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  1. Kaiso depletion attenuates the growth and survival of triple negative breast cancer cells.

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    Bassey-Archibong, Blessing I; Rayner, Lyndsay G A; Hercules, Shawn M; Aarts, Craig W; Dvorkin-Gheva, Anna; Bramson, Jonathan L; Hassell, John A; Daniel, Juliet M

    2017-03-23

    Triple negative breast cancers (TNBC) are highly aggressive and lack specific targeted therapies. Recent studies have reported high expression of the transcription factor Kaiso in triple negative tumors, and this correlates with their increased aggressiveness. However, little is known about the clinical relevance of Kaiso in the growth and survival of TNBCs. Herein, we report that Kaiso depletion attenuates TNBC cell proliferation, and delays tumor onset in mice xenografted with the aggressive MDA-231 breast tumor cells. We further demonstrate that Kaiso depletion attenuates the survival of TNBC cells and increases their propensity for apoptotic-mediated cell death. Notably, Kaiso depletion downregulates BRCA1 expression in TNBC cells expressing mutant-p53 and we found that high Kaiso and BRCA1 expression correlates with a poor overall survival in breast cancer patients. Collectively, our findings reveal a role for Kaiso in the proliferation and survival of TNBC cells, and suggest a relevant role for Kaiso in the prognosis and treatment of TNBCs.

  2. Major depletion of plasmacytoid dendritic cells in HIV-2 infection, an attenuated form of HIV disease.

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    Rita Cavaleiro

    2009-11-01

    Full Text Available Plasmacytoid dendritic cells (pDC provide an important link between innate and acquired immunity, mediating their action mainly through IFN-alpha production. pDC suppress HIV-1 replication, but there is increasing evidence suggesting they may also contribute to the increased levels of cell apoptosis and pan-immune activation associated with disease progression. Although having the same clinical spectrum, HIV-2 infection is characterized by a strikingly lower viremia and a much slower rate of CD4 decline and AIDS progression than HIV-1, irrespective of disease stage. We report here a similar marked reduction in circulating pDC levels in untreated HIV-1 and HIV-2 infections in association with CD4 depletion and T cell activation, in spite of the undetectable viremia found in the majority of HIV-2 patients. Moreover, the same overexpression of CD86 and PD-L1 on circulating pDC was found in both infections irrespective of disease stage or viremia status. Our observation that pDC depletion occurs in HIV-2 infected patients with undetectable viremia indicates that mechanisms other than direct viral infection determine the pDC depletion during persistent infections. However, viremia was associated with an impairment of IFN-alpha production on a per pDC basis upon TLR9 stimulation. These data support the possibility that diminished function in vitro may relate to prior activation by HIV virions in vivo, in agreement with our finding of higher expression levels of the IFN-alpha inducible gene, MxA, in HIV-1 than in HIV-2 individuals. Importantly, serum IFN-alpha levels were not elevated in HIV-2 infected individuals. In conclusion, our data in this unique natural model of "attenuated" HIV immunodeficiency contribute to the understanding of pDC biology in HIV/AIDS pathogenesis, showing that in the absence of detectable viremia a major depletion of circulating pDC in association with a relatively preserved IFN-alpha production does occur.

  3. Oval cell response is attenuated by depletion of liver resident macrophages in the 2-AAF/partial hepatectomy rat.

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    Shuai Xiang

    Full Text Available BACKGROUND/AIMS: Macrophages are known to play an important role in hepatocyte mediated liver regeneration by secreting inflammatory mediators. However, there is little information available on the role of resident macrophages in oval cell mediated liver regeneration. In the present study we aimed to investigate the role of macrophages in oval cell expansion induced by 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH in rats. METHODOLOGY/PRINCIPAL FINDINGS: We depleted macrophages in the liver of 2-AAF/PH treated rats by injecting liposome encapsulated clodronate 48 hours before PH. Regeneration of remnant liver mass, as well as proliferation and differentiation of oval cells were measured. We found that macrophage-depleted rats suffered higher mortality and liver transaminase levels. We also showed that depletion of macrophages yielded a significant decrease of EPCAM and PCK positive oval cells in immunohistochemical stained liver sections 9 days after PH. Meanwhile, oval cell differentiation was also attenuated as a result of macrophage depletion, as large foci of small basophilic hepatocytes were observed by day 9 following hepatectomy in control rats whereas they were almost absent in macrophage depleted rats. Accordingly, real-time polymerase chain reaction analysis showed lower expression of albumin mRNA in macrophage depleted livers. Then we assessed whether macrophage depletion may affect hepatic production of stimulating cytokines for liver regeneration. We showed that macrophage-depletion significantly inhibited hepatic expression of tumor necrosis factor-α and interleukin-6, along with a lack of signal transducer and activator of transcription 3 phosphorylation during the early period following hepatectomy. CONCLUSIONS: These data indicate that macrophages play an important role in oval cell mediated liver regeneration in the 2-AAF/PH model.

  4. B-Cell Depletion Attenuates White and Gray Matter Pathology in Marmoset Experimental Autoimmune Encephalomyelitis

    NARCIS (Netherlands)

    Kap, Yolanda S.; Bauer, Jan; van Driel, Nikki; Bleeker, Wim K.; Parren, Paul W. H. I.; Kooi, Evert-Jan; Geurts, Jeroen J. G.; Laman, Jon D.; Craigen, Jenny L.; Blezer, Erwin; 't Hart, Bert A.

    2011-01-01

    This study investigated the effect of CD20-positive B-cell depletion on central nervous system (CNS) white and gray matter pathology in experimental autoimmune encephalomyelitis in common marmosets, a relevant preclinical model of multiple sclerosis. Experimental autoimmune encephalomyelitis was ind

  5. Major Depletion of Plasmacytoid Dendritic Cells in HIV-2 Infection, an Attenuated Form of HIV Disease

    Science.gov (United States)

    Cavaleiro, Rita; Baptista, António P.; Soares, Rui S.; Tendeiro, Rita; Foxall, Russell B.; Gomes, Perpétua; Victorino, Rui M. M.; Sousa, Ana E.

    2009-01-01

    Plasmacytoid dendritic cells (pDC) provide an important link between innate and acquired immunity, mediating their action mainly through IFN-α production. pDC suppress HIV-1 replication, but there is increasing evidence suggesting they may also contribute to the increased levels of cell apoptosis and pan-immune activation associated with disease progression. Although having the same clinical spectrum, HIV-2 infection is characterized by a strikingly lower viremia and a much slower rate of CD4 decline and AIDS progression than HIV-1, irrespective of disease stage. We report here a similar marked reduction in circulating pDC levels in untreated HIV-1 and HIV-2 infections in association with CD4 depletion and T cell activation, in spite of the undetectable viremia found in the majority of HIV-2 patients. Moreover, the same overexpression of CD86 and PD-L1 on circulating pDC was found in both infections irrespective of disease stage or viremia status. Our observation that pDC depletion occurs in HIV-2 infected patients with undetectable viremia indicates that mechanisms other than direct viral infection determine the pDC depletion during persistent infections. However, viremia was associated with an impairment of IFN-α production on a per pDC basis upon TLR9 stimulation. These data support the possibility that diminished function in vitro may relate to prior activation by HIV virions in vivo, in agreement with our finding of higher expression levels of the IFN-α inducible gene, MxA, in HIV-1 than in HIV-2 individuals. Importantly, serum IFN-α levels were not elevated in HIV-2 infected individuals. In conclusion, our data in this unique natural model of “attenuated” HIV immunodeficiency contribute to the understanding of pDC biology in HIV/AIDS pathogenesis, showing that in the absence of detectable viremia a major depletion of circulating pDC in association with a relatively preserved IFN-α production does occur. PMID:19936055

  6. Kaiso depletion attenuates transforming growth factor-β signaling and metastatic activity of triple-negative breast cancer cells.

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    Bassey-Archibong, B I; Kwiecien, J M; Milosavljevic, S B; Hallett, R M; Rayner, L G A; Erb, M J; Crawford-Brown, C J; Stephenson, K B; Bédard, P-A; Hassell, J A; Daniel, J M

    2016-03-21

    Triple-negative breast cancers (TNBCs) represent a subset of breast tumors that are highly aggressive and metastatic, and are responsible for a disproportionate number of breast cancer-related deaths. Several studies have postulated a role for the epithelial-to-mesenchymal transition (EMT) program in the increased aggressiveness and metastatic propensity of TNBCs. Although EMT is essential for early vertebrate development and wound healing, it is frequently co-opted by cancer cells during tumorigenesis. One prominent signaling pathway involved in EMT is the transforming growth factor-β (TGFβ) pathway. In this study, we report that the novel POZ-ZF transcription factor Kaiso is highly expressed in TNBCs and correlates with a shorter metastasis-free survival. Notably, Kaiso expression is induced by the TGFβ pathway and silencing Kaiso expression in the highly invasive breast cancer cell lines, MDA-MB-231 (hereafter MDA-231) and Hs578T, attenuated the expression of several EMT-associated proteins (Vimentin, Slug and ZEB1), abrogated TGFβ signaling and TGFβ-dependent EMT. Moreover, Kaiso depletion attenuated the metastasis of TNBC cells (MDA-231 and Hs578T) in a mouse model. Although high Kaiso and high TGFβR1 expression is associated with poor overall survival in breast cancer patients, overexpression of a kinase-active TGFβR1 in the Kaiso-depleted cells was insufficient to restore the metastatic potential of these cells, suggesting that Kaiso is a key downstream component of TGFβ-mediated pro-metastatic responses. Collectively, these findings suggest a critical role for Kaiso in TGFβ signaling and the metastasis of TNBCs.

  7. CD4+CD25+Foxp3+ regulatory T cells depletion may attenuate the development of silica-induced lung fibrosis in mice.

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    Fangwei Liu

    Full Text Available BACKGROUND: Silicosis is an occupational lung disease caused by inhalation of silica dust characterized by lung inflammation and fibrosis. Previous study showed that Th1 and Th2 cytokines are involved in silicosis, but Th1/Th2 polarization during the development of silicosis is still a matter of debate. Regulatory T cells (Treg cells represent a crucial role in modulation of immune homeostasis by regulating Th1/Th2 polarization, but their possible implication in silicosis remains to be explored. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the implication of Treg cells in the development of silicosis, we generated the Treg-depleted mice model by administration of anti-CD25 mAbs and mice were exposed to silica by intratracheal instillation to establish experimental model of silica-induced lung fibrosis. The pathologic examinations show that the Treg-depleted mice are susceptive to severer inflammation in the early stage, with enhanced infiltration of inflammatory cells. Also, depletion of Treg cells causes a delay of the progress of silica-induced lung fibrosis in mice model. Further study of mRNA expression of cytokines reveals that depletion of Tregs leads to the increased production of Th1-cytokines and decreased production of Th2-cytokine. The Flow Cytometry and realtime PCR study show that Treg cells exert the modulation function both directly by expressing CTLA-4 at the inflammatory stage, and indirectly by secreting increasing amount of IL-10 and TGF-β during the fibrotic stage in silica-induced lung fibrosis. CONCLUSION/SIGNIFICANCE: Our study suggests that depletion of Tregs may attenuate the progress of silica-induced lung fibrosis and enhance Th1 response and decelerate Th1/Th2 balance toward a Th2 phenotype in silica-induced lung fibrosis. The regulatory function of Treg cells may depend on direct mechanism and indirect mechanism during the inflammatory stage of silicosis.

  8. Wld(S reduces paraquat-induced cytotoxicity via SIRT1 in non-neuronal cells by attenuating the depletion of NAD.

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    Qiujing Yu

    Full Text Available Wld(S is a fusion protein with NAD synthesis activity, and has been reported to protect axonal and synaptic compartments of neurons from various mechanical, genetic and chemical insults. However, whether Wld(S can protect non-neuronal cells against toxic chemicals is largely unknown. Here we found that Wld(S significantly reduced the cytotoxicity of bipyridylium herbicides paraquat and diquat in mouse embryonic fibroblasts, but had no effect on the cytotoxicity induced by chromium (VI, hydrogen peroxide, etoposide, tunicamycin or brefeldin A. Wld(S also slowed down the death of mice induced by intraperitoneal injection of paraquat. Further studies demonstrated that Wld(S markedly attenuated mitochondrial injury including disruption of mitochondrial membrane potential, structural damage and decline of ATP induced by paraquat. Disruption of the NAD synthesis activity of Wld(S by an H112A or F116S point mutation resulted in loss of its protective function against paraquat-induced cell death. Furthermore, Wld(S delayed the decrease of intracellular NAD levels induced by paraquat. Similarly, treatment with NAD or its precursor nicotinamide mononucleotide attenuated paraquat-induced cytotoxicity and decline of ATP and NAD levels. In addition, we showed that SIRT1 was required for both exogenous NAD and Wld(S-mediated cellular protection against paraquat. These findings suggest that NAD and SIRT1 mediate the protective function of Wld(S against the cytotoxicity induced by paraquat, which provides new clues for the mechanisms underlying the protective function of Wld(S in both neuronal and non-neuronal cells, and implies that attenuation of NAD depletion may be effective to alleviate paraquat poisoning.

  9. Glutamine attenuates post-traumatic glutathione depletion in human muscle.

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    Fläring, U B; Rooyackers, O E; Wernerman, J; Hammarqvist, F

    2003-03-01

    Glutathione is quantitatively the most important endogenous scavenger system. Glutathione depletion in skeletal muscle is pronounced following major trauma and sepsis in intensive care unit patients. Also, following elective surgery, glutathione depletion occurs in parallel with a progressive decline in muscle glutamine concentration. The present study was designed to test the hypothesis that glutamine supplementation may counteract glutathione depletion in a human trauma model. A homogeneous group of patients (n = 17) undergoing a standardized surgical procedure were prospectively randomly allocated to receive glutamine (0.56 g x day(-1) x kg(-1)) or placebo as part of isonitrogenous and isocaloric nutrition. Percutaneous muscle biopsies and blood samples were taken pre-operatively and at 24 and 72 h after surgery. The concentrations of muscle glutathione and related amino acids were determined in muscle tissue and plasma. In the control (unsupplemented) subjects, total muscle glutathione had decreased by 47+/-8% and 37+/-11% and reduced glutathione had decreased by 53+/-10% and 45+/-16% respectively at 24 and 72 h after surgery (P glutamine supplementation attenuates glutathione depletion in skeletal muscle in humans following standardized surgical trauma.

  10. X-Ray Attenuation Cell

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    Ryutov, D.; Toor, A.

    2000-03-03

    To minimize the pulse-to-pulse variation, the LCLS FEL must operate at saturation, i.e. 10 orders of magnitude brighter spectral brilliance than 3rd-generation light sources. At this intensity, ultra-high vacuums and windowless transport are required. Many of the experiments, however, will need to be conducted at a much lower intensity thereby requiring a reliable means to reduce the x-ray intensity by many orders of magnitude without increasing the pulse-to-pulse variation. In this report we consider a possible solution for controlled attenuation of the LCLS x-ray radiation. We suggest using for this purpose a windowless gas-filled cell with the differential pumping. Although this scheme is easily realizable in principle, it has to be demonstrated that the attenuator can be made short enough to be practical and that the gas loads delivered to the vacuum line of sight (LOS) are acceptable. We are not going to present a final, optimized design. Instead, we will provide a preliminary analysis showing that the whole concept is robust and is worth further study. The spatial structure of the LCLS x-ray pulse at the location of the attenuator is shown in Fig. 1. The central high-intensity component, due to the FEL, has a FWHM of {approx}100 {micro}m. A second component, due to the undulator's broad band spontaneous radiation is seen as a much lower intensity ''halo'' with a FWHM of 1 mm. We discuss two versions of the attenuation cell. The first is directed towards a controlled attenuation of the FEL up to the 4 orders of magnitude in the intensity, with the spontaneous radiation halo being eliminated by collimators. In the second version, the spontaneous radiation is not sacrificed but the FEL component (as well as the first harmonic of the spontaneous radiation) gets attenuated by a more modest factor up to 100. We will make all the estimates assuming that the gas used in the attenuator is Xenon and that the energy of the FEL is 8.25 keV. At

  11. Polyamine Depletion Attenuates Isoproterenol-Induced Hypertrophy and Endoplasmic Reticulum Stress in Cardiomyocytes

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    Yan Lin

    2014-10-01

    Full Text Available Background/Aim: Polyamines (putrescine, spermidine and spermine play an essential role in cell growth, differentiation and apoptosis. Hypertrophy is accompanied by an increase in polyamine synthesis and endoplasmic reticulum stress (ERS in cardiomyocytes. The present study was undertaken to elucidate the molecular interactions between polyamines, ERS and cardiac hypertrophy. Methods: Myocardial hypertrophy was simulated by incubating cultured neonatal rat cardiomyocytes in 100 nM isoproterenol (ISO. Polyamine deletion was achieved using 0.5 mM difluoromethylornithine (DFMO. Hypertrophy was estimated using cell surface area measurements, total protein concentrations and atrial natriuretic peptide (ANP gene expression. Apoptosis was measured using flow cytometry and transmission electron microscopy. Expression of ornithine decarboxylase (ODC and spermidine/spermine N1-acetyltransferase (SSAT were analyzed via real-time PCR and Western blotting. Protein expression of ERS and apoptosis factors were analyzed using Western blotting. Results: DFMO (0.5 mM and 2 mM treatments significantly attenuated hypertrophy and apoptosis induced by ISO in cardiomyocytes. DFMO also decreased lactate dehydrogenase (LDH and malondialdehyde (MDA level in the culture medium. In addition, DFMO (0.5 mM down regulated the expression of ODC, glucose-regulated protein 78 (GRP78, C/EBP homologous protein (CHOP, cleaved caspase-12, and Bax and up regulated the expression of SSAT and Bcl-2. Finally, these changes were partly reversed by the addition of exogenous putrescine (0.5 mM. Conclusion: The data presented here suggest that polyamine depletion could inhibit cardiac hypertrophy and apoptosis, which is closely related to the ERS pathway.

  12. Intrathecal DSP4 selectively depletes spinal noradrenaline and attenuates morphine analgesia.

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    Zhong, F X; Ji, X Q; Tsou, K

    1985-10-22

    The noradrenergic neurotoxin DSP4 was injected intrathecally into the lumbar spinal subarachnoid space of the rat. After 10 days, dose-response curves for tail-flick inhibition were determined for both intraperitoneal and intraventricular injections of morphine. In rats pretreated with DSP4, the analgesic effect of morphine given either intraventricularly or intraperitoneally was significantly attenuated. The noradrenaline and 5-hydroxytryptamine contents in the brain-stem and the spinal cord were also measured. Intrathecal DSP4 depleted noradrenaline in the spinal cord but not in the brain-stem. Neither brain-stem or spinal cord 5-hydroxytryptamine was affected by DSP4.

  13. Auranofin induces apoptosis and necrosis in HeLa cells via oxidative stress and glutathione depletion.

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    You, Bo Ra; Shin, Hye Rim; Han, Bo Ram; Kim, Suhn Hee; Park, Woo Hyun

    2015-02-01

    Auranofin (Au), an inhibitor of thioredoxin reductase, is a known anti‑cancer drug. In the present study, the anti‑growth effect of Au on HeLa cervical cancer cells was examined in association with levels of reactive oxygen species (ROS) and glutathione (GSH). Au inhibited the growth of HeLa cells with an IC50 of ~2 µM at 24 h. This agent induced apoptosis and necrosis, accompanied by the cleavage of poly (ADP‑ribose) polymerase and loss of mitochondrial membrane potential. The pan‑caspase inhibitor, benzyloxycarbonyl‑Val‑Ala‑Asp‑fluoromethylketone, prevented apoptotic cell death and each of the assessed caspase inhibitors inhibited necrotic cell death induced by Au. With respect to the levels of ROS and GSH, Au increased intracellular O2•- in the HeLa cells and induced GSH depletion. The pan‑caspase inhibitor reduced the levels of O2•- and GSH depletion in Au‑treated HeLa cells. The antioxidant, N‑acetyl cysteine, not only attenuated apoptosis and necrosis in the Au‑treated HeLa cells, but also decreased the levels of O2•- and GSH depletion in the cells. By contrast, L‑buthionine sulfoximine, a GSH synthesis inhibitor, intensified cell death O2•- and GSH depletion in the Au‑treated HeLa cells. In conclusion, Au induced apoptosis and necrosis in HeLa cells via the induction of oxidative stress and the depletion of GSH.

  14. CD20(+) B Cell Depletion Alters T Cell Homing

    NARCIS (Netherlands)

    Kap, Yolanda S.; van Driel, Nikki; Laman, Jon D.; Tak, Paul P.; 't Hart, Bert A.

    2014-01-01

    Depleting mAbs against the pan B cell marker CD20 are remarkably effective in the treatment of autoimmune-mediated inflammatory disorders, but the underlying mechanisms are poorly defined. The primary objective of this study was to find a mechanistic explanation for the remarkable clinical effect of

  15. Mulberry Leaf Extract Attenuates Oxidative Stress-Mediated Testosterone Depletion in Streptozotocin-Induced Diabetic Rats

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    Mohammad Reza Hajizadeh

    2014-03-01

    Full Text Available Background: It has been proposed that oxidative stress may contribute to the development of testicular abnormalities in diabetes. Morus alba leaf extract (MAE has hypoglycemic and antioxidant properties. We, therefore, explored the impact of the administration of MAE on steroidogenesis in diabetic rats. Methods: To address this hypothesis, we measured the serum level of glucose, insulin, and free testosterone (Ts as well as oxidative stress parameters (including glutathione peroxidase, glutathione reductase, total antioxidant capacity, and malondialdehyde in the testis of control, untreated and MAE-treated (1 g/day/kg diabetic rats. In order to determine the likely mechanism of MAE action on Ts levels, we analyzed the quantitative mRNA expression level of the two key steroidogenic proteins, namely steroid acute regulatory protein (StAR and P450 cholesterol side-chain cleavage enzyme (P450scc, by real-time PCR. Results: The MAE-treated diabetic rats had significantly decreased glucose levels and on the other hand increased insulin and free Ts levels than the untreated diabetic rats. In addition, the administration of MAE to the diabetic rats restored the oxidative stress parameters toward control. Induction of diabetes decreased testicular StAR mRNA expression by 66% and MAE treatment enhanced mRNA expression to the same level of the control group. However, the expression of P540scc was not significantly decreased in the diabetic group as compared to the control group. Conclusion: Our findings indicated that MAE significantly increased Ts production in the diabetic rats, probably through the induction of StAR mRNA expression levels. Administration of MAE to experimental models of diabetes can effectively attenuate oxidative stress-mediated testosterone depletion. Please cite this article as: Hajizadeh MR, Eftekhar E, Zal F, Jaffarian A, Mostafavi-Pour Z. Mulberry Leaf Extract Attenuates Oxidative Stress-Mediated Testosterone Depletion in

  16. Acute tryptophan depletion attenuates brain-heart coupling following external feedback

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    Erik M Mueller

    2012-04-01

    Full Text Available External and internal performance feedback triggers neural and visceral modulations such as reactions in the medial prefrontal cortex and insulae or changes of heart period (HP. The functional coupling of neural and cardiac responses following feedback (cortico-cardiac connectivity is not well understood. While linear time-lagged within-subjects correlations of single-trial EEG and HP (cardio-electroencephalographic covariance-tracing, CECT indicate a robust negative coupling of EEG magnitude 300 ms after presentation of an external feedback stimulus with subsequent alterations of heart period (the so-called N300H phenomenon, the neurotransmitter systems underlying feedback-evoked cortico-cardiac connectivity are largely unknown. Because it has been shown that acute tryptophan depletion (ATD, attenuating brain serotonin (5-HT, decreases cardiac but not neural correlates of feedback processing, we hypothesized that 5-HT may be involved in feedback-evoked cortico-cardiac connectivity. In a placebo-controlled double-blind crossover design, twelve healthy participants received a tryptophan-free amino-acid drink at one session and a balanced amino-acid control-drink on another and twice performed a time-estimation task with feedback presented after each trial. N300H magnitude and plasma tryptophan levels were assessed. Results indicated a robust N300H after the control drink, which was significantly attenuated following ATD. Moreover, plasma tryptophan levels during the control session were correlated with N300H amplitude such that individuals with lower tryptophan levels showed reduced N300H. Together, these findings indicate that 5-HT is important for feedback-induced covariation of cortical and cardiac activity. Because individual differences in anxiety have previously been linked to 5-HT, cortico-cardiac coupling and feedback processing, the present findings may be particularly relevant for futures studies linking 5-HT to anxiety.

  17. Light attenuation on Chlorella vulgaris cells

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    Krol, Tadeusz; Lotocka, Maria

    1993-12-01

    The laboratory measurements of spectrum of light attenuation on phytoplankton particles i.e. monoculture of unicellural green algae Chlorella vulgaris are presented. The measurements were carried out for alive culture and the cultures subjected to chemical (NaOH) or physical (ultrasounds) modification. The distinct changes in the light attenuation spectrum were a result of modification of the internal cell structures.

  18. Asymmetric spindle pole formation in CPAP-depleted mitotic cells.

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    Lee, Miseon; Chang, Jaerak; Chang, Sunghoe; Lee, Kyung S; Rhee, Kunsoo

    2014-02-21

    CPAP is an essential component for centriole formation. Here, we report that CPAP is also critical for symmetric spindle pole formation during mitosis. We observed that pericentriolar material between the mitotic spindle poles were asymmetrically distributed in CPAP-depleted cells even with intact numbers of centrioles. The length of procentrioles was slightly reduced by CPAP depletion, but the length of mother centrioles was not affected. Surprisingly, the young mother centrioles of the CPAP-depleted cells are not fully matured, as evidenced by the absence of distal and subdistal appendage proteins. We propose that the selective absence of centriolar appendages at the young mother centrioles may be responsible for asymmetric spindle pole formation in CPAP-depleted cells. Our results suggest that the neural stem cells with CPAP mutations might form asymmetric spindle poles, which results in premature initiation of differentiation.

  19. Treg depletion attenuates irradiation-induced pulmonary fibrosis by reducing fibrocyte accumulation, inducing Th17 response, and shifting IFN-γ, IL-12/IL-4, IL-5 balance.

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    Xiong, Shanshan; Guo, Renfeng; Yang, Zhihua; Xu, Long; Du, Li; Li, Ruoxi; Xiao, Fengjun; Wang, Qianjun; Zhu, Maoxiang; Pan, Xiujie

    2015-11-01

    Irradiation-induced pulmonary fibrosis results from thoracic radiotherapy and severely limits radiotherapy approaches. CD4(+) CD25(+) FoxP3(+) regulatory T cells (Tregs) are involved in experimentally induced murine lung fibrosis. However, the precise contribution of Tregs to irradiation-induced pulmonary fibrosis still remains unclear. We have previously established the mouse model of irradiation-induced pulmonary fibrosis and observed an increased frequency of Tregs during the process. This study aimed to investigate the effects of Treg depletion on irradiation-induced pulmonary fibrosis and on fibrocyte, Th17 cell response and production of multiple cytokines in mice. Treg-depleted mice were generated by intraperitoneal injection with anti-CD25 mAb 2h after 20 Gy (60)CO γ-ray thoracic irradiation and every 7 days thereafter. Pulmonary fibrosis was semi-quantitatively assessed using Masson's trichrome staining. The proportions of Tregs, fibrocyte and Th17 cells were detected by flow cytometry. Th1/Th2 cytokines were assessed by Luminex assays. We found that Treg depletion decelerated the process of irradiation-induced pulmonary fibrosis and hindered fibrocyte recruitment to the lung. In response to Treg depletion, the number of CD4(+) T lymphocytes and Th17 cells increased. Moreover, Th1/Th2 cytokine balance was disturbed into Th1 dominance upon Treg depletion. Our study demonstrates that Tregs are involved in irradiation-induced pulmonary fibrosis by promoting fibrocyte accumulation, attenuating Th17 response and regulating Th1/Th2 cytokine balance in the lung tissues, which suggests that Tregs may be therapeutically manipulated to decelerate the progression of irradiation-induced pulmonary fibrosis.

  20. Apoptosis and T cell depletion during feline infectious peritonitis

    OpenAIRE

    Horzinek, M.C.; Haagmans, B. L.; Egberink, H F

    1996-01-01

    Cats that have succumbed to feline infectious peritonitis, an immune- mediated disease caused by variants of feline coronaviruses, show apoptosis and T-cell depletion in their lymphoid organs. The ascitic fluid that develops in the course of the condition causes apoptosis in vitro but only in activated T cells. Since feline infectious peritonitis virus does not infect T cells, and viral proteins did not inhibit T-cell proliferation, we postulate that soluble mediators released during the infe...

  1. T cell depleted haploidentical transplantation: positive selection

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    Franco Aversa

    2011-06-01

    Full Text Available Interest in mismatched transplantation arises from the fact that a suitable one-haplotype mismatched donor is immediately available for virtually all patients, particularly for those who urgently need an allogenic transplant. Work on one haplotype-mismatched transplants has been proceeding for over 20 years all over the world and novel transplant techniques have been developed. Some centres have focused on the conditioning regimens and post transplant immune suppression; others have concentrated on manipulating the graft which may be a megadose of extensively T celldepleted or unmanipulated progenitor cells. Excellent engraftment rates are associated with a very low incidence of acute and chronic GVHD and regimen-related mortality even in patients who are over 50 years old. Overall, event-free survival and transplant-related mortality compare favourably with reports on transplants from sources of stem cells other than the matched sibling.

  2. Apoptosis and T cell depletion during feline infectious peritonitis

    NARCIS (Netherlands)

    Horzinek, M.C.; Haagmans, B.L.; Egberink, H.F.

    1996-01-01

    Cats that have succumbed to feline infectious peritonitis, an immune- mediated disease caused by variants of feline coronaviruses, show apoptosis and T-cell depletion in their lymphoid organs. The ascitic fluid that develops in the course of the condition causes apoptosis in vitro but only in activa

  3. EZH2 depletion blocks the proliferation of colon cancer cells.

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    Bettina Fussbroich

    Full Text Available The Enhancer of Zeste 2 (EZH2 protein has been reported to stimulate cell growth in some cancers and is therefore considered to represent an interesting new target for therapeutic intervention. Here, we investigated a possible role of EZH2 for the growth control of colon cancer cells. RNA interference (RNAi-mediated intracellular EZH2 depletion led to cell cycle arrest of colon carcinoma cells at the G1/S transition. This was associated with a reduction of cell numbers upon transient transfection of synthetic EZH2-targeting siRNAs and with inhibition of their colony formation capacity upon stable expression of vector-borne siRNAs. We furthermore tested whether EZH2 may repress the growth-inhibitory p27 gene, as reported for pancreatic cancer. However, expression analyses of colon cancer cell lines and colon cancer biopsies did not reveal a consistent correlation between EZH2 and p27 levels. Moreover, EZH2 depletion did not re-induce p27 expression in colon cancer cells, indicating that p27 repression by EZH2 may be cell- or tissue-specific. Whole genome transcriptome analyses identified cellular genes affected by EZH2 depletion in colon cancer cell lines. They included several cancer-associated genes linked to cellular proliferation or invasion, such as Dag1, MageD1, SDC1, Timp2, and Tob1. In conclusion, our results demonstrate that EZH2 depletion blocks the growth of colon cancer cells. These findings might provide benefits for the treatment of colon cancer.

  4. Effective fiber hypertrophy in satellite cell-depleted skeletal muscle.

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    McCarthy, John J; Mula, Jyothi; Miyazaki, Mitsunori; Erfani, Rod; Garrison, Kelcye; Farooqui, Amreen B; Srikuea, Ratchakrit; Lawson, Benjamin A; Grimes, Barry; Keller, Charles; Van Zant, Gary; Campbell, Kenneth S; Esser, Karyn A; Dupont-Versteegden, Esther E; Peterson, Charlotte A

    2011-09-01

    An important unresolved question in skeletal muscle plasticity is whether satellite cells are necessary for muscle fiber hypertrophy. To address this issue, a novel mouse strain (Pax7-DTA) was created which enabled the conditional ablation of >90% of satellite cells in mature skeletal muscle following tamoxifen administration. To test the hypothesis that satellite cells are necessary for skeletal muscle hypertrophy, the plantaris muscle of adult Pax7-DTA mice was subjected to mechanical overload by surgical removal of the synergist muscle. Following two weeks of overload, satellite cell-depleted muscle showed the same increases in muscle mass (approximately twofold) and fiber cross-sectional area with hypertrophy as observed in the vehicle-treated group. The typical increase in myonuclei with hypertrophy was absent in satellite cell-depleted fibers, resulting in expansion of the myonuclear domain. Consistent with lack of nuclear addition to enlarged fibers, long-term BrdU labeling showed a significant reduction in the number of BrdU-positive myonuclei in satellite cell-depleted muscle compared with vehicle-treated muscle. Single fiber functional analyses showed no difference in specific force, Ca(2+) sensitivity, rate of cross-bridge cycling and cooperativity between hypertrophied fibers from vehicle and tamoxifen-treated groups. Although a small component of the hypertrophic response, both fiber hyperplasia and regeneration were significantly blunted following satellite cell depletion, indicating a distinct requirement for satellite cells during these processes. These results provide convincing evidence that skeletal muscle fibers are capable of mounting a robust hypertrophic response to mechanical overload that is not dependent on satellite cells.

  5. Dystroglycan depletion inhibits the functions of differentiated HL-60 cells.

    Science.gov (United States)

    Martínez-Zárate, Alma Delia; Martínez-Vieyra, Ivette; Alonso-Rangel, Lea; Cisneros, Bulmaro; Winder, Steve J; Cerecedo, Doris

    2014-06-01

    Dystroglycan has recently been characterized in blood tissue cells, as part of the dystrophin glycoprotein complex but to date nothing is known of its role in the differentiation process of neutrophils. We have investigated the role of dystroglycan in the human promyelocytic leukemic cell line HL-60 differentiated to neutrophils. Depletion of dystroglycan by RNAi resulted in altered morphology and reduced properties of differentiated HL-60 cells, including chemotaxis, respiratory burst, phagocytic activities and expression of markers of differentiation. These findings strongly implicate dystroglycan as a key membrane adhesion protein involved in the differentiation process in HL-60 cells.

  6. Effect of adoptive transfer or depletion of regulatory T cells on triptolide-induced liver injury

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    Xinzhi eWang

    2016-04-01

    Full Text Available ObjectiveThe aim of this study is to clarify the role of regulatory T cell (Treg in triptolide (TP-induced hepatotoxicity. MethodsFemale C57BL/6 mice received either adoptive transfer of Tregs or depletion of Tregs, then underwent TP administration and were sacrificed 24 hours after TP administration. Liver injury was determined according to ALT and AST levels in serum and histopathological change in liver tissue. Hepatic frequencies of Treg cells and the mRNA expression levles of transcription factor FoxP3 and RORγt, IL-10, SOCS and Notch/Notch ligand were investigated.ResultsDuring TP-induced liver injury, hepatic Treg and IL-10 decreased, while Th17 cell transcription factor RORγt, SOCS signaling and Notch signaling increased, accompanied with liver inflammation. Adoptive transfer of Tregs ameliorated the severity of TP-induced liver injury, accompanied with increased levels of hepatic Treg and IL-10. Adoptive transfer of Tregs remarkably inhibited the expression of RORγt, SOCS3, Notch1 and Notch3. On the contrary, depletion of Treg cells in TP-administered mice resulted in a notable increase of RORγt, SOCS1, SOCS3 and Notch3, while the Treg and IL-10 of liver decreased. Consistent with the exacerbation of liver injury, higher serum levels of ALT and AST were detected in Treg-depleted mice. ConclusionsThese results showed that adoptive transfer or depletion of Tregs attenuated or aggravated TP-induced liver injury, suggesting that Tregs could play important roles in the progression of liver injury. SOCS proteins and Notch signaling affected Tregs, which may contribute to the pathogenesis of TP-induced hepatotoxicity.

  7. Quantification of depletion-induced adhesion of Red Blood Cells

    CERN Document Server

    Steffen, Patrick; Wagner, Christian

    2012-01-01

    Red blood cells (RBC) are known to form aggregates in the forms of rouleaux due to the presence of plasma proteins under physiological conditions. Rouleaux formation can be also induced in vitro by the addition of macromolecules to the RBC solution. Current data on the adhesion strength between red blood cells in their natural discocyte shapes mostly rely on indirect measurements like flow chamber experiments, but on the single cell level data is lacking. Here we present measurements on the dextran induced aggregation of red blood cells by use of atomic force microscopy based single cell force spectroscopy (SCFS). The effects of dextran concentration and molecular weight on the interaction energy of adhering RBCs was determined. The results are in good agreement with a model based on the depletion effect and former experimental studies.

  8. SUZ12 Depletion Suppresses the Proliferation of Gastric Cancer Cells

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    Yingjun Cui

    2013-05-01

    Full Text Available Background/Aims: SUZ12 and EZH2 are two main components of polycomb repressive complex 2 (PRC2 that is known to be of great importance in tumorigenesis. EZH2 has been reported to play a vital role in pathogenesis of human cancer. However, whether SUZ12 has equivalent roles in tumorigenesis has not been demonstrated. Here, we investigated a possible role of SUZ12 for the proliferation of gastric cancer cells. Methods: Western-blot analysis was used to detected the levels of SUZ12, H3K27me3, EZH2 and p27 in ten gastric cell lines. SUZ12 was depleted by RNA interference. Cell cycle was detected by flow cytometry. Luciferase assays was to analyze whether miR-200b directly regulate SUZ12. Results: We found that SUZ12 depletion mediated by RNA interference (RNAi led to a reduction of gastric cell numbers and arrested the cell cycle at G1/S point. As an important G1/S phase inhibitory gene, p27 is re-induced to some extent by SUZ12 knockdown. Furthermore, we demonstrated that SUZ12 was directly downregulated by miR-200b. Conclusion: We provide evidence suggesting that SUZ12 may be a potential therapeutic target for gastric cancer.

  9. Macrophage depletion by clodronate liposome attenuates muscle injury and inflammation following exhaustive exercise

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    Noriaki Kawanishi

    2016-03-01

    Full Text Available Exhaustive exercise promotes muscle injury, including myofiber lesions; however, its exact mechanism has not yet been elucidated. In this study, we tested the hypothesis that macrophage depletion by pretreatment with clodronate liposomes alters muscle injury and inflammation following exhaustive exercise. Male C57BL/6J mice were divided into four groups: rest plus control liposome (n=8, rest plus clodronate liposome (n=8, exhaustive exercise plus control liposome (n=8, and exhaustive exercise plus clodronate liposome (n=8. Mice were treated with clodronate liposome or control liposome for 48 h before undergoing exhaustive exercise on a treadmill. Twenty-four hours after exhaustive exercise, the gastrocnemius muscles were removed for histological and PCR analyses. Exhaustive exercise increased the number of macrophages in the muscle; however, clodronate liposome treatment reduced this infiltration. Although exhaustive exercise resulted in an increase in injured myofibers, clodronate liposome treatment following exhaustive exercise reduced the injured myofibers. Clodronate liposome treatment also decreased the mRNA expression levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6 in the skeletal muscle after exhaustive exercise. These results suggest that macrophages play a critical role in increasing muscle injury by regulating inflammation.

  10. Depleted-Heterojunction Colloidal Quantum Dot Solar Cells

    KAUST Repository

    Pattantyus-Abraham, Andras G.

    2010-06-22

    Colloidal quantum dot (CQD) photovoltaics combine low-cost solution processability with quantum size-effect tunability to match absorption with the solar spectrum. Rapid recent advances in CQD photovoltaics have led to impressive 3.6% AM1.5 solar power conversion efficiencies. Two distinct device architectures and operating mechanisms have been advanced. The first-the Schottky device-was optimized and explained in terms of a depletion region driving electron-hole pair separation on the semiconductor side of a junction between an opaque low-work-function metal and a p-type CQD film. The second-the excitonic device-employed a CQD layer atop a transparent conductive oxide (TCO) and was explained in terms of diffusive exciton transport via energy transfer followed by exciton separation at the type-II heterointerface between the CQD film and the TCO. Here we fabricate CQD photovoltaic devices on TCOs and show that our devices rely on the establishment of a depletion region for field-driven charge transport and separation, and that they also exploit the large bandgap of the TCO to improve rectification and block undesired hole extraction. The resultant depletedheterojunction solar cells provide a 5.1% AM1.5 power conversion efficiency. The devices employ infrared-bandgap size-effect-tuned PbS CQDs, enabling broadband harvesting of the solar spectrum. We report the highest opencircuit voltages observed in solid-state CQD solar cells to date, as well as fill factors approaching 60%, through the combination of efficient hole blocking (heterojunction) and very small minority carrier density (depletion) in the large-bandgap moiety. © 2010 American Chemical Society.

  11. Influence of 5 major Salmonella pathogenicity islands on NK cell depletion in mice infected with Salmonella enterica serovar Enteritidis

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    Ondrackova Petra

    2010-03-01

    Full Text Available Abstract Background In this study we were interested in the colonisation and early immune response of Balb/C mice to infection with Salmonella Enteritidis and isogenic pathogenicity island free mutants. Results The virulence of S. Enteritidis for Balb/C mice was exclusively dependent on intact SPI-2. Infections with any of the mutants harbouring SPI-2 (including the mutant in which we left only SPI-2 but removed SPI-1, SPI-3, SPI-4 and SPI-5 resulted in fatalities, liver injures and NK cell depletion from the spleen. The infection was of minimal influence on counts of splenic CD4 CD8 T lymphocytes and γδ T-lymphocytes although a reduced ability of splenic lymphocytes to respond to non-specific mitogens indicated general immunosuppression in mice infected with SPI-2 positive S. Enteritidis mutants. Further investigations showed that NK cells were depleted also in blood but not in the caecal lamina propria. However, NK cell depletion was not directly associated with the presence of SPI-2 and was rather an indicator of virulence or avirulence of a particular mutant because the depletion was not observed in mice infected with other attenuated mutants such as lon and rfaL. Conclusions The virulence of S. Enteritidis for Balb/C mice is exclusively dependent on the presence of SPI-2 in its genome, and a major hallmark of the infection in terms of early changes in lymphocyte populations is the depletion of NK cells in spleen and blood. The decrease of NK cells in circulation can be used as a marker of attenuation of S. Enteritidis mutants for Balb/C mice.

  12. Conversion of Fibroblasts to Neural Cells by p53 Depletion

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    Di Zhou

    2014-12-01

    Full Text Available Conversion from fibroblasts to neurons has recently been successfully induced. However, the underlying mechanisms are poorly understood. Here, we find that depletion of p53 alone converts fibroblasts into all three major neural lineages. The induced neuronal cells express multiple neuron-specific proteins and generate action potentials and transmitter-receptor-mediated currents. Surprisingly, depletion does not affect the well-known tumorigenic p53 target, p21. Instead, knockdown of p53 upregulates neurogenic transcription factors, which in turn boosts fibroblast-neuron conversion. p53 binds the promoter of the neurogenic transcription factor Neurod2 and regulates its expression during fibroblast-neuron conversion. Furthermore, our method provides a high efficiency of conversion in late-passage fibroblasts. Genome-wide transcriptional analysis shows that the p53-deficiency-induced neurons exhibit an expression profile different from parental fibroblasts and similar to control-induced neurons. The results may help to understand and improve neural conversion mechanisms to develop robust neuron-replacement therapy strategies.

  13. Modeling and Forecasting of Depletion of Additives in Car Engine Oils Using Attenuated Total Reflectance Fast Transform Infrared Spectroscopy

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    Ronald Nguele

    2014-11-01

    Full Text Available On average, additives make up to 7% of a typical lubricant base. Commonly, they are blended with lube oils to enhance specific features thereby improving their qualities. Ultimately, additives participate in the performance of car engine oils. Using an analytical tool, attenuated total reflectance fast transform infrared spectroscopy, various grades of car engine oils, at different mileages, were analyzed. Sulfate oxidation and wear were found to trigger chemical processes which, in the long run, cause lubricant degradation while carbonyl oxidation was observed to occur only at a slow rate. Based upon data obtained from infrared spectra and using a curve fitting technique, mathematical equations predicting the theoretical rates of chemical change due to the aforementioned processes were examined. Additive depletions were found to obey exponential regression rather than polynomial. Moreover, breakpoint (breakpoint is used here to denote the initiation of deterioration of additives and critical mileage (critical mileage defines the distance at which the lubricant is chemically unusable of both samples were determined.

  14. Inhibitory effect of Polo-like kinase 1 depletion on mitosis and apoptosis of gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Xue-Hua Chen; Bin Lan; Ying Qu; Xiao-Qing Zhang; Qu Cai; Bing-Ya Liu; Zheng-Gang Zhu

    2006-01-01

    AIM: Polo-like kinase 1 (PLK1) serine/threonine kinase plays a vital role in multiple phases of mitosis in gastric cancer cells. To investigate the effect of PLK1 depletion on mitosis and apoptosis of gastric cancer cells.METHODS: PLK1 expression was blocked by small RNA interference(siRNA). The expression levels of PLK1, cdc2, cyclin B and caspase 3 were detected by Western blotting. Then, PLK1 depletion, cdc2 activity,cell proliferation, cell cycle phase distribution, mitotic spindle structure, and the rate of apoptosis of the PLK1knockdown cells were observed.RESULTS: PLK1 gene knockdown was associated with increased cyclin B expression, increased cdc2 activity (but not with the expression levels), accumulation of gastric cancer cells at G2/M, improper mitotic spindle formation,delayed chromosome separation and delayed or arrested cytokinesis. Moreover, PLK1 depletion in gastric cancer cells was associated with decreased proliferation,attenuated pro-caspase 3 levels and increased apoptosis.CONCLUSION: Blockage of PLK1 expression may lead to decreased mitosis or even apoptosis in gastric cancer cells, indicating that PLK1 may be a valuable therapeutic target for gastric cancer.

  15. Long-lived plasma cells in autoimmunity: lessons from B-cell depleting therapy.

    Science.gov (United States)

    Mahévas, Matthieu; Michel, Marc; Weill, Jean-Claude; Reynaud, Claude-Agnès

    2013-12-27

    A large number of auto-immune diseases are treated with rituximab, an antibody against CD20 that depletes most of the B-cells in the organism. The response to this treatment depends largely on the disease and the type of lymphoid cells involved in the auto-immune process. We recently reported that B-cell depletion in immune thrombocytopenia induced the appearance of pathogenic long-lived plasma cells in the spleen, which were not present before treatment or in non-auto-immune conditions. The spleen of treated patients produced an excess of the cytokine B-cell activating factor, which in in vitro-cultured splenic cells, could increase the longevity of plasma cells. Our results suggested that, paradoxically, the B-cell depletion itself, by altering the splenic milieu, promoted the differentiation of short-lived auto-immune plasma cells into long-lived ones. We describe the cellular and cytokinic components of the splenic plasma cell niche, notably CD4(+) T cells and discuss possible survival factors that could be targeted simultaneously with rituximab-mediated B-cell depletion to interfere with plasma cell persistence.

  16. Long-Lived Plasma Cells in Autoimmunity: Lessons from B-Cell Depleting Therapy

    OpenAIRE

    2013-01-01

    A large number of autoimmune diseases are treated with rituximab, an antibody against CD20 that depletes most of the B cells in the organism. The response to this treatment depends largely on the disease and the type of lymphoid cells involved in the autoimmune process. We recently reported that B-cell depletion in immune thrombocytopenia induced the appearance of pathogenic long-lived plasma cells in the spleen, which were not present before treatment or in non-autoimmune conditions. The spl...

  17. PCM1 Depletion Inhibits Glioblastoma Cell Ciliogenesis and Increases Cell Death and Sensitivity to Temozolomide

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    Lan B. Hoang-Minh

    2016-10-01

    Full Text Available A better understanding of the molecules implicated in the growth and survival of glioblastoma (GBM cells and their response to temozolomide (TMZ, the standard-of-care chemotherapeutic agent, is necessary for the development of new therapies that would improve the outcome of current GBM treatments. In this study, we characterize the role of pericentriolar material 1 (PCM1, a component of centriolar satellites surrounding centrosomes, in GBM cell proliferation and sensitivity to genotoxic agents such as TMZ. We show that PCM1 is expressed around centrioles and ciliary basal bodies in patient GBM biopsies and derived cell lines and that its localization is dynamic throughout the cell cycle. To test whether PCM1 mediates GBM cell proliferation and/or response to TMZ, we used CRISPR/Cas9 genome editing to generate primary GBM cell lines depleted of PCM1. These PCM1-depleted cells displayed reduced AZI1 satellite protein localization and significantly decreased proliferation, which was attributable to increased apoptotic cell death. Furthermore, PCM1-depleted lines were more sensitive to TMZ toxicity than control lines. The increase in TMZ sensitivity may be partly due to the reduced ability of PCM1-depleted cells to form primary cilia, as depletion of KIF3A also ablated GBM cells' ciliogenesis and increased their sensitivity to TMZ while preserving PCM1 localization. In addition, the co-depletion of KIF3A and PCM1 did not have any additive effect on TMZ sensitivity. Together, our data suggest that PCM1 plays multiple roles in GBM pathogenesis and that associated pathways could be targeted to augment current or future anti-GBM therapies.

  18. Transient Treg-cell depletion in adult mice results in persistent self-reactive CD4(+) T-cell responses.

    Science.gov (United States)

    Nyström, Sofia N; Bourges, Dorothée; Garry, Sarah; Ross, Ellen M; van Driel, Ian R; Gleeson, Paul A

    2014-12-01

    Depletion of Foxp3(+) CD4(+) regulatory T cells (Treg) in adults results in chronic inflammation and autoimmune disease. However, the impact of transient Treg-cell depletion on self-reactive responses is poorly defined. Here, we studied the effect of transient depletion of Treg cells on CD4(+) T-cell responses to endogenous self-antigens. Short-term ablation of Treg cells in mice resulted in rapid activation of CD4(+) T cells, increased percentage of IFN-γ(+) and Th17 cells in lymphoid organs, and development of autoimmune gastritis. To track self-reactive responses, we analyzed the activation of naïve gastric-specific CD4(+) T cells. There was a dramatic increase in proliferation and acquisition of effector function of gastric-specific T cells in the stomach draining LNs of Treg-cell-depleted mice, compared with untreated mice, either during Treg-cell depletion or after Treg-cell reconstitution. Moreover, the hyperproliferation of gastric-specific T cells in the Treg-cell-ablated mice was predominantly antigen-dependent. Transient depletion of Treg cells resulted in a shift in the ratio of peripheral:thymic Treg cells in the reemerged Treg-cell population, indicating an altered composition of Treg cells. These findings indicate that transient Treg-cell depletion results in ongoing antigen-driven self-reactive T-cell responses and emphasize the continual requirement for an intact Treg-cell population.

  19. Accelerated cellular senescence phenotype of GAPDH-depleted human lung carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Phadke, Manali; Krynetskaia, Natalia [Temple University School of Pharmacy, Philadelphia, PA 19140 (United States); Mishra, Anurag [Jayne Haines Center for Pharmacogenomics, Temple University School of Pharmacy, Philadelphia, PA 19140 (United States); Krynetskiy, Evgeny, E-mail: ekrynets@temple.edu [Temple University School of Pharmacy, Philadelphia, PA 19140 (United States); Jayne Haines Center for Pharmacogenomics, Temple University School of Pharmacy, Philadelphia, PA 19140 (United States)

    2011-07-29

    Highlights: {yields} We examined the effect of glyceraldehyde 3-phosphate (GAPDH) depletion on proliferation of human carcinoma A549 cells. {yields} GAPDH depletion induces accelerated senescence in tumor cells via AMPK network, in the absence of DNA damage. {yields} Metabolic and genetic rescue experiments indicate that GAPDH has regulatory functions linking energy metabolism and cell cycle. {yields} Induction of senescence in LKB1-deficient lung cancer cells via GAPDH depletion suggests a novel strategy to control tumor cell proliferation. -- Abstract: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a pivotal glycolytic enzyme, and a signaling molecule which acts at the interface between stress factors and the cellular apoptotic machinery. Earlier, we found that knockdown of GAPDH in human carcinoma cell lines resulted in cell proliferation arrest and chemoresistance to S phase-specific cytotoxic agents. To elucidate the mechanism by which GAPDH depletion arrests cell proliferation, we examined the effect of GAPDH knockdown on human carcinoma cells A549. Our results show that GAPDH-depleted cells establish senescence phenotype, as revealed by proliferation arrest, changes in morphology, SA-{beta}-galactosidase staining, and more than 2-fold up-regulation of senescence-associated genes DEC1 and GLB1. Accelerated senescence following GAPDH depletion results from compromised glycolysis and energy crisis leading to the sustained AMPK activation via phosphorylation of {alpha} subunit at Thr172. Our findings demonstrate that GAPDH depletion switches human tumor cells to senescent phenotype via AMPK network, in the absence of DNA damage. Rescue experiments using metabolic and genetic models confirmed that GAPDH has important regulatory functions linking the energy metabolism and the cell cycle networks. Induction of senescence in LKB1-deficient non-small cell lung cancer cells via GAPDH depletion suggests a novel strategy to control tumor cell proliferation.

  20. Plasmacytoid dendritic cells promote HIV-1-induced group 3 innate lymphoid cell depletion.

    Science.gov (United States)

    Zhang, Zheng; Cheng, Liang; Zhao, Juanjuan; Li, Guangming; Zhang, Liguo; Chen, Weiwei; Nie, Weiming; Reszka-Blanco, Natalia J; Wang, Fu-Sheng; Su, Lishan

    2015-09-01

    Group 3 innate lymphoid cells (ILC3s) have demonstrated roles in promoting antibacterial immunity, maintaining epithelial barrier function, and supporting tissue repair. ILC3 alterations are associated with chronic inflammation and inflammatory disease; however, the characteristics and relevant regulatory mechanisms of this cell population in HIV-1 infection are poorly understood due in part to a lack of a robust model. Here, we determined that functional human ILC3s develop in lymphoid organs of humanized mice and that persistent HIV-1 infection in this model depletes ILC3s, as observed in chronic HIV-1-infected patients. In HIV-1-infected mice, effective antiretroviral therapy reversed the loss of ILC3s. HIV-1-dependent reduction of ILC3s required plasmacytoid dendritic cells (pDCs), IFN-I, and the CD95/FasL pathway, as targeted depletion or blockade of these prevented HIV-1-induced ILC3 depletion in vivo and in vitro, respectively. Finally, we determined that HIV-1 infection induces CD95 expression on ILC3s via a pDC- and IFN-I-dependent mechanism that sensitizes ILC3s to undergo CD95/FasL-mediated apoptosis. We conclude that chronic HIV-1 infection depletes ILC3s through pDC activation, induction of IFN-I, and CD95-mediated apoptosis.

  1. Treg-cell depletion promotes chemokine production and accumulation of CXCR3(+) conventional T cells in intestinal tumors.

    Science.gov (United States)

    Akeus, Paulina; Langenes, Veronica; Kristensen, Jonas; von Mentzer, Astrid; Sparwasser, Tim; Raghavan, Sukanya; Quiding-Järbrink, Marianne

    2015-06-01

    Colorectal cancer (CRC) is one of the most prevalent tumor types worldwide and tumor-infiltrating T cells are crucial for anti-tumor immunity. We previously demonstrated that Treg cells from CRC patients inhibit transendothelial migration of conventional T cells. However, it remains unclear if local Treg cells affect lymphocyte migration into colonic tumors. By breeding APC(Min/+) mice with depletion of regulatory T cells mice, expressing the diphtheria toxin receptor under the control of the FoxP3 promoter, we were able to selectively deplete Treg cells in tumor-bearing mice, and investigate the impact of these cells on the infiltration of conventional T cells into intestinal tumors. Short-term Treg-cell depletion led to a substantial increase in the frequencies of T cells in the tumors, attributed by both increased infiltration and proliferation of T cells in the Treg-cell-depleted tumors. We also demonstrate a selective increase of the chemokines CXCL9 and CXCL10 in Treg-cell-depleted tumors, which were accompanied by accumulation of CXCR3(+) T cells, and increased IFN-γ mRNA expression. In conclusion, Treg-cell depletion increases the accumulation of conventional T cells in intestinal tumors, and targeting Treg cells could be a possible anti-tumor immunotherapy, which not only affects T-cell effector functions, but also their recruitment to tumors.

  2. Effect of arginase II on L-arginine depletion and cell growth in murine cell lines of renal cell carcinoma

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    Patterson John R

    2008-09-01

    Full Text Available Abstract Background L-arginine is the common substrate for the two isoforms of arginase. Arginase I, highly expressed in the liver and arginase II mainly expressed in the kidney. Arginase I-producing myeloid derived suppressor cells have been shown to inhibit T-cell function by the depletion of L-arginine. On the other hand, arginase II has been detected in patients with cancer and is thought to metabolize L-arginine to L-ornithine needed to sustain rapid tumor growth; however its role in L-arginine depletion is unclear. Thus, in tumor biology, L-arginine metabolism may play a dual role in tumor growth and in the induction of T cell dysfunction. Therefore, we studied in murine renal cell carcinoma (RCC cell lines, the effect of arginase II on tumor cell proliferation and L-arginine depletion. The effect of arginase inhibitors on cell proliferation was also tested. Methods Three murine renal cell carcinoma (mRCC cell lines were tested for the presence of arginase. nor-NOHA, an arginase inhibitor was used to substantiate the effect of arginase on cell growth and L-arginine depletion. Amino acid levels were tested by HPLC. Results Our results show that mRCC cell lines express only arginase II and were able to deplete L-arginine from the medium. Cell growth was independent of the amount of arginase activity expressed by the cells. nor-NOHA significantly (P = 0.01 reduced arginase II activity and suppressed cell growth in cells exhibiting high arginase activity. The depletion of L-arginine by mRCC induced the decrease expression of CD3ζ a key element for T-cell function. Conclusion The results of this study show for the first time that arginase II produced by RCC cell lines depletes L-arginine resulting in decreased expression of CD3ζ. These results indicate that RCC cell lines expressing arginase II can modulate the L-arginine metabolic pathway to regulate both cell growth and T-cell function. Blocking arginase may lead to a decrease in RCC cell

  3. Macromolecular depletion modulates the binding of red blood cells to activated endothelial cells.

    Science.gov (United States)

    Yang, Yang; Koo, Stephanie; Lin, Cheryl Shuyi; Neu, Björn

    2010-09-01

    Adhesion of red blood cells (RBCs) to endothelial cells (ECs) is usually insignificant but an enhanced adhesion has been observed in various diseases associated with vascular complications. This abnormal adhesion under pathological conditions such as sickle cell disease has been correlated with increased levels of various plasma proteins but the detailed underlying mechanism(s) remains unclear. Usually it is assumed that the proadhesive effects of plasma proteins originate from ligand interactions cross-linking receptors on adjacent cells, but explicit results detailing binding sites or receptors for some proteins (e.g., fibrinogen) on either RBC or EC surfaces that would support this model are missing. In this study, the authors tested whether there is an alternative mechanism. Their results demonstrate that dextran 2 MDa promotes the adhesion of normal RBCs to thrombin-activated ECs and that this effect becomes more pronounced with increasing thrombin concentration or with prolonged thrombin incubation time. It is concluded that depletion interaction originating from nonadsorbing macromolecules (i.e., dextran) can modulate the adhesion of red blood cells to thrombin-activated EC. This study thereby suggests macromolecular depletion as an alternative mechanism for the adhesion-promoting effects of nonadsorbing plasma proteins. These findings should not only aid in getting a better understanding of diseases associated with vascular complications but should also have many potential applications in biomedical or biotechnological areas that require the control of cell-cell or cell surface interactions.

  4. B cell depletion inhibits spontaneous autoimmune thyroiditis in NOD.H-2h4 mice.

    Science.gov (United States)

    Yu, Shiguang; Dunn, Robert; Kehry, Marilyn R; Braley-Mullen, Helen

    2008-06-01

    B cells are important for the development of most autoimmune diseases. B cell depletion immunotherapy has emerged as an effective treatment for several human autoimmune diseases, although it is unclear whether B cells are necessary for disease induction, autoantibody production, or disease progression. To address the role of B cells in a murine model of spontaneous autoimmune thyroiditis (SAT), B cells were depleted from adult NOD.H-2h4 mice using anti-mouse CD20 mAb. Anti-CD20 depleted most B cells in peripheral blood and cervical lymph nodes and 50-80% of splenic B cells. Flow cytometry analysis showed that marginal zone B cells in the spleen were relatively resistant to depletion by anti-CD20, whereas most follicular and transitional (T2) B cells were depleted after anti-CD20 treatment. When anti-CD20 was administered before development of SAT, development of SAT and anti-mouse thyroglobulin autoantibody responses were reduced. Anti-CD20 also reduced SAT severity and inhibited further increases in anti-mouse thyroglobulin autoantibodies when administered to mice that already had autoantibodies and thyroid inflammation. The results suggest that B cells are necessary for initiation as well as progression or maintenance of SAT in NOD.H-2h4 mice.

  5. Depletion of DNMT3A Suppressed Cell Proliferation and Restored PTEN in Hepatocellular Carcinoma Cell

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    Zhujiang Zhao

    2010-01-01

    Full Text Available Promoter hypermethylation mediated by DNA methyltransferases (DNMTs is the main reason for epigenetic inactivation of tumor suppressor genes (TSGs. Previous studies showed that DNMT1 and DNMT3B play an important role in CpG island methylation in tumorigenesis. Little is known about the role of DNMT3A in this process, especially in hepatocellular carcinoma (HCC. In the present study, increased DNMT3A expression in 3 out of 6 HCC cell lines and 16/25 (64% HCC tissues implied that DNMT3A is involved in hepatocellular carcinogenesis. Depletion of DNMT3A in HCC cell line SMMC-7721 inhibited cell proliferation and decreased the colony formation (about 65%. Microarray data revealed that 153 genes were upregulated in DNMT3A knockdown cells and that almost 71% (109/153 of them contain CpG islands in their 5′ region. 13 of them including PTEN, a crucial tumor suppressor gene in HCC, are genes involved in cell cycle and cell proliferation. Demethylation of PTEN promoter was observed in DNMT3A-depleted cells implying that DNMT3A silenced PTEN via DNA methylation. These results provide insights into the mechanisms of DNMT3A to regulate TSGs by an epigenetic approach in HCC.

  6. Depletion of host cell riboflavin reduces Wolbachia levels in cultured mosquito cells.

    Science.gov (United States)

    Fallon, Ann M; Baldridge, Gerald D; Carroll, Elissa M; Kurtz, Cassandra M

    2014-09-01

    Wolbachia is an obligate intracellular alphaproteobacterium that occurs in arthropod and nematode hosts. Wolbachia presumably provides a fitness benefit to its hosts, but the basis for its retention and spread in host populations remains unclear. Wolbachia genomes retain biosynthetic pathways for some vitamins, and the possibility that these vitamins benefit host cells provides a potential means of selecting for Wolbachia-infected cell lines. To explore whether riboflavin produced by Wolbachia is available to its host cell, we established that growth of uninfected C7-10 mosquito cells decreases in riboflavin-depleted culture medium. A well-studied inhibitor of riboflavin uptake, lumiflavin, further inhibits growth of uninfected C7-10 cells with an LC50 of approximately 12 μg/ml. Growth of C/wStr1 mosquito cells, infected with Wolbachia from the planthopper, Laodelphax striatellus, was enhanced in medium containing low levels of lumiflavin, but Wolbachia levels decreased. Lumiflavin-enhanced growth thus resembled the improved growth that accompanies treatment with antibiotics that deplete Wolbachia, rather than a metabolic advantage provided by the Wolbachia infection. We used the polymerase chain reaction to validate the decrease in Wolbachia abundance and evaluated our results in the context of a proteomic analysis in which we detected nearly 800 wStr proteins. Our data indicate that Wolbachia converts riboflavin to FMN and FAD for its own metabolic needs, and does not provide a source of riboflavin for its host cell.

  7. Depletion of dendritic cells enhances innate anti-bacterial host defense through modulation of phagocyte homeostasis.

    Directory of Open Access Journals (Sweden)

    Stella E Autenrieth

    2012-02-01

    Full Text Available Dendritic cells (DCs as professional antigen-presenting cells play an important role in the initiation and modulation of the adaptive immune response. However, their role in the innate immune response against bacterial infections is not completely defined. Here we have analyzed the role of DCs and their impact on the innate anti-bacterial host defense in an experimental infection model of Yersinia enterocolitica (Ye. We used CD11c-diphtheria toxin (DT mice to deplete DCs prior to severe infection with Ye. DC depletion significantly increased animal survival after Ye infection. The bacterial load in the spleen of DC-depleted mice was significantly lower than that of control mice throughout the infection. DC depletion was accompanied by an increase in the serum levels of CXCL1, G-CSF, IL-1α, and CCL2 and an increase in the numbers of splenic phagocytes. Functionally, splenocytes from DC-depleted mice exhibited an increased bacterial killing capacity compared to splenocytes from control mice. Cellular studies further showed that this was due to an increased production of reactive oxygen species (ROS by neutrophils. Adoptive transfer of neutrophils from DC-depleted mice into control mice prior to Ye infection reduced the bacterial load to the level of Ye-infected DC-depleted mice, suggesting that the increased number of phagocytes with additional ROS production account for the decreased bacterial load. Furthermore, after incubation with serum from DC-depleted mice splenocytes from control mice increased their bacterial killing capacity, most likely due to enhanced ROS production by neutrophils, indicating that serum factors from DC-depleted mice account for this effect. In summary, we could show that DC depletion triggers phagocyte accumulation in the spleen and enhances their anti-bacterial killing capacity upon bacterial infection.

  8. The interaction of manganese nanoparticles with PC-12 cells induces dopamine depletion.

    Science.gov (United States)

    Hussain, Saber M; Javorina, Amanda K; Schrand, Amanda M; Duhart, Helen M; Ali, Syed F; Schlager, John J

    2006-08-01

    This investigation was designed to determine whether nano-sized manganese oxide (Mn-40 nm) particles would induce dopamine (DA) depletion in a cultured neuronal phenotype, PC-12 cells, similar to free ionic manganese (Mn(2+)). Cells were exposed to Mn-40 nm, Mn(2+) (acetate), or known cytotoxic silver nanoparticles (Ag-15 nm) for 24 h. Phase-contrast microscopy studies show that Mn-40 nm or Mn(2+) exposure did not greatly change morphology of PC-12 cells. However, Ag-15 nm and AgNO(3) produce cell shrinkage and irregular membrane borders compared to control cells. Further microscopic studies at higher resolution demonstrated that Mn-40 nm nanoparticles and agglomerates were effectively internalized by PC-12 cells. Mitochondrial reduction activity, a sensitive measure of particle and metal cytotoxicity, showed only moderate toxicity for Mn-40 nm compared to similar Ag-15 nm and Mn(2+) doses. Mn-40 nm and Mn(2+) dose dependently depleted DA and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), while Ag-15 nm only significantly reduced DA and DOPAC at concentrations of 50 mug/ml. Therefore, the DA depletion of Mn-40 nm was most similar to Mn(2+), which is known to induce concentration-dependent DA depletion. There was a significant increase (> 10-fold) in reactive oxygen species (ROS) with Mn-40 nm exposure, suggesting that increased ROS levels may participate in DA depletion. These results clearly demonstrate that nanoscale manganese can deplete DA, DOPAC, and HVA in a dose-dependent manner. Further study is required to evaluate the specific intracellular distribution of Mn-40 nm nanoparticles, metal dissolution rates in cells and cellular matrices, if DA depletion is induced in vivo, and the propensity of Mn nanoparticles to cross the blood-brain barrier or be selectively uptaken by nasal epithelium.

  9. Germinal center B cell depletion diminishes CD4+ follicular T helper cells in autoimmune mice.

    Directory of Open Access Journals (Sweden)

    Isharat Yusuf

    Full Text Available BACKGROUND: Continuous support from follicular CD4(+ T helper (Tfh cells drives germinal center (GC responses, which last for several weeks to produce high affinity memory B cells and plasma cells. In autoimmune Sle1 and NZB/W F1 mice, elevated numbers of Tfh cells persist, promoting the expansion of self-reactive B cells. Expansion of circulating Tfh like cells have also been described in several autoimmune diseases. Although, the signals required for Tfh differentiation have now been well described, the mechanisms that sustain the maintenance of fully differentiated Tfh are less understood. Recent data demonstrate a role for GC B cells for Tfh maintenance after protein immunization. METHODS AND FINDING: Given the pathogenic role Tfh play in autoimmune disease, we explored whether B cells are required for maintenance of autoreactive Tfh. Our data suggest that the number of mature autoreactive Tfh cells is controlled by GC B cells. Depletion of B cells in Sle1 autoimmune mice leads to a dramatic reduction in Tfh cells. In NZB/W F1 autoimmune mice, similar to the SRBC immunization model, GC B cells support the maintenance of mature Tfh, which is dependent mainly on ICOS. The CD28-associated pathway is dispensable for Tfh maintenance in SRBC immunized mice, but is required in the spontaneous NZB/W F1 model. CONCLUSION: These data suggest that mature Tfh cells require signals from GC B cells to sustain their optimal numbers and function in both autoimmune and immunization models. Thus, immunotherapies targeting B cells in autoimmune disease may affect pathogenic Tfh cells.

  10. Cumulative Doses of T-Cell Depleting Antibody and Cancer Risk after Kidney Transplantation.

    Directory of Open Access Journals (Sweden)

    Jenny H C Chen

    Full Text Available T-cell depleting antibody is associated with an increased risk of cancer after kidney transplantation, but a dose-dependent relationship has not been established. This study aimed to determine the association between cumulative doses of T-cell depleting antibody and the risk of cancer after kidney transplantation. Using data from the Australian and New Zealand Dialysis and Transplant Registry between 1997-2012, we assessed the risk of incident cancer and cumulative doses of T-cell depleting antibody using adjusted Cox regression models. Of the 503 kidney transplant recipients with 2835 person-years of follow-up, 276 (55%, 209 (41% and 18 (4% patients received T-cell depleting antibody for induction, rejection or induction and rejection respectively. The overall cancer incidence rate was 1,118 cancers per 100,000 patient-years, with 975, 1093 and 1377 cancers per 100,000 patient-years among those who had received 1-5 doses, 6-10 doses and >10 doses, respectively. There was no association between cumulative doses of T cell depleting antibody and risk of incident cancer (1-5: referent, 6-10: adjusted hazard ratio (HR 1.19, 95%CI 0.48-2.95, >10: HR 1.42, 95%CI 0.50-4.02, p = 0.801. This lack of association is contradictory to our hypothesis and is likely attributed to the low event rates resulting in insufficient power to detect significant differences.

  11. In vivo T cell depletion regulates resistance and morbidity in murine schistosomiasis

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    Phillips, S.M.; Linette, G.P.; Doughty, B.L.; Byram, J.E.; Von Lichtenberg, F.

    1987-08-01

    These studies assessed the roles of subpopulations of T lymphocytes in inducing and modulating resistance to schistosomiasis and thereby influencing subsequent morbidity. C57BL/6 mice were depleted in vivo of Lyt-1+, Lyt-2+, and L3T4+ cells by the daily administration of monoclonal antibodies. The development of protective immunity, induced by exposure to irradiated Schistosoma mansoni cercariae as expressed in depleted animals, was compared to that demonstrated in undepleted, normal, and congenitally athymic C57BL/6 mice. The development of morbidity was determined by spleen weight, portal pressure and reticuloendothelial system activity. The results indicated that depletion of specific subpopulations of T lymphocytes minimally affected the primary development of parasites; however, depletion strongly influenced the development of resistance to the parasite and subsequent morbidity due to infection. Depletion of T lymphocytes by anti-Lyt-1+ or anti-L3T4+ antibody decreased the development of resistance, antibody and delayed-type hypersensitivity directed against schistosome antigens. Morbidity due to disease was increased. Depletion of Lyt-2+ cells produced opposite changes with augmented resistance and reduced morbidity. Congenitally athymic mice developed minimal resistance and morbidity. Moreover, resistance was inversely related to the morbidity shown by a given animal. These studies indicate that the development of protective immunity to S. mansoni cercariae is regulated by discrete subpopulations of T lymphocytes. The feasibility of decreasing morbidity by increasing specific immunologically mediated resistance is suggested.

  12. Depletion of the AP-1 repressor JDP2 induces cell death similar to apoptosis

    DEFF Research Database (Denmark)

    Lerdrup, Mads; Holmberg, Christian Henrik; Dietrich, Nikolaj;

    2005-01-01

    depletion of JDP2 resulted in p53-independent cell death that resembles apoptosis and was evident at 72 h. The death mechanism was caspase dependent as the cells could be rescued by treatment with caspase inhibitor zVAD. Our studies suggest that JDP2 functions as a general survival protein, not only...

  13. Optimising B-cell depletion in autoimmune disease: is obinutuzumab the answer?

    Science.gov (United States)

    Reddy, Venkat; Dahal, Lekh N; Cragg, Mark S; Leandro, Maria

    2016-08-01

    In Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE), B-cell depletion therapy using rituximab results in variable clinical responses between individuals, which likely relates to variable B-cell depletion in the presence of immune defects. Outcomes in clinical trials with other type I anti-CD20 mAbs, ocrelizumab and ofatumumab, are comparable to rituximab. A mechanistically different type II mAb, obinutuzumab (OBZ), with greater capacity for B-cell depletion, has recently entered clinical trials in SLE. Here we consider whether type II anti-CD20 mAbs will provide mechanistic advantages to overcome the disease-related immune defects in autoimmune diseases such as SLE.

  14. Mesenchymal dental pulp cells attenuate dentin resorption in homeostasis.

    Science.gov (United States)

    Zheng, Y; Chen, M; He, L; Marão, H F; Sun, D M; Zhou, J; Kim, S G; Song, S; Wang, S L; Mao, J J

    2015-06-01

    Dentin in permanent teeth rarely undergoes resorption in development, homeostasis, or aging, in contrast to bone that undergoes periodic resorption/remodeling. The authors hypothesized that cells in the mesenchymal compartment of dental pulp attenuate osteoclastogenesis. Mononucleated and adherent cells from donor-matched rat dental pulp (dental pulp cells [DPCs]) and alveolar bone (alveolar bone cells [ABCs]) were isolated and separately cocultured with primary rat splenocytes. Primary splenocytes readily aggregated and formed osteoclast-like cells in chemically defined osteoclastogenesis medium with 20 ng/mL of macrophage colony-stimulating factor (M-CSF) and 50 ng/mL of receptor activator of nuclear factor κB ligand (RANKL). Strikingly, DPCs attenuated osteoclastogenesis when cocultured with primary splenocytes, whereas ABCs slightly but significantly promoted osteoclastogenesis. DPCs yielded ~20-fold lower RANKL expression but >2-fold higher osteoprotegerin (OPG) expression than donor-matched ABCs, yielding a RANKL/OPG ratio of 41:1 (ABCs:DPCs). Vitamin D3 significantly promoted RANKL expression in ABCs and OPG in DPCs. In vivo, rat maxillary incisors were atraumatically extracted (without any tooth fractures), followed by retrograde pulpectomy to remove DPCs and immediate replantation into the extraction sockets to allow repopulation of the surgically treated root canal with periodontal and alveolar bone-derived cells. After 8 wk, multiple dentin/root resorption lacunae were present in root dentin with robust RANKL and OPG expression. There were areas of dentin resoprtion alternating with areas of osteodentin formation in root dentin surface in the observed 8 wk. These findings suggest that DPCs of the mesenchymal compartment have an innate ability to attenuate osteoclastogenesis and that this innate ability may be responsible for the absence of dentin resorption in homeostasis. Mesenchymal attenuation of dentin resorption may have implications in internal

  15. Inhibition of autophagy augments apoptosis in human oral squamous cell carcinoma under nutrient depletion.

    Science.gov (United States)

    Jiang, Li-Cheng; Xin, Zhi-Yuan; Deborah, Baremberg; Zhang, Jun-Sheng; Yuan, Dao-Ying; Xu, Kai; Liu, Xian-Bin; Jiang, Hu-Quan; Fan, Qing-Chun; Zhang, Bin; Li, Ke-Yi

    2015-05-01

    There has been little research conducted regarding autophagy in oral squamous cell carcinoma (OSCC). Given the prevalence of oral cancers which are OSCC and the severe side effects of current treatments, there is a pressing need to develop effective alternative therapies. In this study, we have endeavored to explore the biological characteristics of oral squamous cell carcinoma cell line KB cells, in particular with regard to the role played by autophagy in their survival. Autophagy was activated by nutrient depletion via culturing cells in Earle's balanced salts (EBSS) and was measured via indices relating to Beclin 1, microtubule-associated protein light chain 3 (MAPLC3, LC3), p62, and Green fluorescent protein-light chain 3 plasmid transfection (GFP-LC3). Cell death and apoptosis induced by nutrient depletion was measured using both MTT assay and flow cytometry (FCM). Compared to initial levels at 0 h, Beclin 1 density in EBSS-treated cells was found to have increased at 6, 12, and 18 h in a time-dependent manner and was found to have subsequently declined at 24 and 48 h. p62 levels, LC3-II/LC3-I ratio, and GFP-LC3 levels increased at 6, 12, 18, 24, and 48 h in a time-dependent manner. 3-methyladenine (3-MA) was found to inhibit autophagy and the expression of Beclin 1 and significantly enhanced nutrient depletion-induced apoptosis and death. We concluded that nutrient depletion enhances OSCC cell autophagy in time-course patterns and that the inhibition of autophagy augments apoptosis in OSCC cells. We also deduced that Beclin 1 takes part in the development and progression of autophagy, potentially playing an important role in the crosstalk between apoptosis and autophagy in OSCC cells. These findings suggest that nutrient depletion may be an effective way to explore autophagy and that autophagy inhibitors should be investigated as a potential novel agent for the adjuvant treatment of human OSCC.

  16. Stem cell depletion in Hutchinson-Gilford progeria syndrome.

    Science.gov (United States)

    Rosengardten, Ylva; McKenna, Tomás; Grochová, Diana; Eriksson, Maria

    2011-12-01

    Hutchinson-Gilford progeria syndrome (HGPS or progeria) is a very rare genetic disorder with clinical features suggestive of premature aging. Here, we show that induced expression of the most common HGPS mutation (LMNA c.1824C>T, p.G608G) results in a decreased epidermal population of adult stem cells and impaired wound healing in mice. Isolation and growth of primary keratinocytes from these mice demonstrated a reduced proliferative potential and ability to form colonies. Downregulation of the epidermal stem cell maintenance protein p63 with accompanying activation of DNA repair and premature senescence was the probable cause of this loss of adult stem cells. Additionally, upregulation of multiple genes in major inflammatory pathways indicated an activated inflammatory response. This response has also been associated with normal aging, emphasizing the importance of studying progeria to increase the understanding of the normal aging process.

  17. The telomerase inhibitor imetelstat depletes cancer stem cells in breast and pancreatic cancer cell lines.

    Science.gov (United States)

    Joseph, Immanual; Tressler, Robert; Bassett, Ekaterina; Harley, Calvin; Buseman, Christen M; Pattamatta, Preeti; Wright, Woodring E; Shay, Jerry W; Go, Ning F

    2010-11-15

    Cancer stem cells (CSC) are rare drug-resistant cancer cell subsets proposed to be responsible for the maintenance and recurrence of cancer and metastasis. Telomerase is constitutively active in both bulk tumor cell and CSC populations but has only limited expression in normal tissues. Thus, inhibition of telomerase has been shown to be a viable approach in controlling cancer growth in nonclinical studies and is currently in phase II clinical trials. In this study, we investigated the effects of imetelstat (GRN163L), a potent telomerase inhibitor, on both the bulk cancer cells and putative CSCs. When breast and pancreatic cancer cell lines were treated with imetelstat in vitro, telomerase activity in the bulk tumor cells and CSC subpopulations were inhibited. Additionally, imetelstat treatment reduced the CSC fractions present in the breast and pancreatic cell lines. In vitro treatment with imetelstat, but not control oligonucleotides, also reduced the proliferation and self-renewal potential of MCF7 mammospheres and resulted in cell death after imetelstat, suggesting a mechanism of action independent of telomere shortening for the effects of imetelstat on the CSC subpopulations. Our results suggest that imetelstat-mediated depletion of CSCs may offer an alternative mechanism by which telomerase inhibition may be exploited for cancer therapy.

  18. Anti-CD20 B-cell depletion enhances monocyte reactivity in neuroimmunological disorders

    Directory of Open Access Journals (Sweden)

    Hohlfeld Reinhard

    2011-10-01

    Full Text Available Abstract Background Clinical trials evaluating anti-CD20-mediated B-cell depletion in multiple sclerosis (MS and neuromyelitis optica (NMO generated encouraging results. Our recent studies in the MS model experimental autoimmune encephalomyelitis (EAE attributed clinical benefit to extinction of activated B-cells, but cautioned that depletion of naïve B-cells may be undesirable. We elucidated the regulatory role of un-activated B-cells in EAE and investigated whether anti-CD20 may collaterally diminish regulatory B-cell properties in treatment of neuroimmunological disorders. Methods Myelin oligodendrocyte glycoprotein (MOG peptide-immunized C57Bl/6 mice were depleted of B-cells. Functional consequences for regulatory T-cells (Treg and cytokine production of CD11b+ antigen presenting cells (APC were assessed. Peripheral blood mononuclear cells from 22 patients receiving anti-CD20 and 23 untreated neuroimmunological patients were evaluated for frequencies of B-cells, T-cells and monocytes; monocytic reactivity was determined by TNF-production and expression of signalling lymphocytic activation molecule (SLAM. Results We observed that EAE-exacerbation upon depletion of un-activated B-cells closely correlated with an enhanced production of pro-inflammatory TNF by CD11b+ APC. Paralleling this pre-clinical finding, anti-CD20 treatment of human neuroimmunological disorders increased the relative frequency of monocytes and accentuated pro-inflammatory monocyte function; when reactivated ex vivo, a higher frequency of monocytes from B-cell depleted patients produced TNF and expressed the activation marker SLAM. Conclusions These data suggest that in neuroimmunological disorders, pro-inflammatory APC activity is controlled by a subset of B-cells which is eliminated concomitantly upon anti-CD20 treatment. While this observation does not conflict with the general concept of B-cell depletion in human autoimmunity, it implies that its safety and

  19. Lignin depletion enhances the digestibility of cellulose in cultured xylem cells.

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    Catherine I Lacayo

    Full Text Available Plant lignocellulose constitutes an abundant and sustainable source of polysaccharides that can be converted into biofuels. However, the enzymatic digestion of native plant cell walls is inefficient, presenting a considerable barrier to cost-effective biofuel production. In addition to the insolubility of cellulose and hemicellulose, the tight association of lignin with these polysaccharides intensifies the problem of cell wall recalcitrance. To determine the extent to which lignin influences the enzymatic digestion of cellulose, specifically in secondary walls that contain the majority of cellulose and lignin in plants, we used a model system consisting of cultured xylem cells from Zinniaelegans. Rather than using purified cell wall substrates or plant tissue, we have applied this system to study cell wall degradation because it predominantly consists of homogeneous populations of single cells exhibiting large deposits of lignocellulose. We depleted lignin in these cells by treating with an oxidative chemical or by inhibiting lignin biosynthesis, and then examined the resulting cellulose digestibility and accessibility using a fluorescent cellulose-binding probe. Following cellulase digestion, we measured a significant decrease in relative cellulose content in lignin-depleted cells, whereas cells with intact lignin remained essentially unaltered. We also observed a significant increase in probe binding after lignin depletion, indicating that decreased lignin levels improve cellulose accessibility. These results indicate that lignin depletion considerably enhances the digestibility of cellulose in the cell wall by increasing the susceptibility of cellulose to enzymatic attack. Although other wall components are likely to contribute, our quantitative study exploits cultured Zinnia xylem cells to demonstrate the dominant influence of lignin on the enzymatic digestion of the cell wall. This system is simple enough for quantitative image analysis

  20. Cyclin D1 depletion induces DNA damage in mantle cell lymphoma lines.

    Science.gov (United States)

    Mohanty, Suchismita; Mohanty, Atish; Sandoval, Natalie; Tran, Thai; Bedell, Victoria; Wu, Jun; Scuto, Anna; Murata-Collins, Joyce; Weisenburger, Dennis D; Ngo, Vu N

    2017-03-01

    Elevated cyclin D1 (CCND1) expression levels in mantle cell lymphoma (MCL) are associated with aggressive clinical manifestations related to chemoresistance, but little is known about how this important proto-oncogene contributes to the resistance of MCL. Here, we showed that RNA interference-mediated depletion of CCND1 increased caspase-3 activities and induced apoptosis in the human MCL lines UPN-1 and JEKO-1. In vitro and xenotransplant studies revealed that the toxic effect of CCND1 depletion in MCL cells was likely due to increase in histone H2AX phosphorylation, a DNA damage marker. DNA fiber analysis suggested deregulated replication initiation after CCND1 depletion as a potential cause of DNA damage. Finally, in contrast to depletion or inhibition of cyclin-dependent kinase 4, CCND1 depletion increased chemosensitivity of MCL cells to replication inhibitors hydroxyurea and cytarabine. Our findings have an important implication for CCND1 as a potential therapeutic target in MCL patients who are refractory to standard chemotherapy.

  1. Troxerutin protects against 2,2',4,4'-tetrabromodiphenyl ether (BDE-47)-induced liver inflammation by attenuating oxidative stress-mediated NAD⁺-depletion.

    Science.gov (United States)

    Zhang, Zi-Feng; Zhang, Yan-Qiu; Fan, Shao-Hua; Zhuang, Juan; Zheng, Yuan-Lin; Lu, Jun; Wu, Dong-Mei; Shan, Qun; Hu, Bin

    2015-01-01

    Emerging evidence indicates that 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) induces liver injury through enhanced ROS production and lymphocytic infiltration, which may promote a liver inflammatory response. Antioxidants have been reported to attenuate the cellular toxicity associated with polybrominated diphenyl ethers (PBDEs). In this study, we investigated the effect of troxerutin, a trihydroxyethylated derivative of the natural bioflavonoid rutin, on BDE-47-induced liver inflammation and explored the potential mechanisms underlying this effect. Our results showed that NAD(+)-depletion was involved in the oxidative stress-mediated liver injury in a BDE-47 treated mouse model, which was confirmed by Vitamin E treatment. Furthermore, our data revealed that troxerutin effectively alleviated liver inflammation by mitigating oxidative stress-mediated NAD(+)-depletion in BDE-47 treated mice. Consequently, troxerutin remarkably restored SirT1 protein expression and activity in the livers of BDE-47-treated mice. Mechanistically, troxerutin dramatically repressed the nuclear translocation of NF-κB p65 and the acetylation of NF-κB p65 (Lys 310) and Histone H3 (Lys9) to abate the transcription of inflammatory genes in BDE-47-treated mouse livers. These inhibitory effects of troxerutin were markedly blunted by EX527 (SirT1 inhibitor) treatment. This study provides novel mechanistic insights into the toxicity of BDE-47 and indicates that troxerutin might be used in the prevention and therapy of BDE-47-induced hepatotoxicity.

  2. Blocking TNF-α inhibits angiogenesis and growth of IFIT2-depleted metastatic oral squamous cell carcinoma cells.

    Science.gov (United States)

    Lai, Kuo-Chu; Liu, Chung-Ji; Lin, Tsung-Jen; Mar, Ai-Chung; Wang, Hsiu-Hua; Chen, Chi-Wei; Hong, Zi-Xuan; Lee, Te-Chang

    2016-01-28

    Our previous study demonstrated that the depletion of interferon-induced protein with tetratricopeptide repeats 2 (IFIT2) promoted metastasis and was associated with a poor prognosis in patients with oral squamous cell carcinoma (OSCC). Our current study explores the major downstream signaling involved in IFIT2 depletion-induced OSCC metastasis. To this end, we used two cell lines (designated sh-control-xeno and sh-IFIT2-xeno) derived from human OSCC xenografts expressing sh-control and sh-IFIT2, respectively, and one metastatic OSCC subline (sh-IFIT2-meta) from an IFIT2-depleted metastatic tumor. We found that the sh-IFIT2-meta cells proliferated more slowly than the sh-control-xeno cells but exhibited higher migration and chemoresistance. Using microarray technology and Ingenuity Pathway Analysis, we found that TNF-α was one of the major downstream targets in IFIT2-depleted OSCC cells. Quantitative real-time PCR, western blotting, and ELISA results confirmed that TNF-α was upregulated in the sh-IFIT2-meta cells. Blocking TNF-α abolished the angiogenic activity induced by the sh-IFIT2-meta cells. Furthermore, the human-specific TNF-α antibody golimumab significantly inhibited in vivo angiogenesis, tumor growth and metastasis of sh-IFIT2-meta cells. These results demonstrate that IFIT2 depletion results in TNF-α upregulation, leading to angiogenesis and metastasis of OSCC cells.

  3. B Cell Depletion: Rituximab in Glomerular Disease and Transplantation

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    S. Marinaki

    2013-12-01

    Full Text Available B cells play a central role in the pathogenesis of many autoimmune diseases. Selective targeting can be achieved with the use of the monoclonal antibody rituximab. In addition to being a drug for non-Hodgkin's lymphoma, rituximab is also an FDA-approved treatment for refractory rheumatoid arthritis and, since recently, ANCA vasculitis. It has shown efficacy in many autoimmune diseases. This review will discuss current evidence and the rationale of the use of rituximab in glomerular diseases, including randomized controlled trials. The focus will be on the use of rituximab in idiopathic membranous nephropathy, systemic lupus erythematosus and ANCA-associated vasculitis. The emerging role of rituximab in renal transplantation, where it seems to be important for the desensitization protocols for highly sensitized patients as well as for the preconditioning of ABO-incompatible recipients and the treatment of antibody-mediated rejection, will also be addressed.

  4. Kupffer cell depletion with liposomal clodronate prevents suppression of Ntcp expression in endotoxin-treated rats

    NARCIS (Netherlands)

    Sturm, E; Havinga, R; Baller, JFW; Wolters, H; van Rooijen, N; Kamps, JAAM; Verkade, HJ; Karpen, SJ; Kuipers, F

    2005-01-01

    Background/Aims: In sepsis-associated cholestasis, expression of many genes involved in bile acid transport, including Ntcp, is suppressed by cytokines. Kupffer cells (KC) are an important source of cytokines in sepsis. To assess the consequences of KC depletion on hepatic Ntcp expression in endotox

  5. Radiation-Released Histamine in the Rhesus Monkey as Modified by Mast Cell Depletion and Antihistamine

    Science.gov (United States)

    1975-06-01

    mast cell histamine depleter, compound 48/80 (1mg/kg per day) for four consecutive days and then irradiated (4000 rads), histamine concentrations did not change significantly. When 48/80 was given 20 min after irradiation, histamine concentrations changed from 18 + or - 2 ng/ml to a maximum of 35 + or - 9 ng/ml after 48/80 injection.

  6. Immunity to Schistosoma mansoni in congenitally athymic, irradiated and mast cell-depleted rats

    Energy Technology Data Exchange (ETDEWEB)

    Ford, M.J.; Bickle, Q.D.; Taylor, M.G.

    1987-04-01

    Immunity to Schistosoma mansoni was investigated in congenitally athymic (Nu/Nu) rats, irradiated rats and in mast cell-depleted rats. Nu/Nu rats failed to develop significant resistance following vaccination with irradiated cercariae, although Nu/Nu recipients of serum from vaccinated Fischer rats (VRS) manifested resistance comparable to heterozygous controls, suggesting that T-cells were required in the induction of resistance but were not involved in the efferent arm of antibody-dependent elimination. Radiosensitive cells (including eosinophils, basophils, neutrophils, lymphocytes and mast cells) were apparently not essential for the antibody-dependent elimination of lung or post-lung stages since irradiated (700-750 rad.) recipients of VRS manifested comparable degrees of resistance to unirradiated controls in spite of a greater than 85% reduction in total blood leucocyte counts after irradiation. Depletion of 99% of tissue mast cells by treatment of rats with Compound 48/80 had no significant effect on the attrition of a challenge infection in rats rendered immune by vaccination with irradiated cercariae or by transfer of VRS. However, there was a significant increase in worm recovery in unimmunized and mast cell-depleted or irradiated rats, indicating that mast cells and perhaps other radio-isotope sensitive cells may be involved in innate resistance.

  7. PTPN2 attenuates T-cell lymphopenia-induced proliferation

    Science.gov (United States)

    Wiede, Florian; La Gruta, Nicole L.; Tiganis, Tony

    2014-01-01

    When the peripheral T-cell pool is depleted, T cells undergo homoeostatic expansion. This expansion is reliant on the recognition of self-antigens and/or cytokines, in particular interleukin-7. The T cell-intrinsic mechanisms that prevent excessive homoeostatic T-cell responses and consequent overt autoreactivity remain poorly defined. Here we show that protein tyrosine phosphatase N2 (PTPN2) is elevated in naive T cells leaving the thymus to restrict homoeostatic T-cell proliferation and prevent excess responses to self-antigens in the periphery. PTPN2-deficient CD8+ T cells undergo rapid lymphopenia-induced proliferation (LIP) when transferred into lymphopenic hosts and acquire the characteristics of antigen-experienced effector T cells. The enhanced LIP is attributed to elevated T-cell receptor-dependent, but not interleukin-7-dependent responses, results in a skewed T-cell receptor repertoire and the development of autoimmunity. Our results identify a major mechanism by which homoeostatic T-cell responses are tuned to prevent the development of autoimmune and inflammatory disorders.

  8. In utero depletion of fetal hematopoietic stem cells improves engraftment after neonatal transplantation in mice.

    Science.gov (United States)

    Derderian, S Christopher; Togarrati, P Priya; King, Charmin; Moradi, Patriss W; Reynaud, Damien; Czechowicz, Agnieszka; Weissman, Irving L; MacKenzie, Tippi C

    2014-08-07

    Although in utero hematopoietic cell transplantation is a promising strategy to treat congenital hematopoietic disorders, levels of engraftment have not been therapeutic for diseases in which donor cells have no survival advantage. We used an antibody against the murine c-Kit receptor (ACK2) to deplete fetal host hematopoietic stem cells (HSCs) and increase space within the hematopoietic niche for donor cell engraftment. Fetal mice were injected with ACK2 on embryonic days 13.5 to 14.5 and surviving pups were transplanted with congenic hematopoietic cells on day of life 1. Low-dose ACK2 treatment effectively depleted HSCs within the bone marrow with minimal toxicity and the antibody was cleared from the serum before the neonatal transplantation. Chimerism levels were significantly higher in treated pups than in controls; both myeloid and lymphoid cell chimerism increased because of higher engraftment of HSCs in the bone marrow. To test the strategy of repeated HSC depletion and transplantation, some mice were treated with ACK2 postnatally, but the increase in engraftment was lower than that seen with prenatal treatment. We demonstrate a successful fetal conditioning strategy associated with minimal toxicity. Such strategies could be used to achieve clinically relevant levels of engraftment to treat congenital stem cell disorders.

  9. Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors

    Directory of Open Access Journals (Sweden)

    Toyoaki Natsume

    2016-04-01

    Full Text Available Studying the role of essential proteins is dependent upon a method for rapid inactivation, in order to study the immediate phenotypic consequences. Auxin-inducible degron (AID technology allows rapid depletion of proteins in animal cells and fungi, but its application to human cells has been limited by the difficulties of tagging endogenous proteins. We have developed a simple and scalable CRISPR/Cas-based method to tag endogenous proteins in human HCT116 and mouse embryonic stem (ES cells by using donor constructs that harbor synthetic short homology arms. Using a combination of AID tagging with CRISPR/Cas, we have generated conditional alleles of essential nuclear and cytoplasmic proteins in HCT116 cells, which can then be depleted very rapidly after the addition of auxin to the culture medium. This approach should greatly facilitate the functional analysis of essential proteins, particularly those of previously unknown function.

  10. Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors.

    Science.gov (United States)

    Natsume, Toyoaki; Kiyomitsu, Tomomi; Saga, Yumiko; Kanemaki, Masato T

    2016-04-01

    Studying the role of essential proteins is dependent upon a method for rapid inactivation, in order to study the immediate phenotypic consequences. Auxin-inducible degron (AID) technology allows rapid depletion of proteins in animal cells and fungi, but its application to human cells has been limited by the difficulties of tagging endogenous proteins. We have developed a simple and scalable CRISPR/Cas-based method to tag endogenous proteins in human HCT116 and mouse embryonic stem (ES) cells by using donor constructs that harbor synthetic short homology arms. Using a combination of AID tagging with CRISPR/Cas, we have generated conditional alleles of essential nuclear and cytoplasmic proteins in HCT116 cells, which can then be depleted very rapidly after the addition of auxin to the culture medium. This approach should greatly facilitate the functional analysis of essential proteins, particularly those of previously unknown function.

  11. Intrathecal anti-CD20 efficiently depletes meningeal B cells in CNS autoimmunity

    Science.gov (United States)

    Lehmann-Horn, Klaus; Kinzel, Silke; Feldmann, Linda; Radelfahr, Florentine; Hemmer, Bernhard; Traffehn, Sarah; Bernard, Claude C A; Stadelmann, Christine; Brück, Wolfgang; Weber, Martin S

    2014-01-01

    Clinical trials revealed that systemic administration of B-cell-depleting anti-CD20 antibodies can hold lesion formation in the early relapsing-remitting phase of multiple sclerosis (MS). Throughout the secondary-progressive (SP) course of MS, pathogenic B cells may, however, progressively replicate within the central nervous system (CNS) itself, which is largely inaccessible to systemic anti-CD20 treatment. Utilizing the murine MS model of experimental autoimmune encephalomyelitis, we show that intrathecal (i.t.) administration of anti-CD20 alone very efficiently depletes meningeal B cells from established CNS lesions. In SP-MS patients, adding i.t. administration of anti-CD20 to its systemic use may be a valuable strategy to target pathogenic B-cell function. PMID:25356419

  12. Disruption of microtubule network rescues aberrant actin comets in dynamin2-depleted cells.

    Directory of Open Access Journals (Sweden)

    Yuji Henmi

    Full Text Available A large GTPase dynamin, which is required for endocytic vesicle formation, regulates the actin cytoskeleton through its interaction with cortactin. Dynamin2 mutants impair the formation of actin comets, which are induced by Listeria monocytogenes or phosphatidylinositol-4-phosphate 5-kinase. However, the role of dynamin2 in the regulation of the actin comet is still unclear. Here we show that aberrant actin comets in dynamin2-depleted cells were rescued by disrupting of microtubule networks. Depletion of dynamin2, but not cortactin, significantly reduced the length and the speed of actin comets induced by Listeria. This implies that dynamin2 may regulate the actin comet in a cortactin-independent manner. As dynamin regulates microtubules, we investigated whether perturbation of microtubules would rescue actin comet formation in dynamin2-depleted cells. Treatment with taxol or colchicine created a microtubule-free space in the cytoplasm, and made no difference between control and dynamin2 siRNA cells. This suggests that the alteration of microtubules by dynamin2 depletion reduced the length and the speed of the actin comet.

  13. The depletion of Interleukin-8 causes cell cycle arrest and increases the efficacy of docetaxel in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Shao, Nan [Breast Disease Center, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou (China); Chen, Liu-Hua [Department of Minimally Invasive Surgery Center, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou (China); Ye, Run-Yi [Breast Disease Center, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou (China); Lin, Ying, E-mail: frostlin@hotmail.com [Breast Disease Center, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou (China); Wang, Shen-Ming, E-mail: shenmingwang@hotmail.com [Breast Disease Center, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou (China)

    2013-02-15

    Highlights: ► IL-8 depletion affects cell cycle distribution. ► Intrinsic IL-8 mediates breast cancer cell migration and invasion. ► IL-8 siRNA down regulates key factors that control survival and metastatic pathway. ► IL-8 depletion reduces integrin β3 expression. ► IL-8 depletion increases the chemosensitivity to docetaxel. -- Abstract: IL-8 is a multi-functional pro-inflammatory chemokine, which is highly expressed in cancers, such as ER-negative breast cancer. The present study demonstrates the pervasive role of IL-8 in the malignant progression of ER-negative breast cancer. IL-8 siRNA inhibited proliferation and delayed the G1 to S cell cycle progression in MDA-MB-231 and BT549 cells. IL-8 silencing resulted in the upregulation of the CDK inhibitor p27, the downregulation of cyclin D1, and the reduction of phosphorylated-Akt and NF-κB activities. IL-8 depletion also increased the chemosensitivity to docetaxel. These results indicate a role for IL-8 in promoting tumor cell survival and resistance to docetaxel and highlight the potential therapeutic significance of IL-8 depletion in ER-negative breast cancer patients.

  14. Targeted Type 2 Alveolar Cell Depletion. A Dynamic Functional Model for Lung Injury Repair.

    Science.gov (United States)

    Garcia, Orquidea; Hiatt, Michael J; Lundin, Amber; Lee, Jooeun; Reddy, Raghava; Navarro, Sonia; Kikuchi, Alex; Driscoll, Barbara

    2016-03-01

    Type 2 alveolar epithelial cells (AEC2) are regarded as the progenitor population of the alveolus responsible for injury repair and homeostatic maintenance. Depletion of this population is hypothesized to underlie various lung pathologies. Current models of lung injury rely on either uncontrolled, nonspecific destruction of alveolar epithelia or on targeted, nontitratable levels of fixed AEC2 ablation. We hypothesized that discrete levels of AEC2 ablation would trigger stereotypical and informative patterns of repair. To this end, we created a transgenic mouse model in which the surfactant protein-C promoter drives expression of a mutant SR39TK herpes simplex virus-1 thymidine kinase specifically in AEC2. Because of the sensitivity of SR39TK, low doses of ganciclovir can be administered to these animals to induce dose-dependent AEC2 depletion ranging from mild (50%) to lethal (82%) levels. We demonstrate that specific levels of AEC2 depletion cause altered expression patterns of apoptosis and repair proteins in surviving AEC2 as well as distinct changes in distal lung morphology, pulmonary function, collagen deposition, and expression of remodeling proteins in whole lung that persist for up to 60 days. We believe SPCTK mice demonstrate the utility of cell-specific expression of the SR39TK transgene for exerting fine control of target cell depletion. Our data demonstrate, for the first time, that specific levels of type 2 alveolar epithelial cell depletion produce characteristic injury repair outcomes. Most importantly, use of these mice will contribute to a better understanding of the role of AEC2 in the initiation of, and response to, lung injury.

  15. B-Cell Depletion Reduces the Maturation of Cerebral Cavernous Malformations in Murine Models.

    Science.gov (United States)

    Shi, Changbin; Shenkar, Robert; Zeineddine, Hussein A; Girard, Romuald; Fam, Maged D; Austin, Cecilia; Moore, Thomas; Lightle, Rhonda; Zhang, Lingjiao; Wu, Meijing; Cao, Ying; Gunel, Murat; Louvi, Angeliki; Rorrer, Autumn; Gallione, Carol; Marchuk, Douglas A; Awad, Issam A

    2016-06-01

    Cerebral cavernous malformations (CCMs) are relatively common vascular malformations, characterized by increased Rho kinase (ROCK) activity, vascular hyper-permeability and the presence of blood degradation products including non-heme iron. Previous studies revealed robust inflammatory cell infiltration, selective synthesis of IgG, in situ antigen driven B-cell clonal expansion, and deposition of immune complexes and complement proteins within CCM lesions. We aimed to evaluate the impact of suppressing the immune response on the formation and maturation of CCM lesions, as well as lesional iron deposition and ROCK activity. Two murine models of heterozygous Ccm3 (Pdcd10), which spontaneously develop CCM lesions with severe and milder phenotypes, were either untreated or received anti-mouse BR3 to deplete B cells. Brains from anti-mouse BR3-treated mice exhibited significantly fewer mature CCM lesions and smaller lesions compared to untreated mice. B cell depletion halted the progression of lesions into mature stage 2 lesions but did not prevent their genesis. Non-heme iron deposition and ROCK activity was decreased in lesions of B cell depleted mice. This represents the first report of the therapeutic benefit of B-cell depletion in the development and progression of CCMs, and provides a proof of principle that B cells play a critical role in CCM lesion genesis and maturation. These findings add biologics to the list of potential therapeutic agents for CCM disease. Future studies would characterize the putative antigenic trigger and further define the mechanism of immune response in the lesions.

  16. Calvatia lilacina protein-extract induces apoptosis through glutathione depletion in human colorectal carcinoma cells.

    Science.gov (United States)

    Tsay, Jwu-Guh; Chung, King-Thom; Yeh, Chung-Hung; Chen, Wan-Ling; Chen, Chi-Hung; Lin, Martin Hsiu-Chu; Lu, Fung-Jou; Chiou, Jeng-Fong; Chen, Ching-Hsein

    2009-02-25

    This paper reports that a novel protein extract isolated from Calvatia lilacina (CL) can induce cell death against four types of human colorectal cancer cells. Importantly, CL was shown to be free of apoptotic effects against normal rat liver cells. We have also identified that CL-induced glutathione (GSH) depletion is the major contributor responsible for the apoptotic cell death induction of SW 480 cells, as evidenced by the observation that exogenously added N-acetylcysteine (NAC), or GSH, but not vitamin C, could offer a near complete protection of CL-treated cells against apoptotic cell death. Furthermore, the participation of reactive oxygen species (ROS) evoked a drop in the transmembrane potential (Delta Psi(m)) in the CL-induced apoptotic cell death. This observation can only be deemed as a minor pathway due to the fact that cyclosporine A (CyA) could only partially rescue the CL-treated cells from apoptotic cell death. Likewise, despite the fact that CL could induce the upregulation of Bax, its knockdown via siRNA (48 h) failed to completely mitigate apoptotic cell death, indicating that its role in this apoptotic process was insignificant. To further explore the possible underlying mechanism associated with CL-induced GSH depletion, we proceeded to determine the effect of CL on the cellular gamma-glutamylcysteine synthetase (gamma-GCS), a rate-limiting enzyme responsible for GSH biosynthesis, and demonstrated that indeed gamma-GCS could be repressed by CL. Taken together, we report here for the first time that the anticancer effect of CL on human colorectal cancer cells is mediated through GSH depletion mechanism rather than a ROS-mediated killing process. This functional attribute of CL can thus provide the basis for the strategic design of a treatment of colorectal cancer.

  17. Recent advances in haploidentical hematopoietic stem cell transplantation using ex vivo T cell-depleted graft in children and adolescents.

    Science.gov (United States)

    Im, Ho Joon; Koh, Kyung-Nam; Seo, Jong Jin

    2016-03-01

    Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for children and adolescents with various malignant and non-malignant diseases. While human leukocyte antigen (HLA)-identical sibling donor is the preferred choice, matched unrelated volunteer donor is another realistic option for successful HSCT. Unfortunately, it is not always possible to find a HLA-matched donor for patients requiring HSCT, leading to a considerable number of deaths of patients without undergoing transplantation. Alternatively, allogeneic HSCT from haploidentical family members could provide donors for virtually all patients who need HSCT. Although the early attempts at allogeneic HSCT from haploidentical family donor (HFD) were disappointing, recent advances in the effective ex vivo depletion of T cells or unmanipulated in vivo regulation of T cells, better supportive care, and optimal conditioning regimens have significantly improved the outcomes of haploidentical HSCT. The ex vivo techniques used to remove T cells have evolved from the selection of CD34(+) hematopoietic stem cell progenitors to the depletion of CD3(+) cells, and more recently to the depletion of αβ(+) T cells. The recent emerging evidence for ex vivo T cell-depleted haploidentical HSCT has provided additional therapeutic options for pediatric patients with diseases curable by HSCT but has not found a suitable related or unrelated donor. This review discusses recent advances in haploidentical HSCT, focusing on transplant using ex vivo T cell-depleted grafts. In addition, our experiences with this novel approach for the treatment of pediatric patients with malignant and non-malignant diseases are described.

  18. GFP-specific CD8 T cells enable targeted cell depletion and visualization of T-cell interactions.

    Science.gov (United States)

    Agudo, Judith; Ruzo, Albert; Park, Eun Sook; Sweeney, Robert; Kana, Veronika; Wu, Meng; Zhao, Yong; Egli, Dieter; Merad, Miriam; Brown, Brian D

    2015-12-01

    There are numerous cell types with scarcely understood functions, whose interactions with the immune system are not well characterized. To facilitate their study, we generated a mouse bearing enhanced green fluorescent protein (EGFP)-specific CD8(+) T cells. Transfer of the T cells into EGFP reporter animals can be used to kill EGFP-expressing cells, allowing selective depletion of desired cell types, or to interrogate T-cell interactions with specific populations. Using this system, we eliminate a rare EGFP-expressing cell type in the heart and demonstrate its role in cardiac function. We also show that naive T cells are recruited into the mouse brain by antigen-expressing microglia, providing evidence of an immune surveillance pathway in the central nervous system. The just EGFP death-inducing (Jedi) T cells enable visualization of a T-cell antigen. They also make it possible to utilize hundreds of existing EGFP-expressing mice, tumors, pathogens and other tools, to study T-cell interactions with many different cell types, to model disease states and to determine the functions of poorly characterized cell populations.

  19. Determination of Mother Centriole Maturation in CPAP-Depleted Cells Using the Ninein Antibody

    OpenAIRE

    Miseon Lee; Kunsoo Rhee

    2015-01-01

    Background Mutations in centrosomal protein genes have been identified in a number of genetic diseases in brain development, including microcephaly. Centrosomal P4.1-associated protein (CPAP) is one of the causal genes implicated in primary microcephaly. We previously proposed that CPAP is essential for mother centriole maturation during mitosis. Methods We immunostained CPAP-depleted cells using the ninein antibody, which selectively detects subdistal appendages in mature mother centrioles. ...

  20. In situ depletion of CD4(+) T cells in human skin by Zanolimumab

    DEFF Research Database (Denmark)

    Villadsen, L.S.; Skov, L.; Dam, T.N.

    2007-01-01

    -driving T cells in situ may therefore be a useful approach in the treatment of inflammatory and malignant skin diseases. Depletion of CD4(+) T cells in intact inflamed human skin tissue by Zanolimumab, a fully human therapeutic monoclonal antibody (IgG1, kappa) against CD4, was studied in a human psoriasis......CD4(+) T cells, in activated or malignant form, are involved in a number of diseases including inflammatory skin diseases such as psoriasis, and T cell lymphomas such as the majority of cutaneous T cell lymphomas (CTCL). Targeting CD4 with an antibody that inhibits and/or eliminates disease...... xenograft mouse model. Zanolimumab treatment was shown to induce a significant reduction in the numbers of inflammatory mononuclear cells in upper dermis. This reduction in inflammatory mononuclear cells in situ was primarily due to a significant reduction in the numbers of skin-infiltrating CD4...

  1. Cytotoxicity of tumor antigen specific human T cells is unimpaired by arginine depletion.

    Directory of Open Access Journals (Sweden)

    Markus Munder

    Full Text Available Tumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8(+ T cell activation in a tumor peptide as well as a viral peptide antigen specific system: (i CD8(+ T cells with specificity against the MART-1aa26-35*A27L tumor antigen expanded with in vitro generated dendritic cells, and (ii clonal CMV pp65aa495-503 specific T cells and T cells retrovirally transduced with a CMV pp65aa495-503 specific T cell receptor were analyzed. Our data demonstrate that human CD8(+ T cell antigen specific cytotoxicity and perforin secretion are completely preserved in the absence of arginine, while antigen specific proliferation as well as IFN-γ and granzyme B secretion are severely compromised. These novel results highlight the complexity of antigen specific T cell activation and demonstrate that human T cells can preserve important activation-induced effector functions in the context of arginine deficiency.

  2. HRP-2 determines HIV-1 integration site selection in LEDGF/p75 depleted cells

    Directory of Open Access Journals (Sweden)

    Schrijvers Rik

    2012-10-01

    Full Text Available Abstract Background Lens epithelium–derived growth factor (LEDGF/p75 is a cellular co-factor of HIV-1 integrase (IN that tethers the viral pre-integration complex to the host cell chromatin and determines the genome wide integration site distribution pattern of HIV-1. Recently, we demonstrated that HIV-1 replication was reduced in LEDGF/p75 knockout (KO cells. LEDGF/p75 KO significantly altered the integration site preference of HIV-1, but the pattern remained distinct from a computationally generated matched random control set (MRC, suggesting the presence of an alternative tethering factor. We previously identified Hepatoma-derived growth factor related protein 2 (HRP-2 as a factor mediating LEDGF/p75-independent HIV-1 replication. However, the role of HRP-2 in HIV-1 integration site selection was not addressed. Findings We studied the HIV-1 integration site distribution in the presence and absence of LEDGF/p75 and/or HRP-2, and in LEDGF/p75-depleted cells that overexpress HRP-2. We show that HRP-2 functions as a co-factor of HIV-1 IN in LEDGF/p75-depleted cells. Endogenous HRP-2 only weakly supported HIV-1 replication in LEDGF/p75 depleted cells. However, HRP-2 overexpression rescued HIV-1 replication and restored integration in RefSeq genes to wild-type levels. Additional HRP-2 KD in LEDGF/p75-depleted cells reduces integration frequency in transcription units and shifts the integration distribution towards random. Conclusions We demonstrate that HRP-2 overexpression can compensate for the absence of LEDGF/p75 and indicate that the residual bias in integration targeting observed in the absence of LEDGF/p75 can be ascribed to HRP-2. Knockdown of HRP-2 upon LEDGF/p75 depletion results in a more random HIV-1 integration pattern. These data therefore reinforce the understanding that LEDGF/p75 is the dominant HIV-1 IN co-factor.

  3. CD4 T cell depletion at the cervix during HIV infection is associated with accumulation of terminally differentiated T cells.

    Science.gov (United States)

    Gumbi, P P; Jaumdally, S Z; Salkinder, A L; Burgers, W A; Mkhize, N N; Hanekom, W; Coetzee, D; Williamson, A; Passmore, J S

    2011-12-01

    In blood, the accumulation of terminally differentiated (TD) T cells during HIV infection is associated with CD4 T cell loss and HIV disease progression. Here, we investigated the maintenance and functional characteristics of memory T cells at the cervix. We found that CD4 T cell depletion at the cervix mirrors CD4 depletion in blood. In all women, depletion of CD4 T cells at the cervix was associated with significant reductions in CD45RA- CCR7+ (central memory [CM]) T cells and the accumulation of CD45RA+ CCR7- (TD T cells). We determined whether inflammation in the genital tract was associated with the local differentiation of T cells at the cervix. In uninfected women, genital tract inflammation was associated with the accumulation of CD45RA- CCR7+ CM CD4 T cells and reduced frequencies of CD45RA+ CCR7- TD cells at the cervix. This finding may reflect the fact that, in the absence of HIV infection, TD T cells may be slowly lost in the presence of genital inflammation, while CD45RA- CCR7+ CM T cells are recruited to replenish the diminishing CD4 T cell pool. Following global stimulation with phorbol myristate acetate (PMA)-ionomycin, we noted a significant interleukin 2 (IL-2) deficit in both cervical and blood CD4 T cells from HIV-infected women compared to uninfected women, while gamma interferon (IFN-γ) production was similar, irrespective of HIV status. Few HIV-infected women had detectable IFN-γ and IL-2 HIV-specific T cell responses at the cervix, and these responses were significantly lower in magnitude than the corresponding responses in blood. These data suggest that CD4 depletion was associated with the accumulation of terminally differentiated T cell phenotypes at the cervical mucosa defective in their ability to produce IL-2. CD4 depletion and compromised immunity at the cervix may be accompanied by progressive decline of central memory-like T cells and development of T cells toward terminally differentiated phenotypes.

  4. Apocynin attenuates cholesterol oxidation product-induced programmed cell death by suppressing NF-κB-mediated cell death process in differentiated PC12 cells.

    Science.gov (United States)

    Lee, Da Hee; Nam, Yoon Jeong; Lee, Chung Soo

    2015-10-01

    Cholesterol oxidation products are suggested to be involved in neuronal degeneration. Apocynin has demonstrated to have anti-inflammatory and anti-oxidant effects. We assessed the effect of apocynin on the cholesterol oxidation product-induced programmed cell death in neuronal cells using differentiated PC12 cells in relation to NF-κB-mediated cell death process. 7-Ketocholesterol and 25-hydroxycholesterol decreased the levels of Bid and Bcl-2, increased the levels of Bax and p53, and induced loss of the mitochondrial transmembrane potential, release of cytochrome c and activation of caspases (-8, -9 and -3). 7-Ketocholesterol caused an increase in the levels of cytosolic and nuclear NF-κB p65, cytosolic NF-κB p50 and cytosolic phospho-IκB-α, which was inhibited by the addition of 0.5 μM Bay11-7085 (an inhibitor of NF-κB activation). Apocynin attenuated the cholesterol oxidation product-induced changes in the programmed cell death-related protein levels, NF-κB activation, production of reactive oxygen species, and depletion of GSH. The results show that apocynin appears to attenuate the cholesterol oxidation product-induced programmed cell death in PC12 cells by suppressing the activation of the mitochondrial pathway and the caspase-8- and Bid-dependent pathways that are mediated by NF-κB activation. The preventive effect appears to be associated with the inhibitory effect on the production of reactive oxygen species and depletion of GSH.

  5. Tumor eradication after cyclophosphamide depends on concurrent depletion of regulatory T cells: a role for cycling TNFR2-expressing effector-suppressor T cells in limiting effective chemotherapy.

    Science.gov (United States)

    van der Most, Robbert G; Currie, Andrew J; Mahendran, Sathish; Prosser, Amy; Darabi, Anna; Robinson, Bruce W S; Nowak, Anna K; Lake, Richard A

    2009-08-01

    Tumor cell death potentially engages with the immune system. However, the efficacy of anti-tumor chemotherapy may be limited by tumor-driven immunosuppression, e.g., through CD25+ regulatory T cells. We addressed this question in a mouse model of mesothelioma by depleting or reconstituting CD25+ regulatory T cells in combination with two different chemotherapeutic drugs. We found that the efficacy of cyclophosphamide to eradicate established tumors, which has been linked to regulatory T cell depletion, was negated by adoptive transfer of CD25+ regulatory T cells. Analysis of post-chemotherapy regulatory T cell populations revealed that cyclophosphamide depleted cycling (Ki-67(hi)) T cells, including foxp3+ regulatory CD4+ T cells. Ki-67(hi) CD4+ T cells expressed increased levels of two markers, TNFR2 and ICOS, that have been associated with a maximally suppressive phenotype according to recently published studies. This suggest that cyclophosphamide depletes a population of maximally suppressive regulatory T cells, which may explain its superior anti-tumor efficacy in our model. Our data suggest that regulatory T cell depletion could be used to improve the efficacy of anti-cancer chemotherapy regimens. Indeed, we observed that the drug gemcitabine, which does not deplete cycling regulatory T cells, eradicates established tumors in mice only when CD25+ CD4+ T cells are concurrently depleted. Cyclophosphamide could be used to achieve regulatory T cell depletion in combination with chemotherapy.

  6. Glutathione depletion and carbon ion radiation potentiate clustered DNA lesions, cell death and prevent chromosomal changes in cancer cells progeny.

    Science.gov (United States)

    Hanot, Maïté; Boivin, Anthony; Malésys, Céline; Beuve, Michaël; Colliaux, Anthony; Foray, Nicolas; Douki, Thierry; Ardail, Dominique; Rodriguez-Lafrasse, Claire

    2012-01-01

    Poor local control and tumor escape are of major concern in head-and-neck cancers treated by conventional radiotherapy or hadrontherapy. Reduced glutathione (GSH) is suspected of playing an important role in mechanisms leading to radioresistance, and its depletion should enable oxidative stress insult, thereby modifying the nature of DNA lesions and the subsequent chromosomal changes that potentially lead to tumor escape.This study aimed to highlight the impact of a GSH-depletion strategy (dimethylfumarate, and L-buthionine sulfoximine association) combined with carbon ion or X-ray irradiation on types of DNA lesions (sparse or clustered) and the subsequent transmission of chromosomal changes to the progeny in a radioresistant cell line (SQ20B) expressing a high endogenous GSH content. Results are compared with those of a radiosensitive cell line (SCC61) displaying a low endogenous GSH level. DNA damage measurements (γH2AX/comet assay) demonstrated that a transient GSH depletion in resistant SQ20B cells potentiated the effects of irradiation by initially increasing sparse DNA breaks and oxidative lesions after X-ray irradiation, while carbon ion irradiation enhanced the complexity of clustered oxidative damage. Moreover, residual DNA double-strand breaks were measured whatever the radiation qualities. The nature of the initial DNA lesions and amount of residual DNA damage were similar to those observed in sensitive SCC61 cells after both types of irradiation. Misrepaired or unrepaired lesions may lead to chromosomal changes, estimated in cell progeny by the cytome assay. Both types of irradiation induced aberrations in nondepleted resistant SQ20B and sensitive SCC61 cells. The GSH-depletion strategy prevented the transmission of aberrations (complex rearrangements and chromosome break or loss) in radioresistant SQ20B only when associated with carbon ion irradiation. A GSH-depleting strategy combined with hadrontherapy may thus have considerable advantage in the

  7. Glutathione depletion and carbon ion radiation potentiate clustered DNA lesions, cell death and prevent chromosomal changes in cancer cells progeny.

    Directory of Open Access Journals (Sweden)

    Maïté Hanot

    Full Text Available Poor local control and tumor escape are of major concern in head-and-neck cancers treated by conventional radiotherapy or hadrontherapy. Reduced glutathione (GSH is suspected of playing an important role in mechanisms leading to radioresistance, and its depletion should enable oxidative stress insult, thereby modifying the nature of DNA lesions and the subsequent chromosomal changes that potentially lead to tumor escape.This study aimed to highlight the impact of a GSH-depletion strategy (dimethylfumarate, and L-buthionine sulfoximine association combined with carbon ion or X-ray irradiation on types of DNA lesions (sparse or clustered and the subsequent transmission of chromosomal changes to the progeny in a radioresistant cell line (SQ20B expressing a high endogenous GSH content. Results are compared with those of a radiosensitive cell line (SCC61 displaying a low endogenous GSH level. DNA damage measurements (γH2AX/comet assay demonstrated that a transient GSH depletion in resistant SQ20B cells potentiated the effects of irradiation by initially increasing sparse DNA breaks and oxidative lesions after X-ray irradiation, while carbon ion irradiation enhanced the complexity of clustered oxidative damage. Moreover, residual DNA double-strand breaks were measured whatever the radiation qualities. The nature of the initial DNA lesions and amount of residual DNA damage were similar to those observed in sensitive SCC61 cells after both types of irradiation. Misrepaired or unrepaired lesions may lead to chromosomal changes, estimated in cell progeny by the cytome assay. Both types of irradiation induced aberrations in nondepleted resistant SQ20B and sensitive SCC61 cells. The GSH-depletion strategy prevented the transmission of aberrations (complex rearrangements and chromosome break or loss in radioresistant SQ20B only when associated with carbon ion irradiation. A GSH-depleting strategy combined with hadrontherapy may thus have considerable

  8. Depleted internal store-activated Ca2+ entry can trigger neurotransmitter release in bovine chromaffin cells.

    Science.gov (United States)

    Powis, D A; Clark, C L; O'Brien, K J

    1996-02-09

    A potential role of the intracellular Ca2+ stores in modulating catecholamine release has been investigated in bovine chromaffin cells maintained in tissue culture. Pharmacological depletion of the stores with a combination of caffeine, histamine and thapsigargin in Ca2+-free media resulted in a significantly greater release of catecholamines on re-exposure to Ca2+-containing media compared with that from non-store depleted cells. The increase in catecholamine release was prevented by intracellular BAPTA indicating that the increase was caused by a rise in Ca2+. Measurement of intracellular free Ca2+ concentration with the fluorescent indicator, fura-2, over the same time-course as the catecholamine release experiments showed that upon restoration of external Ca2+ there was an immediate, substantial and maintained increase in cytosolic Ca2+. It is most probable that the increase in catecholamine release was a consequence of an increase in Ca2+ influx triggered by prior depletion of the internal Ca2+ stores. However, the data suggest that capacitative Ca2+ entry is poorly linked to catecholamine release; although Ca2+ entry on restoration of external Ca2+ was immediate and substantial, the increase in catecholamine release, although quantitatively significant, was slowly realised.

  9. RETRACTED: Advances in colloidal quantum dot solar cells: The depleted-heterojunction device

    KAUST Repository

    Kramer, Illan J.

    2011-08-01

    Colloidal quantum dot (CQD) photovoltaics combine low-cost solution processibility with quantum size-effect tunability to match absorption with the solar spectrum. Recent advances in CQD photovoltaics have led to 3.6% AM1.5 solar power conversion efficiencies. Here we report CQD photovoltaic devices on transparent conductive oxides and show that our devices rely on the establishment of a depletion region for field-driven charge transport and separation. The resultant depleted-heterojunction solar cells provide a 5.1% AM1.5 power conversion efficiency. The devices employ infrared-bandgap size-effect-tuned PbS colloidal quantum dots, enabling broadband harvesting of the solar spectrum. © 2010 Elsevier B.V.

  10. Advances in colloidal quantum dot solar cells: The depleted-heterojunction device

    Energy Technology Data Exchange (ETDEWEB)

    Kramer, Illan J.; Pattantyus-Abraham, Andras G.; Barkhouse, Aaron R.; Wang, Xihua [Department of Electrical and Computer Engineering, University of Toronto, 10 King' s College Rd., Toronto, Ontario M5S 3G4 (Canada); Konstantatos, Gerasimos [Department of Electrical and Computer Engineering, University of Toronto, 10 King' s College Rd., Toronto, Ontario M5S 3G4 (Canada); ICFO - Institut de Ciencies Fotoniques, Mediterranean Technology Park, 08860 Castelldefels, Barcelona (Spain); Debnath, Ratan; Levina, Larissa [Department of Electrical and Computer Engineering, University of Toronto, 10 King' s College Rd., Toronto, Ontario M5S 3G4 (Canada); Raabe, Ines; Nazeeruddin, Md. K.; Graetzel, Michael [Laboratory for Photonics and Interfaces, Institute of Chemical Sciences and Engineering, School of Basic Sciences, Swiss Federal Institute of Technology, CH-1015 Lausanne (Switzerland); Sargent, Edward H., E-mail: ted.sargent@utoronto.ca [Department of Electrical and Computer Engineering, University of Toronto, 10 King' s College Rd., Toronto, Ontario M5S 3G4 (Canada)

    2011-08-31

    Colloidal quantum dot (CQD) photovoltaics combine low-cost solution processibility with quantum size-effect tunability to match absorption with the solar spectrum. Recent advances in CQD photovoltaics have led to 3.6% AM1.5 solar power conversion efficiencies. Here we report CQD photovoltaic devices on transparent conductive oxides and show that our devices rely on the establishment of a depletion region for field-driven charge transport and separation. The resultant depleted-heterojunction solar cells provide a 5.1% AM1.5 power conversion efficiency. The devices employ infrared-bandgap size-effect-tuned PbS colloidal quantum dots, enabling broadband harvesting of the solar spectrum.

  11. Oxymatrine liposome attenuates hepatic fibrosis via targeting hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Ning-Li Chai; Qiang Fu; Hui Shi; Chang-Hao Cai; Jun Wan; Shi-Ping Xu; Ben-Yan Wu

    2012-01-01

    AIM:To investigate the potential mechanism of ArgGly-Asp (RGD) peptide-labeled liposome loading oxymatrine (OM) therapy in CCl4-induced hepatic fibrosis in METHODS:We constructed a rat model of CCl4-induced hepatic fibrosis and treated the rats with different formulations of OM.To evaluate the antifibrotic effect of OM,we detected levels of alkaline phosphatase,hepatic histopathology (hematoxylin and eosin stain and Masson staining) and fibrosis-related gene expression of matrix metallopeptidase (MMP)-2,tissue inhibitor of metalloproteinase (TIMP)-1 as well as type Ⅰ procollagen via quantitative real-time polymerase chain reaction.To detect cell viability and apoptosis of hepatic stellate cells (HSCs),we performed 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay and flow cytometry.To reinforce the combination of oxymatrine with HSCs,we constructed fluorescein-isothiocyanate-conjugated Arg-Gly-Asp peptide-labeled liposomes loading OM,and its targeting of HSCs was examined by fluorescent microscopy.RESULTS:OM attenuated CCl4-induced hepatic fibrosis,as defined by reducing serum alkaline phosphatase (344.47 ± 27.52 U/L vs 550.69 ± 43.78 U/L,P < 0.05),attenuating liver injury and improving collagen deposits (2.36% ± 0.09% vs 7.70% ± 0.60%,P < 0.05) and downregulating fibrosis-related gene expression,that is,MMP-2,TIMP-1 and type Ⅰ procollagen (P < 0.05).OM inhibited cell viability and induced apoptosis of HSCs in vitro.RGD promoted OM targeting of HSCs and enhanced the therapeutic effect of OM in terms of serum alkaline phosphatase (272.51 ± 19.55 U/L vs 344.47 ± 27.52 U/L,P < 0.05),liver injury,collagen deposits (0.26% ± 0.09% vs 2.36% ± 0.09%,P < 0.05) and downregulating fibrosis-related gene expression,that is,MMP-2,TIMP-1 and type Ⅰ procollagen (P < 0.05).Moreover,in vitro assay demonstrated that RGD enhanced the effect of OM on HSC viability and apoptosis.CONCLUSION:OM attenuated hepatic fibrosis by

  12. A method for enriching myeloid (CFU-GM) and erythroid (BFU-E) progenitor cells from human cord blood by accessory cell depletion.

    Science.gov (United States)

    Dowton, L A; Ma, D D

    1992-10-01

    Human cord blood provides a convenient alternative to bone marrow as a rich source of hemopoietic progenitor cells. This study reports a simple means for enriching a cord blood progenitor cell population by accessory cell depletion. Two methods of monocyte depletion were tested. A Cytodex 3 microcarrier system using collagen coated dextran beads was compared to the more commonly used method of plastic plate adhesion. The method of plastic plate adhesion gave a significantly higher cell recovery. T cell depletion using a recently characterized rat monoclonal antibody which fixes human complement was also investigated. A combined method of monocyte depletion by plate adhesion and T cell depletion resulted in the removal of > 96% of monocytes and > 98% of T cells. This led to a significant enrichment of myeloid (CFU-GM) and erythroid (BFU-E) colony growth. Such enriched progenitor cell populations provide a useful starting population for any study on hemopoiesis.

  13. B cell depletion in treating primary biliary cirrhosis: Pros and cons

    Institute of Scientific and Technical Information of China (English)

    Yu-Feng Yin; Xuan Zhang

    2012-01-01

    Primary biliary cirrhosis (PBC) is a progressive autoimmune liver disease of unknown etiology that affects almost exclusively women.Ursodeoxycholic acid (UDCA) is currently the only approved drug by Food and Drug Administration for patients with PBC.Although the precise pathogenesis of PBC remains unclear,it has been postulated that many cell populations,including B cells,are involved in the ongoing inflammatory process,which implicates,not surprisingly,a potential therapeutic target of depleting B cell to treat this disorder.Rituximab is a chimeric anti-CD20 monoclonal antibody that has been approved for the treatment of lymphoma and some autoimmune diseases such as rheumatoid arthritis.Whether it is effective in the treatment of PBC has not been evaluated.Recently,Tsuda et al[1] demonstrated that B cell depletion with rituximab significantly reduced the number of anti-mitochondrial antibodies (AMA)-producing B cells,AMA titers,the plasma levels of immunoglobulins (IgA,IgM and IgG) as well as serum alkaline phosphatase,and it was well tolerated by all the treated patients with no serious adverse events.This observation provides a novel treatment option for the patients with PBC who have incomplete response to UDCA.

  14. Norcantharidin induced DU145 cell apoptosis through ROS-mediated mitochondrial dysfunction and energy depletion.

    Science.gov (United States)

    Shen, Bo; He, Pei-Jie; Shao, Chun-Lin

    2013-01-01

    Norcantharidin (NCTD), a demethylated analog of cantharidin derived from blister beetles, has attracted considerable attentions in recent years due to their definitely toxic properties and the noteworthy advantages in stimulating bone marrow and increasing the peripheral leukocytes. Hence, it is worth studying the anti-tumor effect of NCTD on human prostate cancer cells DU145. It was found that after the treatment of NCTD with different concentrations (25-100 μM), the cell proliferation was significantly inhibited, which led to the appearance of micronucleus (MN). Moreover, the cells could be killed in a dose-/time-dependent manner along with the reduction of PCNA (proliferating cell nuclear antigen) expression, destruction of mitochondrial membrane potential (MMP), down-regulation of MnSOD, induction of ROS, depletion of ATP, and activation of AMPK (Adenosine 5'-monophosphate -activated protein kinase) . In addition, a remarkable release of cytochrome c was found in the cells exposed to 100 μM NCTD and exogenous SOD-PEG could eliminate the generation of NCTD-induced MN. In conclusion, our studies indicated that NCTD could induce the collapse of MMP and mitochondria dysfunction. Accumulation of intercellular ROS could eventually switch on the apoptotic pathway by causing DNA damage and depleting ATP.

  15. Norcantharidin induced DU145 cell apoptosis through ROS-mediated mitochondrial dysfunction and energy depletion.

    Directory of Open Access Journals (Sweden)

    Bo Shen

    Full Text Available Norcantharidin (NCTD, a demethylated analog of cantharidin derived from blister beetles, has attracted considerable attentions in recent years due to their definitely toxic properties and the noteworthy advantages in stimulating bone marrow and increasing the peripheral leukocytes. Hence, it is worth studying the anti-tumor effect of NCTD on human prostate cancer cells DU145. It was found that after the treatment of NCTD with different concentrations (25-100 μM, the cell proliferation was significantly inhibited, which led to the appearance of micronucleus (MN. Moreover, the cells could be killed in a dose-/time-dependent manner along with the reduction of PCNA (proliferating cell nuclear antigen expression, destruction of mitochondrial membrane potential (MMP, down-regulation of MnSOD, induction of ROS, depletion of ATP, and activation of AMPK (Adenosine 5'-monophosphate -activated protein kinase . In addition, a remarkable release of cytochrome c was found in the cells exposed to 100 μM NCTD and exogenous SOD-PEG could eliminate the generation of NCTD-induced MN. In conclusion, our studies indicated that NCTD could induce the collapse of MMP and mitochondria dysfunction. Accumulation of intercellular ROS could eventually switch on the apoptotic pathway by causing DNA damage and depleting ATP.

  16. Syntaxin and VAMP association with lipid rafts depends on cholesterol depletion in capacitating sperm cells.

    Science.gov (United States)

    Tsai, Pei-Shiue; De Vries, Klaas J; De Boer-Brouwer, Mieke; Garcia-Gil, Nuria; Van Gestel, Renske A; Colenbrander, Ben; Gadella, Bart M; Van Haeften, Theo

    2007-01-01

    Sperm cells represent a special exocytotic system since mature sperm cells contain only one large secretory vesicle, the acrosome, which fuses with the overlying plasma membrane during the fertilization process. Acrosomal exocytosis is believed to be regulated by activation of SNARE proteins. In this paper, we identified specific members of the SNARE protein family, i.e., the t-SNAREs syntaxin1 and 2, and the v-SNARE VAMP, present in boar sperm cells. Both syntaxins were predominantly found in the plasma membrane whereas v-SNAREs are mainly located in the outer acrosomal membrane of these cells. Under non-capacitating conditions both syntaxins and VAMP are scattered in well-defined punctate structures over the entire sperm head. Bicarbonate-induced in vitro activation in the presence of BSA causes a relocalization of these SNAREs to a more homogeneous distribution restricted to the apical ridge area of the sperm head, exactly matching the site of sperm zona binding and subsequent induced acrosomal exocytosis. This redistribution of syntaxin and VAMP depends on cholesterol depletion and closely resembles the previously reported redistribution of lipid raft marker proteins. Detergent-resistant membrane isolation and subsequent analysis shows that a significant proportion of syntaxin emerges in the detergent-resistant membrane (raft) fraction under such conditions, which is not the case under those conditions where cholesterol depletion is blocked. The v-SNARE VAMP displays a similar cholesterol depletion-dependent lateral and raft redistribution. Taken together, our results indicate that redistribution of syntaxin and VAMP during capacitation depends on association of these SNAREs with lipid rafts and that such a SNARE-raft association may be essential for spatial control of exocytosis and/or regulation of SNARE functioning.

  17. Mechanosensitive channel activity and F-actin organization in cholesterol-depleted human leukaemia cells.

    Science.gov (United States)

    Morachevskaya, Elena; Sudarikova, Anastasiya; Negulyaev, Yuri

    2007-04-01

    This study focuses on the functional role of cellular cholesterol in the regulation of mechanosensitive cation channels activated by stretch in human leukaemia K562 cells. The patch-clamp method was employed to examine the effect of methyl-beta-cyclodextrin (MbetaCD), a synthetic cholesterol-sequestering agent, on stretch-activated single currents. We found that cholesterol-depleting treatment with MbetaCD resulted in a suppression of the activity of mechanosensitive channels without a change in the unitary conductance. The probability that the channel was open significantly decreased after treatment with MbetaCD. Fluorescent microscopy revealed F-actin reorganization, possibly involving actin assembly, after incubation of the cells with MbetaCD. We suggest that suppression of mechanosensitive channel activation in cholesterol-depleted leukaemia cells is due to F-actin rearrangement, presumably induced by lipid raft destruction. Our observations are consistent with the notion that stretch-activated cation channels in eukaryotic cells are regulated by the membrane-cytoskeleton complex rather than by tension developed purely in the lipid bilayer.

  18. SCD1 inhibition causes cancer cell death by depleting mono-unsaturated fatty acids.

    Directory of Open Access Journals (Sweden)

    Paul Mason

    Full Text Available Increased metabolism is a requirement for tumor cell proliferation. To understand the dependence of tumor cells on fatty acid metabolism, we evaluated various nodes of the fatty acid synthesis pathway. Using RNAi we have demonstrated that depletion of fatty-acid synthesis pathway enzymes SCD1, FASN, or ACC1 in HCT116 colon cancer cells results in cytotoxicity that is reversible by addition of exogenous fatty acids. This conditional phenotype is most pronounced when SCD1 is depleted. We used this fatty-acid rescue strategy to characterize several small-molecule inhibitors of fatty acid synthesis, including identification of TOFA as a potent SCD1 inhibitor, representing a previously undescribed activity for this compound. Reference FASN and ACC inhibitors show cytotoxicity that is less pronounced than that of TOFA, and fatty-acid rescue profiles consistent with their proposed enzyme targets. Two reference SCD1 inhibitors show low-nanomolar cytotoxicity that is offset by at least two orders of magnitude by exogenous oleate. One of these inhibitors slows growth of HCT116 xenograft tumors. Our data outline an effective strategy for interrogation of on-mechanism potency and pathway-node-specificity of fatty acid synthesis inhibitors, establish an unambiguous link between fatty acid synthesis and cancer cell survival, and point toward SCD1 as a key target in this pathway.

  19. Hybrid tandem solar cells with depleted-heterojunction quantum dot and polymer bulk heterojunction subcells

    KAUST Repository

    Kim, Taesoo

    2015-10-01

    We investigate hybrid tandem solar cells that rely on the combination of solution-processed depleted-heterojunction colloidal quantum dot (CQD) and bulk heterojunction polymer:fullerene subcells. The hybrid tandem solar cell is monolithically integrated and electrically connected in series with a suitable p-n recombination layer that includes metal oxides and a conjugated polyelectrolyte. We discuss the monolithic integration of the subcells, taking into account solvent interactions with underlayers and associated constraints on the tandem architecture, and show that an adequate device configuration consists of a low bandgap CQD bottom cell and a high bandgap polymer:fullerene top cell. Once we optimize the recombination layer and individual subcells, the hybrid tandem device reaches a VOC of 1.3V, approaching the sum of the individual subcell voltages. An impressive fill factor of 70% is achieved, further confirming that the subcells are efficiently connected via an appropriate recombination layer. © 2015.

  20. The depletion of interleukin-8 causes cell cycle arrest and increases the efficacy of docetaxel in breast cancer cells.

    Science.gov (United States)

    Shao, Nan; Chen, Liu-Hua; Ye, Run-Yi; Lin, Ying; Wang, Shen-Ming

    2013-02-15

    IL-8 is a multi-functional pro-inflammatory chemokine, which is highly expressed in cancers, such as ER-negative breast cancer. The present study demonstrates the pervasive role of IL-8 in the malignant progression of ER-negative breast cancer. IL-8 siRNA inhibited proliferation and delayed the G1 to S cell cycle progression in MDA-MB-231 and BT549 cells. IL-8 silencing resulted in the upregulation of the CDK inhibitor p27, the downregulation of cyclin D1, and the reduction of phosphorylated-Akt and NF-κB activities. IL-8 depletion also increased the chemosensitivity to docetaxel. These results indicate a role for IL-8 in promoting tumor cell survival and resistance to docetaxel and highlight the potential therapeutic significance of IL-8 depletion in ER-negative breast cancer patients.

  1. Selaginellatamariscina attenuates metastasis via Akt pathways in oral cancer cells.

    Directory of Open Access Journals (Sweden)

    Jia-Sin Yang

    Full Text Available BACKGROUND: Crude extracts of Selaginellatamariscina, an oriental medicinal herb, have been evidenced to treat several human diseases. This study investigated the mechanisms by which Selaginellatamariscina inhibits the invasiveness of human oral squamous-cell carcinoma (OSCC HSC-3 cells. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we demonstrate that Selaginellatamariscina attenuated HSC-3 cell migration and invasion in a dose-dependent manner. The anti-metastatic activities of Selaginellatamariscina occurred at least partially because of the down-regulation of matrix metalloproteinases (MMP-2 and MMP-9 gelatinase activity and the down-regulation of protein expression. The expression and function of both MMP-2 and MMP-9 were regulated by Selaginellatamariscina at a transcriptional level, as shown by quantitative real-time PCR and reporter assays. Chromatin immunoprecipitation (ChIP data further indicated that binding of the cAMP response element-binding (CREB protein and activating protein-1 (AP-1 to the MMP-2 promoter diminished at the highest dosage level of Selaginellatamariscina. The DNA-binding activity of specificity protein 1 (SP-1 to the MMP-9 promoter was also suppressed at the same concentration. Selaginellatamariscina did not affect the mitogen-activated protein kinase signaling pathway, but did inhibit the effects of gelatinase by reducing the activation of serine-threonine kinase Akt. CONCLUSIONS: These results demonstrate that Selaginellatamariscina may be a potent adjuvant therapeutic agent in the prevention of oral cancer.

  2. Novel metastasis model of human lung cancer in SCID mice depleted of NK cells.

    Science.gov (United States)

    Yano, S; Nishioka, Y; Izumi, K; Tsuruo, T; Tanaka, T; Miyasaka, M; Sone, S

    1996-07-17

    Metastasis is a critical problem in the treatment of human lung cancer. Thus, a suitable animal model of metastasis of human lung cancer is required for in vivo biological and preclinical studies. In this study, we tried to establish a suitable model for this, using SCID mice. Neither human SCLC H69/VP cells (5 x 10(6)) nor squamous-cell carcinoma RERF-LC-AI cells (1 x 10(6)), injected through a tail vein, formed metastases in untreated SCID mice. Pre-treatment of SCID mice with anti-asialo GM1 serum resulted in only a few metastases of H69/VP cells, but pre-treatment with anti-mouse IL-2 receptor beta chain Ab (TM-beta 1) resulted in numerous lymph-node metastases 56 days after tumor inoculation. H69/VP-M cells, an in vivo-selected variant line, formed significant numbers of lymph-node metastases even in SCID mice pre-treated with anti-asialo GM1 serum. SCID mice depleted of NK cells by treatment with TM-beta 1 showed different patterns of metastasis when inoculated intravenously with the 2 different human lung cancer cell lines (H69/VP and RERF-LC-AI cells): H69/VP cells formed metastases mainly in systemic lymph nodes and the liver, whereas RERF-LC-AI cells formed metastases mainly in the liver and kidneys, with only a few in lymph nodes. A histopathological study showed that the metastatic colonies consisted of cancer cells. The numbers of metastatic colonies formed by the 2 cell lines increased with the number of cells inoculated. TM-beta 1 treatment of SCID mice efficiently removed NK cells from peripheral blood for at least 6 weeks, whereas, after treatment of the mice with anti-asialo GM1 serum, NK cells were recovered within 9 days. These findings suggest that NK-cell-depleted SCID mice may be useful as a model in biological and pre-clinical studies on metastasis of human lung cancer.

  3. Persistent Simian Immunodeficiency Virus Infection Causes Ultimate Depletion of Follicular Th Cells in AIDS.

    Science.gov (United States)

    Xu, Huanbin; Wang, Xiaolei; Malam, Naomi; Lackner, Andrew A; Veazey, Ronald S

    2015-11-01

    CD4(+) T follicular helper (Tfh) cells are critical for the generation of humoral immune responses to pathogenic infections, providing help for B cell development, survival, and affinity maturation of Abs. Although CD4(+) Tfh cells are reported to accumulate in HIV or SIV infection, we found that germinal center Tfh cells, defined in this study as CXCR5(+)PD-1(HIGH)CD4(+) T cells, did not consistently accumulate in chronically SIV-infected rhesus macaques compared with those infected with less pathogenic simian HIV, vaccinated and SIVmac-challenged, or SIVmac-infected Mamu-A*01(+) macaques, all of which are associated with some control of virus replication and slower disease progression. Interestingly, CXCR5(+)PD-1(HIGH) Tfh cells in lymphoid tissues were eventually depleted in macaques with AIDS compared with the other cohorts. Chronic activation and proliferation of CXCR5(+)PD-1(HIGH) Tfh were increased, but PD-L2 expression was downregulated on B cells, possibly resulting in germinal center Tfh cell apoptosis. Together, these findings suggest that changes in CXCR5(+)PD-1(HIGH) Tfh cells in lymph nodes correlate with immune control during infection, and their loss or dysregulation contribute to impairment of B cell responses and progression to AIDS.

  4. Stem cell expansion during carcinogenesis in stem cell-depleted conditional telomeric repeat factor 2 null mutant mice.

    Science.gov (United States)

    Bojovic, B; Ho, H-Y; Wu, J; Crowe, D L

    2013-10-24

    To examine the role of telomeric repeat-binding factor 2 (TRF2) in epithelial tumorigenesis, we characterized conditional loss of TRF2 expression in the basal layer of mouse epidermis. These mice exhibit some characteristics of dyskeratosis congenita, a human stem cell depletion syndrome caused by telomere dysfunction. The epidermis in conditional TRF2 null mice exhibited DNA damage response and apoptosis, which correlated with stem cell depletion. The stem cell population in conditional TRF2 null epidermis exhibited shorter telomeres than those in control mice. Squamous cell carcinomas induced in conditional TRF2 null mice developed with increased latency and slower growth due to reduced numbers of proliferating cells as the result of increased apoptosis. TRF2 null epidermal stem cells were found in both primary and metastatic tumors. Despite the low-grade phenotype of the conditional TRF2 null primary tumors, the number of metastatic lesions was similar to control cancers. Basal cells from TRF2 null tumors demonstrated extreme telomere shortening and dramatically increased numbers of telomeric signals by fluorescence in situ hybridization due to increased genomic instability and aneuploidy in these cancers. DNA damage response signals were detected at telomeres in TRF2 null tumor cells from these mice. The increased genomic instability in these tumors correlated with eightfold expansion of the transformed stem cell population compared with that in control cancers. We concluded that genomic instability resulting from loss of TRF2 expression provides biological advantages to the cancer stem cell population.

  5. Attenuation of oxidative neuronal cell death by coffee phenolic phytochemicals

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Eun Sun; Jang, Young Jin [Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921 (Korea, Republic of); Hwang, Mun Kyung; Kang, Nam Joo [Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921 (Korea, Republic of); Department of Bioscience and Biotechnology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701 (Korea, Republic of); Lee, Ki Won [Department of Bioscience and Biotechnology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701 (Korea, Republic of)], E-mail: kiwon@konkuk.ac.kr; Lee, Hyong Joo [Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921 (Korea, Republic of)], E-mail: leehyjo@snu.ac.kr

    2009-02-10

    Neurodegenerative disorders such as Alzheimer's disease (AD) are strongly associated with oxidative stress, which is induced by reactive oxygen species (ROS) including hydrogen peroxide (H{sub 2}O{sub 2}). Recent studies suggest that moderate coffee consumption may reduce the risk of neurodegenerative diseases such as AD, but the molecular mechanisms underlying this effect remain to be clarified. In this study, we investigated the protective effects of chlorogenic acid (5-O-caffeoylquinic acid; CGA), a major phenolic phytochemical found in instant decaffeinated coffee (IDC), and IDC against oxidative PC12 neuronal cell death. IDC (1 and 5 {mu}g/ml) or CGA (1 and 5 {mu}M) attenuated H{sub 2}O{sub 2}-induced PC12 cell death. H{sub 2}O{sub 2}-induced nuclear condensation and DNA fragmentation were strongly inhibited by pretreatment with IDC or CGA. Pretreatment with IDC or CGA also inhibited the H{sub 2}O{sub 2}-induced cleavage of poly(ADP-ribose) polymerase (PARP), and downregulation of Bcl-X{sub L} and caspase-3. The accumulation of intracellular ROS in H{sub 2}O{sub 2}-treated PC12 cells was dose-dependently diminished by IDC or CGA. The activation of c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) by H{sub 2}O{sub 2} in PC12 cells was also inhibited by IDC or CGA. Collectively, these results indicate that IDC and CGA protect PC12 cells from H{sub 2}O{sub 2}-induced apoptosis by blocking the accumulation of intracellular ROS and the activation of MAPKs.

  6. CD4 T Cell Depletion at the Cervix during HIV Infection Is Associated with Accumulation of Terminally Differentiated T Cells

    OpenAIRE

    Gumbi, P. P.; Jaumdally, S. Z.; Salkinder, A. L.; Burgers, W. A.; Mkhize, N. N.; Hanekom, W; Coetzee, D; Williamson, A; Passmore, J. S.

    2011-01-01

    In blood, the accumulation of terminally differentiated (TD) T cells during HIV infection is associated with CD4 T cell loss and HIV disease progression. Here, we investigated the maintenance and functional characteristics of memory T cells at the cervix. We found that CD4 T cell depletion at the cervix mirrors CD4 depletion in blood. In all women, depletion of CD4 T cells at the cervix was associated with significant reductions in CD45RA− CCR7+ (central memory [CM]) T cells and the accumulat...

  7. Self-assembled, nanowire network electrodes for depleted bulk heterojunction solar cells

    KAUST Repository

    Lan, Xinzheng

    2013-01-06

    Herein, a solution-processed, bottom-up-fabricated, nanowire network electrode is developed. This electrode features a ZnO template which is converted into locally connected, infiltratable, TiO2 nanowires. This new electrode is used to build a depleted bulk heterojunction solar cell employing hybrid-passivated colloidal quantum dots. The new electrode allows the application of a thicker, and thus more light-absorbing, colloidal quantum dot active layer, from which charge extraction of an efficiency comparable to that obtained from a thinner, planar device could be obtained. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Myoferlin depletion in breast cancer cells promotes mesenchymal to epithelial shape change and stalls invasion.

    Directory of Open Access Journals (Sweden)

    Ruth Li

    Full Text Available Myoferlin (MYOF is a mammalian ferlin protein with homology to ancestral Fer-1, a nematode protein that regulates spermatic membrane fusion, which underlies the amoeboid-like movements of its sperm. Studies in muscle and endothelial cells have reported on the role of myoferlin in membrane repair, endocytosis, myoblast fusion, and the proper expression of various plasma membrane receptors. In this study, using an in vitro human breast cancer cell model, we demonstrate that myoferlin is abundantly expressed in invasive breast tumor cells. Depletion of MYOF using lentiviral-driven shRNA expression revealed that MDA-MB-231 cells reverted to an epithelial morphology, suggesting at least some features of mesenchymal to epithelial transition (MET. These observations were confirmed by the down-regulation of some mesenchymal cell markers (e.g., fibronectin and vimentin and coordinate up-regulation of the E-cadherin epithelial marker. Cell invasion assays using Boyden chambers showed that loss of MYOF led to a significant diminution in invasion through Matrigel or type I collagen, while cell migration was unaffected. PCR array and screening of serum-free culture supernatants from shRNA(MYOF transduced MDA-MB-231 cells indicated a significant reduction in the steady-state levels of several matrix metalloproteinases. These data when considered in toto suggest a novel role of MYOF in breast tumor cell invasion and a potential reversion to an epithelial phenotype upon loss of MYOF.

  9. RNA G-quadruplexes are globally unfolded in eukaryotic cells and depleted in bacteria

    Science.gov (United States)

    Guo, Junjie U.; Bartel, David P.

    2017-01-01

    In vitro, some RNAs can form stable four-stranded structures known as G-quadruplexes. Although RNA G-quadruplexes have been implicated in post-transcriptional gene regulation and diseases, direct evidence for their formation in cells has been lacking. Here, we identified thousands of mammalian RNA regions that can fold into G-quadruplexes in vitro, but in contrast to previous assumptions, these regions were overwhelmingly unfolded in cells. Model RNA G-quadruplexes that were unfolded in eukaryotic cells were folded when ectopically expressed in Escherichia coli; however, they impaired translation and growth, which helps explain why we detected few G-quadruplex–forming regions in bacterial transcriptomes. Our results suggest that eukaryotes have a robust machinery that globally unfolds RNA G-quadruplexes, whereas some bacteria have instead undergone evolutionary depletion of G-quadruplex–forming sequences. PMID:27708011

  10. Acute dyskerin depletion triggers cellular senescence and renders osteosarcoma cells resistant to genotoxic stress-induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Ping; Mobasher, Maral E.; Alawi, Faizan, E-mail: falawi@upenn.edu

    2014-04-18

    Highlights: • Dyskerin depletion triggers cellular senescence in U2OS osteosarcoma cells. • Dyskerin-depleted cells are resistant to apoptosis induced by genotoxic stress. • Chromatin relaxation sensitizes dyskerin-depleted cells to apoptosis. - Abstract: Dyskerin is a conserved, nucleolar RNA-binding protein implicated in an increasing array of fundamental cellular processes. Germline mutation in the dyskerin gene (DKC1) is the cause of X-linked dyskeratosis congenita (DC). Conversely, wild-type dyskerin is overexpressed in sporadic cancers, and high-levels may be associated with poor prognosis. It was previously reported that acute loss of dyskerin function via siRNA-mediated depletion slowed the proliferation of transformed cell lines. However, the mechanisms remained unclear. Using human U2OS osteosarcoma cells, we show that siRNA-mediated dyskerin depletion induced cellular senescence as evidenced by proliferative arrest, senescence-associated heterochromatinization and a senescence-associated molecular profile. Senescence can render cells resistant to apoptosis. Conversely, chromatin relaxation can reverse the repressive effects of senescence-associated heterochromatinization on apoptosis. To this end, genotoxic stress-induced apoptosis was suppressed in dyskerin-depleted cells. In contrast, agents that induce chromatin relaxation, including histone deacetylase inhibitors and the DNA intercalator chloroquine, sensitized dyskerin-depleted cells to apoptosis. Dyskerin is a core component of the telomerase complex and plays an important role in telomere homeostasis. Defective telomere maintenance resulting in premature senescence is thought to primarily underlie the pathogenesis of X-linked DC. Since U2OS cells are telomerase-negative, this leads us to conclude that loss of dyskerin function can also induce cellular senescence via mechanisms independent of telomere shortening.

  11. Androgen Depletion Induces Senescence in Prostate Cancer Cells through Down-regulation of Skp2

    Directory of Open Access Journals (Sweden)

    Zuzana Pernicová

    2011-06-01

    Full Text Available Although the induction of senescence in cancer cells is a potent mechanism of tumor suppression, senescent cells remain metabolically active and may secrete a broad spectrum of factors that promote tumorigenicity in neighboring malignant cells. Here we show that androgen deprivation therapy (ADT, a widely used treatment for advanced prostate cancer, induces a senescence-associated secretory phenotype in prostate cancer epithelial cells, indicated by increases in senescence-associated β-galactosidase activity, heterochromatin protein 1β foci, and expression of cathepsin B and insulin-like growth factor binding protein 3. Interestingly, ADT also induced high levels of vimentin expression in prostate cancer cell lines in vitro and in human prostate tumors in vivo. The induction of the senescence-associated secretory phenotype by androgen depletion was mediated, at least in part, by down-regulation of S-phase kinase-associated protein 2, whereas the neuroendocrine differentiation of prostate cancer cells was under separate control. These data demonstrate a previously unrecognized link between inhibition of androgen receptor signaling, down-regulation of S-phase kinase-associated protein 2, and the appearance of secretory, tumor-promoting senescent cells in prostate tumors. We propose that ADT may contribute to the development of androgen-independent prostate cancer through modulation of the tissue microenvironment by senescent cells.

  12. Golgi enlargement in Arf-depleted yeast cells is due to altered dynamics of cisternal maturation

    Science.gov (United States)

    Bhave, Madhura; Papanikou, Effrosyni; Iyer, Prasanna; Pandya, Koushal; Jain, Bhawik Kumar; Ganguly, Abira; Sharma, Chandrakala; Pawar, Ketakee; Austin, Jotham; Day, Kasey J.; Rossanese, Olivia W.; Glick, Benjamin S.; Bhattacharyya, Dibyendu

    2014-01-01

    ABSTRACT Regulation of the size and abundance of membrane compartments is a fundamental cellular activity. In Saccharomyces cerevisiae, disruption of the ADP-ribosylation factor 1 (ARF1) gene yields larger and fewer Golgi cisternae by partially depleting the Arf GTPase. We observed a similar phenotype with a thermosensitive mutation in Nmt1, which myristoylates and activates Arf. Therefore, partial depletion of Arf is a convenient tool for dissecting mechanisms that regulate Golgi structure. We found that in arf1Δ cells, late Golgi structure is particularly abnormal, with the number of late Golgi cisternae being severely reduced. This effect can be explained by selective changes in cisternal maturation kinetics. The arf1Δ mutation causes early Golgi cisternae to mature more slowly and less frequently, but does not alter the maturation of late Golgi cisternae. These changes quantitatively explain why late Golgi cisternae are fewer in number and correspondingly larger. With a stacked Golgi, similar changes in maturation kinetics could be used by the cell to modulate the number of cisternae per stack. Thus, the rates of processes that transform a maturing compartment can determine compartmental size and copy number. PMID:24190882

  13. Depletion of mitochondrial fission factor DRP1 causes increased apoptosis in human colon cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Inoue-Yamauchi, Akane, E-mail: ainoyama@research.twmu.ac.jp [Department of Pathology, Tokyo Women' s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (Japan); Oda, Hideaki [Department of Pathology, Tokyo Women' s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (Japan)

    2012-04-27

    Highlights: Black-Right-Pointing-Pointer DRP1 is required for mitochondrial fission in colon cancer cells. Black-Right-Pointing-Pointer DRP1 participates in inhibition of colon cancer cell apoptosis. Black-Right-Pointing-Pointer DRP1 can inhibit apoptosis through the regulation of cytochrome c release. -- Abstract: Mitochondria play a critical role in regulation of apoptosis, a form of programmed cell death, by releasing apoptogenic factors including cytochrome c. Growing evidence suggests that dynamic changes in mitochondrial morphology are involved in cellular apoptotic response. However, whether DRP1-mediated mitochondrial fission is required for induction of apoptosis remains speculative. Here, we show that siRNA-mediated DRP1 knockdown promoted accumulation of elongated mitochondria in HCT116 and SW480 human colon cancer cells. Surprisingly, DRP1 down-regulation led to decreased proliferation and increased apoptosis of these cells. A higher rate of cytochrome c release and reductions in mitochondrial membrane potential were also revealed in DRP1-depleted cells. Taken together, our present findings suggest that mitochondrial fission factor DRP1 inhibits colon cancer cell apoptosis through the regulation of cytochrome c release and mitochondrial membrane integrity.

  14. B-Cell Depletion Therapy in Systemic Sclerosis: Experimental Rationale and Update on Clinical Evidence

    Directory of Open Access Journals (Sweden)

    Dimitrios Daoussis

    2011-01-01

    Full Text Available Systemic sclerosis (SSc is a systemic rheumatic disease with poor prognosis since therapeutic options are limited. Recent evidence from animal models suggests that B-cells may be actively involved in the fibrotic process. B-cells from tight skin mice, an animal model of scleroderma, display a “hyperresponsive” phenotype; treatment with rituximab (RTX significantly attenuates skin fibrosis in this animal model. In humans, B-cell infiltration is a prominent finding in most lung biopsies obtained from patients with SSc-associated interstitial lung disease. Several open label studies have assessed the clinical efficacy of RTX in SSc. In most patients skin fibrosis improved; lung function either improved or remained stable. Definite conclusions regarding the clinical efficacy of RTX in SSc cannot be drawn but further exploration with a multicenter, randomized study is warranted.

  15. Depletion of CD25+ cells during acute toxoplasmosis does not significantly increase mortality in Swiss OF1 mice

    Directory of Open Access Journals (Sweden)

    Haroon Akbar

    2012-03-01

    Full Text Available The interleukin (IL-2R alpha chain (CD25 is expressed on regulatory T cells (Treg, which constitute more than 85% of the CD25+ T cell population in a naïve mouse. CD25 is also expressed on effector T cells in mice suffering from an acute infection by the obligate intracellular protozoan parasite, Toxoplasma gondii. Lethal toxoplasmosis is accompanied by a significant loss of Treg in mice naturally susceptible to toxoplasmosis. The present study was done to explore the role of Treg cells using an anti-CD25 antibody-mediated depletion in mice naturally resistant to toxoplasmosis. Although a significant decrease in the percentage of Treg cells was observed following anti-CD25 monoclonal antibody injections, the depletion of CD25+ cells during acute toxoplasmosis did not significantly increase the mortality of Swiss OF1 mice and no significant difference was observed in the brain parasitic load between the mice in the depleted-infected and isotype-infected groups. We found no significant difference between the titres of total IgG in the sera of the mice from the two groups in the chronic phase. However, CD25+ cells depletion was followed by significantly higher levels of IL-12 in the serum of depleted mice than in that of mice injected with the isotype control antibody.

  16. Ets Factors Regulate Neural Stem Cell Depletion and Gliogenesis in Ras Pathway Glioma.

    Science.gov (United States)

    Breunig, Joshua J; Levy, Rachelle; Antonuk, C Danielle; Molina, Jessica; Dutra-Clarke, Marina; Park, Hannah; Akhtar, Aslam Abbasi; Kim, Gi Bum; Hu, Xin; Bannykh, Serguei I; Verhaak, Roel G W; Danielpour, Moise

    2015-07-14

    As the list of putative driver mutations in glioma grows, we are just beginning to elucidate the effects of dysregulated developmental signaling pathways on the transformation of neural cells. We have employed a postnatal, mosaic, autochthonous glioma model that captures the first hours and days of gliomagenesis in more resolution than conventional genetically engineered mouse models of cancer. We provide evidence that disruption of the Nf1-Ras pathway in the ventricular zone at multiple signaling nodes uniformly results in rapid neural stem cell depletion, progenitor hyperproliferation, and gliogenic lineage restriction. Abolishing Ets subfamily activity, which is upregulated downstream of Ras, rescues these phenotypes and blocks glioma initiation. Thus, the Nf1-Ras-Ets axis might be one of the select molecular pathways that are perturbed for initiation and maintenance in glioma.

  17. Dystroglycan Depletion Impairs Actin-Dependent Functions of Differentiated Kasumi-1 Cells.

    Directory of Open Access Journals (Sweden)

    Marco Antonio Escárcega-Tame

    Full Text Available Dystroglycan has recently been characterised in blood tissue cells, as part of the dystrophin glycoprotein complex involved in the differentiation process of neutrophils.In the present study we have investigated the role of dystroglycan in the human promyelocytic leukemic cell line Kasumi-1 differentiated to macrophage-like cells.We characterised the pattern expression and subcellular distribution of dystroglycans in non-differentiated and differentiated Kasumi-1 cells.Our results demonstrated by WB and flow cytometer assays that during the differentiation process to macrophages, dystroglycans were down-regulated; these results were confirmed with qRT-PCR assays. Additionally, depletion of dystroglycan by RNAi resulted in altered morphology and reduced properties of differentiated Kasumi-1 cells, including morphology, migration and phagocytic activities although secretion of IL-1β and expression of markers of differentiation are not altered.Our findings strongly implicate dystroglycan as a key membrane adhesion protein involved in actin-based structures during the differentiation process in Kasumi-1 cells.

  18. Continuous-Wave Stimulated Emission Depletion Microscope for Imaging Actin Cytoskeleton in Fixed and Live Cells

    Directory of Open Access Journals (Sweden)

    Bhanu Neupane

    2015-09-01

    Full Text Available Stimulated emission depletion (STED microscopy provides a new opportunity to study fine sub-cellular structures and highly dynamic cellular processes, which are challenging to observe using conventional optical microscopy. Using actin as an example, we explored the feasibility of using a continuous wave (CW-STED microscope to study the fine structure and dynamics in fixed and live cells. Actin plays an important role in cellular processes, whose functioning involves dynamic formation and reorganization of fine structures of actin filaments. Frequently used confocal fluorescence and STED microscopy dyes were employed to image fixed PC-12 cells (dyed with phalloidin- fluorescein isothiocyante and live rat chondrosarcoma cells (RCS transfected with actin-green fluorescent protein (GFP. Compared to conventional confocal fluorescence microscopy, CW-STED microscopy shows improved spatial resolution in both fixed and live cells. We were able to monitor cell morphology changes continuously; however, the number of repetitive analyses were limited primarily by the dyes used in these experiments and could be improved with the use of dyes less susceptible to photobleaching. In conclusion, CW-STED may disclose new information for biological systems with a proper characteristic length scale. The challenges of using CW-STED microscopy to study cell structures are discussed.

  19. A versatile viral system for expression and depletion of proteins in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Eric Campeau

    Full Text Available The ability to express or deplete proteins in living cells is crucial for the study of biological processes. Viral vectors are often useful to deliver DNA constructs to cells that are difficult to transfect by other methods. Lentiviruses have the additional advantage of being able to integrate into the genomes of non-dividing mammalian cells. However, existing viral expression systems generally require different vector backbones for expression of cDNA, small hairpin RNA (shRNA or microRNA (miRNA and provide limited drug selection markers. Furthermore, viral backbones are often recombinogenic in bacteria, complicating the generation and maintenance of desired clones. Here, we describe a collection of 59 vectors that comprise an integrated system for constitutive or inducible expression of cDNAs, shRNAs or miRNAs, and use a wide variety of drug selection markers. These vectors are based on the Gateway technology (Invitrogen whereby the cDNA, shRNA or miRNA of interest is cloned into an Entry vector and then recombined into a Destination vector that carries the chosen viral backbone and drug selection marker. This recombination reaction generates the desired product with >95% efficiency and greatly reduces the frequency of unwanted recombination in bacteria. We generated Destination vectors for the production of both retroviruses and lentiviruses. Further, we characterized each vector for its viral titer production as well as its efficiency in expressing or depleting proteins of interest. We also generated multiple types of vectors for the production of fusion proteins and confirmed expression of each. We demonstrated the utility of these vectors in a variety of functional studies. First, we show that the FKBP12 Destabilization Domain system can be used to either express or deplete the protein of interest in mitotically-arrested cells. Also, we generate primary fibroblasts that can be induced to senesce in the presence or absence of DNA damage

  20. Clinical grade isolation of regulatory T cells from G-CSF mobilized peripheral blood improves with initial depletion of monocytes.

    Science.gov (United States)

    Patel, Pritesh; Mahmud, Dolores; Park, Youngmin; Yoshinaga, Kazumi; Mahmud, Nadim; Rondelli, Damiano

    2015-01-01

    Clinical isolation of circulating CD4(+)CD25(+) regulatory T cells (Tregs) from peripheral blood mononuclear cells is usually performed by CD4(+) cell negative selection followed by CD25(+) cell positive selection. Although G-CSF mobilized peripheral blood (G-PBSC) contains a high number of Tregs, a high number of monocytes in G-PBSC limits Treg isolation. Using a small scale device (MidiMACS, Miltenyi) we initially demonstrated that an initial depletion of monocytes would be necessary to obtaina separation of CD4(+)CD25(+)FoxP3(+)CD127(-) cells from G-PBSC (G-Tregs) with a consistent purity >70% and inhibitory activity of T cell alloreactivity in-vitro. We then validated the same approach in a clinical scale setting by separating G-Tregs with clinically available antibodies to perform a CD8(+)CD19(+)CD14(+) cell depletion followed by CD25(+) cell selection (2-step process) or by adding an initial CD14(+) cell depletion (3-step process) using a CliniMACS column. The 3-step approach resulted in a better purity (81±12% vs. 35±33%) and yield (66% vs. 39%). Clinically isolated G-Tregs were also FoxP3(+)CD127(dim) and functionally suppressive in-vitro. Our findings suggest that a better and more consistent purity of Tregs can be achieved from G-PBSC by an initial single depletion of monocytes prior to selection of CD4(+)CD25(+) cells.

  1. Clinical grade isolation of regulatory T cells from G-CSF mobilized peripheral blood improves with initial depletion of monocytes

    Science.gov (United States)

    Patel, Pritesh; Mahmud, Dolores; Park, Youngmin; Yoshinaga, Kazumi; Mahmud, Nadim; Rondelli, Damiano

    2015-01-01

    Clinical isolation of circulating CD4+CD25+ regulatory T cells (Tregs) from peripheral blood mononuclear cells is usually performed by CD4+ cell negative selection followed by CD25+ cell positive selection. Although G-CSF mobilized peripheral blood (G-PBSC) contains a high number of Tregs, a high number of monocytes in G-PBSC limits Treg isolation. Using a small scale device (MidiMACS, Miltenyi) we initially demonstrated that an initial depletion of monocytes would be necessary to obtaina separation of CD4+CD25+FoxP3+CD127- cells from G-PBSC (G-Tregs) with a consistent purity >70% and inhibitory activity of T cell alloreactivity in-vitro. We then validated the same approach in a clinical scale setting by separating G-Tregs with clinically available antibodies to perform a CD8+CD19+CD14+ cell depletion followed by CD25+ cell selection (2-step process) or by adding an initial CD14+ cell depletion (3-step process) using a CliniMACS column. The 3-step approach resulted in a better purity (81±12% vs. 35±33%) and yield (66% vs. 39%). Clinically isolated G-Tregs were also FoxP3+CD127dim and functionally suppressive in-vitro. Our findings suggest that a better and more consistent purity of Tregs can be achieved from G-PBSC by an initial single depletion of monocytes prior to selection of CD4+CD25+ cells. PMID:27069755

  2. Inhibition of p38-MAPK potentiates cisplatin-induced apoptosis via GSH depletion and increases intracellular drug accumulation in growth-arrested kidney tubular epithelial cells.

    Science.gov (United States)

    Rodríguez-García, Maria Elena; Quiroga, Adoración G; Castro, José; Ortiz, Alberto; Aller, Patricio; Mata, Felicísima

    2009-10-01

    We were interested in analyzing the regulation by mitogen-activated protein kinases (MAPKs) of cisplatin-provoked toxicity in epithelial renal tubule cell lines, when assayed under culture conditions (cell confluence plus serum deprivation), which mimic the characteristics of a nonproliferating epithelium. Under these restrictive growth conditions, cisplatin induced apoptosis with lower efficacy than in exponentially growing cells, and decreased p38-MAPK phosphorylation in NRK-52E and other (LLC-PK1, MDCK, HK2) cell lines. Moreover, cisplatin-provoked apoptosis was potentiated by cotreatment with p38-MAPK-specific inhibitors (SB203580, SB220025) or transfection with a kinase-negative mutant of MKK6, whereas c-Jun NH2-terminal kinase or extracellular signal-regulated kinase/MAPK and ERK Kinase inhibitors were ineffective. By contrast, when applied to exponentially growing cells, cisplatin stimulated p38-MAPK phosphorylation and apoptosis, was attenuated by kinase inhibitors. Treatment of confluent/serum-deprived cells with cisplatin caused mitochondrial transmembrane potential disruption and activated the mitochondrial apoptotic pathway, as indicated by the decrease in Bcl-X(L) expression, increase in Bax expression and cytochrome c release, and these effects were potentiated by cotreatment with SB203580. Treatment of confluent/serum-deprived cells with cisplatin plus SB203580 decreased the intracellular reduced glutathione (GSH) content, and increased intracellular cisplatin accumulation as well as cisplatin binding to DNA. Cotreatment with the GSH-depleting agent D,L-buthionine-R,S-sulfoximine also potentiated cisplatin-provoked apoptosis. In summary, p38-MAPK inhibition potentiates cisplatin-provoked apoptosis in growth-arrested epithelial renal tubule cells, a result that may be explained at least in part by GSH depletion and drug transport alteration.

  3. Depletion of host CCR7(+) dendritic cells prevented donor T cell tissue tropism in anti-CD3-conditioned recipients.

    Science.gov (United States)

    He, Wei; Racine, Jeremy J; Johnston, Heather F; Li, Xiaofan; Li, Nainong; Cassady, Kaniel; Liu, Can; Deng, Ruishu; Martin, Paul; Forman, Stephen; Zeng, Defu

    2014-07-01

    We reported previously that anti-CD3 mAb treatment before hematopoietic cell transplantation (HCT) prevented graft-versus-host disease (GVHD) and preserved graft-versus-leukemia (GVL) effects in mice. These effects were associated with downregulated donor T cell expression of tissue-specific homing and chemokine receptors, marked reduction of donor T cell migration into GVHD target tissues, and deletion of CD103(+) dendritic cells (DCs) in mesenteric lymph nodes (MLN). MLN CD103(+) DCs and peripheral lymph node (PLN) DCs include CCR7(+) and CCR7(-) subsets, but the role of these DC subsets in regulating donor T cell expression of homing and chemokine receptors remain unclear. Here, we show that recipient CCR7(+), but not CCR7(-), DCs in MLN induced donor T cell expression of gut-specific homing and chemokine receptors in a retinoid acid-dependent manner. CCR7 regulated activated DC migration from tissue to draining lymph node, but it was not required for the ability of DCs to induce donor T cell expression of tissue-specific homing and chemokine receptors. Finally, anti-CD3 treatment depleted CCR7(+) but not CCR7(-) DCs by inducing sequential expansion and apoptosis of CCR7(+) DCs in MLN and PLN. Apoptosis of CCR7(+) DCs was associated with DC upregulation of Fas expression and natural killer cell but not T, B, or dendritic cell upregulation of FasL expression in the lymph nodes. These results suggest that depletion of CCR7(+) host-type DCs, with subsequent inhibition of donor T cell migration into GVHD target tissues, can be an effective approach in prevention of acute GVHD and preservation of GVL effects.

  4. Live-attenuated measles virus vaccine targets dendritic cells and macrophages in muscle of nonhuman primates

    NARCIS (Netherlands)

    L.J. Rennick (Linda); R.D. de Vries (Rory); T.J. Carsillo (Thomas J.); K. Lemon (Ken); G. van Amerongen (Geert); M. Ludlow (Martin); D.T. Nguyen (Tien); S. Yüksel (Selma); R.J. Verbugh (Joyce); P. Haddock (Paula); S. McQuaid (Stephen); W.P. Duprex (Paul); R.L. de Swart (Rik)

    2015-01-01

    textabstractAlthough live-attenuated measles virus (MV) vaccines have been used successfully for over 50 years, the target cells that sustain virus replication in vivo are still unknown. We generated a reverse genetics system for the live-attenuated MV vaccine strain Edmonston- Zagreb (EZ), allowing

  5. Depletion of polyamines prevents the neurotrophic activity of the GABA-agonist THIP in cultured rat cerebellar granule cells

    DEFF Research Database (Denmark)

    Abraham, J H; Hansen, Gert Helge; Seiler, N

    1993-01-01

    Effects of polyamine depletion by alpha-difluoromethylornithine (DFMO) were studied on the GABA-agonist mediated enhancement of the morphological development of cultured rat cerebellar granule cells. An increase in the number of neurite extending cells and in the cytoplasmic density of organelles...... endoplasmic reticulum, Golgi apparatus and different types of vesicles was prevented by the exposure to DFMO....

  6. Transfusion of leukocyte-depleted red blood cells is not a risk factor for nosocomial infections in critically ill children

    NARCIS (Netherlands)

    van der Wal, Judith; van Heerde, Marc; Markhorst, Dick G.; Kneyber, Martin C. J.

    2011-01-01

    Objectives: Transfusion of red blood cells is increasingly linked with adverse outcomes in critically ill children. We tested the hypothesis that leukocyte-depleted red blood cell transfusions were independently associated with increased development of bloodstream infections, ventilator-associated p

  7. A single dose of rituximab does not deplete B cells in secondary lymphoid organs but alters phenotype and function

    NARCIS (Netherlands)

    Kamburova, E.G.; Koenen, H.J.P.M.; Borgman, K.J.; Berge, I.J. Ten; Joosten, I.; Hilbrands, L.B.

    2013-01-01

    A single dose of the anti-CD20 monoclonal antibody rituximab induces a nearly complete B cell depletion in peripheral blood, but not in secondary lymphoid organs. Modulation of this remaining B cell population due to rituximab treatment may contribute to the therapeutic effects of rituximab. To asse

  8. Inhibitory effect of Polo-like kinase 1 depletion on mitosis and apoptosis of gastric cancer cells

    OpenAIRE

    Chen, Xue-Hua; Lan, Bin; Qu, Ying; ZHANG, XIAO-QING; Cai, Qu; Liu, Bing-Ya; Zhu, Zheng-Gang

    2006-01-01

    AIM: Polo-like kinase 1 (PLK1) serine/threonine kinase plays a vital role in multiple phases of mitosis in gastric cancer cells. To investigate the effect of PLK1 depletion on mitosis and apoptosis of gastric cancer cells.

  9. Selective depletion of non-specific T cells as an early event in T cell response to bacterial and viral infections

    Institute of Scientific and Technical Information of China (English)

    JIANG Jiu

    2005-01-01

    @@ Early T cell depletion occurs prior to the development of an effective immune response to infections.Both antigen-specific and non-specific T cells are induced to express early activation markers soon after microbial infections.This is followed by massive depletion of non-specific T cells and extensive proliferation of antigen-specific T cells.Proliferating antigen-specific cells exhibit a broad spectrum of late activation markers while non-specific cells exhibit no sign of further activation before succumbing to apoptosis.These results have crucial implications for the understanding of early events in the development of a robust T cell response.

  10. Lycopene Pretreatment Ameliorates Acute Ethanol Induced NAD+ Depletion in Human Astroglial Cells

    Directory of Open Access Journals (Sweden)

    Jade Guest

    2015-01-01

    Full Text Available Excessive alcohol consumption is associated with reduced brain volume and cognition. While the mechanisms by which ethanol induces these deleterious effects in vivo are varied most are associated with increased inflammatory and oxidative processes. In order to further characterise the effect of acute ethanol exposure on oxidative damage and NAD+ levels in the brain, human U251 astroglioma cells were exposed to physiologically relevant doses of ethanol (11 mM, 22 mM, 65 mM, and 100 mM for ≤ 30 minutes. Ethanol exposure resulted in a dose dependent increase in both ROS and poly(ADP-ribose polymer production. Significant decreases in total NAD(H and sirtuin 1 activity were also observed at concentrations ≥ 22 mM. Similar to U251 cells, exposure to ethanol (≥22 mM decreased levels of NAD(H in primary human astrocytes. NAD(H depletion in primary astrocytes was prevented by pretreatment with 1 μM of lycopene for 3.5 hours. Unexpectedly, in U251 cells lycopene treatment at concentrations ≥ 5 μM resulted in significant reductions in [NAD(H]. This study suggests that exposure of the brain to alcohol at commonly observed blood concentrations may cause transitory oxidative damage which may be at least partly ameliorated by lycopene.

  11. A mitotic kinase scaffold depleted in testicular seminomas impacts spindle orientation in germ line stem cells.

    Science.gov (United States)

    Hehnly, Heidi; Canton, David; Bucko, Paula; Langeberg, Lorene K; Ogier, Leah; Gelman, Irwin; Santana, L Fernando; Wordeman, Linda; Scott, John D

    2015-09-25

    Correct orientation of the mitotic spindle in stem cells underlies organogenesis. Spindle abnormalities correlate with cancer progression in germ line-derived tumors. We discover a macromolecular complex between the scaffolding protein Gravin/AKAP12 and the mitotic kinases, Aurora A and Plk1, that is down regulated in human seminoma. Depletion of Gravin correlates with an increased mitotic index and disorganization of seminiferous tubules. Biochemical, super-resolution imaging, and enzymology approaches establish that this Gravin scaffold accumulates at the mother spindle pole during metaphase. Manipulating elements of the Gravin-Aurora A-Plk1 axis prompts mitotic delay and prevents appropriate assembly of astral microtubules to promote spindle misorientation. These pathological responses are conserved in seminiferous tubules from Gravin(-/-) mice where an overabundance of Oct3/4 positive germ line stem cells displays randomized orientation of mitotic spindles. Thus, we propose that Gravin-mediated recruitment of Aurora A and Plk1 to the mother (oldest) spindle pole contributes to the fidelity of symmetric cell division.

  12. Whole Body Irradiation Induces Cutaneous Dendritic Cells Depletion via NF-κB Activation

    Directory of Open Access Journals (Sweden)

    Yanyong Yang

    2013-07-01

    Full Text Available Background: Effect of ionizing radiation on cutaneous dendritic cells (cDC is critical to its influence on immune status of the skin, which plays an important role in the progression and recovery of radiation skin sickness. This study was to study the influence of whole body irradiation (WBI on the cDC. Methods: Density of epidermal and dermal DC was determined with a fluorescent microscopy and the DC numbers in lymph node were measured by flow cytometry. A FITC induced migration assay was also used to study the migration of DC. The expressions of cytokines and chemokines were evaluated by Realtime PCR, and the protein level of was measured by Western blot. Results: WBI caused depletion of cDC in epidermal as well as dermal and augmented FITC-induced migration of DC to the draining lymph node (LN. The number of DC migrated from ear explants to the CCL19-containing medium also increased after exposure to WBI. It was also found that WBI increased mRNA level of CCL19/CCL21 as well as CCR7 in LN and skin tissue. The expressions of TNFa, IL-1a, IL-1ß, and IL-6 in skin tissues were also greatly induced by WBI in a dose dependent manner. Finally, we found that WBI induced translocation of nuclear factor κB (NF-κB and that the radiation-induced migration of DC was blocked by NF-κB inhibitor or TLR4 knockout. Conclusion: WBI caused cDC depletion through induction of DC migration to the draining LN, which might result from the activation of NF-κB and the induction of inflammatory microenvironment within the skin.

  13. Single-cell time-lapse analysis of depletion of the universally conserved essential protein YgjD

    Directory of Open Access Journals (Sweden)

    Ackermann Martin

    2011-05-01

    Full Text Available Abstract Background The essential Escherichia coli gene ygjD belongs to a universally conserved group of genes whose function has been the focus of a number of recent studies. Here, we put ygjD under control of an inducible promoter, and used time-lapse microscopy and single cell analysis to investigate the phenotypic consequences of the depletion of YgjD protein from growing cells. Results We show that loss of YgjD leads to a marked decrease in cell size and termination of cell division. The transition towards smaller size occurs in a controlled manner: cell elongation and cell division remain coupled, but cell size at division decreases. We also find evidence that depletion of YgjD leads to the synthesis of the intracellular signaling molecule (pppGpp, inducing a cellular reaction resembling the stringent response. Concomitant deletion of the relA and spoT genes - leading to a strain that is uncapable of synthesizing (pppGpp - abrogates the decrease in cell size, but does not prevent termination of cell division upon YgjD depletion. Conclusions Depletion of YgjD protein from growing cells leads to a decrease in cell size that is contingent on (pppGpp, and to a termination of cell division. The combination of single-cell timelapse microscopy and statistical analysis can give detailed insights into the phenotypic consequences of the loss of essential genes, and can thus serve as a new tool to study the function of essential genes.

  14. HIV-1 infection and CD4 T cell depletion in the humanized Rag2-/-γc-/- (RAG-hu mouse model

    Directory of Open Access Journals (Sweden)

    Connick Elizabeth

    2006-11-01

    Full Text Available Abstract Background The currently well-established humanized mouse models, namely the hu-PBL-SCID and SCID-hu systems played an important role in HIV pathogenesis studies. However, despite many notable successes, several limitations still exist. They lack multi-lineage human hematopoiesis and a functional human immune system. These models primarily reflect an acute HIV infection with rapid CD4 T cell loss thus limiting pathogenesis studies to a short-term period. The new humanized Rag2-/-γc-/- mouse model (RAG-hu created by intrahepatic injection of CD34 hematopoietic stem cells sustains long-term multi-lineage human hematopoiesis and is capable of mounting immune responses. Thus, this model shows considerable promise to study long-term in vivo HIV infection and pathogenesis. Results Here we demonstrate that RAG-hu mice produce human cell types permissive to HIV-1 infection and that they can be productively infected by HIV-1 ex vivo. To assess the capacity of these mice to sustain long-term infection in vivo, they were infected by either X4-tropic or R5-tropic HIV-1. Viral infection was assessed by PCR, co-culture, and in situ hybridization. Our results show that both X4 and R5 viruses are capable of infecting RAG-hu mice and that viremia lasts for at least 30 weeks. Moreover, HIV-1 infection leads to CD4 T cell depletion in peripheral blood and thymus, thus mimicking key aspects of HIV-1 pathogenesis. Additionally, a chimeric HIV-1 NL4-3 virus expressing a GFP reporter, although capable of causing viremia, failed to show CD4 T cell depletion possibly due to attenuation. Conclusion The humanized RAG-hu mouse model, characterized by its capacity for sustained multi-lineage human hematopoiesis and immune response, can support productive HIV-1 infection. Both T cell and macrophage tropic HIV-1 strains can cause persistent infection of RAG-hu mice resulting in CD4 T cell loss. Prolonged viremia in the context of CD4 T cell depletion seen in this

  15. DNA Damage Follows Repair Factor Depletion and Portends Genome Variation in Cancer Cells after Pore Migration.

    Science.gov (United States)

    Irianto, Jerome; Xia, Yuntao; Pfeifer, Charlotte R; Athirasala, Avathamsa; Ji, Jiazheng; Alvey, Cory; Tewari, Manu; Bennett, Rachel R; Harding, Shane M; Liu, Andrea J; Greenberg, Roger A; Discher, Dennis E

    2017-01-23

    Migration through micron-size constrictions has been seen to rupture the nucleus, release nuclear-localized GFP, and cause localized accumulations of ectopic 53BP1-a DNA repair protein. Here, constricted migration of two human cancer cell types and primary mesenchymal stem cells (MSCs) increases DNA breaks throughout the nucleoplasm as assessed by endogenous damage markers and by electrophoretic "comet" measurements. Migration also causes multiple DNA repair proteins to segregate away from DNA, with cytoplasmic mis-localization sustained for many hours as is relevant to delayed repair. Partial knockdown of repair factors that also regulate chromosome copy numbers is seen to increase DNA breaks in U2OS osteosarcoma cells without affecting migration and with nucleoplasmic patterns of damage similar to constricted migration. Such depletion also causes aberrant levels of DNA. Migration-induced nuclear damage is nonetheless reversible for wild-type and sub-cloned U2OS cells, except for lasting genomic differences between stable clones as revealed by DNA arrays and sequencing. Gains and losses of hundreds of megabases in many chromosomes are typical of the changes and heterogeneity in bone cancer. Phenotypic differences that arise from constricted migration of U2OS clones are further illustrated by a clone with a highly elongated and stable MSC-like shape that depends on microtubule assembly downstream of the transcription factor GATA4. Such changes are consistent with reversion to a more stem-like state upstream of cancerous osteoblastic cells. Migration-induced genomic instability can thus associate with heritable changes.

  16. Rituximab efficiently depletes B cells in lung tumors and normal lung tissue [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Albane Joly-Battaglini

    2016-01-01

    Full Text Available Rituximab is a monoclonal antibody that targets the CD20 B-cell-specific antigen and is widely used as therapy for B-cell lymphoma. Since rituximab depletes both malignant and normal B cells, it is increasingly being used to treat various conditions in which normal B cells have a pathogenic role, such as rheumatoid arthritis and multiple sclerosis. It is well-established that rituximab efficiently eliminates B cells in blood, lymph nodes, and spleen. In contrast, the effect of rituximab in non-lymphoid tissues remains poorly documented and is debated. Here, we report a rheumatoid arthritis patient who was treated with rituximab before receiving thoracic surgery for non-small cell lung cancer. Using flow cytometry and immunohistochemistry, we show that rituximab efficiently depleted CD20-positive B cells in a primary lung tumor, in lung-associated lymph nodes, and in normal lung tissue. We conclude that rituximab may be very efficient at depleting normal B cells in the lungs. This property of rituximab may potentially be exploited for the treatment of conditions in which pathogenic B cells reside in the lungs. On the other hand, the clearance of lung B cells may provide an explanation for the rare cases of severe non-infectious pulmonary toxicity of rituximab.

  17. Separase phosphosite mutation leads to genome instability and primordial germ cell depletion during oogenesis.

    Directory of Open Access Journals (Sweden)

    Juan Xu

    Full Text Available To ensure equal chromosome segregation and the stability of the genome during cell division, Separase is strictly regulated primarily by Securin binding and inhibitory phosphorylation. By generating a mouse model that contained a mutation to the inhibitory phosphosite of Separase, we demonstrated that mice of both sexes are infertile. We showed that Separase deregulation leads to chromosome mis-segregation, genome instability, and eventually apoptosis of primordial germ cells (PGCs during embryonic oogenesis. Although the PGCs of mutant male mice were completely depleted, a population of PGCs from mutant females survived Separase deregulation. The surviving PGCs completed oogenesis but produced deficient initial follicles. These results indicate a sexual dimorphism effect on PGCs from Separase deregulation, which may be correlated with a gender-specific discrepancy of Securin. Our results reveal that Separase phospho-regulation is critical for genome stability in oogenesis. Furthermore, we provided the first evidence of a pre-zygotic mitotic chromosome segregation error resulting from Separase deregulation, whose sex-specific differences may be a reason for the sexual dimorphism of aneuploidy in gametogenesis.

  18. Depletion of SMN by RNA interference in HeLa cells induces defects in Cajal body formation.

    Science.gov (United States)

    Girard, Cyrille; Neel, Henry; Bertrand, Edouard; Bordonné, Rémy

    2006-01-01

    Neuronal degeneration in spinal muscular atrophy (SMA) is caused by reduced expression of the survival of motor neuron (SMN) protein. The SMN protein is ubiquitously expressed and is present both in the cytoplasm and in the nucleus where it localizes in Cajal bodies. The SMN complex plays an essential role for the biogenesis of spliceosomal U-snRNPs. In this article, we have used an RNA interference approach in order to analyse the effects of SMN depletion on snRNP assembly in HeLa cells. Although snRNP profiles are not perturbed in SMN-depleted cells, we found that SMN depletion gives rise to cytoplasmic accumulation of a GFP-SmB reporter protein. We also demonstrate that the SMN protein depletion induces defects in Cajal body formation with coilin being localized in multiple nuclear foci and in nucleolus instead of canonical Cajal bodies. Interestingly, the coilin containing foci do not contain snRNPs but appear to co-localize with U85 scaRNA. Because Cajal bodies represent the location in which snRNPs undergo 2'-O-methylation and pseudouridylation, our results raise the possibility that SMN depletion might give rise to a defect in the snRNA modification process.

  19. Depletion of the actin bundling protein SM22/transgelin increases actin dynamics and enhances the tumourigenic phenotypes of cells

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    Thompson Oliver

    2012-01-01

    Full Text Available Abstract Background SM22 has long been studied as an actin-associated protein. Interestingly, levels of SM22 are often reduced in tumour cell lines, while they are increased during senescence possibly indicating a role for SM22 in cell fate decisions via its interaction with actin. In this study we aimed to determine whether reducing levels of SM22 could actively contribute to a tumourigenic phenotype. Results We demonstrate that in REF52 fibroblasts, decreased levels of SM22 disrupt normal actin organization leading to changes in the motile behaviour of cells. Interestingly, SM22 depletion also led to an increase in the capacity of cells to spontaneously form podosomes with a concomitant increase in the ability to invade Matrigel. In PC3 prostate epithelial cancer cells by contrast, where SM22 is undetectable, re-expression of SM22 reduced the ability to invade Matrigel. Furthermore SM22 depleted cells also had reduced levels of reactive oxygen species when under serum starvation stress. Conclusions These findings suggest that depletion of SM22 could contribute to tumourigenic properties of cells. Reduction in SM22 levels would tend to promote cell survival when cells are under stress, such as in a hypoxic tumour environment, and may also contribute to increases in actin dynamics that favour metastatic potential.

  20. FAS-Based Cell Depletion Facilitates the Selective Isolation of Mouse Induced Pluripotent Stem Cells

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    Warlich, Eva; Schambach, Axel; Lock, Dominik; Wedekind, Dirk; Glage, Silke; Eckardt, Dominik; Bosio, Andreas; Knöbel, Sebastian

    2014-01-01

    Cellular reprogramming of somatic cells into induced pluripotent stem cells (iPSC) opens up new avenues for basic research and regenerative medicine. However, the low efficiency of the procedure remains a major limitation. To identify iPSC, many studies to date relied on the activation of pluripotency-associated transcription factors. Such strategies are either retrospective or depend on genetically modified reporter cells. We aimed at identifying naturally occurring surface proteins in a systematic approach, focusing on antibody-targeted markers to enable live-cell identification and selective isolation. We tested 170 antibodies for differential expression between mouse embryonic fibroblasts (MEF) and mouse pluripotent stem cells (PSC). Differentially expressed markers were evaluated for their ability to identify and isolate iPSC in reprogramming cultures. Epithelial cell adhesion molecule (EPCAM) and stage-specific embryonic antigen 1 (SSEA1) were upregulated early during reprogramming and enabled enrichment of OCT4 expressing cells by magnetic cell sorting. Downregulation of somatic marker FAS was equally suitable to enrich OCT4 expressing cells, which has not been described so far. Furthermore, FAS downregulation correlated with viral transgene silencing. Finally, using the marker SSEA-1 we exemplified that magnetic separation enables the establishment of bona fide iPSC and propose strategies to enrich iPSC from a variety of human source tissues. PMID:25029550

  1. FAS-based cell depletion facilitates the selective isolation of mouse induced pluripotent stem cells.

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    Eva Warlich

    Full Text Available Cellular reprogramming of somatic cells into induced pluripotent stem cells (iPSC opens up new avenues for basic research and regenerative medicine. However, the low efficiency of the procedure remains a major limitation. To identify iPSC, many studies to date relied on the activation of pluripotency-associated transcription factors. Such strategies are either retrospective or depend on genetically modified reporter cells. We aimed at identifying naturally occurring surface proteins in a systematic approach, focusing on antibody-targeted markers to enable live-cell identification and selective isolation. We tested 170 antibodies for differential expression between mouse embryonic fibroblasts (MEF and mouse pluripotent stem cells (PSC. Differentially expressed markers were evaluated for their ability to identify and isolate iPSC in reprogramming cultures. Epithelial cell adhesion molecule (EPCAM and stage-specific embryonic antigen 1 (SSEA1 were upregulated early during reprogramming and enabled enrichment of OCT4 expressing cells by magnetic cell sorting. Downregulation of somatic marker FAS was equally suitable to enrich OCT4 expressing cells, which has not been described so far. Furthermore, FAS downregulation correlated with viral transgene silencing. Finally, using the marker SSEA-1 we exemplified that magnetic separation enables the establishment of bona fide iPSC and propose strategies to enrich iPSC from a variety of human source tissues.

  2. Fibroblastic reticular cells from lymph nodes attenuate T cell expansion by producing nitric oxide.

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    Stefanie Siegert

    Full Text Available Adaptive immune responses are initiated when T cells encounter antigen on dendritic cells (DC in T zones of secondary lymphoid organs. T zones contain a 3-dimensional scaffold of fibroblastic reticular cells (FRC but currently it is unclear how FRC influence T cell activation. Here we report that FRC lines and ex vivo FRC inhibit T cell proliferation but not differentiation. FRC share this feature with fibroblasts from non-lymphoid tissues as well as mesenchymal stromal cells. We identified FRC as strong source of nitric oxide (NO thereby directly dampening T cell expansion as well as reducing the T cell priming capacity of DC. The expression of inducible nitric oxide synthase (iNOS was up-regulated in a subset of FRC by both DC-signals as well as interferon-γ produced by primed CD8+ T cells. Importantly, iNOS expression was induced during viral infection in vivo in both LN FRC and DC. As a consequence, the primary T cell response was found to be exaggerated in Inos(-/- mice. Our findings highlight that in addition to their established positive roles in T cell responses FRC and DC cooperate in a negative feedback loop to attenuate T cell expansion during acute inflammation.

  3. Effects of in vivo injection of anti-chicken CD25 monoclonal antibody on regulatory T cell depletion and CD4+CD25- T cell properties in chickens.

    Science.gov (United States)

    Shanmugasundaram, Revathi; Selvaraj, Ramesh K

    2012-03-01

    Regulatory T cells (Tregs) are defined as CD4(+)CD25(+) cells in chickens. This study examined the effects of an anti-chicken CD25 monoclonal antibody injection (0.5 mg/bird) on in vivo depletion of Tregs and the properties of CD4(+)CD25(-) cells in Treg-depleted birds. The CD4(+)CD25(+) cell percentage in the blood was lower at 8 d post injection than at 0 d. Anti-CD25-mediated CD4(+)CD25(+) cell depletion in blood was maximum at 12 d post injection. The anti-CD25 antibody injection depleted CD4(+)CD25(+) cells in the spleen and cecal tonsils, but not in the thymus, at 12 d post antibody injection. CD4(+)CD25(-) cells from the spleen and cecal tonsils of birds injected with the anti-chicken CD25 antibody had higher proliferation and higher IL-2 and IFNγ mRNA amounts than the controls at 12 d post injection. At 20 d post injection, CD4(+)CD25(+) cell percentages in the blood, spleen and thymus were comparable to that of the 0 d post injection. It could be concluded that anti-chicken CD25 injection temporarily depleted Treg population and increased and IL-2 and IFNγ mRNA amounts in CD4(+)CD25(-) cells at 12d post injection.

  4. Tracking the stochastic fate of cells of the renin lineage after podocyte depletion using multicolor reporters and intravital imaging.

    Science.gov (United States)

    Kaverina, Natalya V; Kadoya, Hiroyuki; Eng, Diana G; Rusiniak, Michael E; Sequeira-Lopez, Maria Luisa S; Gomez, R Ariel; Pippin, Jeffrey W; Gross, Kenneth W; Peti-Peterdi, Janos; Shankland, Stuart J

    2017-01-01

    Podocyte depletion plays a major role in focal segmental glomerular sclerosis (FSGS). Because cells of the renin lineage (CoRL) serve as adult podocyte and parietal epithelial cell (PEC) progenitor candidates, we generated Ren1cCre/R26R-ConfettiTG/WT and Ren1dCre/R26R-ConfettiTG/WT mice to determine CoRL clonality during podocyte replacement. Four CoRL reporters (GFP, YFP, RFP, CFP) were restricted to cells in the juxtaglomerular compartment (JGC) at baseline. Following abrupt podocyte depletion in experimental FSGS, all four CoRL reporters were detected in a subset of glomeruli at day 28, where they co-expressed de novo four podocyte proteins (podocin, nephrin, WT-1 and p57) and two glomerular parietal epithelial cell (PEC) proteins (claudin-1, PAX8). To monitor the precise migration of a subset of CoRL over a 2w period following podocyte depletion, intravital multiphoton microscopy was used. Our findings demonstrate direct visual support for the migration of single CoRL from the JGC to the parietal Bowman's capsule, early proximal tubule, mesangium and glomerular tuft. In summary, these results suggest that following podocyte depletion, multi-clonal CoRL migrate to the glomerulus and replace podocyte and PECs in experimental FSGS.

  5. Survival after T cell-depleted haploidentical stem cell transplantation is improved using the mother as donor.

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    Stern, Martin; Ruggeri, Loredana; Mancusi, Antonella; Bernardo, Maria Ester; de Angelis, Claudia; Bucher, Christoph; Locatelli, Franco; Aversa, Franco; Velardi, Andrea

    2008-10-01

    We hypothesized that transplacental leukocyte trafficking during pregnancy, which induces long-term, stable, reciprocal microchimerism in mother and child, might influence outcome of patients with acute leukemia given parental donor haploidentical hematopoietic stem cell transplantation (HSCT). We analyzed the outcome of 118 patients who received transplants for acute leukemia in 2 centers. Patients received highly T cell-depleted haploidentical grafts after myelo-ablative conditioning. Five-year event-free survival was better in patients who received transplants from the mother than from the father (50.6% +/- 7.6% vs 11.1% +/- 4.2%; P mother = 2.36; P = .003). In contrast, in a control cohort of patients who received transplants from haploidentical siblings, donor sex had no influence on outcome. Although obtained in a retrospective analysis, these data suggest that the mother of the patient should be preferred as donor for haploidentical HSCT.

  6. RNAi mediated acute depletion of Retinoblastoma protein (pRb promotes aneuploidy in human primary cells via micronuclei formation

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    Iovino Flora

    2009-11-01

    Full Text Available Abstract Background Changes in chromosome number or structure as well as supernumerary centrosomes and multipolar mitoses are commonly observed in human tumors. Thus, centrosome amplification and mitotic checkpoint dysfunctions are believed possible causes of chromosomal instability. The Retinoblastoma tumor suppressor (RB participates in the regulation of synchrony between DNA synthesis and centrosome duplication and it is involved in transcription regulation of some mitotic genes. Primary human fibroblasts were transfected transiently with short interfering RNA (siRNA specific for human pRb to investigate the effects of pRb acute loss on chromosomal stability. Results Acutely pRb-depleted fibroblasts showed altered expression of genes necessary for cell cycle progression, centrosome homeostasis, kinetochore and mitotic checkpoint proteins. Despite altered expression of genes involved in the Spindle Assembly Checkpoint (SAC the checkpoint seemed to function properly in pRb-depleted fibroblasts. In particular AURORA-A and PLK1 overexpression suggested that these two genes might have a role in the observed genomic instability. However, when they were post-transcriptionally silenced in pRb-depleted fibroblasts we did not observe reduction in the number of aneuploid cells. This finding suggests that overexpression of these two genes did not contribute to genomic instability triggered by RB acute loss although it affected cell proliferation. Acutely pRb-depleted human fibroblasts showed the presence of micronuclei containing whole chromosomes besides the presence of supernumerary centrosomes and aneuploidy. Conclusion Here we show for the first time that RB acute loss triggers centrosome amplification and aneuploidy in human primary fibroblasts. Altogether, our results suggest that pRb-depleted primary human fibroblasts possess an intact spindle checkpoint and that micronuclei, likely caused by mis-attached kinetochores that in turn trigger

  7. Tolbutamide attenuates diazoxide-induced aggravation of hypoxic cell injury.

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    Pissarek, M; Reichelt, C; Krauss, G J; Illes, P

    1998-11-23

    ATP-dependent potassium (KATP) channels of neurons are closed in the presence of physiological levels of intracellular ATP and open when ATP is depleted during hypoxia or metabolic damage. The present study investigates hypoxic alterations of purine and pyrimidine nucleotide levels supposed to intracellularly modulate KATP channels. In addition, the effects of the KATP channel activator diazoxide and its antagonist tolbutamide were investigated on ATP, GTP, CTP and UTP levels in slices of the parietal cortex. Hypoxia was evoked by saturation of the medium with 95% N2-5% CO2 instead of 95% O2-5% CO2 for 5 min. Nucleotide contents were measured by anion-exchange HPLC in neutralized perchloric acid extracts obtained from slices frozen immediately at the end of incubation. Hypoxia per se decreased purine and pyrimidine nucleoside triphosphate contents. Thus, ATP and GTP contents were reduced to 69.9 and 77.6% of the respective normoxic levels. UTP and CTP contents were even more decreased (to 60.9 and 41.6%),, probably because the salvage pathway of these pyrimidine nucleotides is less effective than that of the purine nucleotides ATP and GTP. While tolbutamide (30 microM) had no effect on the hypoxia-induced decrease of nucleotides, diazoxide at 300, but not 30 microM aggravated the decline of ATP, UTP and CTP to 51.8, 37.5 and 28.5% of the contents observed at normoxia; GTP levels also showed a tendency to decrease after diazoxide application. Tolbutamide (300 microM) antagonized the effects of diazoxide (300 but not 30 microM aggravated the decline of ATP, UTP and CTP to 51.8, 37.5 and 28.5% of the contents observed at normoxia; GTP levels also showed a tendency to decrease after diazoxide application. Tolbutamide (300 microM) antagonized the effects of diazoxide (300 MicroM). Nucleoside diphosphate (ADP, GDP and UDP) levels were uniformly increased by hypoxia. There was no hypoxia-induced increase of ADP contents in the presence of tolbutamide (300 microM). The ATP

  8. CIITA enhances HIV-1 attachment to CD4+ T cells leading to enhanced infection and cell depletion.

    Science.gov (United States)

    Porter, Kristen A; Kelley, Lauren N; Nekorchuk, Michael D; Jones, James H; Hahn, Amy B; de Noronha, Carlos M C; Harton, Jonathan A; Duus, Karen M

    2010-12-01

    Activated CD4(+) T cells are more susceptible to HIV infection than resting T cells; the reason for this remains unresolved. Induction of CIITA and subsequent expression of the MHC class II isotype HLA-DR are hallmarks of CD4(+) T cell activation; therefore, we investigated the role of CIITA expression in T cells during HIV infection. CIITA-expressing SupT1 cells display enhanced virion attachment in a gp160/CD4-dependent manner, which results in increased HIV infection, virus release, and T cell depletion. Although increased attachment and infection of T cells correlated with HLA-DR surface expression, Ab blocking, transient expression of HLA-DR without CIITA, and short hairpin RNA knockdown demonstrate that HLA-DR does not directly enhance susceptibility of CIITA-expressing cells to HIV infection. Further analysis of the remaining MHC class II isotypes, HLA-DP and HLA-DQ, MHC class I isotypes, HLA-A, HLA-B, and HLA-C, and the class II Ag presentation genes, invariant chain and HLA-DM, demonstrate that these proteins likely do not contribute to CIITA enhancement of HIV infection. Finally, we demonstrate that in activated primary CD4(+) T cells as HLA-DR/CIITA expression increases there is a corresponding increase in virion attachment. Overall, this work suggests that induction of CIITA expression upon CD4(+) T cell activation contributes to enhanced attachment, infection, virus release, and cell death through an undefined CIITA transcription product that may serve as a new antiviral target.

  9. Attenuating effect of daidzein on polychlorinated biphenyls-induced oxidative toxicity in mouse testicular cells

    Institute of Scientific and Technical Information of China (English)

    Da-lei ZHANG; Yu-ling MI; Kai-ming WANG; Wei-dong ZENG; Cai-qiao ZHANG

    2008-01-01

    The attenuating effect of daidzein (DAD on oxidative toxicity induced by Aroclor 1254 (A 1254) was investigated in mouse testicular cells. Cells were exposed to A1254 alone or with DAI. The oxidative damage was estimated by measuring malondialdehyde (MDA) formation, superoxide dismutase (SOD) activity and glutathione (GSH) content. Results show that A 1254 induced a decrease of germ cell number, an elevation in thiobarbituric acid reactive substances (TBARS) but a decrease in SOD activity and GSH content. However, simultaneous supplementation with DAI decreased TBARS level and increased SOD activity and GSH content. Consequently, dietary DAI may restore the intracellular antioxidant system to attenuate the oxidative toxicity of A1254 in testicular cells.

  10. Glioblastoma formation from cell population depleted of Prominin1-expressing cells.

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    Kenji Nishide

    Full Text Available Prominin1 (Prom1, also known as CD133 in human has been widely used as a marker for cancer stem cells (CSCs, which self-renew and are tumorigenic, in malignant tumors including glioblastoma multiforme (GBM. However, there is other evidence showing that Prom1-negative cancer cells also form tumors in vivo. Thus it remains controversial whether Prom1 is a bona fide marker for CSCs. To verify if Prom1-expressing cells are essential for tumorigenesis, we established a mouse line, whose Prom1-expressing cells can be eliminated conditionally by a Cre-inducible DTA gene on the Prom1 locus together with a tamoxifen-inducible CreER(TM, and generated glioma-initiating cells (GICs-LD by overexpressing both the SV40 Large T antigen and an oncogenic H-Ras(L61 in neural stem cells of the mouse line. We show here that the tamoxifen-treated GICs-LD (GICs-DTA form tumor-spheres in culture and transplantable GBM in vivo. Thus, our studies demonstrate that Prom1-expressing cells are dispensable for gliomagenesis in this mouse model.

  11. Regulatory T-cell depletion in the gut caused by integrin β7 deficiency exacerbates DSS colitis by evoking aberrant innate immunity.

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    Zhang, H L; Zheng, Y J; Pan, Y D; Xie, C; Sun, H; Zhang, Y H; Yuan, M Y; Song, B L; Chen, J F

    2016-03-01

    Integrin α4β7 controls lymphocyte trafficking into the gut and has essential roles in inflammatory bowel disease (IBD). The α4β7-blocking antibody vedolizumab is approved for IBD treatment; however, high dose of vedolizumab aggravates colitis in a small percentage of patients. Herein, we show that integrin β7 deficiency results in colonic regulatory T (Treg) cell depletion and exacerbates dextran sulfate sodium (DSS) colitis by evoking aberrant innate immunity. In DSS-treated β7-deficient mice, the loss of colonic Treg cells induces excessive macrophage infiltration in the colon via upregulation of colonic epithelial intercellular adhesion molecule 1 and increases proinflammatory cytokine expression, thereby exacerbating DSS-induced colitis. Moreover, reconstitution of the colonic Treg cell population in β7-deficient mice suppresses aberrant innate immune response in the colon and attenuates DSS colitis. Thus, integrin α4β7 is essential for suppression of DSS colitis as it regulates the colonic Treg cell population and innate immunity.

  12. Experimental depletion of CD8+ cells in acutely SIVagm-Infected African Green Monkeys results in increased viral replication

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    Apetrei Cristian

    2010-05-01

    Full Text Available Abstract Background In vivo CD8+ cell depletions in pathogenic SIV infections identified a key role for cellular immunity in controlling viral load (VL and disease progression. However, similar studies gave discordant results in chronically-infected SMs, leading some authors to propose that in natural hosts, SIV replication is independent of cellular immunity. To assess the role of cellular immune responses in the control of SIV replication in natural hosts, we investigated the impact of CD8+ cell depletion during acute SIV infection in AGMs. Results Nine AGMs were infected with SIVagm.sab and were followed up to day 225 p.i. Four were intravenously infused with the cM-T807 antibody on days 0 (50 mg/kg, 6, and 13 (10 mg/kg, respectively post infection (p.i.. CD8+ cells were depleted for up to 28 days p.i. in peripheral blood and LNs in all treated AGMs. Partial CD8+ T cell depletion occurred in the intestine. SIVagm VLs peaked at similar levels in both groups (107-108 RNA copies/ml. However, while VLs were controlled in undepleted AGMs, reaching set-point levels (104-105 RNA copies/ml by day 28 p.i., high VLs (>106 RNA copies/ml were maintained by day 21 p.i. in CD8-depleted AGMs. By day 42 p.i., VLs were comparable between the two groups. The levels of immune activation and proliferation remained elevated up to day 72 p.i. in CD8-depleted AGMs and returned to preinfection levels in controls by day 28 p.i. None of the CD8-depleted animals progressed to AIDS. Conclusion CD8+ cells are responsible for a partial control of postacute viral replication in SIVagm.sab-infected AGMs. In contrast to macaques, the SIVagm-infected AGMs are able to control viral replication after recovery of the CD8+ T cells and avoid disease progression.

  13. Histone deacetylase 3 depletion in osteo/chondroprogenitor cells decreases bone density and increases marrow fat.

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    David F Razidlo

    Full Text Available Histone deacetylase (Hdac3 is a nuclear enzyme that contributes to epigenetic programming and is required for embryonic development. To determine the role of Hdac3 in bone formation, we crossed mice harboring loxP sites around exon 7 of Hdac3 with mice expressing Cre recombinase under the control of the osterix promoter. The resulting Hdac3 conditional knockout (CKO mice were runted and had severe deficits in intramembranous and endochondral bone formation. Calvarial bones were significantly thinner and trabecular bone volume in the distal femur was decreased 75% in the Hdac3 CKO mice due to a substantial reduction in trabecular number. Hdac3-CKO mice had fewer osteoblasts and more bone marrow adipocytes as a proportion of tissue area than their wildtype or heterozygous littermates. Bone formation rates were depressed in both the cortical and trabecular regions of Hdac3 CKO femurs. Microarray analyses revealed that numerous developmental signaling pathways were affected by Hdac3-deficiency. Thus, Hdac3 depletion in osterix-expressing progenitor cells interferes with bone formation and promotes bone marrow adipocyte differentiation. These results demonstrate that Hdac3 inhibition is detrimental to skeletal health.

  14. Selected flavonoids potentiate the toxicity of cisplatin in human lung adenocarcinoma cells: A role for glutathione depletion

    OpenAIRE

    Kachadourian, Remy; LEITNER, VHEATHER M.; Day, Brian J.

    2007-01-01

    Adjuvant therapies that enhance the anti-tumor effects of cisplatin are actively being pursued. Growing evidence supports the involvement of mitochondrial dysfunction in the anti-cancer effect of cis-diammineplatinum(II) dichloride (cisplatin, CDDP). We examined the potential of using selective flavonoids that are effective in depleting tumor cells of glu-tathione (GSH) to potentiate cisplatin-mediated cytotoxicity in human lung adenocarcinoma (A549) cells. We found that cisplatin (40 μM, 48-...

  15. Depletion of the histone chaperone tNASP inhibits proliferation and induces apoptosis in prostate cancer PC-3 cells

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    Tsuruta James K

    2011-04-01

    Full Text Available Abstract Background NASP (Nuclear Autoantigenic Sperm Protein is a histone chaperone that is present in all dividing cells. NASP has two splice variants: tNASP and sNASP. Only cancer, germ, transformed, and embryonic cells have a high level of expression of the tNASP splice variant. We examined the consequences of tNASP depletion for prostate cancer PC-3 cells. Methods tNASP was depleted from prostate cancer PC-3 cells, cervical cancer HeLa cells, and prostate epithelial PWR-1E cells using lentivirus expression of tNASP shRNA. Cell cycle changes were studied by proliferation assay with CFSE labeling and double thymidine synchronization. Gene expression profiles were detected using RT2Profiler PCR Array, Western and Northern blotting. Results PC-3 and HeLa cells showed inhibited proliferation, increased levels of cyclin-dependant kinase inhibitor p21 protein and apoptosis, whereas non-tumorigenic PWR-1E cells did not. All three cell types showed decreased levels of HSPA2. Supporting in vitro experiments demonstrated that tNASP, but not sNASP is required for activation of HSPA2. Conclusions Our results demonstrate that PC-3 and HeLa cancer cells require tNASP to maintain high levels of HSPA2 activity and therefore viability, while PWR-1E cells are unaffected by tNASP depletion. These different cellular responses most likely arise from changes in the interaction between tNASP and HSPA2 and disturbed tNASP chaperoning of linker histones. This study has demonstrated that tNASP is critical for the survival of prostate cancer cells and suggests that targeting tNASP expression can lead to a new approach for prostate cancer treatment.

  16. Preferential infection and depletion of Mycobacterium tuberculosis-specific CD4 T cells after HIV-1 infection.

    Science.gov (United States)

    Geldmacher, Christof; Ngwenyama, Njabulo; Schuetz, Alexandra; Petrovas, Constantinos; Reither, Klaus; Heeregrave, Edwin J; Casazza, Joseph P; Ambrozak, David R; Louder, Mark; Ampofo, William; Pollakis, Georgios; Hill, Brenna; Sanga, Erica; Saathoff, Elmar; Maboko, Leonard; Roederer, Mario; Paxton, William A; Hoelscher, Michael; Koup, Richard A

    2010-12-20

    HIV-1 infection results in the progressive loss of CD4 T cells. In this study, we address how different pathogen-specific CD4 T cells are affected by HIV infection and the cellular parameters involved. We found striking differences in the depletion rates between CD4 T cells to two common opportunistic pathogens, cytomegalovirus (CMV) and Mycobacterium tuberculosis (MTB). CMV-specific CD4 T cells persisted after HIV infection, whereas MTB-specific CD4 T cells were depleted rapidly. CMV-specific CD4 T cells expressed a mature phenotype and produced very little IL-2, but large amounts of MIP-1β. In contrast, MTB-specific CD4 T cells were less mature, and most produced IL-2 but not MIP-1β. Staphylococcal enterotoxin B-stimulated IL-2-producing cells were more susceptible to HIV infection in vitro than MIP-1β-producing cells. Moreover, IL-2 production was associated with expression of CD25, and neutralization of IL-2 completely abrogated productive HIV infection in vitro. HIV DNA was found to be most abundant in IL-2-producing cells, and least abundant in MIP-1β-producing MTB-specific CD4 T cells from HIV-infected subjects with active tuberculosis. These data support the hypothesis that differences in function affect the susceptibility of pathogen-specific CD4 T cells to HIV infection and depletion in vivo, providing a potential mechanism to explain the rapid loss of MTB-specific CD4 T cells after HIV infection.

  17. Human T cell priming assay: depletion of peripheral blood lymphocytes in CD25(+) cells improves the in vitro detection of weak allergen-specific T cells.

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    Vocanson, Marc; Achachi, Amine; Mutez, Virginie; Cluzel-Tailhardat, Magalie; Varlet, Béatrice Le; Rozières, Aurore; Fournier, Philippe; Nicolas, Jean-François

    2014-01-01

    To develop an in vitro assay that recapitulates the key event of allergic contact dermatitis (ACD), that is the priming of effector T cells by hapten-presenting dendritic cells, and then allows for the sensitive detection of chemical allergens represents a major challenge. Classical human T cell priming assays (hTCPA) that have been developed in the past, using hapten-loaded monocyte-derived dendritic cells (MDDCs) as antigen-presenting cells and peripheral blood lymphocytes (PBLs) as responding cells, were not efficient to prime T cells to common allergens with moderate/weak sensitizing properties. Recent progress in the understanding of the effector and regulatory mechanisms of ACD have shown that T cell priming requires efficient uptake of allergens by immunogenic DCs and that it is controlled by several subsets of regulatory cells including CD25(+) Tregs. We therefore analyzed various parameters involved in allergen-specific T cell activation in vitro and showed that priming of allergen-specific T cells is hampered by several subsets of immune cells comprising CD1a(neg) DCs, CD25(+) T cells, and CD56(+) regulatory cells.CD4(+)CD25(+)FoxP3(+) Tregs prevented the in vitro T cell priming to moderate/weak allergens, and depletion of human PBLs in CD25(+) cells significantly increased specific T cell proliferation and IFN-γ secretion. CD56(+) cells exerted an additional control of T cell priming since co-depletion of both CD56(+) and CD25(+) cells improved the magnitude of chemical-specific T cell activation. Finally, CD1a(low) MDDCs were able to inhibit T cell activation obtained by allergen-pulsed CD1a(high) MDDC. Moreover, we showed that uptake by DC of allergen-encapsulated nanoparticles significantly increased their activation status and their ability to prompt specific T cell activation. Hence, by combining the different strategies, i.e., depletion of CD25(+) and CD56(+) cells, use of CD1a(high) MDDC, and nanoparticle encapsulation of allergens, it was

  18. Iron depletion results in Src kinase inhibition with associated cell cycle arrest in neuroblastoma cells.

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    Siriwardana, Gamini; Seligman, Paul A

    2015-03-01

    Iron is required for cellular proliferation. Recently, using systematic time studies of neuroblastoma cell growth, we better defined the G1 arrest caused by iron chelation to a point in mid-G1, where cyclin E protein is present, but the cyclin E/CDK2 complex kinase activity is inhibited. In this study, we again used the neuroblastoma SKNSH cells lines to pinpoint the mechanism responsible for this G1 block. Initial studies showed in the presence of DFO, these cells have high levels of p27 and after reversal of iron chelation p27 is degraded allowing for CDK2 kinase activity. The initial activation of CDK2 kinase allows cells to exit G1 and enter S phase. Furthermore, we found that inhibition of p27 degradation by DFO is directly associated with inhibition of Src kinase activity measured by lack of phosphorylation of Src at the 416 residue. Activation of Src kinase occurs very early after reversal from the DFO G1 block and is temporally associated with initiation of cellular proliferation associated with entry into S phase. For the first time therefore we show that iron chelation inhibits Src kinase activity and this activity is a requirement for cellular proliferation.

  19. In ovo injection of anti-chicken CD25 monoclonal antibodies depletes CD4+CD25+ T cells in chickens.

    Science.gov (United States)

    Shanmugasundaram, Revathi; Selvaraj, Ramesh K

    2013-01-01

    The CD4(+)CD25(+) cells have T regulatory cell properties in chickens. This study investigated the effect of in ovo injection of anti-chicken CD25 monoclonal antibodies (0.5 mg/egg) on CD4(+)CD25(+) cell depletion and on amounts of interleukin-2 mRNA and interferon-γ mRNA in CD4(+)CD25(-) cells posthatch. Anti-chicken CD25 or PBS (control) was injected into 16-d-old embryos. Chicks hatched from eggs injected with anti-chicken CD25 antibodies had a lower CD4(+)CD25(+) cell percentage in the blood until 25 d posthatch. The anti-chicken CD25 antibody injection nearly depleted CD4(+)CD25(+) cells in the blood until 16 d posthatch. At 30 d posthatch, the CD4(+)CD25(+) cell percentage in the anti-CD25-antibody-injected group was comparable with the percentage in the control group. At 16 d posthatch, the anti-chicken CD25 antibody injection decreased CD4(+)CD25(+) cell percentages in the thymus, spleen, and cecal tonsils. Chickens hatched from anti-CD25-antibody-injected eggs had approximately 25% of CD4(+)CD25(+) cells in the cecal tonsils and thymus compared with those in the cecal tonsils and thymus of the control group. The CD4(+)CD25(-) cells from the spleen and cecal tonsils of chicks hatched from anti-chicken-CD25-injected eggs had higher amounts of interferon-γ and interleukin-2 mRNA than CD4(+)CD25(-) cells from the control group. It could be concluded that injecting anti-chicken CD25 antibodies in ovo at 16 d of incubation nearly depleted the CD4(+)CD25(+) cells until 25 d posthatch.

  20. Potentiation of arsenic trioxide-induced apoptosis by 8-bromo-7-methoxychrysin in human leukemia cells involves depletion of intracellular reduced glutathione

    Institute of Scientific and Technical Information of China (English)

    Guangfen Xiao; Xueyuan Tang; Chenjiao Yao; Chenghong Wang

    2011-01-01

    The novel chrysin analog 8-bromo-7-methoxychrysin (BrMC) has been reported to induce apoptosis of various cancer cell lines.Arsenic trioxide (ATO) treatment induces clinical remission in acute promyelocytic leukemia patients.The combination of ATO with other agents has been shown to improve therapeutic effectiveness in vitro and in vivo.In this report,the mechanism of apoptosis induced by treatment with ATO alone or in combination with BrMC was studied in U937,HL-60,and Jurkat cells.Our results demonstrated that BrMC cooperated with ATO to induce apoptosis in human leukemia cells.This co-treatment caused mitochondrial transmembrane potential dissipation and stimulated the mitochondrial apoptotic pathway,as evidenced by cytochrome c release,down-regulation of X-linked inhibitor of apoptosis (XIAP) and Bcl-XL,and up-regulation of Bax.BrMC alone or in combination with ATO,decreased Akt phosphorylation as well as intracellular reduced glutathione (GSH) content.The thiol antioxidant N-acetylcysteine and exogenous GSH restored GSH content and attenuated apoptosis induced by co-treatment with ATO plus BrMC.In contrast,the non-thiol antioxidant butylated hydroxyanisole and mannitol failed to do so.These findings suggest that GSH depletion explains at least in part the potentiation of ATO-induced apoptosis by BrMC.

  1. Depletion of hCINAP by RNA interference causes defects in Cajal body formation, histone transcription, and cell viability.

    Science.gov (United States)

    Zhang, Jinfang; Zhang, Feiyun; Zheng, Xiaofeng

    2010-06-01

    hCINAP is a highly conserved and ubiquitously expressed protein in eukaryotic organisms and its overexpression decreases the average number of Cajal bodies (CBs) with diverse nuclear functions. Here, we report that hCINAP is associated with important components of CBs. Depletion of hCINAP by RNA interference causes defects in CB formation and disrupts subcellular localizations of its components including coilin, survival motor neurons protein, spliceosomal small nuclear ribonucleoproteins, and nuclear protein ataxia-telangiectasia. Moreover, knockdown of hCINAP expression results in marked reduction of histone transcription, lower levels of U small nuclear RNAs (U1, U2, U4, and U5), and a loss of cell viability. Detection of increased caspase-3 activities in hCINAP-depleted cells indicate that apoptosis is one of the reasons for the loss of viability. Altogether, these data suggest that hCINAP is essential for the formation of canonical CBs, histone transcription, and cell viability.

  2. Neutrophil depletion-but not prevention of Kupffer cell activation-decreases the severity of cerulein-induced acute pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Catherine M Pastor; Alain Vonlaufen; Fabianna Georgi; Antoine Hadengue; Philippe Morel; Jean-Louis Frossard

    2006-01-01

    AIM: To determine whether neutrophil depletion and Kupffer cell inhibition might combine their protective effects to decrease the severity of acute pancreatitis.METHODS: Mice had cerulein administration to induce acute pancreatitis and were pretreated with either anti-mouse neutrophil serum or gadolinium chloride (GdCl3) to prevent Kupffer cell activation, or both treatments. Injury was assessed in pancreas and lungs.Myeloperoxidases (MPO) assessed neutrophil infiltration.Interleukin-6 (IL-6) and IL-10 were measured in serum,pancreas, lungs and liver.RESULTS: In mice with acute pancreatitis, neutrophil depletion reduced the severity of pancreatitis and pancreatitis-associated lung injury. Kupffer cell inactivation by GdCl3 had less protective effect, although IL-6 and IL-10 concentrations were significantly decreased. The protective treatment brought by neutrophil depletion was not enhanced by Kupffer cell inactivation and both treatments did not combine their protective effects.CONCLUSION: Our results confirm the role of activated neutrophils in aggravating organ injury in acute pancreatitis while the role of Kupffer cell activation is less obvious.

  3. Depletion of Aurora-A in zebrafish causes growth retardation due to mitotic delay and p53-dependent cell death.

    Science.gov (United States)

    Jeon, Hee-Yeon; Lee, Hyunsook

    2013-03-01

    Aurora-A is a serine/threonine mitotic kinase that is required for centrosome maturation. Many cancer cells over-express Aurora-A, and several reports have suggested that Aurora-A has prognostic value in the clinical treatment of cancer. Therefore, inhibitors for Aurora-A kinase have been developed. However, studies on Aurora-A are largely performed in cancer cell lines and are sometimes controversial. For effective evaluation of Aurora-A inhibitors in cancer treatment, it is essential to understand its function at the organism level. Here, we report the crucial functions of Aurora-A in homeostasis of spindle organization in mitosis using zebrafish embryogenesis as a model system. Using morpholino technology, we show that depletion of Aurora-A in zebrafish embryogenesis results in short bent trunks, accompanied by growth retardation and eventual cell death. Live-imaging and immunofluorescence analyses of the embryos revealed that the developmental defects are due to problems in mitosis, manifested through monopolar and disorganized spindle formation. Aurora-A-depleted cells exhibited mitotic arrest with congression failure, leading to activation of the spindle assembly checkpoint. Cell death in the absence of Aurora-A was partially rescued by co-injection of the p53 morpholino, suggesting that apoptosis after Aurora-A depletion is p53-dependent. The clinical implications of these results relate to the indication that Aurora-A inhibitors may be effective towards cancers with intact p53.

  4. Novel squamosamide derivative (compound FLZ) attenuates Aβ25-35-induced toxicity in SH-SY5Y cells

    Institute of Scientific and Technical Information of China (English)

    Fang FANG; Geng-tao LIU

    2008-01-01

    Aim: The aim of the present study was to investigate the protective effect of compound N-[2-(4-hydroxy-phenyl)-ethyl]-2-(2,5-dimethoxy-phenyl)-3-(3-methoxy-4-hydroxy-phenyl)-acrylamide (compound FLZ), a novel synthetic ana-logue of nature squamosamide, on Aβ25-35-induced toxicity and its active mecha-nism in human neuroblastoma SH-SY5Y cells. Methods: SH-SY5Y cells were pre-incubated with various concentrations of compound FLZ for 30 min and then cultivated with Aβ25-35 (25 μmol/L) for 48 h to induce neurotoxicity. Cell viability, lactate dehydrogenase (LDH) release, and the glutathione (GSH) level were deter-mined by a biochemical analysis. The cell apoptotic ratio and intracellular reactive oxygen species (ROS) level were measured by a flow cytometry analysis. The expression of apoptosis protein (Bcl-2 and Bax) and cytochrome c release were assayed by the Western blot method. Results: The pretreatment of SH-SY5Y cells with FLZ (1 and 10 μmol/L) markedly increased cell viability and decreased LDH release and morphological injury. Also, FLZ attenuated the Aβ25-35-induced apoptotic cell ratio, regulated the apoptosis protein (Bcl-2 and Bax) expression, and decreased the cytochrome c release from mitochondria. FLZ also signifi-cantly inhibited the generation of ROS and the depletion of GSH induced by Aβ25-35 in SH-SY5Y cells. Conclusion: FLZ has protective action against Aβ25-35-in-duced toxicity in SH-SY5Y cells, which might be mediated through its antioxi-dant property.

  5. YB-1 immunization combined with regulatory T-cell depletion induces specific T-cell responses that protect against neuroblastoma in the early stage

    Institute of Scientific and Technical Information of China (English)

    Jin Zheng; Ping Liu; Xiaofeng Yang

    2012-01-01

    Neuroblastoma is the most common extracranial solid cancer in childhood and the most common cancer in infancy.Currently,no effective clinical treatments are available for advanced neuroblastoma.In a previous study,we screened Y Box protein 1 (YB-1) as a potential neuroblastoma-associated antigen from sera of AGN2a-immunized mice by serological analysis of recombinant cDNA expression libraries technique.The aim of this study is to explore if YB-1 immunization in the context of Treg depletion could induce protective immune response against the neuroblastoma in mice.YB-1 was expressed and purified by pET-15b prokaryotic expression system.It was demonstrated that anti-YB-1 CD8+ T-cell responses could be induced by AGN2a immunization,and the strongest CD8+ T-cell responses against AGN2a were induced by YB-1-immunized mice in the context of Treg depletion compared with YB-1 only immunization group and control group.Importantly,the survival rate of mice treated with YB-1 immunization combined with Treg depletion was 80% when challenged by 1 × 104 AGN2a cells,significantly higher than that of mice immunized with YB-1 alone (P< 0.01).Furthermore,T-cell adoptive therapy showed that the neuroblastoma growth was inhibited when T cells or splenic cells from YB-1-immunized mice with Treg depletion were transferred to AGN2a bearing mice.Both CD4+ and CD8+ T cells were involved in the anti-neuroblastoma responses induced by YB-1immunization combined with Treg depletion.These results indicated that YB-1 immunization combined with Treg depletion could induce specific T-cell responses against neuroblastoma and could be a potential strategy for the prevention and treatment of neuroblastoma in the early stage.

  6. Depletion of histone demethylase KDM2A enhanced the adipogenic and chondrogenic differentiation potentials of stem cells from apical papilla

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Rui [Laboratory of Molecular Signaling and Stem Cells Therapy, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing 100050 (China); Yao, Rui [Department of Pediatrics, Stomatological Hospital of Nankai University, Tianjin 300041 (China); Du, Juan [Laboratory of Molecular Signaling and Stem Cells Therapy, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing 100050 (China); Wang, Songlin [Molecular Laboratory for Gene Therapy and Tooth Regeneration, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing 100050 (China); Department of Biochemistry and Molecular Biology, Capital Medical University School of Basic Medical Sciences, Beijing 100069 (China); Fan, Zhipeng, E-mail: zpfan@ccmu.edu.cn [Laboratory of Molecular Signaling and Stem Cells Therapy, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing 100050 (China)

    2013-11-01

    Mesenchymal stem cells (MSCs) are a reliable resource for tissue regeneration, but the molecular mechanism underlying directed differentiation remains unclear; this has restricted potential MSC applications. The histone demethylase, lysine (K)-specific demethylase 2A (KDM2A), is evolutionarily conserved and ubiquitously expressed members of the JmjC-domain-containing histone demethylase family. A previous study determined that KDM2A can regulate the cell proliferation and osteo/dentinogenic differentiation of MSCs. It is not known whether KDM2A is involved in the other cell lineages differentiation of MSCs. Here, we show that depletion of KDM2A by short hairpin RNAs can enhance adipogenic and chondrogenic differentiation potentials in human stem cells from apical papilla (SCAPs). We found that the stemness-related genes, SOX2, and the embryonic stem cell master transcription factor, NANOG were significantly increased after silence of KDM2A in SCAPs. Moreover, we found that knock-down of the KDM2A co-factor, BCOR also up-regulated the mRNA levels of SOX2 and NANOG. Furthermore, Chromatin immunoprecipitation assays demonstrate that silence of KDM2A increased the histone H3 Lysine 4 (H3K4) trimethylation in the SOX2 and NANOG locus and regulates its expression. In conclusion, our results suggested that depletion of KDM2A enhanced the adipogenic and chondrogenic differentiation potentials of SCAPs by up-regulated SOX2 and NANOG, BCOR also involved in this regulation as co-factor, and provided useful information to understand the molecular mechanism underlying directed differentiation in MSCs. - Highlights: • Depletion of KDM2A enhances adipogenic/chondrogenic differentiation in SCAPs. • Depletion of KDM2A enhances the differentiation of SCAPs by activate SOX2 and NANOG. • Silence of KDM2A increases histone H3 Lysine 4 trimethylation in SOX2 and NANOG. • BCOR is co-factor of KDM2A involved in the differentiation regulation.

  7. Depletion of intrinsic expression of Interleukin-8 in prostate cancer cells causes cell cycle arrest, spontaneous apoptosis and increases the efficacy of chemotherapeutic drugs

    Directory of Open Access Journals (Sweden)

    Lokeshwar Bal L

    2009-07-01

    Full Text Available Abstract Background The progression of all cancers is characterized by increased-cell proliferation and decreased-apoptosis. The androgen-independent prostate cancer (AIPC is the terminal stage of the disease. Many chemokines and cytokines are suspects to cause this increased tumor cell survival that ultimately leads to resistance to therapy and demise of the host. The AIPC cells, but not androgen-responsive cells, constitutively express abundant amount of the pro-inflammatory chemokine, Interleukin-8 (IL-8. The mechanism of IL-8 mediated survival and therapeutic resistance in AIPC cells is unclear at present. The purpose of this report is to show the pervasive role of IL-8 in malignant progression of androgen-independent prostate cancer (AIPC and to provide a potential new therapeutic avenue, using RNA interference. Results The functional consequence of IL-8 depletion in AIPC cells was investigated by RNA interference in two IL-8 secreting AIPC cell lines, PC-3 and DU145. The non-IL-8 secreting LNCaP and LAPC-4 cells served as controls. Cells were transfected with RISC-free siRNA (control or validated-pool of IL-8 siRNA. Transfection with 50 nM IL-8 siRNA caused >95% depletion of IL-8 mRNA and >92% decrease in IL-8 protein. This reduction in IL-8 led to cell cycle arrest at G1/S boundary and decreases in cell cycle-regulated proteins: Cyclin D1 and Cyclin B1 (both decreased >50% and inhibition of ERK1/2 activity by >50%. Further, the spontaneous apoptosis was increased by >43% in IL-8 depleted cells, evidenced by increases in caspase-9 activation and cleaved-PARP. IL-8 depletion caused significant decreases in anti-apoptotic proteins, BCL-2, BCL-xL due to decrease in both mRNA and post-translational stability, and increased levels of pro-apoptotic BAX and BAD proteins. More significantly, depletion of intracellular IL-8 increased the cytotoxic activity of multiple chemotherapeutic drugs. Specifically, the cytotoxicity of Docetaxel

  8. Anti-apoptotic protein BRE/BRCC45 attenuates apoptosis through maintaining the expression of caspase inhibitor XIAP in mouse Lewis lung carcinoma D122 cells.

    Science.gov (United States)

    Chui, Yiu-Loon; Ma, Chun-Hung; Li, Wei; Xu, Zhenyu; Yao, Yao; Lin, Frances Ka-Yin; Chan, John Yeuk-Hon; Lee, Kenneth Ka-Ho

    2014-05-01

    Brain and Reproductive Organ Expressed (BRE), or BRCC45, is a death receptor-associated antiapoptotic protein, which is also involved in DNA-damage repair, and K63-specific deubiquitination. BRE overexpression attenuates both death receptor- and stress-induced apoptosis, promotes experimental tumor growth, and is associated with human hepatocellular and esophageal carcinoma. How BRE mediates its antiapoptotic function is unknown. Here we report based on the use of a mouse Lewis lung carcinoma cell line D122 that BRE has an essential role in maintaining the cellular protein level of XIAP, which is the most potent endogenous inhibitor of the caspases functioning in both extrinsic and intrinsic apoptosis. shRNA-mediated exhaustive depletion of BRE sensitized D122 cells to apoptosis induced not only by etopoxide, but also by TNF-α even in the absence of cycloheximide, which blocks the synthesis of antiapoptotic proteins by TNF-α-activated NF-κB pathway. In BRE-depleted cells, protein level of XIAP was downregulated, but not the levels of other antiapoptotic proteins, cIAP-1, 2, and cFLIP, regulated by the same NF-κB pathway. Reconstitution of BRE restored XIAP levels and increased resistance to apoptosis. XIAP mRNA level was also reduced in the BRE-depleted cells, but the level of reduction was less profound than that of the protein level. However, BRE could not delay protein turnover of XIAP. Depletion of BRE also increased tumor cell apoptosis, and decreased both local and metastatic tumor growth. Taken together, these findings indicate that BRE and its XIAP-sustaining mechanism could represent novel targets for anti-cancer therapy.

  9. Regulation of death induction and chemosensitizing action of 3-bromopyruvate in myeloid leukemia cells: energy depletion, oxidative stress, and protein kinase activity modulation.

    Science.gov (United States)

    Calviño, Eva; Estañ, María Cristina; Sánchez-Martín, Carlos; Brea, Rocío; de Blas, Elena; Boyano-Adánez, María del Carmen; Rial, Eduardo; Aller, Patricio

    2014-02-01

    3-Bromopyruvate (3-BrP) is an alkylating, energy-depleting drug that is of interest in antitumor therapies, although the mechanisms underlying its cytotoxicity are ill-defined. We show here that 3-BrP causes concentration-dependent cell death of HL60 and other human myeloid leukemia cells, inducing both apoptosis and necrosis at 20-30 μM and a pure necrotic response at 60 μM. Low concentrations of 3-BrP (10-20 μM) brought about a rapid inhibition of glycolysis, which at higher concentrations was followed by the inhibition of mitochondrial respiration. The combination of these effects causes concentration-dependent ATP depletion, although this cannot explain the lethality at intermediate 3-BrP concentrations (20-30 μM). The oxidative stress caused by exposure to 3-BrP was evident as a moderate overproduction of reactive oxygen species and a concentration-dependent depletion of glutathione, which was an important determinant of 3-BrP toxicity. In addition, 3-BrP caused glutathione-dependent stimulation of p38 mitogen-activated protein kinase (MAPK), mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK), and protein kinase B (Akt)/mammalian target of rapamycin/p70S6K phosphorylation or activation, as well as rapid LKB-1/AMP kinase (AMPK) activation, which was later followed by Akt-mediated inactivation. Experiments with pharmacological inhibitors revealed that p38 MAPK activation enhances 3-BrP toxicity, which is conversely restrained by ERK and Akt activity. Finally, 3-BrP was seen to cooperate with antitumor agents like arsenic trioxide and curcumin in causing cell death, a response apparently mediated by both the generation of oxidative stress induced by 3-BrP and the attenuation of Akt and ERK activation by curcumin. In summary, 3-BrP cytotoxicity is the result of several combined regulatory mechanisms that might represent important targets to improve therapeutic efficacy.

  10. A murine herpesvirus closely related to ubiquitous human herpesviruses causes T-cell depletion.

    Science.gov (United States)

    Patel, Swapneel J; Zhao, Guoyan; Penna, Vinay R; Park, Eugene; Lauron, Elvin J; Harvey, Ian B; Beatty, Wandy L; Plougastel-Douglas, Beatrice; Poursine-Laurent, Jennifer; Fremont, Daved H; Wang, David; Yokoyama, Wayne M

    2017-02-08

    clinical ramifications of roseolovirus infection in humans have been hypothesized, but studies showing definitive causative relationships between infection and disease susceptibility are lacking. Here we show that MRV infects the thymus and causes T-cell depletion, suggesting that other roseoloviruses may have similar properties.

  11. Activation and Genetic Modification of Human Monocyte-Derived Dendritic Cells using Attenuated Salmonella typhimurium

    Directory of Open Access Journals (Sweden)

    Agnieszka Michael

    2010-01-01

    Full Text Available Live attenuated bacterial vectors, such as Salmonella typhimurium, have shown promise as delivery vehicles for DNA. We have examined two new strains of S. typhimurium and their impact on dendritic cell maturation (CD12-sifA/aroC mutant and WT05-ssaV/aroC, both in TML background. Strain WT05 matured dendritic cells in a more efficient way; caused higher release of cytokines TNF-α, IL-12, IL-1β; and was efficient for gene transfer. These findings suggest that the genetic background of the attenuation can influence the pattern of inflammatory immune response to Salmonella infection.

  12. A gene expression atlas of a bicoid-depleted Drosophila embryo reveals early canalization of cell fate.

    Science.gov (United States)

    Staller, Max V; Fowlkes, Charless C; Bragdon, Meghan D J; Wunderlich, Zeba; Estrada, Javier; DePace, Angela H

    2015-02-01

    In developing embryos, gene regulatory networks drive cells towards discrete terminal fates, a process called canalization. We studied the behavior of the anterior-posterior segmentation network in Drosophila melanogaster embryos by depleting a key maternal input, bicoid (bcd), and measuring gene expression patterns of the network at cellular resolution. This method results in a gene expression atlas containing the levels of mRNA or protein expression of 13 core patterning genes over six time points for every cell of the blastoderm embryo. This is the first cellular resolution dataset of a genetically perturbed Drosophila embryo that captures all cells in 3D. We describe the technical developments required to build this atlas and how the method can be employed and extended by others. We also analyze this novel dataset to characterize the degree and timing of cell fate canalization in the segmentation network. We find that in two layers of this gene regulatory network, following depletion of bcd, individual cells rapidly canalize towards normal cell fates. This result supports the hypothesis that the segmentation network directly canalizes cell fate, rather than an alternative hypothesis whereby cells are initially mis-specified and later eliminated by apoptosis. Our gene expression atlas provides a high resolution picture of a classic perturbation and will enable further computational modeling of canalization and gene regulation in this transcriptional network.

  13. Sequential Dysfunction and Progressive Depletion of Candida albicans-Specific CD4 T Cell Response in HIV-1 Infection.

    Directory of Open Access Journals (Sweden)

    Fengliang Liu

    2016-06-01

    Full Text Available Loss of immune control over opportunistic infections can occur at different stages of HIV-1 (HIV disease, among which mucosal candidiasis caused by the fungal pathogen Candida albicans (C. albicans is one of the early and common manifestations in HIV-infected human subjects. The underlying immunological basis is not well defined. We have previously shown that compared to cytomegalovirus (CMV-specific CD4 cells, C. albicans-specific CD4 T cells are highly permissive to HIV in vitro. Here, based on an antiretroviral treatment (ART naïve HIV infection cohort (RV21, we investigated longitudinally the impact of HIV on C. albicans- and CMV-specific CD4 T-cell immunity in vivo. We found a sequential dysfunction and preferential depletion for C. albicans-specific CD4 T cell response during progressive HIV infection. Compared to Th1 (IFN-γ, MIP-1β functional subsets, the Th17 functional subsets (IL-17, IL-22 of C. albicans-specific CD4 T cells were more permissive to HIV in vitro and impaired earlier in HIV-infected subjects. Infection history analysis showed that C. albicans-specific CD4 T cells were more susceptible to HIV in vivo, harboring modestly but significantly higher levels of HIV DNA, than CMV-specific CD4 T cells. Longitudinal analysis of HIV-infected individuals with ongoing CD4 depletion demonstrated that C. albicans-specific CD4 T-cell response was preferentially and progressively depleted. Taken together, these data suggest a potential mechanism for earlier loss of immune control over mucosal candidiasis in HIV-infected patients and provide new insights into pathogen-specific immune failure in AIDS pathogenesis.

  14. Troxerutin protects against 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47)-induced liver inflammation by attenuating oxidative stress-mediated NAD{sup +}-depletion

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Zi-Feng [School of Environment Science and Spatial Informatics, China University of Mining and Technology, Xuzhou 221008, Jiangsu Province (China); Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, School of Life Science, Jiangsu Normal University, 101 Shanghai Road, Xuzhou 221116, Jiangsu Province (China); Zhang, Yan-qiu [School of Environment Science and Spatial Informatics, China University of Mining and Technology, Xuzhou 221008, Jiangsu Province (China); Fan, Shao-Hua [Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, School of Life Science, Jiangsu Normal University, 101 Shanghai Road, Xuzhou 221116, Jiangsu Province (China); Zhuang, Juan [School of Environment Science and Spatial Informatics, China University of Mining and Technology, Xuzhou 221008, Jiangsu Province (China); Zheng, Yuan-Lin, E-mail: ylzheng@jsnu.edu.cn [Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, School of Life Science, Jiangsu Normal University, 101 Shanghai Road, Xuzhou 221116, Jiangsu Province (China); Lu, Jun; Wu, Dong-Mei; Shan, Qun; Hu, Bin [Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, School of Life Science, Jiangsu Normal University, 101 Shanghai Road, Xuzhou 221116, Jiangsu Province (China)

    2015-02-11

    Highlights: • BDE-47 promotes liver inflammation by triggering oxidative stress-induced NAD{sup +} depletion. • Troxerutin inhibits BDE-47-induced liver inflammation via its antioxidant properties. • Troxerutin restores NAD{sup +} level and consequently abates SirT1 loss. • Troxerutin represses acetylation of NF-κB p65 (K310) and H3K9. • Troxerutin is a candidate for prevention and therapy of BDE-47-induced hepatotoxicity. - Abstract: Emerging evidence indicates that 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) induces liver injury through enhanced ROS production and lymphocytic infiltration, which may promote a liver inflammatory response. Antioxidants have been reported to attenuate the cellular toxicity associated with polybrominated diphenyl ethers (PBDEs). In this study, we investigated the effect of troxerutin, a trihydroxyethylated derivative of the natural bioflavonoid rutin, on BDE-47-induced liver inflammation and explored the potential mechanisms underlying this effect. Our results showed that NAD{sup +}-depletion was involved in the oxidative stress-mediated liver injury in a BDE-47 treated mouse model, which was confirmed by Vitamin E treatment. Furthermore, our data revealed that troxerutin effectively alleviated liver inflammation by mitigating oxidative stress-mediated NAD{sup +}-depletion in BDE-47 treated mice. Consequently, troxerutin remarkably restored SirT1 protein expression and activity in the livers of BDE-47-treated mice. Mechanistically, troxerutin dramatically repressed the nuclear translocation of NF-κB p65 and the acetylation of NF-κB p65 (Lys 310) and Histone H3 (Lys9) to abate the transcription of inflammatory genes in BDE-47-treated mouse livers. These inhibitory effects of troxerutin were markedly blunted by EX527 (SirT1 inhibitor) treatment. This study provides novel mechanistic insights into the toxicity of BDE-47 and indicates that troxerutin might be used in the prevention and therapy of BDE-47-induced

  15. Attenuating effect of daidzein on polychlorinated biphenyls-induced oxidative toxicity in mouse testicular cells*

    OpenAIRE

    Zhang, Da-lei; Mi, Yu-ling; Wang, Kai-Ming; Zeng, Wei-dong; Zhang, Cai-qiao

    2008-01-01

    The attenuating effect of daidzein (DAI) on oxidative toxicity induced by Aroclor 1254 (A1254) was investigated in mouse testicular cells. Cells were exposed to A1254 alone or with DAI. The oxidative damage was estimated by measuring malondialdehyde (MDA) formation, superoxide dismutase (SOD) activity and glutathione (GSH) content. Results show that A1254 induced a decrease of germ cell number, an elevation in thiobarbituric acid reactive substances (TBARS) but a decrease in SOD activity and ...

  16. Metformin and phenformin deplete tricarboxylic acid cycle and glycolytic intermediates during cell transformation and NTPs in cancer stem cells.

    Science.gov (United States)

    Janzer, Andreas; German, Natalie J; Gonzalez-Herrera, Karina N; Asara, John M; Haigis, Marcia C; Struhl, Kevin

    2014-07-22

    Metformin, a first-line diabetes drug linked to cancer prevention in retrospective clinical analyses, inhibits cellular transformation and selectively kills breast cancer stem cells (CSCs). Although a few metabolic effects of metformin and the related biguanide phenformin have been investigated in established cancer cell lines, the global metabolic impact of biguanides during the process of neoplastic transformation and in CSCs is unknown. Here, we use LC/MS/MS metabolomics (>200 metabolites) to assess metabolic changes induced by metformin and phenformin in an Src-inducible model of cellular transformation and in mammosphere-derived breast CSCs. Although phenformin is the more potent biguanide in both systems, the metabolic profiles of these drugs are remarkably similar, although not identical. During the process of cellular transformation, biguanide treatment prevents the boost in glycolytic intermediates at a specific stage of the pathway and coordinately decreases tricarboxylic acid (TCA) cycle intermediates. In contrast, in breast CSCs, biguanides have a modest effect on glycolytic and TCA cycle intermediates, but they strongly deplete nucleotide triphosphates and may impede nucleotide synthesis. These metabolic profiles are consistent with the idea that biguanides inhibit mitochondrial complex 1, but they indicate that their metabolic effects differ depending on the stage of cellular transformation.

  17. mtDNA depletion confers specific gene expression profiles in human cells grown in culture and in xenograft

    Directory of Open Access Journals (Sweden)

    Ramaswamy Krishna

    2008-11-01

    Full Text Available Abstract Background Interactions between the gene products encoded by the mitochondrial and nuclear genomes play critical roles in eukaryotic cellular function. However, the effects mitochondrial DNA (mtDNA levels have on the nuclear transcriptome have not been defined under physiological conditions. In order to address this issue, we characterized the gene expression profiles of A549 lung cancer cells and their mtDNA-depleted ρ0 counterparts grown in culture and as tumor xenografts in immune-deficient mice. Results Cultured A549 ρ0 cells were respiration-deficient and showed enhanced levels of transcripts relevant to metal homeostasis, initiation of the epithelial-mesenchymal transition, and glucuronidation pathways. Several well-established HIF-regulated transcripts showed increased or decreased abundance relative to the parental cell line. Furthermore, growth in culture versus xenograft has a significantly greater influence on expression profiles, including transcripts involved in mitochondrial structure and both aerobic and anaerobic energy metabolism. However, both in vitro and in vivo, mtDNA levels explained the majority of the variance observed in the expression of transcripts in glucuronidation, tRNA synthetase, and immune surveillance related pathways. mtDNA levels in A549 xenografts also affected the expression of genes, such as AMACR and PHYH, involved in peroxisomal lipid metabolic pathways. Conclusion We have identified mtDNA-dependent gene expression profiles that are shared in cultured cells and in xenografts. These profiles indicate that mtDNA-depleted cells could provide informative model systems for the testing the efficacy of select classes of therapeutics, such as anti-angiogenesis agents. Furthermore, mtDNA-depleted cells grown culture and in xenografts provide a powerful means to investigate possible relationships between mitochondrial activity and gene expression profiles in normal and pathological cells.

  18. Extracellular vesicles released from mesenchymal stromal cells modulate miRNA in renal tubular cells and inhibit ATP depletion injury.

    Science.gov (United States)

    Lindoso, Rafael S; Collino, Federica; Bruno, Stefania; Araujo, Dayana S; Sant'Anna, Julliana F; Tetta, Ciro; Provero, Paolo; Quesenberry, Peter J; Vieyra, Adalberto; Einicker-Lamas, Marcelo; Camussi, Giovanni

    2014-08-01

    The mechanisms involved in renal repair by mesenchymal stromal cells (MSCs) are not entirely elucidated. The paracrine secretion of bioactive molecules has been implicated in the protective effects. Besides soluble mediators, MSCs have been shown to release extracellular vesicles (EVs), involved in renal repair process for different injury models. EVs have been shown to mediate communication between cells through the transference of several molecules, like protein, bioactive lipids, mRNA, and microRNAs (miRNAs). The miRNAs are noncoding RNAs that posttranscriptionally modulate gene expression and are involved in the regulation of several cellular processes, including those related to repair. The aim of the present study was to investigate the role of MSC-EVs in the modulation of miRNAs inside renal proximal tubular epithelial cells (PTECs) in an in vitro model of ischemia-reperfusion injury induced by ATP depletion. In this model we evaluated whether changes in miRNA expression were dependent on direct miRNA transfer or on transcription induction by MSC-EVs. The obtained results showed an enhanced incorporation of MSC-EVs in injured PTECs with protection from cell death. This biological effect was associated with EV-mediated miRNA transfer and with transcriptional modulation of miRNAs expressed by injured PTECs. Prediction of miRNA targets showed that miRNAs modulated in PTECs are involved in process of renal recovery with downregulation of coding-mRNAs associated with apoptosis, cytoskeleton reorganization, and hypoxia, such as CASP3 and 7, SHC1 and SMAD4. In conclusion, these results indicate that MSC-EVs may transfer and modulate the expression of several miRNAs involved in the repair and recovery process in PTECs.

  19. DNA damage response in renal ischemia-reperfusion and ATP-depletion injury of renal tubular cells.

    Science.gov (United States)

    Ma, Zhengwei; Wei, Qingqing; Dong, Guie; Huo, Yuqing; Dong, Zheng

    2014-07-01

    Renal ischemia-reperfusion leads to acute kidney injury (AKI) that is characterized pathologically by tubular damage and cell death, followed by tubular repair, atrophy and interstitial fibrosis. Recent work suggested the possible presence of DNA damage response (DDR) in AKI. However, the evidence is sketchy and the role and regulation of DDR in ischemic AKI remain elusive. In this study, we demonstrated the induction of phosphorylation of ATM, H2AX, Chk2 and p53 during renal ischemia-reperfusion in mice, suggesting DDR in kidney tissues. DDR was also induced in vitro during the recovery or "reperfusion" of renal proximal tubular cells (RPTCs) after ATP depletion. DDR in RPTCs was abrogated by supplying glucose to maintain ATP via glycolysis, indicating that the DDR depends on ATP depletion. The DDR was also suppressed by the general caspase inhibitor z-VAD and the overexpression of Bcl-2, supporting a role of apoptosis-associated DNA damage in the DDR. N-acetylcysteine (NAC), an antioxidant, suppressed the phosphorylation of ATM and p53 and, to a less extent, Chk2, but NAC increased the phosphorylation and nuclear foci formation of H2AX. Interestingly, NAC increased apoptosis, which may account for the observed H2AX activation. Ku55933, an ATM inhibitor, blocked ATM phosphorylation and ameliorated the phosphorylation of Chk2 and p53, but it increased H2AX phosphorylation and nuclear foci formation. Ku55933 also increased apoptosis in RPTCs following ATP depletion. The results suggest that DDR occurs during renal ischemia-reperfusion in vivo and ATP-depletion injury in vitro. The DDR is partially induced by apoptosis and oxidative stress-related DNA damage. ATM, as a sensor in the DDR, may play a cytoprotective role against tubular cell injury and death.

  20. Dihydroorotate dehydrogenase (DHODH) inhibitors affect ATP depletion, endogenous ROS and mediate S-phase arrest in breast cancer cells.

    Science.gov (United States)

    Mohamad Fairus, A K; Choudhary, B; Hosahalli, S; Kavitha, N; Shatrah, O

    2017-04-01

    Dihydroorotate dehydrogenase (DHODH) is the key enzyme in de novo biosynthesis of pyrimidine in both prokaryotes and eukaryotes. The de novo pathway of pyrimidine biosynthesis is essential in cancer cells proliferation. Leflunomide is an approved DHODH inhibitor that has been widely used for the treatment of arthritis. Similarly, brequinar sodium is another DHODH inhibitor that showed anti-tumour effect in MC38 colon carcinoma cells when used in combination with fluorouracil. Despite the potential role of DHODH inhibitors in cancer therapy, their mechanisms of action remain obscure and await further elucidation. Here, we evaluated the effect of DHODH inhibitors on the production of ATP and ROS in sensitive and non-sensitive breast cancer cells. Subsequently, the effects of DHODH inhibitors on cell cycle as well as on signalling molecules such as p53, p65 and STAT6 were evaluated in sensitive T-47D and non-sensitive MDAMB-436 cells. The correlations between DHODH protein expression, proliferation speed and sensitivity to DHODH inhibitors were also investigated in a panel of cancer cell lines. DHODH inhibitors-sensitive T-47D and MDAMB-231 cells appeared to preserve ROS production closely to endogenous ROS level whereas the opposite was observed in non-sensitive MDAMB-436 and W3.006 cells. In addition, we observed approximately 90% of intracellular ATP depletion in highly sensitive T-47D and MDAMB-231 cells compared to non-sensitive MDAMB-436 cells. There was significant over-expression of p53, p65 and STAT6 signalling molecules in sensitive cells which may be involved in mediating the S-phase arrest in cell cycle progression. The current study suggests that DHODH inhibitors are most effective in cells that express high levels of DHODH enzyme. The inhibition of cell proliferation by these inhibitors appears to be accompanied by ROS production as well as ATP depletion. The increase in expression of signalling molecules observed may be due to pyrimidine depletion

  1. Massive glycosaminoglycan-dependent entry of Trp-containing cell-penetrating peptides induced by exogenous sphingomyelinase or cholesterol depletion.

    Science.gov (United States)

    Bechara, Chérine; Pallerla, Manjula; Burlina, Fabienne; Illien, Françoise; Cribier, Sophie; Sagan, Sandrine

    2015-02-01

    Among non-invasive cell delivery strategies, cell-penetrating peptide (CPP) vectors represent interesting new tools. To get fundamental knowledge about the still debated internalisation mechanisms of these peptides, we modified the membrane content of cells, typically by hydrolysis of sphingomyelin or depletion of cholesterol from the membrane outer leaflet. We quantified and visualised the effect of these viable cell surface treatments on the internalisation efficiency of different CPPs, among which the most studied Tat, R9, penetratin and analogues, that all carry the N-terminal biotin-Gly4 tag cargo. Under these cell membrane treatments, only penetratin and R6W3 underwent a massive glycosaminoglycan (GAG)-dependent entry in cells. Internalisation of the other peptides was only slightly increased, similarly in the absence or the presence of GAGs for R9, and only in the presence of GAGs for Tat and R6L3. Ceramide formation (or cholesterol depletion) is known to lead to the reorganisation of membrane lipid domains into larger platforms, which can serve as a trap and cluster receptors. These results show that GAG clustering, enhanced by formation of ceramide, is efficiently exploited by penetratin and R6W3, which contains Trp residues in their sequence but not Tat, R9 and R6L3. Hence, these data shed new lights on the differences in the internalisation mechanism and pathway of these peptides that are widely used in delivery of cargo molecules.

  2. CERT depletion predicts chemotherapy benefit and mediates cytotoxic and polyploid‐specific cancer cell death through autophagy induction

    DEFF Research Database (Denmark)

    Lee, Alvin J. X.; Roylance, Rebecca; Sander, Jil

    2012-01-01

    and predictor of outcome in adjuvant chemotherapy‐treated patients with primary breast cancer. These data suggest that the induction of LAMP2‐dependent autophagic flux through CERT targeting may provide a rational approach to enhance multidrug sensitization and potentiate the death of polyploid cells following...... to the death of CIN cancer cells. Using an integrative functional genomics approach, we find that CERT‐specific multidrug sensitization is associated with enhanced autophagosome–lysosome flux, resulting from the expression of LAMP2 following CERT silencing in colorectal and HER2+ breast cancer cell lines. Live...... cell microscopy analysis revealed that CERT depletion induces LAMP2‐dependent death of polyploid cells following exit from mitosis in the presence of paclitaxel. We find that CERT is relatively over‐expressed in HER2+ breast cancer and CERT protein expression acts as an independent prognostic variable...

  3. Depletion of alloreactive T-cells in vitro using the proteasome inhibitor bortezomib preserves the immune response against pathogens.

    Science.gov (United States)

    Blanco, Belén; Sánchez-Abarca, Luis Ignacio; Caballero-Velázquez, Teresa; Santamaría, Carlos; Inogés, Susana; Pérez-Simón, José Antonio

    2011-10-01

    Current graft-versus-host disease (GVHD) inhibition approaches lead to abrogation of pathogen-specific T-cell responses. We propose an approach to inhibit GVHD without hampering immunity against pathogens: in vitro depletion of alloreactive T cells with the preoteasome inhibitor bortezomib. We show that PBMCs stimulated with allogeneic cells and treated with bortezomib greatly reduce their ability to produce IFN-γ when re-stimulated with the same allogeneic cells, but mainly preserve their ability to respond to citomegalovirus stimulation. Unlike in vivo administration of immunosuppressive drugs or other strategies of allodepletion, in vitro allodepletion with bortezomib maintains pathogen-specific T cells, representing a promising alternative for GVHD prophylaxis.

  4. CFTR depletion results in changes in fatty acid composition and promotes lipogenesis in intestinal Caco 2/15 cells.

    Directory of Open Access Journals (Sweden)

    Geneviève Mailhot

    Full Text Available BACKGROUND: Abnormal fatty acid composition (FA in plasma and tissue lipids frequently occurs in homozygous and even in heterozygous carriers of cystic fibrosis transmembrane conductance regulator (CFTR mutations. The mechanism(s underlying these abnormalities remained, however, poorly understood despite the potentially CFTR contributing role. METHODOLOGY/PRINCIPAL FINDINGS: The aim of the present study was to investigate the impact of CFTR depletion on FA uptake, composition and metabolism using the intestinal Caco-2/15 cell line. shRNA-mediated cftr gene silencing induced qualitative and quantitative modifications in FA composition in differentiated enterocytes as determined by gas-liquid chromatography. With the cftr gene disruption, there was a 1,5 fold increase in the total FA amount, largely attributable to monounsaturated and saturated FA compared to controls. The activity of delta-7 desaturase, estimated by the 16:1(n-7/16:0, was significantly higher in knockdown cells and consistent with the striking elevation of the n-7 FA family. When incubated with [14C]-oleic acid, CFTR-depleted cells were capable of quick incorporation and export to the medium concomitantly with the high protein expression of L-FABP known to promote intracellular FA trafficking. Accordingly, lipoprotein vehicles (CM, VLDL, LDL and HDL, isolated from CFTR knockdown cells, exhibited higher levels of radiolabeled FA. Moreover, in the presence of [14C]-acetate, knockdown cells exhibited enhanced secretion of newly synthesized phospholipids, triglycerides, cholesteryl esters and free FA, thereby suggesting a stimulation of the lipogenic pathway. Conformably, gene expression of SREBP-1c, a key lipogenic transcription factor, was increased while protein expression of the phosphorylated and inactive form of acetylCoA carboxylase was reduced, confirming lipogenesis induction. Finally, CFTR-depleted cells exhibited lower gene expression of transcription factors (PPARalpha

  5. Local suppression of T cell responses by arginase-induced L-arginine depletion in nonhealing leishmaniasis.

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    Manuel Modolell

    Full Text Available The balance between T helper (Th 1 and Th2 cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized Th1 or Th2 responses are not sufficient to account for healing or nonhealing. Here we show that high arginase activity, a hallmark of nonhealing disease, is primarily expressed locally at the site of pathology. The high arginase activity causes local depletion of L-arginine, which impairs the capacity of T cells in the lesion to proliferate and to produce interferon-gamma, while T cells in the local draining lymph nodes respond normally. Healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. Moreover, competitive inhibition of arginase as well as supplementation with L-arginine restored T cell effector functions and reduced pathology and parasite growth at the site of lesions. These results demonstrate that in nonhealing leishmaniasis, arginase-induced L-arginine depletion results in impaired T cell responses. Our results identify a novel mechanism in leishmaniasis that contributes to the failure to heal persistent lesions and suggest new approaches to therapy.

  6. Partial depletion of regulatory T cells does not influence the inflammation caused by high dose hemi-body irradiation.

    Directory of Open Access Journals (Sweden)

    Shihong Ma

    Full Text Available There is clinical interest in the modulation of regulatory T cells for cancer therapy. The safety of these therapies in combination with conventional anti-cancer therapies, including radiation therapy, can be studied in animal models. The effects of partial depletion of regulatory T (Treg cells with an anti-CD25 antibody in conjunction with ionizing radiation on inflammation and tissue injury were analyzed in C57BL/6 mice. An anti-CD25 antibody (PC61 was administered 3 days prior to 13 Gy lower-half hemi-body irradiation (HBI. The blood, spleen, mesenteric lymph nodes (mLNs and inguinal lymph nodes (iLNs were harvested at various times thereafter. Alterations in the proportion of leukocyte subsets including CD4(+ T cells, CD8(+ T cells, Treg cells, B cells, NK cells, NK1.1(+ T cells, macrophages and granulocytes were analyzed by FACS. The lungs, liver, pancreas, stomach, jejunum, duodenum, ileum, colon and kidney were harvested and studied by H&E staining. Expression of inflammatory mediators in plasma and tissue were investigated by ELISA. HBI significantly decreased the leukocyte pool though the various leukocyte subsets had different sensitivities to HBI. The administration of PC61 significantly decreased the proportion of Treg cells in spleen, iLN, mLN and blood (reduction of approximately 60%. Irradiation significantly increased the proportion of Treg cells in the spleen, iLN and mLN. HBI induced a systemic inflammatory reaction as demonstrated by increased plasma levels of IL-6, KC/CXCL1 and circulating granulocytes in the blood. Neutrophils also infiltrated the small bowel. The same general patterns were observed whether or not Treg cells were partially depleted with PC61 prior to HBI. These data demonstrate that partial depletion of Treg cells in these mice does not influence HBI-induced inflammatory response and tissue injury, and that combining anti-CD25 therapy with radiation may be safe and well tolerated in a clinical setting.

  7. Partial Depletion of Regulatory T Cells Does Not Influence the Inflammation Caused by High Dose Hemi-Body Irradiation

    Science.gov (United States)

    Ma, Shihong; Richardson, James A.; Bitmansour, Andrew; Solberg, Timothy D.; Pidikiti, Rajesh; Song, Kwang; Stojadinovic, Strahinja; Vitetta, Ellen S.; Meyer, Jeffrey J.

    2013-01-01

    There is clinical interest in the modulation of regulatory T cells for cancer therapy. The safety of these therapies in combination with conventional anti-cancer therapies, including radiation therapy, can be studied in animal models. The effects of partial depletion of regulatory T (Treg) cells with an anti-CD25 antibody in conjunction with ionizing radiation on inflammation and tissue injury were analyzed in C57BL/6 mice. An anti-CD25 antibody (PC61) was administered 3 days prior to 13 Gy lower-half hemi-body irradiation (HBI). The blood, spleen, mesenteric lymph nodes (mLNs) and inguinal lymph nodes (iLNs) were harvested at various times thereafter. Alterations in the proportion of leukocyte subsets including CD4+ T cells, CD8+ T cells, Treg cells, B cells, NK cells, NK1.1+ T cells, macrophages and granulocytes were analyzed by FACS. The lungs, liver, pancreas, stomach, jejunum, duodenum, ileum, colon and kidney were harvested and studied by H&E staining. Expression of inflammatory mediators in plasma and tissue were investigated by ELISA. HBI significantly decreased the leukocyte pool though the various leukocyte subsets had different sensitivities to HBI. The administration of PC61 significantly decreased the proportion of Treg cells in spleen, iLN, mLN and blood (reduction of approximately 60%). Irradiation significantly increased the proportion of Treg cells in the spleen, iLN and mLN. HBI induced a systemic inflammatory reaction as demonstrated by increased plasma levels of IL-6, KC/CXCL1 and circulating granulocytes in the blood. Neutrophils also infiltrated the small bowel. The same general patterns were observed whether or not Treg cells were partially depleted with PC61 prior to HBI. These data demonstrate that partial depletion of Treg cells in these mice does not influence HBI-induced inflammatory response and tissue injury, and that combining anti-CD25 therapy with radiation may be safe and well tolerated in a clinical setting. PMID:23409194

  8. Depletion of CD25+CD4+T cells (Tregs) enhances the HBV-specific CD8+ T cell response primed by DNA immunization

    Institute of Scientific and Technical Information of China (English)

    Yoshihiro Furuichi; Hirotake Tokuyama; Satoshi Ueha; Makoto Kurachi; Fuminori Moriyasu; Kazuhiro Kakimi

    2005-01-01

    AIM: Persistent hepatitis B virus (HBV) infection is characterized by a weak CD8+ T cell response to HBV. Immunotherapeutic strategies that overcome tolerance and boost these suboptimal responses may facilitate viral clearance in chronically infected individuals. Therefore, we examined whether CD25+CD4+ regulatory T (Treg) cells might be involved in a inhibition of CD8+T cell priming or in the modulation of the magnitude of the'peak' antiviral CD8+ T cell response primed by DNA immunization. METHODS: B10.D2 mice were immunized once with plasmid pCMV-S. Mice received 500 μg of anti-CD25 mAb injected intraperitoneally 3 d before DNA immunization to deplete CD25+ cells. Induction of HBV-specific CD8+ T ceils in peripheral blood mononuclear cells (PBMCs) was measured by S28-39 peptide loaded DimerX staining and their function was analyzed by intracellular IFN-γ staining.RESULTS: DNA immunization induced HBV-specific CD8+ T cells. At the peak T cell response (d 10), 7.1±2.0% of CD8+ T cells were HBV-specific after DNA immunization, whereas 12.7±3.2% of CD8+ T cells were HBV-specific in Treg-depleted mice, suggesting that DNA immunization induced more antigen-specific CD8+ T cells in the absence of CD25+ Treg cells (n = 6, P<0.05). Similarly, fewer HBVspecific memory T cells were detected in the presence of these cells (1.3±0.4%) in comparison to Treg-depleted mice (2.6±0.9%) on d 30 after DNA immunization (n = 6, P<0.01). Both IFN-γ production and the avidity of the HBV-specific CD8+ T cell response to antigen were higher in HBV-specific CD8+ T cells induced in the absence of Treg cells.CONCLUSION: CD25+ Treg cells suppress priming and/or expansion of antigen-specific CD8+ T cells during DNA immunization and the peak CD8+ T cell response is enhanced by depleting this cell population. Furthermore, Treg cells appear to be involved in the contraction phase of the CD8+ T ceil response and may affect the quality of memory T cell pools. The elimination of Treg

  9. Ordered Nanopillar Structured Electrodes for Depleted Bulk Heterojunction Colloidal Quantum Dot Solar Cells

    KAUST Repository

    Kramer, Illan J.

    2012-03-30

    A bulk heterojunction of ordered titania nanopillars and PbS colloidal quantum dots is developed. By using a pre-patterned template, an ordered titania nanopillar matrix with nearest neighbours 275 nm apart and height of 300 nm is fabricated and subsequently filled in with PbS colloidal quantum dots to form an ordered depleted bulk heterojunction exhibiting power conversion efficiency of 5.6%. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. NK and NKT Cell Depletion Alters the Outcome of Experimental Pneumococcal Pneumonia: Relationship with Regulation of Interferon-γ Production

    Directory of Open Access Journals (Sweden)

    Eirini Christaki

    2015-01-01

    Full Text Available Background. Natural killer (NK and natural killer T (NKT cells contribute to the innate host defense but their role in bacterial sepsis remains controversial. Methods. C57BL/6 mice were infected intratracheally with 5 × 105 cfu of Streptococcus pneumoniae. Animals were divided into sham group (Sham; pretreated with isotype control antibody (CON group; pretreated with anti-asialo GM1 antibody (NKd group; and pretreated with anti-CD1d monoclonal antibody (NKTd group before bacterial challenge. Serum and tissue samples were analyzed for bacterial load, cytokine levels, splenocyte apoptosis rates, and cell characteristics by flow cytometry. Splenocyte miRNA expression was also analyzed and survival was assessed. Results. NK cell depletion prolonged survival. Upon inhibition of NKT cell activation, spleen NK (CD3−/NK1.1+ cells increased compared to all other groups. Inhibition of NKT cell activation led to higher bacterial loads and increased levels of serum and splenocyte IFN-γ. Splenocyte miRNA analysis showed that miR-200c and miR-29a were downregulated, while miR-125a-5p was upregulated, in anti-CD1d treated animals. These changes were moderate after NK cell depletion. Conclusions. NK cells appear to contribute to mortality in pneumococcal pneumonia. Inhibition of NKT cell activation resulted in an increase in spleen NK (CD3−/NK1.1+ cells and a higher IFN-γ production, while altering splenocyte miRNA expression.

  11. Mesenchymal Stem Cell Transplantation Attenuates Brain Injury After Neonatal Stroke

    NARCIS (Netherlands)

    van Velthoven, Cindy T. J.; Sheldon, R. Ann; Kavelaars, Annemieke; Derugin, Nikita; Vexler, Zinaida S.; Willemen, Hanneke L. D. M.; Maas, Mirjam; Heijnen, Cobi J.; Ferriero, Donna M.

    2013-01-01

    Background and Purpose-Brain injury caused by stroke is a frequent cause of perinatal morbidity and mortality with limited therapeutic options. Mesenchymal stem cells (MSC) have been shown to improve outcome after neonatal hypoxic-ischemic brain injury mainly by secretion of growth factors stimulati

  12. Simvastatin Attenuates TGF-β1-Induced Epithelial-Mesenchymal Transition in Human Alveolar Epithelial Cells

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    Tuo Yang

    2013-06-01

    Full Text Available Background: Transforming growth factor-β1 (TGF-β1-induced epithelial-mesenchymal transition (EMT of alveolar epithelial cells (AEC may contribute to idiopathic pulmonary fibrosis (IPF. TGF-β1-induced EMT in A549 cells (a human AEC cell line resulted in the adoption of mesenchymal responses that were predominantly mediated via the TGF-β1-Smad2/3 signaling pathway. Simvastatin (Sim, a 3-hydroxy-3-methylglutaryl CoA (HMG-CoA reductase inhibitor, has been previously reported to inhibit EMT in human proximal tubular epithelial cells and porcine lens epithelial cells and to suppress Smad2/3 phosphorylation in animal models. However, whether Sim can attenuate TGF-β1-induced EMT in A549 cells and its underlying mechanisms remains unknown. Methods: Cells were incubated with TGF-β1 in the presence or absence of Sim. The epithelial marker E-cadherin (E-Cad and the mesenchymal markers, α-smooth muscle actin (α-SMA, vimentin (Vi and fibronectin (FN, were detected using western blotting analyses and immunofluorescence. Phosphorylated Smad2 and Smad3 levels and connective tissue growth factor (CTGF were analyzed using western blotting. In addition, a cell migration assay was performed. Moreover, the levels of matrix metalloproteinase (MMP-2 and -9 in the culture medium were examined using ELISA. Results: Sim significantly attenuated the TGF-β1-induced decrease in E-Cad levels and elevated the levels of α-SMA, Vi and FN via the suppression of Smad2 and Smad3 phosphorylation. Furthermore, Sim inhibited the mesenchymal-like responses in A549 cells, including cell migration, CTGF expression and secretion of MMP-2 and -9. However, Sim failed to reverse the cell morphologial changes induced by TGF-β1 in A549 cells. Conclusion: Sim attenuated TGF-β1-induced EMT in A549 cells and might be a promising therapeutic agent for treating IPF.

  13. Depletion of regulatory T cells augments a vaccine-induced T effector cell response against the liver-stage of malaria but fails to increase memory.

    Directory of Open Access Journals (Sweden)

    Maria del Rosario Espinoza Mora

    Full Text Available Regulatory T cells (T(reg have been shown to restrict vaccine-induced T cell responses in different experimental models. In these studies CD4(+CD25(+ T(reg were depleted using monoclonal antibodies against CD25, which might also interfere with CD25 on non-regulatory T cell populations and would have no effect on Foxp3(+CD25(- T(reg. To obtain more insights in the specific function of T(reg during vaccination we used mice that are transgenic for a bacterial artificial chromosome expressing a diphtheria toxin (DT receptor-eGFP fusion protein under the control of the foxp3 gene locus (depletion of regulatory T cell mice; DEREG. As an experimental vaccine-carrier recombinant Bordetella adenylate cyclase toxoid fused with a MHC-class I-restricted epitope of the circumsporozoite protein (ACT-CSP of Plasmodium berghei (Pb was used. ACT-CSP was shown by us previously to introduce the CD8+ epitope of Pb-CSP into the MHC class I presentation pathway of professional antigen-presenting cells (APC. Using this system we demonstrate here that the number of CSP-specific T cells increases when T(reg are depleted during prime but also during boost immunization. Importantly, despite this increase of T effector cells no difference in the number of antigen-specific memory cells was observed.

  14. Submolecular regulation of cell transformation by deuterium depleting water exchange reactions in the tricarboxylic acid substrate cycle.

    Science.gov (United States)

    Boros, László G; D'Agostino, Dominic P; Katz, Howard E; Roth, Justine P; Meuillet, Emmanuelle J; Somlyai, Gábor

    2016-02-01

    The naturally occurring isotope of hydrogen ((1)H), deuterium ((2)H), could have an important biological role. Deuterium depleted water delays tumor progression in mice, dogs, cats and humans. Hydratase enzymes of the tricarboxylic acid (TCA) cycle control cell growth and deplete deuterium from redox cofactors, fatty acids and DNA, which undergo hydride ion and hydrogen atom transfer reactions. A model is proposed that emphasizes the terminal complex of mitochondrial electron transport chain reducing molecular oxygen to deuterium depleted water (DDW); this affects gluconeogenesis as well as fatty acid oxidation. In the former, the DDW is thought to diminish the deuteration of sugar-phosphates in the DNA backbone, helping to preserve stability of hydrogen bond networks, possibly protecting against aneuploidy and resisting strand breaks, occurring upon exposure to radiation and certain anticancer chemotherapeutics. DDW is proposed here to link cancer prevention and treatment using natural ketogenic diets, low deuterium drinking water, as well as DDW production as the mitochondrial downstream mechanism of targeted anti-cancer drugs such as Avastin and Glivec. The role of (2)H in biology is a potential missing link to the elusive cancer puzzle seemingly correlated with cancer epidemiology in western populations as a result of excessive (2)H loading from processed carbohydrate intake in place of natural fat consumption.

  15. Identification of CETP as a molecular target for estrogen positive breast cancer cell death by cholesterol depleting agents.

    Science.gov (United States)

    Esau, Luke; Sagar, Sunil; Bangarusamy, Dhinoth; Kaur, Mandeep

    2016-09-01

    Cholesterol and its metabolites act as steroid hormone precursors, which promote estrogen receptor positive (ER+) breast cancer (BC) progression. Development of cholesterol targeting anticancer drugs has been hindered due to the lack of knowledge of viable molecular targets. Till now, Cholesteryl ester transfer protein (CETP) has been envisaged as a feasible molecular target in atherosclerosis, but for the first time, we show that CETP contributes to BC cell survival when challenged with cholesterol depleting agents. We show that MCF-7 CETP knockout BC cells pose less resistance towards cytotoxic compounds (Tamoxifen and Acetyl Plumbagin (AP)), and were more susceptible to intrinsic apoptosis. Analysis of differentially expressed genes using Ingenuity Pathway Analysis (IPA), in vivo tumor inhibition, and in vitro phenotypic responses to AP revealed a unique CETP-centric cholesterol pathway involved in sensitizing ER+ BC cells to intrinsic mitochondrial apoptosis. Furthermore, analysis of cell line, tissue and patient data available in publicly available databases linked elevated CETP expression to cancer, cancer relapse and overall poor survival. Overall, our findings highlight CETP as a pharmacologically relevant and unexploited cellular target in BC. The work also highlights AP as a promising chemical entity for preclinical investigations as a cholesterol depleting anticancer therapeutic agent.

  16. Long-term B cell depletion in murine lupus eliminates autoantibody-secreting cells and is associated with alterations in the kidney plasma cell niche.

    Science.gov (United States)

    Wang, Wensheng; Rangel-Moreno, Javier; Owen, Teresa; Barnard, Jennifer; Nevarez, Sarah; Ichikawa, H Travis; Anolik, Jennifer H

    2014-04-01

    Autoantibodies to dsDNA, produced by autoreactive plasma cells (PCs), are a hallmark of systemic lupus erythematosus and play a key role in disease pathogenesis. Recent data suggest that autoreactive PCs accumulate not only in lymphoid tissues, but also in the inflamed kidney in lupus nephritis. We hypothesized that the variable efficacy of anti-CD20 (rituximab)-mediated B cell depletion in systemic lupus erythematosus may be related to the absence of an effect on autoreactive PCs in the kidney. In this article, we report that an enrichment of autoreactive dsDNA Ab-secreting cells (ASCs) in the kidney of lupus-prone mice (up to 40% of the ASCs) coincided with a progressive increase in splenic germinal centers and PCs, and an increase in renal expression for PC survival factors (BAFF, a proliferation-inducing ligand, and IL-6) and PC attracting chemokines (CXCL12). Short-term treatment with anti-CD20 (4 wk) neither decreased anti-dsDNA nor IgG ASCs in different anatomical locations. However, long-term treatment (12 wk) significantly reduced both IgG- and dsDNA-specific ASCs. In addition, long-term treatment substantially decreased splenic germinal center and PC generation, and unexpectedly reduced the expression for PC survival factors in the kidney. These results suggest that prolonged B cell depletion may alter the PC survival niche in the kidney, regulating the accumulation and maintenance of autoreactive PCs.

  17. Plant recombinant erythropoietin attenuates inflammatory kidney cell injury.

    Science.gov (United States)

    Conley, Andrew J; Mohib, Kanishka; Jevnikar, Anthony M; Brandle, Jim E

    2009-02-01

    Human erythropoietin (EPO) is a pleiotropic cytokine with remarkable tissue-protective activities in addition to its well-established role in red blood cell production. Unfortunately, conventional mammalian cell cultures are unlikely to meet the anticipated market demands for recombinant EPO because of limited capacity and high production costs. Plant expression systems may address these limitations to enable practical, cost-effective delivery of EPO in tissue injury prevention therapeutics. In this study, we produced human EPO in tobacco and demonstrated that plant-derived EPO had tissue-protective activity. Our results indicated that targeting to the endoplasmic reticulum (ER) provided the highest accumulation levels of EPO, with a yield approaching 0.05% of total soluble protein in tobacco leaves. The codon optimization of the human EPO gene for plant expression had no clear advantage; furthermore, the human EPO signal peptide performed better than a tobacco signal peptide. In addition, we found that glycosylation was essential for the stability of plant recombinant EPO, whereas the presence of an elastin-like polypeptide fusion had a limited positive impact on the level of EPO accumulation. Confocal microscopy showed that apoplast and ER-targeted EPO were correctly localized, and N-glycan analysis demonstrated that complex plant glycans existed on apoplast-targeted EPO, but not on ER-targeted EPO. Importantly, plant-derived EPO had enhanced receptor-binding affinity and was able to protect kidney epithelial cells from cytokine-induced death in vitro. These findings demonstrate that tobacco plants may be an attractive alternative for the production of large amounts of biologically active EPO.

  18. Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells

    Energy Technology Data Exchange (ETDEWEB)

    Yeh, Lee-Chuan C.; Ford, Jeffery J. [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); Lee, John C. [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); The Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, TX (United States); Adamo, Martin L., E-mail: adamo@biochem.uthscsa.edu [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); The Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, TX (United States)

    2014-07-18

    Highlights: • Palmitate inhibits osteoblast differentiation. • Fatty acid synthase. • PPARγ. • Acetyl Co-A carboxylase inhibitor TOFA. • Fetal rat calvarial cell culture. - Abstract: Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibited the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl CoA carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPARγ in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects.

  19. Haptoglobin attenuates hemoglobin-induced heme oxygenase-1 in renal proximal tubule cells and kidneys of a mouse model of sickle cell disease.

    Science.gov (United States)

    Chintagari, Narendranath Reddy; Nguyen, Julia; Belcher, John D; Vercellotti, Gregory M; Alayash, Abdu I

    2015-03-01

    Sickle cell disease (SCD), a hereditary hemolytic disorder is characterized by chronic hemolysis, oxidative stress, vaso-occlusion and end-organ damage. Hemolysis releases toxic cell-free hemoglobin (Hb) into circulation. Under physiologic conditions, plasma Hb binds to haptoglobin (Hp) and forms Hb-Hp dimers. The dimers bind to CD163 receptors on macrophages for further internalization and degradation. However, in SCD patients plasma Hp is depleted and free Hb is cleared primarily by proximal tubules of kidneys. Excess free Hb in plasma predisposes patients to renal damage. We hypothesized that administration of exogenous Hp reduces Hb-mediated renal damage. To test this hypothesis, human renal proximal tubular cells (HK-2) were exposed to HbA (50μM heme) for 24h. HbA increased the expression of heme oxygenase-1 (HO-1), an enzyme which degrades heme, reduces heme-mediated oxidative toxicity, and confers cytoprotection. Similarly, infusion of HbA (32μM heme/kg) induced HO-1 expression in kidneys of SCD mice. Immunohistochemistry confirmed the increased HO-1 expression in the proximal tubules of the kidney. Exogenous Hp attenuated the HbA-induced HO-1 expression in vitro and in SCD mice. Our results suggest that Hb-mediated oxidative toxicity may contribute to renal damage in SCD and that Hp treatment reduces heme/iron toxicity in the kidneys following hemolysis.

  20. Transient B cell depletion or improved transgene expression by codon optimization promote tolerance to factor VIII in gene therapy.

    Directory of Open Access Journals (Sweden)

    Brandon K Sack

    Full Text Available The major complication in the treatment of hemophilia A is the development of neutralizing antibodies (inhibitors against factor VIII (FVIII. The current method for eradicating inhibitors, termed immune tolerance induction (ITI, is costly and protracted. Clinical protocols that prevent rather than treat inhibitors are not yet established. Liver-directed gene therapy hopes to achieve long-term correction of the disease while also inducing immune tolerance. We sought to investigate the use of adeno-associated viral (serotype 8 gene transfer to induce tolerance to human B domain deleted FVIII in hemophilia A mice. We administered an AAV8 vector with either human B domain deleted FVIII or a codon-optimized transgene, both under a liver-specific promoter to two strains of hemophilia A mice. Protein therapy or gene therapy was given either alone or in conjunction with anti-CD20 antibody-mediated B cell depletion. Gene therapy with a low-expressing vector resulted in sustained near-therapeutic expression. However, supplementary protein therapy revealed that gene transfer had sensitized mice to hFVIII in a high-responder strain but not in mice of a low-responding strain. This heightened response was ameliorated when gene therapy was delivered with anti-murine CD20 treatment. Transient B cell depletion prevented inhibitor formation in protein therapy, but failed to achieve a sustained hypo-responsiveness. Importantly, use of a codon-optimized hFVIII transgene resulted in sustained therapeutic expression and tolerance without a need for B cell depletion. Therefore, anti-CD20 may be beneficial in preventing vector-induced immune priming to FVIII, but higher levels of liver-restricted expression are preferred for tolerance.

  1. Perfluorocarbon emulsion therapy attenuates pneumococcal infection in sickle cell mice.

    Science.gov (United States)

    Helmi, Nawal; Andrew, Peter W; Pandya, Hitesh C

    2015-05-15

    Impaired immunity and tissue hypoxia-ischemia are strongly linked with Streptococcus pneumoniae pathogenesis in patients with sickle cell anemia. Perfluorocarbon emulsions (PFCEs) have high O2-dissolving capacity and can alleviate tissue hypoxia. Here, we evaluate the effects of intravenous PFCE therapy in transgenic sickle cell (HbSS) mice infected with S. pneumoniae. HbSS and C57BL/6 (control) mice intravenously infected with S. pneumoniae were treated intravenously with PFCE or phosphate-buffered saline (PBS) and then managed in either air/O2 (FiO2 proportion, 50%; hereafter referred to as the PFCE-O2 and PBS-O2 groups) or air only (hereafter, the PFCE-air and PBS-air groups) gas mixtures. Lungs were processed for leukocyte and bacterial counts and cytokine measurements. HbSS mice developed severe pneumococcal infection significantly faster than C57BL/6 mice (Kaplan-Maier analysis, P < .05). PFCE-O2-treated HbSS mice had significantly better survival at 72 hours than HBSS mice treated with PFCE-air, PBS-O2, or PBS-air (P < .05). PFCE-O2-treated HbSS mice also had significantly lower pulmonary leukocyte counts, lower interleukin 1β and interferon γ levels, and higher interleukin 10 levels than PFCE-air-treated HbSS mice. Clearance of S. pneumoniae from lungs of HbSS mice or C57BL/6 mice was not altered by PFCE treatment. Improved survival of PFCE-O₂-treated HbSS mice infected with S. pneumoniae is associated with altered pulmonary inflammation but not enhanced bacterial clearance.

  2. Trafficking of high avidity HER-2/neu-specific T cells into HER-2/neu-expressing tumors after depletion of effector/memory-like regulatory T cells.

    Directory of Open Access Journals (Sweden)

    Vivian L Weiss

    Full Text Available Cancer vaccines are designed to activate and enhance cancer-antigen-targeted T cells that are suppressed through multiple mechanisms of immune tolerance in cancer-bearing hosts. T regulatory cell (Treg suppression of tumor-specific T cells is one barrier to effective immunization. A second mechanism is the deletion of high avidity tumor-specific T cells, which leaves a less effective low avidity tumor specific T cell repertoire available for activation by vaccines. Treg depleting agents including low dose cyclophosphamide (Cy and antibodies that deplete CD25-expressing Tregs have been used with limited success to enhance the potency of tumor-specific vaccines. In addition, few studies have evaluated mechanisms that activate low avidity cancer antigen-specific T cells. Therefore, we developed high and low avidity HER-2/neu-specific TCR transgenic mouse colonies specific for the same HER-2/neu epitope to define the tolerance mechanisms that specifically affect high versus low avidity tumor-specific T cells.High and low avidity CD8(+ T cell receptor (TCR transgenic mice specific for the breast cancer antigen HER-2/neu (neu were developed to provide a purified source of naïve, tumor-specific T cells that can be used to study tolerance mechanisms. Adoptive transfer studies into tolerant FVB/N-derived HER-2/neu transgenic (neu-N mice demonstrated that high avidity, but not low avidity, neu-specific T cells are inhibited by Tregs as the dominant tolerizing mechanism. High avidity T cells persisted, produced IFNγ, trafficked into tumors, and lysed tumors after adoptive transfer into mice treated with a neu-specific vaccine and low dose Cy to deplete Tregs. Analysis of Treg subsets revealed a Cy-sensitive CD4(+Foxp3(+CD25(low tumor-seeking migratory phenotype, characteristic of effector/memory Tregs, and capable of high avidity T cell suppression.Depletion of CD25(low Tregs allows activation of tumor-clearing high avidity T cells. Thus, the development

  3. Simultaneous Assessment of Rotavirus-Specific Memory B Cells and Serological Memory after B Cell Depletion Therapy with Rituximab

    Science.gov (United States)

    Herrera, Daniel; Rojas, Olga L.; Duarte-Rey, Carolina; Mantilla, Rubén D.; Ángel, Juana; Franco, Manuel A.

    2014-01-01

    The mechanisms that contribute to the maintenance of serological memory are still unclear. Rotavirus (RV) memory B cells (mBc) are enriched in IgM+ and CD27- subpopulations, which are associated with autoimmune diseases pathogenesis. In patients with autoimmune diseases treated with Rituximab (RTX), some autoantibodies (auto-Abs) decrease after treatment, but other auto-Abs and pathogen-specific IgG Abs remain unchanged. Thus, maintenance of autoimmune and pathogen-specific serological memory may depend on the type of antigen and/or Ab isotype evaluated. Antigen-specific mBc and antigen-specific Abs of different isotypes have not been simultaneously assessed in patients after RTX treatment. To study the relationship between mBc subpopulations and serological memory we characterized total, RV- and tetanus toxoid (TT)-specific mBc by flow cytometry in patients with autoimmune diseases before and after treatment with RTX. We also measured total, RV- and TT-Abs, and some auto-Abs by kinetic nephelometry, ELISA, and EliA tests, respectively. Minor differences were observed between the relative frequencies of RV-mBc in healthy controls and patients with autoimmune disease. After RTX treatment, naïve Bc and total, RV- and TT-specific mBc [IgM+, switched (IgA+/IgG+), IgM+ only, IgD+ only, and CD27- (IgA+/IgG+/IgM+)] were significantly diminished. An important decrease in total plasma IgM and minor decreases in total IgG and IgA levels were also observed. IgM rheumatoid factor, IgG anti-CCP, and IgG anti-dsDNA were significantly diminished. In contrast, RV-IgA, RV-IgG and RV-IgG1, and TT-IgG titers remained stable. In conclusion, in patients with autoimmunity, serological memory against RV and TT seem to be maintained by long-lived plasma cells, unaffected by RTX, and an important proportion of total IgM and serological memory against some auto-antigens seem to be maintained by short-lived plasma cells, dependent on mBc precursors depleted by RTX. PMID:24819618

  4. Differential depletion of total T cells and regulatory T cells and prolonged allotransplant survival in CD3Ɛ humanized mice treated with polyclonal anti human thymocyte globulin

    Science.gov (United States)

    Buszko, Maja; Cardini, Benno; Oberhuber, Rupert; Oberhuber, Lukas; Jakic, Bojana; Beierfuss, Anja; Wick, Georg; Cappellano, Giuseppe

    2017-01-01

    Thymoglobulin (ATG) is a polyclonal rabbit antibody against human thymocytes used as a T cell-depleting agent to prevent or treat allotransplant rejection. The aim of the present study was to investigate the effect of low dose ATG treatment exclusively on T cells using a humanized BALB/c human CD3Ɛ transgenic mouse model expressing both human and murine T cell receptors (TCR). Mice received a single intravenous (i.v.) injection of ATG. Blood and peripheral lymphoid organs were obtained after different time points. We found a significant T cell depletion in this mouse model. In addition, regulatory T cells (Tregs) proved to be less sensitive to depletion than the rest of T cells and the Treg:non-Treg ratio was therefore increased. Finally, we also investigated the effect of ATG in a heterotopic allogenic murine model of heart transplantation. Survival and transplant function were significantly prolonged in ATG-treated mice. In conclusion, we showed (a) an immunosuppressive effect of ATG in this humanized mouse model which is exclusively mediated by reactivity against human CD3Ɛ; (b) provided evidence for a relative resistance of Tregs against this regimen; and (c) demonstrated the immunomodulatory effect of ATG under these experimental circumstances by prolongation of heart allograft survival. PMID:28257450

  5. Leflunomide attenuates hepatocyte injury by inhibiting Kupffer cells

    Institute of Scientific and Technical Information of China (English)

    Hong-Wei Yao; Jun Li; Ji-Qiang Chen; Shu-Yun Xu

    2004-01-01

    AIM: To investigate the importance of direct contact between Kupffer cells (KCs) and hepatocytes (HCs) during hepatic inflammatory responses, and the effect of leflunomide′s active metabolite, A771726, on cytokines in KCs, HCs and KC cocultures (DC cocultures).METHODS: KCs and HCs in liver were isolated by digestion with pronase and collagenase. Lipopolysaccharide (LPS)-induced inflammatory response in monocultures of rat HCs and KCs was compared with that in DC cocultures. Tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1)concentrations in different culture supernatants were measured with ELISA. TNF-α mRNA in KCs of inflammatory liver injury was analyzed with reverse transcriptase polymerase chain reaction (RT-PCR).RESULTS: DC cocultures strongly exhibited the production of TNF-α and IL-1 compared with other cultures, and these cytokines were mainly produced by KCs, especially by activated KCs. Time course studies revealed an increased production of TNF-α preceding the IL-1 production,suggesting that increased TNF-α levels could be involved in the increase of IL-1 production. Leflunomide′s active metabolite, A771726, had significantly inhibitory effect on TNF-αand IL-1 at protein and transcription levels, and the reduced production of IL-1 by A771726 was associated with the inhibitory action of A771726 on TNF-α.

  6. A phase I study of CD25/regulatory T-cell-depleted donor lymphocyte infusion for relapse after allogeneic stem cell transplantation

    Science.gov (United States)

    Nikiforow, Sarah; Kim, Haesook T.; Daley, Heather; Reynolds, Carol; Jones, Kyle Thomas; Armand, Philippe; Ho, Vincent T.; Alyea, Edwin P.; Cutler, Corey S.; Ritz, Jerome; Antin, Joseph H.; Soiffer, Robert J.; Koreth, John

    2016-01-01

    Donor lymphocyte infusions are used to treat relapse after allogeneic hematopoietic stem cell transplantation, but responses are inadequate. In addition to effector cells, infusions contain CD25+ regulatory T cells (Treg) that may suppress graft-versus-tumor responses. We undertook a phase I study of donor lymphocyte infusions depleted of CD25+ T cells in patients with hematologic malignancies who had relapsed after transplantation. Twenty-one subjects received CD25/Treg-depleted infusions following removal of CD25+ cells using antibody-conjugated magnetic beads. Sixteen subjects received prior cytoreductive therapy. Four were in complete remission at the time of infusion. Two dose levels were administered: 1×107 (n=6) and 3×107 CD3+ cells/kg (n=15). A median 2.3 log-depletion of CD4+CD25+FOXP3+ Treg was achieved. Seven subjects (33%) developed clinically significant graft-versus-host disease by 1 year, including one patient who died. At dose level 1, five subjects had progressive disease and one had stable disease. At dose level 2, nine subjects (60%) achieved or maintained responses (8 complete responses, 1 partial response), including seven with active disease at the time of infusion. A shorter period between relapse and infusion was associated with response at dose level 2 (P=0.016). The 1-year survival rate was 53% among patients treated with dose level 2. Four of eight subjects with acute myeloid leukemia remained in remission at 1 year. When compared to unmodified donor lymphocyte infusions in 14 contemporaneous patients meeting study eligibility, CD25/Treg depletion was associated with a better response rate and improved event-free survival. Circulating naïve and central memory CD4+ T cells increased after CD25/Treg-depleted infusion, but no immunophenotypic signature for response was noted. CD25/Treg-depleted donor infusion appears feasible and capable of inducing graft-versus-tumor responses without excessive graft-versus-host disease. (Clinical

  7. Losartan attenuates renal interstitial fibrosis and tubular cell apoptosis in a rat model of obstructive nephropathy.

    Science.gov (United States)

    He, Ping; Li, Detian; Zhang, Beiru

    2014-08-01

    Ureteral obstruction leads to renal injury and progresses to irreversible renal fibrosis, with tubular cell atrophy and apoptosis. There is conflicting evidence concerning whether losartan (an angiotensin II type I receptor antagonist) mitigates renal interstitial fibrosis and renal tubular epithelial cell apoptosis following unilateral ureteral obstruction (UUO) in animal models. The aim of this study was to investigate the effect and mechanism of losartan on renal tubular cell apoptosis and renal fibrosis in a rat model of UUO. The rats were subjected to UUO by ureteral ligation and were treated with dimethyl sulfoxide (control) or losartan. The controls underwent sham surgery. The renal tissues were collected 3, 5, 7 and 14 days after surgery for measurement of various indicators of renal fibrosis. UUO increased the expression levels of α‑smooth muscle actin and collagen I, and the extent of renal tubular fibrosis and apoptosis in a time‑dependent manner. Losartan treatment partially attenuated these responses. Progression of renal interstitial fibrosis was accompanied by phosphorylation of signal transducer and activator of transcription 3 (STAT3) and altered the expression levels of two apoptosis‑related proteins (Bax and Bcl2). Losartan treatment also partially attenuated these responses. The results indicated that losartan attenuated renal fibrosis and renal tubular cell apoptosis in a rat model of UUO. This effect appeared to be mediated by partial blockage of STAT3 phosphorylation.

  8. Depletion region effect of highly efficient hole conductor free CH3NH3PbI3 perovskite solar cells.

    Science.gov (United States)

    Aharon, Sigalit; Gamliel, Shany; El Cohen, Bat; Etgar, Lioz

    2014-06-14

    The inorganic-organic perovskite is currently attracting a lot of attention due to its use as a light harvester in solar cells. The large absorption coefficients, high carrier mobility and good stability of organo-lead halide perovskites present good potential for their use as light harvesters in mesoscopic heterojunction solar cells. This work concentrated on a unique property of the lead halide perovskite, its function simultaneously as a light harvester and a hole conductor in the solar cell. A two-step deposition technique was used to optimize the perovskite deposition and to enhance the solar cell efficiency. It was revealed that the photovoltaic performance of the hole conductor free perovskite solar cell is strongly dependent on the depletion layer width which was created at the TiO2-CH3NH3PbI3 junction. X-ray diffraction measurements indicate that there were no changes in the crystallographic structure of the CH3NH3PbI3 perovskite over time, which supports the high stability of these hole conductor free perovskite solar cells. Furthermore, the power conversion efficiency of the best cells reached 10.85% with a fill factor of 68%, a Voc of 0.84 V, and a Jsc of 19 mA cm(-2), the highest efficiency to date of a hole conductor free perovskite solar cell.

  9. Antigen-Specific Priming is Dispensable in Depletion of Apoptosis-Sensitive T Cells for GvHD Prophylaxis.

    Science.gov (United States)

    Yarkoni, Shai; Stein, Jerry; Yaniv, Isaac; Askenasy, Nadir

    2014-01-01

    Prophylactic approaches to graft versus host disease (GvHD) have employed both phenotypic reduction of T cells and selective elimination of host-primed donor T cells in vitro and in vivo. An additional approach to GvHD prophylaxis by functional depletion of apoptosis-sensitive donor T cells without host-specific sensitization ex vivo showed remarkable reduction in GHD incidence and severity. We address the role and significance of antigen-specific sensitization of donor T cells and discuss the mechanisms of functional T cell purging by apoptosis for GvHD prevention. Host-specific sensitization is dispensable because migration is antigen-independent and donor T cell sensitization is mediated by multiple and redundant mechanisms of presentation of major and minor histocompatibility complex and tissue antigens by donor and host antigen-presenting cells. Our data suggest that potential murine and human GvH effectors reside within subsets of preactivated T cells susceptible to negative regulation by apoptosis prior to encounter of and sensitization to specific antigens.

  10. The calcium current activated by T cell receptor and store depletion in human lymphocytes is absent in a primary immunodeficiency.

    Science.gov (United States)

    Partiseti, M; Le Deist, F; Hivroz, C; Fischer, A; Korn, H; Choquet, D

    1994-12-23

    Stimulation of antigen receptors of lymphocytes triggers a transitory release of Ca2+ from internal stores and the opening of a transmembrane Ca2+ conductive pathway. The latter underlies the sustained increase of intracellular free calcium concentration, and it seems to be a key event in the Ca(2+)-dependent biochemical cascade leading to T cell proliferation. Alternatively, pharmacological depletion of internal stores by itself activates Ca2+ influx. This has led to the hypothesis that antigen-triggered Ca2+ influx is secondary to Ca2+ release from internal stores. However, the precise relationship between antigen and Ca2+ release-activated Ca2+ currents remains unclear, particularly since neither of them has been electrophysiologically recorded in normal lymphocytes. Using the whole-cell and the perforated configurations of the patch clamp technique on peripheral blood lymphocytes, we found that a low amplitude Ca(2+)-selective current was triggered when intracellular stores were depleted by stimuli such as the intracellular perfusion of inositol triphosphate or thapsigargin and the extracellular perfusion of ionomycin. A similar current was elicited by the cross-linking of the T cell receptor-CD3 complex. This current displayed an inward rectification below 0 mV and was completely blocked by the divalent cation Cd2+. It was very selective for Ca2+ over Na+ and insensitive to changes in chloride concentration. The physiological relevance of this conductance was investigated with the analysis of abnormal Ca2+ signaling in lymphocytes from a patient suffering from a primary immunodeficiency associated with a defective T cell proliferation. Using fura-2 video imaging, an absence of Ca2+ influx was established in the patient's lymphocytes, whereas the Ca2+ release from internal stores was normal. This was the case whether cells were stimulated physiologically through their antigen receptors or with store depleting pharmacological agents. Most importantly, no Ca(2

  11. Genetically attenuated, P36p-deficient malarial sporozoites induce protective immunity and apoptosis of infected liver cells.

    NARCIS (Netherlands)

    Dijk, M.R. van; Douradinha, B.; Franke-Fayard, B.; Heussler, V.; Dooren, M.W. van; Schaijk, B.C.L. van; Gemert, G.J.A. van; Sauerwein, R.W.; Mota, M.M.; Waters, A.P.; Janse, C.J.

    2005-01-01

    Immunization with Plasmodium sporozoites that have been attenuated by gamma-irradiation or specific genetic modification can induce protective immunity against subsequent malaria infection. The mechanism of protection is only known for radiation-attenuated sporozoites, involving cell-mediated and hu

  12. Neural Differentiation in HDAC1-Depleted Cells Is Accompanied by Coilin Downregulation and the Accumulation of Cajal Bodies in Nucleoli.

    Science.gov (United States)

    Krejčí, Jana; Legartová, Soňa; Bártová, Eva

    2017-01-01

    Cajal bodies (CBs) are important compartments containing accumulated proteins that preferentially regulate RNA-related nuclear events, including splicing. Here, we studied the nuclear distribution pattern of CBs in neurogenesis. In adult brains, coilin was present at a high density, but CB formation was absent in the nuclei of the choroid plexus of the lateral ventricles. Cells of the adult hippocampus were characterized by a crescent-like morphology of coilin protein. We additionally observed a 70 kDa splice variant of coilin in adult mouse brains, which was different to embryonic brains and mouse pluripotent embryonic stem cells (mESCs), characterized by the 80 kDa standard variant of coilin. Here, we also showed that depletion of coilin is induced during neural differentiation and HDAC1 deficiency in mESCs caused coilin accumulation inside the fibrillarin-positive region of the nucleoli. A similar distribution pattern was observed in adult brain hippocampi, characterized by lower levels of both coilin and HDAC1. In summary, we observed that neural differentiation and HDAC1 deficiency lead to coilin depletion and coilin accumulation in body-like structures inside the nucleoli.

  13. Neural Differentiation in HDAC1-Depleted Cells Is Accompanied by Coilin Downregulation and the Accumulation of Cajal Bodies in Nucleoli

    Directory of Open Access Journals (Sweden)

    Jana Krejčí

    2017-01-01

    Full Text Available Cajal bodies (CBs are important compartments containing accumulated proteins that preferentially regulate RNA-related nuclear events, including splicing. Here, we studied the nuclear distribution pattern of CBs in neurogenesis. In adult brains, coilin was present at a high density, but CB formation was absent in the nuclei of the choroid plexus of the lateral ventricles. Cells of the adult hippocampus were characterized by a crescent-like morphology of coilin protein. We additionally observed a 70 kDa splice variant of coilin in adult mouse brains, which was different to embryonic brains and mouse pluripotent embryonic stem cells (mESCs, characterized by the 80 kDa standard variant of coilin. Here, we also showed that depletion of coilin is induced during neural differentiation and HDAC1 deficiency in mESCs caused coilin accumulation inside the fibrillarin-positive region of the nucleoli. A similar distribution pattern was observed in adult brain hippocampi, characterized by lower levels of both coilin and HDAC1. In summary, we observed that neural differentiation and HDAC1 deficiency lead to coilin depletion and coilin accumulation in body-like structures inside the nucleoli.

  14. Depletion of kinesin 5B affects lysosomal distribution and stability and induces peri-nuclear accumulation of autophagosomes in cancer cells

    DEFF Research Database (Denmark)

    Cardoso, Carla M P; Groth-Pedersen, Line; Høyer-Hansen, Maria

    2009-01-01

    cells. In KIF5B-depleted cells the autophagosomes formed and accumulated in the close proximity to the Golgi apparatus, whereas in the control cells they appeared uniformly distributed in the cytoplasm. CONCLUSIONS/SIGNIFICANCE: Our data identify KIF5B as a cancer relevant lysosomal motor protein...

  15. Deformability of Red Blood Cells and Correlation with ATP Content during Storage as Leukocyte-Depleted Whole Blood.

    Science.gov (United States)

    Karger, Ralf; Lukow, Christian; Kretschmer, Volker

    2012-08-01

    BACKGROUND: Storage duration of red cells has been associated with increased morbidity and mortality following transfusion. This association has been attributed to the loss of deformability of stored red cells leading to deterioration of microvascular perfusion. ATP content is considered a critical determinant of the deformability of stored red cells. METHODS: ATP content and deformability were determined after storage for up to 49 days in 40 leukocyte-depleted whole blood units. Red cell deformability was determined using a laser-assisted optical rotational cell analyzer (LORCA( (®) )) employing shear stress (SS) ranging from 0.3 to 30 Pa. Deformability was expressed as the elongation index (EI). EI was correlated with ATP content. RESULTS: ATP content decreased from 3.5 to 1.7 ?mol/g hemoglobin. EI increased from 0.03 to 0.05 at an SS of 0.3 Pa, and decreased from 0.62 to 0.59 at an SS of 30 Pa. Correlation coefficient (r) of ATP vs. EI at 0.3 Pa ranged from -0.17 to +0.15 during storage. At 30 Pa, r ranged from -0.03 to +0.45. Correlation increased with storage irrespective of SS, and increased with SS irrespective of storage. CONCLUSIONS: ATP content is not a valid surrogate marker for red cell deformability and may not reflect in vivo survival of stored red cells.

  16. Purified T-depleted, CD34+ peripheral blood and bone marrow cell transplantation from haploidentical mother to child with thalassemia.

    Science.gov (United States)

    Sodani, Pietro; Isgrò, Antonella; Gaziev, Javid; Polchi, Paola; Paciaroni, Katia; Marziali, Marco; Simone, Maria Domenica; Roveda, Andrea; Montuoro, Aldo; Alfieri, Cecilia; De Angelis, Gioia; Gallucci, Cristiano; Erer, Buket; Isacchi, Giancarlo; Zinno, Francesco; Adorno, Gaspare; Lanti, Alessandro; Faulkner, Lawrence; Testi, Manuela; Andreani, Marco; Lucarelli, Guido

    2010-02-11

    Fetomaternal microchimerism suggests immunological tolerance between mother and fetus. Thus, we performed primary hematopoietic stem cell transplantation from a mismatched mother to thalassemic patient without an human leukocyte antigen-identical donor. Twenty-two patients with thalassemia major were conditioned with 60 mg/kg hydroxyurea and 3 mg/kg azathioprine from day -59 to -11; 30 mg/m(2) fludarabine from day -17 to -11; 14 mg/kg busulfan starting on day -10; and 200 mg/kg cyclophosphamide, 10 mg/kg thiotepa, and 12.5 mg/kg antithymocyte globulin daily from day -5 to -2. Fourteen patients received CD34(+)-mobilized peripheral blood and bone marrow progenitor cells; 8 patients received marrow graft-selected peripheral blood stem cells CD34(+) and bone marrow CD3/CD19-depleted cells. T-cell dose was adjusted to 2 x 10(5)/kg by fresh marrow cell addback at the time of transplantation. Both groups received cyclosporine for graft-versus-host disease prophylaxis for 2 months after transplantation. Two patients died (cerebral Epstein-Barr virus lymphoma or cytomegalovirus pneumonia), 6 patients reject their grafts, and 14 showed full chimerism with functioning grafts at a median follow-up of 40 months. None of the 14 patients who showed full chimerism developed acute or chronic graft-versus-host disease. These results suggest that maternal haploidentical hematopoietic stem cell transplantation is feasible in patients with thalassemia who lack a matched related donor.

  17. Notch inhibition suppresses nasopharyngeal carcinoma by depleting cancer stem-like side population cells.

    Science.gov (United States)

    Yu, Shudong; Zhang, Ruxin; Liu, Fenye; Wang, Hong; Wu, Jing; Wang, Yanqing

    2012-08-01

    The cancer stem cell (CSC) is responsible for the initiation, proliferation and radiation resistance. Side population (SP) cells are a rare subset of cells enriched with CSCs. The targeting of key signaling pathways that are active in CSCs is a therapeutic approach to treating cancer. Notch signaling is important for the self-renewal and maintenance of stem cells. Our previous studies demonstrated that downregulation of Notch signaling could enhance radiosensitivity of nasopharyngeal carcinoma (NPC) cells. In this study, we found that Notch signaling was highly activated in SP cells compared with that of non-SP (NSP) cells of NPC. Therefore, Notch inhibition could reduce the proportion of SP cells. As SP cells decreased, proliferation, anti-apoptosis and tumorigenesis were also decreased. This study shows that Notch inhibition may be a promising clinical approach in CSC-targeting therapy for NPC.

  18. Gender difference in bone metastasis of human small cell lung cancer, SBC-5 cells in natural killer-cell depleted severe combined immunodeficient mice.

    Science.gov (United States)

    Sakaguchi, Satoshi; Goto, Hisatsugu; Hanibuchi, Masaki; Otsuka, Shinsaku; Ogino, Hirokazu; Kakiuchi, Soji; Uehara, Hisanori; Yano, Seiji; Nishioka, Yasuhiko; Sone, Saburo

    2010-05-01

    Lung cancer frequently develops multiple organ metastases, which thus makes this disease a leading cause of malignancy-related death worldwide. A gender difference is reported to affect the incidence and mortality of lung cancer; however, whether and how the gender difference is involved in lung cancer metastasis is unclear. This study evaluated the gender difference in multiple organ metastases in human small cell lung cancer (SBC-5) cells by using natural killer cell-depleted severe combined immunodeficient mice. Among multiple organ metastases, only bone metastasis formation significantly increased in female mice in comparison to males, while no significant difference was observed in the metastases to the liver and lungs. The suppression of androgen by castration or androgen receptor antagonist treatment in male mice also induced a significant increase of bone metastases. The number of osteoclasts in the bone metastatic lesions was greater in female mice and in mice with androgen suppression than in control male. However, there was no significant difference in the serum concentration of parathyroid hormone-related protein (PTHrP) associated with gender or androgen suppression. An in vitro study also indicated that sex steroid treatment had no effect on the proliferation or PTHrP production in SBC-5 cells. These results indicate that the balance of sex steroids therefore plays an important role in the formation of bone metastasis in small cell lung cancer, and suggests diverse mechanisms of interaction between cancer cells and host cells in the bone microenvironment.

  19. Depletion of regulatory T cells leads to an exacerbation of delayedtype hypersensitivity arthritis in C57BL/6 mice that can be counteracted by IL-17 blockade

    DEFF Research Database (Denmark)

    Atkinson, Sara Marie; Hoffmann, Ute; Bach, Emil;

    2016-01-01

    , this approach could be relevant for advancing the understanding of Tregs in inflammatory arthritis. Selective depletion of Tregs was achieved using a Foxp3-DTR-eGFP mouse, which expresses the diphtheria toxin receptor (DTR) and enhanced green fluorescent protein (eGFP) under control of the Foxp3 gene. Anti...... with synchronised onset, 100% penetrance and low variation. We investigate the role of regulatory T cells (Tregs) in DTHA through selective depletion of Tregs and the role of IL-17 in connection with Treg depletion. Given the relevance of Tregs in RA, and the possibility of developing Treg-directed therapies...

  20. Delayed cell cycle progression in selenoprotein W depleted cells is regulated by a mitogen-activated protein kinase kinase 4–p38–p53 pathway

    Science.gov (United States)

    Selenoprotein W (SEPW1) is a ubiquitous, highly conserved thioredoxin-like protein whose depletion causes a p53- and p21Cip1-dependent G1-phase cell cycle arrest in breast and prostate epithelial cells. SEPW1 depletion increases phosphorylation of Ser33 in p53, which is associated with decreased p53...

  1. GTP depletion synergizes the anti-proliferative activity of chemotherapeutic agents in a cell type-dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Tao; Meng, Lingjun [Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology, Texas A and M Health Science Center, Houston, TX 77030 (United States); Tsai, Robert Y.L., E-mail: rtsai@ibt.tamhsc.edu [Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology, Texas A and M Health Science Center, Houston, TX 77030 (United States)

    2011-10-22

    Highlights: {yields} Strong synergy between mycophenolic acid (MPA) and 5-FU in MDA-MB-231 cells. {yields} Cell type-dependent synergy between MPA and anti-proliferative agents. {yields} The synergy of MPA on 5-FU is recapitulated by RNA polymerase-I inhibition. {yields} The synergy of MPA on 5-FU requires the expression of nucleostemin. -- Abstract: Mycophenolic acid (MPA) depletes intracellular GTP by blocking de novo guanine nucleotide synthesis. GTP is used ubiquitously for DNA/RNA synthesis and as a signaling molecule. Here, we made a surprising discovery that the anti-proliferative activity of MPA acts synergistically with specific chemotherapeutic agents in a cell type-dependent manner. In MDA-MB-231 cells, MPA shows an extremely potent synergy with 5-FU but not with doxorubicin or etoposide. The synergy between 5-FU and MPA works most effectively against the highly tumorigenic mammary tumor cells compared to the less tumorigenic ones, and does not work in the non-breast cancer cell types that we tested, with the exception of PC3 cells. On the contrary, MPA shows the highest synergy with paclitaxel but not with 5-FU in SCC-25 cells, derived from oral squamous cell carcinomas. Mechanistically, the synergistic effect of MPA on 5-FU in MDA-MB-231 cells can be recapitulated by inhibiting the RNA polymerase-I activity and requires the expression of nucleostemin. This work reveals that the synergy between MPA and anti-proliferative agents is determined by cell type-dependent factors.

  2. Depletion of eIF4G from yeast cells narrows the range of translational efficiencies genome-wide

    Directory of Open Access Journals (Sweden)

    Hinnebusch Alan G

    2011-01-01

    Full Text Available Abstract Background Eukaryotic translation initiation factor 4G (eIF4G is thought to influence the translational efficiencies of cellular mRNAs by its roles in forming an eIF4F-mRNA-PABP mRNP that is competent for attachment of the 43S preinitiation complex, and in scanning through structured 5' UTR sequences. We have tested this hypothesis by determining the effects of genetically depleting eIF4G from yeast cells on global translational efficiencies (TEs, using gene expression microarrays to measure the abundance of mRNA in polysomes relative to total mRNA for ~5900 genes. Results Although depletion of eIF4G is lethal and reduces protein synthesis by ~75%, it had small effects (less than a factor of 1.5 on the relative TE of most genes. Within these limits, however, depleting eIF4G narrowed the range of translational efficiencies genome-wide, with mRNAs of better than average TE being translated relatively worse, and mRNAs with lower than average TE being translated relatively better. Surprisingly, the fraction of mRNAs most dependent on eIF4G display an average 5' UTR length at or below the mean for all yeast genes. Conclusions This finding suggests that eIF4G is more critical for ribosome attachment to mRNAs than for scanning long, structured 5' UTRs. Our results also indicate that eIF4G, and the closed-loop mRNP it assembles with the m7 G cap- and poly(A-binding factors (eIF4E and PABP, is not essential for translation of most (if not all mRNAs but enhances the differentiation of translational efficiencies genome-wide.

  3. CD4+CD25+ regulatory T cell depletion modulates anxiety and depression-like behaviors in mice.

    Directory of Open Access Journals (Sweden)

    Soo-Jeong Kim

    Full Text Available Stress has been shown to suppress immune function and increase susceptibility to inflammatory disease and psychiatric disease. CD4(+CD25(+ regulatory T (Treg cells are prominent in immune regulation. This study was conducted to determine if anti-CD25 antibody (Ab mediated depletion of Treg cells in mice susceptibility to stress-induced development of depression-like behaviors, as well as immunological and neurochemical activity. To accomplish this, an elevated plus-maze test (EPM, tail suspension test (TST, and forced swim test (FST were used to examine depression-like behaviors upon chronic immobilization stress. Immune imbalance status was observed based on analysis of serum cytokines using a mouse cytometric bead array in conjunction with flow cytometry and changes in the levels of serotonin (5-HT and dopamine (DA in the brain were measured by high performance liquid chromatography (HPLC. The time spent in the open arms of the EPM decreased significantly and the immobility time in the FST increased significantly in the anti-CD25 Ab-treated group when compared with the non stressed wild-type group. In addition, interlukin-6 (IL-6, tumor necrosis factor-á (TNF-á, interlukin-2 (IL-2, interferon-gamma (IFN-γ, interlukin-4 (IL-4 and interlukin-17A (IL-17A concentrations were significantly upregulated in the stressed anti-CD25 Ab-treated group when compared with the non stressed wild-type group. Furthermore, the non stressed anti-CD25 Ab-treated group displayed decreased 5-HT levels within the hippocampus when compared with the non stressed wild-type group. These results suggest that CD4(+CD25(+ Treg cell depletion modulated alterations in depressive behavior, cytokine and monoaminergic activity. Therefore, controlling CD4(+CD25(+ Treg cell function during stress may be a potent therapeutic strategy for the treatment of depression-like symptoms.

  4. Disruption of NF-κB signaling by fluoxetine attenuates MGMT expression in glioma cells

    Directory of Open Access Journals (Sweden)

    Song T

    2015-08-01

    Full Text Available Tao Song,1 Hui Li,2 Zhiliang Tian,3 Chaojiu Xu,4 Jingfang Liu,1 Yong Guo1 1Department of Neurosurgery, Xiangya Hospital, Central South University, 2Department of Immunology and Microbiology, Medical School of Jishou University, 3Department of Neurosurgery, 4Department of Oncology, The Hospital of Xiangxi Autonomous Prefecture, Jishou, People’s Republic of China Background: Resistance to temozolomide (TMZ in glioma is modulated by the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT. This study aimed to examine the effects of fluoxetine (FLT on MGMT expression in glioma cells and to investigate its underlying mechanisms.Materials and methods: Expression of MGMT, GluR1, or IκB kinase β (IKKβ was attenuated using short hairpin RNA-mediated gene knockdown. The 3-(4,5-dimethylthiazol -2-yl-2,5-diphenyltetrazolium bromide (MTT assay was used to evaluate the growth inhibition induced by FLT or TMZ. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL was conducted to detect apoptotic cells. Western blotting was conducted to analyze the protein expression of MGMT, IKKβ, and NF-κB/p65 following FLT treatment. The murine subcutaneous xenograft model was used to evaluate the combinational effect of TMZ and FLT.Results: FLT markedly reduced MGMT expression in glioma cells, which was independent of GluR1 receptor function. Further, FLT disrupted NF-κB/p65 signaling in glioma cells and consequently attenuated NF-κB/p65 activity in regulating MGMT expression. Importantly, FLT sensitized MGMT-expressing glioma cells to TMZ, as FLT enhanced TMZ’s ability to impair the in vitro tumorigenic potential and to induce apoptosis in glioma cells. Knockdown of MGMT or IKKβ expression abolished the synergistic effect of FLT with TMZ in glioma cells, which suggested that FLT might sensitize glioma cells to TMZ through down-regulation of MGMT expression. Consistently, TMZ combined with FLT markedly attenuated NF

  5. T-cell depletion and autologous stem cell transplantation in the management of tumour stage mycosis fungoides with peripheral blood involvement.

    Science.gov (United States)

    Olavarria, E; Child, F; Woolford, A; Whittaker, S J; Davis, J G; McDonald, C; Chilcott, S; Spittle, M; Grieve, R J; Stewart, S; Apperley, J F; Russell-Jones, R

    2001-09-01

    Nine patients with tumour stage mycosis fungoides (MF) have been entered into a pilot study of T-cell depletion and autologous stem cell transplantation (SCT). Eight patients had detectable rearrangements of the T-cell receptor (TCR) gamma-gene demonstrated by polymerase chain reaction (PCR)/single-stranded conformation polymorphism (SSCP) in the peripheral blood. The median age was 47 years and the median duration of disease before SCT was 61 months; Peripheral blood progenitor cells were mobilized using high-dose etoposide (1.6 g/m2) and granulocyte colony-stimulating factor (G-CSF). The apheresis products underwent rigorous T-cell depletion with immunomagnetic methods. Double CD34-positive and CD4/CD8-negative selection achieved a median reduction of 3.89 log of T cells. All nine patients have been transplanted. Conditioning included carmustine (BCNU), etoposide and melphalan (BEM) in seven patients and total body irradiation plus etoposide or melphalan in two. Eight patients engrafted promptly and one patient died of septicaemia. All survivors entered complete remission. Seven patients have relapsed at a median of 7 months (2-14) post SCT. However, most patients have relapsed into a less aggressive stage, which has responded to conventional therapy. Four out of seven evaluable patients had detectable TCR rearrangements in the T-cell depleted graft. A T-cell clone was also detected in the peripheral blood before relapse in four cases. Autologous SCT is feasible, safe and can result in complete remission in a significant proportion of patients with tumour stage mycosis fungoides. Despite a short relapse-free survival, most patients achieved good disease control at the time of relapse.

  6. In Vitro Immune Toxicity of Depleted Uranium: Effects on Murine Macrophages, CD4+ T Cells, and Gene Expression Profiles

    Science.gov (United States)

    Wan, Bin; Fleming, James T.; Schultz, Terry W.; Sayler, Gary S.

    2006-01-01

    Depleted uranium (DU) is a by-product of the uranium enrichment process and shares chemical properties with natural and enriched uranium. To investigate the toxic effects of environmental DU exposure on the immune system, we examined the influences of DU (in the form of uranyl nitrate) on viability and immune function as well as cytokine gene expression in murine peritoneal macrophages and splenic CD4+ T cells. Macrophages and CD4+ T cells were exposed to various concentrations of DU, and cell death via apoptosis and necrosis was analyzed using annexin-V/propidium iodide assay. DU cytotoxicity in both cell types was concentration dependent, with macrophage apoptosis and necrosis occurring within 24 hr at 100 μM DU exposure, whereas CD4+ T cells underwent cell death at 500 μM DU exposure. Noncytotoxic concentrations for macrophages and CD4+ T cells were determined as 50 and 100 μM, respectively. Lymphoproliferation analysis indicated that macrophage accessory cell function was altered with 200 μM DU after exposure times as short as 2 hr. Microarray and real-time reverse-transcriptase polymerase chain reaction analyses revealed that DU alters gene expression patterns in both cell types. The most differentially expressed genes were related to signal transduction, such as c-jun, NF-κ Bp65, neurotrophic factors (e.g., Mdk), chemokine and chemokine receptors (e.g., TECK/CCL25), and interleukins such as IL-10 and IL-5, indicating a possible involvement of DU in cancer development, autoimmune diseases, and T helper 2 polarization of T cells. The results are a first step in identifying molecular targets for the toxicity of DU and the elucidation of the molecular mechanisms for the immune modulation ability of DU. PMID:16393663

  7. Effects of taurine depletion on cell migration and NCAM expression in cultures of dissociated mouse cerebellum and N2A cells

    DEFF Research Database (Denmark)

    Maar, T E; Lund, Trine Meldgaard; Gegelashvili, G;

    1998-01-01

    Cultures of dissociated cerebellum from 5- to 6-day-old mice as well as of the N2A neuronal cell line were exposed to guanidino ethane sulfonate (GES, 2-5 mM) to reduce the cellular taurine content. Control cultures were kept in culture medium or medium containing 2-5 mM GES plus 2-5 mM taurine...... to restore the intracellular taurine content. Taurine depletion led to changes in the expression of certain splice variants of NCAM mRNA such as the AAG and the VASE containing forms, while no differences were seen in the expression of the three forms of NCAM protein. In the N2A cells taurine depletion led...... to a decreased migration rate of the cells. The results suggest that the reduced migration rate of neurons caused by taurine depletion may be correlated to changes in expression of certain adhesion molecules such as NCAM. Moreover, taurine appears to be involved in regulation of transcription processes....

  8. Depleting Glycine and Sarcosine in Prostate Cancer Cells as a New Treatment for Advanced Prostate Cancer

    Science.gov (United States)

    2015-04-01

    Haasters et al48 has reported that 0.5% bupivacaine has cytotoxic effects on human tendon stem cell/progenitor cells, while morphine had no...ropivacaine, and morphine : comparison of toxicity on human hamstring-derived stem/ progenitor cells. Knee Surg Sports Traumatol Arthrosc. 2011;19(12): 2138...required in mice for lamination of the hippocampus but not the neocortex. J. Neurosci. 2002, 22, 7548–7557. © 2014 by the authors; licensee MDPI

  9. [Effect of endonuclease G depletion on plasmid DNA uptake and levels of homologous recombination in hela cells].

    Science.gov (United States)

    Misic, V; El-Mogy, M; Geng, S; Haj-Ahmad, Y

    2016-01-01

    Endonuclease G (EndoG) is a mitochondrial apoptosis regulator that also has roles outside of programmed cell death. It has been implicated as a defence DNase involved in the degradation of exogenous DNA after transfection of mammalian cells and in homologous recombination of viral and endogenous DNA. In this study, we looked at the effect of EndoG depletion on plasmid DNA uptake and the levels of homologous recombination in HeLa cells. We show that the proposed defence role of EndoG against uptake of non-viral DNA vectors does not extend to the cervical carcinoma HeLa cells, as targeting of EndoG expression by RNA interference failed to increase intracellular plasmid DNA levels. However, reducing EndoG levels in HeLa cells resulted in a statistically significant reduction of homologous recombination between two plasmid DNA substrates. These findings suggest that non-viral DNA vectors are also substrates for EndoG in its role in homologous recombination.

  10. Alefacept promotes immunosuppression-free renal allograft survival in nonhuman primates via depletion of recipient memory T cells.

    Science.gov (United States)

    Lee, S; Yamada, Y; Tonsho, M; Boskovic, S; Nadazdin, O; Schoenfeld, D; Cappetta, K; Atif, M; Smith, R-N; Cosimi, A B; Benichou, G; Kawai, T

    2013-12-01

    Renal allograft tolerance has been achieved in MHC-mismatched primates via nonmyeloablative conditioning beginning 6 days prior to planned kidney and donor bone marrow transplantation (DBMT). To extend the applicability of this approach to deceased donor transplantation, we recently developed a novel-conditioning regimen, the "delayed protocol" in which donor bone marrow (DBM) is transplanted several months after kidney transplantation. However, activation/expansion of donor-reactive CD8(+) memory T cells (TMEM) occurring during the interval between kidney and DBM transplantation impaired tolerance induction using this strategy. In the current study, we tested whether, Alefacept, a fusion protein which targets LFA-3/CD2 interactions and selectively depletes CD2(high) CD8(+) effector memory T cells (TEM) could similarly induce long-term immunosuppression-free renal allograft survival but avoid the deleterious effects of anti-CD8 mAb treatment. We found that Alefacept significantly delayed the expansion of CD2(high) cells including CD8(+) TEM while sparing naïve CD8(+) T and NK cells and achieved mixed chimerism and long-term immunosuppression-free renal allograft survival. In conclusion, elimination of CD2(high) T cells represents a promising approach to prevent electively the expansion/activation of donor-reactive TEM and promotes tolerance induction via the delayed protocol mixed chimerism approach.

  11. Depletion of CD8+ cells does not affect the lifespan of productively infected cells during pathogenic sivmac239 infection of rhesus macaques

    Energy Technology Data Exchange (ETDEWEB)

    Shudo, Emi [Los Alamos National Laboratory; Ribeiro, Ruy M [Los Alamos National Laboratory; Perelson, Alan S [Los Alamos National Laboratory

    2008-01-01

    While CD8+ T cell responses are clearly important in anti-viral immunity during HIV/SIV infection, the mechanisms by which CD8+ T cells induce this effect remain poorly understood, as emphasized by the failure of the Merck adenovirus-based, cytotoxic T lymphocyte (CTL)-inducing AIDS vaccine in a large phase IIb clinical trial. In this study, we measured the in vivo effect of CD8+ lymphocytes on the lifespan of productively infected cells during chronic SIVmac239 infection of rhesus macaques by treating two groups of animals (i.e., CD8+ lymphocyte-depleted or controls) with antiretroviral therapy (PMPA and FTC). The lifespan of productively infected cells was calculated based on the slope of the decline of SIV plasma viremia using a well-accepted mathematical model. We found that, in both early (i.e., day 57 post-inoculation) and late (i.e., day 177 post-inoculation) chronic SIV infection, depletion of CD8+ lymphocytes did not result in an increased lifespan of productively infected cells in vivo. This result indicates that direct killing of cells producing virus is unlikely to be a major mechanism underlying the anti-viral effect of CD8+ T cells during SIV infection. These results have profound implications for the development of AIDS vaccines.

  12. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) as an O2(*-) generator induces apoptosis via the depletion of intracellular GSH contents in Calu-6 cells.

    Science.gov (United States)

    Han, Yong Hwan; Kim, Suhn Hee; Kim, Sung Zoo; Park, Woo Hyun

    2009-02-01

    Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupler of mitochondrial oxidative phosphorylation in eukaryotic cells. Here, we investigated an involvement of O(2)(*-) and GSH in FCCP-induced Calu-6 cell death and examined whether ROS scavengers rescue cells from FCCP-induced cell death. Levels of intracellular O(2)(*-) were markedly increased depending on the concentrations (5-100 microM) of FCCP. A depletion of intracellular GSH content was also observed after exposing cells to FCCP. Stable SOD mimetics, Tempol and Tiron did not change the levels of intracellular O(2)(*-), apoptosis and the loss of mitochondrial membrane potential (DeltaPsi(m)). Treatment with thiol antioxidants, NAC and DTT, showed the recovery of GSH depletion and the reduction of O(2)(*-) levels in FCCP-treated cells, which were accompanied by the inhibition of apoptosis. In contrast, BSO, a well-known inhibitor of GSH synthesis, aggravated GSH depletion, oxidative stress of O(2)(*-) and cell death in FCCP-treated cells. Taken together, our data suggested that FCCP as an O(2)(*-) generator, induces apoptosis via the depletion of intracellular GSH contents in Calu-6 cells.

  13. Attenuated Innate Immunity in Embryonic Stem Cells and Its Implications in Developmental Biology and Regenerative Medicine.

    Science.gov (United States)

    Guo, Yan-Lin; Carmichael, Gordon G; Wang, Ruoxing; Hong, Xiaoxiao; Acharya, Dhiraj; Huang, Faqing; Bai, Fengwei

    2015-11-01

    Embryonic stem cells (ESCs) represent a promising cell source for regenerative medicine. Intensive research over the past 2 decades has led to the feasibility of using ESC-differentiated cells (ESC-DCs) in regenerative medicine. However, increasing evidence indicates that ESC-DCs generated by current differentiation methods may not have equivalent cellular functions to their in vivo counterparts. Recent studies have revealed that both human and mouse ESCs as well as some types of ESC-DCs lack or have attenuated innate immune responses to a wide range of infectious agents. These findings raise important concerns for their therapeutic applications since ESC-DCs, when implanted to a wound site of a patient, where they would likely be exposed to pathogens and inflammatory cytokines. Understanding whether an attenuated immune response is beneficial or harmful to the interaction between host and grafted cells becomes an important issue for ESC-based therapy. A substantial amount of recent evidence has demonstrated that the lack of innate antiviral responses is a common feature to ESCs and other types of pluripotent cells. This has led to the hypothesis that mammals may have adapted different antiviral mechanisms at different stages of organismal development. The underdeveloped innate immunity represents a unique and uncharacterized property of ESCs that may have important implications in developmental biology, immunology, and in regenerative medicine.

  14. Atractylenolide I-mediated Notch pathway inhibition attenuates gastric cancer stem cell traits

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Li; Mao, Rurong; Shen, Ke; Zheng, Yuanhong; Li, Yueqi [State Key Laboratory of Bioreactor Engineering and Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, #268, 130 Meilong Road, Shanghai 200237 (China); Liu, Jianwen, E-mail: liujian@ecust.edu.cn [State Key Laboratory of Bioreactor Engineering and Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, #268, 130 Meilong Road, Shanghai 200237 (China); Ni, Lei, E-mail: nilei625@yahoo.com [Department of Respiration, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, 197 Ruijin Road II, Shanghai 200025 (China)

    2014-07-18

    Highlights: • This paper supports the anti-tumor effects of AT-I on gastric cancer in vitro. • AT-I attenuates gastric cancer stem cell traits. • It is the systematic study regarding AT-I suppression of Notch pathway in GC and GCSLCs. - Abstract: Atractylenolide I (AT-I), one of the main naturally occurring compounds of Rhizoma Atractylodis Macrocephalae, has remarkable anti-cancer effects on various cancers. However, its effects on the treatment of gastric cancer remain unclear. Via multiple cellular and molecular approaches, we demonstrated that AT-I could potently inhibit cancer cell proliferation and induce apoptosis through inactivating Notch pathway. AT-I treatment led to the reduction of expressions of Notch1, Jagged1, and its downstream Hes1/ Hey1. Our results showed that AT-I inhibited the self-renewal capacity of gastric stem-like cells (GCSLCs) by suppression of their sphere formation capacity and cell viability. AT-I attenuated gastric cancer stem cell (GCSC) traits partly through inactivating Notch1, leading to reducing the expressions of its downstream target Hes1, Hey1 and CD44 in vitro. Collectively, our results suggest that AT-I might develop as a potential therapeutic drug for the treatment of gastric cancer.

  15. Replica-moulded polydimethylsiloxane culture vessel lids attenuate osmotic drift in long-term cell cultures

    Indian Academy of Sciences (India)

    Axel Blau; Tanja Neumann; Christiane Ziegler; Fabio Benfenati

    2009-03-01

    An imbalance in medium osmolarity is a determinant that affects cell culture longevity. Even in humidified incubators, evaporation of water leads to a gradual increase in osmolarity overtime. We present a simple replica-moulding strategy for producing self-sealing lids adaptable to standard, small-size cell-culture vessels. They are made of polydimethylsiloxane (PDMS), a flexible, transparent and biocompatible material, which is gas-permeable but largely impermeable to water. Keeping cell cultures in a humidified 5% CO2 incubator at 37°C, medium osmolarity increased by +6.86 mosmol/kg/day in standard 35 mm Petri dishes, while PDMS lids attenuated its rise by a factor of four to changes of +1.72 mosmol/kg/ day. Depending on the lid membrane thickness, pH drifts at ambient CO2 levels were attenuated by a factor of 4 to 9. Comparative evaporation studies at temperatures below 60°C yielded a 10-fold reduced water vapour flux of 1.75 g/day/dm2 through PDMS lids as compared with 18.69 g/day/dm2 with conventional Petri dishes. Using such PDMS lids, about 2/3 of the cell cultures grew longer than 30 days in vitro. Among these, the average survival time was 69 days with the longest survival being 284 days under otherwise conventional cell culture conditions.

  16. Oxygen depletion speeds and simplifies diffusion in HeLa cells.

    Science.gov (United States)

    Edwald, Elin; Stone, Matthew B; Gray, Erin M; Wu, Jing; Veatch, Sarah L

    2014-10-21

    Many cell types undergo a hypoxic response in the presence of low oxygen, which can lead to transcriptional, metabolic, and structural changes within the cell. Many biophysical studies to probe the localization and dynamics of single fluorescently labeled molecules in live cells either require or benefit from low-oxygen conditions. In this study, we examine how low-oxygen conditions alter the mobility of a series of plasma membrane proteins with a range of anchoring motifs in HeLa cells at 37°C. Under high-oxygen conditions, diffusion of all proteins is heterogeneous and confined. When oxygen is reduced with an enzymatic oxygen-scavenging system for ≥ 15 min, diffusion rates increase by > 2-fold, motion becomes unconfined on the timescales and distance scales investigated, and distributions of diffusion coefficients are remarkably consistent with those expected from Brownian motion. More subtle changes in protein mobility are observed in several other laboratory cell lines examined under both high- and low-oxygen conditions. Morphological changes and actin remodeling are observed in HeLa cells placed in a low-oxygen environment for 30 min, but changes are less apparent in the other cell types investigated. This suggests that changes in actin structure are responsible for increased diffusion in hypoxic HeLa cells, although superresolution localization measurements in chemically fixed cells indicate that membrane proteins do not colocalize with F-actin under either experimental condition. These studies emphasize the importance of controls in single-molecule imaging measurements, and indicate that acute response to low oxygen in HeLa cells leads to dramatic changes in plasma membrane structure. It is possible that these changes are either a cause or consequence of phenotypic changes in solid tumor cells associated with increased drug resistance and malignancy.

  17. A novel, blocking, Fc-silent anti-CD40 monoclonal antibody prolongs nonhuman primate renal allograft survival in the absence of B cell depletion.

    Science.gov (United States)

    Cordoba, F; Wieczorek, G; Audet, M; Roth, L; Schneider, M A; Kunkler, A; Stuber, N; Erard, M; Ceci, M; Baumgartner, R; Apolloni, R; Cattini, A; Robert, G; Ristig, D; Munz, J; Haeberli, L; Grau, R; Sickert, D; Heusser, C; Espie, P; Bruns, C; Patel, D; Rush, J S

    2015-11-01

    CD40-CD154 pathway blockade prolongs renal allograft survival in nonhuman primates (NHPs). However, antibodies targeting CD154 were associated with an increased incidence of thromboembolic complications. Antibodies targeting CD40 prolong renal allograft survival in NHPs without thromboembolic events but with accompanying B cell depletion, raising the question of the relative contribution of B cell depletion to the efficacy of anti-CD40 blockade. Here, we investigated whether fully silencing Fc effector functions of an anti-CD40 antibody can still promote graft survival. The parent anti-CD40 monoclonal antibody HCD122 prolonged allograft survival in MHC-mismatched cynomolgus monkey renal allograft transplantation (52, 22, and 24 days) with accompanying B cell depletion. Fc-silencing yielded CFZ533, an antibody incapable of B cell depletion but still able to potently inhibit CD40 pathway activation. CFZ533 prolonged allograft survival and function up to a defined protocol endpoint of 98-100 days (100, 100, 100, 98, and 76 days) in the absence of B cell depletion and preservation of good histological graft morphology. CFZ533 was well-tolerated, with no evidence of thromboembolic events or CD40 pathway activation and suppressed a gene signature associated with acute rejection. Thus, use of the Fc-silent anti-CD40 antibody CFZ533 appears to be an attractive approach for preventing solid organ transplant rejection.

  18. Giardia duodenalis arginine deiminase modulates the phenotype and cytokine secretion of human dendritic cells by depletion of arginine and formation of ammonia.

    Science.gov (United States)

    Banik, Stefanie; Renner Viveros, Pablo; Seeber, Frank; Klotz, Christian; Ignatius, Ralf; Aebischer, Toni

    2013-07-01

    Depletion of arginine is a recognized strategy that pathogens use to evade immune effector mechanisms. Depletion depends on microbial enzymes such as arginases, which are considered virulence factors. The effect is mostly interpreted as being a consequence of successful competition with host enzymes for the substrate. However, both arginases and arginine deiminases (ADI) have been associated with pathogen virulence. Both deplete arginine, but their reaction products differ. An ADI has been implicated in the virulence of Giardia duodenalis, an intestinal parasite that infects humans and animals, causing significant morbidity. Dendritic cells (DC) play a critical role in host defense and also in a murine G. duodenalis infection model. The functional properties of these innate immune cells depend on the milieu in which they are activated. Here, the dependence of the response of these cells on arginine was studied by using Giardia ADI and lipopolysaccharide-stimulated human monocyte-derived DC. Arginine depletion by ADI significantly increased tumor necrosis factor alpha and decreased interleukin-10 (IL-10) and IL-12p40 secretion. It also reduced the upregulation of surface CD83 and CD86 molecules, which are involved in cell-cell interactions. Arginine depletion also reduced the phosphorylation of S6 kinase in DC, suggesting the involvement of the mammalian target of rapamycin signaling pathway. The changes were due to arginine depletion and the formation of reaction products, in particular, ammonium ions. Comparison of NH(4)(+) and urea revealed distinct immunomodulatory activities of these products of deiminases and arginases, respectively. The data suggest that a better understanding of the role of arginine-depleting pathogen enzymes for immune evasion will have to take enzyme class and reaction products into consideration.

  19. Giardia duodenalis Arginine Deiminase Modulates the Phenotype and Cytokine Secretion of Human Dendritic Cells by Depletion of Arginine and Formation of Ammonia

    Science.gov (United States)

    Banik, Stefanie; Renner Viveros, Pablo; Seeber, Frank; Klotz, Christian; Ignatius, Ralf

    2013-01-01

    Depletion of arginine is a recognized strategy that pathogens use to evade immune effector mechanisms. Depletion depends on microbial enzymes such as arginases, which are considered virulence factors. The effect is mostly interpreted as being a consequence of successful competition with host enzymes for the substrate. However, both arginases and arginine deiminases (ADI) have been associated with pathogen virulence. Both deplete arginine, but their reaction products differ. An ADI has been implicated in the virulence of Giardia duodenalis, an intestinal parasite that infects humans and animals, causing significant morbidity. Dendritic cells (DC) play a critical role in host defense and also in a murine G. duodenalis infection model. The functional properties of these innate immune cells depend on the milieu in which they are activated. Here, the dependence of the response of these cells on arginine was studied by using Giardia ADI and lipopolysaccharide-stimulated human monocyte-derived DC. Arginine depletion by ADI significantly increased tumor necrosis factor alpha and decreased interleukin-10 (IL-10) and IL-12p40 secretion. It also reduced the upregulation of surface CD83 and CD86 molecules, which are involved in cell-cell interactions. Arginine depletion also reduced the phosphorylation of S6 kinase in DC, suggesting the involvement of the mammalian target of rapamycin signaling pathway. The changes were due to arginine depletion and the formation of reaction products, in particular, ammonium ions. Comparison of NH4+ and urea revealed distinct immunomodulatory activities of these products of deiminases and arginases, respectively. The data suggest that a better understanding of the role of arginine-depleting pathogen enzymes for immune evasion will have to take enzyme class and reaction products into consideration. PMID:23589577

  20. rRNA maturation in yeast cells depleted of large ribosomal subunit proteins.

    Directory of Open Access Journals (Sweden)

    Gisela Pöll

    Full Text Available The structural constituents of the large eukaryotic ribosomal subunit are 3 ribosomal RNAs, namely the 25S, 5.8S and 5S rRNA and about 46 ribosomal proteins (r-proteins. They assemble and mature in a highly dynamic process that involves more than 150 proteins and 70 small RNAs. Ribosome biogenesis starts in the nucleolus, continues in the nucleoplasm and is completed after nucleo-cytoplasmic translocation of the subunits in the cytoplasm. In this work we created 26 yeast strains, each of which conditionally expresses one of the large ribosomal subunit (LSU proteins. In vivo depletion of the analysed LSU r-proteins was lethal and led to destabilisation and degradation of the LSU and/or its precursors. Detailed steady state and metabolic pulse labelling analyses of rRNA precursors in these mutant strains showed that LSU r-proteins can be grouped according to their requirement for efficient progression of different steps of large ribosomal subunit maturation. Comparative analyses of the observed phenotypes and the nature of r-protein-rRNA interactions as predicted by current atomic LSU structure models led us to discuss working hypotheses on i how individual r-proteins control the productive processing of the major 5' end of 5.8S rRNA precursors by exonucleases Rat1p and Xrn1p, and ii the nature of structural characteristics of nascent LSUs that are required for cytoplasmic accumulation of nascent subunits but are nonessential for most of the nuclear LSU pre-rRNA processing events.

  1. Inebilizumab, a B Cell-Depleting Anti-CD19 Antibody for the Treatment of Autoimmune Neurological Diseases: Insights from Preclinical Studies

    Directory of Open Access Journals (Sweden)

    Ding Chen

    2016-11-01

    Full Text Available Exaggerated or inappropriate responses by B cells are an important feature in many types of autoimmune neurological diseases. The recent success of B-cell depletion in the treatment of multiple sclerosis (MS has stimulated the development of novel B-cell-targeting therapies with the potential for improved efficacy. CD19 has emerged as a promising target for the depletion of B cells as well as CD19-positive plasmablasts and plasma cells. Inebilizumab (MEDI-551, an anti-CD19 antibody with enhanced antibody-dependent cell-mediated cytotoxicity against B cells, is currently being evaluated in MS and neuromyelitis optica. This review discusses the role of B cells in autoimmune neurological disorders, summarizes the development of inebilizumab, and analyzes the recent results for inebilizumab treatment in an autoimmune encephalitis mouse model. The novel insights obtained from these preclinical studies can potentially guide future investigation of inebilizumab in patients.

  2. Radiation-released histamine in the rhesus monkey as modified by mast cell depletion and antihistamine. Scientific report

    Energy Technology Data Exchange (ETDEWEB)

    Doyle, T.F.; Strike, T.A.

    1975-06-01

    Changes in blood histamine concentrations of rhesus monkeys were measured after a 4000-rad dose of mixed gamma-neutron radiation. All animals were pretreated with amino-guanidine to retard histamine catabolism. Histamine concentrations increased from 26 + or - 13.5 to 235 + or - 16 ng/ml after irradiation. When the animals were pretreated with an antihistamine, chlorpheniramine (3 mg/kg), histamine concentrations changed from 25.7 + or - 13.5 to 462 + or - 226 ng/ml after irradiation. When the monkeys were pretreated with a specific mast cell histamine depleter, compound 48/80 (1mg/kg per day) for four consecutive days and then irradiated (4000 rads), histamine concentrations did not change significantly. When 48/80 was given 20 min after irradiation, histamine concentrations changed from 18 + or - 2 ng/ml to a maximum of 35 + or - 9 ng/ml after 48/80 injection. (Author) (GRA)

  3. Attenuated Toxoplasma gondii Stimulates Immunity to Pancreatic Cancer by Manipulation of Myeloid Cell Populations.

    Science.gov (United States)

    Sanders, Kiah L; Fox, Barbara A; Bzik, David J

    2015-08-01

    Suppressive myeloid cells represent a significant barrier to the generation of productive antitumor immune responses to many solid tumors. Eliminating or reprogramming suppressive myeloid cells to abrogate tumor-associated immune suppression is a promising therapeutic approach. We asked whether treatment of established aggressive disseminated pancreatic cancer with the immunotherapeutic attenuated Toxoplasma gondii vaccine strain CPS would trigger tumor-associated myeloid cells to generate therapeutic antitumor immune responses. CPS treatment significantly decreased tumor-associated macrophages and markedly increased dendritic cell infiltration of the pancreatic tumor microenvironment. Tumor-resident macrophages and dendritic cells, particularly cells actively invaded by CPS, increased expression of costimulatory molecules CD80 and CD86 and concomitantly boosted their production of IL12. CPS treatment increased CD4(+) and CD8(+) T-cell infiltration into the tumor microenvironment, activated tumor-resident T cells, and increased IFNγ production by T-cell populations. CPS treatment provided a significant therapeutic benefit in pancreatic tumor-bearing mice. This therapeutic benefit depended on IL12 and IFNγ production, MyD88 signaling, and CD8(+) T-cell populations. Although CD4(+) T cells exhibited activated effector phenotypes and produced IFNγ, CD4(+) T cells as well as natural killer cells were not required for the therapeutic benefit. In addition, CD8(+) T cells isolated from CPS-treated tumor-bearing mice produced IFNγ after re-exposure to pancreatic tumor antigen, suggesting this immunotherapeutic treatment stimulated tumor cell antigen-specific CD8(+) T-cell responses. This work highlights the potency and immunotherapeutic efficacy of CPS treatment and demonstrates the significance of targeting tumor-associated myeloid cells as a mechanism to stimulate more effective immunity to pancreatic cancer.

  4. Targeting Tumor Oct4 to Deplete Prostate Tumor and Metastasis Initiating Cells

    Science.gov (United States)

    2014-10-01

    Recently, Hayashi et al. showed that overexpression of POU5F1B in gastric cancer cells increased cell growth in vitro as well as both tumorigenicity and...POU5F1 is associated with prostate cancer susceptibility. American journal of human genetics 94, 395-404. Hayashi, H., Arao, T., Togashi, Y., Kato, H...promotes an aggressive phenotype in gastric cancer . Oncogene. Kastler, S., Honold, L., Luedeke, M., Kuefer, R., Moller, P., Hoegel, J., Vogel, W., Maier

  5. Comparison of the time courses of selective gene expression and dopaminergic depletion induced by MPP+ in MN9D cells.

    Science.gov (United States)

    Wang, Jianyong; Duhart, Helen M; Xu, Zengjun; Patterson, Tucker A; Newport, Glenn D; Ali, Syed F

    2008-05-01

    Parkinson's disease (PD) is a common neurodegenerative disease characterized by progressive loss of midbrain dopaminergic neurons with unknown etiology. MPP+ (1-methyl-4-phenylpyridinium ion) is the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which induces Parkinson's-like symptoms in humans and animals. MPTP/MPP+ produces selective dopaminergic neuronal degeneration, therefore, these agents are commonly used to study the pathogenesis of PD. However, the mechanisms of their toxicity have not been fully elucidated. Recently, we reported in a microarray study using a midbrain-derived dopaminergic neuronal cell line, MN9D, that MPP+ induced significant changes in a number of genes known to be associated with the dopaminergic system. In this study, we investigated the expression time courses of six genes using real-time RT-PCR, and compared them with the progressive dopaminergic depletion caused by MPP+. Our data showed that dopamine content was significantly decreased after 0.5h of MPP+ (200 microM) exposure and was completely depleted after 40 h. The expression of Gpr37, which is closely related to the pathogenesis of autosomal recessive juvenile Parkinsonism, was up-regulated after 0.5h, and stayed up-regulated up to 48 h. Txnip, which is critical to the adjustment of cellular redox status, was down-regulated after 1h and stayed down-regulated up to 48 h. Ldh1 and Cdo1, which are also involved in oxidative stress, were down-regulated after 16 h and stayed down-regulated up to 48 h. Two pro-apoptotic genes, Egln3 and Bnip3, were down-regulated after 2 and 4h, and stayed down-regulated up to 48 h. These findings suggested that the time course of expression for multiple genes correlated with the dopaminergic depletion; and MPP+-induced neurotoxicity in MN9D cells could be used as a model to further explore the roles of these and other genes in the pathogenesis and possible treatment of PD.

  6. Studying depletion kinetics of circulating prostate cancer cells by in vivo flow cytometer

    Science.gov (United States)

    Liu, Guangda; Gu, Zhengqin; Guo, Jin; Li, Yan; Chen, Yun; Chen, Tong; Wang, Cheng; Wei, Xunbin

    2011-03-01

    Prostate cancer is the most common malignancy in American men and the second leading cause of deaths from cancer, after lung cancer. The tumor usually grows slowly and remains confined to the gland for many years. During this time, the tumor produces little or no symptoms or outward signs. As the cancer advances, however, it can metastasize throughout other areas of the body, such as the bones, lungs, and liver. Surgical resection, hormonal therapy, chemotherapy and radiation therapy are the foundation of current prostate cancer therapies. Treatments for prostate cause both short- and long-term side effects that may be difficult to accept. Molecular mechanisms of prostate cancer metastasis need to be understood better and new therapies must be developed to selectively target to unique characteristics of cancer cell growth and metastasis. We have developed the "in vivo microscopy" to study the mechanisms that govern prostate cancer cell spread through the microenvironment in vivo in real-time confocal near-infrared fluorescence imaging. A recently developed "in vivo flow cytometer" and optical imaging are used to assess prostate cancer cell spreading and the circulation kinetics of prostate cancer cells. A real- time quantitative monitoring of circulating prostate cancer cells by the in vivo flow cytometer will be useful to assess the effectiveness of the potential therapeutic interventions.

  7. Depletion kinetics of circulating prostate cancer cells studied by in vivo flow cytometer

    Science.gov (United States)

    Liu, Guangda; Guo, Jin; Li, Yan; Chen, Yun; Gu, Zhengqin; Chen, Tong; Wang, Cheng; Wei, Xunbin

    2010-11-01

    Prostate cancer is the most common malignancy in American men and the second leading cause of deaths from cancer, after lung cancer. The tumor usually grows slowly and remains confined to the gland for many years. During this time, the tumor produces little or no symptoms or outward signs. As the cancer advances, however, it can metastasize throughout other areas of the body, such as the bones, lungs, and liver. Surgical resection, hormonal therapy, chemotherapy and radiation therapy are the foundation of current prostate cancer therapies. Treatments for prostate cause both short- and long-term side effects that may be difficult to accept. Molecular mechanisms of prostate cancer metastasis need to be understood better and new therapies must be developed to selectively target to unique characteristics of cancer cell growth and metastasis. We have developed the "in vivo microscopy" to study the mechanisms that govern prostate cancer cell spread through the microenvironment in vivo in real-time confocal nearinfrared fluorescence imaging. A recently developed "in vivo flow cytometer" and optical imaging are used to assess prostate cancer cell spreading and the circulation kinetics of prostate cancer cells. A real- time quantitative monitoring of circulating prostate cancer cells by the in vivo flow cytometer will be useful to assess the effectiveness of the potential therapeutic interventions.

  8. Intracellular Glutathione Depletion by Oridonin Leads to Apoptosis in Hepatic Stellate Cells

    Directory of Open Access Journals (Sweden)

    Liang-Mou Kuo

    2014-03-01

    Full Text Available Proliferation of hepatic stellate cells (HSCs plays a key role in the pathogenesis of liver fibrosis. Induction of HSC apoptosis by natural products is considered an effective strategy for treating liver fibrosis. Herein, the apoptotic effects of 7,20-epoxy-ent-kaurane (oridonin, a diterpenoid isolated from Rabdosia rubescens, and its underlying mechanisms were investigated in rat HSC cell line, HSC-T6. We found that oridonin inhibited cell viability of HSC-T6 in a concentration-dependent manner. Oridonin induced a reduction in mitochondrial membrane potential and increases in caspase 3 activation, subG1 phase, and DNA fragmentation. These apoptotic effects of oridonin were completely reversed by thiol antioxidants, N-acetylcysteine (NAC and glutathione monoethyl ester. Moreover, oridonin increased production of reactive oxygen species (ROS, which was also inhibited by NAC. Significantly, oridonin reduced intracellular glutathione (GSH level in a concentration- and time-dependent fashion. Additionally, oridonin induced phosphorylations of extracellular signal-regulated kinase (ERK, c-Jun N-terminal kinase (JNK, and p38 mitogen-activated protein kinase (MAPK. NAC prevented the activation of MAPKs in oridonin-induced cells. However, selective inhibitors of MAPKs failed to alter oridonin-induced cell death. In summary, these results demonstrate that induction of apoptosis in HSC-T6 by oridonin is associated with a decrease in cellular GSH level and increase in ROS production.

  9. Targeted impairment of thymidine kinase 2 expression in cells induces mitochondrial DNA depletion and reveals molecular mechanisms of compensation of mitochondrial respiratory activity

    Energy Technology Data Exchange (ETDEWEB)

    Villarroya, Joan, E-mail: joanvillarroya@gmail.com [Institut de Recerca, Hospital Universitari de la Vall d' Hebron, Barcelona (Spain); Institut de Recerca l' Hospital de la Santa Creu i Sant Pau, Barcelona (Spain); Lara, Mari-Carmen [Institut de Recerca, Hospital Universitari de la Vall d' Hebron, Barcelona (Spain); Department of Neurology, Columbia University Medical Center, New York, NY (United States); Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), ISCIII (Spain); Dorado, Beatriz [Department of Neurology, Columbia University Medical Center, New York, NY (United States); Garrido, Marta [Unitat de Biologia Cel.lular i Molecular, IMIM-Hospital del Mar, Barcelona (Spain); Garcia-Arumi, Elena [Institut de Recerca, Hospital Universitari de la Vall d' Hebron, Barcelona (Spain); Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), ISCIII (Spain); Meseguer, Anna [Institut de Recerca, Hospital Universitari de la Vall d' Hebron, Barcelona (Spain); Hirano, Michio [Department of Neurology, Columbia University Medical Center, New York, NY (United States); Vila, Maya R. [Institut de Recerca, Hospital Universitari de la Vall d' Hebron, Barcelona (Spain)

    2011-04-08

    Highlights: {yields} We impaired TK2 expression in Ost TK1{sup -} cells via siRNA-mediated interference (TK2{sup -}). {yields} TK2 impairment caused severe mitochondrial DNA (mtDNA) depletion in quiescent cells. {yields} Despite mtDNA depletion, TK2{sup -} cells show high cytochrome oxidase activity. {yields} Depletion of mtDNA occurs without imbalance in the mitochondrial dNTP pool. {yields} Nuclear-encoded ENT1, DNA-pol {gamma}, TFAM and TP gene expression is lowered in TK2{sup -} cells. -- Abstract: The mitochondrial DNA (mtDNA) depletion syndrome comprises a clinically heterogeneous group of diseases characterized by reductions of the mtDNA abundance, without associated point mutations or rearrangements. We have developed the first in vitro model to study of mtDNA depletion due to reduced mitochondrial thymidine kinase 2 gene (TK2) expression in order to understand the molecular mechanisms involved in mtDNA depletion syndrome due to TK2 mutations. Small interfering RNA targeting TK2 mRNA was used to decrease TK2 expression in Ost TK1{sup -} cells, a cell line devoid of endogenous thymidine kinase 1 (TK1). Stable TK2-deficient cell lines showed a reduction of TK2 levels close to 80%. In quiescent conditions, TK2-deficient cells showed severe mtDNA depletion, also close to 80% the control levels. However, TK2-deficient clones showed increased cytochrome c oxidase activity, higher cytochrome c oxidase subunit I transcript levels and higher subunit II protein expression respect to control cells. No alterations of the deoxynucleotide pools were found, whereas a reduction in the expression of genes involved in nucleoside/nucleotide homeostasis (human equilibrative nucleoside transporter 1, thymidine phosphorylase) and mtDNA maintenance (DNA-polymerase {gamma}, mitochondrial transcription factor A) was observed. Our findings highlight the importance of cellular compensatory mechanisms that enhance the expression of respiratory components to ensure respiratory activity

  10. Attenuated total reflectance Fourier transform infrared spectroscopy method to differentiate between normal and cancerous breast cells.

    Science.gov (United States)

    Lane, Randy; See, Seong S

    2012-09-01

    Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) is used to find the structural differences between cancerous breast cells (MCF-7 line) and normal breast cells (MCF-12F line). Gold nanoparticles were prepared and the hydrodynamic diameter of the gold nanoparticles found to be 38.45 nm. The Gold nanoparticles were exposed to both MCF-7 and MCF-12F cells from lower to higher concentrations. Spectroscopic studies founds nanoparticles were within the cells, and increasing the nanoparticles concentration inside the cells also resulted in sharper IR peaks as a result of localized surface Plasmon resonance. Asymmetric and symmetric stretching and bending vibrations between phosphate, COO-, CH2 groups were found to give negative shifts in wavenumbers and a decrease in peak intensities when going from noncancerous to cancerous cells. Cellular proteins produced peak assignments at the 1542 and 1644 cm(-1) wavenumbers which were attributed to the amide I and amide II bands of the polypeptide bond of proteins. Significant changes were found in the peak intensities between the cell lines in the spectrum range from 2854-2956 cm(-1). Results show that the concentration range of gold nanoparticles used in this research showed no significant changes in cell viability in either cell line. Therefore, we believe ATR-FTIR and gold nanotechnology can be at the forefront of cancer diagnosis for some time to come.

  11. Silibinin attenuates ionizing radiation-induced pro-angiogenic response and EMT in prostate cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Nambiar, Dhanya K. [Cancer Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi (India); School of Environmental Sciences, Jawaharlal Nehru University, New Delhi (India); Rajamani, Paulraj [School of Environmental Sciences, Jawaharlal Nehru University, New Delhi (India); Singh, Rana P., E-mail: rana_singh@mail.jnu.ac.in [Cancer Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi (India); School of Life Sciences, Central University of Gujarat, Gandhinagar (India)

    2015-01-02

    Graphical abstract: Potential model showing mechanism of silibinin-mediated attenuation of IR-induced angiogenic phenotype and EMT in tumor cells. Silibinin counters radiation induced invasive and migratory phenotype of cancer cells by down-regulating mitogenic pathways activated by IR, leading to inhibition of molecules including VEGF, iNOS, MMPs and N-cadherin. Silibinin also reverses IR mediated E-cadherin down-regulation, inhibiting EMT in tumor cells. Silibinin also radiosensitizes endothelial cells, reduces capillary tube formation by targeting various pro-angiogenic molecules. Further, silibinin may inhibit autocrine and paracrine signaling between tumor and endothelial cells by decreasing the levels of VEGF and other signaling molecules activated in response to IR. - Highlights: • Silibinin radiosensitizes endothelial cells. • Silibinin targets ionization radiation (IR)-induced EMT in PCa cells. • Silibinin is in phase II clinical trial in PCa patients, hence clinically relevant. - Abstract: Radiotherapy of is well established and frequently utilized in prostate cancer (PCa) patients. However, recurrence following therapy and distant metastases are commonly encountered problems. Previous studies underline that, in addition to its therapeutic effects, ionizing radiation (IR) increases the vascularity and invasiveness of surviving radioresistant cancer cells. This invasive phenotype of radioresistant cells is an upshot of IR-induced pro-survival and mitogenic signaling in cancer as well as endothelial cells. Here, we demonstrate that a plant flavonoid, silibinin can radiosensitize endothelial cells by inhibiting expression of pro-angiogenic factors. Combining silibinin with IR not only strongly down-regulated endothelial cell proliferation, clonogenicity and tube formation ability rather it strongly (p < 0.001) reduced migratory and invasive properties of PCa cells which were otherwise marginally affected by IR treatment alone. Most of the pro

  12. Inhibition of envelope-mediated CD4+-T-cell depletion by human immunodeficiency virus attachment inhibitors.

    Science.gov (United States)

    Alexander, Louis; Zhang, Sharon; McAuliffe, Brian; Connors, David; Zhou, Nannon; Wang, Tao; Agler, Michele; Kadow, John; Lin, Pin-Fang

    2009-11-01

    Human immunodeficiency virus type 1 (HIV-1) envelope (Env) binding induces proapoptotic signals in CD4(+) T cells without a requirement of infection. Defective virus particles, which represent the majority of HIV-1, usually contain a functional Env and therefore represent a potentially significant cause of such CD4(+)-T-cell loss. We reasoned that an HIV-1 inhibitor that prohibits Env-host cell interactions could block the destructive effects of defective particles. HIV-1 attachment inhibitors (AIs), which potently inhibit Env-CD4 binding and subsequent downstream effects of Env, display low-nanomolar antiapoptotic potency and prevent CD4(+)-T-cell depletion from mixed lymphocyte cultures, also with low-nanomolar potency. Specific Env amino acid changes that confer resistance to AI antientry activity eliminate AI antiapoptotic effects. We observed that CD4(+)-T-cell destruction is specific for CXCR4-utilizing HIV-1 strains and that the fusion blocker enfuvirtide inhibits Env-mediated CD4(+)-T-cell killing but is substantially less potent than AIs. These observations, in conjunction with observed antiapoptotic activities of soluble CD4 and the CXCR4 blocker AMD3100, suggest that this AI activity functions through a mechanism common to AI antientry activity, e.g., prevention of Env conformation changes necessary for specific interactions with cellular factors that facilitate viral entry. Our study suggests that AIs, in addition to having potent antientry activity, could contribute to immune system homeostasis in individuals infected with HIV-1 that can engage CXCR4, thereby mitigating the increased risk of adverse clinical events observed in such individuals on current antiretroviral regimens.

  13. Depletion of arginine by recombinant arginine deiminase induces nNOS-activated neurotoxicity in neuroblastoma cells.

    Science.gov (United States)

    Lin, Shan-Erh; Wu, Fe-Lin Lin; Wei, Ming-Feng; Shen, Li-Jiuan

    2014-01-01

    The abnormal regulation of inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS) is associated with neurodegenerative disorders. Recombinant arginine deiminase (rADI) is a selective NO modulator of iNOS and eNOS in endothelial cells, and it also exhibits neuroprotective activity in an iNOS-induced neuron-microglia coculture system. However, the effect of rADI on nNOS remains unknown. Addressing this issue is important for evaluating the potential application of rADI in neurodegenerative diseases. SH-SY5Y cells were treated with N-methyl-D-aspartic acid (NMDA) to activate nNOS. NMDA increased NO production by 39.7 ± 3.9% via nNOS under arginine-containing conditions, but there was no significant increase in both arginine-free and rADI pretreated arginine-containing (citrulline) buffer. Subsequently, neither NMDA nor rADI alone caused cytotoxicity, whereas cotreatment with NMDA and rADI resulted in dissipation of the cell mitochondrial membrane potential and decreased cell viability. The mechanism of rADI cytotoxicity in the presence of NMDA is caused by the inhibition of NO production via nNOS mediated by the NMDA receptor, which was abolished when extracellular arginine was absent, even in the presence of citrulline. rADI not only reduced NO production but also caused cellular toxicity in nNOS-activated SH-SY5Y cells, suggesting a dual role for rADI in NOS-mediated neurotoxicity.

  14. Depletion of Arginine by Recombinant Arginine Deiminase Induces nNOS-Activated Neurotoxicity in Neuroblastoma Cells

    Directory of Open Access Journals (Sweden)

    Shan-Erh Lin

    2014-01-01

    Full Text Available The abnormal regulation of inducible nitric oxide synthase (iNOS and neuronal nitric oxide synthase (nNOS is associated with neurodegenerative disorders. Recombinant arginine deiminase (rADI is a selective NO modulator of iNOS and eNOS in endothelial cells, and it also exhibits neuroprotective activity in an iNOS-induced neuron-microglia coculture system. However, the effect of rADI on nNOS remains unknown. Addressing this issue is important for evaluating the potential application of rADI in neurodegenerative diseases. SH-SY5Y cells were treated with N-methyl-D-aspartic acid (NMDA to activate nNOS. NMDA increased NO production by 39.7 ± 3.9% via nNOS under arginine-containing conditions, but there was no significant increase in both arginine-free and rADI pretreated arginine-containing (citrulline buffer. Subsequently, neither NMDA nor rADI alone caused cytotoxicity, whereas cotreatment with NMDA and rADI resulted in dissipation of the cell mitochondrial membrane potential and decreased cell viability. The mechanism of rADI cytotoxicity in the presence of NMDA is caused by the inhibition of NO production via nNOS mediated by the NMDA receptor, which was abolished when extracellular arginine was absent, even in the presence of citrulline. rADI not only reduced NO production but also caused cellular toxicity in nNOS-activated SH-SY5Y cells, suggesting a dual role for rADI in NOS-mediated neurotoxicity.

  15. Solvent-Mediated Crystallization of CH3NH3SnI3 Films for Heterojunction Depleted Perovskite Solar Cells.

    Science.gov (United States)

    Hao, Feng; Stoumpos, Constantinos C; Guo, Peijun; Zhou, Nanjia; Marks, Tobin J; Chang, Robert P H; Kanatzidis, Mercouri G

    2015-09-09

    Organo-lead halide perovskite solar cells have gained enormous significance and have now achieved power conversion efficiencies of ∼20%. However, the potential toxicity of lead in these systems raises environmental concerns for widespread deployment. Here we investigate solvent effects on the crystallization of the lead-free methylammonium tin triiodide (CH3NH3SnI3) perovskite films in a solution growth process. Highly uniform, pinhole-free perovskite films are obtained from a dimethyl sulfoxide (DMSO) solution via a transitional SnI2·3DMSO intermediate phase. This high-quality perovskite film enables the realization of heterojunction depleted solar cells based on mesoporous TiO2 layer but in the absence of any hole-transporting material with an unprecedented photocurrent up to 21 mA cm(-2). Charge extraction and transient photovoltage decay measurements reveal high carrier densities in the CH3NH3SnI3 perovskite device which are one order of magnitude larger than CH3NH3PbI3-based devices but with comparable recombination lifetimes in both devices. The relatively high background dark carrier density of the Sn-based perovskite is responsible for the lower photovoltaic efficiency in comparison to the Pb-based analogues. These results provide important progress toward achieving improved perovskite morphology control in realizing solution-processed highly efficient lead-free perovskite solar cells.

  16. Amniotic fluid stem cells from EGFP transgenic mice attenuate hyperoxia-induced acute lung injury.

    Directory of Open Access Journals (Sweden)

    Shih-Tao Wen

    Full Text Available High concentrations of oxygen aggravate the severity of lung injury in patients requiring mechanical ventilation. Although mesenchymal stem cells have been shown to effectively attenuate various injured tissues, there is limited information regarding a role for amniotic fluid stem cells (AFSCs in treating acute lung injury. We hypothesized that intravenous delivery of AFSCs would attenuate lung injury in an experimental model of hyperoxia-induced lung injury. AFSCs were isolated from EGFP transgenic mice. The in vitro differentiation, surface markers, and migration of the AFSCs were assessed by specific staining, flow cytometry, and a co-culture system, respectively. The in vivo therapeutic potential of AFSCs was evaluated in a model of acute hyperoxia-induced lung injury in mice. The administration of AFSCs significantly reduced the hyperoxia-induced pulmonary inflammation, as reflected by significant reductions in lung wet/dry ratio, neutrophil counts, and the level of apoptosis, as well as reducing the levels of inflammatory cytokine (IL-1β, IL-6, and TNF-α and early-stage fibrosis in lung tissues. Moreover, EGFP-expressing AFSCs were detected and engrafted into a peripheral lung epithelial cell lineage by fluorescence microscopy and DAPI stain. Intravenous administration of AFSCs may offer a new therapeutic strategy for acute lung injury (ALI, for which efficient treatments are currently unavailable.

  17. Gypenoside Attenuates β Amyloid-Induced Inflammation in N9 Microglial Cells via SOCS1 Signaling

    Directory of Open Access Journals (Sweden)

    Hui Cai

    2016-01-01

    Full Text Available Reducing β amyloid- (Aβ- induced microglial activation is believed to be effective in treating Alzheimer’s disease (AD. Microglia can be activated into classic activated state (M1 state or alternative activated state (M2 state, and the former is harmful; in contrast, the latter is beneficial. Gypenoside (GP is the major bioactive constituent of Gynostemma pentaphyllum, a traditional Chinese herb medicine. In this study, we hypothesized that GP attenuates Aβ-induced microglial activation by ameliorating microglial M1/M2 states, and the process may be mediated by suppressor of cell signaling protein 1 (SOCS1. In this study, we found that Aβ exposure increased the levels of microglial M1 markers, including iNOS expression, tumor necrosis factor α (TNF-α, interleukin 1β (IL-1β, and IL-6 releases, and coadministration of GP reversed the increase of M1 markers and enhanced the levels of M2 markers, including arginase-1 (Arg-1 expression, IL-10, brain-derived neurotrophic factor (BDNF, and glial cell-derived neurotrophic factor (GDNF releases in the Aβ-treated microglial cells. SOCS1-siRNA, however, significantly abolished the GP-induced effects on the levels of microglial M1 and M2 markers. These findings indicated that GP attenuates Aβ-induced microglial activation by ameliorating M1/M2 states, and the process may be mediated by SOCS1.

  18. Distinct phenotype, longitudinal changes of numbers and cell-associated virus in blood dendritic cells in SIV-infected CD8-lymphocyte depleted macaques.

    Science.gov (United States)

    Soulas, Caroline; Autissier, Patrick J; Burdo, Tricia H; Piatak, Michael; Lifson, Jeffrey D; Williams, Kenneth C

    2015-01-01

    Loss of circulating CD123+ plasmacytoid dendritic cells (pDCs) during HIV infection is well established. However, changes of myeloid DCs (mDCs) are ambiguous since they are studied as a homogeneous CD11c+ population despite phenotypic and functional heterogeneity. Heterogeneity of CD11c+ mDCs in primates is poorly described in HIV and SIV infection. Using multiparametric flow cytometry, we monitored longitudinally cell number and cell-associated virus of CD123+ pDCs and non-overlapping subsets of CD1c+ and CD16+ mDCs in SIV-infected CD8-depleted rhesus macaques. The numbers of all three DC subsets were significantly decreased by 8 days post-infection. Whereas CD123+ pDCs were persistently depleted, numbers of CD1c+ and CD16+ mDCs rebounded. Numbers of CD1c+ mDCs significantly increased by 3 weeks post-infection while numbers of CD16+ mDCs remained closer to pre-infection levels. We found similar changes in the numbers of all three DC subsets in CD8 depleted animals as we found in animals that were SIV infected animals that were not CD8 lymphocyte depleted. CD16+ mDCs and CD123+ pDCs but not CD1c+ mDCs were significantly decreased terminally with AIDS. All DC subsets harbored SIV RNA as early as 8 days and then throughout infection. However, SIV DNA was only detected in CD123+ pDCs and only at 40 days post-infection consistent with SIV RNA, at least in mDCs, being surface-bound. Altogether our data demonstrate that SIV infection differently affects CD1c+ and CD16+ mDCs where CD16+ but not CD1c+ mDCs are depleted and might be differentially regulated in terminal AIDS. Finally, our data underline the importance of studying CD1c+ and CD16+ mDCs as discrete populations, and not as total CD11c+ mDCs.

  19. Melatonin attenuates 1-methyl-4-phenylpyridinium-induced PC12 cell death

    Institute of Scientific and Technical Information of China (English)

    Jin-feng BAO; Ren-gang WU; Xiao-ping ZHANG; Yan SONG; Chang-ling LI

    2005-01-01

    Aim: To explore the effect of melatonin on PC12 cell death induced by 1-methyl-4-phenylpyridinium (MPP+). Methods: MTT assay, lactate dehydrogenase (LDH)efflux assay, and immunohistochemistry methods were used to measure neurotoxicity of PC 12 cells treated acutely with MPP+ in low glucose and high glucose conditions, and to assess the neuroprotective effect of melatonin on PC 12 cell death induced by MPP+. Results: In a low glucose condition, MPP+ significantly induced PC 12 cell death, which showed time and concentration dependence. In a serum-free low glucose condition, the percentages of viability of cells treated with MPP+ for 12, 24, 48, 72, and 96 h were 85.1%, 75.4%, 64.9%, 28.15%, and 9%, respectively. The level of LDH in the culture medium increased and tyrosine hydroxylase positive (TH+) cell count decreased. However, in a serum-free high glucose condition, MPP+ did not significantly induce PC12 cell death compared with control at various concentrations and time regimens. When the cells were preincubated with melatonin 250 μmol/L for 48, 72, and 96 h in a serum-free low glucose condition, cell survival rate significantly increased to 78.1%, 58.8%, and 31.6%, respectively. Melatonin abolished the LDH leakage of cells treated with MPP+ and increased TH+ cells count. Conclusion: MPP+ caused concentrationdependent PC12 cell death. The level of glucose was an important factor to MPP+induced dopaminergic PC12 cell death. Low glucose level could potentiate MPP+toxicity, while high glucose level could reduce the toxicity. In addition, melatonin attenuated PC12 cell death induced by MPP+.

  20. Partial depletion of CD4(+)CD25(+)Foxp3(+) T regulatory cells significantly increases morbidity during acute phase Toxoplasma gondii infection in resistant BALB/c mice.

    Science.gov (United States)

    Morampudi, Vijay; De Craeye, Stephane; Le Moine, Alain; Detienne, Sophie; Braun, M Y; D'Souza, Sushila

    2011-04-01

    CD4(+)CD25(+)Foxp3(+) T regulatory (Treg) cells, are known to regulate responses to infectious agents. Here we compared disease progression in BALB/c and C57BL/6(B6) mice infected perorally with Toxoplasma gondii for 7 days and examined the affect of partial depletion of Treg cells in these mice. BALB/c mice were seen to be resistant to peroral infection whereas B6 mice were susceptible in terms of mortality. Although the depletion of Treg cells before infection had no effect on the survival of B6 or BALB/c mice, it resulted in increased parasite burdens in BALB/c mice, especially in the lamina propria, but not in B6 mice. Pro-inflammatory cytokines were also increased in Treg cells depleted BALB/c mice as compared to B6 mice. In addition Treg cell depleted BALB/c mice displayed increased ileal histopathology compared to their non-treated counterparts. These findings provide evidence for the contribution of Treg cells, in the resistance of BALB/c mice against peroral T. gondii infection.

  1. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) imaging of tissues and live cells.

    Science.gov (United States)

    Andrew Chan, K L; Kazarian, Sergei G

    2016-04-07

    FTIR spectroscopic imaging is a label-free, non-destructive and chemically specific technique that can be utilised to study a wide range of biomedical applications such as imaging of biopsy tissues, fixed cells and live cells, including cancer cells. In particular, the use of FTIR imaging in attenuated total reflection (ATR) mode has attracted much attention because of the small, but well controlled, depth of penetration and corresponding path length of infrared light into the sample. This has enabled the study of samples containing large amounts of water, as well as achieving an increased spatial resolution provided by the high refractive index of the micro-ATR element. This review is focused on discussing the recent developments in FTIR spectroscopic imaging, particularly in ATR sampling mode, and its applications in the biomedical science field as well as discussing the future opportunities possible as the imaging technology continues to advance.

  2. Inhibition of human copper trafficking by a small molecule significantly attenuates cancer cell proliferation.

    Science.gov (United States)

    Wang, Jing; Luo, Cheng; Shan, Changliang; You, Qiancheng; Lu, Junyan; Elf, Shannon; Zhou, Yu; Wen, Yi; Vinkenborg, Jan L; Fan, Jun; Kang, Heebum; Lin, Ruiting; Han, Dali; Xie, Yuxin; Karpus, Jason; Chen, Shijie; Ouyang, Shisheng; Luan, Chihao; Zhang, Naixia; Ding, Hong; Merkx, Maarten; Liu, Hong; Chen, Jing; Jiang, Hualiang; He, Chuan

    2015-12-01

    Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies.

  3. Inhibition of human copper trafficking by a small molecule significantly attenuates cancer cell proliferation

    Science.gov (United States)

    Wang, Jing; Luo, Cheng; Shan, Changliang; You, Qiancheng; Lu, Junyan; Elf, Shannon; Zhou, Yu; Wen, Yi; Vinkenborg, Jan L.; Fan, Jun; Kang, Heebum; Lin, Ruiting; Han, Dali; Xie, Yuxin; Karpus, Jason; Chen, Shijie; Ouyang, Shisheng; Luan, Chihao; Zhang, Naixia; Ding, Hong; Merkx, Maarten; Liu, Hong; Chen, Jing; Jiang, Hualiang; He, Chuan

    2015-12-01

    Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies.

  4. Bone metastasis model with multiorgan dissemination of human small-cell lung cancer (SBC-5) cells in natural killer cell-depleted SCID mice.

    Science.gov (United States)

    Miki, T; Yano, S; Hanibuchi, M; Sone, S

    2000-01-01

    Lung cancer is commonly associated with multiorgan metastasis, and bone is a frequent metastatic site for lung cancer. Nevertheless, no bone metastasis model of lung cancer with multiorgan dissemination is available, which could provide opportunity to study the molecular pathogenesis. We examined the abilities of eight human lung cancer cell lines injected intravenously into natural killer (NK) cell-depleted SCID mice to generate metastatic nodules in bone and multiple organs, and explored the correlation of the parathyroid hormone-related protein (PTHrP) with the bone metastasis. Although all the small-cell carcinoma cell lines (SBC-5, SBC-3, SBC-3/ADM, H69, H69/VP) formed metastatic nodules in multiple organs (liver, kidney, and lymph nodes), only SBC-5 cells reproducibly developed bone metastases. Squamous cell carcinoma (RERF-LC-AI) cells metastasized mainly into the liver and kidneys, whereas adenocarcinoma (PC-14, A549) mainly produced colonies in the lungs. As assessed by X-ray photography, the osteolytic bone metastases produced by SBC-5 cells were detected as early as on day 28, and all recipient mice developed bone metastasis by day 35. The expression of PTHrP in eight cell lines was directly correlated with the formation of bone metastasis. No correlation was observed between the formation of bone metastasis and the expression of other metastasis-related cytokines (IL-1, IL-6, IL-8, IL-10, IL-11, TNF-alpha, VEGF, M-CSF). Consistent with the formation of bone metastasis by SBC-5 cells, the levels of PTHrP and calcium in the mouse serum were increased in a time-dependent manner, suggesting that PTHrP produced by human lung cancer may play a crucial role in the formation of bone metastasis and hypercalcemia. These findings indicate that a bone metastasis model of SBC-5 cells may be useful for clarifying the molecular aspects of the metastatic processes in different organ microenvironments and the development of therapeutic modalities for lung cancer

  5. APOBEC3G-depleted resting CD4+ T cells remain refractory to HIV1 infection.

    Directory of Open Access Journals (Sweden)

    Francesca R Santoni de Sio

    Full Text Available BACKGROUND: CD4+ T lymphocytes are the primary targets of HIV1 but cannot be infected when fully quiescent, due to a post-entry block preventing the completion of reverse transcription. Chiu et al. recently proposed that this restriction reflects the action of APOBEC3G (A3G. They further suggested that T cell activation abrogates the A3G-mediated block by directing this protein to a high molecular mass complex. METHODOLOGY/PRINCIPAL FINDINGS: In the present work, we sought to explore further this model. However, we found that effective suppression of A3G by combined RNA interference and expression of HIV1 Vif does not relieve the restrictive phenotype of post-activation resting T cells. We also failed to find a correlation between HIV resistance and the presence of A3G in a low molecular complex in primary T cells. CONCLUSIONS/SIGNIFICANCE: We conclude that A3G is unlikely to play a role in the HIV restrictive phenotype of quiescent T lymphocytes.

  6. Deadenylase depletion protects inherited mRNAs in primordial germ cells

    Science.gov (United States)

    Swartz, S. Zachary; Reich, Adrian M.; Oulhen, Nathalie; Raz, Tal; Milos, Patrice M.; Campanale, Joseph P.; Hamdoun, Amro; Wessel, Gary M.

    2014-01-01

    A crucial event in animal development is the specification of primordial germ cells (PGCs), which become the stem cells that create sperm and eggs. How PGCs are created provides a valuable paradigm for understanding stem cells in general. We find that the PGCs of the sea urchin Strongylocentrotus purpuratus exhibit broad transcriptional repression, yet enrichment for a set of inherited mRNAs. Enrichment of several germline determinants in the PGCs requires the RNA-binding protein Nanos to target the transcript that encodes CNOT6, a deadenylase, for degradation in the PGCs, thereby creating a stable environment for RNA. Misexpression of CNOT6 in the PGCs results in their failure to retain Seawi transcripts and Vasa protein. Conversely, broad knockdown of CNOT6 expands the domain of Seawi RNA as well as exogenous reporters. Thus, Nanos-dependent spatially restricted CNOT6 differential expression is used to selectively localize germline RNAs to the PGCs. Our findings support a ‘time capsule’ model of germline determination, whereby the PGCs are insulated from differentiation by retaining the molecular characteristics of the totipotent egg and early embryo. PMID:25100654

  7. Association of radiotherapy with preferential depletion of luminal epithelial cells in a BRCA1 mutation carrier

    Directory of Open Access Journals (Sweden)

    Chiang Huai-Chin

    2012-10-01

    Full Text Available Abstract Radiation therapy (RT after breast conservation therapy has recently been linked with significant reduction in risk of ipsilateral breast cancer among BRCA1 mutation carriers. However, the exact mechanism by which RT reduces incidence of BRCA1-associated cancer remains unclear. Here we studied fresh breast tissue from a BRCA1 mutation carrier who was initially treated with a lumpectomy and RT for a unilateral cancer and two years later chose a prophylactic bilateral mastectomy while remaining cancer-free. Flow cytometry analysis demonstrated a strikingly lower luminal cell population in the irradiated breast as compared to the non-irradiated breast, which was confirmed by immunohistochemistry. Furthermore, the irradiated breast tissue exhibited very low progenitor cell activity in vitro. Given the emerging evidence that BRCA1 tumors originate from luminal progenitor cells, our observations suggest that preferential and long-lasting elimination of luminal ductal epithelium may partly underlie the mechanism of RT-associated reduction in recurrence of BRCA1-associated cancer.

  8. The Vitamin D Analog, MART-10, Attenuates Triple Negative Breast Cancer Cells Metastatic Potential

    Directory of Open Access Journals (Sweden)

    Kun-Chun Chiang

    2016-04-01

    Full Text Available Regarding breast cancer treatment, triple negative breast cancer (TNBC is a difficult issue. Most TNBC patients die of cancer metastasis. Thus, to develop a new regimen to attenuate TNBC metastatic potential is urgently needed. MART-10 (19-nor-2α-(3-hydroxypropyl-1α,25(OH2D3, the newly-synthesized 1α,25(OH2D3 analog, has been shown to be much more potent in cancer growth inhibition than 1α,25(OH2D3 and be active in vivo without inducing obvious side effect. In this study, we demonstrated that both 1α,25(OH2D3 and MART-10 could effectively repress TNBC cells migration and invasion with MART-10 more effective. MART-10 and 1α,25(OH2D3 induced cadherin switching (upregulation of E-cadherin and downregulation of N-cadherin and downregulated P-cadherin expression in MDA-MB-231 cells. The EMT(epithelial mesenchymal transition process in MDA-MB-231 cells was repressed by MART-10 through inhibiting Zeb1, Zeb2, Slug, and Twist expression. LCN2, one kind of breast cancer metastasis stimulator, was also found for the first time to be repressed by 1α,25(OH2D3 and MART-10 in breast cancer cells. Matrix metalloproteinase-9 (MMP-9 activity was also downregulated by MART-10. Furthermore, F-actin synthesis in MDA-MB-231 cells was attenuated as exposure to 1α,25(OH2D3 and MART-10. Based on our result, we conclude that MART-10 could effectively inhibit TNBC cells metastatic potential and deserves further investigation as a new regimen to treat TNBC.

  9. Inhibition of calpain attenuates encephalitogenicity of MBP-specific T cells

    Science.gov (United States)

    Guyton, Mary K.; Das, Arabinda; Inoue, Jun; Azuma, Mitsuyoshi; Ray, Swapan K.; Brahmachari, Saurav; Banik, Naren L.

    2009-01-01

    Multiple sclerosis (MS) is a T cell-mediated autoimmune disease of the CNS, possessing both immune and neurodegenerative events that lead to disability. Adoptive transfer (AT) of myelin basic protein (MBP)-specific T cells into naïve female SJL/J mice results in a relapsing-remitting (RR) form of experimental autoimmune encephalomyelitis (EAE). Blocking the mechanisms by which MBP-specific T cells are activated before AT may help characterize the immune arm of MS and offer novel targets for therapy. One such target is calpain, which is involved in activation of T cells, migration of immune cells into the CNS, degradation of axonal and myelin proteins, and neuronal apoptosis. Thus, the hypothesis that inhibiting calpain in MBP-specific T cells would diminish their encephalitogenicity in RR-EAE mice was tested. Incubating MBP-specific T cells with the calpain inhibitor SJA6017 before AT markedly suppressed the ability of these T cells to induce clinical symptoms of RR-EAE. These reductions correlated with decreases in demyelination, inflammation, axonal damage, and loss of oligodendrocytes and neurons. Also, calpain:calpastatin ratio, production of tBid, and Bax:Bcl-2 ratio, and activities of calpain and caspases, and internucleosomal DNA fragmentation were attenuated. Thus, these data suggest calpain as a promising target for treating EAE and MS. PMID:19627443

  10. Nasal lavage natural killer cell function is suppressed in smokers after live attenuated influenza virus

    Directory of Open Access Journals (Sweden)

    Zhou Haibo

    2011-08-01

    Full Text Available Abstract Background Modified function of immune cells in nasal secretions may play a role in the enhanced susceptibility to respiratory viruses that is seen in smokers. Innate immune cells in nasal secretions have largely been characterized by cellular differentials using morphologic criteria alone, which have successfully identified neutrophils as a significant cell population within nasal lavage fluid (NLF cells. However, flow cytometry may be a superior method to fully characterize NLF immune cells. We therefore characterized immune cells in NLF by flow cytometry, determined the effects of live attenuated influenza virus (LAIV on NLF and peripheral blood immune cells, and compared responses in samples obtained from smokers and nonsmokers. Methods In a prospective observational study, we characterized immune cells in NLF of nonsmokers at baseline using flow cytometry and immunohistochemistry. Nonsmokers and smokers were inoculated with LAIV on day 0 and serial nasal lavages were collected on days 1-4 and day 9 post-LAIV. LAIV-induced changes of NLF cells were characterized using flow cytometry. Cell-free NLF was analyzed for immune mediators by bioassay. Peripheral blood natural killer (NK cells from nonsmokers and smokers at baseline were stimulated in vitro with LAIV followed by flow cytometric and mediator analyses. Results CD45(+CD56(-CD16(+ neutrophils and CD45(+CD56(+ NK cells comprised median 4.62% (range 0.33-14.52 and 23.27% (18.29-33.97, respectively, of non-squamous NLF cells in nonsmokers at baseline. LAIV did not induce changes in total NK cell or neutrophil percentages in either nonsmokers or smokers. Following LAIV inoculation, CD16(+ NK cell percentages and granzyme B levels increased in nonsmokers, and these effects were suppressed in smokers. LAIV inoculation enhanced expression of activating receptor NKG2D and chemokine receptor CXCR3 on peripheral blood NK cells from both nonsmokers and smokers in vitro but did not induce

  11. Bone Marrow-Derived c-kit+ Cells Attenuate Neonatal Hyperoxia-Induced Lung Injury

    Science.gov (United States)

    Ramachandran, Shalini; Suguihara, Cleide; Drummond, Shelley; Chatzistergos, Konstantinos; Klim, Jammie; Torres, Eneida; Huang, Jian; Hehre, Dorothy; Rodrigues, Claudia O.; McNiece, Ian K.; Hare, Joshua M.; Young, Karen C.

    2016-01-01

    Recent studies suggest that bone marrow (BM)-derived stem cells have therapeutic efficacy in neonatal hyperoxia-induced lung injury (HILI). c-kit, a tyrosine kinase receptor that regulates angiogenesis, is expressed on several populations of BM-derived cells. Preterm infants exposed to hyperoxia have decreased lung angiogenesis. Here we tested the hypothesis that administration of BM-derived c-kit+ cells would improve angiogenesis in neonatal rats with HILI. To determine whether intratracheal (IT) administration of BM-derived c-kit+ cells attenuates neonatal HILI, rat pups exposed to either normobaric normoxia (21% O2) or hyperoxia (90% O2) from postnatal day (P) 2 to P15 were randomly assigned to receive either IT BM-derived green fluorescent protein (GFP)+ c-kit− cells (PL) or BM-derived GFP+ c-kit+ cells on P8. The effect of cell therapy on lung angiogenesis, alveolarization, pulmonary hypertension, vascular remodeling, cell proliferation, and apoptosis was determined at P15. Cell engraftment was determined by GFP immunostaining. Compared to PL, the IT administration of BM-derived c-kit+ cells to neonatal rodents with HILI improved alveolarization as evidenced by increased lung septation and decreased mean linear intercept. This was accompanied by an increase in lung vascular density, a decrease in lung apoptosis, and an increase in the secretion of proangiogenic factors. There was no difference in pulmonary vascular remodeling or the degree of pulmonary hypertension. Confocal microscopy demonstrated that 1% of total lung cells were GFP+ cells. IT administration of BM-derived c-kit+ cells improves lung alveolarization and angiogenesis in neonatal HILI, and this may be secondary to an improvement in the lung angiogenic milieu. PMID:23759597

  12. Allogeneic Transplantation of Müller-Derived Retinal Ganglion Cells Improves Retinal Function in a Feline Model of Ganglion Cell Depletion.

    Science.gov (United States)

    Becker, Silke; Eastlake, Karen; Jayaram, Hari; Jones, Megan F; Brown, Robert A; McLellan, Gillian J; Charteris, David G; Khaw, Peng T; Limb, G Astrid

    2016-02-01

    Human Müller glia with stem cell characteristics (hMGSCs) have been shown to improve retinal function upon transplantation into rat models of retinal ganglion cell (RGC) depletion. However, their translational potential may depend upon successful engraftment and improvement of retinal function in experimental models with anatomical and functional features resembling those of the human eye. We investigated the effect of allogeneic transplantation of feline Müller glia with the ability to differentiate into cells expressing RGC markers, following ablation of RGCs by N-methyl-d-aspartate (NMDA). Unlike previous observations in the rat, transplantation of hMGSC-derived RGCs into the feline vitreous formed aggregates and elicited a severe inflammatory response without improving visual function. In contrast, allogeneic transplantation of feline MGSC (fMGSC)-derived RGCs into the vitrectomized eye improved the scotopic threshold response (STR) of the electroretinogram (ERG). Despite causing functional improvement, the cells did not attach onto the retina and formed aggregates on peripheral vitreous remnants, suggesting that vitreous may constitute a barrier for cell attachment onto the retina. This was confirmed by observations that cellular scaffolds of compressed collagen and enriched preparations of fMGSC-derived RGCs facilitated cell attachment. Although cells did not migrate into the RGC layer or the optic nerve, they significantly improved the STR and the photopic negative response of the ERG, indicative of increased RGC function. These results suggest that MGSCs have a neuroprotective ability that promotes partial recovery of impaired RGC function and indicate that cell attachment onto the retina may be necessary for transplanted cells to confer neuroprotection to the retina. Significance: Müller glia with stem cell characteristics are present in the adult human retina, but they do not have regenerative ability. These cells, however, have potential for

  13. Ab initio based Monte Carlo studies of Cu-depleted CIS phases for solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Ludwig, Christian; Gruhn, Thomas; Felser, Claudia [Institut fuer Anorganische and Analytische Chemie, Johannes Gutenberg-Universitaet Mainz (Germany); Windeln, Johannes [IBM Mainz (Germany)

    2011-07-01

    Thin film solar cells with a CuInSe{sub 2} (CIS) absorber layer have an increasing share of the solar cell market because of their low production costs and the high efficiency. One interesting aspect of CIS is the inherent resilience to defects and composition fluctuations. Beside the stable CuInSe{sub 2} phase, there are various Cu-poor phases along the Cu{sub 2}Se-In{sub 2}Se{sub 3} tie line, including the CuIn{sub 3}Se{sub 5} and the CuIn{sub 5}Se{sub 8} phase. We have used ab initio calculations of Cu-poor CIS configurations to make a cluster expansion of the configurational energy. In the configurations, Cu atoms, In atoms, and vacancies are distributed over the Cu and In sites of a CIS cell with fixed Se atoms. With the resulting energy expression, CuIn{sub 3}Se{sub 5} and CuIn{sub 5}Se{sub 8} systems have been studied in the canonical ensemble. By analyzing the free energy landscape the transition temperature between a low-temperature ordered and a high-temperature disordered CuIn{sub 5}Se{sub 8} phase has been determined. Furthermore, grandcanonical ensemble simulations have been carried out, which provide the equilibrium Cu and In concentrations as a function of the chemical potentials {mu}{sub Cu} and {mu}{sub In}. Plateau regions for the CuInSe{sub 2} and the CuIn{sub 5}Se{sub 8} phases have been found and analyzed for different temperatures.

  14. Atracurium Besylate and other neuromuscular blocking agents promote astroglial differentiation and deplete glioblastoma stem cells

    Science.gov (United States)

    Spina, Raffaella; Voss, Dillon M.; Asnaghi, Laura; Sloan, Andrew; Bar, Eli E.

    2016-01-01

    Glioblastoma multiforme (GBM) are the most common primary malignant brain tumor in adults, with a median survival of about one year. This poor prognosis is attributed primarily to therapeutic resistance and tumor recurrence after surgical removal, with the root cause suggested to be found in glioblastoma stem cells (GSCs). Using glial fibrillary acidic protein (GFAP) as a reporter of astrocytic differentiation, we isolated multiple clones from three independent GSC lines which express GFAP in a remarkably stable fashion. We next show that elevated expression of GFAP is associated with reduced clonogenicity in vitro and tumorigenicity in vivo. Utilizing this in vitro cell-based differentiation reporter system we screened chemical libraries and identified the non-depolarizing neuromuscular blocker (NNMB), Atracurium Besylate, as a small molecule which effectively induces astroglial but not neuronal differentiation of GSCs. Functionally, Atracurium Besylate treatment significantly inhibited the clonogenic capacity of several independent patient-derived GSC neurosphere lines, a phenomenon which was largely irreversible. A second NNMB, Vecuronium, also induced GSC astrocytic differentiation while Dimethylphenylpiperazinium (DMPP), a nicotinic acetylcholine receptor (nAChR) agonist, significantly blocked Atracurium Besylate pro-differentiation activity. To investigate the clinical importance of nAChRs in gliomas, we examined clinical outcomes and found that glioma patients with tumors overexpressing CHRNA1 or CHRNA9 (encoding for the AChR-α1 or AChR-α9) exhibit significant shorter overall survival. Finally, we found that ex-vivo pre-treatment of GSCs, expressing CHRNA1 and CHRNA9, with Atracurium Besylate significantly increased the survival of mice xenotransplanted with these cells, therefore suggesting that tumor initiating subpopulations have been reduced. PMID:26575950

  15. Increased basolateral sorting of carcinoembryonic antigen in a polarized colon carcinoma cell line after cholesterol depletion-Implications for treatment of inflammatory bowel disease

    Institute of Scientific and Technical Information of China (English)

    Robert Ehehalt; Markus Krautter; Martin Zorn; Richard Sparla; Joachim Fūllekrug; Hasan Kulaksiz; Wolfgang Stremmel

    2008-01-01

    AIM:To investigate a possible increase of basolateral expression of carcinoembryonic antigen(CEA)by interfering with the apical transport machinery,we studied the effect of cholesterol depletion on CEA sorting and secretion.METHODS:Cholesterol depletion was performed in polarized Caco-2 cells using Iovastatin and methyl-βcyclodextrin.RESULTS:We show that CEA is predominantly expressed and secreted at the apical surface.Reduction of the cholesterol level of the cell by 40%-50% with Iovastatin and methyl-β-cyclodextrin led to a significant change of the apical-to-basolateral transport ratio towards the basolateral membrane.CONCLUSION:As basolateral expression of CEA has been suggested to have anti-inflamatory properties,Cholesterol depletion of enterocytes might be a potential approach to influence the course of inflammatory bowel disease.

  16. Efficacy of a rituximab regimen based on B cell depletion in thrombotic thrombocytopenic purpura with suboptimal response to standard treatment: Results of a phase II, multicenter noncomparative study.

    Science.gov (United States)

    Benhamou, Ygal; Paintaud, Gilles; Azoulay, Elie; Poullin, Pascale; Galicier, Lionel; Desvignes, Céline; Baudel, Jean-Luc; Peltier, Julie; Mira, Jean-Paul; Pène, Frédéric; Presne, Claire; Saheb, Samir; Deligny, Christophe; Rousseau, Alexandra; Féger, Frédéric; Veyradier, Agnès; Coppo, Paul

    2016-12-01

    The standard four-rituximab infusions treatment in acquired thrombotic thrombocytopenic purpura (TTP) remains empirical. Peripheral B cell depletion is correlated with the decrease in serum concentrations of anti-ADAMTS13 and associated with clinical response. To assess the efficacy of a rituximab regimen based on B cell depletion, 24 TTP patients were enrolled in this prospective multicentre single arm phase II study and then compared to patients from a previous study. Patients with a suboptimal response to a plasma exchange-based regimen received two infusions of rituximab 375 mg m(-2) within 4 days, and a third dose at day +15 of the first infusion if peripheral B cells were still detectable. Primary endpoint was the assessment of the time required to platelet count recovery from the first plasma exchange. Three patients died after the first rituximab administration. In the remaining patients, the B cell-driven treatment hastened remission and ADAMTS13 activity recovery as a result of rapid anti-ADAMTS13 depletion in a similar manner to the standard four-rituximab infusions schedule. The 1-year relapse-free survival was also comparable between both groups. A rituximab regimen based on B cell depletion is feasible and provides comparable results than with the four-rituximab infusions schedule. This regimen could represent a new standard in TTP. This trial was registered at www.clinicaltrials.gov (NCT00907751). Am. J. Hematol. 91:1246-1251, 2016. © 2016 Wiley Periodicals, Inc.

  17. Telomerase Cajal body protein 1 depletion inhibits telomerase trafficking to telomeres and induces G1 cell cycle arrest in A549 cells.

    Science.gov (United States)

    Yuan, Ping; Wang, Zhitian; Lv, Wang; Pan, Hui; Yang, Yunhai; Yuan, Xiaoshuai; Hu, Jian

    2014-09-01

    Telomerase Cajal body protein 1 (TCAB1) is a telomerase holoenzyme, which is markedly enriched in Cajal bodies (CBs) and facilitates the recruitment of telomerase to CBs in the S phase of the cell cycle. This recruitment is dependent on TCAB1 binding to a telomerase RNA component. The majority of cancer cells are able to grow indefinitely due to telomerase and its mechanism of trafficking to telomeres. In the present study, a certain level of TCAB1 expression in A549 human lung cells was identified and TCAB1 knockdown exhibited a potent antiproliferative effect on these cells, which was coupled with a decrease in the cell density and activity of the cellular enzymes. In addition, TCAB1-depletion was demonstrated to inhibit telomerase trafficking to telomeres in the A549 cells, leading to subsequent G1 cell cycle arrest without inducing apoptotic cell death. Overall, these observations indicated that TCAB1 may be essential for A549 cell proliferation and cell cycle regulation, and may be a potential candidate for the development of a therapeutic target for lung adenocarcinomas.

  18. Rutin inhibits proliferation, attenuates superoxide production and decreases adhesion and migration of human cancerous cells.

    Science.gov (United States)

    Ben Sghaier, Mohamed; Pagano, Alessandra; Mousslim, Mohamed; Ammari, Youssef; Kovacic, Hervé; Luis, José

    2016-12-01

    Lung and colorectal cancer are the principal causes of death in the world. Rutin, an active flavonoid compound, is known for possessing a wide range of biological activities. In this study, we examined the effect of rutin on the viability, superoxide anion production, adhesion and migration of human lung (A549) and colon (HT29 and Caco-2) cancer cell lines. In order to control the harmlessness of the tested concentrations of rutin, the viability of cancer cell lines was assessed using a 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. ROS generation was measured by lucigenin chemiluminescence detecting superoxide ions. To investigate the effect of rutin on the behavior of human lung and colon cancer cell lines, we performed adhesion assays, using various purified extracellular matrix (ECM) proteins. Finally, in vitro cell migration assays were explored using modified Boyden chambers. The viability of cancerous cells was inhibited by rutin. It also significantly attenuated the superoxide production in HT29 cells. In addition, rutin affected adhesion and migration of A549 and HT29 cell. These findings indicate that rutin, a natural molecule, might have potential as anticancer agent against lung and colorectal carcinogenesis.

  19. Combination Effect of Regulatory T-Cell Depletion and Ionizing Radiation in Mouse Models of Lung and Colon Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Son, Cheol-Hun [Dongnam Institute of Radiological and Medical Sciences, Busan (Korea, Republic of); Department of Biochemistry, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Bae, Jae-Ho [Department of Biochemistry, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Shin, Dong-Yeok; Lee, Hong-Rae; Jo, Wol-Soon; Yang, Kwangmo [Dongnam Institute of Radiological and Medical Sciences, Busan (Korea, Republic of); Park, You-Soo, E-mail: biotek01@hanmail.net [Dongnam Institute of Radiological and Medical Sciences, Busan (Korea, Republic of)

    2015-06-01

    Purpose: To investigate the potential of low-dose cyclophosphamide (LD-CTX) and anti-CD25 antibody to prevent activation of regulatory T cells (Tregs) during radiation therapy. Methods and Materials: We used LD-CTX and anti-CD25 monoclonal antibody as a means to inhibit Tregs and improve the therapeutic effect of radiation in a mouse model of lung and colon cancer. Mice were irradiated on the tumor mass of the right leg and treated with LD-CTX and anti-CD25 antibody once per week for 3 weeks. Results: Combined treatment of LD-CTX or anti-CD25 antibody with radiation significantly decreased Tregs in the spleen and tumor compared with control and irradiation only in both lung and colon cancer. Combinatorial treatments resulted in a significant increase in the effector T cells, longer survival rate, and suppressed irradiated and distal nonirradiated tumor growth. Specifically, the combinatorial treatment of LD-CTX with radiation resulted in outstanding regression of local and distant tumors in colon cancer, and almost all mice in this group survived until the end of the study. Conclusions: Our results suggest that Treg depletion strategies may enhance radiation-mediated antitumor immunity and further improve outcomes after radiation therapy.

  20. Coenzyme depletion by members of the aerolysin family of pore-forming toxins leads to diminished ATP levels and cell death.

    Science.gov (United States)

    Fennessey, Christine M; Ivie, Susan E; McClain, Mark S

    2012-08-01

    Recent studies demonstrated that a variety of bacterial pore-forming toxins induce cell death through a process of programmed necrosis characterized by the rapid depletion of cellular ATP. However, events leading to the necrosis and depletion of ATP are not thoroughly understood. We demonstrate that ATP-depletion induced by two pore-forming toxins, the Clostridium perfringens epsilon-toxin and the Aeromonas hydrophila aerolysin toxin, is associated with decreased mitochondrial membrane potential and opening of the mitochondrial permeability transition pore. To gain further insight into the toxin-induced metabolic changes contributing to necrosis and depletion of ATP, we analyzed the biochemical profiles of 251 distinct compounds by GC/MS or LC/MS/MS following exposure of a human kidney cell line to the epsilon-toxin. As expected, numerous biochemicals were seen to increase or decrease in response to epsilon-toxin. However, the pattern of these changes was consistent with the toxin-induced disruption of major energy-producing pathways in the cell including disruptions to the beta-oxidation of lipids. In particular, treatment with epsilon-toxin led to decreased levels of key coenzymes required for energy production including carnitine, NAD (and NADH), and coenzyme A. Independent biochemical assays confirmed that epsilon-toxin and aerolysin induced the rapid decrease of these coenzymes or their synthetic precursors. Incubation of cells with NADH or carnitine-enriched medium helped protect cells from toxin-induced ATP depletion and cell death. Collectively, these results demonstrate that members of the aerolysin family of pore-forming toxins lead to decreased levels of essential coenzymes required for energy production. The resulting loss of energy substrates is expected to contribute to dissipation of the mitochondrial membrane potential, opening of the mitochondrial permeability transition pore, and ultimately cell death.

  1. Hyperoside attenuates hydrogen peroxide-induced L02 cell damage via MAPK-dependent Keap{sub 1}-Nrf{sub 2}-ARE signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Xing, Hai-Yan; Liu, Yao; Chen, Jian-Hong; Sun, Feng-Jun; Shi, Hui-Qing [Department of Pharmacy, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Xia, Pei-Yuan, E-mail: py_xia@yahoo.com.cn [Department of Pharmacy, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China)

    2011-07-15

    Highlights: {yields} Hyperoside attenuated H{sub 2}O{sub 2}-induced L02 cell damage. {yields} Hyperoside up-regulated HO-1 expression at both mRNA and protein levels. {yields} Hyperoside activated both Nrf{sub 2} nuclear translocation and gene expression. {yields} Hyperoside may inhibit Keap{sub 1} mRNA translation or protein degradation. {yields} Phosphorylation of ERK and p38 is involved in hyperoside-mediated Nrf{sub 2} activation. -- Abstract: The flavonoid hyperoside has been reported to elicit cytoprotection against oxidative stress partly by increasing the activity of antioxidant enzymes, such as glutathione peroxidase, superoxide dismutase and catalase. However, the cellular and molecular mechanisms underlying this effect remain unclear. Here, hepatic L02 cells exposed to H{sub 2}O{sub 2} (100 {mu}M) were used to demonstrate that hyperoside protected cells by significantly inhibiting overproduction of intracellular ROS, depletion of the mitochondrial membrane potential and leakage of lactate dehydrogenase. Hyperoside further enhanced the cellular antioxidant defense system through increasing the activity of heme oxygenase-1 (HO-1), and by up-regulating HO-1 expression. Meanwhile, real time PCR, western blot and immunofluorescence studies revealed that hyperoside stimulated nuclear translocation of the Nrf{sub 2} transcription factor in a dose-dependent manner, and this effect was significantly suppressed by pharmacological inhibition of the mitogen-activated protein kinases (MAPK) p38 and ERK. Collectively, our data provide the first description of the mechanism underlying hyperoside's ability to attenuate H{sub 2}O{sub 2}-induced cell damage, namely this compound interacts with the MAPK-dependent Keap{sub 1}-Nrf{sub 2}-ARE signaling pathway to up-regulate HO-1 expression and enhance intracellular antioxidant activity.

  2. Depletion of histone N-terminal-acetyltransferase Naa40 induces p53-independent apoptosis in colorectal cancer cells via the mitochondrial pathway.

    Science.gov (United States)

    Pavlou, Demetria; Kirmizis, Antonis

    2016-03-01

    Protein N-terminal acetylation is an abundant post-translational modification in eukaryotes implicated in various fundamental cellular and biochemical processes. This modification is catalysed by evolutionarily conserved N-terminal acetyltransferases (NATs) whose deregulation has been linked to cancer development and thus, are emerging as useful diagnostic and therapeutic targets. Naa40 is a highly selective NAT that acetylates the amino-termini of histones H4 and H2A and acts as a sensor of cell growth in yeast. In the present study, we examine the role of Naa40 in cancer cell survival. We demonstrate that depletion of Naa40 in HCT116 and HT-29 colorectal cancer cells decreases cell survival by enhancing apoptosis, whereas Naa40 reduction in non-cancerous mouse embryonic fibroblasts has no effect on cell viability. Specifically, Naa40 knockdown in colon cancer cells activates the mitochondrial caspase-9-mediated apoptotic cascade. Consistent with this, we show that caspase-9 activation is required for the induced apoptosis because treatment of cells with an irreversible caspase-9 inhibitor impedes apoptosis when Naa40 is depleted. Furthermore, the effect of Naa40-depletion on cell-death is mediated through a p53-independent mechanism since p53-null HCT116 cells still undergo apoptosis upon reduction of the acetyltransferase. Altogether, these findings reveal an anti-apoptotic role for Naa40 and exhibit its potential as a therapeutic target in colorectal cancers.

  3. Dicer1 depletion in male germ cells leads to infertility due to cumulative meiotic and spermiogenic defects.

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    Yannick Romero

    Full Text Available BACKGROUND: Spermatogenesis is a complex biological process that requires a highly specialized control of gene expression. In the past decade, small non-coding RNAs have emerged as critical regulators of gene expression both at the transcriptional and post-transcriptional level. DICER1, an RNAse III endonuclease, is essential for the biogenesis of several classes of small RNAs, including microRNAs (miRNAs and endogenous small interfering RNAs (endo-siRNAs, but is also critical for the degradation of toxic transposable elements. In this study, we investigated to which extent DICER1 is required for germ cell development and the progress of spermatogenesis in mice. PRINCIPAL FINDINGS: We show that the selective ablation of Dicer1 at the early onset of male germ cell development leads to infertility, due to multiple cumulative defects at the meiotic and post-meiotic stages culminating with the absence of functional spermatozoa. Alterations were observed in the first spermatogenic wave and include delayed progression of spermatocytes to prophase I and increased apoptosis, resulting in a reduced number of round spermatids. The transition from round to mature spermatozoa was also severely affected, since the few spermatozoa formed in mutant animals were immobile and misshapen, exhibiting morphological defects of the head and flagellum. We also found evidence that the expression of transposable elements of the SINE family is up-regulated in Dicer1-depleted spermatocytes. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that DICER1 is dispensable for spermatogonial stem cell renewal and mitotic proliferation, but is required for germ cell differentiation through the meiotic and haploid phases of spermatogenesis.

  4. Iron depletion in HCT116 cells diminishes the upregulatory effect of phenethyl isothiocyanate on heme oxygenase-1.

    Science.gov (United States)

    Bolloskis, Michael P; Carvalho, Fabiana P; Loo, George

    2016-04-15

    Some of the health-promoting properties of cruciferous vegetables are thought to be partly attributed to isothiocyanates. These phytochemicals can upregulate the expression of certain cytoprotective stress genes, but it is unknown if a particular nutrient is involved. Herein, the objective was to ascertain if adequate iron is needed for enabling HCT116 cells to optimally express heme oxygenase-1 (HO-1) when induced by phenethyl isothiocyanate (PEITC). PEITC increased HO-1 expression and also nuclear translocation of Nrf2, which is a transcription factor known to activate the HO-1 gene. However, in HCT116 cells that were made iron-deficient by depleting intracellular iron with deferoxamine (DFO), PEITC was less able to increase HO-1 expression and nuclear translocation of Nrf2. These suppressive effects of DFO were overcome by replenishing the iron-deficient cells with the missing iron. To elucidate these findings, it was found that PEITC-induced HO-1 upregulation can be inhibited with thiol antioxidants (glutathione and N-acetylcysteine). Furthermore, NADPH oxidase inhibitors (diphenyleneiodonium and apocynin) and a superoxide scavenger (Tiron) each inhibited PEITC-induced HO-1 upregulation. In doing so, diphenyleneiodonium was the most potent and also inhibited nuclear translocation of redox-sensitive Nrf2. Collectively, the results imply that the HO-1 upregulation by PEITC involves an iron-dependent, oxidant signaling pathway. Therefore, it is concluded that ample iron is required to enable PEITC to fully upregulate HO-1 expression in HCT116 cells. As such, it is conceivable that iron-deficient individuals may not reap the full health benefits of eating PEITC-containing cruciferous vegetables that via HO-1 may help protect against multiple chronic diseases.

  5. Thyroiditis in T cell-depleted rats: suppression of the autoallergic response by reconstitution with normal lymphoid cells.

    Science.gov (United States)

    Penhale, W J; Irvine, W J; Inglis, J R; Farmer, A

    1976-07-01

    Qualititive, quantitative and functional differences were found in lymphoid cells of female thymectomized and irradiated (Tx-X) PVG/c strain rats as compared to normal females of the same strain. Tx-X rats were lymphopenic and had reduced numbers of cells within spleen and cervical lymph nodes, depressed transformation responses of peripheral blood lymphocytes to PHA and lower percentage killing of their spleen cells by anti-T-cell serum and complement. There was an increased percentage of immunoglobulin-bearing cells in the lymph nodes. Reconstitution of Tx-X rats by the intravenous route using syngeneic lymph node cells, spleen cells or thymocytes abrogated the autoimmune responses to thyroid components generally observed in this state. Lymph node and spleen cells, but not thymocytes, also prevented thyroid changes when given intraperitoneally. In contrast, bone marrow cells appeared to give enhanced responses. Quntitative studies showed that the relative proportions of the suppressor or autoregulatory cells in various lymphoid tissues were lymph node greater than spleen greater than thymus. Complete abrogation of the autoimmune responses was possible only when cells were administered within a short time of final dose of irradiation and moderate thyroid change was again seen if transfer was delayed for 14 days post-irradiation. At 28 days reconstitution had no influence on the development of the autoimmune responses. Preliminary characterization studies using an anti-T-cell serum and fractionation of lymph node cells on a linear Ficoll gradient suggested that autoregulatory cell is a large T cell.

  6. JMJD2A attenuation affects cell cycle and tumourigenic inflammatory gene regulation in lipopolysaccharide stimulated neuroectodermal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Das, Amitabh, E-mail: amitabhdas.kn@gmail.com [Department of Bionanotechnology, Hanyang University, Seoul 133-791 (Korea, Republic of); Chai, Jin Choul, E-mail: jincchai@gmail.com [Department of Molecular and Life Science, Hanyang University, 1271 Sa 3-dong, Ansan 426-791, Gyeonggi-do (Korea, Republic of); Jung, Kyoung Hwa, E-mail: khjung2@gmail.com [Department of Molecular and Life Science, Hanyang University, 1271 Sa 3-dong, Ansan 426-791, Gyeonggi-do (Korea, Republic of); Das, Nando Dulal, E-mail: nando.hu@gmail.com [Clinical Research Centre, Inha University School of Medicine, Incheon 400-711 (Korea, Republic of); Kang, Sung Chul, E-mail: gujiju11@gmail.com [Department of Molecular and Life Science, Hanyang University, 1271 Sa 3-dong, Ansan 426-791, Gyeonggi-do (Korea, Republic of); Lee, Young Seek, E-mail: yslee@hanyang.ac.kr [Department of Molecular and Life Science, Hanyang University, 1271 Sa 3-dong, Ansan 426-791, Gyeonggi-do (Korea, Republic of); Seo, Hyemyung, E-mail: hseo@hanyang.ac.kr [Department of Molecular and Life Science, Hanyang University, 1271 Sa 3-dong, Ansan 426-791, Gyeonggi-do (Korea, Republic of); Chai, Young Gyu, E-mail: ygchai@hanyang.ac.kr [Department of Bionanotechnology, Hanyang University, Seoul 133-791 (Korea, Republic of); Department of Molecular and Life Science, Hanyang University, 1271 Sa 3-dong, Ansan 426-791, Gyeonggi-do (Korea, Republic of)

    2014-11-01

    JMJD2A is a lysine trimethyl-specific histone demethylase that is highly expressed in a variety of tumours. The role of JMJD2A in tumour progression remains unclear. The objectives of this study were to identify JMJD2A-regulated genes and understand the function of JMJD2A in p53-null neuroectodermal stem cells (p53{sup −/−} NE-4Cs). We determined the effect of LPS as a model of inflammation in p53{sup −/−} NE-4Cs and investigated whether the epigenetic modifier JMJD2A alter the expression of tumourigenic inflammatory genes. Global gene expression was measured in JMJD2A knockdown (kd) p53{sup −/−} NE-4Cs and in LPS-stimulated JMJD2A-kd p53{sup −/−} NE-4C cells. JMJD2A attenuation significantly down-regulated genes were Cdca2, Ccnd2, Ccnd1, Crebbp, IL6rα, and Stat3 related with cell cycle, proliferation, and inflammatory-disease responses. Importantly, some tumour-suppressor genes including Dapk3, Timp2 and TFPI were significantly up-regulated but were not affected by silencing of the JMJD2B. Furthermore, we confirmed the attenuation of JMJD2A also down-regulated Cdca2, Ccnd2, Crebbp, and Rest in primary NSCs isolated from the forebrains of E15 embryos of C57/BL6J mice with effective p53 inhibitor pifithrin-α (PFT-α). Transcription factor (TF) motif analysis revealed known binding patterns for CDC5, MYC, and CREB, as well as three novel motifs in JMJD2A-regulated genes. IPA established molecular networks. The molecular network signatures and functional gene-expression profiling data from this study warrants further investigation as an effective therapeutic target, and studies to elucidate the molecular mechanism of JMJD2A-kd-dependent effects in neuroectodermal stem cells should be performed. - Highlights: • Significant up-regulation of epigenetic modifier JMJD2A mRNA upon LPS treatment. • Inhibition of JMJD2A attenuated key inflammatory and tumourigenic genes. • Establishing IPA based functional genomics in JMJD2A-attenuated p53{sup

  7. Choline Phospholipid Metabolites of Human Vascular Endothelial Cells Altered by Cyclooxygenase Inhibition, Growth Factor Depletion, and Paracrine Factors Secreted by Cancer Cells

    Directory of Open Access Journals (Sweden)

    Noriko Mori

    2003-04-01

    Full Text Available Magnetic resonance studies have previously shown that solid tumors and cancer cells in culture typically exhibit high phosphocholine and total choline. Treatment of cancer cells with the anti-inflammatory agent, indomethacin (INDO, reverted the phenotype of choline phospholipid metabolites in cancer cells towards a less malignant phenotype. Since endothelial cells form a key component of tumor vasculature, in this study, we used MR spectroscopy to characterize the phenotype of choline phospholipid metabolites in human umbilical vein endothelial cells (HUVECs. We determined the effect of growth factors, the anti-inflammatory agent INDO, and conditioned media obtained from a malignant cell line, on choline phospholipid metabolites. Growth factor depletion or treatment with INDO induced similar changes in the choline phospholipid metabolites of HUVECs. Treatment with conditioned medium obtained from MDA-MB-231 cancer cells induced changes similar to the presence of growth factor supplements. These results suggest that cancer cells secrete growth factors and/or other molecules that influence the choline phospholipid metabolism of HUVECs. The ability of INDO to alter choline phospholipid metabolism in the presence of growth factor supplements suggests that the inflammatory response pathways of HUVECs may play a role in cancer cell-HUVEC interaction and in the response of HUVECs to growth factors.

  8. Gis1 and Rph1 regulate glycerol and acetate metabolism in glucose depleted yeast cells.

    Directory of Open Access Journals (Sweden)

    Jakub Orzechowski Westholm

    Full Text Available Aging in organisms as diverse as yeast, nematodes, and mammals is delayed by caloric restriction, an effect mediated by the nutrient sensing TOR, RAS/cAMP, and AKT/Sch9 pathways. The transcription factor Gis1 functions downstream of these pathways in extending the lifespan of nutrient restricted yeast cells, but the mechanisms involved are still poorly understood. We have used gene expression microarrays to study the targets of Gis1 and the related protein Rph1 in different growth phases. Our results show that Gis1 and Rph1 act both as repressors and activators, on overlapping sets of genes as well as on distinct targets. Interestingly, both the activities and the target specificities of Gis1 and Rph1 depend on the growth phase. Thus, both proteins are associated with repression during exponential growth, targeting genes with STRE or PDS motifs in their promoters. After the diauxic shift, both become involved in activation, with Gis1 acting primarily on genes with PDS motifs, and Rph1 on genes with STRE motifs. Significantly, Gis1 and Rph1 control a number of genes involved in acetate and glycerol formation, metabolites that have been implicated in aging. Furthermore, several genes involved in acetyl-CoA metabolism are downregulated by Gis1.

  9. Exosomes released by granulocytic myeloid-derived suppressor cells attenuate DSS-induced colitis in mice.

    Science.gov (United States)

    Wang, Yungang; Tian, Jie; Tang, Xinyi; Rui, Ke; Tian, Xinyu; Ma, Jie; Ma, Bin; Xu, Huaxi; Lu, Liwei; Wang, Shengjun

    2016-03-29

    Myeloid-derived suppressor cells (MDSC) have been described in inflammatory bowel disease (IBD), but their role in the disease remains controversial. We sought to define the effect of granulocytic MDSC-derived exosomes (G-MDSC exo) in dextran sulphate sodium (DSS)-induced murine colitis. G-MDSC exo-treated mice showed greater resistance to colitis, as reflected by lower disease activity index, decreased inflammatory cell infiltration damage. There was a decrease in the proportion of Th1 cells and an increase in the proportion of regulatory T cells (Tregs) in mesenteric lymph nodes (MLNs) from G-MDSC exo-treated colitis mice. Moreover, lower serum levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α were detected in G-MDSC exo-treated colitis mice. Interestingly, inhibition of arginase (Arg)-1 activity in G-MDSC exo partially abrogated the spontaneous improvement of colitis. In addition, G-MDSC exo could suppress CD4+ T cell proliferation and IFN-γ secretion in vitro and inhibit the delayed-type hypersensitivity (DTH) response, and these abilities were associated with Arg-1 activity. Moreover, G-MDSC exo promoted the expansion of Tregs in vitro. Taken together, these results suggest that G-MDSC exo attenuate DSS-induced colitis through inhibiting Th1 cells proliferation and promoting Tregs expansion.

  10. Myeloid cell-derived HIF attenuates inflammation in UUO-induced kidney injury

    Science.gov (United States)

    Kobayashi, Hanako; Gilbert, Victoria; Liu, Qingdu; Kapitsinou, Pinelopi P.; Unger, Travis L.; Rha, Jennifer; Rivella, Stefano; Schlöndorff, Detlef; Haase, Volker H.

    2012-01-01

    Renal fibrosis and inflammation are associated with hypoxia, and tissue pO2 plays a central role in modulating the progression of chronic kidney disease. Key mediators of cellular adaptation to hypoxia are hypoxia-inducible factor (HIF)-1 and -2. In the kidney they are expressed in a cell type-specific manner; to what degree activation of each homolog modulates renal fibrogenesis and inflammation has not been established. To address this issue, we used Cre-loxP recombination to activate or to delete both Hif-1 and Hif-2 either globally or cell type-specifically in myeloid cells. Global activation of Hif suppressed inflammation and fibrogenesis in mice subjected to unilateral ureteral obstruction, while activation of Hif in myeloid cells suppressed inflammation only. Suppression of inflammatory cell infiltration was associated with down-regulation of CC chemokine receptors in renal macrophages. Conversely, global deletion or myeloid-specific inactivation of Hif promoted inflammation. Furthermore, prolonged hypoxia suppressed the expression of multiple inflammatory molecules in non-injured kidneys. Collectively, we provide experimental evidence that hypoxia and/or myeloid cell-specific HIF activation attenuates renal inflammation associated with chronic kidney injury. PMID:22490864

  11. Comparison of different busulfan analogues for depletion of hematopoietic stem cells and promotion of donor-type chimerism in murine bone marrow transplant recipients

    NARCIS (Netherlands)

    G.R. Westerhof; R.E. Ploemacher (Robert); A. Boudewijn (Adrie); I. Blokland (Irene); J.H. Dillingh; A.T. McGown; J.A. Hadfield; M.J. Dawson; J.D. Down (Julian)

    2000-01-01

    textabstractBusulfan (1,4-butanediol dimethanesulfonate, BU) is relatively unique among other standard chemotherapy compounds in its ability to deplete noncycling primitive stem cells in the host and consequently to allow for high levels of long-term, donor-type engraft

  12. Attenuation of eph receptor kinase activation in cancer cells by coexpressed ephrin ligands.

    Directory of Open Access Journals (Sweden)

    Giulia Falivelli

    Full Text Available The Eph receptor tyrosine kinases mediate juxtacrine signals by interacting "in trans" with ligands anchored to the surface of neighboring cells via a GPI-anchor (ephrin-As or a transmembrane segment (ephrin-Bs, which leads to receptor clustering and increased kinase activity. Additionally, soluble forms of the ephrin-A ligands released from the cell surface by matrix metalloproteases can also activate EphA receptor signaling. Besides these trans interactions, recent studies have revealed that Eph receptors and ephrins coexpressed in neurons can also engage in lateral "cis" associations that attenuate receptor activation by ephrins in trans with critical functional consequences. Despite the importance of the Eph/ephrin system in tumorigenesis, Eph receptor-ephrin cis interactions have not been previously investigated in cancer cells. Here we show that in cancer cells, coexpressed ephrin-A3 can inhibit the ability of EphA2 and EphA3 to bind ephrins in trans and become activated, while ephrin-B2 can inhibit not only EphB4 but also EphA3. The cis inhibition of EphA3 by ephrin-B2 implies that in some cases ephrins that cannot activate a particular Eph receptor in trans can nevertheless inhibit its signaling ability through cis association. We also found that an EphA3 mutation identified in lung cancer enhances cis interaction with ephrin-A3. These results suggest a novel mechanism that may contribute to cancer pathogenesis by attenuating the tumor suppressing effects of Eph receptor signaling pathways activated by ephrins in trans.

  13. Bifidobacterium breve attenuates murine dextran sodium sulfate-induced colitis and increases regulatory T cell responses.

    Directory of Open Access Journals (Sweden)

    Bin Zheng

    Full Text Available While some probiotics have shown beneficial effects on preventing or treating colitis development, others have shown no effects. In this study, we have assessed the immunomodulating effects of two probiotic strains, Lactobacillus rhamnosus (L. rhamnosus and Bifidobacterium breve (B. breve on T cell polarization in vitro, using human peripheral blood mononuclear cells (PBMC, and in vivo, using murine dextran sodium sulfate (DSS colitis model. With respect to the latter, the mRNA expression of T cell subset-associated transcription factors and cytokines in the colon was measured and the T helper type (Th 17 and regulatory T cell (Treg subsets were determined in the Peyer's patches. Both L. rhamnosus and B. breve incubations in vitro reduced Th17 and increased Th2 cell subsets in human PBMCs. In addition, B. breve incubation was also able to reduce Th1 and increase Treg cell subsets in contrast to L. rhamnosus. In vivo intervention with B. breve, but not L. rhamnosus, significantly attenuated the severity of DSS-induced colitis. In DSS-treated C57BL/6 mice, intervention with B. breve increased the expression of mRNA encoding for Th2- and Treg-associated cytokines in the distal colon. In addition, intervention with B. breve led to increases of Treg and decreases of Th17 cell subsets in Peyer's patches of DSS-treated mice. B. breve modulates T cell polarization towards Th2 and Treg cell-associated responses in vitro and in vivo. In vivo B. breve intervention ameliorates DSS-induced colitis symptoms and this protective effect may mediated by its effects on the T-cell composition.

  14. Natural killer cell cytokine response to M. bovis BCG Is associated with inhibited proliferation, increased apoptosis and ultimate depletion of NKp44(+CD56(bright cells.

    Directory of Open Access Journals (Sweden)

    Damien Portevin

    Full Text Available Mycobacterium bovis BCG, a live attenuated strain of M. bovis initially developed as a vaccine against tuberculosis, is also used as an adjuvant for immunotherapy of cancers and for treatment of parasitic infections. The underlying mechanisms are thought to rely on its immunomodulatory properties including the recruitment of natural killer (NK cells. In that context, we aimed to study the impact of M. bovis BCG on NK cell functions. We looked at cytotoxicity, cytokine production, proliferation and cell survival of purified human NK cells following exposure to single live particles of mycobacteria. We found that M. bovis BCG mediates apoptosis of NK cells only in the context of IL-2 stimulation during which CD56(bright NK cells are releasing IFN-γ in response to mycobacteria. We found that the presence of mycobacteria prevented the IL-2 induced proliferation and surface expression of NKp44 receptor by the CD56(bright population. In summary, we observed that M. bovis BCG is modulating the functions of CD56(bright NK cells to drive this subset to produce IFN-γ before subsequent programmed cell death. Therefore, IFN-γ production by CD56(bright cells constitutes the main effector mechanism of NK cells that would contribute to the benefits observed for M. bovis BCG as an immunotherapeutic agent.

  15. Pb exposure attenuates hypersensitivity in vivo by increasing regulatory T cells

    Energy Technology Data Exchange (ETDEWEB)

    Fang, Liang [Department of Immunology, Fourth Military Medical University, Xi' an 710032 (China); Zhao, Fang; Shen, Xuefeng [Department of Occupational and Environmental Health and the Ministry of Education Key Lab of Hazard Assessment and Control in Special Operational Environment, School of Public Health, Fourth Military Medical University, Xi' an 710032 (China); Ouyang, Weiming [Laboratory of Cell Biology, Division of Monoclonal Antibodies, Office of Biotechnology Products, Center for Drug Evaluation and Research, United States Food and Drug Administration, Bethesda, MD (United States); Liu, Xinqin; Xu, Yan; Yu, Tao [Department of Occupational and Environmental Health and the Ministry of Education Key Lab of Hazard Assessment and Control in Special Operational Environment, School of Public Health, Fourth Military Medical University, Xi' an 710032 (China); Jin, Boquan [Department of Immunology, Fourth Military Medical University, Xi' an 710032 (China); Chen, Jingyuan, E-mail: jy_chen@fmmu.edu.cn [Department of Occupational and Environmental Health and the Ministry of Education Key Lab of Hazard Assessment and Control in Special Operational Environment, School of Public Health, Fourth Military Medical University, Xi' an 710032 (China); Luo, Wenjing, E-mail: luowenj@fmmu.edu.cn [Department of Occupational and Environmental Health and the Ministry of Education Key Lab of Hazard Assessment and Control in Special Operational Environment, School of Public Health, Fourth Military Medical University, Xi' an 710032 (China)

    2012-12-01

    Pb is a common environmental pollutant affecting various organs. Exposure of the immune system to Pb leads to immunosuppression or immunodysregulation. Although previous studies showed that Pb exposure can modulate the function of helper T cells, Pb immunotoxicity remains incompletely understood. In this study, we investigated the effect of Pb exposure on T cell development, and the underlying mechanism of Pb-induced suppression of the delayed-type hypersensitivity (DTH) response in vivo. Sprague–Dawley rats were exposed to 300 ppm Pb-acetate solution via the drinking water for six weeks, and we found that Pb exposure significantly increased Pb concentrations in the blood by 4.2-fold (p < 0.05) as compared to those in the control rats. In Pb-exposed rats, the amount of thymic CD4{sup +}CD8{sup −} and peripheral CD4{sup +} T cells was significantly reduced, whereas, CD8{sup +} population was not affected. In contrast to conventional CD4{sup +} T cells, Foxp3{sup +} regulatory T cells (Tregs) were increased in both the thymus and peripheral lymphoid organs of Pb-exposed rats. In line with the increase of Tregs, the DTH response of Pb-exposed rats was markedly suppressed. Depletion of Tregs reversed the suppression of DTH response by Pb-exposed CD4{sup +} T cells in an adoptive transfer model, suggesting a critical role of the increased Tregs in suppressing the DTH response. Collectively, this study revealed that Pb-exposure may upregulate Tregs, thereby leading to immunosuppression. -- Highlights: ► Pb exposure impaired CD4{sup +} thymic T cell development. ► Peripheral T lymphocytes were reduced following Pb exposure. ► Pb exposure increases thymic and peripheral Treg cells in rats. ► Tregs played a critical role in Pb-exposure-induced immune suppression.

  16. Repetitive cryotherapy attenuates the in vitro and in vivo mononuclear cell activation response.

    Science.gov (United States)

    Lindsay, Angus; Othman, Mohd Izani; Prebble, Hannah; Davies, Sian; Gieseg, Steven P

    2016-07-01

    What is the central question of this study? Acute and repetitive cryotherapy are routinely used to accelerate postexercise recovery, although the effect on resident immune cells and repetitive exposure has largely been unexplored and neglected. What is the main finding and its importance? Using blood-derived mononuclear cells and semi-professional mixed martial artists, we show that acute and repetitive cryotherapy reduces the in vitro and in vivo T-cell and monocyte activation response whilst remaining independent of the physical performance of elite athletes. We investigated the effect of repetitive cryotherapy on the in vitro (cold exposure) and in vivo (cold water immersion) activation of blood-derived mononuclear cells following high-intensity exercise. Single and repeated cold exposure (5°C) of a mixed cell culture (T cells and monocytes) was investigated using in vitro tissue culture experimentation for total neopterin production (neopterin plus 7,8-dihydroneopterin). Fourteen elite mixed martial art fighters were also randomly assigned to either a cold water immersion (15 min at 10°C) or passive recovery protocol, which they completed three times per week during a 6 week training camp. Urine was collected and analysed for neopterin and total neopterin three times per week, and perceived soreness, fatigue, physical performance (broad jump, push-ups and pull-ups) and training performance were also assessed. Single and repetitive cold exposure significantly (P < 0.001) reduced total neopterin production from the mixed cell culture, whereas cold water immersion significantly (P < 0.05) attenuated urinary neopterin and total neopterin during the training camp without having any effect on physical performance parameters. Soreness and fatigue showed little variation between the groups, whereas training session performance was significantly (P < 0.05) elevated in the cold water immersion group. The data suggest that acute and repetitive cryotherapy

  17. Glucocorticoid-resistant Th17 cells are selectively attenuated by cyclosporine A.

    Science.gov (United States)

    Schewitz-Bowers, Lauren P; Lait, Philippa J P; Copland, David A; Chen, Ping; Wu, Wenting; Dhanda, Ashwin D; Vistica, Barbara P; Williams, Emily L; Liu, Baoying; Jawad, Shayma; Li, Zhiyu; Tucker, William; Hirani, Sima; Wakabayashi, Yoshiyuki; Zhu, Jun; Sen, Nida; Conway-Campbell, Becky L; Gery, Igal; Dick, Andrew D; Wei, Lai; Nussenblatt, Robert B; Lee, Richard W J

    2015-03-31

    Glucocorticoids remain the cornerstone of treatment for inflammatory conditions, but their utility is limited by a plethora of side effects. One of the key goals of immunotherapy across medical disciplines is to minimize patients' glucocorticoid use. Increasing evidence suggests that variations in the adaptive immune response play a critical role in defining the dose of glucocorticoids required to control an individual's disease, and Th17 cells are strong candidate drivers for nonresponsiveness [also called steroid resistance (SR)]. Here we use gene-expression profiling to further characterize the SR phenotype in T cells and show that Th17 cells generated from both SR and steroid-sensitive individuals exhibit restricted genome-wide responses to glucocorticoids in vitro, and that this is independent of glucocorticoid receptor translocation or isoform expression. In addition, we demonstrate, both in transgenic murine T cells in vitro and in an in vivo murine model of autoimmunity, that Th17 cells are reciprocally sensitive to suppression with the calcineurin inhibitor, cyclosporine A. This result was replicated in human Th17 cells in vitro, which were found to have a conversely large genome-wide shift in response to cyclosporine A. These observations suggest that the clinical efficacy of cyclosporine A in the treatment of SR diseases may be because of its selective attenuation of Th17 cells, and also that novel therapeutics, which target either Th17 cells themselves or the effector memory T-helper cell population from which they are derived, would be strong candidates for drug development in the context of SR inflammation.

  18. Loss of Dab2 expression in breast cancer cells impairs their ability to deplete TGF-β and induce Tregs development via TGF-β.

    Directory of Open Access Journals (Sweden)

    Shuguang Xu

    Full Text Available Dab2 is a multifunctional adapter protein which is frequently under-expressed in a variety of cancers. It is implicated in many critical functions, including several signaling pathways, cell arrangement, differentiation of stem cells, and receptor endocytosis. Transforming growth factor-β (TGF-β is a secreted multifunctional protein that controls several developmental processes and pathogenesis of many diseases. It has been documented that Dab2 played an important role in TGF-β receptors endocytosis. Here, we present evidence that re-expression of Dab2 in SK-BR-3 cell partially restored its ability to deplete TGF-β in surrounding medium by normalizing the trafficking of TGF-β receptors. We also demonstrate that the difference in TGF-β depletions produced by Dab2 expression was sufficient to impact on the conversion of naive CD4+ T cells to regulatory T cells (Tregs, and thus inhibited the proliferation of T cells. This work revealed a critical result that breast cancer cell was deficient in Dab2 expression and related receptor endocytosis-mediated TGF-β depletion, which may contribute to the accumulation of TGF-β in tumor microenvironment and the induction of immune tolerance.

  19. Xanthohumol attenuates tumour cell-mediated breaching of the lymphendothelial barrier and prevents intravasation and metastasis.

    Science.gov (United States)

    Viola, Katharina; Kopf, Sabine; Rarova, Lucie; Jarukamjorn, Kanokwan; Kretschy, Nicole; Teichmann, Mathias; Vonach, Caroline; Atanasov, Atanas G; Giessrigl, Benedikt; Huttary, Nicole; Raab, Ingrid; Krieger, Sigurd; Strnad, Miroslav; de Martin, Rainer; Saiko, Philipp; Szekeres, Thomas; Knasmüller, Siegfried; Dirsch, Verena M; Jäger, Walter; Grusch, Michael; Dolznig, Helmut; Mikulits, Wolfgang; Krupitza, Georg

    2013-07-01

    Health beneficial effects of xanthohumol have been reported, and basic research provided evidence for anti-cancer effects. Furthermore, xanthohumol was shown to inhibit the migration of endothelial cells. Therefore, this study investigated the anti-metastatic potential of xanthohumol. MCF-7 breast cancer spheroids which are placed on lymphendothelial cells (LECs) induce "circular chemorepellent-induced defects" (CCIDs) in the LEC monolayer resembling gates for intravasating tumour bulks at an early step of lymph node colonisation. NF-κB reporter-, EROD-, SELE-, 12(S)-HETE- and adhesion assays were performed to investigate the anti-metastatic properties of xanthohumol. Western blot analyses were used to elucidate the mechanisms inhibiting CCID formation. Xanthohumol inhibited the activity of CYP, SELE and NF-kB and consequently, the formation of CCIDs at low micromolar concentrations. More specifically, xanthohumol affected ICAM-1 expression and adherence of MCF-7 cells to LECs, which is a prerequisite for CCID formation. Furthermore, markers of epithelial-to-mesenchymal transition (EMT) and of cell mobility such as paxillin, MCL2 and S100A4 were suppressed by xanthohumol. Xanthohumol attenuated tumour cell-mediated defects at the lymphendothelial barrier and inhibited EMT-like effects thereby providing a mechanistic explanation for the anti-intravasative/anti-metastatic properties of xanthohumol.

  20. Celecoxib attenuates 5-fluorouracil-induced apoptosis in HCT-15 and HT-29 human colon cancer cells

    Institute of Scientific and Technical Information of China (English)

    Yun Jeong Lim; Jong Chul Rhee; Young Mee Bae; Wan Joo Chun

    2007-01-01

    AIM: To investigate the combined chemotherapeutic effects of celecoxib when used with 5-FU in vitro.METHODS: Two human colon cancer cell lines (HCT-15and HT-29) were treated with 5-FU and celecoxib, alone and in combination. The effects of each drug were evaluated using the MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, flow cytometry,and western blotting.RESULTS: 5-FU and celecoxib showed a dosedependent cytotoxic effect. When treated with 10-3mol/L 5-FU (IC50) and celecoxib with its concentration ranging from 10-8 mol/L to 10-4 mol/L of celecoxib,cells showed reduced cytotoxic effect than 5-FU(10-3 mol/L) alone. Flow cytometry showed that celecoxib attenuated 5-FU induced accumulation of cells at subG1 phase. Western blot analyses for caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage showed that celecoxib attenuated 5-FU induced apoptosis. Western blot analyses for cell cycle molecules showed that G2/M arrest might be possible cause of 5-FU induced apoptosis and celecoxib attenuated 5-FU induced apoptosis via blocking of cell cycle progression to the G2/M phase,causing an accumulation of cells at the G1/S phase.CONCLUSION: We found that celecoxib attenuated cytotoxic effect of 5-FU. Celecoxib might act via inhibition of cell cycle progression, thus preventing apoptosis induced by 5-FU.

  1. Hydrogen peroxide attenuates refilling of intracellular calcium store in mouse pancreatic acinar cells

    Science.gov (United States)

    Yoon, Mi Na; Kim, Dong Kwan; Kim, Se Hoon

    2017-01-01

    Intracellular calcium (Ca2+) oscillation is an initial event in digestive enzyme secretion of pancreatic acinar cells. Reactive oxygen species are known to be associated with a variety of oxidative stress-induced cellular disorders including pancreatitis. In this study, we investigated the effect of hydrogen peroxide (H2O2) on intracellular Ca2+ accumulation in mouse pancreatic acinar cells. Perfusion of H2O2 at 300 µM resulted in additional elevation of intracellular Ca2+ levels and termination of oscillatory Ca2+ signals induced by carbamylcholine (CCh) in the presence of normal extracellular Ca2+. Antioxidants, catalase or DTT, completely prevented H2O2-induced additional Ca2+ increase and termination of Ca2+ oscillation. In Ca2+-free medium, H2O2 still enhanced CCh-induced intracellular Ca2+ levels and thapsigargin (TG) mimicked H2O2-induced cytosolic Ca2+ increase. Furthermore, H2O2-induced elevation of intracellular Ca2+ levels was abolished under sarco/endoplasmic reticulum Ca2+ ATPase-inactivated condition by TG pretreatment with CCh. H2O2 at 300 µM failed to affect store-operated Ca2+ entry or Ca2+ extrusion through plasma membrane. Additionally, ruthenium red, a mitochondrial Ca2+ uniporter blocker, failed to attenuate H2O2-induced intracellular Ca2+ elevation. These results provide evidence that excessive generation of H2O2 in pathological conditions could accumulate intracellular Ca2+ by attenuating refilling of internal Ca2+ stores rather than by inhibiting Ca2+ extrusion to extracellular fluid or enhancing Ca2+ mobilization from extracellular medium in mouse pancreatic acinar cells.

  2. Knockout of endothelial cell-derived endothelin-1 attenuates skin fibrosis but accelerates cutaneous wound healing.

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    Katsunari Makino

    Full Text Available Endothelin (ET-1 is known for the most potent vasoconstrictive peptide that is released mainly from endothelial cells. Several studies have reported ET-1 signaling is involved in the process of wound healing or fibrosis as well as vasodilation. However, little is known about the role of ET-1 in these processes. To clarify its mechanism, we compared skin fibrogenesis and wound repair between vascular endothelial cell-specific ET-1 knockout mice and their wild-type littermates. Bleomycin-injected fibrotic skin of the knockout mice showed significantly decreased skin thickness and collagen content compared to that of wild-type mice, indicating that bleomycin-induced skin fibrosis is attenuated in the knockout mice. The mRNA levels of transforming growth factor (TGF-β were decreased in the bleomycin-treated skin of ET-1 knockout mice. On the other hand, skin wound healing was accelerated in ET-1 knockout mice, which was indicated by earlier granulation tissue reduction and re-epithelialization in these mice. The mRNA levels of TGF-β, tumor necrosis factor (TNF-α and connective tissue growth factor (CTGF were reduced in the wound of ET-1 knockout mice. In endothelial ET-1 knockout mouse, the expression of TNF-α, CTGF and TGF-β was down-regulated. Bosentan, an antagonist of dual ET receptors, is known to attenuate skin fibrosis and accelerate wound healing in systemic sclerosis, and such contradictory effect may be mediated by above molecules. The endothelial cell-derived ET-1 is the potent therapeutic target in fibrosis or wound healing, and investigations of the overall regulatory mechanisms of these pathological conditions by ET-1 may lead to a new therapeutic approach.

  3. Cannabidiol protects oligodendrocyte progenitor cells from inflammation-induced apoptosis by attenuating endoplasmic reticulum stress.

    Science.gov (United States)

    Mecha, M; Torrao, A S; Mestre, L; Carrillo-Salinas, F J; Mechoulam, R; Guaza, C

    2012-06-28

    Cannabidiol (CBD) is the most abundant cannabinoid in Cannabis sativa that has no psychoactive properties. CBD has been approved to treat inflammation, pain and spasticity associated with multiple sclerosis (MS), of which demyelination and oligodendrocyte loss are hallmarks. Thus, we investigated the protective effects of CBD against the damage to oligodendrocyte progenitor cells (OPCs) mediated by the immune system. Doses of 1 μM CBD protect OPCs from oxidative stress by decreasing the production of reactive oxygen species. CBD also protects OPCs from apoptosis induced by LPS/IFNγ through the decrease of caspase 3 induction via mechanisms that do not involve CB1, CB2, TRPV1 or PPARγ receptors. Tunicamycin-induced OPC death was attenuated by CBD, suggesting a role of endoplasmic reticulum (ER) stress in the mode of action of CBD. This protection against ER stress-induced apoptosis was associated with reduced phosphorylation of eiF2α, one of the initiators of the ER stress pathway. Indeed, CBD diminished the phosphorylation of PKR and eiF2α induced by LPS/IFNγ. The pro-survival effects of CBD in OPCs were accompanied by decreases in the expression of ER apoptotic effectors (CHOP, Bax and caspase 12), and increased expression of the anti-apoptotic Bcl-2. These findings suggest that attenuation of the ER stress pathway is involved in the 'oligoprotective' effects of CBD during inflammation.

  4. Suppression of the CD8 T cell response by human papillomavirus type 16 E7 occurs in Langerhans cell-depleted mice

    Science.gov (United States)

    Jemon, K.; Leong, C.-M.; Ly, K.; Young, S. L.; McLellan, A. D.; Hibma, M. H.

    2016-01-01

    Human papillomavirus (HPV) is an epitheliotropic virus that is the primary causal agent for cervical cancer. Langerhans cells (LC) are skin antigen presenting cells that are reduced in number in HPV-infected skin. The aim of this study was to understand the immune-modulatory effects of HPV16 E7 on LC and on the CD8 T cell response to a skin-expressed antigen. To test this, HPV16 E7 was expressed in mouse skin keratinocytes with the model antigen ovalbumin (Ova). Similar to what is observed in HPV-infected human skin, LC numbers were significantly reduced in E7-expressing mouse skin. This shows that expression of the E7 protein alone is sufficient to mediate LC depletion. Expression of E7 with Ova in keratinocytes strongly suppressed the Ova-specific CD8+ T cell response in the skin draining lymph node. When tested in LC-ablated mice, the CD8 T cell response to skin-expressed Ova in control mice was not affected, nor was the T cell response to Ova restored in E7-expressing skin. These data indicate a role for E7 in regulation of LC homeostasis in the skin and in suppression of antigen specific CD8 T cell expansion, but suggest that these two effects occur independent of each other. PMID:27708419

  5. B-cell depletion inhibits arthritis in a collagen-induced arthritis (CIA) model, but does not adversely affect humoral responses in a respiratory syncytial virus (RSV) vaccination model.

    Science.gov (United States)

    Dunussi-Joannopoulos, Kyri; Hancock, Gerald E; Kunz, Arthur; Hegen, Martin; Zhou, Xiaochuan X; Sheppard, Barbara J; Lamothe, Jennifer; Li, Evelyn; Ma, Hak-Ling; Hamann, Philip R; Damle, Nitin K; Collins, Mary

    2005-10-01

    We report the development of a mouse B cell-depleting immunoconjugate (anti-CD22 monoclonal antibody [mAb] conjugated to calicheamicin) and its in vivo use to characterize the kinetics of CD22+ B-cell depletion and reconstitution in murine primary and secondary lymphoid tissues. The effect of B-cell depletion was further studied in a murine collagen-induced arthritis (CIA) model and a respiratory syncytial virus (RSV) vaccination model. Our results show that (1) the immunoconjugate has B-cell-specific in vitro and in vivo cytotoxicity; (2) B-cell reconstitution starts in the bone marrow and spleen around day 30 after depletion and is completed in all tissues tested by day 50; (3) B-cell depletion inhibits the development of clinical and histologic arthritis in the CIA model; (4) depletion of type II collagen antibody levels is not necessary for clinical and histologic prevention of CIA; and (5) B-cell depletion does not adversely affect memory antibody responses after challenge nor clearance of infectious virus from lungs in the RSV vaccination model. These results demonstrate for the first time that only B-cell reduction but not type II collagen antibody levels correlate with the prevention of arthritis and represent key insights into the role of CD22-targeted B-cell depletion in mouse autoimmunity and vaccination models.

  6. Macrophage colony-stimulating factor gene transduction into human lung cancer cells differentially regulates metastasis formations in various organ microenvironments of natural killer cell-depleted SCID mice.

    Science.gov (United States)

    Yano, S; Nishioka, Y; Nokihara, H; Sone, S

    1997-02-15

    We investigated whether local production of macrophage colony-stimulating factor (M-CSF), responsible for migration and activation of monocytes/macrophages at a tumor growth site, affected the metastatic pattern of lung cancer. For this, highly metastatic human squamous (RERF-LC-AI) or small (H69/VP) cell lung carcinoma cells were transduced with the human M-CSF gene inserted into pRc/CMV-MCSF to establish M-CSF-producing clones (MCSF-AI-9-18, MCSF-AI-9-24, and MCSF-VP-5). M-CSF gene transduction had no effect on the expression of surface antigen or on in vitro proliferation. After s.c. injection into SCID mice, the growth rates of M-CSF-producing cells were slower than those of parent or mock-transduced cells. In the metastatic model in SCID mice depleted of natural killer cells, RERF-LC-AI cells formed metastases mainly in the liver and kidneys, whereas H69/VP cells metastasized mainly to the liver and systemic lymph nodes. The numbers of metastatic colonies of MCSF-AI-9-18 and MCSF-AI-9-24 cells in the liver but not the kidneys were significantly reduced. The development of lymph node metastases of MCSF-VP-5 cells was also less than that of parent or mock-transduced cells. Treatment of SCID mice with anti-human M-CSF antibody resulted in a significant increase in liver metastases of their M-CSF gene transfectants. No significant differences were observed in the distributions in mice or in the in vitro invasive potentials of MCSF-AI-9-18 cells and Neo-AI-3 cells. These findings indicate that the antimetastatic effect of M-CSF may be specific to particular organs, suggesting the influence of heterogeneity of organ microenvironments on the metastasis of lung cancer.

  7. Screen for abnormal mitochondrial phenotypes in mouse embryonic stem cells identifies a model for succinyl-CoA ligase deficiency and mtDNA depletion

    Directory of Open Access Journals (Sweden)

    Taraka R. Donti

    2014-02-01

    Full Text Available Mutations in subunits of succinyl-CoA synthetase/ligase (SCS, a component of the citric acid cycle, are associated with mitochondrial encephalomyopathy, elevation of methylmalonic acid (MMA, and mitochondrial DNA (mtDNA depletion. A FACS-based retroviral-mediated gene trap mutagenesis screen in mouse embryonic stem (ES cells for abnormal mitochondrial phenotypes identified a gene trap allele of Sucla2 (Sucla2SAβgeo, which was used to generate transgenic mice. Sucla2 encodes the ADP-specific β-subunit isoform of SCS. Sucla2SAβgeo homozygotes exhibited recessive lethality, with most mutants dying late in gestation (e18.5. Mutant placenta and embryonic (e17.5 brain, heart and muscle showed varying degrees of mtDNA depletion (20–60%. However, there was no mtDNA depletion in mutant liver, where the gene is not normally expressed. Elevated levels of MMA were observed in embryonic brain. SCS-deficient mouse embryonic fibroblasts (MEFs demonstrated a 50% reduction in mtDNA content compared with wild-type MEFs. The mtDNA depletion resulted in reduced steady state levels of mtDNA encoded proteins and multiple respiratory chain deficiencies. mtDNA content could be restored by reintroduction of Sucla2. This mouse model of SCS deficiency and mtDNA depletion promises to provide insights into the pathogenesis of mitochondrial diseases with mtDNA depletion and into the biology of mtDNA maintenance. In addition, this report demonstrates the power of a genetic screen that combines gene trap mutagenesis and FACS analysis in mouse ES cells to identify mitochondrial phenotypes and to develop animal models of mitochondrial dysfunction.

  8. Mesenchymal stem cells attenuate peritoneal injury through secretion of TSG-6.

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    Nan Wang

    Full Text Available BACKGROUND: Mesothelial cell injury plays an important role in peritoneal fibrosis. Present clinical therapies aimed at alleviating peritoneal fibrosis have been largely inadequate. Mesenchymal stem cells (MSCs are efficient for repairing injuries and reducing fibrosis. This study was designed to investigate the effects of MSCs on injured mesothelial cells and peritoneal fibrosis. METHODOLOGY/PRINCIPAL FINDINGS: Rat bone marrow-derived MSCs (5 × 10(6 were injected into Sprague-Dawley (SD rats via tail vein 24 h after peritoneal scraping. Distinct reductions in adhesion formation; infiltration of neutrophils, macrophage cells; number of fibroblasts; and level of transforming growth factor (TGF-β1 were found in MSCs-treated rats. The proliferation and repair of peritoneal mesothelial cells in MSCs-treated rats were stimulated. Mechanically injured mesothelial cells co-cultured with MSCs in transwells showed distinct increases in migration and proliferation. In vivo imaging showed that MSCs injected intravenously mainly accumulated in the lungs which persisted for at least seven days. No apparent MSCs were observed in the injured peritoneum even when MSCs were injected intraperitoneally. The injection of serum-starved MSCs-conditioned medium (CM intravenously reduced adhesions similar to MSCs. Antibody based protein array of MSCs-CM showed that the releasing of TNFα-stimulating gene (TSG-6 increased most dramatically. Promotion of mesothelial cell repair and reduction of peritoneal adhesion were produced by the administration of recombinant mouse (rm TSG-6, and were weakened by TSG-6-RNA interfering. CONCLUSIONS/SIGNIFICANCE: Collectively, these results indicate that MSCs may attenuate peritoneal injury by repairing mesothelial cells, reducing inflammation and fibrosis. Rather than the engraftment, the secretion of TSG-6 by MSCs makes a major contribution to the therapeutic benefits of MSCs.

  9. PPARγ antagonist attenuates mouse immune-mediated bone marrow failure by inhibition of T cell function.

    Science.gov (United States)

    Sato, Kazuya; Feng, Xingmin; Chen, Jichun; Li, Jungang; Muranski, Pawel; Desierto, Marie J; Keyvanfar, Keyvan; Malide, Daniela; Kajigaya, Sachiko; Young, Neal S

    2016-01-01

    Acquired aplastic anemia is an immune-mediated disease, in which T cells target hematopoietic cells; at presentation, the bone marrow is replaced by fat. It was reported that bone marrow adipocytes were negative regulators of hematopoietic microenvironment. To examine the role of adipocytes in bone marrow failure, we investigated peroxisomal proliferator-activated receptor gamma, a key transcription factor in adipogenesis, utilizing an antagonist of this factor called bisphenol-A-diglycidyl-ether. While bisphenol-A-diglycidyl-ether inhibited adipogenesis as expected, it also suppressed T cell infiltration of bone marrow, reduced plasma inflammatory cytokines, decreased expression of multiple inflammasome genes, and ameliorated marrow failure. In vitro, bisphenol-A-diglycidyl-ether suppressed activation and proliferation, and reduced phospholipase C gamma 1 and nuclear factor of activated T-cells 1 expression, as well as inhibiting calcium flux in T cells. The in vivo effect of bisphenol-A-diglycidyl-ether on T cells was confirmed in a second immune-mediated bone marrow failure model, using different strains and non-major histocompatibility antigen mismatched: bisphenol-A-diglycidyl-ether ameliorated marrow failure by inhibition of T cell infiltration of bone marrow. Our data indicate that peroxisomal proliferator-activated receptor gamma antagonists may attenuate murine immune-mediated bone marrow failure, at least in part, by suppression of T cell activation, which might hold implications in the application of peroxisomal proliferator-activated receptor gamma antagonists in immune-mediated pathophysiologies, both in the laboratory and in the clinic. Genetically "fatless" mice developed bone marrow failure with accumulation of marrow adipocytes in our model, even in the absence of body fat, suggesting different mechanisms of systematic and marrow adipogenesis and physiologic versus pathophysiologic fat accumulation.

  10. In vitro incubation of bone marrow and peripheral stem cells with vincristine and methylprednisolone: functional T-cell depletion for haploidentical and autologous transplants.

    Science.gov (United States)

    Fragonas, E; Perticarari, S; Presani, G; Rabusin, M; Andolina, M; Mangiarotti, M A

    2000-11-01

    A mismatched bone marrow transplantation is feasible only if the donor's marrow lymphocytes are eliminated from the graft. This can be achieved by several methods, but all have the disadvantage of inducing a long-lasting immune deficiency while the risk of graft rejection and leukemic relapse increase. We use a sort of functional T-cell depletion by treating the cells with vincristine and methylprednisolone. This method is surely the cheapest and has allowed us to perform 60 transplants with a tolerable risk of GVHD. The treatment of the donor's lymphocytes has already been demonstrated to be able to block the mixed lymphocyte culture reaction in vitro. In this experiment Th1 and Th2 activities were almost completely blocked without reduction of lymphocyte viability and apoptosis induction.

  11. Depletion of CABYR-a/b sensitizes lung cancer cells to TRAIL-induced apoptosis through YAP/p73-mediated DR5 upregulation.

    Science.gov (United States)

    Xiao, Qianqian; Qian, Zunlei; Zhang, Weiqing; Liu, Jin; Hu, Enze; Zhang, Jinsan; Li, Mingying; Wang, Junhao; Kong, Fei; Li, Yunguang; Wang, Rui; Tan, Xiaohua; He, Dacheng; Xiao, Xueyuan

    2016-02-23

    Our previous study revealed that knockdown of CABYR-a/b increases the chemosensitivity of lung cancer cells through inactivation of Akt. Here, we demonstrated that depletion of CABYR-a/b significantly increased DR5 expression and sensitized lung cancer cells to TRAIL-induced apoptosis in vitro and/or in vivo. Importantly, treatment with AD5-10, a DR5-specific agonistic monoclonal antibody, was able to mimic TRAIL-induced apoptosis in CABYR-a/b-silenced cells. Strikingly, we identified that depletion of CABYR-a/b not only increased the expressions of p73 and DR5 but also decreased the phosphorylation of YAP S127. Loss- or gain-of-function studies of YAP and p73 revealed that double deletions of YAP and p73 effectively decreased the expression of DR5 and abolished TRAIL-induced apoptosis in CABYR-a/b knockdown cells. Conversely, the co-overexpression of YAP and p73 promoted the expression of DR5 and sensitized cells to TRAIL-induced apoptosis. Taken together, our results demonstrate that depletion of CABYR-a/b sensitizes lung cancer cells to TRAIL-induced apoptosis through YAP/p73-mediated DR5 upregulation.

  12. Interleukin-2/Anti-Interleukin-2 Immune Complex Attenuates Cardiac Remodeling after Myocardial Infarction through Expansion of Regulatory T Cells

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    Zhipeng Zeng

    2016-01-01

    Full Text Available CD4+CD25+Foxp3+ regulatory T cells (Treg cells have protective effects in wound healing and adverse ventricular remodeling after myocardial infarction (MI. We hypothesize that the interleukin- (IL- 2 complex comprising the recombinant mouse IL-2/anti-IL-2 mAb (JES6-1 attenuates cardiac remodeling after MI through the expansion of Treg. Mice were subjected to surgical left anterior descending coronary artery ligation and treated with either PBS or IL-2 complex. The IL-2 complex significantly attenuates ventricular remodeling, as demonstrated by reduced infarct size, improved left ventricular (LV function, and attenuated cardiomyocyte apoptosis. The IL-2 complex increased the percentage of CD4+CD25+Foxp3+ Treg cells, which may be recruited to the infarcted heart, and decreased the frequencies of IFN-γ- and IL-17-producing CD4+ T helper (Th cells among the CD4+Foxp3− T cells in the spleen. Furthermore, the IL-2 complex inhibited the gene expression of proinflammatory cytokines as well as macrophage infiltrates in the infarcted myocardium and induced the differentiation of macrophages from M1 to M2 phenotype in border zone of infarcted myocardium. Our studies indicate that the IL-2 complex may serve as a promising therapeutic approach to attenuate adverse remodeling after MI through expanding Treg cells specifically.

  13. Depletion of Regulatory T Cells Induces High Numbers of Dendritic Cells and Unmasks a Subset of Anti-Tumour CD8+CD11c+ PD-1lo Effector T Cells.

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    Nicolas Goudin

    Full Text Available Natural regulatory T (Treg cells interfere with multiple functions, which are crucial for the development of strong anti-tumour responses. In a model of 4T1 mammary carcinoma, depletion of CD25+Tregs results in tumour regression in Balb/c mice, but the mechanisms underlying this process are not fully understood. Here, we show that partial Treg depletion leads to the generation of a particular effector CD8 T cell subset expressing CD11c and low level of PD-1 in tumour draining lymph nodes. These cells have the capacity to migrate into the tumour, to kill DCs, and to locally regulate the anti-tumour response. These events are concordant with a substantial increase in CD11b+ resident dendritic cells (DCs subsets in draining lymph nodes followed by CD8+ DCs. These results indicate that Treg depletion leads to tumour regression by unmasking an increase of DC subsets as a part of a program that optimizes the microenvironment by orchestrating the activation, amplification, and migration of high numbers of fully differentiated CD8+CD11c+PD1lo effector T cells to the tumour sites. They also indicate that a critical pattern of DC subsets correlates with the evolution of the anti-tumour response and provide a template for Treg depletion and DC-based therapy.

  14. β-Elemonic acid inhibits the cell proliferation of human lung adenocarcinoma A549 cells: The role of MAPK, ROS activation and glutathione depletion.

    Science.gov (United States)

    Wu, Tsu-Tuan; Lu, Chien-Lin; Lin, Hen-I; Chen, Bing-Fang; Jow, Guey-Mei

    2016-01-01

    β-elemonic acid, a known triterpene, exhibits anti-inflammatory effects, yet research on the pharmacological effects of β-elemonic acid is rare. We investigated the anticancer effects and the related molecular mechanisms of β-elemonic acid on human non-small cell lung cancer (NSCLC) A549 cells. The effects of β-elemonic acid on the growth of A549 cells were studied using a 3-(4,5)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was detected using Annexin V staining. The effect of β-elemonic acid on the cell cycle of A549 cells was assessed using the propidium iodide method. The change in reactive oxygen species (ROS) was detected using a dichlorodihydrofluorescein diacetate (DCFH-DA) assay with microscopic examination. The expression levels of Bcl-2 family proteins, mitogen-activated protein kinase (MAPK) family proteins and cyclooxygenase 2 (COX-2) were detected using western blot analysis. Our data revealed that β-elemonic acid strongly induced human A549 lung cancer cell death in a dose- and time-dependent manner as determined by the MTT assay. β-elemonic acid-induced cell death was considered to be apoptotic when the phosphatidylserine exposure was observed using Annexin V staining. The death of human A549 lung cancer cells was caused by apoptosis induced by activation of ROS activity, increase in the sub-G1 proportion, downregulation of Bcl-2 expression, upregulation of Bax expression and inhibition of the MAPK signaling pathways. These results clearly demonstrated that β-elemonic acid inhibits proliferation by inducing hypoploid cells and cell apoptosis. Moreover, the anticancer effects of β-elemonic acid were related to the MAPK signaling pathway, ROS activation and glutathione depletion in human A549 lung cancer cells.

  15. Depletion of OLFM4 gene inhibits cell growth and increases sensitization to hydrogen peroxide and tumor necrosis factor-alpha induced-apoptosis in gastric cancer cells

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    Liu Rui-hua

    2012-04-01

    Full Text Available Abstract Background Human olfactomedin 4 (OLFM4 gene is a secreted glycoprotein more commonly known as the anti-apoptotic molecule GW112. OLFM4 is found to be frequently up-regulated in many types of human tumors including gastric cancer and it was believed to play significant role in the progression of gastric cancer. Although the function of OLFM4 has been indicated in many studies, recent evidence strongly suggests a cell or tissue type-dependent role of OLFM4 in cell growth and apoptosis. The aim of this study is to examine the role of gastric cancer-specific expression of OLFM4 in cell growth and apoptosis resistance. Methods OLFM4 expression was eliminated by RNA interference in SGC-7901 and MKN45 cells. Cell proliferation, anchorage-independent growth, cell cycle and apoptosis were characterized in vitro. Tumorigenicity was analyzed in vivo. The apoptosis and caspase-3 activation in response to hydrogen peroxide (H2O2 or tumor necrosis factor-alpha (TNF α were assessed in the presence or absence of caspase inhibitor Z-VAD-fmk. Results The elimination of OLFM4 protein by RNA interference in SGC-7901 and MKN45 cells significantly inhibits tumorigenicity both in vitro and in vivo by induction of cell G1 arrest (all P 2O2 or TNF α-induced apoptosis and caspase-3 activity (all P 2O2 or TNF α-induced apoptosis in OLFM4 knockdown cells (all P Conclusion Our study suggests that depletion of OLFM4 significantly inhibits tumorigenicity of the gastric cancer SGC-7901 and MKN45 cells. Blocking OLFM4 expression can sensitize gastric cancer cells to H2O2 or TNF α treatment by increasing caspase-3 dependent apoptosis. A combination strategy based on OLFM4 inhibition and anticancer drugs treatment may provide therapeutic potential in gastric cancer intervention.

  16. Human bone marrow mesenchymal stem cell transplantation attenuates axonal injur y in stroke rats

    Institute of Scientific and Technical Information of China (English)

    Yi Xu; Shiwei Du; Xinguang Yu; Xiao Han; Jincai Hou; Hao Guo

    2014-01-01

    Previous studies have shown that transplantation of human bone marrow mesenchymal stem cells promotes neural functional recovery after stroke, but the neurorestorative mechanisms remain largely unknown. We hypothesized that functional recovery of myelinated axons may be one of underlying mechanisms. In this study, an ischemia/reperfusion rat model was established using the middle cerebral artery occlusion method. Rats were used to test the hypothesis that in-travenous transplantation of human bone marrow mesenchymal stem cells through the femoral vein could exert neuroprotective effects against cerebral ischemia via a mechanism associated with the ability to attenuate axonal injury. The results of behavioral tests, infarction volume analysis and immunohistochemistry showed that cerebral ischemia caused severe damage to the myelin sheath and axons. After rats were intravenously transplanted with human bone marrow mesenchymal stem cells, the levels of axon and myelin sheath-related proteins, including mi-crotubule-associated protein 2, myelin basic protein, and growth-associated protein 43, were elevated, infarct volume was decreased and neural function was improved in cerebral ischemic rats. These ifndings suggest that intravenously transplanted human bone marrow mesenchymal stem cells promote neural function. Possible mechanisms underlying these beneifcial effects in-clude resistance to demyelination after cerebral ischemia, prevention of axonal degeneration, and promotion of axonal regeneration.

  17. Rapamycin-mediated mTOR inhibition attenuates survivin and sensitizes glioblastoma cells to radiation therapy

    Institute of Scientific and Technical Information of China (English)

    Arunkumar Anandharaj; Senthilkumar Cinghu; Woo-Yoon Park

    2011-01-01

    Survivin, an antiapoptotic protein, is elevated in most malignancies and attributes to radiation resistance in tumors including glioblastoma multiforme. The downregulation of survivin could sensitize glioblastoma ceils to radiation therapy. In this study, we investigated the effect of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), in attenuating survivin and enhancing the therapeutic efficacy for glioblastoma cells, and elucidated the underlying mechanisms. Here we tested various concentrations of rapamycin (1-8 nM) in combination with radiation dose 4 Gy. Rapamycin effectively modulated the protein kinase B (Akt)/mTOR pathway by inhibiting the phosphorylation of Akt and mTOR proteins, and this inhibition was further enhanced by radiation. The expression level of survivin was decreased in rapamycin pre-treatment glioblastoma ceils followed by radiation; meanwhile, the phosphorylation of H2A histone family member X (H2AX) at serine-139 (γ-H2AX) was increased, p21 protein was also induce on radiation with rapamycin pre-treatment, which enhanced G1 arrest and the accumulation of cells at G0/subG1 phase. Furthermore, the clonogenic cell survival assay revealed a significant dose-dependent decrease in the surviving fraction for all three cell lines pre-treated with rapamycin. Our studies demonstrated that targeting survivin may be an effective approach for radiosensitization of malignant glioblastoma.

  18. Atractylenolide I-mediated Notch pathway inhibition attenuates gastric cancer stem cell traits.

    Science.gov (United States)

    Ma, Li; Mao, Rurong; Shen, Ke; Zheng, Yuanhong; Li, Yueqi; Liu, Jianwen; Ni, Lei

    2014-07-18

    Atractylenolide I (AT-I), one of the main naturally occurring compounds of Rhizoma Atractylodis Macrocephalae, has remarkable anti-cancer effects on various cancers. However, its effects on the treatment of gastric cancer remain unclear. Via multiple cellular and molecular approaches, we demonstrated that AT-I could potently inhibit cancer cell proliferation and induce apoptosis through inactivating Notch pathway. AT-I treatment led to the reduction of expressions of Notch1, Jagged1, and its downstream Hes1/ Hey1. Our results showed that AT-I inhibited the self-renewal capacity of gastric stem-like cells (GCSLCs) by suppression of their sphere formation capacity and cell viability. AT-I attenuated gastric cancer stem cell (GCSC) traits partly through inactivating Notch1, leading to reducing the expressions of its downstream target Hes1, Hey1 and CD44 in vitro. Collectively, our results suggest that AT-I might develop as a potential therapeutic drug for the treatment of gastric cancer.

  19. Estradiol pretreatment attenuated nicotine-induced endothelial cell apoptosis via estradiol functional membrane receptor.

    Science.gov (United States)

    Wang, Li-li; Zhao, Jian-li; Lau, Wayne-Bond; Zhang, Yan-qing; Qiao, Zhong-dong; Wang, Ya-jing

    2011-06-01

    Cigarette smoking is highly associated with increased cardiovascular disease complications. The female population, however, manifests reduced cardiovascular morbidity. We define nicotine's effect upon human umbilical vein endothelial cells (HUVECs), determine whether estradiol might ameliorate endothelial dysfunction via its membrane estrogen receptor (mER), and attempt to elucidate the underlying mechanisms. Endothelial cells were pretreated with estradiol-BSA and measured resultant ion flux across the cells via the patch clamp technique to assess mER is functionality. Estradiol-BSA administration was associated with 30% decreased nicotine-induced apoptosis and also attenuated nicotine-activated phosphorylation of p38 and ERK. Pretreatment of estradiol-BSA triggered a low calcium influx, suggesting ahead low influx calcium played a critical role in the underlying protective mechanisms of estradiol. Furthermore, this estradiol-BSA protection against apoptosis remained effective in the presence of tamoxifen, an intracellular estrogen receptor (iER) inhibitor. Additionally, tamoxifen did not abolish estradiol-BSA's inhibitory effect upon p38 and ERK's activation, giving evidence to the obligatory role of p38 and ERK signaling in the estradiol-BSA's anti-apoptotic action via mER. Our study provides evidence that nicotine enhances endothelial cell apoptosis, but estrogen exerts anti-apoptotic effect through its functional membrane estrogen receptor. Clinically, the nicotine in cigarettes might contribute to endothelial dysfunction, whereas ambient estradiol may provide cellular protection against nicotine-induced injury through its functional membrane receptor via MAPK pathway downregulation.

  20. Panax notoginseng Saponins Attenuate Phenotype Switching of Vascular Smooth Muscle Cells Induced by Notch3 Silencing

    Science.gov (United States)

    Liu, Nan; Shan, Dazhi; Li, Ying; Chen, Hui; Gao, Yonghong; Huang, Yonghua

    2015-01-01

    Panax notoginseng saponins (PNS) could maintain vascular smooth muscle cells (VSMCs) in stable phenotypes so as to keep blood vessel elasticity as well as prevent failing in endovascular treatment with stent. Downregulation of Notch3 expression in VSMCs could influence the phenotype of VSMCs under pathologic status. However, whether PNS is able to attenuate the Notch3 silencing induced phenotype switching of VSMCs remains poorly understood. Primary human VSMCs were transfected with a plasmid containing a small interfering RNA (siRNA) against Notch3 and then exposed to different doses of PNS. The control groups included cells not receiving any treatment and cells transfected with a control siRNA. Phenotypic switching was evaluated by observing cell morphology with confocal microscopy, as well as examining α-SM-actin, SM22α, and OPN using Western blot. Downregulated Notch3 with a siRNA induced apparent phenotype switching, as reflected by morphologic changes, decreased expression of α-SM-actin and SM22α and increased expression of OPN. These changes were inhibited by PNS in a dose-dependent manner. The phenotype switching of VSMCs induced by Notch3 knockdown could be inhibited by PNS in a dose-dependent manner. Our study provided new evidence for searching effective drug for amending stability of atherosclerotic disease. PMID:26539217

  1. Panax notoginseng Saponins Attenuate Phenotype Switching of Vascular Smooth Muscle Cells Induced by Notch3 Silencing

    Directory of Open Access Journals (Sweden)

    Nan Liu

    2015-01-01

    Full Text Available Panax notoginseng saponins (PNS could maintain vascular smooth muscle cells (VSMCs in stable phenotypes so as to keep blood vessel elasticity as well as prevent failing in endovascular treatment with stent. Downregulation of Notch3 expression in VSMCs could influence the phenotype of VSMCs under pathologic status. However, whether PNS is able to attenuate the Notch3 silencing induced phenotype switching of VSMCs remains poorly understood. Primary human VSMCs were transfected with a plasmid containing a small interfering RNA (siRNA against Notch3 and then exposed to different doses of PNS. The control groups included cells not receiving any treatment and cells transfected with a control siRNA. Phenotypic switching was evaluated by observing cell morphology with confocal microscopy, as well as examining α-SM-actin, SM22α, and OPN using Western blot. Downregulated Notch3 with a siRNA induced apparent phenotype switching, as reflected by morphologic changes, decreased expression of α-SM-actin and SM22α and increased expression of OPN. These changes were inhibited by PNS in a dose-dependent manner. The phenotype switching of VSMCs induced by Notch3 knockdown could be inhibited by PNS in a dose-dependent manner. Our study provided new evidence for searching effective drug for amending stability of atherosclerotic disease.

  2. Enhanced Agrobacterium-mediated transformation efficiencies in monocot cells is associated with attenuated defense responses.

    Science.gov (United States)

    Zhang, Wan-Jun; Dewey, Ralph E; Boss, Wendy; Phillippy, Brian Q; Qu, Rongda

    2013-02-01

    Plant defense responses can lead to altered metabolism and even cell death at the sites of Agrobacterium infection, and thus lower transformation frequencies. In this report, we demonstrate that the utilization of culture conditions associated with an attenuation of defense responses in monocot plant cells led to highly improved Agrobacterium-mediated transformation efficiencies in perennial ryegrass (Lolium perenne L.). The removal of myo-inositol from the callus culture media in combination with a cold shock pretreatment and the addition of L-Gln prior to and during Agrobacterium-infection resulted in about 84 % of the treated calluses being stably transformed. The omission of myo-inositol from the callus culture media was associated with the failure of certain pathogenesis related genes to be induced after Agrobacterium infection. The addition of a cold shock and supplemental Gln appeared to have synergistic effects on infection and transformation efficiencies. Nearly 60 % of the stably transformed calluses regenerated into green plantlets. Calluses cultured on media lacking myo-inositol also displayed profound physiological and biochemical changes compared to ones cultured on standard growth media, such as reduced lignin within the cell walls, increased starch and inositol hexaphosphate accumulation, enhanced Agrobacterium binding to the cell surface, and less H(2)O(2) production after Agrobacterium infection. Furthermore, the cold treatment greatly reduced callus browning after infection. The simple modifications described in this report may have broad application for improving genetic transformation of recalcitrant monocot species.

  3. Functional cure of SIVagm infection in rhesus macaques results in complete recovery of CD4+ T cells and is reverted by CD8+ cell depletion.

    Directory of Open Access Journals (Sweden)

    Ivona Pandrea

    2011-08-01

    Full Text Available Understanding the mechanism of infection control in elite controllers (EC may shed light on the correlates of control of disease progression in HIV infection. However, limitations have prevented a clear understanding of the mechanisms of elite controlled infection, as these studies can only be performed at randomly selected late time points in infection, after control is achieved, and the access to tissues is limited. We report that SIVagm infection is elite-controlled in rhesus macaques (RMs and therefore can be used as an animal model for EC HIV infection. A robust acute infection, with high levels of viral replication and dramatic mucosal CD4(+ T cell depletion, similar to pathogenic HIV-1/SIV infections of humans and RMs, was followed by complete and durable control of SIVagm replication, defined as: undetectable VLs in blood and tissues beginning 72 to 90 days postinoculation (pi and continuing at least 4 years; seroreversion; progressive recovery of mucosal CD4(+ T cells, with complete recovery by 4 years pi; normal levels of T cell immune activation, proliferation, and apoptosis; and no disease progression. This "functional cure" of SIVagm infection in RMs could be reverted after 4 years of control of infection by depleting CD8 cells, which resulted in transient rebounds of VLs, thus suggesting that control may be at least in part immune mediated. Viral control was independent of MHC, partial APOBEC restriction was not involved in SIVagm control in RMs and Trim5 genotypes did not impact viral replication. This new animal model of EC lentiviral infection, in which complete control can be predicted in all cases, permits research on the early events of infection in blood and tissues, before the defining characteristics of EC are evident and when host factors are actively driving the infection towards the EC status.

  4. Calpeptin Attenuated Inflammation, Cell Death, and Axonal Damage in Animal Model of Multiple Sclerosis

    Science.gov (United States)

    Guyton, M. Kelly; Das, Arabinda; Samantaray, Supriti; Wallace, Gerald C.; Butler, Jonathan T.; Ray, Swapan K.; Banik, Naren L.

    2011-01-01

    Experimental autoimmune encephalomyelitis (EAE) is an animal model for studying multiple sclerosis (MS). Calpain has been implicated in many inflammatory and neurodegenerative events that lead to disability in EAE and MS. Thus, treating EAE animals with calpain inhibitors may block these events and ameliorate disability. To test this hypothesis, acute EAE Lewis rats were treated dose-dependently with the calpain inhibitor calpeptin (50 – 250 µg/kg). Calpain activity, gliosis, loss of myelin, and axonal damage were attenuated by calpeptin therapy, leading to improved clinical scores. Neuronal and oligodendrocyte death were also decreased with down regulation of pro-apoptotic proteins, suggesting that decreases in cell death were due to decreases in the expression or activity of pro-apoptotic proteins. These results indicate that calpain inhibition may offer a novel therapeutic avenue for treating EAE and MS. PMID:20623621

  5. Regulation of prostaglandin metabolism by calcitriol attenuates growth stimulation in prostate cancer cells.

    Science.gov (United States)

    Moreno, Jacqueline; Krishnan, Aruna V; Swami, Srilatha; Nonn, Larisa; Peehl, Donna M; Feldman, David

    2005-09-01

    Calcitriol exhibits antiproliferative and pro-differentiation effects in prostate cancer. Our goal is to further define the mechanisms underlying these actions. We studied established human prostate cancer cell lines and primary prostatic epithelial cells and showed that calcitriol regulated the expression of genes involved in the metabolism of prostaglandins (PGs), known stimulators of prostate cell growth. Calcitriol significantly repressed the mRNA and protein expression of prostaglandin endoperoxide synthase/cyclooxygenase-2 (COX-2), the key PG synthesis enzyme. Calcitriol also up-regulated the expression of 15-hydroxyprostaglandin dehydrogenase, the enzyme initiating PG catabolism. This dual action was associated with decreased prostaglandin E2 secretion into the conditioned media of prostate cancer cells exposed to calcitriol. Calcitriol also repressed the mRNA expression of the PG receptors EP2 and FP, providing a potential additional mechanism of suppression of the biological activity of PGs. Calcitriol treatment attenuated PG-mediated functional responses, including the stimulation of prostate cancer cell growth. The combination of calcitriol with nonsteroidal anti-inflammatory drugs (NSAIDs) synergistically acted to achieve significant prostate cancer cell growth inhibition at approximately 2 to 10 times lower concentrations of the drugs than when used alone. In conclusion, the regulation of PG metabolism and biological actions constitutes a novel pathway of calcitriol action that may contribute to its antiproliferative effects in prostate cells. We propose that a combination of calcitriol and nonselective NSAIDs might be a useful chemopreventive and/or therapeutic strategy in men with prostate cancer, as it would allow the use of lower concentrations of both drugs, thereby reducing their toxic side effects.

  6. Edaravone protects PC12 cells from ischemic-like injury via attenuating the damage to mitochondria

    Institute of Scientific and Technical Information of China (English)

    SONG Ying; LI Meng; LI Ji-cheng; WEI Er-qing

    2006-01-01

    Background: Edaravone had been validated to effectively protect against ischemic injuries. In this study, we investigated the protective effect of edaravone by observing the effects on anti-apoptosis, regulation of Bcl-2/Bax protein expression and recovering from damage to mitochondria after OGD (oxygen-glucose deprivation)-reperfusion. Methods: Viability of PC 12cells which were injured at different time of OGD injury, was quantified by measuring MTT (2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) staining. In addition, PC 12 cells' viability was also quantified after their preincubation in different concentration of edaravone for 30 min followed by (OGD). Furthermore, apoptotic population of PC12 cells that reinsulted from OGD-reperfusion with or without preincubation with edaravone was determined by flow cytometer analysis,electron microscope and Hoechst/PI staining. Finally, change of Bcl-2/Bax protein expression was detected by Westem blot.Results: (1) The viability of PC 12 cells decreased with time (1~12 h) after OGD. We regarded the model of OGD 2 h, then replacing DMEM (Dulbecco's Modified Eagle's Medium) for another 24 h as an OGD-reperfusion in this research. Furthermore,most PC12 cells were in the state of apoptosis after OGD-reperfusion. (2) The viability of PC12 cells preincubated with edaravone at high concentrations (1,0.1, 0.01 μmol/L) increased significantly with edaravone protecting PC 12 cells from apoptosis after OGD-reperfusion injury. (3) Furthermore, edaravone attenuates the damage of OGD-reperfusion on mitochondria and regulated Bcl-2/Bax protein imbalance expression after OGD-reperfusion. Conclusion: Neuroprotective effects of edaravone on ischemic or other brain injuries may be partly mediated through inhibition of Bcl-2/Bax apoptotic pathways by recovering from the damage of mitochondria.

  7. Contact-dependent depletion of hydrogen peroxide by catalase is a novel mechanism of myeloid-derived suppressor cell induction operating in human hepatic stellate cells.

    Science.gov (United States)

    Resheq, Yazid J; Li, Ka-Kit; Ward, Stephen T; Wilhelm, Annika; Garg, Abhilok; Curbishley, Stuart M; Blahova, Miroslava; Zimmermann, Henning W; Jitschin, Regina; Mougiakakos, Dimitrios; Mackensen, Andreas; Weston, Chris J; Adams, David H

    2015-03-15

    Myeloid-derived suppressor cells (MDSC) represent a unique cell population with distinct immunosuppressive properties that have been demonstrated to shape the outcome of malignant diseases. Recently, human hepatic stellate cells (HSC) have been reported to induce monocytic-MDSC from mature CD14(+) monocytes in a contact-dependent manner. We now report a novel and unexpected mechanism by which CD14(+)HLADR(low/-) suppressive cells are induced by catalase-mediated depletion of hydrogen peroxide (H2O2). Incubation of CD14(+) monocytes with catalase led to a significant induction of functional MDSC compared with media alone, and H2O2 levels inversely correlated with MDSC frequency (r = -0.6555, p Catalase was detected in primary HSC and a stromal cell line, and addition of the competitive catalase inhibitor hydroxylamine resulted in a dose-dependent impairment of MDSC induction and concomitant increase of H2O2 levels. The NADPH-oxidase subunit gp91 was significantly increased in catalase-induced MDSC as determined by quantitative PCR outlining the importance of oxidative burst for the induction of MDSC. These findings represent a so far unrecognized link between immunosuppression by MDSC and metabolism. Moreover, this mechanism potentially explains how stromal cells can induce a favorable immunological microenvironment in the context of tissue oxidative stress such as occurs during cancer therapy.

  8. Temporary CD8(+) T-cell depletion in pigs does not exacerbate infection with porcine reproductive and respiratory syndrome virus (PRRSV)

    DEFF Research Database (Denmark)

    Lohse, Louise; Nielsen, Jens; Eriksen, Lis

    2004-01-01

    , confirmed the depletion effect of specific mAb therapy. Almost complete depletion of cell subsets expressing the CD8(+) antigen was obtained on day 2 and 5 post infection (PI) with nadir less than 1 % of peripheral blood mononuclear cells (PBMC). One week PI, an increase in T-cell subsets was observed...... to test this hypothesis, we examined five 5-week-old pigs, which had been depleted for CD8(+) T-cells by treatment with anti-CD8 mAb injections, starting 2 days before inoculation with PRRSV. Virus-inoculated and sham-inoculated age-matched pigs served as controls. Blood samples were collected...... continuously, together with organ material at necropsy, to study kinetics of leukocyte subpopulations, antibody production and virus persistence in individual pigs. Significant lower CD8(+) T-cell counts on day 0, that is, before virus challenge, in the anti-CD8 mAb treated pigs compared to the control pigs...

  9. Depletion of hepatoma-derived growth factor-related protein-3 induces apoptotic sensitization of radioresistant A549 cells via reactive oxygen species-dependent p53 activation

    Energy Technology Data Exchange (ETDEWEB)

    Yun, Hong Shik; Hong, Eun-Hee [Division of Radiation Cancer Biology, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Lee, Su-Jae [Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Baek, Jeong-Hwa [Division of Radiation Cancer Biology, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746 (Korea, Republic of); Lee, Chang-Woo [Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746 (Korea, Republic of); Yim, Ji-Hye; Um, Hong-Duck [Division of Radiation Cancer Biology, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Hwang, Sang-Gu, E-mail: sgh63@kcch.re.kr [Division of Radiation Cancer Biology, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of)

    2013-09-27

    Highlights: •HRP-3 is a radiation- and anticancer drug-responsive protein in A549 cells. •Depletion of HRP-3 induces apoptosis of radio- and chemoresistant A549 cells. •Depletion of HRP-3 promotes ROS generation via inhibition of the Nrf2/HO-1 pathway. •Depletion of HRP-3 enhances ROS-dependent p53 activation and PUMA expression. -- Abstract: Biomarkers based on functional signaling have the potential to provide greater insight into the pathogenesis of cancer and may offer additional targets for anticancer therapeutics. Here, we identified hepatoma-derived growth factor-related protein-3 (HRP-3) as a radioresistance-related gene and characterized the molecular mechanism by which its encoded protein regulates the radio- and chemoresistant phenotype of lung cancer-derived A549 cells. Knockdown of HRP-3 promoted apoptosis of A549 cells and potentiated the apoptosis-inducing action of radio- and chemotherapy. This increase in apoptosis was associated with a substantial generation of reactive oxygen species (ROS) that was attributable to inhibition of the Nrf2/HO-1 antioxidant pathway and resulted in enhanced ROS-dependent p53 activation and p53-dependent expression of PUMA (p53 upregulated modulator of apoptosis). Therefore, the HRP-3/Nrf2/HO-1/ROS/p53/PUMA cascade is an essential feature of the A549 cell phenotype and a potential radiotherapy target, extending the range of targets in multimodal therapies against lung cancer.

  10. Satureja khuzestanica attenuates apoptosis in hyperglycemic PC12 cells and spinal cord of diabetic rats.

    Science.gov (United States)

    Kaeidi, Ayat; Esmaeili-Mahani, Saeed; Abbasnejad, Mehdi; Sheibani, Vahid; Rasoulian, Bahram; Hajializadeh, Zahra; Pasban-Aliabadi, Hamzeh

    2013-01-01

    Several studies have indicated the involvement of oxidative stress and high glucose-induced cell death in the development of diabetic neuropathy. Satureja khuzestanica has been recommended in the literature as a remedy for the treatment of diabetes, and also contains antioxidant agents. Here, we investigated the possible neuroprotective effects of Satureja khuzestanica extract (SKE) on in vitro and in vivo models of diabetic neuropathy pain. High-glucose-induced damage to pheochromocytoma (PC12) cells and in streptozotocin-induced diabetic rats was studied. Tail-flick and rotarod treadmill tests were used to access nociceptive threshold and motor coordination, respectively. Cell viability was determined by MTT assay. Activated caspase 3 and Bax/Bcl-2 ratio-biochemical markers of apoptosis-were evaluated using immunoblotting. We found that elevating the glucose in the medium (to 4× normal) increased cell toxicity and caspase-3 activation in PC12 cells. Incubation with SKE (200 and 250 μg/ml) decreased cell damage. Furthermore, the diabetic rats developed neuropathy, which was evident from thermal hyperalgesia and motor deficit. Administering SKE at a daily dose of between 50 and 200 mg/kg to the diabetic animals for 3 weeks ameliorated hyperglycemia, weight loss, hyperalgesia, and motor deficit, inhibited caspase 3 activation, and decreased the Bax/Bcl-2 ratio. The results suggest that SKE exerts neuroprotective effects against hyperglycemia-induced cellular damage. The mechanisms of these effects may be related to (at least in part) the prevention of neural apoptosis, and the results suggest that Satureja has the therapeutic potential to attenuate side effects of diabetes, such as neuropathy.

  11. Notch activation by phenethyl isothiocyanate attenuates its inhibitory effect on prostate cancer cell migration.

    Directory of Open Access Journals (Sweden)

    Su-Hyeong Kim

    Full Text Available Phenethyl isothiocyanate (PEITC is a promising cancer chemopreventive component of edible cruciferous vegetables with in vivo efficacy against prostate cancer in experimental rodents. Cancer chemopreventive response to PEITC is characterized by its ability to inhibit multiple oncogenic signaling pathways, including nuclear factor-κB, Akt, and androgen receptor. The present study demonstrates, for the first time, that PEITC treatment activates Notch signaling in malignant as well as normal human prostate cells. Exposure of human prostate cancer cells (LNCaP, PC-3, and DU145 and a normal human prostate epithelial cell line (PrEC to PEITC resulted in cleavage (active form of Notch1 and Notch2, and increased transcriptional activity of Notch. In PC-3 and LNCaP cells, PEITC treatment caused induction of Notch ligands Jagged1 and Jagged2 (PC-3, overexpression of γ-secretase complex components Presenilin1 and Nicastrin (PC-3, nuclear enrichment of cleaved Notch2, and/or up-regulation of Notch1, Notch2, Jagged1, and/or Jagged2 mRNA. PEITC-induced apoptosis in LNCaP and PC-3 cells was significantly attenuated by RNA interference of Notch2, but not by pharmacological inhibition of Notch1. Inhibition of PC-3 and LNCaP cell migration resulting from PEITC exposure was significantly augmented by knockdown of Notch2 protein as well as pharmacological inhibition of Notch1 activation. Nuclear expression of cleaved Notch2 protein was significantly higher in PC-3 xenografts from PEITC-treated mice and dorsolateral prostates from PEITC-fed TRAMP mice compared with respective control. Because Notch signaling is implicated in epithelial-mesenchymal transition and metastasis, the present study suggests that anti-metastatic effect of PEITC may be augmented by a combination regimen involving a Notch inhibitor.

  12. Betahistine attenuates murine collagen-induced arthritis by suppressing both inflammatory and Th17 cell responses.

    Science.gov (United States)

    Tang, Kuo-Tung; Chao, Ya-Hsuan; Chen, Der-Yuan; Lim, Yun-Ping; Chen, Yi-Ming; Li, Yi-Rong; Yang, Deng-Ho; Lin, Chi-Chen

    2016-10-01

    The objective of this study was to evaluate the potential therapeutic effects of betahistine dihydrochloride (betahistine) in a collagen-induced arthritis (CIA) mouse model. CIA was induced in DBA/1 male mice by primary immunization with 100μl of emulsion containing 2mg/ml chicken type II collagen (CII) mixed with complete Freund's adjuvant (CFA) in an 1:1 ratio, and booster immunization with 100μl of emulsion containing 2mg/ml CII mixed with incomplete Freund's adjuvant (IFA) in an 1:1 ratio. Immunization was performed subcutaneously at the base of the tail. After being boosted on day 21, betahistine (1 and 5mg/kg) was orally administered daily for 2weeks. The severity of CIA was determined by arthritic scores and assessment of histopathological joint destruction. Expression of cytokines in the paw and anti-CII antibodies in the serum was evaluated by ELISA. The proliferative response against CII in the lymph node cells was measured by (3)H-thymidine incorporation assay. The frequencies of different CII specific CD4(+) T cell subsets in the lymph node were determined by flow-cytometric analysis. Betahistine treatment attenuated the severity of arthritis and reduced the levels of pro-inflammatory cytokines, including TNF-α, IL-6, IL-23 and IL-17A, in the paw tissues of CIA mice. Lymph node cells from betahistine-treated mice showed a decrease in proliferation, as well as a lower frequency of Th17 cells. In vitro, betahistine suppressed CD4(+) T cell differentiation into Th17 cells. These results indicate that betahistine is effective in suppressing both inflammatory and Th17 responses in mouse CIA and that it may have therapeutic value as an adjunct treatment for rheumatoid arthritis.

  13. Tiotropium attenuates IL-13-induced goblet cell metaplasia of human airway epithelial cells

    NARCIS (Netherlands)

    Kistemaker, Loes E. M.; Hiemstra, Pieter S.; Bos, I. Sophie T.; Bouwman, Susanne; van den Berge, Maarten; Hylkema, Machteld N.; Meurs, Herman; Kerstjens, Huib A. M.; Gosens, Reinoud

    2015-01-01

    BACKGROUND: It has been shown that acetylcholine is both a neurotransmitter and acts as a local mediator, produced by airway cells including epithelial cells. In vivo studies have demonstrated an indirect role for acetylcholine in epithelial cell differentiation. Here, we aimed to investigate direct

  14. IGF-I maintains calpastatin expression and attenuates apoptosis in several models of photoreceptor cell death.

    Science.gov (United States)

    Arroba, Ana I; Wallace, Deborah; Mackey, Ashley; de la Rosa, Enrique J; Cotter, Thomas G

    2009-09-01

    Retinitis pigmentosa is a heterogeneous group of inherited retinal dystrophies in which the loss of photoreceptor cells via apoptosis leads to blindness. In this study we have experimentally mimicked this condition by treating 661W cells and wild-type mouse retinal explants with a Ca(2+) ionophore. Ca(2+) overload induced apoptosis, which was correlated with calpain-2 activation, loss of calpastatin, its endogenous inhibitor, as well as the loss of its transcriptional activator, phospho-cAMP response element binding (CREB). All are similar changes to those observed in the rd1 mouse model of retinitis pigmentosa. Insulin like-growth factor-I (IGF-I) attenuated this Ca(2+)-induced apoptosis, as well as decreased the activation of calpain-2 and maintained calpastatin levels through the activation of the Akt-CREB pathway. Similarly, IGF-I decreased photoreceptor apoptosis in rd1 mouse retinal explants in parallel with reduced activation of calpain-2 and increased levels of calpastatin and activation of phospho-CREB. In conclusion, IGF-I seems to protect neural cells following a physiopathological or an experimental increase in intracellular Ca(2+), an observation that may have therapeutic consequences in neurodegenerative diseases such as retinitis pigmentosa.

  15. Proteasome inhibitors attenuated cholesterol-induced cardiac hypertrophy in H9c2 cells

    Science.gov (United States)

    Lee, Hyunjung; Park, Jinyoung; Kim, Eunice EunKyeong; Yoo, Young Sook; Song, Eun Joo

    2016-01-01

    The Ubiquitin proteasome system (UPS) plays roles in protein degradation, cell cycle control, and growth and inflammatory cell signaling. Dysfunction of UPS in cardiac diseases has been seen in many studies. Cholesterol acts as an inducer of cardiac hypertrophy. In this study, the effect of proteasome inhibitors on the cholesterol-induced hypertrophic growth in H9c2 cells is examined in order to observe whether UPS is involved in cardiac hypertrophy. The treatment of proteasome inhibitors MG132 and Bortezomib markedly reduced cellular surface area and mRNA expression of β-MHC in cholesterol-induced cardiac hypertrophy. In addition, activated AKT and ERK were significantly attenuated by MG132 and Bortezomib in cholesterol-induced cardiac hypertrophy. We demonstrated that cholesterol-induced cardiac hypertrophy was suppressed by proteasome inhibitors. Thus, regulatory mechanism of cholesterol-induced cardiac hypertrophy by proteasome inhibitors may provide a new therapeutic strategy to prevent the progression of heart failure. [BMB Reports 2016; 49(5): 270-275] PMID:26592933

  16. Targeted silencing of DNA-specific B cells combined with partial plasma cell depletion displays additive effects on delaying disease onset in lupus-prone mice.

    Science.gov (United States)

    Nikolova-Ganeva, K A; Gesheva, V V; Todorov, T A; Voll, R E; Vassilev, T L

    2013-11-01

    Targeting autoreactive B lymphocytes at any stage of their differentiation could yield viable therapeutic strategies for treating autoimmunity. All currently used drugs, including the most recently introduced biological agents, lack target specificity. Selective silencing of double-stranded DNA-specific B cells in animals with spontaneous lupus has been achieved previously by the administration of a chimeric antibody molecule that cross-links their DNA-reactive B cell immunoglobulin receptors with inhibitory FcγIIb (CD32) receptors. However, long-lived plasmacytes are resistant to this chimeric antibody as well as to all conventional treatments. Bortezomib (a proteasome inhibitor) depletes most plasma cells and has been shown recently to suppress disease activity in lupus mice. We hypothesized that the co-administration of non-toxic doses of bortezomib, that partially purge long-lived plasma cells, together with an agent that selectively silences DNA-specific B cells, should have additive effects in an autoantibody-mediated disease. Indeed, our data show that the simultaneous treatment of lupus-prone MRL/lpr mice with suboptimal doses of bortezomib plus the chimeric antibody resulted in the prevention or the delayed appearance of the disease manifestations as well as in a prolonged survival. The effect of the combination therapy was significantly stronger than that of the respective monotherapies and was comparable to that observed after cyclophosphamide administration.

  17. The impact of homologous recombination repair deficiency on depleted uranium clastogenicity in Chinese hamster ovary cells: XRCC3 protects cells from chromosome aberrations, but increases chromosome fragmentation

    Energy Technology Data Exchange (ETDEWEB)

    Holmes, Amie L. [Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Maine Center for Toxicology and Environmental Health, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Department of Applied Medical Science, University of Southern Maine, 96 Falmouth Street, P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Joyce, Kellie [Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Maine Center for Toxicology and Environmental Health, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Xie, Hong [Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Maine Center for Toxicology and Environmental Health, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Department of Applied Medical Science, University of Southern Maine, 96 Falmouth Street, P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Falank, Carolyne [Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Maine Center for Toxicology and Environmental Health, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); and others

    2014-04-15

    Highlights: • The role of homologous recombination repair in DU-induced toxicity was examined. • Loss of RAD51D did not affect DU-induced cytotoxicity or genotoxicity. • XRCC3 protects cell from DU-induced chromosome breaks and fusions. • XRCC3 plays a role in DU-induced chromosome fragmentation of the X chromosome. - Abstract: Depleted uranium (DU) is extensively used in both industry and military applications. The potential for civilian and military personnel exposure to DU is rising, but there are limited data on the potential health hazards of DU exposure. Previous laboratory research indicates DU is a potential carcinogen, but epidemiological studies remain inconclusive. DU is genotoxic, inducing DNA double strand breaks, chromosome damage and mutations, but the mechanisms of genotoxicity or repair pathways involved in protecting cells against DU-induced damage remain unknown. The purpose of this study was to investigate the effects of homologous recombination repair deficiency on DU-induced genotoxicity using RAD51D and XRCC3-deficient Chinese hamster ovary (CHO) cell lines. Cells deficient in XRCC3 (irs1SF) exhibited similar cytotoxicity after DU exposure compared to wild-type (AA8) and XRCC3-complemented (1SFwt8) cells, but DU induced more break-type and fusion-type lesions in XRCC3-deficient cells compared to wild-type and XRCC3-complemented cells. Surprisingly, loss of RAD51D did not affect DU-induced cytotoxicity or genotoxicity. DU induced selective X-chromosome fragmentation irrespective of RAD51D status, but loss of XRCC3 nearly eliminated fragmentation observed after DU exposure in wild-type and XRCC3-complemented cells. Thus, XRCC3, but not RAD51D, protects cells from DU-induced breaks and fusions and also plays a role in DU-induced chromosome fragmentation.

  18. Troglitazone inhibits cell proliferation by attenuation of epidermal growth factor receptor signaling independent of peroxisome proliferator-activated receptor γ

    Institute of Scientific and Technical Information of China (English)

    Xiaoqi Li; Xuanming Yang; Youli Xu; Xuejun Jiang; Xin Li; Fajun Nan; Hong Tang

    2009-01-01

    Peroxisome proliferator-activated receptors (PPAR) belong to the nuclear hormone receptor superfamily of ligand-dependent transcription factors. Recent results have shown that agonists of PPARy, such as troglitazone (TGZ), can inhibit cell proliferation and promote cell differentiation independent of PPARγ. In the present study, we provide evidence that TGZ may bind directly to EGFR and trigger its signaling and internalization independent of PPARγ. Detailed studies revealed that prolonged incubation with TGZ effectively attenuated EGFR signaling by target-ing the receptor to the endo-lysosomal degradation machinery. Although the extracellular signal-regulated kinase-signaling pathway was transiently activated by TGZ in EGFR overexpressing cancer cells, inhibition of EGF-induced Akt phosphorylation most likely accounted for the growth arrest of tumor cells caused by TGZ at pharmacologically achievable concentrations. Therefore, we have provided a new line of evidence indicating that TGZ inhibits cell pro-liferation by promoting EGFR degradation and attenuating Akt phosphorylation.

  19. Amiloride attenuates lipopolysaccharide-accelerated atherosclerosis via inhibition of NHE1-dependent endothelial cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    Gui-mei CUI; Yu-xi ZHAO; Na-na ZHANG; Zeng-shan LIU; Wan-chun SUN; Qi-sheng PENG

    2013-01-01

    Aim: To investigate the effects of the potassium-sparing diuretic amiloride on endothelial cell apoptosis during lipopolysaccharide (LPS)-accelerated atherosclerosis.Methods: Human umbilical vein endothelial cells (HUVECs) were exposed to LPS (100 ng/mL) in the presence of drugs tested.The activity of Na+/H+ exchanger 1 (NHE1) and calpain,intracellular free Ca2+ level ([Ca2+]i),as well as the expression of apoptosis-related proteins in the cells were measured.For in vivo study,ApoE-deficient (ApoE-/-) mice were fed high-fat diets with 0.5% (w/w) amiloride for 4 weeks and LPS (10 μg/mouse) infusion into caudal veins.Afterwards,atherosclerotic lesions,NHE1 activity and Bcl-2 expression in the aortic tissues were evaluated.Results: LPS treatment increased NHE1 activity and [Ca2+]i in HUVECs in a time-dependent manner,which was associated with increased activity of the Ca2+-dependent protease calpain.Amiloride (1-10 μmol/L) significantly suppressed LPS-induced increases in NHE1 activity,[Ca2+]i.and calpain activity.In the presence of the Ca2+ chelator BAPTA (0.5 mmol/L),LPS-induced increase of calpain activity was also abolished.In LPS-treated HUVECs,the expression of Bcl-2 protein was significantly decreased without altering its mRNA level.In the presence of amiloride (10 μmol/L) or the calpain inhibitor ZLLal (50 μmol/L),the down-regulation of Bcl-2 protein by LPS was blocked.LPS treatment did not alter the expression of Bax and Bak proteins in HUVECs.In the presence of amiloride,BAPTA or ZLLal,LPS-induced HUVEC apoptosis was significantly attenuated.In ApoE-/-mice,administration of amiloride significantly suppressed LPS-accelerated atherosclerosis and LPS-induced increase of NHE1 activity,and reversed LPS-induced down-regulation of Bcl-2 expression.Conclusion: LPS stimulates NHE1 activity,increases [Ca2+]i,and activates calpain,which leads to endothelial cell apoptosis related to decreased Bcl-2 expression.Amiloride inhibits NHE1 activity,thus attenuates LPS

  20. Endogenous n-3 polyunsaturated fatty acids attenuate T cell-mediated hepatitis via autophagy activation

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    Yanli Li

    2016-09-01

    Full Text Available Omega-3 polyunsaturated fatty acids (n-3 PUFAs exert anti-inflammatory effects in several liver disorders, including cirrhosis, acute liver failure, and fatty liver disease. To date, little is known about their role in immune-mediated liver diseases. In this study, we used fat-1 transgenic mice rich in endogenous n-3 PUFAs to examine the role of n-3 PUFAs in immune-mediated liver injury. Concanavalin A (Con A was administered intravenously to wild-type (WT and fat-1 transgenic mice to induce T cell-mediated hepatitis. Reduced liver damage was shown in Con A-administrated fat-1 transgenic mice, as evidenced by decreased mortality, attenuated hepatic necrosis, lessened serum alanine aminotransferase (ALT activity, and inhibited production of pro-inflammatory cytokines (e.g. TNF-α, IL-6, IL-17A and IFN-γ. In vivo and in vitro studies demonstrated that n-3 PUFAs significantly inhibited the activation of hepatic T cells and the differentiation of Th1 cells after Con A challenge. Further studies showed that n-3 PUFAs markedly increased autophagy level in Con A-treated fat-1 T cells compared with the WT counterparts. Blocking hepatic autophagy activity with chloroquine diminished the differences in T cell activation and liver injury between Con A-injected WT and fat-1 transgenic mice. We conclude that n-3 PUFAs limit Con A-induced hepatitis via an autophagy-dependent mechanism, and could be exploited as a new therapeutic approach for autoimmune hepatitis.

  1. Dimethylfumarate attenuates restenosis after acute vascular injury by cell-specific and Nrf2-dependent mechanisms

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    Chang Joo Oh

    2014-01-01

    Full Text Available Excessive proliferation of vascular smooth muscle cells (VSMCs and incomplete re-endothelialization is a major clinical problem limiting the long-term efficacy of percutaneous coronary angioplasty. We tested if dimethylfumarate (DMF, an anti-psoriasis drug, could inhibit abnormal vascular remodeling via NF−E2-related factor 2 (Nrf2-NAD(PH quinone oxidoreductase 1 (NQO1 activity. DMF significantly attenuated neointimal hyperplasia induced by balloon injury in rat carotid arteries via suppression of the G1 to S phase transition resulting from induction of p21 protein in VSMCs. Initially, DMF increased p21 protein stability through an enhancement in Nrf2 activity without an increase in p21 mRNA. Later on, DMF stimulated p21 mRNA expression through a process dependent on p53 activity. However, heme oxygenase-1 (HO-1 or NQO1 activity, well-known target genes induced by Nrf2, were dispensable for the DMF induction of p21 protein and the effect on the VSMC proliferation. Likewise, DMF protected endothelial cells from TNF-α-induced apoptosis and the dysfunction characterized by decreased eNOS expression. With knock-down of Nrf2 or NQO1, DMF failed to prevent TNF-α-induced cell apoptosis and decreased eNOS expression. Also, CD31 expression, an endothelial specific marker, was restored in vivo by DMF. In conclusion, DMF prevented abnormal proliferation in VSMCs by G1 cell cycle arrest via p21 upregulation driven by Nrf2 and p53 activity, and had a beneficial effect on TNF-α-induced apoptosis and dysfunction in endothelial cells through Nrf2–NQO1 activity suggesting that DMF might be a therapeutic drug for patients with vascular disease.

  2. Endogenous n-3 Polyunsaturated Fatty Acids Attenuate T Cell-Mediated Hepatitis via Autophagy Activation

    Science.gov (United States)

    Li, Yanli; Tang, Yuan; Wang, Shoujie; Zhou, Jing; Zhou, Jia; Lu, Xiao; Bai, Xiaochun; Wang, Xiang-Yang; Chen, Zhengliang; Zuo, Daming

    2016-01-01

    Omega-3 polyunsaturated fatty acids (n-3 PUFAs) exert anti-inflammatory effects in several liver disorders, including cirrhosis, acute liver failure, and fatty liver disease. To date, little is known about their role in immune-mediated liver diseases. In this study, we used fat-1 transgenic mice rich in endogenous n-3 PUFAs to examine the role of n-3 PUFAs in immune-mediated liver injury. Concanavalin A (Con A) was administered intravenously to wild-type (WT) and fat-1 transgenic mice to induce T cell-mediated hepatitis. Reduced liver damage was shown in Con A-administrated fat-1 transgenic mice, as evidenced by decreased mortality, attenuated hepatic necrosis, lessened serum alanine aminotransferase activity, and inhibited production of pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-17A, and IFN-γ). In vivo and in vitro studies demonstrated that n-3 PUFAs significantly inhibited the activation of hepatic T cells and the differentiation of Th1 cells after Con A challenge. Further studies showed that n-3 PUFAs markedly increased autophagy level in Con A-treated fat-1 T cells compared with the WT counterparts. Blocking hepatic autophagy activity with chloroquine diminished the differences in T cell activation and liver injury between Con A-injected WT and fat-1 transgenic mice. We conclude that n-3 PUFAs limit Con A-induced hepatitis via an autophagy-dependent mechanism and could be exploited as a new therapeutic approach for autoimmune hepatitis. PMID:27679638

  3. Secondary cell wall development in cotton fibers as examined with attenuated total reflection Fourier transform infrared spectroscopy

    Science.gov (United States)

    Cotton fibers harvested at 18, 20, 24, 28, 32, 36 and 40 days after flowering were examined using attenuated total reflection Fourier transform-infrared (ATR FT-IR) spectroscopy. The selected harvesting points coincide with secondary cell wall (SCW) development in the fibers. Progressive but moderat...

  4. Protective essential oil attenuates influenza virus infection: An in vitro study in MDCK cells

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    Metcalf Jordan P

    2010-11-01

    viability was only seen with concentrations of oil that were 2 to 6 times greater than the doses that inhibited viral infectivity. RT-PCR and western blotting demonstrated that oil treatment of the virus inhibited viral NP and NS1 protein, but not mRNA expression. Conclusions An essential oil blend significantly attenuates influenza virus PR8 infectivity in vitro without affecting viral binding or cellular internalization in MDCK cells. Oil treated virus continued to express viral mRNAs but had minimal expression of viral proteins, suggesting that the antiviral effect may be due to inhibition of viral protein translation.

  5. Depletion of the cereblon gene activates the unfolded protein response and protects cells from ER stress-induced cell death.

    Science.gov (United States)

    Lee, Kwang Min; Yang, Seung-Joo; Park, Sojung; Choi, Yoo Duk; Shin, Hwa Kyoung; Pak, Jhang Ho; Park, Chul-Seung; Kim, Inki

    2015-02-27

    Previous studies showed that cereblon (CRBN) binds to various cellular target proteins, implying that CRBN regulates a wide range of cell responses. In this study, we found that deletion of the Crbn gene desensitized mouse embryonic fibroblast cells to various cell death-promoting stimuli, including endoplasmic reticulum stress inducers. Mechanistically, deletion of Crbn activates pathways involved in the unfolded protein response prior to ER stress induction. Loss of Crbn activated PKR-like ER kinase (PERK) with enhanced phosphorylation of eIF2α. Following ER stress induction, loss of Crbn delayed dephosphorylation of eIF2α, while reconstitution of Crbn reversed enhanced phosphorylation of PERK and eIF2α. Lastly, we found that activation of the PERK/eIF2α pathway following Crbn deletion is caused by activation of AMP-activated protein kinase (AMPK). We propose that CRBN plays a role in cellular stress signaling, including the unfolded protein response, by controlling the activity of AMPK.

  6. Depletion of elongation initiation factor 4E binding proteins by CRISPR/Cas9 enhances the antiviral response in porcine cells.

    Science.gov (United States)

    Ramírez-Carvajal, Lisbeth; Singh, Neetu; de los Santos, Teresa; Rodríguez, Luis L; Long, Charles R

    2016-01-01

    Type I interferons (IFNs) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor 4E binding proteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF-7), the "master regulator" of IFN transcription. Previous studies have suggested that mouse cells depleted of 4E-BPs are more sensitive to IFNβ treatment and had lower viral loads as compared to wild type (WT) cells. However, such approach has not been tested as an antiviral strategy in livestock species. In this study, we tested the antiviral activity of porcine cells depleted of 4E-BP1 by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome engineering system. We found that 4E-BP1 knockout (KO) porcine cells had increased expression of IFNα and β, IFN stimulated genes, and significant reduction in vesicular stomatitis virus titer as compare to WT cells. No phenotypical changes associated with CRISPR/Cas9 manipulation were observed in 4E-BP1 KO cells. This work highlights the use of the CRISPR/Cas9 system to enhance the antiviral response in porcine cells.

  7. Morphine Attenuates Apically-Directed Cytokine Secretion from Intestinal Epithelial Cells in Response to Enteric Pathogens

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    Amanda J. Brosnahan

    2014-04-01

    Full Text Available Epithelial cells represent the first line of host immune defense at mucosal surfaces. Although opioids appear to increase host susceptibility to infection, no studies have examined opioid effects on epithelial immune functions. We tested the hypothesis that morphine alters vectorial cytokine secretion from intestinal epithelial cell (IPEC-J2 monolayers in response to enteropathogens. Both entero-adherent Escherichia coli O157:H7 and entero-invasive Salmonella enterica serovar Typhimurium increased apically-directed IL-6 secretion and bi-directional IL-8 secretion from epithelial monolayers, but only IL-6 secretion evoked by E. coli was reduced by morphine acting through a naloxone-sensitive mechanism. Moreover, the respective type 4 and 5 Toll-like receptor agonists, lipopolysaccharide and flagellin, increased IL-8 secretion from monolayers, which was also attenuated by morphine pretreatment. These results suggest that morphine decreases cytokine secretion and potentially phagocyte migration and activation directed towards the mucosal surface; actions that could increase host susceptibility to some enteric infections.

  8. Epigallocatechin-3-gallate attenuates head and neck cancer stem cell traits through suppression of Notch pathway.

    Science.gov (United States)

    Lee, Sang Hyuk; Nam, Hyo Jung; Kang, Hyun Jung; Kwon, Hye Won; Lim, Young Chang

    2013-10-01

    Most solid cancers including head and neck squamous carcinoma (HNSC) are believed to be initiated from and maintained by cancer stem cells (CSCs) that are responsible for treatment resistance, resulting in tumour relapse. Epigallocatechin-3-gallate (EGCG), the most abundant polyphenol in green tea, can potently inhibit cancer growth and induce apoptosis in various cancers, including HNSC. However, its effect on HNSC CSCs is not well elucidated. In this study, we examined the anti-tumour effect of EGCG on HNSC CSCs. We demonstrated that EGCG inhibits the self-renewal capacity of HNSC CSCs by suppressing their sphere forming capacity, and attenuates the expression of stem cell markers, such as Oct4, Sox2, Nanog and CD44. EGCG treatment augmented cisplatin-mediated chemosensitivity by suppressing ABCC2 and ABCG2 transporter genes, which are putative molecules of treatment resistance of CSC. In addition, the combination treatment of EGCG and cisplatin inhibited tumour formation and induced apoptosis in a xenograft model. As one of mechanisms of suppression of HNSC CSC traits, EGCG decreased the transcriptional level of Notch, resulting in the inhibition of Notch signalling. Collectively, our data suggest that EGCG in combination with cisplatin can be used for the management of HNSC CSCs.

  9. Cucurbitacin B induces rapid depletion of the G-actin pool through reactive oxygen species-dependent actin aggregation in melanoma cells

    Institute of Scientific and Technical Information of China (English)

    Yanting Zhang; Dongyun Ouyang; Lihui Xu; Yuhua Ji; Qingbing Zha; Jiye Cai; Xianhui He

    2011-01-01

    Cucurbitacin B (CuB), a triterpenoid compound isolated from Cucurbitaceae plants, has been reported as a promising anti-cancer agent, yet its action mechanism is still controversial. In this study, we explored the potential mechanism of CuB in murine B16F10 melanoma cells.Anti-proliferation and anti-invasion effects were assessed in cultured cells, and in vivo anti-tumor activity was evaluated in a murine subcutaneous melanoma model. Flow cytometry was adopted to analyze cell cycle distribution and reactive oxygen species (ROS) levels. Actin levels were determined by western blot analysis, and the profiles of differential expressed proteins were identified by a quantitative proteomic approach. The results showed that CuB exerted inhibitory effects on cell proliferation, colony formation, as well as migration and invasion potential of the melanoma cells. The growth of subcutaneous melanoma was significantly inhibited in mice treated with CuB when compared with control group. Furthermore,CuB treatment caused rapid cell membrane blebbing and deformation, and induced G2/M-phase arrest and formation of multiploid cells. Notably, the G-actin pool was rapidly depleted and actin aggregates were formed quickly after CuB treatment. A number of cytoskeleton-regulatory proteins were differentially regulated. Blockage of ROS production significantly reduced the G-actin depletion ability and the anti-tumor activity of CuB. These findings indicate that CuB induces rapid depletion of the G-actin pool through ROS-dependent actin aggregation in melanoma cells, which may at least partly account for its anti-tumor activity.

  10. iNKT-CELL ACTIVATION INDUCES LATE PRETERM BIRTH THAT IS ATTENUATED BY ROSIGLITAZONE1

    Science.gov (United States)

    St Louis, Derek; Romero, Roberto; Plazyo, Olesya; Arenas-Hernandez, Marcia; Panaitescu, Bogdan; Xu, Yi; Milovic, Tatjana; Xu, Zhonghui; Bhatti, Gaurav; Qing-Sheng, Mi; Drewlo, Sascha; Tarca, Adi L.; Hassan, Sonia S.; Gomez-Lopez, Nardhy

    2015-01-01

    Preterm birth (PTB) is a leading cause of neonatal morbidity and mortality; however, its non-infection-related mechanisms are poorly understood. Herein, we show that the expansion of activated CD1d-restricted invariant NKT (iNKT) cells in the third trimester by administration of α-galactosylceramide (α-GalCer) induces late PTB and neonatal mortality. In vivo imaging revealed that fetuses from mice that underwent α-GalCer-induced late PTB had bradycardia and died shortly after delivery. Yet, administration of α-GalCer in the second trimester did not cause pregnancy loss. PPARγ activation, through rosiglitazone treatment, reduced the rate of α-GalCer-induced late PTB and improved neonatal survival. Administration of α-GalCer in the third trimester suppressed PPARγ activation as shown by the down-regulation of Fabp4 and Fatp4 in myometrial and decidual tissues, respectively; this suppression was rescued by rosiglitazone treatment. Administration of α-GalCer in the third trimester induced an increase in the activation of conventional CD4+ T cells in myometrial tissues and the infiltration of activated macrophages, neutrophils and mature DCs to myometrial and/or decidual tissues. All of these effects were blunted after rosiglitazone treatment. Administration of α-GalCer also up-regulated the expression of inflammatory genes at the maternal-fetal interface and systemically, and rosiglitazone treatment partially attenuated these responses. Finally, an increased infiltration of activated iNKT-like cells in human decidual tissues is associated with non-infection-related preterm labor/birth. Collectively, these results demonstrate that iNKT-cell activation in vivo leads to late PTB by initiating innate and adaptive immune responses and suggest that the PPARγ pathway has potential as a target for prevention of this syndrome. PMID:26740111

  11. Astragalus Polysaccharide Attenuated Iron Overload-Induced Dysfunction of Mesenchymal Stem Cells via Suppressing Mitochondrial ROS

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    Fan Yang

    2016-09-01

    Full Text Available Background/Aims: Bone marrow-derived mesenchymal stem cells (BMSCs have the ability to differentiate into multilineage cells such as osteoblasts, chondrocytes, and cardiomyocytes. Dysfunction of BMSCs in response to pathological stimuli participates in the development of diseases such as osteoporosis. Astragalus polysaccharide (APS is a major active ingredient of Astragalus membranaceus, a commonly used anti-aging herb in traditional Chinese medicine. The aim of this study was to investigate whether APS protects against iron overload-induced dysfunction of BMSCs and its underlying mechanisms. Methods: BMSCs were exposed to ferric ammonium citrate (FAC with or without different concentrations of APS. The viability and proliferation of BMSCs were assessed by CCK-8 assay and EdU staining. Cell apoptosis, senescence and pluripotency were examined utilizing TUNEL staining, β-galactosidase staining and qRT-PCR respectively. The reactive oxygen species (ROS level was assessed in BMSCs with a DCFH-DA probe and MitoSOX Red staining. Results: Firstly, we found that iron overload induced by FAC markedly reduced the viability and proliferation of BMSCs, but treatment with APS at 10, 30 and 100 μg/mL was able to counter the reduction of cell proliferation. Furthermore, exposure to FAC led to apoptosis and senescence in BMSCs, which were partially attenuated by APS. The pluripotent genes Nanog, Sox2 and Oct4 were shown to be downregulated in BMSCs after FAC treatment, however APS inhibited the reduction of Nanog, Sox2 and Oct4 expression. Further study uncovered that APS treatment abrogated the increase of intracellular and mitochondrial ROS level in FAC-treated BMSCs. Conclusion: Treatment of BMSCs with APS to impede mitochondrial ROS accumulation can remarkably inhibit apoptosis, senescence, and the reduction of proliferation and pluripotency of BMSCs caused by FAC-induced iron overload.

  12. Rat adipose tissue-derived stem cells transplantation attenuates cardiac dysfunction post infarction and biopolymers enhance cell retention.

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    Maria E Danoviz

    Full Text Available BACKGROUND: Cardiac cell transplantation is compromised by low cell retention and poor graft viability. Here, the effects of co-injecting adipose tissue-derived stem cells (ASCs with biopolymers on cell cardiac retention, ventricular morphometry and performance were evaluated in a rat model of myocardial infarction (MI. METHODOLOGY/PRINCIPAL FINDINGS: 99mTc-labeled ASCs (1x10(6 cells isolated from isogenic Lewis rats were injected 24 hours post-MI using fibrin a, collagen (ASC/C, or culture medium (ASC/M as vehicle, and cell body distribution was assessed 24 hours later by gamma-emission counting of harvested organs. ASC/F and ASC/C groups retained significantly more cells in the myocardium than ASC/M (13.8+/-2.0 and 26.8+/-2.4% vs. 4.8+/-0.7%, respectively. Then, morphometric and direct cardiac functional parameters were evaluated 4 weeks post-MI cell injection. Left ventricle (LV perimeter and percentage of interstitial collagen in the spare myocardium were significantly attenuated in all ASC-treated groups compared to the non-treated (NT and control groups (culture medium, fibrin, or collagen alone. Direct hemodynamic assessment under pharmacological stress showed that stroke volume (SV and left ventricle end-diastolic pressure were preserved in ASC-treated groups regardless of the vehicle used to deliver ASCs. Stroke work (SW, a global index of cardiac function, improved in ASC/M while it normalized when biopolymers were co-injected with ASCs. A positive correlation was observed between cardiac ASCs retention and preservation of SV and improvement in SW post-MI under hemodynamic stress. CONCLUSIONS: We provided direct evidence that intramyocardial injection of ASCs mitigates the negative cardiac remodeling and preserves ventricular function post-MI in rats and these beneficial effects can be further enhanced by administering co-injection of ASCs with biopolymers.

  13. Rat Adipose Tissue-Derived Stem Cells Transplantation Attenuates Cardiac Dysfunction Post Infarction and Biopolymers Enhance Cell Retention

    Science.gov (United States)

    Danoviz, Maria E.; Nakamuta, Juliana S.; Marques, Fabio L. N.; dos Santos, Leonardo; Alvarenga, Erica C.; dos Santos, Alexandra A.; Antonio, Ednei L.; Schettert, Isolmar T.; Tucci, Paulo J.; Krieger, Jose E.

    2010-01-01

    Background Cardiac cell transplantation is compromised by low cell retention and poor graft viability. Here, the effects of co-injecting adipose tissue-derived stem cells (ASCs) with biopolymers on cell cardiac retention, ventricular morphometry and performance were evaluated in a rat model of myocardial infarction (MI). Methodology/Principal Findings 99mTc-labeled ASCs (1×106 cells) isolated from isogenic Lewis rats were injected 24 hours post-MI using fibrin a, collagen (ASC/C), or culture medium (ASC/M) as vehicle, and cell body distribution was assessed 24 hours later by γ-emission counting of harvested organs. ASC/F and ASC/C groups retained significantly more cells in the myocardium than ASC/M (13.8±2.0 and 26.8±2.4% vs. 4.8±0.7%, respectively). Then, morphometric and direct cardiac functional parameters were evaluated 4 weeks post-MI cell injection. Left ventricle (LV) perimeter and percentage of interstitial collagen in the spare myocardium were significantly attenuated in all ASC-treated groups compared to the non-treated (NT) and control groups (culture medium, fibrin, or collagen alone). Direct hemodynamic assessment under pharmacological stress showed that stroke volume (SV) and left ventricle end-diastolic pressure were preserved in ASC-treated groups regardless of the vehicle used to deliver ASCs. Stroke work (SW), a global index of cardiac function, improved in ASC/M while it normalized when biopolymers were co-injected with ASCs. A positive correlation was observed between cardiac ASCs retention and preservation of SV and improvement in SW post-MI under hemodynamic stress. Conclusions We provided direct evidence that intramyocardial injection of ASCs mitigates the negative cardiac remodeling and preserves ventricular function post-MI in rats and these beneficial effects can be further enhanced by administrating co-injection of ASCs with biopolymers. PMID:20711471

  14. Curcumin Attenuates Rapamycin-induced Cell Injury of Vascular Endothelial Cells.

    Science.gov (United States)

    Guo, Ning; Chen, Fangyuan; Zhou, Juan; Fang, Yuan; Li, Hongbing; Luo, Yongbai; Zhang, Yong

    2015-10-01

    Although drug-eluting stents (DES) effectively improve the clinical efficacy of percutaneous coronary intervention, a high risk of late stent thrombosis and in-stent restenosis also exists after DES implantation. Anti-smooth muscle proliferation drugs, such as rapamycin, coating stents, not only inhibit the growth of vascular smooth muscle cells but also inhibit vascular endothelial cells and delay the reendothelialization. Therefore, the development of an ideal agent that protects vascular endothelial cells from rapamycin-eluting stents is of great importance for the next generation of DES. In this study, we demonstrated that rapamycin significantly inhibited the growth of rat aortic endothelial cells in both dose- and time-dependent manner in vitro. Cell apoptosis was increased and migration was decreased by rapamycin treatments in rat aortic endothelial cells in vitro. Surprisingly, treatment with curcumin, an active ingredient of turmeric, significantly reversed these detrimental effects of rapamycin. Moreover, curcumin increased the expression of vascular nitric oxide synthases (eNOS), which was decreased by rapamycin. Furthermore, caveolin-1, the inhibitor of eNOS, was decreased by curcumin. Knockdown of eNOS by small interfering RNA significantly abrogated the protective effects of curcumin. Taken together, our results suggest that curcumin antagonizes the detrimental effect of rapamycin on aortic endothelial cells in vitro through upregulating eNOS. Therefore, curcumin is a promising combined agent for the rescue of DES-induced reendothelialization delay.

  15. Hybrid pn-junction solar cells based on layers of inorganic nanocrystals and organic semiconductors: optimization of layer thickness by considering the width of the depletion region.

    Science.gov (United States)

    Saha, Sudip K; Guchhait, Asim; Pal, Amlan J

    2014-03-07

    We report the formation and characterization of hybrid pn-junction solar cells based on a layer of copper diffused silver indium disulfide (AgInS2@Cu) nanoparticles and another layer of copper phthalocyanine (CuPc) molecules. With copper diffusion in the nanocrystals, their optical absorption and hence the activity of the hybrid pn-junction solar cells was extended towards the near-IR region. To decrease the particle-to-particle separation for improved carrier transport through the inorganic layer, we replaced the long-chain ligands of copper-diffused nanocrystals in each monolayer with short-ones. Under illumination, the hybrid pn-junctions yielded a higher short-circuit current as compared to the combined contribution of the Schottky junctions based on the components. A wider depletion region at the interface between the two active layers in the pn-junction device as compared to that of the Schottky junctions has been considered to analyze the results. Capacitance-voltage characteristics under a dark condition supported such a hypothesis. We also determined the width of the depletion region in the two layers separately so that a pn-junction could be formed with a tailored thickness of the two materials. Such a "fully-depleted" device resulted in an improved photovoltaic performance, primarily due to lessening of the internal resistance of the hybrid pn-junction solar cells.

  16. ATP Depletion Via Mitochondrial F1F0 Complex by Lethal Factor is an Early Event in B. Anthracis-Induced Sudden Cell Death

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    Mitchell W. Woodberry

    2009-08-01

    Full Text Available Bacillus anthracis’ primary virulence factor is a tripartite anthrax toxin consisting of edema factor (EF, lethal factor (LF and protective antigen (PA. In complex with PA, EF and LF are internalized via receptor-mediated endocytosis. EF is a calmodulin- dependent adenylate cyclase that induces tissue edema. LF is a zinc-metalloprotease that cleaves members of mitogen-activated protein kinase kinases. Lethal toxin (LT: PA plus LF-induced death of macrophages is primarily attributed to expression of the sensitive Nalp1b allele, inflammasome formation and activation of caspase-1, but early events that initiate these processes are unknown. Here we provide evidence that an early essential event in pyroptosis of alveolar macrophages is LF-mediated depletion of cellular ATP. The underlying mechanism involves interaction of LF with F1F0-complex gamma and beta subunits leading to increased ATPase activity in mitochondria. In support, mitochondrial DNA-depleted MH-S cells have decreased F1F0 ATPase activity due to the lack of F06 and F08 polypeptides and show increased resistance to LT. We conclude that ATP depletion is an important early event in LT-induced sudden cell death and its prevention increases survival of toxin-sensitive cells.

  17. Heme-Mediated Induction of CXCL10 and Depletion of CD34+ Progenitor Cells Is Toll-Like Receptor 4 Dependent.

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    Carmen M Dickinson-Copeland

    Full Text Available Plasmodium falciparum infection can cause microvascular dysfunction, cerebral encephalopathy and death if untreated. We have previously shown that high concentrations of free heme, and C-X-C motif chemokine 10 (CXCL10 in sera of malaria patients induce apoptosis in microvascular endothelial and neuronal cells contributing to vascular dysfunction, blood-brain barrier (BBB damage and mortality. Endothelial progenitor cells (EPC are microvascular endothelial cell precursors partly responsible for repair and regeneration of damaged BBB endothelium. Studies have shown that EPC's are depleted in severe malaria patients, but the mechanisms mediating this phenomenon are unknown. Toll-like receptors recognize a wide variety of pathogen-associated molecular patterns generated by pathogens such as bacteria and parasites. We tested the hypothesis that EPC depletion during malaria pathogenesis is a function of heme-induced apoptosis mediated by CXCL10 induction and toll-like receptor (TLR activation. Heme and CXCL10 concentrations in plasma obtained from malaria patients were elevated compared with non-malaria subjects. EPC numbers were significantly decreased in malaria patients (P < 0.02 and TLR4 expression was significantly elevated in vivo. These findings were confirmed in EPC precursors in vitro; where it was determined that heme-induced apoptosis and CXCL10 expression was TLR4-mediated. We conclude that increased serum heme mediates depletion of EPC during malaria pathogenesis.

  18. Depletion of regulatory T lymphocytes reverses the imbalance between pro- and anti-tumor immunities via enhancing antigen-specific T cell immune responses.

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    Yu-Li Chen

    Full Text Available BACKGROUND: The regulatory T cells (Tregs can actively suppress the immune responses. However, literature about detailed changes of host effective and suppressive immunities before and after depletion of Tregs in ovarian carcinomas, is rare. MATERIALS AND METHODS: Ovarian cancer patients and the ascitogenic animal model were employed. Immunologic profiles with flow cytometric analyses, immunohistochemistric staining, RT-PCR, ELISA, and ELISPOT assays were performed. In vivo depletion of Treg cells with the mAb PC61was also performed in the animal model. RESULTS: The cytokines, including IL-4 (p=0.017 and TNF-α (p=0.046, significantly decreased while others such as TGF-β (p=0.013, IL-6 (p=0.016, and IL-10 (p=0.018 were elevated in ascites of ovarian cancer patients, when the disease progressed to advanced stages. The ratio of CD8(+ T cell/Treg cell in ascites was also lower in advanced diseases than in early diseases (advanced 7.37 ± 0.64 vs. early 14.25 ± 3.11, p=0.037. The kinetic low-dose CD25 Ab depletion group had significantly lower intra-peritoneal tumor weight (0.20 ± 0.03 g than the sequential high-dose (0.69 ± 0.06 g and sequential low-dose (0.67 ± 0.07 g CD25 Ab deletion groups (p=0.001 after 49 days of tumor challenge in the animal. The kinetic low-dose CD25 Ab depletion group generated the highest number of IFN-γ-secreting, mesothelin-specific T lymphocytes compared to the other groups (p<0.001. CONCLUSIONS: The imbalance between effective and suppressive immunities becomes more severe as a tumor progresses. The depletion of Treg cells can correct the imbalance of immunologic profiles and generate potent anti-tumor effects. Targeting Treg cells can be a new strategy for the immunotherapy of ovarian carcinoma.

  19. Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells

    Energy Technology Data Exchange (ETDEWEB)

    Genz, Berit [Institute for Experimental Surgery, Rostock University Medical Center, Rostock (Germany); Thomas, Maria [Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart (Germany); Pützer, Brigitte M. [Institute of Experimental Gene Therapy and Cancer Research, Rostock University Medical Center, Rostock (Germany); Siatkowski, Marcin; Fuellen, Georg [Institute for Biostatistics and Informatics in Medicine and Ageing Research, Rostock University Medical Center, Rostock (Germany); Vollmar, Brigitte [Institute for Experimental Surgery, Rostock University Medical Center, Rostock (Germany); Abshagen, Kerstin, E-mail: kerstin.abshagen@uni-rostock.de [Institute for Experimental Surgery, Rostock University Medical Center, Rostock (Germany)

    2014-11-01

    Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. - Highlights: • We performed adenoviral overexpression of Lhx2 in primary hepatic stellate cells. • Hepatic stellate cells expressed stem cell markers during cultivation. • Cell migration and contractility was slightly hampered upon Lhx2 overexpression. • Lhx2 overexpression did not affect stem cell character of hepatic stellate cells.

  20. Heat Shock Factor 1 Depletion Sensitizes A172 Glioblastoma Cells to Temozolomide via Suppression of Cancer Stem Cell-Like Properties

    Directory of Open Access Journals (Sweden)

    Chang-Nim Im

    2017-02-01

    Full Text Available Heat shock factor 1 (HSF1, a transcription factor activated by various stressors, regulates proliferation and apoptosis by inducing expression of target genes, such as heat shock proteins and Bcl-2 (B-cell lymphoma 2 interacting cell death suppressor (BIS. HSF1 also directly interacts with BIS, although it is still unclear whether this interaction is critical in the regulation of glioblastoma stem cells (GSCs. In this study, we examined whether small interfering RNA-mediated BIS knockdown decreased protein levels of HSF1 and subsequent nuclear localization under GSC-like sphere (SP-forming conditions. Consistent with BIS depletion, HSF1 knockdown also reduced sex determining region Y (SRY-box 2 (SOX2 expression, a marker of stemness, accompanying the decrease in SP-forming ability and matrix metalloprotease 2 (MMP2 activity. When HSF1 or BIS knockdown was combined with temozolomide (TMZ treatment, a standard drug used in glioblastoma therapy, apoptosis increased, as measured by an increase in poly (ADP-ribose polymerase (PARP cleavage, whereas cancer stem-like properties, such as colony-forming activity and SOX2 protein expression, decreased. Taken together, our findings suggest that targeting BIS or HSF1 could be a viable therapeutic strategy for GSCs resistant to conventional TMZ treatment.

  1. Heat Shock Factor 1 Depletion Sensitizes A172 Glioblastoma Cells to Temozolomide via Suppression of Cancer Stem Cell-Like Properties

    Science.gov (United States)

    Im, Chang-Nim; Yun, Hye Hyeon; Lee, Jeong-Hwa

    2017-01-01

    Heat shock factor 1 (HSF1), a transcription factor activated by various stressors, regulates proliferation and apoptosis by inducing expression of target genes, such as heat shock proteins and Bcl-2 (B-cell lymphoma 2) interacting cell death suppressor (BIS). HSF1 also directly interacts with BIS, although it is still unclear whether this interaction is critical in the regulation of glioblastoma stem cells (GSCs). In this study, we examined whether small interfering RNA-mediated BIS knockdown decreased protein levels of HSF1 and subsequent nuclear localization under GSC-like sphere (SP)-forming conditions. Consistent with BIS depletion, HSF1 knockdown also reduced sex determining region Y (SRY)-box 2 (SOX2) expression, a marker of stemness, accompanying the decrease in SP-forming ability and matrix metalloprotease 2 (MMP2) activity. When HSF1 or BIS knockdown was combined with temozolomide (TMZ) treatment, a standard drug used in glioblastoma therapy, apoptosis increased, as measured by an increase in poly (ADP-ribose) polymerase (PARP) cleavage, whereas cancer stem-like properties, such as colony-forming activity and SOX2 protein expression, decreased. Taken together, our findings suggest that targeting BIS or HSF1 could be a viable therapeutic strategy for GSCs resistant to conventional TMZ treatment. PMID:28241425

  2. Macrophage depletion improves survival of porcine neonatal pancreatic cell clusters contained in alginate macrocapsules transplanted into rats.

    NARCIS (Netherlands)

    Omer, A; Keegan, M; Czismadia, E; Vos, P De; Rooijen, van N.; Bonner-Weir, S; Weir, GC

    2003-01-01

    BACKGROUND: Macrophages can accumulate on the surface of empty and islet-containing alginate capsules, leading to loss of functional tissue. In this study, the effect of peritoneal macrophage depletion on the biocompatibility of alginate macrocapsules and function of macroencapsulated porcine neonat

  3. Macrophage depletion improves survival of porcine neonatal pancreatic cell clusters contained in alginate macrocapsules transplanted into rats

    NARCIS (Netherlands)

    Omer, A; Keegan, M; Czismadia, E; De Vos, P; Van Rooijen, N; Bonner-Weir, S; Weir, GC

    2003-01-01

    Background: Macrophages can accumulate on the surface of empty and islet-containing alginate capsules, leading to loss of functional tissue. In this study, the effect of peritoneal macrophage depletion on the biocompatibility of alginate macrocapsules and function of macroencapsulated porcine neonat

  4. Secretory clusterin inhibits osteoclastogenesis by attenuating M-CSF-dependent osteoclast precursor cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Bongkun; Kang, Soon-Suk [Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Kang, Sang-Wook [Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Department of Anatomy and Cell Biology, Cell Dysfunction Research Center and BMIT, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Min, Bon-Hong [Department of Pharmacology, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of); Lee, Eun-Jin; Song, Da-Hyun; Kim, Sang-Min [Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Song, Youngsup [Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Department of Anatomy and Cell Biology, Cell Dysfunction Research Center and BMIT, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Yoon, Seung-Yong [Department of Anatomy and Cell Biology, Cell Dysfunction Research Center and BMIT, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Chang, Eun-Ju, E-mail: ejchang@amc.seoul.kr [Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Department of Anatomy and Cell Biology, Cell Dysfunction Research Center and BMIT, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of)

    2014-07-18

    Highlights: • We describe the expression and secretion of clusterin in osteoclasts. • Endogenous clusterin deficiency does not affect osteoclast formation. • Exogenous treatment with secretory clusterin decreases osteoclast differentiation. • Secretory clusterin attenuates osteoclast precursor cell proliferation by inhibiting M-CSF-mediated ERK activation. - Abstract: Secretory clusterin (sCLU)/apolipoprotein J is a multifunctional glycoprotein that is ubiquitously expressed in various tissues. Reduced sCLU in the joints of patients with bone erosive disease is associated with disease activity; however, its exact role has yet to be elucidated. Here, we report that CLU is expressed and secreted during osteoclastogenesis in mouse bone marrow-derived macrophages (BMMs) that are treated with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). CLU-deficient BMMs obtained from CLU{sup −/−} mice exhibited no significant alterations in OC differentiation in comparison with BMMs obtained from wild-type mice. In contrast, exogenous sCLU treatment significantly inhibited OC formation in both BMMs and OC precursor cultures. The inhibitory effect of sCLU was more prominent in BMMs than OC precursor cultures. Interestingly, treating BMMs with sCLU decreased the proliferative effects elicited by M-CSF and suppressed M-CSF-induced ERK activation of OC precursor cells without causing apoptotic cell death. This study provides the first evidence that sCLU reduces OC formation by inhibiting the actions of M-CSF, thereby suggesting its protective role in bone erosion.

  5. Live Cell Analysis and Mathematical Modeling Identify Determinants of Attenuation of Dengue Virus 2'-O-Methylation Mutant.

    Directory of Open Access Journals (Sweden)

    Bianca Schmid

    2015-12-01

    Full Text Available Dengue virus (DENV is the most common mosquito-transmitted virus infecting ~390 million people worldwide. In spite of this high medical relevance, neither a vaccine nor antiviral therapy is currently available. DENV elicits a strong interferon (IFN response in infected cells, but at the same time actively counteracts IFN production and signaling. Although the kinetics of activation of this innate antiviral defense and the timing of viral counteraction critically determine the magnitude of infection and thus disease, quantitative and kinetic analyses are lacking and it remains poorly understood how DENV spreads in IFN-competent cell systems. To dissect the dynamics of replication versus antiviral defense at the single cell level, we generated a fully viable reporter DENV and host cells with authentic reporters for IFN-stimulated antiviral genes. We find that IFN controls DENV infection in a kinetically determined manner that at the single cell level is highly heterogeneous and stochastic. Even at high-dose, IFN does not fully protect all cells in the culture and, therefore, viral spread occurs even in the face of antiviral protection of naïve cells by IFN. By contrast, a vaccine candidate DENV mutant, which lacks 2'-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells. Through mathematical modeling of time-resolved data and validation experiments we show that the primary determinant for attenuation is the accelerated kinetics of IFN production. This rapid induction triggered by mutant DENV precedes establishment of IFN-resistance in infected cells, thus causing a massive reduction of virus production rate. In contrast, accelerated protection of naïve cells by paracrine IFN action has negligible impact. In conclusion, these results show that attenuation of the 2'-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells.

  6. (-)Schisandrin B ameliorates paraquat-induced oxidative stress by suppressing glutathione depletion and enhancing glutathione recovery in differentiated PC12 cells.

    Science.gov (United States)

    Lam, Philip Y; Ko, Kam Ming

    2011-01-01

    Exposure to paraquat (PQ; N,N'-dimethyl-4-4'-bipyridium), a potent herbicide, can lead to neuronal cell death and increased risk of Parkinson's disease because of oxidative stress. In this study, we investigated the effect of (-)schisandrin B [(-)Sch B, a potent enantiomer of schisandrin B] on PQ-induced cell injury in differentiated pheochromocytoma cells (PC12). PQ treatment caused cell injury in PC12 cells, as indicated by the significant increase in lactate dehydrogenase (LDH) leakage. Pretreatment with (-)Sch B (5 μM) protected against PQ-induced toxicity in PC12 cells, as evidenced by the significant decrease in LDH leakage. (-)Sch B induced the cytochrome P-450-mediated reactive oxygen species generation in differentiated PC12 cells. The cytoprotection afforded by (-)Sch B pretreatment was associated with an increase in cellular reduced glutathione (GSH) level as well as the enhancement of γ-glutamylcysteine ligase (GCL) and glutathione reductase (GR) activity in PQ-challenged cells. Both GCL and GR inhibitors abrogated the cytoprotective effect of (-)Sch B in PQ-challenged cells. The biochemical mechanism underlying the GSH-enhancing effect of (-)Sch B was further investigated in PC12 cells subjected to an acute peroxide challenge. Although the initial GSH depletion induced by peroxide was reduced through GR-catalyzed regeneration of GSH in (-)Sch B-pretreated cells, the later enhanced GSH recovery was mainly mediated by GCL-catalyzed GSH synthesis. The results suggest that (-)Sch B treatment may increase the resistance of dopaminergic cells against PQ-induced oxidative stress through reducing the extent of oxidant-induced GSH depletion and enhancing the subsequent GSH recovery.

  7. Reactivation of latent tuberculosis in cynomolgus macaques infected with SIV is associated with early peripheral T cell depletion and not virus load.

    Directory of Open Access Journals (Sweden)

    Collin R Diedrich

    Full Text Available HIV-infected individuals with latent Mycobacterium tuberculosis (Mtb infection are at significantly greater risk of reactivation tuberculosis (TB than HIV-negative individuals with latent TB, even while CD4 T cell numbers are well preserved. Factors underlying high rates of reactivation are poorly understood and investigative tools are limited. We used cynomolgus macaques with latent TB co-infected with SIVmac251 to develop the first animal model of reactivated TB in HIV-infected humans to better explore these factors. All latent animals developed reactivated TB following SIV infection, with a variable time to reactivation (up to 11 months post-SIV. Reactivation was independent of virus load but correlated with depletion of peripheral T cells during acute SIV infection. Animals experiencing reactivation early after SIV infection (<17 weeks had fewer CD4 T cells in the periphery and airways than animals reactivating in later phases of SIV infection. Co-infected animals had fewer T cells in involved lungs than SIV-negative animals with active TB despite similar T cell numbers in draining lymph nodes. Granulomas from these animals demonstrated histopathologic characteristics consistent with a chronically active disease process. These results suggest initial T cell depletion may strongly influence outcomes of HIV-Mtb co-infection.

  8. Inhibition of mixed lineage kinase 3 attenuates MPP+-induced neurotoxicity in SH-SY5Y cells.

    Science.gov (United States)

    Mathiasen, Joanne R; McKenna, Beth Ann W; Saporito, Michael S; Ghadge, Ghanashyam D; Roos, Raymond P; Holskin, Beverly P; Wu, Zhi-Liang; Trusko, Stephen P; Connors, Thomas C; Maroney, Anna C; Thomas, Beth Ann; Thomas, Jeffrey C; Bozyczko-Coyne, Donna

    2004-04-02

    The neuropathology of Parkinson's Disease has been modeled in experimental animals following MPTP treatment and in dopaminergic cells in culture treated with the MPTP neurotoxic metabolite, MPP(+). MPTP through MPP(+) activates the stress-activated c-Jun N-terminal kinase (JNK) pathway in mice and SH-SY5Y neuroblastoma cells. Recently, it was demonstrated that CEP-1347/KT7515 attenuated MPTP-induced nigrostriatal dopaminergic neuron degeneration in mice, as well as MPTP-induced JNK phosphorylation. Presumably, CEP-1347 acts through inhibition of at least one upstream kinase within the mixed lineage kinase (MLK) family since it has been shown to inhibit MLK 1, 2 and 3 in vitro. Activation of the MLK family leads to JNK activation. In this study, the potential role of MLK and the JNK pathway was examined in MPP(+)-induced cell death of differentiated SH-SY5Y cells using CEP-1347 as a pharmacological probe and dominant negative adenoviral constructs to MLKs. CEP-1347 inhibited MPP(+)-induced cell death and the morphological features of apoptosis. CEP-1347 also prevented MPP(+)-induced JNK activation in SH-SY5Y cells. Endogenous MLK 3 expression was demonstrated in SH-SY5Y cells through protein levels and RT-PCR. Adenoviral infection of SH-SY5Y cells with a dominant negative MLK 3 construct attenuated the MPP(+)-mediated increase in activated JNK levels and inhibited neuronal death following MPP(+) addition compared to cultures infected with a control construct. Adenoviral dominant negative constructs of two other MLK family members (MLK 2 and DLK) did not protect against MPP(+)-induced cell death. These studies show that inhibition of the MLK 3/JNK pathway attenuates MPP(+)-mediated SH-SY5Y cell death in culture and supports the mechanism of action of CEP-1347 as an MLK family inhibitor.

  9. Dose-dependent ATP depletion and cancer cell death following calcium electroporation, relative effect of calcium concentration and electric field strength

    DEFF Research Database (Denmark)

    Hansen, Emilie Louise; Sozer, Esin Bengisu; Romeo, Stefania;

    2015-01-01

    death and could be a novel cancer treatment. This study aims at understanding the relationship between applied electric field, calcium concentration, ATP depletion and efficacy. METHODS: In three human cell lines--H69 (small-cell lung cancer), SW780 (bladder cancer), and U937 (leukaemia), viability...... was observed with fluorescence confocal microscopy of quinacrine-labelled U937 cells. RESULTS: Both H69 and SW780 cells showed dose-dependent (calcium concentration and electric field) decrease in intracellular ATP (p...-dependently reduced cell survival and intracellular ATP. Increasing extracellular calcium allows the use of a lower electric field. GENERAL SIGNIFICANCE: This study supports the use of calcium electroporation for treatment of cancer and possibly lowering the applied electric field in future trials....

  10. Intravenous transplantation of mesenchymal stem cells attenuates oleic acid induced acute lung injury in rats

    Institute of Scientific and Technical Information of China (English)

    XU Yu-lin; LIU Ying-long; WANG Qiang; LI Gang; L(U) Xiao-dong; KONG Bo

    2012-01-01

    Background Acute lung injury (ALI) and end-stage acute respiratory distress syndrome (ARDS) were among the most common causes of death in intensive care units.The activation of an inflammatory response and the damage of pulmonary epithelium and endotheliumwerethe hallmark of ALI/ARDS.Recent studies had demonstrated the importance of mesenchymal stem cells (MSCs) in maintaining the normal pulmonary endothelial and epithelial function as well as participating in modulating the inflammatory response and they are involved in epithelial and endothelial repair after injury.Here,our study demonstrates MSCs therapeutic potential in a rat model of ALI/ARDS.Methods Bone marrow derived MSCs were obtained from Sprague-Dawley (SD) rats and their differential potential was verified.ALl was induced in rats byoleic acid (OA),and MSCs were transplanted intravenously.The lung injury and the concentration of cytokines in plasma and lung tissue extracts were assessed at 8 hours,24 hours and 48 hours after OA-injection.Results The histological appearance and water content in rat lung tissue were significantly improved at different time points in rats treated with MSCs.The concentration of tumor necrosis factor-α and intercellular adhesion molecular-1 in rats plasma and lung tissue extracts were significantly inhibited after intravenous transplantation of MSCs,whereas interleukin-10 was significantly higher after MSCs transplantation at 8 hours,24 hours and 48 hours after OA-challenge.Conclusions Intravenous transplantation of MSCs could maintain the integrity of the pulmonary alveolar-capillary barrier and modulate the inflammatory response to attenuate the experimental ALI/ARDS.Transplantation of MSCs could be a novel cell-based therapeutic strategy for prevention and treatment of ALI/ARDS.

  11. CD45 immunoaffinity depletion of vesicles from Jurkat T cells demonstrates that exosomes contain CD45: no evidence for a distinct exosome/HIV-1 budding pathway

    Directory of Open Access Journals (Sweden)

    Ott David E

    2008-07-01

    Full Text Available Abstract The presence of relatively high levels of cellular protein contamination in density-purified virion preparations is a confounding factor in biochemical analyses of HIV and SIV produced from hematopoietic cells. A major source of this contamination is from vesicles, either microvesicles or exosomes, that have similar physical properties as virions. Thus, these particles can not be removed by size or density fractionation. Although virions and vesicles have similar cellular protein compositions, CD45 is excluded from HIV-1 yet is present in vesicles produced from hematopoietic cells. By exploiting this finding, we have developed a CD45 immunoaffinity depletion procedure that removes vesicles from HIV-1 preparations. While this approach has been successfully applied to virion preparations from several different cell types, some groups have concluded that "exosomes" from certain T cell lines, specifically Jurkat, do not contain CD45. If this interpretation is correct, then these vesicles could not be removed by CD45 immunoaffinity depletion. Here we show that dense vesicles produced by Jurkat and SupT1/CCR5 cells contain CD45 and are efficiently removed from preparations by CD45-immunoaffinity depletion. Also, contaminating cellular proteins were removed from virion preparations produced by these lines. Previously, the absence of CD45 from both "exosomes" and virions has been used to support the so called Trojan exosome hypothesis, namely that HIV-1 is simply an exosome containing viral material. The presence of CD45 on vesicles, including exosomes, and its absence on virions argues against a specialized budding pathway that is shared by both exosomes and HIV-1.

  12. Taxifolin synergizes Andrographolide-induced cell death by attenuation of autophagy and augmentation of caspase dependent and independent cell death in HeLa cells.

    Science.gov (United States)

    Alzaharna, Mazen; Alqouqa, Iyad; Cheung, Hon-Yeung

    2017-01-01

    Andrographolide (Andro) has emerged recently as a potential and effective anticancer agent with induction of apoptosis in some cancer cell lines while induction of G2/M arrest with weak apoptosis in others. Few studies have proved that Andro is also effective in combination therapy. The flavonoid Taxifolin (Taxi) has showed anti-oxidant and antiproliferative effects against different cancer cells. Therefore, the present study investigated the cytotoxic effects of Andro alone or in combination with Taxi on HeLa cells. The combination of Andro with Taxi was synergistic at all tested concentrations and combination ratios. Andro alone induced caspase-dependent apoptosis which was enhanced by the combination with Taxi and attenuated partly by using Z-Vad-Fmk. Andro induced a protective reactive oxygen species (ROS)-dependent autophagy which was attenuated by Taxi. The activation of p53 was involved in Andro-induced autophagy where the use of Taxi or pifithrin-α (PFT-α) decreased it while the activation of JNK was involved in the cell death of HeLa cells but not in the induction of autophagy. The mitochondrial outer-membrane permeabilization (MOMP) plays an important role in Andro-induced cell death in HeLa cells. Andro alone increased the MOMP which was further increased in the case of combination. This led to the increase in AIF and cytochrome c release from mitochondria which consequently increased caspase-dependent and independent cell death. In conclusion, Andro induced a protective autophagy in HeLa cells which was reduced by Taxi and the cell death was increased by increasing the MOMP and subsequently the caspase-dependent and independent cell death.

  13. Resveratrol induces cellular senescence with attenuated mono-ubiquitination of histone H2B in glioma cells

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Zhen; Xu, Michael S.; Barnett, Tamara L. [Nevada Cancer Institute, Las Vegas, NV 89135 (United States); Xu, C. Wilson, E-mail: wxu@nvcancer.org [Nevada Cancer Institute, Las Vegas, NV 89135 (United States)

    2011-04-08

    Research highlights: {yields} Resveratrol induces cellular senescence in glioma cell. {yields} Resveratrol inhibits mono-ubiquitination of histone H2B at K120. {yields} Depletion of RNF20, phenocopies the inhibitory effects of resveratrol. {yields} Mono-ubiquitination of histone H2B at K120 is a novel target of resveratrol. {yields} RNF20 inhibits cellular senescence in proliferating glioma cells. -- Abstract: Resveratrol (3,4',5-trihydroxy-trans-stilbene), a polyphenol naturally occurring in grapes and other plants, has cancer chemo-preventive effects and therapeutic potential. Although resveratrol modulates multiple pathways in tumor cells, how resveratrol or its affected pathways converge on chromatin to mediate its effects is not known. Using glioma cells as a model, we showed here that resveratrol inhibited cell proliferation and induced cellular hypertrophy by transforming spindle-shaped cells to enlarged, irregular and flatten-shaped ones. We further showed that resveratrol-induced hypertrophic cells expressed senescence-associated-{beta}-galactosidase, suggesting that resveratrol-induced cellular senescence in glioma cells. Consistent with these observations, we demonstrated that resveratrol inhibited clonogenic efficiencies in vitro and tumor growth in a xenograft model. Furthermore, we found that acute treatment of resveratrol inhibited mono-ubiquitination of histone H2B at K120 (uH2B) in breast, prostate, pancreatic, lung, brain tumor cells as well as primary human cells. Chronic treatment with low doses of resveratrol also inhibited uH2B in the resveratrol-induced senescent glioma cells. Moreover, we showed that depletion of RNF20, a ubiquitin ligase of histone H2B, inhibited uH2B and induced cellular senescence in glioma cells in vitro, thereby recapitulated the effects of resveratrol. Taken together, our results suggest that uH2B is a novel direct or indirect chromatin target of resveratrol and RNF20 plays an important role in inhibiting cellular

  14. Alarming Oxygen Depletion Caused by Hydrogen Combustion and Fuel Cells and their Resolution by Magnegas$^{TM}$

    OpenAIRE

    Santilli, R. M.

    2000-01-01

    We recall that hydrogen combustion does resolve the environmental problems of fossil fuels due to excessive emission of carcinogenic substances and carbon dioxide. However, hydrogen combustion implies the permanent removal from our atmosphere of directly usable oxygen, a serious environmental problem called oxygen depletion, since the combustion turns oxygen into water whose separation to restore the original oxygen is prohibitive due to cost. We then show that a conceivable global use of hyd...

  15. IL-2-targeted therapy ameliorates the severity of graft-versus-host disease: ex vivo selective depletion of host-reactive T cells and in vivo therapy.

    Science.gov (United States)

    Yarkoni, Shai; Prigozhina, Tatyana B; Slavin, Shimon; Askenasy, Nadir

    2012-04-01

    T cell depletion prevents graft-versus-host disease (GVHD) but also removes T cell-mediated support of hematopoietic cell engraftment. A chimeric molecule composed of IL-2 and caspase-3 (IL2-cas) has been evaluated as a therapeutic modality for GVHD and selective ex vivo depletion of host-reactive T cells. IL2-cas does not affect hematopoietic cell engraftment and significantly reduces the clinical and histological severity of GVHD. Early administration of IL2-cas reduced the lethal outcome of haploidentical transplants, and survivor mice displayed markedly elevated levels of X-linked forkhead/winged helix (FoxP3(+); 50%) and CD25(+)FoxP3(+) T cells (35%) in the lymph nodes. The chimeric molecule induces in vitro apoptosis in both CD4(+)CD25(-) and CD4(+)CD25(+) subsets of lymphocytes from alloimmunized mice, and stimulates proliferation of cells with highest levels of CD25 expression. Adoptive transfer of IL2-cas-pretreated viable splenocytes into sublethally irradiated haploidentical recipients resulted in 60% survival after a lethal challenge with lipopolysaccharide, which is associated with elevated fractions of CD25(high)FoxP3(+) T cells in the lymph nodes of survivors. These data demonstrate that ex vivo purging of host-presensitized lymphocytes is effectively achieved with IL2-cas, and that IL-2-targeted apoptotic therapy reduces GVHD severity in vivo. Both approaches promote survival in lethal models of haploidentical GVHD. The mechanism of protection includes direct killing of GVHD effectors, prevention of transition to effector/memory T cells, and induction of regulatory T cell proliferation, which becomes the dominant subset under conditions of homeostatic expansion.

  16. Comparison of use of Vero cell line and suspension culture of murine macrophage to attenuation of virulence of Neospora caninum.

    Science.gov (United States)

    Khordadmehr, Monireh; Namavari, Mehdi; Khodakaram-Tafti, Azizollah; Mansourian, Maryam; Rahimian, Abdollah; Daneshbod, Yahya

    2013-10-01

    In this study the tachyzoite yields of Neospora caninum were compared in two cell lines: Vero (African Green Monkey Kidney) and suspension culture of murine macrophage (J774) cell lines. Then, N. caninum were continuously passaged in these cell lines for 3 months and the effect of host cells on virulence of tachyzoites was assessed by broiler chicken embryonated eggs. Inoculation was performed in the chorioallantoic (CA) liquid of the embryonated eggs with different dilutions (0.5 × 10(4), 1.0 × 10(4), 1.5 × 10(4)) of tachtzoites isolated from these cell cultures. The mortality pattern and pathological changes of the dead embryos and hatched chickens were noted. Tissue samples of brain, liver and heart were examined by histopathological and detection of DNA of parasite by polymerase chain reaction (PCR). Also, consecutive sections of the tissues examined histologically were used for immunohistochemical (IHC) examination. Embryos inoculated with tachyzoites derived from Vero cell line (group V) showed a higher mortality rate (100%) than the embryos that received tachyzoites derived from J774 cell line (group J) (10% mortality rate). The results of this study indicated that the culture of N. caninum in J774 cell led to a marked increase in the number of tachyzoite yields and rapid attenuation in comparison to Vero, so the results were confirmed by IHC and PCR. This study is the first report of the significant effect of host cell on the attenuation of virulence of N. caninum tachyzoites. These findings could potentially provide a practical approach in the mass production of N. caninum tachyzoites, and also in producing live attenuated vaccine.

  17. Cell-permeable Tat-NBD peptide attenuates rat pancreatitis and acinus cell inflammation response

    Institute of Scientific and Technical Information of China (English)

    You-Ming Long; Ken Chen; Xue-Jin Liu; Wen-Rui Xie; Hui Wang

    2009-01-01

    AIM: To investigate the effects of Tat-NEMO-binding domain (NBD) peptide on taurocholate-induced pancreatitis and lipopolysaccharide (LPS)-stimulated AR42J acinus cells inflammatory response in rats. METHODS: Sodium taurocholate (5%) was used to induce the pancreatitis model. Forty-eight rats from the taurocholate group aeceuved an intravenous bolus of 13 mg/kg Tat-NBD (wild-type, WT) peptide, Tat-NBD (mutant-type, MT) peptide, NBD peptide or Tat peptide. The pancreatic histopathology was analyzed by hematoxylin staining. LPS was added to the culture medium to stimulate the AR42J cells. For pretreatment, cells were incubated with different peptides for 2 h before LPS stimulation. Expression of IL-1β and TNF-α mRNA was analyzed using a semi-quantitative reverse-transcript polymerase chain reaction (RT-PCR) method. IL-1β and TNF-α protein in culture medium were detected by enzyme linked immunosorbent assay (ELISA). NF-κB DNA-binding in pancreas was examined by electrophoretic mobility shift assays. P65 expression of AR42J was determined by Strept Actividin-Biotin Complex (SABC) method. RESULTS: Pretreatment with Tat-NBD (WT) peptide at a concentration of 13 mg/kg body wt showed beneficial effect in pancreaitis model. LPS (10 mg/L) resulted in an increase of IL-1β mRNA, IL-1β protein, TNF-α mRNA and TNF-α protein, whereas significantly inhibitory effects were observed when cells were incubated with Tat-NBD (WT). Consisting with p65 expression decrease analyzed by SABC method, NF-κB DNA-binding activity significantly decreased in Tat-NBD (WT) pretreatment group, especially at the largest dose. No significant changes were found in the control peptide group. CONCLUSION: Our result supports that active NF-κB participates in the pathogenesis of STC-induced acute pancreatitis in rats. Tat-NBD (WT) peptide has antiinflammatory effects in this model and inhibits the inflammation of acinus simulated by LPS.

  18. Depletion of sirtuin 1 (SIRT1 leads to epigenetic modifications of telomerase (TERT gene in hepatocellular carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Bin Zhang

    Full Text Available Sirtuin 1 (SIRT1 is a nicotinamide adenine dinucleotide (NAD-dependent deacetylase that is implicated in plethora of biological processes, including metabolism, aging, stress response, and tumorigenesis. Telomerase (TERT is essential for telomere maintenance. Activation of TERT is considered a crucial step in tumorigenesis, and therefore it is a potential therapeutic target against cancer. We have recently found that SIRT1 expression is highly elevated in hepatocellular carcinoma, and the depletion of SIRT1 leads to substantial reduction in TERT mRNA and protein expression. However, the underlying molecular mechanism of SIRT1-dependent TERT expression remains uncharacterized. Here, we elucidated if SIRT1 regulates TERT expression via transcriptional, epigenetic and post-transcriptional mechanisms. We report that depletion of SIRT1 does not lead to significant change in transcriptional activity and CpG methylation patterns of the TERT promoter, nor does it affect mRNA stability or 3'-UTR regulation of TERT. Intriguingly, depletion of SIRT1 is associated with substantial induction of acetylated histone H3-K9 and reduction of trimethyl H3-K9 at the TERT gene, which are known to be associated with gene activation. Our data revealed that SIRT1 regulates histone acetylation and methylation at the TERT promoter. We postulated that SIRT1 may regulate TERT expression via long-range interaction, or via yet unidentified histone modifications.

  19. A splenic marginal zone-like peripheral blood CD27+B220- B cell population is preferentially depleted in HIV type 1-infected individuals.

    Science.gov (United States)

    Morrow, Matthew; Valentin, Antonio; Little, Richard; Yarchoan, Robert; Pavlakis, George N

    2008-04-01

    Peripheral blood CD27(+) B cells are reduced in HIV-1-infected individuals. In healthy individuals, the human peripheral blood CD27(+) B cell pool consists of two subsets defined by the expression, or lack thereof, of the CD45 isoform B220. We investigated the presence of circulating B220(+) and B220(-) memory B cells in HIV(+) individuals and found that the reduction in CD27(+) memory B cells occurs primarily among CD27(+)B220(-) B cells. Studies conducted using healthy controls indicate that CD27(+)B220(-) B cells have a splenic marginal zone like the immunophenotype IgM(hi)IgD(lo)CD21(+)CD23(-), express TLR9, and proliferate and secrete IgG and IgM in response to B cell-specific ODN. CD27(+)B220(+) B cells have the immunophenotype IgM(lo)IgD(hi)CD21(+)CD23(+), express activation-induced cytidine deaminase, and proliferate in response to SAC but do not secrete immunoglobulin. The AICD expression, along with CD86 expression, by CD27(+)B220(+) suggests these cells are of germinal center origin. The preferential depletion of CD27(+)B220(-) B cells mirrors alterations in spleen morphology and resident B cell populations due to HIV infection reported by other investigators and may play an important role in the defective B cell immunity against T-independent pathogens such as pneumococcus observed in HIV-1-infected individuals.

  20. Ba2+- and bupivacaine-sensitive background K+ conductances mediate rapid EPSP attenuation in oligodendrocyte precursor cells.

    Science.gov (United States)

    Chan, Chu-Fang; Kuo, Tzu-Wei; Weng, Ju-Yun; Lin, Yen-Chu; Chen, Ting-Yu; Cheng, Jen-Kun; Lien, Cheng-Chang

    2013-10-01

    Glutamatergic transmission onto oligodendrocyte precursor cells (OPCs) may regulate OPC proliferation, migration and differentiation. Dendritic integration of excitatory postsynaptic potentials (EPSPs) is critical for neuronal functions, and mechanisms regulating dendritic propagation and summation of EPSPs are well understood. However, little is known about EPSP attenuation and integration in OPCs. We developed realistic OPC models for synaptic integration, based on passive membrane responses of OPCs obtained by simultaneous dual whole-cell patch-pipette recordings. Compared with neurons, OPCs have a very low value of membrane resistivity, which is largely mediated by Ba(2+)- and bupivacaine-sensitive background K(+) conductances. The very low membrane resistivity not only leads to rapid EPSP attenuation along OPC processes but also sharpens EPSPs and narrows the temporal window for EPSP summation. Thus, background K(+) conductances regulate synaptic responses and integration in OPCs, thereby affecting activity-dependent neuronal control of OPC development and function.

  1. Antigen-Specific lgA B Memory Cell Responses to Shigella Antigens Elicited in Volunteers Immunized with Live Attenuated Shigella flexneri 2a Oral Vaccine Candidates

    Science.gov (United States)

    2011-01-01

    cell responses to Shigella antigens elicited in volunteers immunized with live attenuated Shigella flexneri 2a oral vaccine candidates J.K. Simona,b... Shigella ;. B cell memory; Immunoglobulin lgA; Mucosal immunity Abstract We studied the induction of antigen-specific lgA memory B cells (BM) in...volunteers who received live attenuated Shigella flexneri 2a vaccines. Subjects ingested a single oral dose of 107 , 108 or 109 CFU of S. flexneri 2a with

  2. Depletion of CD4+CD25+ regulatory T cells can promote local immunity to suppress tumor growth in benzo[a]pyrene-induced forestomach carcinoma

    Institute of Scientific and Technical Information of China (English)

    Yi-Ling Chen; Jung-Hua Fang; Ming-Derg Lai; Yan-Shen Shan

    2008-01-01

    AIM: To elucidate the distribution of CD4+CD25+ regulatory T cells (Tregs) in different lymphoid tissues and its local enhancement on tumor growth before and after depletion of CD4+CD25+ Tregs.METHODS: Female ICR mice were gavaged with benzo[a]pyrene (BaP) to induce forestomach carcinoma. CD4+CD25+ Tregs were intraperitoneally depleted with monoclonal antibody PC61. These mice were divided into BaP-only, BaP+IgG, BaP+PC61, and control groups. The forestomach of mice was dissected for histological analysis, and tunnel test was performed for apoptosis of tumor cells. CD4+CD25+ Tregs were sorted from different lymphoid tissues and expression of Foxp3, IL-10, and chemokine receptors was analyzed by flow cytometry, semi-quantitative and real-time polymerase chain reaction.RESULTS: The mice gavaged with only BaP showed increased forestomach papilloma and carcinoma at wk 16 and 32. The proportion of CD4+CD25+ Tregs was significantly higher in peri-stomach regional lymph nodes than in other lymphoid tissues. These CD4+CD25+ Tregs in regional lymph nodes expressed higher levels of Foxp3 and IL-10, enriched in the CD62L-subset, and CCR1 and CCR5 chemokine receptors. In mice gavaged with BaP+PC61, the number of tumor nodules and tumor volume decreased significantly with massive infiltrating cells and apoptosis of tumor cells. In the draining regional lymph nodes, the number of CD4+CD25+ Tregs also decreased significantly.CONCLUSION: Inducible and activated CD4+CD25+ Tregs in the draining regional lymph nodes suppress host local immunity during tumor growth. Depletion of CD4+CD25+ Tregs can promote host local immunity to suppress tumor growth.

  3. Phenolic antioxidants attenuate hippocampal neuronal cell damage against kainic acid induced excitotoxicity

    Indian Academy of Sciences (India)

    M S Parihar; Taruna Hemnani

    2003-02-01

    Increasing evidence supports the role of excitotoxicity in neuronal cell injury. Thus, it is extremely important to explore methods to retard or reverse excitotoxic neuronal injury. In this regard, certain dietary compounds are begining to receive increased attention, in particular those involving phytochemicals found in medicinal plants in alleviating neuronal injury. In the present study, we examined whether medicinal plant extracts protect neurons against excitotoxic lesions induced by kainic acid (KA) in female Swiss albino mice. Mice were anesthetized with ketamine and xylazine (200 mg and 2 mg/kg body wt. respectively) and KA (0.25 g in a volume of 0.5 l) was administered to mice by intra hippocampal injections. The results showed an impairment of the hippocampus region of brain after KA injection. The lipid peroxidation and protein carbonyl content were significantly ( < 0.05) increased in comparison to controls. Glutathione peroxidase (GPx) activity (EC 1.11.1.9) and reduced glutathione (GSH) content declined after appearance of excitotoxic lesions. As GPx and GSH represent a major pathway in the cell for metabolizing hydrogen peroxide (H2O2), their depletion would be expected to allow H2O2 to accumulate to toxic levels. Dried ethanolic plant extracts of Withania somnifera (WS), Convolvulus pleuricauas (CP) and Aloe vera (AV) dissolved in distilled water were tested for their total antioxidant activity. The diet was prepared in terms of total antioxidant activity of plant extracts. The iron (Fe3+) reducing activity of plant extracts was also tested and it was found that WS and AV were potent reductants of Fe3+ at pH 5.5. CP had lower Fe3+ reducing activity in comparison to WS and AV. Plant extracts given singly and in combination 3 weeks prior to KA injections resulted in a decrease in neurotoxicity. Measures of lipid peroxidation and protein carbonyl declined. GPx activity and GSH content were elevated in hippocampus supplemented with WS and combination of

  4. Magnolol inhibits migration of vascular smooth muscle cells via cytoskeletal remodeling pathway to attenuate neointima formation

    Energy Technology Data Exchange (ETDEWEB)

    Karki, Rajendra [Division of Pharmacology and Toxicology, School of Pharmacy, University of Missouri-Kansas City (United States); Department of Oriental Medicine Resources, Mokpo National University (Korea, Republic of); Kim, Seong-Bin [Jeollanamdo Development Institute for Korean Traditional Medicine, Jangheung gun, Jeollanamdo (Korea, Republic of); Kim, Dong-Wook, E-mail: dbkim@mokpo.ac.kr [Department of Oriental Medicine Resources, Mokpo National University (Korea, Republic of)

    2013-12-10

    Background: Increased proliferation and migration of vascular smooth muscle cells (VSMCs) contribute importantly to the formation of both atherosclerotic and restenotic lesions. The objective of this study was to investigate the effect of magnolol on VSMC migration. Methods: The proteolytic activity of matrix metalloproteinases (MMPs) in tumor necrosis factor alpha (TNF-α) stimulated VSMCs was performed by gelatin zymography. VSMC migration was assessed by wound healing and Boyden chamber methods. Collagen induced VSMC adhesion was determined by spectrofluorimeter and stress fibers formation was evaluated by fluorescence microscope. The expression of signaling molecules involved in stress fibers formation was determined by western blot. The phosphorylation of myosin light chain (MLC20) was determined by urea-glycerol polyacrylamide gel electrophoresis. Immunohistochemistry was performed to determine the expression of β1-integrin and collagen type I in the injured carotid arteries of rats on day 35 after vascular injury. Results: VSMC migration was strongly inhibited by magnolol without affecting MMPs expression. Also, magnolol inhibited β1-integrin expression, FAK phosphorylation and RhoA and Cdc42 activation to inhibit the collagen induced stress fibers formation. Moreover, magnolol inhibited the phosphorylation of MLC20. Our in vivo results showed that magnolol inhibited β1-integrin expression, collagen type I deposition and FAK phosphorylation in injured carotid arteries without affecting MMP-2 activity. Conclusions: Magnolol inhibited VSMC migration via inhibition of cytoskeletal remodeling pathway to attenuate neointima formation. General significance: This study provides a rationale for further evaluation of magnolol for the management of atherosclerosis and restenosis. - Highlights: • Magnolol strongly inhibited migration of VSMCs. • Magnolol inhibited stress fibers formation. • MLC20 phosphorylation was also inhibited by magnolol. • Anti

  5. A calcium-insensitive attenuated nitrosative stress response contributes significantly in the radioresistance of Sf9 insect cells.

    Science.gov (United States)

    Suman, Shubhankar; Seth, Rakesh Kumar; Chandna, Sudhir

    2011-09-01

    Lepidopteran insects/insect cells display 50-100 times higher radioresistance than humans, and are evolutionarily closest to mammals amongst all radioresistant organisms known. Compared to mammalian cells, Lepidopteran cells (TN-368, Sf9) display more efficient antioxidant system and DNA repair and suffer considerably less radiation-induced DNA/cytogenetic damage and apoptosis. Recent studies indicate that a considerably lower radiation-induced oxidative stress may significantly reduce macromolecular damage in Lepidopteran cells. Since nitrosative stress contributes in radiation-induced cellular damage, we investigated its nature in the γ-irradiated Sf9 cells (derived from Spodoptera frugiperda; order Lepidoptera; family Noctuidae) and compared with BMG-1 human cell line having significant NOS expression. Radiation induced considerably less ROS/RNS in Sf9 cells, which remained unchanged on treatment with NOS inhibitor l-NMMA. Surprisingly, growth of Sf9 cultures or irradiation could not induce NO or its metabolites, indicating negligible basal/radiation-induced NOS activity that remained unchanged even after supplementation with arginine. Cytosolic calcium release following high-dose (1000-2000Gy at 61.1cGys(-1)) γ-irradiation or H(2)O(2) (250μM) treatment also failed to generate NO in Sf9 cells having high constitutive levels of calmodulin, whereas BMG-1 cells displayed considerable calcium-dependent NO generation even following 10Gy dose. These results strongly imply the lack of calcium-mediated NOS activity in Sf9 cells. Addition of exogenous NO from GSH-NO caused considerable increase in radiation-induced apoptosis, indicating significant contribution of constitutively attenuated nitrosative stress response into the radioresistance of Lepidopteran cells. Our study demonstrates for the first time that a calcium-insensitive, attenuated nitrosative stress response may contribute significantly in the unusual radioresistance displayed by Lepidopteran insect cells.

  6. CD20+ B Cell Depletion in Systemic Autoimmune Diseases: Common Mechanism of Inhibition or Disease-Specific Effect on Humoral Immunity?

    Directory of Open Access Journals (Sweden)

    Panagiotis Pateinakis

    2014-01-01

    Full Text Available Autoimmunity remains a complex physiologic deviation, enabled and perpetuated by a variety of interplayers and pathways. Simplistic approaches, targeting either isolated end-effectors of more centrally placed interactors of these mechanisms, are continuously tried in an effort to comprehend and halt cascades with potential disabling and deleterious effects in the affected individuals. This review focuses on theoretical and clinically proved effects of rituximab-induced CD20+ B cell depletion on different systemic autoimmune diseases and extrapolates on pathogenetic mechanisms that may account for different interindividual or interdisease responses.

  7. Glucocorticoids induce CCN5/WISP-2 expression and attenuate invasion in oestrogen receptor-negative human breast cancer cells.

    Science.gov (United States)

    Ferrand, Nathalie; Stragier, Emilien; Redeuilh, Gérard; Sabbah, Michèle

    2012-10-01

    CCN5 (cysteine-rich 61/connective tissue growth factor/nephroblastoma overexpressed 5)/WISP-2 [WNT1 (wingless-type MMTV integration site family, member 1)-inducible signalling pathway protein 2] is an oestrogen-regulated member of the CCN family. CCN5 is a transcriptional repressor of genes associated with the EMT (epithelial-mesenchymal transition) and plays an important role in maintenance of the differentiated phenotype in ER (oestrogen receptor)-positive breast cancer cells. In contrast, CCN5 is undetectable in more aggressive ER-negative breast cancer cells. We now report that CCN5 is induced in ER-negative breast cancer cells such as MDA-MB-231 following glucocorticoid exposure, due to interaction of the endogenous glucocorticoid receptor with a functional glucocorticoid-response element in the CCN5 gene promoter. Glucocorticoid treatment of MDA-MB-231 cells is accompanied by morphological alterations, decreased invasiveness and attenuated expression of mesenchymal markers, including vimentin, cadherin 11 and ZEB1 (zinc finger E-box binding homeobox 1). Interestingly, glucocorticoid exposure did not increase CCN5 expression in ER-positive breast cancer cells, but rather down-regulated ER expression, thereby attenuating oestrogen pathway signalling. Taken together, our results indicate that glucocorticoid treatment of ER-negative breast cancer cells induces high levels of CCN5 expression and is accompanied by the appearance of a more differentiated and less invasive epithelial phenotype. These findings propose a novel therapeutic strategy for high-risk breast cancer patients.

  8. Early changes of graft function, cytokines and superoxide dismutase serum levels after donor liver denervation and Kupffer cell depletion in a rat-to-rat liver transplantation model

    Institute of Scientific and Technical Information of China (English)

    Hong Zhu; Catena Marco; Ferla Gianfranco

    2009-01-01

    BACKGROUND:Hepatic reperfusion injury may cause acute inlfammatory damage, producing signiifcant organ dysfunction, and is an important problem in liver transplantation. This experiment aimed to study early changes of hepatic function after donor liver denervation and Kupffer cell depletion in rat-to-rat liver transplantation and to evaluate the effect of pre-treatment on liver reperfusion injury. METHODS:Donor rats were divided into four groups:control group; group G was pre-treated with gadolinium chloride (G), an inhibitor of Kupffer cells; group H with hexamethonium (H), a sympathetic ganglionic blocking agent; and group HG, with combined H and G pre-treatment. Under the same conditions, serum alanine aminotransferase (ALT), arterial ketone body ratio (AKBR), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and superoxide dismutase (SOD) of recipient rats were assessed at 4, 8, 16 and 24 hours after liver transplantation. Histological studies of the grafts were compared. RESULTS:HG pre-treatment signiifcantly decreased ALT, TNF-α, and IL-6 levels, increased AKBR and SOD levels, and demonstrated less pathological damage at 8, 16 and 24 hours compared with the control group. Similar trends were also found in the other groups (G and H). However, the differences among them were not signiifcant at 4 post-operative hours.CONCLUSIONS:Donor denervation and Kupffer cell depletion had preventive effect on liver reperfusion injury. HG pre-treatment is a feasible and reproducible method to protect grafts from reperfusion injury.

  9. An amino acid depleted cell-free protein synthesis system for the incorporation of non-canonical amino acid analogs into proteins.

    Science.gov (United States)

    Singh-Blom, Amrita; Hughes, Randall A; Ellington, Andrew D

    2014-05-20

    Residue-specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue-specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high-efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies.

  10. Thiosemicarbazone p-Substituted Acetophenone Derivatives Promote the Loss of Mitochondrial Δψ, GSH Depletion, and Death in K562 Cells

    Directory of Open Access Journals (Sweden)

    Felipe S. Pessoto

    2015-01-01

    Full Text Available A series of thiosemicarbazone (TSC p-substituted acetophenone derivatives were synthesized and chemically characterized. The p-substituents appended to the phenyl group of the TSC structures were hydrogen, fluor, chlorine, methyl, and nitro, producing compounds named TSC-H, TSC-F, TSC-Cl, TSC-Me, and TSC-NO2, respectively. The TSC compounds were evaluated for their capacity to induce mitochondrial permeability, to deplete mitochondrial thiol content, and to promote cell death in the K562 cell lineage using flow cytometry and fluorescence microscopy. TSC-H, TSC-F, and TSC-Cl exhibited a bell-shaped dose-response curve for the induction of apoptosis in K562 cells due to the change from apoptosis to necrosis as the principal mechanism of cell death at the highest tested doses. TSC-Me and TSC-NO2 exhibited a typical dose-response profile, with a half maximal effective concentration of approximately 10 µM for cell death. Cell death was also evaluated using the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay, which revealed lower toxicity of these compounds for peripheral blood mononuclear cells than for K562 cells. The possible mechanisms leading to cell death are discussed based on the observed effects of the new TSC compounds on the cellular thiol content and on mitochondrial bioenergetics.

  11. Experimentally-induced immune activation in natural hosts of SIV induces significant increases in viral replication and CD4+ T cell depletion

    Energy Technology Data Exchange (ETDEWEB)

    Ribeiro, Ruy M [Los Alamos National Laboratory

    2008-01-01

    Chronically SIVagm-infected African green monkeys (AGMs) have a remarkably stable non-pathogenic disease course, with levels of immune activation in chronic SIVagm infection similar to those observed in uninfected monkeys and stable viral loads (VLs) for long periods of time. In vivo administration of lipopolysaccharide (LPS) or an IL-2/diphtheria toxin fusion protein (Ontak) to chronically SIVagm-infected AGMs triggered increases in immune activation and subsequently of viral replication and depletion of intestinal CD4{sup +} T cells. Our study indicates that circulating microbial products can increase viral replication by inducing immune activation and increasing the number of viral target cells, thus demonstrating that immune activation and T cell prolifeation are key factors in AIDS pathogenesis.

  12. Inhibition of p300 histone acetyltransferase activity in palate mesenchyme cells attenuates Wnt signaling via aberrant E-cadherin expression.

    Science.gov (United States)

    Warner, Dennis R; Smith, Scott C; Smolenkova, Irina A; Pisano, M Michele; Greene, Robert M

    2016-03-01

    p300 is a multifunctional transcriptional coactivator that interacts with numerous transcription factors and exhibits protein/histone acetyltransferase activity. Loss of p300 function in humans and in mice leads to craniofacial defects. In this study, we demonstrated that inhibition of p300 histone acetyltransferase activity with the compound, C646, altered the expression of several genes, including Cdh1 (E-cadherin) in mouse maxillary mesenchyme cells, which are the cells that give rise to the secondary palate. The increased expression of plasma membrane-bound E-cadherin was associated with reduced cytosolic β-catenin, that led to attenuated signaling through the canonical Wnt pathway. Furthermore, C646 reduced both cell proliferation and the migratory ability of these cells. These results suggest that p300 histone acetyltransferase activity is critical for Wnt-dependent palate mesenchymal cell proliferation and migration, both processes that play a significant role in morphogenesis of the palate.

  13. A modified golden gate attenuated total reflection (ATR) cell for monitoring phase transitions in multicomponent fluids at high temperatures.

    Science.gov (United States)

    Novitskiy, Alexander A; Ke, Jie; Comak, Gurbuz; Poliakoff, Martyn; George, Michael W

    2011-08-01

    A new continuous flow method using attenuated total reflection infrared (ATR-IR) spectroscopy has been developed for monitoring phase transitions in multicomponent fluids at high pressures and temperatures. Our approach uses Fourier transform infrared (FT-IR) and a modified Golden Gate attenuated total reflection (ATR) cell and exploits the fact that the absorbance of a vapor is much lower than that of the corresponding liquid to monitor the phase transition between vapor and liquid. We demonstrate that this method can provide quantitative measurements on both the dew point and the bubble point. We have validated our approach using three single-component systems (EtOH, MeOH, and H(2)O) and a binary system of EtOH + H(2)O, monitoring phase transitions at temperature up to 300 °C and pressure up to 10 MPa.

  14. The mechanism by which MEK/ERK regulates JNK and p38 activity in polyamine depleted IEC-6 cells during apoptosis.

    Science.gov (United States)

    Bavaria, Mitul N; Jin, Shi; Ray, Ramesh M; Johnson, Leonard R

    2014-03-01

    Polyamine-depletion inhibited apoptosis by activating ERK1/2, while, preventing JNK1/2 activation. MKP-1 knockdown by SiRNA increased ERK1/2, JNK1/2, and p38 phosphorylation and apoptosis. Therefore, we predicted that polyamines might regulate MKP1 via MEK/ERK and thereby apoptosis. We examined the role of MEK/ERK in the regulation of MKP1 and JNK, and p38 activities and apoptosis. Inhibition of MKP-1 activity with a pharmacological inhibitor, sanguinarine (SA), increased JNK1/2, p38, and ERK1/2 activities without causing apoptosis. However, pre-activation of these kinases by SA significantly increased camptothecin (CPT)-induced apoptosis suggesting different roles for MAPKs during survival and apoptosis. Inhibition of MEK1 activity prevented the expression of MKP-1 protein and augmented CPT-induced apoptosis, which correlated with increased activities of JNK1/2, caspases, and DNA fragmentation. Polyamine depleted cells had higher levels of MKP-1 protein and decreased JNK1/2 activity and apoptosis. Inhibition of MEK1 prevented MKP-1 expression and increased JNK1/2 and apoptosis. Phospho-JNK1/2, phospho-ERK2, MKP-1, and the catalytic subunit of PP2Ac formed a complex in response to TNF/CPT. Inactivation of PP2Ac had no effect on the association of MKP-1 and JNK1. However, inhibition of MKP-1 activity decreased the formation of the MKP-1, PP2Ac and JNK complex. Following inhibition by SA, MKP-1 localized in the cytoplasm, while basal and CPT-induced MKP-1 remained in the nuclear fraction. These results suggest that nuclear MKP-1 translocates to the cytoplasm, binds phosphorylated JNK and p38 resulting in dephosphorylation and decreased activity. Thus, MEK/ERK activity controls the levels of MKP-1 and, thereby, regulates JNK activity in polyamine-depleted cells.

  15. Antagonism of Stem Cell Factor/c-kit Signaling Attenuates Neonatal Chronic Hypoxia-Induced Pulmonary Vascular Remodeling

    Science.gov (United States)

    Young, Karen C; Torres, Eneida; Hehre, Dorothy; Wu, Shu; Suguihara, Cleide; Hare, Joshua M.

    2015-01-01

    Background Accumulating evidence suggests that c-kit positive cells are present in the remodeled pulmonary vasculature bed of patients with pulmonary hypertension (PH). Whether stem cell factor (SCF)/ c-kit regulated pathways potentiate pulmonary vascular remodeling is unknown. Here, we tested the hypothesis that attenuated c-kit signaling would decrease chronic hypoxia-induced pulmonary vascular remodeling by decreasing pulmonary vascular cell mitogenesis. Methods Neonatal FVB/NJ mice treated with non-immune IgG (PL), or c-kit neutralizing antibody (ACK2) as well as c-kit mutant mice (WBB6F1- Kit W− v/ +) and their congenic controls, were exposed to normoxia (FiO2=0.21) or hypoxia (FiO2=0.12) for two weeks. Following this exposure, right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH), pulmonary vascular cell proliferation and remodeling were evaluated. Results As compared to chronically hypoxic controls, c-kit mutant mice had decreased RVSP, RVH, pulmonary vascular remodeling and proliferation. Consistent with these findings, administration of ACK2 to neonatal mice with chronic hypoxia-induced PH decreased RVSP, RVH, pulmonary vascular cell proliferation and remodeling. This attenuation in PH was accompanied by decreased extracellular signal-regulated protein kinase (ERK) 1/2 activation. Conclusion SCF/c-kit signaling may potentiate chronic hypoxia-induced vascular remodeling by modulating ERK activation. Inhibition of c-kit activity may be a potential strategy to alleviate PH. PMID:26705118

  16. Attenuation of PTEN increases p21 stability and cytosolic localization in kidney cancer cells: a potential mechanism of apoptosis resistance

    Directory of Open Access Journals (Sweden)

    Baksh Shairaz

    2007-02-01

    Full Text Available Abstract Background The PTEN (Phosphatase and Tensin homolog deleted on chromosome Ten tumor suppressor gene is frequently mutated or deleted in a wide variety of solid tumors, and these cancers are generally more aggressive and difficult to treat than those possessing wild type PTEN. While PTEN lies upstream of the phosphoinositide-3 kinase signaling pathway, the mechanisms that mediate its effects on tumor survival remain incompletely understood. Renal cell carcinoma (RCC is associated with frequent treatment failures (~90% in metastatic cases, and these tumors frequently contain PTEN abnormalities. Results Using the ACHN cell line containing wild type PTEN, we generated a stable PTEN knockdown RCC cell line using RNA interference. We then used this PTEN knockdown cell line to show that PTEN attenuation increases resistance to cisplatin-induced apoptosis, a finding associated with increased levels of the cyclin kinase inhibitor p21. Elevated levels of p21 result from stabilization of the protein, and they are dependent on the activities of phosphoinositide-3 kinase and Akt. More specifically, the accumulation of p21 occurs preferentially in the cytosolic compartment, which likely contributes to both cell cycle progression and resistance to apoptosis. Conclusion Since p21 regulates a decision point between repair and apoptosis after DNA damage, our data suggest that p21 plays a key role in mechanisms used by PTEN-deficient tumors to escape chemotherapy. This in turn raises the possibility to use p21 attenuators as chemotherapy sensitizers, an area under active continuing investigation in our laboratories.

  17. Intravenous transplantation of mouse embryonic stem cells attenuates demyelination in an ICR outbred mouse model of demyelinating diseases

    Institute of Scientific and Technical Information of China (English)

    Kidsadagon Pringproa; Anucha Sathanawongs; Chananthida Khamphilai; Sarocha Sukkarinprom; Apichart Oranratnachai

    2016-01-01

    Induction of demyelination in the central nervous system (CNS) of experimental mice using cuprizone is widely used as an animal model for studying the pathogenesis and treatment of demyelination. How-ever, different mouse strains used result in different pathological outcomes. Moreover, because current medicinal treatments are not always effective in multiple sclerosis patients, so the study of exogenous cell transplantation in an animal model is of great importance. hTe aims of the present study were to establish an alternative ICR outbred mouse model for studying demyelination and to evaluate the effects of intrave-nous cell transplantation in the present developed mouse model. Two sets of experiments were conducted. Firstly, ICR outbred and BALB/c inbred mice were fed with 0.2% cuprizone for 6 consecutive weeks; then demyelinating scores determined by luxol fast blue stain or immunolabeling with CNPase were evaluated. Secondly, attenuation of demyelination in ICR mice by intravenous injection of mES cells was studied. Scores for demyelination in the brains of ICR mice receiving cell injection (mES cells-injected group) and vehicle (sham-inoculated group) were assessed and compared. hTe results showed that cuprizone signiif-cantly induced demyelination in the cerebral cortex and corpus callosum of both ICR and BALB/c mice. Additionally, intravenous transplantation of mES cells potentially attenuated demyelination in ICR mice compared with sham-inoculated groups. hTe present study is among the earliest reports to describe the cuprizone-induced demyelination in ICR outbred mice. Although it remains unclear whether mES cells or trophic effects from mES cells are the cause of enhanced remyelination, the results of the present study may shed some light on exogenous cell therapy in central nervous system demyelinating diseases.

  18. Impaired recovery of Epstein-Barr virus (EBV)--specific CD8+ T lymphocytes after partially T-depleted allogeneic stem cell transplantation may identify patients at very high risk for progressive EBV reactivation and lymphoproliferative disease

    NARCIS (Netherlands)

    P. Meij; B. Löwenberg (Bob); J.W. Gratama (Jan-Willem); J.J. Cornelissen (Jan); J.W.J. van Esser (Joost); H.G.M. Niesters (Bert); D. van Baarle (Debbie); F. Miedema (Frank); N. Blake; A.B. Rickinson; I. Leiner; E. Pamer

    2003-01-01

    textabstractEpstein-Barr virus (EBV)-specific cytotoxic T lymphocytes are considered pivotal to prevent lymphoproliferative disease (LPD) in allogeneic stem cell transplantation (SCT) recipients. We evaluated the recovery of EBV-specific CD8+ T cells after partially T-cell-depleted

  19. Impaired recovery of Epstein-Barr virus (EBV)--specific CD8+ T lymphocytes after partially T-depleted allogeneic stem cell transplantation may identify patients at very high risk for progressive EBV reactivation and lymphoproliferative disease

    NARCIS (Netherlands)

    Meij, Pauline; van Esser, Joost W J; Niesters, Hubert G M; van Baarle, Debbie; Miedema, Frank; Blake, Neil; Rickinson, Alan B; Leiner, Ingrid; Pamer, Eric; Lowenberg, Bob; Cornelissen, Jan J; Gratama, Jan W

    2003-01-01

    Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes are considered pivotal to prevent lymphoproliferative disease (LPD) in allogeneic stem cell transplantation (SCT) recipients. We evaluated the recovery of EBV-specific CD8+ T cells after partially T-cell-depleted SCT and studied the interacti

  20. Macrophage-stimulating protein attenuates gentamicin-induced inflammation and apoptosis in human renal proximal tubular epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ko Eun [Department of Internal Medicine, Chonnam National University Medical School, Gwangju 501-757 (Korea, Republic of); Kim, Eun Young [Department of Physiology, Chonnam National University Medical School, Gwangju 501-757 (Korea, Republic of); Kim, Chang Seong; Choi, Joon Seok; Bae, Eun Hui; Ma, Seong Kwon [Department of Internal Medicine, Chonnam National University Medical School, Gwangju 501-757 (Korea, Republic of); Kim, Kyung Keun [Department of Pharmacology, Chonnam National University Medical School, Gwangju 501-757 (Korea, Republic of); Lee, Jong Un [Department of Physiology, Chonnam National University Medical School, Gwangju 501-757 (Korea, Republic of); Kim, Soo Wan, E-mail: skimw@chonnam.ac.kr [Department of Internal Medicine, Chonnam National University Medical School, Gwangju 501-757 (Korea, Republic of)

    2013-05-10

    Highlights: •MSP/RON system is activated in rat kidney damaged by gentamicin. •MSP inhibits GM-induced cellular apoptosis and inflammation in HK-2 cells. •MSP attenuates GM-induced activation of MAPKs and NF-κB pathways in HK-2 cells. -- Abstract: The present study aimed to investigate whether macrophage-stimulating protein (MSP) treatment attenuates renal apoptosis and inflammation in gentamicin (GM)-induced tubule injury and its underlying molecular mechanisms. To examine changes in MSP and its receptor, recepteur d’origine nantais (RON) in GM-induced nephropathy, rats were injected with GM for 7 days. Human renal proximal tubular epithelial (HK-2) cells were incubated with GM for 24 h in the presence of different concentrations of MSP and cell viability was measured by MTT assay. Apoptosis was determined by flow cytometry of cells stained with fluorescein isothiocyanate-conjugated annexin V protein and propidium iodide. Expression of Bcl-2, Bax, caspase-3, cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), nuclear factor-kappa B (NF-κB), IκB-α, and mitogen-activated protein kinases (MAPKs) was analyzed by semiquantitative immunoblotting. MSP and RON expression was significantly greater in GM-treated rats, than in untreated controls. GM-treatment reduced HK-2 cell viability, an effect that was counteracted by MSP. Flow cytometry and DAPI staining revealed GM-induced apoptosis was prevented by MSP. GM reduced expression of anti-apoptotic protein Bcl-2 and induced expression of Bax and cleaved caspase 3; these effects and GM-induced expression of COX-2 and iNOS were also attenuated by MSP. GM caused MSP-reversible induction of phospho-ERK, phospho-JNK, and phospho-p38. GM induced NF-κB activation and degradation of IκB-α; the increase in nuclear NF-κB was blocked by inhibitors of ERK, JNK, p-38, or MSP pretreatment. These findings suggest that MSP attenuates GM-induced inflammation and apoptosis by inhibition of the MAPKs

  1. Intravenous multipotent adult progenitor cell therapy attenuates activated microglial/macrophage response and improves spatial learning after traumatic brain injury.

    Science.gov (United States)

    Bedi, Supinder S; Hetz, Robert; Thomas, Chelsea; Smith, Philippa; Olsen, Alex B; Williams, Stephen; Xue, Hasen; Aroom, Kevin; Uray, Karen; Hamilton, Jason; Mays, Robert W; Cox, Charles S

    2013-12-01

    We previously demonstrated that the intravenous delivery of multipotent adult progenitor cells (MAPCs) after traumatic brain injury (TBI) in rodents provides neuroprotection by preserving the blood-brain barrier and systemically attenuating inflammation in the acute time frame following cell treatment; however, the long-term behavioral and anti-inflammatory effects of MAPC administration after TBI have yet to be explored. We hypothesized that the intravenous injection of MAPCs after TBI attenuates the inflammatory response (as measured by microglial morphology) and improves performance at motor tasks and spatial learning (Morris water maze [MWM]). MAPCs were administered intravenously 2 and 24 hours after a cortical contusion injury (CCI). We tested four groups at 120 days after TBI: sham (uninjured), injured but not treated (CCI), and injured and treated with one of two concentrations of MAPCs, either 2 million cells per kilogram (CCI-2) or 10 million cells per kilogram (CCI-10). CCI-10 rats showed significant improvement in left hind limb deficit on the balance beam. On the fifth day of MWM trials, CCI-10 animals showed a significant decrease in both latency to platform and distance traveled compared with CCI. Probe trials revealed a significant decrease in proximity measure in CCI-10 compared with CCI, suggesting improved memory retrieval. Neuroinflammation was quantified by enumerating activated microglia in the ipsilateral hippocampus. We observed a significant decrease in the number of activated microglia in the dentate gyrus in CCI-10 compared with CCI. Our results demonstrate that intravenous MAPC treatment after TBI in a rodent model offers long-term improvements in spatial learning as well as attenuation of neuroinflammation.

  2. Cuprous oxide nanoparticles inhibit prostate cancer by attenuating the stemness of cancer cells via inhibition of the Wnt signaling pathway

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    Wang, Ye; Yang, Qi-Wei; Yang, Qing; Zhou, Tie; Shi, Min-Feng; Sun, Chen-Xia; Gao, Xiu-Xia; Cheng, Yan-Qiong; Cui, Xin-Gang; Sun, Ying-Hao

    2017-01-01

    Disordered copper metabolism plays a critical role in the development of various cancers. As a nanomedicine containing copper, cuprous oxide nanoparticles (CONPs) exert ideal antitumor pharmacological effects in vitro and in vivo. Prostate cancer is a frequently diagnosed male malignancy prone to relapse, and castration resistance is the main reason for endocrine therapy failure. However, whether CONPs have the potential to treat castration-resistant prostate cancer is still unknown. Here, using the castration-resistant PC-3 human prostate cancer cell line as a model, we report that CONPs can selectively induce apoptosis and inhibit the proliferation of cancer cells in vitro and in vivo without affecting normal prostate epithelial cells. CONPs can also attenuate the stemness of cancer cells and inhibit the Wnt signaling pathway, both of which highlight the great potential of CONPs as a new clinical castration-resistant prostate cancer therapy.

  3. β-Elemene-Attenuated Tumor Angiogenesis by Targeting Notch-1 in Gastric Cancer Stem-Like Cells

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    Bing Yan

    2013-01-01

    Full Text Available Emerging evidence suggests that cancer stem cells are involved in tumor angiogenesis. The Notch signaling pathway is one of the most important regulators of these processes. β-Elemene, a naturally occurring compound extracted from Curcumae Radix, has been used as an antitumor drug for various cancers in China. However, its underlying mechanism in the treatment of gastric cancer remains largely unknown. Here, we report that CD44+ gastric cancer stem-like cells (GCSCs showed enhanced proliferation capacity compared to their CD44− counterparts, and this proliferation was accompanied by the high expression of Notch-1 (in vitro. These cells were also more superior in spheroid colony formation (in vitro and tumorigenicity (in vivo and positively associated with microvessel density (in vivo. β-Elemene was demonstrated to effectively inhibit the viability of GCSCs in a dose-dependent manner, most likely by suppressing Notch-1 (in vitro. β-Elemene also contributed to growth suppression and attenuated the angiogenesis capacity of these cells (in vivo most likely by interfering with the expression of Notch-1 but not with Dll4. Our findings indicated that GCSCs play an important role in tumor angiogenesis, and Notch-1 is one of the most likely mediators involved in these processes. β-Elemene was effective at attenuating angiogenesis by targeting the GCSCs, which could be regarded as a potential mechanism for its efficacy in gastric cancer management in the future.

  4. Lycopene attenuates Aβ1-42 secretion and its toxicity in human cell and Caenorhabditis elegans models of Alzheimer disease.

    Science.gov (United States)

    Chen, Wei; Mao, Liuqun; Xing, Huanhuan; Xu, Lei; Fu, Xiang; Huang, Liyingzi; Huang, Dongling; Pu, Zhijun; Li, Qinghua

    2015-11-03

    Growing evidence suggests concentration of lycopene was reduced in plasma of patients with Alzheimer disease (AD). Lycopene, a member of the carotenoid family, has been identified as an antioxidant to attenuate oxidative damage and has neuroprotective role in several AD models. However, whether lycopene is involved in the pathogenesis of AD and molecular underpinnings are elusive. In this study, we found that lycopene can significantly delay paralysis in the Aβ1-42-transgenic Caenorhabditis elegans strain GMC101. Lycopene treatment reduced Aβ1-42 secretion in SH-SY5Y cells overexpressing the Swedish mutant form of human β-amyloid precursor protein (APPsw). Next, we found lycopene can down-regulate expression level of β-amyloid precursor protein(APP) in APPsw cells. Moreover, lycopene treatment can not change endogenous reactive oxygen species level and apoptosis in APPsw cells. However, lycopene treatment protected against H2O2-induced oxidative stress and copper-induced damage in APPsw cells. Collectively, our data support that elevated lycopene contributes to the lower pathogenesis of AD. Our findings suggest that increasing lycopene in neurons may be a novel approach to attenuate onset and development of AD.

  5. Heat Killed Attenuated Leishmania Induces Apoptosis of HepG2 Cells Through ROS Mediated p53 Dependent Mitochondrial Pathway

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    Dipayan Bose

    2016-03-01

    Full Text Available Background/Aims: Cytotoxic effect of attenuated Leishmania on liver cancer cells by inducing ROS generation. Methods: Spectrophotometric study to analyze cell death and levels of different active caspases. Flow cytometric study was done to analyze apoptosis induction and ROS generation and levels of different protein. Western blot analysis was performed to study the levels of protein. Confocal microscopy was done to ascertain the expression of different apoptotic markers. Results: We have now observed that attenuated Leishmania donovani UR6 also has potentiality towards growth inhibition of HepG2 cells and investigated the mechanism of action. The effect is associated with increased DNA fragmentation, rise in number of annexinV positive cells, and cell cycle arrest at G1 phase. The detection of unregulated levels of active PARP, cleaved caspases 3 and 9, cytosolic cytochrome C, Bax, and Bad, along with the observed downregulation of Bcl-2 and loss of mitochondrial membrane potential suggested the involvement of mitochondrial pathway. Enhanced ROS and p53 levels regulate the apoptosis of HepG2 cells. NAC was found to inhibit p53 production but PFT-α has no effect on ROS generation. In conclusion, Leishmania donovani UR6 efficiently induces apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. Conclusion: It has been reported earlier that some parasites show prominent cytotoxic effect and prevent tumor growth. From our study we found that Leishmania donovani UR6 efficiently induced apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. This study has rejuvenated the age old idea of bio-therapy.

  6. Arsenic trioxide depletes cancer stem-like cells and inhibits repopulation of neurosphere derived from glioblastoma by downregulation of Notch pathway.

    Science.gov (United States)

    Wu, Jianing; Ji, Zhiyong; Liu, Huailei; Liu, Yaohua; Han, Dayong; Shi, Chen; Shi, Changbin; Wang, Chunlei; Yang, Guang; Chen, Xiaofeng; Shen, Chen; Li, Huadong; Bi, Yunke; Zhang, Dongzhi; Zhao, Shiguang

    2013-06-20

    Notch signaling has been demonstrated to have a central role in cancer stem-like cells (CSLCs) in glioblastoma multiforme (GBM). We have recently demonstrated the inhibitory effect of arsenic trioxide (ATO) on CSLCs in glioblastoma cell lines. In this study we used neurosphere recovery assay that measured neurosphere formation at three time points to assess the capacity of the culture to repopulate after ATO treatment. Our results provided strong evidence that ATO depleted CSLCs in GBM, and inhibited neurosphere recovery and secondary neurosphere formation. ATO inhibited the phosphorylation and activation of AKT and STAT3 through Notch signaling blockade. These data show that the ATO is a promising new approach to decrease glioblastoma proliferation and recurrence by downregulation of Notch pathway.

  7. Transient B-cell depletion with anti-CD20 in combination with proinsulin DNA vaccine or oral insulin: immunologic effects and efficacy in NOD mice.

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    Ghanashyam Sarikonda

    Full Text Available A recent type 1 diabetes (T1D clinical trial of rituximab (a B cell-depleting anti-CD20 antibody achieved some therapeutic benefit in preserving C-peptide for a period of approximately nine months in patients with recently diagnosed diabetes. Our previous data in the NOD mouse demonstrated that co-administration of antigen (insulin with anti-CD3 antibody (a T cell-directed immunomodulator offers better protection than either entity alone, indicating that novel combination therapies that include a T1D-related autoantigen are possible. To accelerate the identification and development of novel combination therapies that can be advanced into the clinic, we have evaluated the combination of a mouse anti-CD20 antibody with either oral insulin or a proinsulin-expressing DNA vaccine. Anti-CD20 alone, given once or on 4 consecutive days, produced transient B cell depletion but did not prevent or reverse T1D in the NOD mouse. Oral insulin alone (twice weekly for 6 weeks was also ineffective, while proinsulin DNA (weekly for up to 12 weeks showed a trend toward modest efficacy. Combination of anti-CD20 with oral insulin was ineffective in reversing diabetes in NOD mice whose glycemia was controlled with SC insulin pellets; these experiments were performed in three independent labs. Combination of anti-CD20 with proinsulin DNA was also ineffective in diabetes reversal, but did show modest efficacy in diabetes prevention (p = 0.04. In the prevention studies, anti-CD20 plus proinsulin resulted in modest increases in Tregs in pancreatic lymph nodes and elevated levels of proinsulin-specific CD4+ T-cells that produced IL-4. Thus, combination therapy with anti-CD20 and either oral insulin or proinsulin does not protect hyperglycemic NOD mice, but the combination with proinsulin offers limited efficacy in T1D prevention, potentially by augmentation of proinsulin-specific IL-4 production.

  8. Lipoic acid inhibits the DNA repair protein O 6-methylguanine-DNA methyltransferase (MGMT) and triggers its depletion in colorectal cancer cells with concomitant autophagy induction.

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    Göder, Anja; Nagel, Georg; Kraus, Alexander; Dörsam, Bastian; Seiwert, Nina; Kaina, Bernd; Fahrer, Jörg

    2015-08-01

    Alkylating agents are present in food and tobacco smoke, but are also used in cancer chemotherapy, inducing the DNA lesion O (6)-methylguanine. This critical adduct is repaired by O (6)-methylguanine-DNA methyltransferase (MGMT), resulting in MGMT inactivation and degradation. In the present study, we analyzed the effects of the natural disulfide compound lipoic acid (LA) on MGMT in vitro and in colorectal cancer cells. We show that LA, but not its reduced form dihydrolipoic acid, potently inhibits the activity of recombinant MGMT by interfering with its catalytic Cys-145 residue, which was partially reversible by N-acetyl cysteine. Incubation of HCT116 colorectal cancer cells with LA altered their glutathione pool and caused a decline in MGMT activity. This was mirrored by LA-induced depletion of MGMT protein, which was not attributable to changes in MGMT messenger RNA levels. Loss of MGMT protein coincided with LA-induced autophagy, a process resulting in lysosomal degradation of proteins, including presumably MGMT. LA-stimulated autophagy in a p53-independent manner as revealed by the response of isogenic HCT116 cell lines. Knockdown of the crucial autophagy component beclin-1 and chemical inhibitors blocked LA-induced autophagy, but did not abrogate LA-triggered MGMT degradation. Concomitant with MGMT depletion, LA pretreatment resulted in enhanced O (6)-methylguanine levels in DNA. It also increased the cytotoxicity of the alkylating anticancer drug temozolomide in temozolomide-resistant colorectal cancer cells. Taken together, our study showed that the natural compound LA inhibits MGMT and induces autophagy. Furthermore, LA enhanced the cytotoxic effects of temozolomide, which makes it a candidate for a supplement in cancer therapy.

  9. An antisense oligodeoxynucleotide that depletes RI alpha subunit of cyclic AMP-dependent protein kinase induces growth inhibition in human cancer cells.

    Science.gov (United States)

    Yokozaki, H; Budillon, A; Tortora, G; Meissner, S; Beaucage, S L; Miki, K; Cho-Chung, Y S

    1993-02-15

    Enhanced expression of the RI alpha subunit of cyclic AMP-dependent protein kinase type I has been correlated with cancer cell growth. We provide evidence that RI alpha is a growth-inducing protein that may be essential for neoplastic cell growth. Human colon, breast, and gastric carcinoma and neuroblastoma cell lines exposed to a 21-mer human RI alpha antisense phosphorothioate oligodeoxynucleotide (S-oligodeoxynucleotide) exhibited growth inhibition with no sign of cytotoxicity. Mismatched sequence (random) S-oligodeoxynucleotides of the same length exhibited no effect. The growth inhibitory effect of RI alpha antisense oligomer correlated with a decrease in the RI alpha mRNA and protein levels and with an increase in RII beta (the regulatory subunit of protein kinase type II) expression. The growth inhibition was abolished, however, when cells were exposed simultaneously to both RI alpha and RII beta antisense S-oligodeoxynucleotides. The RII beta antisense S-oligodeoxynucleotide alone, exhibiting suppression of RII beta along with enhancement of RI alpha expression, led to slight stimulation of cell growth. These results demonstrate that two isoforms of cyclic AMP receptor proteins, RI alpha and RII beta, are reciprocally related in the growth control of cancer cells and that the RI alpha antisense oligodeoxynucleotide, which efficiently depletes the growth stimulatory RI alpha, is a powerful biological tool toward suppression of malignancy.

  10. Dynamics of Lymphocyte Populations during Trypanosoma cruzi Infection: From Thymocyte Depletion to Differential Cell Expansion/Contraction in Peripheral Lymphoid Organs

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    Alexandre Morrot

    2012-01-01

    Full Text Available The comprehension of the immune responses in infectious diseases is crucial for developing novel therapeutic strategies. Here, we review current findings on the dynamics of lymphocyte subpopulations following experimental acute infection by Trypanosoma cruzi, the causative agent of Chagas disease. In the thymus, although the negative selection process of the T-cell repertoire remains operational, there is a massive thymocyte depletion and abnormal release of immature CD4+CD8+ cells to peripheral lymphoid organs, where they acquire an activated phenotype similar to activated effector or memory T cells. These cells apparently bypassed the negative selection process, and some of them are potentially autoimmune. In infected animals, an atrophy of mesenteric lymph nodes is also observed, in contrast with the lymphocyte expansion in spleen and subcutaneous lymph nodes, illustrating a complex and organ specific dynamics of lymphocyte subpopulations. Accordingly, T- and B-cell activation is seen in subcutaneous lymph nodes and spleen, but not in mesenteric lymph nodes. Lastly, although the function of peripheral CD4+CD8+ T-cell population remains to be defined in vivo, their presence may contribute to the immunopathological events found in both murine and human Chagas disease.

  11. Novel benzoxazole derivatives DCPAB and HPAB attenuate Th1 cell-mediated inflammation through T-bet suppression

    Science.gov (United States)

    Oh, Yeon Ji; Kim, Darong; Oh, Sera; Jang, Eun Jung; Won, Hee Yeon; Jeong, Hana; Jeong, Mi Gyeong; Choo, Hea-Young Park; Hwang, Eun Sook

    2017-01-01

    Interferon-γ (IFN-γ), a critical inflammatory cytokine, is primarily produced by T helper 1 (Th1) cells and accelerates the pathogenesis of inflammatory colitis. Pharmacological suppression of IFN-γ production attenuates dysregulated inflammatory responses and may be beneficial for treating inflammatory disease. In this study, we aimed to discover potent anti-inflammatory compounds that suppress IFN-γ production and found that the novel benzoxazole derivatives, 2-((3,4-dichlorophenyl) amino) benzo[d]xazol-5-ol (DCPAB) and 2-((3,4-hydroxyphenyl) amino) benzo[d]xazol-5-ol (HPAB), suppressed IFN-γ production by T cells. Treatment of CD4+ T cells with DCPAB and HPAB selectively inhibited Th1 cell development, and DCPAB more potently suppressed IFN-γ than HPAB did. Interestingly, DCPAB and HPAB significantly suppressed the expression of T-box containing protein expressed in T cells (T-bet) that activates IFN-γ gene transcription. DCPAB additionally suppressed transcriptional activity of T-bet on IFN-γ gene promoter, whereas HPAB had no effect on T-bet activity. IFN-γ suppressive activity of DCPAB and HPAB was impaired in the absence of T-bet but was retrieved by the restoration of T-bet in T-bet-deficient T cells. Furthermore, DCPAB and HPAB attenuated inflammatory colitis development that was induced by CD4+ T cells in vivo. We suggest that the novel benzoxazole derivatives, DCPAB and HPAB, may have therapeutic effects on inflammatory colitis. PMID:28169371

  12. Hydrogen sulfide attenuates doxorubicin-induced cardiotoxicity by inhibiting the expression of peroxiredoxin III in H9c2 cells.

    Science.gov (United States)

    Liu, Mi-Hua; Lin, Xiao-Long; Yuan, Cong; He, Jun; Tan, Tian-Ping; Wu, Shao-Jian; Yu, Shan; Chen, Li; Liu, Jun; Tian, Wei; Chen, Yu-Dan; Fu, Hong-Yun; Li, Jian; Zhang, Yuan

    2016-01-01

    Doxorubicin (DOX) is a widely used chemotherapeutic agent, which can give rise to severe cardiotoxicity, limiting its clinical use. Preliminary evidence suggests that hydrogen sulfide (H2S) may exert protective effects on DOX‑induced cardiotoxicity. Therefore, the aim of the present study was to investigate whether peroxiredoxin III is involved in the cardioprotection of H2S against DOX‑induced cardiotoxicity. The results demonstrated that DOX not only markedly induced injuries, including cytotoxicity and apoptosis, it also increased the expression levels of peroxiredoxin III. Notably, pretreatment with sodium hydrosulfide significantly attenuated the DOX‑induced decrease in cell viability and increase in apoptosis, and also reversed the increased expression levels of peroxiredoxin III in H9c2 cardiomyocytes. In addition, pretreatment of the H9c2 cells with N‑acetyl‑L‑cysteine, a scavenger of reactive oxygen species, prior to exposure to DOX markedly decreased the expression levels of peroxiredoxin III. In conclusion, the results of the present study suggested that exogenous H2S attenuates DOX‑induced cardiotoxicity by inhibiting the expression of peroxiredoxin III in H9c2 cells. In the present study, the apoptosis of H9c2 cardiomyocytes was assessed using an methyl thiazolyl tetrazolium assay and Hoechst staining. The levels of Prx III and cystathionine-γ-lyase were examined by western blotting.

  13. Pegylated G-CSF Inhibits Blood Cell Depletion, Increases Platelets, Blocks Splenomegaly, and Improves Survival after Whole-Body Ionizing Irradiation but Not after Irradiation Combined with Burn

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    Juliann G. Kiang

    2014-01-01

    Full Text Available Exposure to ionizing radiation alone (radiation injury, RI or combined with traumatic tissue injury (radiation combined injury, CI is a crucial life-threatening factor in nuclear and radiological accidents. As demonstrated in animal models, CI results in greater mortality than RI. In our laboratory, we found that B6D2F1/J female mice exposed to 60Co-γ-photon radiation followed by 15% total-body-surface-area skin burns experienced an increment of 18% higher mortality over a 30-day observation period compared to irradiation alone; that was accompanied by severe cytopenia, thrombopenia, erythropenia, and anemia. At the 30th day after injury, neutrophils, lymphocytes, and platelets still remained very low in surviving RI and CI mice. In contrast, their RBC, hemoglobin, and hematocrit were similar to basal levels. Comparing CI and RI mice, only RI induced splenomegaly. Both RI and CI resulted in bone marrow cell depletion. It was observed that only the RI mice treated with pegylated G-CSF after RI resulted in 100% survival over the 30-day period, and pegylated G-CSF mitigated RI-induced body-weight loss and depletion of WBC and platelets. Peg-G-CSF treatment sustained RBC balance, hemoglobin levels, and hematocrits and inhibited splenomegaly after RI. The results suggest that pegylated G-CSF effectively sustained animal survival by mitigating radiation-induced cytopenia, thrombopenia, erythropenia, and anemia.

  14. Sustained Epigenetic Drug Delivery Depletes Cholesterol-Sphingomyelin Rafts from Resistant Breast Cancer Cells, Influencing Biophysical Characteristics of Membrane Lipids.

    Science.gov (United States)

    Raghavan, Vijay; Vijayaraghavalu, Sivakumar; Peetla, Chiranjeevi; Yamada, Masayoshi; Morisada, Megan; Labhasetwar, Vinod

    2015-10-27

    Cell-membrane lipid composition can greatly influence biophysical properties of cell membranes, affecting various cellular functions. We previously showed that lipid synthesis becomes altered in the membranes of resistant breast cancer cells (MCF-7/ADR); they form a more rigid, hydrophobic lipid monolayer than do sensitive cell membranes (MCF-7). These changes in membrane lipids of resistant cells, attributed to epigenetic aberration, significantly affected drug transport and endocytic function, thus impacting the efficacy of anticancer drugs. The present study's objective was to determine the effects of the epigenetic drug, 5-aza-2'-deoxycytidine (DAC), delivered in sustained-release nanogels (DAC-NGs), on the composition and biophysical properties of membrane lipids of resistant cells. Resistant and sensitive cells were treated with DAC in solution (DAC-sol) or DAC-NGs, and cell-membrane lipids were isolated and analyzed for lipid composition and biophysical properties. In resistant cells, we found increased formation of cholesterol-sphingomyelin (CHOL-SM) rafts with culturing time, whereas DAC treatment reduced their formation. In general, the effect of DAC-NGs was greater in changing the lipid composition than with DAC-sol. DAC treatment also caused a rise in levels of certain phospholipids and neutral lipids known to increase membrane fluidity, while reducing the levels of certain lipids known to increase membrane rigidity. Isotherm data showed increased lipid membrane fluidity following DAC treatment, attributed to decrease levels of CHOL-SM rafts (lamellar beta [Lβ] structures or ordered gel) and a corresponding increase in lipids that form lamellar alpha-structures (Lα, liquid crystalline phase). Sensitive cells showed marginal or insignificant changes in lipid profile following DAC-treatment, suggesting that epigenetic changes affecting lipid biosynthesis are more specific to resistant cells. Since membrane fluidity plays a major role in drug transport

  15. Type 3 innate lymphoid cell depletion is mediated by TLRs in lymphoid tissues of simian immunodeficiency virus-infected macaques.

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    Xu, Huanbin; Wang, Xiaolei; Lackner, Andrew A; Veazey, Ronald S

    2015-12-01

    Innate lymphoid cells (ILCs) type 3, also known as lymphoid tissue inducer cells, plays a major role in both the development and remodeling of organized lymphoid tissues and the maintenance of adaptive immune responses. HIV/simian immunodeficiency virus (SIV) infection causes breakdown of intestinal barriers resulting in microbial translocation, leading to systemic immune activation and disease progression. However, the effects of HIV/SIV infection on ILC3 are unknown. Here, we analyzed ILC3 from mucosal and systemic lymphoid tissues in chronically SIV-infected macaques and uninfected controls. ILC3 cells were defined and identified in macaque lymphoid tissues as non-T, non-B (lineage-negative), c-Kit(+)IL-7Rα(+) (CD117(+)CD127(+)) cells. These ILC3 cells highly expressed CD90 (∼ 63%) and aryl hydrocarbon receptor and produced IL-17 (∼ 63%), IL-22 (∼ 36%), and TNF-α (∼ 72%) but did not coexpress CD4 or NK cell markers. The intestinal ILC3 cell loss correlated with the reduction of total CD4(+) T cells and T helper (Th)17 and Th22 cells in the gut during SIV infection (P lymphoid tissues in SIV-infected macaques, further contributing to the HIV-induced impairment of gut-associated lymphoid tissue structure and function, especially in mucosal tissues.

  16. Graft versus host disease in a rat small bowel transplant model after T-cell depleted donor specific bone marrow infusion

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    Bakonyi Neto Alexandre

    2003-01-01

    Full Text Available Low cytoreductive regimen of irradiation associated to unmodified bone marrow infusion (UBM does not prevent the occurrence of graft versus host disease (GVHD after transplant. PURPOSE: In this study we evaluated the potential advantages of a long-term immunossupression and T-cell depleted bone marrow infusion (TCDBMI in preventing the occurrence of GVHD after small bowel transplantation (SBTx. METHODS: Heterotopic SBTX was performed with Lewis rats as recipients and DA as donors and distributed into 5 groups according to the irradiation, duration of immunossupression and the use of UBM or TCDBMI: G1 (n=6, without irradiation and G2 (n=9, G3 (n=4, G4 (n=5 and G5 (n=6 was given 250 rd of irradiation. Groups 1,2,4 and G3 and 5 were infused with 100 x 10(6 UBM and TCDBM respectively. Animals in G1, 2, 3 were immunossupressed with 1mg/ FK506/Kg/IM for 5 days and G4 and G5 for 15 days. Anti CD3 monoclonal antibodies and immunomagnetic beads were used for T-cell depletion.Animals were examined for rejection, GVHD, chimerism characterization and ileal and skin biopsies. RESULTS: Minimal to mild rejection was observed in all groups; however, GVHD were present only in irradiated groups. Long-term immunossupression changed the severity of GVHD in G4 and G5. Rejection was the cause of death in G1 while GVHD in G2, 3, 4 and 5, not avoided by the use of TCDBMI. Total chimerism and T-cell chimerism was statistically higher in irradiated groups when compared to G1. CONCLUSION: Extended immunossupression associated to low dose of irradiation decrease the severity of GVHD, not avoided by the use of TCDBMI.

  17. Mitochondrial genome depletion dysregulates bile acid- and paracetamol-induced expression of the transporters Mdr1, Mrp1 and Mrp4 in liver cells

    Science.gov (United States)

    Perez, MJ; Gonzalez-Sanchez, E; Gonzalez-Loyola, A; Gonzalez-Buitrago, JM; Marin, JJG

    2011-01-01

    BACKGROUND AND PURPOSE Mitochondria are involved in the toxicity of several compounds, retro-control of gene expression and apoptosis activation. The effect of mitochondrial genome (mtDNA) depletion on changes in ABC transporter protein expression in response to bile acids and paracetamol was investigated. EXPERIMENTAL APPROACH Hepa 1-6 mouse hepatoma cells with 70% decrease in 16S/18S rRNA ratio (Rho cells) were obtained by long-term treatment with ethidium bromide. KEY RESULTS Spontaneous apoptosis and reactive oxygen species (ROS) generation were decreased in Rho cells. Following glycochenodeoxycholic acid (GCDCA) or paracetamol, Rho cells generated less ROS and were more resistant to cell death. Apoptosis induced by GCDCA and Fas was also reduced. The basal expression of Mdr1 was significantly enhanced, but this was not further stimulated by GCDCA or paracetamol, as observed in wild-type (WT) cells. Basal expression of Mrp1 and Mrp4 was similar in WT and Rho cells, whereas they were up-regulated only in WT cells after GCDCA or paracetamol, along with the transcription factors Shp and Nrf2, but not Fxr or Pxr. Increased expression of Nrf2 was accompanied by its enhanced nuclear translocation. Glycoursodeoxycholic acid failed to cause any of the effects observed for GCDCA or paracetamol. CONCLUSIONS AND IMPLICATIONS The Nrf2-mediated pathway is partly independent of ROS production. Nuclear translocation of Nrf2 is insufficient to up-regulate Mdr1, Mrp1 and Mrp4, which requires the participation of other regulatory element(s) whose activation in response to GCDCA and paracetamol is impaired in Rho cells and hence probably sensitive to ROS. PMID:21175587

  18. CYP2S1 depletion enhances colorectal cell proliferation is associated with PGE2-mediated activation of β-catenin signaling

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Chao [Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200030 (China); College of Life Science, Anhui Normal University, Wuhu 241000, Anhui (China); Li, Changyuan [College of Life Science, Anhui Normal University, Wuhu 241000, Anhui (China); Li, Minle; Tong, Xuemei [Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Shanghai Jiao Tong University School of Medicine, Shanghai 200025 (China); Hu, Xiaowen; Yang, Xuhan [Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200030 (China); Yan, Xiaomei [School of Life Sciences & Biotechnology, Shanghai JiaoTong University, Shanghai 200240 (China); He, Lin, E-mail: helinhelin@gmail.com [Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200030 (China); Wan, Chunling, E-mail: clwan@sjtu.edu.cn [Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200030 (China)

    2015-02-15

    Colorectal epithelial cancer is one of the most common cancers in the world and its 5-year survival rate is still relatively low. Cytochrome P450 (CYP) enzymes in epithelial cells lining the alimentary tract play an important role in the oxidative metabolism of a wide range of xenobiotics, including (pro-)carcinogens and endogenous compounds. Although CYP2S1, a member of CYP family, strongly expressed in many extrahepatic tissues, the role of CYP2S1 in cancer remains unclear. To investigate whether CYP2S1 involves in colorectal carcinogenesis, cell proliferation was analyzed in HCT116 cells depleted of CYP2S1 using small hairpin interfering RNA. Our data show that CYP2S1 knockdown promotes cell proliferation through increasing the level of endogenous prostaglandin E2(PGE2). PGE2, in turn, reduces phosphorylation of β-catenin and activates β-catenin signaling, which contributes to the cell proliferation. Furthermore, CYP2S1 knockdown increase tumor growth in xenograft mouse model. In brief, these results demonstrate that CYP2S1 regulates colorectal cancer growth through associated with PGE2-mediated activation of β-catenin signaling. - Highlights: • Knockdown of CYP2S1 expression improve HCT116 cell proliferation in vitro and in vivo. • Elevate PGE2 production in CYP2S1 knockdown cell is associated with its proliferation. • Elevate PGE2 level in CYP2S1 knockdown cells enhance β-catenin accumulation. • β-catenin activate TCF/LEF and target gene expression thus promote cell proliferation.

  19. Sinomenine induces the generation of intestinal Treg cells and attenuates arthritis via activation of aryl hydrocarbon receptor.

    Science.gov (United States)

    Tong, Bei; Yuan, Xusheng; Dou, Yannong; Wu, Xin; Wang, Yuhui; Xia, Yufeng; Dai, Yue

    2016-10-01

    Sinomenine (SIN), an anti-arthritis drug, has previously been proven to exert immunomodulatory activity in rats by inducing intestinal regulatory T-cells (Treg cells). Here, we assessed the effect of SIN on the generation and function of Treg cells in autoimmune arthritis, and the underlying mechanisms in view of aryl hydrocarbon receptor (AhR). The proportions of Treg cells and IL-17-producing T-cells (Th17 cells) differentiated from naive T-cells were analyzed by flow cytometric analysis. The AhR agonistic effect of SIN was tested by analyzing the activation of downstream signaling pathways and target genes. The dependence of intestinal Treg cell induction and arthritis alleviation by SIN on AhR activation was confirmed in a mouse collagen-induced arthritis (CIA) model. SIN promoted the differentiation and function of intestinal Treg cells in vitro. It induced the expression and activity of AhR target gene, promoted AhR/Hsp90 dissociation and AhR nuclear translocation, induced XRE reporter activity, and facilitated AhR/XRE binding in vitro, displaying the potential to be an agonist of AhR. In CIA mice, SIN induced the generation of intestinal Treg cells, and facilitated the immunosuppressive function of these Treg cells as shown by an adoptive transfer test. In addition, the induction of intestinal Treg cells and the anti-arthritic effect of SIN in CIA mice could be largely diminished by the AhR antagonist resveratrol. SIN attenuates arthritis by promoting the generation and function of Treg cells in an AhR-dependent manner.

  20. ARQ 092, an orally-available, selective AKT inhibitor, attenuates neutrophil-platelet interactions in sickle cell disease

    Science.gov (United States)

    Kim, Kyungho; Li, Jing; Barazia, Andrew; Tseng, Alan; Youn, Seock-Won; Abbadessa, Giovanni; Yu, Yi; Schwartz, Brian; Andrews, Robert K.; Gordeuk, Victor R.; Cho, Jaehyung

    2017-01-01

    Previous studies identified the Ser/Thr protein kinase, AKT, as a therapeutic target in thrombo-inflammatory diseases. Here we report that specific inhibition of AKT with ARQ 092, an orally-available AKT inhibitor currently in phase Ib clinical trials as an anti-cancer drug, attenuates the adhesive function of neutrophils and platelets from sickle cell disease patients in vitro and cell-cell interactions in a mouse model of sickle cell disease. Studies using neutrophils and platelets isolated from sickle cell disease patients rev