(Exp Mol Pathol[TA])
- Global and site-specific changes in 5-methylcytosine and 5-hydroxymethylcytosine after extended post-mortem interval. [Journal Article]
- EMExp Mol Pathol 2016 Oct 13
- There has been a growing interest in the study of epigenetic mechanisms to elucidate the molecular bases of human brain-related diseases and disorders. Frequently, researchers utilize post-mortem tis...
There has been a growing interest in the study of epigenetic mechanisms to elucidate the molecular bases of human brain-related diseases and disorders. Frequently, researchers utilize post-mortem tissue with the assumption that post-mortem tissue decay has little or no effect on epigenetic marks. Although previous studies show no effect of post-mortem interval on certain epigenetic marks, no such research has been performed on cytosine modifications. In this study, we use DNA from the brains of adult Sprague Dawley rats subjected to post-mortem intervals at room temperature, ranging from 0 to 96h, to assess the stability of cytosine modifications, namely 5-methycytosine and 5-hydroxymethylcytosine. Our results indicate that neither global nor site-specific levels of 5-methycytosine and 5-hydroxymethylcytosine are affected by the post-mortem intervals we studied. As such, the use of post-mortem tissue to study cytosine modifications in the context of neurological or neuropsychiatric disorders is appropriate.
- Corrigendum to "Prostaglandin F2α receptor silencing attenuates vascular remodeling in rats with type 2 diabetes" (Exp. Mol. Pathol. 2015 99(3) 517-523). [PUBLISHED ERRATUM]
- EMExp Mol Pathol 2016 Oct 14
- Activation of PPAR-γ reduces HPA axis activity in diabetic rats by up-regulating PI3K expression. [Journal Article]
- EMExp Mol Pathol 2016 Oct 8; 101(2):290-301
- Increased hypothalamus-pituitary-adrenal axis (HPA) activity in diabetes is strongly associated with several morbidities noted in patients with the disease. We previously demonstrated that hyperactiv...
Increased hypothalamus-pituitary-adrenal axis (HPA) activity in diabetes is strongly associated with several morbidities noted in patients with the disease. We previously demonstrated that hyperactivity of HPA axis under diabetic conditions is associated with up-regulation of adrenocorticotrophic hormone (ACTH) receptors (MC2R) in adrenal and down-regulation of glucocorticoid receptors (GR and MR) in pituitary. This study investigates the role of peroxisome proliferator-activated receptor (PPAR)-γ in HPA axis hyperactivity in diabetic rats. Diabetes was induced by intravenous injection of alloxan into fasted rats. The PPAR-γ agonist rosiglitazone and/or PI3K inhibitor wortmannin were administered daily for 18 consecutive days, starting 3days after diabetes induction. Plasma ACTH and corticosterone were evaluated by radioimmunoassay, while intensities of MC2R, proopiomelanocortin (POMC), GR, MR, PI3K p110α and PPAR-γ were assessed using immunohistochemistry. Rosiglitazone treatment inhibited adrenal hypertrophy and hypercorticoidism observed in diabetic rats. Rosiglitazone also significantly reversed the diabetes-induced increase in the MC2R expression in adrenal cortex. We noted that rosiglitazone reduced the number of corticotroph cells and inhibited both anterior pituitary POMC expression and plasma ACTH levels. Furthermore, rosiglitazone treatment was unable to restore the reduced expression of GR and MR in the anterior pituitary of diabetic rats. Rosiglitazone increased the number of PPAR-γ(+) cells and expression of PI3K p110α in both anterior pituitary and adrenal cortex of diabetic rats. In addition, wortmannin blocked the ability of rosiglitazone to restore corticotroph cell numbers, adrenal hypertrophy and plasma corticosterone levels in diabetic rats. In conclusion, our findings revealed that rosiglitazone down-regulates HPA axis hyperactivity in diabetic rats via a mechanism dependent on PI3K activation in pituitary and adrenal glands.
- Identification of high-affinity anti-CD16A allotype-independent human antibody domains. [Journal Article]
- EMExp Mol Pathol 2016 Oct 3; 101(2):281-289
- CD16A (FcγRIIIA) is an activating receptor mostly expressed on natural killer (NK) cells and monocytes/macrophages. It can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) through low-aff...
CD16A (FcγRIIIA) is an activating receptor mostly expressed on natural killer (NK) cells and monocytes/macrophages. It can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) through low-affinity interaction with human immunoglobulin G (IgG) Fc. It can also mediate cell lysis if NK cells are guided by bispecific killer cells engagers (BiKEs). BiKEs showed some success in clinical trials of cancer and are promising candidate therapeutics. However, currently reported BiKEs are based on antibody fragments (scFvs) of relatively large size. The CD16A-specific antibodies are also typically from animal origin. Decreasing the BiKE size could result in enhanced penetration into solid tumor and normal tissues, and using fully human antibodies could decrease the likelihood of immunogenicity. Here we report the identification and characterization of two antibody domains, D6 and E11, isolated from a very large human VH antibody domain library displayed on phage. D6 and E11 bound CD16A with EC50 of 4nM and 8nM, respectively, but not other Fc gamma receptors (FcγRs) such as CD64 (FcγRI), CD32 (FcγRII) and CD16B (FcγRIIIB). They bound to both CD16A allotypes (158F,V) with equal affinity and competed with each other as well as with human IgG1 and the mouse anti-CD16A antibody 3G8. These and other results were used to build a molecular docking model predicting that D6 and E11 may bind to the CD16A membrane proximal D2 domain by interacting with its BC, C'E and EF loops. Importantly, cross-linked (bivalent) D6 and E11 induced secretion of IL-2 after binding to CD16A-expressing Jurkat T cells. The small size of these antibody domains combined with their high-affinity, specific, allotype-independent, activating interactions with CD16A could allow generation of novel highly effective BiKEs and other candidate protein therapeutics.
- Long non-coding RNA TUG1 regulates ovarian cancer proliferation and metastasis via affecting epithelial-mesenchymal transition. [Journal Article]
- EMExp Mol Pathol 2016 Sep 28; 101(2):267-273
- Ovarian cancer is the fifth leading cause of cancer-related death in women worldwide, and recent studies have highlighted the role of long non-coding RNAs (lncRNAs) in cancer development. However, th...
Ovarian cancer is the fifth leading cause of cancer-related death in women worldwide, and recent studies have highlighted the role of long non-coding RNAs (lncRNAs) in cancer development. However, the role of lncRNAs in ovarian cancer is largely unclear. In this study, we focused on the taurine up-regulated gene 1 (TUG1) and examined its molecular mechanism in ovarian cancer. Here, we reported that TUG1 was up-regulated in ovarian cancer tissues and ovarian cancer cells, and TUG1 expression was positively correlated with tumor grade and FIGO stage. In vitro functional assays (CCK-8 assay, colony formation assay, and cell invasion assay) revealed that knock-down of TUG1 by small RNA inference significantly inhibited cell proliferation, colony formation and cell invasion in ovarian cancer cells. Further experiment showed that knock-down of TUG1 induced cell apoptosis and altered the protein expression levels of apoptosis-related mediators in ovarian cancer cells. More importantly, knock-down of TUG1 also reversed epithelial-mesenchymal transition in ovarian cancer. In summary, our results suggest that knock-down of TUG1 may represent a novel therapeutic strategy for the treatment of ovarian cancer.
- BMP4 promotes a phenotype change of an esophageal squamous epithelium via up-regulation of KLF4. [Journal Article]
- EMExp Mol Pathol 2016 Sep 28; 101(2):259-266
- CONCLUSIONS: Our results demonstrate that BMP4 promotes a phenotype change of an esophageal squamous epithelium via up-regulation of KLF4.
- Quantitative PCR and unconventional serological methods to evaluate clomipramine treatment effectiveness in experimental Trypanosoma cruzi infection. [Journal Article]
- EMExp Mol Pathol 2016 Sep 24; 101(2):274-280
- Clomipramine (CLO), a tricyclic antidepressant drug, has been used for the treatment of mice infected with Trypanosoma cruzi. In this work we evaluated the effectiveness of CLO treatment upon T. cruz...
Clomipramine (CLO), a tricyclic antidepressant drug, has been used for the treatment of mice infected with Trypanosoma cruzi. In this work we evaluated the effectiveness of CLO treatment upon T. cruzi-infected mice in the chronic phase of the experimental infection using Quantitative polymerase chain reaction (qPCR) and recombinant ELISA. Sixty Swiss albino mice were inoculated with 50 trypomastigote forms of T. cruzi (Tulahuen strain). CLO treatment consisted of 5mg/kg/day during 60days by intraperitoneal injection, beginning on day 90 post infection (p.i) when the mice presented electrocardiographic (ECG) alterations compatible with the chronic phase of the disease. The evolution of experimental infection and the treatment efficacy were studied through survival, electrocardiography, serology using a mixture and individual (1, 2, 13, 30, 36 and SAPA) recombinant proteins from epimastigotes and trypomastigotes of T. cruzi; and qPCR on days 180 and 270 p.i. CLO treatment in the chronic phase decreased the parasite load, reduced the levels of antibodies against antigen 13 throughout 270days p.i and reversed the ECG abnormalities in the treated animals, from 100% of the mice with alterations at the beginning of the treatment to only 20% of the mice with alterations by day 270 p.i. This study shows that qPCR and the use of recombinant antigens are more sensitive to evaluate the effectiveness of the treatment and proves that clomipramine may be considered as a new chemotherapy for the chronic phase of the disease.
- Usp2-69 overexpression slows down the progression of rat anti-Thy1.1 nephritis. [Journal Article]
- EMExp Mol Pathol 2016 Sep 15; 101(2):249-258
- Mesangial proliferative glomerulonephritis is characterized by proliferation of mesangial cells (MCs) and transforming growth factor-β (TGF-β)-dependent stimulation of abnormal extracellular matrix (...
Mesangial proliferative glomerulonephritis is characterized by proliferation of mesangial cells (MCs) and transforming growth factor-β (TGF-β)-dependent stimulation of abnormal extracellular matrix (ECM) accumulation. We previously showed that Decorin--a leucine-rich proteoglycan inhibiting the progression of glomerulonephritis and glomerular sclerosis--can be degraded by the ubiquitin-proteasome pathway and deubiquitinated and stabilized by ubiquitin-specific processing protease 2-69(Usp2-69). Usp2-69 is highly expressed in the kidney and has been implicated in the regulation of cell proliferation and apoptosis. However, its role in mesangial proliferative glomerulonephritis remains unclear. Here, we explored the effect of Usp2-69 on MC proliferation and ECM deposition by transfecting Usp2-69 plasmid into rat anti-Thy1.1 nephritis model and into cultured MCs, as well as detected Usp2-69 and Decorin in rat anti-Thy1.1 nephritis model by western blot. Overexpressing Usp2-69 at the early stage, but not advanced stage, of anti-Thy1.1 nephritis alleviated cell proliferation and ECM deposition, which was shown by decreased Ki-67, Collagen IV and Fibronectin detected by immunohistochemistry. Overexpression also increased Decorin and decreased TGF-β1 and Collagen IV both in vitro and in vivo. In conclusion, our findings suggest that Usp2-69 overexpression alleviates the progression of rat anti-Thy1.1 nephritis and, therefore, that exogenous plasmid injection via the renal artery enhanced by electrotransfer technology could be a promising avenue for glomerular disease research.
- Deregulation of the planar cell polarity genes CELSR3 and FZD3 in Hirschsprung disease. [Journal Article]
- EMExp Mol Pathol 2016 Sep 13; 101(2):241-248
- Hirschsprung disease (HSCR) is a congenital disorder characterized by the absence of intrinsic ganglion cells in the lower intestine. Genetic factors in the pathogenesis of this disease are under act...
Hirschsprung disease (HSCR) is a congenital disorder characterized by the absence of intrinsic ganglion cells in the lower intestine. Genetic factors in the pathogenesis of this disease are under active investigation. As core genes in the planar cell polarity pathway, Celsr3 and Fzd3 are believed to play vital roles in the development of the murine enteric nervous system. The potential association of CELSR3 and FZD3 with the development of HSCR in humans, however, is still unknown. We determined the genotypes of eight CELSR3 and FZD3 polymorphisms in 113 patients. Furthermore, target gene sequencing was used to search for rare mutations in the planar cell polarity genes. The mRNA and protein expression of CELSR3 and FZD3 were explored in patients with HSCR. Class III β-tubulin in colon tissue samples was examined to elucidate enteric innervation patterns. We observed a significant association between the FZD3 rs17059206 polymorphism and HSCR susceptibility (p<0.001). In addition, five rare mutations in CELSR3 were identified in six patients with HSCR. Upregulation of CELSR3 mRNA expression was detected in 80% of aganglionic segments; a similar increase was found for FZD3 protein expression in 81.8% of aganglionic tissues, compared with the ganglionic segments. Immunohistochemical staining on tissue sections revealed obvious excess expression of both molecules in the mucosal layer. The neurite patterns were highly disorganized in the aganglionic bowel segments, with a marked reduction in the prominence of TUJ1 bundles in number, thickness, and length. Our results showed that deregulation of the planar cell polarity genes CELSR3 and FZD3 might disrupt the enteric innervation pattern and consequently contribute to the susceptibility to HSCR.
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- The role of cardiac fibroblasts in post-myocardial heart tissue repair. [Review]
- EMExp Mol Pathol 2016 Sep 9; 101(2):231-240
- The relative resistance of fibroblasts to hypoxia and their remarkable adaptive plasticity in response to rapid changes in local tissue microenvironment made interstitial cardiac fibroblasts to be a ...
The relative resistance of fibroblasts to hypoxia and their remarkable adaptive plasticity in response to rapid changes in local tissue microenvironment made interstitial cardiac fibroblasts to be a key player in post-myocardial infarction myocardial repair. Cardiac fibroblasts are abundantly presented in the interstitial and perivascular extracellular matrix. These cells can be rapidly mobilized in response to cardiac injury. Inflammatory activation of fibroblasts leads to the loss of their quiescent phenotype and inhibition of matrix-producing capacity. Acute inflammation that follows the infarct induces production of inflammatory mediators, matrix-degrading activity, proliferation, and migration of fibroblasts. Fibroblasts migrate to the injured myocardial site where undergo transdifferentiation to myofibroblasts in response to anti-inflammatory and mitogenic stimuli. They acquire capacity to synthesize matrix and contractile proteins. In the infarcted zone, fibroblasts/myofibroblasts actively proliferate, expand, and extensively produce and deposit collagen and other matrix proteins. The proliferative stage of heart healing transits to the scar maturation stage, in which collagen-based scar exhibits formation of intramolecular and extramolecular cross-links, deactivation and apoptosis of fibroblasts/myofibroblasts. Generally, cardiac reparation is strongly controlled. Inability to pass from one repair stage to another in a timely manner can induce detrimental events such as expansion of the infarct area due to advanced inflammation, cardiac fibrosis and adverse remodeling due to the excessive proliferative and profibrotic response, left ventricular hypertrophy, arrhythmogenicity, and heart failure.