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Sample records for murine microglial cells

  1. Enhanced detection and study of murine norovirus-1 using a more efficient microglial cell line

    PubMed Central

    Cox, Courtney; Cao, Shengbo; Lu, Yuanan

    2009-01-01

    Background Human Noroviruses are the predominant cause of non-bacterial gastroenteritis worldwide. To facilitate prevention and control, a norovirus isolated from mice can provide a model to understand human noroviruses. To establish optimal viral infectivity conditions for murine noroviruses, several cell lines of hematopoietic lineage, including murine BV-2, RAW 264.7, and TIB, as well as human CHME-5, were tested comparatively for their sensitivity to murine norovirus-1. Results Except for CHME-5, all three murine-derived cell lines were susceptible to MNV infection. Viral infection of these cells was confirmed by RT-PCR. Using both viral plaque and replication assays, BV-2 and RAW 264.7 cells were determined to have comparable sensitivities to MNV-1 infection. Comparisons of cell growth characteristics, general laboratory handling and potential in-field applications suggest the use of BV-2 to be more advantageous. Conclusion Results obtained from these studies demonstrate that an immortalized microglial cell line can support MNV-1 replication and provides a more efficient method to detect and study murine noroviruses, facilitating future investigations using MNV-1 as a model to study, detect, and control Human Norovirus. PMID:19903359

  2. The origin of microglial cells.

    PubMed Central

    Boya, J; Calvo, J; Prado, A

    1979-01-01

    The rat brain has been studied between 6 hours after birth and 100 days, using histochemical techniques for acid phosphatase and peroxidase, and silver impregnation for microglial cells. The results indicate that microglia come initially from acid phosphatase-positive cells of the meninges. These invade the nervous parenchyma and transform into ramified microglia. At 3 days of age similar cells are present on the outer surface of the large blood vessels, from which site they migrate into the nervous parenchyma. In 100 days old rats the acid phosphatase-positive cells are practically all pericytes. None of the microglial cells or their precursors give a positive reaction for peroxidase. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 PMID:511761

  3. Isolation and analysis of mouse microglial cells.

    PubMed

    Garcia, Jenny A; Cardona, Sandra M; Cardona, Astrid E

    2014-01-01

    Microglia are mononuclear phagocytes that make up about 10% of the central nervous system (CNS). They are known for their surveillant behavior, which involves continuous monitoring of neural tissue by extending and retracting their processes. Microglial cells are derived from myeloid progenitor cells and play important roles in homeostasis as well as inflammatory and immune responses in the brain. This unit describes several microglial cell isolation protocols that can be easily adapted for projects requiring a rapid and efficient analysis of mouse microglial cells by flow cytometry. Methods for visualizing microglial cells using in situ immunohistochemistry and immunochemistry in free-floating sections are also included.

  4. Trimethyltin-Induced Microglial Activation via NADPH Oxidase and MAPKs Pathway in BV-2 Microglial Cells

    PubMed Central

    Kim, Da Jung; Kim, Yong Sik

    2015-01-01

    Trimethyltin (TMT) is known as a potent neurotoxicant that causes neuronal cell death and neuroinflammation, particularly in the hippocampus. Microglial activation is one of the prominent pathological features of TMT neurotoxicity. Nevertheless, it remains unclear how microglial activation occurs in TMT intoxication. In this study, we aimed to investigate the signaling pathways in TMT-induced microglial activation using BV-2 murine microglial cells. Our results revealed that TMT generates reactive oxygen species (ROS) and increases the expression of CD11b and nuclear factor-κB- (NF-κB-) mediated nitric oxide (NO) and tumor necrosis factor- (TNF-) α in BV-2 cells. We also observed that NF-κB activation was controlled by p38 and JNK phosphorylation. Moreover, TMT-induced ROS generation occurred via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in BV-2 cells. Interestingly, treatment with the NADPH oxidase inhibitor apocynin significantly suppressed p38 and JNK phosphorylation and NF-κB activation and ultimately the production of proinflammatory mediators upon TMT exposure. These findings indicate that NADPH oxidase-dependent ROS generation activated p38 and JNK mitogen-activated protein kinases (MAPKs), which then stimulated NF-κB to release proinflammatory mediators in the TMT-treated BV-2 cells. PMID:26221064

  5. Mineralocorticoid and glucocorticoid receptors differentially regulate NF-kappaB activity and pro-inflammatory cytokine production in murine BV-2 microglial cells

    PubMed Central

    2012-01-01

    Background Microglia, the resident macrophage-like cells in the brain, regulate innate immune responses in the CNS to protect neurons. However, excessive activation of microglia contributes to neurodegenerative diseases. Corticosteroids are potent modulators of inflammation and mediate their effects by binding to mineralocorticoid receptors (MR) and glucocorticoid receptors (GR). Here, the coordinated activities of GR and MR on the modulation of the nuclear factor-κB (NF-κB) pathway in murine BV-2 microglial cells were studied. Methods BV-2 cells were treated with different corticosteroids in the presence or absence of MR and GR antagonists. The impact of the glucocorticoid-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) was determined by incubating cells with 11-dehydrocorticosterone, with or without selective inhibitors. Expression of interleukin-6 (IL-6), tumor necrosis factor receptor 2 (TNFR2), and 11β-HSD1 mRNA was analyzed by RT-PCR and IL-6 protein expression by ELISA. NF-κB activation and translocation upon treatment with various corticosteroids were visualized by western blotting, immunofluorescence microscopy, and translocation assays. Results GR and MR differentially regulate NF-κB activation and neuroinflammatory parameters in BV-2 cells. By converting inactive 11-dehydrocorticosterone to active corticosterone, 11β-HSD1 essentially modulates the coordinated action of GR and MR. Biphasic effects were observed for 11-dehydrocorticosterone and corticosterone, with an MR-dependent potentiation of IL-6 and tumor necrosis factor-α (TNF-α) expression and NF-κB activation at low/moderate concentrations and a GR-dependent suppression at high concentrations. The respective effects were confirmed using the MR ligand aldosterone and the antagonist spironolactone as well as the GR ligand dexamethasone and the antagonist RU-486. NF-κB activation could be blocked by spironolactone and the inhibitor of NF-κB translocation Cay-10512

  6. Isolation and Analysis of Mouse Microglial Cells

    PubMed Central

    Garcia, Jenny A.; Cardona, Sandra M.

    2014-01-01

    Microglia are mononuclear phagocytes that make up about 10% of the central nervous system (CNS). They are known for their surveillant behavior which comprises continuously monitoring neural tissue by extending and retracting their processes. Microglial cells are derived from myeloid progenitor cells and play important roles in homeostasis, inflammatory and immune responses in the brain. This Unit describes several microglial cell isolation protocols (Basic Protocol 1, Alternate Protocol, and Basic Protocol 2) that can be easily adapted for projects requiring a rapid and efficient analysis of mouse microglial cells by flow cytometry (Support Protocol 1). Methods for visualizing microglial cells using in situ immunohistochemistry (Basic Protocol 3) and immunochemistry in free-floating sections (Basic Protocol 4) are also included. PMID:24510618

  7. Blue Light Modulates Murine Microglial Gene Expression in the Absence of Optogenetic Protein Expression

    PubMed Central

    Cheng, Kevin P.; Kiernan, Elizabeth A.; Eliceiri, Kevin W.; Williams, Justin C.; Watters, Jyoti J.

    2016-01-01

    Neural optogenetic applications over the past decade have steadily increased; however the effects of commonly used blue light paradigms on surrounding, non-optogenetic protein-expressing CNS cells are rarely considered, despite their simultaneous exposure. Here we report that blue light (450 nm) repetitively delivered in both long-duration boluses and rapid optogenetic bursts gene-specifically altered basal expression of inflammatory and neurotrophic genes in immortalized and primary murine wild type microglial cultures. In addition, blue light reduced pro-inflammatory gene expression in microglia activated with lipopolysaccharide. These results demonstrate previously unreported, off-target effects of blue light in cells not expressing optogenetic constructs. The unexpected gene modulatory effects of blue light on wild type CNS resident immune cells have novel and important implications for the neuro-optogenetic field. Further studies are needed to elucidate the molecular mechanisms and potential therapeutic utility of blue light modulation of the wild type CNS. PMID:26883795

  8. Role of orexin A signaling in dietary palmitic acid-activated microglial cells.

    PubMed

    Duffy, Cayla M; Yuan, Ce; Wisdorf, Lauren E; Billington, Charles J; Kotz, Catherine M; Nixon, Joshua P; Butterick, Tammy A

    2015-10-01

    Excess dietary saturated fatty acids such as palmitic acid (PA) induce peripheral and hypothalamic inflammation. Hypothalamic inflammation, mediated in part by microglial activation, contributes to metabolic dysregulation. In rodents, high fat diet-induced microglial activation results in nuclear translocation of nuclear factor-kappa B (NFκB), and increased central pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). The hypothalamic neuropeptide orexin A (OXA, hypocretin 1) is neuroprotective in brain. In cortex, OXA can also reduce inflammation and neurodegeneration through a microglial-mediated pathway. Whether hypothalamic orexin neuroprotection mechanisms depend upon microglia is unknown. To address this issue, we evaluated effects of OXA and PA on inflammatory response in immortalized murine microglial and hypothalamic neuronal cell lines. We demonstrate for the first time in microglial cells that exposure to PA increases gene expression of orexin-1 receptor but not orexin-2 receptor. Pro-inflammatory markers IL-6, TNF-α, and inducible nitric oxide synthase in microglial cells are increased following PA exposure, but are reduced by pretreatment with OXA. The anti-inflammatory marker arginase-1 is increased by OXA. Finally, we show hypothalamic neurons exposed to conditioned media from PA-challenged microglia have increased cell survival only when microglia were pretreated with OXA. These data support the concept that OXA may act as an immunomodulatory regulator of microglia, reducing pro-inflammatory cytokines and increasing anti-inflammatory factors to promote a favorable neuronal microenvironment. PMID:26306651

  9. Role of orexin A signaling in dietary palmitic acid-activated microglial cells.

    PubMed

    Duffy, Cayla M; Yuan, Ce; Wisdorf, Lauren E; Billington, Charles J; Kotz, Catherine M; Nixon, Joshua P; Butterick, Tammy A

    2015-10-01

    Excess dietary saturated fatty acids such as palmitic acid (PA) induce peripheral and hypothalamic inflammation. Hypothalamic inflammation, mediated in part by microglial activation, contributes to metabolic dysregulation. In rodents, high fat diet-induced microglial activation results in nuclear translocation of nuclear factor-kappa B (NFκB), and increased central pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). The hypothalamic neuropeptide orexin A (OXA, hypocretin 1) is neuroprotective in brain. In cortex, OXA can also reduce inflammation and neurodegeneration through a microglial-mediated pathway. Whether hypothalamic orexin neuroprotection mechanisms depend upon microglia is unknown. To address this issue, we evaluated effects of OXA and PA on inflammatory response in immortalized murine microglial and hypothalamic neuronal cell lines. We demonstrate for the first time in microglial cells that exposure to PA increases gene expression of orexin-1 receptor but not orexin-2 receptor. Pro-inflammatory markers IL-6, TNF-α, and inducible nitric oxide synthase in microglial cells are increased following PA exposure, but are reduced by pretreatment with OXA. The anti-inflammatory marker arginase-1 is increased by OXA. Finally, we show hypothalamic neurons exposed to conditioned media from PA-challenged microglia have increased cell survival only when microglia were pretreated with OXA. These data support the concept that OXA may act as an immunomodulatory regulator of microglia, reducing pro-inflammatory cytokines and increasing anti-inflammatory factors to promote a favorable neuronal microenvironment.

  10. Vitamin d deficiency reduces the immune response, phagocytosis rate, and intracellular killing rate of microglial cells.

    PubMed

    Djukic, Marija; Onken, Marie Luise; Schütze, Sandra; Redlich, Sandra; Götz, Alexander; Hanisch, Uwe-Karsten; Bertsch, Thomas; Ribes, Sandra; Hanenberg, Andrea; Schneider, Simon; Bollheimer, Cornelius; Sieber, Cornel; Nau, Roland

    2014-06-01

    Meningitis and meningoencephalitis caused by Escherichia coli are associated with high rates of mortality and neurological sequelae. A high prevalence of neurological disorders has been observed in geriatric populations at risk of hypovitaminosis D. Vitamin D has potent effects on human immunity, including induction of antimicrobial peptides (AMPs) and suppression of T-cell proliferation, but its influence on microglial cells is unknown. The purpose of the present study was to determine the effects of vitamin D deficiency on the phagocytosis rate, intracellular killing, and immune response of murine microglial cultures after stimulation with the Toll-like receptor (TLR) agonists tripalmitoyl-S-glyceryl-cysteine (TLR1/2), poly(I·C) (TLR3), lipopolysaccharide (TLR4), and CpG oligodeoxynucleotide (TLR9). Upon stimulation with high concentrations of TLR agonists, the release of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) was decreased in vitamin D-deficient compared to that in vitamin D-sufficient microglial cultures. Phagocytosis of E. coli K1 after stimulation of microglial cells with high concentrations of TLR3, -4, and -9 agonists and intracellular killing of E. coli K1 after stimulation with high concentrations of all TLR agonists were lower in vitamin D-deficient microglial cells than in the respective control cells. Our observations suggest that vitamin D deficiency may impair the resistance of the brain against bacterial infections. PMID:24686054

  11. Vitamin D Deficiency Reduces the Immune Response, Phagocytosis Rate, and Intracellular Killing Rate of Microglial Cells

    PubMed Central

    Onken, Marie Luise; Schütze, Sandra; Redlich, Sandra; Götz, Alexander; Hanisch, Uwe-Karsten; Bertsch, Thomas; Ribes, Sandra; Hanenberg, Andrea; Schneider, Simon; Bollheimer, Cornelius; Sieber, Cornel; Nau, Roland

    2014-01-01

    Meningitis and meningoencephalitis caused by Escherichia coli are associated with high rates of mortality and neurological sequelae. A high prevalence of neurological disorders has been observed in geriatric populations at risk of hypovitaminosis D. Vitamin D has potent effects on human immunity, including induction of antimicrobial peptides (AMPs) and suppression of T-cell proliferation, but its influence on microglial cells is unknown. The purpose of the present study was to determine the effects of vitamin D deficiency on the phagocytosis rate, intracellular killing, and immune response of murine microglial cultures after stimulation with the Toll-like receptor (TLR) agonists tripalmitoyl-S-glyceryl-cysteine (TLR1/2), poly(I·C) (TLR3), lipopolysaccharide (TLR4), and CpG oligodeoxynucleotide (TLR9). Upon stimulation with high concentrations of TLR agonists, the release of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) was decreased in vitamin D-deficient compared to that in vitamin D-sufficient microglial cultures. Phagocytosis of E. coli K1 after stimulation of microglial cells with high concentrations of TLR3, -4, and -9 agonists and intracellular killing of E. coli K1 after stimulation with high concentrations of all TLR agonists were lower in vitamin D-deficient microglial cells than in the respective control cells. Our observations suggest that vitamin D deficiency may impair the resistance of the brain against bacterial infections. PMID:24686054

  12. Effects of Elderberry Juice from Different Genotypes on Oxidative and Inflammatory Responses in Microglial Cells

    PubMed Central

    Jiang, J.M.; Zong, Y.; Chuang, D.Y.; Lei, W.; Lu, C.-H.; Gu, Z.; Fritsche, K.L.; Thomas, A.L.; Lubahn, D.B.; Simonyi, A.; Sun, G.Y.

    2016-01-01

    Many species of berries are nutritious food and offer health benefits. However, among the different types of berries, information on health effects of American elderberries (Sambucus nigra subsp. canadensis) has been lacking and little is known about whether elderberry consumption can confer neuroprotective effects on the central nervous system. Microglial cells constitute a unique class of immune cells and exhibit characteristic properties to carry out multifunctional duties in the brain. Activation of microglial cells has been implicated in brain injury and in many types of neurodegenerative diseases. Our recent studies demonstrated the ability for endotoxin (lipopolysaccharide, LPS) and interferon gamma (IFNγ) to induce reactive oxygen species (ROS) and nitric oxide (NO) in murine microglial cells (BV-2) through activating NADPH oxidase and the MAPK pathways. In this study, BV-2 microglial cells were used to examine effects of elderberry juice obtained from different genotypes on oxidative and inflammatory responses induced by LPS and IFNγ. Results show that ‘Wyldewood’ extract demonstrated antioxidant properties by inhibiting IFNγ-induced ROS production and p-ERK1/2 expression. On the other hand, most juice extracts exerted small effects on LPS-induced NO production and some extracts showed an increase in NO production upon stimulation with IFNγ. The disparity of responses on ROS and NO production from different extracts suggests possible presence of unknown endogenous factor(s) in the extract in promoting the IFNγ-induced iNOS synthesis pathway. PMID:27158184

  13. Interferon γ-dependent migration of microglial cells in the retina after systemic cytomegalovirus infection.

    PubMed

    Zinkernagel, Martin S; Chinnery, Holly R; Ong, Monique L; Petitjean, Claire; Voigt, Valentina; McLenachan, Samuel; McMenamin, Paul G; Hill, Geoffrey R; Forrester, John V; Wikstrom, Matthew E; Degli-Esposti, Mariapia A

    2013-03-01

    Microglial cells are the resident macrophages of the central nervous system and participate in both innate and adaptive immune responses but can also lead to exacerbation of neurodegenerative pathologies after viral infections. Microglia in the outer layers of the retina and the subretinal space are thought to be involved in retinal diseases where low-grade chronic inflammation and oxidative stress play a role. This study investigated the effect of systemic infection with murine cytomegalovirus on the distribution and dynamics of retinal microglia cells. Systemic infection with murine cytomegalovirus elicited a significant increase in the number of microglia in the subretinal space and an accumulation of iris macrophages, along with morphological signs of activation. Interferon γ (IFN-γ)-deficient mice failed to induce changes in microglia distribution. Bone marrow chimera experiments confirmed that microglial cells in the subretinal space were not recruited from the circulating monocyte pool, but rather represented an accumulation of resident microglial cells from within the retina. Our results demonstrate that a systemic viral infection can lead to IFN-γ-mediated accumulation of microglia into the outer retinal layers and offer proof of concept that systemic viral infections alter the ocular microenvironment and therefore, may influence the course of diseases such as macular degeneration, diabetic retinopathy, or autoimmune uveitis, where low-grade inflammation is implicated.

  14. [Microglial cells and development of the embryonic central nervous system].

    PubMed

    Legendre, Pascal; Le Corronc, Hervé

    2014-02-01

    Microglia cells are the macrophages of the central nervous system with a crucial function in the homeostasis of the adult brain. However, recent studies showed that microglial cells may also have important functions during early embryonic central nervous system development. In this review we summarize recent works on the extra embryonic origin of microglia, their progenitor niche, the pattern of their invasion of the embryonic central nervous system and on interactions between embryonic microglia and their local environment during invasion. We describe microglial functions during development of embryonic neuronal networks, including their roles in neurogenesis, in angiogenesis and developmental cell death. These recent discoveries open a new field of research on the functions of neural-microglial interactions during the development of the embryonic central nervous system.

  15. A microglial hypothesis of globoid cell leukodystrophy pathology.

    PubMed

    Nicaise, Alexandra M; Bongarzone, Ernesto R; Crocker, Stephen J

    2016-11-01

    Globoid cell leukodystrophy (GLD), also known as Krabbe's disease, is a fatal demyelinating disease accompanied by the formation of giant, multinucleated cells called globoid cells. Previously believed to be a byproduct of inflammation, these cells can be found early in disease before evidence of any damage. The precise mechanism by which these globoid cells cause oligodendrocyte dysfunction is not completely understood, nor is their cell type defined. This Review outlines the idea that microglial cells are transformed into an unknown and undefined novel M3 phenotype in GLD, which is cytotoxic to oligodendrocytes, leading to disease progression. © 2016 Wiley Periodicals, Inc. PMID:27638591

  16. Toll-like receptor 2 ligands promote microglial cell death by inducing autophagy.

    PubMed

    Arroyo, Daniela S; Soria, Javier A; Gaviglio, Emilia A; Garcia-Keller, Constanza; Cancela, Liliana M; Rodriguez-Galan, Maria C; Wang, Ji Ming; Iribarren, Pablo

    2013-01-01

    Microglial cells are phagocytes in the central nervous system (CNS) and become activated in pathological conditions, resulting in microgliosis, manifested by increased cell numbers and inflammation in the affected regions. Thus, controlling microgliosis is important to prevent pathological damage to the brain. Here, we evaluated the contribution of Toll-like receptor 2 (TLR2) to microglial survival. We observed that activation of microglial cells with peptidoglycan (PGN) from Staphylococcus aureus and other TLR2 ligands results in cell activation followed by the induction of autophagy and autophagy-dependent cell death. In C57BL/6J mice, intracerebral injection of PGN increased the autophagy of microglial cells and reduced the microglial/macrophage cell number in brain parenchyma. Our results demonstrate a novel role of TLRs in the regulation of microglial cell activation and survival, which are important for the control of microgliosis and associated inflammatory responses in the CNS. PMID:23073832

  17. Layer 5 Pyramidal Neurons' Dendritic Remodeling and Increased Microglial Density in Primary Motor Cortex in a Murine Model of Facial Paralysis.

    PubMed

    Urrego, Diana; Troncoso, Julieta; Múnera, Alejandro

    2015-01-01

    This work was aimed at characterizing structural changes in primary motor cortex layer 5 pyramidal neurons and their relationship with microglial density induced by facial nerve lesion using a murine facial paralysis model. Adult transgenic mice, expressing green fluorescent protein in microglia and yellow fluorescent protein in projecting neurons, were submitted to either unilateral section of the facial nerve or sham surgery. Injured animals were sacrificed either 1 or 3 weeks after surgery. Two-photon excitation microscopy was then used for evaluating both layer 5 pyramidal neurons and microglia in vibrissal primary motor cortex (vM1). It was found that facial nerve lesion induced long-lasting changes in the dendritic morphology of vM1 layer 5 pyramidal neurons and in their surrounding microglia. Dendritic arborization of the pyramidal cells underwent overall shrinkage. Apical dendrites suffered transient shortening while basal dendrites displayed sustained shortening. Moreover, dendrites suffered transient spine pruning. Significantly higher microglial cell density was found surrounding vM1 layer 5 pyramidal neurons after facial nerve lesion with morphological bias towards the activated phenotype. These results suggest that facial nerve lesions elicit active dendrite remodeling due to pyramidal neuron and microglia interaction, which could be the pathophysiological underpinning of some neuropathic motor sequelae in humans.

  18. Automatic counting of microglial cell activation and its applications

    PubMed Central

    Gallego, Beatriz I.; de Gracia, Pablo

    2016-01-01

    Glaucoma is a multifactorial optic neuropathy characterized by the damage and death of the retinal ganglion cells. This disease results in vision loss and blindness. Any vision loss resulting from the disease cannot be restored and nowadays there is no available cure for glaucoma; however an early detection and treatment, could offer neuronal protection and avoid later serious damages to the visual function. A full understanding of the etiology of the disease will still require the contribution of many scientific efforts. Glial activation has been observed in glaucoma, being microglial proliferation a hallmark in this neurodegenerative disease. A typical project studying these cellular changes involved in glaucoma often needs thousands of images - from several animals - covering different layers and regions of the retina. The gold standard to evaluate them is the manual count. This method requires a large amount of time from specialized personnel. It is a tedious process and prone to human error. We present here a new method to count microglial cells by using a computer algorithm. It counts in one hour the same number of images that a researcher counts in four weeks, with no loss of reliability.

  19. Automatic counting of microglial cell activation and its applications

    PubMed Central

    Gallego, Beatriz I.; de Gracia, Pablo

    2016-01-01

    Glaucoma is a multifactorial optic neuropathy characterized by the damage and death of the retinal ganglion cells. This disease results in vision loss and blindness. Any vision loss resulting from the disease cannot be restored and nowadays there is no available cure for glaucoma; however an early detection and treatment, could offer neuronal protection and avoid later serious damages to the visual function. A full understanding of the etiology of the disease will still require the contribution of many scientific efforts. Glial activation has been observed in glaucoma, being microglial proliferation a hallmark in this neurodegenerative disease. A typical project studying these cellular changes involved in glaucoma often needs thousands of images - from several animals - covering different layers and regions of the retina. The gold standard to evaluate them is the manual count. This method requires a large amount of time from specialized personnel. It is a tedious process and prone to human error. We present here a new method to count microglial cells by using a computer algorithm. It counts in one hour the same number of images that a researcher counts in four weeks, with no loss of reliability. PMID:27651757

  20. Automatic counting of microglial cell activation and its applications.

    PubMed

    Gallego, Beatriz I; de Gracia, Pablo

    2016-08-01

    Glaucoma is a multifactorial optic neuropathy characterized by the damage and death of the retinal ganglion cells. This disease results in vision loss and blindness. Any vision loss resulting from the disease cannot be restored and nowadays there is no available cure for glaucoma; however an early detection and treatment, could offer neuronal protection and avoid later serious damages to the visual function. A full understanding of the etiology of the disease will still require the contribution of many scientific efforts. Glial activation has been observed in glaucoma, being microglial proliferation a hallmark in this neurodegenerative disease. A typical project studying these cellular changes involved in glaucoma often needs thousands of images - from several animals - covering different layers and regions of the retina. The gold standard to evaluate them is the manual count. This method requires a large amount of time from specialized personnel. It is a tedious process and prone to human error. We present here a new method to count microglial cells by using a computer algorithm. It counts in one hour the same number of images that a researcher counts in four weeks, with no loss of reliability.

  1. Automatic counting of microglial cell activation and its applications.

    PubMed

    Gallego, Beatriz I; de Gracia, Pablo

    2016-08-01

    Glaucoma is a multifactorial optic neuropathy characterized by the damage and death of the retinal ganglion cells. This disease results in vision loss and blindness. Any vision loss resulting from the disease cannot be restored and nowadays there is no available cure for glaucoma; however an early detection and treatment, could offer neuronal protection and avoid later serious damages to the visual function. A full understanding of the etiology of the disease will still require the contribution of many scientific efforts. Glial activation has been observed in glaucoma, being microglial proliferation a hallmark in this neurodegenerative disease. A typical project studying these cellular changes involved in glaucoma often needs thousands of images - from several animals - covering different layers and regions of the retina. The gold standard to evaluate them is the manual count. This method requires a large amount of time from specialized personnel. It is a tedious process and prone to human error. We present here a new method to count microglial cells by using a computer algorithm. It counts in one hour the same number of images that a researcher counts in four weeks, with no loss of reliability. PMID:27651757

  2. Fractalkine Attenuates Microglial Cell Activation Induced by Prenatal Stress

    PubMed Central

    Ślusarczyk, Joanna; Trojan, Ewa; Głombik, Katarzyna; Chamera, Katarzyna; Roman, Adam; Budziszewska, Bogusława; Basta-Kaim, Agnieszka

    2016-01-01

    The potential contribution of inflammation to the development of neuropsychiatric diseases has recently received substantial attention. In the brain, the main immune cells are the microglia. As they are the main source of inflammatory factors, it is plausible that the regulation of their activation may be a potential therapeutic target. Fractalkine (CX3CL1) and its receptor CX3CR1 play a crucial role in the control of the biological activity of the microglia. In the present study, using microglial cultures we investigated whether fractalkine is able to reverse changes in microglia caused by a prenatal stress procedure. Our study found that the microglia do not express fractalkine. Prenatal stress decreases the expression of the fractalkine receptor, which in turn is enhanced by the administration of exogenous fractalkine. Moreover, treatment with fractalkine diminishes the prenatal stress-induced overproduction of proinflammatory factors such as IL-1β, IL-18, IL-6, TNF-α, CCL2, or NO in the microglial cells derived from prenatally stressed newborns. In conclusion, the present results revealed that the pathological activation of microglia in prenatally stressed newborns may be attenuated by fractalkine administration. Therefore, understanding of the role of the CX3CL1-CX3CR1 system may help to elucidate the mechanisms underlying the neuron-microglia interaction and its role in pathological conditions in the brain. PMID:27239349

  3. Toll-like receptor 2 ligands promote microglial cell death by inducing autophagy

    PubMed Central

    Arroyo, Daniela S.; Soria, Javier A.; Gaviglio, Emilia A.; Garcia-Keller, Constanza; Cancela, Liliana M.; Rodriguez-Galan, Maria C.; Wang, Ji Ming; Iribarren, Pablo

    2013-01-01

    Microglial cells are phagocytes in the central nervous system (CNS) and become activated in pathological conditions, resulting in microgliosis, manifested by increased cell numbers and inflammation in the affected regions. Thus, controlling microgliosis is important to prevent pathological damage to the brain. Here, we evaluated the contribution of Toll-like receptor 2 (TLR2) to microglial survival. We observed that activation of microglial cells with peptidoglycan (PGN) from Staphylococcus aureus and other TLR2 ligands results in cell activation followed by the induction of autophagy and autophagy-dependent cell death. In C57BL/6J mice, intracerebral injection of PGN increased the autophagy of microglial cells and reduced the microglial/macrophage cell number in brain parenchyma. Our results demonstrate a novel role of TLRs in the regulation of microglial cell activation and survival, which are important for the control of microgliosis and associated inflammatory responses in the CNS.—Arroyo, D. S., Soria, J. A., Gaviglio, E. A., Garcia-Keller, C., Cancela, L. M., Rodriguez-Galan, M. C., Wang, J. M., Iribarren, P. Toll-like receptor 2 ligands promote microglial cell death by inducing autophagy. PMID:23073832

  4. Astrocytes Enhance Streptococcus suis-Glial Cell Interaction in Primary Astrocyte-Microglial Cell Co-Cultures.

    PubMed

    Seele, Jana; Nau, Roland; Prajeeth, Chittappen K; Stangel, Martin; Valentin-Weigand, Peter; Seitz, Maren

    2016-06-13

    Streptococcus (S.) suis infections are the most common cause of meningitis in pigs. Moreover, S. suis is a zoonotic pathogen, which can lead to meningitis in humans, mainly in adults. We assume that glial cells may play a crucial role in host-pathogen interactions during S. suis infection of the central nervous system. Glial cells are considered to possess important functions during inflammation and injury of the brain in bacterial meningitis. In the present study, we established primary astrocyte-microglial cell co-cultures to investigate interactions of S. suis with glial cells. For this purpose, microglial cells and astrocytes were isolated from new-born mouse brains and characterized by flow cytometry, followed by the establishment of astrocyte and microglial cell mono-cultures as well as astrocyte-microglial cell co-cultures. In addition, we prepared microglial cell mono-cultures co-incubated with uninfected astrocyte mono-culture supernatants and astrocyte mono-cultures co-incubated with uninfected microglial cell mono-culture supernatants. After infection of the different cell cultures with S. suis, bacteria-cell association was mainly observed with microglial cells and most prominently with a non-encapsulated mutant of S. suis. A time-dependent induction of NO release was found only in the co-cultures and after co-incubation of microglial cells with uninfected supernatants of astrocyte mono-cultures mainly after infection with the capsular mutant. Only moderate cytotoxic effects were found in co-cultured glial cells after infection with S. suis. Taken together, astrocytes and astrocyte supernatants increased interaction of microglial cells with S. suis. Astrocyte-microglial cell co-cultures are suitable to study S. suis infections and bacteria-cell association as well as NO release by microglial cells was enhanced in the presence of astrocytes.

  5. Astrocytes Enhance Streptococcus suis-Glial Cell Interaction in Primary Astrocyte-Microglial Cell Co-Cultures

    PubMed Central

    Seele, Jana; Nau, Roland; Prajeeth, Chittappen K.; Stangel, Martin; Valentin-Weigand, Peter; Seitz, Maren

    2016-01-01

    Streptococcus (S.) suis infections are the most common cause of meningitis in pigs. Moreover, S. suis is a zoonotic pathogen, which can lead to meningitis in humans, mainly in adults. We assume that glial cells may play a crucial role in host-pathogen interactions during S. suis infection of the central nervous system. Glial cells are considered to possess important functions during inflammation and injury of the brain in bacterial meningitis. In the present study, we established primary astrocyte-microglial cell co-cultures to investigate interactions of S. suis with glial cells. For this purpose, microglial cells and astrocytes were isolated from new-born mouse brains and characterized by flow cytometry, followed by the establishment of astrocyte and microglial cell mono-cultures as well as astrocyte-microglial cell co-cultures. In addition, we prepared microglial cell mono-cultures co-incubated with uninfected astrocyte mono-culture supernatants and astrocyte mono-cultures co-incubated with uninfected microglial cell mono-culture supernatants. After infection of the different cell cultures with S. suis, bacteria-cell association was mainly observed with microglial cells and most prominently with a non-encapsulated mutant of S. suis. A time-dependent induction of NO release was found only in the co-cultures and after co-incubation of microglial cells with uninfected supernatants of astrocyte mono-cultures mainly after infection with the capsular mutant. Only moderate cytotoxic effects were found in co-cultured glial cells after infection with S. suis. Taken together, astrocytes and astrocyte supernatants increased interaction of microglial cells with S. suis. Astrocyte-microglial cell co-cultures are suitable to study S. suis infections and bacteria-cell association as well as NO release by microglial cells was enhanced in the presence of astrocytes. PMID:27304968

  6. A novel synthetic compound MCAP suppresses LPS-induced murine microglial activation in vitro via inhibiting NF-kB and p38 MAPK pathways

    PubMed Central

    Kim, Byung-Wook; More, Sandeep Vasant; Yun, Yo-Sep; Ko, Hyun-Myung; Kwak, Jae-Hwan; Lee, Heesoon; Suk, Kyoungho; Kim, In-Su; Choi, Dong-Kug

    2016-01-01

    Aim: To investigate the anti-neuroinflammatory activity of a novel synthetic compound, 7-methylchroman-2-carboxylic acid N-(2-trifluoromethyl) phenylamide (MCAP) against LPS-induced microglial activation in vitro. Methods: Primary mouse microglia and BV2 microglia cells were exposed to LPS (50 or 100 ng/mL). The expression of iNOS and COX-2, proinflammatory cytokines, NF-κB and p38 MAPK signaling molecules were analyzed by RT-PCR, Western blot and ELISA. The morphological changes of microglia and nuclear translocation of NF-ĸB were visualized using phase contrast and fluorescence microscopy, respectively. Results: Pretreatment with MCAP (0.1, 1, 10 μmol/L) dose-dependently inhibited LPS-induced expression of iNOS and COX-2 in BV2 microglia cells. Similar results were obtained in primary microglia pretreated with MCAP (0.1, 0.5 μmol/L). MCAP dose-dependently abated LPS-induced release of TNF-α, IL-6 and IL-1β, and mitigated LPS-induced activation of NF-κB by reducing the phosphorylation of IκBα in BV2 microglia cells. Moreover, MCAP attenuated LPS-induced phosphorylation of p38 MAPK, whereas SB203580, a p38 MAPK inhibitor, significantly potentiated MCAP-caused inhibition on the expression of MEF-2 (a transcription factor downstream of p38 MAPK). Conclusion: MCAP exerts anti-inflammatory effects in murine microglia in vitro by inhibiting the p38 MAPK and NF-κB signaling pathways and proinflammatory responses. MCAP may be developed as a novel agent for treating diseases involving activated microglial cells. PMID:26838070

  7. Microglial cell dysregulation in brain aging and neurodegeneration

    PubMed Central

    von Bernhardi, Rommy; Eugenín-von Bernhardi, Laura; Eugenín, Jaime

    2015-01-01

    Aging is the main risk factor for neurodegenerative diseases. In aging, microglia undergoes phenotypic changes compatible with their activation. Glial activation can lead to neuroinflammation, which is increasingly accepted as part of the pathogenesis of neurodegenerative diseases, including Alzheimer’s disease (AD). We hypothesize that in aging, aberrant microglia activation leads to a deleterious environment and neurodegeneration. In aged mice, microglia exhibit an increased expression of cytokines and an exacerbated inflammatory response to pathological changes. Whereas LPS increases nitric oxide (NO) secretion in microglia from young mice, induction of reactive oxygen species (ROS) predominates in older mice. Furthermore, there is accumulation of DNA oxidative damage in mitochondria of microglia during aging, and also an increased intracellular ROS production. Increased ROS activates the redox-sensitive nuclear factor kappa B, which promotes more neuroinflammation, and can be translated in functional deficits, such as cognitive impairment. Mitochondria-derived ROS and cathepsin B, are also necessary for the microglial cell production of interleukin-1β, a key inflammatory cytokine. Interestingly, whereas the regulatory cytokine TGFβ1 is also increased in the aged brain, neuroinflammation persists. Assessing this apparent contradiction, we have reported that TGFβ1 induction and activation of Smad3 signaling after inflammatory stimulation are reduced in adult mice. Other protective functions, such as phagocytosis, although observed in aged animals, become not inducible by inflammatory stimuli and TGFβ1. Here, we discuss data suggesting that mitochondrial and endolysosomal dysfunction could at least partially mediate age-associated microglial cell changes, and, together with the impairment of the TGFβ1-Smad3 pathway, could result in the reduction of protective activation and the facilitation of cytotoxic activation of microglia, resulting in the promotion of

  8. Fine-tuning the central nervous system: microglial modelling of cells and synapses.

    PubMed

    Xavier, Anna L; Menezes, João R L; Goldman, Steven A; Nedergaard, Maiken

    2014-10-19

    Microglia constitute as much as 10-15% of all cells in the mammalian central nervous system (CNS) and are the only glial cells that do not arise from the neuroectoderm. As the principal CNS immune cells, microglial cells represent the first line of defence in response to exogenous threats. Past studies have largely been dedicated to defining the complex immune functions of microglial cells. However, our understanding of the roles of microglia has expanded radically over the past years. It is now clear that microglia are critically involved in shaping neural circuits in both the developing and adult CNS, and in modulating synaptic transmission in the adult brain. Intriguingly, microglial cells appear to use the same sets of tools, including cytokine and chemokine release as well as phagocytosis, whether modulating neural function or mediating the brain's innate immune responses. This review will discuss recent developments that have broadened our views of neuro-glial signalling to include the contribution of microglial cells.

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

    PubMed Central

    Cai, Hui; Liang, Qianlei; Ge, Guanqun

    2016-01-01

    Reducing β amyloid- (Aβ-) induced microglial activation is believed to be effective in treating Alzheimer's disease (AD). Microglia can be activated into classic activated state (M1 state) or alternative activated state (M2 state), and the former is harmful; in contrast, the latter is beneficial. Gypenoside (GP) is the major bioactive constituent of Gynostemma pentaphyllum, a traditional Chinese herb medicine. In this study, we hypothesized that GP attenuates Aβ-induced microglial activation by ameliorating microglial M1/M2 states, and the process may be mediated by suppressor of cell signaling protein 1 (SOCS1). In this study, we found that Aβ exposure increased the levels of microglial M1 markers, including iNOS expression, tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-6 releases, and coadministration of GP reversed the increase of M1 markers and enhanced the levels of M2 markers, including arginase-1 (Arg-1) expression, IL-10, brain-derived neurotrophic factor (BDNF), and glial cell-derived neurotrophic factor (GDNF) releases in the Aβ-treated microglial cells. SOCS1-siRNA, however, significantly abolished the GP-induced effects on the levels of microglial M1 and M2 markers. These findings indicated that GP attenuates Aβ-induced microglial activation by ameliorating M1/M2 states, and the process may be mediated by SOCS1. PMID:27213058

  10. Stress Granules Modulate SYK to Cause Microglial Cell Dysfunction in Alzheimer's Disease.

    PubMed

    Ghosh, Soumitra; Geahlen, Robert L

    2015-11-01

    Microglial cells in the brains of Alzheimer's patients are known to be recruited to amyloid-beta (Aβ) plaques where they exhibit an activated phenotype, but are defective for plaque removal by phagocytosis. In this study, we show that microglia stressed by exposure to sodium arsenite or Aβ(1-42) peptides or fibrils form extensive stress granules (SGs) to which the tyrosine kinase, SYK, is recruited. SYK enhances the formation of SGs, is active within the resulting SGs and stimulates the production of reactive oxygen and nitrogen species that are toxic to neuronal cells. This sequestration of SYK inhibits the ability of microglial cells to phagocytose Escherichia coli or Aβ fibrils. We find that aged microglial cells are more susceptible to the formation of SGs; and SGs containing SYK and phosphotyrosine are prevalent in the brains of patients with severe Alzheimer's disease. Phagocytic activity can be restored to stressed microglial cells by treatment with IgG, suggesting a mechanism to explain the therapeutic efficacy of intravenous IgG. These studies describe a mechanism by which stress, including exposure to Aβ, compromises the function of microglial cells in Alzheimer's disease and suggest approaches to restore activity to dysfunctional microglial cells.

  11. Microglial Cells as a Link between Cannabinoids and the Immune Hypothesis of Psychiatric Disorders

    PubMed Central

    Lisboa, Sabrina F.; Gomes, Felipe V.; Guimaraes, Francisco S.; Campos, Alline C.

    2016-01-01

    Psychiatric disorders are one of the leading causes of disability worldwide. Although several therapeutic options are available, the exact mechanisms responsible for the genesis of these disorders remain to be fully elucidated. In the last decade, a body of evidence has supported the involvement of the immune system in the pathophysiology of these conditions. Microglial cells play a significant role in maintaining brain homeostasis and surveillance. Dysregulation of microglial functions has been associated with several psychiatric conditions. Cannabinoids regulate the brain–immune axis and inhibit microglial cell activation. Here, we summarized evidence supporting the hypothesis that microglial cells could be a target for cannabinoid influence on psychiatric disorders, such as anxiety, depression, schizophrenia, and stress-related disorders. PMID:26858686

  12. NitroDIGE analysis reveals inhibition of protein S-nitrosylation by epigallocatechin gallates in lipopolysaccharide-stimulated microglial cells

    PubMed Central

    2014-01-01

    Background Nitric oxide (NO) is a signaling molecule regulating numerous cellular functions in development and disease. In the brain, neuronal injury or neuroinflammation can lead to microglial activation, which induces NO production. NO can react with critical cysteine thiols of target proteins forming S-nitroso-proteins. This modification, known as S-nitrosylation, is an evolutionarily conserved redox-based post-translational modification (PTM) of specific proteins analogous to phosphorylation. In this study, we describe a protocol for analyzing S-nitrosylation of proteins using a gel-based proteomic approach and use it to investigate the modes of action of a botanical compound found in green tea, epigallocatechin-3-gallate (EGCG), on protein S-nitrosylation after microglial activation. Methods/Results To globally and quantitatively analyze NO-induced protein S-nitrosylation, the sensitive gel-based proteomic method, termed NitroDIGE, was developed by combining two-dimensional differential in-gel electrophoresis (2-D DIGE) with the modified biotin switch technique (BST) using fluorescence-tagged CyDye™ thiol reactive agents to label S-nitrosothiols. The NitroDIGE method showed high specificity and sensitivity in detecting S-nitrosylated proteins (SNO-proteins). Using this approach, we identified a subset of SNO-proteins ex vivo by exposing immortalized murine BV-2 microglial cells to a physiological NO donor, or in vivo by exposing BV-2 cells to endotoxin lipopolysaccharides (LPS) to induce a proinflammatory response. Moreover, EGCG was shown to attenuate S-nitrosylation of proteins after LPS-induced activation of microglial cells primarily by modulation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated oxidative stress response. Conclusions These results demonstrate that NitroDIGE is an effective proteomic strategy for “top-down” quantitative analysis of protein S-nitrosylation in multi-group samples in response to nitrosative stress due

  13. Suppression of LPS-induced inflammatory responses by inflexanin B in BV2 microglial cells.

    PubMed

    Lim, Ji-Youn; Sul, Donggeun; Hwang, Bang Yeon; Hwang, Kwang Woo; Yoo, Ki-Yeol; Park, So-Young

    2013-02-01

    Microglia are a type of resident macrophage that functions as an inflammation modulator in the central nervous system. Over-activation of microglia by a range of stimuli disrupts the physiological homeostasis of the brain, and induces inflammatory response and degenerative processes, such as those implicated in neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Therefore, we investigated the possible anti-inflammatory mechanisms of inflexanin B in murine microglial BV2 cells. Lipopolysaccharide (LPS) activated BV2 cells and induced the production of pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E2 (PGE2), and cytokines (interleukins-1β and -6, and tumour necrosis factor α). The LPS-induced production of pro-inflammatory mediators was associated with the enhancement of nuclear factor-kappaB (NF-κB) nuclear translocation and the activation of mitogen-activated protein kinase (MAPK) including ERK1/2 and JNK. Conversely, pretreatment of cells with inflexanin B (10 and 20 μg/mL) significantly reduced the production of pro-inflammatory mediators. This was accompanied with the reduced nuclear translocation of NF-κB and reduced activation of MAPKs. These results suggest that inflexanin B attenuated the LPS-induced inflammatory process by inhibiting the activation of NF-κB and MAPKs. PMID:23458198

  14. Constitutive and functional expression of YB-1 in microglial cells.

    PubMed

    Keilhoff, G; Titze, M; Esser, T; Langnaese, K; Ebmeyer, U

    2015-08-20

    Y-box-binding protein (YB-1) is a member of the cold-shock protein family and participates in a wide variety of DNA/RNA-dependent cellular processes including DNA repair, transcription, mRNA splicing, packaging, and translation. At the cellular level, YB-1 is involved in cell proliferation and differentiation, stress responses, and malignant cell transformation. A general role for YB-1 during inflammation has also been well described; however, there are minimal data concerning YB-1 expression in microglia, which are the immune cells of the brain. Therefore, we studied the expression of YB-1 in a clinically relevant global ischemia model for neurological injury following cardiac arrest. This model is characterized by massive neurodegeneration of the hippocampal CA1 region and the subsequent long-lasting activation of microglia. In addition, we studied YB-1 expression in BV-2 cells, which are an accepted microglia culture model. BV-2 cells were stressed by oxygen/glucose deprivation (OGD), OGD-relevant mediators, lipopolysaccharide (LPS), and phagocytosis-inducing cell debris and nanoparticles. Using quantitative polymerase chain reaction (PCR), we show constitutive expression of YB-1 transcripts in unstressed BV-2 cells. The functional upregulation of the YB-1 protein was demonstrated in microglia in vivo and in BV-2 cells in vitro. All stressors except for LPS were potent enhancers of the level of YB-1 protein, which appears to be regulated primarily by proteasomal degradation and, to a lesser extent, by the activation (phosphorylation) of the translation initiation factor eIF4E. The proteasome of BV-2 cells is impaired by OGD, which results in decreased protein degradation and therefore increased levels of YB-1 protein. LPS induces proteasome activity, which enables the level of YB-1 protein to remain at control levels despite enhanced protein ubiquitination. The proteasome inhibitor MG-132 was able to increase YB-1 protein levels in control and LPS

  15. Phagocytosis-dependent and independent mechanisms underlie the microglial cell damage caused by carbon nanotube agglomerates.

    PubMed

    Shigemoto-Mogami, Yukari; Hoshikawa, Kazue; Hirose, Akihiko; Sato, Kaoru

    2016-01-01

    Although carbon nanotubes (CNTs) are used in many fields, including energy, healthcare, environmental technology, materials, and electronics, the adverse effects of CNTs in the brain are poorly understood. In this study, we investigated the effects of CNTs on cultured microglia, as microglia are the first responders to foreign materials. We compared the effects of sonicated suspensions of 5 kinds of CNTs and their flow-through filtered with a 0.22 µm membrane filter on microglial viability. We found that sonicated suspensions caused microglial cell damage, but their flow-through did not. The number of microglial aggregates was well correlated with the extent of the damage. We also determined that the CNT agglomerates consisted of two groups: one was phagocytosed by microglia and caused microglial cell damage, and the other caused cell damage without phagocytosis. These results suggest that phagocytosis-dependent and independent mechanisms underlie the microglial cell damage caused by CNT agglomerates and it is important to conduct studies about the relationships between physical properties of nanomaterial-agglomerates and cell damage. PMID:27432236

  16. Blockade of microglial KATP -channel abrogates suppression of inflammatory-mediated inhibition of neural precursor cells.

    PubMed

    Ortega, Francisco J; Vukovic, Jana; Rodríguez, Manuel J; Bartlett, Perry F

    2014-02-01

    Microglia positively affect neural progenitor cell physiology through the release of inflammatory mediators or trophic factors. We demonstrated previously that reactive microglia foster K(ATP) -channel expression and that blocking this channel using glibenclamide administration enhances striatal neurogenesis after stroke. In this study, we investigated whether the microglial K(ATP) -channel directly influences the activation of neural precursor cells (NPCs) from the subventricular zone using transgenic Csf1r-GFP mice. In vitro exposure of NPCs to lipopolysaccharide and interferon-gamma resulted in a significant decrease in precursor cell number. The complete removal of microglia from the culture or exposure to enriched microglia culture also decreased the precursor cell number. The addition of glibenclamide rescued the negative effects of enriched microglia on neurosphere formation and promoted a ∼20% improvement in precursor cell number. Similar results were found using microglial-conditioned media from isolated microglia. Using primary mixed glial and pure microglial cultures, glibenclamide specifically targeted reactive microglia to restore neurogenesis and increased the microglial production of the chemokine monocyte chemoattractant protein-1 (MCP-1). These findings provide the first direct evidence that the microglial K(ATP) -channel is a regulator of the proliferation of NPCs under inflammatory conditions.

  17. Tocopherol-mediated modulation of age-related changes in microglial cells: turnover of extracellular oxidized protein material.

    PubMed

    Stolzing, Alexandra; Widmer, Rebecca; Jung, Tobias; Voss, Peter; Grune, Tilman

    2006-06-15

    Proteins accumulate during aging and form insoluble protein aggregates. Microglia are responsible for their removal from the brain. During aging, changes within the microglia might play a crucial role in the malfunctioning of these cells. Therefore, we isolated primary microglial cells from adult rats and compared their activation status and their ability to degrade proteins to that of microglial cells isolated from newborn animals. The ability of adult microglial cells to degrade proteins is substantially decreased. However, the preincubation of microglial cells with vitamin E improves significantly the degradation of such modified proteins. The degradation of proteins from apoptotic vesicles is decreased in microglia isolated from adult rats. This might be the result of a suppression of the CD36 receptor due to vitamin E treatment. We concluded that microglial cells isolated from adult organisms have different metabolic properties and seem to be a more valuable model to study age-related diseases.

  18. Fine-tuning the central nervous system: microglial modelling of cells and synapses

    PubMed Central

    Xavier, Anna L.; Menezes, João R. L.; Goldman, Steven A.; Nedergaard, Maiken

    2014-01-01

    Microglia constitute as much as 10–15% of all cells in the mammalian central nervous system (CNS) and are the only glial cells that do not arise from the neuroectoderm. As the principal CNS immune cells, microglial cells represent the first line of defence in response to exogenous threats. Past studies have largely been dedicated to defining the complex immune functions of microglial cells. However, our understanding of the roles of microglia has expanded radically over the past years. It is now clear that microglia are critically involved in shaping neural circuits in both the developing and adult CNS, and in modulating synaptic transmission in the adult brain. Intriguingly, microglial cells appear to use the same sets of tools, including cytokine and chemokine release as well as phagocytosis, whether modulating neural function or mediating the brain's innate immune responses. This review will discuss recent developments that have broadened our views of neuro-glial signalling to include the contribution of microglial cells. PMID:25225087

  19. Essential Role of MFG-E8 for Phagocytic Properties of Microglial Cells

    PubMed Central

    Guo, Chenying; Nie, Pan; Liu, Yan; Ma, Jie

    2013-01-01

    Milk fat globule factor-E8 (MFG-E8) has been regarded as a key factor involved in the phagocytosis of apoptotic cells. We induced a lentivirus into the microglial cells for the augmentation or abrogation of MFG-E8 expression in mouse microglial cells, and investigated phagocytosis of phosphatidylserine tagged human red blood cells (hRBCs) in co-cultures. Increased MFG-E8 levels were associated with a significant increase in phagocytic activity compared to the controls. Conversely, phagocytosis dramitically decreased due to the abrogation of MFG-E8. In addition, the expression of the inflammatory cytokines, TNF-α and IL-1β, also increased or decreased in the microglial cells with the augmentation or abrogation of MFG-E8, respectively. Our findings indicate that the enhanced expression of MFG-E8 could increase phagocytosis of apoptotic cells; conversely, the rate of phagocytosis and the expression of inflammatory cytokines decreased when MFG-E8 expression was knocked down. Our results confirm that MFG-E8 plays an important role in phagocytosis, and possibly serves as an essential signal molecule for microglial cells. PMID:23405209

  20. Iron uptake of the normoxic, anoxic and postanoxic microglial cell line RAW 264.7.

    PubMed

    Widmer, Rebecca; Grune, Tilman

    2005-01-01

    Iron is one of the trace elements playing a key role in the normal brain metabolism. An excess of free iron on the other hand is catalyzing the iron-mediated oxygen radical production. Such a condition might be a harmful event leading perhaps to serious tissue damage and degeneration. Therefore, during evolution a complex iron sequestering apparatus developed, minimizing the amount of redox-reactive free iron. However, this system might be severely disturbed under pathophysiological conditions including hypoxia or anoxia. Since little is known about the non-transferrin-mediated iron metabolism of the brain during anoxia/reoxygenation, we tested the ability of the microglial cell line RAW 264.7 to take up iron independently of transferrin under various oxygen concentrations. Microglial cells are thought to be the major player in the maintenance of the extracellular homeostasis in the brain. Therefore, we investigated the iron metabolism of microglial cells employing radiolabeled ferric chloride. We tested the uptake of iron under normoxic, anoxic and postanoxic conditions. Furthermore, the amount of ferritin was measured by immunoblotting. We were able to show that iron enters the microglial cell line in the absence of extracellular transferrin under normoxic, anoxic and postanoxic conditions. Interestingly, the amount of ferritin is decreasing in the early reoxygenation phase. Therefore, we concluded that microglia is able to contribute to the brain iron homeostasis under anoxic and postanoxic conditions.

  1. Docosahexaenoic acid modulates inflammatory and antineurogenic functions of activated microglial cells.

    PubMed

    Antonietta Ajmone-Cat, Maria; Lavinia Salvatori, Maria; De Simone, Roberta; Mancini, Melissa; Biagioni, Stefano; Bernardo, Antonietta; Cacci, Emanuele; Minghetti, Luisa

    2012-03-01

    The complex process of microglial activation encompasses several functional activation states associated either with neurotoxic/antineurogenic or with neurotrophic/proneurogenic properties, depending mainly on the extent of activation and the nature of the activating stimuli. Several studies have demonstrated that acute exposure to the prototypical activating agent lipopolysaccharide (LPS) confers antineurogenic properties upon microglial cells. Acutely activated microglia ortheir conditioned media (CM) reduce neural stem progenitor cell (NPC) survival and prevent NPC differentiation into neurons. The present study tested the hypothesis that docosahexaenoic acid (DHA), a long-chain polyunsatured fatty acid (L-PUFA) with potent immunomodulatory properties, could dampen microglial proinflammatory functions and modulate their antineurogenic effect. We demonstrate that DHA dose dependently inhibits the synthesis of inflammatory products in activated microglia without inducing an alternative antiinflammatory phenotype. Among the possible DHA mechanisms of action, we propose the inhibition of p38 MAPK phosphorylation and the activation of the nuclear receptor peroxisome proliferator activated receptor (PPAR)-γ. The attenuation of M1 proinflammatory phenotype has relevant consequences for the survival and differentiation of NPC, because DHA reverses the antineurogenic activities of conditioned media from LPS-activated microglia. Our study identifies new relevant potentially protective and proneurogenic functions of DHA, exerted through the modulation of microglial functions, that could be exploited to sustain or promote neuroregenerative processes in damaged/aged brain. PMID:22057807

  2. Data from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with Withaferin A (WA)

    PubMed Central

    Narayan, Malathi; Seeley, Kent W.; Jinwal, Umesh K.

    2016-01-01

    Mass spectrometry data collected in a study analyzing the effect of withaferin A (WA) on a mouse microglial (N9) cell line is presented in this article. Data was collected from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with either WA or DMSO vehicle control. This article reports all the proteins that were identified in this analysis. The data presented here is related to the published research article on the effect of WA on the differential regulation of proteins in mouse microglial cells [1]. Mass spectrometry data has also been deposited in the ProteomeXchange with the identifier PXD003032. PMID:27054189

  3. Anti-inflammatory effects of thymoquinone in activated BV-2 microglial cells.

    PubMed

    Taka, Equar; Mazzio, Elizabeth A; Goodman, Carl B; Redmon, Natalie; Flores-Rozas, Hernan; Reams, Renee; Darling-Reed, Selina; Soliman, Karam F A

    2015-09-15

    Thymoquinone (TQ), the main pharmacological active ingredient within the black cumin seed (Nigella sativa) is believed to be responsible for the therapeutic effects on chronic inflammatory conditions such as arthritis, asthma and neurodegeneration. In this study, we evaluated the potential anti-inflammatory role of TQ in lipopolysaccharide (LPS)-stimulated BV-2 murine microglia cells. The results obtained indicate that TQ was effective in reducing NO2(-) with an IC50 of 5.04μM, relative to selective iNOS inhibitor LNIL-l-N6-(1-iminoethyl)lysine (IC50 4.09μM). TQ mediated reduction in NO2(-) was found to parallel the decline of iNOS protein expression as confirmed by immunocytochemistry. In addition, we evaluated the anti-inflammatory effects of TQ on ninety-six (96) cytokines using a RayBio AAM-CYT-3 and 4 cytokine antibody protein array. Data obtained establish a baseline protein expression profile characteristic of resting BV-2 cells in the order of osteopontin>MIP-1alpha>MIP-1g>IGF-1 and MCP-I. In the presence of LPS [1ug/ml], activated BV-2 cells produced a sharp rise in specific pro-inflammatory cytokines/chemokine's IL-6, IL-12p40/70, CCL12 /MCP-5, CCL2/MCP-1, and G-CSF which were attenuated by the addition of TQ (10μM). The TQ mediated attenuation of MCP-5, MCP-1 and IL-6 protein in supernatants from activated BV-2 cells were corroborated by independent ELISA. Moreover, the data obtained from the RT(2) PCR demonstrated a similar pattern where the LPS mediated elevation of mRNA for IL-6, CCL12/MCP-5, CCL2/MCP-1 were significantly attenuated by TQ (10μM). Also, in this study, consistent data were obtained for both protein antibody array densitometry and ELISA assays. In addition, TQ was found to reduce LPS mediated elevation in gene expression of Cxcl10 and a number of other cytokines in the panel. These findings demonstrate the significant anti-inflammatory properties of TQ in LPS activated microglial cells. Therefore, the obtained results might indicate

  4. Anti-inflammatory effects of thymoquinone in activated BV-2 microglial cells.

    PubMed

    Taka, Equar; Mazzio, Elizabeth A; Goodman, Carl B; Redmon, Natalie; Flores-Rozas, Hernan; Reams, Renee; Darling-Reed, Selina; Soliman, Karam F A

    2015-09-15

    Thymoquinone (TQ), the main pharmacological active ingredient within the black cumin seed (Nigella sativa) is believed to be responsible for the therapeutic effects on chronic inflammatory conditions such as arthritis, asthma and neurodegeneration. In this study, we evaluated the potential anti-inflammatory role of TQ in lipopolysaccharide (LPS)-stimulated BV-2 murine microglia cells. The results obtained indicate that TQ was effective in reducing NO2(-) with an IC50 of 5.04μM, relative to selective iNOS inhibitor LNIL-l-N6-(1-iminoethyl)lysine (IC50 4.09μM). TQ mediated reduction in NO2(-) was found to parallel the decline of iNOS protein expression as confirmed by immunocytochemistry. In addition, we evaluated the anti-inflammatory effects of TQ on ninety-six (96) cytokines using a RayBio AAM-CYT-3 and 4 cytokine antibody protein array. Data obtained establish a baseline protein expression profile characteristic of resting BV-2 cells in the order of osteopontin>MIP-1alpha>MIP-1g>IGF-1 and MCP-I. In the presence of LPS [1ug/ml], activated BV-2 cells produced a sharp rise in specific pro-inflammatory cytokines/chemokine's IL-6, IL-12p40/70, CCL12 /MCP-5, CCL2/MCP-1, and G-CSF which were attenuated by the addition of TQ (10μM). The TQ mediated attenuation of MCP-5, MCP-1 and IL-6 protein in supernatants from activated BV-2 cells were corroborated by independent ELISA. Moreover, the data obtained from the RT(2) PCR demonstrated a similar pattern where the LPS mediated elevation of mRNA for IL-6, CCL12/MCP-5, CCL2/MCP-1 were significantly attenuated by TQ (10μM). Also, in this study, consistent data were obtained for both protein antibody array densitometry and ELISA assays. In addition, TQ was found to reduce LPS mediated elevation in gene expression of Cxcl10 and a number of other cytokines in the panel. These findings demonstrate the significant anti-inflammatory properties of TQ in LPS activated microglial cells. Therefore, the obtained results might indicate

  5. Immunocytochemical localization of 3-methylcrotonyl-CoA carboxylase in cultured ependymal, microglial and oligodendroglial cells.

    PubMed

    Murín, Radovan; Verleysdonk, Stephan; Rapp, Mirna; Hamprecht, Bernd

    2006-06-01

    To evaluate the ability of ependymal, microglial and oligodendroglial cells to degrade leucine, the presence of 3-methylcrotonyl-CoA carboxylase (MCC) was investigated in cultures of these cells. MCC is a biotin-containing heterodimeric enzyme that is specific for the irreversible part of the leucine catabolic pathway. It has been reported previously that in cell culture MCC is expressed in astrocytes and a subpopulation of neurones. In the present study ependymal, microglial and oligodendroglial cell cultures, derived from the brains of newborn rats, were examined for the expression of MCC by RT-PCR, western blotting and immunocytochemistry. The results of RT-PCR and western blotting showed the presence of mRNA as well as protein of both subunits of MCC in ependymal, microglial and oligodendroglial cell cultures. Immunocytochemical investigation of the cellular and subcellular distribution of MCC demonstrated a mitochondrial location of MCC in all neuroglial cell types investigated. The ubiquitous expression of MCC in glial cells demonstrates the ability of the cells to engage in the catabolism of leucine transported into the brain, mainly for the generation of energy.

  6. Cytomegalovirus Infection of the Rat Developing Brain In Utero Prominently Targets Immune Cells and Promotes Early Microglial Activation

    PubMed Central

    Cloarec, Robin; Bauer, Sylvian; Luche, Hervé; Buhler, Emmanuelle; Pallesi-Pocachard, Emilie; Salmi, Manal; Courtens, Sandra; Massacrier, Annick; Grenot, Pierre; Teissier, Natacha; Watrin, Françoise; Schaller, Fabienne; Adle-Biassette, Homa; Gressens, Pierre; Malissen, Marie; Stamminger, Thomas; Streblow, Daniel N.; Bruneau, Nadine; Szepetowski, Pierre

    2016-01-01

    Background Congenital cytomegalovirus infections are a leading cause of neurodevelopmental disorders in human and represent a major health care and socio-economical burden. In contrast with this medical importance, the pathophysiological events remain poorly known. Murine models of brain cytomegalovirus infection, mostly neonatal, have brought recent insights into the possible pathogenesis, with convergent evidence for the alteration and possible involvement of brain immune cells. Objectives and Methods In order to confirm and expand those findings, particularly concerning the early developmental stages following infection of the fetal brain, we have created a model of in utero cytomegalovirus infection in the developing rat brain. Rat cytomegalovirus was injected intraventricularly at embryonic day 15 (E15) and the brains analyzed at various stages until the first postnatal day, using a combination of gene expression analysis, immunohistochemistry and multicolor flow cytometry experiments. Results Rat cytomegalovirus infection was increasingly seen in various brain areas including the choroid plexi and the ventricular and subventricular areas and was prominently detected in CD45low/int, CD11b+ microglial cells, in CD45high, CD11b+ cells of the myeloid lineage including macrophages, and in CD45+, CD11b– lymphocytes and non-B non-T cells. In parallel, rat cytomegalovirus infection of the developing rat brain rapidly triggered a cascade of pathophysiological events comprising: chemokines upregulation, including CCL2-4, 7 and 12; infiltration by peripheral cells including B-cells and monocytes at E17 and P1, and T-cells at P1; and microglia activation at E17 and P1. Conclusion In line with previous findings in neonatal murine models and in human specimen, our study further suggests that neuroimmune alterations might play critical roles in the early stages following cytomegalovirus infection of the brain in utero. Further studies are now needed to determine which

  7. Calmodulin inhibition regulates morphological and functional changes related to the actin cytoskeleton in pure microglial cells.

    PubMed

    Szabo, Melinda; Dulka, Karolina; Gulya, Karoly

    2016-01-01

    The roles of calmodulin (CaM), a multifunctional intracellular calcium receptor protein, as concerns selected morphological and functional characteristics of pure microglial cells derived from mixed primary cultures from embryonal forebrains of rats, were investigated through use of the CaM antagonists calmidazolium (CALMID) and trifluoperazine (TFP). The intracellular localization of the CaM protein relative to phalloidin, a bicyclic heptapeptide that binds only to filamentous actin, and the ionized calcium-binding adaptor molecule 1 (Iba1), a microglia-specific actin-binding protein, was determined by immunocytochemistry, with quantitative analysis by immunoblotting. In unchallenged and untreated (control) microglia, high concentrations of CaM protein were found mainly perinuclearly in ameboid microglia, while the cell cortex had a smaller CaM content that diminished progressively deeper into the branches in the ramified microglia. The amounts and intracellular distributions of both Iba1 and CaM proteins were altered after lipopolysaccharide (LPS) challenge in activated microglia. CALMID and TFP exerted different, sometimes opposing, effects on many morphological, cytoskeletal and functional characteristics of the microglial cells. They affected the CaM and Iba1 protein expressions and their intracellular localizations differently, inhibited cell proliferation, viability and fluid-phase phagocytosis to different degrees both in unchallenged and in LPS-treated (immunologically challenged) cells, and differentially affected the reorganization of the actin cytoskeleton in the microglial cell cortex, influencing lamellipodia, filopodia and podosome formation. In summary, these CaM antagonists altered different aspects of filamentous actin-based cell morphology and related functions with variable efficacy, which could be important in deciphering the roles of CaM in regulating microglial functions in health and disease.

  8. Infection of brain microglial cells by human immunodeficiency virus type 1 is CD4 dependent.

    PubMed Central

    Jordan, C A; Watkins, B A; Kufta, C; Dubois-Dalcq, M

    1991-01-01

    In the central nervous system of AIDS patients, human immunodeficiency virus (HIV) infects primarily microglia, a cell type of bone marrow origin. Moreover, microglial cells isolated from adult human brain support the replication of macrophage-adapted strains of HIV type 1 (HIV-1) (B.A. Watkins, H.H. Dorn, W.B. Kelly, R.C. Armstrong, B. Potts, F. Michaels, C.V. Kufta, and M. Dubois-Dalcq, Science 249:549-553, 1990). To determine whether the CD4 receptor, which is expressed in brain, mediates the entry of HIV-1 in microglial cells, we analyzed CD4 transcript expression in cultured microglia using highly sensitive polymerase chain reaction detection of cDNAs synthesized from RNA. With this method, CD4 transcripts could be detected in cultured microglia--as well as in various human brain regions and cultured macrophages used as positive controls--along with transcripts for the LDL and Fc receptors which are characteristic of cells of the macrophage lineage. We then attempted to block viral entry into microglial cells using anti-CD4 antibodies or soluble CD4 (sCD4), which recognize binding sites on CD4 and HIV-1 glycoprotein gp120, respectively. Cultures were pretreated with blocking antibodies (Leu-3a, OKT4A) or virus was preincubated with sCD4 prior to infection with HIV-1 strain AD87(M) or BaL. With either viral strain, these treatments resulted in the prevention of infection or significant and dose-dependent reduction in the number of infected cells and in the levels of reverse transcriptase or p24 antigen released in the medium. Thus, brain-derived microglial cells, which are the primary target of HIV-1 infection in the brain, express the CD4 receptor and this receptor is effectively used for viral entry in vitro. Images PMID:1702842

  9. Levo-Tetrahydropalmatine Attenuates Bone Cancer Pain by Inhibiting Microglial Cells Activation

    PubMed Central

    Zhang, Mao-yin; Liu, Yue-peng; Zhang, Lian-yi; Yue, Dong-mei; Qi, Dun-yi; Liu, Gong-jian; Liu, Su

    2015-01-01

    Objective. The present study is to investigate the analgesic roles of L-THP in rats with bone cancer pain caused by tumor cell implantation (TCI). Methods. Thermal hyperalgesia and mechanical allodynia were measured at different time points before and after operation. L-THP (20, 40, and 60 mg/kg) were administrated intragastrically at early phase of postoperation (before pain appearance) and later phase of postoperation (after pain appearance), respectively. The concentrations of TNF-α, IL-1β, and IL-18 in spinal cord were measured by enzyme-linked immunosorbent assay. Western blot was used to test the activation of astrocytes and microglial cells in spinal cord after TCI treatment. Results. TCI treatment induced significant thermal hyperalgesia and mechanical allodynia. Administration of L-THP at high doses significantly prevented and/or reversed bone cancer-related pain behaviors. Besides, TCI-induced activation of microglial cells and the increased levels of TNF-α and IL-18 were inhibited by L-THP administration. However, L-THP failed to affect TCI-induced astrocytes activation and IL-1β increase. Conclusion. This study suggests the possible clinical utility of L-THP in the treatment of bone cancer pain. The analgesic effects of L-THP on bone cancer pain maybe underlying the inhibition of microglial cells activation and proinflammatory cytokines increase. PMID:26819501

  10. Effects of a brain-engraftable microglial cell line expressing anti-prion scFv antibodies on survival times of mice infected with scrapie prions.

    PubMed

    Fujita, Koji; Yamaguchi, Yoshitaka; Mori, Tsuyoshi; Muramatsu, Naomi; Miyamoto, Takahito; Yano, Masashi; Miyata, Hironori; Ootsuyama, Akira; Sawada, Makoto; Matsuda, Haruo; Kaji, Ryuji; Sakaguchi, Suehiro

    2011-10-01

    We first verified that a single chain Fv fragment against prion protein (anti-PrP scFv) was secreted by HEK293T cells and prevented prion replication in infected cells. We then stably expressed anti-PrP scFv in brain-engraftable murine microglial cells and intracerebrally injected these cells into mice before or after infection with prions. Interestingly, the injection before or at an early time point after infection attenuated the infection marginally but significantly prolonged survival times of the mice. These suggest that the ex vivo gene transfer of anti-PrP scFvs using brain-engraftable cells could be a possible immunotherapeutic approach against prion diseases.

  11. Microglial Activation & Chronic Neurodegeneration

    PubMed Central

    Lull, Melinda E.; Block, Michelle L.

    2010-01-01

    Microglia, the resident innate immune cells in the brain, have long been implicated in the pathology of neurodegenerative diseases. Accumulating evidence points to activated microglia as a chronic source of multiple neurotoxic factors, including TNFα, NO, IL1-β, and reactive oxygen species (ROS), driving progressive neuron damage. Microglia can become chronically activated by either a single stimulus (ex. LPS or neuron damage) or multiple stimuli exposures to result in cumulative neuronal loss over time. While the mechanisms driving these phenomena are just beginning to be understood, reactive microgliosis (the microglial response to neuron damage) and ROS have been implicated as key mechanisms of chronic and neurotoxic microglial activation, particularly in the case of Parkinson’s Disease. Here, we review the mechanisms of neurotoxicity associated with chronic microglial activation and discuss the role of neuronal death and microglial ROS driving the chronic and toxic microglial phenotype. PMID:20880500

  12. Two-dimensional zymography differentiates gelatinase isoforms in stimulated microglial cells and in brain tissues of acute brain injuries.

    PubMed

    Chen, Shanyan; Meng, Fanjun; Chen, Zhenzhou; Tomlinson, Brittany N; Wesley, Jennifer M; Sun, Grace Y; Whaley-Connell, Adam T; Sowers, James R; Cui, Jiankun; Gu, Zezong

    2015-01-01

    Excessive activation of gelatinases (MMP-2/-9) is a key cause of detrimental outcomes in neurodegenerative diseases. A single-dimension zymography has been widely used to determine gelatinase expression and activity, but this method is inadequate in resolving complex enzyme isoforms, because gelatinase expression and activity could be modified at transcriptional and posttranslational levels. In this study, we investigated gelatinase isoforms under in vitro and in vivo conditions using two-dimensional (2D) gelatin zymography electrophoresis, a protocol allowing separation of proteins based on isoelectric points (pI) and molecular weights. We observed organomercuric chemical 4-aminophenylmercuric acetate-induced activation of MMP-2 isoforms with variant pI values in the conditioned medium of human fibrosarcoma HT1080 cells. Studies with murine BV-2 microglial cells indicated a series of proform MMP-9 spots separated by variant pI values due to stimulation with lipopolysaccharide (LPS). The MMP-9 pI values were shifted after treatment with alkaline phosphatase, suggesting presence of phosphorylated isoforms due to the proinflammatory stimulation. Similar MMP-9 isoforms with variant pI values in the same molecular weight were also found in mouse brains after ischemic and traumatic brain injuries. In contrast, there was no detectable pI differentiation of MMP-9 in the brains of chronic Zucker obese rats. These results demonstrated effective use of 2D zymography to separate modified MMP isoforms with variant pI values and to detect posttranslational modifications under different pathological conditions.

  13. Contact-independent cell death of human microglial cells due to pathogenic Naegleria fowleri trophozoites.

    PubMed

    Kim, Jong-Hyun; Kim, Daesik; Shin, Ho-Joon

    2008-12-01

    Free-living Naegleria fowleri leads to a fatal infection known as primary amebic meningoencephalitis in humans. Previously, the target cell death could be induced by phagocytic activity of N. fowleri as a contact-dependent mechanism. However, in this study we investigated the target cell death under a non-contact system using a tissue-culture insert. The human microglial cells, U87MG cells, co-cultured with N. fowleri trophozoites for 30 min in a non-contact system showed morphological changes such as the cell membrane destruction and a reduction in the number. By fluorescence-activated cell sorter (FACS) analysis, U87MG cells co-cultured with N. fowleri trophozoites in a non-contact system showed a significant increase of apoptotic cells (16%) in comparison with that of the control or N. fowleri lysate. When U87MG cells were co-cultured with N. fowleri trophozoites in a non-contact system for 30 min, 2 hr, and 4 hr, the cytotoxicity of amebae against target cells was 40.5, 44.2, and 45.6%, respectively. By contrast, the cytotoxicity of non-pathogenic N. gruberi trophozoites was 10.2, 12.4, and 13.2%, respectively. These results suggest that the molecules released from N. fowleri in a contact-independent manner as well as phagocytosis in a contact-dependent manner may induce the host cell death.

  14. Withania somnifera and Its Withanolides Attenuate Oxidative and Inflammatory Responses and Up-Regulate Antioxidant Responses in BV-2 Microglial Cells.

    PubMed

    Sun, Grace Y; Li, Runting; Cui, Jiankun; Hannink, Mark; Gu, Zezong; Fritsche, Kevin L; Lubahn, Dennis B; Simonyi, Agnes

    2016-09-01

    Withania somnifera (L.) Dunal, commonly known as Ashwagandha, has been used in Ayurvedic medicine for promoting health and quality of life. Recent clinical trials together with experimental studies indicated significant neuroprotective effects of Ashwagandha and its constituents. This study is aimed to investigate anti-inflammatory and anti-oxidative properties of this botanical and its two withanolide constituents, namely, Withaferin A and Withanolide A, using the murine immortalized BV-2 microglial cells. Ashwagandha extracts not only effectively inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and reactive oxygen species (ROS) production in BV-2 cells, but also stimulates the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway, leading to induction of heme oxygenase-1 (HO-1), both in the presence and absence of LPS. Although the withanolides were also capable of inhibiting LPS-induced NO production and stimulating Nrf2/HO-1 pathway, Withaferin A was tenfold more effective than Withanolide A. In serum-free culture, LPS can also induce production of long thin processes (filopodia) between 4 and 8 h in BV-2 cells. This morphological change was significantly suppressed by Ashwagandha and both withanolides at concentrations for suppressing LPS-induced NO production. Taken together, these results suggest an immunomodulatory role for Ashwagandha and its withanolides, and their ability to suppress oxidative and inflammatory responses in microglial cells by simultaneously down-regulating the NF-kB and upregulating the Nrf2 pathways. PMID:27209361

  15. Naegleria fowleri lysate induces strong cytopathic effects and pro-inflammatory cytokine release in rat microglial cells.

    PubMed

    Lee, Yang-Jin; Park, Chang-Eun; Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Jinyoung; Jung, Suk-Yul; Shin, Ho-Joon

    2011-09-01

    Naegleria fowleri, a ubiquitous free-living ameba, causes fatal primary amebic meningoencephalitis in humans. N. fowleri trophozoites are known to induce cytopathic changes upon contact with microglial cells, including necrotic and apoptotic cell death and pro-inflammatory cytokine release. In this study, we treated rat microglial cells with amebic lysate to probe contact-independent mechanisms for cytotoxicity, determining through a combination of light microscopy and scanning and transmission electron microscopy whether N. fowleri lysate could effect on both necrosis and apoptosis on microglia in a time- as well as dose-dependent fashion. A (51)Cr release assay demonstrated pronounced lysate induction of cytotoxicity (71.5%) toward microglial cells by 24 hr after its addition to cultures. In an assay of pro-inflammatory cytokine release, microglial cells treated with N. fowleri lysate produced TNF-α, IL-6, and IL-1β, though generation of the former 2 cytokines was reduced with time, and that of the last increased throughout the experimental period. In summary, N. fowleri lysate exerted strong cytopathic effects on microglial cells, and elicited pro-inflammatory cytokine release as a primary immune response. PMID:22072830

  16. Naegleria fowleri lysate induces strong cytopathic effects and pro-inflammatory cytokine release in rat microglial cells.

    PubMed

    Lee, Yang-Jin; Park, Chang-Eun; Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Jinyoung; Jung, Suk-Yul; Shin, Ho-Joon

    2011-09-01

    Naegleria fowleri, a ubiquitous free-living ameba, causes fatal primary amebic meningoencephalitis in humans. N. fowleri trophozoites are known to induce cytopathic changes upon contact with microglial cells, including necrotic and apoptotic cell death and pro-inflammatory cytokine release. In this study, we treated rat microglial cells with amebic lysate to probe contact-independent mechanisms for cytotoxicity, determining through a combination of light microscopy and scanning and transmission electron microscopy whether N. fowleri lysate could effect on both necrosis and apoptosis on microglia in a time- as well as dose-dependent fashion. A (51)Cr release assay demonstrated pronounced lysate induction of cytotoxicity (71.5%) toward microglial cells by 24 hr after its addition to cultures. In an assay of pro-inflammatory cytokine release, microglial cells treated with N. fowleri lysate produced TNF-α, IL-6, and IL-1β, though generation of the former 2 cytokines was reduced with time, and that of the last increased throughout the experimental period. In summary, N. fowleri lysate exerted strong cytopathic effects on microglial cells, and elicited pro-inflammatory cytokine release as a primary immune response.

  17. Naegleria fowleri Lysate Induces Strong Cytopathic Effects and Pro-inflammatory Cytokine Release in Rat Microglial Cells

    PubMed Central

    Lee, Yang-Jin; Park, Chang-Eun; Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Jinyoung; Jung, Suk-Yul

    2011-01-01

    Naegleria fowleri, a ubiquitous free-living ameba, causes fatal primary amebic meningoencephalitis in humans. N. fowleri trophozoites are known to induce cytopathic changes upon contact with microglial cells, including necrotic and apoptotic cell death and pro-inflammatory cytokine release. In this study, we treated rat microglial cells with amebic lysate to probe contact-independent mechanisms for cytotoxicity, determining through a combination of light microscopy and scanning and transmission electron microscopy whether N. fowleri lysate could effect on both necrosis and apoptosis on microglia in a time- as well as dose-dependent fashion. A 51Cr release assay demonstrated pronounced lysate induction of cytotoxicity (71.5%) toward microglial cells by 24 hr after its addition to cultures. In an assay of pro-inflammatory cytokine release, microglial cells treated with N. fowleri lysate produced TNF-α, IL-6, and IL-1β, though generation of the former 2 cytokines was reduced with time, and that of the last increased throughout the experimental period. In summary, N. fowleri lysate exerted strong cytopathic effects on microglial cells, and elicited pro-inflammatory cytokine release as a primary immune response. PMID:22072830

  18. Delayed activation of human microglial cells by high dose ionizing radiation.

    PubMed

    Chen, Hongxin; Chong, Zhao Zhong; De Toledo, Sonia M; Azzam, Edouard I; Elkabes, Stella; Souayah, Nizar

    2016-09-01

    Recent studies have shown that microglia affects the fate of neural stem cells in response to ionizing radiation, which suggests a role for microglia in radiation-induced degenerative outcomes. We therefore investigated the effects of γ-irradiation on cell survival, proliferation, and activation of microglia and explored associated mechanisms. Specifically, we evaluated cellular and molecular changes associated with exposure of human microglial cells (CHME5) to low and high doses of acute cesium-137 γ rays. Twenty-four hours after irradiation, cell cycle analyses revealed dose-dependent decreases in the fraction of cells in S and G2/M phase, which correlated with significant oxidative stress. By one week after irradiation, 20-30% of the cells exposed to high doses of γ rays underwent apoptosis, which correlated with significant concomitant decrease in metabolic activity as assessed by the MTT assay, and microglial activation as judged by both morphological changes and increased expression of Glut-5 and CR43. These changes were associated with increases in the mRNA levels for IL-1α, IL-10 and TNFα. Together, the results show that human CHME5 microglia are relatively resistant to low and moderate doses of γ rays, but are sensitive to acute high doses, and that CHME5 cells are a useful tool for in vitro study of human microglia. PMID:27265419

  19. Effects of aspirin on expression of iron transport and storage proteins in BV-2 microglial cells.

    PubMed

    Xu, Yan Xin; Du, Fang; Jiang, Li Rong; Gong, Jing; Zhou, Yu-Fu; Luo, Qian Qian; Qian, Zhong Ming; Ke, Ya

    2015-12-01

    In the light of recent studies, we hypothesized that aspirin might have the functions to regulate the expression of iron transport proteins and then affect cellular iron levels. To test this hypothesis, we investigated the effects of aspirin on expression of iron uptake protein transferrin receptor 1 (TfR1), iron release protein ferroportin 1 (Fpn1) and iron storage protein ferritin using Western blot analysis and on tumor necrosis factor (TNF)-αlpha, interleukin (IL)-6, interleukin (IL)-10 and hepcidin using quantitative real-time PCR in BV-2 microglial cells treated with lipopolysaccharides (LPS). We found that aspirin significantly down-regulated TfR1, while also up-regulated Fpn1 and ferritin expressions in BV-2 microglial cells in vitro. We also showed that TfR1 and Fpn1 expressions were significantly higher, while ferritin contents, IL-6, TNF-alpha and hepcidin mRNA levels were lower in cells treated with aspirin plus LPS than those in cells treated with LPS only. We concluded that aspirin has a negative effect on cell iron contents under 'normal' conditions and could partly reverse LPS-induced-disruption in cell iron balance under in vitro inflammatory conditions. Our findings also suggested that hepcidin might play a dominant role in the control of TfR1 expression by aspirin in the cells treated with LPS. PMID:26522688

  20. Effects of aspirin on expression of iron transport and storage proteins in BV-2 microglial cells.

    PubMed

    Xu, Yan Xin; Du, Fang; Jiang, Li Rong; Gong, Jing; Zhou, Yu-Fu; Luo, Qian Qian; Qian, Zhong Ming; Ke, Ya

    2015-12-01

    In the light of recent studies, we hypothesized that aspirin might have the functions to regulate the expression of iron transport proteins and then affect cellular iron levels. To test this hypothesis, we investigated the effects of aspirin on expression of iron uptake protein transferrin receptor 1 (TfR1), iron release protein ferroportin 1 (Fpn1) and iron storage protein ferritin using Western blot analysis and on tumor necrosis factor (TNF)-αlpha, interleukin (IL)-6, interleukin (IL)-10 and hepcidin using quantitative real-time PCR in BV-2 microglial cells treated with lipopolysaccharides (LPS). We found that aspirin significantly down-regulated TfR1, while also up-regulated Fpn1 and ferritin expressions in BV-2 microglial cells in vitro. We also showed that TfR1 and Fpn1 expressions were significantly higher, while ferritin contents, IL-6, TNF-alpha and hepcidin mRNA levels were lower in cells treated with aspirin plus LPS than those in cells treated with LPS only. We concluded that aspirin has a negative effect on cell iron contents under 'normal' conditions and could partly reverse LPS-induced-disruption in cell iron balance under in vitro inflammatory conditions. Our findings also suggested that hepcidin might play a dominant role in the control of TfR1 expression by aspirin in the cells treated with LPS.

  1. Ghrelin Inhibits Oligodendrocyte Cell Death by Attenuating Microglial Activation

    PubMed Central

    Lee, Jee Youn

    2014-01-01

    Background Recently, we reported the antiapoptotic effect of ghrelin in spinal cord injury-induced apoptotic cell death of oligodendrocytes. However, how ghrelin inhibits oligodendrocytes apoptosis, is still unknown. Therefore, in the present study, we examined whether ghrelin inhibits microglia activation and thereby inhibits oligodendrocyte apoptosis. Methods Using total cell extracts prepared from BV-2 cells activated by lipopolysaccharide (LPS) with or without ghrelin, the levels of p-p38 phosphor-p38 mitogen-activated protein kinase (p-p38MAPK), phospho-c-Jun N-terminal kinase (pJNK), p-c-Jun, and pro-nerve growth factor (proNGF) were examined by Western blot analysis. Reactive oxygen species (ROS) production was investigated by using dichlorodihydrofluorescein diacetate. To examine the effect of ghrelin on oligodendrocyte cell death, oligodendrocytes were cocultured in transwell chambers of 24-well plates with LPS-stimulated BV-2 cells. After 48 hours incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate nick end labeling staining were assessed. Results Ghrelin treatment significantly decreased levels of p-p38MAPK, p-JNK, p-c-Jun, and proNGF in LPS-stimulated BV-2 cells. ROS production increased in LPS-stimulated BV-2 cells was also significantly inhibited by ghrelin treatment. In addition, ghrelin significantly inhibited oligodendrocyte cell death when cocultured with LPS-stimulated BV-2 cells. Conclusion Ghrelin inhibits oligodendrocyte cell death by decreasing proNGF and ROS production as well as p38MAPK and JNK activation in activated microglia as an anti-inflammatory hormone. PMID:25309797

  2. Tart Cherry Extracts Reduce Inflammatory and Oxidative Stress Signaling in Microglial Cells.

    PubMed

    Shukitt-Hale, Barbara; Kelly, Megan E; Bielinski, Donna F; Fisher, Derek R

    2016-01-01

    Tart cherries contain an array of polyphenols that can decrease inflammation and oxidative stress (OS), which contribute to cognitive declines seen in aging populations. Previous studies have shown that polyphenols from dark-colored fruits can reduce stress-mediated signaling in BV-2 mouse microglial cells, leading to decreases in nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression. Thus, the present study sought to determine if tart cherries-which improved cognitive behavior in aged rats-would be efficacious in reducing inflammatory and OS signaling in HAPI rat microglial cells. Cells were pretreated with different concentrations (0-1.0 mg/mL) of Montmorency tart cherry powder for 1-4 h, then treated with 0 or 100 ng/mL lipopolysaccharide (LPS) overnight. LPS application increased extracellular levels of NO and tumor necrosis factor-alpha (TNF-α), and intracellular levels of iNOS and cyclooxygenase-2 (COX-2). Pretreatment with tart cherry decreased levels of NO, TNF-α, and COX-2 in a dose- and time-dependent manner versus those without pretreatment; the optimal combination was between 0.125 and 0.25 mg/mL tart cherry for 2 h. Higher concentrations of tart cherry powder and longer exposure times negatively affected cell viability. Therefore, tart cherries (like other dark-colored fruits), may be effective in reducing inflammatory and OS-mediated signals. PMID:27669317

  3. Sutherlandia frutescens Ethanol Extracts Inhibit Oxidative Stress and Inflammatory Responses in Neurons and Microglial Cells

    PubMed Central

    Jiang, Jinghua; Chuang, Dennis Y.; Zong, Yijia; Patel, Jayleenkumar; Brownstein, Korey; Lei, Wei; Lu, Chi-Hua; Simonyi, Agnes; Gu, Zezong; Cui, Jiankun; Rottinghaus, George E.; Fritsche, Kevin L.; Lubahn, Dennis B.; Folk, William R.; Sun, Grace Y.

    2014-01-01

    Sutherlandia frutescens (L.) R.Br. (SF) is a medicinal plant indigenous to southern Africa and used in folk and contemporary remedies for stress, chronic diseases, cancer, and HIV/AIDS. While previous studies have focused on physiological effects of SF on cellular and systemic abnormalities associated with these diseases, little is known about its effects in the brain and immune cells in the central nervous system. Results of this study indicate that ethanol extracts of SF (SF-E) suppress NMDA-induced reactive oxygen species (ROS) production in neurons, and LPS- and IFNγ-induced ROS and nitric oxide (NO) production in microglial cells. SF-E’s action on microglial cells appears to be mediated through inhibition of the IFNγ-induced p-ERK1/2 signaling pathway which is central to regulating a number of intracellular metabolic processes including enhancing STAT1α phosphorylation and filopodia formation. The involvement of SF in these pathways suggests the potential for novel therapeutics for stress and prevention, and/or treatment of HIV/AIDS as well as other inflammatory diseases in the brain. PMID:24587007

  4. Anti-inflammatory effects and antioxidant activity of dihydroasparagusic acid in lipopolysaccharide-activated microglial cells.

    PubMed

    Salemme, Adele; Togna, Anna Rita; Mastrofrancesco, Arianna; Cammisotto, Vittoria; Ottaviani, Monica; Bianco, Armandodoriano; Venditti, Alessandro

    2016-01-01

    The activation of microglia and subsequent release of toxic pro-inflammatory factors are crucially associated with neurodegenerative disease, characterized by increased oxidative stress and neuroinflammation, including Alzheimer and Parkinson diseases and multiple sclerosis. Dihydroasparagusic acid is the reduced form of asparagusic acid, a sulfur-containing flavor component produced by Asparagus plants. It has two thiolic functions able to coordinate the metal ions, and a carboxylic moiety, a polar function, which may enhance excretion of the complexes. Thiol functions are also present in several biomolecules with important physiological antioxidant role as glutathione. The aim of this study is to evaluate the anti-inflammatory and antioxidant potential effect of dihydroasparagusic acid on microglial activation in an in vitro model of neuroinflammation. We have used lipopolysaccharide to induce an inflammatory response in primary rat microglial cultures. Our results suggest that dihydroasparagusic acid significantly prevented lipopolysaccharide-induced production of pro-inflammatory and neurotoxic mediators such as nitric oxide, tumor necrosis factor-α, prostaglandin E2, as well as inducible nitric oxide synthase and cyclooxygenase-2 protein expression and lipoxygenase activity in microglia cells. Moreover it effectively suppressed the level of reactive oxygen species and affected lipopolysaccharide-stimulated activation of mitogen activated protein kinase, including p38, and nuclear factor-kB pathway. These results suggest that dihydroasparagusic acid's neuroprotective properties may be due to its ability to dampen induction of microglial activation. It is a compound that can effectively inhibit inflammatory and oxidative processes that are important factors of the etiopathogenesis of neurodegenerative diseases. PMID:26592472

  5. Prenylated Flavonoids from Cudrania tricuspidata Suppress Lipopolysaccharide-Induced Neuroinflammatory Activities in BV2 Microglial Cells

    PubMed Central

    Kim, Dong-Cheol; Yoon, Chi-Su; Quang, Tran Hong; Ko, Wonmin; Kim, Jong-Su; Oh, Hyuncheol; Kim, Youn-Chul

    2016-01-01

    In Korea and China, Cudrania tricuspidata Bureau (Moraceae) is an important traditional medicinal plant used to treat lumbago, hemoptysis, and contusions. The C. tricuspidata methanol extract suppressed both production of NO and PGE2 in BV2 microglial cells. Cudraflavanone D (1), isolated from this extract, remarkably suppressed the protein expression of inducible NO synthase and cyclooxygenase-2, and decreased the levels of NO and PGE2 in BV2 microglial cells exposed to lipopolysaccharide. Cudraflavanone D (1) also decreased IL-6, TNF-α, IL-12, and IL-1β production, blocked nuclear translocation of NF-κB heterodimers (p50 and p65) by interrupting the degradation and phosphorylation of inhibitor of IκB-α, and inhibited NF-κB binding. In addition, cudraflavanone D (1) suppressed the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK pathways. This study indicated that cudraflavanone D (1) can be a potential drug candidate for the cure of neuroinflammation. PMID:26907256

  6. Celecoxib Inhibits Prion Protein 90-231-Mediated Pro-inflammatory Responses in Microglial Cells.

    PubMed

    Villa, Valentina; Thellung, Stefano; Corsaro, Alessandro; Novelli, Federica; Tasso, Bruno; Colucci-D'Amato, Luca; Gatta, Elena; Tonelli, Michele; Florio, Tullio

    2016-01-01

    Activation of microglia is a central event in the atypical inflammatory response occurring during prion encephalopathies. We report that the prion protein fragment encompassing amino acids 90-231 (PrP90-231), a model of the neurotoxic activity of the pathogenic prion protein (PrP(Sc)), causes activation of both primary microglia cultures and N9 microglial cells in vitro. This effect was characterized by cell proliferation arrest and induction of a secretory phenotype, releasing prostaglandin E2 (PGE2) and nitric oxide (NO). Conditioned medium from PrP90-231-treated microglia induced in vitro cytotoxicity of A1 mesencephalic neurons, supporting the notion that soluble mediators released by activated microglia contributes to the neurodegeneration during prion diseases. The neuroinflammatory role of COX activity, and its potential targeting for anti-prion therapies, was tested measuring the effects of ketoprofen and celecoxib (preferential inhibitors of COX1 and COX2, respectively) on PrP90-231-induced microglial activation. Celecoxib, but not ketoprofen significantly reverted the growth arrest as well as NO and PGE2 secretion induced by PrP90-231, indicating that PrP90-231 pro-inflammatory response in microglia is mainly dependent on COX2 activation. Taken together, these data outline the importance of microglia in the neurotoxicity occurring during prion diseases and highlight the potentiality of COX2-selective inhibitors to revert microglia as adjunctive pharmacological approach to contrast the neuroinflammation-dependent neurotoxicity.

  7. Regulation of Macrophage, Dendritic Cell, and Microglial Phenotype and Function by the SOCS Proteins

    PubMed Central

    McCormick, Sarah M.; Heller, Nicola M.

    2015-01-01

    Macrophages are innate immune cells of dynamic phenotype that rapidly respond to external stimuli in the microenvironment by altering their phenotype to respond to and to direct the immune response. The ability to dynamically change phenotype must be carefully regulated to prevent uncontrolled inflammatory responses and subsequently to promote resolution of inflammation. The suppressor of cytokine signaling (SOCS) proteins play a key role in regulating macrophage phenotype. In this review, we summarize research to date from mouse and human studies on the role of the SOCS proteins in determining the phenotype and function of macrophages. We will also touch on the influence of the SOCS on dendritic cell (DC) and microglial phenotype and function. The molecular mechanisms of SOCS function in macrophages and DCs are discussed, along with how dysregulation of SOCS expression or function can lead to alterations in macrophage/DC/microglial phenotype and function and to disease. Regulation of SOCS expression by microRNA is discussed. Novel therapies and unanswered questions with regard to SOCS regulation of monocyte–macrophage phenotype and function are highlighted. PMID:26579124

  8. 2-Arachidonoylglycerol elicits neuroprotective effects on excitotoxically lesioned dentate gyrus granule cells via abnormal-cannabidiol-sensitive receptors on microglial cells.

    PubMed

    Kreutz, Susanne; Koch, Marco; Böttger, Charlotte; Ghadban, Chalid; Korf, Horst-Werner; Dehghani, Faramarz

    2009-02-01

    Endocannabinoids like 2-arachidonoylglycerol (2-AG) exert neuroprotective effects after brain injuries. According to current concepts, these neuroprotective effects are due to interactions between 2-AG and cannabinoid (CB)1 receptors on neurons. Moreover, 2-AG modulates migration and proliferation of microglial cells which are rapidly activated after brain lesion. This effect is mediated via CB2- and abnormal-cannabidiol (abn-CBD)-sensitive receptors. In the present study, we investigated whether the abn-CBD-sensitive receptor on microglial cells contributes to 2-AG-mediated neuroprotection in organotypic hippocampal slice cultures (OHSCs) after excitotoxic lesion induced by NMDA (50 microM) application for 4 h. This lesion caused neuronal damage and accumulation of microglial cells within the granule cell layer. To analyze the role of abn-CBD-sensitive receptors for neuroprotection and microglial cell accumulation, two agonists of the abn-CBD-sensitive receptor, abn-CBD or 2-AG, two antagonists, 1,3-dimethoxy-5-methyl-2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen1-yl]-benzene (O-1918) or cannabidiol (CBD), and the CB1 receptor antagonist AM251, were applied to NMDA-lesioned OHSC. Propidium iodide (PI) labeling was used as a marker of degenerating neurons and isolectin B(4) (IB(4)) as a marker of microglial cells. Application of both, abn-CBD or 2-AG to lesioned OHSC significantly decreased the number of IB(4)(+) microglial cells and PI(+) neurons in the dentate gyrus. In contrast to AM251, application of O-1918 or CBD antagonized these effects. When microglial cells were depleted by preincubation of OHSC with the bisphosphonate clodronate (100 microg/mL) for 5 days before excitotoxic lesion, 2-AG and abn-CBD lost their neuroprotective effects. We therefore propose that the endocannabinoid 2-AG exerts its neuroprotective effects via activation of abn-CBD-sensitive receptors on microglial cells.

  9. Highly permissive infection of microglial cells by Japanese encephalitis virus: a possible role as a viral reservoir.

    PubMed

    Thongtan, Thananya; Cheepsunthorn, Poonlarp; Chaiworakul, Voravasa; Rattanarungsan, Chutima; Wikan, Nitwara; Smith, Duncan R

    2010-01-01

    Japanese encephalitis virus (JEV), a mosquito-borne Flavivirus, is a major cause of acute encephalitis, and neurons have been proposed to be the principle JEV target cells in the central nervous system. However, clinically, infection with JEV leads to increased levels of cytokines and chemokines in the serum and cerebrospinal fluid (CSF) the levels of which correlate with the mortality rate of patients. This research aimed to study the role of microglial cells in JEV infection. Mouse microglial cells (BV-2) supported the replication of JEV with extracellular production of virus by 10h post-infection, and virus titer reached a maximum (2.55x10(10)pfu/ml) by day 3 post-infection. While apoptosis was induced in response to virus infection, no alteration in nitric oxide production was observed. Microglial cells remained productively infected with JEV for up to 16 weeks without significant morphological alterations, and the released virions were infectious to mouse neuroblastoma (NA) cells. The high virus production and long persistence of JEV in microglial cells suggests that these cells may serve as viral reservoirs for the infection of neurons in the CNS.

  10. Inhibition of endotoxin-induced nitric oxide synthase production in microglial cells by the presence of astroglial cells: a role for transforming growth factor beta.

    PubMed

    Vincent, V A; Tilders, F J; Van Dam, A M

    1997-03-01

    In mixed glial cell cultures from cerebral cortices of newborn rats, endotoxin induces inducible nitric oxide (iNOS), nitric oxide (NO), and interleukin-1 beta (IL-1 beta) production in microglial cells. Earlier we demonstrated that endotoxin induced iNOS but not IL-1 beta expression in microglial cells is inhibited by the presence of astroglial cells. In the present paper we describe studies on the mechanism by which astroglial cells exert selective suppressive action on iNOS expression by microglial cells. Expression of iNOS and IL-1 beta was studied by single or double label immunocytochemical techniques and cell identification was performed with GSA-I-B4-isolectin and an antibody against GFAP. Production of IL-1 beta and NO was determined by measurement of IL-1 beta and nitrite concentrations in cell lysates and the culture medium, respectively. TGF beta, a cytokine known to inhibit NO production by endotoxin challenged macrophages, was measured in culture medium of mixed glial cell cultures using a bioassay. Microglial, astroglial, and mixed glial cell cultures produced similar concentrations of TGF beta. The potential effect of TGF beta was studied by using immunoneutralizing antibodies against TGF beta 1 and TGF beta 2 on the induction of iNOS in microglial cells in the presence of astroglial cells. Incubation of the mixed glial cell culture with these TGF beta antibodies (3 micrograms/ml) markedly increased endotoxin-induced NO production and iNOS expression in microglial cells, whereas the production of IL-1 beta was not affected. The antibodies against TGF beta 1 and TGF beta 2 marginally increased NO production in pure microglial cell cultures, nonetheless in cultures of purified microglial cells recombinant TGF beta 1 and TGF beta 2 together with endotoxin inhibited NO production. We conclude that the presence of astroglial cells is essential for the inhibitory effect of TGF beta on NO production by microglial cells (possibly) by activation of TGF beta

  11. Anti-inflammatory effects of cordycepin via suppression of inflammatory mediators in BV2 microglial cells.

    PubMed

    Jeong, Jin-Woo; Jin, Cheng-Yun; Kim, Gi-Young; Lee, Jae-Dong; Park, Cheol; Kim, Gun-Do; Kim, Wun-Jae; Jung, Won-Kyo; Seo, Su Kil; Choi, Il-Whan; Choi, Yung Hyun

    2010-12-01

    Cordyceps militaris, a traditional medicinal mushroom, produces the bioactive compound cordycepin (3'-deoxyadenosine). Although cordycepin has been shown to have pharmacological, immunological stimulating, anti-cancer, and anti-inflammatory activities, its activities and cellular mechanisms during microglial activation have yet to be elucidated. Thus, we evaluated the anti-inflammatory effect of cordycepin on the production of inflammatory mediators in lipopolysaccharide (LPS)-stimulated murine BV2 microglia. We also investigated the effects of cordycepin on LPS-induced nuclear factor-kappaB (NF-κB) activation and on phosphorylation of mitogen-activated protein kinases (MAPKs). After LPS stimulation, nitric oxide (NO), prostaglandin E₂ (PGE₂), and pro-inflammatory cytokine production was detected in BV2 microglia. However, we found that cordycepin significantly inhibited the excessive production of NO, PGE₂, and pro-inflammatory cytokines in a concentration-dependent manner without causing cytotoxicity. In addition, cordycepin suppressed NF-κB translocation by blocking IkappaB-α (IκB-α) degradation and inhibited the phosphorylation of Akt, ERK-1/2, JNK, and p38 kinase. Our results indicate that the inhibitory effect of cordycepin on LPS-stimulated inflammatory mediator production in BV2 microglia is associated with the suppression of the NF-κB, Akt, and MAPK signaling pathways. Therefore, cordycepin may be useful in treating neurodegenerative diseases by inhibiting inflammatory mediator production in activated microglia. PMID:20937401

  12. Anti-neuro-inflammatory effects of Nardostachys chinensis in lipopolysaccharide-and lipoteichoic acid-stimulated microglial cells.

    PubMed

    Park, Sun Young; Kim, Young Hun; Park, Geuntae

    2016-05-01

    Excessive microglial cell activation is related to the progression of chronic neuro-inflammatory disorders. Heme oxygenase-1 (HO-1) expression mediated by the NFE2-related factor (Nrf-2) pathway is a key regulator of neuro-inflammation. Nardostachys chinensis is used as an anti-malarial, anti-nociceptive, and neurotrophic treatment in traditional Asian medicines. In the present study, we examined the effects of an ethyl acetate extract of N. chinensis (EN) on the anti-neuro-inflammatory effects mediated by HO-1 up-regulation in Salmonella lipopolysaccharide (LPS)- or Staphylococcus aureus lipoteichoic acid (LTA)-stimulated BV2 microglial cells. Our results indicated that EN suppressed pro-inflammatory cytokine production and induced HO-1 transcription and translation through Nrf-2/antioxidant response element (ARE) signaling. EN markedly inhibited LPS- and LTA-induced activation of nuclear factor-kappa B (NF-κB) as well as phosphorylation of mitogen-activated protein kinases (MAPKs) and signal transducer and activator of transcription (STAT). Furthermore, EN protected hippocampal HT22 cells from indirect neuronal toxicity mediated by LPS- and LTA-treated microglial cells. These results suggested that EN impairs LPS- and LTA-induced neuro-inflammatory responses in microglial cells and confers protection against indirect neuronal damage to HT22 cells. In conclusion, our findings indicate that EN could be used as a natural anti-neuro-inflammatory and neuroprotective agent. PMID:27478097

  13. Regulatory Effects of Caffeic Acid Phenethyl Ester on Neuroinflammation in Microglial Cells

    PubMed Central

    Tsai, Cheng-Fang; Kuo, Yueh-Hsiung; Yeh, Wei-Lan; Wu, Caren Yu-Ju; Lin, Hsiao-Yun; Lai, Sheng-Wei; Liu, Yu-Shu; Wu, Ling-Hsuan; Lu, Jheng-Kun; Lu, Dah-Yuu

    2015-01-01

    Microglial activation has been widely demonstrated to mediate inflammatory processes that are crucial in several neurodegenerative disorders. Pharmaceuticals that can deliver direct inhibitory effects on microglia are therefore considered as a potential strategy to counter balance neurodegenerative progression. Caffeic acid phenethyl ester (CAPE), a natural phenol in honeybee propolis, is known to possess antioxidant, anti-inflammatory and anti-microbial properties. Accordingly, the current study intended to probe the effects of CAPE on microglia activation by using in vitro and in vivo models. Western blot and Griess reaction assay revealed CAPE significantly inhibited the expressions of inducible nitric oxide synthase (NOS), cyclooxygenase (COX)-2 and the production of nitric oxide (NO). Administration of CAPE resulted in increased expressions of hemeoxygenase (HO)-1and erythropoietin (EPO) in microglia. The phosphorylated adenosine monophosphate-activated protein kinase (AMPK)-α was further found to regulate the anti-inflammatory effects of caffeic acid. In vivo results from immunohistochemistry along with rotarod test also revealed the anti-neuroinflammatory effects of CAPE in microglia activation. The current study has evidenced several possible molecular determinants, AMPKα, EPO, and HO-1, in mediating anti-neuroinflammatory responses in microglial cells. PMID:25768341

  14. Organization of hyperactive microglial cells in trigeminal spinal subnucleus caudalis and upper cervical spinal cord associated with orofacial neuropathic pain.

    PubMed

    Shibuta, Kazuo; Suzuki, Ikuko; Shinoda, Masamichi; Tsuboi, Yoshiyuki; Honda, Kuniya; Shimizu, Noriyoshi; Sessle, Barry J; Iwata, Koichi

    2012-04-27

    The aim of this study was to evaluate spatial organization of hyperactive microglial cells in trigeminal spinal subnucleus caudalis (Vc) and upper cervical spinal cord (C1), and to clarify the involvement in mechanisms underlying orofacial secondary hyperalgesia following infraorbital nerve injury. We found that the head-withdrawal threshold to non-noxious mechanical stimulation of the maxillary whisker pad skin was significantly reduced in chronic constriction injury of the infraorbital nerve (ION-CCI) rats from day 1 to day 14 after ION-CCI. On day 3 after ION-CCI, mechanical allodynia was obvious in the orofacial skin areas innervated by the 1st and 3rd branches of the trigeminal nerve as well as the 2nd branch area. Hyperactive microglial cells in Vc and C1 were observed on days 3 and 7 after ION-CCI. On day 3 after ION-CCI, a large number of phosphorylated extracellular signal-regulated kinase (pERK)-immunoreactive (IR) cells were observed in Vc and C1. Many hyperactive microglial cells were also distributed over a wide area of Vc and C1 innervated by the trigeminal nerve. The intraperitoneal administration of minocycline significantly reduced the activation of microglial cells and the number of pERK-IR cells in Vc and C1, and also significantly attenuated the development of mechanical allodynia. Furthermore, enhanced background activity and mechanical evoked responses of Vc wide dynamic range neurons in ION-CCI rats were significantly reversed following minocycline administration. These findings suggest that activation of microglial cells over a wide area of Vc and C1 is involved in the enhancement of Vc and C1 neuronal excitability in the early period after ION-CCI, resulting in the neuropathic pain in orofacial areas innervated by the injured as well as uninjured nerves.

  15. Titration of murine leukemia viruses with rat cell line RFL.

    PubMed

    Koga, M

    1977-08-01

    Normal rat embryo cell (RFL) from syncytia after infection with murine leukemia virus. The assay for counting the number of syncytium foci produced in RFL cells is a sensitive method for a direct infectivity assay of murine leukemia virus.

  16. Enhanced Cultivation Of Stimulated Murine B Cells

    NASA Technical Reports Server (NTRS)

    Sammons, David W.

    1994-01-01

    Method of in vitro cultivation of large numbers of stimulated murine B lymphocytes. Cells electrofused with other cells to produce hybridomas and monoclonal antibodies. Offers several advantages: polyclonally stimulated B-cell blasts cultivated for as long as 14 days, hybridomas created throughout culture period, yield of hybridomas increases during cultivation, and possible to expand polyclonally in vitro number of B cells specific for antigenic determinants first recognized in vivo.

  17. Quercetin Attenuates Inflammatory Responses in BV-2 Microglial Cells: Role of MAPKs on the Nrf2 Pathway and Induction of Heme Oxygenase-1

    PubMed Central

    Sun, Grace Y.; Chen, Zihong; Jasmer, Kimberly J.; Chuang, Dennis Y.; Gu, Zezong; Hannink, Mark; Simonyi, Agnes

    2015-01-01

    A large group of flavonoids found in fruits and vegetables have been suggested to elicit health benefits due mainly to their anti-oxidative and anti-inflammatory properties. Recent studies with immune cells have demonstrated inhibition of these inflammatory responses through down-regulation of the pro-inflammatory pathway involving NF-κB and up-regulation of the anti-oxidative pathway involving Nrf2. In the present study, the murine BV-2 microglial cells were used to compare anti-inflammatory activity of quercetin and cyanidin, two flavonoids differing by their alpha, beta keto carbonyl group. Quercetin was 10 folds more potent than cyanidin in inhibition of lipopolysaccharide (LPS)-induced NO production as well as stimulation of Nrf2-induced heme-oxygenase-1 (HO-1) protein expression. In addition, quercetin demonstrated enhanced ability to stimulate HO-1 protein expression when cells were treated with LPS. In an attempt to unveil mechanism(s) for quercetin to enhance Nrf2/HO-1 activity under endotoxic stress, results pointed to an increase in phospho-p38MAPK expression upon addition of quercetin to LPS. In addition, pharmacological inhibitors for phospho-p38MAPK and MEK1/2 for ERK1/2 further showed that these MAPKs target different sites of the Nrf2 pathway that regulates HO-1 expression. However, inhibition of LPS-induced NO by quercetin was not fully reversed by TinPPIX, a specific inhibitor for HO-1 activity. Taken together, results suggest an important role of quercetin to regulate inflammatory responses in microglial cells and its ability to upregulate HO-1 against endotoxic stress through involvement of MAPKs. PMID:26505893

  18. Infiltrating cells from host brain restore the microglial population in grafted cortical tissue.

    PubMed

    Wang, Cong; Tao, Sijue; Fang, Yukun; Guo, Jing; Zhu, Lirui; Zhang, Shengxiang

    2016-01-01

    Transplantation of embryonic cortical tissue is considered as a promising therapy for brain injury. Grafted neurons can reestablish neuronal network and improve cortical function of the host brain. Microglia is a key player in regulating neuronal survival and plasticity, but its activation and dynamics in grafted cortical tissue remain unknown. Using two-photon intravital imaging and parabiotic model, here we investigated the proliferation and source of microglia in the donor region by transplanting embryonic cortical tissue into adult cortex. Live imaging showed that the endogenous microglia of the grafted tissue were rapidly lost after transplantation. Instead, host-derived microglia infiltrated and colonized the graft. Parabiotic model suggested that the main source of infiltrating cells is the parenchyma of the host brain. Colonized microglia proliferated and experienced an extensive morphological transition and eventually differentiated into resting ramified morphology. Collectively, these results demonstrated that donor tissue has little contribution to the activated microglia and host brain controls the microglial population in the graft.

  19. Monoclonal antibody to macrophages (EMB/11) labels macrophages and microglial cells in human brain.

    PubMed Central

    Esiri, M M; McGee, J O

    1986-01-01

    Normal and diseased human central nervous system (CNS) tissues were studied immunohistochemically by a monoclonal antibody to human macrophages (EBM/11), antisera to glial fibrillary acidic protein (anti-GFAP), and alpha-1-antichymotrypsin (alpha 1-ACT). EBM/11 reacted with brain macrophages located mainly around blood vessels in normal brain; it also reacted with resting microglia in normal brain and with numerous reactive microglia and macrophages in brain tumours and inflammatory lesions. Microglia did not react with anti-GFAP or alpha 1-ACT. An EBM/11 positive phenotype, therefore, is shared by microglia and macrophages and suggests that microglial cells form a specialised part of the mononuclear phagocyte system. Images PMID:3755142

  20. Activation of microglial cells triggers a release of brain-derived neurotrophic factor (BDNF) inducing their proliferation in an adenosine A2A receptor-dependent manner: A2A receptor blockade prevents BDNF release and proliferation of microglia

    PubMed Central

    2013-01-01

    Background Brain-derived neurotrophic factor (BDNF) has been shown to control microglial responses in neuropathic pain. Since adenosine A2A receptors (A2ARs) control neuroinflammation, as well as the production and function of BDNF, we tested to see if A2AR controls the microglia-dependent secretion of BDNF and the proliferation of microglial cells, a crucial event in neuroinflammation. Methods Murine N9 microglial cells were challenged with lipopolysaccharide (LPS, 100 ng/mL) in the absence or in the presence of the A2AR antagonist, SCH58261 (50 nM), as well as other modulators of A2AR signaling. The BDNF cellular content and secretion were quantified by Western blotting and ELISA, A2AR density was probed by Western blotting and immunocytochemistry and cell proliferation was assessed by BrdU incorporation. Additionally, the A2AR modulation of LPS-driven cell proliferation was also tested in primary cultures of mouse microglia. Results LPS induced time-dependent changes of the intra- and extracellular levels of BDNF and increased microglial proliferation. The maximal LPS-induced BDNF release was time-coincident with an LPS-induced increase of the A2AR density. Notably, removing endogenous extracellular adenosine or blocking A2AR prevented the LPS-mediated increase of both BDNF secretion and proliferation, as well as exogenous BDNF-induced proliferation. Conclusions We conclude that A2AR activation plays a mandatory role controlling the release of BDNF from activated microglia, as well as the autocrine/paracrine proliferative role of BDNF. PMID:23363775

  1. Microglial NADPH oxidase activation mediates rod cell death in the retinal degeneration in rd mice.

    PubMed

    Zeng, H; Ding, M; Chen, X-X; Lu, Q

    2014-09-01

    Accumulating evidence supports that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase contributes to microglia-mediated neurotoxicity in the CNS neurodegenerative diseases. Several studies, including ours, suggest that microglial activation is involved in the retinal degeneration in the animal models of retinitis pigmentosa (RP). In the present study, we investigated the activation of NADPH oxidase in the rod degeneration in rd mice and further explored its role in the microglia-mediated photoreceptor apoptosis. Expression of gp91phox protein, a major subunit of NAPDH oxidase in the whole retina of rd mice at postnatal days (P) 8, 10, 12, 14, 16 and 18 was assessed by western blot analysis. Location of gp91phox in the rd retina at each age group and its cellular source were studied by immunohistochemical analysis and double labeling respectively. The generation of superoxide radicals in the rd retinas was demonstrated by intraperitoneal injection of hydroethidine. Apocynin was applied intraperitoneally in the rd mice from P8 to P14 to inhibit the activity of NAPDH oxidase and the outer nuclear layer (ONL) thickness was measured before and after apocynin treatment. Our results demonstrated that during the rod degenerative process, the expression of gp91phox started to increase in the outer part of rd retina at P10 and reached a peak at P14. Double labeling of gp91phox with CD11b showed co-localization of gp91phox in the retinal microglial cells. Increasing generation of superoxide radicals visualized by hydroethidine was noted at P8 and reached a peak at P14. Apocynin markedly reduced the production of superoxide radicals and preserved the rod cells. The results suggested that NADPH oxidase might play an important role in the rod degeneration in the rd mice. Inhibition of NAPDH oxidase could be a possible approach to treat RP in the early degenerative stage.

  2. Myeloid/Microglial Driven Autologous Hematopoietic Stem Cell Gene Therapy Corrects a Neuronopathic Lysosomal Disease

    PubMed Central

    Sergijenko, Ana; Langford-Smith, Alexander; Liao, Ai Y; Pickford, Claire E; McDermott, John; Nowinski, Gabriel; Langford-Smith, Kia J; Merry, Catherine LR; Jones, Simon A; Wraith, J Edmond; Wynn, Robert F; Wilkinson, Fiona L; Bigger, Brian W

    2013-01-01

    Mucopolysaccharidosis type IIIA (MPSIIIA) is a lysosomal storage disorder caused by mutations in N-sulfoglucosamine sulfohydrolase (SGSH), resulting in heparan sulfate (HS) accumulation and progressive neurodegeneration. There are no treatments. We previously demonstrated improved neuropathology in MPSIIIA mice using lentiviral vectors (LVs) overexpressing SGSH in wild-type (WT) hematopoietic stem cell (HSC) transplants (HSCTs), achieved via donor monocyte/microglial engraftment in the brain. However, neurological disease was not corrected using LVs in autologous MPSIIIA HSCTs. To improve brain expression via monocyte/microglial specificity, LVs expressing enhanced green fluorescent protein (eGFP) under ubiquitous phosphoglycerate kinase (PGK) or myeloid-specific promoters were compared in transplanted HSCs. LV-CD11b-GFP gave significantly higher monocyte/B-cell eGFP expression than LV-PGK-GFP or LV-CD18-GFP after 6 months. Subsequently, autologous MPSIIIA HSCs were transduced with either LV-PGK-coSGSH or LV-CD11b-coSGSH vectors expressing codon-optimized SGSH and transplanted into MPSIIIA mice. Eight months after HSCT, LV-PGK-coSGSH vectors produced bone marrow SGSH (576% normal activity) similar to LV-CD11b-coSGSH (473%), but LV-CD11b-coSGSH had significantly higher brain expression (11 versus 7%), demonstrating improved brain specificity. LV-CD11b-coSGSH normalized MPSIIIA behavior, brain HS, GM2 ganglioside, and neuroinflammation to WT levels, whereas LV-PGK-coSGSH partly corrected neuropathology but not behavior. We demonstrate compelling evidence of neurological disease correction using autologous myeloid driven lentiviral-HSC gene therapy in MPSIIIA mice. PMID:23748415

  3. Glycine modulates membrane potential, cell volume, and phagocytosis in murine microglia.

    PubMed

    Komm, Barbara; Beyreis, Marlena; Kittl, Michael; Jakab, Martin; Ritter, Markus; Kerschbaum, Hubert H

    2014-08-01

    Phagocytes form engulfment pseudopodia at the contact area with their target particle by a process resembling cell volume (CV) regulatory mechanisms. We evaluated whether the osmoregulatory active neutral amino acid glycine, which contributes to CV regulation via activation of sodium-dependent neutral amino acid transporters (SNATs) improves phagocytosis in isotonic and hypertonic conditions in the murine microglial cell line BV-2 and primary microglial cells (pMG). In BV-2 cells and pMG, RT-PCR analysis revealed expression of SNATs (Slc38a1, Slc38a2), but not of GlyRs (Glra1-4). In BV-2 cells, glycine (5 mM) led to a rapid Na(+)-dependent depolarization of membrane potential (V mem). Furthermore, glycine increased CV by about 9%. Visualizing of phagocytosis of polystyrene microspheres by scanning electron microscopy revealed that glycine (1 mM) increased the number of BV-2 cells containing at least one microsphere by about 13%. Glycine-dependent increase in phagocytosis was suppressed by the SNAT inhibitor α-(methylamino)isobutyric acid (MeAIB), by replacing extracellular Na(+) with choline, and under hypertonic conditions, but not by the GlyR antagonist strychnine or the GlyR agonist taurine. Interestingly, hypertonicity-induced suppression of phagocytosis was rescued by glycine. These findings demonstrate that glycine increases phagocytosis in iso- and hypertonic conditions by activation of SNATs.

  4. Phenotypic dynamics of microglial and monocyte-derived cells in glioblastoma-bearing mice

    PubMed Central

    Ricard, Clément; Tchoghandjian, Aurélie; Luche, Hervé; Grenot, Pierre; Figarella-Branger, Dominique; Rougon, Geneviève; Malissen, Marie; Debarbieux, Franck

    2016-01-01

    Inflammatory cells, an integral component of tumor evolution, are present in Glioblastomas multiforme (GBM). To address the cellular basis and dynamics of the inflammatory microenvironment in GBM, we established an orthotopic syngenic model by grafting GL261-DsRed cells in immunocompetent transgenic LysM-EGFP//CD11c-EYFP reporter mice. We combined dynamic spectral two-photon imaging with multiparametric cytometry and multicolor immunostaining to characterize spatio-temporal distribution, morphology and activity of microglia and blood-derived infiltrating myeloid cells in live mice. Early stages of tumor development were dominated by microglial EYFP+ cells invading the tumor, followed by massive recruitment of circulating LysM-EGFP+ cells. Fluorescent invading cells were conventional XCR1+ and monocyte-derived dendritic cells distributed in subpopulations of different maturation stages, located in different areas relative to the tumor core. The lethal stage of the disease was characterized by the progressive accumulation of EGFP+/EYFP+ monocyte-derived dendritic cells. This local phenotypic regulation of monocyte subtypes marked a transition in the immune response. PMID:27193333

  5. β-Arrestin 2 mediates the anti-inflammatory effects of fluoxetine in lipopolysaccharide-stimulated microglial cells.

    PubMed

    Du, Ren-Wei; Du, Ren-Hong; Bu, Wen-Guang

    2014-09-01

    Recent evidence has suggested that microglial activation plays an important role in the pathogenesis of depression. Activated microglia can secrete various pro-inflammatory cytokines, which may contribute to the development and maintenance of depression. Thus, inhibition of microglial activation may have a therapeutic benefit in the treatment of depression. In the present study, we found that fluoxetine significantly inhibited lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-α), interleukin- 6 (IL-6) and nitric oxide (NO) and reduced the phosphorylation of transforming growth factor-beta-activated kinase 1 (TAK1) and nuclear factor-kappa B (NF-κB) p65 nuclear translocation in microglia. We further found that fluoxetine increased the expression of β-arrestin 2 and enhanced the association of β-arrestin 2 with TAK1-binding protein 1 (TAB1) and disrupted TAK1-TAB1 interaction. Moreover, β-arrestin 2 knock-down abolished the anti-inflammatory effects of fluoxetine in lipopolysaccharide-stimulated microglial cells. Collectively, our findings suggest that β-arrestin 2 is necessary for the anti-inflammatory effects of fluoxetine and offers novel drug targets in the convergent fluoxetine/β-arrestin 2 and inflammatory pathways for treating microglial inflammatory neuropathologies like depression.

  6. Interaction between Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium subspecies paratuberculosis with the enteric glia and microglial cells

    PubMed Central

    2011-01-01

    Background We investigated the interaction of Mycobacterium avium subspecies paratuberculosis, M. bovis and M. tuberculosis and different glial cells (enteric glial and microglial cells) in order to evaluate the infecting ability of these microorganisms and the effects produced on these cells, such as the evaluation of cytokines expression. Results Our experiments demonstrated the adhesion of M. paratuberculosis to the enteroglial cells and the induction of IL-1A and IL-6 expression; M. tuberculosis and M. bovis showed a good adhesive capability to the enteric cell line with the expression of the following cytokines: IL-1A and IL-1B, TNF-α, G-CSF and GM-CSF; M. bovis induced the expression of IL-6 too. The experiment performed with the microglial cells confirmed the results obtained with the enteroglial cells after the infection with M. tuberculosis and M. bovis, whereas M. paratuberculosis stimulated the production of IL-1A and IL-1B. Conclusion Enteroglial and microglial cells, could be the target of pathogenic mycobacteria and, even if present in different locations (Enteric Nervous System and Central Nervous System), show to have similar mechanism of immunomodulation. PMID:22151930

  7. Dual RNA Sequencing Reveals the Expression of Unique Transcriptomic Signatures in Lipopolysaccharide-Induced BV-2 Microglial Cells

    PubMed Central

    Kim, Sun Hwa; Park, Kyoung Sun; Lee, Young Seek; Jung, Kyoung Hwa; Chai, Young Gyu

    2015-01-01

    Microglial cells become rapidly activated through interactions with pathogens, and the persistent activation of these cells is associated with various neurodegenerative diseases. Previous studies have investigated the transcriptomic signatures in microglia or macrophages using microarray technologies. However, this method has numerous restrictions, such as spatial biases, uneven probe properties, low sensitivity, and dependency on the probes spotted. To overcome this limitation and identify novel transcribed genes in response to LPS, we used RNA Sequencing (RNA-Seq) to determine the novel transcriptomic signatures in BV-2 microglial cells. Sequencing assessment and quality evaluation showed that approximately 263 and 319 genes (≥ 1.5 log2-fold), such as cytokines and chemokines, were strongly induced after 2 and 4 h, respectively, and the induction of several genes with unknown immunological functions was also observed. Importantly, we observed that previously unidentified transcription factors (TFs) (irf1, irf7, and irf9), histone demethylases (kdm4a) and DNA methyltransferases (dnmt3l) were significantly and selectively expressed in BV-2 microglial cells. The gene expression levels, transcription start sites (TSS), isoforms, and differential promoter usage revealed a complex pattern of transcriptional and post-transcriptional gene regulation upon infection with LPS. In addition, gene ontology, molecular networks and pathway analyses identified the top significantly regulated functional classification, canonical pathways and network functions at each activation status. Moreover, we further analyzed differentially expressed genes to identify transcription factor (TF) motifs (−950 to +50 bp of the 5’ upstream promoters) and epigenetic mechanisms. Furthermore, we confirmed that the expressions of key inflammatory genes as well as pro-inflammatory mediators in the supernatants were significantly induced in LPS treated primary microglial cells. This

  8. Brucella outer membrane protein Omp25 induces microglial cells in vitro to secrete inflammatory cytokines and inhibit apoptosis

    PubMed Central

    Ma, Qiao-Li; Liu, Ai-Cui; Ma, Xiao-Juan; Wang, Yan-Bai; Hou, Yu-Ting; Wang, Zhen-Hai

    2015-01-01

    Omp25 protein, an outer membrane protein of Brucella, can cause damage to the central nervous system. As one type of macrophage, microglial cells play a role in immune surveillance and immune protection in the central nervous system; therefore, they are major targets of bacterial attack. The present study examined BV2 mouse microglial cells that were stimulated with different concentrations of Omp25 recombinant protein, and the secretion of inflammatory cytokines by the BV2 cells as well as their level of apoptosis were observed. The objective of the study was to preliminarily illustrate the possible mechanism that Omp25 uses to damage the central nervous system. Mouse BV2 microglial cells were incubated with different concentrations of Omp25 for 24 h, and an enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of the inflammatory cytokines interleukin (IL)-6, tumour necrosis factor (TNF)-α and HMGB1 (high mobility group box-1 protein); reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of TLR4 (Toll-like receptor 4) mRNA; Annexin V-fluorescein isothiocyanate (FITC) double staining was used to detect apoptosis in the BV2 cells. After the BV2 cells were stimulated with different concentrations of Omp25, the levels of IL-6, TNF-α and HMGB1 was increased, and the difference was statistically significant compared with the control group (P<0.05). The secretion of TNF-α and HMGB1 showed a trend toward an initial increase followed by a decrease. The expression level of TLR4 mRNA was increased. Omp25 protein can inhibit apoptosis in BV2 cells. The outer membrane protein Omp25 of Brucella promotes microglial cells to secrete inflammatory cytokines and inhibit apoptosis. TLR4 may be involved in the immune response of the central nervous system to Brucella infection. PMID:26770344

  9. In situ Synthesis of Fluorescent Gold Nanoclusters by Nontumorigenic Microglial Cells.

    PubMed

    West, Abby L; Schaeublin, Nicole M; Griep, Mark H; Maurer-Gardner, Elizabeth I; Cole, Daniel P; Fakner, Alexis M; Hussain, Saber M; Karna, Shashi P

    2016-08-24

    To date, the directed in situ synthesis of fluorescent gold nanoclusters (AuNCs) has only been demonstrated in cancerous cells, with the theorized synthesis mechanism prohibiting AuNC formation in nontumorigenic cell lines. This limitation hinders potential biostabilized AuNC-based technology in healthy cells involving both chemical and mechanical analysis, such as the direct sensing of protein function and the elucidation of local mechanical environments. Thus, new synthesis strategies are required to expand the application space of AuNCs beyond cancer-focused cellular studies. In this contribution, we have developed the methodology and demonstrated the direct in situ synthesis of AuNCs in the nontumorigenic neuronal microglial line, C8B4. The as-synthesized AuNCs form in situ and are stabilized by cellular proteins. The clusters exhibit bright green fluorescence and demonstrate low (<10%) toxicity. Interestingly, elevated ROS levels were not required for the in situ formation of AuNCs, although intracellular reductants such as glutamate were required for the synthesis of AuNCs in C8B4 cells. To our knowledge, this is the first-ever demonstration of AuNC synthesis in nontumorigenic cells and, as such, it considerably expands the application space of biostabilized fluorescent AuNCs. PMID:27328035

  10. In situ Synthesis of Fluorescent Gold Nanoclusters by Nontumorigenic Microglial Cells.

    PubMed

    West, Abby L; Schaeublin, Nicole M; Griep, Mark H; Maurer-Gardner, Elizabeth I; Cole, Daniel P; Fakner, Alexis M; Hussain, Saber M; Karna, Shashi P

    2016-08-24

    To date, the directed in situ synthesis of fluorescent gold nanoclusters (AuNCs) has only been demonstrated in cancerous cells, with the theorized synthesis mechanism prohibiting AuNC formation in nontumorigenic cell lines. This limitation hinders potential biostabilized AuNC-based technology in healthy cells involving both chemical and mechanical analysis, such as the direct sensing of protein function and the elucidation of local mechanical environments. Thus, new synthesis strategies are required to expand the application space of AuNCs beyond cancer-focused cellular studies. In this contribution, we have developed the methodology and demonstrated the direct in situ synthesis of AuNCs in the nontumorigenic neuronal microglial line, C8B4. The as-synthesized AuNCs form in situ and are stabilized by cellular proteins. The clusters exhibit bright green fluorescence and demonstrate low (<10%) toxicity. Interestingly, elevated ROS levels were not required for the in situ formation of AuNCs, although intracellular reductants such as glutamate were required for the synthesis of AuNCs in C8B4 cells. To our knowledge, this is the first-ever demonstration of AuNC synthesis in nontumorigenic cells and, as such, it considerably expands the application space of biostabilized fluorescent AuNCs.

  11. TREM2 regulates microglial cell activation in response to demyelination in vivo

    PubMed Central

    Cantoni, Claudia; Bollman, Bryan; Licastro, Danilo; Xie, Mingqiang; Mikesell, Robert; Schmidt, Robert; Yuede, Carla M.; Galimberti, Daniela; Olivecrona, Gunilla; Klein, Robyn S.; Cross, Anne H.; Otero, Karel; Piccio, Laura

    2015-01-01

    Microglia are phagocytic cells that survey the brain and perform neuroprotective functions in response to tissue damage, but their activating receptors are largely unknown. Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglial immunoreceptor whose loss-of-function mutations in humans cause presenile dementia, while genetic variants are associated with increased risk of neurodegenerative diseases. In myeloid cells, TREM2 has been involved in the regulation of phagocytosis, cell proliferation and inflammatory responses in vitro. However, it is unknown how TREM2 contributes to microglia function in vivo. Here, we identify a critical role for TREM2 in the activation and function of microglia during cuprizone (CPZ)-induced demyelination. TREM2-deficient (TREM2−/−) mice had defective clearance of myelin debris and more axonal pathology, resulting in impaired clinical performances compared to wild-type (WT) mice. TREM2−/− microglia proliferated less in areas of demyelination and were less activated, displaying a more resting morphology and decreased expression of the activation markers MHC II and inducible nitric oxide synthase as compared to WT. Mechanistically, gene expression and ultrastructural analysis of microglia suggested a defect in myelin degradation and phagosome processing during CPZ intoxication in TREM2−/− microglia. These findings place TREM2 as a key regulator of microglia activation in vivo in response to tissue damage. PMID:25631124

  12. Cholinergic modulation of microglial activation by alpha 7 nicotinic receptors.

    PubMed

    Shytle, R Douglas; Mori, Takashi; Townsend, Kirk; Vendrame, Martina; Sun, Nan; Zeng, Jin; Ehrhart, Jared; Silver, Archie A; Sanberg, Paul R; Tan, Jun

    2004-04-01

    Almost all degenerative diseases of the CNS are associated with chronic inflammation. A central step in this process is the activation of brain mononuclear phagocyte cells, called microglia. While it is recognized that healthy neurons and astrocytes regulate the magnitude of microglia-mediated innate immune responses and limit excessive CNS inflammation, the endogenous signals governing this process are not fully understood. In the peripheral nervous system, recent studies suggest that an endogenous 'cholinergic anti-inflammatory pathway' regulates systemic inflammatory responses via alpha 7 nicotinic acetylcholinergic receptors (nAChR) found on blood-borne macrophages. These data led us to investigate whether a similar cholinergic pathway exists in the brain that could regulate microglial activation. Here we report for the first time that cultured microglial cells express alpha 7 nAChR subunit as determined by RT-PCR, western blot, immunofluorescent, and immunohistochemistry analyses. Acetylcholine and nicotine pre-treatment inhibit lipopolysaccharide (LPS)-induced TNF-alpha release in murine-derived microglial cells, an effect attenuated by alpha 7 selective nicotinic antagonist, alpha-bungarotoxin. Furthermore, this inhibition appears to be mediated by a reduction in phosphorylation of p44/42 and p38 mitogen-activated protein kinase (MAPK). Though preliminary, our findings suggest the existence of a brain cholinergic pathway that regulates microglial activation through alpha 7 nicotinic receptors. Negative regulation of microglia activation may also represent additional mechanism underlying nicotine's reported neuroprotective properties.

  13. Microglial derived tumor necrosis factor-α drives Alzheimer's disease-related neuronal cell cycle events.

    PubMed

    Bhaskar, Kiran; Maphis, Nicole; Xu, Guixiang; Varvel, Nicholas H; Kokiko-Cochran, Olga N; Weick, Jason P; Staugaitis, Susan M; Cardona, Astrid; Ransohoff, Richard M; Herrup, Karl; Lamb, Bruce T

    2014-02-01

    Massive neuronal loss is a key pathological hallmark of Alzheimer's disease (AD). However, the mechanisms are still unclear. Here we demonstrate that neuroinflammation, cell autonomous to microglia, is capable of inducing neuronal cell cycle events (CCEs), which are toxic for terminally differentiated neurons. First, oligomeric amyloid-beta peptide (AβO)-mediated microglial activation induced neuronal CCEs via the tumor-necrosis factor-α (TNFα) and the c-Jun Kinase (JNK) signaling pathway. Second, adoptive transfer of CD11b+ microglia from AD transgenic mice (R1.40) induced neuronal cyclin D1 expression via TNFα signaling pathway. Third, genetic deficiency of TNFα in R1.40 mice (R1.40-Tnfα(-/-)) failed to induce neuronal CCEs. Finally, the mitotically active neurons spatially co-exist with F4/80+ activated microglia in the human AD brain and that a portion of these neurons are apoptotic. Together our data suggest a cell-autonomous role of microglia, and identify TNFα as the responsible cytokine, in promoting neuronal CCEs in the pathogenesis of AD.

  14. Intracellular delivery of dendrimer triamcinolone acetonide conjugates into microglial and human retinal pigment epithelial cells

    PubMed Central

    Kambhampati, Siva P.; Mishra, Manoj K.; Mastorakos, Panagiotis; Oh, Yumin; Lutty, Gerard A.; Kannan, Rangaramanujam M.

    2016-01-01

    Triamcinolone acetonide (TA) is a potent, intermediate-acting, steroid that has anti-inflammatory and anti-angiogenic activity. Intravitreal administration of TA has been used for diabetic macular edema, proliferative diabetic retinopathy and exudative age-related macular degeneration (AMD). However, the hydrophobicity, lack of solubility, and the side effects limit its effectiveness in the treatment of retinal diseases. In this study, we explore a PAMAM dendrimer-TA conjugate (D-TA) as a potential strategy to improve intracellular delivery and efficacy of TA to target cells. The conjugates were prepared with a high drug payload (~21%) and were readily soluble in saline. Compared to free TA, D-TA demonstrated a significantly improved toxicity profile in two important target [microglial and human retinal pigment epithelium (RPE)] cells. The D-TA was ~100-fold more effective than free TA in its anti-inflammatory activity (measured in microglia), and in suppressing VEGF production (in hypoxic RPE cells). Dendrimer-based delivery may improve the efficacy of TA towards both its key targets of inflammation and VEGF production, with significant clinical implications. PMID:25701805

  15. Maternal immune activation evoked by polyinosinic:polycytidylic acid does not evoke microglial cell activation in the embryo

    PubMed Central

    Smolders, Silke; Smolders, Sophie M. T.; Swinnen, Nina; Gärtner, Annette; Rigo, Jean-Michel; Legendre, Pascal; Brône, Bert

    2015-01-01

    Several studies have indicated that inflammation during pregnancy increases the risk for the development of neuropsychiatric disorders in the offspring. Morphological brain abnormalities combined with deviations in the inflammatory status of the brain can be observed in patients of both autism and schizophrenia. It was shown that acute infection can induce changes in maternal cytokine levels which in turn are suggested to affect fetal brain development and increase the risk on the development of neuropsychiatric disorders in the offspring. Animal models of maternal immune activation reproduce the etiology of neurodevelopmental disorders such as schizophrenia and autism. In this study the poly (I:C) model was used to mimic viral immune activation in pregnant mice in order to assess the activation status of fetal microglia in these developmental disorders. Because microglia are the resident immune cells of the brain they were expected to be activated due to the inflammatory stimulus. Microglial cell density and activation level in the fetal cortex and hippocampus were determined. Despite the presence of a systemic inflammation in the pregnant mice, there was no significant difference in fetal microglial cell density or immunohistochemically determined activation level between the control and inflammation group. These data indicate that activation of the fetal microglial cells is not likely to be responsible for the inflammation induced deficits in the offspring in this model. PMID:26300736

  16. NG2 expression in microglial cells affects the expression of neurotrophic and proinflammatory factors by regulating FAK phosphorylation

    PubMed Central

    Zhu, Lie; Su, Qing; Jie, Xiang; Liu, Antang; Wang, Hui; He, Beiping; Jiang, Hua

    2016-01-01

    Neural/glial antigen 2 (NG2), a chondroitin sulfate proteoglycan, is significantly upregulated in a subset of glial cells in the facial motor nucleus (FMN) following CNS injury. NG2 is reported to promote the resulting inflammatory reaction, however, the mechanism by which NG2 mediates these effects is yet to be determined. In this study, we examined the changes in NG2 expressing microglial cells in the FMN in response to facial nerve axotomy (FNA) in mice. Our findings indicated that NG2 expression was progressively induced and upregulated specifically in the ipsilateral facial nucleus following FNA. To further investigate the effects of NG2 expression, in vivo studies in NG2-knockout mice and in vitro studies in rat microglial cells transfected with NG2 shRNAs were performed. Abolition of NG2 expression both in vitro and in vivo resulted in increased expression of neurotrophic factors (nerve growth factor and glial derived neurotrophic factor), decreased expression of inflammatory mediators (tumor necrosis factor-α and interleukin-1β) and decreased apoptosis in the ipsilateral facial nucleus in response to FNA. Furthermore, we demonstrated the role of FAK in these NG2-induced effects. Taken together, our findings suggest that NG2 expression mediates inflammatory reactions and neurodegeneration in microglial cells in response to CNS injury, potentially by regulating FAK phosphorylation. PMID:27306838

  17. Differential Effects of Stress on Microglial Cell Activation in Male and Female Medial Prefrontal Cortex

    PubMed Central

    Bollinger, Justin L.; Bergeon Burns, Christine M.; Wellman, Cara L.

    2016-01-01

    Susceptibility to stress-linked psychological disorders, including post-traumatic stress disorder and depression, differs between men and women. Dysfunction of medial prefrontal cortex (mPFC) has been implicated in many of these disorders. Chronic stress affects mPFC in a sex-dependent manner, differentially remodeling dendritic morphology and disrupting prefrontally mediated behaviors in males and females. Chronic restraint stress induces microglial activation, reflected in altered microglial morphology and immune factor expression, in mPFC in male rats. Unstressed females exhibit increased microglial ramification in several brain regions compared to males, suggesting both heightened basal activation and a potential for sex-dependent effects of stress on microglial activation. Therefore, we assessed microglial density and ramification in the prelimbic region of mPFC, and immune-associated genes in dorsal mPFC in male and female rats following acute or chronic restraint stress. Control rats were left unstressed. On the final day of restraint, brains were collected for either qPCR or visualization of microglia using Iba-1 immunohistochemistry. Microglia in mPFC were classified as ramified, primed, reactive, or amoeboid, and counted stereologically. Expression of microglia-associated genes (MHCII, CD40, IL6, CX3CL1, and CX3CR1) was also assessed using qPCR. Unstressed females showed a greater proportion of primed to ramified microglia relative to males, alongside heightened CX3CL1-CX3CR1 expression. Acute and chronic restraint stress reduced the proportion of primed to ramified microglia and microglial CD40 expression in females, but did not significantly alter microglial activation in males. This sex difference in microglial activation could contribute to the differential effects of stress on mPFC structure and function in males versus females. PMID:26441134

  18. Vaccinium bracteatum Thunb. Exerts Anti-Inflammatory Activity by Inhibiting NF-κB Activation in BV-2 Microglial Cells

    PubMed Central

    Kwon, Seung-Hwan; Ma, Shi-Xun; Ko, Yong-Hyun; Seo, Jee-Yeon; Lee, Bo-Ram; Lee, Taek Hwan; Kim, Sun Yeou; Lee, Seok-Yong; Jang, Choon-Gon

    2016-01-01

    This study was designed to evaluate the pharmacological effects of Vaccinium bracteatum Thunb. methanol extract (VBME) on microglial activation and to identify the underlying mechanisms of action of these effects. The anti-inflammatory properties of VBME were studied using lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. We measured the production of nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase (COX)-2, prostaglandin E2 (PGE2), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) as inflammatory parameters. We also examined the effect of VBME on intracellular reactive oxygen species (ROS) production and the activity of nuclear factor-kappa B p65 (NF-κB p65). VBME significantly inhibited LPS-induced production of NO and PGE2 and LPS-mediated upregulation of iNOS and COX-2 expression in a dose-dependent manner; importantly, VBME was not cytotoxic. VBME also significantly reduced the generation of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6. In addition, VBME significantly dampened intracellular ROS production and suppressed NF-κB p65 translocation by blocking IκB-α phosphorylation and degradation in LPS-stimulated BV2 cells. Our findings indicate that VBME inhibits the production of inflammatory mediators in BV-2 microglial cells by suppressing NF-κB signaling. Thus, VBME may be useful in the treatment of neurodegenerative diseases due to its ability to inhibit inflammatory mediator production in activated BV-2 microglial cells. PMID:27169820

  19. Neuroprotective and anti-inflammatory effects of flavonoids isolated from Rhus verniciflua in neuronal HT22 and microglial BV2 cell lines.

    PubMed

    Cho, Namki; Choi, Ji Hoon; Yang, Heejung; Jeong, Eun Ju; Lee, Ki Yong; Kim, Young Choong; Sung, Sang Hyun

    2012-06-01

    The neuroprotective and anti-inflammatory activities of the methanolic extract of Rhus verniciflua Stokes (Anacardiaceae) were investigated with mouse hippocampal and microglial cells. Bioactivity-guided isolation yielded 10 flavonoids including fustin (1), fisetin (2), sulfuretin (3), butein (4), butin (5), eriodictyol (6), morin hydrate (7), quercetin (8), kaempferol (9) and isoliquiritigenin (10). Among the isolated flavonoids, compounds 2-5 significantly protected the murine hippocampal HT22 cells against glutamate-induced neurotoxicity and attenuated reactive oxygen species (ROS) generations. In addition, these flavonoids significantly maintained antioxidative defense systems preserving the activities of superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GSH-Px) and the content of glutathione (GSH) decreased by glutamate insult. These compounds also showed significant inhibitory effects on LPS-induced nitric oxide (NO) production in BV2 cells. Especially, compound 4 dose-dependently suppressed the expression of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). These results suggest that these flavonoids possess therapeutic potentials as a multipotent agent against neurodegenerative diseases related to oxidative stress and pathological inflammatory responses.

  20. Immunodetection of Murine Lymphotoxins in Eukaryotic Cells.

    PubMed

    Boitchenko, Veronika E.; Korobko, Vyacheslav G.; Prassolov, Vladimir S.; Kravchenko, Vladimir V.; Kuimov, Alexander N.; Turetskaya, Regina L.; Kuprash, Dmitry V.; Nedospasov, Sergei A.

    2000-10-01

    Lymphotoxins alpha and beta (LTalpha and LTbeta) are members of tumor necrosis factor superfamily. LT heterotrimers exist on the surface of lymphocytes and signal through LTbeta receptor while soluble LTalpha homotrimer can signal through TNF receptors p55 and p75. LT-, as well as TNF-mediated signaling are important for the organogenesis and maintenance of microarchitecture of secondary lymphoid organs in mice and has been implicated in the mechanism of certain inflammatory syndromes in humans. In this study we describe the generation of eukaryotic expression plasmids encoding murine LTalpha and LTbeta genes and a prokaryotic expression construct for murine LTalpha. Using recombinant proteins expressed by these vectors as tools for antisera selection, we produced and characterized several polyclonal antibodies capable of detecting LT proteins in eukaryotic cells.

  1. Infiltrating cells from host brain restore the microglial population in grafted cortical tissue

    PubMed Central

    Wang, Cong; Tao, Sijue; Fang, Yukun; Guo, Jing; Zhu, Lirui; Zhang, Shengxiang

    2016-01-01

    Transplantation of embryonic cortical tissue is considered as a promising therapy for brain injury. Grafted neurons can reestablish neuronal network and improve cortical function of the host brain. Microglia is a key player in regulating neuronal survival and plasticity, but its activation and dynamics in grafted cortical tissue remain unknown. Using two-photon intravital imaging and parabiotic model, here we investigated the proliferation and source of microglia in the donor region by transplanting embryonic cortical tissue into adult cortex. Live imaging showed that the endogenous microglia of the grafted tissue were rapidly lost after transplantation. Instead, host-derived microglia infiltrated and colonized the graft. Parabiotic model suggested that the main source of infiltrating cells is the parenchyma of the host brain. Colonized microglia proliferated and experienced an extensive morphological transition and eventually differentiated into resting ramified morphology. Collectively, these results demonstrated that donor tissue has little contribution to the activated microglia and host brain controls the microglial population in the graft. PMID:27615195

  2. Infiltrating cells from host brain restore the microglial population in grafted cortical tissue.

    PubMed

    Wang, Cong; Tao, Sijue; Fang, Yukun; Guo, Jing; Zhu, Lirui; Zhang, Shengxiang

    2016-01-01

    Transplantation of embryonic cortical tissue is considered as a promising therapy for brain injury. Grafted neurons can reestablish neuronal network and improve cortical function of the host brain. Microglia is a key player in regulating neuronal survival and plasticity, but its activation and dynamics in grafted cortical tissue remain unknown. Using two-photon intravital imaging and parabiotic model, here we investigated the proliferation and source of microglia in the donor region by transplanting embryonic cortical tissue into adult cortex. Live imaging showed that the endogenous microglia of the grafted tissue were rapidly lost after transplantation. Instead, host-derived microglia infiltrated and colonized the graft. Parabiotic model suggested that the main source of infiltrating cells is the parenchyma of the host brain. Colonized microglia proliferated and experienced an extensive morphological transition and eventually differentiated into resting ramified morphology. Collectively, these results demonstrated that donor tissue has little contribution to the activated microglia and host brain controls the microglial population in the graft. PMID:27615195

  3. Cannabinoids and neuronal damage: differential effects of THC, AEA and 2-AG on activated microglial cells and degenerating neurons in excitotoxically lesioned rat organotypic hippocampal slice cultures.

    PubMed

    Kreutz, Susanne; Koch, Marco; Ghadban, Chalid; Korf, Horst-Werner; Dehghani, Faramarz

    2007-01-01

    Cannabinoids (CBs) are attributed neuroprotective effects in vivo. Here, we determined the neuroprotective potential of CBs during neuronal damage in excitotoxically lesioned organotypic hippocampal slice cultures (OHSCs). OHSCs are the best characterized in vitro model to investigate the function of microglial cells in neuronal damage since blood-borne monocytes and T-lymphocytes are absent and microglial cells represent the only immunocompetent cell type. Excitotoxic neuronal damage was induced by NMDA (50 microM) application for 4 h. Neuroprotective properties of 9-carboxy-11-nor-delta-9-tetrahydrocannabinol (THC), N-arachidonoylethanolamide (AEA) or 2-arachidonoylglycerol (2-AG) in different concentrations were determined after co-application with NMDA by counting degenerating neurons identified by propidium iodide labeling (PI(+)) and microglial cells labeled by isolectin B(4) (IB(4)(+)). All three CBs used significantly decreased the number of IB(4)(+) microglial cells in the dentate gyrus but the number of PI(+) neurons was reduced only after 2-AG treatment. Application of AM630, antagonizing CB2 receptors highly expressed by activated microglial cells, did not counteract neuroprotective effects of 2-AG, but affected THC-mediated reduction of IB(4)(+) microglial cells. Our results indicate that (1) only 2-AG exerts neuroprotective effects in OHSCs; (2) reduction of IB(4)(+) microglial cells is not a neuroprotective event per se and involves other CB receptors than the CB2 receptor; (3) the discrepancy in the neuroprotective effects of CBs observed in vivo and in our in vitro model system may underline the functional relevance of invading monocytes and T-lymphocytes that are absent in OHSCs. PMID:17010339

  4. Serotonin 5-HT7 receptors coupled to induction of interleukin-6 in human microglial MC-3 cells.

    PubMed

    Mahé, Cécile; Loetscher, Erika; Dev, Kumlesh K; Bobirnac, Ionel; Otten, Uwe; Schoeffter, Philippe

    2005-07-01

    Brain serotonin 5-HT(7) receptors are known to be expressed in neurons and astrocytes. We now report the presence of these receptors in a third type of cell, microglial cells. 5-Hydroxytryptamine (5-HT), 5-carboxamidotryptamine (5-CT), 5-methoxytryptamine (5-MeOT) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) induced concentration-dependent stimulations of cAMP accumulation in the human microglial MC-3 cell line. The maximal effect of 5-HT was 3.4+/-0.3-fold stimulation (mean+/-S.E.M., n=5) above basal levels. The rank order of agonist potency (pEC50 values) was 5-CT (7.09)>5-HT (6.13)>or=5-MeOT (5.78)>8-OH-DPAT (ca. 5). The effect of 5-CT was inhibited in a concentration-dependent manner by the selective 5-HT7 receptor antagonist SB-269970 (pA2 value 9.03). Western blot analysis revealed the presence of immunoreactive bands corresponding to the human 5-HT7 receptor in extracts of MC-3 cells. The presence of two splice variants of the 5-HT7 receptor (5-HT7(a/b)) was visualized by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis with specific primers. In real-time PCR studies, the mRNA for interleukin-6 (IL-6) was found to be increased by 2.5-fold in MC-3 cells after 1 h incubation with 5-CT (1 microM) and this effect was fully blocked by the 5-HT7 receptor antagonist SB-269970 (1 microM). These data show that functional 5-HT7 receptors are present in human microglial MC-3 cells, suggesting that they are involved in neuroinflammatory processes. PMID:15992579

  5. Single episode of mild murine malaria induces neuroinflammation, alters microglial profile, impairs adult neurogenesis, and causes deficits in social and anxiety-like behavior.

    PubMed

    Guha, Suman K; Tillu, Rucha; Sood, Ankit; Patgaonkar, Mandar; Nanavaty, Ishira N; Sengupta, Arjun; Sharma, Shobhona; Vaidya, Vidita A; Pathak, Sulabha

    2014-11-01

    Cerebral malaria is associated with cerebrovascular damage and neurological sequelae. However, the neurological consequences of uncomplicated malaria, the most prevalent form of the disease, remain uninvestigated. Here, using a mild malaria model, we show that a single Plasmodium chabaudi adami infection in adult mice induces neuroinflammation, neurogenic, and behavioral changes in the absence of a blood-brain barrier breach. Using cytokine arrays we show that the infection induces differential serum and brain cytokine profiles, both at peak parasitemia and 15days post-parasite clearance. At the peak of infection, along with the serum, the brain also exhibited a definitive pro-inflammatory cytokine profile, and gene expression analysis revealed that pro-inflammatory cytokines were also produced locally in the hippocampus, an adult neurogenic niche. Hippocampal microglia numbers were enhanced, and we noted a shift to an activated profile at this time point, accompanied by a striking redistribution of the microglia to the subgranular zone adjacent to hippocampal neuronal progenitors. In the hippocampus, a distinct decline in progenitor turnover and survival was observed at peak parasitemia, accompanied by a shift from neuronal to glial fate specification. Studies in transgenic Nestin-GFP reporter mice demonstrated a decline in the Nestin-GFP(+)/GFAP(+) quiescent neural stem cell pool at peak parasitemia. Although these cellular changes reverted to normal 15days post-parasite clearance, specific brain cytokines continued to exhibit dysregulation. Behavioral analysis revealed selective deficits in social and anxiety-like behaviors, with no change observed in locomotor, cognitive, and depression-like behaviors, with a return to baseline at recovery. Collectively, these findings indicate that even a single episode of mild malaria results in alterations of the brain cytokine profile, causes specific behavioral dysfunction, is accompanied by hippocampal microglial

  6. Olfactory Sensory Activity Modulates Microglial-Neuronal Interactions during Dopaminergic Cell Loss in the Olfactory Bulb

    PubMed Central

    Grier, Bryce D.; Belluscio, Leonardo; Cheetham, Claire E. J.

    2016-01-01

    The mammalian olfactory bulb (OB) displays robust activity-dependent plasticity throughout life. Dopaminergic (DA) neurons in the glomerular layer (GL) of the OB are particularly plastic, with loss of sensory input rapidly reducing tyrosine hydroxylase (TH) expression and dopamine production, followed by a substantial reduction in DA neuron number. Here, we asked whether microglia participate in activity-dependent elimination of DA neurons in the mouse OB. Interestingly, we found a significant reduction in the number of both DA neurons and their synapses in the OB ipsilateral to the occluded naris (occluded OB) within just 7 days of sensory deprivation. Concomitantly, the volume of the occluded OB decreased, resulting in an increase in microglial density. Microglia in the occluded OB also adopted morphologies consistent with activation. Using in vivo 2-photon imaging and histological analysis we then showed that loss of olfactory input markedly altered microglial-neuronal interactions during the time that DA neurons are being eliminated: both microglial process motility and the frequency of wrapping of DA neuron somata by activated microglia increased significantly in the occluded OB. Furthermore, we found microglia in the occluded OB that had completely engulfed components of DA neurons. Together, our data provide evidence that loss of olfactory input modulates microglial-DA neuron interactions in the OB, thereby suggesting an important role for microglia in the activity-dependent elimination of DA neurons and their synapses. PMID:27471450

  7. Anti-neuroinflammatory effect of a novel caffeamide derivative, KS370G, in microglial cells.

    PubMed

    Lu, Dah-Yuu; Huang, Bor-Ren; Yeh, Wei-Lan; Lin, Hsiao-Yun; Huang, Shiang-Suo; Liu, Yu-Shu; Kuo, Yueh-Hsiung

    2013-12-01

    Accumulating evidence suggests that inflammatory processes in the central nervous system that are mediated by microglial activation play important roles in several neurodegenerative disorders. Therefore, development of methods for microglial inhibition is considered an important strategy in the search for neuroprotective agents. Caffeic acid phenethyl ester (CAPE) is distributed wildly in nature, but rapid decomposition by esterase leads to its low bioavailability. In this study, we investigated the effects of KS370G, a novel caffeic acid phenylethyl amide, on microglial activation. KS370G significantly inhibited the release of nitric oxide (NO) and the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Treatment with KS370G also induced heme oxygenase (HO)-1 and suppressors of cytokine signaling (SOCS)-3 expression in the microglia. Furthermore, the anti-inflammatory effects of KS370G were found to be regulated by phosphorylated adenosine monophosphate-activated protein kinase-α (AMPK-α) translocated to the nucleus. Moreover, KS370G showed significant anti-neuroinflammatory effects on microglial activation in vivo and on motor behavior as well. The protective effect of KS370G was weakened by an AMPK inhibitor Compound C. This study focuses on the importance of key molecular determinants of inflammatory homeostasis, AMPK, HO-1, and SOCS-3, and their possible involvement in anti-neuroinflammatory responses.

  8. Olfactory Sensory Activity Modulates Microglial-Neuronal Interactions during Dopaminergic Cell Loss in the Olfactory Bulb.

    PubMed

    Grier, Bryce D; Belluscio, Leonardo; Cheetham, Claire E J

    2016-01-01

    The mammalian olfactory bulb (OB) displays robust activity-dependent plasticity throughout life. Dopaminergic (DA) neurons in the glomerular layer (GL) of the OB are particularly plastic, with loss of sensory input rapidly reducing tyrosine hydroxylase (TH) expression and dopamine production, followed by a substantial reduction in DA neuron number. Here, we asked whether microglia participate in activity-dependent elimination of DA neurons in the mouse OB. Interestingly, we found a significant reduction in the number of both DA neurons and their synapses in the OB ipsilateral to the occluded naris (occluded OB) within just 7 days of sensory deprivation. Concomitantly, the volume of the occluded OB decreased, resulting in an increase in microglial density. Microglia in the occluded OB also adopted morphologies consistent with activation. Using in vivo 2-photon imaging and histological analysis we then showed that loss of olfactory input markedly altered microglial-neuronal interactions during the time that DA neurons are being eliminated: both microglial process motility and the frequency of wrapping of DA neuron somata by activated microglia increased significantly in the occluded OB. Furthermore, we found microglia in the occluded OB that had completely engulfed components of DA neurons. Together, our data provide evidence that loss of olfactory input modulates microglial-DA neuron interactions in the OB, thereby suggesting an important role for microglia in the activity-dependent elimination of DA neurons and their synapses. PMID:27471450

  9. Proteomic analysis of the effects of aged garlic extract and its FruArg component on lipopolysaccharide-induced neuroinflammatory response in microglial cells.

    PubMed

    Zhou, Hui; Qu, Zhe; Mossine, Valeri V; Nknolise, Dineo L; Li, Jilong; Chen, Zhenzhou; Cheng, Jianlin; Greenlief, C Michael; Mawhinney, Thomas P; Brown, Paula N; Fritsche, Kevin L; Hannink, Mark; Lubahn, Dennis B; Sun, Grace Y; Gu, Zezong

    2014-01-01

    Aged garlic extract (AGE) is widely used as a dietary supplement, and is claimed to promote human health through anti-oxidant/anti-inflammatory activities with hypolipidemic, antiplatelet and neuroprotective effects. Prior studies of AGE have mainly focused on its organosulfur compounds, with little attention paid to its carbohydrate derivatives, such as N-α-(1-deoxy-D-fructos-1-yl)-L-arginine (FruArg). The goal of this study is to investigate actions of AGE and FruArg on antioxidative and neuroinflammatory responses in lipopolysaccharide (LPS)-activated murine BV-2 microglial cells using a proteomic approach. Our data show that both AGE and FruArg can significantly inhibit LPS-induced nitric oxide (NO) production in BV-2 cells. Quantitative proteomic analysis by combining two dimensional differential in-gel electrophoresis (2D-DIGE) with mass spectrometry revealed that expressions of 26 proteins were significantly altered upon LPS exposure, while levels of 20 and 21 proteins exhibited significant changes in response to AGE and FruArg treatments, respectively, in LPS-stimulated BV-2 cells. Notably, approximate 78% of the proteins responding to AGE and FruArg treatments are in common, suggesting that FruArg is a major active component of AGE. MULTICOM-PDCN and Ingenuity Pathway Analyses indicate that the proteins differentially affected by treatment with AGE and FruArg are involved in inflammatory responses and the Nrf2-mediated oxidative stress response. Collectively, these results suggest that AGE and FruArg attenuate neuroinflammatory responses and promote resilience in LPS-activated BV-2 cells by suppressing NO production and by regulating expression of multiple protein targets associated with oxidative stress.

  10. Proteomic analysis of the effects of aged garlic extract and its FruArg component on lipopolysaccharide-induced neuroinflammatory response in microglial cells.

    PubMed

    Zhou, Hui; Qu, Zhe; Mossine, Valeri V; Nknolise, Dineo L; Li, Jilong; Chen, Zhenzhou; Cheng, Jianlin; Greenlief, C Michael; Mawhinney, Thomas P; Brown, Paula N; Fritsche, Kevin L; Hannink, Mark; Lubahn, Dennis B; Sun, Grace Y; Gu, Zezong

    2014-01-01

    Aged garlic extract (AGE) is widely used as a dietary supplement, and is claimed to promote human health through anti-oxidant/anti-inflammatory activities with hypolipidemic, antiplatelet and neuroprotective effects. Prior studies of AGE have mainly focused on its organosulfur compounds, with little attention paid to its carbohydrate derivatives, such as N-α-(1-deoxy-D-fructos-1-yl)-L-arginine (FruArg). The goal of this study is to investigate actions of AGE and FruArg on antioxidative and neuroinflammatory responses in lipopolysaccharide (LPS)-activated murine BV-2 microglial cells using a proteomic approach. Our data show that both AGE and FruArg can significantly inhibit LPS-induced nitric oxide (NO) production in BV-2 cells. Quantitative proteomic analysis by combining two dimensional differential in-gel electrophoresis (2D-DIGE) with mass spectrometry revealed that expressions of 26 proteins were significantly altered upon LPS exposure, while levels of 20 and 21 proteins exhibited significant changes in response to AGE and FruArg treatments, respectively, in LPS-stimulated BV-2 cells. Notably, approximate 78% of the proteins responding to AGE and FruArg treatments are in common, suggesting that FruArg is a major active component of AGE. MULTICOM-PDCN and Ingenuity Pathway Analyses indicate that the proteins differentially affected by treatment with AGE and FruArg are involved in inflammatory responses and the Nrf2-mediated oxidative stress response. Collectively, these results suggest that AGE and FruArg attenuate neuroinflammatory responses and promote resilience in LPS-activated BV-2 cells by suppressing NO production and by regulating expression of multiple protein targets associated with oxidative stress. PMID:25420111

  11. Proteomic Analysis of the Effects of Aged Garlic Extract and Its FruArg Component on Lipopolysaccharide-Induced Neuroinflammatory Response in Microglial Cells

    PubMed Central

    Mossine, Valeri V.; Nknolise, Dineo L.; Li, Jilong; Chen, Zhenzhou; Cheng, Jianlin; Greenlief, C. Michael; Mawhinney, Thomas P.; Brown, Paula N.; Fritsche, Kevin L.; Hannink, Mark; Lubahn, Dennis B.; Sun, Grace Y.; Gu, Zezong

    2014-01-01

    Aged garlic extract (AGE) is widely used as a dietary supplement, and is claimed to promote human health through anti-oxidant/anti-inflammatory activities with hypolipidemic, antiplatelet and neuroprotective effects. Prior studies of AGE have mainly focused on its organosulfur compounds, with little attention paid to its carbohydrate derivatives, such as N-α-(1-deoxy-D-fructos-1-yl)-L-arginine (FruArg). The goal of this study is to investigate actions of AGE and FruArg on antioxidative and neuroinflammatory responses in lipopolysaccharide (LPS)-activated murine BV-2 microglial cells using a proteomic approach. Our data show that both AGE and FruArg can significantly inhibit LPS-induced nitric oxide (NO) production in BV-2 cells. Quantitative proteomic analysis by combining two dimensional differential in-gel electrophoresis (2D-DIGE) with mass spectrometry revealed that expressions of 26 proteins were significantly altered upon LPS exposure, while levels of 20 and 21 proteins exhibited significant changes in response to AGE and FruArg treatments, respectively, in LPS-stimulated BV-2 cells. Notably, approximate 78% of the proteins responding to AGE and FruArg treatments are in common, suggesting that FruArg is a major active component of AGE. MULTICOM-PDCN and Ingenuity Pathway Analyses indicate that the proteins differentially affected by treatment with AGE and FruArg are involved in inflammatory responses and the Nrf2-mediated oxidative stress response. Collectively, these results suggest that AGE and FruArg attenuate neuroinflammatory responses and promote resilience in LPS-activated BV-2 cells by suppressing NO production and by regulating expression of multiple protein targets associated with oxidative stress. PMID:25420111

  12. Isolation of Murine Embryonic Hemogenic Endothelial Cells

    PubMed Central

    Marcelo, Kathrina L.; Hirschi, Karen K.

    2016-01-01

    The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level. PMID:27341393

  13. A label-free impedance-based whole cell assay revealed a new G protein-coupled receptor ligand for mouse microglial cell migration.

    PubMed

    Fukano, Yasufumi; Okino, Nozomu; Furuya, Shigeki; Ito, Makoto

    2016-09-16

    We report the usefulness of an impedance-based label-free whole cell assay to identify new ligands for G protein-coupled receptors (GPCRs) involved in microglial cell migration. Authentic GPCR ligands were subjected to the impedance-based cell assay in order to examine the responses of ligands for MG5 mouse microglial cells. Complement component 5 (C5a), adenosine 5'-diphosphate (ADP), uridine 5'-triphosphate (UTP), lysophosphatidic acid (LPA), and lysophosphatidylserine (LysoPS) were found to elicit different cellular impedance patterns, i.e. C5a, ADP, and UTP caused a transient increase in cellular impedance, while LPA and LysoPS decreased it. The responses for C5a and ADP were abolished by pertussis toxin (PTX), but not rho-associated protein kinase inhibitor, Y-27632, indicating that C5a and ADP elicited responses through the Gαi pathway. On the other hand, the response for UTP, LPA or LysoPS was not cancelled by PTX or Y-27632. In a modified Boyden chamber assay, C5a and ADP, but not UTP, LPA, or LysoPS, induced the migration of MG5 cells. These results suggest that PTX-sensitive increase in cellular impedance with the assay is characteristic for ligands of GPCRs involved in microglial cell migration. We found using this assay that 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) is a new chemoattractant inducing microglial cell migration through the activation of Gαi. PMID:27480930

  14. Curcumin Ameliorates the Reduction Effect of PGE2 on Fibrillar β-Amyloid Peptide (1-42)-Induced Microglial Phagocytosis through the Inhibition of EP2-PKA Signaling in N9 Microglial Cells

    PubMed Central

    Yang, Ju; Shen, Ting-ting; Chen, Yi; Yang, Xue-Sen

    2016-01-01

    Inflammatory activation of microglia and β amyloid (Aβ) deposition are considered to work both independently and synergistically to contribute to the increased risk of Alzheimer’s disease (AD). Recent studies indicate that long-term use of phenolic compounds provides protection against AD, primarily due to their anti-inflammatory actions. We previously suggested that phenolic compound curcumin ameliorated phagocytosis possibly through its anti-inflammatory effects rather than direct regulation of phagocytic function in electromagnetic field-exposed N9 microglial cells (N9 cells). Here, we explored the prostaglandin-E2 (PGE2)-related signaling pathway that involved in curcumin-mediated phagocytosis in fibrillar β-amyloid peptide (1–42) (fAβ42)-stimulated N9 cells. Treatment with fAβ42 increased phagocytosis of fluorescent-labeled latex beads in N9 cells. This increase was attenuated in a dose-dependent manner by endogenous and exogenous PGE2, as well as a selective EP2 or protein kinase A (PKA) agonist, but not by an EP4 agonist. We also found that an antagonist of EP2, but not EP4, abolished the reduction effect of PGE2 on fAβ42-induced microglial phagocytosis. Additionally, the increased expression of endogenous PGE2, EP2, and cyclic adenosine monophosphate (AMP), and activation of vasodilator-stimulated phosphoprotein, cyclic AMP responsive element-binding protein, and PKA were depressed by curcumin administration. This reduction led to the amelioration of the phagocytic abilities of PGE2-stimulated N9 cells. Taken together, these data suggested that curcumin restored the attenuating effect of PGE2 on fAβ42-induced microglial phagocytosis via a signaling mechanism involving EP2 and PKA. Moreover, due to its immune modulatory effects, curcumin may be a promising pharmacological candidate for neurodegenerative diseases. PMID:26824354

  15. Curcumin Ameliorates the Reduction Effect of PGE2 on Fibrillar β-Amyloid Peptide (1-42)-Induced Microglial Phagocytosis through the Inhibition of EP2-PKA Signaling in N9 Microglial Cells.

    PubMed

    He, Gen-Lin; Luo, Zhen; Yang, Ju; Shen, Ting-Ting; Chen, Yi; Yang, Xue-Sen

    2016-01-01

    Inflammatory activation of microglia and β amyloid (Aβ) deposition are considered to work both independently and synergistically to contribute to the increased risk of Alzheimer's disease (AD). Recent studies indicate that long-term use of phenolic compounds provides protection against AD, primarily due to their anti-inflammatory actions. We previously suggested that phenolic compound curcumin ameliorated phagocytosis possibly through its anti-inflammatory effects rather than direct regulation of phagocytic function in electromagnetic field-exposed N9 microglial cells (N9 cells). Here, we explored the prostaglandin-E2 (PGE2)-related signaling pathway that involved in curcumin-mediated phagocytosis in fibrillar β-amyloid peptide (1-42) (fAβ42)-stimulated N9 cells. Treatment with fAβ42 increased phagocytosis of fluorescent-labeled latex beads in N9 cells. This increase was attenuated in a dose-dependent manner by endogenous and exogenous PGE2, as well as a selective EP2 or protein kinase A (PKA) agonist, but not by an EP4 agonist. We also found that an antagonist of EP2, but not EP4, abolished the reduction effect of PGE2 on fAβ42-induced microglial phagocytosis. Additionally, the increased expression of endogenous PGE2, EP2, and cyclic adenosine monophosphate (AMP), and activation of vasodilator-stimulated phosphoprotein, cyclic AMP responsive element-binding protein, and PKA were depressed by curcumin administration. This reduction led to the amelioration of the phagocytic abilities of PGE2-stimulated N9 cells. Taken together, these data suggested that curcumin restored the attenuating effect of PGE2 on fAβ42-induced microglial phagocytosis via a signaling mechanism involving EP2 and PKA. Moreover, due to its immune modulatory effects, curcumin may be a promising pharmacological candidate for neurodegenerative diseases.

  16. Curcumin Ameliorates the Reduction Effect of PGE2 on Fibrillar β-Amyloid Peptide (1-42)-Induced Microglial Phagocytosis through the Inhibition of EP2-PKA Signaling in N9 Microglial Cells.

    PubMed

    He, Gen-Lin; Luo, Zhen; Yang, Ju; Shen, Ting-Ting; Chen, Yi; Yang, Xue-Sen

    2016-01-01

    Inflammatory activation of microglia and β amyloid (Aβ) deposition are considered to work both independently and synergistically to contribute to the increased risk of Alzheimer's disease (AD). Recent studies indicate that long-term use of phenolic compounds provides protection against AD, primarily due to their anti-inflammatory actions. We previously suggested that phenolic compound curcumin ameliorated phagocytosis possibly through its anti-inflammatory effects rather than direct regulation of phagocytic function in electromagnetic field-exposed N9 microglial cells (N9 cells). Here, we explored the prostaglandin-E2 (PGE2)-related signaling pathway that involved in curcumin-mediated phagocytosis in fibrillar β-amyloid peptide (1-42) (fAβ42)-stimulated N9 cells. Treatment with fAβ42 increased phagocytosis of fluorescent-labeled latex beads in N9 cells. This increase was attenuated in a dose-dependent manner by endogenous and exogenous PGE2, as well as a selective EP2 or protein kinase A (PKA) agonist, but not by an EP4 agonist. We also found that an antagonist of EP2, but not EP4, abolished the reduction effect of PGE2 on fAβ42-induced microglial phagocytosis. Additionally, the increased expression of endogenous PGE2, EP2, and cyclic adenosine monophosphate (AMP), and activation of vasodilator-stimulated phosphoprotein, cyclic AMP responsive element-binding protein, and PKA were depressed by curcumin administration. This reduction led to the amelioration of the phagocytic abilities of PGE2-stimulated N9 cells. Taken together, these data suggested that curcumin restored the attenuating effect of PGE2 on fAβ42-induced microglial phagocytosis via a signaling mechanism involving EP2 and PKA. Moreover, due to its immune modulatory effects, curcumin may be a promising pharmacological candidate for neurodegenerative diseases. PMID:26824354

  17. Dissecting the effects of endogenous brain IL-2 and normal versus autoreactive T lymphocytes on microglial responsiveness and T cell trafficking in response to axonal injury.

    PubMed

    Huang, Zhi; Meola, Danielle; Petitto, John M

    2012-09-27

    IL-2 is essential for T-helper regulatory (Treg) cell function and self-tolerance, and dysregulation of both endogenous brain and peripheral IL-2 gene expression may have important implications for neuronal injury and repair. We used an experimental approach combining mouse congenic breeding and immune reconstitution to test the hypothesis that the response of motoneurons to injury is modulated by the combined effects of IL2-mediated processes in the brain that modulate its endogenous neuroimmunological milieu, and IL2-mediated processes in the peripheral immune system that regulate T cell function (i.e., normal versus autoreactive Treg-deficient T cells). This experimental strategy enabled us to test our hypothesis by disentangling the effect of normal versus autoreactive T lymphocytes from the effect of endogenous brain IL-2 on microglial responsiveness (microglial phagocytic clusters normally associated with dead motoneurons and MHC2(+) activated microglia) and T cell trafficking, using the facial nerve axotomy model of injury. The results demonstrate that the loss of both brain and peripheral IL-2 had an additive effect on numbers of microglial phagocytic clusters at day 14 following injury, whereas the autoreactive status of peripheral T cells was the primary factor that determined the degree to which T cells entered the injured brain and contributed to increased microglial phagocytic clusters. Changes in activated MHC2(+) microglial in the injured FMN were associated with loss of endogenous brain IL-2 and/or peripheral IL-2. This model may provide greater understanding of the mechanisms involved in determining if T cells entering the injured central nervous system (CNS) have damaging or proregenerative effects.

  18. Anthocyanin-rich acai (Euterpe oleracea mart.) fruit pulp fractions attenuate inflammatory stress signaling in mouse brain BV-2 microglial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Age-related increases in oxidative stress and inflammation are associated with loss of cognitive and motor functions. Previous research has shown that supplementation with berry fruits can modulate signaling in primary hippocampal neurons or BV-2 mouse microglial cells. Because of the high polypheno...

  19. Cytopathic changes and pro-inflammatory cytokines induced by Naegleria fowleri trophozoites in rat microglial cells and protective effects of an anti-Nfa1 antibody.

    PubMed

    Oh, Y-H; Jeong, S-R; Kim, J-H; Song, K-J; Kim, K; Park, S; Sohn, S; Shin, H-J

    2005-12-01

    Naegleria fowleri, a free-living amoeba, causes fatal primary amoebic meningoencephalitis in experimental animals and humans. The nfa1 gene (360 bp) was previously cloned from a cDNA library of pathogenic N. fowleri by immunoscreening, and produced a 13.1-kDa recombinant protein that showed pseudopodia-specific localization by immunocytochemistry. On the basis of an idea that the pseudopodia-specific Nfa1 protein seems to be involved in the pathogenicity of N. fowleri, the cytopathic activity of N. fowleri trophozoites co-cultured with rat microglial cells was observed, and the effects of an anti-Nfa1 antibody in a co-culture system were elucidated. Using light, scanning and transmission electron microscopy, it was seen that N. fowleri trophozoites in contact with microglial cells produced vigorous pseudopodia and a food-cup structure. Microglial cells were destroyed by N. fowleri trophozoites as seen from necrotic cell death in a time-dependent manner. In a(51)Cr release assay, N. fowleri showed 17.8%, 24.9%, 54.6% and 98% cytotoxicity against microglial cells at 3, 6, 12 and 24 h post-incubation, respectively. However, when anti-Nfa1 antibody was added in a coculture system, N. fowleri cytotoxicity was reduced to 15.5%, 20.3%, 46.7% and 66.9%, respectively. Moreover, microglial cells co-cultured with N. fowleri trophozoites secreted the pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-6. In the presence of anti-Nfa1 antibody, the secretion of TNF-alpha was slightly, but not significantly, decreased. PMID:16255744

  20. Cytopathic changes and pro-inflammatory cytokines induced by Naegleria fowleri trophozoites in rat microglial cells and protective effects of an anti-Nfa1 antibody.

    PubMed

    Oh, Y-H; Jeong, S-R; Kim, J-H; Song, K-J; Kim, K; Park, S; Sohn, S; Shin, H-J

    2005-12-01

    Naegleria fowleri, a free-living amoeba, causes fatal primary amoebic meningoencephalitis in experimental animals and humans. The nfa1 gene (360 bp) was previously cloned from a cDNA library of pathogenic N. fowleri by immunoscreening, and produced a 13.1-kDa recombinant protein that showed pseudopodia-specific localization by immunocytochemistry. On the basis of an idea that the pseudopodia-specific Nfa1 protein seems to be involved in the pathogenicity of N. fowleri, the cytopathic activity of N. fowleri trophozoites co-cultured with rat microglial cells was observed, and the effects of an anti-Nfa1 antibody in a co-culture system were elucidated. Using light, scanning and transmission electron microscopy, it was seen that N. fowleri trophozoites in contact with microglial cells produced vigorous pseudopodia and a food-cup structure. Microglial cells were destroyed by N. fowleri trophozoites as seen from necrotic cell death in a time-dependent manner. In a(51)Cr release assay, N. fowleri showed 17.8%, 24.9%, 54.6% and 98% cytotoxicity against microglial cells at 3, 6, 12 and 24 h post-incubation, respectively. However, when anti-Nfa1 antibody was added in a coculture system, N. fowleri cytotoxicity was reduced to 15.5%, 20.3%, 46.7% and 66.9%, respectively. Moreover, microglial cells co-cultured with N. fowleri trophozoites secreted the pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-6. In the presence of anti-Nfa1 antibody, the secretion of TNF-alpha was slightly, but not significantly, decreased.

  1. Redefining Myeloid Cell Subsets in Murine Spleen

    PubMed Central

    Hey, Ying-Ying; Tan, Jonathan K. H.; O’Neill, Helen C.

    2016-01-01

    Spleen is known to contain multiple dendritic and myeloid cell subsets, distinguishable on the basis of phenotype, function and anatomical location. As a result of recent intensive flow cytometric analyses, splenic dendritic cell (DC) subsets are now better characterized than other myeloid subsets. In order to identify and fully characterize a novel splenic subset termed “L-DC” in relation to other myeloid cells, it was necessary to investigate myeloid subsets in more detail. In terms of cell surface phenotype, L-DC were initially characterized as a CD11bhiCD11cloMHCII−Ly6C−Ly6G− subset in murine spleen. Their expression of CD43, lack of MHCII, and a low level of CD11c was shown to best differentiate L-DC by phenotype from conventional DC subsets. A complete analysis of all subsets in spleen led to the classification of CD11bhiCD11cloMHCII−Ly6CloLy6G− cells as monocytes expressing CX3CR1, CD43 and CD115. Siglec-F expression was used to identify a specific eosinophil population, distinguishable from both Ly6Clo and Ly6Chi monocytes, and other DC subsets. L-DC were characterized as a clear subset of CD11bhiCD11cloMHCII−Ly6C−Ly6G− cells, which are CD43+, Siglec-F− and CD115−. Changes in the prevalence of L-DC compared to other subsets in spleens of mutant mice confirmed the phenotypic distinction between L-DC, cDC and monocyte subsets. L-DC development in vivo was shown to occur independently of the BATF3 transcription factor that regulates cDC development, and also independently of the FLT3L and GM-CSF growth factors which drive cDC and monocyte development, so distinguishing L-DC from these commonly defined cell types. PMID:26793192

  2. Murine Mueller cells are progenitor cells for neuronal cells and fibrous tissue cells

    SciTech Connect

    Florian, Christian; Langmann, Thomas; Weber, Bernhard H.F.; Morsczeck, Christian

    2008-09-19

    Mammalian Mueller cells have been reported to possess retinal progenitor cell properties and generate new neurons after injury. This study investigates murine Mueller cells under in vitro conditions for their capability of dedifferentiation into retinal progenitor cells. Mueller cells were isolated from mouse retina, and proliferating cells were expanded in serum-containing medium. For dedifferentiation, the cultured cells were transferred to serum-replacement medium (SRM) at different points in time after their isolation. Interestingly, early cell passages produced fibrous tissue in which extracellular matrix proteins and connective tissue markers were differentially expressed. In contrast, aged Mueller cell cultures formed neurospheres in SRM that are characteristic for neuronal progenitor cells. These neurospheres differentiated into neuron-like cells after cultivation on laminin/ornithine cell culture substrate. Here, we report for the first time that murine Mueller cells can be progenitors for both, fibrous tissue cells and neuronal cells, depending on the age of the cell culture.

  3. Anti-inflammatory properties of tianeptine on lipopolysaccharide-induced changes in microglial cells involve toll-like receptor-related pathways.

    PubMed

    Slusarczyk, Joanna; Trojan, Ewa; Glombik, Katarzyna; Piotrowska, Anna; Budziszewska, Boguslawa; Kubera, Marta; Popiolek-Barczyk, Katarzyna; Lason, Wladyslaw; Mika, Joanna; Basta-Kaim, Agnieszka

    2016-03-01

    Accumulating evidence suggests that activation of microglia plays a key role in the pathogenesis of depression. Activated microglia produce a wide range of factors whose prolonged or excessive release may lead to brain disorders. Thus, the inhibition of microglial cells may be beneficial in the treatment of depressive diseases. Tianeptine is an atypical antidepressant drug with proven clinical efficacy, but its mechanism of action remains still not fully understood. In the present study, using microglial cultures we investigated whether tianeptine modifies microglial activation after lipopolysaccharide (LPS) stimulation and which intracellular pathways are involved in the activity of this antidepressant. Our study shows that tianeptine attenuated the LPS-evoked inflammatory activation of microglia by decreasing the expression of proinflammatory cytokines such as IL-1β, IL-18, IL-6 and tumor necrosis factor α (TNF-α), the release of nitric oxide (NO) and reactive oxygen species (ROS) as well as the expression of inducible nitric oxide synthase. Analyses of signaling pathways demonstrate that tianeptine led to the suppression of LPS-induced TLR4 expression and ERK1/2 phosphorylation. Furthermore, our study reveals the inhibitory impact of tianeptine on caspase-3-induced PKCδ degradation and consequently on the activation of NF-κB factor in microglial cells. Taken together, present results show anti-inflammatory properties of tianeptine in microglial cultures stimulated by LPS. This study provides evidence that the inhibition of microglial activation may underlie the therapeutic activity of tianeptine. Our findings show the anti-inflammatory effect of tianeptine (TIA) in lipopolisaccharide (LPS)-stimulated microglial cells. The beneficial tianeptine action is mediated through the inhibition of Toll-like receptor 4 (TLR4) expression as well as the TLR4-related pathways: extracellular signal-regulated kinase 1/2 (ERK1/2), caspase-3-dependent protein kinase δ (PKC

  4. Anti-inflammatory properties of tianeptine on lipopolysaccharide-induced changes in microglial cells involve toll-like receptor-related pathways.

    PubMed

    Slusarczyk, Joanna; Trojan, Ewa; Glombik, Katarzyna; Piotrowska, Anna; Budziszewska, Boguslawa; Kubera, Marta; Popiolek-Barczyk, Katarzyna; Lason, Wladyslaw; Mika, Joanna; Basta-Kaim, Agnieszka

    2016-03-01

    Accumulating evidence suggests that activation of microglia plays a key role in the pathogenesis of depression. Activated microglia produce a wide range of factors whose prolonged or excessive release may lead to brain disorders. Thus, the inhibition of microglial cells may be beneficial in the treatment of depressive diseases. Tianeptine is an atypical antidepressant drug with proven clinical efficacy, but its mechanism of action remains still not fully understood. In the present study, using microglial cultures we investigated whether tianeptine modifies microglial activation after lipopolysaccharide (LPS) stimulation and which intracellular pathways are involved in the activity of this antidepressant. Our study shows that tianeptine attenuated the LPS-evoked inflammatory activation of microglia by decreasing the expression of proinflammatory cytokines such as IL-1β, IL-18, IL-6 and tumor necrosis factor α (TNF-α), the release of nitric oxide (NO) and reactive oxygen species (ROS) as well as the expression of inducible nitric oxide synthase. Analyses of signaling pathways demonstrate that tianeptine led to the suppression of LPS-induced TLR4 expression and ERK1/2 phosphorylation. Furthermore, our study reveals the inhibitory impact of tianeptine on caspase-3-induced PKCδ degradation and consequently on the activation of NF-κB factor in microglial cells. Taken together, present results show anti-inflammatory properties of tianeptine in microglial cultures stimulated by LPS. This study provides evidence that the inhibition of microglial activation may underlie the therapeutic activity of tianeptine. Our findings show the anti-inflammatory effect of tianeptine (TIA) in lipopolisaccharide (LPS)-stimulated microglial cells. The beneficial tianeptine action is mediated through the inhibition of Toll-like receptor 4 (TLR4) expression as well as the TLR4-related pathways: extracellular signal-regulated kinase 1/2 (ERK1/2), caspase-3-dependent protein kinase δ (PKC

  5. Possible impact of microglial cells and the monocyte-macrophage system on suicidal behavior.

    PubMed

    Steiner, Johann; Gos, Tomasz; Bogerts, Bernhard; Bielau, Hendrik; Drexhage, Hemmo A; Bernstein, Hans-Gert

    2013-11-01

    Immune dysfunction, including monocytosis, increased blood levels of interleukin-1 (IL-1), interleukin-6 (IL- 6) and tumor necrosis factor-alpha (TNF-alpha), as well as an increased microglial density in certain brain areas, have been described in schizophrenia and depression. Interestingly, similar immune alterations have been observed in suicide patients regardless of their underlying psychiatric diagnosis. This review summarizes relevant data from previous studies that have examined peripheral blood, cerebrospinal fluid and human brains (using postmortem histology and in vivo positron emission tomography) to investigate immune mechanisms in suicidal patients. We discuss whether the observed findings indicate that microgliosis and monocyte-macrophage system activation may be a useful marker of disease acuity/severity or whether they instead indicate a distinct neurobiology of suicide. Notably, pathophysiological mechanisms could change during the long-term course of psychiatric diseases. Therefore, different patterns of immune activation may be observed when comparing newly diseased patients with those who are chronically ill.

  6. Microglial P2Y12 Receptors Regulate Microglial Activation and Surveillance during Neuropathic Pain

    PubMed Central

    Gu, Nan; Eyo, Ukpong B.; Murugan, Madhuvika; Peng, Jiyun; Matta, Sanjana; Dong, Hailong; Wu, Long-Jun

    2016-01-01

    Microglial cells are critical in the pathogenesis of neuropathic pain and several microglial receptors have been proposed to mediate this process. Of these receptors, the P2Y12 receptor is a unique purinergic receptor that is exclusively expressed by microglia in the central nervous system (CNS). In this study, we set forth to investigate the role of P2Y12 receptors in microglial electrophysiological and morphological (static and dynamic) activation during spinal nerve transection (SNT)-induced neuropathic pain in mice. First, we found that a genetic deficiency of the P2Y12 receptor (P2Y12−/− mice) ameliorated pain hypersensitivities during the initiation phase of neuropathic pain. Next, we characterized both the electrophysiological and morphological properties of microglia in the superficial spinal cord dorsal horn following SNT injury. We show dramatic alterations including a peak at 3 days post injury in microglial electrophysiology while high resolution two-photon imaging revealed significant changes of both static and dynamic microglial morphological properties by 7 days post injury. Finally, in P2Y12−/− mice, these electrophysiological and morphological changes were ameliorated suggesting roles for P2Y12 receptors in SNT-induced microglial activation. Our results therefore indicate that P2Y12 receptors regulate microglial electrophysiological as well as static and dynamic microglial properties after peripheral nerve injury, suggesting that the microglial P2Y12 receptor could be a potential therapeutic target for the treatment of neuropathic pain. PMID:26576724

  7. Telomere sister chromatid exchange in telomerase deficient murine cells

    SciTech Connect

    Wang, Yisong; Giannone, Richard J; Liu, Yie

    2005-01-01

    We have recently demonstrated that several types of genomic rearrangements (i.e., telomere sister chromatid exchange (T-SCE), genomic-SCE, or end-to-end fusions) were more often detected in long-term cultured murine telomerase deficient embryonic stem (ES) cells than in freshly prepared murine splenocytes, even through they possessed similar frequencies of critically short telomeres. The high rate of genomic rearrangements in telomerase deficient ES cells, when compared to murine splenocytes, may reflect the cultured cells' gained ability to protect chromosome ends with eroded telomeres allowing them to escape 'end crisis'. However, the possibility that ES cells were more permissive to genomic rearrangements than other cell types or that differences in the microenvironment or genetic background of the animals might consequentially determine the rate of T-SCEs or other genomic rearrangements at critically short telomeres could not be ruled out.

  8. Active immunotherapy facilitates Aβ plaque removal following through microglial activation without obvious T cells infiltrating the CNS.

    PubMed

    Sha, Sha; Xing, Xiao-Na; Cao, Yun-Peng

    2014-09-15

    Immunization of AD mouse models with Aβ reduced Aβ deposits and improved memory and learning deficits, but some clinical trials of immunization with Aβ were halted due to brain inflammation which was presumably induced by a T cell-mediated autoimmune response. We have developed a "possibly safer" vaccine. Our results demonstrate that pcDNA3.1 vector encoding ten repeats of Aβ3-10 fragments elicited high titers of antibodies which reacted well with not only monomeric but also oligomeric and fibrillar forms of Aβ42 peptide. Induced antibodies strongly reacted with amyloid plaques in the brain, demonstrating functional activity of the antibodies. Immunohistochemical and immunofluorescence showed there was significantly less plaque deposition accomplied with less microglia activation as detected both in the frontal cortex and hippocampus. These data suggested that microglial activation is necessary for efficient removal of compact amyloid deposits with immunotherapy. No obvious inflammation T cell and Prussian blue positive cell was found indicated that inflammation T cell infiltration and microhemmorage can be avoided or at least reduced to the minimum level. PMID:25087756

  9. Vaccination with heat-shocked mononuclear cells as a strategy for treating neurodegenerative disorders driven by microglial inflammation.

    PubMed

    McCarty, Mark F; Al-Harbi, Saleh A

    2013-11-01

    Naturally occurring T regulatory cells targeting epitopes derived from various heat shock proteins escape thymic negative selection and can be activated by vaccination with heat shock proteins; hence, vaccination with such proteins has exerted favorable effects on rodent models of autoimmune disorders. A more elegant way to achieve such vaccination, first evaluated clinically by Al-Harbi in the early 1990s, is to subject mononuclear cells to survivable heat shock ex vivo, incubate them at physiological temperature for a further 24-48 h, and then inject them subcutaneously; anecdotally, beneficial effects were observed with this strategy in a wide range of autoimmune and inflammatory conditions. There is growing evidence that M1-activated microglia play a primary or secondary role in the pathogenesis of numerous neurodegenerative diseases, as well as in major depression. T regulatory cells, by polarizing microglial toward a reparative M2 phenotype, have the potential to aid control of such disorders. It would be appropriate to test the heat-shocked mononuclear cell vaccination strategy in animal models of neurodegeneration and major depression, and to evaluate this approach clinically if such studies yield encouraging results. PMID:23968572

  10. Neurotoxicity effects of atrazine-induced SH-SY5Y human dopaminergic neuroblastoma cells via microglial activation.

    PubMed

    Ma, Kun; Wu, Hao-Yu; Zhang, Bo; He, Xi; Li, Bai-Xiang

    2015-11-01

    Atrazine (2-chloro-4-ethytlamino-6-isopropylamine-1,3,5-triazine; ATR) is a broad-spectrum herbicide with a wide range of applications worldwide. However, ATR is neurotoxic; it reduces dopamine levels in the substantia nigra and corpus striatum in the midbrain, affects the absorption of synaptic vesicles and synaptic bodies, and interferes with dopamine storage and uptake in synaptic vesicles, leading to neurodegenerative disorders. Microglia are resident immunocompetent and phagocytic cells that regulate and participate in the microenvironment in the central nervous system. They demonstrate macrophage characteristics after activation by releasing inflammatory cytokines and neurotoxic substances to increase the inflammatory response, and are thus involved in neurodegeneration. The aim of this study was to investigate the neurotoxic effects of ATR-activated microglia-mediated neuronal damage in terms of human dopaminergic neuroblastoma SH-SY5Y cell death. ATR was administered to BV-2 microglial cells at 12.5, 25, and 50 μM for 1, 6, 12, 24 and 48 h, respectively. ATR increased activated-microglia-induced overexpression of reactive oxygen species, inducible nitric oxide synthase, nitric oxide, gp91(phox), p47(phox), and the inflammatory cytokines tumor necrosis factor α and interleukin-1β, thus reducing SH-SY5Y cell viability. These results suggest that activated microglia may play a critical role in inflammation-mediated dopaminergic neuronal death, and provide the basis for further studies on the mechanisms of ATR-induced dopaminergic system toxicity. PMID:26256823

  11. Production of Nfa1-specific monoclonal antibodies that influences the in vitro cytotoxicity of Naegleria fowleri trophozoites on microglial cells.

    PubMed

    Lee, Yang-Jin; Kim, Jong-Hyun; Jeong, Seok-Ryoul; Song, Kyoung-Ju; Kim, Kyongmin; Park, Sun; Park, Moon-Sung; Shin, Ho-Joon

    2007-10-01

    Naegleria fowleri, agent of fatal primary amoebic meningoencephalitis, appears to induce cytotoxicity mechanically through its contact with the cell. The nfa1 gene cloned from a cDNA library of pathogenic N. fowleri by immunoscreening consists of 360 bp and expresses a 13.1-kDa recombinant protein (rNfa1) that demonstrated localization in the pseudopodia when examined using immunocytochemistry. To study the mechanisms involved in N. fowleri cytotoxicity, we developed a large volume of rNfa1-specific monoclonal antibody (McAb) against a 17-kDa His-tag fusion rNfa1 protein using a cell fusion technique. We established eight McAb-producing hybridoma cells. The antibodies were all immunoglobulin G2b and reacted strongly with a 17-kDa band representing the rNfa1 fusion protein in Western blotting, demonstrating immunoreactivity to the Nfa1 protein in pseudopodia (especially in the food cups) of N. fowleri trophozoites. A 51Cr-release assay indicated N. fowleri cytotoxicity by demonstrating that it eliminated 37.8, 60.6, and 98.8% of the target (microglial) cells 6, 12, and 24 h after co-incubation, respectively. When an anti-Nfa1 McAb was added to the coculture system, N. fowleri cytotoxicity decreased to 29.8, 44.1, and 66.3%, respectively.

  12. Neurotoxicity effects of atrazine-induced SH-SY5Y human dopaminergic neuroblastoma cells via microglial activation.

    PubMed

    Ma, Kun; Wu, Hao-Yu; Zhang, Bo; He, Xi; Li, Bai-Xiang

    2015-11-01

    Atrazine (2-chloro-4-ethytlamino-6-isopropylamine-1,3,5-triazine; ATR) is a broad-spectrum herbicide with a wide range of applications worldwide. However, ATR is neurotoxic; it reduces dopamine levels in the substantia nigra and corpus striatum in the midbrain, affects the absorption of synaptic vesicles and synaptic bodies, and interferes with dopamine storage and uptake in synaptic vesicles, leading to neurodegenerative disorders. Microglia are resident immunocompetent and phagocytic cells that regulate and participate in the microenvironment in the central nervous system. They demonstrate macrophage characteristics after activation by releasing inflammatory cytokines and neurotoxic substances to increase the inflammatory response, and are thus involved in neurodegeneration. The aim of this study was to investigate the neurotoxic effects of ATR-activated microglia-mediated neuronal damage in terms of human dopaminergic neuroblastoma SH-SY5Y cell death. ATR was administered to BV-2 microglial cells at 12.5, 25, and 50 μM for 1, 6, 12, 24 and 48 h, respectively. ATR increased activated-microglia-induced overexpression of reactive oxygen species, inducible nitric oxide synthase, nitric oxide, gp91(phox), p47(phox), and the inflammatory cytokines tumor necrosis factor α and interleukin-1β, thus reducing SH-SY5Y cell viability. These results suggest that activated microglia may play a critical role in inflammation-mediated dopaminergic neuronal death, and provide the basis for further studies on the mechanisms of ATR-induced dopaminergic system toxicity.

  13. Fasudil inhibits LPS-induced migration of retinal microglial cells via regulating p38-MAPK signaling pathway

    PubMed Central

    Xu, Fan; Xu, Yue; Zhu, Liqiong; Rao, Pinhong; Wen, Jiamin; Sang, Yunyun; Shang, Fu

    2016-01-01

    Purpose To investigate the effect and possible molecular mechanisms of fasudil on retinal microglial (RMG) cell migration. Methods Primary cultured RMG cells were incubated with lipopolysaccharide (LPS), fasudil, and/or SB203580 (a p38 inhibitor). RMG cell motility was determined with the scratch wound assay and the Transwell migration assay. The phosphorylation of p38 and levels of matrix metalloproteinase 2 (MMP-2) and MMP-9 were measured with western blot. Results In the scratch-induced migration assay, as well as in the Transwell migration assay, the results indicated that LPS stimulated the migratory potential of RMG cells and fasudil significantly reduced LPS-stimulated RMG cell migration in a concentration-dependent manner. However, fasudil had no effect on RMG cell migration in the absence of LPS stimulation. Moreover, fasudil reduced the level of phosphor-p38 mitogen-activated protein kinase (p-p38-MAPK) in a concentration-dependent manner, without effects on the levels of phospho-p44/42 (p-ERK1/2) and phospho-c-Jun N-terminal kinase (p-JNK). Cotreatment with SB203580 (a p38 inhibitor) and fasudil resulted in the synergistic reduction of MMP-2, MMP-9, and p-p38-MAPK, as well as a reduction in the LPS-stimulated migration capabilities of the RMG cells, suggesting fasudil suppresses the LPS-stimulated migration of RMG cells via directly downregulating the p38-MAPK signaling pathway. Conclusions Our studies indicated that fasudil inhibited LPS-stimulated RMG cell migration via suppression of the p38-MAPK signaling pathway. PMID:27441000

  14. Involvement of PKA and HO-1 signaling in anti-inflammatory effects of surfactin in BV-2 microglial cells

    SciTech Connect

    Park, Sun Young; Kim, Ji-Hee; Lee, Sang Joon; Kim, YoungHee

    2013-04-01

    Surfactin, one of the most powerful biosurfactants, is a bacterial cyclic lipopeptide. Here, we investigated the anti-neuroinflammatory properties of surfactin in lipoteichoic acid (LTA)-stimulated BV-2 microglial cells. Surfactin significantly inhibited excessive production of the pro-inflammatory mediators TNF-α, IL-1β, IL-6, monocyte chemoattractant protein-1 (MCP-1), prostaglandin E{sub 2} (PGE{sub 2}), nitric oxide (NO) and reactive oxygen species (ROS), and suppressed the expression of matrix metalloproteinase-9 (MMP-9), inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). Subsequent mechanistic studies revealed that surfactin inhibited LTA-induced nuclear factor-kappaB (NF-κB) and signal transducer and activator of transcription-1 (STAT-1) activation. However, surfactin increases the phosphorylation of the STAT-3, a component of the homeostatic mechanism causing anti-inflammatory events. We also demonstrated that surfactin induces heme oxygenase-1 (HO-1) expression and nuclear factor-regulated factor-2 (Nrf-2) activation, and that the anti-inflammatory effects of surfactin are abrogated by small interfering RNA-mediated knock-down of HO-1 or Nrf-2. Interestingly, we found that surfactin increased the level of cAMP and induced phosphorylation of cAMP responsive element binding protein (CREB) in microglial cells. Furthermore, treatment with the protein kinase A (PKA) inhibitor, H-89, blocked HO-1 induction by surfactin and abolished surfactin's suppressive effects on ROS and NO production. These results indicate that HO-1 and its upstream effector, PKA, play a pivotal role in the anti-neuroinflammatory response of surfactin in LTA-stimulated microglia. Therefore, surfactin might have therapeutic potential for neuroprotective agents to treat inflammatory and neurodegenerative diseases. - Highlights: ► Surfactin inhibits proinflammatory mediator synthesis in LTA-activated BV-2 cells. ► Surfactin suppresses NF-κB and STAT-1, but potentiates

  15. Neutralization capacity of measles virus H protein specific IgG determines the balance between antibody-enhanced infectivity and protection in microglial cells

    PubMed Central

    Iankov, Ianko D.; Penheiter, Alan R.; Griesmann, Guy E.; Carlson, Stephanie K.; Federspiel, Mark J.; Galanis, Evanthia

    2013-01-01

    Neutralizing antibodies directed against measles virus (MV) surface glycoproteins prevent viral attachment and entry through the natural receptors. H protein specific IgG can enhance MV infectivity in macrophages via Fcγ receptor (FcγR)-dependent mechanism. H-specific IgM, anti-F antibodies and complement cascade activation are protective against antibody-mediated enhancement of MV infection. However, protective role of anti-H IgG against antibody-enhanced infection is not well understood. Here we designed a set of experiments to test the protective effect of H-specific IgG against FcγR-mediated infection in microglial cells. Microglial cells are also potential target of the antibody-mediated enhancement and spread of MV infection in the central nervous system. A partially neutralizing IgG monoclonal antibody (MAb) CL55, specific for MV H protein, at 10 μg/ml enhanced MV infection in mouse microglial cells by 13-14-fold. Infection-enhancing antibody concentrations induced large multinucleated syncytia formation 48-72 h post inoculation. We generated anti-H IgG MAb 20H6 with a strong neutralization capacity >1:80,000 at 1 mg/ml concentration in MV plaque-reduction neutralization assay. In contrast to the partially protective MAb CL55, enhancement of MV infectivity by MAb 20H6 required dilutions below the 1:120 serum titer considered protective against measles infection in humans. At a concentration of 10 μg/ml MAb 20H6 exhibited a dominant protective effect and prevented MAb CL55-mediated enhancement of MV infection and virus-mediated fusion. These results indicate that neutralization capacity of the H-specific IgG determines the balance between antibody enhancement and protection against MV infection in microglial cells. PMID:23266401

  16. Differential transcriptional profiles mediated by exposure to the cannabinoids cannabidiol and Δ9-tetrahydrocannabinol in BV-2 microglial cells

    PubMed Central

    Juknat, Ana; Pietr, Maciej; Kozela, Ewa; Rimmerman, Neta; Levy, Rivka; Coppola, Giovanni; Geschwind, Daniel; Vogel, Zvi

    2012-01-01

    BACKGROUND AND PURPOSE Apart from their effects on mood and reward, cannabinoids exert beneficial actions such as neuroprotection and attenuation of inflammation. The immunosuppressive activity of cannabinoids has been well established. However, the underlying mechanisms are largely unknown. We previously showed that the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) and the non-psychoactive cannabidiol (CBD) differ in their anti-inflammatory signalling pathways. EXPERIMENTAL APPROACH To characterize the transcriptional effects of CBD and THC, we treated BV-2 microglial cells with these compounds and performed comparative microarray analysis using the Illumina MouseRef-8 BeadChip platform. Ingenuity Pathway Analysis was performed to identify functional subsets of genes and networks regulated by CBD and/or THC. KEY RESULTS Overall, CBD altered the expression of many more genes; from the 1298 transcripts found to be differentially regulated by the treatments, 680 gene probe sets were up-regulated by CBD and 58 by THC, and 524 gene products were down-regulated by CBD and only 36 by THC. CBD-specific gene expression profile showed changes associated with oxidative stress and glutathione depletion, normally occurring under nutrient limiting conditions or proteasome inhibition and involving the GCN2/eIF2α/p8/ATF4/CHOP-TRIB3 pathway. Furthermore, CBD-stimulated genes were shown to be controlled by nuclear factors known to be involved in the regulation of stress response and inflammation, mainly via the (EpRE/ARE)-Nrf2/ATF4 system and the Nrf2/Hmox1 axis. CONCLUSIONS AND IMPLICATIONS These observations indicated that CBD, but much less than THC, induced a cellular stress response in microglial cells and suggested that this effect could underlie its anti-inflammatory activity. LINKED ARTICLES This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012

  17. Minocycline Effects on IL-6 Concentration in Macrophage and Microglial Cells in a Rat Model of Neuropathic Pain

    PubMed Central

    Moini-Zanjani, Taraneh; Ostad, Seyed-Nasser; Labibi, Farzaneh; Ameli, Haleh; Mosaffa, Nariman; Sabetkasaei, Masoumeh

    2016-01-01

    Background: Evidence indicates that neuropathic pain pathogenesis is not confined to changes in the activity of neuronal systems but involves interactions between neurons, inflammatory immune and immune-like glial cells. Substances released from immune cells during inflammation play an important role in development and maintenance of neuropathic pain. It has been found that minocycline suppresses the development of neuropathic pain. Here, we evaluated the analgesic effect of minocycline in a chronic constriction injury (CCI) model of neuropathic pain in rat and assessed IL-6 concentration from cultured macrophage and microglia cells. Methods: Male Wistar rat (n=6, 150-200 g) were divided into three different groups: 1) CCI+vehicle, 2) sham+vehicle, and 3) CCI+drug. Minocycline (10, 20, and 40 mg/kg) was injected one hour before surgery and continued daily to day 14 post ligation. Von Frey filaments and acetone, as pain behavioral tests, were used for mechanical allodynia and cold allodynia, respectively. Experiments were performed on day 0 (before surgery) and days 1, 3, 5, 7, 10, and 14 post -injury. At day 14, rats were killed and monocyte-derived macrophage from right ventricle and microglia from lumbar part of the spinal cord were isolated and cultured in RPMI and Leibovitz’s media, respectively. IL-6 concentration was evaluated in cell culture supernatant after 24 h. Results: Minocycline (10, 20, and 40 mg/kg) attenuated pain behavior, and a decrease in IL-6 concentration was observed in immune cells compared to CCI vehicle-treated animals. Conclusion: Minocycline reduced pain behavior and decreased IL-6 concentration in macrophage and microglial cells. PMID:27221523

  18. Morphology and growth of murine cell lines on model biomaterials.

    PubMed

    Godek, Marisha L; Duchsherer, Nichole L; McElwee, Quinn; Grainger, David W

    2004-01-01

    All biomaterial implants are assaulted by the host "foreign body" immune response. Understanding the complex, dynamic relationship between cells, biomaterials and milieu is an important first step towards controlling this reaction. Material surface chemistry dictates protein adsorption, and thus subsequent cell interactions. The cell-implant is a microenvironment involving 1) proteins that coat the surface and 2) cells that interact with these proteins. Macrophages and fibroblasts are two cell types that interact with proteins on biomaterials surfaces and play different related, but equally important, roles in biomaterials rejection and implant failure. Growth characteristics of four murine cell lines on model biomaterials surfaces were examined. Murine monocyte-macrophages (RAW 264.7 and J774A.1), murine macrophage (IC-21) and murine fibroblast (NIH 3T3) cell lines were tested to determine whether differences exist in adhesion, proliferation, differentiation, spreading, and fusion (macrophage lineages only) on these surfaces. Differences were observed in the ability of cells to adhere to and subsequently proliferate on polymer surfaces. (Monocyte-) macrophages grew well on all surfaces tested and growth rates were measured on three representative polymer biomaterials surfaces: tissue culture polystyrene (TCPS), polystyrene, and Teflon-AF. J774A.1 cultures grown on TCPS and treated with exogenous cytokines IL-4 and GM-CSF were observed to contain multinucleate cells with unusual morphologies. Thus, (monocyte-) macrophage cell lines were found to effectively attach to and interrogate each surface presented, with evidence of extensive spreading on Teflon-AF surfaces, particularly in the IC-21 cultures. The J774A.1 line was able to proliferate and/or differentiate to more specialized cell types (multinucleate/dendritic-like cells) in the presence of soluble chemokine cues. PMID:15133927

  19. Differential plasticity of microglial cells in the rostrocaudal neuraxis of the accessory olfactory bulb of female mice following mating and stud male exposure.

    PubMed

    Okere, Chuma O

    2012-04-11

    The formation of an olfactory recognition memory by female mice for the stud male pheromones requires two fundamental conditions: incidence of mating and retention of the stud male with the female for a critical 6h interval following mating. This biologically critical recognition memory results from plasticity of reciprocal dendrodendritic synapses in the accessory olfactory bulb (AOB). In this study, a microglia marker antibody (ionized calcium-binding adaptor protein, Iba1) was used to determine how mating and stud pheromones affect microglia in the AOB rostrocaudal axis in female mice. The results showed that compared with estrus and mating only, mating and pheromone exposure significantly increased Iba1 immunoreactivity in the AOB evidenced by increased complexity of ramified microglial processes characteristic of resting microglial morphological phenotype, particularly in the rostral AOB. The density of Iba1 staining after mating and stud pheromone exposure was higher in the rostral - compared to caudal - AOB and was most prevalent in the external plexiform layer, the site of reciprocal mitral-granule dendrodendritic synapses. While cells with activated phenotype were observed in caudal AOB during estrus, mating/pheromone exposure appeared to induce a morphological transformation to the resting microglia phenotype. Since previous evidence implicate the rostral AOB in processing pheromonal signals and microglial cells monitor active synapses, these observations have important functional implications for a potential role for microglia in processing pheromonal signals in the AOB during the formation of olfactory memory.

  20. Neochlorogenic Acid Inhibits Lipopolysaccharide-Induced Activation and Pro-inflammatory Responses in BV2 Microglial Cells.

    PubMed

    Kim, Mina; Choi, Sang-Yoon; Lee, Pyeongjae; Hur, Jinyoung

    2015-09-01

    Microglia is the resident innate immune cells that sense pathogens and tissue injury in the central nervous system. Microglia becomes activated in response to injury, infection, and other stimuli that threaten neuronal survival. Microglia activation plays an important role in neurodegenerative diseases. Neochlorogenic acid (NCA) is a natural polyphenolic compound found in dried fruits and other plants. Although previous studies have shown that phenolic acids including NCA have outstanding antioxidant, antibacterial, antiviral, and antipyretic activities, there has not yet been investigated for anti-inflammatory effects. Therefore, for the first time we have examined the potential of NCA to inhibit microglial activation and pro-inflammatory responses in the brain. We found that lipopolysaccharide-induced inducible nitric oxide synthase, and cyclooxygenase-2 expression, and nitric oxide formation was suppressed by NCA in a dose-dependent manner in BV2 microglia. NCA also inhibited the production of pro-inflammatory mediators, tumor necrosis factor-α and interleukin-1 beta. Furthermore, phosphorylated nuclear factor-kappa B p65 and p38 mitogen-activated protein kinase activation were blocked by NCA. Taken together, these results suggest that NCA exerts neuroprotective effects through the inhibition of pro-inflammatory pathways in activated microglia.

  1. Autologous bone marrow mononuclear cells therapy attenuates activated microglial/macrophage response and improves spatial learning after traumatic brain injury

    PubMed Central

    Bedi, Supinder S.; Walker, Peter A.; Shah, Shinil K.; Jimenez, Fernando; Thomas, Chelsea P.; Smith, Philippa; Hetz, Robert A.; Xue, Hasen; Pati, Shibani; Dash, Pramod K.; Cox, Charles S.

    2014-01-01

    Background Autologous bone marrow-derived mononuclear cells (AMNC) have shown therapeutic promise for central nervous system insults such as stroke and traumatic brain injury (TBI). We hypothesized that intravenous injection of AMNC provides neuroprotection which leads to cognitive improvement after TBI. Methods A controlled cortical impact (CCI) rodent traumatic brain injury (TBI) model was used to examine blood-brain barrier permeability (BBB), neuronal and glial apoptosis and cognitive behavior. Two groups of rats underwent CCI with (CCI-Autologous) or without AMNC treatment (CCI-Alone), consisting of 2 million AMNC/kilogram body weight harvested from the tibia and intravenously injected 72 hr after injury. CCI-Alone animals underwent sham harvests and received vehicle injections. Results 96 hr after injury, AMNC significantly reduced the BBB permeability in injured animals, and there was an increase in apoptosis of pro-inflammatory activated microglia in the ipsilateral hippocampus. At 4 weeks after injury, we examined changes in spatial memory after TBI due to AMNC treatment. There was a significant improvement in probe testing of CCI-Autologous group in comparison to CCI-Alone in the Morris Water Maze paradigm. Conclusions Our data demonstrate that the intravenous injection of AMNC after TBI leads to neuroprotection by preserving early BBB integrity and increasing activated microglial apoptosis. In addition, AMNC also improves cognitive function. PMID:23928737

  2. Methamphetamine activates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and induces human immunodeficiency virus (HIV) transcription in human microglial cells.

    PubMed

    Wires, Emily S; Alvarez, David; Dobrowolski, Curtis; Wang, Yun; Morales, Marisela; Karn, Jonathan; Harvey, Brandon K

    2012-10-01

    Human immunodeficiency virus (HIV) primarily infects glial cells in the central nervous system (CNS). Recent evidence suggests that HIV-infected individuals who abuse drugs such as methamphetamine (METH) have higher viral loads and experience more severe neurological complications than HIV-infected individuals who do not abuse drugs. The aim of this study was to determine the effect of METH on HIV expression from the HIV long terminal repeat (LTR) promoter and on an HIV integrated provirus in microglial cells, the primary host cells for HIV in the CNS. Primary human microglial cells immortalized with SV40 T antigen (CHME-5 cells) were cotransfected with an HIV LTR reporter and the HIV Tat gene, a key regulator of viral replication and gene expression, and exposed to METH. Our results demonstrate that METH treatment induced LTR activation, an effect potentiated in the presence of Tat. We also found that METH increased the nuclear translocation of the nuclear factor kappa B (NF-κB), a key cellular transcriptional regulator of the LTR promoter, and the activity of an NF-κB-specific reporter plasmid in CHME-5 cells. The presence of a dominant-negative regulator of NF-κB blocked METH-related activation of the HIV LTR. Furthermore, treatment of HIV-latently infected CHME-5 (CHME-5/HIV) cells with METH induced HIV expression and nuclear translocation of the p65 subunit of NF-κB. These results suggest that METH can stimulate HIV gene expression in microglia cells through activation of the NF-κB signaling pathway. This mechanism may outline the initial biochemical events leading to the observed increased neurodegeneration in HIV-positive individuals who use METH.

  3. Intravenous multipotent adult progenitor cell therapy attenuates activated microglial/macrophage response and improves spatial learning after traumatic brain injury.

    PubMed

    Bedi, Supinder S; Hetz, Robert; Thomas, Chelsea; Smith, Philippa; Olsen, Alex B; Williams, Stephen; Xue, Hasen; Aroom, Kevin; Uray, Karen; Hamilton, Jason; Mays, Robert W; Cox, Charles S

    2013-12-01

    We previously demonstrated that the intravenous delivery of multipotent adult progenitor cells (MAPCs) after traumatic brain injury (TBI) in rodents provides neuroprotection by preserving the blood-brain barrier and systemically attenuating inflammation in the acute time frame following cell treatment; however, the long-term behavioral and anti-inflammatory effects of MAPC administration after TBI have yet to be explored. We hypothesized that the intravenous injection of MAPCs after TBI attenuates the inflammatory response (as measured by microglial morphology) and improves performance at motor tasks and spatial learning (Morris water maze [MWM]). MAPCs were administered intravenously 2 and 24 hours after a cortical contusion injury (CCI). We tested four groups at 120 days after TBI: sham (uninjured), injured but not treated (CCI), and injured and treated with one of two concentrations of MAPCs, either 2 million cells per kilogram (CCI-2) or 10 million cells per kilogram (CCI-10). CCI-10 rats showed significant improvement in left hind limb deficit on the balance beam. On the fifth day of MWM trials, CCI-10 animals showed a significant decrease in both latency to platform and distance traveled compared with CCI. Probe trials revealed a significant decrease in proximity measure in CCI-10 compared with CCI, suggesting improved memory retrieval. Neuroinflammation was quantified by enumerating activated microglia in the ipsilateral hippocampus. We observed a significant decrease in the number of activated microglia in the dentate gyrus in CCI-10 compared with CCI. Our results demonstrate that intravenous MAPC treatment after TBI in a rodent model offers long-term improvements in spatial learning as well as attenuation of neuroinflammation.

  4. Diphtheria toxin-based recombinant murine IL-2 fusion toxin for depleting murine regulatory T cells in vivo.

    PubMed

    Wei, Min; Marino, Jose; Trowell, Aaron; Zhang, Huiping; Stromp Peraino, Jaclyn; Rajasekera, Priyani V; Madsen, Joren C; Sachs, David H; Huang, Christene A; Benichou, Gilles; Wang, Zhirui

    2014-09-01

    Regulatory T cells (Tregs) are a subpopulation of CD4(+) T cells which suppress immune responses of effector cells and are known to play a very important role in protection against autoimmune disease development, induction of transplantation tolerance and suppression of effective immune response against tumor cells. An effective in vivo Treg depletion agent would facilitate Treg-associated studies across many research areas. In this study, we have developed diphtheria toxin-based monovalent and bivalent murine IL-2 fusion toxins for depleting murine IL-2 receptor positive cells including CD25(+) Treg in vivo. Their potencies were assessed by in vitro protein synthesis inhibition and cell proliferation inhibition assays using a murine CD25(+) CTLL-2 cell line. Surprisingly, in contrast to our previously developed recombinant fusion toxins, the monovalent isoform (DT390-mIL-2) was approximately 4-fold more potent than its bivalent counterpart (DT390-bi-mIL-2). Binding analysis by flow cytometry demonstrated that the monovalent isoform bound stronger than the bivalent version. In vivo Treg depletion with the monovalent murine IL-2 fusion toxin was performed using C57BL/6J (B6) mice. Spleen Treg were significantly depleted with a maximum reduction of ∼70% and detectable as early as 12 h after the last injection. The spleen Treg numbers were reduced until Day 3 and returned to control levels by Day 7. We believe that this monovalent murine IL-2 fusion toxin will be an effective in vivo murine Treg depleter. PMID:25147093

  5. Reticulocalbin-1 Facilitates Microglial Phagocytosis

    PubMed Central

    Ding, Ying; Caberoy, Nora B.; Guo, Feiye; LeBlanc, Michelle E.; Zhang, Chenming; Wang, Weiwen; Wang, Feng; Chen, Rui; Li, Wei

    2015-01-01

    Phagocytosis is critical to the clearance of apoptotic cells, cellular debris and deleterious metabolic products for tissue homeostasis. Phagocytosis ligands directly recognizing deleterious cargos are the key to defining the functional roles of phagocytes, but are traditionally identified on a case-by-case basis with technical challenges. As a result, extrinsic regulation of phagocytosis is poorly defined. Here we demonstrate that microglial phagocytosis ligands can be systematically identified by a new approach of functional screening. One of the identified ligands is reticulocalbin-1 (Rcn1), which was originally reported as a Ca2+-binding protein with a strict expression in the endoplasmic reticulum. Our results showed that Rcn1 can be secreted from healthy cells and that secreted Rcn1 selectively bound to the surface of apoptotic neurons, but not healthy neurons. Independent characterization revealed that Rcn1 stimulated microglial phagocytosis of apoptotic but not healthy neurons. Ingested apoptotic cells were targeted to phagosomes and co-localized with phagosome marker Rab7. These data suggest that Rcn1 is a genuine phagocytosis ligand. The new approach described in this study will enable systematic identification of microglial phagocytosis ligands with broad applicability to many other phagocytes. PMID:25992960

  6. Reticulocalbin-1 facilitates microglial phagocytosis.

    PubMed

    Ding, Ying; Caberoy, Nora B; Guo, Feiye; LeBlanc, Michelle E; Zhang, Chenming; Wang, Weiwen; Wang, Feng; Chen, Rui; Li, Wei

    2015-01-01

    Phagocytosis is critical to the clearance of apoptotic cells, cellular debris and deleterious metabolic products for tissue homeostasis. Phagocytosis ligands directly recognizing deleterious cargos are the key to defining the functional roles of phagocytes, but are traditionally identified on a case-by-case basis with technical challenges. As a result, extrinsic regulation of phagocytosis is poorly defined. Here we demonstrate that microglial phagocytosis ligands can be systematically identified by a new approach of functional screening. One of the identified ligands is reticulocalbin-1 (Rcn1), which was originally reported as a Ca2+-binding protein with a strict expression in the endoplasmic reticulum. Our results showed that Rcn1 can be secreted from healthy cells and that secreted Rcn1 selectively bound to the surface of apoptotic neurons, but not healthy neurons. Independent characterization revealed that Rcn1 stimulated microglial phagocytosis of apoptotic but not healthy neurons. Ingested apoptotic cells were targeted to phagosomes and co-localized with phagosome marker Rab7. These data suggest that Rcn1 is a genuine phagocytosis ligand. The new approach described in this study will enable systematic identification of microglial phagocytosis ligands with broad applicability to many other phagocytes.

  7. Murine somatic cell nuclear transfer using reprogrammed donor cells expressing male germ cell-specific genes.

    PubMed

    Kang, Hoin; Park, Jong Im; Roh, Sangho

    2016-01-01

    In vivo-matured mouse oocytes were enucleated, and a single murine embryonic fibroblast (control or reprogrammed by introducing extracts from murine testis tissue, which showed expression of male germ cell-specific genes) was injected into the cytoplasm of the oocytes. The rate of blastocyst development and expression levels of Oct-4, Eomes and Cdx-2 were not significantly different in both experimental groups. However, the expression levels of Nanog, Sox9 and Glut-1 were significantly increased when reprogrammed cells were used as donor nuclei. Increased expression of Nanog can be supportive of complete reprogramming of somatic cell nuclear transfer murine embryos. The present study suggested that donor cells expressing male germ cell-specific genes can be reconstructed and can develop into embryos with normal high expression of developmentally essential genes. PMID:26369430

  8. High resolution and dynamic imaging of biopersistence and bioreactivity of extra and intracellular MWNTs exposed to microglial cells

    PubMed Central

    Gonzalez Carter, Daniel A.; Motskin, Michael; Pienaar, Ilse S.; Chen, Shu; Hu, Sheng; Ruenraroengsak, Pakatip; Ryan, Mary P.; Shaffer, Milo S. P.; Dexter, David T.

    2016-01-01

    Multi-walled carbon nanotubes (MWNTs) are increasingly being developed both as neuro-therapeutic drug delivery systems to the brain and as neural scaffolds to drive tissue regeneration across lesion sites. MWNTs with different degrees of acid oxidation may have different bioreactivities and propensities to aggregate in the extracellular environment, and both individualised and aggregated MWNTs may be expected to be found in the brain. Before practical application, it is vital to understand how both aggregates and individual MWNTs will interact with local phagocytic immune cells, the microglia, and ultimately to determine their biopersistence in the brain. The processing of extra- and intracellular MWNTs (both pristine and when acid oxidised) by microglia was characterised across multiple length scales by correlating a range of dynamic, quantitative and multi-scale techniques, including: UV-vis spectroscopy, light microscopy, focussed ion beam scanning electron microscopy and transmission electron microscopy. Dynamic, live cell imaging revealed the ability of microglia to break apart and internalise micron-sized extracellular agglomerates of acid oxidised MWNT, but not pristine MWNTs. The total amount of MWNTs internalised by, or strongly bound to, microglia was quantified as a function of time. Neither the significant uptake of oxidised MWNTs, nor the incomplete uptake of pristine MWNTs affected microglial viability, pro-inflammatory cytokine release or nitric oxide production. However, after 24 hrs exposure to pristine MWNTs, a significant increase in the production of reactive oxygen species was observed. Small aggregates and individualised oxidised MWNTs were present in the cytoplasm and vesicles, including within multilaminar bodies, after 72 hours. Some evidence of morphological damage to oxidised MWNT structure was observed including highly disordered graphitic structures, suggesting possible biodegradation. This work demonstrates the utility of dynamic

  9. Miglustat Improves Purkinje Cell Survival and Alters Microglial Phenotype in Feline Niemann-Pick Disease Type C

    PubMed Central

    Stein, Veronika M.; Crooks, Alexandra; Ding, Wenge; Prociuk, Maria; O’Donnell, Patricia; Bryan, Caroline; Sikora, Tracey; Dingemanse, Jasper; Vanier, Marie T.; Walkley, Steven U.; Vite, Charles H.

    2012-01-01

    Niemann-Pick disease type C (NPC disease) is an incurable cellular lipid trafficking disorder characterized by neurodegeneration and intralysosomal accumulation of cholesterol and glycosphingolipids. Treatment with miglustat, a small imino sugar that reversibly inhibits glucosylceramide synthase, which is necessary for glycosphingolipid synthesis, has been shown to benefit patients with NPC disease. The mechanism(s) and extent of brain cellular changes underlying this benefit are not understood. To investigate the basis of the efficacy of miglustat, cats with disease homologous to the juvenile-onset form of human NPC disease received daily miglustat orally beginning at 3 weeks of age. The plasma half-life of miglustat was 6.6 ± 1.1 hours, with a tmax, Cmax, and area under the plasma concentration-time curve of 1.7 ± 0.6 hours, 20.3 ± 4.6 μg/ml, and 104.1 ± 16.6 μg hours/ml, respectively. Miglustat delayed the onset of neurological signs and increased the lifespan of treated cats, and was associated with decreased GM2 ganglioside accumulation in the cerebellum and improved Purkinje cell survival. Ex vivo examination of microglia from the brains of treated cats revealed normalization of CD1c and class II major histocompatibility complex expression, as well as generation of reactive oxygen species. Together, these results suggest that prolonged Purkinje cell survival, reduced glycosphingolipid accumulation, and/or the modulation of microglial immunophenotype and function contribute to miglustat-induced neurological improvement in treated cats. PMID:22487861

  10. High resolution and dynamic imaging of biopersistence and bioreactivity of extra and intracellular MWNTs exposed to microglial cells.

    PubMed

    Goode, Angela E; Gonzalez Carter, Daniel A; Motskin, Michael; Pienaar, Ilse S; Chen, Shu; Hu, Sheng; Ruenraroengsak, Pakatip; Ryan, Mary P; Shaffer, Milo S P; Dexter, David T; Porter, Alexandra E

    2015-11-01

    Multi-walled carbon nanotubes (MWNTs) are increasingly being developed both as neuro-therapeutic drug delivery systems to the brain and as neural scaffolds to drive tissue regeneration across lesion sites. MWNTs with different degrees of acid oxidation may have different bioreactivities and propensities to aggregate in the extracellular environment, and both individualised and aggregated MWNTs may be expected to be found in the brain. Before practical application, it is vital to understand how both aggregates and individual MWNTs will interact with local phagocytic immune cells, the microglia, and ultimately to determine their biopersistence in the brain. The processing of extra- and intracellular MWNTs (both pristine and when acid oxidised) by microglia was characterised across multiple length scales by correlating a range of dynamic, quantitative and multi-scale techniques, including: UV-vis spectroscopy, light microscopy, focussed ion beam scanning electron microscopy and transmission electron microscopy. Dynamic, live cell imaging revealed the ability of microglia to break apart and internalise micron-sized extracellular agglomerates of acid oxidised MWNTs, but not pristine MWNTs. The total amount of MWNTs internalised by, or strongly bound to, microglia was quantified as a function of time. Neither the significant uptake of oxidised MWNTs, nor the incomplete uptake of pristine MWNTs affected microglial viability, pro-inflammatory cytokine release or nitric oxide production. However, after 24 h exposure to pristine MWNTs, a significant increase in the production of reactive oxygen species was observed. Small aggregates and individualised oxidised MWNTs were present in the cytoplasm and vesicles, including within multilaminar bodies, after 72 h. Some evidence of morphological damage to oxidised MWNT structure was observed including highly disordered graphitic structures, suggesting possible biodegradation. This work demonstrates the utility of dynamic

  11. The role of the JAK2-STAT3 pathway in pro-inflammatory responses of EMF-stimulated N9 microglial cells

    PubMed Central

    2010-01-01

    Background In several neuropathological conditions, microglia can become overactivated and cause neurotoxicity by initiating neuronal damage in response to pro-inflammatory stimuli. Our previous studies have shown that exposure to electromagnetic fields (EMF) activates cultured microglia to produce tumor necrosis factor (TNF)-α and nitric oxide (NO) through signal transduction involving the activator of transcription STAT3. Here, we investigated the role of STAT3 signaling in EMF-induced microglial activation and pro-inflammatory responses in more detail than the previous study. Methods N9 microglial cells were treated with EMF exposure or a sham treatment, with or without pretreatment with an inhibitor (Pyridone 6, P6) of the Janus family of tyrosine kinases (JAK). The activation state of microglia was assessed via immunoreaction using the microglial marker CD11b. Levels of inducible nitric oxide synthase (iNOS), TNF-α and NO were measured using real-time reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and the nitrate reductase method. Activation of JAKs and STAT3 proteins was evaluated by western blotting for specific tyrosine phosphorylation. The ability of STAT3 to bind to DNA was detected with an electrophoresis mobility shift assay (EMSA). Results EMF was found to significantly induce phosphorylation of JAK2 and STAT3, and DNA-binding ability of STAT3 in N9 microglia. In addition, EMF dramatically increased the expression of CD11b, TNF-α and iNOS, and the production of NO. P6 strongly suppressed the phosphorylation of JAK2 and STAT3 and diminished STAT3 activity in EMF-stimulated microglia. Interestingly, expression of CD11b as well as gene expression and production of TNF-α and iNOS were suppressed by P6 at 12 h, but not at 3 h, after EMF exposure. Conclusions EMF exposure directly triggers initial activation of microglia and produces a significant pro-inflammatory response. Our findings confirm that

  12. Measles virus persistence in an immortalized murine macrophage cell line.

    PubMed

    Goldman, M B; Buckthal, D J; Picciotto, S; O'Bryan, T A; Goldman, J N

    1995-02-20

    Persistent infection with the Edmonston strain of measles virus (MV) has been established in IC-21 cells, an immortalized murine macrophage cell line. Persistence was established immediately without syncytia formation or cytopathic effects. MV was expressed in the majority of the cells as evidenced by immunofluorescence microscopy, flow cytometry, infectious centers assays, and limiting dilution analysis. Hemagglutinin (H) and phosphoprotein expressed in persistently infected IC-21 cells had retarded migration in SDS-PAGE gels when compared to these proteins expressed in Vero cells. H protein differences were also found between freshly infected IC-21 cells and persistently infected IC-21 cells passaged for over 2 years. Six sublines of IC-21 cells, infected at different times, have maintained these characteristics for 2 years of passage. During this time period the intensity of immunofluorescence and the number of infectious virus particles recoverable fluctuated in five of the six cell lines. In one cell line virus expression remained at a consistent high level. The ability to establish a persistent MV infection in murine macrophages allows studies using a cell important in disseminating the infection. It facilitates experiments on immunological aspects of viral immunity by enabling cell mixing experiments with histocompatible cell populations and by making available the wide array of cellular and humoral reagents in the mouse. PMID:7871720

  13. Development of the microglial phenotype in culture.

    PubMed

    Szabo, M; Gulya, K

    2013-06-25

    Selected morphological, molecular and functional aspects of various microglial cell populations were characterized in cell cultures established from the forebrains of E18 rat embryos. The mixed primary cortical cultures were maintained for up to 28days using routine culturing techniques when the microglial cells in the culture were not stimulated or immunologically challenged. During culturing, expansion of the microglial cell populations was observed, as evidenced by quantitative assessment of selected monocyte/macrophage/microglial cell-specific markers (human leukocyte antigen (HLA) DP, DQ, DR, CD11b/c and Iba1) via immunocyto- and histochemistry and Western blot analysis. The Iba1 immunoreactivity in Western blots steadily increased about 750-fold, and the number of Iba1-immunoreactive cells rose at least 67-fold between one day in vitro (DIV1) and DIV28. Morphometric analysis on binary (digital) silhouettes of the microglia revealed their evolving morphology during culturing. Microglial cells were mainly ameboid in the early stages of in vitro differentiation, while mixed populations of ameboid and ramified cell morphologies were characteristic of older cultures as the average transformation index (TI) increased from 1.96 (DIV1) to 15.17 (DIV28). Multiple immunofluorescence labeling of selected biomarkers revealed different microglial phenotypes during culturing. For example, while HLA DP, DQ, DR immunoreactivity was present exclusively in ameboid microglia (TI<3) between DIV1 and DIV10, CD11b/c- and Iba1-positive microglial cells were moderately (TI<13) and progressively (TI<81) more ramified, respectively, and always present throughout culturing. Regardless of the age of the cultures, proliferating microglia were Ki67-positive and characterized by low TI values (TI<3). The microglial function was assessed by an in vitro phagocytosis assay. Unstimulated microglia with low TI values were significantly more active in phagocytosing fluorescent microspheres than

  14. Pheromone-induced cell proliferation in the murine subventricular zone.

    PubMed

    Koyama, Sachiko; Soini, Helena A; Foley, John; Novotny, Milos V; Lai, Cary

    2014-08-01

    Enhancement of adult neurogenesis in female mice was previously demonstrated through exposure to soiled bedding from males, although the identity of relevant chemosignals has remained unknown. The farnesenes and SBT (2-sec-butyl-4,5-dihydrothiazole) are male murine pheromones that dominant males secrete at higher levels. Previous studies have shown that they induce oestrus in female mice. We have recently shown that these pheromones strongly increase cell proliferation in the SVZ (subventricular zone) of adult female mice. In addition, we found that a female murine pheromone, 2,5-dimethylpyrazine, facilitates similar changes in males. 2,5-dimethylpyrazine is a female pheromone that is secreted when females are housed in large groups and it was originally found to suppress oestrus in females. We found that it does not have suppressive effect on the cell proliferation in the SVZ of females. Similarly, male murine pheromones, SBT and the farnesenes, do not show a suppressive effect on the cell proliferation in the SVZ of males. Our results demonstrated that pheromonal communication between males and females has strong stimulatory effect on both the reproductive physiology and brain cell proliferation, but intrasex pheromonal exchanges do not reduce progenitor proliferation in these brain regions.

  15. Modulation of cell-surface antigens of a murine neuroblastoma.

    PubMed

    Akeson, R; Herschman, H R

    1974-01-01

    Antisera were produced in rabbits to morphologically differentiated cells from the C1300 murine neuroblastoma (i.e., cells in which process formation was induced by maintenance on serum-free medium for 5 days). These antisera reacted more strongly in the complement fixation reaction with such "differentiated" cells than with "undifferentiated" (nonprocess-bearing) neuroblastoma cells. Adsorption of the antisera with undifferentiated cells removed the reactivity to cells without processes, while the reactivity with serum-free cells which possess processes was retained. Indirect immunofluorescence studies confirmed the results obtained by complement fixation and demonstrated that antibodies to the surface antigens of process-bearing cells could be adsorbed by particulate preparations from brain but not liver, spleen, or kidney. This is the first description of neural-associated cell-surface changes that correlate with the morphological differentiation in culture of neuroblastoma cells.

  16. The antiviral drug ganciclovir does not inhibit microglial proliferation and activation

    PubMed Central

    Skripuletz, Thomas; Salinas Tejedor, Laura; Prajeeth, Chittappen K.; Hansmann, Florian; Chhatbar, Chintan; Kucman, Valeria; Zhang, Ning; Raddatz, Barbara B.; Detje, Claudia N.; Sühs, Kurt-Wolfram; Pul, Refik; Gudi, Viktoria; Kalinke, Ulrich; Baumgärtner, Wolfgang; Stangel, Martin

    2015-01-01

    Ganciclovir is effective in the treatment of human infections with viruses of the Herpesviridae family. Beside antiviral properties, recently ganciclovir was described to inhibit microglial proliferation and disease severity of experimental autoimmune encephalomyelitis, an inflammatory model of multiple sclerosis. Microglial activation and proliferation are main characteristics of neuroinflammatory CNS diseases and inhibition of microglial functions might be beneficial in autoimmune diseases, or detrimental in infectious diseases. The objective of this study was to determine potential inhibitory effects of ganciclovir in three different murine animal models of CNS neuroinflammation in which microglia play an important role: Theiler´s murine encephalomyelitis, the cuprizone model of de- and remyelination, and the vesicular stomatitis virus encephalitis model. In addition, in vitro experiments with microglial cultures were performed to test the hypothesis that ganciclovir inhibits microglial proliferation. In all three animal models, neither microglial proliferation or recruitment nor disease activity was changed by ganciclovir. In vitro experiments confirmed that microglial proliferation was not affected by ganciclovir. In conclusion, our results show that the antiviral drug ganciclovir does not inhibit microglial activation and proliferation in the murine CNS. PMID:26447351

  17. The antiviral drug ganciclovir does not inhibit microglial proliferation and activation.

    PubMed

    Skripuletz, Thomas; Salinas Tejedor, Laura; Prajeeth, Chittappen K; Hansmann, Florian; Chhatbar, Chintan; Kucman, Valeria; Zhang, Ning; Raddatz, Barbara B; Detje, Claudia N; Sühs, Kurt-Wolfram; Pul, Refik; Gudi, Viktoria; Kalinke, Ulrich; Baumgärtner, Wolfgang; Stangel, Martin

    2015-01-01

    Ganciclovir is effective in the treatment of human infections with viruses of the Herpesviridae family. Beside antiviral properties, recently ganciclovir was described to inhibit microglial proliferation and disease severity of experimental autoimmune encephalomyelitis, an inflammatory model of multiple sclerosis. Microglial activation and proliferation are main characteristics of neuroinflammatory CNS diseases and inhibition of microglial functions might be beneficial in autoimmune diseases, or detrimental in infectious diseases. The objective of this study was to determine potential inhibitory effects of ganciclovir in three different murine animal models of CNS neuroinflammation in which microglia play an important role: Theiler´s murine encephalomyelitis, the cuprizone model of de- and remyelination, and the vesicular stomatitis virus encephalitis model. In addition, in vitro experiments with microglial cultures were performed to test the hypothesis that ganciclovir inhibits microglial proliferation. In all three animal models, neither microglial proliferation or recruitment nor disease activity was changed by ganciclovir. In vitro experiments confirmed that microglial proliferation was not affected by ganciclovir. In conclusion, our results show that the antiviral drug ganciclovir does not inhibit microglial activation and proliferation in the murine CNS. PMID:26447351

  18. Flow Cytometric Analysis of Immune Cells Within Murine Aorta.

    PubMed

    Gjurich, Breanne N; Taghavie-Moghadam, Parésa L; Galkina, Elena V

    2015-01-01

    The immune system plays a critical role in the modulation of atherogenesis at all stages of the disease. However, there are many technical difficulties when studying the immune system within murine aortas. Common techniques such as PCR and immunohistochemistry have answered many questions about the presence of immune cells and mediators of inflammation within the aorta yet many questions remain unanswered due to the limitations of these techniques. On the other hand, cumulatively the flow cytometry approach has propelled the immunology field forward but it has been challenging to apply this technique to aortic tissues. Here, we describe the methodology to isolate and characterize the immune cells within the murine aorta and provide examples of functional assays for aortic leukocytes using flow cytometry. The method involves the harvesting and enzymatic digestion of the aorta, extracellular and intracellular protein staining, and a subsequent flow cytometric analysis. PMID:26445788

  19. Maternal separation activates microglial cells and induces an inflammatory response in the hippocampus of male rat pups, independently of hypothalamic and peripheral cytokine levels.

    PubMed

    Roque, Angélica; Ochoa-Zarzosa, Alejandra; Torner, Luz

    2016-07-01

    Adult animals subjected to chronic stress show an inflammatory response in the hippocampus which has been related to cognitive dysfunction and psychopathology. However the immediate consequences of early life stress on hippocampal glial cells have not been studied. Here we analyzed the effects of maternal separation (MS) on astrocyte and microglial cell morphology in the hippocampal hilus, compared the expression of cytokines in the hippocampus and hypothalamus, and the peripheral response of cytokines, on postnatal day (PD) 15. Male rat pups of MS (3h/day, PD1-PD14) and Control (CONT) pups showed similar microglial cell densities in the hilus, but MS pups presented more activated microglia. MS decreased astrocyte density and the number of processes in the hilus. Cytokine mRNA expression (qPCR) was analyzed in MS and CONT groups, sacrificed (i) under basal (B) conditions or (ii) after a single stress event (SS) at PN15. In hippocampal extracts, MS increased IL-1β mRNA, under B and SS conditions while IL-6 and TNF-α did not change. In hypothalamic tissue, MS increased TNF-α and IL-6 mRNA, but not IL-1b, after SS. Peripheral concentrations of IL-1β were decreased under B and SS conditions in MS; IL-6 concentration increased after SS in MS pups, and TNF-α concentration was unchanged. In conclusion, MS activates microglial cells and decreases astrocyte density in the hippocampus. A differential cytokine expression is observed in the hippocampus and the hypothalamus after MS, and after SS. Also, MS triggers an independent response of peripheral cytokines. These specific responses together could contribute to decrease hippocampal neurogenesis and alter the neuroendocrine axis.

  20. Subneurotoxic copper(II)-induced NF-κB-dependent microglial activation is associated with mitochondrial ROS

    SciTech Connect

    Hu, Zhuqin; Yu, Fengxiang; Gong, Ping; Qiu, Yu; Zhou, Wei; Cui, Yongyao; Li, Juan Chen, Hongzhuan

    2014-04-15

    Microglia-mediated neuroinflammation and the associated neuronal damage play critical roles in the pathogenesis of neurodegenerative disorders. Evidence shows an elevated concentration of extracellular copper(II) in the brains of these disorders, which may contribute to neuronal death through direct neurotoxicity. Here we explored whether extracellular copper(II) triggers microglial activation. Primary rat microglia and murine microglial cell line BV-2 cells were cultured and treated with copper(II). The content of tumor necrosis factor-α (TNF-α) and nitric oxide in the medium was determined. Extracellular hydrogen peroxide was quantified by a fluorometric assay with Amplex Red. Mitochondrial superoxide was measured by MitoSOX oxidation. At subneurotoxic concentrations, copper(II) treatment induced a dose- and time-dependent release of TNF-α and nitric oxide from microglial cells, and caused an indirect, microglia-mediated neurotoxicity that was blocked by inhibition of TNF-α and nitric oxide production. Copper(II)-initiated microglial activation was accompanied with reduced IkB-α expression as well as phosphorylation and translocation of nuclear factor-κB (NF-κB) p65 and was blocked by NF-κB inhibitors (BAY11-7082 and SC-514). Moreover, copper(II) treatment evoked a rapid release of hydrogen peroxide from microglial cells, an effect that was not affected by NADPH oxidase inhibitors. N-acetyl-cysteine, a scavenger of reactive oxygen species (ROS), abrogated copper(II)-elicited microglial release of TNF-α and nitric oxide and subsequent neurotoxicity. Importantly, mitochondrial production of superoxide, paralleled to extracellular release of hydrogen peroxide, was induced after copper(II) stimulation. Our findings suggest that extracellular copper(II) at subneurotoxic concentrations could trigger NF-κB-dependent microglial activation and subsequent neurotoxicity. NADPH oxidase-independent, mitochondria-derived ROS may be involved in this activation

  1. Acrylamide induces mitochondrial dysfunction and apoptosis in BV-2 microglial cells.

    PubMed

    Liu, Zhigang; Song, Ge; Zou, Chen; Liu, Gongguan; Wu, Wanqiang; Yuan, Tian; Liu, Xuebo

    2015-07-01

    Acrylamide (ACR), a potent neurotoxin, can be produced during food processing at high temperature. This study examined the redox-dependent apoptotic and inflammatory responses of ACR in an immortalized mouse microglia cell line BV2. The exposure of BV2 cells to ACR reduced cell viability and induced apoptosis in a concentration-dependent manner. ACR impaired cell energy metabolism by decreasing mitochondrial respiration, anaerobic glycolysis, and lowering expression of the complex I, III, and IV subunits. Mitochondrial dysfunction was associated with a decrease of the mitochondrial membrane potential and the Bcl-2/Bax ratio, thus resulting in activation of the mitochondrion-driven apoptotic signaling. This was accompanied by (a) the modulation of redox-sensitive signaling, suppressed Akt activation and increased JNK and p38 activation, and (b) increased expression of NFκB and downstream inducible nitric oxide synthase (iNOS) and nitric oxide generation, thus supporting indirectly a proinflammatory effect of ACR. Nrf2 expression was also increased but not its translocation to the nucleus. Expectedly, the electrophilic attack of ACR on GSH resulted in substantial loss of GSH with a minor GSSG formation. These changes in the cell׳s redox status elicited by ACR resulted in increased H2O2 formation. The changes in mitochondrial functionality and complex subunit expression caused by ACR were reversed by N-acetyl-L-cysteine (NAC). Likewise, NAC restored the cell׳s redox status by increasing GSH levels with concomitant attenuation of H2O2 generation; these effects resulted in decreased apoptotic cell death and inflammatory responses. ACR-mediated mitochondrial dysfunction along with a more oxidized redox status seems to be critical events leading to activation of the intrinsic apoptotic pathway and inflammatory responses.

  2. Chronically active: activation of microglial proteolysis in ageing and neurodegeneration.

    PubMed

    Stolzing, Alexandra; Sethe, Sebastian; Grune, Tilman

    2005-01-01

    One of the microglial cell functions is the removal of modified extracellular proteins in the brain. The connection between protein oxidation, proteolysis, and microglial activation is the topic of this review. The effect of various activation agents on microglial cells with regard to changes in substrate uptake, proteolytic capacity and degradation efficiency of different types of oxidized protein materials is reviewed. It is shown that different activation stimuli initiate substrate-specific modulation for uptake and proteolysis, influencing an array of factors including receptor expression, lysosomal pH, and proteasome subunit composition. Age-related alterations in activation and proteolytic capacity in microglial cells are also discussed. In ageing, proteolytic effectiveness is diminished, while microglial cells are chronically activated and lose the oxidative burst ability, possibly supporting a 'vicious circle' of macrophage-induced neurodegeneration.

  3. Nanoelectroablation therapy for murine basal cell carcinoma

    SciTech Connect

    Nuccitelli, Richard; Tran, Kevin; Athos, Brian; Kreis, Mark; Nuccitelli, Pamela; Chang, Kris S.; Epstein, Ervin H.; Tang, Jean Y.

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Nanoelectroablation is a new, non-thermal therapy that triggers apoptosis in tumors. Black-Right-Pointing-Pointer Low energy, ultrashort, high voltage pulses ablate the tumor with little or no scar. Black-Right-Pointing-Pointer Nanoelectroablation eliminates 99.8% of the BCC but may leave a few remnants behind. Black-Right-Pointing-Pointer Pilot clinical trials on human BCCs are ongoing and leave no remnants in most cases. -- Abstract: When skin tumors are exposed to non-thermal, low energy, nanosecond pulsed electric fields (nsPEF), apoptosis is initiated both in vitro and in vivo. This nanoelectroablation therapy has already been proven effective in treating subdermal murine allograft tumors. We wanted to determine if this therapy would be equally effective in the treatment of autochthonous BCC tumors in Ptch1{sup +/-}K14-Cre-ER p53 fl/fl mice. These tumors are similar to human BCCs in histology and in response to drug therapy . We have treated 27 BCCs across 8 mice with either 300 pulses of 300 ns duration or 2700 pulses of 100 ns duration, all at 30 kV/cm and 5-7 pulses per second. Every nsPEF-treated BCC began to shrink within a day after treatment and their initial mean volume of 36 {+-} 5 (SEM) mm{sup 3} shrunk by 76 {+-} 3% over the ensuing two weeks. After four weeks, they were 99.8% ablated if the size of the treatment electrode matched the tumor size. If the tumor was larger than the 4 mm wide electrode, multiple treatments were needed for complete ablation. Treated tumors were harvested for histological analysis at various times after treatment and exhibited apoptosis markers. Specifically, pyknosis of nuclei was evident as soon as 2 days after nsPEF treatment, and DNA fragmentation as detected via TUNEL staining was also evident post treatment. Nanoelectroablation is effective in triggering apoptosis and remission of radiation-induced BCCs with a single 6 min-long treatment of 2700 pulses.

  4. Lonicera japonica THUNB. Extract Inhibits Lipopolysaccharide-Stimulated Inflammatory Responses by Suppressing NF-κB Signaling in BV-2 Microglial Cells.

    PubMed

    Kwon, Seung-Hwan; Ma, Shi-Xun; Hong, Sa-Ik; Lee, Seok-Yong; Jang, Choon-Gon

    2015-07-01

    In the current study, we evaluated the anti-inflammatory effects of Lonicera japonica THUNB. (LJ) and its underlying molecular mechanism in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. Our results indicated that LJ significantly inhibits LPS-stimulated production of nitric oxide (NO) and prostaglandin E2 (PGE2). In addition, LJ inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at both the protein and mRNA levels. In LPS-stimulated BV-2 microglial cells, LJ inhibited proinflammatory cytokines and chemokines, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase-9 (MMP-9) enzymatic activities, and/or mRNA expression, as well as reactive oxygen species (ROS) production. LJ significantly suppressed activation of nuclear factor-κB (NF-κB) and its translocation from the cytosol to the nucleus and suppressed the DNA-binding activity of NF-κB. Furthermore, LJ significantly inhibited phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK 1/2), p38 mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinases (PI3K)/Akt, and Janus kinase 1 (JAK1)/signal transducer and activator of transcription (STAT)1/3. Collectively, our findings indicated that the antineuroinflammatory properties of LJ in LPS-induced BV-2 microglial cells is due to downregulation of proinflammatory cytokines and chemokines downstream of inhibition of NF-κB activation.

  5. Modulation of Lipopolysaccharide Stimulated Nuclear Factor kappa B Mediated iNOS/NO Production by Bromelain in Rat Primary Microglial Cells

    PubMed Central

    Abbasi Habashi, Soraya; Sabouni, Farzaneh; Moghimi, Ali; Ansari Majd, Saeed

    2016-01-01

    Background: Microglial cells act as the sentinel of the central nervous system .They are involved in neuroprotection but are highly implicated in neurodegeneration of the aging brain. When over-activated, microglia release pro-inflammatory factors, such as nitric oxide (NO) and cytokines, which are critical in eliciting neuroinflammatory responses associated with neurodegenerative diseases. This study examined whether bromelain, the pineapple-derived extract, may exert an anti-inflammatory effect in primary microglia and may be neuroprotective by regulating microglial activation. Methods: Following the isolation of neonatal rat primary microglial cells, the activation profile of microglia was investigated by studying the effects of bromelain (5, 10, 20, and 30 µg/ml) on the levels of NO, inducible nitric oxide synthase (iNOS), and nuclear factor kappa B (NF-κB) in microglia treated with lipopolysaccharide (LPS) (1 µg/ml). Data were analyzed using Student's t-test. P values less than 0.05 were considered to be statistically significant, compared with the LPS-treated group without bromelain. Results: Results showed that pretreatment of rat primary microglia with bromelain, decreased the production of NO induced by LPS (1 µg/ml) treatment in a dose-dependent manner. Bromelain (30 µg/ml) also significantly reduced the expression of iNOS at mRNA level and NF-κB at protein level. Moreover, the study of mitochondrial activity in microglia indicated that bromelain had no cytotoxicity at any of the applied doses, suggesting that the anti-inflammatory effects of bromelain are not due to cell death. Conclusion: Bromelain can be of potential use as an agent for alleviation of symptoms in neurodegenerative diseases. PMID:26459398

  6. Lonicera japonica THUNB. Extract Inhibits Lipopolysaccharide-Stimulated Inflammatory Responses by Suppressing NF-κB Signaling in BV-2 Microglial Cells

    PubMed Central

    Kwon, Seung-Hwan; Ma, Shi-Xun; Hong, Sa-Ik; Lee, Seok-Yong

    2015-01-01

    Abstract In the current study, we evaluated the anti-inflammatory effects of Lonicera japonica THUNB. (LJ) and its underlying molecular mechanism in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. Our results indicated that LJ significantly inhibits LPS-stimulated production of nitric oxide (NO) and prostaglandin E2 (PGE2). In addition, LJ inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at both the protein and mRNA levels. In LPS-stimulated BV-2 microglial cells, LJ inhibited proinflammatory cytokines and chemokines, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase-9 (MMP-9) enzymatic activities, and/or mRNA expression, as well as reactive oxygen species (ROS) production. LJ significantly suppressed activation of nuclear factor-κB (NF-κB) and its translocation from the cytosol to the nucleus and suppressed the DNA-binding activity of NF-κB. Furthermore, LJ significantly inhibited phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK 1/2), p38 mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinases (PI3K)/Akt, and Janus kinase 1 (JAK1)/signal transducer and activator of transcription (STAT)1/3. Collectively, our findings indicated that the antineuroinflammatory properties of LJ in LPS-induced BV-2 microglial cells is due to downregulation of proinflammatory cytokines and chemokines downstream of inhibition of NF-κB activation. PMID:25897683

  7. Changes in phagocytosis and expression of microglial cells in craniocerebral injury mice models.

    PubMed

    Guo, F Y; Liu, T; Chen, J J; Gao, W; Yang, F; Zhou, X Y; Liao, Y L

    2016-01-01

    The objective of this study was to investigate the changes in phagocytic function and expression quantities of CD11b and tumor necrosis factor-α (TNF-α) among microglia cells of craniocerebral injury mice. Modified Feeney method was used to establish the craniocerebral injury mice models. Twenty-one male SPF mice were divided into a control group and a trauma group. The scalp was incised and a bone window was opened in the control group without cerebral injury. In the trauma group, the mice were sacrificed after the craniocerebral injury at 1, 3, 6, 12, 24 and 48 h to make frozen sections of cerebral tissues. The phagocytic rate of microglia cells was observed by using fluorescent microsphere. The changes in the expression quantities of CD11b and TNF-α were detected by enzyme-linked immuno sorbent assay (ELISA). The phagocytic ability of the microglia cells after the craniocerebral injury increased at 1 h after injury compared with that of the control group (P less than 0.01). The expression of surface antigen CD11b of the microglia cells and the expression of TNF-α increased at 1, 3, 6, 12, 24 and 48 h after the injury compared with those of the control group (P less than 0.01). The phagocytic ability of the microglia cells increased. The expressions of CD11b and TNF-α were also gradually enhanced in the acute phase after craniocerebral injury, and then gradually decreased to the normal level. The expressions of CD11b and TNF-α indicated a high consistency with the changing trend of the phagocytic ability, suggesting that the microglia cells may participate in the regulation of the inflammatory process of the central nervous system through absorbing apoptotic cells and increasing and secreting inflammatory and anti-inflammatory factors.

  8. Changes in phagocytosis and expression of microglial cells in craniocerebral injury mice models.

    PubMed

    Guo, F Y; Liu, T; Chen, J J; Gao, W; Yang, F; Zhou, X Y; Liao, Y L

    2016-01-01

    The objective of this study was to investigate the changes in phagocytic function and expression quantities of CD11b and tumor necrosis factor-α (TNF-α) among microglia cells of craniocerebral injury mice. Modified Feeney method was used to establish the craniocerebral injury mice models. Twenty-one male SPF mice were divided into a control group and a trauma group. The scalp was incised and a bone window was opened in the control group without cerebral injury. In the trauma group, the mice were sacrificed after the craniocerebral injury at 1, 3, 6, 12, 24 and 48 h to make frozen sections of cerebral tissues. The phagocytic rate of microglia cells was observed by using fluorescent microsphere. The changes in the expression quantities of CD11b and TNF-α were detected by enzyme-linked immuno sorbent assay (ELISA). The phagocytic ability of the microglia cells after the craniocerebral injury increased at 1 h after injury compared with that of the control group (P less than 0.01). The expression of surface antigen CD11b of the microglia cells and the expression of TNF-α increased at 1, 3, 6, 12, 24 and 48 h after the injury compared with those of the control group (P less than 0.01). The phagocytic ability of the microglia cells increased. The expressions of CD11b and TNF-α were also gradually enhanced in the acute phase after craniocerebral injury, and then gradually decreased to the normal level. The expressions of CD11b and TNF-α indicated a high consistency with the changing trend of the phagocytic ability, suggesting that the microglia cells may participate in the regulation of the inflammatory process of the central nervous system through absorbing apoptotic cells and increasing and secreting inflammatory and anti-inflammatory factors. PMID:27358141

  9. Chronotopographical distribution patterns of cell death and of lectin-positive macrophages/microglial cells during the visual system ontogeny of the small-spotted catshark Scyliorhinus canicula

    PubMed Central

    Bejarano-Escobar, Ruth; Blasco, Manuel; Durán, Ana Carmen; Martín-Partido, Gervasio; Francisco-Morcillo, Javier

    2013-01-01

    The patterns of distribution of TUNEL-positive bodies and of lectin-positive phagocytes were investigated in the developing visual system of the small-spotted catshark Scyliorhinus canicula, from the optic vesicle stage to adulthood. During early stages of development, TUNEL-staining was mainly found in the protruding dorsal part of the optic cup and in the presumptive optic chiasm. Furthermore, TUNEL-positive bodies were also detected during detachment of the embryonic lens. Coinciding with the developmental period during which ganglion cells began to differentiate, an area of programmed cell death occurred in the distal optic stalk and in the retinal pigment epithelium that surrounds the optic nerve head. The topographical distribution of TUNEL-positive bodies in the differentiating retina recapitulated the sequence of maturation of the various layers and cell types following a vitreal-to-scleral gradient. Lectin-positive cells apparently entered the retina by the optic nerve head when the retinal layering was almost complete. As development proceeded, these labelled cells migrated parallel to the axon fascicles of the optic fiber layer and then reached more external layers by radial migration. In the mature retina, lectin-positive cells were confined to the optic fiber layer, ganglion cell layer and inner plexiform layer. No evident correlation was found between the chronotopographical pattern of distribution of TUNEL-positive bodies and the pattern of distribution of lectin-labelled macrophages/microglial cells during the shark′s visual system ontogeny. PMID:23758763

  10. Small-Ruminant Lentivirus Enhances PrP-Sc Accumulation in Cultured Sheep Microglial Cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Scrapie is the prototype member of the family of transmissible spongiform encephalopathies, fatal neurodegenerative diseases associated with conversion and accumulation of prion proteins in a number of neural and extraneural cell types. Although scrapie has been the focus of research investigations...

  11. Amyloid β25-35 induced ROS-burst through NADPH oxidase is sensitive to iron chelation in microglial Bv2 cells.

    PubMed

    Part, Kristin; Künnis-Beres, Kai; Poska, Helen; Land, Tiit; Shimmo, Ruth; Zetterström Fernaeus, Sandra

    2015-12-10

    Iron chelation therapy and inhibition of glial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can both represent possible routes for Alzheimer's disease modifying therapies. The metal hypothesis is largely focused on direct binding of metals to the N-terminal hydrophilic 1-16 domain peptides of Amyloid beta (Aβ) and how they jointly give rise to reactive oxygen species (ROS) production. The cytotoxic effects of Aβ through ROS and metals are mainly studied in neuronal cells using full-length Aβ1-40/42 peptides. Here we study cellularly-derived ROS during 2-60min in response to non-metal associated mid domain Aβ25-35 in microglial Bv2 cells by fluorescence based spectroscopy. We analyze if Aβ25-35 induce ROS production through NADPH oxidase and if the production is sensitive to iron chelation. NADPH oxidase inhibitor diphenyliodonium (DPI) is used to confirm the production of ROS through NADPH oxidase. We modulate cellular iron homeostasis by applying cell permeable iron chelators desferrioxamine (DFO) and deferiprone (DFP). NADPH oxidase subunit gp91-phox level was analyzed by Western blotting. Our results show that Aβ25-35 induces strong ROS production through NADPH oxidase in Bv2 microglial cells. Intracellular iron depletion resulted in restrained Aβ25-35 induced ROS.

  12. Anti-inflammatory effects of three-dimensional graphene foams cultured with microglial cells.

    PubMed

    Song, Qin; Jiang, Ziyun; Li, Ning; Liu, Ping; Liu, Liwei; Tang, Mingliang; Cheng, Guosheng

    2014-08-01

    One of the key goals in nerve tissue engineering is to develop new materials which cause less or no neuroinflammation. Despite the rapid advances of using graphene as a neural interface material, it still remains unknown whether graphene could provoke neuroinflammation or not, and whether and how the topographical features of graphene influence the neuroinflammation induction. By immunofluorescence, Elisa technique, western blot, scanning electron microscope (SEM) methods, we investigated the pro- and/or anti-inflammatory responses of microglia in the graphene films (2D-graphene) or graphene foams (3D-graphene) culturing systems. Furthermore, the growth situations of the neural stem cells (NSCs) in the conditioned culture medium produced in the graphene substrates were evaluated. The results show that: 1) neither 2D nor 3D graphene induced distinct neuroinflammation when compared to the tissue culture polystyrene (TCPS) substrates; 2) the topographical structures of the graphene might affect the material/cell interactions, leading to disparate effects on lipopolysaccharide (LPS)-induced neuroinflammation; 3) 3D graphene exhibited a remarkable capability of rescuing LPS-induced neuroinflammation probably through the restriction of microglia morphological transformation by the unique topographical features on the surface, showing the ability of anti-inflammation against external insults, while 2D graphene failed to. These results provide insights into the diverse biological effects of the material's topographical structures and open new opportunity for the applications of graphene in neuroscience. PMID:24875763

  13. Resolvin D1 and E1 promote resolution of inflammation in microglial cells in vitro.

    PubMed

    Rey, C; Nadjar, A; Buaud, B; Vaysse, C; Aubert, A; Pallet, V; Layé, S; Joffre, C

    2016-07-01

    Sustained inflammation in the brain together with microglia activation can lead to neuronal damage. Hence limiting brain inflammation and activation of microglia is a real therapeutic strategy for inflammatory disease. Resolvin D1 (RvD1) and resolvin E1 (RvE1) derived from n-3 long chain polyunsaturated fatty acids are promising therapeutic compounds since they actively turn off the systemic inflammatory response. We thus evaluated the anti-inflammatory activities of RvD1 and RvE1 in microglia cells in vitro. BV2 cells were pre-incubated with RvD1 or RvE1 before lipopolysaccharide (LPS) treatment. RvD1 and RvE1 both decreased LPS-induced proinflammatory cytokines (TNF-α, IL-6 and IL-1β) gene expression, suggesting their proresolutive activity in microglia. However, the mechanisms involved are distinct as RvE1 regulates NFκB signaling pathway and RvD1 regulates miRNAs expression. Overall, our findings support that pro-resolving lipids are involved in the resolution of brain inflammation and can be considered as promising therapeutic agents for brain inflammation. PMID:26718448

  14. Safflower Yellow regulates microglial polarization and inhibits inflammatory response in LPS-stimulated Bv2 cells.

    PubMed

    Yang, Xing-Wang; Li, Yan-Hua; Zhang, Hui; Zhao, Yong-Fei; Ding, Zhi-Bin; Yu, Jie-Zhong; Liu, Chun-Yun; Liu, Jian-Chun; Jiang, Wei-Jia; Feng, Qian-Jin; Xiao, Bao-Guo; Ma, Cun-Gen

    2016-03-01

    Activated microglia, especially polarized M1 cells, produce pro-inflammatory cytokines and free radicals, thereby contributing directly to neuroinflammation and various brain disorders. Given that excessive or chronic neuroinflammation within the central nervous system (CNS) exacerbates neuronal damage, molecules that modulate neuroinflammation are candidates as neuroprotective agents. In this study, we provide evidence that Safflor yellow (SY), the main active component in the traditional Chinese medicine safflower, modulates inflammatory responses by acting directly on BV2 microglia. LPS stimulated BV2 cells to upregulate expression of TLR4-Myd88 and MAPK-NF-κB signaling pathways and to release IL-1β, IL-6, TNF-α, and COX-2. However, SY treatment inhibited expression of TLR4-Myd88 and p-38/p-JNK-NF-κB, downregulated expression of iNOS, CD16/32, and IL-12, and upregulated CD206 and IL-10. In conclusion, our results demonstrate that SY exerts an anti-inflammatory effect on BV2 microglia, possibly through TLR-4/p-38/p-JNK/NF-κB signaling pathways and the conversion of microglia from inflammatory M1 to an anti-inflammatory M2 phenotype. PMID:26634402

  15. TAM receptors affect adult brain neurogenesis by negative regulation of microglial cell activation.

    PubMed

    Ji, Rui; Tian, Shifu; Lu, Helen J; Lu, Qingjun; Zheng, Yan; Wang, Xiaomin; Ding, Jixiang; Li, Qiutang; Lu, Qingxian

    2013-12-15

    TAM tyrosine kinases play multiple functional roles, including regulation of the target genes important in homeostatic regulation of cytokine receptors or TLR-mediated signal transduction pathways. In this study, we show that TAM receptors affect adult hippocampal neurogenesis and loss of TAM receptors impairs hippocampal neurogenesis, largely attributed to exaggerated inflammatory responses by microglia characterized by increased MAPK and NF-κB activation and elevated production of proinflammatory cytokines that are detrimental to neuron stem cell proliferation and neuronal differentiation. Injection of LPS causes even more severe inhibition of BrdU incorporation in the Tyro3(-/-)Axl(-/-)Mertk(-/-) triple-knockout (TKO) brains, consistent with the LPS-elicited enhanced expression of proinflammatory mediators, for example, IL-1β, IL-6, TNF-α, and inducible NO synthase, and this effect is antagonized by coinjection of the anti-inflammatory drug indomethacin in wild-type but not TKO brains. Conditioned medium from TKO microglia cultures inhibits neuron stem cell proliferation and neuronal differentiation. IL-6 knockout in Axl(-/-)Mertk(-/-) double-knockout mice overcomes the inflammatory inhibition of neurogenesis, suggesting that IL-6 is a major downstream neurotoxic mediator under homeostatic regulation by TAM receptors in microglia. Additionally, autonomous trophic function of the TAM receptors on the proliferating neuronal progenitors may also promote progenitor differentiation into immature neurons.

  16. Mesenchymal Stem Cells Reduce Murine Atherosclerosis Development

    PubMed Central

    Frodermann, Vanessa; van Duijn, Janine; van Pel, Melissa; van Santbrink, Peter J.; Bot, Ilze; Kuiper, Johan; de Jager, Saskia C. A.

    2015-01-01

    Mesenchymal stem cells (MSCs) have regenerative properties, but recently they were also found to have immunomodulatory capacities. We therefore investigated whether MSCs could reduce atherosclerosis, which is determined by dyslipidaemia and chronic inflammation. We adoptively transferred MSCs into low-density lipoprotein-receptor knockout mice and put these on a Western-type diet to induce atherosclerosis. Initially after treatment, we found higher levels of circulating regulatory T cells. In the long-term, overall numbers of effector T cells were reduced by MSC treatment. Moreover, MSC-treated mice displayed a significant 33% reduction in circulating monocytes and a 77% reduction of serum CCL2 levels. Most strikingly, we found a previously unappreciated effect on lipid metabolism. Serum cholesterol was reduced by 33%, due to reduced very low-density lipoprotein levels, likely a result of reduced de novo hepatic lipogenesis as determined by a reduced expression of Stearoyl-CoA desaturase-1 and lipoprotein lipase. MSCs significantly affected lesion development, which was reduced by 33% in the aortic root. These lesions contained 56% less macrophages and showed a 61% reduction in T cell numbers. We show here for the first time that MSC treatment affects not only inflammatory responses but also significantly reduces dyslipidaemia in mice. This makes MSCs a potent candidate for atherosclerosis therapies. PMID:26490642

  17. Quantitative image analysis of cell colocalization in murine bone marrow.

    PubMed

    Mokhtari, Zeinab; Mech, Franziska; Zehentmeier, Sandra; Hauser, Anja E; Figge, Marc Thilo

    2015-06-01

    Long-term antibody production is a key property of humoral immunity and is accomplished by long-lived plasma cells. They mainly reside in the bone marrow, whose importance as an organ hosting immunological memory is becoming increasingly evident. Signals provided by stromal cells and eosinophils may play an important role for plasma cell maintenance, constituting a survival microenvironment. In this joint study of experiment and theory, we investigated the spatial colocalization of plasma cells, eosinophils and B cells by applying an image-based systems biology approach. To this end, we generated confocal fluorescence microscopy images of histological sections from murine bone marrow that were subsequently analyzed in an automated fashion. This quantitative analysis was combined with computer simulations of the experimental system for hypothesis testing. In particular, we tested the observed spatial colocalization of cells in the bone marrow against the hypothesis that cells are found within available areas at positions that were drawn from a uniform random number distribution. We find that B cells and plasma cells highly colocalize with stromal cells, to an extent larger than in the simulated random situation. While B cells are preferentially in contact with each other, i.e., form clusters among themselves, plasma cells seem to be solitary or organized in aggregates, i.e., loosely defined groups of cells that are not necessarily in direct contact. Our data suggest that the plasma cell bone marrow survival niche facilitates colocalization of plasma cells with stromal cells and eosinophils, respectively, promoting plasma cell longevity.

  18. [Microglial Phagocytosis in the Neurodegenerative Diseases].

    PubMed

    Cao, Sheng-nan; Bao, Xiu-qi; Sun, Hua; Zhang, Dan

    2016-04-01

    Microglia are the resident innate immune cells in the brain. Under endogenous or exogenous stimulates, they become activated and play an important role in the neurodegenerative diseases. Microglial phagocytosis is a process of receptor-mediated engulfment and degradation of apoptotic cells. In addition, microglia can phagocyte brain-specific cargo, such as myelin debris and abnormal protein aggregation. However, recent studies have shown that microglia can also phagocyte stressed-but-viable neurons, causing loss of neurons in the brain. Thus, whether microglial phagocytosis is beneficial or not in neurodegenerative disease remains controversial. This article reviews microglial phagocytosis related mechanisms and its potential roles in neurodegenerative diseases, with an attempt to provide new insights in the treatment of neurodegenerative diseases. PMID:27181903

  19. [Microglial Phagocytosis in the Neurodegenerative Diseases].

    PubMed

    Cao, Sheng-nan; Bao, Xiu-qi; Sun, Hua; Zhang, Dan

    2016-04-01

    Microglia are the resident innate immune cells in the brain. Under endogenous or exogenous stimulates, they become activated and play an important role in the neurodegenerative diseases. Microglial phagocytosis is a process of receptor-mediated engulfment and degradation of apoptotic cells. In addition, microglia can phagocyte brain-specific cargo, such as myelin debris and abnormal protein aggregation. However, recent studies have shown that microglia can also phagocyte stressed-but-viable neurons, causing loss of neurons in the brain. Thus, whether microglial phagocytosis is beneficial or not in neurodegenerative disease remains controversial. This article reviews microglial phagocytosis related mechanisms and its potential roles in neurodegenerative diseases, with an attempt to provide new insights in the treatment of neurodegenerative diseases.

  20. Exposure to electromagnetic field attenuates oxygen-glucose deprivation-induced microglial cell death by reducing intracellular Ca(2+) and ROS.

    PubMed

    Duong, Cao Nguyen; Kim, Jae Young

    2016-01-01

    Purpose The aim of this research was to demonstrate the protective effects of electromagnetic field (EMF) exposure on the human microglial cell line, HMO6, against ischemic cell death induced by in vitro oxygen-glucose deprivation (OGD). Materials and methods HMO6 cells were cultured for 4 h under OGD with or without exposure to EMF with different combinations of frequencies and intensities (10, 50, or 100 Hz/1 mT and 50 Hz/0.01, 0.1, or 1 mT). Cell survival, intracellular calcium and reactive oxygen species (ROS) levels were measured. Results OGD caused significant HMO6 cell death as well as elevation of intracellular Ca(2+) and ROS levels. Among different combinations of EMF frequencies and intensities, 50 Hz/1 mT EMF was the most potent to attenuate OGD-induced cell death and intracellular Ca(2+) and ROS levels. A significant but less potent protective effect was also found at 10 Hz/1 mT, whereas no protective effect was found at other combinations of EMF. A xanthine oxidase inhibitor reversed OGD-induced ROS production and cell death, while NADPH oxidase and mitochondrial respiration chain complex II inhibitors did not affect cell death. Conclusions 50 Hz/1 mT EMF protects human microglial cells from OGD-induced cell death by interfering with OGD-induced elevation of intracellular Ca(2+) and ROS levels, and xanthine oxidase is one of the main mediators involved in OGD-induced HMO6 cell death. Non-invasive treatment of EMF radiation may be clinically useful to attenuate hypoxic-ischemic brain injury.

  1. Dye-mediated photosensitization of murine neuroblastoma cells

    SciTech Connect

    Sieber, F.; Sieber-Blum, M.

    1986-04-01

    The purpose of this study was to determine if photosensitization mediated by the fluorescent dye, merocyanine 540, could be used to preferentially kill murine neuroblastoma cells in simulated autologous remission marrow grafts. Simultaneous exposure of Neuro 2a or NB41A3 neuroblastoma cells to merocyanine 540 and white light reduced the concentration of in vitro-clonogenic tumor cells 50,000-fold. By contrast, the same treatment had little effect on the graft's ability to rescue lethally irradiated syngeneic hosts. Lethally irradiated C57BL/6J X A/J F1 mice transplanted with photosensitized mixtures of neuroblastoma cells and normal marrow cells (1:100 or 1:10) survived without developing neuroblastomas. It is conceivable that merocyanine 540-mediated photosensitization will prove useful for the extracorporeal purging of residual neuroblastoma cells from human autologous remission marrow grafts.

  2. Flow cytometric quantification of radiation responses of murine peritoneal cells

    SciTech Connect

    Tokita, N.; Raju, M.R.

    1982-01-01

    Methods have been developed to distinguish subpopulations of murine peritoneal cells, and these were applied to the measurement of early changes in peritoneal cells after irradiation. The ratio of the two major subpopulations in the peritoneal fluid, lymphocytes and macrophages, was measured rapidly by means of cell volume distribution analysis as well as by hypotonic propidium iodide (PI) staining. After irradiation, dose and time dependent changes were noted in the cell volume distributions: a rapid loss of peritoneal lymphocytes, and an increase in the mean cell volume of macrophages. The hypotonic PI staining characteristics of the peritoneal cells showed two or three distinctive G/sub 1/ peaks. The ratio of the areas of these peaks was also found to be dependent of the radiation dose and the time after irradiation. These results demonstrate that these two parameters may be used to monitor changes induced by irradiation (biological dosimetry), and to sort different peritoneal subpopulations.

  3. Theiler's murine encephalomyelitis virus induces tumour necrosis factor-alpha in murine astrocyte cell cultures.

    PubMed Central

    Sierra, A; Rubio, N

    1993-01-01

    Cytokines have been postulated to exert an important modulatory and recruiting role in demyelination induced by Theiler's murine encephalomyelitis virus (TMEV) in SJL/J mice. Using a cytolytic bioassay and ELISA, we have detected and quantified a cytokine, tumour necrosis factor-alpha (TNF-alpha), in supernatants from astrocyte cultures infected in vitro with TMEV. TNF was detected only after TMEV-specific infection of astrocyte cultures (approximately 200-400 U/ml). In vitro TNF synthesis appeared in a dose- and time-dependent manner and was produced by both SJL/J (a strain susceptible to TMEV-induced demyelination) and BALB/c (a resistant strain) astrocytes. The precise nature of TNF activity was further assessed by fast protein liquid chromatography (FPLC) and antibody neutralization. These results indicate an active role for astrocytes as accessory immune cells in our experimental model for multiple sclerosis. PMID:8478023

  4. Myelin-specific T cells induce interleukin-1beta expression in lesion-reactive microglial-like cells in zones of axonal degeneration.

    PubMed

    Grebing, Manuela; Nielsen, Helle H; Fenger, Christina D; T Jensen, Katrine; von Linstow, Christian U; Clausen, Bettina H; Söderman, Martin; Lambertsen, Kate L; Thomassen, Mads; Kruse, Torben A; Finsen, Bente

    2016-03-01

    Infiltration of myelin-specific T cells into the central nervous system induces the expression of proinflammatory cytokines in patients with multiple sclerosis (MS). We have previously shown that myelin-specific T cells are recruited into zones of axonal degeneration, where they stimulate lesion-reactive microglia. To gain mechanistic insight, we used RNA microarray analysis to compare the transcript profile in hippocampi from perforant pathway axonal-lesioned mice with and without adoptively transferred myelin-specific T cells 2 days postlesion, when microglia are clearly lesion reactive. Pathway analysis revealed that, among the 1,447 differently expressed transcripts, the interleukin (IL)-1 pathway including all IL-1 receptor ligands was upregulated in the presence of myelin-specific T cells. Quantitative polymerase chain reaction showed increased mRNA levels of IL-1β, IL-1α, and IL-1 receptor antagonist in the T-cell-infiltrated hippocampi from axonal-lesioned mice. In situ hybridization and immunohistochemistry showed a T-cell-enhanced lesion-specific expression of IL-1β mRNA and protein, respectively, and induction of the apoptosis-associated speck-like protein, ASC, in CD11b(+) cells. Double in situ hybridization showed colocalization of IL-1β mRNA in a subset of CD11b mRNA(+) cells, of which many were part of cellular doublets or clusters, characteristic of proliferating, lesion-reactive microglia. Double-immunofluorescence showed a T-cell-enhanced colocalization of IL-1β to CD11b(+) cells, including lesion-reactive CD11b(+) ramified microglia. These results suggest that myelin-specific T cells stimulate lesion-reactive microglial-like cells to produce IL-1β. These findings are relevant to understand the consequences of T-cell infiltration in white and gray matter lesions in patients with MS.

  5. Index sorting resolves heterogeneous murine hematopoietic stem cell populations.

    PubMed

    Schulte, Reiner; Wilson, Nicola K; Prick, Janine C M; Cossetti, Chiara; Maj, Michal K; Gottgens, Berthold; Kent, David G

    2015-09-01

    Recent advances in the cellular and molecular biology of single stem cells have uncovered significant heterogeneity in the functional properties of stem cell populations. This has prompted the development of approaches to study single cells in isolation, often performed using multiparameter flow cytometry. However, many stem cell populations are too rare to test all possible cell surface marker combinations, and virtually nothing is known about functional differences associated with varying intensities of such markers. Here we describe the use of index sorting for further resolution of the flow cytometric isolation of single murine hematopoietic stem cells (HSCs). Specifically, we associate single-cell functional assay outcomes with distinct cell surface marker expression intensities. High levels of both CD150 and EPCR associate with delayed kinetics of cell division and low levels of differentiation. Moreover, cells that do not form single HSC-derived clones appear in the 7AAD(dim) fraction, suggesting that even low levels of 7AAD staining are indicative of less healthy cell populations. These data indicate that when used in combination with single-cell functional assays, index sorting is a powerful tool for refining cell isolation strategies. This approach can be broadly applied to other single-cell systems, both to improve isolation and to acquire additional cell surface marker information.

  6. Ulinastatin suppresses lipopolysaccharide-induced prostaglandin E2 synthesis and nitric oxide production through the downregulation of nuclear factor‑κB in BV2 mouse microglial cells.

    PubMed

    Sung, Yun-Hee; Shin, Mal-Soon; Ko, Il-Gyu; Kim, Sung-Eun; Kim, Chang-Ju; Ahn, Hyun-Jong; Yoon, Hye-Sun; Lee, Bong-Jae

    2013-05-01

    Ulinastatin is an intrinsic serine-protease urinary trypsin inhibitor that can be extracted and purified from human urine. Urinary trypsin inhibitors are widely used to treat patients with acute inflammatory disorders, such as shock and pancreatitis. However, although the anti-inflammatory activities of urinary trypsin inhibitors have been investigated, the mechanisms underlying their actions are not yet fully understood. In the present study, we evaluated the effect of ulinastatin on lipopolysaccharide (LPS)-induced inflammation in relation with nuclear factor-κB (NF-κB) activation using BV2 mouse microglial cells. To accomplish this, we performed a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, reverse transcription-polymerase chain reaction (RT-PCR), western blot analysis, electrophoretic mobility gel shift assay (EMSA), prostaglandin E(2) (PGE(2)) immunoassay and nitric oxide (NO) detection. The results demonstrated that ulinastatin suppressed PGE2 synthesis and NO production by inhibiting the LPS-induced mRNA and protein expression of cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS) in BV2 mouse microglial cells. Ulinastatin suppressed the activation of NF-κB in the nucleus. These findings demonstrate that ulinastatin exerts analgesic and anti-inflammatory effects that possibly occur via the suppression of COX-2 and iNOS expression through the downregulation of NF-κB activity.

  7. Involvement of mitogen-activated protein kinase and NF-κB signaling pathways in perfluorooctane sulfonic acid-induced inflammatory reaction in BV2 microglial cells.

    PubMed

    Zhu, Jingying; Qian, Wenyi; Wang, Yixin; Gao, Rong; Wang, Jun; Xiao, Hang

    2015-12-01

    Microglial activation is closely related to the pathogenesis of neurodegenerative diseases by producing proinflammatory cytokines. Perfluorooctane sulfonic acid (PFOS), known as an emerging persistent organic pollutant, is reported to disturb human immune homeostasis; however, whether it affects cytokine production or the immune response in the central nervous system remains unclear. The present study was aimed to explore whether PFOS contributed to inflammatory action and to investigate the corresponding mechanisms in BV2 microglia. PFOS-mediated morphologic changes, cytokine responses and signaling events were examined by light microscopy, real-time polymerase chain reaction, enzyme-linked immunosorbent assay and Western blot assays. Our results indicated that PFOS increased BV2 cells activation and simultaneously increased tumor necrosis factor alpha and interleukin-6 expression. In addition, the c-Jun N-terminal protein kinase inhibitor (SP600125), as well as ERK1/2 blocker (PD98059), transcriptionally at least, displayed anti-inflammatory properties on PFOS-elicited cytokine responses. Moreover, the inflammatory transcription factor NF-κB was specifically activated by PFOS as well. These results, taken together, suggested that PFOS exerts its functional effects on the response of microglial cell activation via, in part, the c-Jun N-terminal protein kinase, ERK and NF-κB signaling pathways with its subsequent influence on proinflammatory action.

  8. Auraptene and Other Prenyloxyphenylpropanoids Suppress Microglial Activation and Dopaminergic Neuronal Cell Death in a Lipopolysaccharide-Induced Model of Parkinson’s Disease

    PubMed Central

    Okuyama, Satoshi; Semba, Tomoki; Toyoda, Nobuki; Epifano, Francesco; Genovese, Salvatore; Fiorito, Serena; Taddeo, Vito Alessandro; Sawamoto, Atsushi; Nakajima, Mitsunari; Furukawa, Yoshiko

    2016-01-01

    In patients with Parkinson’s disease (PD), hyperactivated inflammation in the brain, particularly microglial hyperactivation in the substantia nigra (SN), is reported to be one of the triggers for the delayed loss of dopaminergic neurons and sequential motor functional impairments. We previously reported that (1) auraptene (AUR), a natural prenyloxycoumain, suppressed inflammatory responses including the hyperactivation of microglia in the ischemic brain and inflamed brain, thereby inhibiting neuronal cell death; (2) 7-isopentenyloxycoumarin (7-IP), another natural prenyloxycoumain, exerted anti-inflammatory and neuroprotective effects against excitotoxicity; and (3) 4′-geranyloxyferulic acid (GOFA), a natural prenyloxycinnamic acid, also exerted anti-inflammatory effects. In the present study, using an intranigral lipopolysaccharide (LPS)-induced PD-like mouse model, we investigated whether AUR, 7-IP, and GOFA suppress microglial activation and protect against dopaminergic neuronal cell death in the SN. We successfully showed that these prenyloxyphenylpropanoids exhibited these prospective abilities, suggesting the potential of these compounds as neuroprotective agents for patients with PD. PMID:27763495

  9. Surface Markers for the Murine Oval Cell Response

    PubMed Central

    Dorrell, Craig; Erker, Laura; Lanxon-Cookson, Kelsea M.; Abraham, Stephanie L.; Victoroff, Tristan; Ro, Simon; Canaday, Pamela S.; Streeter, Philip R.; Grompe, Markus

    2011-01-01

    The biology of progenitor activation in the liver is of considerable medical and scientific interest. The powerful genetic tools available for the mouse make it an ideal model system to study this complex process involving many different cell types. However, reagents for the isolation and study of distinct hepatic subpopulations have been quite limited compared to those available for hematopoietic cells. To produce cell surface reactive reagents more specific for the oval cell response, we generated a new collection of monoclonal antibodies by immunization of Fischer rats with enzymatically dispersed nonparenchymal cells from the livers of adult mice treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Each of the resulting antibodies recognized a surface antigen present on a liver cell subset and permitted the viable isolation of the associated subpopulation by fluorescence-activated cell sorting. Differential activity was observed on normal liver cells and at different stages of oval cell activation, indicating potential utility for progenitor cell identification. The subdivision of liver cells using these tools should facilitate the study of the biology of ductal and periductal hepatic cell types, including progenitors. Conclusion A new panel of surface reactive monoclonal antibodies to support investigation of the murine oval cell response has been developed. PMID:18726953

  10. Effects of caffeine and paracetamol alone or in combination with acetylsalicylic acid on prostaglandin E(2) synthesis in rat microglial cells.

    PubMed

    Fiebich, B L; Lieb, K; Hüll, M; Aicher, B; van Ryn, J; Pairet, M; Engelhardt, G

    2000-08-23

    Paracetamol has mild analgesic and antipyretic properties and is, along with acetylsalicylic acid, one of the most popular "over the counter" analgesic agents. However, the mechanism underlying its clinical effects is unknown. Another drug whose mechanism of action is unknown is caffeine, which is often used in combination with other analgesics, augmenting their effect. We investigated the inhibitory effect of paracetamol and caffeine on lipopolysaccharide (LPS)-induced cyclooxygenase (COX)- and prostaglandin (PG)E(2)-synthesis in primary rat microglial cells and compared it with the effect of acetylsalicylic acid, salicylic acid, and dipyrone. Furthermore, combinations of these drugs were used to investigate a possible synergistic inhibitory effect on PGE(2)-synthesis. Both paracetamol (IC(50)=7.45 microM) and caffeine (IC(50)=42.5 microM) dose-dependently inhibited microglial PGE(2) synthesis. In combination with acetylsalicylic acid (IC(50)=3.12 microM), both substances augmented the inhibitory effect of acetylsalicylic acid on LPS-induced PGE(2)-synthesis. Whereas paracetamol inhibited only COX enzyme activity, caffeine also inhibited COX-2 protein synthesis. These results are compatible with the view that the clinical activity of paracetamol and caffeine is due to inhibition of COX. Furthermore, these results may help explain the clinical experience of an adjuvant analgesic effect of caffeine and paracetamol when combined with acetylsalicylic acid.

  11. Murine fertilized ovum, blastomere and morula cells lacking SP phenotype.

    PubMed

    Xu, YiXin; He, ZhiYing; Zhu, HaiYing; Chen, XueSong; Li, JianXiu; Zhang, HongXia; Pan, XingHua; Hu, YiPing

    2007-12-01

    In the field of stem cell research, SP (side population) phenotype is used to define the property that cells maintain a high efflux capability for some fluorescent dye, such as Hoechst 33342. Recently, many researches proposed that SP phenotype is a phenotype shared by some stem cells and some progenitor cells, and that SP phenotype is regarded as a candidate purification marker for stem cells. In this research, murine fertilized ova (including conjugate and single nucleus fertilized ova), 2-cell stage and 8-cell stage blastomeres, morulas and blastocysts were isolated and directly stained by Hoechst 33342 dye. The results show that fertilized ovum, blastomere and morula cells do not demonstrate any ability to efflux the dye. However, the inner cell mass (ICM) cells of blastocyst exhibit SP phenotype, which is consistent with the result of embryonic stem cells (ESCs) in vitro. These results indicate that the SP phenotype of ICM-derived ESCs is an intrinsic property and independent of the culture condition in vitro, and that SP phenotype is one of the characteristics of at least some pluripotent stem cells, but is not shared by totipotent stem cells. In addition, the result that the SP phenotype of ICM cells disappeared when the inhibitor verapamil was added into medium implies that the SP phenotype is directly associated with ABCG2. These results suggest that not all the stem cells demonstrate SP phenotype, and that SP phenotype might act as a purification marker for partial stem cells such as some pluripotent embryonic stem cells and multipotent adult stem cells, but not for all stem cells exampled by the totipotent stem cells in the very early stage of mouse embryos.

  12. T cell-activating protein on murine lymphocytes.

    PubMed

    Yeh, E T; Reiser, H; Benacerraf, B; Rock, K L

    1986-12-01

    A functional T cell surface molecule, T cell-activating protein (TAP), has been identified on murine lymphocytes. TAP is a protein with an apparent molecular mass of 10-12 kilodaltons (kDa) nonreduced, 16-17 kDa reduced. Cross-linking of TAP can result in profound activation of T lymphocytes to produce lymphokines and to enter the cell cycle. Furthermore, antibody binding to TAP can modulate antigen-driven T cell stimulation. Current data suggest that TAP is physically distinct from the T cell receptor complex. On unstimulated lymphocytes, TAP is expressed on T cells and defines heterogeneity within the major T cell subsets. Its profile of expression is rapidly altered on cell activation. Ontologically, it is first detected in the thymus, where it is found on both the most immature and the most mature cell subsets, and it is functional on both. Together, these TAP+ cells constitute a small fraction of thymocytes. TAP expression, however, defines the immunocompetent compartment of the thymus, and thus could be involved in functional maturation. Finally, the gene controlling TAP expression has been mapped, and is tightly linked to a locus of hematopoietic antigens (Ly-6). TAP is molecularly distinct from these antigens. Furthermore, all of these proteins show a markedly distinct developmental regulation in their cell surface expression.

  13. Characterization of murine lung dendritic cells: similarities to Langerhans cells and thymic dendritic cells

    PubMed Central

    1990-01-01

    Dendritic cells (DC) are potent accessory cells (AC) for the initiation of primary immune responses. Although murine lymphoid DC and Langerhans cells have been extensively characterized, DC from murine lung have been incompletely described. We isolated cells from enzyme-digested murine lungs and bronchoalveolar lavages that were potent stimulators of a primary mixed lymphocyte response (MLR). The AC had a low buoyant density, were loosely adherent and nonphagocytic. AC function was unaffected by depletion of cells expressing the splenic DC marker, 33D1. In addition, antibody and complement depletion of cells bearing the macrophage marker F4/80, or removal of phagocytic cells with silica also failed to decrease AC activity. In contrast, AC function was decreased by depletion of cells expressing the markers J11d and the low affinity interleukin 2 receptor (IL-2R), both present on thymic and skin DC. AC function was approximately equal in FcR+ and FcR- subpopulations, indicating there was heterogeneity within the AC population. Consistent with the functional data, a combined two-color immunofluorescence and latex bead uptake technique revealed that lung cells high in AC activity were enriched in brightly Ia+ dendritic- shaped cells that (a) were nonphagocytic, (b) lacked specific T and B lymphocyte markers and the macrophage marker F4/80, but (c) frequently expressed C3biR, low affinity IL-2R, FcRII, and the markers NLDC-145 and J11d. Taken together, the functional and phenotypic data suggest the lung cells that stimulate resting T cells in an MLR and that might be important in local pulmonary immune responses are DC that bear functional and phenotypic similarity to other tissues DC, such as Langerhans cells and thymic DC. PMID:2162904

  14. Murine Norovirus Transcytosis across an In Vitro Polarized Murine Intestinal Epithelial Monolayer Is Mediated by M-Like Cells

    PubMed Central

    Gonzalez-Hernandez, Mariam B.; Liu, Thomas; Blanco, Luz P.; Auble, Heather; Payne, Hilary C.

    2013-01-01

    Noroviruses (NoVs) are the causative agent of the vast majority of nonbacterial gastroenteritis worldwide. Due to the inability to culture human NoVs and the inability to orally infect a small animal model, little is known about the initial steps of viral entry. One particular step that is not understood is how NoVs breach the intestinal epithelial barrier. Murine NoV (MNV) is the only NoV that can be propagated in vitro by infecting murine macrophages and dendritic cells, making this virus an attractive model for studies of different aspects of NoV biology. Polarized murine intestinal epithelial mICcl2 cells were used to investigate how MNV interacts with and crosses the intestinal epithelium. In this in vitro model of the follicle-associated epithelium (FAE), MNV is transported across the polarized cell monolayer in the absence of viral replication or disruption of tight junctions by a distinct epithelial cell with microfold (M) cell properties. In addition to transporting MNV, these M-like cells also transcytose microbeads and express an IgA receptor. Interestingly, B myeloma cells cultured in the basolateral compartment underlying the epithelial monolayer did not alter the number of M-like cells but increased their transcytotic activity. Our data demonstrate that MNV can cross an intact intestinal epithelial monolayer in vitro by hijacking the M-like cells' intrinsic transcytotic pathway and suggest a potential mechanism for MNV entry into the host. PMID:24049163

  15. A murine-ES like state facilitates transgenesis and homologous recombination in human pluripotent stem cells

    PubMed Central

    Buecker, Christa; Chen, Hsu-Hsin; Polo, Jose; Daheron, Laurence; Bu, Lei; Barakat, Tahsin Stefan; Okwieka, Patricia; Porter, Andrew; Gribnau, Joost; Hochedlinger, Konrad; Geijsen, Niels

    2010-01-01

    Murine embryonic stem cells have been shown to exist in two functionally distinct pluripotent states, embryonic stem cells (ES cell)- and epiblast stem cells (EpiSCs), which are defined by the culture growth factor conditions. Human ES cells appear to exist in an epiblast-like state, which in comparison to their murine counterparts, is relatively difficult to propagate and manipulate. As a result, gene targeting is difficult and to-date only a handful of human knock-in or knock-out cell lines exist. We explored whether an alternative stem cell state exists for human stem cells as well, and demonstrate that manipulation of the growth factor milieu allows the derivation of a novel human stem cell type that displays morphological, molecular and functional properties of murine ES cells and facilitates gene targeting. As such, the murine ES-like state provides a powerful tool for the generation of recombinant human pluripotent stem cell lines. PMID:20569691

  16. The dynamics of murine mammary stem/progenitor cells

    PubMed Central

    DONG, Qiaoxiang; SUN, Lu-Zhe

    2014-01-01

    The stem/progenitor cells in the murine mammary gland are a highly dynamic population of cells that are responsible for ductal elongation in puberty, homeostasis maintenance in adult, and lobulo-alveolar genesis during pregnancy. In recent years understanding the epithelial cell hierarchy within the mammary gland is becoming particularly important as these different stem/progenitor cells were perceived to be the cells of origin for various subtypes of breast cancer. Although significant advances have been made in enrichment and isolation of stem/progenitor cells by combinations of antibodies against cell surface proteins together with flow cytometry, and in identification of stem/progenitor cells with multi-lineage differentiation and self-renewal using mammary fat pad reconstitution assay and in vivo genetic labeling technique, a clear understanding of how these different stem/progenitors are orchestrated in the mammary gland is still lacking. Here we discuss the different in vivo and in vitro methods currently available for stem/progenitor identification, their associated caveats, and a possible new hierarchy model to reconcile various putative stem/progenitor cell populations identified by different research groups. PMID:25580105

  17. Depletion of B cells in murine lupus: efficacy and resistance.

    PubMed

    Ahuja, Anupama; Shupe, Jonathan; Dunn, Robert; Kashgarian, Michael; Kehry, Marilyn R; Shlomchik, Mark J

    2007-09-01

    In mice, genetic deletion of B cells strongly suppresses systemic autoimmunity, providing a rationale for depleting B cells to treat autoimmunity. In fact, B cell depletion with rituximab is approved for rheumatoid arthritis patients, and clinical trials are underway for systemic lupus erythematosus. Yet, basic questions concerning mechanism, pathologic effect, and extent of B cell depletion cannot be easily studied in humans. To better understand how B cell depletion affects autoimmunity, we have generated a transgenic mouse expressing human CD20 on B cells in an autoimmune-prone MRL/MpJ-Fas(lpr) (MRL/lpr) background. Using high doses of a murine anti-human CD20 mAb, we were able to achieve significant depletion of B cells, which in turn markedly ameliorated clinical and histologic disease as well as antinuclear Ab and serum autoantibody levels. However, we also found that B cells were quite refractory to depletion in autoimmune-prone strains compared with non-autoimmune-prone strains. This was true with multiple anti-CD20 Abs, including a new anti-mouse CD20 Ab, and in several different autoimmune-prone strains. Thus, whereas successful B cell depletion is a promising therapy for lupus, at least some patients might be resistant to the therapy as a byproduct of the autoimmune condition itself.

  18. Murine mammary stem/progenitor cell isolation: Different method matters?

    PubMed

    Gao, Hui; Dong, Qiaoxiang; Chen, Yuanhong; Zhang, Fuchuang; Wu, Anqi; Shi, Yuanshuo; Bandyopadhyay, Abhik; Daniel, Benjamin J; Huang, Changjiang; Sun, Lu-Zhe

    2016-01-01

    Murine mammary stem/progenitor cell isolation has been routinely used in many laboratories, yet direct comparison among different methods is lacking. In this study, we compared two frequently used digestion methods and three sets of frequently used surface markers for their efficiency in enriching mammary stem and progenitor cells in two commonly used mouse strains, C57BL/6J and FVB. Our findings revealed that the slow overnight digestion method using gentle collagenase/hyaluronidase could be easily adopted and yielded reliable and consistent results in different batches of animals. In contrast, the different fast digestion protocols, as described in published studies, yielded high percent of non-epithelial cells with very few basal epithelial cells liberated in our hands. The three sets of markers tested in our hands reveal rather equally efficiency in separating luminal and basal cells if same fluorochrome conjugations were used. However, the tendency of non-epithelial cell inclusion in the basal cell gate was highest in samples profiled by CD24/CD29 and lowest in samples profiled by CD49f/EpCAM, this is especially true in mammary cells isolated from C57BL/6J mice. This finding will have significant implication when sorted basal cells are used for subsequent gene expression analysis. PMID:26933638

  19. T cells in murine lupus: propagation and regulation of disease.

    PubMed

    Peng, S L; Craft, J

    1996-01-01

    MRL/Mp-lpr/lpr mice develop a spontaneous lupus syndrome, including hypergammaglobulinemia, autoantibodies, glomerulonephritis, and lymphadenopathy. To investigate the role of lymphocytes subsets in the pathogenesis of disease, lupus-prone MRL mice deficient in alpha beta T cells, gamma delta T cells, or both were generated. Mice deficient in alpha beta T cells developed a partially penetrant lupus syndrome, characterized by lymphadenopathy, elevated levels of class-switched immunoglobulins, an increased incidence of antinuclear antibodies, and immune deposits in kidneys which progressed to renal insufficiency over time. In comparison to wild type animals, gamma delta T cell-deficient animals developed an accelerated and exacerbated disease phenotype, characterized by accelerated hypergammaglobulinemia and enhanced autoantibody production and mortality. Repertoire analysis of these latter animals identified polyclonal expansion (V beta) of alpha beta CD4+ B220-cells. Mice lacking both alpha beta and gamma delta T cells failed to generate class-switched autoantibodies and immune complex renal disease. First, these findings demonstrate that murine lupus in the setting of Fas-deficiency does not absolutely require the presence of alpha beta T cells, and they also suggest that a significant basis for MRL/lpr disease, including renal disease, involves alpha beta T cell-independent, gamma delta T cell dependent, polyreactive B cell autoimmunity, upon which alpha beta T cell-dependent mechanisms aggravate specific autoimmune responses. Second, these data indicate that gamma delta T cells partake in the regulation of systemic autoimmunity, presumably via their effects on alpha beta CD4+ B220-T cells that provide B cell help. Finally, these results demonstrate that MRL/lpr B cells, despite their intrinsic abnormalities, cannot per se cause tissue injury without T cell help.

  20. Permissive and restricted virus infection of murine embryonic stem cells

    PubMed Central

    Wash, Rachael; Calabressi, Sabrina; Franz, Stephanie; Griffiths, Samantha J.; Goulding, David; Tan, E-Pien; Wise, Helen; Digard, Paul; Haas, Jürgen; Efstathiou, Stacey

    2012-01-01

    Recent RNA interference (RNAi) studies have identified many host proteins that modulate virus infection, but small interfering RNA ‘off-target’ effects and the use of transformed cell lines limit their conclusiveness. As murine embryonic stem (mES) cells can be genetically modified and resources exist where many and eventually all known mouse genes are insertionally inactivated, it was reasoned that mES cells would provide a useful alternative to RNAi screens. Beyond allowing investigation of host–pathogen interactions in vitro, mES cells have the potential to differentiate into other primary cell types, as well as being used to generate knockout mice for in vivo studies. However, mES cells are poorly characterized for virus infection. To investigate whether ES cells can be used to explore host–virus interactions, this study characterized the responses of mES cells following infection by herpes simplex virus type 1 (HSV-1) and influenza A virus. HSV-1 replicated lytically in mES cells, although mES cells were less permissive than most other cell types tested. Influenza virus was able to enter mES cells and express some viral proteins, but the replication cycle was incomplete and no infectious virus was produced. Knockdown of the host protein AHCYL1 in mES cells reduced HSV-1 replication, showing the potential for using mES cells to study host–virus interactions. Transcriptional profiling, however, indicated the lack of an efficient innate immune response in these cells. mES cells may thus be useful to identify host proteins that play a role in virus replication, but they are not suitable to determine factors that are involved in innate host defence. PMID:22815272

  1. Licochalcone E activates Nrf2/antioxidant response element signaling pathway in both neuronal and microglial cells: therapeutic relevance to neurodegenerative disease.

    PubMed

    Kim, Sa Suk; Lim, Juhee; Bang, Yeojin; Gal, Jiyeong; Lee, Sang-Uk; Cho, Young-Chang; Yoon, Goo; Kang, Bok Yun; Cheon, Seung Hoon; Choi, Hyun Jin

    2012-10-01

    Oxidative stress and neuroinflammation are hallmarks of neurodegenerative diseases, which do not play independently but work synergistically through complex interactions exacerbating neurodegeneration. Therefore, the mechanism that is directly implicated in controlling oxidative stress and inflammatory response could be an attractive strategy to prevent the onset and/or delay the progression of neurodegenerative diseases. The transcription factor nuclear factor-E2-related factor-2 (Nrf2) is the guardian of redox homeostasis by regulating a battery of antioxidant and phase II detoxification genes, which are relevant to defense mechanism against oxidative stress and inflammatory responses. In this study, we show that a recently identified Glycyrrhiza-inflata-derived chalcone, licochalcone E (Lico-E), attenuates lipopolysaccharide-induced inflammatory responses in microglial BV2 cells and protects dopaminergic SH-SY5Y cells from 6-hydroxydopamine cytotoxicity. Lico-E activates Nrf2-antioxidant response element (ARE) system and up-regulates downstream NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1). Anti-inflammatory and cytoprotective effects of Lico-E are attenuated in siRNA-mediated Nrf2-silencing cells as well as in the presence with specific inhibitor of HO-1 or NQO1, respectively. Lico-E also has neuroprotective effect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced nigrostriatal dopaminergic neurodegeneration in mice, with up-regulation of HO-1 and NQO1 in the substantia nigra of the brain. This study demonstrates that Lico-E is a potential activator of the Nrf2/ARE-dependent pathway and is therapeutically relevant not only to oxidative-stress-related neurodegeneration but also inflammatory responses of microglial cells both in vitro and in vivo.

  2. HNO suppresses LPS-induced inflammation in BV-2 microglial cells via inhibition of NF-κB and p38 MAPK pathways.

    PubMed

    Zhou, Yebo; Wu, Zhiyuan; Cao, Xu; Ding, Lei; Wen, ZhengShun; Bian, Jin-Song

    2016-09-01

    Both hydrogen sulfide (H2S) and nitric oxide (NO) are important gaseous mediators. We and others previously reported that these two gases react with each other to generate a new mediator, nitroxyl (HNO), and regulate cardiovascular functions. In this study, we demonstrated for the first time that the interaction between the two gases also existed in microglia. The biological functions of HNO in microglial cells were further studied with Angeli's salt (AS), an HNO donor. We found that AS attenuated lipopolysaccharide (LPS)-evoked production of reactive oxygen species (ROS) and pro-inflammatory cytokines (e.g. IL-1β and TNFα) through downregulating the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). HNO significantly reduced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and the activation of nuclear factor-κB (NF-κB) through suppression of phosphorylation p65 and IκBα. The above effects were abolished by l-cysteine, an HNO scavenger, but were not mimicked by nitrite, another product of AS during generating HNO. A Cys-179-to-Ala mutation in inhibitory κB kinase β (IKKβ) mimicked the effect of HNO on LPS-induced NF-κB activation. Interestingly, AS abolished the inflammation in cells overexpressing WT-IKKβ, but had no significant effect in cells overexpressing C179A-IKKβ. These data suggest that HNO may act on C179 to prevent IKKβ-dependent inflammation. Taken together, our data demonstrated for the first time that H2S interacts with NO to generate HNO in microglial cells. HNO produces anti-inflammatory effects through suppressing the IKKβ dependent NF-κB activation and p38 MAPK pathways. PMID:27507578

  3. Anti-Inflammatory Activity of Bee Venom in BV2 Microglial Cells: Mediation of MyD88-Dependent NF-κB Signaling Pathway.

    PubMed

    Im, Eun Ju; Kim, Su Jung; Hong, Seung Bok; Park, Jin-Kyu; Rhee, Man Hee

    2016-01-01

    Bee venom has long been used as a traditional folk medicine in Korea. It has been reportedly used for the treatment of arthritis, cancer, and inflammation. Although its anti-inflammatory activity in lipopolysaccharide- (LPS-) stimulated inflammatory cells has been reported, the exact mechanism of its anti-inflammatory action has not been fully elucidated. Therefore, the aim of this study was to investigate the anti-inflammatory mechanism of bee venom in BV2 microglial cells. We first investigated whether NO production in LPS-activated BV2 cells was inhibited by bee venom, and further iNOS mRNA and protein expressions were determined. The mRNA and protein levels of proinflammatory cytokines were examined using semiquantitative RT-PCR and immunoblotting, respectively. Moreover, modulation of the transcription factor NF-κB by bee venom was also investigated using a luciferase assay. LPS-induced NO production in BV2 microglial cells was significantly inhibited in a concentration-dependent manner upon pretreatment with bee venom. Bee venom markedly reduced the mRNA expression of COX-2, TNF-α, IL-1β, and IL-6 and suppressed LPS-induced activation of MyD88 and IRAK1 and phosphorylation of TAK1. Moreover, NF-κB translocation by IKKα/β phosphorylation and subsequent IκB-α degradation were also attenuated. Thus, collectively, these results indicate that bee venom exerts its anti-inflammatory activity via the IRAK1/TAK1/NF-κB signaling pathway. PMID:27563334

  4. Anti-Inflammatory Activity of Bee Venom in BV2 Microglial Cells: Mediation of MyD88-Dependent NF-κB Signaling Pathway

    PubMed Central

    Kim, Su Jung; Hong, Seung Bok; Park, Jin-Kyu

    2016-01-01

    Bee venom has long been used as a traditional folk medicine in Korea. It has been reportedly used for the treatment of arthritis, cancer, and inflammation. Although its anti-inflammatory activity in lipopolysaccharide- (LPS-) stimulated inflammatory cells has been reported, the exact mechanism of its anti-inflammatory action has not been fully elucidated. Therefore, the aim of this study was to investigate the anti-inflammatory mechanism of bee venom in BV2 microglial cells. We first investigated whether NO production in LPS-activated BV2 cells was inhibited by bee venom, and further iNOS mRNA and protein expressions were determined. The mRNA and protein levels of proinflammatory cytokines were examined using semiquantitative RT-PCR and immunoblotting, respectively. Moreover, modulation of the transcription factor NF-κB by bee venom was also investigated using a luciferase assay. LPS-induced NO production in BV2 microglial cells was significantly inhibited in a concentration-dependent manner upon pretreatment with bee venom. Bee venom markedly reduced the mRNA expression of COX-2, TNF-α, IL-1β, and IL-6 and suppressed LPS-induced activation of MyD88 and IRAK1 and phosphorylation of TAK1. Moreover, NF-κB translocation by IKKα/β phosphorylation and subsequent IκB-α degradation were also attenuated. Thus, collectively, these results indicate that bee venom exerts its anti-inflammatory activity via the IRAK1/TAK1/NF-κB signaling pathway. PMID:27563334

  5. Regulatory effects of fisetin on microglial activation.

    PubMed

    Chuang, Jing-Yuan; Chang, Pei-Chun; Shen, Yi-Chun; Lin, Chingju; Tsai, Cheng-Fang; Chen, Jia-Hong; Yeh, Wei-Lan; Wu, Ling-Hsuan; Lin, Hsiao-Yun; Liu, Yu-Shu; Lu, Dah-Yuu

    2014-06-26

    Increasing evidence suggests that inflammatory processes in the central nervous system that are mediated by microglial activation play a key role in neurodegeneration. Fisetin, a plant flavonol commonly found in fruits and vegetables, is frequently added to nutritional supplements due to its antioxidant properties. In the present study, treatment with fisetin inhibited microglial cell migration and ROS (reactive oxygen species) production. Treatment with fisetin also effectively inhibited LPS plus IFN-γ-induced nitric oxide (NO) production, and inducible nitric oxide synthase (iNOS) expression in microglial cells. Furthermore, fisetin also reduced expressions of iNOS and NO by stimulation of peptidoglycan, the major component of the Gram-positive bacterium cell wall. Fisetin also inhibited the enhancement of LPS/IFN-γ- or peptidoglycan-induced inflammatory mediator IL (interlukin)-1 β expression. Besides the antioxidative and anti-inflammatory effects of fisetin, our study also elucidates the manner in fisetin-induced an endogenous anti-oxidative enzyme HO (heme oxygenase)-1 expression. Moreover, the regulatory molecular mechanism of fisetin-induced HO-1 expression operates through the PI-3 kinase/AKT and p38 signaling pathways in microglia. Notably, fisetin also significantly attenuated inflammation-related microglial activation and coordination deficit in mice in vivo. These findings suggest that fisetin may be a candidate agent for the development of therapies for inflammation-related neurodegenerative diseases.

  6. Time-dependent retinal ganglion cell loss, microglial activation and blood-retina-barrier tightness in an acute model of ocular hypertension.

    PubMed

    Trost, A; Motloch, K; Bruckner, D; Schroedl, F; Bogner, B; Kaser-Eichberger, A; Runge, C; Strohmaier, C; Klein, B; Aigner, L; Reitsamer, H A

    2015-07-01

    Glaucoma is a group of neurodegenerative diseases characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, and is the second leading cause of blindness worldwide. Elevated intraocular pressure is a well known risk factor for the development of glaucomatous optic neuropathy and pharmacological or surgical lowering of intraocular pressure represents a standard procedure in glaucoma treatment. However, the treatment options are limited and although lowering of intraocular pressure impedes disease progression, glaucoma cannot be cured by the currently available therapy concepts. In an acute short-term ocular hypertension model in rat, we characterize RGC loss, but also microglial cell activation and vascular alterations of the retina at certain time points. The combination of these three parameters might facilitate a better evaluation of the disease progression, and could further serve as a new model to test novel treatment strategies at certain time points. Acute ocular hypertension (OHT) was induced by the injection of magnetic microbeads into the rat anterior chamber angle (n = 22) with magnetic position control, leading to constant elevation of IOP. At certain time points post injection (4d, 7d, 10d, 14d and 21d), RGC loss, microglial activation, and microvascular pericyte (PC) coverage was analyzed using immunohistochemistry with corresponding specific markers (Brn3a, Iba1, NG2). Additionally, the tightness of the retinal vasculature was determined via injections of Texas Red labeled dextran (10 kDa) and subsequently analyzed for vascular leakage. For documentation, confocal laser-scanning microscopy was used, followed by cell counts, capillary length measurements and morphological and statistical analysis. The injection of magnetic microbeads led to a progressive loss of RGCs at the five time points investigated (20.07%, 29.52%, 41.80%, 61.40% and 76.57%). Microglial cells increased in number and displayed an activated morphology

  7. Effects of trichostatins on differentiation of murine erythroleukemia cells

    SciTech Connect

    Yoshida, M.; Nomura, S.; Beppu, T.

    1987-07-15

    The fungistatic antibiotics trichostatins (TS) A and C were isolated from culture broth of Streptomyces platensis No. 145 and were found to be potent inducers of differentiation in murine erythroleukemia (Friend and RV133) cells at concentrations of 1.5 X 10(-8) M for TSA and 5 X 10(-7) M for TSC. Differentiation induced by TS was cooperatively enhanced by UV irradiation but not by treatment with dimethyl sulfoxide. This enhanced activity was completely inhibited by adding cycloheximide to the culture medium 2 h after exposure to TS, suggesting that TS are dimethyl sulfoxide-type inducers of erythroid differentiation. No inhibitory effect of TS was observed on macromolecular synthesis in cultured cells.

  8. Nicotine contributes to the neural stem cells fate against toxicity of microglial-derived factors induced by Aβ via the Wnt/β-catenin pathway.

    PubMed

    Jiang, De-Qi; Wei, Mei-Dan; Wang, Ke-Wan; Lan, Yan-Xian; Zhu, Ning; Wang, Yong

    2016-01-01

    Recent studies have demonstrated that the molecules secreted from microglias play important roles in the cell fate determination of neural stem cells (NSCs), and nicotinic acetylcholine receptor agonist treatment could reduce neuroinflammation in some neurodegenerative disease models, such as Alzheimer's disease (AD). However, it is not clear how nicotine plays a neuroprotective role in inflammation-mediated central nervous diseases, and its possible mechanisms in the process remain largely elusive. The aim of this study is to improve the survival microenvironment of NSCs co-cultured with microglias in vitro by weakening inflammation that mediated by accumulation of β-amyloid peptide (Aβ). The viability, proliferation, differentiation, apoptosis of NSCs and underlying mechanisms associated with Wnt signaling pathway were investigated. The results showed that Aβ could directly damage NSCs. Furthermore, concomitant to elevated levels of TNF-α, IL-1β derived from microglias, the NSCs had been damaged more severely with the upregulation of Axin 2, p-β-catenin and the downregulation of β-catenin, p-GSK-3β, microtubule-associated protein-2, choline acetyltransferase. However, addition of 10 μmol/L nicotine before microglias treated with Aβ was beneficial to protect the NSCs against neurotoxicity of microglial-derived factors induced by Aβ, which partially rescued proliferation, differentiation and inhibited apoptosis of NSCs via activation of Wnt/β-catenin pathway. Taken together, these data imply that low concentration nicotine attenuates NSCs injury induced by microglial-derived factors via Wnt signaling pathway. Thus, treatment with nicotinic acetylcholine receptor agonist provides a promising research field for neural stem cell fate and therapeutic intervention in neuroinflammation diseases.

  9. Paeonol attenuates inflammation-mediated neurotoxicity and microglial activation☆

    PubMed Central

    Nam, Kyong Nyon; Woo, Byung-Cheol; Moon, Sang-Kwan; Park, Seong-Uk; Park, Joo-young; Hwang, Jae-Woong; Bae, Hyung-Sup; Ko, Chang-Nam; Lee, Eunjoo Hwang

    2013-01-01

    Chronic activation of microglial cells endangers neuronal survival through the release of various proinflammatory and neurotoxic factors. The root of Paeonia lactiflora Pall has been considered useful for the treatment of various disorders in traditional oriental medicine. Paeonol, found in the root of Paeonia lactiflora Pall, has a wide range of pharmacological functions, including anti-oxidative, anti-inflammatory and neuroprotective activities. The objective of this study was to examine the efficacy of paeonol in the repression of inflammation-induced neurotoxicity and microglial cell activation. Organotypic hippocampal slice cultures and primary microglial cells from rat brain were stimulated with bacterial lipopolysaccharide. Paeonol pretreatment was performed for 30 minutes prior to lipopolysaccharide addition. Cell viability and nitrite (the production of nitric oxide), tumor necrosis factor-alpha and interleukin-1beta products were measured after lipopolysaccharide treatment. In organotypic hippocampal slice cultures, paeonol blocked lipopolysaccharide-related hippocampal cell death and inhibited the release of nitrite and interleukin-1beta. Paeonol was effective in inhibiting nitric oxide release from primary microglial cells. It also reduced the lipopolysaccharide-stimulated release of tumor necrosis factor-alpha and interleukin-1β from microglial cells. Paeonol possesses neuroprotective activity in a model of inflammation-induced neurotoxicity and reduces the release of neurotoxic and proinflammatory factors in activated microglial cells. PMID:25206460

  10. Toxicity of Calcium Hydroxide Nanoparticles on Murine Fibroblast Cell Line

    PubMed Central

    Dianat, Omid; Azadnia, Sina; Mozayeni, Mohammad Ali

    2015-01-01

    Introduction: One of the major contributing factors, which may cause failure of endodontic treatment, is the presence of residual microorganisms in the root canal system. For years, most dentists have been using calcium hydroxide (CH) as the intracanal medicament between treatment sessions to eliminate remnant microorganisms. Reducing the size of CH particles into nanoparticles enhances the penetration of this medicament into dentinal tubules and increases their antimicrobial efficacy. This in vitro study aimed to compare the cytotoxicity of CH nanoparticles and conventional CH on fibroblast cell line using the Mosmann’s Tetrazolium Toxicity (MTT) assay. Methods and Materials: This study was conducted on L929 murine fibroblast cell line by cell culture and evaluation of the direct effect of materials on the cultured cells. Materials were evaluated in two groups of 10 samples each at 24, 48 and 72 h. At each time point, 10 samples along with 5 positive and 5 negative controls were evaluated. The samples were transferred into tubes and exposed to fibroblast cells. The viability of cells was then evaluated. The Two-way ANOVA was used for statistical analysis and the level of significance was set at 0.05. Results: Cytotoxicity of both materials decreased over time and for conventional CH was lower than that of nanoparticles. However, this difference was not statistically significant (P>0.05). Conclusion: The cytotoxicity of CH nanoparticles was similar to that of conventional CH. PMID:25598810

  11. Dietary Sutherlandia and Elderberry Mitigate Cerebral Ischemia-Induced Neuronal Damage and Attenuate p47phox and Phospho-ERK1/2 Expression in Microglial Cells

    PubMed Central

    Chuang, Dennis Y.; Cui, Jiankun; Simonyi, Agnes; Engel, Victoria A.; Chen, Shanyan; Fritsche, Kevin L.; Thomas, Andrew L.; Applequist, Wendy L.; Folk, William R.; Lubahn, Dennis B.; Sun, Albert Y.; Sun, Grace Y.

    2014-01-01

    Sutherlandia (Sutherlandia frutescens) and elderberry (Sambucus spp.) are used to promote health and for treatment of a number of ailments. Although studies with cultured cells have demonstrated antioxidative and anti-inflammatory properties of these botanicals, little is known about their ability to mitigate brain injury. In this study, C57BL/6 J male mice were fed AIN93G diets without or with Sutherlandia or American elderberry for 2 months prior to a 30-min global cerebral ischemia induced by occlusion of the bilateral common carotid arteries (BCCAs), followed by reperfusion for 3 days. Accelerating rotarod assessment at 24 h after BCCA occlusion showed amelioration of sensorimotor impairment in the mice fed the supplemented diets as compared with the ischemic mice fed the control diet. Quantitative digital pathology assessment of brain slides stained with cresyl violet at 3 days after ischemia/reperfusion (I/R) revealed significant reduction in neuronal cell death in both dietary groups. Immunohistochemical staining for ionized calcium-binding adapter molecule-1 demonstrated pronounced activation of microglia in the hippocampus and striatum in the ischemic brains 3 days after I/R, and microglial activation was significantly reduced in animals fed supplemented diets. Mitigation of microglial activation by the supplements was further supported by the decrease in expression of p47phox, a cytosolic subunit of NADPH oxidase, and phospho-ERK1/2, a mitogen-activated protein kinase known to mediate a number of cytoplasmic processes including oxidative stress and neuroinflammatory responses. These results demonstrate neuroprotective effect of Sutherlandia and American elderberry botanicals against oxidative and inflammatory responses to cerebral I/R. PMID:25324465

  12. The Anti-Inflammatory Activity of Eucommia ulmoides Oliv. Bark. Involves NF-κB Suppression and Nrf2-Dependent HO-1 Induction in BV-2 Microglial Cells

    PubMed Central

    Kwon, Seung-Hwan; Ma, Shi-Xun; Hwang, Ji-Young; Ko, Yong-Hyun; Seo, Ji-Yeon; Lee, Bo-Ram; Lee, Seok-Yong; Jang, Choon-Gon

    2016-01-01

    In the present study, we investigated the anti-inflammatory properties of Eucommia ulmoides Oliv. Bark. (EUE) in lipopolysaccharide (LPS)-stimulated microglial BV-2 cells and found that EUE inhibited LPS-mediated up-regulation of pro-inflammatory response factors. In addition, EUE inhibited the elevated production of pro-inflammatory cytokines, mediators, and reactive oxygen species (ROS) in LPS-stimulated BV-2 microglial cells. Subsequent mechanistic studies revealed that EUE suppressed LPS-induced phosphorylation of mitogen-activated protein kinases (MAPKs), phosphoinositide-3-kinase (PI3K)/Akt, glycogen synthase kinase-3β (GSK-3β), and their downstream transcription factor, nuclear factor-kappa B (NF-κB). EUE also blocked the nuclear translocation of NF-κB and inhibited its binding to DNA. We next demonstrated that EUE induced the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and upregulated heme oxygenase-1 (HO-1) expression. We determined that the significant up-regulation of HO-1 expression by EUE was a consequence of Nrf2 nuclear translocation; furthermore, EUE increased the DNA binding of Nrf2. In contrast, zinc protoporphyrin (ZnPP), a specific HO-1 inhibitor, blocked the ability of EUE to inhibit NO and PGE2 production, indicating the vital role of HO-1. Overall, our results indicate that EUE inhibits pro-inflammatory responses by modulating MAPKs, PI3K/Akt, and GSK-3β, consequently suppressing NF-κB activation and inducing Nrf2-dependent HO-1 activation. PMID:27068259

  13. Dependence on exogenous methionine of rat sarcoma and murine leukemia cells in culture.

    PubMed

    Koziorowska, J; Pieńkowska, K; Tautt, J

    1980-01-01

    A comparative study was performed on methionine auxotrophy of rat sarcoma and murine leukemia cells taken directly from the organism and grown in culture in media lacking methionine or in which methionine was substituted by homocysteine. Methionine auxotrophy was observed in both kinds of cells. At low levels of methionine in the media containing homocysteine rat sarcoma cells showed an increase in growth. Addition of homocysteine to the media with low levels of methionine did not influence the survival of murine leukemia cells.

  14. Microglial neurotransmitter receptors trigger superoxide production in microglia; consequences for microglial-neuronal interactions.

    PubMed

    Mead, Emma L; Mosley, Angelina; Eaton, Simon; Dobson, Lucianne; Heales, Simon J; Pocock, Jennifer M

    2012-04-01

    Microglia express three isoforms of the NADPH oxidase, Nox1, Nox2 and Nox4, with the potential to produce superoxide (O(2) ˙(-) ). Microglia also express neurotransmitter receptors, which can modulate microglial responses. In this study, microglial activity of Nox1, Nox2 and Nox4 in primary rat cultured microglia or the rodent BV2 cell line were altered by microglial neurotransmitter receptor modulation. Glutamate, GABA or ATP triggered microglial O(2) ˙(-) production via Nox activation. Nox activation was elicited by agonists of metabotropic mGlu3 receptors and by group III receptors, by GABA(A) but not GABA(B) receptors, and by purinergic P2X(7) or P2Y(2/4) receptors but not P2Y(1) receptors, and inhibited by metabotropic glutamate receptor 5 antagonists. The neurotransmitters also modulated Nox mRNA expression and NADPH activity. The activation of Nox by BzATP or GABA promoted a neuroprotective phenotype whilst the activation of Nox by glutamate promoted a neurotoxic phenotype. Taken together, these data indicate that microglial neurotransmitter receptors can signal via Nox to promote neuroprotection or neurotoxicity. This has implications for the subsequent neurotoxic profile of microglia when neurotransmitter levels may become skewed in neurodegeneration. PMID:22243365

  15. B CELL DEPLETION THERAPY EXACERBATES MURINE PRIMARY BILIARY CIRRHOSIS

    PubMed Central

    Dhirapong, Amy; Lleo, Ana; Yang, Guo-Xiang; Tsuneyama, Koichi; Dunn, Robert; Kehry, Marilyn; Packard, Thomas A.; Cambier, John C.; Liu, Fu-Tong; Lindor, Keith; Coppel, Ross L.; Ansari, Aftab A.; Gershwin, M. Eric

    2010-01-01

    Primary biliary cirrhosis (PBC) is considered a model autoimmune disease due to the clinical homogeneity of patients and the classic hallmark of anti-mitochondrial antibodies (AMAS). Indeed, the presence of AMAS is the most highly directed and specific autoantibody in autoimmune diseases. However, the contribution of B cells to the pathogenesis of PBC is unclear. Thus, although AMAs appear to interact with the biliary cell apotope and contribute to biliary pathology, there is no correlation of disease severity and titer of AMA. The recent development of well characterized mAbs specific for the B cell populations, anti-CD20 and anti-CD79, and the development of a well defined xenobiotic induced model of autoimmune cholangitis, prompted us to utilize these reagents and the model to address the contribution of B cells in the pathogenesis of murine PBC. Prior to the induction of autoimmune cholangitis, mice were treated with either anti-CD20, anti-CD79, or isotype matched control mAb and followed for B cell development, the appearance of AMAs, liver pathology and cytokine production. Results of the studies reported herein show that the in vivo depletion of B cells using either anti-CD20 or anti-CD79 led to the development of a more severe form of cholangitis than control mice which is in contrast with results from a number of other autoimmune models which have documented an important therapeutic role of B cell specific depletion. The anti-CD20/CD79 treated mice have increased liver T cell infiltrates and higher levels of pro-inflammatory cytokines. In conclusion, our results reflect a novel disease protective role of B cells in PBC and suggest that B cell depletion therapy in humans with PBC should be approached with caution. PMID:21274873

  16. Definition of a serum marker panel for glioblastoma discrimination and identification of Interleukin 1β in the microglial secretome as a novel mediator of endothelial cell survival induced by C-reactive protein.

    PubMed

    Nijaguna, Mamatha B; Schröder, Christoph; Patil, Vikas; Shwetha, Shivayogi D; Hegde, Alangar S; Chandramouli, Bangalore A; Arivazhagan, Arimappamagan; Santosh, Vani; Hoheisel, Jörg D; Somasundaram, Kumaravel

    2015-10-14

    Glioblastoma (GBM) is the most common malignant adult primary brain tumor. We profiled 724 cancer-associated proteins in sera of healthy individuals (n=27) and GBM (n=28) using antibody microarray. While 69 proteins exhibited differential abundance in GBM sera, a three-marker panel (LYAM1, BHE40 and CRP) could discriminate GBM sera from that of healthy donors with an accuracy of 89.7% and p<0.0001. The high abundance of C-reactive protein (CRP) in GBM sera was confirmed in 264 independent samples. High levels of CRP protein was seen in GBM but without a change in transcript levels suggesting a non-tumoral origin. Glioma-secreted Interleukin 6 (IL6) was found to induce hepatocytes to secrete CRP, involving JAK-STAT pathway. The culture supernatant from CRP-treated microglial cells induced endothelial cell survival under nutrient-deprivation condition involving CRP-FcγRIII signaling cascade. Transcript profiling of CRP-treated microglial cells identified Interleukin 1β (IL1β) present in the microglial secretome as the key mediator of CRP-induced endothelial cell survival. IL1β neutralization by antibody-binding or siRNA-mediated silencing in microglial cells reduced the ability of the supernatant from CRP-treated microglial cells to induce endothelial cell survival. Thus our study identifies a serum based three-marker panel for GBM diagnosis and provides leads for developing targeted therapies. Biological significance A complex antibody microarray based serum marker profiling identified a three-marker panel - LYAM1, BHE40 and CRP as an accurate discriminator of glioblastoma sera from that of healthy individuals. CRP protein is seen in high levels without a concomitant increase of CRP transcripts in glioblastoma. Glioma-secreted IL6 induced hepatocytes to produce CRP in a JAK-STAT signaling dependent manner. CRP induced microglial cells to release IL1β which in turn promoted endothelial cell survival. This study, besides defining a serum panel for glioblastoma

  17. Complementation analysis of the murine scid cell line

    SciTech Connect

    Zdzienicka, M.Z. |; Priestly, A.; Jeggo, P.A.

    1995-09-01

    It has been shown that several X-ray-sensitive Chinese hamster cell mutants defective in repair of DNA double-strand breaks (DSBs) are also impaired in the process of V(D)J recombination. The hamster mutants with this phenotype represent three distinct complementation groups, represented by the xrs series, XR-1 and V-3. The murine scid cell line also shows the same phenotype, and therefore we examined whether the scid mutant represents a new complementation group or belongs to one of the existing groups. Scid cells were fused with hamster cell mutants representing the three complementation groups. Hybrids between V-3 and scid cells were only partially complemented for X-ray sensitivity, whereas hybrids derived from fusions with the other mutants were resistant to X rays. These results suggest that V-3 and scid cells are defective in the same gene. To confirm this finding, a single human chromosome 8, which is known to carry the scid gene, was introduced into V-3 cells by microcell-mediated chromosome transfer. Nine hybrid clones derived from V-3 and carrying human chromosome 8 were obtained, and seven were found to be partially complemented for X-ray sensitivity. When human chromosome 8 was introduced into scid cells, seven of eight hybrid clones became resistant to X rays. The results indicate that the defective genes in V-3 and scid are both localized on human chromosome 8. This supports the results from the fusion analysis that V-3 and scid cells are defective in the same gene. 53 refs., 4 figs., 1 tab.

  18. High-dimensional analysis of the murine myeloid cell system.

    PubMed

    Becher, Burkhard; Schlitzer, Andreas; Chen, Jinmiao; Mair, Florian; Sumatoh, Hermi R; Teng, Karen Wei Weng; Low, Donovan; Ruedl, Christiane; Riccardi-Castagnoli, Paola; Poidinger, Michael; Greter, Melanie; Ginhoux, Florent; Newell, Evan W

    2014-12-01

    Advances in cell-fate mapping have revealed the complexity in phenotype, ontogeny and tissue distribution of the mammalian myeloid system. To capture this phenotypic diversity, we developed a 38-antibody panel for mass cytometry and used dimensionality reduction with machine learning-aided cluster analysis to build a composite of murine (mouse) myeloid cells in the steady state across lymphoid and nonlymphoid tissues. In addition to identifying all previously described myeloid populations, higher-order analysis allowed objective delineation of otherwise ambiguous subsets, including monocyte-macrophage intermediates and an array of granulocyte variants. Using mice that cannot sense granulocyte macrophage-colony stimulating factor GM-CSF (Csf2rb(-/-)), which have discrete alterations in myeloid development, we confirmed differences in barrier tissue dendritic cells, lung macrophages and eosinophils. The methodology further identified variations in the monocyte and innate lymphoid cell compartment that were unexpected, which confirmed that this approach is a powerful tool for unambiguous and unbiased characterization of the myeloid system. PMID:25306126

  19. Simvastatin induces osteogenic differentiation of murine embryonic stem cells.

    PubMed

    Pagkalos, Joseph; Cha, Jae Min; Kang, Yunyi; Heliotis, Manolis; Tsiridis, Eleftherios; Mantalaris, Athanasios

    2010-11-01

    Statins are potent inhibitors of cholesterol synthesis. Several statins are available with different molecular and pharmacokinetic properties. Simvastatin is more lipophilic than pravastatin and has a higher affinity to phospholipid membranes than atorvastatin, allowing its passive diffusion through the cell membrane. In vitro studies on bone marrow stromal cells, osteoblast-like cells, and embryonic stem cells have shown statins to have cholesterol-independent anabolic effects on bone metabolism; alas, statins were supplemented in osteogenic medium, which does not facilitate elucidation of their potential osteoinductive properties. Embryonic stem cells (ESCs), derived from the inner cell mass of the blastocyst, are unique in that they enjoy perpetual self-proliferation, are pluripotent, and are able to differentiate toward all the cellular lineages composing the body, including the osteogenic lineage. Consequently, ESCs represent a potentially potent cell source for future clinical cellular therapies of various bone diseases, even though there are several hurdles that still need to be overcome. Herein we demonstrate, for the first time to our knowledge, that simvastatin induces murine ESC (mESC) differentiation toward the osteogenic lineage in the absence of osteoinductive supplements. Specifically, we found that a simvastatin concentration in the micromolar range and higher was toxic to the cells and that an effective concentration for osteoinduction is 0.1 nM, as shown by increased alizarin red staining as well as increased osteocalcin and osetrix gene expression. These results suggest that in the future, lipophilic simvastatin may provide a novel pharmacologic agent for bone tissue engineering applications. PMID:20564244

  20. Interleukin 6 promotes vasculogenesis of murine brain microvessel endothelial cells.

    PubMed

    Fee, D; Grzybicki, D; Dobbs, M; Ihyer, S; Clotfelter, J; Macvilay, S; Hart, M N; Sandor, M; Fabry, Z

    2000-06-01

    Interleukin 6 (IL-6) is a cytokine that acts on a wide range of tissues influencing cell growth and differentiation. Here we show that IL-6 plays a role in the early vascular development (vasculogenesis) in the central nervous system (CNS). We report that IL-6 induces the proliferation of brain microvascular endothelial cells in vitro. Furthermore, IL-6 significantly accelerates the formation of tube-like structures by these cells in Matrigel basement matrix. Moreover, IL-6 mRNA is expressed in vivo in two physiological conditions in which vascularization in the CNS is important: (1) during normal brain development, (2) during the healing process of a traumatic brain injury. Expression of IL-6 mRNA coincides with the expression of vascular endothelial growth factor (VEGF) mRNA in the developing brain with decreasing expression following birth. However, IL-6 mRNA can be detected in the healing adult murine brain tissue by in situ hybridization coinciding with the period of intense tissue reorganization. The transient upregulation of IL-6 mRNA during normal brain development and at brain injury site and the effect of IL-6 on in vitro vasculogenesis suggest that IL-6 may play a role in normal physiology of vascularization in the CNS.

  1. DNA repair in murine embryonic stem cells and differentiated cells

    SciTech Connect

    Tichy, Elisia D. Stambrook, Peter J.

    2008-06-10

    Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells.

  2. Murine macrophage heparanase: inhibition and comparison with metastatic tumor cells

    SciTech Connect

    Savion, N.; Disatnik, M.H.; Nevo, Z.

    1987-01-01

    Circulating macrophages and metastatic tumor cells can penetrate the vascular endothelium and migrate from the circulatory system to extravascular compartments. Both activated murine macrophages and different metastatic tumor cells attach, invade, and penetrate confluent vascular endothelial cell monolayer in vitro, by degrading heparan sulfate proteoglycans in the subendothelial extracellular matrix. The sensitivity of the enzymes from the various sources degrading the heparan sulfate proteoglycan was challenged and compared by a series of inhibitors. Activated macrophages demonstrate a heparanase with an endoglycosidase activity that cleaves from the (/sup 35/S)O/sub 4//sup -/-labeled heparan sulfate proteoglycans of the extracellular matrix 10 kDa glycosaminoglycan fragments. The degradation of (/sup 35/S)O/sub 4//sup -/-labeled extracellular matrix proteoglycans by the macrophages' heparanase is significantly inhibited in the presence of heparan sulfate (10..mu..g/ml), arteparon (10..mu..g/ml), and heparin at a concentration of 3 ..mu..g/ml. Degradation of this heparan sulfate proteoglycan is a two-step sequential process involving protease activity followed by heparanase activity. B16-BL6 metastatic melanoma cell heparanase, which is also a cell-associated enzyme, was inhibited by heparin to the same extent as the macrophage haparanase. On the other hand, heparanase of the highly metastatic variant (ESb) of a methylcholanthrene-induced T lymphoma, which is an extracellular enzyme released by the cells to the incubation medium, was more sensitive to heparin and arteparon than the macrophages' heparanase. These results may indicate the potential use of heparin or other glycosaminoglycans as specific and differential inhibitors for the formation in certain cases of blood-borne tumor metastasis.

  3. TREM2-mediated early microglial response limits diffusion and toxicity of amyloid plaques

    PubMed Central

    Wang, Yaming; Ulland, Tyler K.; Ulrich, Jason D.; Song, Wilbur; Tzaferis, John A.; Hole, Justin T.; Yuan, Peng; Mahan, Thomas E.; Shi, Yang; Gilfillan, Susan; Cella, Marina; Grutzendler, Jaime; DeMattos, Ronald B.; Cirrito, John R.; Holtzman, David M.

    2016-01-01

    Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglial receptor that recognizes changes in the lipid microenvironment, which may occur during amyloid β (Aβ) accumulation and neuronal degeneration in Alzheimer’s disease (AD). Rare TREM2 variants that affect TREM2 function lead to an increased risk of developing AD. In murine models of AD, TREM2 deficiency prevents microglial clustering around Aβ deposits. However, the origin of myeloid cells surrounding amyloid and the impact of TREM2 on Aβ accumulation are a matter of debate. Using parabiosis, we found that amyloid-associated myeloid cells derive from brain-resident microglia rather than from recruitment of peripheral blood monocytes. To determine the impact of TREM2 deficiency on Aβ accumulation, we examined Aβ plaques in the 5XFAD model of AD at the onset of Aβ-related pathology. At this early time point, Aβ accumulation was similar in TREM2-deficient and -sufficient 5XFAD mice. However, in the absence of TREM2, Aβ plaques were not fully enclosed by microglia; they were more diffuse, less dense, and were associated with significantly greater neuritic damage. Thus, TREM2 protects from AD by enabling microglia to surround and alter Aβ plaque structure, thereby limiting neuritic damage. PMID:27091843

  4. TAM receptors regulate multiple features of microglial physiology.

    PubMed

    Fourgeaud, Lawrence; Través, Paqui G; Tufail, Yusuf; Leal-Bailey, Humberto; Lew, Erin D; Burrola, Patrick G; Callaway, Perri; Zagórska, Anna; Rothlin, Carla V; Nimmerjahn, Axel; Lemke, Greg

    2016-04-14

    Microglia are damage sensors for the central nervous system (CNS), and the phagocytes responsible for routine non-inflammatory clearance of dead brain cells. Here we show that the TAM receptor tyrosine kinases Mer and Axl regulate these microglial functions. We find that adult mice deficient in microglial Mer and Axl exhibit a marked accumulation of apoptotic cells specifically in neurogenic regions of the CNS, and that microglial phagocytosis of the apoptotic cells generated during adult neurogenesis is normally driven by both TAM receptor ligands Gas6 and protein S. Using live two-photon imaging, we demonstrate that the microglial response to brain damage is also TAM-regulated, as TAM-deficient microglia display reduced process motility and delayed convergence to sites of injury. Finally, we show that microglial expression of Axl is prominently upregulated in the inflammatory environment that develops in a mouse model of Parkinson's disease. Together, these results establish TAM receptors as both controllers of microglial physiology and potential targets for therapeutic intervention in CNS disease. PMID:27049947

  5. Characterization of iron uptake from transferrin by murine endothelial cells.

    PubMed

    Hallmann, R; Savigni, D L; Morgan, E H; Baker, E

    2000-01-01

    Iron is required by the brain for normal function, however, the mechanisms by which it crosses the blood-brain barrier (BBB) are poorly understood. The uptake and efflux of transferrin (Tf) and Fe by murine brain-derived (bEND3) and lymph node-derived (m1END1) endothelial cell lines was compared. The effects of iron chelators, metabolic inhibitors and the cellular activators, lipopolysaccharide (LPS) and tumour necrosis factor-alpha (TNF-alpha), on Tf and Fe uptake were investigated. Cells were incubated with 59Fe-125I-Tf; Fe uptake was shown to increase linearly over time for both cell lines, while Tf uptake reached a plateau within 2 h. Both Tf and Fe uptake were saturable. bEND3 cells were shown to have half as many Tf receptors as m1END1 cells, but the mean cycling times of a Tf molecule were the same. Tf and Fe efflux from the cells were measured over time, revealing that after 2 h only 25% of the Tf but 80% of the Fe remained associated with the cells. Of 7 iron chelators, only deferriprone (L1) markedly decreased Tf uptake. However, Fe uptake was reduced by more than 50% by L1, pyridoxal isonicotinoyl hydrazone (PIH) and desferrithiocin (DFT). The cellular activators TNF-alpha or LPS had little effect on Tf turnover, but they accelerated Fe uptake in both endothelial cell types. Phenylarsenoxide (PhAsO) and N-ethyl maleimide (NEM), inhibitors of Tf endocytosis, reduced both Tf and Fe uptake in both cell lines, while bafilomycin A1, an inhibitor of endosomal acidification, reduced Fe uptake but did not affect Tf uptake. The results suggest that Tf and Fe uptake by both bEND3 and m1END1 is via receptor-mediated endocytosis with release of Fe from Tf within the cell and recycling of apo-Tf. On the basis of Tf- and Fe-metabolism both cell lines are similar and therefore well suited for use in in vitro models for Fe transport across the BBB.

  6. Characterization of iron uptake from transferrin by murine endothelial cells.

    PubMed

    Hallmann, R; Savigni, D L; Morgan, E H; Baker, E

    2000-01-01

    Iron is required by the brain for normal function, however, the mechanisms by which it crosses the blood-brain barrier (BBB) are poorly understood. The uptake and efflux of transferrin (Tf) and Fe by murine brain-derived (bEND3) and lymph node-derived (m1END1) endothelial cell lines was compared. The effects of iron chelators, metabolic inhibitors and the cellular activators, lipopolysaccharide (LPS) and tumour necrosis factor-alpha (TNF-alpha), on Tf and Fe uptake were investigated. Cells were incubated with 59Fe-125I-Tf; Fe uptake was shown to increase linearly over time for both cell lines, while Tf uptake reached a plateau within 2 h. Both Tf and Fe uptake were saturable. bEND3 cells were shown to have half as many Tf receptors as m1END1 cells, but the mean cycling times of a Tf molecule were the same. Tf and Fe efflux from the cells were measured over time, revealing that after 2 h only 25% of the Tf but 80% of the Fe remained associated with the cells. Of 7 iron chelators, only deferriprone (L1) markedly decreased Tf uptake. However, Fe uptake was reduced by more than 50% by L1, pyridoxal isonicotinoyl hydrazone (PIH) and desferrithiocin (DFT). The cellular activators TNF-alpha or LPS had little effect on Tf turnover, but they accelerated Fe uptake in both endothelial cell types. Phenylarsenoxide (PhAsO) and N-ethyl maleimide (NEM), inhibitors of Tf endocytosis, reduced both Tf and Fe uptake in both cell lines, while bafilomycin A1, an inhibitor of endosomal acidification, reduced Fe uptake but did not affect Tf uptake. The results suggest that Tf and Fe uptake by both bEND3 and m1END1 is via receptor-mediated endocytosis with release of Fe from Tf within the cell and recycling of apo-Tf. On the basis of Tf- and Fe-metabolism both cell lines are similar and therefore well suited for use in in vitro models for Fe transport across the BBB. PMID:10865941

  7. Generation of Murine Sympathoadrenergic Progenitor-Like Cells from Embryonic Stem Cells and Postnatal Adrenal Glands

    PubMed Central

    Saxena, Shobhit; Wahl, Joachim; Huber-Lang, Markus S.; Stadel, Dominic; Braubach, Peter; Debatin, Klaus-Michael; Beltinger, Christian

    2013-01-01

    Sympathoadrenergic progenitor cells (SAPs) of the peripheral nervous system (PNS) are important for normal development of the sympathetic PNS and for the genesis of neuroblastoma, the most common and often lethal extracranial solid tumor in childhood. However, it remains difficult to isolate sufficient numbers of SAPs for investigations. We therefore set out to improve generation of SAPs by using two complementary approaches, differentiation from murine embryonic stem cells (ESCs) and isolation from postnatal murine adrenal glands. We provide evidence that selecting for GD2 expression enriches for ESC-derived SAP-like cells and that proliferating SAP-like cells can be isolated from postnatal adrenal glands of mice. These advances may facilitate investigations about the development and malignant transformation of the sympathetic PNS. PMID:23675538

  8. Generation of murine sympathoadrenergic progenitor-like cells from embryonic stem cells and postnatal adrenal glands.

    PubMed

    Saxena, Shobhit; Wahl, Joachim; Huber-Lang, Markus S; Stadel, Dominic; Braubach, Peter; Debatin, Klaus-Michael; Beltinger, Christian

    2013-01-01

    Sympathoadrenergic progenitor cells (SAPs) of the peripheral nervous system (PNS) are important for normal development of the sympathetic PNS and for the genesis of neuroblastoma, the most common and often lethal extracranial solid tumor in childhood. However, it remains difficult to isolate sufficient numbers of SAPs for investigations. We therefore set out to improve generation of SAPs by using two complementary approaches, differentiation from murine embryonic stem cells (ESCs) and isolation from postnatal murine adrenal glands. We provide evidence that selecting for GD2 expression enriches for ESC-derived SAP-like cells and that proliferating SAP-like cells can be isolated from postnatal adrenal glands of mice. These advances may facilitate investigations about the development and malignant transformation of the sympathetic PNS.

  9. Malignant Potential of Murine Stromal Cells after Transplantation of Human Tumors into Nude Mice

    NASA Astrophysics Data System (ADS)

    Goldenberg, David M.; Pavia, Rose A.

    1981-04-01

    Human malignant cancer tumors grafted into nude mice produce tumors containing both human cancer cells and the host's stromal cells. After short-term propagation of these tumors in vitro, the murine mesenchymal cells appear transformed and are tumorigenic in nude mice. However, established human cancer cell lines fail to similarly alter adjacent murine stromal cells when used to produce tumors in nude mice. These experiments suggest that cancer cells may recruit normal cells to become malignant, qualifying the view of the clonal (unicellular) origin of cancer.

  10. Analysis of Cell Cycle Status of Murine Hematopoietic Stem Cells.

    PubMed

    Szade, Krzysztof; Bukowska-Strakova, Karolina; Zukowska, Monika; Jozkowicz, Alicja; Dulak, Józef

    2016-01-01

    Hematopoietic stem cells (HSC) act as paradigmatic tissue-specific adult stem cells. While they are quiescent in steady-state conditions, they enter the cell cycle and proliferate in stress conditions and during tissue regeneration. Therefore, analysis of cell cycle status of HSC is crucial for understanding their biology. However, due to low number of HSC in tissue and need to use many surface markers for their identification, analysis of their cycle status is technically complicated. Here, we presented our simple strategy to analyze cell cycle of strictly defined LKS CD48(-)CD150(+)CD34(-) HSC, together with Ki67 and DAPI staining by flow cytometry.

  11. Effects of aged garlic extract and FruArg on gene expression and signaling pathways in lipopolysaccharide-activated microglial cells

    PubMed Central

    Song, Hailong; Lu, Yuan; Qu, Zhe; Mossine, Valeri V.; Martin, Matthew B.; Hou, Jie; Cui, Jiankun; Peculis, Brenda A.; Mawhinney, Thomas P.; Cheng, Jianlin; Greenlief, C. Michael; Fritsche, Kevin; Schmidt, Francis J.; Walter, Ronald B.; Lubahn, Dennis B.; Sun, Grace Y.; Gu, Zezong

    2016-01-01

    Aged garlic extract (AGE) is widely used as a dietary supplement on account of its protective effects against oxidative stress and inflammation. But less is known about specific molecular targets of AGE and its bioactive components, including N-α-(1-deoxy-D-fructos-1-yl)-L-arginine (FruArg). Our recent study showed that both AGE and FruArg significantly attenuate lipopolysaccharide (LPS)-induced neuroinflammatory responses in BV-2 microglial cells. This study aims to unveil effects of AGE and FruArg on gene expression regulation in LPS stimulated BV-2 cells. Results showed that LPS treatment significantly altered mRNA levels from 2563 genes. AGE reversed 67% of the transcriptome alteration induced by LPS, whereas FruArg accounted for the protective effect by reversing expression levels of 55% of genes altered by LPS. Key pro-inflammatory canonical pathways induced by the LPS stimulation included toll-like receptor signaling, IL-6 signaling, and Nrf2-mediated oxidative stress pathway, along with elevated expression levels of genes, such as Il6, Cd14, Casp3, Nfkb1, Hmox1, and Tnf. These effects could be modulated by treatment with both AGE and FruArg. These findings suggests that AGE and FruArg are capable of alleviating oxidative stress and neuroinflammatory responses stimulated by LPS in BV-2 cells. PMID:27734935

  12. Inhibitory effect of ent-Sauchinone on amyloidogenesis via inhibition of STAT3-mediated NF-κB activation in cultured astrocytes and microglial BV-2 cells

    PubMed Central

    2014-01-01

    Background ent-Sauchinone is a polyphenolic compound found in plants belonging to the lignan family. ent-Sauchinone has been shown to modulate the expression of inflammatory factors through the nuclear factor-kappa B (NF-κB) signaling pathway. It is well known that neuroinflammation is associated with amyloidogenesis. Thus, in the present study, we investigated whether ent-Sauchinone could have anti-amyloidogenic effects through the inhibition of NF-κB pathways via its anti-inflammatory property. Methods To investigate the potential effect of ent-Sauchinone on anti-neuroinflammation and anti-amyloidogenesis in in vitro studies, we used microglial BV-2 cells and cultured astrocytes treated with ent-Sauchinone (1, 5, and 10 μM) for 24 hours. For the detection of anti-neuro-inflammatory responses, reative oxygen species (ROS) and Nitric oxide (NO) generation and inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression were measured with assay kits and western blotting. β-secretase and β-secretase activities and β-amyloid levels were determined for measuring the anti-amyloidogenic effects of ent-Sauchinone by enzyme assay kits. NF-κB and STAT3 signals were detected with electromobility shift assay (EMSA) to study the related signaling pathways. The binding of ent-Sauchinone to STAT3 was evaluated by a pull-down assay and by a docking model using Autodock VINA software (Hoover’s Inc., Texas, United states). Results ent-Sauchinone (1, 5, and 10 μM) effectively decreased lipopolysaccharide (LPS)-(1 μg/ml) induced inflammatory responses through the reduction of ROS and NO generations and iNOS and COX-2 expressions in cultured astrocytes and microglial BV-2 cells. ent-Sauchinone also inhibited LPS-induced amyloidogenesis through the inhibition of β-secretase and β-secretase activity. NF- κB amyloid and STAT3, critical transcriptional factors regulating not only inflammation but also amyloidogenesis, were also inhibited in a

  13. Tubby regulates microglial phagocytosis through MerTK.

    PubMed

    Caberoy, Nora B; Alvarado, Gabriela; Li, Wei

    2012-11-15

    Immunologically-silent microglial phagocytosis of apoptotic cells and cellular debris is critical for CNS homeostasis and innate immune balance. The beneficial and detrimental effects of microglial phagocytosis on neurons remain controversial. Phagocytosis ligands are the key to selecting extracellular cargos, initiating the engulfment process, defining phagocyte functional roles and regulating phagocyte activities with therapeutic potentials. Here we characterized tubby as a new ligand to regulate microglial phagocytosis through MerTK receptor, which is well known for its immunosuppressive signaling. Tubby at 0.1nM significantly induced microglial phagocytosis of apoptotic cells with a maximal activity at 10nM. Tubby activated MerTK with receptor autophosphorylation in a similar dose range. Excessive soluble MerTK extracellular domain blocked tubby-mediated microglial phagocytosis of plasma membrane vesicles as cellular debris. Immunocytochemistry revealed that the ingested cargos were co-localized with MerTK-dependent non-muscle myosin II, whose rearrangement is necessary for cargo engulfment. Phagosome biomarker Rab7 was colocalized with cargos, suggesting that internalized cargos were targeted to phagocytic pathway. Tubby stimulated phagocytosis by neonatal and aged microglia with similar activities, but not by MerTK(-/-) microglia. These results suggest that tubby is a ligand to facilitate microglial phagocytosis through MerTK for the maintenance of CNS homeostasis.

  14. HAC stability in murine cells is influenced by nuclear localization and chromatin organization

    PubMed Central

    Moralli, Daniela; Chan, David YL; Jefferson, Andrew; Volpi, Emanuela V; Monaco, Zoia L

    2009-01-01

    Background Human artificial chromosomes (HAC) are small functional extrachromosomal elements, which segregate correctly during each cell division. In human cells, they are mitotically stable, however when the HAC are transferred to murine cells they show an increased and variable rate of loss. In some cell lines the HAC are lost over a short period of time, while in others the HAC become stable without acquiring murine DNA. Results In this study, we linked the loss rate to the position of the HAC in the murine cell nucleus with respect to the chromocenters. HAC that associated preferentially with the chromocenter displayed a lower loss rate compared to the HAC that are less frequently associated. The chromocenter acts as a hub for the deposition of heterochromatic markers, controlling centromeric and pericentromeric DNA replication timing and chromosome segregation. The HAC which localized more frequently outside the chromocenters bound variable amounts of histone H3 tri-methylated at lysine 9, and the high level of intraclonal variability was associated with an increase in HAC segregation errors and delayed DNA replication timing. Conclusion This is a novel result indicating that HAC segregation is closely linked to the position in the murine nucleus and gives important insight for HAC gene expression studies in murine cells and establishing murine models of human genetic disease. PMID:19267891

  15. The murine coronavirus mouse hepatitis virus strain A59 from persistently infected murine cells exhibits an extended host range.

    PubMed Central

    Schickli, J H; Zelus, B D; Wentworth, D E; Sawicki, S G; Holmes, K V

    1997-01-01

    In murine 17 Cl 1 cells persistently infected with murine coronavirus mouse hepatitis virus strain A59 (MHV-A59), expression of the virus receptor glycoprotein MHVR was markedly reduced (S. G. Sawicki, J. H. Lu, and K. V. Holmes, J. Virol. 69:5535-5543, 1995). Virus isolated from passage 600 of the persistently infected cells made smaller plaques on 17 Cl 1 cells than did MHV-A59. Unlike the parental MHV-A59, this variant virus also infected the BHK-21 (BHK) line of hamster cells. Virus plaque purified on BHK cells (MHV/BHK) grew more slowly in murine cells than did MHV-A59, and the rate of viral RNA synthesis was lower and the development of the viral nucleocapsid (N) protein was slower than those of MHV-A59. MHV/BHK was 100-fold more resistant to neutralization with the purified soluble recombinant MHV receptor glycoprotein (sMHVR) than was MHV-A59. Pretreatment of 17 Cl 1 cells with anti-MHVR monoclonal antibody CC1 protected the cells from infection with MHV-A59 but only partially protected them from infection with MHV/BHK. Thus, although MHV/BHK could still utilize MHVR as a receptor, its interactions with the receptor were significantly different from those of MHV-A59. To determine whether a hemagglutinin esterase (HE) glycoprotein that could bind the virions to 9-O-acetylated neuraminic acid moieties on the cell surface was expressed by MHV/BHK, an in situ esterase assay was used. No expression of HE activity was detected in 17 Cl 1 cells infected with MHV/BHK, suggesting that this virus, like MHV-A59, bound to cell membranes via its S glycoprotein. MHV/BHK was able to infect cell lines from many mammalian species, including murine (17 Cl 1), hamster (BHK), feline (Fcwf), bovine (MDBK), rat (RIE), monkey (Vero), and human (L132 and HeLa) cell lines. MHV/BHK could not infect dog kidney (MDCK I) or swine testis (ST) cell lines. Thus, in persistently infected murine cell lines that express very low levels of virus receptor MHVR and which also have and may

  16. Role of Cytosolic Calcium Diffusion in Murine Cardiac Purkinje Cells

    PubMed Central

    Limbu, Bijay; Shah, Kushal; Weinberg, Seth H.; Deo, Makarand

    2016-01-01

    Cardiac Purkinje cells (PCs) are morphologically and electrophysiologically different from ventricular myocytes and, importantly, exhibit distinct calcium (Ca2+) homeostasis. Recent studies suggest that PCs are more susceptible to action potential (AP) abnormalities than ventricular myocytes; however, the exact mechanisms are poorly understood. In this study, we utilized a detailed biophysical mathematical model of a murine PC to systematically examine the role of cytosolic Ca2+ diffusion in shaping the AP in PCs. A biphasic spatiotemporal Ca2+ diffusion process, as recorded experimentally, was implemented in the model. In this study, we investigated the role of cytosolic Ca2+ dynamics on AP and ionic current properties by varying the effective Ca2+ diffusion rate. It was observed that AP morphology, specifically the plateau, was affected due to changes in the intracellular Ca2+ dynamics. Elevated Ca2+ concentration in the sarcolemmal region activated inward sodium–Ca2+ exchanger (NCX) current, resulting in a prolongation of the AP plateau at faster diffusion rates. Artificially clamping the NCX current to control values completely reversed the alterations in the AP plateau, thus confirming the role of NCX in modifying the AP morphology. Our results demonstrate that cytosolic Ca2+ diffusion waves play a significant role in shaping APs of PCs and could provide mechanistic insights in the increased arrhythmogeneity of PCs. PMID:27478391

  17. Dynamic changes in global microRNAome and transcriptome reveal complex miRNA-mRNA regulated host response to Japanese Encephalitis Virus in microglial cells

    PubMed Central

    Kumari, Bharti; Jain, Pratistha; Das, Shaoli; Ghosal, Suman; Hazra, Bibhabasu; Trivedi, Ashish Chandra; Basu, Anirban; Chakrabarti, Jayprokas; Vrati, Sudhanshu; Banerjee, Arup

    2016-01-01

    Microglia cells in the brain play essential role during Japanese Encephalitis Virus (JEV) infection and may lead to change in microRNA (miRNA) and mRNA profile. These changes may together control disease outcome. Using Affymetrix microarray platform, we profiled cellular miRNA and mRNA expression at multiple time points during viral infection in human microglial (CHME3) cells. In silico analysis of microarray data revealed a phased pattern of miRNAs expression, associated with JEV replication and provided unique signatures of infection. Target prediction and pathway enrichment analysis identified anti correlation between differentially expressed miRNA and the gene expression at multiple time point which ultimately affected diverse signaling pathways including Notch signaling pathways in microglia. Activation of Notch pathway during JEV infection was demonstrated in vitro and in vivo. The expression of a subset of miRNAs that target multiple genes in Notch signaling pathways were suppressed and their overexpression could affect JEV induced immune response. Further analysis provided evidence for the possible presence of cellular competing endogenous RNA (ceRNA) associated with innate immune response. Collectively, our data provide a uniquely comprehensive view of the changes in the host miRNAs induced by JEV during cellular infection and identify Notch pathway in modulating microglia mediated inflammation. PMID:26838068

  18. [Antibody adsorption to tetrodotoxin-sensitive cytoplasmic proteins by murine neuroblastoma cells].

    PubMed

    Pinchuk, G V; Pinchuk, L N; Gerasimenko, O V

    1988-01-01

    The study was aimed to examine properties of polyclonal antibodies elicited after immunization by cytoplasmic nerve cell glycoproteins forming sodium channels in liposomes. It was shown that intact murine neuroblastoma cells can absorb these antibodies. Absorbing cell dose-effect curves were found to have a characteristic form able to shift when the cell culture time or serum concentration in the growth medium varied.

  19. Murine granulated metrial gland cells are susceptible to Chlamydia psittaci infection in vivo.

    PubMed

    Sánchez, J; Buendía, A J; Salinas, J; Bernabé, A; Rodolakis, A; Stewart, I J

    1996-09-01

    Granulated metrial gland (GMG) cells are the most numerous lymphoid cells in the uteroplacental unit in rodent pregnancy. In an experimental murine model of abortion-causing infection, we have studied the responses of GMG cells to Chlamydia psittaci. Chlamydial inclusions have been found within GMG cells, both in apparently healthy cells and in cells with degenerative changes. Establishing the existence of GMG cells infected by C. psittaci opens a new and interesting chapter in the study of these cells.

  20. Murine granulated metrial gland cells are susceptible to Chlamydia psittaci infection in vivo.

    PubMed Central

    Sánchez, J; Buendía, A J; Salinas, J; Bernabé, A; Rodolakis, A; Stewart, I J

    1996-01-01

    Granulated metrial gland (GMG) cells are the most numerous lymphoid cells in the uteroplacental unit in rodent pregnancy. In an experimental murine model of abortion-causing infection, we have studied the responses of GMG cells to Chlamydia psittaci. Chlamydial inclusions have been found within GMG cells, both in apparently healthy cells and in cells with degenerative changes. Establishing the existence of GMG cells infected by C. psittaci opens a new and interesting chapter in the study of these cells. PMID:8751945

  1. Loss of Interleukin Receptor Associated Kinase 4 Signaling Suppresses Amyloid Pathology and Alters Microglial Phenotype in a Mouse Model of Alzheimer’s Disease

    PubMed Central

    Cameron, Brent; Tse, Wayne; Lamb, Raza; Li, Xiaoxia; Lamb, Bruce T.; Landreth, Gary E.

    2012-01-01

    Alzheimer’s disease (AD) typified the deposition of amyloid in the brain which elicits a robust microglial-mediated inflammatory response that is associated with disease exacerbation and accelerated progression. Microglia are the principal immune effector cells in the brain and interact with fibrillar forms of Aβ (fAβ) through a receptor complex that includes Toll-Like Receptors (TLR) 2/4/6 and their coreceptors. Interleukin receptor-associated kinases (IRAKs) are essential intracellular signaling molecules for transduction of TLR signals. Studies of mouse models of AD in which the individual TLRs are knocked out have produced conflicting results on roles of TLR signaling in amyloid homeostasis. Therefore, we disrupted a common downstream TLR signaling element, IRAK4. We report that microglial IRAK4 is necessary in vitro for fAβ to activate the canonical proinflammatory signaling pathways leading to activation of p38, JNK, and ERK MAP kinases and to generate reactive oxygen species. In vivo the loss of IRAK4 function results in decreased Aβ levels in a murine model of AD. This was associated with diminished microgliosis and astrogliosis in aged mice. Analysis of microglia isolated from the adult mouse brain revealed an altered pattern of gene expression associated with changes in microglial phenotype that were associated with expression of IRF transcription factors that govern microglial phenotype. Further, loss of IRAK4 function also promoted amyloid clearance mechanisms, including elevated expression of insulin degrading enzyme. Finally, blocking IRAK function restored olfactory behavior. These data demonstrate that IRAK4 activation acts normally to regulate microglial activation status and influence amyloid homeostasis in the brain. PMID:23100432

  2. Effects of oxaliplatin and oleic acid Gc-protein-derived macrophage-activating factor on murine and human microglia.

    PubMed

    Branca, Jacopo J V; Morucci, Gabriele; Malentacchi, Francesca; Gelmini, Stefania; Ruggiero, Marco; Pacini, Stefania

    2015-09-01

    The biological properties and characteristics of microglia in rodents have been widely described, but little is known about these features in human microglia. Several murine microglial cell lines are used to investigate neurodegenerative and neuroinflammatory conditions; however, the extrapolation of the results to human conditions is frequently met with criticism because of the possibility of species-specific differences. This study compares the effects of oxaliplatin and of oleic acid Gc-protein-derived macrophage-activating factor (OA-GcMAF) on two microglial cell lines, murine BV-2 cells and human C13NJ cells. Cell viability, cAMP levels, microglial activation, and vascular endothelial growth factor (VEGF) expression were evaluated. Our data demonstrate that oxaliplatin induced a significant decrease in cell viability in BV-2 and in C13NJ cells and that this effect was not reversed with OA-GcMAF treatment. The signal transduction pathway involving cAMP/VEGF was activated after treatment with oxaliplatin and/or OA-GcMAF in both cell lines. OA-GcMAF induced a significant increase in microglia activation, as evidenced by the expression of the B7-2 protein, in BV-2 as well as in C13NJ cells that was not associated with a concomitant increase in cell number. Furthermore, the effects of oxaliplatin and OA-GcMAF on coculture morphology and apoptosis were evaluated. Oxaliplatin-induced cell damage and apoptosis were nearly completely reversed by OA-GcMAF treatment in both BV-2/SH-SY5Y and C13NJ/SH-SY5Y cocultures. Our data show that murine and human microglia share common signal transduction pathways and activation mechanisms, suggesting that the murine BV-2 cell line may represent an excellent model for studying human microglia. PMID:25782915

  3. Effects of oxaliplatin and oleic acid Gc-protein-derived macrophage-activating factor on murine and human microglia.

    PubMed

    Branca, Jacopo J V; Morucci, Gabriele; Malentacchi, Francesca; Gelmini, Stefania; Ruggiero, Marco; Pacini, Stefania

    2015-09-01

    The biological properties and characteristics of microglia in rodents have been widely described, but little is known about these features in human microglia. Several murine microglial cell lines are used to investigate neurodegenerative and neuroinflammatory conditions; however, the extrapolation of the results to human conditions is frequently met with criticism because of the possibility of species-specific differences. This study compares the effects of oxaliplatin and of oleic acid Gc-protein-derived macrophage-activating factor (OA-GcMAF) on two microglial cell lines, murine BV-2 cells and human C13NJ cells. Cell viability, cAMP levels, microglial activation, and vascular endothelial growth factor (VEGF) expression were evaluated. Our data demonstrate that oxaliplatin induced a significant decrease in cell viability in BV-2 and in C13NJ cells and that this effect was not reversed with OA-GcMAF treatment. The signal transduction pathway involving cAMP/VEGF was activated after treatment with oxaliplatin and/or OA-GcMAF in both cell lines. OA-GcMAF induced a significant increase in microglia activation, as evidenced by the expression of the B7-2 protein, in BV-2 as well as in C13NJ cells that was not associated with a concomitant increase in cell number. Furthermore, the effects of oxaliplatin and OA-GcMAF on coculture morphology and apoptosis were evaluated. Oxaliplatin-induced cell damage and apoptosis were nearly completely reversed by OA-GcMAF treatment in both BV-2/SH-SY5Y and C13NJ/SH-SY5Y cocultures. Our data show that murine and human microglia share common signal transduction pathways and activation mechanisms, suggesting that the murine BV-2 cell line may represent an excellent model for studying human microglia.

  4. Phenotypic, Morphological and Adhesive Differences of Human Hematopoietic Progenitor Cells Cultured on Murine versus Human Mesenchymal Stromal Cells

    PubMed Central

    Reichert, Doreen; Friedrichs, Jens; Ritter, Steffi; Käubler, Theresa; Werner, Carsten; Bornhäuser, Martin; Corbeil, Denis

    2015-01-01

    Xenogenic transplantation models have been developed to study human hematopoiesis in immunocompromised murine recipients. They still have limitations and therefore it is important to delineate all players within the bone marrow that could account for species-specific differences. Here, we evaluated the proliferative capacity, morphological and physical characteristics of human CD34+ hematopoietic stem and progenitor cells (HSPCs) after co-culture on murine or human bone marrow-derived mesenchymal stromal cells (MSCs). After seven days, human CD34+CD133– HSPCs expanded to similar extents on both feeder layers while cellular subsets comprising primitive CD34+CD133+ and CD133+CD34– phenotypes are reduced fivefold on murine MSCs. The number of migrating HSPCs was also reduced on murine cells suggesting that MSC adhesion influences cellular polarization of HSPC. We used atomic force microscopy-based single-cell force spectroscopy to quantify their adhesive interactions. We found threefold higher detachment forces of human HSPCs from murine MSCs compared to human ones. This difference is related to the N-cadherin expression level on murine MSCs since its knockdown abolished their differential adhesion properties with human HSPCs. Our observations highlight phenotypic, morphological and adhesive differences of human HSPCs when cultured on murine or human MSCs, which raise some caution in data interpretation when xenogenic transplantation models are used. PMID:26498381

  5. Specific lysis of murine cells expressing HLA molecules by allospecific human and murine H-2-restricted anti-HLA T killer lymphocytes.

    PubMed

    Achour, A; Begue, B; Gomard, E; Paul, P; Sayagh, B; Van Pel, A; Levy, J P

    1986-06-01

    The lysis by human and murine anti-HLA cytolytic T lymphocytes (CTL) of murine cells expressing class I HLA molecule after gene transfection has been studied using two different murine cells: LMTK- and P815-HTR-TK-. Weak but significant HLA-A11-specific lysis was found occasionally with human CTL on the HLA-A11+ L cells. On the contrary, P815-A11 or P815-A2 cells were lysed strongly and specifically by HLA-A11 or HLA-A2-specific human CTL. The T8+T4- phenotype of the effector cells was confirmed and the reaction was inhibited by anti-HLA class I monoclonal antibodies. Despite their higher sensitivity to human CTL, the P815-HLA+ cells did not express higher levels of HLA antigens than L cells, and the presence or the absence of human beta 2 microglobulin was irrelevant. Anti-human LFA-1 antibodies abrogated the lysis of P815-A11+ cells showing that the LFA-1 receptor which is apparently lacking on the L cell surface was on the contrary expressed on P815 cells. On the other hand, murine anti-HLA CTL have been prepared by immunizing mice against syngeneic HLA-A11+ L cells. They lysed very efficiently and specifically these cells, but appeared completely devoid of activity against human HLA-A11 target cells. This barrier was apparently due to the H-2 restriction of these H-2k anti-HLA murine CTL, as shown by their inability to lyse allogeneic H-2d cells expressing HLA-A11, and by the blocking of their activity by anti H-2k antibodies. By contrast, xenogeneic anti-HLA CTL obtained by immunizing murine lymphocytes against human cells lysed both human and murine HLA+ cells but they reacted with a monomorphic epitope of the HLA molecule in a nonrestricted way. These results show that human cells lyse very efficiently P815 murine cells expressing HLA class I antigens; the higher sensitivity of P815 cells compared to L cells is probably due to the presence of a LFA-1 receptor on these cells; a class I molecule of human origin can be seen as an H-2-restricted minor

  6. Pseudolaric Acid B Induced Cell Cycle Arrest, Autophagy and Senescence in Murine Fibrosarcoma L929 Cell

    PubMed Central

    hua Yu, Jing; yu Liu, Chun; bin Zheng, Gui; Zhang, Li Ying; hui Yan, Ming; yan Zhang, Wen; ying Meng, Xian; fang Yu, Xiao

    2013-01-01

    Objective: PAB induced various cancer cell apoptosis, cell cycle arrest and senescence. But in cell line murine fibrosarcoma L929, PAB did not induce apoptosis, but autophagy, therefore it was thought by us as a good model to research the relationship of cell cycle arrest, autophagy and senescence bypass apoptosis. Methods: Inhibitory ratio was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. Phase contrast microscopy visualized cell morphology. Hoechst 33258 staining for nuclear change, propidium iodode (PI) staining for cell cycle, monodansylcadaverine (MDC) staining for autophagy, and rodanmine 123 staining for mitochondrial membrane potential (MMP) were measured by fluorescence microscopy or flowcytometry. Apoptosis was determined by DNA ladder test. Protein kinase C (PKC) activity was detected by PKC assay kit. SA-β-galactosidase assay was used to detect senescence. Protein expression was examined by western blot. Results: PAB inhibited L929 cell growth in time-and dose-dependent manner. At 12 h, 80 μmol/L PAB induced obvious mitotic arrest; at 24 h, PAB began to induce autophagy; at 36 h, cell-treated with PAB slip into G1 cell cycle; and 3 d PAB induced senescence. In time sequence PAB induced firstly cell cycle arrest, then autophagy, then slippage into G1 phase, lastly senescence. Senescent cells had high level of autophagy, inhibiting autophagy led to apoptosis, and no senescence. PAB activated PKC activity to induce cell cycle arrest, autophagy and senescence, inhibiting PKC activity suppressed cell cycle arrest, autophagy and senescence. Conclusion: PAB induced cell cycle arrest, autophagy and senescence in murine fibrosarcoma L929 cell through PKC. PMID:23630435

  7. Identification and characterization of the inducible murine mast cell gene, imc-415.

    PubMed

    Cho, S H; Cho, J J; Kim, I S; Vliagoftis, H; Metcalfe, D D; Oh, C K

    1998-11-01

    Activation of mast cells results in the generation and release of bioactive mediators which in turn initiate allergic inflammation. Mast cell function is enhanced following stimulation in part because of the induction of specific genes and their products. To identify additional genes induced in mast cells that support this process, we thus constructed an activation-specific mast cell subtraction library. To date, we have isolated 26 novel inducible murine mast cell (imc) cDNA clones. Among them, a full-coding region of the murine gene imc-415 was found to have a greater than 90% nucleotide sequence homology and a 97.5% amino acid sequence homology to both a human beta4 integrin-binding protein (p27(BBP)) and a human translation initiation factor 6 (eIF6), which in turn are identical. In vitro translation of the imc-415 gene yielded a band of an approximately 26 kDa. This is the same as the calculated molecular weight of murine IMC-415 protein based on the predicted amino acid sequence and is the molecular weight of p27(BBP)/eIF6. Murine imc-415 message was also induced in inflamed lung tissues in a mouse model of asthma. These results suggest a role for murine imc-415 in allergic inflammation where it may enhance protein synthesis. Human eIF6/p27(BBP) may also play a role in allergic diseases based on the similarities in sequence and in gene expression patterns.

  8. Plasmacytoid dendritic cells promote rotavirus-induced human and murine B cell responses.

    PubMed

    Deal, Emily M; Lahl, Katharina; Narváez, Carlos F; Butcher, Eugene C; Greenberg, Harry B

    2013-06-01

    B cell-dependent immunity to rotavirus, an important intestinal pathogen, plays a significant role in viral clearance and protects against reinfection. Human in vitro and murine in vivo models of rotavirus infection were used to delineate the role of primary plasmacytoid DCs (pDCs) in initiating B cell responses. Human pDCs were necessary and sufficient for B cell activation induced by rotavirus. Type I IFN recognition by B cells was essential for rotavirus-mediated B cell activation in vitro and murine pDCs and IFN-α/β-mediated B cell activation after in vivo intestinal rotavirus infection. Furthermore, rotavirus-specific serum and mucosal antibody responses were defective in mice lacking functional pDCs at the time of infection. These data demonstrate that optimal B cell activation and virus-specific antibody secretion following mucosal infection were a direct result of pDC-derived type I IFN. Importantly, viral shedding significantly increased in pDC-deficient mice, suggesting that pDC-dependent antibody production influences viral clearance. Thus, mucosal pDCs critically influence the course of rotavirus infection through rotavirus recognition and subsequent IFN production and display powerful adjuvant properties to initiate and enhance humoral immunity.

  9. Human allospecific cytolytic T lymphocyte lysis of a murine cell transfected with HLA-A2.

    PubMed

    Koller, T D; Clayberger, C; Maryanski, J L; Krensky, A M

    1987-04-01

    A variety of molecules are involved in the interaction of human allospecific cytolytic T lymphocytes (CTL) with target cells. Monoclonal antibodies specific for these molecules inhibit CTL-target conjugate formation and/or lysis. To further study recognition and lysis of targets by human CTL, we used a murine mastocytoma cell line transfected with the histocompatibility leukocyte antigen (HLA)-A2 gene (P815-A2+) as a target for human HLA-A2-specific CTL. We find that only a subset of human HLA-A2-specific CTL can lyse murine P815-A2+ cells, suggesting that the murine cells may lack one or more accessory molecules needed for CTL recognition and lysis. PMID:3549894

  10. Rabies virus replication in primary murine bone marrow macrophages and in human and murine macrophage-like cell lines: implications for viral persistence.

    PubMed

    Ray, N B; Ewalt, L C; Lodmell, D L

    1995-02-01

    To determine whether rabies viruses replicate in macrophage or macrophage-like cells, several human and murine macrophage-like cell lines, as well as primary cultures of murine bone marrow macrophages, were incubated with the Evelyn-Rokitnicki-Abelseth (ERA) virus and several different street rabies viruses (SRV). ERA rabies virus replicated well in human monocytic U937 and THP-1 cells and murine macrophage IC-21 cells, as well as primary cultures of murine macrophages. Minimal replication was detected in murine monocytic WEHI-3BD- and PU5-1R cells, and ERA virus did not replicate in murine monocytic P388D1 or J774A.1 cells. A tissue culture-adapted SRV of bat origin also replicated in IC-21 and U937 cells. Non-tissue culture-adapted SRV isolated from different animal species, particularly bats, replicated minimally in U937, THP-1, IC-21 cells and primary murine bone marrow macrophages. To determine whether rabies virus replication is dependent upon the state of differentiation of the macrophage-like cell, human promyelocytic HL-60 cells were differentiated with 12-O-tetradecanoylphorbol-13-acetate (TPA). ERA rabies virus replicated in the differentiated HL-60 cells but not in undifferentiated HL-60 cells. Persistent infections were established in macrophage-like U937 cells with ERA rabies virus and SRV, and infectious SRV was isolated from adherent bone marrow cells of mice that had been infected 96 days previously. Virus harvested from persistently infected U937 cells and the adherent bone marrow cells had specifically adapted to each cell. This specificity was shown by the inability of the viruses to infect macrophages other than U937 cells and primary bone marrow macrophages, respectively. Virus titers of the persistently infected U937 cells fluctuated with extended cell passage. After 30 passages, virus released from the cells had lost virulence as shown by its inability to kill intracranially inoculated mice. However, the avirulent virus released from the

  11. Microglial action in glioma: a boon turns bane.

    PubMed

    Ghosh, Anirban; Chaudhuri, Swapna

    2010-06-15

    Microglia has the potential to shape the neuroimmune defense with vast array of functional attributes. The cells prime infiltrated lymphocytes to retain their effector functions, play crucial role in controlling microenvironmental milieu and significantly participate in glioma. Reports demonstrate microglial accumulation in glioma and predict their assistance in glioma growth and spreading. Clarification of the 'double-edged' appearance of microglia is necessary to unfold its role in glioma biology. In this article the interpretation of microglial activities has been attempted to reveal their actual function in glioma. Contrary to the trendy acceptance of its glioma promoting infamy, accumulated evidences make an effort to view the state of affairs in favor of the cell. Critical scrutiny indicates that microglial immune assaults are intended to demolish the neoplastic cells in brain. But the weaponry of microglia has been tactically utilized by glioma in their favor as the survival strategy. Hence the defender appears as enemy in advanced glioma. PMID:20338195

  12. Microglial phagocytosis of living photoreceptors contributes to inherited retinal degeneration.

    PubMed

    Zhao, Lian; Zabel, Matthew K; Wang, Xu; Ma, Wenxin; Shah, Parth; Fariss, Robert N; Qian, Haohua; Parkhurst, Christopher N; Gan, Wen-Biao; Wong, Wai T

    2015-07-02

    Retinitis pigmentosa, caused predominantly by mutations in photoreceptor genes, currently lacks comprehensive treatment. We discover that retinal microglia contribute non-cell autonomously to rod photoreceptor degeneration by primary phagocytosis of living rods. Using rd10 mice, we found that the initiation of rod degeneration is accompanied by early infiltration of microglia, upregulation of phagocytic molecules in microglia, and presentation of "eat-me" signals on mutated rods. On live-cell imaging, infiltrating microglia interact dynamically with photoreceptors via motile processes and engage in rapid phagocytic engulfment of non-apoptotic rods. Microglial contribution to rod demise is evidenced by morphological and functional amelioration of photoreceptor degeneration following genetic ablation of retinal microglia. Molecular inhibition of microglial phagocytosis using the vitronectin receptor antagonist cRGD also improved morphological and functional parameters of degeneration. Our findings highlight primary microglial phagocytosis as a contributing mechanism underlying cell death in retinitis pigmentosa and implicate microglia as a potential cellular target for therapy.

  13. Type I collagen gel protects murine fibrosarcoma L929 cells from TNFα-induced cell death

    SciTech Connect

    Wang, Hong-Ju; He, Wen-Qi; Chen, Ling; Liu, Wei-Wei; Xu, Qian; Xia, Ming-Yu; Hayashi, Toshihiko; Fujisaki, Hitomi; Hattori, Shunji; Tashiro, Shin-ichi; Onodera, Satoshi; Ikejima, Takashi

    2015-02-20

    Murine fibrosarcoma L929 cells have been used to test efficacy of proinflammatory cytokine TNFα. In the present study, we reported on protective effect of type I collagen gel used as L929 cell culture. L929 cell grew and proliferated well on collagen gel. However, the L929 cells exhibited cobblestone-like morphology which was much different from the spread fusiform shape when cultured on conventional cell dishes as well as the cells tended to aggregate. On conventional cell culture dishes, the cells treated with TNFα became round in shape and eventually died in a necroptotic manner. The cells cultured on collagen gel, however, were completely unaffected. TNFα treatment was reported to induce autophagy in L929 cells on the plastic dish, and therefore we investigated the effect of collagen gel on induction of autophagy. The results indicated that autophagy induced by TNFα treatment was much reduced when the cells were cultured on collagen gel. In conclusion, type I collagen gel protected L929 cell from TNFα-induced cell death. - Highlights: • Collagen gel culture changed the morphology of L929 cells. • L929 cell cultured on collagen gel were resistant to TNFα-induced cell death. • Collagen gel culture inhibited TNFα-induced autophagy in L929 cells.

  14. Reactive oxygen species and phosphatidylserine externalization in murine sickle red cells.

    PubMed

    Banerjee, Tinku; Kuypers, Frans A

    2004-02-01

    Due to their role in oxygen transport and the presence of redox active haemoglobin molecules, red blood cells (RBC) generate relatively high levels of reactive oxygen species (ROS). To counteract the potential deleterious effects of ROS, RBCs have a well-integrated network of anti-oxidant mechanisms to combat this oxidative stress. ROS formation is increased in sickle-cell disease (SCD) and our studies in a murine SCD model showed a significant increase in the generation of ROS when compared with normal mice. Our data also indicated that murine sickle RBCs exhibit a significantly increased ATP catabolism, partly due to the increased activity of glucose-6-phosphate dehydrogenase and glutathione reductase to regenerate intracellular glutathione (GSH) levels to neutralize the adverse milieu of oxidative stress. Higher ATP consumption by the murine sickle RBCs, together with the increased ROS formation and impairment of the aminophospholipid translocase or flipase may underlie the exposure of phosphatidylserine on the surface of these cells.

  15. Tiagabine Protects Dopaminergic Neurons against Neurotoxins by Inhibiting Microglial Activation

    PubMed Central

    Liu, Jie; Huang, Dongping; Xu, Jing; Tong, Jiabin; Wang, Zishan; Huang, Li; Yang, Yufang; Bai, Xiaochen; Wang, Pan; Suo, Haiyun; Ma, Yuanyuan; Yu, Mei; Fei, Jian; Huang, Fang

    2015-01-01

    Microglial activation and inflammation are associated with progressive neuronal apoptosis in neurodegenerative disorders such as Parkinson’s disease (PD). γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system, has recently been shown to play an inhibitory role in the immune system. Tiagabine, a piperidine derivative, enhances GABAergic transmission by inhibiting GABA transporter 1 (GAT 1). In the present study, we found that tiagabine pretreatment attenuated microglial activation, provided partial protection to the nigrostriatal axis and improved motor deficits in a methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. The protective function of tiagabine was abolished in GAT 1 knockout mice that were challenged with MPTP. In an alternative PD model, induced by intranigral infusion of lipopolysaccharide (LPS), microglial suppression and subsequent neuroprotective effects of tiagabine were demonstrated. Furthermore, the LPS-induced inflammatory activation of BV-2 microglial cells and the toxicity of conditioned medium toward SH-SY5Y cells were inhibited by pretreatment with GABAergic drugs. The attenuation of the nuclear translocation of nuclear factor κB (NF-κB) and the inhibition of the generation of inflammatory mediators were the underlying mechanisms. Our results suggest that tiagabine acts as a brake for nigrostriatal microglial activation and that it might be a novel therapeutic approach for PD. PMID:26499517

  16. Comparative Analysis of Protein Tyrosine Phosphatases Regulating Microglial Activation

    PubMed Central

    Song, Gyun Jee; Kim, Jaehong; Kim, Jong-Heon; Song, Seungeun; Park, Hana; Zhang, Zhong-Yin

    2016-01-01

    Protein tyrosine phosphatases (PTPs) are key regulatory factors in inflammatory signaling pathways. Although PTPs have been extensively studied, little is known about their role in neuroinflammation. In the present study, we examined the expression of 6 different PTPs (PTP1B, TC-PTP, SHP2, MEG2, LYP, and RPTPβ) and their role in glial activation and neuroinflammation. All PTPs were expressed in brain and glia. The expression of PTP1B, SHP2, and LYP was enhanced in the inflamed brain. The expression of PTP1B, TC-PTP, and LYP was increased after treating microglia cells with lipopolysaccharide (LPS). To examine the role of PTPs in microglial activation and neuroinflammation, we used specific pharmacological inhibitors of PTPs. Inhibition of PTP1B, TC-PTP, SHP2, LYP, and RPTPβ suppressed nitric oxide production in LPS-treated microglial cells in a dose-dependent manner. Furthermore, intracerebroventricular injection of PTP1B, TC-PTP, SHP2, and RPTPβ inhibitors downregulated microglial activation in an LPS-induced neuroinflammation model. Our results indicate that multiple PTPs are involved in regulating microglial activation and neuroinflammation, with different expression patterns and specific functions. Thus, PTP inhibitors can be exploited for therapeutic modulation of microglial activation in neuroinflammatory diseases. PMID:27790059

  17. Tiagabine Protects Dopaminergic Neurons against Neurotoxins by Inhibiting Microglial Activation.

    PubMed

    Liu, Jie; Huang, Dongping; Xu, Jing; Tong, Jiabin; Wang, Zishan; Huang, Li; Yang, Yufang; Bai, Xiaochen; Wang, Pan; Suo, Haiyun; Ma, Yuanyuan; Yu, Mei; Fei, Jian; Huang, Fang

    2015-10-26

    Microglial activation and inflammation are associated with progressive neuronal apoptosis in neurodegenerative disorders such as Parkinson's disease (PD). γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system, has recently been shown to play an inhibitory role in the immune system. Tiagabine, a piperidine derivative, enhances GABAergic transmission by inhibiting GABA transporter 1 (GAT 1). In the present study, we found that tiagabine pretreatment attenuated microglial activation, provided partial protection to the nigrostriatal axis and improved motor deficits in a methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. The protective function of tiagabine was abolished in GAT 1 knockout mice that were challenged with MPTP. In an alternative PD model, induced by intranigral infusion of lipopolysaccharide (LPS), microglial suppression and subsequent neuroprotective effects of tiagabine were demonstrated. Furthermore, the LPS-induced inflammatory activation of BV-2 microglial cells and the toxicity of conditioned medium toward SH-SY5Y cells were inhibited by pretreatment with GABAergic drugs. The attenuation of the nuclear translocation of nuclear factor κB (NF-κB) and the inhibition of the generation of inflammatory mediators were the underlying mechanisms. Our results suggest that tiagabine acts as a brake for nigrostriatal microglial activation and that it might be a novel therapeutic approach for PD.

  18. Retinoic acid receptor agonist Am80 inhibits CXCL2 production from microglial BV-2 cells via attenuation of NF-κB signaling.

    PubMed

    Takaoka, Yuichiro; Takahashi, Moeka; Kurauchi, Yuki; Hisatsune, Akinori; Seki, Takahiro; Shudo, Koichi; Katsuki, Hiroshi

    2016-09-01

    Accumulating lines of evidence suggest that retinoic acid receptor agonists such as Am80 exerts anti-inflammatory actions in the central nervous system, although detailed mechanisms of the action remain largely unknown. Our previous findings suggest that Am80 provides therapeutic effect on intracerebral hemorrhage in mice via suppression of expression of chemokine (C-X-C motif) ligand 2 (CXCL2). Here we investigated the mechanisms of inhibitory action of Am80 on expression of CXCL2 and other pro-inflammatory factors in microglial BV-2 cells. Pretreatment with Am80 markedly suppressed lipopolysaccharide (LPS)-induced expression of CXCL2 mRNA and release of CXCL2 protein. Am80 had no effect on LPS-induced activation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinase. On the other hand, Am80 prevented LPS-induced nuclear translocation of p65 subunit of NF-κB complex. In addition, total expression levels of p65 and IκBα proteins, as well as of mRNAs encoding p65 and IκBα, were lowered by Am80. Dependence of CXCL2 expression on NF-κB was confirmed by the effect of an NF-κB inhibitor caffeic acid phenethyl ester that abolished LPS-induced CXCL2 expression. Caffeic acid phenethyl ester also abolished LPS-induced expression of inducible nitric oxide synthase, interleukin-1β and tumor necrosis factor α, which may be relevant to the inhibitory effect of Am80 on expression of these pro-inflammatory factors. We additionally found that Am80 attenuated LPS-induced up-regulation of CD14, a co-receptor for Toll-like receptor 4 (TLR4). These results suggest that inhibitory effect on TLR4 signaling mediated by NF-κB pathway underlies the anti-inflammatory action of retinoic acid receptor agonists in microglia. PMID:27351827

  19. Multispectral Imaging of T and B Cells in Murine Spleen and Tumor

    PubMed Central

    Feng, Zipei; Jensen, Shawn M.; Messenheimer, David J.; Farhad, Mohammed; Neuberger, Michael; Bifulco, Carlo B.

    2016-01-01

    Recent advances in multiplex immunohistochemistry techniques allow for quantitative, spatial identification of multiple immune parameters for enhanced diagnostic and prognostic insight. However, applying such techniques to murine fixed tissues, particularly sensitive epitopes, such as CD4, CD8α, and CD19, has been difficult. We compared different fixation protocols and Ag-retrieval techniques and validated the use of multiplex immunohistochemistry for detection of CD3+CD4+ and CD3+CD8+ T cell subsets in murine spleen and tumor. This allows for enumeration of these T cell subsets within immune environments, as well as the study of their spatial distribution. PMID:26994219

  20. A B-Cell Superantigen Induces the Apoptosis of Murine and Human Malignant B Cells

    PubMed Central

    Lorenzo, Daniela; Duarte, Alejandra; Mundiñano, Juliana; Berguer, Paula; Nepomnaschy, Irene; Piazzon, Isabel

    2016-01-01

    B-cell superantigens (Sags) bind to conserved sites of the VH or VL regions of immunoglobulin molecules outside their complementarity-determining regions causing the apoptosis of normal cognate B cells. No attempts to investigate whether B-cell Sags are able to induce the apoptosis of cognate malignant B cells were reported. In the present study we show that protein L (PpL), secreted by Finegoldia magna, a B-cell Sag which interacts with κ+ bearing cells, induces the apoptosis of murine and human κ+ lymphoma B cells both in vitro and in vivo. Apoptosis was not altered by caspase-8 inhibitor. No alterations in the levels of Bid, Fas and Fas-L were found suggesting that PpL does not activate the extrinsic pathway of apoptosis. The involvement of the intrinsic pathway was clearly indicated by: i) alterations in mitochondrial membrane potential (ΔΨm) both in murine and human lymphoma cells exposed to PpL; ii) decreased levels of apoptosis in the presence of caspase-9 inhibitor; iii) significant increases of Bim and Bax protein levels and downregulation of Bcl-2; iv) the translocation from the cytoplasm to the mitochondria of Bax and Bim pro-apoptotic proteins and its inhibition by caspase-9 inhibitor but not by caspase-8 inhibitor and v) the translocation of Bcl-2 protein from the mitochondria to the cytosol and its inhibition by caspase-9 inhibitor but not by caspase-8 inhibitor. The possibility of a therapeutic use of Sags in lymphoma/leukemia B cell malignancies is discussed. PMID:27603942

  1. A B-Cell Superantigen Induces the Apoptosis of Murine and Human Malignant B Cells.

    PubMed

    Lorenzo, Daniela; Duarte, Alejandra; Mundiñano, Juliana; Berguer, Paula; Nepomnaschy, Irene; Piazzon, Isabel

    2016-01-01

    B-cell superantigens (Sags) bind to conserved sites of the VH or VL regions of immunoglobulin molecules outside their complementarity-determining regions causing the apoptosis of normal cognate B cells. No attempts to investigate whether B-cell Sags are able to induce the apoptosis of cognate malignant B cells were reported. In the present study we show that protein L (PpL), secreted by Finegoldia magna, a B-cell Sag which interacts with κ+ bearing cells, induces the apoptosis of murine and human κ+ lymphoma B cells both in vitro and in vivo. Apoptosis was not altered by caspase-8 inhibitor. No alterations in the levels of Bid, Fas and Fas-L were found suggesting that PpL does not activate the extrinsic pathway of apoptosis. The involvement of the intrinsic pathway was clearly indicated by: i) alterations in mitochondrial membrane potential (ΔΨm) both in murine and human lymphoma cells exposed to PpL; ii) decreased levels of apoptosis in the presence of caspase-9 inhibitor; iii) significant increases of Bim and Bax protein levels and downregulation of Bcl-2; iv) the translocation from the cytoplasm to the mitochondria of Bax and Bim pro-apoptotic proteins and its inhibition by caspase-9 inhibitor but not by caspase-8 inhibitor and v) the translocation of Bcl-2 protein from the mitochondria to the cytosol and its inhibition by caspase-9 inhibitor but not by caspase-8 inhibitor. The possibility of a therapeutic use of Sags in lymphoma/leukemia B cell malignancies is discussed. PMID:27603942

  2. Systemic inflammation regulates microglial responses to tissue damage in vivo

    PubMed Central

    Gyoneva, Stefka; Davalos, Dimitrios; Biswas, Dipankar; Swanger, Sharon A.; Garnier-Amblard, Ethel; Loth, Francis; Akassoglou, Katerina; Traynelis, Stephen F.

    2015-01-01

    Microglia, the resident immune cells of the central nervous system, exist in either a “resting” state associated with physiological tissue surveillance or an “activated” state in neuroinflammation. We recently showed that ATP is the primary chemoattractor to tissue damage in vivo and elicits opposite effects on the motility of activated microglia in vitro through activation of adenosine A2A receptors. However, whether systemic inflammation affects microglial responses to tissue damage in vivo remains largely unknown. Using in vivo two-photon imaging of mice, we show that injection of lipopolysaccharide (LPS) at levels that can produce both clear neuroinflammation and some features of sepsis significantly reduced the rate of microglial response to laser-induced ablation injury in vivo. Under pro-inflammatory conditions, microglial processes initially retracted from the ablation site, but subsequently moved toward and engulfed the damaged area. Analyzing the process dynamics in 3D cultures of primary microglia indicated that only A2A, but not A1 or A3 receptors, mediate process retraction in LPS-activated microglia. The A2A receptor antagonists caffeine and preladenant reduced adenosine-mediated process retraction in activated microglia in vitro. Finally, administration of preladenant before induction of laser ablation in vivo accelerated the microglial response to injury following systemic inflammation. The regulation of rapid microglial responses to sites of injury by A2A receptors could have implications for their ability to respond to the neuronal death occurring under conditions of neuroinflammation in neurodegenerative disorders. PMID:24807189

  3. Microglial activation and progressive brain changes in schizophrenia.

    PubMed

    Laskaris, L E; Di Biase, M A; Everall, I; Chana, G; Christopoulos, A; Skafidas, E; Cropley, V L; Pantelis, C

    2016-02-01

    Schizophrenia is a debilitating disorder that typically begins in adolescence and is characterized by perceptual abnormalities, delusions, cognitive and behavioural disturbances and functional impairments. While current treatments can be effective, they are often insufficient to alleviate the full range of symptoms. Schizophrenia is associated with structural brain abnormalities including grey and white matter volume loss and impaired connectivity. Recent findings suggest these abnormalities follow a neuroprogressive course in the earliest stages of the illness, which may be associated with episodes of acute relapse. Neuroinflammation has been proposed as a potential mechanism underlying these brain changes, with evidence of increased density and activation of microglia, immune cells resident in the brain, at various stages of the illness. We review evidence for microglial dysfunction in schizophrenia from both neuroimaging and neuropathological data, with a specific focus on studies examining microglial activation in relation to the pathology of grey and white matter. The studies available indicate that the link between microglial dysfunction and brain change in schizophrenia remains an intriguing hypothesis worthy of further examination. Future studies in schizophrenia should: (i) use multimodal imaging to clarify this association by mapping brain changes longitudinally across illness stages in relation to microglial activation; (ii) clarify the nature of microglial dysfunction with markers specific to activation states and phenotypes; (iii) examine the role of microglia and neurons with reference to their overlapping roles in neuroinflammatory pathways; and (iv) examine the impact of novel immunomodulatory treatments on brain structure in schizophrenia. PMID:26455353

  4. Microglial polarization and plasticity: evidence from organotypic hippocampal slice cultures.

    PubMed

    Ajmone-Cat, Maria Antonietta; Mancini, Melissa; De Simone, Roberta; Cilli, Piera; Minghetti, Luisa

    2013-10-01

    Increasing evidence indicates that "functional plasticity" is not solely a neuronal attribute but a hallmark of microglial cells, the main brain resident macrophage population. Far from being a univocal phenomenon, microglial activation can originate a plethora of functional phenotypes, encompassing the classic M1 proinflammatory and the alternative M2 anti-inflammatory phenotypes. This concept overturns the popular view of microglial activation as a synonym of neurotoxicity and neurogenesis failure in brain disorders. The characterization of the alternative programs is a matter of intense investigation, but still scarce information is available on the course of microglial activation, on the reversibility of the different commitments and on the capability of preserving molecular memory of previous priming stimuli. By using organotypic hippocampal slice cultures as a model, we developed paradigms of stimulation aimed at shedding light on some of these aspects. We show that persistent stimulation of TLR4 signaling promotes an anti-inflammatory response and microglial polarization toward M2-like phenotype. Moreover, acute and chronic preconditioning regimens permanently affect the capability to respond to a later challenge, suggesting the onset of mechanisms of molecular memory. Similar phenomena could occur in the intact brain and differently affect the vulnerability of mature and newborn neurons to noxious signals. PMID:23918452

  5. Quantitating the subtleties of microglial morphology with fractal analysis

    PubMed Central

    Karperien, Audrey; Ahammer, Helmut; Jelinek, Herbert F.

    2013-01-01

    It is well established that microglial form and function are inextricably linked. In recent years, the traditional view that microglial form ranges between “ramified resting” and “activated amoeboid” has been emphasized through advancing imaging techniques that point to microglial form being highly dynamic even within the currently accepted morphological categories. Moreover, microglia adopt meaningful intermediate forms between categories, with considerable crossover in function and varying morphologies as they cycle, migrate, wave, phagocytose, and extend and retract fine and gross processes. From a quantitative perspective, it is problematic to measure such variability using traditional methods, but one way of quantitating such detail is through fractal analysis. The techniques of fractal analysis have been used for quantitating microglial morphology, to categorize gross differences but also to differentiate subtle differences (e.g., amongst ramified cells). Multifractal analysis in particular is one technique of fractal analysis that may be useful for identifying intermediate forms. Here we review current trends and methods of fractal analysis, focusing on box counting analysis, including lacunarity and multifractal analysis, as applied to microglial morphology. PMID:23386810

  6. The selective mGluR5 agonist CHPG attenuates SO2-induced oxidative stress and inflammation through TSG-6/NF-κB pathway in BV2 microglial cells.

    PubMed

    Qiu, Jun-Ling; Zhu, Wen-Li; Lu, Yu-Jie; Bai, Zheng-Fa; Liu, Zhi-Gang; Zhao, Pei; Sun, Chao; Zhang, Ya-Bin; Li, Hua; Liu, Wei

    2015-01-01

    Sulfur dioxide (SO2) is a common air pollutant and can cause harmful insults on neurons. Microglial activation has been implicated in the signaling cascades that contribute to neuronal cell death in various neurological disorders. In the present study, we found that SO2 derivatives decreased cell viability via inducing oxidative stress, inflammatory responses and apoptotic cell death in BV2 microglial cells. Pretreatment with (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), an mGluR5 agonist, significantly attenuated the SO2-induced cytotoxicity, which was fully prevented by the mGluR5 antagonist MPEP. CHPG increased the expression of TNF-α stimulated gene/protein 6 (TSG-6), but decreased the activation of nuclear factor-κB (NF-κB) after SO2 derivatives treatment in BV2 cells. In addition, knockdown of TSG-6 expression by specific targeted short interfering RNA (siRNA) partially reversed the protection induced by CHPG. Therefore, our findings reveal a mechanistic basis for exploring the association between SO2 exposure and neurological disorders, and also for opening up therapeutic approaches of ameliorating neuronal injury resulting from exposure in atmospheric polluting environment.

  7. A murine model for genetic manipulation of the T cell compartment.

    PubMed

    Gu, J; Kuo, M L; Rivera, A; Sutkowski, N; Ron, Y; Dougherty, J P

    1996-10-01

    The expression of exogenous genes in long-lived primary T cells is potentially beneficial for the treatment of various diseases including cancer, AIDS, genetic defects of the lymphoid compartment, and systemic enzyme deficiencies such as hemophilia. One approach for genetic modification of T cells is to introduce therapeutic genes into hematopoietic stem cells that would give rise to cells of the lymphoid lineage. Efficient gene transfer and expression in stem cells is often problematic, however. A more direct approach is to introduce the genes into mature primary T lymphocytes since the transferred genes can be maintained and expressed for long periods by long-lived peripheral T cells. In this report, we describe the adoptive transfer into SCID mice of both murine and human primary T cells that have been efficiently transduced with exogenous genes. Primary murine T cells transduced with a retroviral vector containing the human adenosine deaminase (ADA) gene persisted for at least 5 months in lymphoid organs of SCID mice, continuously expressing the exogenous gene. Primary human T cells were also used as target cells for transfer of the beta-galactosidase (lacZ) gene. Expression of the lacZ gene could be detected in over 20% of the transduced primary T cells before adoptive transfer into SCID mice. Transduced human T cells were injected into SCID mice intraperitoneally (ip), and the beta-galactosidase activity could be detected in cells collected from peritoneal exudate washes of recipient mice 6 weeks post-injection. These results demonstrate the availability of a murine model in which the long-term effects of expression of exogenous genes in both murine and human T cells can be tested. PMID:8913290

  8. Tomato lectin histochemistry for microglial visualization.

    PubMed

    Villacampa, Nàdia; Almolda, Beatriz; González, Berta; Castellano, Bernardo

    2013-01-01

    The use of different lectins for the study of microglial cells in the central nervous system (CNS) is a valuable tool that has been extensively used in the last years for the selective staining of this glial cell population, not only in normal physiological conditions, but also in a wide range of pathological situations where the normal homeostasis of the parenchyma is disturbed. In this chapter we accurately describe the methodology for the selective labelling of microglial cells by using the tomato lectin (TL), a protein lectin obtained from Lycopersicum esculentum with specific affinity for poly-N-acetyl lactosamine sugar residues which are found on the plasma membrane and in the cytoplasm of microglia. Here we describe how to perform this technique on vibratome, frozen, and paraffin sections for optical microscopy, as well as for transmission electron microscopy (TEM) studies. Using this methodology it is possible to visualize amoeboid microglia in the developing brain, ramified microglia in the adult, and activated/reactive microglia in the experimentally damaged brain. In addition, as TL also recognized sugar residues in endothelial cells, this technique is very useful for the study of the relationship established between microglia and the CNS vasculature. PMID:23813385

  9. The multidrug resistance protein 1: a functionally important activation marker for murine Th1 cells.

    PubMed

    Prechtl, S; Roellinghoff, M; Scheper, R; Cole, S P; Deeley, R G; Lohoff, M

    2000-01-15

    Previously, we described the expression of an energy-dependent pump in resting murine Th2 (but not resting Th1) cells which extruded the fluorescent dye Fluo-3. After stimulation with Ag and APCs, Th1 cells also expressed this pump. Furthermore, expression of the murine multidrug resistance protein 1 (mrp1) correlated with the presence of the pump. In this study, we report that Fluo-3 is indeed transported by murine mrp1 or its human ortholog MRP1, as revealed by transfection of HEK 293 cells with mrp1 or MRP1 cDNA. Like antigenic activation, IL-2 dose-dependently enhanced the Fluo-3-extruding activity in murine Th1 cells. Although TNF-alpha and IL-12 by themselves only weakly enhanced Fluo-3 extrusion, each of them did so in strong synergism with IL-2. An Ab directed against mrp1 was used to quantify the expression of mrp1 protein in T cells at the single-cell level. Like the Fluo-3 pump, mrp1 protein expression was enhanced by IL-2. Immunohistochemical studies using confocal laser microscopy indicated that mrp1 is localized mainly at the plasma membrane. In addition, protein expression of mrp1 was induced in Vbeta8+CD4+ T cells 12 h after in vivo application of Staphylococcal enterotoxin B. Finally, mrp1 was functionally relevant during the activation process of Th1 cells, because T cell activation could be suppressed by exposure of cells to the mrp1 inhibitor MK571. Thus, we present mrp1 as a novel, functionally important activation marker for Th1 cells and short-term in vivo activated CD4+ T cells, whereas its expression seems to be constitutive in Th2 cells.

  10. Transduction of Murine Hematopoietic Stem Cells with Tetracycline-regulated Lentiviral Vectors.

    PubMed

    Stahlhut, Maike; Schambach, Axel; Kustikova, Olga S

    2016-01-01

    Tetracycline-regulated integrating vectors allow pharmacologically controlled genetic modification of murine and human hematopoietic stem cells (HSCs). This approach combines the stable transgene insertion into a host genome with the opportunity for time- and dose-controlled reversible transgene expression in HSCs. Here, we describe the step-by-step protocol for transduction of murine stem-cell enriched populations of bone marrow cells, such as lineage negative cells (Lin(-)), with a lentiviral vector expressing the enhanced green fluorescent protein (EGFP) under the control of the tetracycline-regulated promoter. This chapter explains how to establish in vitro and in vivo systems to study transgene dose-dependent mechanisms affecting cell fate decisions of genetically modified hematopoietic cells. PMID:27317173

  11. Production of Virus by Mammalian Cells Transformed by Rous Sarcoma and Murine Sarcoma Viruses

    PubMed Central

    Valentine, Artrice F.; Bader, John P.

    1968-01-01

    Cultured cells of mammalian tumors induced by ribonucleic acid (RNA)-containing oncogenic viruses were examined for production of virus. The cell lines were established from tumors induced in rats and hamsters with either Rous sarcoma virus (Schmidt-Ruppin or Bryan strains) or murine sarcoma virus (Moloney strain). When culture fluids from each of the cell lines were examined for transforming activity or production of progeny virus, none of the cell lines was found to be infectious. However, electron microscopic examination of the various cell lines revealed the presence of particles in the rat cells transformed by either Rous sarcoma virus or murine sarcoma virus. These particles, morphologically similar to those associated with murine leukemias, were found both in the extracellular fluid concentrates and in whole-cell preparations. In the latter, they were seen budding from the cell membranes or lying in the intercellular spaces. No viruslike particles were seen in preparations from hamster tumors. Exposure of the rat cells to 3H-uridine resulted in the appearance of labeled particles with densities in sucrose gradients typical of virus (1.16 g/ml.). RNA of high molecular weight was extracted from these particles, and double-labeling experiments showed that this RNA sedimented at the same rate as RNA extracted from Rous sarcoma virus. None of the hamster cell lines gave radioactive peaks in the virus density range, and no extractable high molecular weight RNA was found. These studies suggest that the murine sarcoma virus produces an infection analogous to certain “defective” strains of Rous sarcoma virus, in that particles produced by infected cells have a low efficiency of infection. The control of the host cell over the production and properties of the RNA-containing tumorigenic viruses is discussed. Images PMID:4316021

  12. COMPARATIVE TOXICITY OF DIFFERENT EMISSION PARTICLES IN MURINE PULMONARY EPITHELIAL CELLS AND MACROPHAGES

    EPA Science Inventory

    Comparative Toxicity of Different Emission Particles in Murine Pulmonary Epithelial Cells and Macrophages. T Stevens1, M Daniels2, P Singh2, M I Gilmour2. 1 UNC, Chapel Hill 27599 2Experimental Toxicology Division, NHEERL, RTP, NC 27711

    Epidemiological studies have shown ...

  13. C1q, the recognition subcomponent of the classical pathway of complement, drives microglial activation.

    PubMed

    Färber, Katrin; Cheung, Giselle; Mitchell, Daniel; Wallis, Russell; Weihe, Eberhard; Schwaeble, Wilhelm; Kettenmann, Helmut

    2009-02-15

    Microglia, central nervous system (CNS) resident phagocytic cells, persistently police the integrity of CNS tissue and respond to any kind of damage or pathophysiological changes. These cells sense and rapidly respond to danger and inflammatory signals by changing their cell morphology; by release of cytokines, chemokines, or nitric oxide; and by changing their MHC expression profile. We have shown previously that microglial biosynthesis of the complement subcomponent C1q may serve as a reliable marker of microglial activation ranging from undetectable levels of C1q biosynthesis in resting microglia to abundant C1q expression in activated, nonramified microglia. In this study, we demonstrate that cultured microglial cells respond to extrinsic C1q with a marked intracellular Ca(2+) increase. A shift toward proinflammatory microglial activation is indicated by the release of interleukin-6, tumor necrosis factor-alpha, and nitric oxide and the oxidative burst in rat primary microglial cells, an activation and differentiation process similar to the proinflammatory response of microglia to exposure to lipopolysaccharide. Our findings indicate 1) that extrinsic plasma C1q is involved in the initiation of microglial activation in the course of CNS diseases with blood-brain barrier impairment and 2) that C1q synthesized and released by activated microglia is likely to contribute in an autocrine/paracrine way to maintain and balance microglial activation in the diseased CNS tissue. PMID:18831010

  14. Comparative study of artificial chromosome centromeres in human and murine cells

    PubMed Central

    Moralli, Daniela; Jefferson, Andrew; Valeria Volpi, Emanuela; Larin Monaco, Zoia

    2013-01-01

    Human artificial chromosomes (HAC) are a valuable tool in the analysis of complex chromatin structures such as the human centromere because of their small size and relative simplicity compared with normal human chromosomes. This report includes a comprehensive study of the centromere and chromatin composition of HAC, expressing human genes, generated in human cells and transferred to murine cells. The analysis involved chromatin immuno-precipitation and immuno-FISH on metaphase chromosomes and chromatin fibres. In both the cell types, the HAC consisted of alphoid and non-alphoid DNA and were mainly euchromatic in composition, although a pericentromeric heterochromatic region was present on all the HAC. Fibre-FISH and chromatin immuno-precipitation data indicated that the position of the centromere differed between HAC in human cells and in murine cells. Our work highlights the importance and utilisation of HAC for understanding the epigenetic aspects of chromosome biology. PMID:23403904

  15. Microglial VPAC1R mediates a novel mechanism of neuroimmune-modulation of hippocampal precursor cells via IL-4 release

    PubMed Central

    Nunan, Robert; Sivasathiaseelan, Harri; Khan, Damla; Zaben, Malik; Gray, William

    2014-01-01

    Neurogenesis, the production of new neurons from neural stem/progenitor cells (NSPCs), occurs throughout adulthood in the dentate gyrus of the hippocampus, where it supports learning and memory. The innate and adaptive immune systems are increasingly recognized as important modulators of hippocampal neurogenesis under both physiological and pathological conditions. However, the mechanisms by which the immune system regulates hippocampal neurogenesis are incompletely understood. In particular, the role of microglia, the brains resident immune cell is complex, as they have been reported to both positively and negatively regulate neurogenesis. Interestingly, neuronal activity can also regulate the function of the immune system. Here, we show that depleting microglia from hippocampal cultures reduces NSPC survival and proliferation. Furthermore, addition of purified hippocampal microglia, or their conditioned media, is trophic and proliferative to NSPCs. VIP, a neuropeptide released by dentate gyrus interneurons, enhances the proliferative and pro-neurogenic effect of microglia via the VPAC1 receptor. This VIP-induced enhancement is mediated by IL-4 release, which directly targets NSPCs. This demonstrates a potential neuro-immuno-neurogenic pathway, disruption of which may have significant implications in conditions where combined cognitive impairments, interneuron loss, and immune system activation occurs, such as temporal lobe epilepsy and Alzheimer's disease. PMID:24801739

  16. Microglial VPAC1R mediates a novel mechanism of neuroimmune-modulation of hippocampal precursor cells via IL-4 release.

    PubMed

    Nunan, Robert; Sivasathiaseelan, Harri; Khan, Damla; Zaben, Malik; Gray, William

    2014-08-01

    Neurogenesis, the production of new neurons from neural stem/progenitor cells (NSPCs), occurs throughout adulthood in the dentate gyrus of the hippocampus, where it supports learning and memory. The innate and adaptive immune systems are increasingly recognized as important modulators of hippocampal neurogenesis under both physiological and pathological conditions. However, the mechanisms by which the immune system regulates hippocampal neurogenesis are incompletely understood. In particular, the role of microglia, the brains resident immune cell is complex, as they have been reported to both positively and negatively regulate neurogenesis. Interestingly, neuronal activity can also regulate the function of the immune system. Here, we show that depleting microglia from hippocampal cultures reduces NSPC survival and proliferation. Furthermore, addition of purified hippocampal microglia, or their conditioned media, is trophic and proliferative to NSPCs. VIP, a neuropeptide released by dentate gyrus interneurons, enhances the proliferative and pro-neurogenic effect of microglia via the VPAC1 receptor. This VIP-induced enhancement is mediated by IL-4 release, which directly targets NSPCs. This demonstrates a potential neuro-immuno-neurogenic pathway, disruption of which may have significant implications in conditions where combined cognitive impairments, interneuron loss, and immune system activation occurs, such as temporal lobe epilepsy and Alzheimer's disease.

  17. Murine B7 antigen provides an efficient costimulatory signal for activation of murine T lymphocytes via the T-cell receptor/CD3 complex.

    PubMed Central

    Reiser, H; Freeman, G J; Razi-Wolf, Z; Gimmi, C D; Benacerraf, B; Nadler, L M

    1992-01-01

    We demonstrate that the murine B7 (mB7) protein is a potent costimulatory molecule for the activation of resting murine CD4+ T cells through the T-cell receptor (TCR)/CD3 complex. Stable mB7-transfected Chinese hamster ovary cells, but not vector-transfected controls, synergize with anti-CD3 monoclonal antibody and Con A-induced T-cell activation, resulting ultimately in proliferation. mB7 exerted its effect by inducing production of interleukin 2 and expression of the interleukin 2 receptor. Thus, mB7 costimulates T-cell activation through the TCR/CD3 complex by positively modulating the normal pathway of T-cell expansion. In contrast to the pronounced effect of mB7 on the activation of T cells through the TCR/CD3 complex, the mB7-transfected CHO cell line costimulated T-cell activation via the glycosylphosphatidylinositol-anchored proteins Thy-1 and Ly-6A.2 only inefficiently. Finally, the combination of a calcium ionophore and mB7 is not sufficient to cause T-cell proliferation, while the combination of a calcium ionophore and phorbol 12-myristate 13-acetate (PMA) stimulates T cells efficiently. The signals that mB7 and PMA provide for murine T lymphocyte activation are therefore not interchangeable, although both costimulate activation through the TCR/CD3 complex. Images PMID:1370349

  18. Differential detection of impact site versus rotational site injury by magnetic resonance imaging and microglial morphology in an unrestrained mild closed head injury model

    PubMed Central

    Grinberg, Yelena Y.; Obenaus, Andre; Carson, Monica J.

    2016-01-01

    Seventy-five percent of all traumatic brain injuries are mild and do not cause readily visible abnormalities on routine medical imaging making it difficult to predict which individuals will develop unwanted clinical sequelae. Microglia are brain-resident macrophages and early responders to brain insults. Their activation is associated with changes in morphology or expression of phenotypic markers including P2Y12 and major histocompatibility complex class II. Using a murine model of unrestrained mild closed head injury (mCHI), we used microglia as reporters of acute brain injury at sites of impact versus sites experiencing rotational stress 24 h post-mCHI. Consistent with mild injury, a modest 20% reduction in P2Y12 expression was detected by quantitative real-time PCR (qPCR) analysis but only in the impacted region of the cortex. Furthermore, neither an influx of blood-derived immune cells nor changes in microglial expression of CD45, TREM1, TREM2, major histocompatibility complex class II or CD40 were detected. Using magnetic resonance imaging (MRI), small reductions in T2 weighted values were observed but only near the area of impact and without overt tissue damage (blood deposition, edema). Microglial morphology was quantified without cryosectioning artifacts using ScaleA2 clarified brains from CX3CR1-green fluorescence protein (GFP) mice. The cortex rostral to the mCHI impact site receives greater rotational stress but neither MRI nor molecular markers of microglial activation showed significant changes from shams in this region. However, microglia in this rostral region did display signs of morphologic activation equivalent to that observed in severe CHI. Thus, mCHI-triggered rotational stress is sufficient to cause injuries undetectable by routine MRI that could result in altered microglial surveillance of brain homeostasis. PMID:26806371

  19. Cell differentiation mediated by co-culture of human umbilical cord blood stem cells with murine hepatic cells.

    PubMed

    Stecklum, Maria; Wulf-Goldenberg, Annika; Purfürst, Bettina; Siegert, Antje; Keil, Marlen; Eckert, Klaus; Fichtner, Iduna

    2015-02-01

    In the present study, purified human cord blood stem cells were co-cultivated with murine hepatic alpha mouse liver 12 (AML12) cells to compare the effect on endodermal stem cell differentiation by either direct cell-cell interaction or by soluble factors in conditioned hepatic cell medium. With that approach, we want to mimic in vitro the situation of preclinical transplantation experiments using human cells in mice. Cord blood stem cells, cultivated with hepatic conditioned medium, showed a low endodermal differentiation but an increased connexin 32 (Cx32) and Cx43, and cytokeratin 8 (CK8) and CK19 expression was monitored by reverse transcription polymerase chain reaction (RT-PCR). Microarray profiling indicated that in cultivated cord blood cells, 604 genes were upregulated 2-fold, with the highest expression for epithelial CK19 and epithelial cadherin (E-cadherin). On ultrastructural level, there were no major changes in the cellular morphology, except a higher presence of phago(ly)some-like structures observed. Direct co-culture of AML12 cells with cord blood cells led to less incisive differentiation with increased sex-determining region Y-box 17 (SOX17), Cx32 and Cx43, as well as epithelial CK8 and CK19 expressions. On ultrastructural level, tight cell contacts along the plasma membranes were revealed. FACS analysis in co-cultivated cells quantified dye exchange on low level, as also proved by time relapse video-imaging of labelled cells. Modulators of gap junction formation influenced dye transfer between the co-cultured cells, whereby retinoic acid increased and 3-heptanol reduced the dye transfer. The study indicated that the cell-co-cultured model of human umbilical cord blood cells and murine AML12 cells may be a suitable approach to study some aspects of endodermal/hepatic cell differentiation induction. PMID:25270685

  20. The preparation of primary hematopoietic cell cultures from murine bone marrow for electroporation.

    PubMed

    Kroeger, Kelly; Collins, Michelle; Ugozzoli, Luis

    2009-01-01

    It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed. PMID:19229174

  1. Redox Control of Microglial Function: Molecular Mechanisms and Functional Significance

    PubMed Central

    McBean, Gethin; Cindric, Marina; Egea, Javier; López, Manuela G.; Rada, Patricia; Zarkovic, Neven

    2014-01-01

    Abstract Neurodegenerative diseases are characterized by chronic microglial over-activation and oxidative stress. It is now beginning to be recognized that reactive oxygen species (ROS) produced by either microglia or the surrounding environment not only impact neurons but also modulate microglial activity. In this review, we first analyze the hallmarks of pro-inflammatory and anti-inflammatory phenotypes of microglia and their regulation by ROS. Then, we consider the production of reactive oxygen and nitrogen species by NADPH oxidases and nitric oxide synthases and the new findings that also indicate an essential role of glutathione (γ-glutamyl-l-cysteinylglycine) in redox homeostasis of microglia. The effect of oxidant modification of macromolecules on signaling is analyzed at the level of oxidized lipid by-products and sulfhydryl modification of microglial proteins. Redox signaling has a profound impact on two transcription factors that modulate microglial fate, nuclear factor kappa-light-chain-enhancer of activated B cells, and nuclear factor (erythroid-derived 2)-like 2, master regulators of the pro-inflammatory and antioxidant responses of microglia, respectively. The relevance of these proteins in the modulation of microglial activity and the interplay between them will be evaluated. Finally, the relevance of ROS in altering blood brain barrier permeability is discussed. Recent examples of the importance of these findings in the onset or progression of neurodegenerative diseases are also discussed. This review should provide a profound insight into the role of redox homeostasis in microglial activity and help in the identification of new promising targets to control neuroinflammation through redox control of the brain. Antioxid. Redox Signal. 21, 1766–1801. PMID:24597893

  2. Redox control of microglial function: molecular mechanisms and functional significance.

    PubMed

    Rojo, Ana I; McBean, Gethin; Cindric, Marina; Egea, Javier; López, Manuela G; Rada, Patricia; Zarkovic, Neven; Cuadrado, Antonio

    2014-10-20

    Neurodegenerative diseases are characterized by chronic microglial over-activation and oxidative stress. It is now beginning to be recognized that reactive oxygen species (ROS) produced by either microglia or the surrounding environment not only impact neurons but also modulate microglial activity. In this review, we first analyze the hallmarks of pro-inflammatory and anti-inflammatory phenotypes of microglia and their regulation by ROS. Then, we consider the production of reactive oxygen and nitrogen species by NADPH oxidases and nitric oxide synthases and the new findings that also indicate an essential role of glutathione (γ-glutamyl-l-cysteinylglycine) in redox homeostasis of microglia. The effect of oxidant modification of macromolecules on signaling is analyzed at the level of oxidized lipid by-products and sulfhydryl modification of microglial proteins. Redox signaling has a profound impact on two transcription factors that modulate microglial fate, nuclear factor kappa-light-chain-enhancer of activated B cells, and nuclear factor (erythroid-derived 2)-like 2, master regulators of the pro-inflammatory and antioxidant responses of microglia, respectively. The relevance of these proteins in the modulation of microglial activity and the interplay between them will be evaluated. Finally, the relevance of ROS in altering blood brain barrier permeability is discussed. Recent examples of the importance of these findings in the onset or progression of neurodegenerative diseases are also discussed. This review should provide a profound insight into the role of redox homeostasis in microglial activity and help in the identification of new promising targets to control neuroinflammation through redox control of the brain.

  3. Apoptosis and cell-cycle arrest in human and murine tumor cells are initiated by isoprenoids.

    PubMed

    Mo, H; Elson, C E

    1999-04-01

    Diverse classes of phytochemicals initiate biological responses that effectively lower cancer risk. One class of phytochemicals, broadly defined as pure and mixed isoprenoids, encompasses an estimated 22,000 individual components. A representative mixed isoprenoid, gamma-tocotrienol, suppresses the growth of murine B16(F10) melanoma cells, and with greater potency, the growth of human breast adenocarcinoma (MCF-7) and human leukemic (HL-60) cells. beta-Ionone, a pure isoprenoid, suppresses the growth of B16 cells and with greater potency, the growth of MCF-7, HL-60 and human colon adenocarcinoma (Caco-2) cells. Results obtained with diverse cell lines differing in ras and p53 status showed that the isoprenoid-mediated suppression of growth is independent of mutated ras and p53 functions. beta-Ionone suppressed the growth of human colon fibroblasts (CCD-18Co) but only when present at three-fold the concentration required to suppress the growth of Caco-2 cells. The isoprenoids initiated apoptosis and, concomitantly arrested cells in the G1 phase of the cell cycle. Both suppress 3-hydroxy-3-methylglutaryl CoA reductase activity. beta-Ionone and lovastatin interfered with the posttranslational processing of lamin B, an activity essential to assembly of daughter nuclei. This interference, we postulate, renders neosynthesized DNA available to the endonuclease activities leading to apoptotic cell death. Lovastatin-imposed mevalonate starvation suppressed the glycosylation and translocation of growth factor receptors to the cell surface. As a consequence, cells were arrested in the G1 phase of the cell cycle. This rationale may apply to the isoprenoid-mediated G1-phase arrest of tumor cells. The additive and potentially synergistic actions of these isoprenoids in the suppression of tumor cell proliferation and initiation of apoptosis coupled with the mass action of the diverse isoprenoid constituents of plant products may explain, in part, the impact of fruit, vegetable

  4. Differential detection of impact site versus rotational site injury by magnetic resonance imaging and microglial morphology in an unrestrained mild closed head injury model.

    PubMed

    Hernandez, Alfredo; Donovan, Virgina; Grinberg, Yelena Y; Obenaus, Andre; Carson, Monica J

    2016-01-01

    Seventy-five percent of all traumatic brain injuries are mild and do not cause readily visible abnormalities on routine medical imaging making it difficult to predict which individuals will develop unwanted clinical sequelae. Microglia are brain-resident macrophages and early responders to brain insults. Their activation is associated with changes in morphology or expression of phenotypic markers including P2Y12 and major histocompatibility complex class II. Using a murine model of unrestrained mild closed head injury (mCHI), we used microglia as reporters of acute brain injury at sites of impact versus sites experiencing rotational stress 24 h post-mCHI. Consistent with mild injury, a modest 20% reduction in P2Y12 expression was detected by quantitative real-time PCR (qPCR) analysis but only in the impacted region of the cortex. Furthermore, neither an influx of blood-derived immune cells nor changes in microglial expression of CD45, TREM1, TREM2, major histocompatibility complex class II or CD40 were detected. Using magnetic resonance imaging (MRI), small reductions in T2 weighted values were observed but only near the area of impact and without overt tissue damage (blood deposition, edema). Microglial morphology was quantified without cryosectioning artifacts using ScaleA(2) clarified brains from CX3CR1-green fluorescence protein (GFP) mice. The cortex rostral to the mCHI impact site receives greater rotational stress but neither MRI nor molecular markers of microglial activation showed significant changes from shams in this region. However, microglia in this rostral region did display signs of morphologic activation equivalent to that observed in severe CHI. Thus, mCHI-triggered rotational stress is sufficient to cause injuries undetectable by routine MRI that could result in altered microglial surveillance of brain homeostasis. Acute changes in microglial morphology reveal brain responses to unrestrained mild traumatic brain injury In areas subjected to

  5. Differential detection of impact site versus rotational site injury by magnetic resonance imaging and microglial morphology in an unrestrained mild closed head injury model.

    PubMed

    Hernandez, Alfredo; Donovan, Virgina; Grinberg, Yelena Y; Obenaus, Andre; Carson, Monica J

    2016-01-01

    Seventy-five percent of all traumatic brain injuries are mild and do not cause readily visible abnormalities on routine medical imaging making it difficult to predict which individuals will develop unwanted clinical sequelae. Microglia are brain-resident macrophages and early responders to brain insults. Their activation is associated with changes in morphology or expression of phenotypic markers including P2Y12 and major histocompatibility complex class II. Using a murine model of unrestrained mild closed head injury (mCHI), we used microglia as reporters of acute brain injury at sites of impact versus sites experiencing rotational stress 24 h post-mCHI. Consistent with mild injury, a modest 20% reduction in P2Y12 expression was detected by quantitative real-time PCR (qPCR) analysis but only in the impacted region of the cortex. Furthermore, neither an influx of blood-derived immune cells nor changes in microglial expression of CD45, TREM1, TREM2, major histocompatibility complex class II or CD40 were detected. Using magnetic resonance imaging (MRI), small reductions in T2 weighted values were observed but only near the area of impact and without overt tissue damage (blood deposition, edema). Microglial morphology was quantified without cryosectioning artifacts using ScaleA(2) clarified brains from CX3CR1-green fluorescence protein (GFP) mice. The cortex rostral to the mCHI impact site receives greater rotational stress but neither MRI nor molecular markers of microglial activation showed significant changes from shams in this region. However, microglia in this rostral region did display signs of morphologic activation equivalent to that observed in severe CHI. Thus, mCHI-triggered rotational stress is sufficient to cause injuries undetectable by routine MRI that could result in altered microglial surveillance of brain homeostasis. Acute changes in microglial morphology reveal brain responses to unrestrained mild traumatic brain injury In areas subjected to

  6. Comparison of the gating behaviour of human and murine cystic fibrosis transmembrane conductance regulator Cl− channels expressed in mammalian cells

    PubMed Central

    Lansdell, K A; Delaney, S J; Lunn, D P; Thomson, S A; Sheppard, D N; Wainwright, B J

    1998-01-01

    To investigate the function of the murine cystic fibrosis transmembrane conductance regulator (CFTR), a full-length cDNA encoding wild-type murine CFTR was assembled and stably expressed in Chinese hamster ovary (CHO) cells. Like human CFTR, murine CFTR formed Cl− channels that were regulated by cAMP-dependent phosphorylation and intracellular ATP. However, murine CFTR Cl− channels had a reduced single-channel conductance and decreased open probability (Po) compared with those of human CFTR. Analysis of the dwell time distributions of single channels suggested that the reduced Po of murine CFTR was caused by both decreased residence in the open state and transitions to a new closed state, described by an intermediate closed time constant. For both human and murine CFTR, ATP and ADP regulated the rate of exit from the long-lived closed state. 5′-Adenylylimidodiphosphate (AMP-PNP) and pyrophosphate, two compounds that disrupt cycles of ATP hydrolysis, stabilized the open state of human CFTR. However, neither agent locked murine CFTR Cl− channels open, although AMP-PNP increased the Po of murine CFTR. The data indicate that although human and murine CFTR have many properties in common, some important differences in function are observed. These differences could be exploited in future studies to provide new understanding about CFTR. PMID:9508803

  7. Neuronal Regulation of Neuroprotective Microglial Apolipoprotein E Secretion in Rat In Vitro Models of Brain Pathophysiology.

    PubMed

    Polazzi, Elisabetta; Mengoni, Ilaria; Peña-Altamira, Emiliano; Massenzio, Francesca; Virgili, Marco; Petralla, Sabrina; Monti, Barbara

    2015-08-01

    Apolipoprotein E (ApoE) is mainly secreted by glial cells and is involved in many brain functions, including neuronal plasticity, β-amyloid clearance, and neuroprotection. Microglia--the main immune cells of the brain--are one source of ApoE, but little is known about the physiologic regulation of microglial ApoE secretion by neurons and whether this release changes under inflammatory or neurodegenerative conditions. Using rat primary neural cell cultures, we show that microglia release ApoE through a Golgi-mediated secretion pathway and that ApoE progressively accumulates in neuroprotective microglia-conditioned medium. This constitutive ApoE release is negatively affected by microglial activation both with lipopolysaccharide and with ATP. Microglial ApoE release is stimulated by neuron-conditioned media and under coculture conditions. Neuron-stimulated microglial ApoE release is mediated by serine and glutamate through N-methyl-D-aspartate receptors and is differently regulated by activation states (i.e. lipopolysaccharide vs ATP) and by 6-hydroxydopamine. Microglial ApoE silencing abrogated protection of cerebellar granule neurons against 6-hydroxydopamine toxicity in cocultures, indicating that microglial ApoE release is neuroprotective. Our findings shed light on the reciprocal cross-talk between neurons and microglia that is crucial for normal brain functions. They also open the way for the identification of possible pharmacologic targets that can modulate neuroprotective microglial ApoE release under pathologic conditions.

  8. Phenotypic non-equivalence of murine (monocyte-) macrophage cells in biomaterial and inflammatory models.

    PubMed

    Chamberlain, Lisa M; Godek, Marisha L; Gonzalez-Juarrero, Mercedes; Grainger, David W

    2009-03-15

    Cells of the mononuclear phagocytic system including monocytes and macrophages (e.g., pooled human monocytes, bone marrow-derived macrophages, etc.) are often employed for in vitro assessment of novel biomaterials and to assay anti-inflammatory drug activity. In this context, numerous macrophage cells are treated interchangeably in the literature despite a lack of demonstrated equivalence among immortalized cell lines and further, between cell lines and primary-derived macrophages of different species. Three murine (monocyte-) macrophage cell lines (IC-21, J774A.1, and RAW 264.7), commonly utilizedin biomaterial and pharmaceutical screening research, have been compared with primary-derived murine bone marrow macrophages. Significant differences were discovered in the expression of cell surface proteins requisite for cell adhesion and activation among cell lines and primary-derived cells as well as between the different cell lines. Results demonstrate activation but with reduced cytokine expression to chemical stimulus (lipopolysaccharide) by cell lines compared with that of primary-derived macrophages. Limited correlation between cultured primary and immortalized cells in cytokine production, phenotype and intrinsic activation states has relevance to fidelity for in vitro testing. These differences warrant justification for selection of various cell lines for specific assay purposes, and merit caution if comparisons to primary cell types (i.e., for biocompatibility) are required. PMID:18357567

  9. Development and characterization of Histoplasma capsulatum-reactive murine T-cell lines and clones

    NASA Technical Reports Server (NTRS)

    Deepe, George S., Jr.; Smith, James G.; Denman, David; Bullock, Ward E.; Sonnenfeld, Gerald

    1986-01-01

    Several Histoplasma capsulatum-reactive murine cloned T-cell lines (TCLs) were isolated from spleens of C57BL/6 mice immunized with viable H. capsulatum yeast cells, using the methodology of Kimoto and Fathman (1980). These T-cells were characterized phenotypically as Thy-1.2(+) Lyt-1(+) L3T4(+) Lyt-2(-), that is, as the helper/inducer phenotype. The cloned T cells proliferate in response to histoplasmin and, in some cases, to heterologous fungal anigens. Upon injection of mice with the antigen, the T-cells mediate local delayed-type hypersensitivity responses and, after stimulation, release regulatory lymphokines.

  10. Purified murine granulocyte/macrophage progenitor cells express a high-affinity receptor for recombinant murine granulocyte/macrophage colony-stimulating factor

    SciTech Connect

    Williams, D.E.; Bicknell, D.C.; Park, L.S.; Straneva, J.E.; Cooper, S.; Broxmeyer, H.E.

    1988-01-01

    Purified recombinant murine granulocyte/macrophage colony-stimulating factor (GM-CSF) was labeled with /sup 125/I and used to examine the GM-CSF receptor on unfractionated normal murine bone marrow cells, casein-induced peritoneal exudate cells, and highly purified murine granulocyte/macrophage progenitor cells (CFU-GM). CFU-GM were isolated from cyclophosphamide-treated mice by Ficoll-Hypaque density centrifugation followed by counterflow centrifugal elutriation. The resulting population had a cloning efficiency of 62-99% in cultures containing conditioned medium from pokeweed mitogen-stimulated spleen cells and 55-86% in the presence of a plateau concentration of purified recombinant murine GM-CSF. Equilibrium binding studies with /sup 125/I-labeled GM-CSF showed that normal bone marrow cells, casein-induced peritoneal exudate cells, and purified CFU-GM had a single class of high-affinity receptor. Affinity crosslinking studies demonstrated that /sup 125/I-labeled GM-CSF bound specifically to two species of M/sub r/ 180,000 and 70,000 on CFU-GM, normal bone marrow cells, and peritoneal exudate cells. The M/sub r/ 70,000 species is thought to be a proteolytic fragment of the intact M/sub r/ 180,000 receptor. The present studies indicate that the GM-CSF receptor expressed on CFU-GM and mature myeloid cells are structurally similar. In addition, the number of GM-CSF receptors on CFU-GM is twice the average number of receptors on casein-induced mature myeloid cells, suggesting that receptor number may decrease as CFU-GM mature.

  11. Stimulation of cell proliferation in the subventricular zone by synthetic murine pheromones.

    PubMed

    Koyama, Sachiko; Soini, Helena A; Foley, John; Novotny, Milos V; Lai, Cary

    2013-01-01

    Adult neurogenesis in female mice is known to be enhanced by exposure to soiled bedding from males, although the identity of the relevant chemosignals has remained unknown. Here we show that the previously recognized male murine pheromones, the farnesenes and 2-sec-butyl-4,5-dihydrothiazole (SBT), strongly increase cell proliferation in the subventricular zone (SVZ) of adult female mice, but not younger female mice. In addition, we found that a unique female murine pheromone, 2,5-dimethylpyrazine, facilitates similar changes in males. SBT stimulated cell proliferation in the SVZ of only adult females and not in young adult or pre- and post-puberty females. Our study suggests that pheromonal communication between males and females is enhancing reproductive success by controlling the estrous cycle and by promoting cell proliferation in a reciprocal manner.

  12. Neuronal Hyperactivity Disturbs ATP Microgradients, Impairs Microglial Motility, and Reduces Phagocytic Receptor Expression Triggering Apoptosis/Microglial Phagocytosis Uncoupling

    PubMed Central

    Nadjar, Agnes; Layé, Sophie; Leyrolle, Quentin; Gómez-Nicola, Diego; Domercq, María; Pérez-Samartín, Alberto; Sánchez-Zafra, Víctor; Savage, Julie C.; Hui, Chin-Wai; Deudero, Juan J. P.; Brewster, Amy L.; Anderson, Anne E.; Zaldumbide, Laura; Galbarriatu, Lara; Marinas, Ainhoa; Vivanco, Maria dM.; Matute, Carlos; Maletic-Savatic, Mirjana

    2016-01-01

    Phagocytosis is essential to maintain tissue homeostasis in a large number of inflammatory and autoimmune diseases, but its role in the diseased brain is poorly explored. Recent findings suggest that in the adult hippocampal neurogenic niche, where the excess of newborn cells undergo apoptosis in physiological conditions, phagocytosis is efficiently executed by surveillant, ramified microglia. To test whether microglia are efficient phagocytes in the diseased brain as well, we confronted them with a series of apoptotic challenges and discovered a generalized response. When challenged with excitotoxicity in vitro (via the glutamate agonist NMDA) or inflammation in vivo (via systemic administration of bacterial lipopolysaccharides or by omega 3 fatty acid deficient diets), microglia resorted to different strategies to boost their phagocytic efficiency and compensate for the increased number of apoptotic cells, thus maintaining phagocytosis and apoptosis tightly coupled. Unexpectedly, this coupling was chronically lost in a mouse model of mesial temporal lobe epilepsy (MTLE) as well as in hippocampal tissue resected from individuals with MTLE, a major neurological disorder characterized by seizures, excitotoxicity, and inflammation. Importantly, the loss of phagocytosis/apoptosis coupling correlated with the expression of microglial proinflammatory, epileptogenic cytokines, suggesting its contribution to the pathophysiology of epilepsy. The phagocytic blockade resulted from reduced microglial surveillance and apoptotic cell recognition receptor expression and was not directly mediated by signaling through microglial glutamate receptors. Instead, it was related to the disruption of local ATP microgradients caused by the hyperactivity of the hippocampal network, at least in the acute phase of epilepsy. Finally, the uncoupling led to an accumulation of apoptotic newborn cells in the neurogenic niche that was due not to decreased survival but to delayed cell clearance

  13. Role of Microglial Activation in the Pathophysiology of Bacterial Meningitis.

    PubMed

    Barichello, Tatiana; Generoso, Jaqueline S; Simões, Lutiana R; Goularte, Jessica A; Petronilho, Fabricia; Saigal, Priyanka; Badawy, Marwa; Quevedo, João

    2016-04-01

    Bacterial meningitis is a life-threatening infection associated with cognitive impairment in many survivors. The pathogen invades the central nervous system (CNS) by penetrating through the luminal side of the cerebral endothelium, which is an integral part of the blood-brain barrier. The replication of bacteria within the subarachnoid space occurs concomitantly with the release of their compounds that are highly immunogenic. These compounds known as pathogen-associated molecular patterns (PAMPs) may lead to both an increase in the inflammatory response in the host and also microglial activation. Microglia are the resident macrophages of the CNS which, when activated, can trigger a host of immunological pathways. Classical activation increases the production of pro-inflammatory cytokines, chemokines, and reactive oxygen species, while alternative activation is implicated in the inhibition of inflammation and restoration of homeostasis. The inflammatory response from classical microglial activation can facilitate the elimination of invasive microorganisms; however, excessive or extended microglial activation can result in neuronal damage and eventually cell death. This review aims to discuss the role of microglia in the pathophysiology of bacterial meningitis as well as the process of microglial activation by PAMPs and by endogenous constituents that are normally released from damaged cells known as danger-associated molecular patterns (DAMPs). PMID:25744564

  14. Magnetic field-magnetic nanoparticle culture system used to grow in vitro murine embryonic stem cells.

    PubMed

    de Freitas, Erika Regina Leal; Soares, Paula Roberta Otaviano; de Santos, Rachel Paula; dos Santos, Regiane Lopes; Porfírio, Elaine Paulucio; Báo, Sônia N; Lima, Emília Celma Oliveira; Guillo, Lídia Andreu

    2011-01-01

    The in vitro growth of embryonic stem cells (ESCs) is usually obtained in the presence of murine embryonic fibroblasts (MEF), but new methods for in vitro expansion of ESCs should be developed due to their potential clinical use. This study aims to establish a culture system to expand and maintain ESCs in the absence of MEF by using murine embryonic stem cells (mECS) as a model of embryonic stem cell. Magnetic nanoparticles (MNPs) were used for growing mESCs in the presence of an external magnetic field, creating the magnetic field-magnetic nanoparticle (MF-MNP) culture system. The growth characteristics were evaluated showing a doubling time slightly higher for mESCs cultivated in the presence of the system than in the presence of the MEF. The undifferentiated state was characterized by RT-PCR, immunofluorescence, alkaline phosphatase activity and electron microscopy. Murine embryonic stem cells cultivated in presence of the MF-MNP culture system exhibited Oct-4 and Nanog expression and high alkaline phosphatase activity. Ultrastructural morphology showed that the MF-MNP culture system did not interfere with processes that cause structural changes in the cytoplasm or nucleus. The MF-MNP culture system provides a tool for in vitro expansion of mESCs and could contribute to studies that aim the therapeutic use of embryonic stem cells. PMID:21446404

  15. Secretion of N- and O-linked Glycoproteins from 4T1 Murine Mammary Carcinoma Cells.

    PubMed

    Phang, Wai-Mei; Tan, Aik-Aun; Gopinath, Subash C B; Hashim, Onn H; Kiew, Lik Voon; Chen, Yeng

    2016-01-01

    Breast cancer is one of the most common cancers that affect women globally and accounts for ~23% of all cancers diagnosed in women. Breast cancer is also one of the leading causes of death primarily due to late stage diagnoses and a lack of effective treatments. Therefore, discovering protein expression biomarkers is mandatory for early detection and thus, critical for successful therapy. Two-dimensional electrophoresis (2D-E) coupled with lectin-based analysis followed by mass spectrometry were applied to identify potential biomarkers in the secretions of a murine mammary carcinoma cell line. Comparisons of the protein profiles of the murine 4T1 mammary carcinoma cell line and a normal murine MM3MG mammary cell line indicated that cadherin-1 (CDH), collagenase 3 (MMP-13), Viral envelope protein G7e (VEP), Gag protein (GAG) and Hypothetical protein LOC433182 (LOC) were uniquely expressed by the 4T1 cells, and pigment epithelium-derived factor (PEDF) was exclusively secreted by the MM3MG cells. Further analysis by a lectin-based study revealed that aberrant O-glycosylated CDH, N-glycosylated MMP-13 and LOC were present in the 4T1 medium. These differentially expressed N- and O-linked glycoprotein candidates, which were identified by combining lectin-based analysis with 2D-E, could serve as potential diagnostic and prognostic markers for breast cancer. PMID:27226773

  16. Secretion of N- and O-linked Glycoproteins from 4T1 Murine Mammary Carcinoma Cells

    PubMed Central

    Phang, Wai-Mei; Tan, Aik-Aun; Gopinath, Subash C.B.; Hashim, Onn H.; Kiew, Lik Voon; Chen, Yeng

    2016-01-01

    Breast cancer is one of the most common cancers that affect women globally and accounts for ~23% of all cancers diagnosed in women. Breast cancer is also one of the leading causes of death primarily due to late stage diagnoses and a lack of effective treatments. Therefore, discovering protein expression biomarkers is mandatory for early detection and thus, critical for successful therapy. Two-dimensional electrophoresis (2D-E) coupled with lectin-based analysis followed by mass spectrometry were applied to identify potential biomarkers in the secretions of a murine mammary carcinoma cell line. Comparisons of the protein profiles of the murine 4T1 mammary carcinoma cell line and a normal murine MM3MG mammary cell line indicated that cadherin-1 (CDH), collagenase 3 (MMP-13), Viral envelope protein G7e (VEP), Gag protein (GAG) and Hypothetical protein LOC433182 (LOC) were uniquely expressed by the 4T1 cells, and pigment epithelium-derived factor (PEDF) was exclusively secreted by the MM3MG cells. Further analysis by a lectin-based study revealed that aberrant O-glycosylated CDH, N-glycosylated MMP-13 and LOC were present in the 4T1 medium. These differentially expressed N- and O-linked glycoprotein candidates, which were identified by combining lectin-based analysis with 2D-E, could serve as potential diagnostic and prognostic markers for breast cancer. PMID:27226773

  17. Potentiated cytotoxic effects of statins and ajoene in murine melanoma cells.

    PubMed

    Ledezma, Eliades; Wittig, Olga; Alonso, Jose; Cardier, Jose E

    2009-04-01

    Because statins and ajoene inhibit the 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, we evaluated the hypothesis that the cytotoxic effect of these compounds may be potentiated when both are used in combination on tumor cells. We showed that cotreatment of the murine melanoma B16F10 cell with statins (atorvastatin and pravastatin) and ajoene, all at nontoxic doses, dramatically increased their cytotoxicity. B16F10 cell death induced by statins, but not by ajoene, was prevented by mevalonate and geranylgeranylpyrophosphate. To our knowledge, this is the first report that the combination of statins and ajoene, which alters the mevalonate pathway, might potentiate their cytotoxic effects on tumor cells.

  18. Neonatal CD71+ erythroid cells do not modify murine sepsis mortality

    PubMed Central

    Wynn, James L.; Scumpia, Philip O.; Stocks, Blair T.; Romano-Keeler, Joann; Alrifai, Mhd Wael; Liu, Jin-Hua; Kim, Annette S.; Alford, Catherine E.; Matta, Pranathi; Weitkamp, Jörn-Hendrik; Moore, Daniel J.

    2015-01-01

    Sepsis is a major cause of neonatal mortality and morbidity worldwide. A recent report suggested murine neonatal host defense against infection could be compromised by immunosuppressive CD71+ erythroid splenocytes. We examined the impact of CD71+ erythroid splenocytes on murine neonatal mortality to endotoxin challenge or polymicrobial sepsis and characterized circulating CD71+ erythroid (CD235a+) cells in human neonates. Adoptive transfer or antibody-mediated reduction of neonatal CD71+ erythroid splenocytes did not alter murine neonatal survival to endotoxin challenge or polymicrobial sepsis challenge. Ex vivo immunosuppression of stimulated adult CD11b+ cells was not limited to neonatal splenocytes as it also occurred with adult and neonatal bone marrow. Animals treated with anti-CD71 antibody showed reduced splenic bacterial load following bacterial challenge compared to isotype-treated mice. However, adoptive transfer of enriched CD71+ erythroid splenocytes to CD71+-reduced animals did not reduce bacterial clearance. Human CD71+CD235a+ cells were common among cord blood mononuclear cells and were shown to be reticulocytes. In summary, a lack of effect on murine survival to polymicrobial sepsis following adoptive transfer or diminution of CD71+ erythroid splenocytes under these experimental conditions suggests the impact of these cells on neonatal infection risk and progression may be limited. An unanticipated immune priming effect of anti-CD71 antibody treatment was likely responsible for the reported enhanced bacterial clearance, rather than a reduction of immunosuppressive CD71+ erythroid splenocytes. In humans, the well-described rapid decrease in circulating reticulocytes after birth suggests they may have a limited role in reducing inflammation secondary to microbial colonization. PMID:26101326

  19. Identification of Replication Competent Murine Gammaretroviruses in Commonly Used Prostate Cancer Cell Lines

    PubMed Central

    Sfanos, Karen Sandell; Aloia, Amanda L.; Hicks, Jessica L.; Esopi, David M.; Steranka, Jared P.; Shao, Wei; Sanchez-Martinez, Silvia; Yegnasubramanian, Srinivasan; Burns, Kathleen H.; Rein, Alan; De Marzo, Angelo M.

    2011-01-01

    A newly discovered gammaretrovirus, termed XMRV, was recently reported to be present in the prostate cancer cell line CWR22Rv1. Using a combination of both immunohistochemistry with broadly-reactive murine leukemia virus (MLV) anti-sera and PCR, we determined if additional prostate cancer or other cell lines contain XMRV or MLV-related viruses. Our study included a total of 72 cell lines, which included 58 of the 60 human cancer cell lines used in anticancer drug screens and maintained at the NCI-Frederick (NCI-60). We have identified gammaretroviruses in two additional prostate cancer cell lines: LAPC4 and VCaP, and show that these viruses are replication competent. Viral genome sequencing identified the virus in LAPC4 and VCaP as nearly identical to another known xenotropic MLV, Bxv-1. We also identified a gammaretrovirus in the non-small-cell lung carcinoma cell line EKVX. Prostate cancer cell lines appear to have a propensity for infection with murine gammaretroviruses, and we propose that this may be in part due to cell line establishment by xenograft passage in immunocompromised mice. It is unclear if infection with these viruses is necessary for cell line establishment, or what confounding role they may play in experiments performed with these commonly used lines. Importantly, our results suggest a need for regular screening of cancer cell lines for retroviral “contamination”, much like routine mycoplasma testing. PMID:21698104

  20. Potent inhibition of Junín virus infection by interferon in murine cells.

    PubMed

    Huang, Cheng; Walker, Aida G; Grant, Ashley M; Kolokoltsova, Olga A; Yun, Nadezhda E; Seregin, Alexey V; Paessler, Slobodan

    2014-06-01

    The new world arenavirus Junín virus (JUNV) is the causative agent of Argentine hemorrhagic fever, a lethal human infectious disease. Adult laboratory mice are generally resistant to peripheral infection by JUNV. The mechanism underlying the mouse resistance to JUNV infection is largely unknown. We have reported that interferon receptor knockout mice succumb to JUNV infection, indicating the critical role of interferon in restricting JUNV infection in mice. Here we report that the pathogenic and vaccine strains of JUNV were highly sensitive to interferon in murine primary cells. Treatment with low concentrations of interferon abrogated viral NP protein expression in murine cells. The replication of both JUNVs was enhanced in IRF3/IRF7 deficient cells. In addition, the vaccine strain of JUNV displayed impaired growth in primary murine cells. Our data suggested a direct and potent role of host interferon response in restricting JUNV replication in mice. The defect in viral growth for vaccine JUNV might also partially explain its attenuation in mice.

  1. Potent Inhibition of Junín Virus Infection by Interferon in Murine Cells

    PubMed Central

    Huang, Cheng; Walker, Aida G.; Grant, Ashley M.; Kolokoltsova, Olga A.; Yun, Nadezhda E.; Seregin, Alexey V.; Paessler, Slobodan

    2014-01-01

    The new world arenavirus Junín virus (JUNV) is the causative agent of Argentine hemorrhagic fever, a lethal human infectious disease. Adult laboratory mice are generally resistant to peripheral infection by JUNV. The mechanism underlying the mouse resistance to JUNV infection is largely unknown. We have reported that interferon receptor knockout mice succumb to JUNV infection, indicating the critical role of interferon in restricting JUNV infection in mice. Here we report that the pathogenic and vaccine strains of JUNV were highly sensitive to interferon in murine primary cells. Treatment with low concentrations of interferon abrogated viral NP protein expression in murine cells. The replication of both JUNVs was enhanced in IRF3/IRF7 deficient cells. In addition, the vaccine strain of JUNV displayed impaired growth in primary murine cells. Our data suggested a direct and potent role of host interferon response in restricting JUNV replication in mice. The defect in viral growth for vaccine JUNV might also partially explain its attenuation in mice. PMID:24901990

  2. Leptin enhances endothelial cell differentiation and angiogenesis in murine embryonic stem cells.

    PubMed

    Kurtovic, Silvia; Ng, Tina T; Gupta, Ankur; Arumugaswami, Vaithilingaraja; Chaiboonma, Kira L; Aminzadeh, Mohammad Amin; Makkar, Raj; Dafoe, Donald C; Talavera-Adame, Dodanim

    2015-01-01

    The metabolic regulation of leptin and its angiogenic effects have been well characterized in adult mammals. However, the role of leptin in the differentiation of embryonic stem cells (ESCs) to endothelial cells (ECs) has not been characterized. We hypothesized that leptin enhances the generation of ECs derived from ESCs and, in this way, promotes angiogenesis in embryonic vessels. To address this hypothesis, we utilized an in vitro model consisting of murine ESCs-derived embryoid bodies (EBs). Vascular density, EC and angiogenesis markers as well as phosphorylation levels of signal transducer and activator of transcription 3 (pSTAT3) were investigated in leptin-treated EBs and in untreated EBs as controls. ESC-derived ECs were isolated by magnetic sorting based on the expression of platelet endothelial cell adhesion molecule (PECAM-1/CD31). Significant upregulation of EC and angiogenic markers as well as higher vessel density were found in leptin-treated EBs compared to controls. CD31 positive enriched cells derived from leptin-treated EBs had improved proliferation and survival rate and showed higher levels of pSTAT3. These results suggested that leptin promotes EC differentiation and angiogenesis in mouse EBs and that janus tyrosine kinase (JAK)/STAT pathway can play a role in this biological process. Leptin-mediated EC differentiation and angiogenesis in ESCs can be a useful application towards regenerative medicine and tissue engineering. PMID:25250519

  3. Natural cytotoxicity of haemopoietic cell populations against murine lymphoid tumours.

    PubMed Central

    Burton, R. C.; Grail, D.; Warner, N. L.

    1978-01-01

    Homozygous nude and normal mice of 3 strains, BALB/c, CBA and C57BL, were used as sources of nucleated haemopoietic "natural killer" (NK) cells. These killer cells could lyse a wide range of syngeneic and allogeneic lymphoid tumour cell lines in vitro, and it was found that cell suspensions from nude mice were always significantly more active than those from normal mice, and that the most active effector population was a polymorph-enriched peritoneal-exudate cell suspension. Eosinophils did not appear to be involved in the phenomenon, and mononuclear peritoneal-exudate cell suspensions were actually highly inhibitory. Three non-lymphoid tumours, a carcinoma, a fibrosarcoma and a mastocytoma, were totally resistant to in vitro lysis. Although all susceptible tumour cell lines were C-type virus-associated, not all of these tumours were killed by all strain sources of spleen cells, indicating a specificity of killing. PMID:656308

  4. Conserved signaling through vascular endothelial growth (VEGF) receptor family members in murine lymphatic endothelial cells.

    PubMed

    Coso, Sanja; Zeng, Yiping; Sooraj, Dhanya; Williams, Elizabeth D

    2011-10-15

    Lymphatic vessels guide interstitial fluid, modulate immune responses by regulating leukocyte and antigen trafficking to lymph nodes, and in a cancer setting enable tumor cells to track to regional lymph nodes. The aim of the study was to determine whether primary murine lymphatic endothelial cells (mLECs) show conserved vascular endothelial growth factor (VEGF) signaling pathways with human LECs (hLECs). LECs were successfully isolated from murine dermis and prostate. Similar to hLECs, vascular endothelial growth factor (VEGF) family ligands activated MAPK and pAkt intracellular signaling pathways in mLECs. We describe a robust protocol for isolation of mLECs which, by harnessing the power of transgenic and knockout mouse models, will be a useful tool to study how LEC phenotype contributes to alterations in lymphatic vessel formation and function.

  5. Sorting and Expansion of Murine Embryonic Stem Cell Colonies using Micropallet Arrays

    PubMed Central

    Shadpour, Hamed; Sims, Christopher E.; Thresher, Randy J.; Allbritton, Nancy L.

    2008-01-01

    Background Isolation of cell colonies is an essential task in most stem cell studies. Conventional techniques for colony selection and isolation require significant time, labor, and consumption of expensive reagents. New microengineered technologies hold the promise for improving colony manipulation by reducing the required manpower and reagent consumption. Methods Murine embryonic stem cells were cultured on arrays composed of releasable elements termed micropallets created from a biocompatible photoresist. Micropallets containing undifferentiated colonies were released using a laser-based technique followed by cell collection and expansion in culture. Results The micropallet arrays provided a biocompatible substrate for maintaining undifferentiated murine stem cells in culture. A surface coating of 0.025% gelatin was shown to be optimal for cell culture and collection. Arrays composed of surface roughened micropallets provided further improvements in culture and isolation. Colonies of viable stem cells were efficiently isolated and collected. Colonies sorted in this manner were shown to remain undifferentiated even after collection and further expansion in culture. Conclusions Qualitative and quantitative analyses of sorting, collection efficiency and cell viability after release and expansion of stem cell colonies demonstrated that the micropallet array technology is a promising alternative to conventional sorting methods for stem cell applications. PMID:19012319

  6. Sesquiterpenes inhibiting the microglial activation from Laurus nobilis.

    PubMed

    Chen, Hongqiang; Xie, Chunfeng; Wang, Hao; Jin, Da-Qing; Li, Shen; Wang, Meicheng; Ren, Quanhui; Xu, Jing; Ohizumi, Yasushi; Guo, Yuanqiang

    2014-05-21

    The inhibitory reagents to inhibit the activation of microglial cells may be potentially useful for the treatment of neurodegenerative diseases. The leaves of the plant Laurus nobilis belonging to the family Lauraceae, namely, bay leaves, have been used as a popular spice, and their extract showed moderate inhibition on microglial activation. A further phytochemical investigation of the leaves led to the isolation of two new (1, 2) and eight known (3-10) sesquiterpenes. Their structures were elucidated on the basis of extensive 1D and 2D NMR (HMQC, HMBC, (1)H-(1)H COSY, and NOESY) spectroscopic data analyses and Chem3D modeling. The following biological studies disclosed that these isolated compounds showed inhibitory activities on LPS-induced microglial activation. The results of our phytochemical investigation, including two new sesquiterpenes (1 and 2) and the first report of two compounds (3 and 4) from this species, further revealed the chemical composition of bay leaves as a popular spice, and the biological studies implied that bay leaves, containing bioactive substances with the inhibition of microglial activation, were potentially beneficial to human health. PMID:24801989

  7. Sesquiterpenes inhibiting the microglial activation from Laurus nobilis.

    PubMed

    Chen, Hongqiang; Xie, Chunfeng; Wang, Hao; Jin, Da-Qing; Li, Shen; Wang, Meicheng; Ren, Quanhui; Xu, Jing; Ohizumi, Yasushi; Guo, Yuanqiang

    2014-05-21

    The inhibitory reagents to inhibit the activation of microglial cells may be potentially useful for the treatment of neurodegenerative diseases. The leaves of the plant Laurus nobilis belonging to the family Lauraceae, namely, bay leaves, have been used as a popular spice, and their extract showed moderate inhibition on microglial activation. A further phytochemical investigation of the leaves led to the isolation of two new (1, 2) and eight known (3-10) sesquiterpenes. Their structures were elucidated on the basis of extensive 1D and 2D NMR (HMQC, HMBC, (1)H-(1)H COSY, and NOESY) spectroscopic data analyses and Chem3D modeling. The following biological studies disclosed that these isolated compounds showed inhibitory activities on LPS-induced microglial activation. The results of our phytochemical investigation, including two new sesquiterpenes (1 and 2) and the first report of two compounds (3 and 4) from this species, further revealed the chemical composition of bay leaves as a popular spice, and the biological studies implied that bay leaves, containing bioactive substances with the inhibition of microglial activation, were potentially beneficial to human health.

  8. Expression and function of the murine B7 antigen, the major costimulatory molecule expressed by peritoneal exudate cells.

    PubMed Central

    Razi-Wolf, Z; Freeman, G J; Galvin, F; Benacerraf, B; Nadler, L; Reiser, H

    1992-01-01

    The murine B7 (mB7) protein is a potent costimulatory molecule for the T-cell receptor (TCR)-mediated activation of murine CD4+ T cells. We have previously shown that stable mB7-transfected Chinese hamster ovary (CHO) cells but not vector-transfected controls synergize with either anti-CD3 monoclonal antibody-induced or concanavalin A-induced T-cell activation, resulting ultimately in lymphokine production and proliferation. We now have generated a hamster anti-mB7 monoclonal antibody. This reagent recognizes a protein with an apparent molecular mass of 50-60 kDa. The mB7 antigen is expressed on activated B cells and on peritoneal exudate cells (PECs). Antibody blocking experiments demonstrate that mB7 is the major costimulatory molecule expressed by PECs for the activation of murine CD4+ T cells. This suggests an important role for mB7 during immune-cell interactions. We have also surveyed a panel of murine cell lines capable of providing costimulatory activity. Our results indicate that mB7 is the major costimulatory molecule on some but not all cell lines and that there may be additional molecules besides mB7 that can costimulate the activation of murine CD4+ T cells. Images PMID:1373896

  9. Optimization of Gene Transfection in Murine Myeloma Cell Lines using Different Transfection Reagents

    PubMed Central

    Shabani, Mahdi; Hemmati, Sheyda; Hadavi, Reza; Amirghofran, Zahra; Jeddi-Tehrani, Mahmood; Rabbani, Hodjatallah; Shokri, Fazel

    2010-01-01

    Purification and isolation of cellular target proteins for monoclonal antibody (MAb) production is a difficult and time-consuming process. Immunization of mice with murine cell lines stably transfected with genes coding for xenogenic target molecules is an alternative method for mouse immunization and MAb production. Here we present data on transfection efficiency of some commercial reagents used for transfection of murine myeloma cell lines. Little is known about transfectability of murine myeloma cell lines by different transfection reagents. Mouse myeloma cell lines (SP2/0, NS0, NS1, Ag8, and P3U1) were transfected with pEGFP-N1 vector using Lipofectamine 2000, jetPEI and LyoVec commercial transfection reagents in different combinations. The transfection permissible HEK293-FT cell line was used as a control in transfection procedure. Transfected cells, expressing the Enhanced Green Fluorescent Protein (EGFP), were analyzed by flow cytometry 48 hrs post transfection. Our results showed transfection efficiency of 71%, 57% and 22% for HEK293-FT, 5.5%, 3.4% and 1% for SP2/0, 55.7%, 21.1% and 9.3% for NS0, 8.2%, 6% and 5.5% for NS1, 22%, 49.2% and 5.5% for Ag8 and 6.3%, 21.5% and 4.6% for P3U1 cell lines after transfection with Lipofectamine 2000, jetPEI and LyoVec reagents, respectively. Our data indicate that NS0 and Ag8 are efficiently transfected by Lipofectamine 2000 and jetPEI reagents. Finally, we propose Ag8 and NS0 cell lines as suitable host cells for efficient expression of target genes which can be used for mouse immunization and MAb production. PMID:23408356

  10. SuperSILAC Quantitative Proteome Profiling of Murine Middle Ear Epithelial Cell Remodeling with NTHi

    PubMed Central

    Val, Stéphanie; Burgett, Katelyn; Brown, Kristy J.; Preciado, Diego

    2016-01-01

    Background Chronic Otitis Media with effusion (COME) develops after sustained inflammation and is characterized by secretory middle ear epithelial metaplasia and effusion, most frequently mucoid. Non-typeable Haemophilus influenzae (NTHi), the most common acute Otitis Media (OM) pathogen, is postulated to promote middle ear epithelial remodeling in the progression of OM from acute to chronic. The goals of this study were to examine histopathological and quantitative proteomic epithelial effects of NTHi challenge in a murine middle ear epithelial cell line. Methods NTHi lysates were generated and used to stimulate murine epithelial cells (mMEEC) cultured at air-liquid interface over 48 hours– 1 week. Conditional quantitative Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) of cell lysates was performed to interrogate the global protein production in the cells, using the SuperSILAC technique. Histology of the epithelium over time was done to measure bacterial dependent remodeling. Results Mass spectrometry analysis identified 2,565 proteins across samples, of which 74 exhibited differential enrichment or depletion in cell lysates (+/-2.0 fold-change; p value<0.05). The key molecular functions regulated by NTHi lysates exposure were related to cell proliferation, death, migration, adhesion and inflammation. Finally, chronic exposure induced significant epithelial thickening of cells grown at air liquid interface. Conclusions NTHi lysates drive pathways responsible of cell remodeling in murine middle ear epithelium which likely contributes to observed epithelial hyperplasia in vitro. Further elucidation of these mediators will be critical in understanding the progression of OM from acute to chronic at the molecular level. PMID:26859300

  11. Cytoplasmic superoxide dismutase and catalase activity and resistance to radiation lethality in murine tumor cells

    SciTech Connect

    Davy, C.A.; Tesfay, Z.; Jones, J.; Rosenberg, R.C.; McCarthy, C.; Rosenberg, S.O.

    1986-05-01

    Reduced species of molecular oxygen are produced by the interaction of ionizing radiation with aqueous solutions containing molecular oxygen. The enzymes catalase and superoxide dismutase (SOD) are thought to function in vivo as scavengers of metabolically produced peroxide and superoxide respectively. SOD has been shown to protect against the lethal effects of ionizing radiation in vitro and in vivo. The authors have investigated the relationship between the cytosolic SOD catalase content and the sensitivity to radiation lethality of a number of murine cell lines (402AX, EL-4, MB-2T3, MB-4, MEL, P-815, SAI, SP-2, and SV-3T3). K/sub i/(CN/sup -/) for murine Cu-Zn-SOD was determined to be 6.8 x 10/sup -6/ M. No cytosolic Mn-SOD activity was found in any of the cell lines studied. No correlation was found between the cytosolic Cu-Zn-SOD or cytosolic catalase activity and the resistance to radiation lethality or the murine cell lines studied.

  12. Cholinergic epithelial cell with chemosensory traits in murine thymic medulla.

    PubMed

    Panneck, Alexandra Regina; Rafiq, Amir; Schütz, Burkhard; Soultanova, Aichurek; Deckmann, Klaus; Chubanov, Vladimir; Gudermann, Thomas; Weihe, Eberhard; Krasteva-Christ, Gabriela; Grau, Veronika; del Rey, Adriana; Kummer, Wolfgang

    2014-12-01

    Specialized epithelial cells with a tuft of apical microvilli ("brush cells") sense luminal content and initiate protective reflexes in response to potentially harmful substances. They utilize the canonical taste transduction cascade to detect "bitter" substances such as bacterial quorum-sensing molecules. In the respiratory tract, most of these cells are cholinergic and are approached by cholinoceptive sensory nerve fibers. Utilizing two different reporter mouse strains for the expression of choline acetyltransferase (ChAT), we observed intense labeling of a subset of thymic medullary cells. ChAT expression was confirmed by in situ hybridization. These cells showed expression of villin, a brush cell marker protein, and ultrastructurally exhibited lateral microvilli. They did not express neuroendocrine (chromogranin A, PGP9.5) or thymocyte (CD3) markers but rather thymic epithelial (CK8, CK18) markers and were immunoreactive for components of the taste transduction cascade such as Gα-gustducin, transient receptor potential melastatin-like subtype 5 channel (TRPM5), and phospholipase Cβ2. Reverse transcription and polymerase chain reaction confirmed the expression of Gα-gustducin, TRPM5, and phospholipase Cβ2. Thymic "cholinergic chemosensory cells" were often in direct contact with medullary epithelial cells expressing the nicotinic acetylcholine receptor subunit α3. These cells have recently been identified as terminally differentiated epithelial cells (Hassall's corpuscle-like structures in mice). Contacts with nerve fibers (identified by PGP9.5 and CGRP antibodies), however, were not observed. Our data identify, in the thymus, a previously unrecognized presumptive chemosensitive cell that probably utilizes acetylcholine for paracrine signaling. This cell might participate in intrathymic infection-sensing mechanisms.

  13. Regulation of Glycan Structures in Murine Embryonic Stem Cells

    PubMed Central

    Nairn, Alison V.; Aoki, Kazuhiro; dela Rosa, Mitche; Porterfield, Mindy; Lim, Jae-Min; Kulik, Michael; Pierce, J. Michael; Wells, Lance; Dalton, Stephen; Tiemeyer, Michael; Moremen, Kelley W.

    2012-01-01

    The abundance and structural diversity of glycans on glycoproteins and glycolipids are highly regulated and play important roles during vertebrate development. Because of the challenges associated with studying glycan regulation in vertebrate embryos, we have chosen to study mouse embryonic stem (ES) cells as they differentiate into embryoid bodies (EBs) or into extraembryonic endodermal (ExE) cells as a model for cellular differentiation. We profiled N- and O-glycan structures isolated from these cell populations and examined transcripts encoding the corresponding enzymatic machinery for glycan biosynthesis in an effort to probe the mechanisms that drive the regulation of glycan diversity. During differentiation from mouse ES cells to either EBs or ExE cells, general trends were detected. The predominance of high mannose N-glycans in ES cells shifted to an equal abundance of complex and high mannose structures, increased sialylation, and increased α-Gal termination in the differentiated cell populations. Whereas core 1 O-glycan structures predominated in all three cell populations, increased sialylation and increased core diversity characterized the O-glycans of both differentiated cell types. Increased polysialylation was also found in both differentiated cell types. Differences between the two differentiated cell types included greater sialylation of N-glycans in EBs, whereas α-Gal-capped structures were more prevalent in ExE cells. Changes in glycan structures generally, but not uniformly, correlated with alterations in transcript abundance for the corresponding biosynthetic enzymes, suggesting that transcriptional regulation contributes significantly to the regulation of glycan expression. Knowledge of glycan structural diversity and transcript regulation should provide greater understanding of the roles of protein glycosylation in vertebrate development. PMID:22988249

  14. Poly I:C induced microglial activation impairs motor activity in adult rats.

    PubMed

    Patro, I K; Amit; Shrivastava, M; Bhumika, S; Patro, N

    2010-02-01

    Polyinosinic:polycytidic acid (poly I:C) is a synthetic double stranded RNA, which mimics with viral genome and mediates immune activation response similar to double stranded RNA virus infection into the brain. Microglial cells are the immune competent cells of the central nervous system having Toll like receptors-3 on their surface. Upon establishing that poly I:C infusion into the brain causes microgliosis by creating a viral infection model, the present study was designed to evaluate the effects of microglial activation following poly I:C infusion on motor activity. We infused 100 microl of 1% solution of Poly I:C in TBE buffer directly into the lateral ventricle and TBE buffer as vehicle to controls. A significantly higher microglial cell count as compared to control on 2, 3 and 7 days post infusion was recorded. Motor activity and microglial cell count was assessed in both controls and poly I:C infused rats on 1, 2, 3, 7, 14, 21 and 28 days post infusion. A significant decrease in motor activity and motor coordination occurred with respect to control. The results clearly demonstrate that microglial activation has a direct relevance with decreased motor activity. Findings could also have their importance in understanding the role of microglial cells on behavioral aspects in viral diseases.

  15. Effects of benzene inhalation on murine pluripotent stem cells.

    PubMed

    Cronkite, E P; Inoue, T; Carsten, A L; Miller, M E; Bullis, J E; Drew, R T

    1982-03-01

    Effects of benzene inhalation on mouse pluripotent hematopoietic stem cells have been evaluated. Male mice 8--12 wk old were exposed to 400 ppm benzene for 6 h/d, 5 d/wk, for up to 9 1/2 wk. At various time intervals exposed and control animals were killed, and cardiac blood was evaluated for changes in white blood cell (WBC) and red blood cell (RBC) content. In addition, femora and tibiae were evaluated for total marrow cellularity, stem cell content (as measured by the spleen colony technique), and the percent of stem cells in DNA synthesis (as determined by the tritiated thymidine cytocide technique). Exogenous spleen colonies grown from marrow of exposed animals were counted, identified, and scored by histological type. Exposure to benzene caused significant depressions of RBCs and WBCs throughout the exposure period, which continued for at least 14 d after exposure. Bone marrow cellularity and stem cell content were also depressed in exposed animals throughout the study. Tritiated thymidine cytocide of spleen colony-forming cells was generally increased in exposed animals, perhaps indicating a compensatory response to the reduction of circulating cells. Spleen colonies of all types were depressed after exposure to benzene. The significance of the reduction in cellularity, stem cell content, and changes in morphology of spleen colonies is discussed in relation to cellular toxicity and residual injury.

  16. Extracortical origin of some murine subplate cell populations

    PubMed Central

    Pedraza, María; Hoerder-Suabedissen, Anna; Albert-Maestro, María Amparo; Molnár, Zoltán; De Carlos, Juan A.

    2014-01-01

    The subplate layer, the deepest cortical layer in mammals, has important roles in cerebral cortical development. The subplate contains heterogeneous cell populations that are morphologically diverse, with several projection targets. It is currently assumed that these cells are generated in the germinative zone of the earliest cortical neuroepithelium. Here we identify a pallial but extracortical area located in the rostromedial telencephalic wall (RMTW) that gives rise to several cell populations. Postmitotic neurons migrate tangentially from the RMTW toward the cerebral cortex. Most RMTW-derived cells are incorporated into the subplate layer throughout its rostrocaudal extension, with others contributing to the GABAergic interneuron pool of cortical layers V and VI. PMID:24778253

  17. Foxp3+ cells control Th2 responses in a murine model of atopic dermatitis.

    PubMed

    Fyhrquist, Nanna; Lehtimäki, Sari; Lahl, Katharina; Savinko, Terhi; Lappeteläinen, Anna-Mari; Sparwasser, Tim; Wolff, Henrik; Lauerma, Antti; Alenius, Harri

    2012-06-01

    The role of Foxp3+ regulatory T (Treg) cells in atopic dermatitis (AD) is still unclear. In a murine AD model, the number of Foxp3+ cells increased in the allergen-exposed skin area and in the secondary lymphoid organs. Both Foxp3+ and Foxp3- IL-10+ T cells accumulated at the site of allergen exposure, and CD103+ effector/memory Foxp3+ Treg cells expanded gradually in the lymph nodes throughout the sensitization protocol. The depletion of Foxp3+ Treg cells led to significantly exacerbated skin inflammation, including increased recruitment of inflammatory cells and expression of T helper type 2 cytokines, as well as elevated serum IgE levels. The effect of depleting Treg cells during epicutaneous sensitization was mirrored off by a stronger inflammatory response also in the lungs following airway challenge. Thus, Treg cells have an important role in controlling AD-like inflammation and the transfer of allergic skin inflammation to the lungs. PMID:22402436

  18. The effects of simulated hypogravity on murine bone marrow cells

    NASA Technical Reports Server (NTRS)

    Lawless, Desales

    1989-01-01

    Mouse bone marrow cells grown in complete medium at unit gravity were compared with a similar population cultured in conditions that mimic some aspects of microgravity. After the cells adjusted to the conditions that simulated microgravity, they proliferated as fetal or oncogenic populations; their numbers doubled in twelve hour periods. Differentiated subpopulations were depleted from the heterogeneous mixture with time and the undifferentiated hematopoietic stem cells increased in numbers. The cells in the control groups in unit gravity and those in the bioreactors in conditions of microgravity were monitored under a number of parameters. Each were phenotyped as to cell surface antigens using a panel of monoclonal antibodies and flow cytometry. Other parameters compared included: pH, glucose uptake, oxygen consumption and carbon-dioxide production. Nuclear DNA was monitored by flow cytometry. Functional responses were studied by mitogenic stimulation by various lectins. The importance of these findings should have relevance to the space program. Cells should behave predictably in zero gravity; specific populations can be eliminated from diverse populations and other populations isolated. The availability of stem cell populations will enhance both bone marrow and gene transplant programs. Stem cells will permit developmental biologists study the paths of hematopoiesis.

  19. AN IN VITRO MODEL FOR MURINE URETERIC EPITHELIAL CELLS

    EPA Science Inventory

    This report presents a model developed to study growth and differentiation of primary cultures of ureteric epithelial cells from embryonic C57BL/6N mouse urinary tracts. Single cells were resuspended in medium and plated onto transwells coated with collagen IV and laminin. Basa...

  20. Compactin enhances osteogenesis in murine embryonic stem cells.

    PubMed

    Phillips, B W; Belmonte, N; Vernochet, C; Ailhaud, G; Dani, C

    2001-06-01

    Embryonic stem (ES) cells have the capacity to differentiate into various cell types in vitro. In this study, we show that retinoic acid is important for the commitment of ES cells into osteoblasts. Culturing retinoic acid treated ES cells in the presence of the osteogenic supplements ascorbic acid and beta-glycerophosphate resulted in the expression of several osteoblast marker genes, osteocalcin, alkaline phosphatase, and osteopontin. However, there was only a slight amount of mineralized matrix secretion. Addition of bone morphogenic protein-2 or compactin, a drug of the statin family of HMG-CoA reductase inhibitors, resulted in a greatly enhanced formation of bone nodules. Compactin did not modify the expression of osteogenic markers, but at the late stage of differentiation promoted an increase in BMP-2 expression. These results establish ES-cell derived osteogenesis as an effective model system to study the molecular mechanisms by which the statin compactin promotes osteoblastic differentiation and bone nodule formation. PMID:11394905

  1. Hypoxia promotes thyroid differentiation of native murine induced pluripotent stem cells.

    PubMed

    Yang, Yipeng; Lu, Yunshu; Chen, Tong; Zhang, Shenglai; Chu, Bingfeng; Gong, Yurong; Zhao, Weixin; Zhu, Jian; Liu, Yingbin

    2016-01-01

    Hypothyroidism is a very common hormonal deficiency and the stem cell technology which developed in the recent years may offer a therapeutic strategy for treating this disorder. Hypoxia has been demonstrated to play an important role in embryonic formation and development and to modulate stem cell differentiation. However, the influence of oxygen tension on thyroid differentiation has not been studied. In this study, we used murine induced pluripotent stem (iPS) cells for thyroid cell differentiation under normoxic and hypoxic conditions and compared differentiation efficiency in morphology, function, gene and protein expression under both conditions. We found that hypoxia promoted adhesion and outgrowth of embryoid bodies (EBs) derived from murine iPS cells. Expression of endodermal markers (Foxa2 and Gata4) and thyroid transcription factors (Pax8 and Nkx2.1) was increased by hypoxia at both gene and protein levels during early-mid differentiation stages (p<0.05). And so were the thyroid specific markers NIS and TSHR at the end of the experiment (p<0.05). In addition, functional iodide uptake by differentiated cells was also increased after hypoxia. Thyroid differentiation from iPS cells is enhanced under hypoxia and this may involve hypoxia inducible factors (HIFs) and their downstream gene FGF2. Our data offer a foundation for understanding thyroid development and provide a potentially more efficient way to use cell therapy for treating thyroid deficiency. PMID:27389981

  2. Characterization of Long-Term Cultured Murine Submandibular Gland Epithelial Cells

    PubMed Central

    Tsunoda, Kazuyuki; Nakagawa, Taneaki; Tsubota, Kazuo

    2016-01-01

    Purpose Human and rat salivary gland cell lines derived from tumors or genetic modification are currently available for research. Here, we attempted to culture and characterize long-term cultured cells spontaneously derived from wild type murine submandibular glands (SGs). Methods SGs were removed from 3-week-old C57B/6J female mice and dissociated by collagenase type 1 and hyaluronidase digestion. Isolated SG epithelial cells were cultured in low calcium, serum-free growth media in the presence of cholera toxin (CT) during early passages. Single-cell colonies were isolated by limiting dilution culture after 25 passages. Early- and late-stage cell cultures were characterized for keratin 14, keratin 18, α-smooth muscle actin, and p63 by immunostaining and quantitative real-time PCR analysis. Results SG epithelial cells cultured in optimized media maintained their proliferative ability and morphology for over 80 passages. Long-term cultured cells expressed keratin 14, keratin 18, and p63, indicative of an epithelial phenotype. Conclusions Epithelial cells originating from wild type murine SGs could be cultured for longer periods of time and remain phenotypically similar to ductal basal epithelium. PMID:26800086

  3. Identification and Analysis of Natural Killer Cells in Murine Nasal Passages

    PubMed Central

    Okada, Kazunari; Sato, Shintaro; Sato, Ayuko; Mandelboim, Ofer; Yamasoba, Tatsuya; Kiyono, Hiroshi

    2015-01-01

    Background Natural killer (NK) cells in the upper respiratory airways are not well characterized. In the current study, we sought to characterize and functionally assess murine nasal NK cells. Methods Using immunohistochemistry and flow cytometry, we compared the nasal NK cells of Ncr1GFP/+ knock-in mice, whose NK cells produced green fluorescent protein, with their splenic and pulmonary counterparts. In addition, we functionally analyzed the nasal NK cells of these mice in vitro. To assess the in vivo functions of nasal NK cells, C57BL/6 mice depleted of NK cells after treatment with PK136 antibody were nasally infected with influenza virus PR8. Results Immunohistochemical analysis confirmed the presence of NK cells in the lamina propria of nasal mucosa, and flow cytometry showed that these cells were of NK cell lineage. The expression patterns of Ly49 receptor, CD11b/CD27, CD62L and CD69 revealed that nasal NK cells had an immature and activated phenotype compared with that of their splenic and pulmonary counterparts. Effector functions including degranulation and IFN(interferon)-γ production after in vitro stimulation with phorbol 12-myristate-13-acetate plus ionomycin or IL(interleukin)-12 plus IL-18 were dampened in nasal NK cells, and the depletion of NK cells led to an increased influenza virus titer in nasal passages. Conclusions The NK cells of the murine nasal passage belong to the conventional NK cell linage and characteristically demonstrate an immature and activated phenotype. Despite their hyporesponsiveness in vitro, nasal NK cells play important roles in the host defense against nasal influenza virus infection. PMID:26575399

  4. Hexane fraction of Zingiberis Rhizoma Crudus extract inhibits the production of nitric oxide and proinflammatory cytokines in LPS-stimulated BV2 microglial cells via the NF-kappaB pathway.

    PubMed

    Jung, Hyo Won; Yoon, Cheol-Ho; Park, Kwon Moo; Han, Hyung Soo; Park, Yong-Ki

    2009-06-01

    Excessive production of inflammatory mediators such as nitric oxide (NO), prostaglandin E(2) (PGE2), and proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) from activated microglia contributes to uncontrolled inflammation in neurodegenerative diseases. It seems possible that treatment with anti-inflammatory agents, including plants used in Oriental medicine, might delay the progression of neurodegeneration through the inhibition of microglial activation. The present study is focused on the inhibitory effect of the rhizome hexane fraction extract of Zingiber officinale Roscoe (ginger hexan extract; GHE) on the production of inflammatory mediators such as NO, PGE(2), and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated BV-2 cells, a mouse microglial cell line. GHE significantly inhibited the excessive production of NO, PGE(2), TNF-alpha, and IL-1beta in LPS-stimulated BV2 cells. In addition, GHE attenuated the mRNA expressions and protein levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and proinflammatory cytokines. The molecular mechanisms that underlie GHE-mediated attenuation are related to the inhibition of the phosphorylation of three mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase (JNK), and the activation of nuclear factor-kappaB (NF-kappaB). Our results indicate that GHE exhibits anti-inflammatory properties by suppressing the transcription of inflammatory mediator genes through the MAPK and NF-kappaB signaling pathways. The anti-inflammatory properties of GHE may make it useful as a therapeutic candidate for the treatment of human neurodegenerative diseases. PMID:19233241

  5. Deciphering Resting Microglial Morphology and Process Motility from a Synaptic Prospect

    PubMed Central

    Hristovska, Ines; Pascual, Olivier

    2016-01-01

    Microglia, the resident immune cells of the central nervous system (CNS), were traditionally believed to be set into action only in case of injury or disease. Accordingly, microglia were assumed to be inactive or resting in the healthy brain. However, recent studies revealed that microglia carry out active tissue sampling in the intact brain by extending and retracting their ramified processes while periodically contacting synapses. Microglial morphology and motility as well as the frequency and duration of physical contacts with synaptic elements were found to be modulated by neuronal activity, sensory experience and neurotransmission; however findings have not been straightforward. Microglial cells are the most morphologically plastic element of the CNS. This unique feature confers them the possibility to locally sense activity, and to respond adequately by establishing synaptic contacts to regulate synaptic inputs by the secretion of signaling molecules. Indeed, microglial cells can hold new roles as critical players in maintaining brain homeostasis and regulating synaptic number, maturation and plasticity. For this reason, a better characterization of microglial cells and cues mediating neuron-to-microglia communication under physiological conditions may help advance our understanding of the microglial behavior and its regulation in the healthy brain. This review highlights recent findings on the instructive role of neuronal activity on microglial motility and microglia-synapse interactions, focusing on the main transmitters involved in this communication and including newly described communication at the tripartite synapse. PMID:26834588

  6. Donepezil Regulates 1-Methyl-4-phenylpyridinium-Induced Microglial Polarization in Parkinson's Disease.

    PubMed

    Chen, Teng; Hou, Ruihua; Xu, Shujun; Wu, Chengyuan

    2015-10-21

    1-Methyl-4-phenylpyridinium (MPP+) induces microglial activation and degeneration of dopaminergic (DAergic) neurons. Donepezil is a well-known acetylcholinesterase inhibitor used clinically to treat cognitive dysfunction in Alzheimer's disease (AD). In the present study, we tested the hypothesis that MPP+ promotes microglial M1 polarization and suppresses M2 polarization and that this can be restored by donepezil. Results indicate that MPP+ treatment in microglial BV2 cells promotes microglial polarization toward the M1 state. However, pretreatment with donepezil inhibited MPP+-induced M1 polarization in microglia by suppressing the release of interleukin (IL)-6, IL-1β, or tumor necrosis factor (TNF)-α. Importantly, we found that MPP+ inhibited microglial M2 polarization by suppressing expression of Arg-1, Fizz1, and Ym1, which was also rescued by pretreatment with donepezil. In addition, IL-4-mediated induction of anti-inflammatory marker genes IL-10, IL-13, and transforming growth factor-β2 (TGF-β2) were significantly attenuated by MPP+ in BV2 cells, which was restored by pretreatment with donepezil in a concentration-dependent manner. Mechanistically, we found that the addition of MPP+ reduced the intensity of phosphorylated signal transducer and activator of transcription 6 (STAT6) but not total STAT6 in IL-4-stimulated BV2 cells. Importantly, pretreatment of microglial BV2 cells with donepezil 3 h prior to administration of MPP+ rescued the reduction of STAT6 phosphorylation induced by MPP+.

  7. H4 Histamine Receptors Mediate Cell Cycle Arrest in Growth Factor-Induced Murine and Human Hematopoietic Progenitor Cells

    PubMed Central

    Petit-Bertron, Anne-France; Machavoine, François; Defresne, Marie Paule; Gillard, Michel; Chatelain, Pierre; Mistry, Prakash

    2009-01-01

    The most recently characterized H4 histamine receptor (H4R) is expressed preferentially in the bone marrow, raising the question of its role during hematopoiesis. Here we show that both murine and human progenitor cell populations express this receptor subtype on transcriptional and protein levels and respond to its agonists by reduced growth factor-induced cell cycle progression that leads to decreased myeloid, erythroid and lymphoid colony formation. H4R activation prevents the induction of cell cycle genes through a cAMP/PKA-dependent pathway that is not associated with apoptosis. It is mediated specifically through H4R signaling since gene silencing or treatment with selective antagonists restores normal cell cycle progression. The arrest of growth factor-induced G1/S transition protects murine and human progenitor cells from the toxicity of the cell cycle-dependent anticancer drug Ara-C in vitro and reduces aplasia in a murine model of chemotherapy. This first evidence for functional H4R expression in hematopoietic progenitors opens new therapeutic perspectives for alleviating hematotoxic side effects of antineoplastic drugs. PMID:19662098

  8. Cell-type-resolved quantitative proteomics of murine liver.

    PubMed

    Azimifar, S Babak; Nagaraj, Nagarjuna; Cox, Juergen; Mann, Matthias

    2014-12-01

    Mass spectrometry (MS)-based proteomics provides a powerful approach to globally investigate the biological function of individual cell types in mammalian organs. Here, we applied this technology to the in-depth analysis of purified hepatic cell types from mouse. We quantified 11,520 proteins, making this the most comprehensive proteomic resource of any organ to date. Global protein copy number determination demonstrated that a large proportion of the hepatocyte proteome is dedicated to fatty acid and xenobiotic metabolism. We identified as-yet-unknown components of the TGF-β signaling pathway and extracellular matrix in hepatic stellate cells, uncovering their regulative role in liver physiology. Moreover, our high-resolution proteomic data set enabled us to compare the distinct functional roles of hepatic cell types in cholesterol flux, cellular trafficking, and growth factor receptor signaling. This study provides a comprehensive resource for liver biology and biomedicine.

  9. Altered transcription of genes coding for class I histocompatibility antigens in murine tumor cells

    PubMed Central

    1983-01-01

    Three murine tumors induced by Moloney murine leukemia virus (M-MLV) which exhibited loss of some or all H-2 class I antigens at the cell surface were analyzed at the DNA and RNA level with molecular probes specific of H-2 heavy chains and beta 2-microglobulin sequences. No observable difference could be detected at the DNA level between the tumors and the parent animals. However, a decrease in H-2 mRNA was observed, especially in phenotypically H-2 negative tumor, BM5R, where H-2 transcripts were at least 30-fold less abundant. These results show that an H-2-negative character may result from a general alteration in the transcription of H-2 genes, which could reflect some kind of regulatory process. PMID:6311935

  10. Systemic Administration of Tolerogenic Dendritic Cells Ameliorates Murine Inflammatory Arthritis

    PubMed Central

    Healy, Louise J; Collins, Helen L; Thompson, Stephen J

    2008-01-01

    The expression of various cell surface molecules and the production of certain cytokines are important mechanisms by which dendritic cells (DC) are able to bias immune responses. This paper describes the effects of the inflammatory cytokine tumor necrosis factor (TNF)-α on DC phenotype and function. TNF-α treatment resulted in upregulation of MHC class II and CD86 in the absence of increased cell surface CD40 and CD80 or the production of IL-12. Additionally TNF-α treated cells were able to bias T cell responses towards an anti-inflammatory profile. On a note of caution this tolerogenic phenotype of the DC was not stable upon subsequent TLR-4 ligation as a 4 hour pulse of the TNF-α treated DC with lipopolysaccharide (LPS) resulted in the restoration of IL-12 production and an enhancement of their T cell stimulatory capacity which resulted in an increased IFN-γ production. However, TNF-α treated DC, when administered in vivo, were shown to ameliorate disease in collagen induced arthritis, an experimental model of inflammatory joint disease. Mice receiving TNF-α treated DC but not LPS matured DC had a delayed onset, and significantly reduced severity, of arthritis. Disease suppression was associated with reduced levels of collagen specific IgG2a and decreased inflammatory cell infiltration into affected joints. In summary the treatment of DC with TNF-α generates an antigen presenting cell with a phenotype that can reduce the pro-inflammatory response and direct the immune system towards a disease modifying, anti-inflammatory state. PMID:19156221

  11. Schistosoma mansoni larvicidal activity of murine bronchoalveolar lavage cells.

    PubMed

    Lewis, F A; White-Ziegler, C A; Ball, J E; Niemann, G M

    1990-12-01

    We have investigated the ability of cells obtained from both normal and immune mice by bronchoalveolar lavage (BACs) to kill Schistosoma mansoni larvae in vitro. In cultures with mechanically derived schistosomules, high levels of larvicidal activity were displayed by BACs from both normal and irradiated cercaria-immunized C57BL/6 mice. Based on effector-to-target-cell ratios, BAC-mediated killing was two- to threefold more efficient than killing mediated by macrophage-rich cell populations obtained from the peritoneal cavity. BACs from normal A/J mice were essentially as larvicidal as normal C57BL/6 cells. However, BACs from a strain of mouse (P/J) with a known macrophage defect possessed negligible larvicidal activity. Macrophages made up 85 to 95% of BACs from all three strains tested. In contrast to cells of the IC-21 macrophage cell line, B6 BACs did not show enhanced killing activity when preincubated with lymphokine-containing supernatants. Lung schistosomules harvested 10 days after cercarial penetration were refractory to BAC-mediated killing. PMID:2254018

  12. Schistosoma mansoni larvicidal activity of murine bronchoalveolar lavage cells.

    PubMed Central

    Lewis, F A; White-Ziegler, C A; Ball, J E; Niemann, G M

    1990-01-01

    We have investigated the ability of cells obtained from both normal and immune mice by bronchoalveolar lavage (BACs) to kill Schistosoma mansoni larvae in vitro. In cultures with mechanically derived schistosomules, high levels of larvicidal activity were displayed by BACs from both normal and irradiated cercaria-immunized C57BL/6 mice. Based on effector-to-target-cell ratios, BAC-mediated killing was two- to threefold more efficient than killing mediated by macrophage-rich cell populations obtained from the peritoneal cavity. BACs from normal A/J mice were essentially as larvicidal as normal C57BL/6 cells. However, BACs from a strain of mouse (P/J) with a known macrophage defect possessed negligible larvicidal activity. Macrophages made up 85 to 95% of BACs from all three strains tested. In contrast to cells of the IC-21 macrophage cell line, B6 BACs did not show enhanced killing activity when preincubated with lymphokine-containing supernatants. Lung schistosomules harvested 10 days after cercarial penetration were refractory to BAC-mediated killing. PMID:2254018

  13. Measuring ATP Concentration in a Small Number of Murine Hematopoietic Stem Cells.

    PubMed

    Szade, Krzysztof; Zukowska, Monika; Jozkowicz, Alicja; Dulak, Jozef

    2016-01-01

    The metabolism of quiescent adult stem cells differs from the metabolism of differentiated cells. The metabolic processes are tightly regulated and their alterations disturb function of stem cells. One of the indicators of metabolic status of cells is the ATP level. While the method of measuring the ATP levels has been known for many years, estimating ATP levels in small population of defined stem cells isolated directly from the tissue has remained challenging. Here, we show our method of measuring the ATP levels in hematopoietic stem cells sorted from murine bone marrow. We used magnetic sorting as well as cell sorter and adopted the commonly used bioluminescence-based detection kits in described protocol. Our strategy allows to measure ATP levels in 1000 highly purified HSC.

  14. Measuring ATP Concentration in a Small Number of Murine Hematopoietic Stem Cells.

    PubMed

    Szade, Krzysztof; Zukowska, Monika; Jozkowicz, Alicja; Dulak, Jozef

    2016-01-01

    The metabolism of quiescent adult stem cells differs from the metabolism of differentiated cells. The metabolic processes are tightly regulated and their alterations disturb function of stem cells. One of the indicators of metabolic status of cells is the ATP level. While the method of measuring the ATP levels has been known for many years, estimating ATP levels in small population of defined stem cells isolated directly from the tissue has remained challenging. Here, we show our method of measuring the ATP levels in hematopoietic stem cells sorted from murine bone marrow. We used magnetic sorting as well as cell sorter and adopted the commonly used bioluminescence-based detection kits in described protocol. Our strategy allows to measure ATP levels in 1000 highly purified HSC. PMID:27138010

  15. 1.8 Astroms Structure of Murine GITR Ligand Dimer Expressed in Drosophila Melanogaster S2 Cells

    SciTech Connect

    Chattopadhyay, K.; Ramagopal, U; Nathenson, S; Almo, S

    2009-01-01

    Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure, murine GITRL demonstrated a unique 'strand-exchanged' dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8 {angstrom} resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli-expressed proteins. The S2 cell-expressed murine GITRL adopts an identical 'strand-exchanged' dimeric structure to that observed for the E. coli-expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL.

  16. Specific uptake of serotonin by murine lymphoid cells

    SciTech Connect

    Jackson, J.C.; Walker, R.F.; Brooks, W.H.; Roszman, T.L.

    1986-03-01

    Recently the authors confirmed and extended earlier observations that serotonin (5-hydroxytryptamine, 5HT) can influence immune function. Both 5HT and its precursor, 5-hydroxytryptophan inhibit the primary, in vivo antibody response to sheep red blood cells, in mice. Here, the authors report specific in vitro association of this amine with mouse splenocytes. Spleen cells from 6-8 week old CBA/J mice incorporated /sup 3/H-5HT(10/sup -8/ to 2.5 x 10/sup -6/M) in a saturable manner, at 37/sup 0/C. Specificity of uptake was indicated by competition with excess (10/sup -5/M) unlabelled 5HT and with 10/sup -5/M fluoxetine, a selective inhibitor of active 5HT reuptake in rat brain. The 5HT receptor antagonists, methysergide and cyproheptadine, also blocked 5HT uptake. Cell lysis and displacement studies revealed largely intracellular accumulation of /sup 3/H-5HT with little membrane association, in splenocytes. Hofstee analysis of uptake kinetics yielded an apparent Km of 0.82 +/- 0.22 x 10/sup -7/M and Vmax of 501 +/- 108 pM/3 x 10/sup 6/ cells/10 min. Spleen cells fractionated on Sephadex G10 showed virtually no specific 5HT uptake while peritoneal exudate cells from thioglycollate treated mice displayed 5HT uptake kinetics similar to those of splenocytes. The site of specific /sup 3/H-5HT incorporation within a population of spleen cells and the functional significance of this phenomenon to immunomodulation by 5HT remain to be elucidated.

  17. KLN205--a murine lung carcinoma cell line.

    PubMed

    Kaneko, T; LePage, G A; Shnitka, T K

    1980-10-01

    KLN205 cells, a cloned cell line established from the Nettesheim lung carcinoma, grow in various synthetic media such as MEM, Fisher's or Roswell Park Memorial Institute Medium (RPMI) with the addition of 5 to 20% fetal bovine serum (FBS), calf-serum (CS) or horse serum (HS). They grow optimally in minimum Eagle's medium plus nonessential amino acids (NEAA) plus 5 to 10% FBS or HS. The cells are transplantable to DBA/2, BDF1, AKD2F1, and BALB/c, but not to C3H/He or ICR mice. The growth curves, plating efficiency, ultrastructural characteristics, modal number of chromosomes and transplantability to mice of various strains are almost the same for early and late passage of cells passaged in vitro. These parameters for 16th and 36th passages were: doubling time, 31 and 33 hr; plating efficiency, 12.4 +/- 1.2 and 14.6 +/- 2.6%; modal number of chromosomes, 73 and 76; lung colony formation in DBA/2, 50 and 45.9/mouse; and subcutaneous tumor diameter 24.5 and 27.4 mm, respectively. Only the numbers of lung colonies formed in BDF1 mice were different: 24.4/mouse with 16th passage cells, and 10.2/mouse with 36th passage cells. The results suggest that KLN205 is a relatively stable cultured cell line through 36 passages. As was expected, immunosuppression by higher concentrations of triaminolone acetonide (TA) enhanced lung colony formation in BDF1 mice. On the other hand, a low concentration of TA inhibited lung colony formation in DBA/2 mice, which was unexpected. These results suggest that KLN205 offers a model for investigations on metastases to lungs as well as chemotherapy for lung carcinoma.

  18. Hinokitiol induces autophagy in murine breast and colorectal cancer cells.

    PubMed

    Wang, Wei-Kuang; Lin, Song-Tao; Chang, Wen-Wei; Liu, Li-Wen; Li, Tom Yu-Tung; Kuo, Chun-Yu; Hsieh, Jeng-Long; Lee, Che-Hsin

    2016-01-01

    Hinokitiol is found in the heartwood of cupressaceous plants and possesses several biological activities. Hinokitiol may play an important role in anti-inflammation and antioxidant processes, making it potentially useful in therapies for inflammatory-mediated disease. Previously, the suppression of tumor growth by hinokitiol has been shown to occur through apoptosis. Programmed cell death can also occur through autophagy, but the mechanism of hinokitiol-induced autophagy in tumor cells is poorly defined. We used an autophagy inhibitor (3-methyladenine) to demonstrate that hinokitiol can induce cell death via an autophagic pathway. Further, we suggest that hinokitiol induces autophagy in a dose-dependent manner. Markers of autophagy were increased after tumor cells were treated with hinokitiol. In addition, immunoblotting revealed that the levels of phosphoprotein kinase B (P-AKT), phosphomammalian target of rapamycin (P-mTOR), and phospho-p70 ribosomal s6 kinase (P-p70S6K) in tumor cells were decreased after hinokitiol treatment. In conclusion, our results indicate that hinokitiol induces the autophagic signaling pathway via downregulation of the AKT/mTOR pathway. Therefore, our findings show that hinokitiol may control tumor growth by inducing autophagic signaling. PMID:25044443

  19. Poliovirus type 1 infection of murine PRNP-knockout neuronal cells.

    PubMed

    Baj, Andreina; Bettaccini, Alessia; Nishimura, Takuya; Onodera, Takashi; Toniolo, Antonio

    2005-07-01

    Transfection of the prion protein gene (Prnp) into prion-deficient mouse cells was shown to reduce the replication of coxsackievirus B3, an enterovirus. Because mice can be susceptible to poliovirus infection by parenteral routes, the authors tested the susceptibility to poliovirus-1 (PV-1) of a panel of murine neuronal cell lines differing in their ability to express Prnp. The investigated cell lines (prionless HpL3.4 cells, HpL3.4 cells transfected with a Prnp vector, HpL3.4 cells transfected with a void vector, wild-type Hw3.5 Prnp(+/+) cells) expressed the murine homologue (Tage4) of human poliovirus receptor (CD155/hPVR). PV-1 infection of Prnp(-/-) HpL3.4 cells resulted in the production of high viral titers, though viral antigens could be detected in only 0.5% to 2% of cells. Wild-type Prnp(+/+) cells and prionless cells transfected with the Prnp gene were not permissive to PV-1. Results of viral titration and immunofluorescence were confirmed by conventional polymerase chain reaction (PCR) and quantitative real-time PCR. Exposure to PV-1 had no influence on the gene expression profile of Prnp(+/+) cells. In contrast, PV-1 infection was associated with upregulation of several genes in permissive Prnp(-/-) cell cultures: type I interferon (IFN) genes, IFN-related developmental regulator 1 (IFNRD1), tumor necrosis factor superfamily member 13b (TNFSF13b), interleukin (IL) - 7, granulocyte/macrophage colony-stimulating factors (CSFs), hepatocyte growth factor (HGF), vascular endothelial growth factor-A, transforming growth factors beta1 and beta3 (TGFb1, TGFb3), as well as a variety of bone morphogenetic proteins endowed with neuroprotective activity. Distinction of permissive from nonpermissive neuronal cells on the basis of Prnp expression suggests that prion-deficient mice could represent an extraordinarily sensitive animal model for poliovirus infection.

  20. Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

    PubMed Central

    van der Schaft, Daisy W. J.; van Spreeuwel, Ariane C. C.; Boonen, Kristel J. M.; Langelaan, Marloes L. P.; Bouten, Carlijn V. C.; Baaijens, Frank P. T.

    2013-01-01

    Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a

  1. Engineering skeletal muscle tissues from murine myoblast progenitor cells and application of electrical stimulation.

    PubMed

    van der Schaft, Daisy W J; van Spreeuwel, Ariane C C; Boonen, Kristel J M; Langelaan, Marloes L P; Bouten, Carlijn V C; Baaijens, Frank P T

    2013-03-19

    Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative (1). The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues (2,3). Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts (4), neonatal muscle derived progenitor cells (5), cells derived from adult muscle tissues from other species such as human (6) or even induced pluripotent stem cells (iPS cells) (7). Cell contractility causes alignment of the cells along the long axis of the construct (8,9) and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent (8). Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while

  2. Morin downregulates nitric oxide and prostaglandin E2 production in LPS-stimulated BV2 microglial cells by suppressing NF-κB activity and activating HO-1 induction.

    PubMed

    Dilshara, Matharage Gayani; Jayasooriya, Rajapaksha Gedara Prasad Tharanga; Lee, Seungheon; Choi, Yung Hyun; Kim, Gi-Young

    2016-06-01

    Morin possesses anti-inflammatory activity against septic shock and allergic responses, and prevents acute liver damage. However, the biological mechanism of action of morin in neuroinflammation remains largely unknown. Therefore, the present study investigated whether morin has the ability to attenuate expression of proinflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Morin inhibited the expression of LPS-induced proinflammatory mediators such as NO and PGE2, without any cytotoxic effects. Furthermore, LPS-induced inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) were inhibited both at the mRNA and protein levels in response to morin. Morin also attenuated LPS-induced DNA-binding activity of nuclear transcription factor-κB (NF-κB) and its promoter activity. Pyrrolidine dithiocarbamate (PDTC), a specific NF-κB inhibitor, downregulated the expression of LPS-induced iNOS and COX-2, which suggests that morin-mediated NF-κB inhibition is the main signaling pathway responsible for the inhibition of iNOS and COX-2 expression. Additionally, morin increased induction of heme oxygenase-1 (HO-1) activity, leading to the suppression of NO and PGE2 production. Our results indicate that morin downregulates the expression of proinflammatory genes, such as iNOS and COX-2, involved in the synthesis of NO and PGE2 in LPS-stimulated BV2 microglial cells by suppressing NF-κB activity and activation of HO-1. Taken together, the findings of the present study suggest that morin may have potential as a therapeutic for the prevention of neuroinflammation.

  3. Characterization of tumor cell lines derived from murine gammaherpesvirus-68-infected mice.

    PubMed Central

    Usherwood, E J; Stewart, J P; Nash, A A

    1996-01-01

    Cell lines were derived from mice with murine gammaherpesvirus-68 (MHV-68)-associated lymphoproliferative disease. Four were of an ambiguous phenotype and were MHV-68 negative. One, S11, was a B lymphocyte that contained MHV-68 genomes in both linear and episomal forms and released virus. The line was clonable and grew into tumors in nude mice. This is the first naturally occurring MHV-68-positive B-cell line to be generated, and it will be an invaluable tool for the study of MHV-68 latency. PMID:8709292

  4. Hippo/MST1 signaling mediates microglial activation following acute cerebral ischemia-reperfusion injury.

    PubMed

    Zhao, Siqi; Yin, Jie; Zhou, Lujun; Yan, Feng; He, Qing; Huang, Li; Peng, Shengyi; Jia, Junying; Cheng, Jinbo; Chen, Hong; Tao, Wufan; Ji, Xunming; Xu, Yun; Yuan, Zengqiang

    2016-07-01

    Cerebral ischemia-reperfusion injury is a major public health concern that causes high rates of disability and mortality in adults. Microglial activation plays a crucial role in ischemic stroke-induced alteration of the immune microenvironment. However, the mechanism underlying the triggering of microglial activation by ischemic stroke remains to be elucidated. Previously, we demonstrated that the protein kinase Hippo/MST1 plays an important role in oxidative stress-induced cell death in mammalian primary neurons and that the protein kinase c-Abl phosphorylates MST1 at Y433, which increases MST1 kinase activity. Microglial activation has been implicated as a secondary detrimental cellular response that contributes to neuronal cell death in ischemic stroke. Here, we are the first, to our knowledge, to demonstrate that MST1 mediates stroke-induced microglial activation by directly phosphorylating IκBα at residues S32 and S36. We further demonstrate that Src kinase functions upstream of MST1-IκB signaling during microglial activation. Specific deletion of MST1 in microglia mitigates stroke-induced brain injury. Therefore, we propose that Src-MST1-IκB signaling plays a critical role in stroke-induced microglial activation. Together with our previous work demonstrating that MST1 is important for oxidative stress-induced neuronal cell death, our results indicate that MST1 could represent a potent therapeutic target for ischemic stroke.

  5. Chloroquine stimulates nitric oxide synthesis in murine, porcine, and human endothelial cells.

    PubMed Central

    Ghigo, D; Aldieri, E; Todde, R; Costamagna, C; Garbarino, G; Pescarmona, G; Bosia, A

    1998-01-01

    Nitric oxide (NO) is a free radical involved in the regulation of many cell functions and in the expression of several diseases. We have found that the antimalarial and antiinflammatory drug, chloroquine, is able to stimulate NO synthase (NOS) activity in murine, porcine, and human endothelial cells in vitro: the increase of enzyme activity is dependent on a de novo synthesis of some regulatory protein, as it is inhibited by cycloheximide but is not accompanied by an increased expression of inducible or constitutive NOS isoforms. Increased NO synthesis is, at least partly, responsible for chloroquine-induced inhibition of cell proliferation: indeed, NOS inhibitors revert the drug-evoked blockage of mitogenesis and ornithine decarboxylase activity in murine and porcine endothelial cells. The NOS-activating effect of chloroquine is dependent on its weak base properties, as it is exerted also by ammonium chloride, another lysosomotropic agent. Both compounds activate NOS by limiting the availability of iron: their stimulating effects on NO synthesis and inhibiting action on cell proliferation are reverted by iron supplementation with ferric nitrilotriacetate, and are mimicked by incubation with desferrioxamine. Our results suggest that NO synthesis can be stimulated in endothelial cells by chloroquine via an impairment of iron metabolism. PMID:9691096

  6. IAP antagonists sensitize murine osteosarcoma cells to killing by TNFα

    PubMed Central

    Shekhar, Tanmay M.; Miles, Mark A.; Gupte, Ankita; Taylor, Scott; Tascone, Brianna; Walkley, Carl R.; Hawkins, Christine J.

    2016-01-01

    Outcomes for patients diagnosed with the bone cancer osteosarcoma have not improved significantly in the last four decades. Only around 60% of patients and about a quarter of those with metastatic disease survive for more than five years. Although DNA-damaging chemotherapy drugs can be effective, they can provoke serious or fatal adverse effects including cardiotoxicity and therapy-related cancers. Better and safer treatments are therefore needed. We investigated the anti-osteosarcoma activity of IAP antagonists (also known as Smac mimetics) using cells from primary and metastatic osteosarcomas that arose spontaneously in mice engineered to lack p53 and Rb expression in osteoblast-derived cells. The IAP antagonists SM-164, GDC-0152 and LCL161, which efficiently target XIAP and cIAPs, sensitized cells from most osteosarcomas to killing by low levels of TNFα but not TRAIL. RIPK1 expression levels and activity correlated with sensitivity. RIPK3 levels varied considerably between tumors and RIPK3 was not required for IAP antagonism to sensitize osteosarcoma cells to TNFα. IAP antagonists, including SM-164, lacked mutagenic activity. These data suggest that drugs targeting XIAP and cIAP1/2 may be effective for osteosarcoma patients whose tumors express abundant RIPK1 and contain high levels of TNFα, and would be unlikely to provoke therapy-induced cancers in osteosarcoma survivors. PMID:27129149

  7. Reproducible establishment of hemopoietic supportive stromal cell lines from murine bone marrow

    SciTech Connect

    Itoh, K.; Tezuka, H.; Sakoda, H.; Konno, M.; Nagata, K.; Uchiyama, T.; Uchino, H.; Mori, K.J.

    1989-02-01

    Stromal cell lines, designated MS-1, -2, -3, -4, -5, -6, and -7 were established by irradiating the adherent cells in long-term bone marrow cultures with 900-rad x-rays. Two of the cell lines, MS-1 and MS-5, have the capacity to support the growth of hemopoietic stem cells (spleen colony-forming cells and granulocyte-macrophage colony-forming cells) for greater than 2 months in vitro. These two cell lines were alkaline phosphatase-, peroxidase-, and factor VIII-negative and positive for periodic acid-Schiff and nonspecific esterase. Extracellular matrix proteins such as fibronectin, laminin, and collagen type I were produced by these two cell lines. Neither MS-1 cell- nor MS-5 cell-conditioned medium supported the growth of hemopoietic stem cells, and hemopoietic stem cells were found preferentially to be under and on MS-1 and MS-5 layers rather than in suspension. Close contact with the MS-1 cell layer or the MS-5 cell layer appears to be essential in maintaining hemopoiesis in vitro. Conditioned media from MS-1 cells and MS-5 cells stimulated granulocyte colony formation from murine bone marrow cells in semisolid culture.

  8. Disruption of canonical TGFβ-signaling in murine coronary progenitor cells by low level arsenic

    SciTech Connect

    Allison, Patrick; Huang, Tianfang; Broka, Derrick; Parker, Patti; Barnett, Joey V.; Camenisch, Todd D.

    2013-10-01

    Exposure to arsenic results in several types of cancers as well as heart disease. A major contributor to ischemic heart pathologies is coronary artery disease, however the influences by environmental arsenic in this disease process are not known. Similarly, the impact of toxicants on blood vessel formation and function during development has not been studied. During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types including smooth muscle cells which contribute to the coronary vessels. The TGFβ family of ligands and receptors is essential for developmental cardiac epithelial to mesenchymal transition (EMT) and differentiation into coronary smooth muscle cells. In this in vitro study, 18 hour exposure to 1.34 μM arsenite disrupted developmental EMT programming in murine epicardial cells causing a deficit in cardiac mesenchyme. The expression of EMT genes including TGFβ2, TGFβ receptor-3, Snail, and Has-2 are decreased in a dose-dependent manner following exposure to arsenite. TGFβ2 cell signaling is abrogated as detected by decreases in phosphorylated Smad2/3 when cells are exposed to 1.34 μM arsenite. There is also loss of nuclear accumulation pSmad due to arsenite exposure. These observations coincide with a decrease in vimentin positive mesenchymal cells invading three-dimensional collagen gels. However, arsenite does not block TGFβ2 mediated smooth muscle cell differentiation by epicardial cells. Overall these results show that arsenic exposure blocks developmental EMT gene programming in murine coronary progenitor cells by disrupting TGFβ2 signals and Smad activation, and that smooth muscle cell differentiation is refractory to this arsenic toxicity. - Highlights: • Arsenic blocks TGFβ2 induced expression of EMT genes. • Arsenic blocks TGFβ2 triggered Smad2/3 phosphorylation and nuclear translocation. • Arsenic blocks epicardial cell differentiation into cardiac mesenchyme.

  9. Pomegranate Polyphenols and Extract Inhibit Nuclear Factor of Activated T-Cell Activity and Microglial Activation In Vitro and in a Transgenic Mouse Model of Alzheimer Disease123

    PubMed Central

    Rojanathammanee, Lalida; Puig, Kendra L.; Combs, Colin K.

    2013-01-01

    cell line (P < 0.05) and decreased Aβ-stimulated TNF-α secretion by murine microglia (P < 0.05). These data indicate that dietary pomegranate produces brain antiinflammatory effects that may attenuate AD progression. PMID:23468550

  10. Differential expression of Ran GTPase during HMBA-induced differentiation in murine erythroleukemia cells.

    PubMed

    Vanegas, N; García-Sacristán, A; López-Fernández, L A; Párraga, M; del Mazo, J; Hernández, P; Schvartzman, J B; Krimer, D B

    2003-07-01

    Murine erythroleukemia (MEL) cells undergo erythroid differentiation in vitro when treated with hexamethylene bisacetamide (HMBA). To identify genes involved in the commitment of MEL cells to differentiate, we screened a cDNA library constructed from HMBA-induced cells by differential hybridization and isolated GTPase Ran as a down-regulated gene. We observed that Ran was expressed in a biphasic mode. Following a decrease in mRNA level during the initial hours of induction, Ran re-expressed at 24-48 h, and gradually declined again. To investigate the role of Ran during MEL differentiation we constructed MEL transfectants capable to express or block Ran mRNA production constitutively. No effects were observed on cell growth and proliferation. Blockage of Ran, however, interfered with MEL cell differentiation resulting in a decrease of cell survival in the committed population.

  11. Xenogenic transfer of isolated murine mitochondria into human rho0 cells can improve respiratory function.

    PubMed

    Katrangi, Eyad; D'Souza, Gerard; Boddapati, Sarathi V; Kulawiec, Mariola; Singh, Keshav K; Bigger, Brian; Weissig, Volkmar

    2007-12-01

    Mitochondrial DNA mutations are the direct cause of several physiological disorders and are also associated with the aging process. The modest progress made over the past two decades towards manipulating the mitochondrial genome and understanding its function within living mammalian cells means that cures for mitochondrial DNA mutations are still elusive. Here, we report that transformed mammalian cells internalize exogenous isolated mitochondria upon simple co-incubation. We first demonstrate the physical presence of internalized mitochondria within recipient cells using fluorescence microscopy. Second, we show that xenogenic transfer of murine mitochondria into human cells lacking functional mitochondria can functionally restore respiration in cells lacking mtDNA. Third, utilizing the natural competence of isolated mitochondria to take up linear DNA molecules, we demonstrate the feasibility of using cellular internalization of isolated exogenous mitochondria as a potential tool for studying mitochondrial genetics in living mammalian cells. PMID:18069915

  12. Oatp-associated uptake and toxicity of microcystins in primary murine whole brain cells

    SciTech Connect

    Feurstein, D.; Holst, K.; Fischer, A.; Dietrich, D.R.

    2009-01-15

    Microcystins (MCs) are naturally occurring cyclic heptapeptides that exhibit hepato-, nephro- and possibly neurotoxic effects in mammals. Organic anion transporting polypeptides (rodent Oatp/human OATP) appear to be specifically required for active uptake of MCs into hepatocytes and kidney epithelial cells. Based on symptoms of neurotoxicity in MC-intoxicated patients and the presence of Oatp/OATP at the blood-brain-barrier (BBB) and blood-cerebrospinal-fluid-barrier (BCFB) it is hypothesized that MCs can be transported across the BBB/BCFB in an Oatp/OATP-dependent manner and can induce toxicity in brain cells via inhibition of protein phosphatase (PP). To test these hypotheses, the presence of murine Oatp (mOatp) in primary murine whole brain cells (mWBC) was investigated at the mRNA and protein level. MC transport was tested by exposing mWBCs to three different MC-congeners (MC-LR, -LW, -LF) with/without co-incubation with the OATP/Oatp-substrates taurocholate (TC) and bromosulfophthalein (BSP). Uptake of MCs and cytotoxicity was demonstrated via MC-Western blot analysis, immunocytochemistry, cell viability and PP inhibition assays. All MC congeners bound covalently and inhibited mWBC PP. MC-LF was the most cytotoxic congener followed by -LW and -LR. The lowest toxin concentration significantly reducing mWBC viability after 48 h exposure was 400 nM (MC-LF). Uptake of MCs into mWBCs was inhibited via co-incubation with excess TC (50 and 500 {mu}M) and BSP (50 {mu}M). MC-Western blot analysis demonstrated a concentration-dependent accumulation of MCs. In conclusion, the in vitro data support the assumed MC-congener-dependent uptake in a mOatp-associated manner and cytotoxicity of MCs in primary murine whole brain cells.

  13. Differential effects of protoporphyrin and uroporphyrin on murine mast cells

    SciTech Connect

    Lim, H.W.; Gigli, I.; Wasserman, S.I.

    1987-03-01

    To investigate the mechanisms responsible for the distinct cutaneous manifestations of erythropoietic protoporphyria and porphyria cutanea tarda, the effects of protoporphyrin (PP) and uroporphyrin (URO), the predominant porphyrins in the respective disease, on mast cells were examined. Release of preformed and generated mediators was assessed by the release of radioactivity from cells labeled with (/sup 3/H)serotonin and (/sup 14/C)arachidonic acid, respectively. Clinically relevant doses of PP (25-500 ng/ml) and 396-407 nm irradiation (3-16 X 10(2)J/m2) induced maximal net release of preformed mediators ,f 44.52 +/- 6.6 to 58.01 +/- 4.0% (mean +/- SE). In contrast, irradiation in the presence of URO (50-5000 ng/ml) resulted in less than 5% net release. (3H)Serotonin release induced by PP and irradiation was calcium-independent, and was not enhanced by phorbol 12-myristate 13-acetate, a known activator of protein kinase C. This release was suppressed by catalase, a scavenger of hydrogen peroxide. Furthermore, irradiation in the presence of PP, but not in the presence of URO, resulted in perturbation of cell membrane. Irradiation in the presence of PP also resulted in a maximal net release of generated mediators of 9.98 +/- 3.5% (mean +/- SE), whereas similar treatment in the presence of URO induced less than 0.5% net release. These results suggested that the burning, stinging, erythema, and edema experienced by patients with erythropoietic protoporphyria following sun exposure, and the lack of such findings in patients with porphyria cutanea tarda, may be explained, at least in part, by the differential effects of PP and URO on mast cells.

  14. Endothelial cell proliferation associated with lesions of murine systemic candidiasis.

    PubMed Central

    Ashman, R B; Papadimitriou, J M

    1994-01-01

    Neovascularization is associated with tumor growth and some inflammatory diseases but has not been reported to be induced by infectious agents. In a mouse model of systemic Candida albicans infection, extensive endothelial cell proliferation was seen in the periphery of brain abscesses and in the areas of fungal pyelonephritis in the kidney. This finding is important for an understanding of the pathogenesis of fungal infections and may contribute to an analysis of the mechanisms of angiogenesis. Images PMID:7523305

  15. Functional screen identifies regulators of murine hematopoietic stem cell repopulation

    PubMed Central

    Holmfeldt, Per; Ganuza, Miguel; Marathe, Himangi; He, Bing; Hall, Trent; Kang, Guolian; Moen, Joseph; Pardieck, Jennifer; Saulsberry, Angelica C.; Cico, Alba; Gaut, Ludovic; McGoldrick, Daniel; Finkelstein, David; Tan, Kai

    2016-01-01

    Understanding the molecular regulation of hematopoietic stem and progenitor cell (HSPC) engraftment is paramount to improving transplant outcomes. To discover novel regulators of HSPC repopulation, we transplanted >1,300 mice with shRNA-transduced HSPCs within 24 h of isolation and transduction to focus on detecting genes regulating repopulation. We identified 17 regulators of HSPC repopulation: Arhgef5, Armcx1, Cadps2, Crispld1, Emcn, Foxa3, Fstl1, Glis2, Gprasp2, Gpr56, Myct1, Nbea, P2ry14, Smarca2, Sox4, Stat4, and Zfp521. Knockdown of each of these genes yielded a loss of function, except in the cases of Armcx1 and Gprasp2, whose loss enhanced hematopoietic stem cell (HSC) repopulation. The discovery of multiple genes regulating vesicular trafficking, cell surface receptor turnover, and secretion of extracellular matrix components suggests active cross talk between HSCs and the niche and that HSCs may actively condition the niche to promote engraftment. We validated that Foxa3 is required for HSC repopulating activity, as Foxa3−/− HSC fails to repopulate ablated hosts efficiently, implicating for the first time Foxa genes as regulators of HSPCs. We further show that Foxa3 likely regulates the HSC response to hematologic stress. Each gene discovered here offers a window into the novel processes that regulate stable HSPC engraftment into an ablated host. PMID:26880577

  16. Altered microglial phagocytosis in GPR34-deficient mice.

    PubMed

    Preissler, Julia; Grosche, Antje; Lede, Vera; Le Duc, Diana; Krügel, Katja; Matyash, Vitali; Szulzewsky, Frank; Kallendrusch, Sonja; Immig, Kerstin; Kettenmann, Helmut; Bechmann, Ingo; Schöneberg, Torsten; Schulz, Angela

    2015-02-01

    GPR34 is a Gi/o protein-coupled receptor (GPCR) of the nucleotide receptor P2Y12 -like group. This receptor is highly expressed in microglia, however, the functional relevance of GPR34 in these glial cells is unknown. Previous results suggested an impaired immune response in GPR34-deficient mice infected with Cryptococcus neoformans. Here we show that GPR34 deficiency results in morphological changes in retinal and cortical microglia. RNA sequencing analysis of microglia revealed a number of differentially expressed transcripts involved in cell motility and phagocytosis. We found no differences in microglial motility after entorhinal cortex lesion and in response to laser lesion. However, GPR34-deficient microglia showed reduced phagocytosis activity in both retina and acutely isolated cortical slices. Our study identifies GPR34 as an important signaling component controlling microglial function, morphology and phagocytosis.

  17. Altered microglial phagocytosis in GPR34-deficient mice.

    PubMed

    Preissler, Julia; Grosche, Antje; Lede, Vera; Le Duc, Diana; Krügel, Katja; Matyash, Vitali; Szulzewsky, Frank; Kallendrusch, Sonja; Immig, Kerstin; Kettenmann, Helmut; Bechmann, Ingo; Schöneberg, Torsten; Schulz, Angela

    2015-02-01

    GPR34 is a Gi/o protein-coupled receptor (GPCR) of the nucleotide receptor P2Y12 -like group. This receptor is highly expressed in microglia, however, the functional relevance of GPR34 in these glial cells is unknown. Previous results suggested an impaired immune response in GPR34-deficient mice infected with Cryptococcus neoformans. Here we show that GPR34 deficiency results in morphological changes in retinal and cortical microglia. RNA sequencing analysis of microglia revealed a number of differentially expressed transcripts involved in cell motility and phagocytosis. We found no differences in microglial motility after entorhinal cortex lesion and in response to laser lesion. However, GPR34-deficient microglia showed reduced phagocytosis activity in both retina and acutely isolated cortical slices. Our study identifies GPR34 as an important signaling component controlling microglial function, morphology and phagocytosis. PMID:25142016

  18. Label-Retaining Cells in the Adult Murine Salivary Glands Possess Characteristics of Adult Progenitor Cells

    PubMed Central

    Chibly, Alejandro M.; Querin, Lauren; Harris, Zoey; Limesand, Kirsten H.

    2014-01-01

    Radiotherapy is the primary treatment for patients with head and neck cancer, which account for roughly 500,000 annual cases worldwide. Dysfunction of the salivary glands and associated conditions like xerostomia and dysphagia are often developed by these patients, greatly diminishing their life quality. Current preventative and palliative care fail to deliver an improvement in the quality of life, thus accentuating the need for regenerative therapies. In this study, a model of label retaining cells (LRCs) in murine salivary glands was developed, in which LRCs demonstrated proliferative potential and possessed markers of putative salivary progenitors. Mice were labeled with 5-Ethynyl-2′-deoxyuridine (EdU) at postnatal day 10 and chased for 8 weeks. Tissue sections from salivary glands obtained at the end of chase demonstrated co-localization between LRCs and the salivary progenitor markers keratin 5 and keratin 14, as well as kit mRNA, indicating that LRCs encompass a heterogeneous population of salivary progenitors. Proliferative potential of LRCs was demonstrated by a sphere assay, in which LRCs were found in primary and secondary spheres and they co-localized with the proliferation marker Ki67 throughout sphere formation. Surprisingly, LRCs were shown to be radio-resistant and evade apoptosis following radiation treatment. The clinical significance of these findings lie in the potential of this model to study the mechanisms that prevent salivary progenitors from maintaining homeostasis upon exposure to radiation, which will in turn facilitate the development of regenerative therapies for salivary gland dysfunction. PMID:25238060

  19. Label-retaining cells in the adult murine salivary glands possess characteristics of adult progenitor cells.

    PubMed

    Chibly, Alejandro M; Querin, Lauren; Harris, Zoey; Limesand, Kirsten H

    2014-01-01

    Radiotherapy is the primary treatment for patients with head and neck cancer, which account for roughly 500,000 annual cases worldwide. Dysfunction of the salivary glands and associated conditions like xerostomia and dysphagia are often developed by these patients, greatly diminishing their life quality. Current preventative and palliative care fail to deliver an improvement in the quality of life, thus accentuating the need for regenerative therapies. In this study, a model of label retaining cells (LRCs) in murine salivary glands was developed, in which LRCs demonstrated proliferative potential and possessed markers of putative salivary progenitors. Mice were labeled with 5-Ethynyl-2'-deoxyuridine (EdU) at postnatal day 10 and chased for 8 weeks. Tissue sections from salivary glands obtained at the end of chase demonstrated co-localization between LRCs and the salivary progenitor markers keratin 5 and keratin 14, as well as kit mRNA, indicating that LRCs encompass a heterogeneous population of salivary progenitors. Proliferative potential of LRCs was demonstrated by a sphere assay, in which LRCs were found in primary and secondary spheres and they co-localized with the proliferation marker Ki67 throughout sphere formation. Surprisingly, LRCs were shown to be radio-resistant and evade apoptosis following radiation treatment. The clinical significance of these findings lie in the potential of this model to study the mechanisms that prevent salivary progenitors from maintaining homeostasis upon exposure to radiation, which will in turn facilitate the development of regenerative therapies for salivary gland dysfunction.

  20. An ES-Like Pluripotent State in FGF-Dependent Murine iPS cells

    PubMed Central

    Ungaro, Federica; Prigione, Alessandro; Chen, Hsu-Hsin; Welling, Maaike; Eijpe, Maureen; Mostoslavsky, Gustavo; Tesar, Paul; Adjaye, James; Geijsen, Niels; Broccoli, Vania

    2010-01-01

    Recent data demonstrates that stem cells can exist in two morphologically, molecularly and functionally distinct pluripotent states; a naïve LIF-dependent pluripotent state which is represented by murine embryonic stem cells (mESCs) and an FGF-dependent primed pluripotent state represented by murine and rat epiblast stem cells (EpiSCs). We find that derivation of induced pluripotent stem cells (iPSCs) under EpiSC culture conditions yields FGF-dependent iPSCs from hereon called FGF-iPSCs) which, unexpectedly, display naïve ES-like/ICM properties. FGF-iPSCs display X-chromosome activation, multi-lineage differentiation, teratoma competence and chimera contribution in vivo. Our findings suggest that in 129 and Bl6 mouse strains, iPSCs can dominantly adopt a naive pluripotent state regardless of culture growth factor conditions. Characterization of the key molecular signalling pathways revealed FGF-iPSCs to depend on the Activin/Nodal and FGF pathways, while signalling through the JAK-STAT pathway is not required for FGF-iPS cell maintenance. Our findings suggest that in 129 and Bl6 mouse strains, iPSCs can dominantly adopt a naive pluripotent state regardless of culture growth factor conditions. PMID:21209851

  1. R-phycoerythrin-conjugated antibodies are inappropriate for intracellular staining of murine plasma cells.

    PubMed

    Kim, Myun Soo; Kim, Tae Sung

    2013-05-01

    Phycoerythrin (PE) is a type of phycobiliproteins found in cyanobacteria and red algae. PE-conjugated antibodies are broadly used for flow cytometry and immunofluorescence microscopy. Because nonspecific binding of antibodies results in decreased analytic accuracy, numerous efforts have been made to unveil cases and mechanisms of nonspecific bindings. However, nonspecific binding of specific cell types by a fluorescent dye-conjugated form of antibody has been rarely reported. In the present study, we discovered that PE-conjugated antibodies, but not FITC- or APC-antibodies, selectively stained lamina propria plasma cells (LP-PCs) from the murine small intestine after membrane permeabilization. We demonstrated that LP-PC-selective staining with PE-antibodies was not due to interactions of antibody-epitope or antibody-Fc receptor. This unexpected staining by PE-antibody was not dependent on the mouse strain of LP-PCs, experimental methods, or origin species of the antibody, but dependent on PE itself. This phenomenon was also observed in plasma cells isolated from bone marrow, spleen, and mesenteric lymph nodes. Furthermore, in vitro activated B cells and in vivo generated LP-PCs were also selectively stained by PE-conjugated antibodies. Taken together, these results show that PE-conjugated antibodies are inappropriate for intracellular staining of murine plasma cells.

  2. Quantitative Analysis of Exosomes From Murine Lung Cancer Cells by Flow Cytometry

    PubMed Central

    Rim, Kyung-Taek; Kim, Soo-Jin

    2016-01-01

    In vivo studies regarding biochemical, molecular biological, and histopathological changes in cancer tissues have been widely performed by the administration of carcinogens in rodents. In these established methods, dissection of the animal following sacrifice must be carried out. Exosomes are cell-derived vesicles that are present in all body fluids and these vesicles have specific roles within cells. Thus, much attention is given to the clinical application of exosomes that can possibly be used for prediction and therapy and as biomarkers related to cancer. To develop a new tool for monitoring in vivo genetic alterations, as a result of carcinogenesis, without the need for frequent euthanasia, we performed quantitative measurement of exosomes in Mlg2908 murine lung fibroblasts and LA-4 and KLN 205 murine lung cancer cells using fluorescence-activated cell sorting. We detected an increase in CD63-specific exosomes in LA-4 lung cancer cells. This result is able to be applied to the classification of cancer-specific proteins and miRNA as diagnostic markers. PMID:27722146

  3. Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow.

    PubMed

    Chen, Yonghong; Maeda, Azusa; Bu, Jiachuan; DaCosta, Ralph

    2016-01-01

    Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer. PMID:27500928

  4. Evaluation of continuous cell lines in antiviral studies with murine cytomegalovirus.

    PubMed

    Smee, D F; Colletti, A; Alaghamandan, H A; Allen, L B

    1989-01-01

    Cell culture systems were developed for rapid antiviral drug screening, using murine cytomegalovirus (MCMV) as an alternative to the slower growing human CMV. Since previous assay methods with MCMV employed mouse embryo fibroblasts (MEF cells), which are labor intensive to prepare and die off after 3-4 passages from primary culture, identification of virus-susceptible continuous cell lines was desirable. Three cell lines were found useful for assaying MCMV: C127I, SC-1, and 3T3. The antiviral agents acyclovir, ganciclovir, 5-fluoroarabinofuranosylcytosine, and 2'-fluoro-2'-deoxy-5-iodoarabinofuranosylcytosine were evaluated in the 3 continuous cell lines and in MEF cells. The 50% virus- or cell-inhibitory concentration values determined for each compound did not vary much from cell to cell. MEF cells were 10-fold more sensitive than the other cell lines to quantify virus from mouse organs, however. Virus propagated in 3T3 and SC-1 cells were as virulent to mice as salivary gland virus, whereas virus from MEF and C127I cells was more attenuated. Overall, C127I cells were judged to be the best for large scale antiviral screening in vitro, but MEF was the cell type of choice for titration of viruses from mouse organs and tissues.

  5. Index sorting resolves heterogeneous murine hematopoietic stem cell populations

    PubMed Central

    Schulte, Reiner; Wilson, Nicola K.; Prick, Janine C.M.; Cossetti, Chiara; Maj, Michal K.; Gottgens, Berthold; Kent, David G.

    2015-01-01

    Recent advances in the cellular and molecular biology of single stem cells have uncovered significant heterogeneity in the functional properties of stem cell populations. This has prompted the development of approaches to study single cells in isolation, often performed using multiparameter flow cytometry. However, many stem cell populations are too rare to test all possible cell surface marker combinations, and virtually nothing is known about functional differences associated with varying intensities of such markers. Here we describe the use of index sorting for further resolution of the flow cytometric isolation of single murine hematopoietic stem cells (HSCs). Specifically, we associate single-cell functional assay outcomes with distinct cell surface marker expression intensities. High levels of both CD150 and EPCR associate with delayed kinetics of cell division and low levels of differentiation. Moreover, cells that do not form single HSC-derived clones appear in the 7AADdim fraction, suggesting that even low levels of 7AAD staining are indicative of less healthy cell populations. These data indicate that when used in combination with single-cell functional assays, index sorting is a powerful tool for refining cell isolation strategies. This approach can be broadly applied to other single-cell systems, both to improve isolation and to acquire additional cell surface marker information. PMID:26051918

  6. Ex vivo 3D osteocyte network construction with primary murine bone cells

    PubMed Central

    Sun, Qiaoling; Gu, Yexin; Zhang, Wenting; Dziopa, Leah; Zilberberg, Jenny; Lee, Woo

    2015-01-01

    Osteocytes reside as three-dimensionally (3D) networked cells in the lacunocanalicular structure of bones and regulate bone and mineral homeostasis. Despite of their important regulatory roles, in vitro studies of osteocytes have been challenging because: (1) current cell lines do not sufficiently represent the phenotypic features of mature osteocytes and (2) primary cells rapidly differentiate to osteoblasts upon isolation. In this study, we used a 3D perfusion culture approach to: (1) construct the 3D cellular network of primary murine osteocytes by biomimetic assembly with microbeads and (2) reproduce ex vivo the phenotype of primary murine osteocytes, for the first time to our best knowledge. In order to enable 3D construction with a sufficient number of viable cells, we used a proliferated osteoblastic population of healthy cells outgrown from digested bone chips. The diameter of microbeads was controlled to: (1) distribute and entrap cells within the interstitial spaces between the microbeads and (2) maintain average cell-to-cell distance to be about 19 µm. The entrapped cells formed a 3D cellular network by extending and connecting their processes through openings between the microbeads. Also, with increasing culture time, the entrapped cells exhibited the characteristic gene expressions (SOST and FGF23) and nonproliferative behavior of mature osteocytes. In contrast, 2D-cultured cells continued their osteoblastic differentiation and proliferation. This 3D biomimetic approach is expected to provide a new means of: (1) studying flow-induced shear stress on the mechanotransduction function of primary osteocytes, (2) studying physiological functions of 3D-networked osteocytes with in vitro convenience, and (3) developing clinically relevant human bone disease models. PMID:26421212

  7. A novel cholinergic epithelial cell with chemosensory traits in the murine conjunctiva.

    PubMed

    Wiederhold, Stephanie; Papadakis, Tamara; Chubanov, Vladimir; Gudermann, Thomas; Krasteva-Christ, Gabriela; Kummer, Wolfgang

    2015-11-01

    We recently identified a specialized cholinergic cell type in tracheal and urethral epithelium that utilizes molecules of the canonical taste transduction signaling cascade to sense potentially harmful substances in the luminal content. Upon stimulation, this cell initiates protective reflexes. Assuming a sentinel role of such cells at mucosal surfaces exposed to bacteria, we hypothesized their occurrence also in ocular mucosal surfaces. Utilizing a mouse strain expressing eGFP under the promoter of the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT-eGFP), we observed a cholinergic cell in the murine conjunctiva. Singular cholinergic cells reaching the epithelial surface with slender processes were detected in fornical, but neither in bulbar nor palpebral epithelia. These cells were found neither in the lacrimal canaliculi, nor in the lacrimal sac and the nasolacrimal duct. Cholinergic conjunctival epithelial cells were immunoreactive for components of the canonical taste transduction signaling cascade, i.e. α-gustducin, phospholipase Cβ2 and the monovalent cation channel TRPM5. Calcitonin gene-related peptide- and substance P-immunoreactive sensory nerve fibers were observed extending into the conjunctival epithelium approaching slender ChAT-eGFP-positive cells. In addition, we noted both ChAT-eGFP expression and α-gustducin-immunoreactivity, albeit in different cell populations, in occasionally occurring lymphoid follicles of the nictitating membrane. The data show a previously unidentified cholinergic cell in murine conjunctiva with chemosensory traits that presumably utilizes acetylcholine for signaling. In analogy to similar cells described in the respiratory and urethral epithelium, it might serve to detect bacterial products and to initiate protective reflexes. PMID:26119492

  8. Circadian Mechanisms in Murine and Human Bone Marrow Mesenchymal Stem Cells Following Dexamethasone Exposure

    PubMed Central

    Wu, Xiying; Yu, Gang; Parks, Helen; Hebert, Teddi; Goh, Brian C.; Dietrich, Marilyn A.; Pelled, Gadi; Izadpanah, Reza; Gazit, Dan; Bunnell, Bruce A.; Gimble, Jeffrey M.

    2008-01-01

    A core group of transcriptional regulatory factors regulate circadian rhythms in mammalian cells. While the suprachiasmatic nucleus in the brain serves as the central core circadian oscillator, circadian clocks also exist within peripheral tissues and cells. A growing body of evidence has demonstrated that >20% of expressed mRNAs in bone and adipose tissues oscillate in a circadian manner. The current manuscript reports evidence of the core circadian transcriptional apparatus within primary cultures of murine and human bone marrow-derived mesenchymal stem cells (BMSCs). Exposure of confluent, quiescent BMSCs to dexamethasone synchronized the oscillating expression of the mRNAs encoding the albumin D binding protein (dbp), brain-muscle arnt-like 1 (bmal1), period 3 (per3), rev-erb α, and rev-erb β. The genes displayed a mean oscillatory period of 22.2 to 24.3 hours. The acrophase or peak expression of mRNAs encoding “positive” (bmal1) and “negative” (per3) transcriptional regulatory factors were out of phase with each other by ∼8-12 hours, consistent with in vivo observations. In vivo, glycogen synthase kinase 3β (GSK3β) mediated phosphorylation regulates the turnover of per3 and core circadian transcriptional apparatus. In vitro addition of lithium chloride, a GSK3β inhibitor, significantly shifted the acrophase of all genes by 4.2-4.7 hours oscillation in BMSCs; however, only the male murine BMSCs displayed a significant increase in the length of the period of oscillation. We conclude that human and murine BMSCs represent a valid in vitro model for the analysis of circadian mechanisms in bone metabolism and stem cell biology. PMID:18302991

  9. Total body irradiation selectively induces murine hematopoietic stem cell senescence.

    PubMed

    Wang, Yong; Schulte, Bradley A; LaRue, Amanda C; Ogawa, Makio; Zhou, Daohong

    2006-01-01

    Exposure to ionizing radiation (IR) and certain chemotherapeutic agents not only causes acute bone marrow (BM) suppression but also leads to long-term residual hematopoietic injury. This latter effect has been attributed to damage to hematopoietic stem cell (HSC) self-renewal. Using a mouse model, we investigated whether IR induces senescence in HSCs, as induction of HSC senescence can lead to the defect in HSC self-renewal. It was found that exposure of C57BL/6 mice to a sublethal dose (6.5 Gy) of total body irradiation (TBI) resulted in a sustained quantitative and qualitative reduction of LKS+ HSCs. In addition, LKS+ HSCs from irradiated mice exhibited an increased expression of the 2 commonly used biomarkers of cellular senescence, p16(Ink4a) and SA-beta-gal. In contrast, no such changes were observed in irradiated LKS- hematopoietic progenitor cells. These results provide the first direct evidence demonstrating that IR exposure can selectively induce HSC senescence. Of interest, the induction of HSC senescence was associated with a prolonged elevation of p21(Cip1/Waf1), p19(Arf), and p16(Ink4a) mRNA expression, while the expression of p27(Kip1) and p18(Ink4c) mRNA was not increased following TBI. This suggests that p21(Cip1/Waf1), p19(Arf), and p16(Ink4a) may play an important role in IR-induced senescence in HSCs.

  10. DAZL regulates Tet1 translation in murine embryonic stem cells

    PubMed Central

    Welling, Maaike; Chen, Hsu-Hsin; Muñoz, Javier; Musheev, Michael U; Kester, Lennart; Junker, Jan Philipp; Mischerikow, Nikolai; Arbab, Mandana; Kuijk, Ewart; Silberstein, Lev; Kharchenko, Peter V; Geens, Mieke; Niehrs, Christof; van de Velde, Hilde; van Oudenaarden, Alexander; Heck, Albert JR; Geijsen, Niels

    2015-01-01

    Embryonic stem cell (ESC) cultures display a heterogeneous gene expression profile, ranging from a pristine naïve pluripotent state to a primed epiblast state. Addition of inhibitors of GSK3β and MEK (so-called 2i conditions) pushes ESC cultures toward a more homogeneous naïve pluripotent state, but the molecular underpinnings of this naïve transition are not completely understood. Here, we demonstrate that DAZL, an RNA-binding protein known to play a key role in germ-cell development, marks a subpopulation of ESCs that is actively transitioning toward naïve pluripotency. Moreover, DAZL plays an essential role in the active reprogramming of cytosine methylation. We demonstrate that DAZL associates with mRNA of Tet1, a catalyst of 5-hydroxylation of methyl-cytosine, and enhances Tet1 mRNA translation. Overexpression of DAZL in heterogeneous ESC cultures results in elevated TET1 protein levels as well as increased global hydroxymethylation. Conversely, null mutation of Dazl severely stunts 2i-mediated TET1 induction and hydroxymethylation. Our results provide insight into the regulation of the acquisition of naïve pluripotency and demonstrate that DAZL enhances TET1-mediated cytosine hydroxymethylation in ESCs that are actively reprogramming to a pluripotent ground state. PMID:26077710

  11. Slow and steady cell shrinkage reduces osmotic stress in bovine and murine oocyte and zygote vitrification

    PubMed Central

    Lai, D.; Ding, J.; Smith, G.W.; Smith, G.D.; Takayama, S.

    2015-01-01

    STUDY QUESTION Does the use of a new cryoprotectant agent (CPA) exchange protocol designed to minimize osmotic stress improve oocyte or zygote vitrification by reducing sublethal cryodamage? SUMMARY ANSWER The use of a new CPA exchange protocol made possible by automated microfluidics improved oocyte and zygote vitrification with superior morphology as indicated by a smoother cell surface, higher sphericity, higher cytoplasmic lipid retention, less cytoplasmic leakage and higher developmental competence compared with conventional methods. WHAT IS KNOWN ALREADY The use of more ‘steps’ of CPA exposure during the vitrification protocol increases cryosurvival and development in the bovine model. However, such an attempt to eliminate osmotic stress is limited by the practicality of performing numerous precise pipetting steps in a short amount of time. STUDY DESIGN, SIZE, DURATION Murine meiotically competent germinal vesicle intact oocytes and zygotes were harvested from the antral follicles in ovaries and ampulla, respectively. Bovine ovaries were obtained from a local abattoir at random stages of the estrous cycle. A total of 110 murine oocytes, 802 murine zygotes and 52 bovine oocytes were used in this study. PARTICIPANTS/MATERIALS, SETTING, METHODS Microfluidic devices were fabricated using conventional photo- and soft-lithography. CPAs used were 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for equilibration solution and 15% EG, 15% DMSO and 0.5 M sucrose for vitrification solution. End-point analyses include mathematical modeling using Kedem–Katchalsky equations, morphometrics assessed by conventional and confocal microscopy, cytoplasmic lipid quantification by nile red staining, cytoplasmic leakage quantification by fluorescent dextran intercalation and developmental competence analysis by 96 h embryo culture and blastomere quantification. MAIN RESULTS AND THE ROLE OF CHANCE The automated microfluidics protocol decreased the shrinkage rate of

  12. An In Vitro Murine Model of Vascular Smooth Muscle Cell Mineralization.

    PubMed

    Kelynack, Kristen J; Holt, Stephen G

    2016-01-01

    Vascular calcification (VC) is seen ubiquitously in aging blood vessels and prematurely in disease states like renal failure. It is thought to be driven by a number of systemic and local factors that lead to extra-osseous deposition of mineral in the vascular wall and valves as a common endpoint. The response of resident vascular smooth muscle cell to these dystrophic signals appears to be important in this process. Whilst in vivo models allow the observation of global changes in a pro-calcific environment, identifying the specific cells and mechanisms involved has been largely garnered from in vitro experiments, which provide added benefits in terms of reproducibility, cost, and convenience. Here we describe a 7-21 day cell culture model of calcification developed using immortalized murine vascular smooth muscle cells (MOVAS-1). This model provides a method by which vascular smooth muscle cell involvement and manipulation within a mineralizing domain can be studied.

  13. Butyrate suppresses murine mast cell proliferation and cytokine production through inhibiting histone deacetylase.

    PubMed

    Zhang, Hanying; Du, Min; Yang, Qiyuan; Zhu, Mei-Jun

    2016-01-01

    Beyond their nutritional impact to colonic epithelial cells, the intestinal microbiota metabolite butyrate has pleotropic effects to host cells and is known for its beneficial effects on intestinal homeostasis and metabolism. However, it remains unclear how it modulates mast cell function. Here, we demonstrate that butyrate profoundly inhibited proliferation of mouse mastocytoma P815 cells through inducing cell cycle arrest and apoptosis, as well as decreasing c-Kit activation. In addition, butyrate increased early- and late-stage apoptotic P815 cells. In murine bone marrow-derived mast cells (BMMC), butyrate-suppressed FcεRI-dependent tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) release without affecting β-Hexosaminidase, but that was associated with decreased mitogen-activated protein kinase extracellular signal-regulated kinase 1/2, p38 and c-Jun N-terminal kinases activation. Butyrate treatment substantially enhanced histone 3 acetylation in both P815 and BMMC and decreased FcεRI-dependent mRNA expression of tnf-α and il-6 in BMMC, mimicking the effect of Trichostatin A, a known histone deacetylase inhibitor. Chromatin immunoprecipitation revealed that butyrate enhanced acetylation of the tnf-α and il-6 promoter regions but blocked RNA polymerase II binding to the promoters of tnf-α and il-6 genes, indicating suppressed transcription initiation. These phenotypes mimicked those of Trichostatin A treatment. In conclusion, butyrate inhibits cell proliferation and increases cell apoptosis in mastocytoma P815 cells and suppresses FcεRI-dependent cytokine production in murine primary BMMC, which are likely mediated by HDAC inhibition.

  14. MUTYH promotes oxidative microglial activation and inherited retinal degeneration

    PubMed Central

    Nakatake, Shunji; Ikeda, Yasuhiro; Morioka, Noriko; Tachibana, Takashi; Fujiwara, Kohta; Yoshida, Noriko; Notomi, Shoji; Hisatomi, Toshio; Yoshida, Shigeo; Ishibashi, Tatsuro; Sonoda, Koh-Hei

    2016-01-01

    Oxidative stress is implicated in various neurodegenerative disorders, including retinitis pigmentosa (RP), an inherited disease that causes blindness. The biological and cellular mechanisms by which oxidative stress mediates neuronal cell death are largely unknown. In a mouse model of RP (rd10 mice), we show that oxidative DNA damage activates microglia through MutY homolog–mediated (MUYTH-mediated) base excision repair (BER), thereby exacerbating retinal inflammation and degeneration. In the early stage of retinal degeneration, oxidative DNA damage accumulated in the microglia and caused single-strand breaks (SSBs) and poly(ADP-ribose) polymerase activation. In contrast, Mutyh deficiency in rd10 mice prevented SSB formation in microglia, which in turn suppressed microglial activation and photoreceptor cell death. Moreover, Mutyh-deficient primary microglial cells attenuated the polarization to the inflammatory and cytotoxic phenotype under oxidative stress. Thus, MUTYH-mediated BER in oxidative microglial activation may be a novel target to dampen the disease progression in RP and other neurodegenerative disorders that are associated with oxidative stress.

  15. Microglial phagocytosis of living photoreceptors contributes to inherited retinal degeneration

    PubMed Central

    Zhao, Lian; Zabel, Matthew K; Wang, Xu; Ma, Wenxin; Shah, Parth; Fariss, Robert N; Qian, Haohua; Parkhurst, Christopher N; Gan, Wen-Biao; Wong, Wai T

    2015-01-01

    Retinitis pigmentosa, caused predominantly by mutations in photoreceptor genes, currently lacks comprehensive treatment. We discover that retinal microglia contribute non-cell autonomously to rod photoreceptor degeneration by primary phagocytosis of living rods. Using rd10 mice, we found that the initiation of rod degeneration is accompanied by early infiltration of microglia, upregulation of phagocytic molecules in microglia, and presentation of “eat-me” signals on mutated rods. On live-cell imaging, infiltrating microglia interact dynamically with photoreceptors via motile processes and engage in rapid phagocytic engulfment of non-apoptotic rods. Microglial contribution to rod demise is evidenced by morphological and functional amelioration of photoreceptor degeneration following genetic ablation of retinal microglia. Molecular inhibition of microglial phagocytosis using the vitronectin receptor antagonist cRGD also improved morphological and functional parameters of degeneration. Our findings highlight primary microglial phagocytosis as a contributing mechanism underlying cell death in retinitis pigmentosa and implicate microglia as a potential cellular target for therapy. PMID:26139610

  16. MUTYH promotes oxidative microglial activation and inherited retinal degeneration

    PubMed Central

    Nakatake, Shunji; Ikeda, Yasuhiro; Morioka, Noriko; Tachibana, Takashi; Fujiwara, Kohta; Yoshida, Noriko; Notomi, Shoji; Hisatomi, Toshio; Yoshida, Shigeo; Ishibashi, Tatsuro; Sonoda, Koh-Hei

    2016-01-01

    Oxidative stress is implicated in various neurodegenerative disorders, including retinitis pigmentosa (RP), an inherited disease that causes blindness. The biological and cellular mechanisms by which oxidative stress mediates neuronal cell death are largely unknown. In a mouse model of RP (rd10 mice), we show that oxidative DNA damage activates microglia through MutY homolog–mediated (MUYTH-mediated) base excision repair (BER), thereby exacerbating retinal inflammation and degeneration. In the early stage of retinal degeneration, oxidative DNA damage accumulated in the microglia and caused single-strand breaks (SSBs) and poly(ADP-ribose) polymerase activation. In contrast, Mutyh deficiency in rd10 mice prevented SSB formation in microglia, which in turn suppressed microglial activation and photoreceptor cell death. Moreover, Mutyh-deficient primary microglial cells attenuated the polarization to the inflammatory and cytotoxic phenotype under oxidative stress. Thus, MUTYH-mediated BER in oxidative microglial activation may be a novel target to dampen the disease progression in RP and other neurodegenerative disorders that are associated with oxidative stress. PMID:27699246

  17. Diminished organelle motion in murine Kupffer cells during the erythrocytic stage of malaria

    PubMed Central

    Bellows, Charles F.; Molina, Ramon M.; Brain, Joseph D.

    2011-01-01

    Parasitized erythrocytes are ingested by murine hepatic macrophages during malaria infection. We non-invasively monitored how this altered the motion of intracellular phagosomes in Kupffer cells using magnetometry. Submicrometric γFe2O3 particles were injected prior to malaria infection. They were cleared from the blood, primarily by Kupffer cells, and retained within their phagosomes. The mice were periodically magnetized. After removing this external magnet, the aligned iron particles created a remnant magnetic field (RMF) which then decayed (relaxation), reflecting the motion of particle-containing phagosomes. After baseline measurements of relaxation, the mice were injected intravenously with Plasmodium chabaudi-parasitized or normal murine red blood cells (RBCs). During the next 15 days, relaxation measurements, parasitaemia and haematocrit values were monitored. At 6 days post injection with 3 × 107 parasitized RBCs, relaxation rates had decreased. At this time, all mice had parasitaemias greater than 58 per cent and haematocrits less than 20 per cent. At day 7, while the parasitaemias were declining, the rate of relaxation continued to decrease. Throughout the experiment, relaxation remained constant in animals injected with normal RBCs. Electron microscopy revealed Kupffer cells filled with damaged and parasitized erythrocytes, and haemoglobin degradation pigment. We conclude that ingestion and metabolism of parasitized erythrocytes by liver macrophages during malaria infection decreases their organelle motion with likely consequences of compromised host defences. PMID:21068031

  18. Murine coronavirus nonstructural protein ns2 is not essential for virus replication in transformed cells.

    PubMed Central

    Schwarz, B; Routledge, E; Siddell, S G

    1990-01-01

    Two isolates of the murine hepatitis virus (MHV) strain JHM, which differed in their ability to express the nonstructural gene product ns2, were characterized. The MHV Wb3 isolate encodes a 30,000-molecular-weight ns2 protein that can be readily detected in infected cells by using a specific monoclonal antibody, MAb 2A. The MHV Wb1 isolate is a deletion mutant that lacks a functional ns2 gene and the transcriptional signals required for the synthesis of an ns2 mRNA. However, there are no obviously significant differences in the growth of the MHV Wb1 and MHV Wb3 isolates in continuous cell lines or in the synthesis of viral mRNAs or proteins in infected cells. These results demonstrate that the ns2 gene product is not essential for MHV replication in transformed murine cells and suggests that the function of the ns2 gene may only be manifest in vivo. Images PMID:2168966

  19. The interaction of Naegleria fowleri amoebae with murine macrophage cell lines.

    PubMed

    Fischer-Stenger, K; Cabral, G A; Marciano-Cabral, F

    1990-01-01

    The present study was undertaken to determine whether murine macrophage cell lines exhibited in vitro amoebicidal activity comparable to that elicited by activated murine peritoneal macrophages. Peritoneal macrophages activated in vivo by bacillus Calmette-Guérin or Propionibacterium acnes demonstrated significant cytolysis of Naegleria fowleri amoebae. The macrophage cell line RAW264.7 also effected cytolysis of amoebae, but to a lesser extent than that elicited by activated peritoneal macrophages. However, the macrophage cell lines, J774A.1 and P388D1, did not exhibit amoebicidal activity. Macrophage conditioned medium prepared from RAW264.7 macrophages mediated cytolysis of L929 tumor cells but had no effect on N. fowleri amoebae. In addition, neither recombinant tumor necrosis factor nor recombinant interleukin-1 exhibited amoebicidal activity. Scanning electron microscopy of co-cultures revealed that N. fowleri bound to activated peritoneal macrophages and RAW264.7 macrophages. These results suggest that RAW264.7 macrophages treated in vitro with lipopolysaccharide are similar to macrophages activated in vivo in that they effect contact-dependent cytolysis of Naegleria fowleri amoebae. The RAW264.7 macrophages are unlike primary macrophage cultures in that they either do not release soluble amoebicidal factors into the conditioned medium or they release insufficient quantities.

  20. Toll-like receptor ligand activation of murine bone marrow-derived dendritic cells

    PubMed Central

    Dearman, Rebecca J; Cumberbatch, Marie; Maxwell, Gavin; Basketter, David A; Kimber, Ian

    2009-01-01

    Dendritic cells (DCs) are required for the initiation of primary immune responses. The pattern of Toll-like receptor (TLR) expression on various subsets of these cells has been shown to differ, suggestive of distinct roles in influencing immune responses. We have examined here the responses of immature DCs derived from murine bone marrow (BMDCs) to a range of TLR ligands. BMDCs cultured for 6 days in the presence of granulocyte–macrophage colony-stimulating factor were stimulated for 24 hr with ligands to TLR1-2 [Pam3Cys-Ser-(Lys)4 (PAM)], TLR2-6 (macrophage-activating lipopeptide-2 (MALP-2); zymosan or peptidoglycan (PG)], TLR3 (polyinosinic-polycytidylic acid), TLR4 [lipopolysaccharide R515 (LPS)], TLR5 (flagellin), TLR7 (polyuridylic acid) and TLR9 [CpG ODN2395 (CpG)]. DC activation was monitored using membrane marker expression and analysis of culture supernatants for cytokine/chemokine release. Ligands to TLR3 and TLR7 failed to activate BMDCs. All other TLR ligands caused elevated expression of membrane markers. PAM, MALP-2 and LPS induced high-level expression of proinflammatory cytokines and chemokines. Treatment with CpG was associated with a preferential type 1 cytokine and chemokine profile. Zymosan and PG were proinflammatory but also skewed towards a type 2 pattern of cytokines and chemokines. In contrast, flagellin did not cause marked secretion by BMDCs of cytokines or chemokines. These data for BMDCs are largely consistent with the reported TLR repertoire of freshly isolated murine Langerhans cells. In addition, murine BMDCs show selective responses to TLR ligands with respect to general activation, with differentiated cytokine patterns suggestive of potential priming for divergent immune responses. PMID:18778283

  1. Cinnamon extract reduces symptoms, inflammatory mediators and mast cell markers in murine IL-10(-/-) colitis.

    PubMed

    Hagenlocher, Yvonne; Hösel, Angela; Bischoff, Stephan C; Lorentz, Axel

    2016-04-01

    Inflammatory bowel disease (IBD) shows an increasing prevalence and harm in western countries. Conventional therapies are associated with bad compliance and adverse side effects. Natural substances like cinnamon extract (CE) could be an additional therapy. We found recently that CE acts anti-inflammatory on mast cells - discussed of being relevant in IBD. Here, we analysed the effects of CE on murine IL-10(-/-) colitis as model for IBD. Mice were treated 12 weeks with or without CE in drinking water. Clinical scores and disease activity index were assessed. Colonic tissue samples were analysed for infiltration, tissue damage, bowel wall thickness, expression of pro-inflammatory mediators, mast cell proteases, tight junction proteins, and NF-κB signaling. Following treatment with CE, symptoms of murine colitis as well as increased infiltration of immune cells, tissue damage and bowel wall thickness in colon tissue of IL-10(-/-) mice were diminished significantly. MIP-2, TNF, IFNγ, CCL2, CCL3, CCL4 and IL-1β as well as MC-CPA, MCP-1 and MCP-4 were strongly upregulated in IL-10(-/-) mice compared to WT, but noteworthy not in CE group. Expression of tight junction proteins was not influenced by CE. Phosphorylation of IκB was slightly down-regulated in CE treated IL-10(-/-) mice compared to IL-10(-/-) controls. In summary, CE decreases inflammatory symptoms and expression of inflammatory markers in murine IL-10(-/-) colitis. CE has no influence on tight junction proteins, but seems acting via reducing pro-inflammatory mediators and recruitment of neutrophil granulocytes probably by inhibiting NF-κB signaling. PMID:27012624

  2. Effects of Wnt-10b on proliferation and differentiation of murine melanoma cells

    SciTech Connect

    Misu, Masayasu; Ouji, Yukiteru; Kawai, Norikazu; Nishimura, Fumihiko; Nakamura-Uchiyama, Fukumi; Yoshikawa, Masahide

    2015-08-07

    In spite of the strong expression of Wnt-10b in melanomas, its role in melanoma cells has not been elucidated. In the present study, the biological effects of Wnt-10b on murine B16F10 (B16) melanoma cells were investigated using conditioned medium from Wnt-10b-producing COS cells (Wnt-CM). After 2 days of culture in the presence of Wnt-CM, proliferation of B16 melanoma cells was inhibited, whereas tyrosinase activity was increased. An in vitro wound healing assay demonstrated that migration of melanoma cells to the wound area was inhibited with the addition of Wnt-CM. Furthermore, evaluation of cellular senescence revealed prominent induction of SA-β-gal-positive senescent cells in cultures with Wnt-CM. Finally, the growth of B16 melanoma cell aggregates in collagen 3D-gel cultures was markedly suppressed in the presence of Wnt-CM. These results suggest that Wnt-10b represses tumor cell properties, such as proliferation and migration of B16 melanoma cells, driving them toward a more differentiated state along a melanocyte lineage. - Highlights: • Wnt-10b inhibited proliferation and migration of melanoma cells. • Wnt-10b induced tyrosinase activity and senescence of melanoma cells. • Wnt-10b suppressed growth of cell aggregates in collagen 3D-gel cultures. • Wnt-10b represses tumor cell properties, driving them toward a more differentiated state along a melanocyte lineage.

  3. Murine cytomegalovirus stimulates natural killer cell function but kills genetically resistant mice treated with radioactive strontium

    SciTech Connect

    Masuda, A.; Bennett, M.

    1981-12-01

    Treatment of C3H/St mice with 100 microCi of 89Sr weakened their genetic resistance to murine cytomegalovirus (MCMV) infection. The criteria utilized to detect increased susceptibility were: (i) survival of mice; (ii) numbers of MCMV-infected cells in the spleens and liver; and (iii) serum glutamic pyruvic transaminase levels. The natural killer (NK) cell activity of spleen cells from mice treated with 89Sr is very low. However, the NK activities of spleen cells of both normal and 89Sr-treated mice were greatly augmented 3 days after infection with MCMV. These NK cells lysed a variety of tumor cells and shared several features with conventional NK cells, but were not lysed by anti-Nk-1.2 serum (specific for NK cells) plus complement. Splenic adherent cells did not lyse tumor cells themselves but were necessary for the stimulation of NK cells by MCMV. The paradox of high NK cell function and poor survival in 89Sr-treated mice infected with MCMV was a surprise. We conclude that these augmented NK cells, of themselves, cannot account for the genetic resistance of C3H/St mice to infection with MCMV.

  4. Interaction of Enteric Bacterial Pathogens with Murine Embryonic Stem Cells ▿ †

    PubMed Central

    Yu, Jun; Rossi, Raffaella; Hale, Christine; Goulding, David; Dougan, Gordon

    2009-01-01

    Embryonic stem (ES) cells are susceptible to genetic manipulation and retain the potential to differentiate into diverse cell types, which are factors that make them potentially attractive cells for studying host-pathogen interactions. Murine ES cells were found to be susceptible to invasion by Salmonella enterica serovar Typhimurium and Shigella flexneri and to the formation of attaching and effacing lesions by enteropathogenic Escherichia coli. S. enterica serovar Typhimurium and S. flexneri cell entry was dependent on the Salmonella pathogenicity island 1 and Shigella mxi/spa type III secretion systems, respectively. Microscopy studies indicated that both S. enterica serovar Typhimurium and S. flexneri were located in intracellular niches in ES cells that were similar to the niches occupied in differentiated cells. ES cells were eventually killed following bacterial invasion, but no evidence of activation of classical caspase-associated apoptotic or innate immune pathways was found. To demonstrate the potential of mutant ES cells, we employed an ES cell line defective in cholesterol synthesis and found that the mutant cells were less susceptible to infection by Salmonella and Shigella than the parental ES cells. Thus, we highlighted the practical use of genetically modified ES cells for studying microbe-host interactions. PMID:19029302

  5. CD166 regulates human and murine hematopoietic stem cells and the hematopoietic niche

    PubMed Central

    Chitteti, Brahmananda Reddy; Kobayashi, Michihiro; Cheng, Yinghua; Zhang, Huajia; Poteat, Bradley A.; Broxmeyer, Hal E.; Pelus, Louis M.; Hanenberg, Helmut; Zollman, Amy; Kamocka, Malgorzata M.; Carlesso, Nadia; Cardoso, Angelo A.; Kacena, Melissa A.

    2014-01-01

    We previously showed that immature CD166+ osteoblasts (OB) promote hematopoietic stem cell (HSC) function. Here, we demonstrate that CD166 is a functional HSC marker that identifies both murine and human long-term repopulating cells. Both murine LSKCD48−CD166+CD150+ and LSKCD48−CD166+CD150+CD9+ cells, as well as human Lin−CD34+CD38−CD49f+CD166+ cells sustained significantly higher levels of chimerism in primary and secondary recipients than CD166− cells. CD166−/− knockout (KO) LSK cells engrafted poorly in wild-type (WT) recipients and KO bone marrow cells failed to radioprotect lethally irradiated WT recipients. CD166−/− hosts supported short-term, but not long-term WT HSC engraftment, confirming that loss of CD166 is detrimental to the competence of the hematopoietic niche. CD166−/− mice were significantly more sensitive to hematopoietic stress. Marrow-homed transplanted WT hematopoietic cells lodged closer to the recipient endosteum than CD166−/− cells, suggesting that HSC-OB homophilic CD166 interactions are critical for HSC engraftment. STAT3 has 3 binding sites on the CD166 promoter and STAT3 inhibition reduced CD166 expression, suggesting that both CD166 and STAT3 may be functionally coupled and involved in HSC competence. These studies illustrate the significance of CD166 in the identification and engraftment of HSC and in HSC-niche interactions, and suggest that CD166 expression can be modulated to enhance HSC function. PMID:24740813

  6. ANTIGENIC PROPERTIES OF MURINE SARCOMA VIRUS-TRANSFORMED BALB/3T3 NONPRODUCER CELLS

    PubMed Central

    Stephenson, John R.; Aaronson, Stuart A.

    1972-01-01

    The isolation of clonal lines of murine sarcoma virus-transformed, non-producer BALB/3T3 cells has provided a model system for determining whether RNA tumor virus-transformed cells possess virus-specific transplantation antigens. MSV nonproducer cells (K-234) were clonally derived from an inbred mouse cell line, BALB/3T3. A parallel virus-producing cell line was obtained by infection of the MSV nonproducer cells with Rauscher leukemia virus. K-234 was much more tumorigenic than K-234(R). Preimmunization of syngeneic mice with either K-234(R) or with UV-inactivated Rauscher leukemia virus induced transplantation resistance to subsequent challenge with K-234(R), but not with K-234. In contrast, mice preimmunized with nonproducer cells were not made resistant to subsequent challenge with the homologous cells. Antisera prepared from mice immunized with K-234(R) were specifically cytotoxic and positive by fluorescent antibody staining for K-234(R) target cells, but not to either BALB/3T3 or K-234. The results show that MSV nonproducer cells lack detectable transplantation antigens and suggest that the transplantation resistance to the producing cells is attributable to maturing virus at the cell surface. PMID:4550769

  7. Simultaneous Isolation of Three Different Stem Cell Populations from Murine Skin.

    PubMed

    Forni, Maria Fernanda; Ramos Maia Lobba, Aline; Pereira Ferreira, Alexandre Hamilton; Sogayar, Mari Cleide

    2015-01-01

    The skin is a rich source of readily accessible stem cells. The level of plasticity afforded by these cells is becoming increasingly important as the potential of stem cells in Cell Therapy and Regenerative Medicine continues to be explored. Several protocols described single type stem cell isolation from skin; however, none of them afforded simultaneous isolation of more than one population. Herein, we describe the simultaneous isolation and characterization of three stem cell populations from the dermis and epidermis of murine skin, namely Epidermal Stem Cells (EpiSCs), Skin-derived Precursors (SKPs) and Mesenchymal Stem Cells (MSCs). The simultaneous isolation was possible through a simple protocol based on culture selection techniques. These cell populations are shown to be capable of generating chondrocytes, adipocytes, osteocytes, terminally differentiated keratinocytes, neurons and glia, rendering this protocol suitable for the isolation of cells for tissue replenishment and cell based therapies. The advantages of this procedure are far-reaching since the skin is not only the largest organ in the body, but also provides an easily accessible source of stem cells for autologous graft.

  8. Simultaneous Isolation of Three Different Stem Cell Populations from Murine Skin

    PubMed Central

    Forni, Maria Fernanda; Ramos Maia Lobba, Aline; Pereira Ferreira, Alexandre Hamilton; Sogayar, Mari Cleide

    2015-01-01

    The skin is a rich source of readily accessible stem cells. The level of plasticity afforded by these cells is becoming increasingly important as the potential of stem cells in Cell Therapy and Regenerative Medicine continues to be explored. Several protocols described single type stem cell isolation from skin; however, none of them afforded simultaneous isolation of more than one population. Herein, we describe the simultaneous isolation and characterization of three stem cell populations from the dermis and epidermis of murine skin, namely Epidermal Stem Cells (EpiSCs), Skin-derived Precursors (SKPs) and Mesenchymal Stem Cells (MSCs). The simultaneous isolation was possible through a simple protocol based on culture selection techniques. These cell populations are shown to be capable of generating chondrocytes, adipocytes, osteocytes, terminally differentiated keratinocytes, neurons and glia, rendering this protocol suitable for the isolation of cells for tissue replenishment and cell based therapies. The advantages of this procedure are far-reaching since the skin is not only the largest organ in the body, but also provides an easily accessible source of stem cells for autologous graft. PMID:26462205

  9. T cell receptor transgenic lymphocytes infiltrating murine tumors are not induced to express foxp3

    PubMed Central

    2011-01-01

    Regulatory T cells (Treg) that express the transcription factor Foxp3 are enriched within a broad range of murine and human solid tumors. The ontogeny of these Foxp3 Tregs - selective accumulation or proliferation of natural thymus-derived Treg (nTreg) or induced Treg (iTreg) converted in the periphery from naïve T cells - is not known. We used several strains of mice in which Foxp3 and EGFP are coordinately expressed to address this issue. We confirmed that Foxp3-positive CD4 T cells are enriched among tumor-infiltrating lymphocytes (TIL) and splenocytes (SPL) in B16 murine melanoma-bearing C57BL/6 Foxp3EGFP mice. OT-II Foxp3EGFP mice are essentially devoid of nTreg, having transgenic CD4 T cells that recognize a class II-restricted epitope derived from ovalbumin; Foxp3 expression could not be detected in TIL or SPL in these mice when implanted with ovalbumin-transfected B16 tumor (B16-OVA). Likewise, TIL isolated from B16 tumors implanted in Pmel-1 Foxp3EGFP mice, whose CD8 T cells recognize a class I-restricted gp100 epitope, were not induced to express Foxp3. All of these T cell populations - wild-type CD4, pmel CD8 and OTII CD4 - could be induced in vitro to express Foxp3 by engagement of their T cell receptor (TCR) and exposure to transforming growth factor β (TGFβ). B16 melanoma produces TGFβ and both pmel CD8 and OTII CD4 express TCR that should be engaged within B16 and B16-OVA respectively. Thus, CD8 and CD4 transgenic T cells in these animal models failed to undergo peripheral induction of Foxp3 in a tumor microenvironment. PMID:22112546

  10. Paradoxical antiproliferative effect by a murine mammary tumor-derived epithelial cell line

    PubMed Central

    Gurzov, Esteban N; Nabha, Sanaa M; Yamamoto, Hamilto; Meng, Hong; Scharovsky, O Graciela; Bonfil, R Daniel

    2007-01-01

    Background Despite significant advancement in breast cancer therapy, there is a great need for a better understanding of the mechanisms involved in breast carcinogenesis and progression, as well as of the role of epigenetic contributions from stromal cells in mammary tumorigenesis. In this study, we isolated and characterized murine mammary tumor-derived epithelial and myofibroblast cell lines, and investigated the in vitro and in vivo effect of cellular soluble factors produced by the epithelial cell line on tumor cells. Methods Morphology, immunophenotype, cytogenetics, invasiveness, and tumorigenicity of epithelial (LM-234ep) and myofibroblast (LM-234mf) cell lines isolated from two murine mammary adenocarcinomas with common ancestor were studied. The in vitro effects of LM-234ep conditioned medium on proliferation, cell cycle distribution, and expression of cell cycle proteins, were investigated in LM-234mf cells, mouse melanoma cells (B16-F10), and human cervical adenocarcinoma cells (HeLa). The in vivo anti-tumor activity of LM-234ep conditioned media was evaluated in subcutaneous tumors formed in nude mice by B16-F10 and HeLa cells. Results LM-234ep cells were found to be cytokeratin positive and hipertriploid, whereas LM-234mf cells were α-smooth muscle actin positive and hypohexaploid. Chromosome aberrations were found in both cases. Only LM-234mf revealed to be invasive in vitro and to secrete active MMP-2, though neither of the cell types were able to produce progressing tumors. LM-234ep-derived factors were able to inhibit the in vitro growth of LM-234mf, B16-F10, and HeLa cells, inducing cell cycle arrest in G0/G1 phase. The administration of LM-234ep conditioned medium inhibited the growth of B16-F10 and HeLa tumors in nude mice. Conclusion Our data suggest the existence of epithelial cell variants with tumor suppressive properties within mammary tumors. To our knowledge, this is the first report showing antiproliferative and antineoplastic

  11. Microglial Priming and Alzheimer's Disease: A Possible Role for (Early) Immune Challenges and Epigenetics?

    PubMed

    Hoeijmakers, Lianne; Heinen, Yvonne; van Dam, Anne-Marie; Lucassen, Paul J; Korosi, Aniko

    2016-01-01

    Neuroinflammation is thought to contribute to Alzheimer's disease (AD) pathogenesis that is, to a large extent, mediated by microglia. Given the tight interaction between the immune system and the brain, peripheral immune challenges can profoundly affect brain function. Indeed, both preclinical and clinical studies have indicated that an aberrant inflammatory response can elicit behavioral impairments and cognitive deficits, especially when the brain is in a vulnerable state, e.g., during early development, as a result of aging, or under disease conditions like AD. However, how exactly peripheral immune challenges affect brain function and whether this is mediated by aberrant microglial functioning remains largely elusive. In this review, we hypothesize that: (1) systemic immune challenges occurring during vulnerable periods of life can increase the propensity to induce later cognitive dysfunction and accelerate AD pathology; and (2) that "priming" of microglial cells is instrumental in mediating this vulnerability. We highlight how microglia can be primed by both neonatal infections as well as by aging, two periods of life during which microglial activity is known to be specifically upregulated. Lasting changes in (the ratios of) specific microglial phenotypes can result in an exaggerated pro-inflammatory cytokine response to subsequent inflammatory challenges. While the resulting changes in brain function are initially transient, a continued and/or excess release of such pro-inflammatory cytokines can activate various downstream cellular cascades known to be relevant for AD. Finally, we discuss microglial priming and the aberrant microglial response as potential target for treatment strategies for AD. PMID:27555812

  12. Activation of serotonin receptors promotes microglial injury-induced motility but attenuates phagocytic activity.

    PubMed

    Krabbe, Grietje; Matyash, Vitali; Pannasch, Ulrike; Mamer, Lauren; Boddeke, Hendrikus W G M; Kettenmann, Helmut

    2012-03-01

    Microglia, the brain immune cell, express several neurotransmitter receptors which modulate microglial functions. In this project we studied the impact of serotonin receptor activation on distinct microglial properties as serotonin deficiency not only has been linked to a number of psychiatric disease like depression and anxiety but may also permeate from the periphery through blood-brain barrier openings seen in neurodegenerative disease. First, we tested the impact of serotonin on the microglial response to an insult caused by a laser lesion in the cortex of acute slices from Cx3Cr1-GFP-/+ mice. In the presence of serotonin the microglial processes moved more rapidly towards the laser lesion which is considered to be a chemotactic response to ATP. Similarly, the chemotactic response of cultured microglia to ATP was also enhanced by serotonin. Quantification of phagocytic activity by determining the uptake of microspheres showed that the amoeboid microglia in slices from early postnatal animals or microglia in culture respond to serotonin application with a decreased phagocytic activity whereas we could not detect any significant change in ramified microglia in situ. The presence of microglial serotonin receptors was confirmed by patch-clamp experiments in culture and amoeboid microglia and by qPCR analysis of RNA isolated from primary cultured and acutely isolated adult microglia. These data suggest that microglia express functional serotonin receptors linked to distinct microglial properties. PMID:22198120

  13. Isolation, characterization, and long-term cultivation of porcine and murine cerebral capillary endothelial cells.

    PubMed

    Tontsch, U; Bauer, H C

    1989-03-01

    We present a simple method for isolation and long-term cultivation of porcine and murine cerebral capillary endothelial cells (cEC). Two major points are made. First, that the "characteristic" morphology of the endothelial cells depends mainly on the presence of endothelial cell growth factors in the culture medium and second, that the identification of the cells as endothelial cells requires a special lectin instead of criteria used for large vessel endothelial cells, such as factor VIII staining or LDL uptake. Pure cerebral capillaries were isolated by means of a series of centrifugation steps; endothelial cells were released by collagenase treatment and cultivated on plastic petri dishes, which proved to be better for cell attachment than collagen or gelatin coating. The microvascular cells were cultivated in either the presence or absence of growth factors. Medium 199 + 10% FCS produced mainly spindle-shaped cells, growing in the "hills and valleys" pattern, which, if not passaged for weeks, showed three dimensional tubular structures. Cells of the "cobblestone" phenotype were promoted in medium 199 + 10% FCS, enriched with endothelial cell growth supplement (ECGS) and heparin (referred to as complete medium). These cells retained their phenotype for months and could be passaged up to 35 times till now. If ECGS and heparin were omitted from these cultures, the cells became elongated and resembled smooth muscle cells. This effect was reversible when the cells were transferred to complete medium. With cEC, cloned by limiting dilution, we noticed this reversal phenomenon as well. We used several markers to characterize the microvascular cells and could show that the lectin of Bandeiraea simplicifolia is a highly reliable marker for endothelial cells and that the monoclonal antibody alpha-sm-1 (anti-smooth muscle cell actin) is excellent for determining smooth muscle cells.

  14. MMP-3 secreted from endothelial cells of blood vessels after spinal cord injury activates microglia, leading to oligodendrocyte cell death.

    PubMed

    Lee, Jee Y; Choi, Hae Y; Yune, Tae Y

    2015-10-01

    The activation of microglia after spinal cord injury (SCI) contributes to secondary damage by producing pro-inflammatory cytokines and mediators, leading to cell death of oligodendrocytes and neurons. Here, we show that matrix metalloprotease-3 (MMP-3) produced and secreted in the endothelial cells of blood vessels after SCI mediates microglial activation. MMP-3 was produced and secreted in bEnd.3 cells, a mouse brain-derived endothelial cell line, by oxygen-glucose deprivation/reoxygenation (OGD/RO). OGD/RO-induced MMP-3 expression and activity was also significantly inhibited by ghrelin, which was dependent on the ghrelin receptor GHS-R1a. Furthermore, the secreted MMP-3 from OGD/RO-induced bEnd.3 cells activated BV-2 cells, a murine microglial cell line. We also found that microglial activation after SCI was attenuated in MMP-3 knockout (KO) mice compared with wild type (WT) mice. Both p38 mitogen-activated protein kinase (MAPK) activation and pro-nerve growth factor (proNGF) production were more inhibited in MMP-3 KO than WT mice at 5d after injury. When WT mice were treated with Mmp-3 siRNA after injury, MMP-3 activity, microglial activation, p38MAPK activation and proNGF expression were significantly inhibited. Ghrelin treatment also significantly inhibited MMP-3 expression and activation after SCI, which was dependent on GHS-R1a. Finally, RhoA activation and oligodendrocyte cell death after injury were attenuated by Mmp-3 siRNA or ghrelin treatment compared with vehicle control. Thus, our study indicates that MMP-3 produced in blood vessel endothelial cells after SCI serves as an endogenous molecule for microglial activation followed by p38MAPK activation and proNGF production, and further indicates that the protective effect of ghrelin on oligodendrocytes cell death may be at least partly mediated by the inhibition of MMP-3-induced microglial activation after SCI.

  15. Identification and enrichment of colony-forming cells from the adult murine pituitary

    SciTech Connect

    Lepore, D.A.; Roeszler, K.; Wagner, J.; Ross, S.A.; Bauer, K.; Thomas, P.Q. , E-Mail: paul.thomas@mcri.edu.au

    2005-08-01

    Stem and progenitor cells have been identified in many adult tissues including bone marrow, the central nervous system, and skin. While there is direct evidence to indicate the activity of a progenitor cell population in the pituitary gland, this putative subpopulation has not yet been identified. Herein we describe the isolation and characterization of a novel clonogenic cell type in the adult murine pituitary, which we have termed Pituitary Colony-Forming Cells (PCFCs). PCFCs constitute 0.2% of pituitary cells, and generate heterogeneous colonies from single cells. PCFCs exhibit variable proliferative potential, and may exceed 11 population doublings in 14 days. Enrichment of PCFCs to 61.5-fold with 100% recovery can be obtained through the active uptake of the fluorescent dipeptide, {beta}-Ala-Lys-N{epsilon}-AMCA. PCFCs are mostly contained within the large, agranular subpopulation of AMCA{sup +} cells, and constitute 28% of this fraction, corresponding to 140.5-fold enrichment. Interestingly, the AMCA{sup +} population contains rare cells that are GH{sup +} or PRL{sup +}. GH{sup +} cells were also identified in PCFC single cell colonies, suggesting that PCFCs have the potential to differentiate into GH{sup +} cells. Together, these data show that the pituitary contains a rare clonogenic population which may correspond to the somatotrope/lactotrope progenitors suggested by previous experiments.

  16. Differentiation of murine embryonic stem and induced pluripotent stem cells to renal lineage in vitro

    SciTech Connect

    Morizane, Ryuji; Monkawa, Toshiaki; Itoh, Hiroshi

    2009-12-25

    Embryonic stem (ES) cells which have the unlimited proliferative capacity and extensive differentiation potency can be an attractive source for kidney regeneration therapies. Recent breakthroughs in the generation of induced pluripotent stem (iPS) cells have provided with another potential source for the artificially-generated kidney. The purpose of this study is to know how to differentiate mouse ES and iPS cells into renal lineage. We used iPS cells from mouse fibroblasts by transfection of four transcription factors, namely Oct4, Sox2, c-Myc and Klf4. Real-time PCR showed that renal lineage markers were expressed in both ES and iPS cells after the induction of differentiation. It also showed that a tubular specific marker, KSP progressively increased to day 18, although the differentiation of iPS cells was slower than ES cells. The results indicated that renal lineage cells can be differentiated from both murine ES and iPS cells. Several inducing factors were tested whether they influenced on cell differentiation. In ES cells, both of GDNF and BMP7 enhanced the differentiation to metanephric mesenchyme, and Activin enhanced the differentiation of ES cells to tubular cells. Activin also enhanced the differentiation of iPS cells to tubular cells, although the enhancement was lower than in ES cells. ES and iPS cells have a potential to differentiate to renal lineage cells, and they will be an attractive resource of kidney regeneration therapy. This differentiation is enhanced by Activin in both ES and iPS cells.

  17. Murine T-cell response to native and recombinant protein antigens of Rickettsia tsutsugamushi.

    PubMed Central

    Hickman, C J; Stover, C K; Joseph, S W; Oaks, E V

    1993-01-01

    A polyclonal T-cell line with TH1 characteristics was used to assess the murine cellular immune response to native and recombinant Rickettsia tsutsugamushi antigens. Proliferation of this T-cell line was observed in response to numerous native antigen fractions, which indicates that the murine T-helper-cell response is directed at multiple scrub typhus antigens with no apparent antigenic immunodominance. Subsequent analysis of recombinant R. tsutsugamushi antigens made it possible to identify a 47-kDa scrub typhus antigen (Sta47) that was stimulatory for the polyclonal T-cell line. Recombinant clones encoding 56-, 58-, and 110-kDa antigens (Sta56, Sta58, and Sta110, respectively) were unable to induce proliferation of this T-cell line. DNA sequence analysis of the cloned rickettsial insert encoding the Sta47 protein revealed the presence of four open reading frames potentially encoding proteins of 47, 30, 18, and 13 kDa. Analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated and eluted fractions of lysates from the recombinant HB101(pRTS47B4.3) demonstrated that the fractions containing the 47-kDa protein as well as those containing proteins less than 18 kDa were stimulatory. Selected synthetic amphipathic peptides derived from the Sta47 antigen sequence identified a 20-amino-acid peptide that gave a 10-fold increase in T-cell proliferation over a control malarial peptide of similar length. Recognition of the 47-kDa antigen by a T-cell line with TH1 characteristics implicates this protein as one of potential importance in protection studies and future vaccine development. Images PMID:8478055

  18. Frataxin Deficiency Promotes Excess Microglial DNA Damage and Inflammation that Is Rescued by PJ34

    PubMed Central

    Shen, Yan; McMackin, Marissa Z.; Shan, Yuxi; Raetz, Alan; David, Sheila; Cortopassi, Gino

    2016-01-01

    An inherited deficiency in the frataxin protein causes neurodegeneration of the dorsal root ganglia and Friedreich's ataxia (FA). Frataxin deficiency leads to oxidative stress and inflammatory changes in cell and animal models; however, the cause of the inflammatory changes, and especially what causes brain microglial activation is unclear. Here we investigated: 1) the mechanism by which frataxin deficiency activates microglia, 2) whether a brain-localized inflammatory stimulus provokes a greater microglial response in FA animal models, and 3) whether an anti-inflammatory treatment improves their condition. Intracerebroventricular administration of LPS induced higher amounts of microglial activation in the FA mouse model vs controls. We also observed an increase in oxidative damage in the form of 8-oxoguanine (8-oxo-G) and the DNA repair proteins MUTYH and PARP-1 in cerebellar microglia of FA mutant mice. We hypothesized that frataxin deficiency increases DNA damage and DNA repair genes specifically in microglia, activating them. siRNA-mediated frataxin knockdown in microglial BV2 cells clearly elevated DNA damage and the expression of DNA repair genes MUTYH and PARP-1. Frataxin knockdown also induced a higher level of PARP-1 in MEF cells, and this was suppressed in MUTYH-/- knockout cells. Administration of the PARP-1 inhibitor PJ34 attenuated the microglial activation induced by intracerebroventricular injection of LPS. The combined administration of LPS and angiotensin II provoke an even stronger activation of microglia and neurobehavioral impairment. PJ34 treatment attenuated the neurobehavioral impairments in FA mice. These results suggest that the DNA repair proteins MUTYH and PARP-1 may form a pathway regulating microglial activation initiated by DNA damage, and inhibition of microglial PARP-1 induction could be an important therapeutic target in Friedreich's ataxia. PMID:26954031

  19. Determination of the differentiation capacities of murines' primary mononucleated cells and MC3T3-E1 cells

    PubMed Central

    2010-01-01

    Background The main morphological features of primitive cells, such as stem and progenitor cells, are that these cells consists only one nucleus. The main purpose of this study was to determine the differentiation capacities of stem and progenitor cells. This study was performed using mononucleated cells originated from murine peripheral blood and MC3T3-E1 cells. Three approaches were used to determine their differentiation capacities: 1) Biochemical assays, 2) Gene expression analysis, and 3) Morphological observations. Results We found that both cells were able to differentiate into mature osteoblasts, as assayed by ALP activity. RT-PCR analysis showed the activation of the Opn gene after osteoblast differentiation. Morphological observations of both cells revealed the formation of black or dark-brown nodules after von Kossa staining. Nevertheless, only mononucleated cells showed the significant increase in TRAP activity characteristic of mature osteoclasts. The osteoclast-specific CatK gene was only upregulated in mononucleated cells. Morphological observations indicated the existence of multinucleated osteoclasts. Sca-1 was activated only in undifferentiated mononucleated cells, indicating that the cells were hematopoietic stem cells. In both cell lines, the housekeeping Gapdh gene was activated before and after differentiation. Conclusion The isolated mononucleated cells were able to differentiate into both osteoblasts and osteoclasts; indicating that they are stem cells. On the other hand, MC3T3-E1 cells can only differentiate into osteoblasts; a characteristic of progenitor cells. PMID:20979664

  20. Effects of ethanol on cAMP production in murine embryonic palate mesenchymal cells

    SciTech Connect

    Weston, W.M.; Greene, R.M. )

    1991-01-01

    Ethanol affected the ability of murine embryonic palate mesenchymal (MEPM) cells to produce cAMP in response to hormone treatment. Acute exposure to ethanol resulted in an increase in hormone-stimulated cAMP levels, while chronic ethanol treatment led to decreased sensitivity to hormone. Forskolin-stimulated cAMP levels were decreased by both acute and chronic ethanol treatment, while the cells' response to cholera toxin was unchanged by ethanol treatment. The lack of sensitivity of the cholera toxin response to ethanol suggests that,in contrast to what has been observed in other systems, ethanol does not affect the production or activity of G{alpha}s in MEPM cells. These results suggest a possible explanation for the molecular basis for the craniofacial abnormalities observed in the fetal alcohol syndrome.

  1. T helper cells in murine germinal centers are antigen-specific emigrants that downregulate Thy-1

    PubMed Central

    1996-01-01

    After immunization, activated splenic T cells proliferate in periarteriolar lymphoid sheaths (PALS) and subsequently migrate to the lymphoid follicle where they enter nascent germinal centers. Analysis of TCR V(D)J gene rearrangements indicates extensive emigration, frequently involving more than a single white pulp region. These migrants constitute a unique set of T helper cells that express antigen- specific alpha beta TCR, CD3, and CD4, but little or no Thy-1, a differentiation antigen present on the great majority of peripheral murine T lymphocytes. The origin of CD4+ Thy-1 follicular T cells appears to be the Thy+ population in the PALS, as both sets commonly share identical V(D)J rearrangements. PMID:9064325

  2. An essential developmental function for murine phosphoglycolate phosphatase in safeguarding cell proliferation

    PubMed Central

    Segerer, Gabriela; Hadamek, Kerstin; Zundler, Matthias; Fekete, Agnes; Seifried, Annegrit; Mueller, Martin J.; Koentgen, Frank; Gessler, Manfred; Jeanclos, Elisabeth; Gohla, Antje

    2016-01-01

    Mammalian phosphoglycolate phosphatase (PGP) is thought to target phosphoglycolate, a 2-deoxyribose fragment derived from the repair of oxidative DNA lesions. However, the physiological role of this activity and the biological function of the DNA damage product phosphoglycolate is unknown. We now show that knockin replacement of murine Pgp with its phosphatase-inactive PgpD34N mutant is embryonically lethal due to intrauterine growth arrest and developmental delay in midgestation. PGP inactivation attenuated triosephosphate isomerase activity, increased triglyceride levels at the expense of the cellular phosphatidylcholine content, and inhibited cell proliferation. These effects were prevented under hypoxic conditions or by blocking phosphoglycolate release from damaged DNA. Thus, PGP is essential to sustain cell proliferation in the presence of oxygen. Collectively, our findings reveal a previously unknown mechanism coupling a DNA damage repair product to the control of intermediary metabolism and cell proliferation. PMID:27731369

  3. Lentiviral gene transfer into human and murine hematopoietic stem cells: size matters.

    PubMed

    Canté-Barrett, Kirsten; Mendes, Rui D; Smits, Willem K; van Helsdingen-van Wijk, Yvette M; Pieters, Rob; Meijerink, Jules P P

    2016-01-01

    Contemporary biomedical research increasingly depends on techniques to induce or to inhibit expression of genes in hematopoietic stem cells (HSCs) or other primary cells to assess their roles on cellular processes including differentiation, apoptosis and migration. Surprisingly little information is available to optimize lentiviral transduction of HSCs. We have therefore carefully optimized transduction of murine and human HSCs by optimizing vector design, serum-free virus production and virus quantitation. We conclude that the viral RNA length, even in relatively small vectors, is an important factor affecting the lentiviral gene transfer on the level of both the virus production and the cellular transduction efficiency. Efficient transfer of large gene sequences into difficult-to-transduce primary cells will benefit from reducing the lentiviral construct size. PMID:27306375

  4. Perforin gene transfer into hematopoietic stem cells improves immune dysregulation in murine models of perforin deficiency.

    PubMed

    Carmo, Marlene; Risma, Kimberly A; Arumugam, Paritha; Tiwari, Swati; Hontz, Adrianne E; Montiel-Equihua, Claudia A; Alonso-Ferrero, Maria E; Blundell, Michael P; Schambach, Axel; Baum, Christopher; Malik, Punam; Thrasher, Adrian J; Jordan, Michael B; Gaspar, H Bobby

    2015-04-01

    Defects in perforin lead to the failure of T and NK cell cytotoxicity, hypercytokinemia, and the immune dysregulatory condition known as familial hemophagocytic lymphohistiocytosis (FHL). The only curative treatment is allogeneic hematopoietic stem cell transplantation which carries substantial risks. We used lentiviral vectors (LV) expressing the human perforin gene, under the transcriptional control of the ubiquitous phosphoglycerate kinase promoter or a lineage-specific perforin promoter, to correct the defect in different murine models. Following LV-mediated gene transfer into progenitor cells from perforin-deficient mice, we observed perforin expression in mature T and NK cells, and there was no evidence of progenitor cell toxicity when transplanted into irradiated recipients. The resulting perforin-reconstituted NK cells showed partial recovery of cytotoxicity, and we observed full recovery of cytotoxicity in polyclonal CD8(+) T cells. Furthermore, reconstituted T cells with defined antigen specificity displayed normal cytotoxic function against peptide-loaded targets. Reconstituted CD8(+) lymphoblasts had reduced interferon-γ secretion following stimulation in vitro, suggesting restoration of normal immune regulation. Finally, upon viral challenge, mice with >30% engraftment of gene-modified cells exhibited reduction of cytokine hypersecretion and cytopenias. This study demonstrates the potential of hematopoietic stem cell gene therapy as a curative treatment for perforin-deficient FHL.

  5. Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells.

    PubMed

    Kubaczka, Caroline; Schorle, Hubert

    2016-01-01

    Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo. PMID:27500445

  6. Perforin gene transfer into hematopoietic stem cells improves immune dysregulation in murine models of perforin deficiency.

    PubMed

    Carmo, Marlene; Risma, Kimberly A; Arumugam, Paritha; Tiwari, Swati; Hontz, Adrianne E; Montiel-Equihua, Claudia A; Alonso-Ferrero, Maria E; Blundell, Michael P; Schambach, Axel; Baum, Christopher; Malik, Punam; Thrasher, Adrian J; Jordan, Michael B; Gaspar, H Bobby

    2015-04-01

    Defects in perforin lead to the failure of T and NK cell cytotoxicity, hypercytokinemia, and the immune dysregulatory condition known as familial hemophagocytic lymphohistiocytosis (FHL). The only curative treatment is allogeneic hematopoietic stem cell transplantation which carries substantial risks. We used lentiviral vectors (LV) expressing the human perforin gene, under the transcriptional control of the ubiquitous phosphoglycerate kinase promoter or a lineage-specific perforin promoter, to correct the defect in different murine models. Following LV-mediated gene transfer into progenitor cells from perforin-deficient mice, we observed perforin expression in mature T and NK cells, and there was no evidence of progenitor cell toxicity when transplanted into irradiated recipients. The resulting perforin-reconstituted NK cells showed partial recovery of cytotoxicity, and we observed full recovery of cytotoxicity in polyclonal CD8(+) T cells. Furthermore, reconstituted T cells with defined antigen specificity displayed normal cytotoxic function against peptide-loaded targets. Reconstituted CD8(+) lymphoblasts had reduced interferon-γ secretion following stimulation in vitro, suggesting restoration of normal immune regulation. Finally, upon viral challenge, mice with >30% engraftment of gene-modified cells exhibited reduction of cytokine hypersecretion and cytopenias. This study demonstrates the potential of hematopoietic stem cell gene therapy as a curative treatment for perforin-deficient FHL. PMID:25523759

  7. Rhesus rotavirus VP4 sequence-specific activation of mononuclear cells is associated with cholangiopathy in murine biliary atresia.

    PubMed

    Walther, Ashley; Mohanty, Sujit K; Donnelly, Bryan; Coots, Abigail; Lages, Celine S; Lobeck, Inna; Dupree, Phylicia; Meller, Jaroslaw; McNeal, Monica; Sestak, Karol; Tiao, Greg

    2015-09-15

    Biliary atresia (BA), a neonatal obstructive cholangiopathy, remains the most common indication for pediatric liver transplantation in the United States. In the murine model of BA, Rhesus rotavirus (RRV) VP4 surface protein determines biliary duct tropism. In this study, we investigated how VP4 governs induction of murine BA. Newborn mice were injected with 16 strains of rotavirus and observed for clinical symptoms of BA and mortality. Cholangiograms were performed to confirm bile duct obstruction. Livers and bile ducts were harvested 7 days postinfection for virus titers and histology. Flow cytometry assessed mononuclear cell activation in harvested cell populations from the liver. Cytotoxic NK cell activity was determined by the ability of NK cells to kill noninfected cholangiocytes. Of the 16 strains investigated, the 6 with the highest homology to the RRV VP4 (>87%) were capable of infecting bile ducts in vivo. Although the strain Ro1845 replicated to a titer similar to RRV in vivo, it caused no symptoms or mortality. A Ro1845 reassortant containing the RRV VP4 induced all BA symptoms, with a mortality rate of 89%. Flow cytometry revealed that NK cell activation was significantly increased in the disease-inducing strains and these NK cells demonstrated a significantly higher percentage of cytotoxicity against noninfected cholangiocytes. Rotavirus strains with >87% homology to RRV's VP4 were capable of infecting murine bile ducts in vivo. Development of murine BA was mediated by RRV VP4-specific activation of mononuclear cells, independent of viral titers.

  8. Rhesus rotavirus VP4 sequence-specific activation of mononuclear cells is associated with cholangiopathy in murine biliary atresia.

    PubMed

    Walther, Ashley; Mohanty, Sujit K; Donnelly, Bryan; Coots, Abigail; Lages, Celine S; Lobeck, Inna; Dupree, Phylicia; Meller, Jaroslaw; McNeal, Monica; Sestak, Karol; Tiao, Greg

    2015-09-15

    Biliary atresia (BA), a neonatal obstructive cholangiopathy, remains the most common indication for pediatric liver transplantation in the United States. In the murine model of BA, Rhesus rotavirus (RRV) VP4 surface protein determines biliary duct tropism. In this study, we investigated how VP4 governs induction of murine BA. Newborn mice were injected with 16 strains of rotavirus and observed for clinical symptoms of BA and mortality. Cholangiograms were performed to confirm bile duct obstruction. Livers and bile ducts were harvested 7 days postinfection for virus titers and histology. Flow cytometry assessed mononuclear cell activation in harvested cell populations from the liver. Cytotoxic NK cell activity was determined by the ability of NK cells to kill noninfected cholangiocytes. Of the 16 strains investigated, the 6 with the highest homology to the RRV VP4 (>87%) were capable of infecting bile ducts in vivo. Although the strain Ro1845 replicated to a titer similar to RRV in vivo, it caused no symptoms or mortality. A Ro1845 reassortant containing the RRV VP4 induced all BA symptoms, with a mortality rate of 89%. Flow cytometry revealed that NK cell activation was significantly increased in the disease-inducing strains and these NK cells demonstrated a significantly higher percentage of cytotoxicity against noninfected cholangiocytes. Rotavirus strains with >87% homology to RRV's VP4 were capable of infecting murine bile ducts in vivo. Development of murine BA was mediated by RRV VP4-specific activation of mononuclear cells, independent of viral titers. PMID:26206856

  9. Loss of interstitial cells of Cajal and development of electrical dysfunction in murine small bowel obstruction

    PubMed Central

    Chang, In-Youb; Glasgow, Nichola J; Takayama, Ichiro; Horiguchi, Kazuhide; Sanders, Kenton M; Ward, Sean M

    2001-01-01

    Partial obstruction of the murine ileum led to changes in the gross morphology and ultrastructure of the tunica muscularis. Populations of interstitial cells of Cajal (ICC) decreased oral, but not aboral, to the site of obstruction. Since ICC generate and propagate electrical slow waves in gastrointestinal muscles, we investigated whether the loss of ICC leads to loss of function in partial bowel obstruction. Changes in ICC networks and electrical activity were monitored in the obstructed murine intestine using immunohistochemistry, electron microscopy and intracellular electrophysiological techniques. Two weeks following the onset of a partial obstruction, the bowel increased in diameter and hypertrophy of the tunica muscularis was observed oral to the obstruction site. ICC networks were disrupted oral to the obstruction, and this disruption was accompanied by the loss of electrical slow waves and responses to enteric nerve stimulation. These defects were not observed aboral to the obstruction. Ultrastructural analysis revealed no evidence of cell death in regions where the lesion in ICC networks was developing. Cells with a morphology intermediate between smooth muscle cells and fibroblasts were found in locations that are typically populated by ICC. These cells may have been the redifferentiated remnants of ICC networks. Removal of the obstruction led to the redevelopment of ICC networks and recovery of slow wave activity within 30 days. Neural responses were partially restored in 30 days. These data describe the plasticity of ICC networks in response to partial obstruction. After obstruction the ICC phenotype was lost, but these cells regenerated when the obstruction was removed. This model may be an important tool for evaluating the cellular/molecular factors responsible for the regulation and maintenance of the ICC phenotype. PMID:11600689

  10. Chemically linked phage idiotype vaccination in the murine B cell lymphoma 1 model

    PubMed Central

    2013-01-01

    Background B cell malignancies are characterized by clonal expansion of B cells expressing tumor-specific idiotypes on their surface. These idiotypes are ideal target antigens for an individualized immunotherapy. However, previous idiotype vaccines mostly lacked efficiency due to a low immunogenicity of the idiotype. The objective of the present study was the determination of the feasibility, safety and immunogenicity of a novel chemically linked phage idiotype vaccine. Methods In the murine B cell lymphoma 1 model, tumor idiotypes were chemically linked to phage particles used as immunological carriers. For comparison, the idiotype was genetically expressed on the major phage coat protein g8 or linked to keyhole limpet hemocynanin. After intradermal immunizations with idiotype vaccines, tolerability and humoral immune responses were assessed. Results Feasibility and tolerability of the chemically linked phage idiotype vaccine was demonstrated. Vaccination with B cell lymphoma 1 idiotype expressing phage resulted in a significant survival benefit in the murine B cell lymphoma 1 protection model (60.2 ± 23.8 days vs. 41.8 ± 1.6 days and 39.8 ± 3.8 days after vaccination with wild type phage or phosphate buffered saline, respectively). Superior immunogenicity of the chemically linked phage idiotype vaccine compared to the genetically engineered phage idiotype and keyhole limpet hemocynanin-coupled idiotype vaccine was demonstrated by significantly higher B cell lymphoma 1 idiotype-specific IgG levels after vaccination with chemically linked phage idiotype. Conclusion We present a novel, simple, time- and cost-efficient phage idiotype vaccination strategy, which represents a safe and feasible therapy and may produce a superior immune response compared to previously employed idiotype vaccination strategies. PMID:24152874

  11. Characterization of the Murine Myeloid Precursor Cell Line MuMac-E8

    PubMed Central

    Fricke, Stephan; Riemschneider, Sina; Kohlschmidt, Janine; Hilger, Nadja; Fueldner, Christiane; Knauer, Jens; Sack, Ulrich; Emmrich, Frank; Lehmann, Jörg

    2014-01-01

    Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies. PMID:25546418

  12. Characterization of the murine myeloid precursor cell line MuMac-E8.

    PubMed

    Fricke, Stephan; Pfefferkorn, Cathleen; Wolf, Doris; Riemschneider, Sina; Kohlschmidt, Janine; Hilger, Nadja; Fueldner, Christiane; Knauer, Jens; Sack, Ulrich; Emmrich, Frank; Lehmann, Jörg

    2014-01-01

    Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies.

  13. [Long-term subculture and biological characterization of the murine bone marrow endothelial cell line].

    PubMed

    Huang, Chang; Zhu, Wen-Biao; Zhu, Hai-Ling; Wang, Bao-He; Wang, Qi-Ru

    2007-12-01

    The murine bone marrow endothelial cell line (mBMEC) has been maintained by means of subculture and cryopreservation for over 10 years since it was established in our laboratory. This study was aimed to newly identify biological characteristics of this cell line for further study. The cultured mBMEC cells were observed by inverted microscopy and transmission electron microscopy (TEM). PECAM-1 (CD31) and von Willebrand factor (vWF) were detected by immunofluorescent staining. The phagocytotic activity of the cells in culture was tested by using fluorescent acetylated low-density lipoprotein (Dil-Ac-LDL). The cell growth kinetics analysis and karyotype analysis were performed. The results showed that the adherent cells were mostly elliptical, rounded and spindle-shaped, and some of them connected to each other to form cord- and network-like arrangements in mBMEC cultures at subconfluence. The adherent cells grew up to confluence as a cobblestone-like monolayer. Several ultrastructural features of the endothelial cells could be observed in TEM sections of the cultured cells. More than 94% of mBMEC cells were positive for either CD31 or vWF. The phagocytotic ingestion of Dil-Ac-LDL occurred in 98.5% of cells. In normal culture conditions, the cells grew with a mean population doubling time of 54.6 hours and the maximal mitotic index was 38 per thousand in the rapid growth period. The colony yields were 4.33% to 7.40% depending on the plating density of cells. Karyotypes of all the cells were aneuploidy with a greater percentage of hyperdiploid. It is concluded that mBMEC cells retain the fundamental properties of endothelial cells, but the growth kinetics and biological behaviors are slightly different from those in the early days after the establishment of this cell line.

  14. Protein Tyrosine Phosphatase PTPRS Is an Inhibitory Receptor on Human and Murine Plasmacytoid Dendritic Cells

    PubMed Central

    Bunin, Anna; Sisirak, Vanja; Ghosh, Hiyaa S.; Grajkowska, Lucja T.; Hou, Z. Esther; Miron, Michelle; Yang, Cliff; Ceribelli, Michele; Uetani, Noriko; Chaperot, Laurence; Plumas, Joel; Hendriks, Wiljan; Tremblay, Michel L.; Haecker, Hans; Staudt, Louis M.; Green, Peter H.; Bhagat, Govind; Reizis, Boris

    2015-01-01

    SUMMARY Plasmacytoid dendritic cells (pDCs) are primary producers of type I interferon (IFN) in response to viruses. The IFN-producing capacity of pDCs is regulated by specific inhibitory receptors, yet none of the known receptors are conserved in evolution. We report that within the human immune system, receptor protein tyrosine phosphatase sigma (PTPRS) is expressed specifically on pDCs. Surface PTPRS was rapidly downregulated after pDC activation, and only PTPRS– pDCs produced IFN-α. Antibody-mediated PTPRS crosslinking inhibited pDC activation, whereas PTPRS knockdown enhanced IFN response in a pDC cell line. Similarly, murine Ptprs and the homologous receptor phosphatase Ptprf were specifically co-expressed in murine pDCs. Haplodeficiency or DC-specific deletion of Ptprs on Ptprf-deficient background were associated with enhanced IFN response of pDCs, leukocyte infiltration in the intestine and mild colitis. Thus, PTPRS represents an evolutionarily conserved pDC-specific inhibitory receptor, and is required to prevent spontaneous IFN production and immune-mediated intestinal inflammation. PMID:26231120

  15. Efficient nucleic acid delivery to murine regulatory T cells by gold nanoparticle conjugates

    PubMed Central

    Gamrad, Lisa; Rehbock, Christoph; Westendorf, Astrid M.; Buer, Jan; Barcikowski, Stephan; Hansen, Wiebke

    2016-01-01

    Immune responses have to be tightly controlled to guarantee maintenance of immunological tolerance and efficient clearance of pathogens and tumorigenic cells without induction of unspecific side effects. CD4+ CD25+ regulatory T cells (Tregs) play an important role in these processes due to their immunosuppressive function. Genetic modification of Tregs would be helpful to understand which molecules and pathways are involved in their function, but currently available methods are limited by time, costs or efficacy. Here, we made use of biofunctionalized gold nanoparticles as non-viral carriers to transport genetic information into murine Tregs. Confocal microscopy and transmission electron microscopy revealed an efficient uptake of the bioconjugates by Tregs. Most importantly, coupling eGFP-siRNA to those particles resulted in a dose and time dependent reduction of up to 50% of eGFP expression in Tregs isolated from Foxp3eGFP reporter mice. Thus, gold particles represent a suitable carrier for efficient import of nucleic acids into murine CD4+ CD25+ Tregs, superior to electroporation. PMID:27381215

  16. Efficient nucleic acid delivery to murine regulatory T cells by gold nanoparticle conjugates.

    PubMed

    Gamrad, Lisa; Rehbock, Christoph; Westendorf, Astrid M; Buer, Jan; Barcikowski, Stephan; Hansen, Wiebke

    2016-01-01

    Immune responses have to be tightly controlled to guarantee maintenance of immunological tolerance and efficient clearance of pathogens and tumorigenic cells without induction of unspecific side effects. CD4(+) CD25(+) regulatory T cells (Tregs) play an important role in these processes due to their immunosuppressive function. Genetic modification of Tregs would be helpful to understand which molecules and pathways are involved in their function, but currently available methods are limited by time, costs or efficacy. Here, we made use of biofunctionalized gold nanoparticles as non-viral carriers to transport genetic information into murine Tregs. Confocal microscopy and transmission electron microscopy revealed an efficient uptake of the bioconjugates by Tregs. Most importantly, coupling eGFP-siRNA to those particles resulted in a dose and time dependent reduction of up to 50% of eGFP expression in Tregs isolated from Foxp3eGFP reporter mice. Thus, gold particles represent a suitable carrier for efficient import of nucleic acids into murine CD4(+) CD25(+) Tregs, superior to electroporation. PMID:27381215

  17. Histamine suppresses regulatory T cells mediated by TGF-β in murine chronic allergic contact dermatitis.

    PubMed

    Tamaka, Kyoko; Seike, Masahiro; Hagiwara, Tamio; Sato, Atsushi; Ohtsu, Hiroshi

    2015-04-01

    Regulatory T cells (Tregs) suppress effector T cells and ameliorate contact hypersensitivity (CH); however, the role of Tregs in chronic allergic contact dermatitis (CACD) has not been assessed. Repeated elicitation of CH has been used to produce CACD models in mice. We previously showed that the presence of histamine facilitates the creation of eczematous lesions in this model using histidine decarboxylase (HDC) (-/-) mice. Therefore, the effects of histamine on Tregs in the CACD model were investigated in this study. CACD was developed by repeated epicutaneous application of 2, 4, 6-trinitro-1-chlorobenzene (TNCB) on HDC (+/+) and HDC (-/-) murine skin to assess the effects of histamine in CACD. Histamine aggravated CACD in the murine model and suppressed the number of Tregs in the skin. Histamine also suppressed the level of TGF-β1 in this model. Recombinant TGF-β1 or anti-TGF-β1 antibody was injected into the dorsal dermis of HDC (+/+) mice daily just before TNCB challenge to determine the effects of histamine-regulated TGF-β on the Treg population in CACD. Recombinant TGF-β1 injection promoted the infiltration of Tregs in the skin and the production of IL-10; however, anti-TGF-β1 antibody injection suppressed the number of Tregs in the skin and the production of IL-10. Histamine suppresses the number of Tregs in CACD, and this effect is mediated by TGF-β.

  18. Propagation and titration of murine cytomegalovirus in a continuous bone marrow-derived stromal cell line (M2-10B4).

    PubMed

    Lutarewych, M A; Quirk, M R; Kringstad, B A; Li, W; Verfaillie, C M; Jordan, M C

    1997-11-01

    Murine cytomegalovirus (MCMV) can only be propagated effectively in mouse embryo fibroblast (MEF) cells. We demonstrate that MCMV replicates significantly better in M2-10B4 cells, a continuous line of murine bone marrow stromal cells. M2-10B4 cells were also comparable to MEF cells for detection of small amounts of MCMV reactivating from latently infected spleen explants. M2-10B4 cells will be very useful for studies of MCMV pathogenesis.

  19. 1.8 Å structure of murine GITR ligand dimer expressed in Drosophila melanogaster S2 cells

    SciTech Connect

    Chattopadhyay, Kausik; Ramagopal, Udupi A.; Nathenson, Stanley G.; Almo, Steven C.

    2009-05-01

    1.8 Å X-ray crystal structure of mouse GITRL expressed in D. melanogaster S2 cells shows an identical ‘strand-exchanged’ dimeric assembly similar to that observed previously for the E. coli-expressed protein. Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure, murine GITRL demonstrated a unique ‘strand-exchanged’ dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8 Å resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli-expressed proteins. The S2 cell-expressed murine GITRL adopts an identical ‘strand-exchanged’ dimeric structure to that observed for the E. coli-expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL.

  20. Spermine uptake is necessary to induce haemoglobin synthesis in murine erythroleukaemia cells.

    PubMed Central

    Clément, S; Delcros, J G; Feuerstein, B G

    1995-01-01

    To determine whether intracellular uptake of spermine is necessary to induce haemoglobin synthesis in murine erythroleukaemia (MEL) DS 19 cells, we used single-step selection for resistance to N1,N12-bis(ethyl)spermine (BESM), a cytotoxic spermine analogue, to isolate clones deficient in polyamine transport. The cells were approximately 500-fold more resistant to BESM than parental cells and were unable to accumulate BESM, putrescine, spermidine or spermine. Addition of spermine to the polyamine-transport-deficient cells failed to induce haemoglobin synthesis. Hexamethylene-1,6-bisacetamide, a well-known differentiating agent, induced haemoglobin synthesis in both parental and resistant cells. Polyamine-transport-deficient cells transfected with DNA purified from the parental cell line were further selected for their ability to grow in the presence of alpha-difluoromethylornithine and putrescine. The transfectants had an active transport system for polyamines, and spermine added to their culture medium accumulated inside the cells and induced haemoglobin production. These findings indicate that intracellular spermine uptake is required to induce haemoglobin production in MEL cells. PMID:8554541

  1. Dynamic Immune Cell Recruitment After Murine Pulmonary Aspergillus fumigatus Infection under Different Immunosuppressive Regimens

    PubMed Central

    Kalleda, Natarajaswamy; Amich, Jorge; Arslan, Berkan; Poreddy, Spoorthi; Mattenheimer, Katharina; Mokhtari, Zeinab; Einsele, Hermann; Brock, Matthias; Heinze, Katrin Gertrud; Beilhack, Andreas

    2016-01-01

    Humans are continuously exposed to airborne spores of the saprophytic fungus Aspergillus fumigatus. However, in healthy individuals pulmonary host defense mechanisms efficiently eliminate the fungus. In contrast, A. fumigatus causes devastating infections in immunocompromised patients. Host immune responses against A. fumigatus lung infections in immunocompromised conditions have remained largely elusive. Given the dynamic changes in immune cell subsets within tissues upon immunosuppressive therapy, we dissected the spatiotemporal pulmonary immune response after A. fumigatus infection to reveal basic immunological events that fail to effectively control invasive fungal disease. In different immunocompromised murine models, myeloid, notably neutrophils, and macrophages, but not lymphoid cells were strongly recruited to the lungs upon infection. Other myeloid cells, particularly dendritic cells and monocytes, were only recruited to lungs of corticosteroid treated mice, which developed a strong pulmonary inflammation after infection. Lymphoid cells, particularly CD4+ or CD8+ T-cells and NK cells were highly reduced upon immunosuppression and not recruited after A. fumigatus infection. Moreover, adoptive CD11b+ myeloid cell transfer rescued cyclophosphamide immunosuppressed mice from lethal A. fumigatus infection but not cortisone and cyclophosphamide immunosuppressed mice. Our findings illustrate that CD11b+ myeloid cells are critical for anti-A. fumigatus defense under cyclophosphamide immunosuppressed conditions. PMID:27468286

  2. Isolation and (111)In-Oxine Labeling of Murine NK Cells for Assessment of Cell Trafficking in Orthotopic Lung Tumor Model.

    PubMed

    Malviya, Gaurav; Nayak, Tapan; Gerdes, Christian; Dierckx, Rudi A J O; Signore, Alberto; de Vries, Erik F J

    2016-04-01

    A noninvasive in vivo imaging method for NK cell trafficking is essential to gain further understanding of the pathogenesis of NK cell mediated immune response to the novel cancer treatment strategies, and to discover the homing sites and physiological distribution of NK cells. Although human NK cells can be labeled for in vivo imaging, little is known about the murine NK cell labeling and its application in animal models. This study describes the isolation and ex vivo radiolabeling of murine NK cells for the evaluation of cell trafficking in an orthotopic model of human lung cancer in mice. Scid-Tg(FCGR3A)Blt transgenic SCID mice were used to isolate NK cells from mouse splenocytes using the CD49b (DX5) MicroBeads positive selection method. The purity and viability of the isolated NK cells were confirmed by FACS analysis. Different labeling buffers and incubation times were evaluated to optimize (111)In-oxine labeling conditions. Functionality of the radiolabeled NK cell was assessed by (51)Cr-release assay. We evaluated physiological distribution of (111)In-oxine labeled murine NK cells in normal SCID mice and biodistribution in irradiated and nonirradiated SCID mice with orthotopic A549 human lung tumor lesions. Imaging findings were confirmed by histology. Results showed that incubation with 0.011 MBq of (111)In-oxine per million murine NK cells in PBS (pH 7.4) for 20 min is the best condition that provides optimum labeling efficiency without affecting cell viability and functionality. Physiological distribution in normal SCID mice demonstrated NK cells homing mainly in the spleen, while (111)In released from NK cells was excreted via kidneys into urine. Biodistribution studies demonstrated a higher lung uptake in orthotopic lung tumor-bearing mice than control mice. In irradiated mice, lung tumor uptake of radiolabeled murine NK cells decreased between 24 h and 72 h postinjection (p.i.), which was accompanied by tumor regression, while in nonirradiated mice

  3. Third-party CD4+ invariant natural killer T cells protect from murine GVHD lethality.

    PubMed

    Schneidawind, Dominik; Baker, Jeanette; Pierini, Antonio; Buechele, Corina; Luong, Richard H; Meyer, Everett H; Negrin, Robert S

    2015-05-28

    Graft-versus-host disease (GVHD) is driven by extensive activation and proliferation of alloreactive donor T cells causing significant morbidity and mortality following allogeneic hematopoietic cell transplantation (HCT). Invariant natural killer T (iNKT) cells are a potent immunoregulatory T-cell subset in both humans and mice. Here, we explored the role of adoptively transferred third-party CD4(+) iNKT cells for protection from lethal GVHD in a murine model of allogeneic HCT across major histocompatibility barriers. We found that low numbers of CD4(+) iNKT cells from third-party mice resulted in a significant survival benefit with retained graft-versus-tumor effects. In vivo expansion of alloreactive T cells was diminished while displaying a T helper cell 2-biased phenotype. Notably, CD4(+) iNKT cells from third-party mice were as protective as CD4(+) iNKT cells from donor mice although third-party CD4(+) iNKT cells were rejected early after allogeneic HCT. Adoptive transfer of third-party CD4(+) iNKT cells resulted in a robust expansion of donor CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) that were required for protection from lethal GVHD. However, in vivo depletion of myeloid-derived suppressor cells abrogated both Treg expansion and protection from lethal GVHD. Despite the fact that iNKT cells are a rare cell population, the almost unlimited third-party availability and feasibility of in vitro expansion provide the basis for clinical translation.

  4. Effects of ionizing radiation on bone cell differentiation in an experimental murine bone cell model

    NASA Astrophysics Data System (ADS)

    Baumstark-Khan, Christa; Lau, Patrick; Hellweg, Christine; Reitz, Guenther

    During long-term space travel astronauts are exposed to a complex mixture of different radiation types under conditions of dramatically reduced weight-bearing activity. It has been validated that astronauts loose a considerable amount of bone mass at a rate up to one to two percent each month in space. Therapeutic doses of ionizing radiation cause bone damage and increase fracture risks after treatment for head-and-neck cancer and in pelvic irradiation. For low radiation doses, the possibility of a disturbed healing potential of bone was described. Radiation induced damage has been discussed to inflict mainly on immature and healing bone. Little is known about radiation effects on bone remodelling and even less on the combined action of microgravity and radiation. Bone remodelling is a life-long process performed by balanced action of cells from the osteoblast and osteoclast lineages. While osteoblasts differentiate either into bone-lining cells or into osteocytes and play a crucial role in bone matrix synthesis, osteoclasts are responsible for bone resorption. We hypothesize that the balance between bone matrix assembly by osteocytes and bone degradation by osteoclasts is modulated by microgravity as well as by ionizing radiation. To address this, a cell model consisting of murine cell lines with the potential to differentiate into bone-forming osteoblasts (OCT-1, MC3T3-E1 S24, and MC3T3-E1 S4) was used for studying radiation response after exposure to simulated components of cosmic radiation. Cells were exposed to graded doses of 150 kV X-rays, α particles (0.525 MeV/u, 160 keV/µm; PTB, Braunschweig, Germany) and accelerated heavy ions (75 MeV/u carbon, 29 keV/µm; 95 MeV/u argon, 230 keV/µm; GANIL, Caen, France). Cell survival was measured as colony forming ability; cell cycle progression was analyzed via fluorescence-activated cell scanning (FACS) by measurement of the content of propidium iodide-stained DNA, DNA damage was visualized by γH2AX

  5. Virus-specific RNA synthesis in interferon-treated mouse cells productively infected with Moloney murine leukemia virus.

    PubMed Central

    Fan, H; MacIsaac, P

    1978-01-01

    Mouse cells productively infected with Moloney murine leukemia virus were treated with interferon, and intracellular virus-specific RNA was studied by hybridization with complementary DNA. The steady-state concentration of virus-specific RNA in interferon-treated cells was somewhat greater than that in untreated cells, and the rates of virus-specific RNA synthesis were approximately equal in treated and untreated cells. PMID:691118

  6. Selective erythroid replacement in murine beta-thalassemia using fetal hematopoietic stem cells.

    PubMed Central

    Bethel, C. A.; Murugesh, D.; Harrison, M. R.; Mohandas, N.; Rubin, E. M.

    1993-01-01

    We have explored the application of fetal hematopoietic stem cell (HSC) transplants for cellular replacement in a murine model of beta-thalassemia. Liver-derived HSCs from nonthalassemic syngeneic murine fetal donors were transplanted into nonirradiated neonatal beta-thalassemic recipients. Significant erythrocyte chimerism (9-27%) was demonstrated in the majority of recipients at 1 month and remained stable or increased (up to 55%) during long-term follow-up in almost all cases. Chimeras had improved phenotypes, as evidenced by decreased reticulocyte counts, increased mean erythrocyte deformability, and decreased iron deposits in comparison to controls. To investigate whether the high degree of peripheral blood chimerism was predominantly a feature of erythroid elements or was a general feature of all hematopoietic elements, chimeras were created using donor HSCs "tagged" with a DNA transgene. Whereas donor hemoglobin comprised > 30% of total hemoglobin, nucleated tagged nonerythroid donor cells comprised < 1% of peripheral blood elements. Explanations for the observed selective increase in erythroid chimerism include longer survival of normal donor red cells compared to that of thalassemic red cells and the effective maturation of the donor erythroid elements in the bone marrow in chimeric animals. The latter explanation bears consideration because it is consistent with the process of ineffective erythropoiesis, well documented to occur in thalassemia, in which the majority of thalassemic erythroid cells are destroyed during erythropoiesis prior to release from the bone marrow. Overall, these data demonstrate the potential for significant erythroid chimerism and suggest that fetal HSC transplantation may play a significant role in future treatment. Images Fig. 1 Fig. 4 Fig. 7 PMID:7980734

  7. Selective erythroid replacement in murine beta-thalassemia using fetal hematopoietic stem cells.

    PubMed

    Bethel, C A; Murugesh, D; Harrison, M R; Mohandas, N; Rubin, E M

    1993-11-01

    We have explored the application of fetal hematopoietic stem cell (HSC) transplants for cellular replacement in a murine model of beta-thalassemia. Liver-derived HSCs from nonthalassemic syngeneic murine fetal donors were transplanted into nonirradiated neonatal beta-thalassemic recipients. Significant erythrocyte chimerism (9-27%) was demonstrated in the majority of recipients at 1 month and remained stable or increased (up to 55%) during long-term follow-up in almost all cases. Chimeras had improved phenotypes, as evidenced by decreased reticulocyte counts, increased mean erythrocyte deformability, and decreased iron deposits in comparison to controls. To investigate whether the high degree of peripheral blood chimerism was predominantly a feature of erythroid elements or was a general feature of all hematopoietic elements, chimeras were created using donor HSCs "tagged" with a DNA transgene. Whereas donor hemoglobin comprised > 30% of total hemoglobin, nucleated tagged nonerythroid donor cells comprised < 1% of peripheral blood elements. Explanations for the observed selective increase in erythroid chimerism include longer survival of normal donor red cells compared to that of thalassemic red cells and the effective maturation of the donor erythroid elements in the bone marrow in chimeric animals. The latter explanation bears consideration because it is consistent with the process of ineffective erythropoiesis, well documented to occur in thalassemia, in which the majority of thalassemic erythroid cells are destroyed during erythropoiesis prior to release from the bone marrow. Overall, these data demonstrate the potential for significant erythroid chimerism and suggest that fetal HSC transplantation may play a significant role in future treatment.

  8. Anti-inflammatory effects of natural product formulations on murine dendritic cells.

    PubMed

    Miller, Andrea K; Benson, Jenna M; Muanza, Dave N; Smith, Jerry R; Shepherd, David M

    2011-03-01

    The popularity and availability of herbal extracts has increased dramatically over the last decade, providing an inexpensive manner of self-medication. Although the efficacy of individual extracts is currently being studied intensively, research regarding complex mixtures is limited. Therefore, we evaluated the effects of three complex formulations, including BRC-301, a polyherbal extract; BRC-304, a mixture of vitamins, minerals, antioxidant enzymes, botanical extracts, and carotenoids; and BRC-306, a proprietary blend of Uncaria tomentosa (cat's claw) and Phytolens(®) on murine dendritic cells (DCs). We hypothesized that these formulations would decrease the inflammatory responsiveness and innate function of DCs. In order to address this hypothesis, we evaluated the effects of BRC-301, BRC-304, and BRC-306 on DC2.4 cells and assessed the effects of BRC-301 on bone marrow-derived DCs (bmDCs). Lipopolysaccharide (LPS) stimulation of DC2.4 cells and bmDCs induced production of nitric oxide (NO), TNF-α, and IL-6, a response that was modulated by concomitant treatment with non-cytotoxic concentrations of BRC-301. In contrast, only the production of NOor IL-6 by LPS-activated DC2.4 cells was affected by BRC-304 or BRC-306, respectively. Flow cytometric evaluation following concurrent BRC-301 and LPS treatment revealed an increased relative expression of CD11c, CD86, and CD54 on bmDCs and an increased frequency of bmDCs expressing MHC II. Finally, BRC-301 enhanced the uptake of fluorescein isothiocyanate-conjugated ovalbumin by bmDCs. Taken together, these results suggest that these commercially available formulations modulate the innate responsiveness of murine DCs and may enhance their ability to initiate T cell-mediated immunity.

  9. Anti-inflammatory effects of natural product formulations on murine dendritic cells

    PubMed Central

    Miller, Andrea K.; Benson, Jenna M.; Muanza, Dave N.; Smith, Jerry R.; Shepherd, David M.

    2011-01-01

    The popularity and availability of herbal extracts has increased dramatically over the last decade, providing an inexpensive manner of self-medication. Although the efficacy of individual extracts is currently being intensively studied, research regarding complex mixtures is limited. Therefore, we evaluated the effects of three complex formulations including BRC-301, a polyherbal extract; BRC-304, a mixture of vitamins, minerals, antioxidant enzymes, botanical extracts and carotenoids; and BRC-306, a proprietary blend of Uncaria tomentosa (cat’s claw) and Phytolens® on murine dendritic cells (DCs). We hypothesized that these formulations would decrease the inflammatory responsiveness and innate function of DCs. To address this hypothesis, we evaluated the effects of BRC-301, 304, and 306 on DC2.4 cells and further assessed the effects of BRC-301 on bone marrow-derived DCs (bmDCs). LPS stimulation of DC2.4 cells and bmDCs induced production of NO, TNF-α, and IL-6, a response that was modulated by concomitant treatment with non-cytotoxic concentrations of BRC-301. In contrast, only the production of NO or IL-6 by LPS-activated DC2.4 cells was affected by BRC-304 or BRC-306, respectively. Flow cytometric evaluation following concurrent BRC-301 and LPS treatment revealed an increased relative expression of CD11c, CD86, and CD54 on bmDCs and an increased frequency of bmDCs expressing MHC II. Finally, BRC-301 enhanced the uptake of FITC-conjugated ovalbumin by bmDCs. Taken together, these results suggest that these commercially available formulations modulate the innate responsiveness of murine DCs and may enhance their ability to initiate T cell-mediated immunity. PMID:21399725

  10. Microglial AGE-albumin is critical in promoting alcohol-induced neurodegeneration in rats and humans.

    PubMed

    Byun, Kyunghee; Bayarsaikhan, Delger; Bayarsaikhan, Enkhjargal; Son, Myeongjoo; Oh, Seyeon; Lee, Jaesuk; Son, Hye-In; Won, Moo-Ho; Kim, Seung U; Song, Byoung-Joon; Lee, Bonghee

    2014-01-01

    Alcohol is a neurotoxic agent, since long-term heavy ingestion of alcohol can cause various neural diseases including fetal alcohol syndrome, cerebellar degeneracy and alcoholic dementia. However, the molecular mechanisms of alcohol-induced neurotoxicity are still poorly understood despite numerous studies. Thus, we hypothesized that activated microglial cells with elevated AGE-albumin levels play an important role in promoting alcohol-induced neurodegeneration. Our results revealed that microglial activation and neuronal damage were found in the hippocampus and entorhinal cortex following alcohol treatment in a rat model. Increased AGE-albumin synthesis and secretion were also observed in activated microglial cells after alcohol exposure. The expressed levels of receptor for AGE (RAGE)-positive neurons and RAGE-dependent neuronal death were markedly elevated by AGE-albumin through the mitogen activated protein kinase pathway. Treatment with soluble RAGE or AGE inhibitors significantly diminished neuronal damage in the animal model. Furthermore, the levels of activated microglial cells, AGE-albumin and neuronal loss were significantly elevated in human brains from alcoholic indivisuals compared to normal controls. Taken together, our data suggest that increased AGE-albumin from activated microglial cells induces neuronal death, and that efficient regulation of its synthesis and secretion is a therapeutic target for preventing alcohol-induced neurodegeneration. PMID:25140518

  11. Effects of ionizing radiation on bone cell differentiation in an experimental murine bone cell model

    NASA Astrophysics Data System (ADS)

    Baumstark-Khan, Christa; Lau, Patrick; Hellweg, Christine; Reitz, Guenther

    During long-term space travel astronauts are exposed to a complex mixture of different radiation types under conditions of dramatically reduced weight-bearing activity. It has been validated that astronauts loose a considerable amount of bone mass at a rate up to one to two percent each month in space. Therapeutic doses of ionizing radiation cause bone damage and increase fracture risks after treatment for head-and-neck cancer and in pelvic irradiation. For low radiation doses, the possibility of a disturbed healing potential of bone was described. Radiation induced damage has been discussed to inflict mainly on immature and healing bone. Little is known about radiation effects on bone remodelling and even less on the combined action of microgravity and radiation. Bone remodelling is a life-long process performed by balanced action of cells from the osteoblast and osteoclast lineages. While osteoblasts differentiate either into bone-lining cells or into osteocytes and play a crucial role in bone matrix synthesis, osteoclasts are responsible for bone resorption. We hypothesize that the balance between bone matrix assembly by osteocytes and bone degradation by osteoclasts is modulated by microgravity as well as by ionizing radiation. To address this, a cell model consisting of murine cell lines with the potential to differentiate into bone-forming osteoblasts (OCT-1, MC3T3-E1 S24, and MC3T3-E1 S4) was used for studying radiation response after exposure to simulated components of cosmic radiation. Cells were exposed to graded doses of 150 kV X-rays, α particles (0.525 MeV/u, 160 keV/µm; PTB, Braunschweig, Germany) and accelerated heavy ions (75 MeV/u carbon, 29 keV/µm; 95 MeV/u argon, 230 keV/µm; GANIL, Caen, France). Cell survival was measured as colony forming ability; cell cycle progression was analyzed via fluorescence-activated cell scanning (FACS) by measurement of the content of propidium iodide-stained DNA, DNA damage was visualized by γH2AX

  12. Expression of TCR-Vβ peptides by murine bone marrow cells does not identify T-cell progenitors

    PubMed Central

    Abbey, Janice L; Karsunky, Holger; Serwold, Thomas; Papathanasiou, Peter; Weissman, Irving L; O’Neill, Helen C

    2015-01-01

    Germline transcription has been described for both immunoglobulin and T-cell receptor (TCR) genes, raising questions of their functional significance during haematopoiesis. Previously, an immature murine T-cell line was shown to bind antibody to TCR-Vβ8.2 in absence of anti-Cβ antibody binding, and an equivalent cell subset was also identified in the mesenteric lymph node. Here, we investigate whether germline transcription and cell surface Vβ8.2 expression could therefore represent a potential marker of T-cell progenitors. Cells with the TCR phenotype of Vβ8.2+Cβ− are found in several lymphoid sites, and among the lineage-negative (Lin−) fraction of hematopoietic progenitors in bone marrow (BM). Cell surface marker analysis of these cells identified subsets reflecting common lymphoid progenitors, common myeloid progenitors and multipotential progenitors. To assess whether the Lin−Vβ8.2+Cβ− BM subset contains hematopoietic progenitors, cells were sorted and adoptively transferred into sub-lethally irradiated recipients. No T-cell or myeloid progeny were detected following introduction of cells via the intrathymic or intravenous routes. However, B-cell development was detected in spleen. This pattern of restricted in vivo reconstitution disputes Lin−Vβ8.2+Cβ− BM cells as committed T-cell progenitors, but raises the possibility of progenitors with potential for B-cell development. PMID:25754612

  13. Stem cell transplantation with S-59 photochemically treated T-cell add-backs to establish allochimerism in murine thalassemia.

    PubMed

    Kuypers, Frans A; Watson, Gordon; Sage, Ezra; Walters, Mark C; Hamrick, James; Hearst, John E

    2005-01-01

    Hematopoietic cell transplantation (HCT) from HLA-identical sibling donors has curative potential for beta-thalassemia. The probability of surviving free of thalassemia under these conditions is approximately 85%. The application of this therapy is limited because many patients lack an HLA-identical sibling donor. HLA-mismatched stem cell transplantation for thalassemia is severely restricted by graft rejection and the risks for graft-versus-host disease (GVHD). Thus, the development of a novel method that facilitates immunological tolerance and improves the safety of HLA-mismatched HCT would greatly expand the opportunity of HCT for thalassemia patients. We hypothesized that removal of T cells from the donor hematopoietic stem cell preparation and subsequent add-back after photochemical treatment with S-59, a psoralen, would promote and stabilize the engraftment and significantly reduce the risk of GVHD. This was tested in a MHC-mismatched HCT model of murine thalassemia. S-59-treated T cells were infused simultaneously with bone marrow-derived stem cells into mice with a heterozygous deletion of one beta-globin alleles that had been conditioned with a sublethal dose of total body irradiation. Mice that received treated T cells showed increased engraftment compared to those that did not receive T cells. T-cell treatment improved survival without GVHD compared to recipients that received untreated T cells. We conclude that photochemical treatment of T cells facilitates engraftment and minimizes GVHD in allo-HCT for murine thalassemia, and sets the stage for further development of such protocols for the treatment of patients with thalassemia.

  14. Stimulation of Mesothelial Cell Proliferation by Exudate Macrophages Enhances Serosal Wound Healing in a Murine Model

    PubMed Central

    Mutsaers, Steven E.; Whitaker, Darrel; Papadimitriou, John M.

    2002-01-01

    Examination of thermally induced serosal lesions in mice displayed collections of inflammatory cells, predominantly macrophages, on and surrounding the wound within 48 hours of injury. Furthermore, by 2 days a large number of uninjured mesothelial cells adjacent to the wound were synthesizing DNA. From these findings, it was hypothesized that macrophages play a major role in serosal repair by stimulating mesothelial cell proliferation. Again, using a murine model of mesothelial regeneration, depletion of circulating monocytes significantly delayed serosal healing whereas addition of peritoneal exudate cells to the wound site 36 hours before injury increased the healing rate. In vivo assessment of mesothelial cell proliferation using tritiated thymidine incorporation and autoradiography demonstrated that peritoneal exudate cells stimulated mesothelial cell proliferation (12.44 ± 1.63% labeling index, compared with controls in which medium only was used 4.48 ± 0.71%). The mesothelial proliferation was predominantly because of macrophage-secreted products with molecular weights of 36 to 53 kd or 67 to 100 kd. These data support the hypothesis that macrophages play an important role in serosal healing by stimulating mesothelial cell proliferation. PMID:11839589

  15. Cytotoxicity characterization of catanionic vesicles in RAW 264.7 murine macrophage-like cells.

    PubMed

    Kuo, Jung-Hua Steven; Jan, Ming-Shiou; Chang, Chien-Hsiang; Chiu, Hsuan-Wen; Li, Cih-Ta

    2005-03-25

    In comparison with cationic liposomes, catanionic vesicles possess more attractive properties such as stability and lower cost, and these characteristics may make them suitable as a non-viral vehicle and for other biomedical applications such as vaccine adjuvants. However, very little is known about their possible cytotoxic mechanisms in cellular system. Also, this information is vital for the future development of safe biomedical systems. In the current study, the cytotoxic effect of catanionic vesicles, consisting of anionic surfactant (SDS), cationic surfactant (HTMAB), and cholesterol, in cultured RAW 264.7 murine macrophage-like cells was determined. The treatment of catanionic vesicles produced a dose-dependent effect on macrophage cells. RAW 264.7 cells exposed to catanionic vesicles exhibited morphological features of apoptosis such as chromatin condensation. Typical apoptotic ladders were observed in DNA extracted from RAW 264.7 cells treated by catanionic vesicles. Analysis from flow cytometry demonstrated an increase of hypodiploid DNA population (sub-G1) and a simultaneous decrease of diploid DNA content, indicating that DNA cleavage occurred after exposure of the cells with catanionic vesicles. In addition, it was shown that pretreatment of RAW 264.7 cells with the general caspase inhibitor (zVAD-fmk) did not prevent apoptosis induced by catanionic vesicles, suggesting that apoptosis in macrophage cells followed a casp