R. Laschi

Detection of mucus glycoconjugates in human conjunctiva by using the lectin‐colloidal gold technique in TEM

Abstract The mucus glycoconjugates produced by conjunctival goblet cells in dry‐eye patients were studied by a specific cytochemical reaction in Transmission Electron Microscopy (TEM). Four lectins, proteins of vegetal origin which specifically bind glycosidic residues, (WGA, PNA, SBA and ConA) were used conjugated with colloidal gold as ultrastructural marker. We performed a quantitative analysis by counting the colloidal gold particles present on mucus granules. The results were compared with normal conditions. We found a decrease in sialic acid, N‐acetyl‐glucosamine, N‐acetyl‐galactosamine and galactose‐N‐acetyl‐galacto‐samine and an increase in mannose. The different content of glycoconjugates in goblet cells may reflect in the change of physical and functional properties of mucus. We think these data may be useful in the search for a therapeutic mucomimetic drug.

Detection of mucus glycoconjugates in human conjunctiva by using the lectin-colloidal gold technique in TEM. II. A quantitative study in dry-eye patients.

Abstract We applied a specific cytochemical reaction to characterize the glycoconjugates produced by goblet and non‐goblet epithelial cells of normal human conjunctiva. For this purpose we utilized the lectins, proteins of vegetal origin, which are extremely sensitive in binding glycosidic residues. In particular, we used WGA, PNA, SBA and ConA conjugated with colfoidal gold as ultrastructural marker for Transmission Electron Microscopy. This technique allowed us also to perform a quantitative analysis, by counting colloidal gold particles present on mucus granules. In this way we analyzed the content both of goblet and non‐goblet epithelial cells. In the former, WGA, PNA, SBA and ConA receptors, here reported in decreasing density, were present. In the latter WGA was always positive, SBA and PNA sometimes were negative, ConA was always negative. We speculate the different contribution to mucus production by these two sources may be important in evaluating tear film stability alterations occurring in those diseases in which non‐goblet epithelial cell vesicles increase.

Relationship between connective tissue cells and fibronectin in a sequential model of experimental hepatic fibrosis

SummaryThe cellular and non-cellular components of fibrous septa formed at early and late stages in a sequential model of experimental hepatic fibrosis have been investigated using ultrastructural and immunocytochemical techniques. In the early septa, cells with intermediate features between lobular Ito cells and active fibroblasts were formed. These cells frequently displayed subplasmalemmal microfilaments (myofibroblast-like cells). Macrophages were also present. Scanty typical fibroblasts were present in the late septa. This cellular recruitment might be related to an extracellular glycoprotein-fibronectin-which is at present under investigation as a chemotactic factor for fibroblasts. Strong positivity for fibronectin in early septa and its sharp decrease in late septa seems to support this view. Fibroblasts and/or macrophages are the likely source of fibronectin synthesis.

Mucus alteration and eye dryness A possible relationship

Abstract. From a very early phase we studied 15 patients suffering from a dry‐eye condition not associated to systemic diseases. Conjunctival biopsies were studied in Transmission (TEM) and Scanning (SEM) electron microscopy. Moreover, the lectin‐gold cytochemistry at ultrastructural level was applied to investigate the distribution of some glycosidic receptors produced by both the goblet cells and the vesicles belonging to the Second Mucus System (SMS). No evidence of epithelial stratification and only a decrease in the goblet cell population was observed. The SMS vesicles and the superficial cell microvilli did not appear greatly reduced in number. A difference in the mucus composition in terms of content of glycosidic residues was detected in dry‐eye patients compared to the normal subjects. The role of the mucus produced by both the goblet cells and the SMS vesicles is debated. A possible correlation between the alteration of the mucus content and the failure of the tear film stability is proposed. On the basis of these data, a new therapeutic approach for eye dryness is suggested.

Ultrastructural and immunohistochemical contribution to the histogenesis of human cardiac myxoma.

The ultrastructural features of 8 human cardiac myxomas were analyzed and correlated with immunohistochemical data, with the aim to clarify the characteristics of the cell lines involved in the tumor genesis. Immunohistochemical studies were performed to detect the presence and the distribution of intracytoplasmic filaments (vimentin, desmin, actin, myosin) as well as myoglobin and factor VIII-related antigen, albumin, and lysozyme. Eighty percent of myxoma cells were simultaneously positive for vimentin, desmin, and actin, whereas 30% of them stained with antifactor VIII and antivimentin antibodies. The submicroscopic analysis revealed two main cell populations: (1) one composed of stellate-shaped cells with scanty organelles and sparse hyaloplasmic filaments scattered throughout the myxoid stroma and forming a loose network with their projections; (2) another one included cells with more cytoplasmic organelles, intermediate filaments, and myofilaments arranged either singly or in both solid and hollow cord-like structures. Our results support the hypothesis that cardiac myxoma may originate from a reserve multipotent mesenchymal cell able to differentiate more or less completely along two major evolutional lines: myoid and endothelial. The tumor tissue thus seems to be involved in vessel formation, suggesting a growth pattern akin to that manifested in other forms of endocardial pathological reactivity in which reserve mesenchymal cells are engaged.

An Ultrastructural and Immunocytochemical Analysis of Human Endothelial Cell Adhesion on Coated Vascular Grafts

Human adult endothelial cells were enzymatically harvested from adipose tissue. Cell viability was established by Trypan blue exclusion and transmission and scanning electron microscopy. Endothelial cells were identified by immunocytochemical investigation at light microscopy, transmission electron microscopy, and scanning electron microscopy. Isolated cells were positive for actin and vimentin, negative for desmin. Factor VIII RA was mainly expressed at cell surface and occasionally disclosed in the cytoplasm. Reactivity for UEA I and J15 was weak or undetectable. Human endothelial cells were seeded and left to adhere for one hour onto different nonvascular substrates (glass, poly-l-lysine, formvar-carbon, fibronectin, Teflon). Scanning electron microscopy defined surface features, suggesting tenacious cell adhesion on the substrate. Different vascular substrates were tested (preclotted Dacron, albumin Dacron, Hemashield Dacron, Gelseal Dacron, ePTFE, fibronectin-ePTFE). Commercially available coated grafts showed qualitative and quantitative differences in cell adhesion. In particular, Gelseal Dacron provided the best quantitative results, even though a wide variability was observed. In contrast, fibronectin-coated ePTFE gave more reliable results and high spreading efficiency. In the short term, coated grafts do not seem to offer greater advantages than fibronectin-coated ePTFE. However, specific incubation times for each coated graft should be selected and the long-term approach (graft culture) should also be attempted.

Dry eye before and after therapy with hydroxypropyl methylcellulose. Ultrastructural and cytochemical study in 20 patients.

We have utilized hydroxypropyl methylcellulose in the therapy of 20 patients suffering from dry eye in the initial stage. The effects of the substance have been evaluated both from a clinical and a morphological standpoint. In particular, transmission and scanning electron microscopy were applied to investigate the ultrastructural features of the conjunctival epithelium before and after treatment. In addition, lectin cytochemistry was carried out to analyse semiquantitatively the presence of glycoconjugates in the conjunctival mucus produced by goblet and non-goblet epithelial cells. Hydroxypropyl methylcellulose did not improve the tear film stability as demonstrated by the clinical tests. The normal surface of the conjunctival epithelium did not appear recovered as well. Only a minimal increase in the density of the second mucus system vesicles was observed. From a cytochemical point of view, the content of glycosidic residues appeared altered in dry-eye patients both before and after treatment. This alteration can lead to changes in the physical and functional properties of the mucus, impairing its role as a surfactant. We conclude that the common treatment by means of lubrificant-like substances palliates the dryness condition. The search for a drug which reintegrates the correct composition of the conjunctival mucus should be seriously considered as a valid alternative.

Detection of mucus glycoconjugates in human conjunctiva by using the lectin-colloidal gold technique in TEM. III. A quantitative study in asymptomatic contact lens wearers.

Abstract We characterized the mucus glycoconjugates produced by goblet and non‐goblet epithelial cells in asymptomatic contact lens (CL) wearers. We employed four lectins (proteins of vegetal origin which specifically recognize glycosidic residues: WGA, PNA, SBA and ConA) conjugated with colloidal gold as ultra‐structural marker, at Transmission Electron Microscopy. A computerized quantitative analysis was carried out in order to compare the results from the CL wearers to those from the control patients. Goblet cells produce different amount of glycosidic residues, in particular, a significant decrease in the distribution of sialic acid, N‐acetylglucosamine, N‐acetylgalactosamine, galactose‐N‐acetylgalactosamine and mannose was observed. The content of glycosidic residues in the mucus vesicles of the non‐goblet epithelial cells appeared unchanged as to the normal situation. We speculate that the CL could possibly contribute to the failure of the tear film stability by altering the production of mucus.

Immunocytochemical analysis of contact lens surface deposits in transmission electron microscopy

We studied 8 soft contact lenses from asymptomatic wearers by means of an immunocytochemical method, in transmission electron microscopy. In particular, the presence of IgA, IgG, IgE, Clq complement fraction within the surface deposits was analyzed. All the lenses were found positive for the immunoglobulins and the Clq, being the tarsal side more heavily coated than the corneal one. IgA was the predominant Ig, followed by IgG, IgE, and Clq in this descending order. New, never worn lenses were found completely negative for any of the proteins under investigation. We conclude that the Igs come from the tear fluid and speculate about the Clq as a possible sign of involvement of the host immuno-defense mechanism against the prosthesis.

Ependymoma of the Foramen of Monro: Ultrastructural Characterization

The characteristics of clear cells of an ependymoma of the foramen of Monro have been studied by electron microscopy to precisely define its organellar composition and to establish the tumor histogenesis. Our data confirm that the once-thought oligodendroglial is, in fact, an ependymal tumor. Both the scarce number of organelles, owing to the low degree of differentiation, and the abundance of hyaloplasmic lipid vacuoles can account for the clear appearance of these tumor cells.