European Journal of Histochemistry <p>The <strong>European Journal of Histochemistry&nbsp;</strong>has been an influential cytology journal for over 60 years, publishing research articles on functional cytology and histology in animals and plants. The&nbsp;<strong>European Journal of Histochemistry&nbsp;</strong>offers original research articles investigating on structural and molecular components performed by histochemical and immunohistochemical methods, at light and electron microscopy, cytometry and imaging techniques.</p> <p>Areas of particular interest include cell differentiation, senescence and death, and cell-cell interactions in normal and pathological tissues; attention is also given to articles on newly developed or originally applied histochemical and microscopical techniques.</p> <p>Since its foundation in 1954,&nbsp;the <strong>European Journal of Histochemistry&nbsp;</strong>is the official organ of the Italian Society of Histochemistry.</p> <h3>The Proceedings of the 29th National Conference of the Italian Group for the Study of Neuromorphology "Gruppo Italiano per lo Studio della Neuromorfologia" G.I.S.N. are available <a href="" target="_blank" rel="noopener">here</a>.</h3> PAGEPress Scientific Publications, Pavia, Italy en-US European Journal of Histochemistry 1121-760X <p><strong>PAGEPress</strong> has chosen to apply the&nbsp;<a href="" target="_blank" rel="noopener"><strong>Creative Commons Attribution NonCommercial 4.0 International License</strong></a>&nbsp;(CC BY-NC 4.0) to all manuscripts to be published.<br><br>An Open Access Publication is one that meets the following two conditions:</p> <ol> <li class="show">the author(s) and copyright holder(s) grant(s) to all users a free, irrevocable, worldwide, perpetual right of access to, and a license to copy, use, distribute, transmit and display the work publicly and to make and distribute derivative works, in any digital medium for any responsible purpose, subject to proper attribution of authorship, as well as the right to make small numbers of printed copies for their personal use.</li> <li class="show">a complete version of the work and all supplemental materials, including a copy of the permission as stated above, in a suitable standard electronic format is deposited immediately upon initial publication in at least one online repository that is supported by an academic institution, scholarly society, government agency, or other well-established organization that seeks to enable open access, unrestricted distribution, interoperability, and long-term archiving.</li> </ol> <p>Authors who publish with this journal agree to the following terms:</p> <ol> <li class="show">Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.</li> <li class="show">Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.</li> <li class="show">Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work.</li> </ol> Expression of E-cadherin, Ki-67, and p53 in urinary bladder cancer in relation to progression, survival, and recurrence <p>Although the incidence varies with age and gender, urothelial bladder cancer is a relatively frequently occurring malignancy with variable clinical behavior that often has high recurrence rates. In this study, we analyzed the tumor tissues of 224 patients with pTa, pT1, and pT2 urinary bladder cancer. We performed a histomorphologic analysis and immunohistochemistry for p53, Ki-67, and E-cadherin, which were selected as markers of the malignant process. For pTa and pT1, univariate analyses of cancer-specific survival (CSS), progression-free survival (PFS), and recurrence-free survival (RFS) were calculated using the Kaplan-Meier method, the log-rank test and Cox regression. Multivariate analysis was performed by a Cox regression analysis. Ki-67 (P&lt;0.001) was significantly associated with CSS, but the highest association was shown for E-cadherin (P&lt;0.001). For pT1 and pTa, the Kaplan-Meier analysis and the log-rank test revealed significantly worse PFS for patients with higher levels of Ki-67 (P&lt;0.001) and lower levels of E-cadherin (P&lt;0.001). Based on these obtained results, it can be clearly stated that Ki-67 and E-cadherin expression levels are associated with CSS, PFS and RFS. The clinical utility of these markers is valuable for pTa and pT1 urinary bladder cancer and should be further verified with prospective multi-center trials.</p> Stanislav Ziaran Stefan Harsanyi Katarina Bevizova Zuzana Varchulova Novakova Branislav Trebaticky Peter Bujdak Stefan Galbavy Lubos Danisovic Copyright (c) 2020 The Author(s) 2020-03-26 2020-03-26 64 2 10.4081/ejh.2020.3098 Defense and protection mechanisms in lung exposed to asbestiform fiber: the role of macrophage migration inhibitory factor and heme oxygenase-1 <p>Fluoro-edenite (FE), an asbestiform fiber, is responsible for many respiratory pathologies: chronic obstructive diseases, pleural plaques, fibrosis, and malignant mesothelioma. Macrophage migration inhibitory factor (MIF) is one of the first cytokines produced in response to lung tissue damage. Heme oxygenase-1 (HO-1) is a protein with protective effects against oxidative stress. It is up regulated by several stimuli including pro-inflammatory cytokines and factors that promote oxidative stress. In this research, the <em>in vivo</em> model of sheep lungs naturally exposed to FE was studied in order to shed light on the pathophysiological events sustaining exposure to fibers, by determining immunohistochemical lung expression of MIF and HO-1. Protein levels expression of HO-1 and MIF were also evaluated in human primary lung fibroblasts after exposure to FE fibers <em>in vitro</em>. In exposed sheep lungs, MIF and HO-1 immunoexpression were spread involving the intraparenchymal stroma around bronchioles, interstitium between alveoli, alveolar epithelium and macrophages. High MIF immunoexpression prevails in macrophages. Similar results were obtained <em>in vitro</em>, but significantly higher values were only detected for HO-1 at concentrations of 50 and 100 μg/mL of FE fibers. MIF and HO-1 expressions seem to play a role in lung self-protection against uncontrolled chronic inflammation, thus counteracting the strong link with cancer development, induced by exposure to FE. Further studies will be conducted in order to add more information about the role of MIF and HO-1 in the toxicity FE-induced.</p> Carla Loreto Rosario Caltabiano Adriana Carol Eleonora Graziano Sergio Castorina Claudia Lombardo Vera Filetti Ermanno Vitale Giuseppe Rapisarda Venera Cardile Caterina Ledda Venerando Rapisarda Copyright (c) 2020 The Author(s) 2020-04-16 2020-04-16 64 2 10.4081/ejh.2020.3073 Thrombin regulates the ability of Schwann cells to support neuritogenesis and to maintain the integrity of the nodes of Ranvier <p>Schwann cells (SC) are characterized by a remarkable plasticity that enables them to promptly respond to nerve injury promoting axonal regeneration. In peripheral nerves after damage SC convert to a repair-promoting phenotype activating a sequence of supportive functions that drive myelin clearance, prevent neuronal death, and help axon growth and guidance. Regeneration of peripheral nerves after damage correlates inversely with thrombin levels. Thrombin is not only the key regulator of the coagulation cascade but also a protease with hormone-like activities that affects various cells of the central and peripheral nervous system mainly through the protease-activated receptor 1 (PAR1). Aim of the present study was to investigate if and how thrombin could affect the axon supportive functions of SC. In particular, our results show that the activation of PAR1 in rat SC cultures with low levels of thrombin or PAR1 agonist peptides induces the release of molecules, which favor neuronal survival and neurite elongation. Conversely, the stimulation of SC with high levels of thrombin or PAR1 agonist peptides drives an opposite effect inducing SC to release factors that inhibit the extension of neurites. Moreover, high levels of thrombin administered to sciatic nerve <em>ex vivo</em> explants induce a dramatic change in SC morphology causing disappearance of the Cajal bands, enlargement of the Schmidt-Lanterman incisures and calcium-mediated demyelination of the paranodes. Our results indicate thrombin as a novel modulator of SC plasticity potentially able to favor or inhibit SC pro-regenerative properties according to its level at the site of lesion.</p> Elena Pompili Viviana Ciraci Stefano Leone Valerio De Franchis Pietro Familiari Roberto Matassa Giuseppe Familiari Ada Maria Tata Lorenzo Fumagalli Cinzia Fabrizi Copyright (c) 2020 The Author(s) 2020-03-30 2020-03-30 64 2 10.4081/ejh.2020.3109 A new method for the direct tracking of <em>in vivo</em> lignin nanocapsules in <em>Eragrostis tef</em> (Poaceae) tissues <p class="western" style="margin-bottom: 0cm; line-height: 200%;" align="justify">Environmental concerns have driven scientists to research new eco-friendly approaches for the preparation of nanosystems. For this purpose, novel bio-polymers have been selected. Among these, one of the most promising is lignin, which is biodegradable and biocompatible. Additionally, lignin is one of the main by-products of the paper industry and can be re-used in nanosystems building. Lignin-based nanosystems could be used in agriculture, to improve the uptake of bioactive compounds, thus avoiding soil pollution. However, the mechanism of penetration in the plant and the route of transportation within the internal plant tissues are unknown and need to be clearly elucidated. Here we present a method of lignin nanocapsules staining and tracking by fluorochrome: Fluoral Yellow 088, which is a well-suited dye for the tracking of lipids and other oil phases. Two different applications were applied: in the first one fourteen-day plants were soaked with fluorescent nanocapsules (fNCs) pure solution and in the second one,<em> Eragrostis tef</em> plants were laid down on blotting paper and soaked with diluted fNCs solution. Wetting the roots of Teff plantlets with the pure fNCs solution resulted in the most efficient way of nanocapsule entrance. The dyeing of lignin nanocapsules allowed us to track them in <em>Eragrostis tef</em> plant tissues through microscopic observations. In particular, fNCs were proven to be able to permeate roots, reaching xylem vessels where, through water pressure, they reached the leaf.</p> Sara Falsini Corrado Tani Silvia Schiff Cristina Gonnelli Ilaria Clemente Sandra Ristori Alessio Papini Copyright (c) 2020 The Author(s) 2020-03-26 2020-03-26 64 2 10.4081/ejh.2020.3112 Seasonal expressions of SPAG11A and androgen receptor in the epididymis of the wild ground squirrels (<em>Citellus dauricus</em> Brandt) <p>Sperm-associated antigen 11A (SPAG11A) is expressed exclusively in the epididymis, which can specifically regulate sperm maturation. The aim of this study was to investigate the seasonal expressions of beta-defensin (SPAG11A) and androgen receptor (AR) in the epididymis of the wild ground squirrels (<em>Citellus dauricus </em>Brandt). Morphologically, the results showed that epididymis length and weight in the breeding season were significantly higher than those of the non-breeding season. Histologically, the results revealed that enlarged lumen diameters, thickened epithelial and abundant sperm in the breeding season while reduced lumen diameters and epithelial with no sperm in the non-breeding season. SPAG11A was intensely expressed in cytoplasm and nucleus of epithelial cells in the breeding season, and weaker staining in the non-breeding season. The immunostaining of AR was only presented in nucleus of smooth muscle cells and epithelial cells in the whole epididymis with stronger staining in the breeding season. The results of real-time quantitative PCR also showed that the mRNA levels of SPAG11A and AR in the epididymis during the breeding season were significantly higher than those of the non-breeding season. In addition, the levels of testosterone (T), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the serum were higher during the breeding season. Taken together, these results suggested that SPAG11A might play an important role to regulate seasonal changes in epididymal function of the wild ground squirrels.</p> Wenyang Yu Ziwen Zhang Pei Liu Xiaoying Yang Haolin Zhang Zhengrong Yuan Yingying Han Qiang Weng Copyright (c) 2020 The Author(s) 2020-04-21 2020-04-21 64 2 10.4081/ejh.2020.3111 The psoriatic shift induced by interleukin 17 is promptly reverted by a specific anti-IL-17A agent in a three-dimensional organotypic model of normal human skin culture <p>Interleukin 17A (IL-17A), mainly produced by the T helper subclass Th17, plays a key role in the psoriatic plaque formation and progression. The clinical effectiveness of anti-IL-17A agents is documented, but the early and specific mechanisms of their protection are not identified yet. The challenge of the present study is to investigate the possible reversal exerted by a specific anti-IL-17A agent on the psoriatic events induced by IL-17A in a three-dimensional organotypic model of normal human skin. Bioptic skin fragments obtained after aesthetic surgery of healthy women (n=5) were incubated with i) IL-17A biological inhibitor (anti-IL-17A), ii) IL-17A, iii) a combination of IL-17A and its specific IL-17A biological inhibitor (COMBO). A Control group was in parallel cultured and incubation lasted for 24 and 48 h epidermal-side-up at the air-liquid interface. All subjects were represented in all experimental groups at all considered time-points. Keratinocyte proliferation and the presence of epidermal Langerhans cells were quantitatively estimated. In parallel with transmission electron microscopy analysis, immunofluorescence studies for the epidermal distribution of keratin (K)10, K14, K16, K17, filaggrin/occludin, Toll-like Receptor 4, and Nuclear Factor kB were performed. IL-17A inhibited cell proliferation and induced K17 expression, while samples incubated with the anti-IL-17A agent were comparable to controls. In the COMBO group the IL-17A-induced effects were almost completely reverted. Our study, for the first time, elucidates the most specific psoriatic cellular events that can be partially affected or completely reverted by a specific anti-IL-17A agent during the early phases of the plaque onset and progression. On the whole, this work contributes to expand the knowledge of the psoriatic tableau.</p> Elena Donetti Giulia Lombardo Serena Indino Laura Cornaghi Francesca Arnaboldi Leonardo Pescitelli Franz Baruffaldi Preis Francesca Prignano Copyright (c) 2020 The Author(s) 2020-04-16 2020-04-16 64 2 10.4081/ejh.2020.3115 The substance P and neurokinin-1 receptor system in human thyroid cancer: an immunohistochemical study <p>To develop a new therapeutic strategy against thyroid cancer (TC), the expression of both substance P (SP) and neurokinin-1 receptor (NK-1R) must be demonstrated in TC cells. This study aims to examine by immunohistochemistry, the localization of SP and the NK-1R in human TC samples (papillary, follicular, medullary, anaplastic), in metastasis and in healthy thyroid samples. SP and the NK-1R were expressed in all normal and TC samples. In healthy glands, SP was located in follicular cells (nucleus) and colloid and NK-1R in follicular cells (cytoplasm) and stroma. In TC samples, SP was visualized in follicular cells (nucleus and cytoplasm), stroma and colloid and NK-1R in follicular cells (cytoplasm), stroma and colloid. A semiquantitative scoring system (Allred Unit Scoring System) was applied. The expression (Allred total score) of SP and NK-1R was weaker in normal thyroid glands than in TC. In comparison with TC samples, a lower intensity/proportion of SP (nucleus and cytoplasm of follicular cells; stroma) was observed in normal samples. By contrast, in the colloid of TC samples the presence of SP was lower than in normal samples. In comparison with TC samples, the presence of the NK-1R in the cytoplasm of follicular cells and colloid was lower in normal thyroid samples, whereas the expression of this receptor in the stroma was higher. The results reported in this study suggest that the NK-1R could be a new target for the treatment of TC and use of the NK-1R antagonists could serve as a new anti-TC therapeutic strategy.</p> Inmaculada Isorna Francisco Esteban Juan Solanellas Rafael Coveñas Miguel Muñoz Copyright (c) 2020 The Author(s) 2020-04-28 2020-04-28 64 2 10.4081/ejh.2020.3117 Presence of N-acetylneuraminic acid in the lung during postnatal development <p>Sialic acids, particularly N-acetylneuraminic acid (Neu5Ac), are present as terminal components of rich and complex oligosaccharide chains, which are termed glycans, and are exhibited on the cell surfaces, especially on epithelial cells. Crucial in the ‘social behavior’ of the cell, sialic acids play vital roles in many physiological and pathological phenomena. The aim of the present study was to separate, identify, and quantify Neu5Ac in purified lung membranes from 4-, 14-, and 21-day-old animals, followed by the statistical analysis of these results with our previously reported data (0-day-old and adult results). Complementary, ultrastructural methodologies were used. The differences in the Neu5Ac values obtained across the examined postnatal-lung development relevant ages studied were found to be statistically significant. A substantial increase in the mean level of this compound was found during the period of ‘bulk’ alveolarization, which takes place from postnatal day 4 to 14 (P4-P14). The comparison of the mean levels of Neu5Ac, during microvascular maturation (mainly between P12 and P21), reveals that the difference, although statistically significant, is the least significant difference among all the pair-wise differences between the developmental stages. The presence of sub-terminal N-acetylgalactosamine (GalNAc)/Galactose (Gal) residues with terminal sialic acids on the bronchioloalveolar cell surfaces was confirmed using lung ultra-thin sections of adult and 0-day-old animals. These results showed that, although Neu5Ac levels increase throughout postnatal lung development, this sialic acid was substantially added to epithelial cell surfaces during the “bulk” alveolarization period, while its presence was less important during the microvascular maturation period. Bearing in mind that sialic acids are negatively charged and create charge repulsions between adjacent cells, we hypothesized that they can substantially contribute to postnatal alveolar formation and maturation.</p> Maria de Fátima Martins Marco S. Freitas Ana Honório-Ferreira Carlos Alberto Gonçalves Copyright (c) 2020 The Author(s) 2020-05-06 2020-05-06 64 2 10.4081/ejh.2020.3124 Morphological and ultrastructural characteristics of the tongue of wild boar <p>The present study aimed to describe the structural and ultrastructural morphological characteristics of the lingual epithelium and the connective tissue cores (CTCs) of wild boar (<em>Sus scrofa</em>). The tongues were processed for light microscopy, scanning electron microscopy, and transmission electron microscopy. In this study, we revealed the filiform, fungiform, foliate, and vallate papillae. The filiform papilla is elongated with a conical shape and its CTC has a conical shape; the fungiform papilla is rounded with a dome-shape and its CTC is flower bud; the foliate papilla is formed by four pairs of epithelial folds and irregular grooves, and its CTC is thin with adjacent conjunctive projections, and taste buds and serous glands in the epithelial layer have been evidenced; and the vallate papilla is oval surrounded by a groove with increases of epithelium surface, and the CTC is formed by numerous connective projections lined. Also noted were serous gland and taste buds on the medial wall of the vallate papilla. The epithelium has the keratinized, granular, spinous, basal, and lamina propria layers. In conclusion, we found new descriptions and shapes of the CTCs of the lingual papillae. In addition, we demonstrated the epithelium structural characteristics, the nuclear distribution between the epithelial layers, and the ultrastructural aspects of the dorsal epithelium of the tongue.</p> Gabriela de Souza Reginato Gabriela Klein Barbosa Amanda Olivotti Ferreira Bruno Gomes Vasconcelos Rose Eli Grassi Rici Ii-sei Watanabe Adriano Polican Ciena Copyright (c) 2020 The Author(s) 2020-05-05 2020-05-05 64 2 10.4081/ejh.2020.3128 Ozone at low concentrations does not affect motility and proliferation of cancer cells in vitro <p>Exposure to low ozone concentrations is used in medicine as an adjuvant/complementary treatment for a variety of diseases. The therapeutic potential of low ozone concentrations relies on their capability to increase the nuclear translocation of the Nuclear factor erythroid 2-related factor 2 (Nrf2), thus inducing the transcription of Antioxidant Response Elements (ARE)-driven genes and, through a cascade of events, a general cytoprotective response. However, based on the controversial role of Nrf2 in cancer initiation, progression and resistance to therapies, possible negative effects of ozone therapy may be hypothesised in oncological patients. With the aim to elucidate the possible changes in morphology, migration capability and proliferation of cancer cells following mild ozone exposure, we performed wound healing experiments <em>in vitro</em> on HeLa cells treated with low ozone concentrations currently used in the clinical practice. By combining a multimodal microscopy approach (light and fluorescence microscopy, scanning electron microscopy, atomic force microscopy) with morphometric analyses, we demonstrated that, under our experimental conditions, exposure to low ozone concentrations does not alter cytomorphology, motility and proliferation features, thus supporting the notion that ozone therapy should not positively affect tumour cell growth and metastasis.</p> Manuela Costanzo Alessandro Romeo Barbara Cisterna Laura Calderan Paolo Bernardi Viviana Covi Gabriele Tabaracci Manuela Malatesta Copyright (c) 2020 The Author(s) 2020-04-02 2020-04-02 64 2 10.4081/ejh.2020.3119 MMP-7 and MMP-9 are overexpressed in the synovial tissue from severe temporomandibular joint dysfunction <p>Matrix metalloproteinases (MMPs) are tissue-enzymes that play a key role during the remodeling process, such as in inflammatory diseases. MMP-7 and MMP-9 have been shown to be implicated in extracellular matrix homeostasis and in joint disc remodeling. The objective of this study was to determine the relation of MMP-7 and MMP-9 expression with severe temporomandibular joint dysfunction, in particular with anterior disk displacement without reduction (ADDwoR), using an immunohistochemical approach. Therefore, twenty human temporomandibular synovia in the test group and ten in the control group were collected. The results showed there was a statistically significant difference (P&lt;0.001) for morphometric and densitometric analysis of both detected MMPs in control and test groups. In conclusion, MMP-7 and MMP-9 were overexpressed in the synovial tissue of patients with ADDwoR.</p> Carla Loreto Vera Filetti Luis Eduardo Almeida Giusy Rita Maria La Rosa Rosalia Leonardi Cristina Grippaudo Antonino Lo Giudice Copyright (c) 2020 The Author(s) 2020-04-16 2020-04-16 64 2 10.4081/ejh.2020.3113 Isolation of rat hepatocytes for pharmacological studies on metabotropic glutamate receptor (mGluR) subtype 5: a comparison between collagenase I <em>versus</em> collagenase IV <p>Isolated hepatocytes can be obtained from the liver by collagenase infusion, a procedure that could affect cell isolation as well as the integrity of membrane receptors. In this respect we compared metabotropic glutamate subtype 5 receptor (mGluR5) protein expression and activity in rat hepatocytes isolated by two collagenases, type I and type IV. Hepatocytes were isolated from male Wistar rats (200-250 g) using collagenase I or collagenase IV and after isolation, viability and morphology of rat hepatocytes were assessed measuring mGluR5 protein expression by Western blot analyses. mGluR5 activation was evaluated by inositol-1-phosphate (IP-1) accumulation after treatment with the mGluR5 orthosteric agonist ACPD or the selective antagonist MPEP. No difference in cellular viability and morphology was observed using collagenase I when compared with collagenase IV. An increase in mGluR5 protein expression was observed in hepatocytes isolated using collagenase IV with respect to collagenase I. Moreover, hepatocytes treated with ACPD and with MPEP presented higher levels of IP-1 when isolated using collagenase IV compared to collagenase I. These results indicate that collagenase IV better preserves the activity of surface proteins such as mGluR5 in isolated rat hepatocytes, an <em>in vitro</em> model useful to reduce the use of experimental animals, in line with the 3R ethical framework.</p> Clarissa Berardo Andrea Ferrigno Veronica Siciliano Plinio Richelmi Mariapia Vairetti Laura Giuseppina Di Pasqua Copyright (c) 2020 The Author(s) 2020-03-26 2020-03-26 64 2 10.4081/ejh.2020.3123