Current Issue : July - September Volume : 2011 Issue Number : 3 Articles : 9 Articles
Thyroglobulin (Tg) represents one of the largest known self-antigens involved in autoimmunity. Numerous studies have implicated it in triggering and perpetuating the autoimmune response in autoimmune thyroid diseases (AITD). Indeed, traditional models of autoimmune thyroid disease, experimental autoimmune thyroiditis (EAT), are generated by immunizing mice with thyroglobulin protein in conjunction with an adjuvant, or by high repeated doses of Tg alone, without adjuvant. These extant models are limited in their experimental flexibility, i.e. the ability to make modifications to the Tg used in immunizations. In this study, we have immunized mice with a plasmid cDNA encoding the full-length human Tg (hTG) protein, in order to generate a model of Hashimoto's thyroiditis which is closer to the human disease and does not require adjuvants to breakdown tolerance. Human thyroglobulin cDNA was injected and subsequently electroporated into skeletal muscle using a square wave generator. Following hTg cDNA immunizations, the mice developed both B and T cell responses to Tg, albeit with no evidence of lymphocytic infiltration of the thyroid. This novel model will afford investigators the means to test various hypotheses which were unavailable with the previous EAT models, specifically the effects of hTg sequence variations on the induction of thyroiditis....
Background\nThough long known to affect smooth muscle biology, recent studies indicate that phosphodiesterase 5 (PDE5) is also expressed in myocardium. Recognizing that the regulation of PDE5 in hypertrophy is not well understood, we assessed the response of PDE5 expression and the level of cGMP-dependent kinase I (cGKI) in the left and right ventricles of feline hypertrophy models.\nMethodology/Principal Findings\nUsing a cDNA library of feline aortic smooth muscle cells, we identified and cloned PDE5 cDNA for the first time in this species. The sequence shares 98% identity with its human orthologue at the amino acid level. E. coli expression of the cloned allele allowed selection of antibodies with appropriate specificity, facilitating the analysis of PDE5 expression in feline models created by selective proximal aortic (Ao) or pulmonary artery (PA) banding that resulted in hypertrophy of the left ventricle (LV) and right ventricle (RV), respectively. We demonstrated that PDE5 expression responded differentially with a decreased expression in the LV and an increased expression in the RV in the Ao-banded model. Similarly, in the PA-banded model, LV showed reduced expression while the RV expression was unaltered. In addition, the expression of cGKI was significantly decreased in the RV of Ao-banded group, correlating inversely with the increase in PDE5 expression.\nConclusions/Significance\nThe differential regulation of PDE5 and cGKI expression suggests that the mechanisms involved in hypertrophy could be different in RV vs. LV. Reciprocal PDE5 and cGKI expression in the RV of Ao-banded model suggests functional significance for PDE5 up-regulation....
Endocrine resistance is a major problem with anti-estrogen treatments and how to overcome resistance is a major concern in the clinic. Reliable measurement of cell viability, proliferation, growth inhibition and death is important in screening for drug treatment efficacy in vitro. This report describes and compares commonly used proliferation assays for induced estrogen-responsive MCF-7 breast cancer cell cycle arrest including: determination of cell number by direct counting of viable cells; or fluorescence SYBR�®Green (SYBR) DNA labeling; determination of mitochondrial metabolic activity by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carbox?ymethoxyphenyl)-2-(4-sulfophenyl)-2H-tet?razolium(MTS) assay; assessment of newly synthesized DNA using 5-ethynyl-2'-deoxyuridine (EdU) nucleoside analog binding and Alexa Fluor�® azide visualization by fluorescence microscopy; cell-cycle phase measurement by flow cytometry. Treatment of MCF-7 cells with ICI 182780 (Faslodex), FTY720, serum deprivation or induction of the tumor suppressor p14ARF showed inhibition of cell proliferation determined by the Trypan Blue exclusion assay and SYBR DNA labeling assay. In contrast, the effects of treatment with ICI 182780 or p14ARF-induction were not confirmed using the MTS assay. Cell cycle inhibition by ICI 182780 and p14ARF-induction was further confirmed by flow cytometric analysis and EdU-DNA incorporation. To explore this discrepancy further, we showed that ICI 182780 and p14ARF-induction increased MCF-7 cell mitochondrial activity by MTS assay in individual cells compared to control cells thereby providing a misleading proliferation readout. Interrogation of p14ARF-induction on MCF-7 metabolic activity using TMRE assays and high content image analysis showed that increased mitochondrial activity was concomitant with increased mitochondrial biomass with no loss of mitochondrial membrane potential, or cell death. We conclude that, whilst p14ARF and ICI 182780 stop cell cycle progression, the cells are still viable and potential treatments utilizing these pathways may contribute to drug resistant cells. These experiments demonstrate how the combined measurement of metabolic activity and DNA labeling provides a more reliable interpretation of cancer cell response to treatment regimens....
Parkinson's disease (PD) is a common neurodegenerative disease that appears essentially as a sporadic condition. PD is characterized by massive degeneration of dopaminergic neurons in the substantia nigra, the loss of striatal dopaminergic fibers and a dramatic reduction of the striatal dopamine levels. Over the years, a broad variety of experimental models of PD were developed and applied in diverse species. By using various standardized procedures, neurological disorders in humans can be modeled in animals that recreate specific pathogenic events and their behavioral outcomes. Most insights into PD pathogenesis come from investigations performed in experimental models of PD, especially those produced by neurotoxins. The classical animal models of PD rely on the use of neurotoxins, including 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and more recently, the agricultural chemicals paraquat and rotenone, to deplete dopamine (DA). These toxin induced models have been used to study the pathophysiology of the degenerating nigrostriatal system and to evaluate novel therapeutic strategies. In this review, we mainly discuss about the mechanisms of above most popular parkinsonian neurotoxins and also to provide an updated summary of the important characteristics and mechanism of each of these neurotoxins....
Background\r\nNeurotrophic factors may be future therapeutic agents for neurodegenerative disease. In the screening of biologically active molecules for neurotrophic potency, we found that a photosensitizing cyanine dye, NK-4, had remarkable neurotrophic activities and was a potent radical scavenger.\r\nMethodology/Principal Findings\r\nIn this study, we evaluated the effect of NK-4 on the protection of neurons against oxidative damage and investigated the associated intracellular signaling pathways. Subsequently, we evaluated the effect of NK-4 in an animal model of neurodegeneration. In vitro, NK-4 showed dose-dependent protection of PC12 cells from toxicity induced by oxidative stress caused by hydrogen peroxide (H2O2) or 6-hydroxydopamine (6-OHDA). Comparison of extracellular signal-regulated kinase signaling pathways between treatment with NK-4 and nerve growth factor (NGF) using K252a, an inhibitor of the NGF receptor TrkA, revealed that NK-4 activity occurs independently of NGF receptors. LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, blocked the protective effect of NK-4, and NK-4 caused activation of Akt/protein kinase B, a downstream effector of PI3K. These results suggest that the neuroprotective effects of NK-4 are mediated by the PI3K-Akt signaling pathway. NK-4 treatment also attenuated stress-induced activation of SAPK/JNK, which suggests that NK-4 activates a survival signaling pathway and inhibits stress-activated apoptotic pathways independently of the TrkA receptor in neuronal cells. In vivo, administration of NK-4 improved motor coordination in genetic ataxic hamsters, as assessed by rota-rod testing. Histological analysis showed that cerebellar atrophy was significantly attenuated by NK-4 treatment. Notably, the Purkinje cell count in the treated group was threefold higher than that in the vehicle group.\r\nConclusions/Significance\r\nThese results suggest that NK-4 is a potential agent for therapy for neurodegenerative disorders based on the activation of survival signaling pathways....
An increasing body of evidence indicates that accumulation of soluble oligomeric assemblies of �Ÿ-amyloid polypeptide (A�Ÿ) play a key role in Alzheimer's disease (AD) pathology. Specifically, 56 kDa oligomeric species were shown to be correlated with impaired cognitive function in AD model mice. Several reports have documented the inhibition of A�Ÿ plaque formation by compounds from natural sources. Yet, evidence for the ability of common edible elements to modulate A�Ÿ oligomerization remains an unmet challenge. Here we identify a natural substance, based on cinnamon extract (CEppt), which markedly inhibits the formation of toxic A�Ÿ oligomers and prevents the toxicity of A�Ÿ on neuronal PC12 cells. When administered to an AD fly model, CEppt rectified their reduced longevity, fully recovered their locomotion defects and totally abolished tetrameric species of A�Ÿ in their brain. Furthermore, oral administration of CEppt to an aggressive AD transgenic mice model led to marked decrease in 56 kDa A�Ÿ oligomers, reduction of plaques and improvement in cognitive behavior. Our results present a novel prophylactic approach for inhibition of toxic oligomeric A�Ÿ species formation in AD through the utilization of a compound that is currently in use in human diet....
Iron may play an important role in Parkinson's disease (PD) since it can induce oxidative stress-dependent neurodegeneration. The objective of this study was to determine whether the iron chelator, phytic acid (IP6) can protect against 6-hydroxydopamine- (6-OHDA-) induced apoptosis in immortalized rat mesencephalic dopaminergic cells under normal and iron-excess conditions. Caspase-3 activity was increased about 6-fold after 6-OHDA treatment (compared to control; ?? < . 0 0 1) and 30?�µmol/L IP6 pretreatment decreased it by 38% (?? < . 0 5). Similarly, a 63% protection (?? < . 0 0 1) against 6-OHDA induced DNA fragmentation was observed with IP6 pretreatment. Under iron-excess condition, a 6-fold increase in caspase-3 activity (?? < . 0 0 1) and a 42% increase in DNA fragmentation (?? < . 0 5) with 6-OHDA treatment were decreased by 41% (?? < . 0 1) and 27% (?? < . 0 5), respectively, with 30?�µmol/L IP6. Together, our data suggest that IP6 protects against 6-OHDA-induced cell apoptosis in both normal and iron-excess conditions, and IP6 may offer neuroprotection in PD....
Background\r\nOvarian cancer remains the leading cause of death from gynaecological malignancy. More than 60% of the patients are presenting the disease in stage III or IV. In spite of combination of chemotherapy and surgery the prognosis stays poor for therapy regimen.\r\nMethods\r\nThe leaves of a plant endemic to Australia, Calomeria amaranthoides, were extracted and then fractionated by column chromatography. In vitro cytotoxicity tests were performed with fractions of the plant extract and later with an isolated compound on ovarian cancer cell lines, as well as normal fibroblasts at concentrations of 1-100 �µg/mL (crude extract) and 1-10 �µg/mL (compound). Cytotoxicity was measured after 24, 48 and 72 hours by using a non-fluorescent substrate, Alamar blue.\r\nIn vivo cytotoxicity was tested on ascites, developed in the abdomen of nude mice after inoculation with human OVCAR3 cells intraperitoneally. The rate of change in abdomen size for the mice was determined by linear regression and statistically evaluated for significance by the unpaired t test.\r\nResults\r\nTwo compounds were isolated by chromatographic fractionation and identified by 1H-NMR, 13C-NMR and mass spectrometry analyses, EPD, an a-methylene sesquiterpene lactone of the eremophilanolide subtype, and EPA, an a-methylene carboxylic acid.\r\nCytotoxicity of EPD for normal fibroblasts at all time points IC50 was greater than 10 �µg/mL, whereas, for OVCAR3 cells at 48 hours IC50 was 5.3 �µg/mL (95% confidence interval 4.3 to 6.5 �µg/mL).\r\nBoth, the crude plant extract as well as EPD killed the cancer cells at a final concentration of 10 �µg/mL and 5 �µg/mL respectively, while in normal cells only 20% cell killing effect was observed. EPA had no cytotoxic effects.\r\nChanges in abdomen size for control versus Cisplatin treated mice were significantly different, P = 0.023, as were control versus EPD treated mice, P = 0.025, whereas, EPD versus Cisplatin treated mice were not significantly different, P = 0.13.\r\nConclusions\r\nFor the first time both crude plant extract from Calomeria amaranthoides and EPD have been shown to have potent anti-cancer effects against ovarian cancer....
Replication-competent adenovirus type 5 (Ad5) vectors promise to be more efficient gene delivery vehicles than their replication-deficient counterparts, and chimeric Ad5 vectors that are capable of targeting CD46 are more effective than Ad5 vectors with native fibers. Although several strategies have been used to improve gene transduction and oncolysis, either by modifying their tropism or enhancing their replication capacity, some tumor cells are still relatively refractory to infection by chimeric Ad5. The oncolytic effects of the vectors are apparent in certain tumors but not in others. Here, we report the biological and oncolytic profiles of a replication-competent adenovirus 11p vector (RCAd11pGFP) in colon carcinoma cells. CD46 was abundantly expressed in all cells studied; however, the transduction efficiency of RCAd11pGFP varied. RCAd11pGFP efficiently transduced HT-29, HCT-8, and LS174T cells, but it transduced T84 cells, derived from a colon cancer metastasis in the lung, less efficiently. Interestingly, RCAd11p replicated more rapidly in the T84 cells than in HCT-8 and LS174T cells and as rapidly as in HT-29 cells. Cell toxicity and proliferation assays indicated that RCAd11pGFP had the highest cell-killing activities in HT29 and T84 cells, the latter of which also expressed the highest levels of glycoproteins of the carcinoma embryonic antigen (CEA) family. In vivo experiments showed significant growth inhibition of T84 and HT-29 tumors in xenograft mice treated with either RCAd11pGFP or Ad11pwt compared to untreated controls. Thus, RCAd11pGFP has a potent cytotoxic effect on colon carcinoma cells....
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