Effect of detergent solutions upon distal lung phospholipids.

Intratracheally injected acid detergent solutions induce in rabbits a predominantly bronchiolo-lobular process evolving in two steps, an epithelial and an epitheliomesenchymal one. When the acid detergent solution reached prominently the alveolar structures it determined a rapid release by exocytosis of phospholipid lamellated bodies from the large granular alveolocytes and the following engulfment by macrophages of released products (Eskenasy, 1982 b). Histochemically, the cells containing phospholipids increased in cellularized alveolar walls, the alveolar lining film appeared thickened and denser, and the accumulated free macrophages filled with phospholipid reactive granules (Landing et al. 's method). Glucose-6-phosphate-dehydrogenase activity is very intense in the cellularly thickened alveolar walls, in macrophages and in the newly formed (regenerated) bronchiolar cells. Lactate dehydrogenase activity becomes very intense in macrophages. These data complete the morphogenic events leading to the lobular pathology.

Morphogenesis and pathogenesis of chronic lung diseases. XX. Effects of repeated hypersensitivity reactions upon experimental immuno-granulomas of the lung.

In order to analyse the effects of repeated hypersensitivity reactions upon chronic lung lesions, lung granulomas were experimentally induced in rabbits by an intravenous injection of 1 ml of the complete Freund's adjuvant added with 10 per cent human gammaglobulin. Groups of rabbits with 14- and 21-day-granulomas were intratracheally challenged with 1.000 I.U. human gammaglobulin injected two or three times at 7-9 days interval. The responses of the lung tissue were of the Arthus type with neutrophil granulocytic accumulations and proteasic action; the intensity of cell accumulations was decreasing after the 2nd and 3rd challenge. The lesional evolution after challenge showed intra- and perigranulomatous infiltrations with alkaline-phosphatase positive lymphocytes and plasma cells. The density of the intragranulomatous reticulin network developed after the hypersensitivity reactions increased progressively and was morphometrically assessed as statistically significant. Cellular and fibrillar lesions led to the restructuring of the lung tissue.

Isolation, culture, and some morphohistochemical features of endothelial cells of the pulmonary microvasculature.

Taking into account the quantitative and qualitative importance of endothelial cells of the pulmonary microvasculature (40 per cent of all lung mesenchymal cells, 26 per cent of all lung cells) the authors attempted their isolation, culture in vitro and specification of some morphohistochemical characters, using the method of Habliston et al. (1979). There were used guinea pigs and rabbits and a 0.5 and 1 per cent trypsin solution in a retrograde perfusion for the detachment of endothelial cells from their rbasement membranes. The obtained cells were maintained in culture for5 7--9 days and morphologically characterized; their caveolar enzymes were evidenced (Mg-dependent ATP-ase, acid and alkaline phosphatases, a.o.).

Bronchiolar pathology. IV. a morphometric analysis of the incidence and location of lesions induced in rabbits by intratracheally injected detergent solutions.

An experimental model of bronchiolar lesions was elaborated. It consisted of three intratracheal administrations in rabbits of 2 ml of a 3 per cent detergent solution, pH 2.5-3; the adequate development of the process was induced when the second injection was given 48 h after the first and the third on days 5-6. The induced process evolved during three phases: a) hypertrophy with hyperfunction of Clara cells, cell beheading by apo- and merocrinia and denudation of basement membranes; b) lesions of subepithelial components, mainly of capillaries, with granulative response and restructuration of the peribronchiolar tissue, and c) tardy regenerative response of epithelial cells, sometimes with neoformation of bronchioli. The morphometric analysis of this experimental process asserted the validity of the model which led to damages involving at less 70 per cent of the bronchioli (under 200 epithelial nuclei on a circumferential section) and of the small bronchi (with 200-500 epithelia nuclei); the other small and large bronchi were never involved. The morphometric analysis also showed the relations of the intervals between injections to the lesional intensity and stereotyping.

Alveolar macrophagic reactions. III. Stereotyped development of alveolar responses to intratracheally administered agents.

Kinetics of alveolar macrophagic reactions was quantitatively studied at the endoalveolar and parietoalveolar levels after challenge by a single intratracheal administration of several agents, such as mineral dusts, antigenic particles, hormones and drugs. The follow up of all the induced alveolar reactions evidenced their basic character and their stereotyped development irrespective of the challenge. From among the tried substances, heparin and euphylline were chosen for the following experiment due to their stimulatory effects towards the alveolar macrophagic reactions in which they seem to act like chemical mediators. Rabbits with alveolar macrophagic reactions induced by 3 intratracheal injections of aluminum silicate were daily treated with heparin or euphylline: the stimulatory effects of the added drugs obviously contributed to the promotion and prolongation of the increased macrophagic reactions both at the endoalveolar and parietoalveolar levels. The phases of these stereotyped alveolar macrophagic reactions and the involvement of circulation monocytes and of mesenchymal interstitial cells are discussed considering the alveolus as a whole.

Histoenzymology of the lung. V. Histoenzymatic analysis of sixty lung tumors.

A series of sixty lung tumors operated during 1977 were histoenzymatically investigated (12 macrocellular carcionomas, 4 microcellular, 18 epidermoid, 18 cylindrocubic, 2 bronchiolo-alveolar cell carcinomas, 4 lung fibroblastic sarcomas, and 2 benign lung tumors: a haemangioma and a carcinoid tumor). There were tested 25 enzymes, hydrolases and oxidoreductases of different metabolic pathways. The histoenzymatic analysis of the neoplastic lung tissue compared with the normal adult human lung tissue pointed to a shifting of the metabolic balance, with the decrease of enzyme activities belonging to the Krebs tricarboxylic acid cycle and with the increase or maintenance of the level of enzymic activities belonging to the Embden-Meyerhoff-Parnas pathway and to the pentose shunt. The enzymes intervening in the protidic activity in all the malignant tumors of the lung. Some hydrolases were also more active lung tissue pointed out the existence of close relations between the normal epithelia and the cylindrocubic and bronchiolo-alveolar types, and of distant ones with the epidermoid and microcellular carcinomas which suffered more accentuated ecogenetic influences (stopping of differentiation/maturation, metaplasia) during the complex oncogenetic process.

Morphogenesis and pathogenesis of chronic lung diseases. XVI. The influence of drugs on the stereotyped development and cellular kinetics of experimental lung granulomas.

The stereotyped development and the cellular kinetics of lung granulomas experimentally-induced in rabbits by an intravenous injection of the complete Freund adjuvant with addition of human gammaglobulin, were studied under the influence of hydrocortisone acetate, rifampicin, heparin and phenobarbital. As in a previous study using actinomycin and puromycin, the analysed drugs delayed up to inhibition (hydrocortisone) the development of lung granulomas which remained small, unfused, without epithelioidosis; the primary and secondary immune responses were more or less influenced up to their suppression. The delivery of macrophages was reduced by hydrocortisone, but enhanced by heparin and phenobarbital. The latter induced a persistent intragranulomatous granulocytosis, expressing the repair of decreased activities of dehydrogenases and especially of NADP-NADPH-oxidases. The action of these drugs was phasic, their intervention being active only during the cellular phase of granulomas; fibrillogenesis was influenced only indirectly. Extrapolation to humans of these results suggests the use of these drugs during the cellular phase of chronic pneumonitic lesions and suppression of the immune component of the inflammatory processes.

Experimental chronic obstructive lung disease. I. Bronchopulmonary changes induced in rabbits by prolonged exposure to formaldehyde.

The prolonged exposure to formaldehyde induces in the rabbit lung reactional and dystrophic changes involving the intrapulmonary bronchi, the bronchioli and the lung tissue. These changes are represented by bronchial cell hyperplasia with hypermucigenesis, extrusion of bronchial cells, bronchiolar hypermucigenesis, parcellary squamous metaplasia or necrobiosis of epithelia, thickening of bronchial and bronchiolar walls by subepithelial cell accumulations, destruction of musculo-elastic structures with stenosis or ectasia; the vascular reactions are hyperhaemic and proliferative with an obstructive and fibrous tendency; the parenchymal lesions are atelectasias, intralobular emphysema, and cellular thickening of alveolar walls and interlobular areas. The acid phosphatase, Tween-60-esterase, naphthol-AS-D-acetate-esterase, proline-oxidase and hydroxyproline-2-epimerase activities are increasing, while the leucyl-aminopeptidase and beta-glucuronidase ones are decreasing. The qualitative observations are completed and sustained by quanitative studies of mucous cell kinetics, of cell accumulations and differentiations.

Histoenzymology of the lung. Some enzymic activities of capillary endothelia of the rabbit alveolar walls.

Mg-dependent ATP-ase, alkaline phosphatase and carbonic anhydrase activities were tested at the level of capillary endothelia of alveolar walls in normal rabbits. A constant and relatively intense activity within these endothelia appeared to be that of the Mg-dependent ATP-ase. The carbonic anhydrase activity marked some endothelia, and the alkaline phosphatase was unequally demonstrable at this level. These different histoenzymic aspects might reflect the unequal activity during time and space of these enzymes at the level of capillary endothelia of alveolar walls.

Histoenzymology of the lung. I. Enzyme activities of the lung tissue of acult humans; relationships between structure and functions.

Human adult lung fragments removed from macroscopically undamaged and anthracosis exempted zones of lungs of 20 pneumonectomies made for cancer, were tested for 25 enzymic activities. The location and intensities of these enzymic activities were different in the lung tissue components; The bronchial epithelia contained highly active LDH, MDH, SDH, NADH-TR and NADPH-TR, glucose-6-phosphate dehydrogenase, active hydroxyproline-2-epimerase, alkaline phosphatase. Ca2+-activated ATP-ase, and beta-galactosidase. Bronchial and vascular muscles presented intense activities of LDH, MDH and SDH of alkalinephosphatase, AMP-ase and Ca2+-activated ATP-ase, as well as of beta-galactosidase. The alveolar walls presented high activities of SDH, MDH and LDH, of alkaline and acid phosphatases, of beta-galactosidase and of Tween-40 and 60-esterases, of HEP, cytochrome-oxidase and peroxidase. The free alveolar macrophages were active for LDH, MDH, SDH, NADH-TR and NADPH-TR, G1-6-ph-DH, acid and alkaline phosphatase, cytochrome-oxidase and peroxidase, HEP, AMP-ase and Mg2+-activated ATP-ase, Tween-esterases, naphthol-ASD-acetate esterase, and beta-galactosidase. The endothelia contained high activities of alkaline phosphatase, of AMP-ase and Mg2+-activated ATPase, of LDH, MDH and SDH, and of beta-galactosidase. In bronchial lymphoid nodules it was the LDH, MDH, SDH, cytochrome-oxidase and peroxidase, HEP, alkaline phosphatase and AMP-ase, Tween-60-esterase and beta-galactosidase that were active. The interlobular areas of the lung presented intense activities of SDH, MDH, LDH, HEP and cytochrome-oxidase. The activities of the other tested enzymes were weaker or absent in the adult human lung components, the same as those of aminopeptidases which were present only in some free alveolar macrophages. The discussion of some relationships between these enzymic actitivies and the morphology of the human adult lung tissue asserted that the latter could not be considered as a "normal" tissue but as one overstrained by the components of blood and polluted air.

Proline-oxidase and hydroxypyroline-2-epimerase activities in experimental fibrosing granulomatous processes of the lung.

Three experimental fibrosing granulomatous processes of the lung (pulmonary granulomas induced by complete Freund adjuvant, viral pneumonitis induced by the A2 influenza virus, and tuberculosis induced in animals exposed to tobacco smoke) were produced in rabbits and the results confronted in view of establishing some relationships between mesenchymal cell accumulations, reticulin fibrillogenesis, and the enzymic activities of proline-oxidase (PO) and of hydroxyproline-2-epimerase (HEP), enzymes intervening in the control of hydroxyproline incorporation in the procollagen molecule. Histopathologic, histochemical and histoenzymic methods, quantification of cells and fibrils, statistical analyses, including the regression lines method, were used. The confrontation of the three processes made obvious that the cell accumulation invariably preceded the fibrillogenetic process, that the immune nature of the process was accompanied by larger cell accumulations than the non-immune one, that between cell accumulations and fibrillogenesis there existed highly significant correlations, and that the increase of the enzymic activities of proline-oxidase and of hydroxyproline-2-epimerase always accompanied these tissular changes, being topographically coincident with them (interlobular areas, alveolar walls).

The foetal development of the rabbit lung: a cytologic, cytochemical and histoenzymatic study.

The developmental peculiarities of the rabbit lung were analyzed in foetuses of 14 and 23 days, and in newborns having respired 30 min. and 48 hrs. Cytochemical, histoenzymatic and quantiative cytologic methods were used. The parallel evolution of epithelial and mesenchymal cells was quantified using conventional fields. The development of air spaces was morphometrically appreciated. Acid and neutral mucopolysaccarides, nucleic acids, and enzymic activities (AcPh-ase, AlkPh-ase, ATP-ase, AMP-ase, SDH, MDH, LDG, G1-6-ph-DH, proline-oxydase, hydroxyproline-2-epimerase, unspecific esterase, TwE-ase, beta-gal-ase and beta-gluc-ase, alanyl- and leucineaminopeptidase) were investigated. This complex analysis showed that in a first phase the development mainly involved the epithelial cells, while the proliferation of mesenchymal ones remained constant. In a second phase, the epithelial cell increase became slower, and the mesenchymal cells were decreasing. At the same time the air spaces were continuously increasing. During this process, neutral mucopolysaccharides were synthesized in epithelial cells and in cartilaginous nodules, and sometimes in mesenchymal cells. The RNA was continuously increasing both in epithelial and mesenchymal cells. The high enzymic activities in the 14-day foetuses appeared to be limited to AcPh-ase, AlkPh-ase, and SDH in both epithelial and mesenchymal cells, the LDH in epithelial and the ATP-ase and AMP-ase mainly in mesenchymal cells. At the same time, the G1-6-ph-DH obviously marked the epithelial cell differentiation. In the other foetal and newborn lungs, the enzymic activities appeared to be more various by limitation of AcPh-ase to epithelial elements and of AlkPh-ase to mesenchymal and vascular ones, by activation of proline-oxydase and especially of hydroxyproline-2-epimerase in pleura and peribronchovascularly, by intensification of the unspecific esterase: the other enzymes active in the 14-day foetuses were now weaker. The activity of beta-gal-ase, beta-gluc-ase, and of peptidases was missing during the entire development of the foetal rabbit lung. The corroboration of these data suggested the relation between the differentiation of enzymic activities and the development of foetal rabbit lung, the strong relations between AcPh-ase activity and the epithelial elements, and of AlkPh-ase and ATP-ase with the mesodermo-mesenchymal ones, the marking of epithelial cell differentiation by the G1-6-ph-DH activity, the presence of SDH in the basal corpuscles of differentiating cili, the increase of enzymes making inactive the hydroxyproline in zones in which connective tissue is developing, the low differentiation of hydrolases (related to the absence of air and blood transport of products) and the lack of peptidase activity corresponding to the reduced pulmonary degradation of proteins (as in adult lungs).