissues where a 6.5 Kb species is prevalent. Transformed human cancer cell lines express the 1.8 Kb NOTCH-4/INT-3 RNA species. We show that this RNA species encodes a truncated form of the NOTCH-4/INT-3 intracellular domain (ICD). This novel NOTCH-4/INT-3 protein includes the CDC10 repeats and amino acid residues C-terminal to them, but is missing the CBF-1 binding region of the NOTCH-4/INT-3 ICD. This suggests that it has a different mode of action. Furthermore, we show that a transgene which expresses the 1.8 Kb NOTCH-4/INT-3 RNA species in the 'normal' human mammary epithelial cell line MCF-10A enables these cells to grow in soft agar.

P3G176 - In vitro modelizatlon of InhalBtion toxicology: Effects of diesel exhaust on organotypic culture of peripheral lung tissue

P3G176 - In vitro modelizatlon of InhalBtion toxicology: Effects of diesel exhaust on organotypic culture of peripheral lung tissue