Ultrastructural and electron immunohistochemical features of medullary thyroid carcinoma

Summary An ultrastructural study, both morphological and immunohistochemical, has been carried out on eight thyroglobulin-positive and nine thyroglobulin-negative medullary carcinomas of the thyroid. The morphometric analysis of granule size showed that all tumours contained cells with small granules and cells with medium size granules, whereas eight tumours had additional cells with large granules. The small granules had an electron dense core, while the medium and large sized granules were both pale-cored and dense-cored. The cells with small, medium or large secretory granules were all immunoreactive for calcitonin and CGRP. No ultrastructural differences were observed between thyroglobulin-positive and thyroglobulin-negative cases of medullary carcinoma of the thyroid.     

Morphological characteristics and stimulus-secretion coupling in bovine adrenal chromaffin cell cultures

Chromaffin cells isolated from adult bovine adrenal medullae were plated on collagen-coated dishes in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. After 8–16 h in culture, the chromaffin cells had started to develop one, two or, more rarely, three neurites. The neurites grew at a rate of7.25 ± 0.07μm/day showing a linear correlation between process length and age of the culture. The neurites displayed one or more varicosity-like structures. Some neurites made long lasting contacts with other cell bodies and processes. Cytochemical tests indicated positive catecholamine fluorescence in boti cell bodies and processes. Fluorometric determinations indicated that by day 7 the catecholamine content was75.7 ± 1% of that found on day 1 and that noradrenaline and adrenaline represented24.6 ±1% and75.3 ± 1% of the total amine content respectively. Catecholamines were released from these cultured chromaffin cells in response to stimulation by either 56 mm KCl or acetylcholine. The stimulation-induced release of amines was Ca²⁺-dependent and was blocked by increasing the extracellular c oncentration of Mg²⁺. Substitution of (a) extracellular Na²⁺ by either choline or sucrose or (b) Ca²⁺ by Ba²⁺ produced a sharp increase in catecholamine output. Acetylcholine produced a doserelated increase in catecholamine release; half maximal release was produced by3.5 × 10^?5m acetylcholine and maximal release by 10^?4m acetylcholine. The cholinergic receptor of cultured chromaffin cells appears to be nicotinic since catecholamine release was stimulated by nicotine and carbamylcholine but not by pilocarpine. Acetylcholine-evoked catecholamine release was blocked by d-tubocurarine (ID₅₀ = 5.5 × 10^?7m), and by hexamethonium (ID₅₀ = 8 × 10^?6m). However, the acetylcholine-evoked amine release was not blocked by α-bungarotoxin at a concentration (1.25 × 10^?7m) which blocked frog rectus abdominus responses to acetylcholine (10^?6–10^?3m). Tetrodotoxin (5 × 10^?6m) produced47 ± 2% inhibition of the secretory response to acetylcholine (10^?4m) but it did not modify the amine release in response to 56 mm KCl.Adult bovine chromaffin cells in culture display some of the morphological and functional characteristics of sympathetic neurones. Consequently, the bovine chromaffin cell in culture appears to be a promising model for studies on development and secretion.     

Ultrastructural characteristics of thyroid medullary carcinoma

17 thyroid medullary carcinomas (TMC) were studied electron microscopically (EM). Two cell groups were revealed regardless of their histological structure: EM differentiated (with signs of C-cell differentiation) and non-differentiated (without these signs). There is no correlation between histological structure of the tumor and the degree of its EM differentiation. In patients who lived 5 years maximum after the diagnosis there were different histological types but EM the tumors had relatively monomorphous structure with domination of non-differentiated cells (more than 70%), high content of dark cells (50% and more) and low content of desmosomes (less than 30%). The prognosis is more favourable the higher is content of EM differentiated cells, the better desmosomes are developed and the lower is content of dark cells. The scope of surgery and local metastases do not influence the prognosis while distant metastases (stage IV) deteriorate the prognosis.     

Ultrastructural study of thyroid medullary carcinoma.

This study deals with 11 cases of thyroid medullary carcinoma of which 7 were familial cases including 3 cases of Sipple's syndrome and 4 cases of sporadic cases. There were also 2 cases of Cushing's syndrome. In addition to the previously described English literatures about human medullary carcinoma of the thyroid, the rod-shaped body with cristae and an abundant glycogen particles in the cytoplasm, nuclear inclusion bodies of cytoplasmic invagination and microvilli at the surface membrane of gland formation were found in our cases. The mean and mode diameters of secretory granules of all familial cases with only calcitonin secretion were larger than those of the sporadic cases with ectopic ACTH and beta-MSH in addition to calcitonin secretion. Ultrastructural study on non-cancerous follicles of grossly normal thyroid of two cases of early familial medullary carcinoma disclosed apparently increased C-cells which were not intrathyroidal metastases. It is supposed that the increased C-cells in the thyroid of the familial cases are multicentric C-cell hyperplasia.     

Potentiation of mitogenesis in adult rat chromaffin cell cultures by immunosuppressive agent FK506

Mild cognitive impairment (MCI) is a transitional state between normal aging and Alzheimer's disease (AD) and is a high-risk condition for dementia. The endothelial nitric oxide synthase (NOS3) gene encodes endothelial NOS, an enzyme that regulates the production of the vasodilatory nitric oxide associated with the cerebral small vessel pathology observed in early AD. We studied the distribution of genotype and allele frequencies of the NOS3 Glu/Asp polymorphism in a sample of 62 MCI subjects and 136 controls. Though no association between NOS3 gene variation and MCI status was observed, MCI cases carrying the Asp variant (T+) performed worse in the Mini-Mental State Examination, Wechsler Memory Scale (Revised) long-term visual memory and the phonetic verbal fluency tests. These results suggest that the T allele is a genetic risk factor for cognitive impairment in the elderly.     

Characterization of catecholamine release from deer adrenal medullary chromaffin cells

Isolated adrenal medullary chromaffin cells maintained in culture have been widely used to study neurosecretory events. Many of these studies have been conducted using cells obtained from the bovine adrenal. In this study we have cultured chromaffin cells from an alternative large animal model, the deer, and have conducted the first characterization of secretion from this preparation. Cervine chromaffin cells, preloaded with [3H]noradrenalin, displayed a strong secretory response to the cholinergic agonist carbachol, with a maximal secretion of approximately 28% cell content over 15 min. This response was reproduced by nicotinic but not muscarinic agonists and was similarly inhibited by nicotinic but not muscarinic antagonists. Nicotine-evoked secretion measured over a 15 min time period was inhibited approximately 50% by the L-type Ca2+-channel antagonist nifedipine and approximately 20% by N-type (omega-conotoxin GVIA) or N, P/Q-type (omega-conotoxin MVIIC) antagonists. In contrast the response was unaffected by omega-agatoxin IVA, a P/Q-type antagonist. In addition to nicotinic receptor stimulation, activation of PACAP or histamine H1 receptors resulted in a concentration-dependent increase in secretion. PACAP was approximately two-fold more effective than histamine although both were weaker secretagogues than nicotine. In contrast, cervine chromaffin cells did not respond to angiotensin II or bradykinin, two agents known to stimulate secretion from bovine chromaffin cells. These data provide an initial characterization of the secretory response from cervine adrenal medullary chromaffin cells indicating that there are marked similarities but also potentially significant differences between them and their far more extensively described bovine counterparts.     

Ultrastructural criteria of malignancy of adrenal medullary tumor

Histological analysis and electron microscopy of 12 benign pheochromocytomas and 9 malignant pheochromoblastomas showed that there are no reliable histological differences between cells of benign and malignant tumor of the adrenal medulla. The ratio of ultrastructurally differentiated and undifferentiated cells in the tumor can reflect their maturity; the more ultrastructurally undifferentiated and less differentiated cells in the tumor, the higher is the malignant potential of this tumor.     

Ultrastructural studies of the replication of fowl adenoviruses in primary cell cultures

The replication of four fowl adenovirus strains (FAV-1, strain Phelps; FAV-5, strains 340 and TR-22; and FAV-7, strain YR-36), in primary chick kidney cell cultures, is described. Differences were found in the distribution of virus particles and virus associated inclusions between viruses belonging to different cytopathology subgroups. Thus in cells infected with FAV-1 (Phelps) and FAV-5 (340) (i.e. subgroup 1) virus particles, as they increased in number, tended to become distributed peripherally, close to the nuclear membranes, with the virus associated inclusions in the centre of the nucleus. With FAV-5 (TR-22) and FAV-7 (YR-36) (i.e. subgroup 2) virus particles and associated inclusions became concentrated initially in the central nuclear area later increasing to fill the whole nucleus, with virus particles and inclusions completely intermixed. The virus-associated inclusions were found to be identical to those described in human adenovirus infected cells and the same nomenclature was adopted. Other inclusions found in infected nuclei, included tubular structures and inclusions composed of granular particles.     

Vasoactive intestinal peptide increases tyrosine hydroxylase activity in normal and neoplastic rat chromaffin cell cultures

Vasoactive intestinal peptide (VIP) acutely increases tyrosine hydroxylase (TH) activity in cultures of dispersed normal adult rat chromaffin cells and of PC12 rat pheochromocytoma cells. High concentrations of VIP (10 microM) produce about 3-fold increases in TH activity in both cell types. VIP also increases the content of cyclic adenosine 3':5'-monophosphate (cAMP) in PC12 cells. VIP may increase TH activity by promoting the cAMP-dependent phosphorylation of the enzyme. Nerve fibers containing VIP-like immunoreactive material have been reported in the adrenal medulla and in other catecholamine (CA)- storing tissues. This VIPergic innervation may regulate CA synthesis and other cAMP-dependent processes in these tissues.     

Ultrastructural features of a human neuroblastoma cell line treated with retinoic acid

This report examines the morphological changes that occur in a line of human neuroblastoma cells (LA-N-5) following treatment with retinoic acid, in vitro. The results demonstrate that retinoic acid induces pronounced differentiation of these cells. Perikarya aggregate into tight clusters and extend long processes that are frequently fasciculated. Growth cones appear at the ends of these processes. Transmission electron microscopy reveals that after 10 days of treatment these long neurites give rise to varicosities which contain clusters of large dense-core vesicles and smaller clear vesicles. After 18 days of treatment the cultures cease to differentiate further. The pattern of neurite outgrowth is very complex by this point and the frequency of growth cones and vesicle-containing varicosities is greatly increased compared with shorter treatments. Most of these varicosities contain a mix of large dense-core vesicles and smaller clear vesicles and in some profiles the clear vesicles are round while in others they are pleomorphic. Despite this increase in the number of vesicle-containing profiles no membrane specializations were seen that resemble mature synapses. The present results demonstrate that retinoic acid can produce morphological changes in these cells in culture, and that these changes closely mimic those of normal differentiating neurons in culture. Considered with previous studies, these findings suggest that this cell line might provide a useful model system for studying neural differentiation.     

Enkephalin-like immunoreactivity in human adrenal medullary cultures

Normal adult human chromaffin cells in culture contain abundant immunoreactive enkephalin and exhibit spontaneous and nerve growth factor-induced outgrowth of processes. In cultures of adrenals from two age-matched white males, approximately 70 per cent of chromaffin cells from one case and 50 per cent from the other contained immunocytochemically demonstrable leuenkephalin-like material. There was no apparent correlation between enkephalin-like immunoreactivity and capacity for process outgrowth. Cells with and without processes stained for enkephalin, and the percentages of stained cells with processes appeared to change in proportion of overall process outgrowth. Numerous cells with and without processes also failed to stain for enkephalin. Marked variations in sizes of stained cells and in distribution and intensity of staining were observed in all of the cultures. These findings suggest that adult chromaffin cells are heterogeneous at a number of levels.     

Ultrastructural study of poorly differentiated medullary carcinoma of the thyroid

Summary Five cases of sporadic medullary carcinoma of the thyroid (MTC) with rapidly progressive disease were studied ultrastructurally. The tumour cells had poorly differentiated C cell characteristics. They exhibited smaller secretory granules in their narrow cytoplasm. Morphometric analysis disclosed that the average diameter of the secretory granules of the cases with a poor prognosis was 173.0 nm in comparison with 254.2 nm of well differentiated cases. The granules were fewer in the poor prognostic group (1.31/µ²) than the well differentiated group (2.75/µ²). Increased free ribosomes and polysomes were noted in the cytoplasm and dispersed chromatin in the nuclei. These cases should be therefore classified as poorly differentiated MTC rather than atypical or anaplastic MTC.     

Ultrastructural analysis of primary human urethral epithelial cell cultures infected with Neisseria gonorrhoeae.

In men with gonococcal urethritis, the urethral epithelial cell is a site of infection. To study the pathogenesis of gonorrhea in this cell type, we have developed a method to culture primary human urethral epithelial cells obtained at the time of urologic surgery. Fluorescent analysis demonstrated that 100% of the cells stained for keratin. Microscopic analyses indicated that these epithelial cells arrayed in a pattern similar to that seen in urethral epithelium. Using immunoelectron and confocal microscopy, we compared the infection process seen in primary cells with events occurring during natural infection of the same cell type in men with gonococcal urethritis. Immunoelectron microscopy studies of cells infected with Neisseria gonorrhoeae 1291 Opa+ P+ showed adherence of organisms to the epithelial cell membrane, pedestal formation with evidence of intimate association between the gonococcal and the epithelial cell membranes, and intracellular gonococci present in vacuoles. Confocal studies of primary urethral epithelial cells showed actin polymerization upon infection. Polyclonal antibodies to the asialoglycoprotein receptor (ASGP-R) demonstrated the presence of this receptor on infected cells in the primary urethral cell culture. In situ hybridization using a fluorescent-labeled probe specific to the ASGP-R mRNA demonstrated this message in uninfected and infected cells. These features were identical to those seen in urethral epithelial cells in exudates from males with gonorrhea. Infection of primary urethral cells in culture mimics events seen in natural infection and will allow detailed molecular analysis of gonococcal pathogenesis in a human epithelial cell which is commonly infected.     

Ultrastructural features that relate to tumor cell behaviour in human breast carcinomas

Human breast tumor biopsies were studied by transmission electron microscopy for ultrastructural characteristics that might be associated with tumor cell function and behaviour. Features related to the state of cell differentiation and tumor cell detachment were evaluated and discussed. The ultrastructure of loose cells at the tumor periphery is compared with that of coherent cells in interior regions. Structural information has been analysed by taking into account available biochemical data for interpretation of cell function. The value of the parameters chosen for study is discussed. This study shows that each tumor needs to be evaluated independently for its specific ultrastructural features. Electron microscopic data becomes more relevant when interpreted in conjunction with histological diagnosis and clinical findings.