Histology and histopathology Vol. 9, nº 4 (1994)

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Open Access
Morphogenesis of normal human salivary gland cells in vitro
(Murcia : F. Hernández, 1994) Azuma, M.; Sato, M.
Primary cultured human salivary gland cells were transfected with ori-defective mutant DNA of SV40. Following 2-3 weeks of transfection, slowly expanding colonies consisting of small compact cells emerged, while mock-transfected cells did not grow any more and eventually entered crisis, followed by cell death. Using limited dilution technique, we isolated 4 cell clones with distinct morphology from a single colony. Morphological observation of cells cultured on plastic dishes precisely revealed the characteristics of the constituent cells of salivary gland; i.e., three cell clones showing cuboidal- (NS-SV-DC), spindle- (NS-SV-MC), and flattened morphology (NS-SV-SC) were similar to duct-, myoepithelial-, and squamous phenotype, respectively. A remaining cell clone, polygonal in shape and with numerous secretory granules (NS-SV-AC), resembled an acinar cell. Characterization of cell clones by ultrastructural exarnination and search for specific antigens showed the similarity of NS-SV-DC, NS-SV-MC, NS-SV-AC, and NS-SV-SC to duct, myoepithelial, acinar, and squamous cells, respectively. Anchorage-independent growth in soft agar and tumorigenicity in nude mice were not recognized in al1 cell clones. These results demonstrate that establishment of cell clones with duct-, myoepithelial-, acinar-, or squamous phenotype was accomplished in the in vitro system, and that based on the evaluation of colony-forming ability in soft agar and tumorigenicity in nude mice these cell clones can be considered to be non-tumorigenic. Using the above in vitro system, we examined the effect of a reconstituted basement membrane extract, Matrigel, on the morphogenesis of cultured normal human salivary gland cells. When NS-SV-DC or NS-SV-MC were seeded on Matrigel in serum-free culture conditions, they formed round or zona1 clusters on day 1; failing however, to develop into a salivary gland morphogenesis. Semithin sections of cell clones cultured on Matrigel exhibited multicellular aggregates on day 1, while on days 2 and 3 these cells lost both cell-cell and cell-Matrigel interactions and eventually entered crisis. In an attempt to understand the mechanism involved in this phenomenon, we have investigated proteolytic enzymes and their inhibitors secreted by cell clones. Although cell clones produced almost identical levels of gelatinases, they released increased amounts of plasminogen activators (PAs) as compared with a neoplastic human salivary gland cell line (HSG), which had already been demonstrated to differentiate into acinar cells when cultured on Matrigel. Obvious difference of expression leve1 of tissue inhibitor of metalloproteinases-1 (TIMP- 1) was not observed in these cells. However, secretion of PA inhibitors was elevated in NS-SV-MC when compared to NS-SV-DC. Neutralization of excess PAs by exogenously-added serine protease inhibitors corrected the aberrant morphogenesis of NS-SV-DC, but not that of NS-SV-MC, and allowed NS-SV-DC to form glandular-like structures. Thus, these results may suggest that a tightly regulated proteolytic activity is, in part, essential for in vitro morphogenesis of normal salivary gland duct cells.
Open Access
Variety of sialic acids occurring in the bovine sublingual gland
(Murcia : F. Hernández, 1994) Accili, Daniela; Gabrielli, M.G.; Menghi, Giovanna
Sialoglycoconjugates were investigated in the bovine sublingual gland by direct visualization of sialic acid with specific lectins (LPA, SNA) and by histochemical procedures combined with sialidase digestion and lectins. The most reactive histological sructures were found to be acini which contained glycoconjugates with terminal disaccharides consisting of sialic acid linked to galactose or N-acetylgalactosamine. Resistance to periodate oxidation was interpreted as demonstrating a relevant presence of C7, C8 and Cg acetylated sialic acids. KOH-Sialidase-DBA and KOHAlcian blue sequences allowed the identification of C4 acetylated sialic acids.
Open Access
Angiogenesis: an update
(Murcia : F. Hernández, 1994) Díaz-Flores, Lucio; Gutiérrez, Ricardo; Varela, H.
Angiogenesis is the neovascularization or formation of new blood vessels from the established microcirculation. It is particularly important and indispensable in a large number of normal and pathological processes during pre- and post-natal life, including neoplasia, inflammation, wound repair and collaterization in response to ischemic stimuli. The current interest in the role of neovascularization in the transition from hyperplasia to neoplasia, as well as in the tumour growth and metastasis, has brought about a large number of studies on angiogenesis. The complex processes of neovascularization, quiescent in the adult organism, may occur rapidly in several circumstances, with the implication of the following events: a) endothelial cell (EC) and pericyte activation; b) basal lamina degradation; c) migration and proliferation of EC and pericytes; d) formation of a new capillary vessel lumen; e) appearance of pericytes around the new capillaries; f) development of a new basal lamina; g) capillary loop formation; h) persistence or involution, and differentiation of the new vessels; and i) capillary network formation and, eventually, organization into larger microvessels. The use of numerous "in vivo" and "in vitro" systems has facilitated the assessment of angiogenesis control, in which angiogenic (fibroblast growth factors, vascular endothelial growth factor, platelet endothelial growth factor, E series prostaglandin, angiogenin, monobutyrin) and antiangiogenic (cartilagederived angiogenic inhibitor, thrombospondin, protamine, platelet factor-4, interferon, angiostatic antibiotics, steroids) substances intervene. Heparin and heparin sulphate also play a key role in these mechanisms. A greater knowledge of angiogenesis control may lead to the development of a potential therapy in angiogenesis-related processes.
Open Access
Cell types in the central nervous system infected by murine retroviruses: implications for the mechanisms of neurodegeneration
(Murcia : F. Hernández, 1994) Wong, P.K.Y.; Yuen, P.H.
Retroviruses are an important cause of neurologic disease in humans but the pathogenic mechanisms are poorly understood. To delineate pathogenic mechanisms in any neurologic disease in humans is extremely difficult and will continue to rely on the use of animal models. This review presents severa1 murine models to study the pathogenic mechanisms of neurodegenerative disease which manifest noninflammatory spongiform lesions in the CNS. The cell types in the CNS infected by these murine retroviruses and their role in disease induction are discussed.
Open Access
Production and characterization of a new monoclonal antibody, GR-ICOR-2, recognizing sarcomeric actin: analysis of the expression in the developing chick heart
(Murcia : F. Hernández, 1994) Fernández, J.E.; Melguizo, C.; Prados, J.; Marchal, J.A.; Alvarez, L.; Aránega, A.
We produced and characterized a specific monoclonal antibody (mAB) designated GR-ICOR-2. This mAb recognizes sarcomeric actin molecules (43 kDa) and was used in an immunohistochemical analysis of staining pattems in Harnburger and Hamilton's stages 18, 22 and 25 (HH 18, 22 and 25) embryonic chick hearts. Staining showed a mainly cytoplasmic distrubition in three regions: the atrioventricular (AV) canal cushion tissue, the primitive ventricle, and conal crests. In addition, this mAb-cross-reacted with rabbit and human cardiac and skeletal muscle tissue; but not with smooth muscle tissue.
Open Access
Ischemia-reperfusion of human skeletal muscle during aortoiliac surgery: effects of acetylcarnitine
(Murcia : F. Hernández, 1994) Adembri, C.; Lombardo Domenici, L.; Formigli, L.; Brunelleschi, S.; Ferrari, E.; Novelli, G.P.
Our previous study on human skeletal muscle undergoing ischemia and reperfusion has revealed that granulocytes, which infiltrate the muscle tissue in large numbers, play an important role in mediating fibre injuries by producing superoxide anion (O?) which is responsible for membrane lipid peroxidation. In the current study, five patients undergoing aortic reconstructive surgery were given acetyl-carnitine (2 mglkg i.v. plus 1 mglkglmin for 30 min) prior to the induction of ischemia. Muscle biopsies and blood samples were examined: a) after anaesthesia; b) at the end of ischemia; and c) 30 min after reperfusion, with the aim of elucidating whether acetylcarnitine could prevent the infiltration andlor the activation of granulocytes and eventually skeletal muscle injuries. During ischemia and reperfusion complement activation recmited numerous granulocytes into the muscle tissue, but, contrary to the untreated samples, the ability for O2 -generation of these cells remained at low levels and was comparable to that of ischemia even when molecular 02 was reintroduced to the tissue. Accordingly, the morphological changes of the postischemic muscle fibers were substantially reduced when compared to the untreated samples; in fact, the mitochondrial swelling was only moderate and the intramitochondrial dense bodies were small and scarce. The current findings support a positive role of acetyl-carnitine in ameliorating the ischernia-reperfusion (1-R)-induced damage of human skeletal muscle.
Open Access
ZIO impregnation and cytochemical localization of thiamine pyrophosphatase and acid phosphatase activities in small granule-containing (SGC) cells of rat superior cervical ganglia
(Murcia : F. Hernández, 1994) Chau, Y.P.; Lu, K.S.
Cytochemical relationship between Golgi complex and dense-cored granules (DCGs) of small granule-containing (SGC) cells in rat superior cervical ganglia was examined in electron microscopy by zinc-iodide-osmium tetroxide (ZIO) method and by enzyme cytochemistry for thiamine pyrophosphatase (TPPase) and acid phosphatase (ACPase). After ZIO impregnation, al1 the saccules of Golgi apparatus and some of tubular rough endoplasmic reticulum (rER) were stained. DCGs in periphery of SGC cells were not stained, but varying degrees of dense deposits occurred in the DCGs in vicinity of Golgi trans-saccules. Both TPPase and ACPase activities were localized in one or two stacked layers of saccules on the trans side of the Golgi complex. No reaction products were demonstrated in the DCGs. From these results, we suggest that the DCGs of SGC cells in rat superior cervical ganglia are derived from the Golgi complex, and that lysosomal cleavage of protein contents in the DCGs may occur in the trans Golgi saccules.
Open Access
Cytogenetic analysis and morphology of malignant nuclear vlimata. The life cycle of malignant cells
(Murcia : F. Hernández, 1994) Logothetou-Rella, H.
The karyotype of malignant nuclear vlimata (NVs) was investigated with the in situ and chromosomal spreading techniques. NV metaphases were recognised by the head with tail morphology, kept in situ and evaluated by the chromosomal spreading technique. It was shown that malignant NVs were produced by random, uncontrolled meiosis. NVs contain and carry single, hypodiploid, haploid, hypohaploid, hyperdiploid and atypical sets of chromosomes. NVs are unstable parasitic cellular elements, invading the cytoplasm or the nucleus of host cells, extruding and implanting their chromosomes in the host cell upon contact. Within a malignant cell culture, NVs play the role of chromosomal donors and host cells of chromosomal recipients. NVs were identified as episomatic on host cell nuclei or incorporated into host cell metaphases. The observations are discusssed in terms of fertilization, vira1 infection and apoptosis. Comparison of malignant NVs with phytohaemagglutinin (PHA)-activated lymphocytic NVs is provided, as well as the life cycle of the malignant cells as follows: meiosis invasion Malignant cell - NVs - recipient host cell - hybrid cells NVs formation
Open Access
Postnatal development of the Ammon's horn (CA1 and CA3 fields). A karyometric and topographic study
(Murcia : F. Hernández, 1994) Pérez Delgado, M.M.; Serrano Aguilar, P.G.; Castañeyra Perdomo, A.; Ferres Torres, R.
We have performed a karyometric study of the pyramidal neurons of CA1 and CA3 fields of the Ammon's horn, in male mice aged from the 5th to the 190th postnatal day. Nuclear sizes were measured with the aid of a Magiscan Analysis System, used in an interactive form, in both superficial and deep layers of the stratum pyramidal in those fields. The measurements were made at three different topographic levels: rostral; intermediate; and caudal, to detec any possible difference related to the topography of the neuron in the same field. We have found that both CAI and CA3 fields are correlated in the postnatal development of their nuclear pyramidal sizes and that al1 topographic levels of the hippocampus reach their highest karyometric sizes at the 10th-15th postnatal day. Caudal levels show higher karyometric values than the other levels and some differences between neurons of the superficial and deep layers of both fields are also described here and analysed in relation to the different ontogenetic gradients of these cells.
Open Access
Modifications of the dermis during scale regeneration in the lizard tail
(Murcia : F. Hernández, 1994) Alibardi, Lorenzo
During scale morphogenesis in the regenerating tail of lizards (Anolis and Lampropholis) the structure of the dermis undergoes changes in relation to the ingrowth of epidermal papillae to form the new scales. Cell proliferation in the dermis, as revealed by the uptake of 3~-thymidinei,s high in the prescaling region of the regenerating tail but lower than the proliferation in the epidermis. Under the epidermis of the scaling region dermal cell proliferation rapidly drops down under the distal (apical) and proximal (caudal) sides of the infoldin epidermal papillae. Dermal fibroblasts take up g ~ - p r o l i n ein high amounts, especially in the forming deep dermal layer, where many collagen fibrils are laid down forming dense connective. Electron microscopic study revealed that ((anchoring filaments~li nk the basement membrane of the epidermis with the deep dermis, in particular in the sinking hnge region. As a result of the higher proliferation of the epidermis with respect to the dermis (heterochrony) and the presence of dermo-epithelial eanchoring filarnents», the superficial laminar epidermis sinks into the dermis to produce new scales. The epidermal downpushing is evidenced by a characteristic distortion of the dermal fibrils under the distal and the proximal sides, and in the hinge region of the forming scales
Open Access
Morphological and physiological bases of crayfish local circuit neurones
(Murcia : F. Hernández, 1994) Nagayama, T.; Namba, H.; Aonuma, H.
As the subject of neuroethological studies, arthropods offer several advantages to elucidate the neural processes that generate and control behavioural acts. The relative simplicity of their neural organization and their identifiable neurones are particularly advantageous. The crayfish, Procambarus clarkii, uropods are paired appendages of the last abdominal segment that show bilateral closing movement in response to mechanical stimulation. This movement, the avoidance «dart» response, is mediated by a cascade and parallel organization of local circuit neurones in the terminal abdominal ganglion. Thirty intersegmental ascending interneurones and 20 spiking local interneurones have been identified both morphologically and physiologically. They receive exteroceptive inputs directly from the sensory afferents. Two complementary groups of unilateral nonspiking local interneurones have also been functionally identified. They exert fine control over the wide range of activity of motor neurones in a graded and sustained manner. These nonspiking intemeurones form opposing and parallel pathways that are essential in modulating the pattern of movement of the uropods.
Open Access
A novel approach to the growth analysis of hamster secondary palate by histone 3 m RNA in situ hybridiration
(Murcia : F. Hernández, 1994) Shah, R.M.; Young, A.V.; Song, B.Z.; Wong, D.T.W.
A study was undertaken to determine the cell proliferation kinetics during the development of hamster vertical palatal shelf ad initium. Harnster embryo heads, obtained at different times between days 10 and 12 of gestation (which is the period of vertical shelf development) were processed and sectioned to localize histone 3 mRNA, a cell cycle specific gene, by in situ hybridization. Sense and antisense 35~-labelledh istone 3 riboprobes were used as hybridization probes. Percent labelled cells were determined. The results showed that a high rate of random proliferation of both epithelial and mesenchymal cells was a major component of early vertical palatal growth. Subsequently, during the latter half of vertical shelf development, the proliferation rates of the epithelial and mesenchymal cells were different in a region specific manner. It was suggested that the spatio-temporal changes in the distribution of cycling mesenchymal and epithelial cells during vertical palate development may indicate their heterogeneity for subsequent segregation into appropnate phenotypes.
Open Access
Prostatic sequestration of Cryptococcus neoformans in immunocompromised persons treated for cryptococcal meningoencephalitis
(Murcia : F. Hernández, 1994) Ndimbie, O.K.; Dekker, A.; Martinez, A.J.; Dixon, B.
We report a case of a patient with acquired immune deficiency syndrome who was successfully treated for cryptococcal meningoencephalitis with amphotericin B and 5-flucytosine. He died from other sequelae of acquired immune deficiency syndrome two years later. An autopsy revealed prominent cryptococcal prostatitis. Cryptococci were neither found in the central nervous system nor in other anatomic sites. The autopsy files yielded seven other cases of men with a history of cryptococcal meningoencephalitis. The possibility that the prostate sequesters Cryptococcus neoformans thereby contributing to systemic relapse is explored. To qualify as a sequestration, cyptococci must be cultured from the prostate, or from a midstream voided specimen after prostatic massage, and the prostate must be the only focus of infection.
Open Access
C-erb-B-2 oncoprotein and epidermal growth factor receptor in human hepatocellular carcinoma: An immunohistochemical study
(Murcia : F. Hernández, 1994) Nakopoulou, Lydia; Stefanaki, K.; Filaktopoulos, D.; Giannopoulou, I.
The aim of this study was to evaluate the irnmunohistochemical expression of epidermal growth factor (EGFR) and c-erb-B-2 oncoprotein in a series of 71 hepatocellular carcinomas as well as in the adjacent hepatic tissue and to assess any correlation with HBsAg expression. The total of the 71 hepatocellular carcinomas (HCCs) was classified into 17 low grade and 54 high grade cases with adjacent non-neoplastic liver parenchyma, observed in 14 and 28 cases respectively. Coexisting cirrhosis or fibrosis was noticed in the adjacent non-neoplastic parenchyma in 12 cases of low grade and 22 cases of high grade HCC. The immunohistochemical avidin-biotin-peroxidase complex (ABC) method was performed on formalin-fixed paraffin sections for the detection of EGFR, c-erb-B-2 oncoprotein and HBsAg using monoclonal antibodies. The expression of c-erb-B-2 was observed in 29.5% (21171) of the HCCs showing no statistically significant correlation with histological grade. The c-erb-B-2 was also detected in the adjacent non-neoplastic parenchyma in 7/14 low grade HCCs, and in 9/28 high grade HCCs. No statistically significant differences in c-erb-B-2 oncoprotein expression were observed between the HCCs and the adjacent non-neoplastic parenchyma. In addition, HBsAg was detected in 10142 examined cases of HCC with adjacent non-neoplastic parenchyma, while only 4 cases of HCCs were simultaneously positive for c-erb-B-2 and HBsAg. EGFR was detected in only 3171 cases of HCC, while the antigen was not detected at al1 in the adjacent non neoplastic parenchyma. HBsAg expression was not observed in any of the EGFR-positive HCCs. Our results suggest that both c-erb-B-2-oncoprotein and EGFR do not seem to be predorninantly involved in the transformation of hepatocytes to the malignant phenotype.
Open Access
Analysis of neogenesis in rabbit skeletal muscles after chronic traction
(Murcia : F. Hernández, 1994) Sun, J.S.; Hou, S.M.; Liu, T,K.; Lu, K.S.
After application of a modified Orthofix mini-extraskeletal fixator, the hind limb of New Zealand White rabbits was osteotomized and then slowly lengthened at a rate of 1 mmlday. After a 20 mm gain in length, the net weight and the length of muscular and tendinous portions were measured and histological examination was carried out in triceps surae muscles. Quantitative analysis showed a significant increase in the gained length of the muscular portion (28.05% to 30.65%). Histological studies of these lengthened muscles showed a generalized increase in cellularity with scanty inflammatory cell infiltration near the myotendinous junction. The increased cellularity is due to the presence of muscle precursor cells characterized by large, oval and pale-stained vesicular nuclei and two prominent nucleoli. The nuclei of these precursor cells were larger and more numerous near the myotendinous junction, and gradually changed into a flattened and more condensed form at a distance from the junction. Occasionally, chains of centrally-located nuclei of primitive myoblasts were also visible. It is concluded that traction neogenesis of the skeletal muscle during limb lengthening does exist and occurs mainly near the myotendinous junction.
Open Access
Three-dimensional reconstruction in microscopical morphology
(Murcia : F. Hernández, 1994) Salisbury, J.R.
This article reviews the current status of three-dimensional reconstruction as a tool in the understanding of microscopical morphology. Many microscopical structures have a three-dimensional shape that canñot readily be appreciated by the study bf sections done. Three-dimensional reconstructions often provide novel information about such structures and enable researchers to formulate new hypotheses about the subject of their study. This review concentrates primarily on the methodology involved and how it is used to generate three-dimensional images and to provide three-dimensional quantitative data.
Open Access
Products of unusual microtubule dynamics in monocyte-derived giant cells. An immunohistochemical study
(Murcia : F. Hernández, 1994) Gadde, P.S.; Moscovic, E.A.
We have studied asteroid bodies (ABs) of multinucleated giant cells (MGCs) in a series of sarcoid and foreign body granulomas with a standard streptavidin-biotin peroxidase technique, using commercial antibodies against collagen, vimentin and tubulin on routinely processed tissue as well as, in one case, on fresh frozen sections (FS). Our findings clearly indicate that ABs are products of the microtubule (MT) system and lack collagen. The tubulin in them stains in fresh FS but is «masked» in formalin-fixed tissue. It can be fully «unmasked» by dephosphorylation and partially by trypsinization. Compared to single microtubule organizing centers (MTOCs) in mononuclear cells serving as interna1 controls, ABs are obvious replicas of centrosome-nucleated MT assemblies from which they differ principally by the disproportionate size of their components and by the invariable vacuolation of the surrounding cytoplasm. Relying on bits of relevant information gleaned from the literature, our observations support the following preliminary conclusions: 1) spokes are massive bundles of MTs nch in tyrosinated a-tubulin coassembled in phosphorylated linkages with yet unidentified microtubule associated proteins (MAPs) and probably microfilament proteins; cores are masses of pericentriolar material including amorphous tubulins, MAPs, phosphoproteins and phospholipids; 2) their size, at least in some ABs, appears to indicate the presence of overlapping centrosome-nucleated MTOCs which in monocyte-derived MGCs are known to be multiple; 3) the cytoplasmic vacuolations around them reflect a collapse and retraction of intermediate filaments (IFs), indicating substantial ongoing MT depolymerization with disruption of MT-IF interactions; 4) ABs are products of unusual MTOC dynamics characterized by simultaneous MT assembly and depolymerization; such a phenomenon, temed «microtubule catastrophec, has been recognized in vitro with centrosome-nucleated MT assemblies under conditions of low tubulin concentrations.
Open Access
Androgen status and expression of glycoconjugates and lectins in the epithelial cells of the mouse ventral prostate. A glycohistochemical approach
(Murcia : F. Hernández, 1994) Akif, F.; Gabius, H.J.; Danguy, A.
We investigated histochemically the expression of glycans and endogenous sugar-binding proteins (endolectins) in the ventral prostate of normal and androgen-deprived mice. The avidin-biotinperoxidase complex technique was used with a panel of biotinylated lectins and neoglycoproteins, respectively, on paraffin sections. Characteristic binding patterns depending upon the type of lectin were observed in the glandular epithelium and basement membranes. This report also initiates the histochemical study of endogenous glycan-binding proteins in this maleaccessory sex organ. Remarkably, the endocrine status was found to affect the distribution of lectin-reactive determinants of glycoconjugates as well as endolectins. The histophysiological significance of these modifications in the glandular cells are discussed in the light of previous biochemical findings obtained mainly in humans and the rat.
Open Access
The breakdown of glycogen in the lysosomes of newborn rat hepatocytes: The effects of glucose, cyclic 3',5'-AMP and caffeine
(Murcia : F. Hernández, 1994) Kalamidas, Stefanos; Kotoulas, Othon B.; Kotoulas, Angeliki O.; Maintas, D.B.
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Open Access
Scanning electron microscopy of the wild boar and pig lingual papillae
(Murcia : F. Hernández, 1994) Chamorro, C.A.; Fernández, J.G.; Paz, P. de; Pelaez, B.; Anel, L.
Lingual papillae of wild boar and pig were studied by means of scanning electron microscopy (SEM). Vallate papillae appear with the typical circumvallate morphology. Their papillary bodies show conical or fungiform-like and spicule-like pseudopapillae in both animals. Taste pores were seen in the papillary grooves. Microplicae or pits are visible at high magnification. In pig and wild boar similar foliate papillae were observed. Pig has less but wider leaves than wild boar. Taste pores on papillary walls were viewed. At high magnification microplicae were seen. Morphologically, fungiform papillae correspond with their denomination. Taste pores open onto the upper surface and they are easily identifiable by SEM. The rostral and lateral regions contain the major number of fungiform papillae. The lateral papillae of wild boar and pig show a high number of pores per papilla. These regions must be considered important in taste sensitivity. Lateral papillae in both animals could provide a source of taste buds for study. In both animals the fungiform papillary epithelium showed a pitted appearance as a consequence of keratinization by food environmental stress. The filiform papillae can be both simple and compound (with body and hairs). Large conical papillae are located caudally and curved in the same direction. Filiform and conical papillae have a function in food mastication, handling and deglutition.