ABSTRACT
Acute lung injury (ALI) is a prevalent and severe clinical condition characterized by inflammatory damage to the lung endothelial and epithelial barriers, resulting in high incidence and mortality rates. Currently, there is a lack of safe and effective drugs for the treatment of ALI. In a previous clinical study, we observed that Jinyinqingre oral liquid (JYQR), a Traditional Chinese Medicine formulation prepared by the Taihe Hospital, Affiliated Hospital of Hubei University of Medicine, exhibited notable efficacy in treating inflammation-related hepatitis and cholecystitis in clinical settings. However, the potential role of JYQR in ALI/acute respiratory distress syndrome (ARDS) and its anti-inflammatory mechanism remains unexplored. Thus, the present study aimed to investigate the therapeutic effects and underlying molecular mechanisms of JYQR in ALI using a mouse model of lipopolysaccharide (LPS)-induced ALI and an in vitro RAW264.7 cell model. JYQR yielded substantial improvements in LPS-induced histological alterations in lung tissues. Additionally, JYQR administration led to a noteworthy reduction in total protein levels within the BALF, a decrease in MPAP, and attenuation of pleural thickness. These findings collectively highlight the remarkable efficacy of JYQR in mitigating the deleterious effects of LPS-induced ALI. Mechanistic investigations revealed that JYQR pretreatment significantly inhibited NF-κB activation and downregulated the expressions of the downstream proteins, namely NLRP3 and GSDMD, as well as proinflammatory cytokine levels in mice and RAW2647 cells. Consequently, JYQR alleviated LPS-induced ALI by inhibiting the NF-κB/NLRP3/GSDMD pathway. JYQR exerts a protective effect against LPS-induced ALI in mice, and its mechanism of action involves the downregulation of the NF-κB/NLRP3/GSDMD inflammatory pathway.
Subject(s)
Humans , NF-kappa B/metabolism , Lipopolysaccharides/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Acute Lung Injury/metabolism , Lung , Phosphate-Binding Proteins/therapeutic use , Pore Forming Cytotoxic Proteins/therapeutic useABSTRACT
This paper was aimed to investigate the effect of voluntary wheel running exercise on depression-like behavior induced by chronic water immersion restraint stress (CWIRS) and the underlying mechanism. Sprague-Dawley (SD) rats received CWIRS to induce depression-like behavior and 4-week voluntary wheel running exercise. Meanwhile, the rats were treated with lipopolysaccharide (LPS) or STAT3 over-expression vector (pcDNA-STAT3) by intracerebroventricular injection. Behavioral tests were used to detect depression-like behavior. ELISA assay was used to detect levels of various inflammatory factors in the rat hippocampus. Western blot was used to detect protein expression levels of ionized calcium binding adaptor molecule 1 (Iba1), inducible nitric oxide synthase (iNOS), arginase 1 (Arg1), phosphorylated STAT3 (p-STAT3) and total STAT3 (t-STAT3). The results showed that, compared with stress group, stress + exercise group exhibited improved depression-like behavior, decreased interleukin-1β (IL-1β) and IL-6 levels, increased IL-4 and IL-10 levels, down-regulated Iba-1 and iNOS protein expression levels, up-regulated Arg1 protein expression level, and decreased p-STAT3/t-STAT3 ratio in hippocampal tissue. LPS reversed the improving effect of voluntary wheel running exercise on depression-like behavior in rats, and the over-expression of STAT3 reversed the promoting effects of voluntary wheel running on M2 polarization of microglial cells in rat hippocampus and depression-like behavior. These results suggest that voluntary wheel running ameliorates the depression-like behavior induced by CWIRS in rats, and the mechanism may be related to regulating hippocampal microglia polarization via STAT3 signaling pathway.
Subject(s)
Animals , Rats , Depression/etiology , Hippocampus/metabolism , Lipopolysaccharides/metabolism , Microglia/metabolism , Motor Activity , Rats, Sprague-Dawley , Signal TransductionABSTRACT
Abstract Purpose To evaluate the kinetics of apical periodontitis development in vivo , induced either by contamination of the root canals by microorganisms from the oral cavity or by inoculation of bacterial lipopolysaccharide (LPS) and the regulation of major enzymes and receptors involved in the arachidonic acid metabolism. Methodology Apical periodontitis was induced in C57BL6 mice (n=96), by root canal exposure to oral cavity (n=48 teeth) or inoculation of LPS (10 µL of a suspension of 0.1 µg/µL) from E. coli into the root canals (n= 48 teeth). Healthy teeth were used as control (n=48 teeth). After 7, 14, 21 and 28 days the animals were euthanized and tissues removed for histopathological and qRT-PCR analyses. Histological analysis data were analyzed using two-way ANOVA followed by Sidak's test, and qRT-PCR data using two-way ANOVA followed by Tukey's test (α=0.05). Results Contamination by microorganisms led to the development of apical periodontitis, characterized by the recruitment of inflammatory cells and bone tissue resorption, whereas inoculation of LPS induced inflammatory cells recruitment without bone resorption. Both stimuli induced mRNA expression for cyclooxygenase-2 and 5-lipoxygenase enzymes. Expression of prostaglandin E 2 and leukotriene B 4 cell surface receptors were more stimulated by LPS. Regarding nuclear peroxisome proliferator-activated receptors (PPAR), oral contamination induced the synthesis of mRNA for PPARδ, differently from inoculation of LPS, that induced PPARα and PPARγ expression. Conclusions Contamination of the root canals by microorganisms from oral cavity induced the development of apical periodontitis differently than by inoculation with LPS, characterized by less bone loss than the first model. Regardless of the model used, it was found a local increase in the synthesis of mRNA for the enzymes 5-lipoxygenase and cyclooxygenase-2 of the arachidonic acid metabolism, as well as in the surface and nuclear receptors for the lipid mediators prostaglandin E2 and leukotriene B4.
Subject(s)
Animals , Male , Periapical Periodontitis/microbiology , Dinoprostone/metabolism , Lipopolysaccharides/metabolism , Leukotriene B4/metabolism , Dental Pulp Cavity/microbiology , Periapical Periodontitis/metabolism , Periapical Periodontitis/pathology , Time Factors , Bone Resorption/metabolism , Bone Resorption/microbiology , Arachidonate 5-Lipoxygenase/analysis , Arachidonate 5-Lipoxygenase/metabolism , RNA, Messenger/analysis , RNA, Messenger/metabolism , Dinoprostone/analysis , Random Allocation , Gene Expression , Leukotriene B4/analysis , Reverse Transcriptase Polymerase Chain Reaction , Dental Pulp Cavity/metabolism , Dental Pulp Cavity/pathology , Cyclooxygenase 2/analysis , Cyclooxygenase 2/metabolism , Mice, Inbred C57BLABSTRACT
Inflammation is one of the most prominent characteristic features of chronic liver disease, liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Most of HCC cases develop in patients with cirrhosis and cirrhosis develops in patients with chronic liver inflammation. Therefore, there is no doubt that there exist some strong connection among inflammation, fibrosis, and cancer. In fact, chronic unresolved inflammation is associated with persistent hepatic injury and concurrent regeneration, leading to sequential development of fibrosis, cirrhosis, and eventually HCC. This review will discuss the common mechanism of inflammation and fibrosis in chronic liver diseases, and then demonstrate why HCC develops in inflammatory and fibrotic conditions.
Subject(s)
Humans , Carcinoma, Hepatocellular/etiology , Gram-Negative Bacteria/growth & development , Hepatitis, Chronic/complications , Hypoxia , Inflammation , Lipopolysaccharides/metabolism , Liver/metabolism , Liver Cirrhosis/complications , Liver Neoplasms/etiology , Toll-Like Receptors/metabolismABSTRACT
A microbiota intestinal, adquirida no período pós-natal, é composta por grande diversidade de bactérias que desempenham diferentes funções no hospedeiro humano, entre elas a absorção de nutrientes, proteção contra patógenos e modulação do sistema imune. O conteúdo bacteriano intestinal ainda não é totalmente conhecido, mas sabe-se que é influenciado por fatores internos e principalmente externos que modulam sua composição e função. Estudos indicam que a microbiota intestinal difere em indivíduos magros e obesos e ainda naqueles que mantêm hábitos alimentares diferentes. Há evidências de que as relações entre dieta, inflamação, resistência à insulina e risco cardiometabólico são em parte mediadas pela composição de bactérias intestinais. Conhecimentos sobre a microbiota poderão reverter em diferentes estratégias para manipular as populações bacterianas e promover saúde. Esta revisão aborda a relevância do conhecimento sobre o papel de fatores ou padrões alimentares na composição da microbiota, assim como mecanismos fisiopatológicos de doenças metabólicas crônicas e as potencialidades de prebióticos e probióticos sobre o perfil de risco cardiometabólico.
The gut microbiota obtained after birth is composed of a large range of bacteria that play different roles in the human host, such as nutrient uptake, protection against pathogens and immune modulation. The intestinal bacterial content is not completely known, but it is influenced by internal, and mainly by external factors, which modulate its composition and function. Studies indicate that the gut microbiota differs in lean and obese individuals, and in individuals with different food habits. There is evidence that the relationship between diet, inflammation, insulin resistance, and cardiometabolic risk are, in part, mediated by the composition of intestinal bacteria. Knowledge about the gut microbiota may result in different strategies to manipulate bacterial populations and promote health. This review discusses the relevance of understanding the role of dietary factors or patterns in the composition of the microbiota, as well as pathophysiological mechanisms of chronic metabolic diseases, and the potential of prebiotics and probiotics on the cardiometabolic risk profile.
Subject(s)
Animals , Humans , Feeding Behavior/physiology , Intestines/microbiology , Microbiota/physiology , Angiopoietins/metabolism , Diet, High-Fat/adverse effects , Glucose Metabolism Disorders/etiology , Hypertension/etiology , Lipid Metabolism Disorders/etiology , Lipopolysaccharides/metabolism , Obesity/etiology , Prebiotics , Probiotics , Risk FactorsABSTRACT
Obesity is a pandemic which has been rapidly developing for three decades. When a population is submitted to the same nutritional stress, some individuals are less susceptible to diet-induced weight gain and hyperglycemia. This observation suggests that other mechanisms are involved which are not directly related to the human genome. The human gut contains an immense number of microorganisms, collectively known as the microbiota. Evidence that gut microbiota composition can differ between obese and lean humans has led to the speculation that gut microbiota can participate in the pathophysiology of obesity. Different mechanisms have been proposed to explain the link between gut flora and obesity. The first mechanism consists in the role of the gut microbiota to increase energy extraction from indigestible dietary polysaccharides. The second, consists in the role of gut flora to modulate plasma lipopolysaccharide levels which triggers chronic low-grade inflammation leading to obesity and diabetes. A third mechanism proposes that gut microbiota may induce regulation of host genes that modulate how energy is expended and stored. However, further studies are needed to clarify a number of issues related to the relationship between the gut microbiota and obesity.
A obesidade é uma pandemia que afeta milhões de pessoas em todo o mundo. Quando uma população é submetida ao mesmo estresse nutricional, alguns indivíduos são menos suscetíveis ao ganho de peso induzido pela dieta e à hiperglicemia. Essa observação sugere que outros mecanismos não diretamente relacionados ao genoma humano estejam envolvidos. O intestino humano é colonizado por milhões de bactérias, que coletivamente constituem a flora comensal normal. A evidência de que a composição da flora intestinal pode ser diferente em humanos magros e obesos levou à especulação de que a flora intestinal pode participar na fisiopatologia da obesidade. Diferentes mecanismos foram propostos para tentar explicar a correlação entre flora intestinal e obesidade. O primeiro mecanismo consiste no papel da flora intestinal na extração de energia de polissacarídeos não digeríveis. O segundo mecanismo envolve a modulação dos níveis de lipopolissacarídeo pela flora intestinal, o que desencadeia uma inflamação crônica subclínica que acarreta obesidade e diabetes. Um terceiro mecanismo propõe que a flora intestinal pode induzir a regulação de genes do hospedeiro que modulam como a energia é gasta e armazenada. Entretanto, estudos adicionais são necessários para estabelecer o papel da flora intestinal no desenvolvimento da obesidade.
Subject(s)
Animals , Humans , Bacterial Physiological Phenomena , Intestines/microbiology , Metagenome , Obesity/microbiology , Dietary Fats/administration & dosage , Energy Intake , Energy Metabolism , Intestines/metabolism , Lipopolysaccharides/metabolism , Obesity/etiology , Obesity/physiopathology , Obesity/therapy , Polysaccharides/chemistry , Translational Research, BiomedicalABSTRACT
Granulocyte macrophage-colony stimulating factor (GM-CSF) has immuno-stimulatory effects. We hypothesized that GM-CSF plays an important role both in lipopolysaccharide (LPS)- and hemorrhage-induced acute lung injury (ALI). We also postulated that GM-CSF augments LPS-induced inflammation by priming neutrophils. ALI was induced in GM-CSF-/- or control C57BL mice either by LPS injection or by hemorrhage. Lung inflammation (by lung expression for tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2), interleukin-1beta (IL-1beta), interleukin- 6 (IL-6), and keratinocyte-derived chemokine) and lung injury (by myeloperoxidase and evans blue dye assay) were evaluated after ALI. Incremental doses of LPS (0, 1, 10, and 100 ng/mL) and GM-CSF (0, 1, 10, and 100 ng/mL) were added to bone marrow neutrophils. The expression of TNF-alpha, MIP-2, and IL-1beta was evaluated with enzyme linked immunosorbent assay. The mRNA expression of three cytokines, and the nuclear translocation of nuclear factor kappa B (NF kappa-B) were evaluated by reverse transcriptase-polymerase chain reaction and electropnoretic mobility shift assay, respectively. GM-CSF -/- mice showed decreased neutrophil infiltration, less leakage, and lower expression of cytokines in the lung after LPS or hemorrhage. GM-CSF augmented LPS-induced protein and mRNA expression of TNF-alpha, MIP-2 and IL-1beta, which was mediated by increased intra-nuclear translocation of NF-kappa B. GM-CSF plays an important role in high-dose LPS and hemorrhage-induced ALI, which appears to be mediated by its priming effect on neutrophils.
Subject(s)
Animals , Male , Mice , Bone Marrow Cells/cytology , Chemokine CXCL2/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Interleukin-1beta/metabolism , Lipopolysaccharides/metabolism , Lung/metabolism , Lung Injury , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Neutrophils/cytology , Peroxidase/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Phage therapy has been used in the past as an alternative therapy against bacterial pathogens. However, phage-resistant bacterial strains can emerge. Some studies show that these phage-resistant strains are avirulent. In this study, we report that phage-resistant strains of Salmonella enterica serovar Enteritidis (hereafter S. Enteritidis) were avirulent in the Caenorhabditis elegans animal model. We isolated phage-resistant strains of S. Enteritidis ATCC 13076 by using three lytic phages (f2aSE, f3aSE and f18aSE). In these mutants, we explored different virulence factors like lipopolysaccharide (LPS), virulence plasmid (Pla), motility and type I fimbriae, all of which may have effects on virulence and could furthermore be related to phage resistance. The phage-resistant strains of S. Enteritidis showed loss of O-Polysaccharide (O-PS) and auto-agglutination, present a rough phenotype and consequently they are avirulent in the C. elegans animal model. We speculate that the O-PS is necessary for phage attachment to the S. Enteritidis cell surface.
Subject(s)
Bacteriophages , Lipopolysaccharides/metabolism , Salmonella enteritidis/pathogenicity , Salmonella enteritidis/virology , Caenorhabditis elegans/microbiology , Drug Resistance, Microbial , MutationABSTRACT
El lipopolisacarido bacteriano (LPS), tambien denominado endotoxina, es el constituyente mayoritario de la membrana externa de bacterias Gram negativas. Esta molecula es liberada de la bacteria a la circulacion exhibiendo una amplia variedad de efectos toxicos y pro-inflamatorios, los cuales estan asociados al lipido A y a su vez estan relacionados a la patogenesis de la sepsis. Muchos de los fenomenos fisiologicos producidos por el LPS resultan de la capacidad de esta molecula de activar las celulas del sistema inmune del huesped, entre ellas monocitos, macrofagos y leucocitos polimorfonucleares. Este proceso produce una inflamacion local, proceso beneficioso para el huesped. Sin embargo, si la cantidad de LPS liberado excede cierta concentracion critica umbral, la exacerbada liberacion de citoquinas inflamatorias como Factor de Necrosis Tumoral (TNF-alfa) e interleuquinas (IL) resulta en sepsis grave, lo que hace necesario encontrar nuevas opciones terapeuticas capaces de neutralizar la endotoxina circulante. En este articulo se presenta una revision actualizada de los resultados experimentales obtenidos in vivo e in vitro empleando proteinas y peptidos sinteticos con la finalidad de neutralizar el LPS, y las perspectivas que en este area ofrece el uso de lipoproteinas, en particular la apolipoproteina A-I y formas mutantes o peptidos derivados de esta proteina.
Lipopolisaccharide (LPS), also called endotoxin, is the major component of the external membrane in Gram negative bacteria. This molecule is released to circulation by the bacteria, producing a large variety of toxic and pro-inflammatory effects which are associated with lipid A as well as with sepsis pathogenesis. Many physiological henomena produced by LPS arise from this molecule's capacity to activate cells in the host immune system such as monocytes, macrophages and polymorphonuclear leukocytes. This process leads to a local inflammation, and it is beneficial for the host. However, if the amount of LPS released exceeds the critical concentration thresholdan augmented release of inflammatory cytokines as TNF-alfa, and interleukines (IL) produce a severe sepsis. This fact led us to find therapeutical alternatives able to neutralize circulating endotoxin. This work is focused on the experimental results obtained in vivo and in vitro using synthetic proteins and peptides in order to neutralizeLPS, and on future perpectives in this research area that offer the use of lipoprotein and in particular apolipoprotein A-I and mutants or peptides derived from this protein.
Subject(s)
Humans , Endotoxins/antagonists & inhibitors , Gram-Negative Bacteria , Lipopolysaccharides/antagonists & inhibitors , Peptides/pharmacology , Sepsis/drug therapy , Anti-Infective Agents/therapeutic use , Apolipoprotein A-I/metabolism , Endotoxins/chemistry , Endotoxins/metabolism , Gram-Negative Bacteria/drug effects , Gram-Negative Bacteria/metabolism , Inflammation , Interleukins/metabolism , Lipopolysaccharides/chemistry , Lipopolysaccharides/metabolism , Lipoproteins, HDL/chemistry , Lipoproteins, HDL/physiology , Peptides/metabolism , Recombinant Proteins , Sepsis/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
OBJECTIVES: The goals of these studies were to characterize the interaction of the P22 phage particle with the Salmonella cell surface and to determine the phage elements involved in this interaction by mutational analysis. BACKGROUND: The phage P22 has been characterized extensively. The gene and protein for the phage P22 tailspike, which is the phage adsorption organelle, have been intensively studied. The kinetics of the interaction of the tailspike protein with the cell surface has been studied in detail, surprisingly no mutational analysis has ever been reported that has defined these components and their interaction between themselves and the cell surface. The main and perhaps only component needed for this cell surface interaction is the tailspike protein. METHODS: Adsorption to the cell surface has been measured in the wild type phage and in mutant derivatives, isolated in this study. Phage mutants have been isolated after hydroxylamine mutagenesis. RESULTS: The adsorption of P22 to the cell surface is a temperature-independent event. Forty putative phage adsorption mutants have been isolated. A sample of them have been further analyzed. These divide the adsorption process into at least two stages. One stage contains mutants that absorb with essential wild type phage kinetics to the cell surface while the other stage with delayed adsorption kinetics. CONCLUSIONS: The interaction of the phage P22 with the Salmonella cell surface has been shown to be a complicated one which is temperature-independent and multi-stage. Mutants isolated in this study may help dissect this process even further
Subject(s)
Humans , Adsorption , /metabolism , Salmonella typhimurium/virology , /ultrastructure , Lipopolysaccharides/metabolism , Viral Tail Proteins/metabolism , Salmonella typhimurium/metabolism , Salmonella typhimurium/ultrastructure , TemperatureABSTRACT
Mammalian epithelia produce the various antimicrobial peptides against the bacterial or viral infection, thereby acting as the active immune modulators in the innate immunity. In this study, we examined the effects of the various proinflammatory cytokines or LPS on cell viability and antimicrobial beta-defensin gene expressions in human corneal epithelial cells. Results showed that the cytokines or LPS did not exert severe cytotoxic effects on the cells, and that beta-defensin 1 was constitutively expressed, while beta-defensin 2 was specifically induced by IL-1beta, supporting the idea that these cytokines or LPS involve the defense mechanism in the cornea. Furthermore, the reporter and gel shift assay to define the induction mechanism of beta-defensin 2 by IL-1beta demonstrated that the most proximal NF-kB site on the promoter region of beta-defensin 2 was not critical for the process. Data obtained from the normal or patients with the varying ocular diseases showed that our in vitro results were relevant in the clinical settings. Our results clearly demonstrated that beta-defensin 1 and 2 are important antimicrobial peptides in the corneal tissues, and that the mechanistic induction process of beta-defensin 2 by IL-1beta is not solely dependent on proximal NF-kB site activation, thus suggesting that the long distal portion of the promoter is needed for the full responsiveness toward IL-1beta.
Subject(s)
Humans , Binding, Competitive , Cell Survival , Cells, Cultured , Corneal Diseases/metabolism , Electrophoretic Mobility Shift Assay , Epithelium, Corneal/drug effects , Gene Expression , Interferon-gamma/metabolism , Interleukin-1/pharmacology , Lipopolysaccharides/metabolism , NF-kappa B/metabolism , Promoter Regions, Genetic/drug effects , Tumor Necrosis Factor-alpha/metabolism , beta-Defensins/biosynthesisABSTRACT
Legume-Rhizobium symbiosis is a multistep process characterized by the formation of root nodules on the host plant. A number of genes from both symbiotic partners share information during the interaction process. Nodulation genes (nod, nol and noe) have been classified as common nodulation genes and host specific (hsn) nodulation genes. Though common nodulation genes are enough to form root nodules, host specific nodulation genes are needed for specific interaction leading to formation of functional nodules. Core lipochitooligosaccharides (LCOs), the products of common nodulation genes are modified by the action of host specific nodulation genes. LCOs seem to be present in legumes as well as nonlegume and are known to act as a morphogen by acting as auxin-transport inhibitor. The understanding of Nod factor may contribute to reveal complex biological functions such as developmental regulation, signal transduction and plant morphogenesis.
Subject(s)
Fabaceae/microbiology , Genes, Bacterial , Lipopolysaccharides/metabolism , Plant Growth Regulators/physiology , Plants, Medicinal , Rhizobium/genetics , Signal Transduction , SymbiosisABSTRACT
To investigate the mechanism of pregnancy termination following immuno-neutralization of riboflavin carrier protein (RCP) and to use acceptable adjuvants, we actively immunized female rats with reduced and carboxymethylated RCP (RCM-RCP) using various adjuvants (during primary immunization) such as sodium phthalylated lipopolysaccharide (SPLPS), purified S. typhi outer membrane proteins (porins) and a combination of them. Rats (5-14 per group) were immunized with alugel adsorbed RCM-RCP (100 microg/dose) either alone or with SPLPS or porins or SPLPS+porins. Control animals received RCM-RCP emulsified with Fruend's completelincomplete adjuvants (FCA/FIA). All animals received five boosters at intervals of 21 days. The lowest (4 X 10(-3)) and the highest (> 70 X 10(-3)) anti-RCM-RCP antibody titers were observed in alugel adsorbed-RCM-RCP group and control groups, respectively. Immunized animals showed reduced fertility following 3rd, 4th and 5th boosters. Reduction in fertility was 30-60% in alugel adsorbed RCM-RCP group, 90-100% in FCA-RCM-RCP group and 80-90% in SPLPS+porins group. Fertility reduction was not strictly correlatable with the serum antibody titers. RCP-specific IgG could be localized in the uterine endometrial glands and luminal epithelial cells in the immunized animals. Animals in the FCA/FIA group showed abnormal implantation/resorption sites and their histological sections showed degenerated embryos. But, day 5 preimplantation embryos were normal. These results show that (a) SPLPS+porins can be used as adjuvants in place of FCA/FIA for active immunization against RCM-RCP and (b) early termination of pregnancy in the immunized animals is due largely to the failure of normal embryo implantation.
Subject(s)
Abortifacient Agents/pharmacology , Abortion, Veterinary/chemically induced , Adjuvants, Immunologic/pharmacology , Animals , Azo Compounds/diagnosis , Blastocyst , Carrier Proteins/immunology , Endometrium/pathology , Female , Fetal Death/chemically induced , Immunoglobulin G/immunology , Lipopolysaccharides/metabolism , Male , Membrane Transport Proteins , Methylation , Peptide Fragments/immunology , Porins/metabolism , Pregnancy , Pregnancy, Animal/blood , Rats , Rats, Wistar , Riboflavin/metabolism , Trypan Blue , VaccinationABSTRACT
CD14 is a leukocyte surface molecule expressed on monocytes but not on lymphocytes. Recently, CD14 molecule was demonstrated to function as a receptor for endotoxin. CD14 specific monoclonal antibody (MAb), therefore, can be used to identify monocytes and study the host defense mechanism to bacterial endotoxin. To produce MAb against CD14 protein, in this study cDNA encoding CD14 protein and COS cell expression systems were used to prepare CD14 expressing COS cells. The CD14 transfectants were then used as antigen for mouse immunization. The spleen cells of the immunized mouse were then fused with myeloma cells by conventional hybridoma technique. By using this strategy, 5 hybridroma clones secreting antibody specific for CD14 molecule were generated within one fusion. The generated CD14 MAbs were strongly positive with monocytes, weakly positive with neutrophils but negative with lymphocytes. In addition, the generated CD14 MAb blocked the binding of lipopolysaccharide (LPS) to the CD14 molecules. These CD14 MAbs could be used to enumerate peripheral blood monocytes as well as using referent CD14 MAb. We, therefore, introduce an alternative method for preparation of antigen for production of monoclonal antibody. This type of antigen is a very effective antigen for the production of monoclonal antibodies against cell surface molecules.
Subject(s)
Animals , Antibodies, Monoclonal/biosynthesis , Antibody Specificity , Lipopolysaccharide Receptors/genetics , COS Cells , DNA, Complementary/genetics , Flow Cytometry , Gene Expression , Immunization , Immunophenotyping , Lipopolysaccharides/metabolism , Lymphocytes/immunology , Mice , Monocytes/immunology , Neutrophils/immunology , Recombinant Proteins/genetics , TransfectionABSTRACT
En los últimos años el óxido nítrico (ON), una molécula sencilla pero altamente reactiva, ha sido el centro de una gran cantidad de investigaciones. Es sintetizado en los tejidos mamíferos a partir del aminoácido L-arginina por una reacción enzimática catalizada por la sintasa de óxido nítrico (SON), produciendo L-citrulina y, ON. El ON tiene una media vida muy corta, es liposoluble, reacciona fácilmente con un gran número de sistemas enzimáticos, y es producido por una amplia variedad de células. La enzima SON existe en, al menos, tres formas: dos isoformas son calcio dependientes y están continuamente presentes en células específicas, llamadas SON constitutivas (CSON). De éstas, una isoforma está presente en el citosol de las células neuronales (nSON), mientras la otra isoforma está presente en forma de proteína ligada a las membranas en las células endoteliales (eSON). Las cSON producen pequeñas cantidades de ON, después de que ocurre una estimulación por agonistas específicos. El ON producido por las cSON frecuentemente media señales celulares y comunicación celulas. Una tercera isofroma de SON es calcio-independientemente, no está presente en células no estimuladas y produce grandes cantidades de ON siguiendo a la estimulación de células apropiadas con citoquinas o lipopolisacáridos. Esta isoforma es llamada SON inducible (iSON). El ON es un mediador tanto de procesos fisiológicos como patológicos. Actúa directamente en sus blancos celulares, de los cuales el más importante es la guanilatociclasa, y produce una gran variedad de efectos biológicos, que van desde citoprotección a citotoxicidad. Es motivo de la presente revisión un análisis de la bio-química y fisiología del ON, así como de sus acciones biológicas y posibles implicaciones terapéuticas
Subject(s)
Animals , Amino Acids/biosynthesis , Lipopolysaccharides/metabolism , Lipopolysaccharides/chemical synthesis , Nitric Oxide/biosynthesisABSTRACT
The pathophysiology of organ system failure in sepsis, in particular the effects of septic shock on the central nervous system, are still incompletely understood. Lipopolysaccharide (LPS) from Gram-negative bacteria affects the permeability of the blood-brain barrier and causes the activation of brain microglia. A growing body of research supports involvement of activated brain microglia in brain pathologies caused by infectious diseases, trauma, tumors, ischemia, Alzheimer's disease, Parkinson's disease, Down's syndrome, multiple sclerosis and AIDS. Those seminal studies that have contributed to the characterization of the in vivo and in vitro effects of LPS on microglia function, mediator generation and receptor expression are presented within a historical perspective. In particular, all those in vitro studies on O2-, H2O2 and NO. generation by either unprimed or primed microglia have been extensively reviewed. The apparent controversial effect of LPS on microglia O2- is discussed. Because treatment modalities for septic shock have not significantly affected the current high mortality, alternative strategies with antioxidants are currently being investigated. Reduction of microglia O2- generation is proposed as a possible complementary strategy to antioxidative therapy for septic shock and CNS pathologies that involve activated microglia.
Subject(s)
Rats , Mice , Animals , Cricetinae , Antioxidants/therapeutic use , Blood-Brain Barrier/physiology , Central Nervous System Diseases/etiology , Inflammation Mediators/metabolism , Lipopolysaccharides/metabolism , Microglia/metabolism , Reactive Oxygen Species/metabolism , Shock, Septic/therapy , Central Nervous System Diseases/therapy , Shock, Septic/complicationsABSTRACT
Los lipopolisacáridos bacterianos (LPS) son los principales componentes de las membranas de las bacterias gram negativas. Los LPS constituyen poderosos estímulos para las células del sistema inmune y están asociados, frecuentemente, al desencadenamiento del síndrome séptico o al shock séptico. Los polimorfonucleares neutrófilos humanos (PMN) son una de las células más importantes involucradas en la depuración de los LPS. El conocimiento de los mecanismos de detoxificación de los LPS es crucial para el control del síndrome séptico o del shock séptico causados por bacterias Gram negativas. En este trabajo estudiamos la capacidad de los PMN en la detoxificación de los LPS en dos situaciones diferentes: a) cuando el LPS es ofertado a los PMN como una molécula aislada; b) cuando el LPS ofrecido a los PMN es parte constitutiva de las bacterias Gram negativas (E. coli 0111:B4). Nuestros resultados demuestran que los PMN son capaces de inhibir la actividad biológica de los LPS de generar el factor de necrosis tumoral alfa (TNF-alpha). Sin embargo, cuando los LPS se ofrecen como componentes de las bacterias enteras, los PMN inducen la liberación de los LPS bacterianos sin modificar su actividad biológica. Esto determina que la capacidad depuradora de los PMN esté condicionada por la forma en la cual los LPS son presentados a estas células.
Subject(s)
Gram-Negative Bacteria/metabolism , Lipopolysaccharides/metabolism , Neutrophils/metabolism , Sepsis , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
The objectives of the present investigation were 1) to study the effect of bacterial lipopolysaccharide (LPS) on rat gastric emptying (GE) and 2) to investigate a possible involvement of the vagus nerve in the gastric action of LPS. Endotoxin from E. coli (strain 055:B5) was administered sc, ip or iv to male Wistar rats (220-280 g body weight) at a maximum dose of 50 mug/kg animal weight. Control animals received an equivalent volume of sterile saline solution. At a given time period after LPS administration, GE was evaluated by measuring gastric retention 10 min after the orogastric infusion of a test meal (2 ml/l00 g animal weight), which consisted of 0.9 per cent NaCl plus the marker phenol red (6 mg/dl). One group of animals was subjected to bilateral subdiaphragmatic vagotomy or sham operation 15 days before the test. A significant delay in GE of the test meal was observed 5 h after iv administration of the endotoxin at the dose of 50 mug/kg animal weight. The LPS-induced delay of GE was detected as early as 30 min and up to 8 h after endotoxin administration. The use of different doses of LPS ranging from 5 to 50 mug/kg animal weight showed that the alteration of GE was dose dependent. In addition, vagotomized animals receiving LPS displayed a GE that was not significantly different from that of the sham control group. However, a participation of the vagus nerve in LPS-induced delay in GE could not be clearly demonstrated by these experiments since vagotomy itself induced changes in this gastric parameter. The present study provides a suitable model for identifying the mechanisms underlying the effects of LPS on gastric emptying.
Subject(s)
Rats , Animals , Male , Bacterial Infections/metabolism , Escherichia coli/pathogenicity , Gastric Emptying/physiology , Lipopolysaccharides/metabolism , Vagotomy , Rats, WistarABSTRACT
Diverse conditions for stimulating human mononuclear cells to release thymocyte costimulatory factors were tested for their contribution to the generation of supernatants high titers of these monokines. Activity titers increased with LPS concentration, reaching a plateau between 1 and 10 microng/ml. Indomethacin did not modify the monokine, but the assay for thymocyte costimulatory activity was substantially affected by inhibitory substances produced by the monocytes in the absence of indomethacin. The use of nylon wool columns to trap the cells was shown to be effective in raising cellular densities without decreasing activity titers. As result, the yield per cell could be maintained even in the absence of serum, an important step toward the goal of purifiying bioactive from crude broths
Subject(s)
In Vitro Techniques , Lipopolysaccharides/metabolism , Monocytes/isolation & purification , Monokines/metabolism , Thymus Gland/cytology , Culture Media , Mice, Inbred BALB C , Mice, Inbred C3H , Microscopy, Phase-Contrast , Monocytes/physiologyABSTRACT
La producción de IL-1 por células esplénicas mononucleares adherentes (CMA) de ratones BALB/c inoculados con Sarcoma 180(S180) fue estudiada como uno de los posibles mecanismos responsables de la inmunosupresión observada durante el crecimiento tumoral. Como agentes estimulantes se utilizó un polisacárido químicamente definido, PCj3, y un lipopolisacárido de E. coli (LPS). La actividad de IL-1 se valoró en base al efecto estimulante de los sobrenadantes de cultivos en CMA sobre la respuesta proliferativa de timocitos murinos frente a la fitohemaglutina (PHA). Ambos esdtimulantes indujeron niveles comparables de IL-1 tanto en ratones normales como en portadores de S180 de 10 días. A los 20 y 30 días de desarrollo tumoral, las CMA disminuyeron significativamente la producción de IL-1 en respuesta a ambos estimulantes. Esos resultados permiten suponer que la inmunosupresión observada en los ultimos estadíos del crecimientos tumoral podría ser consecuencia de la inhibición de la producción de IL-1