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Coral diseases have been increasingly reported over the past few decades and are a major contributor to coral decline worldwide. The Caribbean, in particular, has been noted as a hotspot for coral disease, and the aptly named white syndromes have caused the decline of the dominant reef building corals throughout their range. White band disease (WBD) has been implicated in the dramatic loss of Acropora cervicornis and Acropora palmata since the 1970s, resulting in both species being listed as critically endangered on the International Union for Conservation of Nature Red list. The causal agent of WBD remains unknown, although recent studies based on challenge experiments with filtrate from infected hosts concluded that the disease is probably caused by bacteria. Here, we report an experiment using four different antibiotic treatments, targeting different members of the disease-associated microbial community. Two antibiotics, ampicillin and paromomycin, arrested the disease completely, and by comparing with community shifts brought about by treatments that did not arrest the disease, we have identified the likely candidate causal agent or agents of WBD. Our interpretation of the experimental treatments is that one or a combination of up to three specific bacterial types, detected consistently in diseased corals but not detectable in healthy corals, are likely causal agents of WBD. In addition, a histophagous ciliate (Philaster lucinda) identical to that found consistently in association with white syndrome in Indo-Pacific acroporas was also consistently detected in all WBD samples and absent in healthy coral. Treatment with metronidazole reduced it to below detection limits, but did not arrest the disease. However, the microscopic disease signs changed, suggesting a secondary role in disease causation for this ciliate. In future studies to identify a causal agent of WBD via tests of Henle-Koch's postulates, it will be vital to experimentally control for populations of the other potential pathogens identified in this study.
Assuntos
Antozoários/microbiologia , Antibacterianos/farmacologia , Archaea/efeitos dos fármacos , Bactérias/efeitos dos fármacos , Cilióforos/efeitos dos fármacos , Animais , Antibacterianos/administração & dosagem , Archaea/classificação , Archaea/genética , Archaea/isolamento & purificação , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Região do Caribe , Cilióforos/classificação , Cilióforos/genética , Cilióforos/isolamento & purificação , Eletroforese em Gel de Gradiente Desnaturante , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Silver nanoparticles (NPs) are used for a wide range of commercial reasons to restrict microbial growth. The increasing use of silver NPs in modern materials ensures they will find their way into environmental systems. The mode of action which makes them desirable as an antimicrobial tool could also pose a severe threat to the natural microbial balance existing in these systems. Research into the potential environmental threats of silver NPs has mainly focused on particular areas, such as their influence in rivers and estuaries or their effect on organisms such as earthworms and plants. There is a need to focus studies on all aspects of the microbial world and to highlight potential risks and methods of overcoming problems before significant damage is done. This review focuses on the antimicrobial uses, mechanisms of toxicity, and effects on the environment (mainly soil) of silver NPs, illustrating gaps in current knowledge.
Assuntos
Nanopartículas , Prata , Animais , Meio Ambiente , Nanopartículas Metálicas , Oligoquetos , SoloRESUMO
The receptor for macrophage colony stimulating factor (CSF-1), the c-fms gene product, is a key determinant in the differentiation of monocytic phagocytes. Dissection of the human and mouse c-fms proximal promoters revealed opposing roles for nuclear protooncogenes in the transcriptional regulation of this gene. On the one hand, c-ets-1, c-ets-2, and the macrophage-specific factor PU.1, but not the ets-factor PEA3, trans-activated the c-fms proximal promoter. On the other hand c-myb repressed proximal promoter activity in macrophages and blocked the action of c-ets-1 and c-ets-2. Basal c-fms promoter activity was almost undetectable in the M1 leukaemia line, which expressed high levels of c-myb, but was activated as cells differentiated in response to leukemia inhibitory factor and expressed c-fms mRNA. The repressor function of c-myb depended on the COOH-terminal domain of the protein. We propose that ets-factors are necessary for the tissue-restricted expression of c-fms and that c-myb acts to ensure correct temporal expression of c-fms during myeloid differentiation.
Assuntos
Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Proto-Oncogenes , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Células 3T3 , Animais , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Chlorocebus aethiops , Primers do DNA , Proteínas de Ligação a DNA/metabolismo , Genes fms , Humanos , Camundongos , Dados de Sequência Molecular , Oncogenes , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-ets , Proteínas Proto-Oncogênicas c-myb , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
Vincristine is an antineoplastic substance that is part of many chemotherapy regimens, used especially for the treatment of a variety of pediatric cancers including leukemias and brain tumors. Unfortunately, many vincristine-treated patients develop peripheral neuropathy, a side effect characterized by sensory, motoric, and autonomic symptoms. The sensory symptoms include pain, in particular hypersensitivity to light touch, as well as loss of sensory discrimination to detect vibration and touch. The symptoms of vincristine-induced neuropathy are only poorly controlled by currently available analgesics and therefore often necessitate dose reductions or even cessation of treatment. The aim of this study was to identify new therapeutic targets for the treatment of vincristine-induced peripheral neuropathy (VIPN) by combining behavioral experiments, histology, and pharmacology after vincristine treatment. Local intraplantar injection of vincristine into the hind paw caused dose- and time-dependent mechanical hypersensitivity that developed into mechanical hyposensitivity at high doses, and lead to a pronounced, dose-dependent infiltration of immune cells at the site of injection. Importantly, administration of minocycline effectively prevented the development of mechanical hypersensitivity and infiltration of immune cells in mouse models of vincristine induce peripheral neuropathy (VIPN) based on intraperitoneal or intraplantar administration of vincristine. Similarly, Toll-like receptor 4 knockout mice showed diminished vincristine-induced mechanical hypersensitivity and immune cell infiltration, while treatment with the anti-inflammatory meloxicam had no effect. These results provide evidence for the involvement of Toll-like receptor 4 in the development of VIPN and suggest that minocycline and/or direct Toll-like receptor 4 antagonists may be an effective preventative treatment for patients receiving vincristine.
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With the launch of the teaching excellence framework, teaching in higher education (HE) is under greater scrutiny than ever before. Didactic lecture delivery is still a core element of many HE programmes but there is now a greater expectation for academics to incorporate alternative approaches into their practice to increase student engagement. These approaches may include a large array of techniques from group activities, problem-based learning, practical experience and mock scenarios to newly emerging approaches such as flipped learning practices and the use of gamification. These participatory forms of learning encourage students to become more absorbed within a topic that may otherwise be seen as rather 'dry' and reduce students engagement with, and therefore retention of, material. Here we use participatory-based teaching approaches in microbiology as an example to illustrate to University undergraduate students the potentially devastating effects that a disease can have on a population. The 'threat' that diseases may pose and the manner in which they may spread and/or evolve can be challenging to communicate, especially in relation to the timescales associated with these factors in the case of an epidemic or pandemic.
Assuntos
Surtos de Doenças , Educação Médica/métodos , Epidemiologia/educação , Pandemias , Terapia Comportamental , Humanos , Estudantes/psicologiaRESUMO
Uropathogenic Escherichia coli (UPEC) is the main etiological agent of urinary tract infections (UTIs). Little is known about interactions between UPEC and the inflammasome, a key innate immune pathway. Here we show that UPEC strains CFT073 and UTI89 trigger inflammasome activation and lytic cell death in human macrophages. Several other UPEC strains, including two multidrug-resistant ST131 isolates, did not kill macrophages. In mouse macrophages, UTI89 triggered cell death only at a high multiplicity of infection, and CFT073-mediated inflammasome responses were completely NLRP3-dependent. Surprisingly, CFT073- and UTI89-mediated responses only partially depended on NLRP3 in human macrophages. In these cells, NLRP3 was required for interleukin-1ß (IL-1ß) maturation, but contributed only marginally to cell death. Similarly, caspase-1 inhibition did not block cell death in human macrophages. In keeping with such differences, the pore-forming toxin α-hemolysin mediated a substantial proportion of CFT073-triggered IL-1ß secretion in mouse but not human macrophages. There was also a more substantial α-hemolysin-independent cell death response in human vs. mouse macrophages. Thus, in mouse macrophages, CFT073-triggered inflammasome responses are completely NLRP3-dependent, and largely α-hemolysin-dependent. In contrast, UPEC activates an NLRP3-independent cell death pathway and an α-hemolysin-independent IL-1ß secretion pathway in human macrophages. This has important implications for understanding UTI in humans.
Assuntos
Proteínas de Transporte/imunologia , Inflamassomos/efeitos dos fármacos , Interleucina-1beta/imunologia , Macrófagos/imunologia , Escherichia coli Uropatogênica/imunologia , Animais , Toxinas Bacterianas/toxicidade , Proteínas de Transporte/genética , Morte Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Proteínas Hemolisinas/toxicidade , Interações Hospedeiro-Patógeno , Humanos , Inflamassomos/imunologia , Interleucina-1beta/genética , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Cultura Primária de Células , Transdução de Sinais , Especificidade da Espécie , Escherichia coli Uropatogênica/patogenicidadeRESUMO
Macrophage/dendritic cells and B cells remain the only cell types where direct responses to CpG DNA are well established. The role of macrophages in vivo in DNA clearance and the potent cytokine induction in macrophages and dendritic cells places them in the central role in the in vivo response to foreign DNA. Although responses to DNA are unlikely to evolve and be retained if they are not significant in the immune response to infection, the relative contributions of DNA and other stimulators of the innate immune recognition of foreign organisms is difficult to assess. Although CpG DNA and LPS have similar actions, significant differences are emerging that make the use of DNA as a therapeutic immunostimulatory molecule feasible. The macrophage response to DNA generates cytokines favouring the development of Th1-type immunity, and active oligonucleotides now show promise as Th1-promoting adjuvants and as allergy treatments.
Assuntos
Ilhas de CpG/imunologia , DNA/imunologia , Ativação de Macrófagos/imunologia , Animais , Células Dendríticas/imunologia , HumanosRESUMO
Many bacterial pathogens including Salmonella and Listeria replicate within macrophages. The susceptibility of these organisms to various antibiotics is dependent on the ability of macrophages to take up, retain, and deliver the antibiotic to the correct intracellular compartment. In this context, macrophages are known to express proteins that are involved in efflux of antibiotics and cytotoxic drugs, thereby reducing intracellular accumulation of such compounds. In our studies on the action of bacterial lipopolysaccharide (LPS) on the macrophage-like cell line, RAW264 we found that LPS treatment of these cells conferred resistance to the neomycin-related aminoglycoside G418 (geneticin). This phenotype was stable and was specific to LPS since colony-stimulating factor 1 and phorbol myristate acetate had no effect on G418 resistance. We have extended this observation to show that LPS induces transient resistance to the cytotoxic drugs taxol and doxorubicin. Macrophage resistance to cytotoxic drugs and antibiotics may have a number of important clinical consequences.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Doxorrubicina/farmacologia , Gentamicinas/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Paclitaxel/farmacologia , Animais , Antibióticos Antineoplásicos/toxicidade , Antineoplásicos Fitogênicos/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/toxicidade , Interações Medicamentosas , Resistência Microbiana a Medicamentos , Resistencia a Medicamentos Antineoplásicos , Camundongos , Paclitaxel/toxicidade , Succinato Desidrogenase/metabolismoRESUMO
Through its action on macrophages, bacterial lipopolysaccharide (LPS) or endotoxin can trigger responses that are protective or injurious to the host. This review examines the effects of LPS on macrophages by following events from the cell surface to the nucleus. The involvement of protein tyrosine kinases, mitogen-activated protein kinases, protein kinase C, G proteins, protein kinase A, ceramide-activated protein kinase, and microtubules in this process are reviewed. At the nuclear level, rel, C/EBP, Ets, Egr, fos, and jun family members have been implicated in activation of LPS-inducible gene expression.
Assuntos
Endotoxinas/farmacologia , Proteínas Imediatamente Precoces , Lipopolissacarídeos/farmacologia , Macrófagos/fisiologia , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Proteínas Estimuladoras de Ligação a CCAAT , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Membrana Celular/fisiologia , Núcleo Celular/fisiologia , Sequência Consenso , Proteínas de Ligação a DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce , Proteínas de Ligação ao GTP/metabolismo , Humanos , Receptores de Lipopolissacarídeos/fisiologia , Macrófagos/efeitos dos fármacos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ets , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Expression of reporter genes under the control of the HIV-1 long terminal repeat (LTR) was up-regulated in the murine macrophage cell line RAW264 by cotransfection of a plasmid coding for the viral regulatory protein Nef. To determine if a discrete section of the LTR was exclusively responsive to Nef, a series of promoters was produced by successive 5' deletions from the LTR up to the boundary of the enhancer region. These truncated promoters were as active as the full-length sequence in the RAW264 cells, but elimination of the direct repeats and one of the three Sp1 sites reduced promoter activity to minimal levels. Transcription driven by all constructs was equally susceptible to the trans-activating effect of Nef and could be increased further by the addition of a Tat-expressing plasmid to the cotransfection. Open reading frames of nef from NL4-3, from HXB2, which has a premature stop, and a fully functional hybrid of the two under the control of the SR alpha artificial promoter (SV40 early promoter plus HTLV-I R-U5') were able to transactivate the LTR in RAW264 cells to the same degree as HXB3 nef under the control of the cytomegalovirus (CMV) immediate-early promoter. A frameshift mutation of Nef at the XhoI site at position 8475 did not abrogate trans-activation of the LTR in macrophages. To further define the effective trans-activation region of Nef, internal deletions were made. Changes downstream of the XhoI site at amino acid 35 resulted in little or no reduction in trans-activation, whereas a deletion between the CMV promoter of the expression plasmid and the XhoI site largely abolished activity. Nef trans-activation of the LTR may be restricted to macrophages. Parallel cotransfection experiments in COS-1 simian fibroblast-like cells showed repression of reporter expression by Nef. Results suggested that the section of nef responsible for transactivation of the LTR in macrophages differed slightly from that sufficient for trans-repression in fibroblasts. Translation of the protein from the first translation start site (Met-1) rather than from the second in-frame ATG (Met-20) appears to be necessary for the trans-activating effect of Nef in RAW264 cells. Mutation of the initial ATG to ATA led to loss of trans-activating activity. Expression of Nef also has a cytostatic/cytotoxic effect on RAW264 cells indicated by a reduced rate of establishment of stably transfected clones. The cytostatic effect of Nef was not relieved by internal deletions in the coding sequence.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Produtos do Gene nef/fisiologia , HIV-1/fisiologia , Macrófagos/citologia , Macrófagos/microbiologia , Síndrome da Imunodeficiência Adquirida/genética , Síndrome da Imunodeficiência Adquirida/patologia , Síndrome da Imunodeficiência Adquirida/fisiopatologia , Alelos , Animais , Sequência de Bases , Linhagem Celular , DNA Viral/análise , DNA Viral/genética , Fibroblastos/química , Fibroblastos/citologia , Fibroblastos/microbiologia , Regulação Viral da Expressão Gênica , Produtos do Gene nef/análise , Produtos do Gene nef/genética , Genes Reporter , HIV-1/genética , HIV-1/isolamento & purificação , Macrófagos/química , Camundongos , Dados de Sequência Molecular , Fenótipo , Regiões Promotoras Genéticas , Ativação Transcricional , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
Murine macrophages are able to distinguish bacterial from mammalian DNA. The response is mimicked by single-stranded oligonucleotides containing unmethylated CG dinucleotides ("CpG" motifs) in specific sequence contexts. The dose-response curve for activation is influenced by variation in the sequence flanking the core CpG motif. CpG or bacterial DNA activates several signaling pathways in common with bacterial lipopolysaccharide (LPS), leading to induction of cytokine genes such as tumor necrosis factor alpha. Pretreatment with LPS causes desensitization to subsequent activation by CpG DNA. Both stimuli also cause cell cycle arrest in macrophages proliferating in response to the macrophage growth factor colony-stimulating factor-1 (CSF-1), but prevent apoptosis caused by growth factor removal. In part, cell cycle arrest by CpG DNA and LPS may be linked to rapid down-modulation of the CSF-1 receptor from the cell surface, a response that occurs in an all-or-nothing manner. The response of macrophages to CpG DNA has aspects in common with the DNA damage response in other cell types, which may provide clues to the underlying mechanism.
Assuntos
Ilhas de CpG/imunologia , DNA Bacteriano/imunologia , Ativação de Macrófagos , Macrófagos/imunologia , Animais , Diferenciação Celular , Citocinas/imunologia , Regulação para Baixo , Humanos , Lipopolissacarídeos/imunologia , Ativação de Macrófagos/imunologia , Camundongos , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Células Th1/citologia , Células Th1/imunologiaRESUMO
Soil contamination by silver nanoparticles (AgNP) is of potential environmental concern but little work has been carried out on the effect of such contamination on ectomycorrhizal fungi (EMF). EMF are essential to forest ecosystem functions as they are known to enhance growth of trees by nutrient transfer. In this study, soil was experimentally contaminated with AgNP (0, 350 and 790 mg Ag/kg) and planted with Bishop pine seedlings. The effect of AgNP was subsequently measured, assessing variation in pine growth and ectomycorrhizal diversity associated with the root system. After only 1 month, the highest AgNP level had significantly reduced the root length of pine seedlings, which in turn had a small effect on above ground plant biomass. However, after 4 months growth, both AgNP levels utilised had significantly reduced both pine root and shoot biomass. For example, even the lower levels of AgNP (350 mg Ag/kg) soil, reduced fresh root biomass by approximately 57 %. The root systems of the plants grown in AgNP-contaminated soils lacked the lateral and fine root development seen in the control plants (no AgNP). Although, only five different genera of EMF were found on roots of the control plants, only one genus Laccaria was found on roots of plants grown in soil containing 350 mg AgNP/kg. At the higher levels of AgNP contamination, no EMF were observed. Furthermore, extractable silver was found in soils containing AgNP, indicating potential dissolution of silver ions (Ag+) from the solid AgNP.
RESUMO
BACKGROUND: Macrophages sense microorganisms through activation of members of the Toll-like receptor family, which initiate signals linked to transcription of many inflammation associated genes. In this paper we examine whether the signal from Toll-like receptors [TLRs] is sustained for as long as the ligand is present, and whether responses to different TLR agonists are additive. RESULTS: RAW264 macrophage cells were doubly-transfected with reporter genes in which the IL-12p40, ELAM or IL-6 promoter controls firefly luciferase, and the human IL-1beta promoter drives renilla luciferase. The resultant stable lines provide robust assays of macrophage activation by TLR stimuli including LPS [TLR4], lipopeptide [TLR2], and bacterial DNA [TLR9], with each promoter demonstrating its own intrinsic characteristics. With each of the promoters, luciferase activity was induced over an 8 hr period, and thereafter reached a new steady state. Elevated expression required the continued presence of agonist. Sustained responses to different classes of agonist were perfectly additive. This pattern was confirmed by measuring inducible cytokine production in the same cells. While homodimerization of TLR4 mediates responses to LPS, TLR2 appears to require heterodimerization with another receptor such as TLR6. Transient expression of constitutively active forms of TLR4 or TLR2 plus TLR6 stimulated IL-12 promoter activity. The effect of LPS, a TLR4 agonist, was additive with that of TLR2/6 but not TLR4, whilst that of lipopeptide, a TLR2 agonist, was additive with TLR4 but not TLR2/6. Actions of bacterial DNA were additive with either TLR4 or TLR2/6. CONCLUSIONS: These findings indicate that maximal activation by any one TLR pathway does not preclude further activation by another, suggesting that common downstream regulatory components are not limiting. Upon exposure to a TLR agonist, macrophages enter a state of sustained activation in which they continuously sense the presence of a microbial challenge.
Assuntos
Lipopolissacarídeos/farmacologia , Ativação de Macrófagos , Macrófagos/imunologia , Glicoproteínas de Membrana/agonistas , Receptores de Superfície Celular/agonistas , Animais , Linhagem Celular , Células Cultivadas , Citocinas/biossíntese , Relação Dose-Resposta a Droga , Genes Reporter , Cinética , Luciferases/genética , Luciferases/metabolismo , Macrófagos/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Superfície Celular/metabolismo , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-LikeRESUMO
Macrophages recognize and are activated by unmethylated CpG motifs in bacterial DNA. Here we demonstrate that production of nitric oxide (NO) from murine RAW 264 macrophages and bone marrow-derived macrophages (BMM) in response to bacterial DNA is absolutely dependent on interferon-gamma (IFN-gamma) priming. Similarly, arginine uptake and expression of the inducible nitric oxide synthase (iNOS) gene in response to bacterial DNA in BMM occurred only after IFN-gamma priming. In contrast, mRNA for the cationic amino acid transporter, CAT2, was induced by plasmid DNA alone, and priming with IFN-gamma had no effect on this response. Tumor necrosis factor-alpha (TNF-alpha) release from RAW 264 and BMM in response to bacterial DNA was augmented by IFN-gamma pretreatment. In a stably transfected HIV-1 long terminal repeat (LTR) luciferase RAW 264 cell line, IFN-gamma and bacterial DNA synergized in activation of the HIV-1 LTR. Bacterial DNA has been shown to induce IFN-gamma production in vivo as an indirect consequence of interleukin-12 (IL-12) and TNF-alpha production from macrophages. The results herein suggest the existence of a self-amplifying loop that may have implications for therapeutic applications of bacterial DNA.
Assuntos
DNA Bacteriano/genética , Interferon gama/farmacologia , Macrófagos/efeitos dos fármacos , Óxido Nítrico Sintase/genética , Animais , Arginina/metabolismo , Catalase/genética , Linhagem Celular , HIV-1/genética , Camundongos , Camundongos Endogâmicos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II , RNA Mensageiro/biossíntese , Sequências Repetitivas de Ácido NucleicoRESUMO
Bacterial lipopolysaccharide (LPS) modulates expression of a variety of genes in macrophages, and additionally activates viral promoters including the HIV-1 LTR. The HIV-1 LTR driving the luciferase reporter gene was stably transfected into the murine macrophage cell line, RAW264. In stably transfected cells, luciferase activity was LPS-dependent. As little as 0.01 ng/ml LPS was sufficient to increase luciferase activity over basal levels with maximal stimulation resulting in a 10- to 20-fold response. The cells also responded to human and murine tumour necrosis factor (TNF alpha). Endogenous TNF alpha was not involved in LPS responses, since pretreatment with alpha-TNF alpha antibody did not affect activation. Induction of HIV-1 LTR activity by LPS occurred independently of phorbol myristate acetate (PMA) sensitive protein kinase C (PKC), since depletion of PKC by prolonged exposure to PMA blocked TNF alpha and PMA responses but was not able to abolish LPS action on these cells. Taxol (5-20 micrograms/ml), a chemotherapeutic agent which mimics LPS action on macrophages, was also able to increase expression of the reporter gene driven by the HIV-1 LTR. However, lower doses of taxol that were not sufficient to trans-activate the LTR or to induce TNF alpha expression were cytotoxic to RAW264 cells suggesting that the cytotoxic and LPS-like activities of taxol were not linked. This cell line provides a convenient method for detecting LPS-like activity and is a useful tool for examining LPS and TNF alpha signalling pathways.
Assuntos
Repetição Terminal Longa de HIV/genética , HIV-1/genética , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Paclitaxel/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Linhagem Celular , Expressão Gênica , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Camundongos , Proteína Quinase C/metabolismo , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , TransfecçãoRESUMO
The HIV-1 promoter was used as a model to identify transcription factors involved in LPS-dependent transcription in RAW 264 murine macrophages. Expression plasmids for Ets-2 and PU.1 trans-activated the HIV-1 LTR and recombinant PU.1 and an Ets-2 DNA binding domain/GST fusion protein bound to the 5' kappa B site of the HIV-1 enhancer. Ets-2 mRNA was LPS-inducible in RAW 264 cells and LPS stimulated phosphorylation of threonine 72 residue within the Ets-2 pointed domain. Induction of Ets-2 and other LPS-responsive transcription factors was also observed upon addition of plasmid DNA, which complicates interpretation of transient transfections. The proximal promoter region, containing two Sp1 sites, was also LPS-responsive. We propose that the kappa B elements and the tandem Sp1 sites act as LPS response elements and that kappa B-mediated LPS action involves Ets and rel factors.
Assuntos
Proteínas de Ligação a DNA , Repetição Terminal Longa de HIV/genética , Macrófagos/metabolismo , Proteínas Proto-Oncogênicas/farmacologia , Proteínas Repressoras , Fator de Transcrição Sp1/farmacologia , Transativadores/farmacologia , Fatores de Transcrição , Animais , Sítios de Ligação , Linhagem Celular , DNA/metabolismo , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Camundongos , NF-kappa B/farmacologia , Plasmídeos , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-ets-2 , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/genética , Transativadores/metabolismo , Ativação Transcricional , TransfecçãoRESUMO
Recent evidence suggests that bacterial DNA activates immune responses. Here we showed that TNF-alpha mRNA was induced in bone marrow-derived macrophages and the macrophage cell line RAW 264 by plasmid DNA, but not by DNaseI-digested plasmid, plasmid methylated on CpG dinucleotides, or by vertebrate genomic DNA, which is naturally largely methylated on these sequences. Synthetic polynucleotides poly d(I-C) and poly I x poly C also induced TNF-alpha. IL-1 beta and plasminogen activator inhibitor-2 mRNAs were induced by plasmid DNA, and IFN-gamma-pretreated macrophages responded to DNA with induction of inducible nitric oxide synthase. The HIV-1 long terminal repeat was activated by exogenous DNA in a manner similar to TNF-alpha, and was also activated by a CpG-containing oligonucleotide. Transcription factor nuclear factor-kappa B (NF-kappa B) is involved in regulation of the HIV-1 long terminal repeat and many inflammatory response genes. NF-kappa B binding activity was increased by plasmid DNA. An important question is whether these effects involve DNA binding to a cell surface receptor that signals to the interior, or whether internalization is necessary. Here we found that plasmid was taken up by RAW 264 cells and remained sufficiently intact to code for luciferase protein. Results suggest that DNA is taken up by macrophages and characteristic bacterial DNA sequences, which include an unmethylated CpG sequence, activate a signaling cascade leading to activation of NF-kappa B and inflammatory gene induction. Relevance to DNA vaccination, gene therapy, antisense, and transfection studies is discussed.
Assuntos
DNA Bacteriano/metabolismo , DNA Bacteriano/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Fagocitose/efeitos dos fármacos , Animais , Células da Medula Óssea , Linhagem Celular , Ativação de Macrófagos/genética , Macrófagos/microbiologia , Camundongos , NF-kappa B/biossíntese , NF-kappa B/genética , Fagocitose/genética , RNA Mensageiro/biossínteseRESUMO
The RAW264 murine macrophage cell line was used as a model to examine the role of the tat and nef gene products in the transcription regulation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in macrophages. Contrary to claims that the activity of the HIV-1 LTR responds poorly in rodent cells to trans activation by the viral tat gene product, cotransfection of RAW264 cells with a tat expression plasmid in transient transfection assays caused a > 20-fold increase in reporter gene expression that was inhibited by mutations in the TAR region. RAW264 cells stably transfected with the tat plasmid displayed similarly elevated HIV-1 LTR-driven reporter gene activity. By contrast to previous reports indicating a negative role for nef in HIV transcription, cotransfection of RAW264 cells with a nef expression plasmid trans activated the HIV-1 LTR driving either a chloramphenicol acetyltransferase or a luciferase reporter gene. The action of nef was specific to the LTR, as expression of nef had no effect on the activity of the simian virus 40, c-fms, urokinase plasminogen activator, or type 5 acid phosphatase promoter. trans-activating activity was also manifested by a frameshift mutant expressing only the first 35 amino acids of the protein. The effects of nef were multiplicative with those of tat gene product and occurred even in the presence of bacterial lipopolysaccharide, which itself activated LTR-directed transcription. Examination of the effects of selected mutations in the LTR revealed that neither the kappa B sites in the direct repeat enhancer nor the TAR region was required as a cis-acting element in nef action. The action of nef was not species restricted; it was able to trans activate in the human monocyte-like cell line Mono Mac 6. The presence of a nef expression cassette in a neomycin phosphotransferase gene expression plasmid greatly reduced the number of G418-resistant colonies generated in stable transfection of RAW264 cells, and many of the colonies that were formed exhibited very slow growth. The frameshift mutant was also active in reducing colony generation. Given the absence of any effect of the frameshift mutation on nef function, its actions on macrophage growth and HIV transcription are discussed in terms of the role of the N-terminal 30 amino acids and of stable secondary structures in the mRNA.
Assuntos
Repetição Terminal Longa de HIV/genética , HIV-1/genética , Macrófagos/microbiologia , Transcrição Gênica/efeitos dos fármacos , Proteínas Virais Reguladoras e Acessórias/farmacologia , Animais , Linhagem Celular , Mutação da Fase de Leitura , Regulação Viral da Expressão Gênica , Produtos do Gene nef/farmacologia , Produtos do Gene tat/farmacologia , Genes Reporter , HIV-1/crescimento & desenvolvimento , Haplorrinos , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Conformação de Ácido Nucleico , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Nucleico/genética , Vírus 40 dos Símios/genética , Especificidade da Espécie , Transfecção , Produtos do Gene nef do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Activated macrophages require l-arginine uptake to sustain NO synthesis. Several transport systems could mediate this l-arginine influx. Using competition analysis and gene-expression studies, amino acid transport system y+ was identified as the major carrier responsible for this activity. To identify which of the four known y+ transport-system genes is involved in macrophage-induced l-arginine uptake, we used a hybrid-depletion study in Xenopus oocytes. Cationic amino acid transporter (CAT) 2 antisense oligodeoxyribonucleotides abolished the activated-macrophage-mRNA-induced l-arginine transport. Together with expression studies documenting that CAT2 mRNA and protein levels are elevated with increased l-arginine uptake, our data demonstrate that CAT2 mediates the l-arginine transport that is required for the raised NO production in activated J774 macrophages.
Assuntos
Arginina/metabolismo , Proteínas de Transporte/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Óxido Nítrico/fisiologia , Sistemas de Transporte de Aminoácidos Básicos , Animais , Sequência de Bases , Transporte Biológico , Proteínas de Transporte/genética , Linhagem Celular , Primers do DNA , Ativação de Macrófagos , Proteínas de Membrana/genética , RNA Mensageiro/metabolismo , Xenopus laevisRESUMO
T1/ST2L, an IL-1 receptor homologue, is selectively expressed on murine Th2 cells and specific anti-ST2L antibodies can profoundly modulate the Th1/Th2 balance in vivo. Naive CD4+ T cells do not express ST2L but do so on activation with specific antigen in the presence of IL-4 or when stimulated with low doses of antigen in the absence of exogenously added IL-4. Similarly enhanced ST2L expression occurred after stimulation of Th2 cells with antigen or the mitogen ConA in the presence of APC. Restimulation of Th2 cells in the presence of IFN-gamma led to a decreased expression of ST2L to below basal levels. Conversely, Th2 cells cultured with IL-4 led to increased ST2L expression. The reduced expression of ST2L in response to high doses of antigen is also reversed by the neutralization of IFN-gamma. Using an ST2L promoter/luciferase reporter gene construct, we show that the distal but not proximal ST2L promoter is responsible for specific gene expression in Th2 cells. IL-4 enhances, whereas IFN-gamma suppresses ST2L expression via direct modulation of the distal promoter of the ST2L gene. These data provide a mechanistic explanation for the selective expression of ST2L on Th2 cells.