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Acute ozone (O 3 ) exposure is associated with multiple adverse cardiorespiratory outcomes, the severity of which varies across individuals in human populations and inbred mouse strains. However, molecular determinants of response, including susceptibility biomarkers that distinguish who will develop severe injury and inflammation, are not well characterized. We and others have demonstrated that airway macrophages (AMs) are an important resident immune cell type that are functionally and transcriptionally responsive to O 3 inhalation. Here, we sought to explore influences of strain, exposure, and strain-by-O 3 exposure interactions on AM gene expression and identify transcriptional correlates of O 3 -induced inflammation and injury across six mouse strains, including five Collaborative Cross (CC) strains. We exposed adult mice of both sexes to filtered air (FA) or 2 ppm O 3 for 3 h and measured inflammatory and injury parameters 21 h later. Mice exposed to O 3 developed airway neutrophilia and lung injury with strain-dependent severity. In AMs, we identified a common core O 3 transcriptional response signature across all strains, as well as a set of genes exhibiting strain-by-O 3 exposure interactions. In particular, a prominent gene expression contrast emerged between a low- (CC017/Unc) and high-responding (CC003/Unc) strain, as reflected by cellular inflammation and injury. Further inspection indicated that differences in their baseline gene expression and chromatin accessibility profiles likely contribute to their divergent post-O 3 exposure transcriptional responses. Together, these results suggest that aspects of O 3 -induced respiratory responses are mediated through altered AM transcriptional signatures and further confirm the importance of gene-environment interactions in mediating differential responsiveness to environmental agents.

The global spread of SARS-CoV-2 has made millions ill with COVID-19 and even more from the economic fallout of this pandemic. Our quest to test new therapeutics and vaccines require small animal models that replicate disease phenotypes seen in COVID-19 cases. Rodent models of SARS-CoV-2 infection thus far have shown mild to moderate pulmonary disease; mortality, if any, has been associated with prominent signs of central nervous system (CNS) infection and dysfunction. Here we describe the isolation of SARS-CoV-2 variants with propensity for either pulmonary or CNS infection. Using a wild-type SARS-CoV-2 isolated from a COVID-19 patient, we first found that infection was lethal in transgenic mice expressing the human angiotensin I-converting enzyme 2 (hACE2). Fortuitously, full genome sequencing of SARS-CoV-2 from the brain and lung of these animals showed genetic differences. Likewise, SARS-CoV-2 isolates from brains and lungs of these also showed differences in plaque morphology. Inoculation of these brain and lung SARS-CoV-2 isolates into new batch of hACE2 mice intra-nasally resulted in lethal CNS and pulmonary infection, respectively. Collectively, our study suggests that genetic variants of SARS-CoV-2 could be used to replicate specific features of COVID-19 for the testing of potential vaccines or therapeutics., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Inhalation exposure to ozone (O 3 ) causes adverse respiratory health effects that result from airway inflammation, a complex response mediated in part by changes to airway cellular transcriptional programs. These programs may be regulated by microRNAs transferred between cells (e.g., epithelial cells and macrophages) via extracellular vesicles (EV miRNA). To explore this, we exposed female C57BL/6J mice to filtered air (FA), 1, or 2 ppm O 3 by inhalation and collected bronchoalveolar lavage fluid (BALF) 21 h later for markers of airway inflammation, EVs, and EV miRNA. Both concentrations of O 3  significantly increased markers of inflammation (neutrophils), injury (total protein), and the number of EV-sized particles in the BALF. Imagestream analysis indicated a substantial portion of particles was positive for canonical EV markers (CD81, CD51), and Siglec-F, a marker of alveolar macrophages. Using high-throughput small RNA sequencing, we identified several differentially expressed (DE) BALF EV miRNAs after 1 ppm (16 DE miRNAs) and 2 ppm (99 DE miRNAs) O 3 versus FA exposure. O 3 concentration-response patterns in EV miRNA expression were apparent, particularly for miR-2137, miR-126-3p, and miR-351-5p. Integrative analysis of EV miRNA expression and airway cellular mRNA expression identified EV miR-22-3p as a candidate regulator of transcriptomic responses to O 3 in airway macrophages. In contrast, we did not identify candidate miRNA regulators of mRNA expression data from conducting airways (predominantly composed of epithelial cells). In summary, our data show that O 3 exposure alters EV release and EV miRNA expression, suggesting that further investigation of EVs may provide insight into their effects on airway macrophage function and other mechanisms of O 3 -induced respiratory inflammation., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Objective: To describe the clinical, laboratory, radiologic, and histopathologic features of methotrexate (MTX)-induced lung injury in a combined cohort of selected patients with rheumatoid arthritis (RA) and all cases reported in the English-language literature., Methods: Retrospective combined cohort review and abstraction from the medical literature. Case reports were obtained from 6 centers that had 4 or more cases of potential MTX lung injury per site. RA patients who were seen between 1981 and 1993 and who satisfied predetermined criteria for the presence of MTX lung injury were identified., Results: Twenty-seven patients satisfied the criteria for definite MTX lung injury, and 2 for probable MTX lung injury. Predominant clinical features of MTX lung injury included shortness of breath in 27 patients (93.1%), which was present for 23.5 +/- 22.3 days (mean +/- SD), cough in 24 (82.8%), present for 26.9 +/- 28.5 days, and fever in 20 (69.0%), present for 10.4 +/- 12.8 days. Five patients (17.2%) died, compared with 12 of 68 (17.6%) reported in the medical literature. Four of the 6 patients who were re-treated with MTX after an initial pulmonary event developed recurrent lung toxicity, resulting in 2 deaths, compared with a recurrence rate of 3 of 6 in the literature., Conclusion: MTX lung injury is most often a subacute process, in which symptoms are commonly present for several weeks before diagnosis. Approximately 50% of the cases are diagnosed within 32 weeks from initiation of MTX treatment. A patient who recovers from MTX lung injury should not be re-treated. Earlier recognition and drug withdrawal may avoid the serious and sometimes fatal outcome that has been observed in this and other studies.

Background: Toxicity limits the use of methotrexate., Objective: To identify risk factors for methotrexate-induced lung injury in patients with rheumatoid arthritis., Design: Case-control study., Setting: One private and five academic rheumatology practices., Participants: Methotrexate recipients with rheumatoid arthritis with and without lung injury., Measurements: Potential risk factors examined were sociodemographic and lifestyle characteristics, medical history, clinical and ancillary features and treatment of rheumatoid arthritis before methotrexate therapy, and characteristics of methotrexate therapy. Cases of lung injury were defined according to the modified criteria of Searles and McKendry., Results: Ninety-four percent of the study participants were white, and 67% were women. Case-patients (n = 29) were older than controls (n = 82) (61.5 compared with 54.5 years of age). The strongest predictors of lung injury, after adjustment for other variables, were older age (odds ratio [OR], 5.1 [95% CI, 1.2 to 21.1]), diabetes (OR, 35.6 [CI, 1.3 to infinity]), rheumatoid pleuropulmonary involvement (OR, 7.1 [CI, 1.1 to 45.4]), previous use of disease-modifying antirheumatic drugs (OR, 5.6 [CI, 1.2 to 27.0]), and hypoalbuminemia (OR, 19.5 [CI, 3.5 to 109.7]). Previous use of disease-modifying antirheumatic drugs and hypoalbuminemia had very large attributable risks., Conclusion: Knowledge of the risk factors that predispose patients with rheumatoid arthritis to the toxic effects of methotrexate on the lung may provide a rationale for monitoring high-risk patients and may facilitate their management.

Donor pretreatment with prostaglandin E1 as a pulmonary vasodilator has developed as a simple, effective means to provide excellent preservation in heart-lung transplantation. This study was undertaken to investigate the degree of ultrastructural preservation of the lung with prostaglandin E1 and other pulmonary vasodilators in a primate heart-lung transplantation model. Heart-lung transplantation was performed in 14 African green monkeys. Donor cardiac preservation was achieved with cold crystalloid cardioplegic solution (10 ml/kg). Lung preservation was achieved with cold, modified Euro-Collins solution delivered into the main pulmonary artery (60 ml/kg total). Vasodilator agents were administered intravenously 15 minutes before aortic crossclamping. The heart-lung grafts were stored at 4 degrees C for 6 hours. Three groups of animals were studied: five donors with prostaglandin E1 (0.1 to 4.0 micrograms/kg per minute), five donors with prostacyclin (0.1 to 0.35 micrograms/kg per minute), and four donors with nitroprusside (0.8 to 5.0 micrograms/kg per minute). After transplantation, arterial blood gas measurements and lung biopsies were performed at 1- and 3-hour intervals. Five formalin blocks per specimen were sectioned for hematoxylin and eosin staining. Cellular preservation and endothelial cell swelling were evaluated with electron microscopy. The specimens were graded for alveolar hemorrhage, endothelial cell swelling, and cellular preservation (grade 0, minimal, to grade 3, severe) and a mean score was obtained for each preservative agent. Prostaglandin E1-treated specimens demonstrated the least amount of endothelial swelling (mean score of 1.0) compared with prostacyclin- and nitroprusside-treated specimens (mean scores of 1.4 and 2.7, respectively). All nitroprusside-treated specimens demonstrated moderate to severe endothelial cell swelling. Interstitial and alveolar hemorrhage was noted in poorly preserved specimens, but there were no significant differences between groups. We conclude that prostaglandin E1 provides improved cellular preservation by decreasing the extent of endothelial cell swelling as observed on electron microscopy.

Protein phosphorylation and protein kinase C (PKC) levels were analyzed in intact cultures of spontaneously transformed, chemically transformed, and untransformed mouse pulmonary epithelial cell lines. It was found that although the transformed cell lines contained about 80% less protein kinase C, measured as total enzyme activity or binding of [3H]phorbol ester, phosphorylation events after phorbol ester treatment could still be easily detected. A commonly described Mr 80-kDa protein kinase C substrate (p80, 80 K, MARKS) was identified using 2D-PAGE, following phosphorylation in intact cells, and found to have reduced availability for phosphorylation in the transformed cell lines C4SE9, C1SA5 and NULB5 in comparison to the untransformed C4E10 and C1C10. Available levels of p80 were further analyzed in heat-denatured extracts from all cell lines using partially purified bovine brain PKC and correlated well with changes seen in intact cells. It was also noted that all transformed cell lines contained large amounts of a family of phosphoproteins of Mr 55-65 kDa, that could not be detected in the untransformed cell lines and whose phosphorylation state was increased by protein kinase C activation. This protein was found to be located in the nucleus. Hence, spontaneously and chemically transformed mouse pulmonary epithelial cells exhibit reduced levels of PKC, along with an altered pattern of PKC-mediated phosphorylation.

Hydrogen peroxide production in organotypic cultures of fetal and newborn rat lung cells has been investigated using an ultrastructural histochemical method, in which the quantity and location of an electron-dense reaction product derived from the interaction of cerium chloride and hydrogen peroxide was detected. Hydrogen peroxide was present in fetal cell cultures exposed to hyperoxia (50% oxygen) compared to controls maintained in 10% oxygen. This increase could be limited by incubation of cultures with ascorbic acid and preincubation with dexamethasone. On the other hand, in newborn rat lung cell cultures, less hydrogen peroxide was identified in cultures including those exposed to hyperoxia (50% oxygen).

Many pathologists and pulmonologists have trained and practiced in an era during which therapeutic options for patients have increased manyfold with regard to pharmacologic agents. Toxicity to the lung may be recognized as data accumulate in the form of case reports and clinical reviews to document the clinical and pathologic manifestations. For the histopathologist, paradoxically the more we learn the more difficult problems of interpretation become. Drug reactions previously described extensively at the autopsy now can be relatively easily interpreted in living patients with tissue obtained by open lung biopsy. This latter procedure is becoming less common with diffuse infiltrative lung disease in favor of the transbronchial biopsy in which sampling error abounds. There may be no disease or minimal change. The pathologic lesions may be duly noted and described, but then defy interpretation. An accurate differential diagnosis following biopsy may not solve the clinical problems for which a tissue diagnosis was sought. A specific diagnosis may be made that does not fit the clinical picture, or the real "answer" may be revealed distressingly later in the clinical-course or, more distressingly, at autopsy. The prudent histopathologist deals with such limitation by objectively stating the observable facets of tissue injury and synthesizing an interpretation. Notwithstanding the burgeoning literature on mechanisms of injury of toxic substances, the "state of the art" in 1990 for interpretation of pulmonary drug reactions still lies with the exacting judgment of experienced clinical physicians. Proper evaluation can result in exclusion of important mimickers of interstitial lung disease, usually infection and neoplasia; accurate categorization of tissue injury and comparison with known injurious responses to the drug in question may lead to a relatively specific diagnosis of drug-induced injury. Most drug reactions in the lung are classified as an interstitial pneumonia. This article illustrates and describes several interstitial reactions to injury that have few histologic features in common. However, because the lung can only react to injury in a limited number of ways, lesions that were thought at one time to be a specific clinicopathologic entity, for example, DIP, now evoke an ever-expanding differential diagnosis. In applying diagnostic criteria emphasis must be placed on the areas of overlap among tissue reactions as well. Specific agents may cause changes that are described as common for that agent, but a given patient may react with a variation or in a distinctly uncommon manner. There is enormous potential in this field for the laboratory investigation of drug injury.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of parenteral vitamin E treatment on aspects of the pulmonary biochemical and morphologic response to 100% oxygen were studied in newborn rabbits manifesting chemical evidence of vitamin E deficiency. Pups treated with 2 mg/100 g body weight increased serum vitamin E levels from 0.39 to 2.17 mg/dl by 72 hr and lung tissue vitamin E content from 3.52 to 17 mg/mg wet weight of lung. In vitro lipid peroxidation in lung homoginates of animals in 100% oxygen for 72 hr was inhibited by approximately 80% in animals receiving 100% oxygen plus vitamin E. Hyperoxia-induced increases in the pulmonary antioxidant enzymes, superoxide dismutase, glutathione peroxidase, and glutathione reductase were diminished by vitamin E administration. Lungs from vitamin E-treated animals did not show the early lung epithelial injury seen in animals exposed to 100% oxygen but not treated with vitamin E. Mophometric analysis of lungs of animals in room air for 72 hr showed 81.6% of lung to be normal as compared with 43.3% normal lung in the group maintained in 100% oxygen for 72 hr. In the group treated with oxygen plus vitamin E, the lungs were similar to room air controls (82.6% normal). This study thus provides further evidence for a direct antioxident affect of vitamin E in lung.

Supernatants from concanavalin A-stimulated spleen cells contain a factor which induces morphological alterations, inhibition of replication, and altered phospholipid biosynthesis in cells of a type 2 pneumocyte-related strain designated NAL 1A. This study has demonstrated that the active factor is generated by nonadherent spleen cells in the presence of supernatants from cultures of stimulated adherent cells. The active factor inhibits proliferation of NAL 1A cells in a dose-dependent fashion, exerts an irreversible effect following overnight exposure of these cells to stimulated supernatants, and appears to be continuously synthesized by stimulated spleen cells in culture. It is stable at pH 2 for 4 hours, does not bind to Blue Sepharose CL-6B, and exhibits molecular heterogeneity on gel filtration chromatography, with maximal activity recovered in fractions of molecular size 34,000 and 43,000. Comparable inhibition of growth was induced by exposure of NAL 1A cells to recombinant murine and human interleukin 2, as well as by recombinant murine interferon-gamma. These findings provide evidence for an interaction between type 2 pneumocytes and lymphokines such as interleukin-2 and interferon-gamma. The possible implications of a similar interaction occurring in vivo and its effects on type 2 pneumocyte morphology and function are discussed in the context of chronic interstitial inflammatory lung disease.

17 beta-Estradiol (0.44 to 4.4 micrograms/kg) was intramuscularly administered to pregnant rabbits on day 25 or 26 of gestation, and the fetuses were delivered by cesarean section 24 hr later. On light microscopy, the lungs from the treated group had larger alveoli and thinner interalveolar septa than did those from the controls at the same gestational age. The lumen:septa ratio was 0.62 +/- 0.06 in the control group and 0.88 +/- 0.05 in the treated group (p less than 0.01). Blood vessels in the lungs of the treated group were also more mature than were those in the control group. Alveolar epithelial cells consisted of 52% undifferentiated, 21% type II, and 27% type I cells in the control group. In the estrogen-treated group, the corresponding distribution was 25, 29, and 45%. There were 0.82 +/- 0.16 lamellar bodies per alveolar cell in the treated group compared to 0.38 +/- 0.06 in the controls (P less than 0.05). Estrogen decreased fetal lung glycogen content from 247 +/- 15 micrograms/mg protein to 70 +/- 9 on day 26 and from 103 +/- 13 to 13 +/- 2 on day 27 (P less than 0.001). Estrogen administration increased the rate of incorporation of choline into phosphatidylcholine in fetal lung slices, decreased the rate of thymidine incorporation into DNA, but had no effect on the rates of incorporation of ethanolamine into phosphatidylethanolamine or of leucine into protein. These data indicate that estrogen accelerates the rate of fetal lung maturation. It appears to stimulate lung differentiation at the expense of lung growth.

We have developed a short-term organ culture model for the study of the biochemical and morphological development of late gestation fetal rat lung. Explants (1 mm3) of 19-day lung were cultured in an oxygen enriched environment in the presence of synthetic serum-free medium for 3 days. Morphological maturation continued in culture. The rate of incorporation of choline into disaturated phosphatidylcholine and the content of this phospholipid in the explants increased in vitro in a pattern very similar to that which occurs in vivo. The activities of choline kinase and cholinephosphotransferase were also similar in cultured lung and in vivo. Studies of glucose oxidation to CO2 provided additional evidence that the explants remained viable in culture. The explants retained the sensitivity of fetal lung to hormonal action. This was demonstrated by the stimulation of choline incorporation into phospholipid by cyclic AMP and an increase in the glycogen content after exposure to insulin.

Type 2 pneumocyte responses were studied in the rat after experimental right apical lobe collapse induced by surgical occlusion of the apical bronchus. After initial degranulation, type 2 pneumocytes underwent proliferation, and exhibited large numbers of well formed and densely staining intracytoplasmic surfactant lamellar bodies. The proliferative response, which was diffuse in the initial 3 to 25 days, became focal by 50 days and thereafter subsided. These results are discussed in relationship to the responses of the type 2 pneumocyte to toxic injury as previously reported.

Delipidated proteins from a fraction of mouse lung homogenate enriched for lamellar bodies of type 2 pneumocytes were characterized by a combination of gel filtration and enzyme immunoassay for the presence of a surfactant-associated antigen. Immunoreactivity was associated with a protein of Mr 250-270,000 as well as its multimers and proteolytic fragments. The characteristics of the antigenic protein closely resembled those of a surfactant-associated protein termed alveolyn which is reported to be secreted by type 2 pneumocytes of various other species. Surfactant-associated proteins of M 32-38,000 in bronchoalveolar lavage fluid may be derived from this molecule by proteolysis.

Pulmonary infection in immunocompromised patients is frequently difficult to diagnose. Therapy for the more common pathogens differs greatly from that for infection with unusual opportunistic organisms. However, neither of these infectious agents offers specific radiographic signs. The authors report on 4 patients with acute leukemia and invasive aspergillosis whose radiographs demonstrated a distinctive feature of one or more air crescents within an area of pulmonary infiltrate. Autopsy studies correlated the radiographic changes with an infection due to Aspergillus species fungi. While the sign is not pathognomonic for Aspergillus infection, seen in a suitable host, it would suggest the possibility of invasive aspergillosis.

The mouse lung epithelial cell strain NAL 1A has been confirmed as type 2 pneumocyte-related by immunostaining with a type 2 pneumocyte-specific antiserum. Cytoplasmic immunoreactivity with the antiserum paralleled the appearance of phospholipid-containing osmiophilic cytoplasmic inclusions, which were much more abundant in confluent cell cultures at low passage number than in exponentially growing cultures. However, phospholipid analysis indicated that NAL 1A cells were impoverished in phosphatidylglycerol as compared to lung type 2 pneumocytes. Confluent cultures of the neoplastic cell lines NAL 1AM and NUL 1 did not reveal any reactivity with the specific antiserum.

NAL1A is a murine type 2 pneumocyte-related cell line cultured from normal BALB/c adult mouse lung. In vitro spontaneous transformation of 3 out of 7 clones of NAL1A has led to the isolation and establishment in continuous cell culture of sibling-related non-neoplastic (NAL1A) and spontaneously arising neoplastic (NAL1As) cell strains. NAL1As cells exhibited a similar phenotype to cloned NUL1 cells cultured from urethane-induced mouse lung adenomas. All NAL1As and NUL1 clones grew vigorously in 0.3% agar and formed invasive, poorly differentiated carcinomas following subcutaneous inoculation into immunesuppressed mice. Several subcutaneous nodules metastasised preferentially to the lung. All spontaneous and chemically-derived malignant clones were less differentiated than the non-malignant clones as assessed by staining with a type 2 pneumocyte-specific polyclonal antiserum. The clones described in this report form a useful model in the study of spontaneous and chemically-induced neoplastic transformation in mouse epithelial lung cells.

Despite studies of the pathologic and microbiologic aspects of the lung in cystic fibrosis (CF), there is a lack of information on the lung localization of bacterial pathogens. Bacteriologic data come from cultures of sputum or accessible lung effluent from bronchoscopy. Our objective was to localize Pseudomonas aeruginosa (PA) in situ in order to provide descriptive data on the relationship between the presence of PA and disease of the surrounding tissue. Using stored, uncut blocks of preserved lung, we deparaffinized, cut, mounted, and reacted them with high-titer rabbit sera made to a mucoid strain of PA. The tissues were then reacted with a second antibody, biotinylated goat antirabbit immunoglobulin, developed using a peroxidase technique and counterstained with hematoxylin. PA organisms stained with heavy brown deposits and this was species-specific. In five patients with CF chronically colonized with PA, the organisms could easily be localized in multiple sections. Microscopic study demonstrated that location was generally endobronchiolar and associated with bronchiolar obliterative changes, mainly in small (less than 1 mm) airways. Extraluminal PA organisms were rare even when there was chronic interstitial inflammation. This study demonstrated the presence of PA at the location where physiologic damage in CF is most severe--the small bronchioles--strengthening the association between PA and pulmonary deterioration in CF.

Effects of hyperoxia in an organotypic model consisting of well-differentiated fetal rat type 2 pneumocytes have been studied by light and electron microscopy. In cultures exposed to 50% oxygen for 48 h, hyperoxia caused necrosis of cultured lung cells derived from 18- to 19-day gestation fetal rats, less damage in cells derived from 20- to 21-day gestation fetal rats, and no detectable damage of cells derived from newborn rats. After exposure to hyperoxia in organotypic cultures cocultured with fibroblast monolayers, ultrastructural abnormalities of surfactant (large lamellar bodies with disordered lamellae and abnormal shape) were detected in cells from 18- to 19-day gestation fetuses. These abnormalities were not noted when fibroblast monolayers were absent. Fibroblast conditioned medium from fibroblasts exposed to hyperoxia did not cause significant surfactant abnormalities at the ultrastructural level. These changes were less marked in cultures incubated with glutathione's constituent amino acids and with ascorbic acid during exposure to hyperoxia, and in cultures pretreated with dexamethasone (20 nM) before exposure to hyperoxia.

The clinical course and radiographs of 30 patients with fat embolism syndrome were reviewed. In all cases the classic triad of neurologic dysfunction, respiratory insufficiency, and petechiae were present. Three responses to embolized fat were noted. The hyperacute response was seen in two patients with paradoxical embolization of fat to the systemic circulation. A "classic response" was noted in 18 patients with transient respiratory compromise and variable radiographic findings. The two deaths in the group responding in the classical manner were attributed to massive pulmonary emboli. The third response, noted in ten patients, consisted of a chest radiograph compatible with pulmonary edema in the clinical setting of the adult respiratory distress syndrome. In this group the degree of respiratory dysfunction and pulmonary damage correlated with the development of disseminated intravascular coagulation. Pathologic correlations are presented and the mechanisms by which embolic fat produces tissue damage are discussed.

Pulmonary maturation was studied in fetuses in streptozotocin-diabetic rats on the final four days of gestation. Diabetes was induced prior to conception by the intravenous injection of streptozotocin. Fetuses were hyperglycemic but did not manifest hyperinsulinemia. Whole lung total phospholipid, phosphatidylcholine, and disaturated phosphatidylcholine were significantly decreased in the diabetic group on day 21 (term = 22 days), but not prior to or after that point in gestation. Morphologic analysis also revealed a decreased number of type II cells and lamellar bodies per alveolar lining cell in the diabetic group only on day 21, coincident with the changes in phospholipid analysis. Activities of enzymes involved in fetal pulmonary phospholipid synthesis were measured to see if differences could account for the observed developmental delay. No significant differences between diabetic and control lungs were noted in any of the enzymes studied from days 20-22, with the exception of an increase in cholinephosphate cytidylyltransferase activity in the diabetic fetuses on day 22. Immaturity in both biochemical and morphologic indices of lung development was present at a specific time late in the diabetic rat gestation. This maturational delay could not be accounted for by changes in the activities of enzymes involved in phospholipid synthesis. The fetus of the streptozotocin-diabetic rat provides a useful model to study the effects of hyperglycemia on fetal lung development.

Cell lines which exhibit epithelial morphology with surface microvilli and inclusion bodies characteristic of type 2 pneumocytes have been derived from normal adult mouse lung by a simple procedure involving enzymatic dispersal and mechanical elimination of other cell types. One of these cell lines designated NAL 1A, examined in detail, shows features consistent with its being related to type 2 pneumocytes of mouse lung. These features include desmosomes, dense lamellar bodies as well as phospholipid profiles related to immature surface active material, the inhibition of cell growth rate by dexamethasone, and the close similarity of the cytoskeletal protein patterns of this cell line to those of a metastatic type 2 pneumocyte-related cell line of mouse lung. The cell line from normal lung demonstrated near diploid chromosome number at low passage number with some evidence of karyotype instability at high passage number.

Epithelial cell strains, designated NAL, have been established from normal lungs of adult female Balb/c mice. The ultrastructural characteristics, effect of dexamethasone on cellular morphology and proliferation rate, and the cytoskeletal protein pattern of NAL suggests that these cell strains may be related to a urethane-induced mouse lung adenoma cell strain NUL and to a metastasizing mouse lung tumour cell line CMT. NAL exhibited no anchorage-independent growth under normal conditions, however extensive passaging in the presence of 5 X 10(-6)M dexamethasone resulted in a colony forming efficiency in soft agar of 8.4% at passage number 30.

The epithelial cell strain NAL1A cultured from normal adult mouse lung has been transformed by culturing in dexamethasone into an invasive neoplastic cell strain. The criteria for neoplastic transformation include the capacity for anchorage independent growth in soft agar as well as the formation of invasive neoplastic nodules after subcutaneous transplantation in thymectomized irradiated newborn mice. The cells of the invasive neoplastic nodules induced by dexamethasone culturing of NAL1A were indistinguishable histopathologically and by electron microscopy from invasive nodules evoked by the subcutaneous inoculation of CMT64, a cell line cultured from a metastasizing mouse lung tumour and cell strain NUL1 derived from mouse pulmonary adenomata induced by urethane. Cells of the nodules derived from all three cultured strains possessed desmosomes, surface microvilli and phospholipid lamellar bodies characteristic of the type 2 pneumocyte. It is concluded that cultured cell strains NAL1A, cultured in dexamethasone, NUL1 and CMT64 evoke invasive subcutaneous neoplasms derived from a common ancestor, presumably a type 2 pneumocyte related stem cell.