The Effect of a Unique Region of Parvovirus B19 Capsid Protein VP1 on Endothelial Cells.

Parvovirus B19 (B19V) is a widespread human pathogen possessing a high tropism for erythroid precursor cells. However, the persistence or active replication of B19V in endothelial cells (EC) has been detected in diverse human pathologies. The VP1 unique region (VP1u) of the viral capsid has been reported to act as a major determinant of viral tropism for erythroid precursor cells. Nevertheless, the interaction of VP1u with EC has not been studied. We demonstrate that recombinant VP1u is efficiently internalized by rats' pulmonary trunk blood vessel-derived EC in vitro compared to the human umbilical vein EC line. The exposure to VP1u was not acutely cytotoxic to either human- or rat-derived ECs, but led to the upregulation of cellular stress signaling-related pathways. Our data suggest that high levels of circulating B19V during acute infection can cause endothelial damage, even without active replication or direct internalization into the cells.

Anti-inflammatory effect of different curcumin preparations on adjuvant-induced arthritis in rats.

BACKGROUND: Curcumin, a natural polyphenolic substance, has been known for more than two millennia as having strong anti-inflammatory activity towards multiple ailments, including arthritis. The main drawback of curcumin is its poor solubility in water, which leads to low intestinal absorption and minimal bioavailability. In this study, we aimed to compare the anti-arthritic in vivo effect of different curcumin preparations - basic curcumin extract, micellar curcumin, curcumin mixture with piperine, and microencapsulated curcumin. METHODS: Arthritis was induced in Wistar rats by complete Freund's adjuvant, and the severity of arthritis was evaluated daily using the arthritis score system. Curcumin preparations were given to animals per os daily for 20 consecutive days, starting at 6th day after arthritis induction. To determine the inflammatory background, pro-inflammatory cytokines were determined using the ELISA test. In addition, hematologic test, weight change, and limb swelling were tracked. RESULTS: Our results indicate that curcumin had a rather weak effect on arthritis progression in the Wistar rat model, microencapsulated curcumin effectively prevented the progression of arthritis - the disease stabilized after 10 days of supplementation. It also reduced the levels of immune cells (neutrophils and leukocytes), as well as pro-inflammatory cytokines - TNFα, IL-1, and IL-6, which levels were close to arthritis-free control. Other formulations of curcumin had lower or no effect on arthritis progression. CONCLUSION: Our study shows that the same concentrations of curcumin had a distinctly expressed positive anti-inflammatory effect depending on the form of its delivery. Specifically, we found that microencapsulated curcumin had the most promising effect for treatment.

Slow-Freezing Cryopreservation Ensures High Ovarian Tissue Quality Followed by In Vivo and In Vitro Methods and Is Safe for Fertility Preservation.

Background and objectives: Cancer incidence is growing with younger patients diagnosed with this disease every year. Improved cancer diagnostics and treatment lead to better survival of cancer patients. However, after aggressive chemo- or radiotherapy, cancer survivors suffer from various degrees of subfertility or infertility. Several fertility preservation technologies have been developed for young cancer patients: cryopreservation of germ cells, embryos, or reproductive tissues. The best results have been shown by cryopreservation of sperm and embryos. Yet the success of using cryopreserved oocytes or reproductive tissues (ovarian and testicular) is still insufficient. Therefore, this study was designed to assess the vitality, viability, general quality, and safety of frozen-thawed human ovarian tissue for retransplantation using modern molecular tests. Materials and Methods: The new miRNA array test was used to evaluate miRNA expression in thawed ovarian tissue in combination with standard xenotransplantation and pathological examination of microslides. Results: Our results demonstrated that slow freezing is an efficient way (80%) to cryopreserve ovarian tissue with no structural damage afterwards. We have shown that xenotransplantation into immunodeficient mice, histology, and immunohistochemistry could be potentially replaced by more recent molecular methods. Conclusions: The latter method has shown that altered expression of miRNAs might be used as identifiers of normal/damaged tissue after further analysis. Newer, safer, and more specific approaches need to be developed in order to eliminate the risk of disease reoccurrence.

Salinomycin and dichloroacetate synergistically inhibit Lewis lung carcinoma cell proliferation, tumor growth and metastasis.

Drug combination is considered to be the cornerstone of cancer treatment. Simultaneous administration of two or more drugs but at lower doses not only increases cytotoxic effects on tumor cells, but also reduces side effects and possibly overcomes drug resistance. Salinomycin is a well-known cancer stem cell killer, and dichloroacetate is a pyruvate dehydrogenase kinase inhibitor that exclusively targets cells with altered mitochondrial activity, a characteristic being common to most of the cancer cells. In our recent study, we have demonstrated that salinomycin exerted a cytotoxic effect on colorectal carcinoma cells in the 2D and 3D cultures and provided evidence that the mechanism of their synergy was mediated by dichloroacetate-dependent inhibition of the activity of multidrug resistance proteins. In the current work, we confirmed the synergistic cytotoxic properties of salinomycin and dichloroacetate in the 2D and 3D cultures of Lewis lung carcinoma (LLC1) cells. To verify if a synergistic effect of these compounds persisted in vivo, we performed series of experiments using a syngeneic LLC1-C57BL/6 mouse model and demonstrated that combination therapy with salinomycin and DCA increased the survival rate of allografted mice, inhibited metastatic site formation and reduced the populations of cancer stem cells as well as cells that underwent the epithelial-to-mesenchymal transition. Our results demonstrate that a synergistic effect of salinomycin and dichloroacetate exists not only in vitro but also in vivo and suggest their benefits in the treatment of metastatic cancers.

The Role of Cardiac T-Cadherin in the Indicating Heart Failure Severity of Patients with Non-Ischemic Dilated Cardiomyopathy.

Background and objectives: T-cadherin (T-cad) is one of the adiponectin receptors abundantly expressed in the heart and blood vessels. Experimental studies show that T-cad sequesters adiponectin in cardiovascular tissues and is critical for adiponectin-mediated cardio-protection. However, there are no data connecting cardiac T-cad levels with human chronic heart failure (HF). The aim of this study was to assess whether myocardial T-cad concentration is associated with chronic HF severity and whether the T-cad levels in human heart tissue might predict outcomes in patients with non-ischemic dilated cardiomyopathy (NI-DCM). Materials and Methods: 29 patients with chronic NI-DCM and advanced HF were enrolled. Patients underwent regular laboratory investigations, echocardiography, coronary angiography, and right heart catheterization. TNF-α and IL6 in serum were detected by enzyme-linked immunosorbent assay (ELISA). Additionally, endomyocardial biopsies were obtained, and the levels of T-cad were assessed by ELISA and CD3, CD45Ro, CD68, and CD4- immunohistochemically. Mean pulmonary capillary wedge pressure (PCWP) was used as a marker of HF severity, subdividing patients into two groups: mean PCWP > 19 mmHg vs. mean PCWP < 19 mmHg. Patients were followed-up for 5 years. The study outcome was composite: left ventricular assist device implantation, heart transplantation, or death from cardiovascular causes. Results: T-cad shows an inverse correlation with the mean PCWP (rho = -0.397, p = 0.037). There is a tendency towards a lower T-cad concentration in patients with more severe HF, as indicated by the mean PCWP > 19 mmHg compared to those with mean PCWP ≤ 19 mmHg (p = 0.058). Cardiac T-cad levels correlate negatively with myocardial CD3 cell count (rho = -0.423, p = 0.028). Conclusions: Univariate Cox regression analysis did not prove T-cad to be an outcome predictor (HR = 1, p = 0.349). However, decreased T-cad levels in human myocardium can be an additional indicator of HF severity. T-cad in human myocardium has an anti-inflammatory role. More studies are needed to extend the role of T-cad in the outcome prediction of patients with NI-DCM.

Study of the cytotoxic effects of 2,5-diaziridinyl-3,6-dimethyl-1,4-benzoquinone (MeDZQ) in mouse hepatoma cells.

A number of quinones have been shown to be efficient anticancer agents. However, some mechanisms of their action, in particular cell signaling are not well understood. The aim of this study was to partly fill this gap by characterizing the mode of cytotoxicity of 2,5-diaziridinyl-3,6-dimethyl-1,4-benzoquinone (MeDZQ) in malignant mouse hepatoma cells (MH-22A) with regard to the expression and activation of main molecules in MAPK cell signaling pathway. The study revealed unequal roles of MAP kinases in MeDZQ-induced cell death: the compound did not induce significant changes in ERK expression or its phosphorylation; JNK appeared to be responsible for cell survival, however, p38 kinase was shown to be involved in cell death. In order to assess the enzymatic activation mechanisms responsible for the action of MeDZQ, we have also found that the antioxidant N,N'-diphenyl-p-phenylene diamine, the iron-chelating agent desferrioxamine, and DT-diaphorase inhibitor, dicoumarol, partly protected the cells from MeDZQ cytotoxicity. It points to parallel oxidative stress and bioreductive alkylation modes of the cytotoxicity of MeDZQ.

A Novel Murine Model of Parvovirus Associated Dilated Cardiomyopathy Induced by Immunization with VP1-Unique Region of Parvovirus B19.

Background. Parvovirus B19 (B19V) is a common finding in endomyocardial biopsy specimens from myocarditis and dilated cardiomyopathy patients. However, current understanding of how B19V is contributing to cardiac damage is rather limited due to the lack of appropriate mice models. In this work we demonstrate that immunization of BALB/c mice with the major immunogenic determinant of B19V located in the unique sequence of capsid protein VP1 (VP1u) is an adequate model to study B19V associated heart damage. Methods and Results. We immunized mice in the experimental group with recombinant VP1u; immunization with cardiac myosin derived peptide served as a positive reference and phosphate buffered saline served as negative control. Cardiac function and dimensions were followed echocardiographically 69 days after immunization. Progressive dilatation of left ventricle and decline of ejection fraction were observed in VP1u- and myosin-immunized mice. Histologically, severe cardiac fibrosis and accumulation of heart failure cells in lungs were observed 69 days after immunization. Transcriptomic profiling revealed ongoing cardiac remodeling and immune process in VP1u- and myosin-immunized mice. Conclusions. Immunization of BALB/c mice with VP1u induces dilated cardiomyopathy in BALB/c mice and it could be used as a model to study clinically relevant B19V associated cardiac damage.

Histone modifications patterns in tissues and tumours from acute promyelocytic leukemia xenograft model in response to combined epigenetic therapy.

Xenograft models are suitable for in vivo study of leukemia's pathogenesis and the preclinical development of anti-leukemia agents but understanding of epigenetic regulatory mechanisms linking to adult cell functions in pathological conditions during different in vivo treatments is yet unknown. In this study, for the first time epigenetic chromatin modifications were characterized in tissues and tumours from murine xenograft model generated using the human acute promyelocytic leukemia (APL) NB4 cells engrafted in immunodeficient NOG mice. Xenografts were subjected to combined epigenetic treatment by histone deacetylase inhibitor Belinostat, histone methyltransferase inhibitor 3-DZNeaplanocin A and all-trans-retinoic acid based on in vitro model, where such combination inhibited NB4 cell growth and enhanced retinoic acid-induced differentiation to granulocytes. Xenotransplantation was assessed by peripheral blood cells counts, the analysis of cell surface markers (CD15, CD33, CD45) and the expression of certain genes (PML-RAR alpha, CSF3, G-CSFR, WT1). The combined treatment prolonged APL xenograft mice survival and prevented tumour formation. The analysis of the expression of histone marks such as acetylation of H4, trimethylation of H3K4, H3K9 and H3K27 in APL xenograft mice tumours and tissues demonstrated tissue-specific changes in the level of histone modifications and the APL prognostic mark, WT1 protein. In summary, the effects of epigenetic agents used in this study were positive for leukemia prevention and linked to a modulation of the chromatin epigenetic environment in adult tissues of malignant organism.

Long-term muscle-derived cell culture: multipotency and susceptibility to cell death stimuli.

Improvement in the yield of adult organism stem cells, and the ability to manage their differentiation and survival potential are the major goals in their application in regenerative medicine and in the adult stem cell research. We have demonstrated that adult rabbit muscle-derived cell lines with an unlimited proliferative potential in vitro can differentiate into myogenic, osteogenic, adipogenic and neurogenic lineages. Studies of cell survival in vitro showed that differentiated cells, except neurogenic ones, are more resistant to apoptosis inducers compared to proliferating cells. Resistance to death signals correlated with the level of protein kinase AKT phosphorylation. Skeletal muscle-derived cell lines can be multipurpose tools in therapy. Enhanced resistance of differentiated cells to certain types of damage shows their potential for long-term survival and maintenance in an organism. This article was published online on 29 January 2013. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected 6 March 2013.

Recovery of infarcted myocardium in an in vivo experiment.

UNLABELLED: Acute myocardial infarction leads to the loss of functional cardiomyocytes and structural integrity. The adult heart cannot repair the damaged tissue due to inability of mature cardiomyocytes to divide and lack of stem cells. The aim of this study was to evaluate the efficiency of introduced autologous skeletal musclederived stem cells to recover the function of acutely infarcted rabbit heart in the early postoperative period. MATERIAL AND METHODS: As a model for myocardium restoration in vivo, experimental rabbit heart infarct was used. Autologic adult myogenic stem cells were isolated from skeletal muscle and propagated in culture. Before transplantation, the cells were labeled with 4',6-diamidino-2-phenylindole and then, during heart surgery, introduced into the rabbit acutely infarcted myocardium. Postoperative cardiac function was monitored by recording electrocardiograms and echocardiograms. At the end of the experiment, the efficiency of cell integration was evaluated histologically. RESULTS: Rabbit cardiac function recovered after 1 month after the induction of experimental infarction both in the control and experimental groups. Therefore, the first month after the infarction was the most significant for the assessment of cell transplantation efficacy. Transplanted cell integration into infarcted myocardium was time- and individual-dependent. Evaluation of changes in left ventricular ejection fraction after the induction of myocardial infarction revealed better recovery in the experimental group; however, the difference among animals in the experimental and control groups varied and was not significant. CONCLUSIONS. Autologous myogenic stem cells repopulated infarcted myocardium with different efficiency in each individual. This variability may account for the observed difference in postoperative cardiac recovery in a rabbit model.

Induction of apoptosis and activation of JNK and p38 MAPK pathways in deoxynivalenol-treated cell lines.

Deoxynivalenol (DON) is a mycotoxin produced by what are thought to be the most prevalent toxin-producing fungi of the Fusarium genus. Here, we present the results of apoptosis induction, phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs), and expression of the c-Jun protein after DON treatment, in a pre-B lymphocyte REH cell line. In addition, human pre-T lymphocyte Jurkat, hamster kidney-derived BHK21 and mouse hepatoma MH-22a cells were used in comparative experiments in vitro. We found that the DON effect was cell origin-dependent and dose-dependent, with a significant slow-down of cell proliferation and increase of apoptotic cells in blood cell lines. BHK21 and MH-22a cells were less sensitive to the DON effect. In blood-derived REH and Jurkat cells, DON-induced apoptotic changes were preceded by an increase in JNK and p38 MAPKs phosphorylation, as well as in c-Jun expression. However, the activation of JNK phosphorylation and c-Jun expression were transient, but did not coincide with each other. An inhibitor of JNK1/2, SP600125, had a negligible negative effect on REH cell viability after DON treatment, demonstrating that JNK does not contribute to DON-induced apoptosis. In contrast, studies on the role of p38 MAPK revealed that p38 signalling is required for DON-induced apoptosis in REH cells.

Synthesis and antitumour activity of a series of cyclic amidines of 2,3-dihydro-1H-1,5-benzodiazepines.

2,3-Dihydro-1H-1,5-benzodiazepine amidines were prepared by nucleophilic substitution of 2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-2-thiones. Evaluation of the 13 synthesised amidines as antitumour agents was carried out in vitro against 60 human tumour cell lines at the National Cancer Institute, Bethesda, USA. The screening revealed a moderate cell growth inhibition of two derivatives on all cell lines at concentrations ranging from 10(-5) to 10(-4) mol/l. Log P values were theoretically calculated. The more active derivatives were found to exhibit a higher lipophilicity.