The Synergistic Effect of Reduced Graphene Oxide and Proteasome Inhibitor in the Induction of Apoptosis through Oxidative Stress in Breast Cancer Cell Lines.

Reduced graphene oxide (rGO) and a proteasome inhibitor (MG-132) are some of the most commonly used compounds in various biomedical applications. However, the mechanisms of rGO- and MG-132-induced cytotoxicity remain unclear. The aim of this study was to investigate the anticancer effect of rGO and MG-132 against ZR-75-1 and MDA-MB-231 breast cancer cell lines. The results demonstrated that rGO, MG-132 or a mix (rGO + MG-132) induced time- and dose-dependent cytotoxicity in ZR-75-1 and MDA-MB-231 cells. Apart from that, we found that treatment with rGO and MG-132 or the mix increased apoptosis, necrosis and induction of caspase-8 and caspase-9 activity in both breast cancer cell lines. Apoptosis and caspase activation were accompanied by changes in the ultrastructure of mitochondria in ZR-75-1 and MDA-MB-231 cells incubated with rGO. Additionally, in the analyzed cells, we observed the induction of oxidative stress, accompanied by increased apoptosis and cell necrosis. In conclusion, oxidative stress induces apoptosis in the tested cells. At the same time, both mitochondrial and receptor apoptosis pathways are activated. These studies provided new information on the molecular mechanisms of apoptosis in the ZR-75-1 and MDA-MB-231 breast cancer cell lines.

Aquaporins in human platelets: intracellular localization and possible role in granule and lysosome secretion.

This study was undertaken to establish the presence and the role of aquaporins (AQPs) in human platelets. Immunodetection with polyclonal antibodies and fluorescent microscopy suggest the presence of AQP isoforms - 0-7 and 9-12 - localized (in resting platelets) in the plasma membrane and in the dense and alpha granules. In thrombin- or monensin-treated platelets, the granules' AQPs become visible in the whole cell body, indicating the granules' swelling. In our studies on the role of AQPs in platelet responses we used tetrachloroauric acid (HAuCl4), a classical water channel blocker. We found that 10-100 µM of Au(III) inhibited the hypotonicity-, monensin (simulating the action of Na+/H+ exchanger)-, and collagen-evoked platelet swelling and reduced tritiated water uptake by platelets treated by collagen or monensin, indicating its ability to block water channels in these cells. HAuCl4, at the concentrations reducing water influx, did not induce cell lysis, alter the plasma membrane shape or the -SH group content. The inhibitor also failed to affect Na+ and Cl--related osmotic gradient formation and protein kinase D2 phosphorylation. In platelets activated by threshold concentrations of collagen, the thrombin receptor activating peptide, ADP, calcium ionophore A23187, phorbol ester and arachidonic acid, HAuCl4 (100 µM) completely inhibited secretion of ATP from dense granules but failed to reduce platelet aggregation. In collagen-stimulated platelets, HAuCl4 (10-100 µM) reduced secretion from dense and alpha granules, as well as lysosomes, in a dose-dependent manner. We conclude that human platelets possess numerous AQPs subtypes localized in the plasma and granule membranes. AQP-mediated water fluxes may be crucial for platelet volume regulation as well as secretion from dense and alpha granules and lysosomes.

Where are we with Polish pathology at the turn of the centuries in the light of the Congresses of the Polish Society of Pathologists?

The aim of this study is to depict the current and past research directions in Polish pathology at the turn of the centuries. The analysis was based on the abstracts of the congresses of the Polish Society of Pathologists organized in 1992-2016 and concerned 1,824 presentations. It has been proven that oncology (1,090 presentations, 59.76%) was the most commonly discussed topic and dominated the dispute. Organ pathology was the area of research covered with over » of all papers (464 presentations, 25.44%), while subsequent topics played a marginal role: varia (86 presentations, 4.71%), infectious and parasitic diseases (84 presentations, 4.61%) and toxicopathology (56 presentations, 3.07%). A special, multidisciplinary category of veterinary pathology was particularized (44 presentations, 2.41%). Positive trend was revealed for oncology, while a downward one for the organ pathology, toxicopathology as well as pathology of infectious and parasitic diseases.

Interleukin-6 Affects Aging-Related Changes of the PPARα-PGC-1α Axis in the Myocardium.

Aging is related to gradual increase of interleukin 6 (IL-6) plasma level that affects peroxisome proliferator-activated receptor (PPAR) expression. We evaluated age-related changes in cardiac expression of PPARα, its coactivator PGC-1α, and selected downstream proteins in mice with systemic IL-6 knockout (IL6KO). Male C57BL6/J wild-type (WT) and IL6KO mice were used at the age of 16-20 weeks (young) and 24-30 months (senescent). Echocardiography and electron microscopy were applied to assess the function and ultrastructure of the heart. Western blotting and quantitative real-time PCR were used to estimate protein and mRNA levels of selected genes. PPARα expression in the myocardium of young IL6KO animals is lower and remains unchanged with aging, whereas in WT mice it declines with age and in both senescent groups it is equal. We observed aging-related upregulation of PGC-1α and less pronounced decline of Sirt3 in IL6KO animals; the level of cytochrome C was significantly decreased in IL6KO group only, suggesting disturbed mitochondrial function, which was not sufficient to evoke obvious changes in cardiac performance and function assessed by echocardiography. IL-6 and aging are involved in regulation of PPARα and PGC-1α expression and may influence the mitochondrial function.

The Effects of Silica Nanoparticles on Apoptosis and Autophagy of Glioblastoma Cell Lines.

Silica nanoparticles (SiNPs) are one of the most commonly used nanomaterials in various medical applications. However, possible mechanisms of the toxicity caused by SiNPs remain unclear. The study presented here provides novel information on molecular and cellular effects of SiNPs in glioblastoma LBC3 and LN-18 cells. It has been demonstrated that SiNPs of 7 nm, 5-15 nm and 10-20 nm induce time- and dose-dependent cytotoxicity in LBC3 and LN-18 cell lines. In contrast to glioblastoma cells, we observed only weak reduction in viability of normal skin fibroblasts treated with SiNPs. Furthermore, in LBC3 cells treated with 5-15 nm SiNPs we noticed induction of apoptosis and necrosis, while in LN-18 cells only necrosis. The 5-15 nm SiNPs were also found to cause oxidative stress, a loss in mitochondrial membrane potential, and changes in the ultrastructure of the mitochondria in LBC3 cells. Quantitative real-time PCR results showed that in LBC3 cells the mRNA levels of pro-apoptotic genes Bim, Bax, Puma, and Noxa were significantly upregulated. An increase in activity of caspase-9 in these cells was also observed. Moreover, the activation of SiNP-induced autophagy was demonstrated in LBC3 cells as shown by an increase in LC3-II/LC3-I ratio, the upregulation of Atg5 gene and an increase in AVOs-positive cells. In conclusion, this research provides novel information concerning molecular mechanisms of apoptosis and autophagy in LBC3 cells.

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.

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.

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 ß-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.

IN VITRO ANTIFUNGAL ACTIVITY OF 2,4-DIHYDROXY-N-(3-THIOXO-3H- 1,2,4-DITHIAZOL-5-YL)BENZENECARBOTHIOAMIDE.

An increase in infections due to non albicans species of Candida has been observed in the recent years. The aims of this study were to determine the antifungal activity of 2,4-dihydroxy-N-(3-thioxo-3H-l1,2,4-dithiazol-5-yl)benzenecarbothioamide (DNTDB) against C. albicans, non-C. albicans, dermatophytes, and molds and to evaluate the enzymatic activity of C. albicans strains. We used reference strans C. albicans 10231 ATCC, 200 C. albicans strains, 100 non-C. albicans, 19 dermatophyte strains, and 21 mold strains isolated from different ontocenoses from patients. DNTDB revealed a mean minimum inhibitory concentration (MIC) of 12.5 pg/mL against the reference C. albicans 10231 ATCC strain on Sabouraud agar (SA) and of 6.5 pg/mL on Roswell Park Memorial Institute (RPMI) medium. The mean MIC for C. albicans isolates was of 22.01 ± 7.5 µg/mL on SA, 17.8 ± 7.4 µg/mL on yeast nitrogen base (YNB), and 16.9 ± 7.9 µg/mL on RPMI medium. The mean MIC for non-C. albicans isolates was of 22.4 ± 12.4 µg/mL on SA, 18.2 ± 8.6 µg/mL on YNB and 15.2 ± 9.03 sg/mL on RPMI. Against Trichophyton mentagrophytes v. granulosum, the mean MIC was 10.9 ± 2.04 µg/mL after 5 days of incubation and 21.9 ± 3.8 µg/mL after 15 days, while Trichophyton mentagrophytes . inteiligitale showed a mean MIC of 13.3 ± 5.5 µg/mL and of 20.3 ± 6.1. µg/mL after the same incubation periods, respectively. DNTDB manifested a MIC over the test range of 25-100 µg/mL for molds after 5 days of incubation and inhibited the enzymatic activity of Candida strains. It seems, the new DNTDB demonstrates potential antifungal activity against yeast-like fungus strains, dermatophytes, and molds in vitro.

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.

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.

IgA nephropathy in a girl with psoriasis and seronegative arthritis.

IgA nephropathy is one of the most common forms of glomerulonephritis, but the coexistence of IgA nephropathy and psoriasis is very rare, especially in children. Herein we report the case of an 8-year-old girl with both psoriasis and IgA nephropathy who responded to treatment with cyclosporine A for both conditions.

Alteration of parafollicular (C) cells activity in the experimental model of hypothyroidism in rats.

Our previous study has shown the alteration of C cells activity in rats with experimental model of hyperthyroidism. The aim of the present study was the evaluation of parafollicular cells activity in rats with hypothyroidism evoked by propylthiouracil (PTU) given in drinking water over 21 days. Histological, ultrastructural and immunocytochemical studies using specific antibodies against calcitonin and CGRP were performed on thyroid glands taken from experimental and control groups of rats. Moreover, in all animals the calcitonin plasma levels were evaluated by radioimmunoassay. After chronic administration of PTU, thyroid image showed predominant microfollicular hyperplasia and attenuated density of parafollicular cells. The intensity of immunocytochemical reactions for CT and CGRP were weaker in the majority of C cells in comparison to the control rats, in which strong immunocytochemical reaction was observed. Examination in the electron microscope reveals the features of hypoactivity both in follicular and parafollicular cells, in which the quantity and electron density of secretory granules were smaller in comparison to the control group. These microscopic changes were accompanied by a significant decrease of calcitonin plasma concentration. Alteration of C cells activity in the experimental model of hypothyroidism, accompanied by microfollicular hypertrophy, may point to the mutual cooperation between parafollicular and follicular cells.

Ultrastructural observations of intermediate cells in chronic pancreatitis.

BACKGROUND/AIMS: The intermediate cells exhibiting both exo- and endocrine features occur in small numbers in the normal and pathologic pancreas of many animal species. In human pathology their presence has been reported in children with hyperinsulinemia, in hypoglycemia, and chronic hypergastrinemia. METHODOLOGY: Fragments of the pancreas collected from 27 patients operated on for chronic pancreatitis of various intensity fibrosis (I degree-IV degree) was subjected to ultrastructural analysis. RESULTS: All chronic pancreatitis cases revealed the presence of intermediate cells that were found outside Langerhans islets, separately or in small groups. They were more common in III degree and IV degree chronic pancreatitis. All forms of chronic pancreatitis with I degree-IV degree fibrosis showed, apart from undamaged cells, numerous intermediate cells with features of destruction. Some intermediate cells contained fibrillate cytoplasm inclusion bodies. Intermediate cells took part in the formation of acini and were also found among ductural cells. CONCLUSIONS: The presence of intermediate cells both within the acinar texture and among proliferating ductular cells confirms the common origin of these cellular elements of the pancreas. In chronic pancreatitis intermediate cells appear during pancreatic texture regeneration, being rather abnormal cell forms produced in this process than an intermediate stage in precursor cell differentiation.

Mast cells and pancreatic stellate cells in chronic pancreatitis with differently intensified fibrosis.

BACKGROUND/AIMS: The pathogenesis of pancreatic fibrosis is unknown. Pathogenic analyses take into account the effect of oxidant stress and the increase in free radicals leading to degranulation of mast cells, which may cause inflammation and activate fibrosis. Activated pancreatic stellate cells produce abnormal components of intercellular substance and are responsible for pancreatic fibrogenesis. The aim of the study was to determine the correlation between the presence and state of mast cells and pancreatic stellate cells activation in chronic pancreatitis with varied intensity fibrosis. METHODOLOGY: We studied 27 patients with chronic pancreatitis of varied fibrosis intensity. Immunohistochemical reactions for mast cells (anti-human mast cell tryptase) and ultrastructural analyses of mast cells and pancreatic stellate cells were performed. The number of degranulated mast cells in the ultrastructural picture and the number of pancreatic stellate cells stained positive for vimentin and alpha-smooth muscle actin were counted. RESULTS: A significant increase was revealed in the number of degranulated mast cells--to 35.6% in chronic pancreatitis I degree fibrosis, 68.3% in II degree, 75.1% in III degree and IV degree (control 10.2%) and a parallel increase in the number of activated pancreatic stellate cells (stained positive for alpha-smooth muscle actin) to 328 +/- 29/mm2 in I degree fibrosis, to 978 +/- 67/mm2 in II degree and 2355 +/- 331/mm2 in III degree and IV degree (control 38.8 +/- 9/mm2). CONCLUSIONS: The increase in the number of activated pancreatic stellate cells parallel to the increase in degranulated mast cells in more pronounced pancreatic fibrosis suggests that mast cell-released chemical mediators are involved in pancreatic stellate cells activation.

Endocrine and intermediate cells in pancreatic ductal carcinoma and in chronic pancreatitis.

As most pancreatic ductal carcinomas have the genotype of ductal epithelial cells it is believed that they originate from the epithelium of these structures. It has been proved that endocrine cells are frequently present in ductal carcinoma. Morphological, immunohistochemical, ultrastructural evaluation and statistical analysis revealed the presence of one or a few types of endocrine cells in 46.9% of ductal carcinomas (mainly of I degree or II degree malignancy). These cells were found to secrete insulin. Intermediate cells were not observed in ductal carcinoma. The structure of chronic pancreatitis, mainly the so called tubular complexes, showed various types of endocrine cells and numerous intermediate cells, often with features of destruction. The present study seems to prove that pancreatic cells have a common origin-acinar cells as well as islet cells arise from ductular cells. The intermediate cells observed in chronic pancreatitis are not just a transitory form in the differentiation of precursor cells, but rather the abnormal forms of cells that are produced in that process.

Ultrustructural and enzymatic studies of rat liver after acute cadmium exposure.

The aim of the study was to examine morphological and enzymatic changes in the rat liver following single i.v. administration of CdCl2 (5 and 10 mumol/kg b.w.). In the rats given 5 and 10 mumol CdCl2/kg b.w., a correlation was found between the dose and the intensity of changes in the liver both in pathomorphological, ultrastructural and enzymatic analyses. Pathomorphological and ultrastructural investigations showed that a single dose of 10 mumol CdCl2/kg b.w. caused vacuolar degeneration, and fatty degeneration in single hepatocytes. Structural changes were accompanied by an increase in the activity of indicatory enzymes of the liver-AST and ALT. At the same time, stimulation and activation of B-K cells were observed. However, a single 5 mumol CdCl2/kg b.w. dose induced only blurring of cell structure in single hepatocytes within the peripheral zone of the lobules, which looked as if they had been filled with pinkish "mush".