RESUMO
Trapping/separating bio-entities via magnetic field gradients created a vast number of possibilities to develop biosensors for the early detection of diseases without the need for expensive equipment or physician/lab technicians. Thus, opening a window for at-home disposable rapid test kits. In the scope of the current work, an innovative and cost-effective technique to form well-organized arrays of Nd-Fe-B patterns was successfully developed. High aspect ratio Nd-Fe-B flakes were synthesized by surfactant-assisted ball milling technique. Nd-Fe-B flakes were distributed and patterned into a PDMS matrix by the aforementioned technique. A microfluidic channel was integrated on the fabricated Nd-Fe-B/PDMS patch with a high magnetic field gradient to form a microfluidic device. Fe nanoparticles, suspended in hexane, were flowed through the microfluidic channel, and trapping of the magnetic nanoparticles was observed. More experiments would be needed to quantitatively study efficiency. Ergo, the microfluidic device with high trapping efficiency was developed. The established technique has the potential to outperform the precedents in trapping efficiency, cost, and ease of production. The developed device could be integrated into disposable test kits for the early detection of various diseases.
RESUMO
We describe novel features of the induced magnetic anisotropy in Co nanoclusters coupled with a CoO(111) layer. Individual cluster magnetism was studied using new microbridge superconducting quantum interference devices. Intrinsically, the Co clusters are single domains with an effective anisotropy constant K(F)≈1.5×10(6) erg·cm(-3). A bistable state of the ferromagnetic-antiferromagnetic coupling is revealed, with a maximum bias systematically observed along CoO[10 Ì 1] and an interfacial coupling energy of 0.9 erg·cm(-2). The small bias observed in cluster assembly results from an averaging over the two opposite stable states.
RESUMO
LPS-binding protein (LBP) and CD14 potentiate cell activation by LPS, contributing to lethal endotoxemia. We analyzed the contribution of LBP/CD14 in models of bacterial infection. Mice pretreated with mAbs neutralizing CD14 or LBP showed a delay in TNF-alpha production and died of overwhelming infection within 24 h, after a challenge with 250 CFU of virulent Klebsiella pneumoniae. Blockade of TNF-alpha also increased lethality, whereas pretreatment with TNF-alpha protected mice, even in the presence of LBP and CD14 blockade. Anti-LBP or anti-CD14 mAbs did not improve or decrease lethality with a higher inoculum (10(5) K. pneumoniae) and did not affect outcome following injections of low or high inocula of Escherichia coli O111. These results point to the essential role of LBP/CD14 in innate immunity against virulent bacteria.
Assuntos
Proteínas de Fase Aguda , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Bactérias Gram-Negativas/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/metabolismo , Glicoproteínas de Membrana , Animais , Anticorpos Monoclonais , Bacteriemia/etiologia , Bacteriemia/imunologia , Proteínas de Transporte/antagonistas & inibidores , Endotoxemia/imunologia , Escherichia coli/imunologia , Escherichia coli/patogenicidade , Feminino , Bactérias Gram-Negativas/patogenicidade , Infecções por Bactérias Gram-Negativas/etiologia , Infecções por Bactérias Gram-Negativas/imunologia , Klebsiella pneumoniae/imunologia , Klebsiella pneumoniae/patogenicidade , Lipopolissacarídeos/toxicidade , Camundongos , Testes de Neutralização , Neutrófilos/imunologia , Sepse/etiologia , Sepse/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/farmacologia , VirulênciaRESUMO
Plasma lipopolysaccharide (LPS)-binding protein (LBP) and membrane CD14 function to enhance the responses of monocytes to low concentrations of endotoxin. Surprisingly, recent reports have suggested that LBP or CD14 may be dispensable for macrophage responses to low concentrations of LPS or may even exert an inhibitory effect in the case of LBP. We therefore investigated whether LBP and CD14 participated in the response of mouse peritoneal exudate macrophages (PEM) to LPS stimulation. In the presence of a low amount of plasma (<1%) or of recombinant mouse or human LBP, PEM were found to respond to low concentrations of LPS (<5 to 10 ng/ml) in an LBP- and CD14-dependent manner. However, tumor necrosis factor production (not interleukin-6 production) by LPS-stimulated PEM was reduced when cells were stimulated in the presence of higher concentrations of plasma or serum (5 or 10%). Yet, the inhibitory effect of plasma or serum was not mediated by LBP. Taken together with previous results obtained with LBP and CD14 knockout mice in models of experimental endotoxemia, the present data confirm a critical part for LBP and CD14 in innate immune responses of both blood monocytes and tissue macrophages to endotoxins.
Assuntos
Proteínas de Fase Aguda , Proteínas de Transporte/fisiologia , Receptores de Lipopolissacarídeos/fisiologia , Lipopolissacarídeos/toxicidade , Macrófagos Peritoneais/efeitos dos fármacos , Glicoproteínas de Membrana , Plasma/fisiologia , Animais , Feminino , Humanos , Ativação de Macrófagos , Macrófagos Peritoneais/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Cellular responses to LPS, the major lipid component of the outer membrane of Gram-negative bacteria, are enhanced markedly by the LPS-binding protein (LBP), a plasma protein that transfers LPS to the cell surface CD14 present on cells of the myeloid lineage. LBP has been shown previously to potentiate the host response to LPS. However, experiments performed in mice with a disruption of the LBP gene have yielded discordant results. Whereas one study showed that LBP knockout mice were resistant to endotoxemia, another study did not confirm an important role for LBP in the response of mice challenged in vivo with low doses of LPS. Consequently, we generated rat mAbs to murine LBP to investigate further the contribution of LBP in experimental endotoxemia. Three classes of mAbs were obtained. Class 1 mAbs blocked the binding of LPS to LBP; class 2 mAbs blocked the binding of LPS/LBP complexes to CD14; class 3 mAbs bound LBP but did not suppress LBP activity. In vivo, class 1 and class 2 mAbs suppressed LPS-induced TNF production and protected mice from lethal endotoxemia. These results show that the neutralization of LBP accomplished by blocking either the binding of LPS to LBP or the binding of LPS/LBP complexes to CD14 protects the host from LPS-induced toxicity, confirming that LBP is a critical component of innate immunity.
Assuntos
Proteínas de Fase Aguda , Anticorpos Bloqueadores/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Apresentação de Antígeno/imunologia , Proteínas de Transporte/imunologia , Endotoxemia/prevenção & controle , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/metabolismo , Glicoproteínas de Membrana , Animais , Anticorpos Bloqueadores/química , Anticorpos Monoclonais/química , Sítios de Ligação/imunologia , Células CHO , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Linhagem Celular , Cricetinae , Endotoxemia/imunologia , Feminino , Fluoresceína-5-Isotiocianato/metabolismo , Receptores de Lipopolissacarídeos/imunologia , Lipopolissacarídeos/antagonistas & inibidores , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos BALB C , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Overproduction of nitric oxide (NO) upon expression of inducible NO synthase (iNOS) may be responsible for refractory hypotension in septic shock. Whereas high levels of NOS activity have been documented in experimental models of endotoxemia or intravenous challenge with Escherichia coil, much less is known concerning tissue models of Gram-negative infection. We examined NO production (measured as the accumulation of plasma NO3- + NO2-) in a murine model of Gram-negative peritonitis. Plasma NO3- + NO2- increased progressively from 25 microM to peak levels of 50-150 microM 24 h after intraperitoneal challenge with E. coli 0111:B4, similar to values reported for septic shock patients. Treatment of infected mice with NG-monomethyl-L-arginine, an inhibitor of NOS activity, resulted in the efficient inhibition of NO3- + NO2- production. In order to evaluate the roles of interferon-gamma (IFN-gamma) and tumor necrosis factor (TNF-alpha) in the induction of NO synthesis in murine peritonitis, mice deficient in the respective cytokine receptors were studied. In control in vitro experiments, macrophages from IFN-gammaR- or TNFR55-deficient mice, while failing to respond to IFN-gamma or TNF-alpha, respectively, produced high levels of NO under appropriate stimulation. When challenged intraperitoneally with E. coli, IFN-gammaR- or TNFR55-deficient mice exhibited similar levels of bacteremia and NO production as their wild-type controls. These data thus suggest that enhanced NO production during focal Gram-negative infection may occur in the absence of signaling through either IFN-gammaR or TNFR55.
Assuntos
Infecções por Bactérias Gram-Negativas/metabolismo , Óxido Nítrico/metabolismo , Peritonite/metabolismo , Receptores de Citocinas/deficiência , Sepse/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Feminino , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Mutantes , Peritonite/microbiologia , Receptores de Citocinas/genética , Receptores de Interferon/deficiência , Receptores de Interferon/genética , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral , Receptor de Interferon gamaRESUMO
We have previously described cross-reactive antilipopolysaccharide (anti-LPS), or anti-endotoxin, monoclonal antibodies (MAbs) which provide cross-protection in several systems of endotoxin bioactivity. The protective effects of the murine cross-reactive MAb WN1 222-5 (immunoglobulin G2a(kappa) [IgG/2a(kappa)]) and of its chimerized version, SDZ 219-800 [human IgG1(kappa)], have now been evaluated in lethality models against LPS from three different serotypes and in bacterial infection models. We confirmed the protective activity of the two MAbs in D-galactosamine-sensitized mice challenged with LPS of other E. coli serotypes (O18, O127, and O111). The protective effect correlated with the suppression of tumor necrosis factor formation. Furthermore, WN1 222-5 enhanced bacterial clearance of intravenously administered E. coli O111 bacteria, thus protecting mice from death. However, the MAbs were unable to provide protection in a peritonitis model (intraperitoneal inoculation). Our study, therefore, shows that LPS cross-reactive antibodies are capable of mediating cross-protection against LPS and bacteria but that the selected models have a clear influence on the results.
Assuntos
Anticorpos Monoclonais/química , Bacteriemia/imunologia , Endotoxemia/imunologia , Epitopos/imunologia , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Animais , Especificidade de Anticorpos , Reações Cruzadas , Modelos Animais de Doenças , Infecções por Escherichia coli/imunologia , Feminino , Galactosamina/imunologia , CamundongosRESUMO
Purified cells walls of Streptococcus mitis induced tumor necrosis factor in vitro in whole blood of both lipopolysaccharide (LPS)-sensitive OF1 and LPS-resistant C3H/HeJ mice. They were as effective as heat-killed bacteria in inducing death in both strains of mice sensitized with D-galactosamine. Lethality was suppressed by anti-tumor necrosis factor antibodies. The histopathophysiological findings in mice after challenge with LPS or gram-positive cell walls were indistinguishable.
Assuntos
Parede Celular/imunologia , Galactosamina/imunologia , Lipopolissacarídeos/toxicidade , Streptococcus/imunologia , Streptococcus/patogenicidade , Fator de Necrose Tumoral alfa/fisiologia , Animais , Antígenos de Bactérias/administração & dosagem , Imunização , Técnicas In Vitro , Fígado/patologia , Camundongos , Camundongos Endogâmicos C3H , Neutrófilos/patologia , Neutrófilos/fisiologia , Choque Séptico/etiologia , Infecções Estreptocócicas/etiologia , VirulênciaRESUMO
OBJECTIVE: To determine levels of lipopolysaccharide binding protein (LBP) in serum and in synovial fluid (SF) of patients presenting with various articular disorders [degenerative arthritis, rheumatoid arthritis (RA), reactive arthritis (ReA)] and to correlate these levels with C-reactive protein (CRP) and interleukin 6 (IL-6), 2 markers of the acute phase response. METHODS: LBP was measured by a radioimmunoassay made up of lipopolysaccharide (LPS) to capture LBP and radiolabelled anti-LBP antibodies to detect LBP. LBP was also measured for its ability to present fluorescein isothiocyanate LPS (FITC-LPS) to human monocytes. CRP was measured by nephelometry and IL-6 bioassay. RESULTS: Levels of LBP in serum and in SF were significantly higher in patients with RA and ReA than in the control group of degenerative arthropathies. In the latter group, LBP values were similar to those found in controls. Serum LBP values correlated positively with SF LBP values. LBP values also correlated with CRP and IL-6 levels measured in SF. Functionally, LBP was found to be active and able to present LPS to monocytes, resulting in tumor necrosis factor-alpha (TNF-alpha) release upon LPS challenge. CONCLUSION: These in vitro data support the observation that LBP could play a major role in local joint disorders. Our results also strengthen the view that LBP may be a new marker of synovial inflammation.
Assuntos
Proteínas de Fase Aguda/metabolismo , Artrite/metabolismo , Proteína C-Reativa/metabolismo , Proteínas de Transporte/metabolismo , Interleucina-6/metabolismo , Glicoproteínas de Membrana , Sinovite/metabolismo , Biomarcadores/análise , Humanos , Monócitos/metabolismo , Proibitinas , Líquido Sinovial/química , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Production of aerobactin has been reported to be a virulence factor in members of the family Enterobacteriaceae. To investigate the protection afforded by humoral immunity directed towards aerobactin in infectious diseases caused by aerobactin-producing strains, we tested the efficacy of mAbAERO1, a murine monoclonal antibody directed to ferric aerobactin, which, in vitro, was found to impair the growth of aerobactin-dependent strains of Enterobacteriaceae under iron-limited conditions. The mortality of mice experimentally infected with the aerobactin-producing strains Escherichia coli V2019 (LD50 = 3.5 x 10(5) CFU/mice) or Klebsiella pneumoniae Caroli (LD50 = 1.3 CFU/mice) was not reduced when 1 mg of mAbAERO1 was injected intravenously 1 h before or 1 h after bacterial challenge. Nor was mortality reduced after challenge with either E. coli V2019 or K. pneumoniae Caroli, even though the active immunization of mice with purified FeAero (ferric aerobactin) conjugated with thyroglobulin as followed by a rise in systemic anti-FeAero antibodies. Lastly, chicks born of hens immunized with FeAero showed evidence of antibody transmission towards FeAero, but were not protected when challenged with E. coli MT78, an aerobactin-producing strain highly virulent for chickens. Therefore, under the experimental conditions tested, humoral immunity against aerobactin appeared to play only a minor role in protection against infections caused by aerobactin-producing members of the family Enterobacteriaceae. However, other experimental models should be tested to confirm these observations.
Assuntos
Vacinas Bacterianas/uso terapêutico , Escherichia coli/imunologia , Ácidos Hidroxâmicos/imunologia , Klebsiella pneumoniae/imunologia , Vacinas Conjugadas/uso terapêutico , Animais , Formação de Anticorpos , Galinhas , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/prevenção & controle , Imunidade Ativa , Infecções por Klebsiella/imunologia , Infecções por Klebsiella/prevenção & controle , CamundongosRESUMO
The serum lipopolysaccharide (LPS) binding protein, LBP, has been shown to greatly enhance cellular responses to low concentrations of LPS. Purified LBP facilitates the transfer of LPS to membrane-bound or soluble CD14; the CD14/LPS complex then triggers a signal in responsive cells. We have cloned and sequenced a cDNA encoding murine LBP, and produced recombinant murine LBP using a baculovirus expression system. Using either a solid-phase or a cytofluorometric assay, recombinant murine and human LBP were found to bind avidly to free LPS, but only weakly to live bacteria from most LPS-containing Gram negative strains. Binding correlated loosely with the length and composition of the polysaccharide O chains. However, recombinant LBP did bind well to all heat-killed bacterial preparations. These findings suggest that LBP could be implicated in the response to killed but not live Gram negative bacteria.
Assuntos
Proteínas de Fase Aguda , Proteínas de Transporte/metabolismo , Bactérias Gram-Negativas/metabolismo , Lipopolissacarídeos/metabolismo , Glicoproteínas de Membrana , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Sequência de Bases , Proteínas de Transporte/genética , Linhagem Celular , Clonagem Molecular , DNA Bacteriano/genética , DNA Complementar/genética , Escherichia coli/metabolismo , Expressão Gênica , Temperatura Alta , Humanos , Técnicas In Vitro , Lipopolissacarídeos/química , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , SpodopteraRESUMO
Fas/Fas ligand and TNF/TNF receptors are involved in apoptosis. Whether both systems are involved in septic shock has not been determined so far. We investigated the role of TNF/TNFR and Fas/Fas ligand in models of endotoxemia and of speticemia in mice. Upon LPS challenge, TNF and TNFR p55 were involved in the process inducing lethality. FasL did not contribute to enhance lethality, as evidenced in gld mice, lacing FasL. Following an intraperitoneal injection of live E. coli, TNF and TNFR p55 were necessary to combat infection. Disruption of either gene was associated with enhanced lethality and failure to clear the bacteria. No effect observed in gld mice in this peritonitis model. Thus, these observations confirmed the pathogenic role of TNF/TNFR in endotoxemia and its beneficial role in local bacterial infections. In addition the data ruled out a major role for Fas/FasL in septic shock in mice.
Assuntos
Infecções por Escherichia coli/etiologia , Glicoproteínas de Membrana/fisiologia , Peritonite/etiologia , Toxemia/etiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Antígenos CD/fisiologia , Apoptose , Modelos Animais de Doenças , Endotoxinas/toxicidade , Infecções por Escherichia coli/terapia , Proteína Ligante Fas , Galactosamina/imunologia , Lipopolissacarídeos/toxicidade , Linfotoxina-alfa/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Peritonite/terapia , Receptores do Fator de Necrose Tumoral/fisiologia , Receptores Tipo I de Fatores de Necrose Tumoral , Choque Séptico/etiologia , Choque Séptico/terapia , Toxemia/terapia , Fator de Necrose Tumoral alfa/antagonistas & inibidoresAssuntos
Proteínas de Fase Aguda , Proteínas de Transporte/fisiologia , Lipopolissacarídeos/metabolismo , Glicoproteínas de Membrana , Choque Séptico/metabolismo , Toxemia/metabolismo , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/imunologia , Galactosamina/farmacologia , Imunoglobulina G/farmacologia , Técnicas In Vitro , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/toxicidade , Camundongos , Monócitos/fisiologia , Testes de Neutralização , Neutrófilos/fisiologia , Choque Séptico/terapia , Toxemia/terapia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Lipopolysaccharide (LPS)-binding protein (LBP) has been shown to regulate the response of monocytes to LPS in vitro. In a previous study, polyclonal anti-LBP IgGs were found to protect D-galactosamine-sensitized mice against a lethal endotoxemic shock induced by a low challenge of LPS or lipid A when administered simultaneously with endotoxin. In the present study, we investigated the mode of action of these anti-LBP IgGs. In vitro, we demonstrated that they interfere with LPS binding to monocytes or polymorphonuclear cells in different ways: by the mere prevention of binding of LPS to LBP thus preventing the binding of LPS to CD14, or by reacting with LPS-LBP complexes thus mediating their binding to complement or Fc receptors on monocytes and on polymorphonuclear cells. In vivo, we demonstrated that anti-LBP IgGs afforded protection against lethal endotoxemic shock by one of two mechanisms. First, LBP blockade by pretreatment with anti-LBP IgG allowed protection against a low dose of LPS (100 ng). This protection occurred despite LPS levels in blood similar to those in control mice but in the absence of detectable tumor necrosis factor (TNF). This demonstrated that anti-LBP IgG could block the LBP-mediated TNF release upon LPS challenge. In contrast, anti-LBP IgG did not afford protection in mice not sensitized with D-galactosamine and challenged with high-dose LPS (1 mg), confirming that LPS at high concentrations could stimulate cells independently of the LBP pathway. Second, anti-LBP treatment administered simultaneously with LPS challenge protected mice against both low and high doses of LPS. Unlike after pretreatment with anti-LBP IgG, this protection was accompanied by a decrease of circulating LPS, suggesting that anti-LBP IgG in these conditions facilitated clearance of LPS probably by clearing LPS-LBP complexes. These data and the fact that LBP binds to all LPS through lipid A suggest that antibody directed to LBP could be a candidate for therapeutic strategies in endotoxemic shock.
Assuntos
Proteínas de Transporte/imunologia , Imunoglobulina G/uso terapêutico , Lipopolissacarídeos/toxicidade , Glicoproteínas de Membrana , Choque Séptico/prevenção & controle , Proteínas de Fase Aguda/imunologia , Animais , Biomarcadores/sangue , Endotoxinas/toxicidade , Feminino , Galactosamina/toxicidade , Imunoglobulina G/farmacologia , Lipídeo A/toxicidade , Lipopolissacarídeos/metabolismo , Camundongos , Camundongos Endogâmicos , Monócitos/metabolismo , Neutrófilos/metabolismo , Fator de Necrose Tumoral alfa/análiseRESUMO
Mouse monoclonal antibodies (MAbs) were generated against a 76-kDa IutA receptor of pathogenic avian Escherichia coli 15972. Six of the eight IutA-specific MAbs isolated (AB1 to AB6) were shown to be directed toward membrane-exposed conformational epitopes, although they did not interfere with the uptake of ferric aerobactin and cloacin DF13 as assessed by competition experiments with purified ligands. The two remaining IutA MAbs (AB9 and AB10) recognized linear epitopes buried in the IutA molecule. The panel of IutA MAbs was used to characterize IutA variants occurring in strains of E. coli, Klebsiella pneumoniae, Enterobacter spp., and Shigella spp., resulting in the identification of four immunological groups of IutAs. MAb AB9 defined an epitope conserved in all IutA variants. In addition, the panel of IutA MAbs served to identify the presence of IutA in wild-type bacteria grown in the presence of diphenylamine to reduce the expression of O-specific polysaccharide.
Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Bacteriocinas , Enterobacteriaceae/imunologia , Proteínas de Escherichia coli , Anticorpos Monoclonais , Especificidade de Anticorpos , Proteínas da Membrana Bacteriana Externa/classificação , Proteínas de Bactérias/metabolismo , Ensaio de Imunoadsorção Enzimática , Escherichia coli/patogenicidade , Compostos Férricos/metabolismo , Variação Genética , Ácidos Hidroxâmicos/metabolismo , Especificidade da EspécieRESUMO
LPS-binding protein (LBP) is an acute phase protein present in plasma, that has been shown to play a major role in sensitizing monocytes to LPS. We describe here two assays to quantify LBP in plasma. The first assay made use of monophosphoryl lipid A to capture LBP in human plasma, and LBP was detected by radiolabeled anti-LBP IgG. The second assay measured LBP by flow cytometry, using LBPs ability to present LPS to the CD14 receptor present on monocytes. Both assays had a similar level of sensitivity, that allowed the quantification of LBP in human plasma.
Assuntos
Proteínas de Fase Aguda/análise , Proteínas de Transporte/sangue , Glicoproteínas de Membrana , Especificidade de Anticorpos , Citometria de Fluxo , Humanos , Imunoglobulina G , Lipídeo A/análogos & derivados , Radioimunoensaio , Sensibilidade e EspecificidadeRESUMO
Because lipopolysaccharide (LPS)-binding protein (LBP) sensitizes monocytes to LPS in vitro, it has been suggested that LBP initiates host defenses against Gram-negative bacteria. The role of LBP in vivo, and particularly in endotoxemic shock, is unknown, however. Therefore an IgG against murine LBP was prepared. It was found to neutralize binding of LPS and subsequent activation of murine macrophages in vitro. This anti-LBP protected mice against the lethal effect of LPS when given at the same time as LPS challenge, but it failed to protect mice when delayed 15 min after LPS challenge. The same preparation was also effective after challenge with lipid A but not after challenge with Staphylococcus aureus enterotoxin. The protection was correlated with a strong decrease of circulating tumor necrosis factor. These data demonstrate that in vivo LBP is a major mediator of the lethal effects of endotoxemia.
Assuntos
Proteínas de Fase Aguda/metabolismo , Proteínas de Transporte/sangue , Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Glicoproteínas de Membrana , Choque Séptico/sangue , Animais , Medula Óssea , Proteínas de Transporte/isolamento & purificação , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Células-Tronco Hematopoéticas , Immunoblotting , Imunoglobulina G/farmacologia , Cinética , Camundongos , Camundongos Endogâmicos , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
In order to determine the best strategy for detection of aerobactin in members of the family Enterobacteriaceae, we compared the results of three phenotypic assays, including a chemical assay, a cross-feeding bioassay, and an enzyme-linked immunosorbent assay (ELISA), with the results of a dot blot hybridization assay using a specific probe for the aerobactin genes. The sensitivity and specificity of the ELISA were better than those of the chemical and cross-feeding assays, but the results of dot blot hybridization were the most reproducible. However, none of the Serratia and Enterobacter cloacae strains which produced aerobactin hybridized with the probe. We concluded that the best strategy for aerobactin detection is a two-step procedure that combines screening by dot blot hybridization with an ELISA for negative strains.
Assuntos
Enterobacteriaceae/química , Ácidos Hidroxâmicos/análise , Sideróforos/análise , Bioensaio , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , Ensaio de Imunoadsorção Enzimática , Ácidos Hidroxâmicos/metabolismo , Immunoblotting , Hibridização de Ácido Nucleico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Sideróforos/genética , Sideróforos/metabolismoRESUMO
We isolated a monoclonal antibody directed against the ferric complex of aerobactin purified from Escherichia coli KH576. This antibody, which we designated MAb AERO1, was identified as an immunoglobulin G, subtype 2. A competitive enzyme-linked immunosorbent assay with MAb AERO1 had a limit of 10 nM for the detection of purified ferric aerobactin and allowed detection of the crude aerobactin produced by various members of the family Enterobacteriaceae isolated from cancer patients with bacteremia. The only two other structurally related siderophores recognized by MAb AERO1 were ferric arthrobactin and ferrioxamine B. These results suggest that the epitope recognized by MAb AERO1 was the lysyl moiety of ferric aerobactin. We also showed that MAb AERO1 reduced the growth of an aerobactin-producing strain of E. coli in newborn calf serum, which indicates that it might be effective in reducing the severity of infections caused by bacteria for which the production of aerobactin is an important virulence factor.