Lamellar Inclusion Bodies Research Articles

Toxicity and clearance of intravitreal cefotetan

Direct intravitreal injection of antibiotics plays an important role in the management of bacterial endophthalmitis. In the present study we investigated the toxicity and clearance of intravitreally injected cefotetan in a rabbit model. No toxic ocular side effects could be detected by electroretinography (ERG) or light and electron microscopy up to and including a single intravitreal dose of 1000 micrograms. Intravitreal injection of 2000 micrograms cefotetan resulted in mild degeneration of photoreceptor outer segments and, sporadically, in cataract formation. After intravitreal injection of 4000 micrograms, moderate toxic degeneration of photoreceptors occurred, with displacement and mitochondrial swelling of inner segments. In addition, lysosomal lamellar inclusion bodies could be detected in pigment epithelial cells. After a single intravitreal injection of 1000 micrograms cefotetan, concentrations greater than the minimum necessary for the inhibition of most commonly occurring intraocular pathogens (except Pseudomonas aeruginosa and Staphylococcus epidermidis) were maintained in the vitreous humor for greater than 48 h. Cefotetan may be a potentially important drug for intravitreal injection, especially in cases of gram-negative and suspected anaerobic endophthalmitis.

Hepatic changes produced by 30-day administration of a novel aminocyclitol antibiotic, trospectomycin sulfate, to laboratory animals

The studies described here were done to characterize the hepatic response to a new aminocyclitol antibiotic, trospectomycin sulfate, administered intravenously (beagle dog) or subcutaneously (Sprague-Dawley rat) at a variety of dose levels, to investigate reversibility of observed changes, and to document any untoward effects of subchronic trospectomycin sulfate administration. Both species showed significant elevations in serum levels of alanine and aspartate transaminases in higher dose groups. In the dog only, a transient neuromuscular blockade was also observed within higher dose groups. No other functional, morphological, or serum chemical changes were observed. Examination of liver by electron microscopy revealed the presence of cytoplasmic lamellar inclusion bodies, concentrated in the bile canalicular region of the hepatocytes. Occurrence of the lamellar bodies and coincident transaminase increases were found to be reversible upon discontinuance of treatment (studied in the dog). Electron microscopy of acid phosphatase cytochemistry in the rat indicated that most, but not all, of the lamellar bodies contained this enzyme. This observation suggests that they may be derived from the lysosome, or once formed become lysosomal.

Glucocorticoids regulate surfactant protein synthesis in a pulmonary adenocarcinoma cell line.

Synthesis of pulmonary surfactant proteins SP-A, SP-B, and SP-C was demonstrated in a cell line derived from a human adenocarcinoma of the lung. The cells contained numerous lamellar inclusion bodies and formed organized groups of cells containing well-developed junctional complexes and apical microvillous membranes. Synthesis of SP-A was detected in the cells by enzyme-linked immunoabsorbent assay and by immunoprecipitation of [35S]methionine-labeled protein. SP-A was identified as an Mr 31,000-36,000 polypeptide containing asparagine-linked carbohydrate. Northern blot analysis detected SP-A mRNA of 2.2 kb. Dexamethasone (1-10 nM) enhanced the relative abundance of SP-A mRNA. Despite stimulation of SP-A mRNA, intracellular SP-A content was unaltered or inhibited by dexamethasone. SP-B and SP-C mRNAs and synthesis of the SP-B and SP-C precursors were markedly induced by dexamethasone. ProSP-B was synthesized and secreted primarily as an Mr 42,000-46,000 polypeptide. Proteolysis of the proSP-B resulted in the generation of endoglycosidase F-sensitive Mr = 19,000-21,000 and 25,000-27,000 peptides, which were detected both intra- and extracellularly. SP-C proprotein of Mr = 22,000 and smaller SP-C fragments were detected intracellularly but were not detected in the media. Mature forms of SP-B (Mr = 8,000) and SP-C (Mr = 4,000) were not detected. Glucocorticoids directly enhance the relative synthesis and mRNA of the surfactant proteins SP-A, SP-B, and SP-C. Discrepancies among SP-A mRNA, its de novo synthesis, and cell content suggest that glucocorticoid may alter both pre- and posttranslational factors modulating SP-A expression.

Tissue drug accumulation and ultrastructural changes during amiodarone administration in rats

Pulmonary and hepatotoxicity are the two major side effects of chronic amiodarone therapy. We studied the accumulation of amiodarone and its principal metabolite, desethylamiodarone, in lung and liver of rats treated ip for 21 to 23 days with either 40 or 80 mg/kg/day amiodarone. The ultrastructural changes in liver, lung, and alveolar macrophages in saline controls and in rats on the two amiodarone dosage regimens were investigated. There was a dose-dependent increase in amiodarone and desethylamiodarone levels in serum and in tissues. The desethylamiodarone/amiodarone ratios in liver and lung, but not in serum, increased significantly with increasing dose. Serum also contained another metabolite, monodeiodinated desethylamiodarone. Increase in vacuolization and presence of whorled lamellar inclusion bodies in alveolar macrophages occurred with an increase in dose and higher lung amiodarone and desethylamiodarone levels. Electron microscopy of the liver of amiodarone-treated rats revealed the presence of large inclusion bodies partially filled with amorphous material in the cytoplasm. The quantitative relationship of the above changes to organ toxicity and to phospholipidosis that accompanies amiodarone administration remains to be established.

Lamellar body formation precedes pulmonary surfactant apoprotein expression during embryonic mouse lung development in vivo and in vitro

Abstract The purpose of this investigation was to determine whether lamellar inclusion body (LB) formation and surfactant apoprotein (SP-35) production are directly coordinated by temporal and positional information during development. In the present study we report a comparison between embryonic B10.A mouse lung morphogenesis and cytodifferentiation in vivo with that observed during organ culture in serumless medium. Precursor LB were first detected at embryonic day 12 (E12d), and progressively larger numbers and forms were produced during subsequent differentiation of respiratory alveolar duct epithelium. SP-35 was first detected during the canalicular period (E16.5d). Lung cultures (E12 d) showed pseudoglandular and canalicular periods of morphogenesis, and both ciliated epithelial and type II cell differentiation. Nonciliated cells produced increasing numbers of lamellar inclusion bodies throughout the culture period. SP-35 was detected at 9 days in vitro (d.i.v.). These observations indicate (i) precursor LB formation precedes SP-35 expression and is not dependent on apoprotein synthesis; (ii) E12d lung development in vitro using serumless medium proceeds at a rate equivalent to 0.5 days in vivo through 11 d.i.v.; and (iii) morphogenesis and differentiation occur in the absence of exogenous hormones and growth factors. The cell-cell interactions that play a role in morphogenesis and cell differentiation appear to be intrinsic to the developmental program for embryonic lung development and are likely to be mediated by autocrine and/or paracrine factors.

Tissue Drug Accumulation and Ultrastructural Changes during Amiodarone Administration in Rats

Pulmonary and hepatotoxicity arc the two major side effects of chronic amiodarone therapy. We studied the accumulation of amiodarone and its principal metabolite, desethylamiodarone, in lung and liver of rats treated ip for 21 to 23 days with either 40 or 80 mg/kg/day amiodarone. The ultrastructural changes in liver, lung, and alveolar macro-phages in saline controls and in rats on the two amiodarone dosage regimens were investigated. There was a dose-dependent increase in amiodarone and desethylamiodarone levels in serum and in tissues. The desethylamiodarone/amiodarone ratios in liver and lung, but not in serum, increased significantly with increasing dose. Serum also contained another metabolite, mono-deiodinated desethylamiodarone. Increase in vacuolization and presence of whorled lamellar inclusion bodies in alveolar macrophages occurred with an increase in dose and higher lung amiodarone and desethylamiodarone levels. Electron microscopy of the liver of amiodarone-treated rats revealed the presence of large inclusion bodies partially filled with amorphous material in the cytoplasm. The quantitative relationship of the above changes to organ toxicity and to phospholipidosis that accompanies amiodarone administration remains to be established.

Correlated ultrastructural damage between cerebellum cells after early anticonvulsant treatment in mice

The anticonvulsants phenobarbital and diphenylhydantoin administered early in life to mice resulted in significant and long-lasting ultrastructural damage, including abnormalities of mitochondria, myelin sheaths and lamellar inclusion bodies inside identified cells throughout the cortical layers of the cerebellum in treated vs control mice. The magnitude, distribution and duration of damage was age and treatment specific. No differences were detected in density of parallel fiber processes nor in synapse density within the molecular layer. Neuron profiles containing damaged organelles were not homogeneously distributed but made up only a small fraction of the total cell population examined. In our experiments, there was an overall within-animal correlation explaining 45% of the magnitude of damage in different cerebellar regions, but between synaptically connected cells, specifically mossy fiber axon varicosities and granule cell dendrite profiles, the subset population ratio of damaged-to-total mitochondria was highly significantly correlated (70–90%; P<0.001). We hypothesized that some correlated transneuronal degeneration and death in the central nervous system may have a transynaptically regulated component that first appears as correlated damage between synaptically connected cells, perhaps regardless of the degree of toxicity. The orderly cytoarchitecture and cell connections of the cerebellar cortex can be used to study these patterns of degeneration.

Pulmonary adenoma and endocrine cell hyperplasia in Syrian golden hamster treated with 4-nitroquinoline 1-oxide.

Chronic effects of 4-nitroquinoline 1-oxide (4 NQO) on the lungs of Syrian golden hamsters were studied. 4 NQO was subcutaneously injected weekly for 3 weeks at a dose of 20 mg/kg body weight. The animals were sacrificed at the 65th and 80th experimental weeks. Two cases of pulmonary adenomas were demonstrated in the 10 4 NQO-treated animals at the 80th week, and the tumor cells contained cytoplasmic lamellar inclusion bodies. In a previous study, we reported 4 NQO- induced pulmonary endocrine cell hyperplasias in the 4 NQO-treated hamster after the 20th experimental week (Jpn. J. Cancer Res., 77, 1986). In the present study, 12 pulmonary endocrine cell hyperplasias were recognized in serial sections of the 24 treated animals. The hyperplastic lesions showed positive immunoreactivity to calcitonin. The hyperplastic lesion did not develop to pulmonary endocrine cell neoplasm.

Suramin Keratopathy

Vortex keratopathy occurred in six patients given high-dose intravenous suramin for adrenocortical carcinoma. The corneal findings were bilateral and symmetric. Two patients complained of foreign-body sensation. One patient developed opacities of the anterior lens epithelium. Histopathologic studies of the eyes in one patient showed intraepithelial apical deposits in the cornea, conjunctiva, and lens epithelia. By electron microscopy, these deposits were found to be membranous lamellar inclusion bodies. These findings, as demonstrated by osmium-PAS staining and electron microscopy, were similar to those in other drug-induced lipid storage diseases.

Modification of surfactant metabolizing cells in rat lung by clofibrate, a hypolipidemic peroxisome proliferating agent. Evidence to suggest that clofibrate influences pulmonary surfactant metabolism.

The influence of clofibrate (ethyl-alpha-p-chlorophenoxy-isobutyrate), a hypolipidemic peroxisome proliferating agent, has been tested on the lungs of adult male rats. Drug administration for 7 days caused structural changes in two types of lung cells, both of which are involved in the metabolism of the pulmonary surfactant. By light microscopy the prominent features were the presence of enlarged type II alveolar epithelial cells and foamy intraalveolar macrophages. Compared with controls, type II cells in treated rats apparently contained more numerous surfactant-containing lamellar bodies, as visualized in semi-thin sections of Epon-embedded tissue. This difference was quantified morphometrically by light microscopy: the number of lamellar bodies was estimated as the profile number per individual type II alveolar cell, transsected at its nucleus. Clofibrate administration for 7 days resulted in a significant increase in the number of the lamellar inclusions. In contrast the number of type II alveolar cells per area of lung remained unchanged. There was no evidence of atelectasis or inflammatory infiltration in the drug-treated lungs, a finding confirmed in sections of perfusion-fixed, paraffin-embedded whole lung-lobes. By electron microscopy the lamellar inclusion bodies in the type II alveolar cells in treated rats, apart from being more numerous and sometimes smaller, were morphologically identical to those in controls. The vacuolated alveolar macrophages seen in treated rats also contained various lamellar phospholipid inclusions.(ABSTRACT TRUNCATED AT 250 WORDS)

Microprobe analysis of inclusion bodies related to two benzofuran derivatives

Two benzofuran derivatives, chlorpromazine and amiodarone, are known to produce inclusion bodies in human tissues. Prolonged high dose chlorpromazine therapy causes hyperpigmentation of the skin with electron-dense inclusion bodies present in dermal histiocytes and endothelial cells ultrastructurally. The nature of the deposits is not known although a drug-melanin complex has been hypothesized. Amiodarone may also cause cutaneous hyperpigmentation and lamellar lysosomal inclusion bodies have been demonstrated within the cells of multiple organ systems. These lamellar bodies are believed to be the product of an amiodarone-induced phospholipid storage disorder. We performed transmission electron microscopy (TEM) and energy dispersive x-ray microanalysis (EDXA) on tissue samples from patients treated with these drugs, attempting to detect the sulfur atom of chlorpromazine and the iodine atom of amiodarone within their respective inclusion bodies.A skin biopsy from a patient with hyperpigmentation due to prolonged chlorpromazine therapy was fixed in 4% glutaraldehyde and processed without osmium tetroxide or en bloc uranyl acetate for Epon embedding.

Cerebellar lamellar bodies in two strains of murine scrapie

Lamellar inclusion bodies have been described by other authors in the granular layer of the cerebellum of people suffering from the unconventional slow-virus disease of kuru and were thought to be associated with the disease. In this study, these bodies were also found in mice infected with scrapie, which belongs to the same group of diseases, as well as their age-matched controls.

Silica-induced alveolar cell tumors in rats.

Min-U-Sil5, a form of alpha quartz, has been shown to induce peripheral lung tumors in rats exposed to the dust by inhalation. The animals were exposed to a nominal particle concentration of 12.4 mg/m3 for 8 hr/day, 4 days/week, for 2 years. The induced tumors were large and peripheral, and, when examined by electron microscopy, were found to be composed predominantly of alveolar type II cells. These cells were found in papillary, acinar, and solid forms of the tumors and were characterized by lamellar inclusion bodies. This is in contrast to the mouse, in which the papillary form was associated with Clara cells and the acinar form was linked with the type II cell. In this study, the Clara cell was a minor component of the tumor mass. No clear risk is established in man linking silica exposure to increased lung tumor rates.

An In vitro lung epithelial cell system for evaluating the potential toxicity of inhalable materials

Assays have been developed using a lung epithelial cell strain, LEC, for evaluating the cytotoxicity and genotoxicity of gases, vapours, particles and fibres. LEC was derived from an adult male Fischer 344 rat and was cultured continuously in vitro for over 60 passages. The cells had an epithelial morphology, a near-diploid modal chromosome number of 43–44, and properties of untransformed cells. Lamellar inclusion bodies were found in the cytoplasm, suggesting that LEC originated from the type II alveolar cells. The ability to LEC to metabolize xenobiotics was demonstrated by the formation of polar metabolites from benzo[ a]pyrene, the induction of gene mutation by promutagens in a metabolically incompetent cell line (Chinese hamster ovary cells) after co-cultivation with LEC, and the induction of sister chromatid exchange in LEC by promutagens. LEC will grow on collagen gel in the absence of an overlying medium, thereby constituting an in vitro exposure system closely resembling lung epithelium in vivo. The cytotoxic potential of inhalable agents, including nitrogen dioxide, phenol vapours, formaldehyde, automobile exhaust, titanium dioxide and chrysotile and crocidolite forms of asbestos was studied. The in vitro cytotoxicity appeared to correlate well with the known in vivo pulmonary toxicity of the substances studied. Using chromosomal-aberration induction as an endpoint, chrysotile asbestos was found to be genotoxic to LEC. Our results suggest that LEC may be a useful in vitro system for the evaluation of the toxic potential of inhalable materials.

Ultrastructural and immunofluorescent studies of acute and chronic lactate dehydrogenase elevating virus-induced nonparalytic poliomyelitis in mice.

Light microscopic, electron microscopic, and immunofluorescent studies have been performed on young C58/J mice following administration of cyclophosphamide and peripheral inoculation of the C strain of lactate dehydrogenase-elevating virus (LDV-C). Using special fixation techniques, all three types of studies were performed on the spinal cord tissues obtained from the same mice. LDV-C infection-specific immunofluorescence was detected in anterior horn neurons. Ultrastructurally, anterior horn neurons during the acute inflammatory phase of the disease were characterized by various degrees of chromatolysis, an increase in the number of lamellar inclusion bodies, and extensive membrane proliferation of perinuclear endocytoplasmic reticulum. A chronic vacuolar appearance of the anterior horn was due to intraneuronal vacuolation as well as vacuolation of the neuropil; these changes may have been due to expansion of proliferated intracytoplasmic membranes and/or focal ischemia secondary to the acute inflammatory response. Direct evidence was obtained that indicated the cytopathology was not immune mediated in that highly poliomyelitis-susceptible 18-month-old C58/J mice developed severe paralysis without inflammation of the spinal cord when treated with cyclophosphamide before infection.

The presence of abnormal lysosomes in lymphocytes and neutrophils during chloroquine therapy: a quantitative ultrastructural study.

Lysosomal ultrastructure in circulating lymphocytes and neutrophils from the blood of 22 patients with rheumatoid arthritis or systemic lupus erythematosus undergoing chloroquine therapy has been examined and compared with that of rheumatoid cases receiving alternative forms of treatment and non-rheumatoid controls. The lymphocytes of all 22 patients receiving chloroquine showed a highly significant number of lamellar inclusion bodies (mean 9.3%) compared with only occasional inclusions in six of the 14 rheumatoid and four of the 12 non-rheumatoid controls (means 0.86% and 0.83% respectively). Neutrophils from eight chloroquine-treated patients also showed abnormal granules which were absent from both control groups. These findings confirm our earlier in vitro work which indicated that lymphocytes, compared with neutrophils, had a greater susceptibility to chloroquine action with respect to alterations in lysosomal structure.

Alveolar pre-type II cells from the fetal rabbit lung isolation and characterization

A method for preparing a homogeneous population of undifferentiated cells from the fetal rabbit lung is described. This method utilizes enzymatic digestion, differential adhesion to remove fibroblasts and centrifugation on a discontinuous metrizamide gradient. Cells isolated by this procedure replicate in vitro in medium supplemented with carbon-stripped fetal bovine serum. Mitosis can also be stimulated by heat-inactivated medium conditioned by fetal lung fibroblasts. After confluence, exposure of these cells to 0.55 or 55 nM dexamethasone significantly increased the incorporation of [ 14C]choline into phosphatidylcholine. Lower concentrations of the drug also increased incorporation, but not significantly so. Addition of heat-inactivated fibroblast-conditioned medium produced a 25% increase in choline incorporation, but this was not significant. Furthermore, the presence of conditioned medium tended to reduced the response of the cells to dexamethasone. After confluence, lamellar inclusion bodies were present in more than 90% of those cells exposed to dexamethasone. These organelles were not observed in cell monolayers not exposed to the steroid. These cells did contain a few small electron-dense bodies. The latter may be immature multivesicular bodies.

Malignant mesothelioma

Mesotheliomas and metastatic adenocarcinomas involving the pleura are frequently difficult to distinguish by light-microscopic and histochemical methods. In a double-blind study, we have compared ultrastructural features of 10 mesotheliomas of epithelial type and 10 adenocarcinomas from the lung, breast, and upper GI tract, i.e., sites known to give rise to metastases which mimic mesothelioma. Mesotheliomas were observed to have a significantly greater microvillus length/diameter ratio (LDR) than adenocarcinomas (p less than 0.01) and more abundant intermediate filaments (p less than 0.001). Mesotheliomas had more complex microvilli than adenocarcinomas, whereas adenocarcinomas had rootlets (2/10 cases) and lamellar inclusion bodies (2/10 cases), both of which were absent in the mesotheliomas. This study provides quantitative and qualitative ultrastructural features of potential utility in the differential diagnosis of pleural mesotheliomas and adenocarcinomas.

A simple method for obtaining cultures enriched for Type II pneumonocytes from fetal rabbit

A differential plating method permitted preparation of cultures significantly enriched for Type II pneumocytes. These cells were maintained in a differentiated state for at least 12 d, identifiable morphologically (by presence of osmiophilic lamellar inclusion bodies) and bio-chemically (by demonstration of synthesis of phosphatidyl choline and production of disaturated lecithin).

Assessment of fetal pulmonary maturity by phospholipid analysis of amniotic fluid lamellar bodies

Lamellar inclusion bodies, when present in amniotic fluid, can be quantitatively harvested as a 10,000 × g pellet fraction. Phospholipid analysis of this fraction obtained from human amniotic fluid at various gestational ages revealed a developmental profile which correlated with individual stages in fetal pulmonary maturation. A presurfactant stage (i.e., indicating that the fetal lung has not yet begun to secrete surfactant), the first appearance of lamellar bodies in amniotic fluid, and the accumulation and biochemical maturation of lamellar bodies (as indicated by the appearance and accumulation of phosphatidylglycerol [PG]) were identified by the phospholipid composition of this fraction. Further investigations indicated that the earlier stages of development could be identified simply by measurement of the lecithin content of this fraction, and later stages, by the presence of PG. Since lecithin consistently represented 80% of the total phospholipids when PG was present, this factor could be accurately used in estimating the percentage of PG from the measurement of lecithin and PG alone.