Showing papers on "Ultrastructure published in 1982"

Epithelia suspended in collagen gels can lose polarity and express characteristics of migrating mesenchymal cells.

Abstract: This study of epithelial-mesenchymal transformation and epithelial cell polarity in vitro reveals that environmental conditions can have a profound effect on the epithelial phenotype, cell shape, and polarity as expressed by the presence of apical and basal surfaces. A number of different adult and embryonic epithelia were suspended within native collagen gels. Under these conditions, cells elongate, detach from the explants, and migrate as individual cells within the three-dimensional lattice, a previously unknown property of well-differentiated epithelia. Epithelial cells from adult and embryonic anterior lens were studied in detail. Elongated cells derived from the apical surface develop pseudopodia and filopodia characteristic of migratory cells and acquire a morphology and ultrastructure virtually indistinguishable from that of mesenchymal cells in vivo. It is concluded from these experiments that the three-dimensional collagen gel can promote dissociation, migration, and acquisition of secretory organelles by differentiated epithelial cells, and can abolish the apical-basal cell polarity characteristic of the original epithelium.

Ultrastructure of the human intervertebral disc: II. Cells of the nucleus pulposus.

Abstract: The cells of the intervertebral disc exist in a unique environment; not only are discs subject to large mechanical loads, they are the largest avascular structures in the body. To describe the ultrastructure and age changes in cells from human nucleus pulposus, we studied these cells in individuals ranging in age from the 26th week of fetal life to 91 years. Viable chondrocyte-like cells existed in specimens from all ages. The presence of Golgi cisternae and well-developed endoplasmic reticulum in these cells suggests that they are capable of producing and maintaining the extracellular matrix. Necrotic cells were also present in all samples, and many cells which appeared viable when examined by light microscopy proved to be necrotic when examined by electron microscopy. The percentage of necrotic cells increased with age from 2% or less in fetal specimens to over 50% in adults. In addition, with age, a distinct pericellular matrix or "nest," consisting of collagen fibrils, fine filaments, dense particles, and banded structures, formed around most cells with no apparent preference for viable or necrotic cells. Nest formation and increasing density of the cell nests may reflect accumulation of cell products.

Ultrastructure of human natural killer cells: nature of the cytolytic contacts in relation to cellular secretion.

Abstract: The ultrastructure of human NK cells, NK/target cell conjugates, and the effect of monensin, a secretion inhibitor, were studied. Human peripheral blood lymphocytes, highly enriched (75 to 85%) in large granular lymphocytes (LGL), which are known to be the mediators of human NK activity, were used as effectors, and K562 cells as targets. LGL-type of cells were characterized in electron microscopy by a low nucleocytoplasmic ratio, indented nucleus, several large mitochondria, prominent Golgi apparatus, and cytoplasmic osmiophilic granules bound by a unit membrane. Their surface was of intermediate villous type. Two types of effector/target contacts were seen: either effector cell protrusions were pushed deep into pouches and lacunae of the target cell surface, or a wide area of intimate cell-to-cell contact was formed. The contact formation was followed by polarization of the effector cell Golgi apparatus towards the contact area. Monensin, a carboxylic ionophore, which interrupts the vesicular traffic of Golgi-derived vacuoles to the cell surface, caused an accumulation of cytoplasmic vesicles in the LGL but had no effect on the effector/target contact formation. Monensin inhibited the NK lysis apparently via cessation of secretion by the LGL. It did not affect the viability of the effector cells. The lytic steps of human NK cells thus involve binding to the target cell, polarization of cytoplasmic organelles towards the target, and apparently a precisely directed exocytosis of secretory material.

Ultrastructure and Cytochemistry of the Cell Types in the Larval Hematopoietic Organs and Hemolymph of Drosophila Melanogaster. (drosophila/hematopoiesis/blool cells/ultrastructure/cytochemistry)

Abstract: The ultrastructure of the primordial blood cells in the first and second hematopoietic lobes of the late second and third instar larva and prepupa of Drosophila melanogaster was compared with the ultrastructure of the blood cells found freely in the larval hemolymph. Within the hematopoietic lobes two principal cell-types were detected: (i) the prohemocytes and proplasmatocytes, and (ii) different developmental stages of crystal cells., Prohemocytes are characterized by a ribsome-rich cytoplasm, showing small amounts of mitochondria, rough ER and Golgi complexes and few primary lyosomes. Prohemocytes differentiate into proplasmatocytes. When released into the hemolymph they transform further into plasmato-, podo-, and lamellocytes. This differentiation pathway is characterized by a gradual, numerical increase of cytoplasmic organelles, the development of the lysosomal system and the aquisition of the capacity for phagocytosis and melanin formation. The differentiation of a procrystal cell into a crystal cell involves a number of intermediate stages, during which the crystalline material is produced, accumulated, and crystallized. Primary and secondary lysosomes in the primordial blood cells of the hematopoietic organs as well as the free blood cells in the hemolymph were identified cytochemically with the help of the acid phosphatase test. The capacity for melanin synthesis was studied with the phenol- and polyphenol oxidase test.

Translocation in the staminal hairs ofSetcreasea purpurea: I. A study of cell ultrastructure and cell-to-cell passage of molecular probes

Abstract: SummaryInvestigations into plant intercellular communication were initiated through an examination of plasmodesmata and cell-to-cell passage of molecular probes in the staminal hairs ofSetcreasea purpurea. Plasmodesmata connecting staminal hair cells of small buds are filled with an electron-opaque homogenous material. To examine the permeation selectivity of plasmodesmata, molecular probes made up of fluorescein isothiocyanate (FITC) complexed with amino acids and peptides were injected into the staminal hair cells and the spread of these fluorescent molecules through the symplast, was monitored. Molecules composed of FITC complexed to single amino acids with polar and aliphatic R groups travel rapidly, while those which include peptides travel slowly. Dye molecules composed of an amino acid with an aromatic side group do not pass from cell to cell at all. It is hypothesized that the material occluding the plasmodesmata constitutes the diffusion barrier, by presenting a hydrophilic environment which allows passage of molecules with maximum molecular weights of 700–800 daltons, but which retains those with aromatic side groups.

Comparative ultrastructure of hatched human, mouse and bovine blastocysts.

Abstract: A hatched human blastocyst obtained after in-vitro fertilization and culture was examined by transmission electron microscopy and the ultrastructural features compared with hatched mouse and bovine blastocysts. The human blastocyst contained a continuous layer of trophoblast cells with apical junctional complexes, an inner cell mass and the beginning of a primitive endoderm layer. Certain ultrastructural features were common to the blastocysts of all 3 species; these included characteristic junction regions between adjacent trophoblast cells, an abundance of microvilli on the external surfaces of the blastocysts and the presence of well developed mitochondria and numerous ribosomes in the trophoblast cells. The features that were dissimilar included the extent of development of the endoderm layer, the appearance of the inner cell mass and the nature and extent of vesicular inclusions in the trophoblast cells.

Ultrastructure of swarmers in the laminariales (phaeophyceae). ii. sperm1

Abstract: Zoospores of 17 species in 14 genera of Laminariales, collected in the northeast Pacific Ocean, were studied by electron microscopy. These zoospores are unique in the brown algae in lacking both an eyespot in the single chloroplast and any associated swelling at the base of the shorter, posterior flagellum. Spores of all species examined possess a distal whiplash portion on the longer, mastigoneme-bearing anterior flagellum. This appendage may sometimes be as long as the mastigoneme-bearing portion of the flagellum, but it is only seldom preserved in the preparations for electron microscopy. A microtubular cytoskeleton is probably responsible for maintaining the shape of the spore. It consists of a short band of about 10 microtubules between the two basal bodies, scattered tubules converging at the anterior of the spore, a band of 7–9 tubules directed anteriorly from the anterior basal body, and a band directed posteriorly from the posterior basal body. These anterior and posterior bands may form one continuous band looping around the periphery of the spore. Variation with possible taxonomic significance was found in the ultrastructure of vesicles which apparently contain adhesive material, and which are extruded through the plasmalemma when the zoospores settle.

The preservation of ultrastructure and antigenicity

Abstract: Post-fixation in osmium with complete dehydration and embedding in a tightly bonded, hydrophobic epoxide or polyester is the standard electron microscopic technique. The use of osmium ensures excellent ultrastructural detail, but impairs antigenicity and destroys enzyme activity. I t s omission, however, leads to distortion, contraction, and extraction of tissue elements, particularly if polyesters and epoxides are used (Pease, 1964). Methaerylates have been used as water-soluble, polar embedding agents with unosmicated tissue (Leduc, E.H., Bernhard W., 1967), but poor ultrastructure and retarded enzymatic or antigenic activity reduces their value. More recently the plastic 'Lowicryl K4M' (Roth e t al, 1981) has been used a t -3OOC to improve structural detail. The antigenic yield, however, is not increased, although it is claimed that the accuracy of immunolocalisation is improved. We have studied the problems of fixation and embedding in electron microscope immunocytochemistry using a versatile and sensitive immunolocalisation technique (Lasani e t al, 1980). A variety of tissues were fixed in various combinations and concentrations of glutaraldehyde, formaldehyde, and osmium, together in some instances with picric acid. Epon, HEMA, and L R White (a polar acrylic plastic) were used as embedding agents. With LR White complete or partial tissue dehydration was used. The immunolocalisation procedure was carried out on unsupported ultrathin sections on nickel grids. DAB has a high affinity for osmium, which was used, tpnly at the final stage, to increase the electron density of this immunocytochemical reaction product. Excellent antigenicity was retained after short fixation in buffered formalin-based fixatives, but ultrastructural preservation was poor. The best results were obtained when tissues were fixed for 2 3 hours in a solution of 0.1 M phosphate buffer (83 ml), 50% purified glutaraldehyde (2 ml) and picric acid (sat.aq. 15 ml) followed by embedding into LR White directly from 70% alcohol. The ultrastructure was well preserved (Plate I), particularly after perfusion-fixation, with very accurate localisation of the reaction product (Plate 2). Electron microscope immunocytochemistry involves a compromise between loss of antigenicity through fixation or antigen diffusion, and preservation of ultrastructure. Our technique uses a fixative that preserves adequate antigenicity and ultrastructure. The use of a rapid dehydration step and a polar acrylic plastic embedding agent avoids major tissue disorganisation perhaps by preserving unosmicated lipid. This simple technique allows excellent localisation of a variety of hormones and immunoglobulins; we are currently exploring i t s uses for other antigens.

Thermophilic biomethanation of acetic acid: morphology and ultrastructure of a granular consortium

Abstract: The ultrastructure of a thermophilic methanogenic consortium appearing as morphologically distinct bacterial granules (up to approximately 3 mm in diameter) has been studied. The consortium was enriched and maintained at 60 °C in continuous culture in a defined mineral – vitamin – acetic acid medium at a hydraulic retention time of 44 h. Thin-section electron microscopy showed three morphologically distinct layers of the granules. (1) The outer division zone bears resemblance to a pseudoparenchyma and consists of "macrocysts" and coccoid cells, resembling Methanosarcina cells. (2) The inner zone is built up of loosely packed ovoid cells and (3) internal cavities containing rods.The cavities were the exclusive site of gas formation, as shown with a specially designed minifermenter allowing direct light microscopic observation of gassing granules. Based on labeling studies with cationic ferritin and scanning electron microscopy a working model for methane formation from acetate by the consortium is presented.

Salivary gland of the tick vector of East Coast fever. III. The ultrastructure of sporogony in Theileria parva.

Abstract: Sporogony of the sporozoan Theileria parva in the salivary gland of the tick vector of East Coast fever was studied in electron micrographs. The findings differ in several respects from previous interpretations based upon light microscopy. Cytokinesis of the primary sporoblast to form secondary and tertiary sporoblasts is not substantiated. Instead it is suggested that the parasite develops as a ramifying, multinucleate syncytium rapidly increasing in size and complexity until it gives rise to myriad sporozoites in a terminal episode of cytoplasmic fission. The proliferating nuclei initially occupy peripheral lobules that are continuous with a central labyrinth of branching and anastomosing processes which present a very large surface area for interchange of metabolites with the host cell cytoplasm. The membrane of the labyrinth is rich in cytostomes, but no evidence is found of bulk uptake of host cytoplasmic matrix or organelles into food vacuoles. Rhoptries are the first of the polar organelles of the parasite to develop and are associated with dense plaques irregularly distributed on the inner aspect of the parasite membrane. Micronemes form independently of the rhoptries at a later stage. After 3–4 days of tick feeding, sporogeny is complete and the infected salivary gland cell contains up to 50,000 spherical or ovoid sporozoites about 1 μm in diameter. These are limited by a simple plasma membrane. The inner layer of the ‘pellicle’, the polar ring, and the conoid described for zoites of other Apicomplexa are lacking. Maturational changes are noted in sporozoites after sporogony is completed. Micronemes appear to increase in size, and possibly in number, from days 3–5 and the majority take up positions immediately subjacent to the plasmalemma.

Ultrastructure of 40-Million-Year-Old Insect Tissue

Abstract: Examination of the ultrastructure of preserved tissue in the abdomen of a fossil fly (Mycetophilidae: Diptera) entombed in Baltic amber revealed recognizable cell organelles. Structures that corresponded to muscle fibers, nuclei, ribosomes, lipid droplets, endoplasmic reticulum, and mitochondria were identified with the transmission electron microscope. Preservation was attributed to inert dehydration as well as the presence of compounds in the original sap which functioned as natural fixatives. This evidence of cell organelles in fossilized soft tissues represent an extreme form of mummification since Baltic amber is considered to have formed about 40 million years ago.

Ultrastructure of terminally differentiated adult rat cardiac muscle cells in culture

Abstract: Ventricular cardiac muscle cells isolated from adult rats were maintained in culture for 28 to 60 days and examined by transmission electron microscopy. In order to elucidate the detailed ultrastructure of the cultured myocytes, several different electron-dense stains were used. These included tannic acid, osmium ferrocyanide, osmium tetroxide (applied as a primary fixative), and lanthanum chloride, as well as more widely used stains such as osmium tetroxide, uranyl acetate, and lead citrate. Our results show that, compared to cultured neonatal rat myocytes, cultured myocytes derived from adult rats more closely resemble in vivo adult ventricular cells. The cultured adult myocytes contained typically distributed organelles such as nuclei, mitochondria, and elements of the sarcoplasmic reticulum. Myofilaments were well organized, and typical intercalated disks were observed between adjacent cells. Unlike cultured neonatal myocytes, the adult cells contained numerous residual bodies and a relatively well developed transverse tubular system. The transverse tubular system was identified by its continuity with the extracellular space (as indicated by the penetration of electron-dense extracellular tracers), location at or near the Z line, large lumenal diameter, and frequent participation in couplings with elements of the sarcoplasmic reticulum.

The ultrastructure of the larval malpighian tubules of a saline-water mosquito

Abstract: The larval Malpighian tubules of the saline-water mosquito Aedes taeniorhynchus were examined using light and electron microscopy. The tubules contain two cell types: primary cells and stellate cells. Primary cells are characterized by their size (70 μm × 70 μm × 10 μm) and an abundance of intracellular membranebound crystals. Two types of microvilli are found on the luminal surface of the primary cells: (1) small microvilli containing core microfilaments and extensions of endoplasmic reticulum, and (2) larger microvilli (≈3 μm in length) which in addition to the above components contain a mitochondrion along their entire length. Both microvillar types have abundant knobs lining the cytoplasmic surface of the microvillar membrane. These knobs, which are often found in insect ion transporting tissues, have been termed ‘portasomes’ by Harvey (1980). The possible role of these structures in ion transport and mitochondrial positioning is discussed. The stellate cells are much smaller than the primary cells, and lack intracellular crystals. Their microvilli are smaller as well (≈0.6 μm in length) and contain no endoplasmic reticulum. mitochondria or knobs. The cells types found in the saline-water mosquito larva, Aedes taeniorhynchus , are identical to those found in Aedes aegypti , indicating that the unique capacity of saline-water mosquito larvae to transport Mg 2+ and SO 4 |post|staggered|2− is not associated with the presence of an additional cell type.

Distribution and ultrastructure of taste buds in the oropharyngeal cavity of the rainbow trout, Salmo gairdneri Richardson

Abstract: The distribution, external surface morphology and ultrastructure of taste buds in the oropharyngeal cavity of the rainbow trout, Salmo gairdneri Richardson, were studied using scanning and transmission electron microscopes (SEM and TEM). The SEM revealed three taste bud types, varying only in their degree of elevation from the general level of the epithelium. Types I and II were located on elevated papillae associated with teeth on the dentary, maxilla, palate, tongue and pharyngeal pads while the unelevated Type III were mainly found in the anterior (branchial) pharynx. Each taste bud was composed of four cell types: basal, dark, intermediate and light cells, the apical processes of the last three filling the taste pores. The intermediate and light cells appeared similar in ultrastructure, varying only in the amount and organization of smooth endoplasmic reticulum (SER) in their cytoplasm. In addition to its contacts with the processes of intragemmal nerves distally, the basal cells established independent contacts with processes of extragemmal nerves basally. It is suggested that the distribution of the taste buds and their close association with teeth are adaptations to the predatory feeding habit of the rainbow trout. Age differences may account for the existence of two types of gustatory cells and the manner of innervation of the taste bud suggests the existence of two pathways for the transmission of gustatory sensation to the central nervous system (CNS).

Monolayer culture of human endometrium: Methods of culture and identification of cell types

Abstract: Monolayer cultures can be established from human endometrial tissue after enzymatic dispersal into isolated glands or single cells. Three cell types that have distinct morphology by light and electron microscopy are observed in the resulting primary cultures. One cell type, an elongated spindle cell, is similar in appearance to fibroblasts derived from other tissues. A second cell type forms colonies of tightly cohesive cells, ranging in shape from oval to polygonal. These cells have typical organelles and junctional complexes characteristic of epithelial cells from the endometrium. The third cell type assumes a pavement-like appearance composed of polygonal cells when viewed by phase contrast microscopy, but lacks distinctive ultrastructural features of epithelial cells. These cells in culture resemble the endometrial stromal cell, the predominant cell type of the human endometrium in vivo. The epithelial cell does not survive subculturing but the other two cell types can be passaged through several generations and can be stored in liquid nitrogen and subsequently returned to culture.

Salivary gland of the tick vector of East Coast fever. IV. Cell type selectivity and host cell responses to Theileria parva.

Abstract: Responses of cells in the tick salivary gland to parasitism by Theileria parva were studied by electron microscopy. The gland is composed of three distinct types of acini (I, II, III) which together include ten or more different cell types. Of some 30 infected cells observed in the present study, all were E-cells of acinus III. The parasite thus exhibits a high degree of selectivity for acinus and cell type. The glandular cell invaded undergoes massive hypertrophy and accumulates glycogen deposits in its cytoplasm which may serve as an energy source for the growing intracellular parasite. As synthesis of its secretory material declines the product is packaged in progressively smaller secretory granules. The extensive arrays of endoplasmic reticulum are dismantled and eliminated in autophagic vacuoles. Excess secretory granules are also broken down by crinophagy. After 4 days, sporogony is completed and the host cell contains 30,000-50,000 sporozoites in an electron-lucent cytoplasm largely devoid of cytomembranes and secretory granules. Mitochondria are still present and normal in appearance. The loss of basophilia and secretory granules observed heretofore by light microscopy have been attributed to ingestion and destruction of host organelles by the parasite. The pallid appearance of the cytoplasm has been interpreted as a sign of impending degeneration of the host cell. In electron micrographs no ingestion of organelles by the parasite or degenerative changes were found. The host cell clearly remains viable and metabolically active throughout sporogony. The striking changes in its ultrastructure result from active elimination of organelles and inclusions by the host cell itself in response to parasitism.

The ultrastructure of anthocyanoplasts in red-cabbage

Abstract: A study of the ultrastructure of anthocyanoplasts from the seedling hypocotyl and leaves from the heart of red-cabbage plants has been made. Evidence is presented that the anthocyanoplast does not represent a hydrophobic droplet but is bounded by a single tripartite membrane approximately 10 nm in thickness. The results provide further support for the view that the anthocyanoplast is an intracellular compartment containing at least the later enzymes of anthocyanin biosynthesis, which are thereby separated from the hostile acidic environment of the vacuolar sap.

Ultrastructure of mucocartilage in the larval anadromous sea lamprey, Petromyzon marinus L.

Abstract: The fine structure of mucocartilage, a tissue unique to larval lampreys, was examined in Petromyzon marinus L. This tissue is surrounded by a perichondrium of vascularized, dense connective tissue composed of fibroblasts, collagen fibrils, and elastic-like microfibrils, but it is avascular itself and consists of elastic-like microfibrils, ground substance, and a few diffusely scattered fibroblasts. Fibroblasts possess rough endoplasmic reticulum, many free ribosomes, a well-developed Golgi apparatus, a tubulo-vesicular network, and a number of secondary lysosomes containing crystalline material. The appearance of the organelles suggests the involvement of the cell in the synthesis and secretion of the ground substance and microfibrils. Tubular microfibrils, 11–13 nm in diameter, comprise the major portion of the matrix, and they are similar to those described in developing mammalian elastic tissue (Ross and Bornstein, 1969). The retention of the microfibrils may represent either a primitive form of elastic fiber in this “primitive” vertebrate or reflect the larval condition of the lampreys under examination. Scattered spherical to polyhedral-shaped matrix granules and inter-granular filaments make up the remainder of the matrix. It was concluded that mucocartilage in larval lampreys is not a conventional type of vertebrate connective tissue.

The cellular structure of the leydig organ in the shark, etmopterus spinax (l.)

Abstract: In Etmopterus spinax, a small deep water shark, the predominating lymphomyeloid tissue is the so called Leydig organ. This consists of bone marrow-like tissue situated between the muscularis and the mucosa of the esophagus. Examination by light and electron microscopy shows that the Leydig organ produces large numbers of granulocytes and lymphocytes. Two main types of granulocytes occur, tentatively called heterophilic and eosinophilic. The heterophilic cells may be subdivided into three types which differ in the ultrastructure of the granules. Cells structurally resembling mammalian plasma cells are common. The presence of these cells indicates that the tissue is part of the shark immune system.

The ultrastructure of germinating barley seeds. I. Changes in the scutellum and the aleurone layer in Nordal barley

Abstract: The ultrastructure of the scutellum and the aleurone layer has been examined in seeds of Nordal barley following malting for 0, 30, 72, 120 and 162 hours at 15°C. Thick sections from the seeds were stained with Calcofluor White M2R New and examined in the light microscope to determine the extent of cell wall degradation in the endosperm. The changes in the scutellar parenchyma cells include degradation of the protein bodies and the appearance of vacuoles and prominent amyloplasts. The analysis of a serially sectioned epithelial cell from the scutellum 72 hours after the start of malting revealed a large number of Golgi apparatuses, preferentially located near the cell wall. It is suggested that the presence and location of the Golgi apparatuses are related to enzyme secretion. Following malting for 72 hours or more, the epithelial cells contain lomasomes and membrane aggregates, termed membrane bodies. The analysis indicated that lomasomes originate from the membrane bodies and are involved in the growth of the cell walls of the epithelium. Organelles indicating cellular activity, i.e., rough endoplasmic reticulum, mitochondria and Golgi apparatuses, first appear in the epithelial cells of the scutellum, and later in the cells of the aleurone layer. Furthermore, the degradation of the endosperm starts in the vicinity of the scutellum as judged by the calcofluor staining. Therefore, it is concluded that the scutellar epithelium provides the enzymes during the initial degradation of the endosperm. In seeds malted for 162 hours, aleurone cells with a cytoplasmic organisation indicative of active metabolism are located in the embryo part of the seed, whereas aleurone cells adjacent to unmodified endosperm in the distal end of the same seed show little or no structural sign of activity.

Ocular Adnexal Lymphoid Tumors: Correlative Ultrastructural and Immunologic Marker Studies

Abstract: • Twenty-two ocular adnexal lymphoid infiltrates were analyzed by electron microscopy as well as immunologically and cytochemically. Five reactive polyclonal lesions were found to be preponderantly composed of small mature lymphocytes (presumably T cells) with clumped nuclear chromatin, sparse cytoplasmic organelles, and numerous monoribosomes. In 11 monoclonal B-cell lesions, both the 1-μm plastic sections examined by light microscopy and the electron micrographs disclosed immature cells, with more dispersed nuclear chromatin, prominent nucleoli, abundant cytoplasmic polyribosomes, and increased numbers of mitochondria and strands of endoplasmic reticulum (particularly in plasmacytoid lesions). The remaining six monoclonal B-cell lesions were composed of comparatively well-differentiated cells requiring electron microscopy to show somewhat more prominent nucleoli, slightly less dense clumping of the nuclear chromatin, increased numbers of mitochondria and short segments of rough-surfaced endoplasmic reticulum, and monoribosomes rather than polyribosomes. The importance of distinguishing this group of well-differentiated monoclonal lesions from the less well-differentiated ones was underscored by the results of the follow-up examinations, in that no evidence of extraorbital disease has been discovered in the former group, while a 50% incidence occurred in the latter.