Journal articles on the topic 'Murine SIM'
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Oh, Jun-Sung, and Eun-Jung Lee. "Enhanced Effect of Polyethyleneimine-Modified Graphene Oxide and Simvastatin on Osteogenic Differentiation of Murine Bone Marrow-Derived Mesenchymal Stem Cells." Biomedicines 9, no. 5 (May 2, 2021): 501. http://dx.doi.org/10.3390/biomedicines9050501.
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Statin derivatives traditionally have been used for the treatment of hyperlipidemia, but recent studies have shown their ability to regulate bone metabolism and promote bone growth. In this study, simvastatin (Sim), a new therapeutic candidate for bone regeneration, was combined with graphene oxide (GO), which has recently attracted much interest as a drug delivery method, to produce a compound substance effective for bone regeneration. To create a stable and homogenous complex with Sim, GO was modified with polyethylenimine, and the effect of modification was analyzed using Fourier transform infrared spectroscopy, zeta potential, and cytotoxicity testing. More specifically, the osteogenic differentiation potential expected by the combination of the two effective materials for osteogenic differentiation, GO and Sim, was evaluated in mesenchymal stem cells. Compared with control groups with GO and Sim used separately, the GO/Sim complex showed excellent osteogenic differentiation properties, with especially enhanced effects in the complex containing < 1 μM Sim.
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Barbălată, Cristina Ioana, Alina Silvia Porfire, Alina Sesarman, Valentin-Florian Rauca, Manuela Banciu, Dana Muntean, Rareș Știufiuc, Alin Moldovan, Cristian Moldovan, and Ioan Tomuță. "A Screening Study for the Development of Simvastatin-Doxorubicin Liposomes, a Co-Formulation with Future Perspectives in Colon Cancer Therapy." Pharmaceutics 13, no. 10 (September 22, 2021): 1526. http://dx.doi.org/10.3390/pharmaceutics13101526.
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An increasing number of studies published so far have evidenced the benefits of Simvastatin (SIM) and Doxorubicin (DOX) co-treatment in colorectal cancer. In view of this, the current study aimed to investigate the pharmaceutical development of liposomes co-encapsulating SIM and DOX, by implementing the Quality by Design (QbD) concept, as a means to enhance the antiproliferative effect of the co-formulation on C26 murine colon cancer cells co-cultured with macrophages. It is known that the quality profile of liposomes is dependent on the critical quality attributes (CQAs) of liposomes (drug entrapped concentration, encapsulation efficiency, size, zeta potential, and drug release profile), which are, in turn, directly influenced by various formulation factors and processing parameters. By using the design of experiments, it was possible to outline the increased variability of CQAs in relation to formulation factors and identify by means of statistical analysis the material attributes that are critical (phospholipids, DOX and SIM concentration) for the quality of the co-formulation. The in vitro studies performed on a murine colon cancer cell line highlighted the importance of delivering the optimal drug ratio at the target site, since the balance antiproliferative vs. pro-proliferative effects can easily be shifted when the molar ratio between DOX and SIM changes.
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Moffett, P., M. Reece, and J. Pelletier. "The murine Sim-2 gene product inhibits transcription by active repression and functional interference." Molecular and Cellular Biology 17, no. 9 (September 1997): 4933–47. http://dx.doi.org/10.1128/mcb.17.9.4933.
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The Drosophila single-minded (Dsim) gene encodes a master regulatory protein involved in cell fate determination during midline development. This protein is a member of a rapidly expanding family of gene products possessing basic helix-loop-helix (bHLH) and hydrophobic PAS (designated a conserved region among PER, ARNT [aryl hydrocarbon receptor nuclear translocator] and SIM) protein association domains. Members of this family function as central transcriptional regulators in cellular differentiation and in the response to environmental stimuli such as xenobiotics and hypoxia. We have previously identified a murine member of this family, called mSim-2, showing sequence homology to the bHLH and PAS domains of Dsim. Immunoprecipitation experiments with recombinant proteins indicate that mSIM-2 associates with the arnt gene product. In the present work, by using fine-structure mapping we found that the HLH and PAS motifs of both proteins are required for optimal association. Forced expression of GAL4/mSIM-2 fusion constructs in mammalian cells demonstrated the presence of two separable repression domains within the carboxy terminus of mSIM-2. We found that mSIM-2 is capable of repressing ARNT-mediated transcriptional activation in a mammalian two-hybrid system. This effect (i) is dependent on the ability of mSIM-2 and ARNT to heterodimerize, (ii) is dependent on the presence of the mSIM-2 carboxy-terminal repression domain, and (iii) is not specific to the ARNT activation domain. These results suggest that mSIM-2 repression activity can dominantly override the activation potential of adjacent transcription factors. We also demonstrated that mSIM-2 can functionally interfere with hypoxia-inducible factor 1alpha (HIF-1alpha)/ARNT transcription complexes, providing a second mechanism by which mSIM-2 may inhibit transcription.
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Aitola, Marjo, Christine M. Sadek, Jan-Åke Gustafsson, and Markku Pelto-Huikko. "Aint/Tacc3 Is Highly Expressed in Proliferating Mouse Tissues During Development, Spermatogenesis, and Oogenesis." Journal of Histochemistry & Cytochemistry 51, no. 4 (April 2003): 455–69. http://dx.doi.org/10.1177/002215540305100407.
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Aint was originally identified on the basis of its interaction in vitro with the aryl hydrocarbon nuclear receptor translocator (Arnt). Arnt is a common heterodimerization partner in the basic helix-loop–helix (bHLH)-PER-ARNT-SIM (PAS) protein family and is involved in diverse biological functions. These include xenobiotic metabolism, hypoxic response, and circadian rhythm. In addition, Arnt has a crucial role during development. Aint is a member of a growing family of transforming acidic coiled-coil (TACC) proteins and is the murine homologue of human TACC3. Here we report the spatiotemporal expression of Tacc3 mRNA and protein in embryonic, postnatally developing, and adult mouse tissues using in situ hybridization and immunocytochemistry. Tacc3 mRNA was highly expressed in proliferating cells of several organs during murine development. However, the only adult tissues expressing high levels were testis and ovary. Immunocytochemistry revealed that Tacc3 is a nuclear protein. Our results suggest that Tacc3 has an important role in murine development, spermatogenesis, and oogenesis.
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Ema, M., M. Morita, S. Ikawa, M. Tanaka, Y. Matsuda, O. Gotoh, Y. Saijoh, et al. "Two new members of the murine Sim gene family are transcriptional repressors and show different expression patterns during mouse embryogenesis." Molecular and Cellular Biology 16, no. 10 (October 1996): 5865–75. http://dx.doi.org/10.1128/mcb.16.10.5865.
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From a cDNA library of mouse skeletal muscle, we have isolated mouse Sim1 (mSim1) cDNA encoding a polypeptide of 765 amino acids with striking amino acid identify in basic helix-loop-helix (89% identify) and PAS (89 % identify) domains to previously identified mSim2, although the carboxy-terminal third of the molecule did not show any similarity to mSim2 or Drosophila Sim (dSim). Yeast two-hybrid analysis and coimmunoprecipitation experiments demonstrated that both of the mSim gene products interacted with Arnt even more efficiently than AhR, a natural partner of Arnt, suggesting a functional cooperativity with Arnt. In sharp contrast with dSim having transcriptional-enhancing activity in the carboxy-terminal region, the two mSims possessed a repressive activity toward Arnt in the heterodimer complex. This is the first example of bHLH-PAS proteins with transrepressor activity, although some genetic data suggest that dSim plays a repressive role in gene expression (Z. Chang, D. Price, S. Bockheim, M. J. Boedigheimer, R. Smith, and A. Laughon, Dev. Biol. 160:315-322, 1993; D. M. Mellerick and M. Nirenberg, Dev. Biol. 171:306-316, 1995). Whole-mount in situ hybridization showed restricted and characteristic expression patterns of the two mSim mRNAs in various tissues and organs during embryogenesis, such as those for the somite, the nephrogenic cord, and the mesencephalon (for mSim1) and those for the diencephalon, branchial arches, and limbs (for mSim2). From sequence similarity and chromosomal localization, it is concluded that mSim2 is an ortholog of hSim2, which is proposed to be a candidate gene responsible for Down's syndrome. The sites of mSim2 expression showed an overlap with the affected regions of the syndrome, further strengthening involvement of mSim2 in Down's syndrome.
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Aldhshan, Muhammad S., Gursagar Jhanji, Nicole J. Poritsanos, and Tooru M. Mizuno. "Glucose Stimulates Glial Cell Line-Derived Neurotrophic Factor Gene Expression in Microglia through a GLUT5-Independent Mechanism." International Journal of Molecular Sciences 23, no. 13 (June 25, 2022): 7073. http://dx.doi.org/10.3390/ijms23137073.
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Feeding-regulating neurotrophic factors are expressed in both neurons and glial cells. However, nutritional regulation of anorexigenic glial cell line-derived neurotrophic factor (GDNF) and orexigenic mesencephalic astrocyte-derived neurotrophic factor (MANF) expression in specific cell types remains poorly understood. Hypothalamic glucose sensing plays a critical role in the regulation of food intake. It has been theorized that local glucose concentration modulates microglial activity partially via glucose transporter 5 (GLUT5). We hypothesized that an increased local glucose concentration stimulates GDNF expression while inhibiting MANF expression in the hypothalamus and microglia via GLUT5. The present study investigated the effect of glucose on Gdnf and Manf mRNA expression in the mouse hypothalamus and murine microglial cell line SIM-A9. Intracerebroventricular glucose treatment significantly increased Gdnf mRNA levels in the hypothalamus without altering Manf mRNA levels. Exposure to high glucose caused a significant increase in Gdnf mRNA expression and a time-dependent change in Manf mRNA expression in SIM-A9 cells. GLUT5 inhibitor treatment did not block glucose-induced Gdnf mRNA expression in these cells. These findings suggest that microglia are responsive to changes in the local glucose concentration and increased local glucose availability stimulates the expression of microglial GNDF through a GLUT5-independent mechanism, contributing to glucose-induced feeding suppression.
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Mizuno, Tooru M., Pei San Lew, and Gursagar Jhanji. "Regulation of the Fructose Transporter Gene Slc2a5 Expression by Glucose in Cultured Microglial Cells." International Journal of Molecular Sciences 22, no. 23 (November 23, 2021): 12668. http://dx.doi.org/10.3390/ijms222312668.
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Microglia play a role in the regulation of metabolism and pathogenesis of obesity. Microglial activity is altered in response to changes in diet and the body’s metabolic state. Solute carrier family 2 member 5 (Slc2a5) that encodes glucose transporter 5 (GLUT5) is a fructose transporter primarily expressed in microglia within the central nervous system. However, little is known about the nutritional regulation of Slc2a5 expression in microglia and its role in the regulation of metabolism. The present study aimed to address the hypothesis that nutrients affect microglial activity by altering the expression of glucose transporter genes. Murine microglial cell line SIM-A9 cells and primary microglia from mouse brain were exposed to different concentrations of glucose and levels of microglial activation markers and glucose transporter genes were measured. High concentration of glucose increased levels of the immediate-early gene product c-Fos, a marker of cell activation, Slc2a5 mRNA, and pro-inflammatory cytokine genes in microglial cells in a time-dependent manner, while fructose failed to cause these changes. Glucose-induced changes in pro-inflammatory gene expression were partially attenuated in SIM-A9 cells treated with the GLUT5 inhibitor. These findings suggest that an increase in local glucose availability leads to the activation of microglia by controlling their carbohydrate sensing mechanism through both GLUT5-dependent and –independent mechanisms.
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Zapotoczny, Bartlomiej, Karolina Szafranska, Malgorzata Lekka, Balpreet Singh Ahluwalia, and Peter McCourt. "Tuning of Liver Sieve: The Interplay between Actin and Myosin Regulatory Light Chain Regulates Fenestration Size and Number in Murine Liver Sinusoidal Endothelial Cells." International Journal of Molecular Sciences 23, no. 17 (August 30, 2022): 9850. http://dx.doi.org/10.3390/ijms23179850.
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Liver sinusoidal endothelial cells (LSECs) facilitate the efficient transport of macromolecules and solutes between the blood and hepatocytes. The efficiency of this transport is realized via transcellular nanopores, called fenestrations. The mean fenestration size is 140 ± 20 nm, with the range from 50 nm to 350 nm being mostly below the limits of diffraction of visible light. The cellular mechanisms controlling fenestrations are still poorly understood. In this study, we tested a hypothesis that both Rho kinase (ROCK) and myosin light chain (MLC) kinase (MLCK)-dependent phosphorylation of MLC regulates fenestrations. We verified the hypothesis using a combination of several molecular inhibitors and by applying two high-resolution microscopy modalities: structured illumination microscopy (SIM) and scanning electron microscopy (SEM). We demonstrated precise, dose-dependent, and reversible regulation of the mean fenestration diameter within a wide range from 120 nm to 220 nm and the fine-tuning of the porosity in a range from ~0% up to 12% using the ROCK pathway. Moreover, our findings indicate that MLCK is involved in the formation of new fenestrations—after inhibiting MLCK, closed fenestrations cannot be reopened with other agents. We, therefore, conclude that the Rho-ROCK pathway is responsible for the control of the fenestration diameter, while the inhibition of MLCK prevents the formation of new fenestrations.
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Reinicke, Madlen, Judith Leyh, Silke Zimmermann, Soroth Chey, Ilijana Begcevic Brkovic, Christin Wassermann, Julia Landmann, et al. "Plant Sterol-Poor Diet Is Associated with Pro-Inflammatory Lipid Mediators in the Murine Brain." International Journal of Molecular Sciences 22, no. 24 (December 8, 2021): 13207. http://dx.doi.org/10.3390/ijms222413207.
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Plant sterols (PSs) cannot be synthesized in mammals and are exclusively diet-derived. PSs cross the blood-brain barrier and may have anti-neuroinflammatory effects. Obesity is linked to lower intestinal uptake and blood levels of PSs, but its effects in terms of neuroinflammation—if any—remain unknown. We investigated the effect of high-fat diet-induced obesity on PSs in the brain and the effects of the PSs campesterol and β-sitosterol on in vitro microglia activation. Sterols (cholesterol, precursors, PSs) and polyunsaturated fatty acid-derived lipid mediators were measured in the food, blood, liver and brain of C57BL/6J mice. Under a PSs-poor high-fat diet, PSs levels decreased in the blood, liver and brain (>50%). This effect was reversible after 2 weeks upon changing back to a chow diet. Inflammatory thromboxane B2 and prostaglandin D2 were inversely correlated to campesterol and β-sitosterol levels in all brain regions. PSs content was determined post mortem in human cortex samples as well. In vitro, PSs accumulate in lipid rafts isolated from SIM-A9 microglia cell membranes. In summary, PSs levels in the blood, liver and brain were associated directly with PSs food content and inversely with BMI. PSs dampen pro-inflammatory lipid mediators in the brain. The identification of PSs in the human cortex in comparable concentration ranges implies the relevance of our findings for humans.
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Lin, Xionghao, Elena Afia Adjei, Santosh L. Saraf, Victor R. Gordeuk, Sergei A. Nekhai, and Marina Jerebtsova. "Elevated Levels of Hgfl Protein in Sickle Cell Disease Urine Samples That Induce Glomerular Permeability." Blood 128, no. 22 (December 2, 2016): 4841. http://dx.doi.org/10.1182/blood.v128.22.4841.4841.
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Abstract Background: Chronic kidney disease (CKD) is a prevalent complication of sickle cell disease (SCD) associated with early mortality. Hemoglobinuria is a risk factor for the development of albuminuria and CKD. Currently, there are no biomarkers that predict outcome of CKD. Mass-spectrometry analysis of patient urine is a highly potent modern method for biomarker discovery. An in vitro glomerular permeability assay has been used as a non-invasive test for glomerular disease prognosis. Application of this assay to urinary samples collected before kidney disease onset provides a unique opportunity for differential proteomics of a limited number of urinary proteins. In combination with high resolution/selected ion monitoring (HR/SIM) mass-spectrometry methodology, this experimental platform provides an opportunity for biomarker discovery and validation of proteins whose concentration is too low to be detected by an immunological assay. Objectives: We aimed to determine a biomarker of early stage kidney disease using samples collected from the patients of the Center for Sickle Cell Disease at Howard University. We then used HR/SIM method to validate this biomarker in the cohort of SCD patients with and without CKD from University of Illinois at Chicago (UIC). Methods:Urinary protein, creatinine, albumin were measured by ELISA. The pH and specific gravity (SG) were determined by Multistix. Glomeruli were isolated from the murine kidney (FVB/N strain) and albumin permeability (Palb) activity was determined using urine samples collected from patients of the Center for Sickle Cell Disease, Howard University. Mass-spectrometry analysis was performed and Protein Discovery 1.4 and SIEVE 2.0 programs were used for protein analysis and label-free quantification. Heavy isotope labeled peptide EDQTSPAPGLR(13C6, 15N) was used as an internal standard for HR/SIM analysis of the samples from UIC. Results: Glomerular permeability assay was performed using six urinary samples collected from patients of the Center for Sickle Cell Disease, Howard University. Mass-spectrometry assay was performed for all samples. Samples with similar values of protein, albumin, creatinine, pH and SG, but different Palb activity were compared by SIEVE 2.0. Higher levels of hepatocyte growth factor-like (HGFL) protein were observed in three samples that induced glomerular permeability compared to three samples that did not. Since our attempts to produce antibodies to HGFL peptide for an ELISA assay were unsuccessful, we developed a HR/SIM method to measure HGFL peptide in urine. HR/SIM was performed for eight urine samples from the UIC cohort by measuring the ratio of ion peaks of HGFL peptide (m/z 585.79) and internal standard (IS) (m/z 590.80) (Fig. 1A). HGFL levels were found to be significantly increased (1.72-fold, p=0.015) in the urine samples of two patients at risk of developing renal disease based on the presence of hemoglobinuria compared to six samples without hemoglobinuria (Fig. 1B). Conclusions: Combination of in vitro glomerular permeability assay with mass-spectrometry method of protein discovery and HR/SIM validation assay may be a useful platform for discovery of biomarker of early stage of CKD. Urinary level of HGFL may serve as a prognostic marker for development of CKD in SCD patients. A limitation of our study is the small number of samples used for validation. Acknowledgments: This work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005 and 5G12MD007597. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH. Figure 1 HR/SIM analysis of HGFL peptide in human urine. (A)The ion peaks of HGFL peptide m/z 585.79 and internal standard (IS) m/z 590.80 were chosen for the high resolution/selective ion monitoring (HR/SIM) analysis using LTQ Orbitrap XL™ mass spectrometer. Extracted ion chromatograms (EICs) were based on a ±0.01 Da mass extraction window (MEW) centered on the theoretical m/z. (B) SCD patients at risk for developing renal disease based on the presence of hemoglobinuria showed significantly increased HGFL levels (1.72-fold, p=0.015). Figure 1. HR/SIM analysis of HGFL peptide in human urine. (A)The ion peaks of HGFL peptide m/z 585.79 and internal standard (IS) m/z 590.80 were chosen for the high resolution/selective ion monitoring (HR/SIM) analysis using LTQ Orbitrap XL™ mass spectrometer. Extracted ion chromatograms (EICs) were based on a ±0.01 Da mass extraction window (MEW) centered on the theoretical m/z. (B) SCD patients at risk for developing renal disease based on the presence of hemoglobinuria showed significantly increased HGFL levels (1.72-fold, p=0.015). Disclosures No relevant conflicts of interest to declare.
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Ngwa, Conelius, Abdullah Al Mamun, Yan Xu, Romana Sharmeen, and Fudong Liu. "Phosphorylation of Microglial IRF5 and IRF4 by IRAK4 Regulates Inflammatory Responses to Ischemia." Cells 10, no. 2 (January 30, 2021): 276. http://dx.doi.org/10.3390/cells10020276.
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Background: Interferon Regulatory Factor (IRF) 5 and 4 play a determinant role in regulating microglial pro- and anti-inflammatory responses to cerebral ischemia. How microglial IRF5 and IRF4 signaling are activated has been elusive. We hypothesized that interleukin-1 receptor associated kinase 4 (IRAK4) phosphorylates and activates IRF5 and IRF4 in ischemic microglia. We aimed to explore the upstream signals of the two IRFs, and to determine how the IRAK4-IRF signaling regulates the expression of inflammatory mediators, and impacts neuropathology. Methods: Spontaneously Immortalized Murine (SIM)-A9 microglial cell line, primary microglia and neurons from C57BL/6 WT mice were cultured and exposed to oxygen-glucose deprivation (OGD), followed by stimulation with LPS or IL-4. An IRAK4 inhibitor (ND2158) was used to examine IRAK4′s effects on the phosphorylation of IRF5/IRF4 and the impacts on neuronal morphology by co-immunoprecipitation (Co-IP)/Western blot, ELISA, and immunofluorescence assays. Results: We confirmed that IRAK4 formed a Myddosome with MyD88/IRF5/IRF4, and phosphorylated both IRFs, which subsequently translocated into the nucleus. Inhibition of IRAK4 phosphorylation quenched microglial pro-inflammatory response primarily, and increased neuronal viability and neurite lengths after ischemia. Conclusions: IRAK4 signaling is critical for microglial inflammatory responses and a potential therapeutic target for neuroinflammatory diseases including cerebral ischemia.
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Rios, Kariana E., Ming Zhou, Nathaniel M. Lott, Chelsi R. Beauregard, Dennis P. McDaniel, Thomas P. Conrads, and Brian C. Schaefer. "CARD19 Interacts with Mitochondrial Contact Site and Cristae Organizing System Constituent Proteins and Regulates Cristae Morphology." Cells 11, no. 7 (March 31, 2022): 1175. http://dx.doi.org/10.3390/cells11071175.
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CARD19 is a mitochondrial protein of unknown function. While CARD19 was originally reported to regulate TCR-dependent NF-κB activation via interaction with BCL10, this function is not recapitulated ex vivo in primary murine CD8+ T cells. Here, we employ a combination of SIM, TEM, and confocal microscopy, along with proteinase K protection assays and proteomics approaches, to identify interacting partners of CARD19 in macrophages. Our data show that CARD19 is specifically localized to the outer mitochondrial membrane. Through deletion of functional domains, we demonstrate that both the distal C-terminus and transmembrane domain are required for mitochondrial targeting, whereas the CARD is not. Importantly, mass spectrometry analysis of 3×Myc-CARD19 immunoprecipitates reveals that CARD19 interacts with the components of the mitochondrial intermembrane bridge (MIB), consisting of mitochondrial contact site and cristae organizing system (MICOS) components MIC19, MIC25, and MIC60, and MICOS-interacting proteins SAMM50 and MTX2. These CARD19 interactions are in part dependent on a properly folded CARD. Consistent with previously reported phenotypes upon siRNA silencing of MICOS subunits, absence of CARD19 correlates with irregular cristae morphology. Based on these data, we propose that CARD19 is a previously unknown interacting partner of the MIB and the MIC19–MIC25–MIC60 MICOS subcomplex that regulates cristae morphology.
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Vitale, Candida, Valentina Griggio, Chiara Riganti, Ivana Campia, Marta Robino, Micol Rigoni, Patrizia Sciancalepore, et al. "The Mevalonate Metabolic Pathway and the CXCL12/CXCR4 Axis Reciprocally Interact and Are Implicated in Fludarabine Resistance of Chronic Lymphocytic Leukemia Cells." Blood 124, no. 21 (December 6, 2014): 833. http://dx.doi.org/10.1182/blood.v124.21.833.833.
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Abstract BACKGROUND: Treatment of fludarabine-resistant chronic lymphocytic leukemia (CLL) patients is an unmet clinical need. Fludarabine resistance in CLL depends on intrinsic molecular features of the tumor cells, and on bidirectional interactions occurring between CLL cells and stromal cells (SC) of the tumor microenvironment. One of the main players of SC-induced fludarabine resistance is the CXCL12/CXCR4 axis. CXCR4 is a G protein-coupled receptor constitutively expressed on CLL cells. The binding of CXCR4 with CXCL12 activates the Ras/ERK1-2/Akt and the RhoA-dependent signalling pathways. To be active transducers Ras and RhoA need to undergo a post-translational modification (i.e. isoprenylation) by means of small molecules produced by the mevalonate (Mev) pathway. We have recently demonstrated that the Mev pathway is more active in IGHV unmutated than in mutated CLL cells, and is amenable to pharmacological manipulation by statins (i.e. simvastatin [Sim]). It is currently unknown whether the Mev pathway and its pharmacological targeting are implicated in the modulation of the CXCL12/CXCR4 axis and in the SC-induced fludarabine resistance of CLL cells. AIM: The aim of this study was to investigate the reciprocal interactions between the Mev pathway and the CXCL12/CXCR4 axis, in order to identify potential targets to counteract the constitutive and SC-induced fludarabine resistance of CLL cells. METHODS: Immuno-magnetically purified patient-derived CLL cells were cultured alone or with murine SC (M2-10B4 cell line). In selected experiments, cell cultures were exposed to human recombinant CXCL12 (100 μg/ml), CXCR4 inhibitor AMD3100 (5 μg/ml), fludarabine (F-ara-A, 10 μM), Sim (1 μM), ERK1-2 kinase inhibitor PD98059 (10 μM), RhoA kinase inhibitor Y276 (10 μM), HIF-1α inhibitor YC-1 (10 μM). The activity of the Mev pathway was measured by the quantification of metabolites [i.e. cholesterol and farnesyl pyrophosphate (FPP)] produced by CLL cells after 24 h incubation with 1 μCi of [3H]acetate. Ras and RhoA activities were evaluated measuring their GTP-bound fraction, taken as an index of the G-protein activation, respectively by pull-down assay and by an ELISA based assay. ERK1-2 and HIF-1α phosphorylation were evaluated by Western Blot. RhoA kinase, Akt and HIF-1α activities were measured with specific immunoassay kit. The amount of CXCL12 in culture supernatants was assessed by ELISA assay. Cell viability was determined by Annexin-V/propidium Iodide immunostaining and flow cytometry analysis. RESULTS: Co-culture with SC upregulated the Mev pathway activity of CLL cells, as shown by the increased production of cholesterol and FPP. This SC-induced increase in the Mev pathway activity was followed by the activation of the downstream Ras/ERK1-2 and RhoA/RhoA kinase signalling, the upregulation of the pro-survival factor Akt, and an increase in the transcriptional activity of HIF-1α. These biological and molecular effects were identically observed when CLL cells were exposed to recombinant CXCL12, and were completely abrogated by the CXCR4 antagonist AMD3100, thus showing the key role of the CXCL12/CXCR4 axis in the SC-induced modulation of the Mev pathway and the downstream Ras/ERK1-2 and RhoA/RhoA kinase signalling. On the other hand, blocking the Mev pathway by Sim and targeting ERK1-2 kinases, RhoA kinase and HIF-1α by specific small-molecule inhibitors significantly reduced the constitutive activity and the SC-induced upregulation of the Ras/ERK1-2 and RhoA/RhoA kinase signal transduction. A regulatory role of the Mev pathway on the CXCL12/CXCR4 axis was observed not only in CLL cells but also in SC, as shown by the significant reduction in CXCL12 secretion by SC exposed to Sim. In the last set of experiments, we found that the inhibition of the Mev pathway by Sim potentiated the direct cytotoxic effect of fludarabine against CLL cells. Even more importantly, both Sim and the downstream HIF-1α inhibitor YC-1 were capable of counteracting the SC-mediated protection of CLL cells from fludarabine-induced cytotoxicity. CONCLUSIONS: Our data demonstrate that the Mev pathway has a regulatory role on the CXCL12/CXCR4 axis and on the SC-mediated protective effects toward spontaneous and fludarabine-induced CLL cell death. The upstream inhibition of the Mev pathway and the downstream targeting of HIF-1α are promising strategies to circumvent fludarabine resistance in CLL. Disclosures Boccadoro: Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees.
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Jaśkiewicz, Anna, Beata Pająk, Anna Litwiniuk, Kaja Urbańska, and Arkadiusz Orzechowski. "Geranylgeraniol Prevents Statin-Dependent Myotoxicity in C2C12 Muscle Cells through RAP1 GTPase Prenylation and Cytoprotective Autophagy." Oxidative Medicine and Cellular Longevity 2018 (2018): 1–22. http://dx.doi.org/10.1155/2018/6463807.
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The present study investigated the cytotoxic effects of statins (atorvastatin (ATR) and simvastatin (SIM), resp.) and methyl-beta-cyclodextrin (MβCD), at their respective IC50concentrations, on muscle regeneration in the in vitro model of murine C2C12 myoblasts. Cotreatment with mevalonate (MEV), farnesol (FOH), geranylgeraniol (GGOH), or water-soluble cholesterol (Chol-PEG) was employed to determine whether the statin-dependent myotoxicity resulted from the lower cholesterol levels or the attenuated synthesis of intermediates of mevalonate pathway. Our findings demonstrated that while GGOH fully reverted the statin-mediated cell viability in proliferating myoblasts, Chol-PEG exclusively rescued MβCD-induced toxicity in myocytes. Statins caused loss of prenylated RAP1, whereas the GGOH-dependent positive effect was accompanied by loss of nonprenylated RAP1. Geranylgeranyltransferases are essential for muscle cell survival as inhibition with GGTI-286 could not be reversed by GGOH cotreatment. The increase in cell viability correlated with elevated AKT 1(S463) and GSK-3β(S9) phosphorylations. Slight increase in the levels of autophagy markers (Beclin 1, MAP LC-3IIb) was found in response to GGOH cotreatment. Autophagy rose time-dependently during myogenesis and was inhibited by statins and MβCD. Statins and MβCD also suppressed myogenesis and neither nonsterol isoprenoids nor Chol-PEG could reverse this effect. These results point to GGOH as the principal target of statin-dependent myotoxicity, whereas plasma membrane cholesterol deposit is ultimately essential to restore viability of MβCD-treated myocytes. Overall, this study unveils for the first time a link found between the GGOH- and Chol-PEG-dependent reversal of statin- or MβCD-mediated myotoxicity and cytoprotective autophagy, respectively.
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Hasegawa, Hideo, and Kartika Dewi. "LARVAE AND ADULTS OF ASCAROPS SP. (NEMATODA: SPIROCERCIDAE) COLLECTED FROM THE STOMACH OF MAXOMYS WHITEHEADI (RODENTIA: MURIDAE) IN KALIMANTAN, INDONESIA." TREUBIA 48, no. 1 (June 30, 2021): 69–80. http://dx.doi.org/10.14203/treubia.v48i1.4078.
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Third-stage larvae and adults of spiruroid nematodes were found from the stomach wall and stomach lumen, respectively, of Maxomys whiteheadi (Rodentia: Murinae) captured in Bukit Soeharto, Kalimantan, Indonesia. Close observation using light microscope and scanning electron microscope (SEM) revealed that it belongs to the genus Ascarops (Nematoda: Spirocercidae), possibly to Ascarops strongylina (Rudolphi, 1819). It is presumed that this species is parasitic in wild boars, Sus barbatus, in the forest of Kalimantan, and utilizes the murine as a paratenic host, in which it usually remains as third larval stage but can occasionally develop to adult stage.
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TAKEDA, Toshio. "Development of a Murine Model of Accelerated Senescence, SAM." Proceedings of The Japanese Association of Animal Models for Human Diseases 5 (1989): 1–6. http://dx.doi.org/10.1538/expanim1985.5.1.
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Yoshida, Tetsuya, Dai Izawa, Takashi Nakayama, Koichiro Nakahara, Mayumi Kakizaki, Toshio Imai, Ryuji Suzuki, Masayuki Miyasaka, and Osamu Yoshie. "Molecular cloning of mXCR1, the murine SCM-1/lymphotactin receptor." FEBS Letters 458, no. 1 (September 3, 1999): 37–40. http://dx.doi.org/10.1016/s0014-5793(99)01114-x.
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Takeda, T. "Senescence-accelerated mouse (SAM): A novel murine model of senescence." Experimental Gerontology 32, no. 1-2 (April 1997): 105–9. http://dx.doi.org/10.1016/s0531-5565(96)00036-8.
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Takeda, Toshio, Masanori Hosokawa, Keiichi Higuchi, Masamichi Hosono, Ichiro Akiguchi, and Hideki Katoh. "A novel murine model of aging, Senescence-Accelerated Mouse (SAM)." Archives of Gerontology and Geriatrics 19, no. 2 (September 1994): 185–92. http://dx.doi.org/10.1016/0167-4943(94)90039-6.
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Huang, Jing, Hongqi Huo, Mulan Chen, Lili Wang, Nani Li, Zhijian Huang, and Lin Yang. "Nanoparticle Albumin-Bound Paclitaxel is More Effective Than Paclitaxel in Experimental Endometrial Cancer." Science of Advanced Materials 14, no. 5 (May 1, 2022): 829–35. http://dx.doi.org/10.1166/sam.2022.4251.
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Nab-paclitaxel is a water-soluable formulation of paclitaxel. It is more effective and less toxic than paclitaxel in treating cancer. Here we compared nab-paclitaxel with paclitaxel for their antitumor efficacy and toxicity in vitro and in a murine endometrial cancer model. Nab-paclitaxel was more effective than paclitaxel in inhibiting cancer cell growth. Nab-paclitaxel also caused cell apoptosis and cell cycle G1 arrest more effectively than paclitaxel. Similar effect was observed in mice model of endometrial cancer. In summary this preliminary study shows nab-paclitaxel is a promising therapeutic agent for endometrial cancer and deserves further studies.
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Dennis, G. J., and J. J. Mond. "Corticosteroid-induced suppression of murine B cell immune response antigens." Journal of Immunology 136, no. 5 (March 1, 1986): 1600–1604. http://dx.doi.org/10.4049/jimmunol.136.5.1600.
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Abstract The quantitative variation in expression of B cell surface immune response-associated antigens (sIa) that is induced by in vivo i.v. administration of dexamethasone was studied by flow microfluorometry. Injection of 40 micrograms of dexamethasone resulted in a 35 to 40% reduction in the expression of sIa within 3 hr, reached its maximum effect within 6 hr, which on average resulted in 75% suppression of control values of sIa, and by 12 hr after injection began returning towards baseline levels. The suppressive effect of dexamethasone on B cell sIa was dose dependent with respect to the length of time required to reach maximal suppression, as well as with respect to the duration of suppression that was attained. When injections of dexamethasone were repeated on consecutive days, no additional increase in the level of sIa suppression achieved was observed. B cell sIa was also diminished after injection of dexamethasone into athymic nude mice, which suggests that the suppressive effect of dexamethasone on B cell expression of sIa is not a T cell-dependent phenomenon. Taken together, these data suggest that the suppression of B cell sIa by corticosteroids may be a means whereby endogenous or exogenous corticosteroids are able to influence the normal as well as abnormal immunologic state.
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Torres-Lopez, Ernesto, Nora Elizondo, Luz H. Verastegui, Jose J. Quijano, Rosa María Estrada-Martinez, Celia Mendiburu, and Víctor M. Castaño. "Smart Antibody-Conjugated Gold Nanoparticles for Bioengineered Polymers." Science of Advanced Materials 13, no. 2 (February 1, 2021): 217–21. http://dx.doi.org/10.1166/sam.2021.3887.
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Au and Ag nanoparticles (NP) were synthesized using a green method that allows control of both particle size and surface chemistry. The gold and silver nanoparticles were coated with a fluorescent goat anti-body IgG that chemically incorporated the nanoparticles and the internalization behavior was studied by phagocytosis in murine peritoneal macrophages. Despite that, in principle, the presence of a simple metal induces a greater degree of cell death following the particle uptake, our results suggest that a large part of the silver and gold nanoparticles enter cells by means other than endocytosis and phagocytosis, as truly intelligent nanoparticles. This represents a potential for immunotherapy and studies to modulate the innate immune response as truly smart nanoparticles.
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Takeda, Toshio, Masanori Hosokawa, and Keiichi Higuchi. "Senescence-Accelerated Mouse (SAM): A Novel Murine Model of Accelerated Senescence." Journal of the American Geriatrics Society 39, no. 9 (September 1991): 911–19. http://dx.doi.org/10.1111/j.1532-5415.1991.tb04460.x.
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Cornax, Ingrid, Jacob Zulk, Joshua Olson, Marcus Fulde, Victor Nizet, and Kathryn A. Patras. "Novel Models of Streptococcus canis Colonization and Disease Reveal Modest Contributions of M-Like (SCM) Protein." Microorganisms 9, no. 1 (January 16, 2021): 183. http://dx.doi.org/10.3390/microorganisms9010183.
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Streptococcus canis is a common colonizing bacterium of the urogenital tract of cats and dogs that can also cause invasive disease in these animal populations and in humans. Although the virulence mechanisms of S. canis are not well-characterized, an M-like protein, SCM, has recently identified been as a potential virulence factor. SCM is a surface-associated protein that binds to host plasminogen and IgGs suggesting its possible importance in host-pathogen interactions. In this study, we developed in vitro and ex vivo blood component models and murine models of S. canis vaginal colonization, systemic infection, and dermal infection to compare the virulence potential of the zoonotic S. canis vaginal isolate G361 and its isogenic SCM-deficient mutant (G361∆scm). We found that while S. canis establishes vaginal colonization and causes invasive disease in vivo, the contribution of the SCM protein to virulence phenotypes in these models is modest. We conclude that SCM is dispensable for invasive disease in murine models and for resistance to human blood components ex vivo, but may contribute to mucosal persistence, highlighting a potential contribution to the recently appreciated genetic diversity of SCM across strains and hosts.
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Wetzel, G., J. J. Clarke, and J. Hudson. "Three-Dimensional Visualization of Murine Cardiac Tissue Using FIB-SEM Segmentation Techniques." Microscopy and Microanalysis 19, S2 (August 2013): 914–15. http://dx.doi.org/10.1017/s1431927613006569.
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Jain, Shashank, Susan Russell, and Jerry Ware. "Platelet Glycoprotein VI Contributes to Murine Experimental Metastasis." Blood 112, no. 11 (November 16, 2008): 2862. http://dx.doi.org/10.1182/blood.v112.11.2862.2862.
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Abstract A key receptor supporting the normal role of platelets in hemostasis and thrombosis is the collagen receptor, glycoprotein (GP)VI. GPVI is a member of the immunoglobulin super family and is uniquely expressed on the surface of platelets where it is assembled with the ITAM-bearing activation subunit, FcR-g. We have previously reported the generation of a murine model of GP VI deficiency that revealed profound defects in collagen-induced platelet aggregation and major defects in platelet activation following adhesion under flow to fibrillar collagen. More recently, we have generated congenic GPVI deficient animals through an extensive breeding scheme to the inbred C57BL/6J strain. The relevance of specific platelet receptors in experimental metastasis is emerging with work from our laboratory establishing a key role for the platelet glycoprotein Ib- IX complex. We have now performed similar studies using congenic GPVI deficient animals. In this experimental model, murine tumor cells are placed in the tail vein of mice and establishment of lung tumor burden or lung tumor foci is examined two weeks following injection. This model represents a syngeneic model where animals are fully immunocompetent and the injected tumor cells were originally derived from animals of the C57BL/6J strain. Experiments comparing B16F10.1 cells (murine melanoma) and D121 cells (murine Lewis lung carcinoma) have been performed revealing a consistent and statistically significant reduction in tumor foci as a consequence of GPVI absence. In the case of melanoma cell injections into wild type C57BL/6J mice, a mean of 270 foci (SEM 44.0, n=6) is reduced to a mean of 134 foci (SEM 11.6, n=7) in the absence of platelet GPVI (p = 0.013). Similar reductions were observed using Lewis lung carcinoma cells with wild type animals revealing a mean of 132 surface foci (SEM 13.4, n=15) and GPVI deficient animals having a mean of 69 foci (SEM 7.9, n=12; p = 0.0003). Using either cell line an approximate reduction of 50% in the number of visible tumor foci was observed. Additional studies have been performed to compare the size and growth rate of subcutaneously implanted tumor cells, i.e., primary tumor growth. Here, we observed no noticeable size difference in primary tumors harvested 17 days following subcutaneous injection of D121 cells comparing the presence or absence of platelet GPVI. These results demonstrate in the platelet GPVI facilitates experimental tumor metastasis but does not contribute in the growth of primary tumors. These studies underscore the well-established paradigm of platelet adhesion and activation in hemostasis and thrombosis being applicable to mechanisms participating in the spread of cancer. The importance of metastasis in the prognosis for recovery from cancer can not be under emphasized. Indeed, the spread of metastatic disease represents a fundamental change in significantly shortening the life span of the cancer patient. Thus, understanding the molecules that regulate metastasis identifies potential targets for therapeutic intervention that could significantly improve the patient’s prognosis.
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UEJIMA, Y., Y. FUKUCHI, T. NAGASE, R. TABATA, and H. ORIMO. "A new murine model of aging lung: The senescence accelerated mouse (SAM)-P." Mechanisms of Ageing and Development 61, no. 3 (December 31, 1991): 223–36. http://dx.doi.org/10.1016/0047-6374(91)90057-7.
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de Harven, E., and H. Christensen. "High-resolution immunogold labeling of cell surfaces: The hypothetical homodimeric nature of the cd5 receptor." Proceedings, annual meeting, Electron Microscopy Society of America 46 (1988): 380–81. http://dx.doi.org/10.1017/s0424820100103966.
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Molecules exposed on cell surfaces and labeled with colloidal gold markers can be optimally demonstrated using the backscattered electron imaging (BEI) mode of the scanning electron microscope (SEM). Steric hindrance, however, limits labeling efficiency, making it necessary to use gold markers of small size for labeling at the molecular level. Using a JEOL 840 SEM equipped with a lanthanum hexaboride (LaB6) cathode, 13 nm gold particles were demonstrated. This, however, seems to represent the limit of the resolution of this type of instrument in the BEI mode. Fortunately, it has been demonstrated by Walther and Muller that 5 nm gold particles can be seen in the BEI mode, using field emission SEM.We have confirmed this observation, using the JEOL 890 field emission SEM and a solid state backscattered electron detector. Human peripheral blood lymphocytes prefixed with 0.1% glutaraldehyde, incubated with the murine monoclonal antibody LEU-1 (CD5), were labeled with a goat anti-murine IgG adsorbed on 5 nm gold particles (GAM-G5, Janssen Pharmaceutica, Beerse, Belgium) according to previously described procedures.
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Coscia, Marta, Micol Rigoni, Chiara Riganti, Candida Vitale, Ivana Campia, Valentina Griggio, Myriam Foglietta, et al. "The Mevalonate Pathway and Downstream Signal Transducers As Therapeutic Targets to Overcome Multidrug Resistance in Chronic Lymphocytic Leukemia (CLL)." Blood 120, no. 21 (November 16, 2012): 3881. http://dx.doi.org/10.1182/blood.v120.21.3881.3881.
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Abstract Abstract 3881 Background: The mutational status of tumor immunoglobulin heavy chain variable region (IGHV) is a reliable prognosticator in chronic lymphocytic leukemia (CLL): patients with unmutated (UM) IGHV have a worse prognosis than patients with mutated (M) IGHV. The tumor microenvironment actively supports the survival of CLL cells and confers a multidrug resistance (MDR) phenotype to CLL cells. MDR is due to the over-expression of membrane transporters, like P-glycoprotein (Pgp), which actively extrudes several anticancer drugs. Pgp is under the positive control of the transcription factor Hypoxia-Inducible-Factor-1-alfa (HIF-1α) which is activated by isoprenylated Ras/Rho-dependent downstream signaling pathways. Ras and Rho isoprenylation are regulated by the mevalonate (Mev) pathway activity suggesting that this pathway can be exploited as a metabolic checkpoint to regulate chemresistance. Aim: The aim of this study was twofold: 1) to investigate the correlation between chemoresistance and the activity of the Mev pathway and Ras/Rho-A downstream signaling pathways in purified M and UM CLL cells under basal conditions and after incubation with stromal cells; 2) to evaluate the chemosensitizing effects of agents specifically targeting the Mev pathway and downstream signaling pathways under the same culture conditions. Methods: M and UM CLL cells were cultured in the presence and in the absence of murine stromal cells (M210B4) and exposed to Zoledronic acid (ZA) (1 μmol/L), Simvastatine (Sim) (1 μmol/L), ERK1/2 kinase inhibitor PD98059 (10 μmol/L), HIF-1α inhibitor YC-1 (10 μmol/L) and Doxorubicine (Doxo) (1 μmol/L). The Mev pathway activity was measured by cells radiolabelling with [14C]-mevalonic acid and thin layer chromatography. Ras, ERK1/2 and Akt activity were detected by Western blot. Rho, Rho Kinase and HIF-1α activity were assessed by ELISA. Mdr1 expression was measured by Real Time-PCR. PgP activity was evaluated by measuring Doxo intracellular accumulation. Doxo cytotoxicity was assessed by annexin V and propidium iodide staining. Results: The Mev pathway is significantly more active in UM than in M CLL cells. This hypermetabolic activity translates into a higher activation of Ras/Akt and Rho/Rho kinase signaling pathways and higher expression of the phosphorylated active form of HIF-1α. HIF-1α activation positively regulates mdr1 gene expression in UM CLL cells leading to a more effective Doxo extrusion and therefore better survival upon Doxo exposure. M210B4 stromal cells further protect UM CLL cells from Doxo induced cell death by upregulating Mev pathway activity, HIF-1α/mdr1/PgP axis activation, and Doxo extrusion. Targeting the Mev pathway of UM cells with ZA and Mev reduces the basal activity of HIF-1α/mdr1/PgP axis and significantly increases Doxo retention and cytotoxicity. Similar effects are obtained with PD85 and YC1–10 which are specific inhibitors of the downstream molecules ERK-1/2 and HIF-1α, respectively. All these agents are able to overcome the protective effect exerted by stromal cells by significantly increasing PgP activity and Doxo-induced cell death. Conclusions: Our data demonstrate that the Ras- and Rho-dependent HIF-1α/mdr1/PgP axis is more active and associated with higher levels of MDR in UM compared with M CLL cells. Targeting the Mev pathway and/or downstream signalling pathways is a promising strategy to circumvent basal and stroma-mediated chemoresistance especially in UM CLL cells. Disclosures: Massaia: Novartis Farma S.p.A: Honoraria, Research Funding.
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Hayakawa, Eri H., and Hiroyuki Matsuoka. "Detailed methodology for high resolution scanning electron microscopy (SEM) of murine malaria parasitized-erythrocytes." Parasitology International 65, no. 5 (October 2016): 539–44. http://dx.doi.org/10.1016/j.parint.2016.03.006.
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Zhu, Yuan Xiao, Sally Benn, Zhi Hua Li, Ellen Wei, Esther Masih-Khan, Young Trieu, Meenakshi Bali, C. Jane McGlade, Jaime O. Claudio, and A. Keith Stewart. "The SH3–SAM Adaptor HACS1 is Up-regulated in B Cell Activation Signaling Cascades." Journal of Experimental Medicine 200, no. 6 (September 20, 2004): 737–47. http://dx.doi.org/10.1084/jem.20031816.
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HACS1 is a Src homology 3 and sterile alpha motif domain–containing adaptor that is preferentially expressed in normal hematopoietic tissues and malignancies including myeloid leukemia, lymphoma, and myeloma. Microarray data showed HACS1 expression is up-regulated in activated human B cells treated with interleukin (IL)-4, CD40L, and anti–immunoglobulin (Ig)M and clustered with genes involved in signaling, including TNF receptor–associated protein 1, signaling lymphocytic activation molecule, IL-6, and DEC205. Immunoblot analysis demonstrated that HACS1 is up-regulated by IL-4, IL-13, anti-IgM, and anti-CD40 in human peripheral blood B cells. In murine spleen B cells, Hacs1 can also be up-regulated by lipopolysaccharide but not IL-13. Induction of Hacs1 by IL-4 is dependent on Stat6 signaling and can also be impaired by inhibitors of phosphatidylinositol 3-kinase, protein kinase C, and nuclear factor κB. HACS1 associates with tyrosine-phosphorylated proteins after B cell activation and binds in vitro to the inhibitory molecule paired Ig-like receptor B. Overexpression of HACS1 in murine spleen B cells resulted in a down-regulation of the activation marker CD23 and enhancement of CD138 expression, IgM secretion, and Xbp-1 expression. Knock down of HACS1 in a human B lymphoma cell line by small interfering ribonucleic acid did not significantly change IL-4–stimulated B cell proliferation. Our study demonstrates that HACS1 is up-regulated by B cell activation signals and is a participant in B cell activation and differentiation.
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Popmihajlov, Zoran, and Kendall A. Smith. "The Importance of IL-2 for FOXP3 expression (88.8)." Journal of Immunology 178, no. 1_Supplement (April 1, 2007): S139. http://dx.doi.org/10.4049/jimmunol.178.supp.88.8.
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Abstract To examine the role of TCR-induced Interleukin-2 (IL2) in TCR-regulated FOXP3 expression, PBMC from normals were stimulated in vitro with anti-CD3 monoclonal antibody (OKT3) +/− anti-CD25 or anti-IL2, and/or human rIL-2. 4-color flow cytometry was used for the expression of FOXP3. OKT3 increased FOXP3 expression in both CD4+ and CD8+ T cells. Peak occurred at 24h (CD4+ 13.63% ± 0.45 (SEM)) vs. (CD8+ 5.62% ± 0.27 (SEM) (n=26), & by 96h was back to baseline levels (CD4+ 1.82% ± 0.24 (SEM) vs. CD8+ 0.13% ± 0.05 (SEM))(n=18). Both anti-CD25 and anti-IL2 inhibited FOXP3 expression, to a maximal suppression of ~ 60% in both CD4+ and CD8+ T cells. Human rIL-2 restored and prolonged FOXP3 expression. By comparison, anti CD28/CD3 stimulation +/− rIL2 of murine T cells does not activate FOXP3 expression by CD4+ cells and CD8+ cells do not express FOXP3 +/− stimulation. T cells from IL2 KO mice expressed ~ 50% FOXP3 vs. WT mice. Therefore, TCR-activated FOXP3 expression by mature human T cells is regulated in part by IL-2 signaling, and only occurs transiently in a subset of both CD4+ and CD8+ cells. In contrast, murine T cell FOXP3 expression cannot be activated via the TCR and IL2 deficiency reduces, but does not fully abrogate FOXP3 expression. Supported by NIAID grant # RO1-AI-51181 and the Belfer Foundation.
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Guo, Wenzhong, Diana Smith, Katja Aviszus, Thiago Detanico, Ryan A. Heiser, and Lawrence J. Wysocki. "Somatic hypermutation as a generator of antinuclear antibodies in a murine model of systemic autoimmunity." Journal of Experimental Medicine 207, no. 10 (August 30, 2010): 2225–37. http://dx.doi.org/10.1084/jem.20092712.
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Systemic lupus erythematosus (SLE) is characterized by high-avidity IgG antinuclear antibodies (ANAs) that are almost certainly products of T cell–dependent immune responses. Whether critical amino acids in the third complementarity-determining region (CDR3) of the ANA originate from V(D)J recombination or somatic hypermutation (SHM) is not known. We studied a mouse model of SLE in which all somatic mutations within ANA V regions, including those in CDR3, could be unequivocally identified. Mutation reversion analyses revealed that ANA arose predominantly from nonautoreactive B cells that diversified immunoglobulin genes via SHM. The resolution afforded by this model allowed us to demonstrate that one ANA clone was generated by SHM after a VH gene replacement event. Mutations producing arginine substitutions were frequent and arose largely (66%) from base changes in just two codons: AGC and AGT. These codons are abundant in the repertoires of mouse and human V genes. Our findings reveal the predominant role of SHM in the development of ANA and underscore the importance of self-tolerance checkpoints at the postmutational stage of B cell differentiation.
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Fabian, I., D. Douer, L. Levitt, Y. Kletter, and PL Greenberg. "Human spleen cell generation of factors stimulating human pluripotent stem cell, erythroid, and myeloid progenitor cell growth." Blood 65, no. 4 (April 1, 1985): 990–96. http://dx.doi.org/10.1182/blood.v65.4.990.990.
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Abstract Mitogen-stimulated murine spleen cells produce humoral substances capable of supporting murine hematopoiesis and pluripotent stem cell proliferation in vitro. Thus, we evaluated conditioned media generated by human spleen cells (SCM) in the presence or absence of mitogens for factors stimulatory for human pluripotent (CFU-GEMM), erythroid (BFU- E), and myeloid (CFU-GM) precursors. Two and one half percent to 10% SCM stimulated proliferation of all three types of precursor cells from nonadherent buoyant human marrow target cells. Mitogen-stimulated SCM augmented CFU-GM (175% to 225%), whereas CFU-GEMM and BFU-E growth was essentially unchanged. Cell separation procedures used to determine which cells provided these microenvironmental stimuli indicated that nonadherent mononuclear spleen cells provided the bulk of the CSF-GM, whereas adherent cells (95% nonspecific esterase + monocyte- macrophages) and nonadherent cells provided similar proportions of CSF- mix and erythroid burst-promoting activity (BPA). The nonadherent cells generating high levels of CSF-mix, BPA, and CSF-GM were predominantly Leu-1-negative, ie, non-T, cells. In the presence or absence of mitogens, SCM was a more potent source (1.3- to 3.8-fold) than peripheral leukocyte CM of the growth factors for the three progenitor cell types. Specific in situ cytochemical stains for analyzing morphology of myeloid colonies demonstrated that SCM stimulated the proliferation of the same types and proportions of colonies as human placental CM, suggesting that these CMs may contain similar CSF-GMs. These data show the contribution of spleen cell subsets to the generation of hematopoietic growth factors and the responsiveness of these cells to various mitogenic stimuli.
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Fabian, I., D. Douer, L. Levitt, Y. Kletter, and PL Greenberg. "Human spleen cell generation of factors stimulating human pluripotent stem cell, erythroid, and myeloid progenitor cell growth." Blood 65, no. 4 (April 1, 1985): 990–96. http://dx.doi.org/10.1182/blood.v65.4.990.bloodjournal654990.
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Mitogen-stimulated murine spleen cells produce humoral substances capable of supporting murine hematopoiesis and pluripotent stem cell proliferation in vitro. Thus, we evaluated conditioned media generated by human spleen cells (SCM) in the presence or absence of mitogens for factors stimulatory for human pluripotent (CFU-GEMM), erythroid (BFU- E), and myeloid (CFU-GM) precursors. Two and one half percent to 10% SCM stimulated proliferation of all three types of precursor cells from nonadherent buoyant human marrow target cells. Mitogen-stimulated SCM augmented CFU-GM (175% to 225%), whereas CFU-GEMM and BFU-E growth was essentially unchanged. Cell separation procedures used to determine which cells provided these microenvironmental stimuli indicated that nonadherent mononuclear spleen cells provided the bulk of the CSF-GM, whereas adherent cells (95% nonspecific esterase + monocyte- macrophages) and nonadherent cells provided similar proportions of CSF- mix and erythroid burst-promoting activity (BPA). The nonadherent cells generating high levels of CSF-mix, BPA, and CSF-GM were predominantly Leu-1-negative, ie, non-T, cells. In the presence or absence of mitogens, SCM was a more potent source (1.3- to 3.8-fold) than peripheral leukocyte CM of the growth factors for the three progenitor cell types. Specific in situ cytochemical stains for analyzing morphology of myeloid colonies demonstrated that SCM stimulated the proliferation of the same types and proportions of colonies as human placental CM, suggesting that these CMs may contain similar CSF-GMs. These data show the contribution of spleen cell subsets to the generation of hematopoietic growth factors and the responsiveness of these cells to various mitogenic stimuli.
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Coleman, R., S. Davidson, M. Amira, C. Khouri, and H. Ginsburg. "Murine lymphokine-activated killer (LAK) cells and granular metrial gland cells: TEM and SEM studies." Ultramicroscopy 26, no. 4 (January 1988): 434–35. http://dx.doi.org/10.1016/0304-3991(88)90296-3.
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Moon, Jong Wook, Eui Ri Na, and Young Joon Kim. "Surface Characteristics of Dentin Derived Hydroxyapatite Coated Layer on Titanium by RF Magnetron Sputtering." Key Engineering Materials 773 (July 2018): 349–53. http://dx.doi.org/10.4028/www.scientific.net/kem.773.349.
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The aim of this study was to evaluate surface characteristics and biological properties of the dentin derived hydroxyapatite (DDHA) coating on titanium substrate. DDHA was derived from extracted human teeth with calcination method at 850°C. The commercially pure titanium was used as a metallic substrate and a RF magnetron sputtering method was used as a coating method. Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray analysis (EDX) were performed to investigate the morphology and composition of coating surface. Atomic Forced Microscopy (AFM) and surface profiler were used to assess the surface morphology and roughness. Corrosion tests were performed in phosphate buffered saline at a 36.5±1°C in order to determine the corrosion behavior of the uncoated and coated surface samples. The biocompatibility of DDHA coating surface samples with murine osteoblastic cells was assessed by SEM. As a results, thin coating layer was observed on SEM images and uniformly cover the surfaces without change of titanium substrate. The EDX analysis of this coating surface indicated the presence of Ca, P elements. The mean surface roughness of cp-Ti and DDHA coating samples was 0.27μm, 1.7μm, respectively. The corrosion test indicated the stable passive film on coating samples. SEM observations of murine osteoblastic cells on coating surface showed that cells have proliferated and developed a network of dense interconnections. These results suggest that DDHA coating with RF magnetron sputtering method has good surface characteristics and biocompatibility.
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Kothapalli, Naga Rama, Darrell Norton, and Sebastian Fugmann. "IDENTIFICATION OF CIS-REGULATORY ELEMENTS TARGETING AID-MEDIATED SEQUENCE DIVERSIFICATION TO THE CHICKEN IMMUNOGLOBULIN LIGHT CHAIN GENE (88.2)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 88.2. http://dx.doi.org/10.4049/jimmunol.184.supp.88.2.
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Abstract Somatic hypermutation (SHM) and gene conversion (GCV) are two closely related processes that increase the diversity of the primary immunoglobulin (Ig) repertoire. Both processes are initiated by the activation-induced cytidine deaminase (AID) that converts cytosine residues to uracils in a transcription-dependent manner and processed by direct replication and error-prone DNA repair. It is unknown how this mutagenic activity is targeted almost exclusively to Ig loci. To identify cis-acting elements involved in targeting SHM and GCV to the chicken Ig light chain (IgL), we used DT40 cells and systematically deleted non-coding sequences from the IgL locus using standard gene targeting strategies. We identified a novel as of yet uncharacterized regulatory region (named 3′RR) in the chicken IgL gene, containing a classical transcriptional enhancer and also cis-acting DNA elements essential for targeting AID-mediated sequence diversification. We are continuing our systematic deletion strategy to identify the minimal sequence required for targeting and to determine the trans-acting factors that bind to it. To identify similar targeting elements in the murine Igκ locus, we use a cross-complementation approach replacing the 3′RR in DT40 cells with murine Igκ sequences. Previous mouse transgenic studies indicated that both iEκ and 3'Eκ were necessary for the SHM of Igκ transgenes, and we are currently testing the ability of these enhancers to restore SHM/GCV in the chicken locus.
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Barr, Justin, Anil Chauhan, and David G. Motto. "Endothelial Injury and Thrombus Formation In the Ferric Chloride Murine Model of Hemostasis." Blood 116, no. 21 (November 19, 2010): 650. http://dx.doi.org/10.1182/blood.v116.21.650.650.
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Abstract Abstract 650 Laboratory mice are used in hemostasis and thrombosis research in experiments ranging from the investigation of basic mechanisms of thrombus formation to the generation of preclinical data regarding potential therapeutic anti-thrombotic agents. With the common ferric chloride (FeCl3) model of vascular thrombosis, FeCl3 is applied directly to outside of the carotid artery or mesenteric vessels to induce thrombus formation. However, despite its widespread use, surprisingly little is understood regarding the mechanisms by which FeCl3 induces endothelial injury and subsequent thrombus formation, although it is believed that endothelial denudation and collagen exposure occur, and trigger the onset of thrombosis. Similarly, very little is known regarding the nature of the thrombi that are generated by this method, and importantly, whether these experimental thrombi resemble bona-fide thrombi that occur as a result of “true” vascular injury. To address these issues, we developed a scanning electron microscopy (SEM) protocol to visualize endothelial damage and thrombus formation that occur in situ. Briefly, thrombus formation is initiated by direct application of FeCl3 (or vehicle control) to the carotid artery, and subsequently at defined time points, the circulation is flushed, and then internally aldehyde-fixed. The affected section of carotid artery is removed, externally fixed, sectioned, processed for SEM, and visualized. With this procedure, we have obtained high-quality and high-magnification images of FeCl3-induced endothelial damage and thrombus formation through approximately 5 minutes of injury, in multiple samples and uninjured controls (N>100). Interestingly, we found that through these time points, FeCl3 induces little, if any, endothelial damage or subendothelial exposure. Rather, the endothelium rapidly assumes an “activated” phenotype, clearly changed from baseline, but non-denuded and intact. For verification, we found that mechanical injury of the murine carotid results in endothelial denudation which is easily identified by SEM. Perhaps more surprisingly, we also found that the first cells to adhere to the endothelium following FeCl3 application actually are red blood cells (RBCs), and not platelets. Following binding to the vascular surface, RBCs become misshapen and elongated in the direction of blood flow, or break off, leaving fragments associated with the endothelial surface. Subsequently, surface-bound RBCs and fragments bind additional RBCs and platelets from the circulation, forming large and characteristic-appearing complexes. From this point forward, large numbers of additional platelets accumulate on the RBC and platelet/RBC complexes, and the thrombus grows inward rapidly until the vessel becomes occluded. Next, to investigate formation of these potential RBC and platelet/RBC complexes with an independent system, we turned to intravital fluorescent microscopy in mesenteric venules. We found that FeCl3 rapidly induces structures consistent in size and shape with the “early” platelet/RBC complexes observed by SEM. With further time points, these small/early lesions subsequently bound more labeled platelets, and increased rapidly in size until occlusion of the vessel. As these platelet lesions grew, they also resembled in size and shape the “later” platelet/RBC complexes observed by SEM, including possession of a characteristic trailing “tail” of platelets. It is interesting that similar structures appeared to form in both the carotid artery and mesenteric venules, given the marked difference in shear stress between the two (1000-1500 s-1 vs. 100–200 s-1, respectively). To our knowledge, the existence of such platelet/RBC complexes has not been documented in any system. In summary, we have developed a technique to investigate in situ endothelial damage and thrombus formation in mice by SEM, and demonstrated that thrombus formation in the commonly-used FeCl3 murine model occurs in the absence of endothelial denudation, and appears to involve the active participation of RBCs. Further work will be necessary to confirm whether or not RBCs are necessary for thrombus formation in this model, and whether RBCs participate in any type of naturally-occurring hemostasis or thrombosis. Additionally, these results will likely have strong impact on future interpretation of experiments resulting from the use of FeCl3 in mice. Disclosures: No relevant conflicts of interest to declare.
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Kletter, Y., I. Riklis, I. Shalit, and I. Fabian. "Enhanced repopulation of murine hematopoietic organs in sublethally irradiated mice after treatment with ciprofloxacin." Blood 78, no. 7 (October 1, 1991): 1685–91. http://dx.doi.org/10.1182/blood.v78.7.1685.1685.
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Abstract We analyzed the effect of ciprofloxacin, fleroxacin, and ceftazidime on production of colony-stimulating factors (CSF) by cultured murine spleen cells in the presence of pokeweed mitogen (PWM). Ciprofloxacin at concentrations of 5 to 10 micrograms/mL in concert with PWM stimulated CSF production by cultured spleen cells. A 3.5-fold increase in the number of CFU-C was observed in the presence of ciprofloxacin- PWM spleen conditioned medium (SCM) as compared with control cultures exposed to PWM-SCM only. Antimurine GM-CSF and antimurine interleukin-3 (IL-3) antibodies inhibited colony formation stimulated by PWM-SCM or ciprofloxacin-PWM-SCM. Fleroxacin and ceftazidime at concentrations of 1 to 100 micrograms/mL and ciprofloxacin at high concentration (greater than 10 micrograms/mL) either did not affect CSF production by spleen cells or had an inhibitory effect. In vivo treatment of sublethally irradiated (650 rad) mice with ciprofloxacin (15 mg/kg per dose three times daily for 5 days) resulted in an increased number of myeloid progenitors in the spleen and bone marrow (BM) of treated mice. In contrast, treatment with ceftazidime did not affect progenitor cell numbers. On days 4 and 8 postirradiation ciprofloxacin-treated mice had a 2.3- and 3.8-fold increase, respectively, in the number of CFU-C in the BM. The number of CFU-C in the spleen did not increase on day 4 postirradiation, but on day 8, the number increased 1.7-fold. On day 4 postirradiation, sublethally irradiated mice treated with ciprofloxacin had a higher WBC count, RBC count, and hemoglobin level as compared with ceftazidime- and saline-treated mice. Twenty-four days postirradiation, 45% of saline-treated mice (20 of 44), and 35% of ceftazidime-treated mice (8 of 23) died, as compared with 13% (5 of 38) of ciprofloxacin-treated mice (P less than .05). These studies indicate that ciprofloxacin may have an immune-enhancing effect on the hematopoietic system in neutropenic mice.
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Kletter, Y., I. Riklis, I. Shalit, and I. Fabian. "Enhanced repopulation of murine hematopoietic organs in sublethally irradiated mice after treatment with ciprofloxacin." Blood 78, no. 7 (October 1, 1991): 1685–91. http://dx.doi.org/10.1182/blood.v78.7.1685.bloodjournal7871685.
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We analyzed the effect of ciprofloxacin, fleroxacin, and ceftazidime on production of colony-stimulating factors (CSF) by cultured murine spleen cells in the presence of pokeweed mitogen (PWM). Ciprofloxacin at concentrations of 5 to 10 micrograms/mL in concert with PWM stimulated CSF production by cultured spleen cells. A 3.5-fold increase in the number of CFU-C was observed in the presence of ciprofloxacin- PWM spleen conditioned medium (SCM) as compared with control cultures exposed to PWM-SCM only. Antimurine GM-CSF and antimurine interleukin-3 (IL-3) antibodies inhibited colony formation stimulated by PWM-SCM or ciprofloxacin-PWM-SCM. Fleroxacin and ceftazidime at concentrations of 1 to 100 micrograms/mL and ciprofloxacin at high concentration (greater than 10 micrograms/mL) either did not affect CSF production by spleen cells or had an inhibitory effect. In vivo treatment of sublethally irradiated (650 rad) mice with ciprofloxacin (15 mg/kg per dose three times daily for 5 days) resulted in an increased number of myeloid progenitors in the spleen and bone marrow (BM) of treated mice. In contrast, treatment with ceftazidime did not affect progenitor cell numbers. On days 4 and 8 postirradiation ciprofloxacin-treated mice had a 2.3- and 3.8-fold increase, respectively, in the number of CFU-C in the BM. The number of CFU-C in the spleen did not increase on day 4 postirradiation, but on day 8, the number increased 1.7-fold. On day 4 postirradiation, sublethally irradiated mice treated with ciprofloxacin had a higher WBC count, RBC count, and hemoglobin level as compared with ceftazidime- and saline-treated mice. Twenty-four days postirradiation, 45% of saline-treated mice (20 of 44), and 35% of ceftazidime-treated mice (8 of 23) died, as compared with 13% (5 of 38) of ciprofloxacin-treated mice (P less than .05). These studies indicate that ciprofloxacin may have an immune-enhancing effect on the hematopoietic system in neutropenic mice.
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Lee, Ju Kyung, Han Na Suh, Sung Hoon Yoon, Kyu Hong Lee, Sae Young Ahn, Hyung Jin Kim, and Sang Hee Kim. "Non-Destructive Monitoring via Electrochemical NADH Detection in Murine Cells." Biosensors 12, no. 2 (February 10, 2022): 107. http://dx.doi.org/10.3390/bios12020107.
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Nicotinamide adenine dinucleotide (NADH) is an important cofactor involved in metabolic redox reactions in living cells. The detection of NADH in living animal cells is a challenge. We developed a one-step monitoring method for NADH via an electrocatalytic reaction that uses a surface-modified, screen-printed electrode (SPE) having a redox active monolayer 4′-mercapto-N-phenlyquinone diamine (NPQD) formed by a self-assembled monolayer (SAM) of an aromatic thiol, 4-aminothiophenol (4-ATP). This electrode has a limit of detection (LOD) of 0.49 μM and a sensitivity of 0.0076 ± 0.0006 μM/μA in cell culture media, which indicates that it retains its selectivity. The applicability of this NADH sensor was demonstrated for the first time by cell viability monitoring via NADH-sensing in cell culture supernatants.
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WANG, Naidong, Anwen YUAN, Jun MA, Zhibang DENG, and Liqun XUE. "Sexing murine embryos with an indirect immunofluorescence assay using phage antibody B9-Fab against SDM antigen." Journal of Veterinary Medical Science 77, no. 6 (2015): 711–14. http://dx.doi.org/10.1292/jvms.14-0650.
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Öhman, Miina, Laura Oksanen, Katariina Kainulainen, Olli A. Jänne, Jaakko Kaprio, Markku Koskenvuo, Pertti Mustajoki, Kimmo Kontula, and Leena Peltonen. "Testing of human homologues of murine obesity genes as candidate regions in Finnish obese sib pairs." European Journal of Human Genetics 7, no. 2 (March 1999): 117–24. http://dx.doi.org/10.1038/sj.ejhg.5200256.
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Bentley, Stuart A., Suzanne L. Kirby, Pervin Anklesaria, and Joel S. Greenberger. "Biochemical and functional characterization of proteoglycans produced by SI/SId murine bone marrow stromal cell lines." Journal of Cellular Physiology 145, no. 1 (October 1990): 53–59. http://dx.doi.org/10.1002/jcp.1041450109.
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Thoene, Silvia, Vijay P. S. Rawat, Vegi M. Naidu, Wolfgang Hiddemann, Michaela Feuring-Buske, and Christian Buske. "Cdx4 Differs from Cdx2 in Its Oncogenic Potential and Its Regulation of Hox Gene Expression in the Murine Bone Marrow Transplantation Model." Blood 112, no. 11 (November 16, 2008): 1187. http://dx.doi.org/10.1182/blood.v112.11.1187.1187.
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Abstract Cdx4 is known to be of importance for specification of cell fate in embryonic hematopoiesis with defects leading to severe perturbation of blood formation. When overexpressed in a murine hematopoietic stem cell line, Cdx4 is capable to enhance progenitor formation in vitro and promote lymphoid reconstitution of lethally irradiated, transplanted mice in vivo. In line with this important function of Cdx4 in early hematopoiesis, we analyzed expression of Cdx4 in highly purified subpopulations isolated from murine bone marrow (BM) cells by TaqMan qPCR. Cdx4 showed an expression profile known from other stem cell regulatory genes with high expression in early hematopoietic progenitors followed by decreasing expression towards the more differentiated stages of hematopoiesis, with a more than 1200-fold lower expression in total BM cells compared to progenitor enriched 5-FU BM cells (n=3). To test the impact of Cdx4 on murine progenitors, we retrovirally transduced 5-FU BM cells with Cdx4. Overexpression of Cdx4 induced growth of BM cells in liquid expansion assay (Cdx4 5.7×108±2.2×108 SEM, EGFP 2.6×106±9×105 SEM, p=0.020; cell numbers after 14 days in cytokine supplemented medium, n=5). In addition, expression of Cdx4 conferred serial replating capacity to murine BM progenitors compared to empty vector control (CFU total after 3rd replating: 4.5×109±1.3×109 SEM/500 input cells in 1st CFC, n=5). This effect was significantly stronger compared to hematopoietic progenitors overexpressing the leukemogenic Cdx2 (p=0.008). Immunophenotyping of cells after 3rd replating showed expression of mainly myeloid antigens and cytospin preparation revealed a mature myeloid morphology. Interestingly, these colonies were able to engraft lethally irradiated mice and showed multilineage engraftment (lymphoid:myloid ratio week 16 after transplantation: 0.5:1, n=2), indicating the ability of Cdx4 expressing colonies to maintain stem cell properties in vitro. In contrast to Cdx2-transplanted mice which showed a severe myeloid bias, regular peripheral blood analysis of mice transplanted with Cdx4 overexpressing BM cells showed multilineage engraftment confirmed by immunophenotyping and normal hematological parameters (RBC 6.7×109±4.2×108, WBC 5.8×106±5.19×105; lymphoid:myeloid ratio 1.4:1; week 8–28). Of note, with a median latency of 309 days after transplantation, nine out of ten mice transplanted with Cdx4-transduced BM cells died of transplantable leukemia. In six out of seven cases we found single retroviral integration sites, indicating a monoclonal origin of the disease. We could determine three different integration sites located between 200 and 700 bp upstream of coding sequences (n=4; Opa3, Akap1, Sema4d). The integration sites of two other mice were located intragenic (Zfyve2, Zfp407), indicating that insertional mutagenesis might be a necessary factor for Cdx4 induced leukemogenesis. Moreover, qRT-PCR revealed that Cdx4 in contrast to Cdx2 did not induce ectopic expression of the leukemogenic Hoxb8 and was associated with a significant lower (7.8-fold) expression of the leukemogenic Hoxb6 in transduced murine BM cells. Taken together, these data indicate that Cdx4 plays a major role in the regulation of early hematopoiesis. Its expression profile and its hematopoietic activity in different hematopoietic assays clearly differs from Cdx2, which was shown to be highly leukemogenic in mice and to be ectopically expressed in human AML. Murine models analyzing the impact of Cdx4 and Cdx2 expression on hematopoietic development will help to delineate critical differences between the two related genes.
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Campbell, Andrew J., Kamel Ait-Tahar, Suketu D. Patel, Martin Barnardo, Amanda P. Liggins, Maite Caibes, Alison Banham, Graham Collins, Chris S. R. Hatton, and Karen Pulford. "Cytotoxic T-Cell Response to the Cancer Testis Antigen PASD1 In Multiple Myeloma: PASD1 Peptides for a Generic Vaccine to Treat PASD1-Positive Haematological Malignancies." Blood 116, no. 21 (November 19, 2010): 4294. http://dx.doi.org/10.1182/blood.v116.21.4294.4294.
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Abstract Abstract 4294 Multiple myeloma (MM) is a bone marrow localized plasma cell tumor comprising 1% of all cancers and 10–15% of hematological malignancies. Despite significant advances in treatment, such as bortezomib, patients currently only have a 5-year survival rate of approximately 35%. The identification of improved therapeutic options therefore remains a priority. There is increasing evidence for a role of the immune system in tumor progression. Examples include the remission of some leukemias and lymphomas in immunocompetent patients while allogeneic stem cell transplantation also has a graft-versus-tumor effect in MM. The immunotherapeutic targeting of tumor-associated antigens (TAAs) on MM cells therefore represents an important approach for improved treatment of this disease. MM cells express a number of TAAs, of which cancer testis antigens (CTAs) are of particular interest. Their restricted normal tissue distribution combined with widespread expression in tumors makes them attractive immunotherapeutic targets whilst minimizing potential problems with autoimmunity. Reports of cytotoxic T cells (CTLs, the major effector cells in tumor immunity) and CD4-positive T-helper cells recognizing the NY-ESO-1 and MAGE CTAs in patients with MM suggests the presence of a spontaneous immune response to these molecules, which can be boosted through vaccination with CTAs such as NY-ESO-1. We previously identified PAS (Per ARNT Sim) domain containing 1 (PASD1) protein as a novel diffuse large B-cell lymphoma (DLBCL)-associated CTA. Importantly, PASD1 has a restricted normal tissue distribution but is present in a range of hematological malignancies, including MM. Subsequent in vitro studies have identified immunogenic PASD1 peptides that elicit PASD1-driven CTL or CD4-positive T-helper responses in peripheral blood mononuclear cells from DLBCL patients. Studies using a pre-clinical in vivo murine model have confirmed the immunogenicity of the PASD1 CTL peptides. These critical steps support the use of PASD1 as a potential immunotherapeutic target in DLBCL. The current study was performed to ascertain whether the PASD1 CTL peptides were immunogenic in MM patients and thus have utility for immunotherapy in this disease. Blood samples were obtained from 9 post-treatment myeloma patients attending the John Radcliffe Hospital following informed consent. Peripheral blood mononuclear cells were incubated with the PASD1 CTL peptides PASD1(1)38-47 (QLLDGFMITL) and PASD1(2)167-175 (YLVGNVCIL). A gamma-interferon ELISPOT release assay was performed after 8–10 days. The results are summarized in Table 1.Table 1:Gamma-IFN response to PASD1 in patients with MM.PatientsMHC Class I*Gamma-IFN response to peptides/50,000 cellsPASD1(1)PASD1(2)HIV-1PHADSA*0201+2+/−112+/−41+/−1100+/−14MWA*0201+52+/−244+/−220+/−266+/−8DMA*0201− A*2601+28+/−246+/−68+/−298+/−10JBA*0201+0+/−00+/−02+/−088+/−12JYA*0201+–––72+/−10DAA*0201−2+/−20+/−00+/−082+/−10GGA*0201−58+/−268+/−244+/−288+/−10MDA*0201−52+/−228+/−242+/−292+/−12RSA*0201––––*Results were considered positive if the number of spots in the test wells were at least twice that found in the irrelevant HIV-1 cultures. A significant gamma-interferon response was detected to one or both of the PASD1 peptides in 2/4 A*0201-positive evaluable patients. Analysis of the SYPETHI web-based algorithm predicted the PASD1 peptides used here to be immunogenic in the context of A*2601 and this was confirmed in the one A*2601+ patient studied here. No significant response was detected in the 3 A*0201 and A*2601-negative patients. Double immunolabeling studies using antibodies to PASD1 and CD138 showed PASD1 to be present in a subset of tumor cells in all 7 patients with evaluable ELISPOT data. Our findings demonstrate the immunogenicity of both the PASD1(1) and PASD1(2) peptides in patients with MM. These ‘generic’ peptides therefore represent vaccine candidates for inclusion in a vaccine targeting multiple PASD1-positive hematological malignancies. Disclosures: Banham: University of Oxford: Patents & Royalties. Pulford:Leukaemia and Lymphoma Research: Patents & Royalties.
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Hiroshige, Tasuku, Kei-Ichiro Uemura, Shingo Hirashima, Kiyosato Hino, Akinobu Togo, Keisuke Ohta, Tsukasa Igawa, and Kei-Ichiro Nakamura. "Three-Dimensional Analysis of Interstitial Cells in the Smooth Muscle Layer of Murine Vas Deferens Using Confocal Laser Scanning Microscopy and FIB/SEM." Microscopy and Microanalysis 28, no. 2 (January 26, 2022): 567–75. http://dx.doi.org/10.1017/s1431927622000058.
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The smooth muscle contraction of the vas deferens has the important function of transporting sperm. Interstitial cells (ICs) play a critical role in the pacing and modulation of various smooth muscle organs by interactions with nerves and smooth muscle. Elucidating the three-dimensional (3D) architecture of ICs is important for understanding their spatial relationship on the mesoscale between ICs, smooth muscle cells (SMCs), and nerves. In this study, the 3D ultrastructure of ICs in the smooth muscle layer of murine vas deferens and the spatial relationships between ICs, nerves, and smooth muscles were observed using confocal laser scanning microscopy and focused ion beam/scanning electron microscopy. ICs have sheet-like structures as demonstrated by 3D observation using modern analytical techniques. Sheet-like ICs have two types of 3D structures, one flattened and the other curled. Multiple extracellular vesicle (EV)-like structures were frequently observed in ICs. Various spatial relations were observed in areas between ICs, nerves, and SMCs, which formed a complex 3D network with each other. These results suggest that ICs in the smooth muscle layer of murine vas deferens may have two subtypes with different sheet-like structures and may be involved in neuromuscular signal transmission via physical interaction and EVs.
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Soares, Mônica R. P. S., Rafael O. Corrêa, Pedro Henrique F. Stroppa, Flávia C. Marques, Gustavo F. S. Andrade, Charlane C. Corrêa, Marcos Antônio F. Brandão, and Nádia R. B. Raposo. "Biosynthesis of silver nanoparticles using Caesalpinia ferrea (Tul.) Martius extract: physicochemical characterization, antifungal activity and cytotoxicity." PeerJ 6 (March 19, 2018): e4361. http://dx.doi.org/10.7717/peerj.4361.
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Background Green synthesis is an ecological technique for the production of well characterized metallic nanoparticles using plants. This study investigated the synthesis of silver nanoparticles (AgNPs) using a Caesalpinia ferrea seed extract as a reducing agent. Methods The formation of AgNPs was identified by instrumental analysis, including ultraviolet–visible (UV–Vis) spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD) of the AgNPs, and surface-enhanced Raman scattering (SERS) spectra of rhodamine-6G (R6G). We studied the physicochemical characterization of AgNPs, evaluated them as an antifungal agent against Candida albicans, Candida kruzei, Candida glabrata and Candida guilliermondii, and estimated their minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values. Lastly, this study evaluated the cytotoxicity of the AgNPs in murine L929 fibroblasts cells using an MTT assay. Results The UV–Vis spectroscopy, SERS, SEM and XRD results confirmed the rapid formation of spheroidal 30–50 nm AgNPs. The MIC and MFC values indicated the antifungal potential of AgNPs against most of the fungi studied and high cell viability in murine L929 fibroblasts. In addition, this study demonstrated that C. ferrea seed extracts may be used for the green synthesis of AgNPs at room temperature for the treatment of candidiasis.
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Podar, Klaus, Alexander Zimmerhackl, Ursula Hainz, Mariateresa Fulciniti, Sonia Vallet, Dian Olsen, Yu-Tzu Tai, Teru Hideshima, Dharminder Chauhan, and Kenneth C. Anderson. "Potential Therapeutic Role of the Selective Adhesion Molecule (SAM) Inhibitor Natalizumab in Multiple Myeloma." Blood 114, no. 22 (November 20, 2009): 1850. http://dx.doi.org/10.1182/blood.v114.22.1850.1850.
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Abstract Abstract 1850 Poster Board I-876 Multiple Myeloma (MM) is characterized by the clonal proliferation of malignant plasma cells in the bone marrow. Despite current therapeutic approach and prolongation of the median survival, new therapies are urgently needed. Integrins are cell surface receptors which mediate both cell-cell adhesion and cell-extracellular matrix (ECM) protein adhesion. beta1-integrins, including very-late antigen-4 (VLA-4;á4β1), are typically expressed on MM cells. In MM, VLA-4-mediated binding to ECMS and bone marrow stromal cells (BMSCs) confers protection against drug-induced apoptosis and triggers transcription and secretion of IL-6, the major MM growth and survival factor. In addition to up-regulation of cell surface-clustering, integrin activity can also be triggered by multiple agonists through ‘inside-out’ signaling, independent of changes in integrin expression levels. Importantly, VEGF-induced migration of MM cells on fibronectin is also associated with β1-integrin- and PI3-kinase- dependent PKC activation. Targeting VLA-4 is therefore of potential high therapeutic interest in MM. Indeed, an antibody against murine á4 induces inhibition of MM growth in a murine model. Natalizumab is a recombinant humanized IgG4 monoclonal antibody, which belongs to a new class of molecules known as selective adhesion molecule (SAM) inhibitors and binds to á4-integrin. Clinically, Natalizumab has demonstrated activity in patients with multiple sclerosis and Crohn's disease. Here we tested the potential therapeutic role of Natalizumab on MM cell survival, and migration in the BM microenvironment. VLA-4 is expressed by all MM cell lines investigated (NCIH929, RPMI8226, INA-6, MM.1S, and OPM2). Functionally, Natalizumab but not a control antibody, triggered dose-dependent inhibition of MM cell adhesion to fibronectin, BMSCs, and endothelial cells (ECs). Importantly, inhibition of adhesion to fibronectin, BMSCs, or ECs was observed in MM cells pretreated with Natalizumab. Moreover, inhibition of MM cell adhesion to fibronectin, BMSCs, or ECs was also observed when Natalizumab was added to already adherent MM cells. Taken together, Natalizumab decreases adhesion of non-adherent MM cells as well as binding of already adherent MM cells to non-cellular and cellular components of the microenvironment. Given the protective role of the microenvironment on MM cell survival, we next sought to evaluate the chemosensitizing activity of Natalizumab. Specifically, we investigated dose- and time- dependent effects of Natalizumab, alone and when combined with conventional and novel therapies, on MM cells. Our results show that Natalizumab alone did not inhibit growth or survival of MM cells when cultured without components of the microenvironment. However, Natalizumab enhanced sensitivity of tumor cells to both bortezomib and dexamethasone in MM-BMSC and, MM-EC co-cultures. These data indicate a potential role of Natalizumab in bortezomib- and dexamethasone-containing treatment regimens including MPV. Moreover, Natalizumab decreases IL-6 and VEGF secretion triggered in MM-BMSC co-cultures. Consequently, angiogenesis triggered by supernatants of Natalizumab- treated MM-BMSC co-cultures was inhibited. Moreover, Natalizumab blocked MM cell migration on fibronectin triggered by both VEGF and IGF-1. Finally, our previous results implicate an PKC signaling in MM cell migration on fibronectin, and our current results show that Natalizumab inhibits phosphorylation of á4 integrins and PKC induced by co-stimulation with VEGF/ fibronectin, IGF-1/ fibronectin, and patient serum. Taken together, our data indicate a potential therapeutic role of Natalizumab in MM. Ongoing studies evaluating the effect of Natalizumab in a SCID-hu murine model of MM will also be reported. Disclosures: Podar: Biogen Idec: Research Funding. Off Label Use: natalizumab, integrin inhibitor. Zimmerhackl:Biogen Idec: Research Funding. Olsen:Biogen Idec: Employment.