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International Journal of Clinical... Jan 2014Toxoplasmosis is a common cause of foodborne, gastrointestinal and congenital syndrome with particularly severe or unknown health consequences. There is no safe and...
BACKGROUND
Toxoplasmosis is a common cause of foodborne, gastrointestinal and congenital syndrome with particularly severe or unknown health consequences. There is no safe and effective preventive or therapeutic modality against congenital toxoplasmosis or to eliminate the persistent chronic infection.
HYPOTHESIS
Diclazuril to be safe in pregnancy and effective against gastrointestinal toxoplasmosis.
METHODS
CD1 programmed pregnant mice were divided into groups and administered a diet containing diclazuril, or sham control. Treatments were initiated on Day 5 of pregnancy and continued until Day 16 when dams were euthanatized. On Day 8 of pregnancy dams were infected intraperitoneally with escalating doses of tachyzoites (0, 100, 300, 600) from Type II strain. Dams were monitored daily for distress, pain, and abortion and samples collected at the end of the experiments.
RESULTS
Infected dams developed moderate to severe related complications in tachyzoites dose dependent manner. Animals became anemic and showed hydrothorax, and ascities. Diclazuril effectively protected dams from ascities and anemia (p < 0.05). Infected dams showed splenomegaly, with massive infiltration of epithelioid cells compared with the protective effect of diclazuril in treated animals. Infected dams exhibited severe hepatitis (score 0 to 4 scale = 3.5 ± 0.01) with influx of inflammatory and plasma cells, dysplastic hepatocytes, multinucleated giant cell transformation and hepatic cells necrosis. Diclazuril treatment significantly protected dams from hepatitis, also in tachyzoites dose (100, 300, 600) dependent manner (respectively infected-treated versus infected controls, p < 0.001, p < 0.01 and p < 0.05). Colonic tissues were significantly shortened in length, with infiltration of lymphocytes, and macrophages and microabscess formations in the cryptic structures, with significant improvement in diclazuril treated animals. Additionally, the number of fetuses, fetal length and fetal weight were preserved in diclazuril treated dams.
CONCLUSIONS
This is the first report describing of diclazuril safety in pregnancy as well as efficacy against mild to moderate hepato-gastrointestinal syndrome in dams and fetal toxoplasmosis (Special issue, "Treatment of Liver Diseases").
PubMed: 24851194
DOI: 10.4236/ijcm.2014.53017 -
Veterinary Pathology Nov 2014Multinucleated hepatocytes (MNHs) have been occasionally reported in macaques, as well as chimpanzees and gorillas, as an incidental finding. However, information is...
Multinucleated hepatocytes (MNHs) have been occasionally reported in macaques, as well as chimpanzees and gorillas, as an incidental finding. However, information is sparse on variations in incidence in the cynomolgus macaque (Macaca fascicularis). A survey was conducted to assess the occurrence of MNHs in the liver of stock (nonstudy) animals from SNBL SRC (Alice, TX) and SNBL USA (Everett, WA) submitted for diagnostic purposes. A total of 215 cynomolgus monkeys originally from Cambodia (61), China (5), Indonesia (125), and Mauritius (24) were used for this investigation. From each animal, usually 2 liver samples were processed for histopathology with 2 sections in each slide. An MNH was defined as a hepatocyte with 3 or more nuclei. A threshold of 3 MNHs was selected for the Multinucleated Hepatocyte Grading System: 0 = not remarkable (≤3 MNHs counted from 2-4 liver sections), minimal = 4 to 15 MNHs, mild = 16 to 30 MNHs, moderate = 31 to 59 MNHs, and severe ≥60 MNHs. The incidence of MNHs was 60 of 86 (70%) in males and 72 of 129 (56%) in females for a total overall incidence of 132 of 215 animals (61%). Affected hepatocytes were frequently observed close to the capsule and generally had 3 to 8 nuclei per hepatocyte but as many as 15 occurred in a single cell. Awareness of the incidence of MNHs in cynomolgus monkeys is important for potential use as background data in preclinical safety and toxicity evaluation studies.
Topics: Animals; Cell Nucleus; Female; Hepatocytes; Liver; Macaca fascicularis; Male
PubMed: 24395914
DOI: 10.1177/0300985813516641 -
FASEB Journal : Official Publication of... Feb 2014The Notch signaling pathway is involved in liver development and regeneration. Here, we investigate the role of the 4 mammalian Notch paralogs in the regulation of...
The Notch signaling pathway is involved in liver development and regeneration. Here, we investigate the role of the 4 mammalian Notch paralogs in the regulation of hepatoblast proliferation and hepatocytic differentiation. Our model is based on bipotential mouse embryonic liver (BMEL) progenitors that can differentiate into hepatocytes or cholangiocytes in vitro and in vivo. BMEL cells were subjected to Notch antagonists or agonists. Blocking Notch activation with a γ-secretase inhibitor, at 50 μM for 48 h, reduced cell growth by 50%. S-phase entry was impaired, but no apoptosis was induced. A systematic paralog-specific strategy was set using lentiviral transduction with constitutively active forms of each Notch receptor along with inhibition of endogenous Notch signaling. This assay demonstrates that proliferation of BMEL cells requires Notch2 and Notch4 activity, resulting in significant down-regulation of p27(Kip1) and p57(Kip2) cyclin-dependent kinase inhibitors. Conversely, Notch3-expressing cells proliferate less and express 3-fold higher levels of p57(Kip2). The Notch3 cells present a hepatocyte-like morphology, enhanced multinucleation, and a ploidy shift. Moreover, Notch3 activity is conducive to hepatocytic differentiation in vitro, while its paralogs impede this fate. Our study provides the first evidence of a functional diversity among the mammalian Notch homologues in the proliferation and hepatocytic-lineage commitment of liver progenitors.
Topics: Animals; Cell Differentiation; Cell Line; Cell Proliferation; Fluorescent Antibody Technique; Hepatocytes; Liver; Mice; Real-Time Polymerase Chain Reaction; Receptors, Notch; Stem Cells
PubMed: 24145721
DOI: 10.1096/fj.13-235903 -
International Journal of Nanomedicine 2013Silica nanoparticles (SNPs) are one of the most important nanomaterials, and have been widely used in a variety of fields. Therefore, their effects on human health and...
Silica nanoparticles (SNPs) are one of the most important nanomaterials, and have been widely used in a variety of fields. Therefore, their effects on human health and the environment have been addressed in a number of studies. In this work, the effects of amorphous SNPs were investigated with regard to multinucleation in L-02 human hepatic cells. Our results show that L-02 cells had an abnormally high incidence of multinucleation upon exposure to silica, that increased in a dose-dependent manner. Propidium iodide staining showed that multinucleated cells were arrested in G2/M phase of the cell cycle. Increased multinucleation in L-02 cells was associated with increased generation of cellular reactive oxygen species and mitochondrial damage on flow cytometry and confocal microscopy, which might have led to failure of cytokinesis in these cells. Further, SNPs inhibited cell growth and induced apoptosis in exposed cells. Taken together, our findings demonstrate that multinucleation in L-02 human hepatic cells might be a failure to undergo cytokinesis or cell fusion in response to SNPs, and the increase in cellular reactive oxygen species could be responsible for the apoptosis seen in both mononuclear cells and multinucleated cells.
Topics: Apoptosis; Cell Cycle; Cell Line; Cell Survival; Hepatocytes; Humans; Metal Nanoparticles; Particle Size; Reactive Oxygen Species; Silver
PubMed: 24092974
DOI: 10.2147/IJN.S46732 -
Mutation Research Aug 2013Hepatocellular carcinoma (HCC) is non-responsive to many chemotherapeutic agents including etoposide. The aim of this study was to examine the survival strategy of the...
Hepatocellular carcinoma (HCC) is non-responsive to many chemotherapeutic agents including etoposide. The aim of this study was to examine the survival strategy of the HCC cell line HepG2 after etoposide treatment. Here we analyzed and compared spontaneous and etoposide-induced DNA damage in HepG2 (α-fetoprotein (AFP)-positive) and Chang Liver (AFP-negative) cell lines. Compared to Chang Liver cells, HepG2 cells exhibited a significantly higher degree of micronucleation and a higher nuclear division index, as determined by the cytokinesis-block micronucleus assay, following exposure to etoposide. HepG2 cells were also more resistant to etoposide-induced cytotoxicity compared to Chang Liver cells. We also establish that increased etoposide-induced multinucleation in HepG2 cells is dependent on the catalytic activity of Akt, as phosphatidylinositol-3-kinase inhibitors as well as the overexpression of kinase-defective Akt reversed this phenotype. Moreover, ectopic expression of wild type PTEN reduced the frequency of etoposide-induced multinucleated HepG2 cells, and restored HepG2 etoposide sensitivity. Taken together, these results implicate the Akt/PTEN cellular axis as a major determinant of the etoposide resistance of HCC cells.
Topics: Antineoplastic Agents, Phytogenic; Cell Fusion; Cell Survival; DNA Damage; DNA Repair; DNA, Neoplasm; Drug Resistance, Neoplasm; Etoposide; Giant Cells; HeLa Cells; Hep G2 Cells; Humans; Micronucleus Tests; Neoplasm Proteins; PTEN Phosphohydrolase; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Recombinant Proteins; Signal Transduction; Topoisomerase II Inhibitors
PubMed: 23796964
DOI: 10.1016/j.mrgentox.2013.06.009 -
PloS One 2013Cell-in-cell structures refer to a unique phenomenon that one living cell enters into another living cell intactly, occurring between homotypic tumor cells or tumor (or...
Cell-in-cell structures refer to a unique phenomenon that one living cell enters into another living cell intactly, occurring between homotypic tumor cells or tumor (or other tissue cells) and immune cells (named as heterotypic cell-in-cell structure). In the present study, through a large scale of survey we observed that heterotypic cell-in-cell structure formation occurred commonly in vitro with host cells derived from different human carcinomas as well as xenotypic mouse tumor cell lines. Most of the lineages of human immune cells, including T, B, NK cells, monocytes as well as in vitro activated LAK cells, were able to invade tumor cell lines. Poorly differentiated stem cells were capable of internalizing immune cells as well. More significantly, heterotypic tumor/immune cell-in-cell structures were observed in a higher frequency in tumor-derived tissues than those in adjacent tissues. In mouse hepatitis models, heterotypic immune cell/hepatocyte cell-in-cell structures were also formed in a higher frequency than in normal controls. After in vitro culture, different forms of internalized immune cells in heterotypic cell-in-cell structures were observed, with one or multiple immune cells inside host cells undergoing resting, degradation or mitosis. More strikingly, some internalized immune cells penetrated directly into the nucleus of target cells. Multinuclear cells with aneuploid nucleus were formed in target tumor cells after internalizing immune cells as well as in situ tumor regions. Therefore, with the prevalence of heterotypic cell-in-cell structures observed, we suggest that shielding of immune cells inside tumor or inflammatory tissue cells implies the formation of aneuploidy with the increased multinucleation as well as fine-tuning of microenvironment under pathological status, which may define distinct mechanisms to influence the etiology and progress of tumors.
Topics: Aneuploidy; Animals; B-Lymphocytes; Carcinoma; Cell Communication; Cell Differentiation; Cell Transformation, Neoplastic; Giant Cells; Hepatocytes; Humans; Killer Cells, Lymphokine-Activated; Killer Cells, Natural; Mice; Mitosis; Neoplasms; Stem Cells; T-Lymphocytes; Tumor Cells, Cultured
PubMed: 23555668
DOI: 10.1371/journal.pone.0059418 -
Genes, Chromosomes & Cancer Feb 2013Histone lysine methylation plays a fundamental role in chromatin organization. Although a set of histone methyltransferases have been identified and biochemically...
Histone lysine methylation plays a fundamental role in chromatin organization. Although a set of histone methyltransferases have been identified and biochemically characterized, the pathological roles of their dysfunction in human cancers are still not well understood. In this study, we demonstrate important roles of WHSC1L1 in human carcinogenesis. Expression levels of WHSC1L1 transcript were significantly elevated in various human cancers including bladder carcinoma. Immunohistochemical analysis of bladder, lung, and liver cancers confirmed overexpression of WHSC1L1. WHSC1L1-specific small interfering RNAs significantly knocked down its expression and resulted in suppression of proliferation of bladder and lung cancer cell lines. WHSC1L1 knockdown induced cell cycle arrest at the G(2)/M phase followed by multinucleation of cancer cells. Expression profile analysis using Affymetrix GeneChip(®) showed that WHSC1L1 affected the expression of a number of genes including CCNG1 and NEK7, which are known to play crucial roles in the cell cycle progression at mitosis. As WHSC1L1 expression is significantly low in various normal tissues including vital organs, WHSC1L1 could be a good candidate molecule for development of novel treatment for various types of cancer.
Topics: Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin G1; Female; Flow Cytometry; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; HEK293 Cells; HeLa Cells; Hep G2 Cells; Histone-Lysine N-Methyltransferase; Humans; Immunohistochemistry; Liver Neoplasms; Lung Neoplasms; Male; NIMA-Related Kinases; Neoplasms; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Protein Serine-Threonine Kinases; RNA Interference; Reverse Transcriptase Polymerase Chain Reaction; Urinary Bladder Neoplasms
PubMed: 23011637
DOI: 10.1002/gcc.22012 -
Methods in Molecular Biology (Clifton,... 2013Exoerythrocytic Plasmodium parasites infect hepatocytes and develop to huge multinucleated schizonts inside a parasitophorous vacuole. Finally, thousands of merozoites...
Exoerythrocytic Plasmodium parasites infect hepatocytes and develop to huge multinucleated schizonts inside a parasitophorous vacuole. Finally, thousands of merozoites are formed and released into the host cell cytoplasm by complete disintegration of the parasitophorous vacuole membrane. This, in turn, results in death and detachment of the infected hepatocyte, followed by the formation of merosomes. The fast growth of the parasite and host cell detachment are hallmarks of liver stage development and can easily be monitored. Here, we describe how to translate these observations into assays for characterizing parasite development. Additionally, other recently introduced techniques and tools to analyze and manipulate liver stage parasites are also discussed.
Topics: Animals; Anopheles; Cell Culture Techniques; Genes, Reporter; Hep G2 Cells; Hepatocytes; Humans; Malaria; Merozoites; Plasmids; Plasmodium; Transfection
PubMed: 22990795
DOI: 10.1007/978-1-62703-026-7_29 -
The FEBS Journal Nov 2012Nuclear factor erythroid-derived 2-related factor 1 (Nrf1) regulates cellular stress response genes, and has also been suggested to play a role in other cellular...
Nuclear factor erythroid-derived 2-related factor 1 (Nrf1) regulates cellular stress response genes, and has also been suggested to play a role in other cellular processes. We previously demonstrated that hepatocyte-specific deletion of Nrf1 in mice resulted in spontaneous apoptosis, inflammation, and development of liver tumors. Here, we showed that both fibroblasts derived from Nrf1 null mouse embryos and fibroblasts expressing a conditional Nrf1 allele showed increased micronuclei and formation of abnormal nuclei. Lentiviral shRNA-mediated knockdown of Nrf1 in SAOS-2 cells also resulted in increased micronuclei, abnormal mitosis and multi-nucleated cells. Metaphase analyses showed increased aneuploidy in Nrf1(-/-) embryonic fibroblasts. Nuclear defects in Nrf1-deficient cells were associated with decreased expression of various genes encoding kinetochore and mitotic checkpoint proteins. Our findings suggest that Nrf1 may play a role in maintaining genomic integrity, and that Nrf1 dysregulation may induce tumorigenesis.
Topics: Aneuploidy; Animals; Apoptosis; Blotting, Western; Bone Neoplasms; Cell Nucleus; Cell Proliferation; Cells, Cultured; Colonic Neoplasms; Fibroblasts; Genomic Instability; Humans; Kinetochores; Mice; Mice, Knockout; Micronuclei, Chromosome-Defective; NF-E2 Transcription Factor, p45 Subunit; NF-E2-Related Factor 1; Osteosarcoma; RNA, Messenger; RNA, Small Interfering; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 22971132
DOI: 10.1111/febs.12005 -
Medical Science Monitor : International... Sep 2012Toxoplasma is an important source of foodborne hospitalization with no safe and effective therapy against chronic or congenital Toxopalsmosis. Atovaquone is a drug of...
BACKGROUND
Toxoplasma is an important source of foodborne hospitalization with no safe and effective therapy against chronic or congenital Toxopalsmosis. Atovaquone is a drug of choice but not approved for use in congenital Toxoplasmosis. We hypothesized atovaquone to be safe and effective against feto-maternal Toxoplasmosis.
MATERIAL/METHODS
Programmed pregnant mice were i.p. infected with 50-2400 Tachyzoites from Type II strain (clone PTG). Dams were treated daily with atovaquone or sham and monitored for pain, and complications.
RESULTS
Dams developed pain related abdominal hypersensitivity (allodynia) to mechanical stimuli in a Tachyzoites dose dependent manner. Infected dams were anemic and exhibited ascities and severe hepatitis (score 3.6±0.01 on scale 0--normal to 4--severe) with influx of inflammatory and plasma cells, multinucleated dysplastic hepatocytes and necrosis. In addition, dams expressed mild to severe pancreatitis with mononuclear cell invasion, loss of islets and necrosis. This was consistent with splenomegaly (X3 Fold), and massive infiltration of epithelioid cells and loss of germinal structure. Colon became significantly shortened in length (p<0.01) with semi-normal content. Pathological manifestation included, shortening of crypts with numerous microabscess formations, infiltration of lymphocytes, and macrophages. The severe clinical complications led to abortion (50%), early birth (25%) or still birth (25%) consistent with the high dose of Tachyzoites inoculation. Atovaquone treatment partially but significantly protected the dams from the severity of hepatitis, splenomegaly, colitis, myocarditis, and pain related responses as well as fetal demise.
CONCLUSIONS
This is a valuable model for therapeutic evaluation of feto-maternal Toxoplasmosis and gastrointestinal complications. Atovaquone protects dams and their fetuses against some infectious/inflammatory aspects of the disease.
Topics: Analysis of Variance; Animals; Anti-Infective Agents; Atovaquone; Disease Models, Animal; Female; Gastrointestinal Tract; Hepatitis; Hyperalgesia; Immunohistochemistry; Mice; Pancreatitis; Pregnancy; Toxoplasmosis, Congenital
PubMed: 22936182
DOI: 10.12659/msm.883342