Lysosomes Of Epithelial Cells Research Articles

Intracellular sorting of differently charged chitosan derivatives and chitosan-based nanoparticles.

Chitosan (Chi) is a biodegradable nontoxic polycation with multiple reactive groups that is easily used to obtain derivatives with a desired charge and hydrophobic properties. The aim of this work was to study the intracellular traffic of positively charged hexanoyl-chitosan (HC) or HC-based nanoparticles (HCNPs) and negatively charged succinoyl-chitosan (SC) and SCNPs in epithelial and macrophage cell lines. By using flow cytometry we demonstrated that positively charged HC adhered to cell membranes quicker and more efficiently than negatively charged SC or NPs. However confocal studies showed that SC and SCNPs penetrated cells much more efficiently than HC while HCNPs did not enter the epithelial cells. Macrophages also phagocyted better negatively charged material but were able to engulf both HC and HCNPs. Upon entering the cells, SC and SCNPs were co-localized with endosomes and lysosomes while HC was found in mitochondria and, to a lesser extent, in lysosomes of epithelial cells. Macrophages, RAW264.7, more efficiently transported all Chi samples to the lysosomal compartment while some positively charged material was still found in mitochondria. Incubation of Chi derivatives and ChiNPs at pH specific to mitochondria (8.0) and lysosomes (4.5) demonstrated the neutralization of Chi charge. We concluded that epithelial cells and, to a lesser extent, macrophages sort charged material to the organelles neutralizing Chi charge.

Cholera Toxin Suppresses Expression of Ubiquitin Editing Enzyme A20 and Enhances Transcytosis

Background: The ubiquitin editing enzyme A20 plays an important role in maintaining the homeostasis in the body Microbe-derived adjuvants are commonly used in animal models of intestinal allergy. Objective: This study aims to investigate the role of cholera toxin-induced A20 suppression in compromising intestinal barrier function. Methods: Human intestinal epithelial cells were cultured into monolayers as an in vitro epithelial barrier model. Ovalbumin (OVA) was used as a specific allergen to test the degrading capability of intestinal epithelial cells for the endocytic allergens. The fusion of endosomes and lysosomes in epithelial cells was observed by immunocytochemistry. The antigenicity of OVA was tested by T cell proliferation assay. Results: A20 was detectable in the intestinal cell lines and mouse intestinal epithelialum. A20 was required in the degradation of endocytic allergens in HT-29 cells. The allergen, OVA, could pass through A20-deficient HT-29 monolayer barrier. Exposure to microbial adjuvant, cholera toxin, suppressed the expression of A20 in HT-29 cells, which compromised the epithelial barrier function. Conclusion: The microbial product, cholera toxin, interferes with the expression of A20 in intestinal epithelial cells, which compromises the intestinal epithelial barrier function.

Renal Lipofuscinosis in Danish Slaughter Cattle

A study was performed to characterize dark brown or black discoloured kidneys ("black kidneys") in Danish slaughter cattle and to investigate the aetiology and pathogenesis. In 133 939 cattle entering four abattoirs, 359 cases of "black kidneys" were recorded. Of these, 57 cases were submitted for macroscopical, microscopical, and ultrastructural examination. A pigment with characteristics similar to those of lipofuscin was found in secondary lysosomes in epithelial cells of the proximal tubules. Pigment accumulation was the cause of discoloration, with a positive correlation between the discoloration of the renal cortex and the degree of pigment accumulation. Cases occurred only in cattle of the Holstein breed or the Red Danish Dairy breed and mainly in animals aged 3 years or older. In these breeds, prevalences of 0.44% and 2.51% were found, respectively. Epidemiological analyses indicated that affected animals aged 4.5 to 6.5 years or 7.5 to 8.5 years were culled more frequently than unaffected cattle. Epidemiological and genealogical analyses strongly indicated a genetic aetiology with simple autosomal recessive inheritance.

LIMP-2/LGP85 deficiency causes ureteric pelvic junction obstruction, deafness and peripheral neuropathy in mice

In previous overexpression studies we revealed a role for the lysosomal membrane protein LIMP-2/LGP85 in lysosomal biogenesis. LIMP-2-deficient mice show an increased postnatal mortality which is associated with a development of a uni- or bilateral hydronephrosis caused by an obstruction of the ureteropelvic junction. An accumulation of lysosomes in epithelial cells of the ureter adjacent to the ureteral lumen and a disturbed apical expression of uroplakin was observed, suggesting an impairment of membrane transport processes. Serious hearing impairment in LIMP-2-deficient animals was indicated by deficits in acoustic startle responses, in brainstem evoked auditory potentials and a reduced endochondral potential. LIMP-2-deficient mice suffer from a massive decline of spiral ganglia in the cochlea concomitant with that of the inner and outer hair cells. These pathological changes begin at the age of 3 months and are probably secondary to a degeneration of the stria vascularis. LIMP-2-deficient mice are also characterized by a peripheral demyelinating neuropathy. Demyelinization was found to be associated with a massive loss of peripheral myelin proteins and an increased activity and expression of lysosomal proteins highlighting a hitherto unknown role of the lysosomal compartment in the development of this myelination disorder. The phenotype of LIMP-2-deficient mice stimulates the search for mutations in human disorders associated with degeneration of the stria vascularis and/or demyelinization of peripheral nerves.

Immunolocalization of cathepsin D in pneumocytes of normal human lung and in pulmonary fibrosis.

Cathepsin D expression has been assessed by immunohistochemistry and immunoelectron microscopy in fetal, normal adult and injured lungs of human beings. In addition to the well known positivity of alveolar macrophages and the bronchial epithelial cells, normal type I and to a lesser extent type II pneumocytes showed a granular, cytoplasmic staining pattern. Using immunogold labelling of lowicryl embedded human lung, cathepsin D was present in lysosomes of epithelial cells. Double immunofluorescence labelling employing type I and type II specific antibodies or lectins confirmed the epithelial staining for cathepsin D. At the terminal sac period during lung development cathepsin D appears in the alveolar epithelium. In fibrotic specimens, enhanced immunoreactivity was found in epithelial and non-epithelial cells. Proliferative epithelial formations were strongly stained with cathepsin D antibodies, whereas detached, desquamated epithelial cells were weakly positive or negative. We suggest that cathepsin D plays a role in the remodelling process during fibrogenesis.

Immunocytochemical localisation of some lysosomal hydrolases, their presence in luminal fluid and their directional secretion by human epididymal cells in culture

The way in which the human epididymis modifies spermatozoa during their sojourn in this structure might be clarified by knowledge of the nature of its secretions. We have examined the presence of several lysosomal hydrolases in human epididymal tissue and fluids, and their synthesis and secretion by monolayer cultures. Tissues were obtained from men undergoing orchidectomy for prostatic carcinoma. The enzymes cathepsin D and acid alpha-glucosidase were localised in the lysosomes of epithelial cells from the corpus epididymidis, by an immunocytochemical technique. Cathepsin D was also found in epithelial cells of the efferent ducts within lysosomes, apical vesicles and multivesicular bodies. No immunolocalisation of acid glucosidase in the efferent ducts or on the microvilli of the corpus was demonstrable. Cathepsin D, beta-hexosaminidase (N-acetylglucosaminidase) and alpha-glucosidase were measurable in the luminal fluid from the human corpus epididymidis; beta-hexosaminidase was secreted into the culture medium by confluent monolayers of epididymal and efferent duct cells. Immunoprecipitation of cell extracts and culture medium of these cultures incubated with 35S-methionine revealed that the precursors of cathepsin D and beta-hexosaminidase were synthesized and secreted by such monolayers. Thus, active lytic enzymes are secreted by the human epididymis and could modify sperm membranes.

Thiamine pyrophosphatase cytochemistry in rat endometrium during the oestrous cycle.

The functional morphology of the Golgi apparatus was studied in various types of cells in the rat endometrium during the oestrous cycle. A cerium-based enzyme-cytochemical method was used for the ultrastructural visualization of the activity of thiamine pyrophosphatase (TPPase). The cerium-based method was evidently superior to the classical lead technique, which was used for comparison. TPPase activity in luminal and glandular epithelial cells displayed cyclical modulation and redistribution. It was restricted to only one or two narrow trans lamellae during dioestrus but extended during proestrus and oestrus into nearly all trans-to-cis lamellae of the well-compartmentated Golgi apparatus. A homogeneous staining reaction, which was particularly intense during the latter two phases and only partly due to unspecific alkaline phosphatase, was confined to the apical and basolateral plasma membranes of luminal epithelial cells. In the stromal fibroblasts, only one short Golgi saccule was positive at dioestrus, whereas three or more trans Golgi lamellae were filled with reaction product during oestradiol-dominated oestrus. TPPase activity was furthermore observed in the lysosomes in epithelial cells, stromal fibroblasts, capillary endothelial cells and pericytes. The present findings of cyclic changes in TPPase activity in epithelial cells and stromal fibroblasts provide the first evidence of cyclic modulation and redistribution of this enzyme in the endometrium.

Capillary Permeability and Basolateral Endocytic Pathway of the Epithelium in the Mouse Endolymphatic Sac in vivo

The capillary permeability and the basolateral endocytic pathway of the mouse endolymphatic sac (ES) epithelium were examined in vivo using intravenous injection of horseradish peroxidase (HRP). The capillaries of the ES were classified as either fenestrated or non-fenestrated. Because dense reaction products were observed soon after injection of HRP in macrophages near both types of capillaries, they were both considered to be permeable to macromolecules. In non-fenestrated capillaries, the basement membrane and small vesicles of the endothelium of the ES were stained with reaction product. These non-fenestrated capillaries were considered to be of muscle type. After 15 min, ES epithelial cells absorbed HRP basolaterally, and the multivesicular bodies and lysosomes of epithelial cells were stained with reaction product. The process of basolateral absorption in the ES epithelium was similar to that in the intestinal epithelium. Our results provide further evidence that the ES is a metabolically active organ which plays an important role in fluid transport in the inner ear.

The State of Differentiation of Embryonic Chicken Lens Cells Determines Insulin-Like Growth Factor I Internalization*

Microdissected epithelial cells from chicken embryonic lens differentiate into fiber cells during primary culture. Binding of IGF-I to both cell types has been documented biochemically. This report describes differences in the internalization of insulin-like growth factor I (IGF-I) in the two cell types using three electron microscopic approaches. Cells were incubated with a biologically active colloidal gold-labeled-IGF-I complex or [125I]IGF-I and prepared for electron microscopy. Other cells were incubated with unlabeled IGF-I and prepared for immunoelectron microscopy, using a colloidal gold-labeled anti-IGF-I antibody to detect the IGF-I. Each technique demonstrated binding of IGF-I on the surface of epithelial and differentiated fiber cells. IGF-I was internalized in epithelial cells. In contrast, the ligand exhibited little endocytosis in fiber cells during a 5 h incubation. Intracellular IGF-I was observed in the endosomes, Golgi apparatus, and lysosomes of epithelial cells, in addition, it was shown for the first time that IGF-I was translocated to the nucleus of epithelial cells. The differences between epithelial and fiber cells regarding internalization and nuclear translocation of IGF-I suggest that there are cell-specific itineraries of the hormone, depending on the differentiation stage of the cell. These differences may relate to the specific biological actions of the growth factor on each of these cells, particularly in allowing proliferation and differentiation of the epithelial cells.

Ultrastructural changes in the kidneys of rabbits treated with lead acetate.

Electron microscopical studies were carried out on the kidneys of rabbits given s.c. injections of 0 (control), 0.25 or 0.50 mg lead acetate/kg b.w. 3 times a week during 14 weeks. At the end of the experimental period the animals had lead blood levels of 60, 500 and 600 micrograms/l whole blood respectively. Treatment-related renal changes were found in the proximal tubules; they consisted of a dose-related increase in the amount of lysosomes in epithelial cells of the convoluted part, and of severely damaged cells and loss of brush border in the straight part. There was also an increase in lysosomal tubular inclusions, which are considered characteristic of lysosomes of the proximal tubular cells of the rabbit kidney. The significance of these findings for assessing the risk of occupational exposure to lead is briefly discussed.

Intestinal fine structure in Crohn's disease. Lysosomal inclusions in epithelial cells and macrophages.

Resected intestines from eight patients with Crohn's disease and three control cases were investigated by transmission electron microscopy. Characteristic changes were observed in the mucosa of all Crohn's disease specimens, most typically an infiltration of numerous macrophages into the propria. These cells displayed large lysosomes with inclusions which were mainly dense, irregularly shaped and composed of aggregated particles and bizarre-shaped, myelin-like figures. Similar inclusions were also found in the lysosomes of the surface epithelial cells and the macrophages in the submucosa. This latter layer otherwise consisted of an oedematous, collagenous connective tissue. The muscularis appeared structurally unaffected. Qualitatively, the findings were almost identical in all patients with Crohn's disease, but varied quantitatively without any clear correlation with the clinical histories. Moreover, in all cases typical alterations were found not only in the macroscopically most clearly affected parts of the intestines, but also in grossly normal regions, close to the margins of resection. The analogy of the fine structural findings with those of granulomas produced by injection of bacteria into experimental animals suggests that a microbial invasion of the intestinal wall may have initiated the disease. It therefore seems reasonable to assume that the lysosomal inclusions we observed represent partly degraded bacteria. More occasionally, virus-like particles were found within the lysosomes of the epithelial cells and the macrophages in the underlying propria. In view of the diffuse spread of the alterations it seems possible that there exists a generalized defect in the barrier function of the intestine in Crohn's disease. This could lead to passage of bacteria and/or other agents into the mucosa, followed by an influx of inflammatory cells from the blood. Storage of non-degradable microbial components in macrophages could then be responsible for the initiation and propagation of a chronic inflammatory process, similar to that of other granulomatous disorders.

Final stages of erythrophagocytosis in the sheep placenta

In trophoblastic epithelial cells of the sheep placenta the final stages of erythrocyte breakdown within the lysosomal apparatus were studied at the ultrastructural level. As a result of hemoglobin digestion lysosomes containing hemoglobin-derived pigments (HDP) were formed. The HDP-lysosomes were acid phosphatase-positive, highly electron-dense bodies of round to irregular shape containing whorled membranous formations. The accumulation of these lysosomes in epithelial cells led to fusion resulting in the formation of conglomerates. At the end of the gestation period the amount of HDP-lysosomes and their conglomerates markedly increased. In addition to erythrocytes the trophoblastic epithelial cells in the erythrophagocytic regions phagocytosed maternal leukocytes and neighbouring epithelial cells and giant cells. By gradual accumulation of HDP-lysosomes and remnants of phagocytosed cells, highly electron-dense acid phosphatase-positive residual bodies of variable appearance were formed within the epithelial cells. At the end of pregnancy the spaces between juxtaposed villi of the trophoblastic epithelium in the erythrophagocytic zones were occluded by apposition of the epithelial cells. In these occluded regions an increase in highly electron-dense large-sized residual bodies (15-22 micrometer of dimension) occurred as a result of multiple cell phagocytosis in combination with fusion. In these residual bodies the numerous incorporated HDP-lysosomes and the remnants of phagocytosed cells could still be recognized.