RESUMO
A set of 43 strains corresponding to 20 classified and unclassified genomic Acinetobacter species was analyzed for the production of typical N-acyl homoserine lactone quorum sensing molecules in culture broths. A large percentage of the strains (74%) displayed quorum sensing signals that could be separated into three statistically significantly different chromatographic groups (p < 0.001) based on their retention factor in TLC, i.e., Rf1 (0.22 +/- 0.02); Rf2 (0.40 +/- 0.02) and Rf3 (0.54 +/- 0.02). Noteworthy, 63% of the strains tested produced more than one quorum signal. The frequency of signal appearance was Rf3 > Rf2 > Rf1. None of the three signals could be specifically assigned to a particular species in the genus; furthermore, no distinction could be made between the quorum sensing signals secreted by typical opportunistic strains of the A. calcoaceticus-A. baumannii complex, isolated from patients, with respect to the other species of the genus, except for the Rf1 signal which was present in all the QS positive strains belonging to this complex and DNA group 13 TU. In conclusion, quorum sensors in Acinetobacter are not homogenously distributed among species and one of them is present in most of the A. calcoaceticus-baumannii complex.
Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter/fisiologia , Acil-Butirolactonas/análise , Infecção Hospitalar/microbiologia , Microbiologia Ambiental , Percepção de Quorum/fisiologia , Acinetobacter/química , Acinetobacter/genética , Acinetobacter/isolamento & purificação , Cromatografia em Camada Fina , Humanos , Especificidade da EspécieRESUMO
The role of serum factors in the intracellular killing of bacteria by monocytes was studied on the basis of an assay independent of phagocytosis. After 3 min of phagocytosis of preopsonized bacteria and removal of noningested bacteria, the monocytes containing bacteria are reincubated for various periods and the number of unkilled bacteria is determined by a microbiological method after lysis of the cells. Evidence that this assay measures the killing of ingested bacteria was provided by scanning electron microscopy, lysostaphin treatment, and the effect on the rate of intracellular killing of inactivated serum lacking specific opsonic activity. Intracellular killing of Staphylococcus aureaus, S. epidermidis, and Escherichia coli by human monocytes does not occur or is low in the absence of serum, and maximal killing is only reached when fresh serum is present; intermediate values are obtained in the presence of heat-inactivated serum. These findings indicate that complement stimulates intracellular killing. Isolated heterogeneous immunoglobulin (Ig)G, pFc fragments of heterogeneous IgG, and both IgG1 and IgG3 stimulate intracellular killing of S. aureaus by monocytes to the same degree as heat-inactivated serum. Sphingomyelinase, which decreases the number of Fc receptors, and neuraminidase, which increases these receptors, respectively, decreased and increased the intracellular killing, whereas anti-monocyte serum completely abolished the stimulation of intracellular killing by inactivated serum. These results prove that interaction of the Fc receptor with the Fc part of IgG is required for the intracellular killing. Inhibition of the activation of complement components via the alternative pathway gave a considerable reduction in the intracellular killing of S. aureaus; impairment of the activation via the classical pathway had no effect. The addition of complement components to heat-inactivated serum showed that intracellular killing is maximal only when C3b is generated. Reduction of the number of C3b receptors in the membrane by trypsin or pronase decreased intracellular killing in the presence of fresh serum; anti-monocyte serum completely abolished the stimulation of intracellular killing by fresh serum. These results lead to the conclusion that intracellular killing is also dependent on the interaction between C3b and its receptor in the membrane.
Assuntos
Atividade Bactericida do Sangue , Proteínas do Sistema Complemento/fisiologia , Imunoglobulina G/fisiologia , Imunoglobulina M/fisiologia , Monócitos/fisiologia , Fagocitose , Complemento C3/fisiologia , Escherichia coli , Humanos , Fragmentos Fab das Imunoglobulinas/fisiologia , Fragmentos Fc das Imunoglobulinas/fisiologia , Cinética , Monócitos/imunologia , Proteínas Opsonizantes/fisiologia , StaphylococcusRESUMO
A young man suffering from recurrent Neisseria infections was shown to lack detectable serum complement factor D hemolytic activity. Addition to the patient's serum of purified factor D to a final concentration of 1 microgram/ml resulted in full restoration of the activity of the alternative pathway. Using an enzyme-linked immunosorbent assay, it was shown that the patient's serum did not contain measurable amounts of factor D antigen either. The sister, the father, as well as the parents of the mother had factor D levels within the normal range, and the factor D level of the mother was decreased. The capacity of the patient's serum, at concentrations up to 5%, to promote phagocytosis of Escherichia coli by normal human granulocytes was low when compared to normal serum. Substitution of the patient's serum with purified factor D resulted in a full restoration of opsonic activity. This study describes the first complete deficiency of factor D, and demonstrates its possible relation to recurrent Neisseria infections.
Assuntos
Enzimas Ativadoras do Complemento/deficiência , Fator D do Complemento/deficiência , Gonorreia/etiologia , Meningite Meningocócica/etiologia , Adulto , Fator D do Complemento/genética , Fator D do Complemento/uso terapêutico , Ensaio de Atividade Hemolítica de Complemento , Via Alternativa do Complemento , Escherichia coli , Humanos , Masculino , Linhagem , Fagocitose , Sepse/etiologiaRESUMO
Neutrophil defensins (or human neutrophil peptides-HNP) are major constituents of the azurophilic granules of human neutrophils and have been shown to display broad-spectrum antimicrobial activity. Other activities of these defensins, which are released from stimulated neutrophils, include cytotoxic, stimulatory, and chemotactic activities toward a variety of target cells. We studied the potential use of HNP-1 for antibacterial therapy of experimental bacterial infections in mice. In experimental peritoneal Klebsiella pneumoniae infections in mice, HNP-1 injection was shown to markedly reduce bacterial numbers in the infected peritoneal cavity 24 h after infection. This antibacterial effect was found to be associated with an increased influx of macrophages, granulocytes, and lymphocytes into the peritoneal cavity. These leukocytes appeared to be a requirement for the antibacterial effect, since in leukocytopenic mice administration of HNP-1 did not display antibacterial activity. HNP-1 treatment also reduced bacterial numbers in experimental K. pneumoniae or Staphylococcus aureus thigh muscle infections. In this model, radiolabeled HNP-1 was found to accumulate at the site of infection, whereas most of the injected HNP-1 was rapidly removed from the circulation via renal excretion. These results demonstrate that neutrophil defensins display marked in vivo antibacterial activity in experimental infections in mice and that this activity appears to be mediated, at least in part, by local leukocyte accumulation.
Assuntos
Antibacterianos/uso terapêutico , Infecções por Klebsiella/tratamento farmacológico , Klebsiella pneumoniae , Proteínas/uso terapêutico , Infecções Estafilocócicas/tratamento farmacológico , alfa-Defensinas , Animais , Antibacterianos/farmacocinética , Antivirais/farmacocinética , Antivirais/uso terapêutico , Defensinas , Humanos , Masculino , Camundongos , Doenças Musculares/tratamento farmacológico , Doenças Peritoneais/tratamento farmacológico , Proteínas/farmacocinética , Coxa da Perna , Distribuição TecidualRESUMO
A set of 18 Acinetobacter baumannii isolates, collected prospectively in a Bulgarian hospital during episodes of increased A. baumannii occurrence during 2000-2002, was investigated for genotypic diversity and antibiotic susceptibility. Four genotypes were identified by amplified fragment length polymorphism genomic fingerprinting, one of which (type 1) accounted for 13 isolates, indicating that a specific strain was predominant. The single isolate allocated to type 2 was identified to European clone I. All isolates were resistant to multiple antibiotics, but most retained susceptibility to tobramycin and colistin, and all except one were susceptible to imipenem.
Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Bulgária , Análise por Conglomerados , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla , Variação Genética , Hospitais Militares , Humanos , Testes de Sensibilidade Microbiana , Polimorfismo de Fragmento de Restrição , Estudos ProspectivosRESUMO
Previously we have identified and characterized three murine microbicidal proteins purified from the granule fraction of cells from the murine macrophage cell line RAW264.7. During these studies evidence was obtained for the presence of an additional antimicrobial protein in the cytosolic fraction of RAW264.7 cells that had been activated with interferon-gamma (IFN-gamma). In this study we have purified this protein, designated ubiquicidin, to apparent homogeneity and demonstrated that it is a cationic, small (Mr 6654) protein. Ubiquicidin displayed marked antimicrobial activity against Listeria monocytogenes and Salmonella typhimurium. Using a gel overlay procedure evidence was obtained that the protein also displays activity against Escherichia coli, Staphylococcus aureus, and an avirulent strain of Yersinia enterocolitica. Aminoterminal amino acid sequencing and mass spectrometry analysis of purified ubiquicidin indicated that it is most likely identical to the ribosomal protein S30. This protein is produced by posttranslational processing of the Fau protein, a 133-amino-acid fusion protein consisting of S30 linked to an unusual peptide with significant homology to ubiquitin. The fau gene has been reported to be expressed in a variety of tissues in humans and various animal species. The presence of ubiquicidin in the cytosol of macrophages may serve to restrict the intracellular growth of microorganisms. In addition, because macrophage disintegration will likely lead to release of ubiquicidin into the extracellular environment, it may contribute to host defense after macrophage death.
Assuntos
Bactérias/efeitos dos fármacos , Macrófagos/química , Sequência de Aminoácidos , Animais , Citosol/química , Defensinas , Escherichia coli/efeitos dos fármacos , Interferon gama/farmacologia , Listeria monocytogenes/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Espectrometria de Massas , Camundongos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Peso Molecular , Proteínas de Neoplasias/isolamento & purificação , Proteínas de Neoplasias/metabolismo , Proteínas/farmacologia , Coelhos , Ratos , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Salmonella typhimurium/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Células Tumorais Cultivadas , Yersinia enterocolitica/efeitos dos fármacosRESUMO
The effect of recombinant human interferon-gamma (rHuIFN-gamma) and interferon-alpha (rHuIFN-alpha) as in vivo stimuli for the activation of human monocytes was investigated on the basis of the bactericidal activity of peripheral blood monocytes in 11 patients with metastatic melanoma before and during treatment with interferons. Patients received increasing doses of rHuIFN-gamma and a fixed dose of rHuIFN-alpha, both administered subcutaneously three times a week. The rates of intracellular killing of Listeria monocytogenes and Salmonella typhimurium after in vitro phagocytosis by monocytes collected from melanoma patients before interferon treatment were increased (P less than 0.01) by a factor of 1.7 and 1.4, respectively, relative to the rate constants in blood monocytes of healthy donors. During treatment with the interferons, the rates of intracellular killing of the bacteria by patients' monocytes did not further increase. The findings underscore the immunogenicity of malignant melanoma and put into question the macrophage activating activity of IFN-gamma with respect to the bactericidal activity of monocytes.
Assuntos
Atividade Bactericida do Sangue/imunologia , Interferon Tipo I/uso terapêutico , Interferon gama/uso terapêutico , Melanoma/secundário , Monócitos/imunologia , Adulto , Idoso , Feminino , Humanos , Listeria monocytogenes/imunologia , Masculino , Melanoma/imunologia , Melanoma/terapia , Pessoa de Meia-Idade , Proteínas Recombinantes , Salmonella typhimurium/imunologiaRESUMO
An outbreak of nosocomial meningitis caused by Acinetobacter baumannii, which developed postoperatively in seven neurosurgical patients is described. The clinical isolates of A. baumannii were typed by biochemical profiles and antibiogram patterns, and by random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) and amplified fragment length polymorphism (AFLP) fingerprinting. The implicated strain was multi-drug resistant, however, susceptibility to imipenem and netilmicin was detected. An extensive search for the environmental source of the epidemic strain was carried out. Two of several isolates from hospital environment, corresponded to the A. baumannii outbreak strain, one being cultured from the suctioning equipment used in the care of these patients. The introduction of multiresistant epidemic A. baumannii into a neurosurgical unit is a severe risk factor for patients undergoing neurosurgical procedures. Genotypic typing methods are important for definitive identification of these strains in patients and their environment.
Assuntos
Infecções por Acinetobacter/etiologia , Acinetobacter baumannii , Infecção Hospitalar/etiologia , Surtos de Doenças/estatística & dados numéricos , Meningites Bacterianas/etiologia , Procedimentos Neurocirúrgicos/efeitos adversos , Infecções por Acinetobacter/diagnóstico , Infecções por Acinetobacter/epidemiologia , Infecções por Acinetobacter/transmissão , Acinetobacter baumannii/genética , Técnicas de Tipagem Bacteriana , Líquido Cefalorraquidiano/microbiologia , Análise por Conglomerados , Infecção Hospitalar/diagnóstico , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/transmissão , Impressões Digitais de DNA , DNA Bacteriano/análise , DNA Bacteriano/genética , Surtos de Doenças/prevenção & controle , Farmacorresistência Bacteriana Múltipla , Monitoramento Ambiental , Monitoramento Epidemiológico , Contaminação de Equipamentos/prevenção & controle , Contaminação de Equipamentos/estatística & dados numéricos , Genótipo , Mortalidade Hospitalar , Humanos , Controle de Infecções/métodos , Meningites Bacterianas/diagnóstico , Meningites Bacterianas/epidemiologia , Meningites Bacterianas/transmissão , Testes de Sensibilidade Microbiana , Polimorfismo de Fragmento de Restrição , Técnica de Amplificação ao Acaso de DNA Polimórfico , Estudos Retrospectivos , Fatores de Risco , Sucção/instrumentaçãoRESUMO
The effect of cytostatic treatment on the cellular defence and the efficacy of treatment with ceftriaxone in Klebsiella pneumoniae pneumonia was studied. Mice, made monocytopenic and granulocytopenic by cyclophosphamide or monocytopenic by etoposide, were infected intratracheally with K. pneumoniae (approximately 10(4) CFU) and then treated with ceftriaxone. At various intervals, the numbers of bacteria in the broncho-alveolar lavage (BAL) fluid and in lungs homogenised after lavage were determined. Cyclophosphamide reduced the numbers of granulocytes in the BAL fluid significantly but reduced only slightly the number of alveolar macrophages at the time of inoculation, 12 and 15 h later. The number of CFU in cyclophosphamide-treated mice was higher than that in controls, being significant in the homogenised lungs at 15 h after infection. In etoposide-treated mice, the numbers of alveolar phagocytes in BAL did not differ from those in control mice, whereas the number of bacteria was lower (only significantly in BAL fluid at 15 h after infection) than that in the controls. In this short experimental infection cytostatic treatment did not affect the outgrowth of Klebsiella pneumoniae substantially or the efficacy of treatment with ceftriaxone.
Assuntos
Antineoplásicos/farmacologia , Ceftriaxona/farmacologia , Ciclofosfamida/farmacologia , Infecções por Klebsiella/imunologia , Klebsiella pneumoniae , Pneumopatias/imunologia , Macrófagos Alveolares/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Animais , Líquido da Lavagem Broncoalveolar/citologia , Modelos Animais de Doenças , Etoposídeo/farmacologia , Granulócitos/efeitos dos fármacos , Imunossupressores/farmacologia , Leucopenia/induzido quimicamente , Masculino , Camundongos , Organismos Livres de Patógenos EspecíficosRESUMO
OBJECTIVES: The effect of cyclophosphamide-induced leukocytopenia on the cellular defence and on the efficacy of penicillin treatment in a Streptococcus pneumoniae pneumonia model in mice was studied. METHODS: The number of alveolar phagocytes was determined in broncho-alveolar lavage (BAL) fluid as well as the number of bacteria in both BAL fluid and homogenized lung tissue. RESULTS: Eighteen and 21 h after infection, leukocytopenic animals had significantly lower numbers of alveolar phagocytes than controls, while the numbers of bacteria in both BAL fluid and lungs were significantly higher. The number of bacteria was inversely related to the dose of penicillin and the number of alveolar macrophages. The number of alveolar granulocytes was inversely related to the dose of penicillin. CONCLUSIONS: Leukocytopenia due to cyclophosphamide impairs the cellular defence in the lung against Streptococcus pneumoniae and the dose of penicillin must be increased to compensate for the higher outgrowth of bacteria in these leukocytopenic mice, compared to normal animals.
Assuntos
Ciclofosfamida/efeitos adversos , Imunossupressores/efeitos adversos , Leucopenia/induzido quimicamente , Penicilinas/farmacologia , Fagócitos/efeitos dos fármacos , Pneumonia Pneumocócica/imunologia , Streptococcus pneumoniae/efeitos dos fármacos , Animais , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Camundongos , Penicilinas/administração & dosagem , Pneumonia Pneumocócica/tratamento farmacológico , Resultado do TratamentoRESUMO
In a child with Down's syndrome (DS) and her sibling, host immune responses were evaluated under experimental gingivitis conditions. The children live in the same environment under identical conditions. In the DS child an earlier and more extensive gingival inflammation than in her sibling had been observed. Investigation of nonspecific host defense mechanisms revealed identical results in both children for the phagocytosis and intracellular killing of Candida albicans by polymorphonuclear leukocytes in crevicular washings (CR-PMNs), in blood (PB-PMNs) and blood monocytes. Furthermore, CR- and PB-PMNs were able to secrete identical amounts of hydrogen peroxide upon stimulation. The chemotactic response of PB-PMNs in the DS child was impaired, however. The results of the studies performed on parameters of specific host defense mechanisms showed low blastogenic responses to phytohemagglutinin (PHA) and pokeweed (PWM) by lymphocytes of the DS child as compared with her sibling. Also a lack of immune regulation leading to prolonged helper/inducer cell activation on a local (gingival) and circulation level and a less pronounced T-cell depression in PB were shown. Together, these differences observed in specific and nonspecific host response mechanisms may be responsible for the earlier and more extensive gingival inflammation found in the DS child.
Assuntos
Síndrome de Down/genética , Líquido do Sulco Gengival/imunologia , Gengivite/imunologia , Neutrófilos/imunologia , Quimiotaxia de Leucócito , Criança , Síndrome de Down/imunologia , Feminino , Humanos , Ativação Linfocitária , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
Mycobacteria preferentially reside in resident macrophages whereas activated macrophages are presumed to eliminate the bacteria effectively. The aim of the present study was to determine the antibacterial activities of resident and activated murine peritoneal macrophages against Mycobacterium fortuitum and the intracellular mechanisms involved. After phagocytosis M. fortuitum could not be killed by either BCG/PPD-activated and IFN-gamma-activated macrophages and resident macrophages. The mycobacteria did not multiply in BCG/PPD-activated macrophages and the rate of proliferation of M. fortuitum in IFN-gamma-activated macrophages was only slightly inhibited compared to that in resident macrophages. Experiments with selective inhibitors of the production of reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI) demonstrated that these factors are not essential for the mycobacteriostatic activity of BCG/PPD-activated macrophages. After phagocytosis of M. fortuitum, BCG/PPD-activated and IFN-gamma-activated macrophages produced substantial amounts of both RNI and ROI. No correlation was found between the levels of these intermediates and the proliferation of M. fortuitum in the macrophages. In conclusion, BCG/PPD-activated macrophages are bacteriostatic, but not bacteriocidal for M. fortuitum and the former does not involve reactive nitrogen and oxygen intermediates.
Assuntos
Ativação de Macrófagos/fisiologia , Óxido Nítrico/fisiologia , Micobactérias não Tuberculosas/imunologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Divisão Celular , Masculino , Camundongos , Mycobacterium bovis/imunologia , Óxido Nítrico/antagonistas & inibidores , Fagocitose/fisiologia , Tuberculina/imunologia , ômega-N-MetilargininaRESUMO
The study of the phagocytosis and intracellular killing of Candida albicans by granulocytes and monocytes has been hampered by the budding and pseudomycelium formation of this yeast during a relatively short incubation period at 37 degrees C and by the similar density of candida cells and phagocytes, which makes differential centrifugation impossible. In the present study, C. albicans was used after 5 days of preculture at 30 degrees C, after which the number of candida cells remained constant during incubation at 37 degrees C for 90 min. On this basis, phagocytosis and intracellular killing were limited to a period of 60 min. Phagocytosis of C. albicans by granulocytes and monocytes was measured with a hemocytometer, the number of extracellular candida being a measure of the ingestion of these microorganisms. After 60 min, 96% of the candida cells were ingested by normal human granulocytes and monocytes. This process was dependent on the opsonin concentration and temperature and was inhibited by mono-iodoacetic acid. Heat-inactivated serum was less active than fresh serum, reflecting the role of complement factors with respect to opsonization. Intracellular killing was measured by a microbiological assay. After 60 min of incubation of phagocytes together with C. albicans and serum, human granulocytes and monocytes killed 58 and 50% of the ingested candida, respectively. This process was inhibited by phenylbutazone. Phagocytes from patients with chronic granulomatous disease showed impaired intracellular killing.
Assuntos
Candida albicans/imunologia , Granulócitos/imunologia , Leucócitos/imunologia , Monócitos/imunologia , Fagocitose , Candida albicans/isolamento & purificação , Células Cultivadas , Granulócitos/microbiologia , Humanos , Iodoacetatos/farmacologia , Cinética , Monócitos/microbiologia , Proteínas Opsonizantes/análise , Disfunção de Fagócito Bactericida/imunologia , Fagocitose/efeitos dos fármacos , Fenilbutazona/farmacologia , TemperaturaRESUMO
Human monocytes require serum components immunoglobulin G, C3/C3b, and B/Bb to exert optimal microbicidal action against ingested microorganisms. The present study was performed to find out whether these factors act by enhancing oxygen-dependent antimicrobial mechanisms. Serum enhanced oxygen consumption and superoxide production by monocytes before phagocytosis, but did not further increase these processes in monocytes that had recently ingested bacteria. Furthermore, serum did not boost iodination during intracellular killing by monocytes. Phorbol myristate acetate, N-formyl-methyonyl-leucyl-phenylalanine, concanavalin A, and concanavalin A-Sephadex all stimulated the conversion of O2 to H2O2 by monocytes, but only concanavalin A augmented intracellular killing. Reactive oxygen intermediates generated by cell-free enzymes (xanthine oxidase or glucose oxidase) in concentrations comparable to those accumulating extracellularly during incubation of monocytes containing bacteria with phorbol myristate acetate did not promote intracellular killing. The presence of catalase during phagocytosis inhibited killing, but had no effect on killing in the postphagocytic state. Monocytes deprived of glucose for 24 h showed markedly impaired O2 consumption, O2- generation, and bacterial killing; all of these effects were rapidly reversed by restoration of glucose. It is concluded that both an intact respiratory burst and extracellular serum factors are necessary for optimal killing of intracellular Staphylococcus aureus by human monocytes. Serum does not appear to act by enhancing the respiratory burst, but rather to have a separate, synergistic role, the biochemical basis of which is unknown.
Assuntos
Monócitos/fisiologia , Oxigênio/fisiologia , Fagocitose , Membrana Celular/fisiologia , Espaço Extracelular , Glucose/farmacologia , Humanos , Peróxido de Hidrogênio/metabolismo , Monócitos/metabolismo , Consumo de Oxigênio , Fagocitose/efeitos dos fármacos , Staphylococcus aureus , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The intracellular killing of Staphylococcus aureus by human monocytes requires continuous stimulation by extracellular serum factors interacting with the cells via membrane binding sites. At least 75% of the intracellular killing in the presence of fresh serum was accounted for by the combined stimulatory activities of IgG, C3/C3b, and B/Bb. C3b is a more potent stimulator than C3, and Bb stimulates the killing to the same degree as B. The stimulation of intracellular killing by C3 and C3b occurs by interaction of the stable binding site of this molecule with the C3b receptor in the monocyte membrane. The stimulation of intracellular killing by B and Bb is most probably mediated via a binding site on these proteins interacting with a receptor site in the monocyte membrane. Both complement receptors, i.e., for C3b and B, are pronase sensitive. However, only the C3b receptor can be inhibited by (Fab')2 fragments of anti-C3 receptor antibodies, indicating that the binding sites for C3/C3b and B/bb are different.
Assuntos
Atividade Bactericida do Sangue , Complemento C3b/metabolismo , Imunoglobulina G/metabolismo , Monócitos/imunologia , Infecções Estafilocócicas/imunologia , Ativação do Complemento , Complemento C3/metabolismo , Complemento C3a , Fator B do Complemento/metabolismo , Humanos , Monócitos/microbiologia , Pronase/farmacologia , Receptores de ComplementoRESUMO
The kinetic patterns of the phagocytosis and intracellular killing of Staphylococcus and Escherichia coli by monocytes were investigated separately to acquire more insight into the total process, i.e. from the ingestion to the death of the micro-organisms. Phagocytosis proved to be dependent on: (1) both the bacteria-to-monocyte ratio and the monocyte concentration; a concentration of at least 5 x 10(5) monocytes/ml proved necessary for the measurement of ingestion, whereas the rate of ingestion was found to be proportional to the number of extracellular bacteria until a maximum rate is reached, (2) the serum concentration in the incubation medium, which influenced both the rate of phagocytosis and the maximum number of bacteria taken up by one monocyte, and (3) the temperature, the highest rate of phagocytosis being reached at 37-41 degrees C. The intracellular killing proved to be dependent on: (1) the number of bacteria ingested; the rate of killing was proportional to the number of ingested bacteria until a maximum rate was reached; (2) the temperature, since a maximum rate of killing is only reached at 37-41 degrees C; at lower and higher temperatures the rate of killing is lower, in the latter case due to inactivation of extracellular stimuli. These separate data on the ingestion and killing processes made it possible to compute the theoretical numbers of extracellular, viable intracellular, and total intracellular bacteria for a model system consisting of 5 x 10(6) monocytes, 5 x 10(6) bacteria, and 10% serum. These calculated values are in agreement with the experimental data.
Assuntos
Escherichia coli , Monócitos/imunologia , Fagocitose , Staphylococcus aureus , Contagem de Células , Humanos , Temperatura , Fatores de TempoRESUMO
Immunoglobulins and complement components are required for optimal ingestion and optimal killing of microorganisms by granulocytes. The degree of opsonization of microorganisms necessary for their ingestion was lower than that required for the killing of these bacteria during the ingestion phase. Killing during this phase was found to depend mainly on the presence of heat-labile opsonins, probably C3b, present on the microorganisms. Extracellular immunoglobulin G (IgG) and C3b were indispensable for optimal intracellular killing after ingestion was complete. This was established with an assay permitting assessment of the course of the number of viable intracellular bacteria independent of the ingestion of new live bacteria. Maximal intracellular killing by human granulocytes of ingested catalase-positive (Staphylococcus aureus and Escherichia coli) or catalase-negative (Streptococcus pyogenes and S. pneumoniae) microorganisms was found only when fresh serum was present extracellularly. Killing was suboptimal in the absence of serum. With heat-inactivated serum, the killing index lay between the indices obtained in the presence and absence of fresh serum. The stimulatory activity of heat-inactivated serum was most probably due to the interaction of IgG with the Fc receptor on the granulocyte membrane, since IgG subclasses IgG1 and IgG3 as well as pFc fragments of IgG stimulated the intracellular killing to the same degree as heat-inactivated serum did. In addition, (Fab1)2 fragments of IgG did not stimulate killing, and reduced killing was observed in the presence of heat-inactivated serum after reduction of the number of Fc receptors. The extra stimulation of the killing process in the presence of fresh serum compared with heat-inactivated serum was due to the interaction between membrane receptors and complement--most probably C3b generated by both the classical and the alternative pathways of complement activation. This conclusion is based on results obtained with sera in which one or both complement pathways were blocked, on the restoration of the killing-stimulatory activity of C3-deficient serum after addition of fresh C3, and on the reduced killing observed in the presence of fresh serum after reduction of the number of C3 receptors by the use of pronase or antigranulocyte serum.
Assuntos
Atividade Bactericida do Sangue , Proteínas do Sistema Complemento/fisiologia , Granulócitos/microbiologia , Imunoglobulinas/fisiologia , Ativação do Complemento , Escherichia coli/crescimento & desenvolvimento , Humanos , Imunoglobulina G/fisiologia , Proteínas Opsonizantes/fisiologia , Fagocitose , Receptores de Complemento/fisiologia , Receptores Fc/fisiologia , Staphylococcus aureus/crescimento & desenvolvimento , Streptococcus/crescimento & desenvolvimentoRESUMO
A set of 43 strains corresponding to 20 classified and unclassified genomic Acinetobacter species was analyzed for the production of typical N-acyl homoserine lactone quorum sensing molecules in culture broths. A large percentage of the strains (74%) displayed quorum sensing signals that could be separated into three statistically significantly different chromatographic groups (p < 0.001) based on their retention factor in TLC, i.e. Rf1 (0.22 ± 0.02); Rf2 (0.40 ± 0.02) and Rf3 (0.54 ± 0.02). Noteworthy, 63% of the strains tested produced more than one quorum signal. The frequency of signal appearance was Rf3 > Rf2 > Rf1. None of the three signals could be specifically assigned to a particular species in the genus; furthermore, no distinction could be made between the quorum sensing signals secreted by typical opportunistic strains of the A. calcoaceticus-A. baumannii complex, isolated from patients, with respect to the other species of the genus, except for the Rf1 signal which was present in all the QS positive strains belonging to this complex and DNA group 13 TU. In conclusion, quorum sensors in Acinetobacter are not homogenously distributed among species and one of them is present in most of the A. calcoaceticus-baumannii complex.
Se analizó la producción de moléculas típicas de N-acil homoserina lactona con actividad de quorum sensing en cultivos líquidos de un grupo de 43 cepas correspondientes a 20 especies genómicas clasificadas y no clasificadas de Acinetobacter. Un porcentaje alto de las cepas (74%) mostraron señales de quorum sensing que pudieron ser separadas en tres grupos cromatográficos significativamente diferentes entre sí (p < 0,001) sobre la base de sus factores de retención en TLC, a saber: Rf1 (0.22 ± 0.02); Rf2 (0.40 ± 0.02) y Rf3 (0.54 ± 0.02). Es de notar que 63% de las cepas ensayadas produjeron más de una señal de quorum. La frecuencia de aparición de las señales fue Rf3 > Rf2 > Rf1. Ninguna de las tres señales pudo ser asignada a una especie en particular dentro del género; es más, no se encontró diferencia entre las señales producidas por las cepas típicamente oportunistas (complejo A. calcoaceticus-A. baumannii) aisladas de pacientes respecto de las producidas por otras cepas del mismo género, excepto para el caso de Rf1, que se encontró presente en todos los aislamientos quorum sensing positivos del mencionado complejo y en las cepas del grupo de DNA 13TU. En conclusión, los sensores de quórum en Acinetobacter no están homogéneamente distribuidos entre especies y uno de ellos (Rf1) está presente en la mayoría de los miembros del complejo calcoaceticus-baumannii.
Assuntos
Humanos , Infecções por Acinetobacter/microbiologia , Acinetobacter/fisiologia , Acil-Butirolactonas/análise , Infecção Hospitalar/microbiologia , Microbiologia Ambiental , Percepção de Quorum/fisiologia , Acinetobacter/química , Acinetobacter/genética , Acinetobacter/isolamento & purificação , Cromatografia em Camada Fina , Especificidade da EspécieRESUMO
The present in vitro study concerned the phagocytosis and intracellular killing by polymorphonuclear cells (PMN) of 5 patients with rheumatoid arthritis (RA) and 12 patients with Felty's syndrome (FS). PMN phagocytosis was assessed by microbiologic and morphologic methods, and intracellular killing was measured independently of continuous phagocytosis of viable bacteria (Staphylococcus aureus). PMN from patients with RA or FS ingested S aureus opsonized with immunoglobulins and complement as effectively as did PMN from healthy donors. However, the capacity of patient PMN to ingest S aureus opsonized with sera lacking complement activity, e.g., heat-inactivated donor serum and the sera of 2 patients with FS, was lower than that of healthy donor PMN. This decreased ingestion is associated with diminished expression of Fc receptors on the membrane of PMN from patients who have RA or FS. As with sera lacking complement activity, decreased capacity to ingest S aureus was observed after preloading donor PMN with immune aggregates, which also decreased the expression of Fc receptors. PMN from patients with RA or FS were found to be as active in killing S aureus as cells from healthy donors.