DETECTION OF CELL-PROLIFERATION IN TISSUE-SECTIONS

1. Cell proliferation is of interest since abnormal cell proliferation appears to be a precursor of tumorigenesis and also because the quantitative description of cell proliferation in tumors can be used to predict the biological behavior of a particular neoplasia.2. Them am several reliable methods of studying cell proliferation in tissues. One of the most important is the detection of the Ki67 defined antigen in frozen sections. The number of cells expressing Ki67 correlates with histological grades of tumors and can also be predictive of clinical outcome. The Ki67 can be localized in tissue sections using monoclonal antibodies in association with the immunoperoxidase technique.3. Proliferating cell nuclear antigen (PCNA) is a component of DNA polymerase-delta and is another important cell proliferation marker manifesting a striking increase in concentration during the S phase of the cell cycle. 19A2 and PC10 are two different monoclonal antibodies which can be employed to detect PCNA in paraffin-embedded tissues.4. Molecular biology has also been making a great contribution to the study of cell proliferation. The most recent innovation in tissue identification of proliferating cells is the use of in situ hybridization for the localization of histone H3 and/or H4 mRNA. H3 mRNA-positive cells appear to be present in basal cells of the skin and in crypt cells of the intestine which are sites with high proliferation rate.

Calcifications in a clear cell mucoepidermoid carcinoma: a case report with histological and immunohistochemical findings

The aim of this study was to report an unusual case of mucoepidermoid carcinoma (MEC) in a 39-year-old woman. The tumor showed a prominent population of clear and intermediate basal cells. Clear cells rarely predominate over other cell types. Such cases are called clear cell variant of MEC. The case also revealed a variable amount of calcified material in the tumor mass. Calcifications are rare in clear cell MEC. These structures were periodic acid- Schiff positive and diastase resistant, excluding glycogen origin. Immunohistochemistry was performed, and the epidermoid component was positive for cytokeratin (CK) 7, CK13, CK14, and CK19. The mucous and clear cells presented mild staining for CK7. Cytokeratins 7, 13, and 19 stained luminal cells, and intermediate cells exhibited positivity for CK7, CK14, and vimentin. The origin of the calcifications is speculated to be the result of dystrophic calcification of the amorphous eosinophilic material secreted by intermediate basal cells.

Immunohistochemical Characterization of Canine Prostatic Intraepithelial Neoplasia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cell death in ovarioles causes permanent sterility in Frieseomelitta varia worker bees

Frieseomelitta varia worker bees do not lay eggs even when living in queenless colonies, a condition that favors ovary development and oviposition in the majority of highly social bees. The permanent sterility of these worker bees was initially attributed to a failure in ovary morphogenesis and differentiation. Using transmission electron microscopy we found that at the beginning of the pupal phase the ovaries of F. varia workers are formed by four ovarioles, each of them composed of 1) a terminal filament at the apex of the ovarioles, containing juxtaposed and irregularly shaped cells, 2) a germarium with clusters of cystocytes and prefollicular cells showing long cytoplasmic projections that envelop the cystocyte clusters, 3) fusiform interfollicular and basal stalk precursor cells, and 4) globular, irregularly contoured basal cells with large nuclei. However, during the pupal phase an accentuated and progressive process of cell death takes place in the ovarioles. The dying cells are characterized by large membrane bodies, electron-dense apoptotic bodies, vacuoles, vesiculation, secondary lysosomes, enlarged rough endoplasmic reticulum cisternae, swollen mitochondria, pycnotic nuclei, masses of chromatin adjacent to the convoluted nuclear envelope, and nucleoli showing signs of fragmentation. Cell death continues in ovarioles even after the emergence of the workers. Once they become nurse bees, the ovaries have become transformed into a cell mass in which structurally organized ovarioles can no longer be identified. In F. varia workers, ovariole cell death most certainly is part of the program of caste differentiation.

Intraepithelial alterations in the Guinea pig lateral prostate at different ages after estradiol treatment

The prostate is an accessory gland of the mammal reproductive system with great volume and high functional importance. Many works infer that, in addition to the androgenic ones, the estrogen can be associated with benign prostatic hyperplasia and prostatic cancer, but no conclusive evidence exists on the role of estrogen in normal prostatic and neoplastic tissue. The objective of this work was to evaluate the effects of chronic administration of estradiol benzoate on the lateral prostate of guinea pigs in the pre-pubescent, pubescent, post-pubescent and adult phases, with emphasis on the modifications provoked by this hormone on the glandular epithelium. The analyses of the estradiol-treated and control groups were investigated using histological procedures and transmission electron microscopy. The histopathological analysis of the lateral prostate in the treated group revealed areas where epithelial dysplasia was observed, assuming at some places a pattern of epithelial stratification characteristic of prostatic intraepithelial neoplasia. After ultrastructural analysis, the following were observed: enlargement of the internal membranes, heterogeneity in the cellular types, hypertrophy of the basal cells and apparent decrease of cytoplasmic organelles in some cells of the prostatic intraepithelial neoplasia. Still, a loss of cellular polarity was observed, along with nuclei of various forms, sizes and heights - as well as irregular chromatin distribution patterns. Such alterations were found mainly in pubescent, post-pubescent and adult animals subject to the chronic administration of estradiol. These findings reinforce the already existent data in understanding the role of estrogen in the etiology of prostatic diseases.

Ultrastructural features of the lining epithelium of the vas deferens of Agouti paca

The epididymal epithelium of Agouti paca, a wild South American rodent, was basically formed by principal and basal cells. Principal cells were closely related to processes of adsorptive endocytosis, phasefluid endocytosis and also secretion originating from their cytoplasmic ultrastructural features. Principal cells were also characterized by the presence of vesicles of several shapes, sizes and internalized content occurring in smaller pits, pale small vesicles next to the apical brush border of microvillus, as well as coated vesicles, smooth surface vesicles and great vesicles. Multivesicular bodies, endosomes and lysosomes were mainly observed in supranuclear position. Moreover, presence of an apocrine secretory process was demonstrated by the occurrence of apical cytoplasmic expansions projecting into the vas deferens luminal compartment. Basal flattened cells without luminal surface contact occurred next to the basement membrane of the ductus, and did no exhibit special ultrastructural features.

Effect of topical application of 5-FU on the corneoscleral limbus of rabbits

• Aim: To observe the effects of topical application of 5-fluorouracil (5-FU) on the rabbits corneal limbus. • Methods: 5-FU was applied topically to the corneoscleral region of rabbits eyes. The animals were sacrificed immediately or 7, 15, 30 and 60 days later, and tissues were submitted to histological examination. • Results: All animals presented corneoscleral deepithelization close to the site of application during the immediate post-operative period, whereas on the 4th postoperative day ulceration was no longer present. Histological examination showed absence of epithelium and slight edema. After one week (G2), 2 animals presented epithelial defects, thickened epithelium with larger basal cells and loose chromatin, and slight subepithelial edema. The remaining groups showed no alterations. • Conclusion: The 5-FU topically on the corneoscleral limbus postpone the epitelization.

Morfologia do epidídimo de cobaio (Cavia porcellus) em diferentes fases do desenvolvimento pós-natal

The morphological evolution of the epididymal duct of guinea pigs (Cavia porcellus, L.) was studied on 10, 20, 30, 45, 60, 70, 90 and 100 days of age, being complex, which is due to the proper differentiation postnatal in the epididymal epithelium. Thus, it was observed that the initial segment of the epididymis reveals an increase of epithelial height corresponding to the average height of the main tubular epithelium cells, generally progressive after 45 days of age. The epithelial height in the middle segment were higher in younger stages (10 to 45 days) than in the prepubertal age (60 days), and have a decrease among 70 to 100 days of age. The ductular terminal segment starts a gradual decrease of the epithelial heights from puberal age (70 days), until adult age (90 to 100 days). In addition, this segment showed epithelial waviness that disappeared after 70 days of age, when the lumen fills it with sperm and cellular exfoliation. The cell types of the epididymal tubular epithelium, principal cells, basal cells and apical cells, were observed in all ages. In the epididymis of very young animals, there was a predominance of undifferentiated columnar cells. After 20 days of age, there was natural prevalence of the principal cells on other cell types, what is a feature similar to other mammals.

Estrogen receptors alpha and beta in male and female gerbil prostates

The Mongolian gerbil (Meriones unguiculatus, Gerbilinae: Muridae) is useful for prostate studies, because both males and females spontaneously develop prostatic disorders with age. Estrogens regulate prostate homeostasis via two estrogen receptors, ER alpha (ESR1) and ER beta (ESR2), but the cellular distribution and regulation of these receptors in the gerbil prostate has not been described. Both receptors were localized by immunohistochemistry in the ventral prostate of intact male and female gerbils, in males 7 and 21 days after castration, and in females treated with testosterone for 7 and 21 days. In male and female adult gerbils, ER alpha was detected mainly in prostatic stromal cells, whereas ER beta was present mostly in secretory and basal cells. More ER alpha-positive stromal cells were found in females than in males, as was a reduction toward the male value in females treated with testosterone. Castration did not alter ER alpha expression. Testosterone was necessary for maintenance of ER beta in the male prostate epithelium: ER beta expression declined markedly in prostates of males older than 1 yr, and castration of 4-mo-old males caused a reduction in ER beta to levels seen in 1-yr-old males. Because ER beta is an antiproliferative receptor, its loss with age may predispose the aging gerbil to proliferative diseases of the prostate. © 2013 by the Society for the Study of Reproduction, Inc.

Structure, histochemistry and ultrastructure of the male reproductive accessory glands in the neotropical flat-faced fruit-eating bat Artibeus planirostris (Chiroptera: Phyllostomidae)

Chiroptera, the second largest mammalian order, presents different reproductive strategies and unique reproductive features. However, there are few reports regarding male reproductive accessory glands (RAGs) in Chiroptera. Thus, the aim of the present study was to characterise the RAGs of the exclusively neotropical bat Artibeus planirostris (Chiroptera: Phyllostomidae) macroscopically, microscopically and ultrastructurally. The RAGs were composed of a prostatic complex with two regions (ventral and dorsal) and paraurethral and bulbourethral glands, but no seminal vesicles. The ventral region had an undefined epithelium, with secretory and basal cells, and its secretions were periodic acid-Schiff (PAS) positive. The dorsal region received both deferens ducts, had a columnar pseudostratified epithelium with secretory and basal cells. There were two types of secretions from the dorsal region: one that was basophilic and another that was mixed PAS positive and PAS negative. The paraurethral glands were dispersed in the connective tissue of the urethra, whereas the bulbourethral glands were located in the penile root. Histological and ultrastructural data confirmed the prostatic nature of the ventral and dorsal regions and the holocrine nature of the ventral region, with the latter finding never having been described previously for the prostate gland. Our findings demonstrate the wide discrepancy of RAGs between A. planirostris and other mammals in terms of their composition, structure and morphology. © CSIRO 2013.

NF-kB overexpression and decreased immunoexpression of AR in the muscular layer is related to structural damages and apoptosis in cimetidine-treated rat vas deferens

Background: Cimetidine, histamine H2 receptors antagonist, has caused adverse effects on the male hormones and reproductive tract due to its antiandrogenic effect. In the testes, peritubular myoid cells and muscle vascular cells death has been associated to seminiferous tubules and testicular microvascularization damages, respectively. Either androgen or histamine H2 receptors have been detected in the mucosa and smooth muscular layer of vas deferens. Thus, the effect of cimetidine on this androgen and histamine-dependent muscular duct was morphologically evaluated.Methods: The animals from cimetidine group (CMTG; n=5) received intraperitoneal injections of 100 mg/kg b.w. of cimetidine for 50 days; the control group (CG) received saline solution. The distal portions of vas deferens were fixed in formaldehyde and embedded in paraffin. Massońs trichrome-stained sections were subjected to morphological and the following morphometrical analyzes: epithelial perimeter and area of the smooth muscular layer. TUNEL (Terminal deoxynucleotidyl-transferase mediated dUTP Nick End Labeling) method, NF-kB (nuclear factor kappa B) and AR (androgen receptors) immunohistochemical detection were also carried out. The birefringent collagen of the muscular layer was quantified in picrosirius red-stained sections under polarized light. The muscular layer was also evaluated under Transmission Electron Microscopy (TEM).Results: In CMTG, the mucosa of vas deferens was intensely folded; the epithelial cells showed numerous pyknotic nuclei and the epithelial perimeter and the area of the muscular layer decreased significantly. Numerous TUNEL-labeled nuclei were found either in the epithelial cells, mainly basal cells, or in the smooth muscle cells which also showed typical features of apoptosis under TEM. While an enhanced NF-kB immunoexpression was found in the cytoplasm of muscle cells, a weak AR immunolabeling was detected in these cells. In CMTG, no significant difference was observed in the birefringent collagen content of the muscular layer in comparison to CG.Conclusions: Cimetidine induces significant damages in the epithelium; a possible antiandrogenic effect on the basal cells turnover should be considered. The cimetidine-induced muscle cells apoptosis confirms the susceptibility of these cells to this drug. The parallelism between enhanced cytoplasmic NF-kB immunolabeling in the damaged muscular tissue and muscle cell apoptosis suggests that this drug may avoid the translocation of NF-kB to the nucleus and interfere in the control of NF-kB-mediated smooth muscle cell apoptosis. The decreased immunoexpression of ARs verified in the damaged muscular tissue reinforces this possibility. © 2013 Koshimizu et al.; licensee BioMed Central Ltd.

Keratocyst of the buccal mucosa: case report and immunohistochemical comparative study with sporadic intraosseous keratocystic odontogenic tumor

Objectives: Describe a new case of keratocyst of the buccal mucosa and compare its immunohistochemical features with 13 sporadic intraosseous keratocystic odontogenic tumors (KOT). Case Report and Study Design: A male complaining about an enlargement on the left buccal mucosa was referred to the Stomatology Clinic. Clinical examination revealed a solitary nodule posterior to the parotid papilla. An excisional biopsy was performed following clinical diagnosis of epidermoid cyst. Microscopically, the lesion was characterized by a lining of five cell layers, with columnar basal cells and a corrugated parakeratinized surface. Immunohistochemical reactions for PTCH-1, Smo, Shh, mTOR, bcl-2, Ck17, and Ck19 were performed. PTCH-1 was not expressed in the keratocyst of the buccal mucosa, but was observed in suprabasal layers of eight (61.5%) cases of sporadic intraosseous KOT. Shh, mTOR, bcl-2, Ck17, and Ck19 expression was observed in all the cases investigated. Conclusions: The morphology and immunoprofile of this lesion are similar to sporadic intraosseous KOT. © 2013 Elsevier Inc. All rights reserved.

Light microscope observations on the epididymis of paca (Agouti paca)

The features of paca epididymis, based on its appearance in light microscope, is described in this paper. The cellular population of the epithelial lining comprises principal cells, basal cells, apical cells, narrows cells, and hallo cells. The epididymis is divided in five distinct and continuous regions, Zone I, or initial segment, and zone II, are both localized into the head. Zone III comprises the distal head and all the body. Zones IV and V are restricted to the tail, in the proximal and distal cauda epididymis respectively. Each zone can be readily distinguished on the basis of morphological characteristics. The height of epididymal epithelium is greater in zone I. There is a progressive increase in the diameter of the tubular lumen through the different areas, with the maximum in the zone V. The presence of a high epithelium, and the virtual absence of sperm in zone I suggest fast transit of spermatozoa in this region. Zone V comprises the distal tail, has smaller epithelial lining, greater luminal diameter, shorter stereocilia than the other zones, and contains spermatozoa packed inside the lumen, that characterizes this zone as a place of sperm storage. The findings are compared with other reports in rodents and other domestic animals, to contribute to the understanding of epididymal morphophysiology. © 2013 Firenze University Press.

A new proposed rodent model of chemically induced prostate carcinogenesis: Distinct time-course prostate cancer progression in the dorsolateral and ventral lobes

Background. Characterization of novel rodent models for prostate cancer studies requires evaluation of either spontaneous and carcinogen-induced tumors as well as tumor incidence in different prostatic lobes. We propose a new short-term rodent model of chemically induced prostate carcinogenesis in which prostate cancer progression occurs differentially in the dorsolateral and ventral lobes. Methods. Adult gerbils were treated with MNU alone or associated with testosterone for 3 or 6 months of treatment. Tumor incidence, latency, localization, and immunohistochemistry (AR, PCNA, smooth muscle α-actin, p63, MGMT, and E-cadherin) were studied in both lobes. Results. Comparisons between both lobes revealed that lesions developed first in the DL while the VL presented longer tumor latency. However, after 6 months, there was a dramatic increase in tumor multiplicity in the VL, mainly in MNU-treated groups. Lesions clearly progressed from a premalignant to a malignant phenotype over time and tumor latency was decreased by MNU + testosterone administration. Three-dimensional reconstruction of the prostatic complex showed that the DL developed tumors exclusively in the periurethral area and showed intense AR, PCNA, and MGMT immunostaining. Moreover, VL lesions emerged throughout the entire lobe. MNU-induced lesions presented markers indicative of an aggressive phenotype: lack of basal cells, rupture of the smooth muscle cell layer, loss of E-cadherin, and high MGMT staining. Conclusions. There are distinct pathways involved in tumor progression in gerbil prostate lobes. This animal provides a good model for prostate cancer since it allows the investigation of advanced steps of carcinogenesis with shorter latency periods in both lobes. Copyright © 2013 Wiley Periodicals, Inc.

Morfofisiologia das glândulas reprodutivas acessórias masculinas de morcegos: estudo nas espécies Artibeus planirostris e Platyrrhinus lineatus (Chiroptera: Phylostomidae )

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)