Application of M. Heidenhain’s ferric haematoxylin for the study of semi-thin sections (human adenohypophysis)

Heidenhain´s iron hematoxylin has been one of the most useful methods of classical cytology. Its ability to stain simultaneously many cell and extracellular structures makes it a general purpose histological technique. This current work describes its application to semithin sections, using human adenohypophysis, discussing the staining mechanism and showing his usefulness to evidence cytological variability and to perform image analysis.

Combined lectin- and immuno-histochemistry (CLIH) for applications in cell biology and cancer diagnosis: Analysis of human urothelial carcinomas

Lectin histochemistry (LHC) and immunohistochemistry (IHC), which demonstrate the composition and localisation of sugar residues and proteins in cell membranes, respectively, are generally used separately. Using these two methods, we previously demonstrated that malignant transformation of urothelial cells results in the alterations of protein glycosylation and reduced expression of urothelium-specific integral membrane proteins uroplakins (UPs). However, the correlation between these changes was not studied yet. To evaluate this correlation, we developed innovative method, which we named combined lectin- and immuno- histochemistry (CLIH). We used human biopsies of 6 normal urothelia and 9 papillary urothelial carcinomas, i.e. 3 papillary urothelial neoplasms of low malignant potential (PUNLMP), 3 non-invasive papillary urothelial carcinomas of low grade (pTa, l.g.), and 3 invasive papillary urothelial carcinomas of high grade (pT1, h.g.). We tested five different protocols (numbered 1-5) of CLIH on paraffin and cryo-semithin sections and compared them with LHC and IHC performed separately. Additionally, we carried out western and lectin blotting with antibodies against UPs and lectins Amaranthus caudatus agglutinin (ACA), Datura stramonium agglutinin (DSA), and jacalin, respectively. We showed that incubation with primary antibodies first, followed by the mixture of secondary antibodies and lectins is the most efficient CLIH method (protocol number 5). Additionally, 300 nm thick cryo-semithin sections enabled better resolution of co-localisation between sugar residues and proteins than 5 µm thick paraffin sections. In the normal urothelium, CLIH showed co-localisation of lectins ACA and jacalin with UPs in the apical plasma membrane (PM) of superficial umbrella cells. In papillary urothelial carcinomas, all three lectins (ACA, DSA and jacalin) labelled regions of apical PM, where they occasionally co-localised with UPs. Western and lectin blotting confirmed the differences between normal urothelium and papillary urothelial carcinomas. Our results show that CLIH, when used with various sets of lectins and antigens, is a useful, quick, and reliable method that could be applied for basic cell biology research as well as detailed subtyping of human urothelial carcinomas.

Evaluation of Piper marginatum (Piperales: Piperaceae) Oil and Geraniol on the Embryonic Development of Spodoptera frugiperda (Lepidoptera: Noctuidae) in Comparison to Formulated Products

Essential oils and their isolated constituents are constantly being studied for the control of insect pests. In this context, the present research reports the chemical composition of Piper marginatum (Jacq.) oil aiming to: 1) establish lethal concentrations LC30 and LC50 for this oil and the compound geraniol, 2) histologically examine the embryonic development of Spodoptera frugiperda (J.E. Smith) through light microscopy and scanning electron microscopy (SEM), as well as 3) compare the efficacy of the P. marginatum oil with that of the botanical insecticide azadirachtin, the synthetic insecticide deltamethrin, and acetone as a negative control. Semithin sections of S. frugiperda eggs revealed that the oil, geraniol, azadirachtin, and deltamethrin affected embryonic development at both concentrations. However, geraniol and the oil were more efficient because they caused more significant damage, even at lower concentrations. SEM revealed that all products altered the morphology of the eggs, modifying the structure of the chorion and making the eggs nonviable. Thus, this work demonstrates that P. marginatum oil is effective in the control of S. frugiperda because it results in embryonic damage even at the lowest concentrations.

Intranuclear inclusions in macrophages of lizards Lacerta agilis, experimentally infected by acanthocephalan Corynosoma strumosum

The purpose of the research: to study the cell response of non-natural paratenic host and encapsulation process of acanthocephalan Corynosoma strumosum in experiment for further comparison with encapsulation mechanism of this acanthocephalan in natural paratenic host. Materials and methods. Experiments were carried out on 24 lizards Lacerta agilis and one L. viridis. 17 encapsulated acanthocephalans were received from 13 of them. Acanthocephalans with capsules were prepared for electron microscopic analysis according to standard methods and examined in light (semithin sections) and under electron (in ultrathin sections) microscopes. Semithin sections were stained with methylene blue or a mixture of methylene blue and crystal violet. Ultrathin sections were stained with lead citrate. All capsules received in the experiment were investigated with the use of the light microscope; 1,5 and 10 day capsules were examined under electron microscope. Results and discussion. All acanthocephalans studied in this paper including those discovered one and half day after the start of experiment were enclosed in the thick cellular capsule with prevailing mononuclear and multinuclear macrophages. Single electron-dense inclusions of regular rounded shape surrounded by hallo of moderately dense material were found in approximately half of both types of nuclei. Nature of inclusions remained unknown. In the interpretation of results, it is necessary to take into account: 1) the presence of these inclusions in macrophage nuclei only; 2) their strictly ordinary positioning in the nucleus; 3) strictly spherical shape; 4) very high electronic density of their material, that exceeds the density of the nucleolus and chromatin; 5) presence of halo; 6) absence of visible pathological signs in nuclei and cell’s cytoplasm where these inclusions had been found. Their appearance is supposed to be connected with the overactivity of lizard macrophages caused by invasion of a parasitic worm.

Inhibition ofPasteurella multocidaAdhesion to Rabbit Respiratory Epithelium Using Lectins

This study aimed to evaluate the ability of a panel of lectins to inhibit the ability ofPasteurella multocidato adhere to and affect the rabbit respiratory epithelium. Nasal septa from rabbit fetuses were cultured with various lectins before the addition ofP. multocida. The percentage of bacteria adhering to the epithelium was evaluated semiquantitatively by indirect immunoperoxidase (IIP) staining. The goblet cells (GCs) were counted in semithin sections stained with toluidine blue and served as the main morphological criterion to evaluate the inhibitory effect of the lectins. The lectins PNA, WGA, RCA120, and DBA significantly inhibited the adhesion ofP. multocidato the ciliated epitheliumP<0.05and prevented the pathogen-induced increase in the number of GCsP<0.05compared with those of positive control tissues. In addition, VVA, SJA, UEA I, DSL, SBA, and ECL significantly inhibited the increase in GCs compared with that of the control tissues. The results suggest that less aggressive therapeutic strategies, such as treatment with lectins, may represent alternative approaches to control bacterial respiratory infections.