Beata Szynaka

ULTRASTRUCTURE OF THE TUNICA ALBUGINEA IN CONGENITAL PENILE CURVATURE

PURPOSE We investigated the ultrastructure of the tunica albuginea in individuals with congenital penile curvature to explain the pathology of this disease. MATERIALS AND METHODS Included in our study were 15 patients 17 to 24 years old with congenital penile curvature. Study material consisted of samples of the tunica albuginea excised from the greater curvature of the corpus cavernosum during surgical correction. Control samples were obtained from the lesser curvature on the side opposite the study material during the same operation. The 2 types of tissue were analyzed using transmitter electron microscopy. RESULTS Ultrastructural examination of the control material revealed numerous collagen fibers that were homogenous in size and organization on cross section. Periodic striation was typical in collagen that produced fibers. In the study group the tunica albuginea structure had a chaotic pattern of collagen fibers that formed bundles with disrupted 3-dimensional organization. Diameter of the fibers differed greatly on cross section. We observed periodic widening and fragmentation of collagen fibers with the complete disappearance of striation and transformation into electron dense, fibrous granulated material. Disrupted fibroblasts without cell membrane and cellular organelles between collagen fibers were also visible. There was elastin accumulation without any morphological differences in the control and study groups. CONCLUSIONS Our results show that ultrastructural changes in the tunica albuginea may cause congenital penile curvature, possibly by altering mechanical properties.

Growth arrest and rapid capture of select pathogens following magnetic nanoparticle treatment.

Thorough understanding of magnetic nanoparticle (MNP) properties is essential for developing new theranostics. In this study, we provide evidence that non-modified magnetic iron oxide nanoparticles and their functionalized derivatives may be used to restrict growth and capture different pathogens. Coprecipitation of Fe(2+) and Fe(3+) ions in an alkaline solution was used to synthesize MNPs that subsequently were functionalized by gold and aminosilane coating. Transmission electron microscopy (TEM), differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FT-IR) were used to assess their physicochemical properties. A significant decrease of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans outgrown from medium after addition of MNPs or their derivatives was observed during 24h culture. Measurement of optical density revealed that using MNPs, these pathogens can be quickly captured and removed (with efficiency reaching almost 100%) from purposely infected saline buffer and body fluids such as human blood plasma, serum, abdominal fluids and cerebrospinal fluids. These effects depend on nanoparticle concentration, surface chemistry, the type of pathogen, as well as the surrounding environment.

Remodeling of the intercalated disc related to aging in the mouse heart

BACKGROUND Aging is related to declined cardiac hemodynamic function. As pumping performance may be significantly related to slowed ventricular depolarization and non-synchronous contraction, we hypothesized that aging may cause dysfunction of intercalated disc (ID), which is the structure responsible for intercellular electrical communication between cardiomyocytes. METHODS Male C57BL/6J mice were used for the study at two ages: 4 and 24 months. Electrocardiographic recording was made to analyze the time of ventricular depolarization. Then mice were killed, and the hearts were harvested for examination in transmission electron microscopy (TEM) and immunofluorescence imaging. The expression of connexin 43 (Cx43), N-cadherin, and β-catenin in the myocardium of the left ventricle was evaluated using Western blotting. RESULTS In senescent mice, analysis of averaged QRS complex showed its significant prolongation. At the ultrastructural level, we found frequent disruptions of the ID (affecting 29±5% of them), mainly at the site of adherens junction, with relatively preserved desmosomal intercellular connections and diminished number of gap junctions. Western blotting revealed significantly decreased abundance of Cx43 protein in aged animals, which may cause slowed impulse propagation through the gap junctions and contribute to the observed electrocardiographic alterations. The level of RNA for Cx43 is similar between young and old animals, which suggests a post-transcriptional mechanism of Cx43 protein downregulation. CONCLUSIONS Our study shows age-related disorganization of ID, which may be responsible for slowed conduction of the depolarization wave within the heart, and supports the hypothesis of cardiac dysfunction in senescence.

Ethanolic extract of propolis, chrysin, CAPE inhibit human astroglia cells

PURPOSE The aim of the present study was to examine the effect of freeze dried ethanolic extract of propolis (EEP), chrysin and caffeic acid phenethyl ester (CAPE) dependently on their concentrations on the viability and morphology of human astroglia cells line (SVGp12). MATERIAL AND METHODS Using gas chromatography - mass spectroscopy (GC-MS) we have established the composition of lyophilisate of EEP collected in Podlasie region (Poland). After 24 h, 48 h and 72 h of exposition to EEP or its ingredients we evaluated the survivability of human astroglia cells (SVGp12) using MTT test. Morphological analysis of human astroglia cells was defined by transmission electron microscope. RESULTS About 70 ingredients of EEP were evaluated by GC-MS. We obtained the strong decline of viability of astroglia cells SVGp12 approximately to 16% after EEP; 33% after chrysin and 25% after CAPE application. Condensed form of mitochondria observed in transmission electron microscope may indicate activation of intrinsic pathway of apoptosis induced by EEP, chrysin and CAPE in SVGp12 cell line. CONCLUSION This study showed that EEP, chrysin and CAPE reduced viability of human astroglia cells probably due to apoptosis process.

Interleukin 6 modulates PPARα and PGC-1α and is involved in high-fat diet induced cardiac lipotoxicity in mouse.

BACKGROUND Interleukin 6 (IL-6) may be involved in regulation of cardiac lipid metabolism and mitochondrial function through its influence on peroxisome proliferator-activated receptors (PPARs). In this study we evaluated the impact of the physiological level of IL-6 on the expression of PPARα and PGC-1α in the heart and the effect of lack of this cytokine on high-fat diet (HFD) induced lipotoxicity. METHODS Male C57BL6/J wild type (WT) and IL-6 knock-out (IL-6KO) mice were used. 20 animals of each genotype were fed with HFD for 15-18weeks. Cardiac function was assessed using echocardiography and cardiomyocyte ultrastructure was examined using electron microscopy. QT-PCR and Western blotting were applied to estimate the expression of PPARα and PGC-1α at the transcriptional and protein levels. RESULTS At baseline WT and IL-6KO mice had similar size and function of the left ventricle. HFD induced similar left ventricular hypertrophic response in both groups without causing heart failure, but only WT animals had increased resting ejection fraction of the LV. Ultrastructure of HFD groups showed markers of lipotoxicity, that were more pronounced in IL-6KO group. In basal conditions IL-6KO animals had lower PPARα and similar PGC-1α expression as compared to WT. HFD induced downregulation of both PPARα and PGC-1α in WT animals, while in IL-6KO mice this effect was constrained. CONCLUSION IL-6 is involved in basal regulation of PPARα and PGC-1α expression in cardiomyocytes. The lack of this cytokine promotes high-fat diet induced lipotoxicity but without overt manifestations of cardiac failure.

Alveolar epithelial cells in experimental lung emphysema Ultrastructural analysis of cells in situ in TEM

A study was made of ultrastructural changes in alveolar epithelial cells after a single intratracheal papain injection. The experiment was carried out on male Wistar rats, of 180-220 g body weight. The animals were sacrificed after 1, 7, 14 and 28 days that followed proteolytic enzyme administration. The study revealed that the development of emphysematous changes in the rat lungs was accompanied by alterations in the epithelium of alveoli. In early stages of emphysema (up to 1 week), destructing changes dominated within the epithelial cells. Similar changes were observed in the endothelium of adjacent blood vessels. In later periods an increased number of type II pneumocytes were seen and focal accumulation of elastic and collagenic fibres was noticed in alveolar septa. Correlation was found between changes in the number of type II cells and their ultrastructure as well as between alveolar epithelium damage and alveolar septal interstitium alterations.

Sporicidal activity of ceragenin CSA-13 against Bacillus subtilis

Spore-forming bacteria are a class of microorganisms that possess the ability to survive in extreme environmental conditions. Morphological features of spores assure their resistance to stress factors such as high temperature, radiation, disinfectants, and drying. Consequently, spore elimination in industrial and medical environments is very challenging. Ceragenins are a new class of cationic lipids characterized by a broad spectrum of bactericidal activity resulting from amphipathic nature and membrane-permeabilizing properties. To assess the impact of ceragenin CSA-13 on spores formed by Bacillus subtilis (ATCC 6051), we performed the series of experiments confirming that amphipathic and membrane-permeabilizing properties of CSA-13 are sufficient to disrupt the structure of B. subtilis spores resulting in decreased viability. Raman spectroscopy analysis provided evidence that upon CSA-13 treatment the number of CaDPA-positive spores was clearly diminished. As a consequence, a loss of impermeability of the inner membranes of spores, accompanied by a decrease in spore resistance and killing take place. In addition to their broad antimicrobial spectrum, ceragenins possess great potential for development as new sporicidal agents.

Exocrine cell mitochondria of the rat pancreas after lead intoxication.

The aim of the study was to evaluate alterations in exocrine cell mitochondria of the rat pancreas after lead acetate intoxication. The experiment used 45 rats divided into 2 experimental groups receiving lead acetate to drink, of lead concentration 50 and 500 mg/dm3 (ppm), and a control group given tap water. The animals from the experimental group were decapitated after 2, 4, 6 and 8 weeks, 5 rats from the control group after 8 weeks of the experiment. Rats from experimental groups decapitated after 8 weeks had lead administration stopped after six weeks and then, for two weeks tap water was given. Pancreatic sections were examined with biochemical methods for the activity of cytochrome oxidase and succinic dehydrogenase. Ultrastructural and morphometric examinations were also performed. It was demonstrated that: a) exocrine cell mitochondria are particularly predisposed to lead effect, b) intoxication of rats with lower lead doses (50 ppm) causes reversible adaptative or compensatory changes in these organelles, c) intoxication of rats with higher lead doses (500 ppm) induces irreversible ultrastructural alterations in numerous mitochondria, including damage to inner and to outer mitochondrial membranes, d) structural changes in the mitochondria in the course of lead intoxication are the morphological expression of the impairment of metabolic processes, associated with the inhibited activity of the respiratory enzymes: succinic dehydrogenase and cytochrome oxidase.

Metronidazole Decreases Viability of DLD-1 Colorectal Cancer Cell Line

The aim of our study was to evaluate the impact of metronidazole (MTZ) on DLD-1 colorectal cancer cell (CRC) line. Toxicity of MTZ was determined by MTT test. Cells were incubated with MTZ used in different concentrations for 24, 48, and 72 hours. The effect of MTZ on DNA synthesis was measured as [3H]-thymidine incorporation. The morphological changes in human DLD-1 cell line were defined by transmission electron microscope OPTON 900. The influence of MTZ on the apoptosis of DLD-1 cell lines was detected by flow cytometry and fluorescence microscopy, while cell concentration, volume, and diameter were displayed by Scepter Cell Counter from Millipore. Our results show that cell viability was diminished in all experimental groups in comparison with the control, and the differences were statistically significant. We did not find any significant differences in [3H]-thymidine incorporation in all experimental groups and times of observation. Cytofluorimetric assays demonstrated a statistically significant increase of apoptotic rate in MTZ concentrations 10 and 50 μg/mL after 24 hours; 0.1, 10, 50, and 250 μg/mL after 48 hours; and in all concentrations after 72 hours compared with control groups. In the ultrastructural studies, necrotic or apoptotic cells were occasionally seen. In conclusion, MTZ affects human CRC cell line viability. The reduction of cell viability was consistent with the apoptotic test.

Expression of macrophage markers in cryoglobulinemic glomerulonephritis - a possible role of CXCL9

PURPOSE Cryoglobulinemic glomerulonephritis (CGGN) is a type of membranoproliferative glomerulonephritis (MPGN) that develops in patients with systemic cryoglobulinemia. To date the exact pathogenesis of CGGN remains unclear. It has been suggested that macrophages may be significant contributors to the glomerular injury in this disease. In our study we attempt to characterize the macrophages in human CGGN using classical activation and regulatory macrophage markers. MATERIAL AND METHOD We searched our database for renal biopsy cases of CGGN. Macrophages were detected using a monoclonal anti-CD68 antibody. Two groups of macrophage markers were used: classical activation markers, including iNOS, CXCL9 and CCL20, and regulatory markers: SPHK1 and LIGHT. The stains were performed using immunohistochemical method. RESULTS Five patients with CGGN were identified. Four patients had systemic cryoglobulinemia and two had a serological evidence of hepatitis C virus infection. In all cases the glomeruli contained numerous macrophages. Staining for activatory macrophage markers revealed a strong nuclear staining for CXCL9 in numerous cells, including those corresponding to the macrophage location. Staining for the other activatory markers, as well as staining for regulatory markers, was not significant. CONCLUSION In this study of human CGGN we showed a striking expression of cytokine CXCL9, a classical macrophage activation marker, by the macrophages and possibly other cell types within the glomeruli. This observation points to the possible role of classically activated macrophages in the pathogenesis of MPGN. If this observation is confirmed on a larger group of patients, the cytokine CXCL9 could become a potential therapeutic target for human CGGN.