Ultrastructure of lamellar bodies in congenital surfactant deficiency

Congenital surfactant deficiency (CSD) is a newly identified neonatal lung disorder associated with a variety of molecular defects affecting surfactant synthesis and secretion in alveolar type II cells. The authors present ultrastructural findings of abnormal lamellar bodies in lung biopsies from 4 infants with CSD. All were term infants presenting shortly after birth with severe respiratory failure that was unresponsive to conventional therapy and all died within the first month of life. Lung biopsies were performed between 8 and 25 days of age. Biochemical and molecular studies in 2 unrelated male infants identified SP-B deficiency, one case with 121 ins 2 mutation and the second with a 209 + 4 A > G mutation. Light microscopy in both cases showed features of alveolar proteinosis. Ultrastructurally, alveolar type II cells lacked mature lamellar bodies, and their cytoplasm contained numerous pleomorphic inclusions with membranous and vesicular structures not seen in normal type II cells. The other 2 infants were a pair of siblings in whom molecular studies identified mutations in ABCA3 transporter gene. Light microscopy showed features of acinar dysplasia and desquamative interstitial pneumonitis. TEM studies revealed absence of mature lamellar bodies in type II cells and instead showed a mixture of cytoplasmic electron-dense inclusions with concentric membranes and distinctive electron dense aggregates. The ultrastructural changes in alveolar type II cells correlated well with specific gene defect. In SP-B deficiency, the absence of mature lamellar bodies is consistent with the postulated role for this protein in the formation of lamellar bodies. The lack of mature lamellar bodies in the ABCA3 gene mutations is due to the dysfunction of this endogenous lipid transporter that targets surfactant lipid moieties to the lamellar bodies. The findings demonstrate the importance of TEM studies of lung biopsies from infants with CSD as it is a critical adjunct in the diagnosis of neonatal lung disease and in defining the underlying cellular defects.     

Light and electron microscopic studies of the pulmonary alveolar surfactant.

22 methods of light and electron microscopy have been tested for the identification of the pulmonary alveolar surfactant. Among the non electron microscopic methods Romhányi's anisotropic staining with toluidine blue was the most serviceable. In addition to simple performance it allows the identification of the smallest alteration -- not to be identified by other methods -- of the alveolar surfactant. Among the electron microscopic technics Ruthenium Red staining and Dermer's tricomplex method yielded the best results. Given that different chemical components of the alveolar surfactant will be identified by these 2 methods they supplement each other favourably. In the study of human cadaver material Baker's phospholipid reaction and Romhányi's anisotropic toluidine blue reaction is being used.     

Electron microscopic studies on the formation of vesicular bodies during cell wall degradation and regeneration in yeast

Vesicular bodies are observed during enzymatic degradation of the yeast cell wall and in the course of wall regeneration. In both cases various types of vesicles are formed and exocytated by different mechanisms. They are characterized cytochemically and their possible role in cell wall regeneration is discussed.     

Electron microscopic studies of the antigen-antibody complex

Electron micrographs of the ferritin antibody (rabbit) and ferritin (horse) complex have been obtained. The high iron content of the ferritin molecule (23 per cent Fe) allows its molecules to be recognized within the particles of precipitate.

Three methods of visualizing the molecular distribution have been developed: (a) small particles of the precipitated complex have been dried on to electron microscope grids and either examined directly or first shadowed with metal and then examined, (b) the precipitate has been centrifuged to a plug which was embedded and thin sections cut from it for examination, (c) the bands formed by allowing antibody and antigen to diffuse together in agar gels have been fixed, embedded and sectioned.

All methods have yielded pictures of the distribution of the ferritin within the complex which are broadly similar to what might have been expected from a somewhat irregular lattice as pictured in the Marrack-Heidelberger Lattice Theory.

The antibody molecules are not clearly defined but appear as a halo of low density enveloping the ferritin clusters. The distance, centre to centre, between the ferritin molecules is variable, but is, on the average, in the range 200–400 Å. This is greater than the ferritin-ferritin contact distance (100 Å) and is thought to mean that the ferritin molecules are bridged by antibody molecules as pictured in the Lattice Theory.

The bands produced in the gel-diffusion test contain islands of ferritin-antibody complex. When equivalent concentrations of reagents are used a single band of precipitate is formed. When excess of either antigen or antibody is used multiple bands of precipitate are formed which contain islands of ferritin antibody complex indistinguishable from those formed in the single band at equivalent concentrations, providing direct evidence for the formation of multiple bands from a single antigen.

Ferritin-ferritin contacts have been observed within the complex.

Under all the conditions of relative concentration of the two components used here, the particles of precipitated complex seem to be superficially covered with antibody which is seen as a halo about 300–400 Å thick around each cluster. This distance may correspond to the length of the antibody molecule which is deduced from other measurements to be about 300 Å.

     

Electron microscopic studies of human haemosiderin and ferritin.

Ferritin and haemosiderin were isolated from fresh frozen human spleens that had been removed from patients with secondary iron overload due to multiple transfusions. Haemosiderin was solubilised by a novel technique that maintains its integrity. Unstained preparations of haemosiderin and ferritin were visualised and quantitative measurements made of the volumes of iron core. The mean diameter of the ferritin core (6.4 nm) was larger than that of haemosiderin (5.7 nm). In addition, haemosiderin, in contrast to ferritin, showed a large number of cores of less than 5 nm in diameter. Negatively stained preparations of haemosiderin and ferritin were visualised, confirming the small core size of the haemosiderin. The protein shell of haemosiderin, unlike that of ferritin, was thinner and irregular. These findings are consistent with the suggestion that haemosiderin is derived from ferritin by partial proteolysis and partial solubilisation of the iron core, presumably by lysosomal action.     

Electron microscopic studies of the developing amphibian thymus

Electron-microscopic studies of larval Xenopus laevis thymus at three different stages of development are presented. Studies of thymus from 5 and 8 day old larvae (ages when early thymectomy is performed) reveal a relatively undifferentiated rudiment composed of epithelial cells and lymphoid cell precursors. These findings are in marked contrast to ultrastructural observations of 30 day old larval thymus, where there exists a well defined cortico—medullary differentiation with an abundance of small lymphocytes in the cortical region.     

Electron microscopic studies on granulocytopoiesis in the slender salamander

The morphology of differentiating heterophils and eosinophils of the slender salamander, Batrachoseps attenuatus, was studied with electron microscopy. Enzymes of the granules of both cell types appear to arise from the Golgi cisternae. An agranular stem cell was not observed and the least differentiated cell type encountered was an “early” promyelocyte having small granules which are morphologically distinct from mature granules of either the heterophil or eosinophil series. Heterophil myelocytes and later stages contain only one population of granule which is fibrous in content. Eosinophils likewise possess but one type of granule; the granules are larger than those of heterophils, have a homogeneous content, and lack the crystallin core so characteristic of mammalian eosinophils.     

Electron microscopic studies of pseudointima in synthetic prostheses

Thirty one prostheses made of Lavsan (therylene) or teflon-lavsan that had been functioning in human patients for 16 months to 21 years were examined. No continuous endothelial lining was found present on the pseudointimal surface of any of the prostheses. The major cell types in a formed, well-vascularized pseudointima were fibroblasts and smooth-muscle cells. Cells of the macrophage series were involved in forming the granulation tissue seen around fibers of the prostheses.