RESUMEN
Interleukin-13 (IL-13) sequentially binds to IL-13Ralpha1 and IL-4Ralpha forming a high affinity signalling complex. This receptor complex is expressed on multiple cell types in the airway and signals through signal transducer and activator of transcription factor-6 (STAT-6) to stimulate the production of chemokines, cytokines and mucus. Antibodies have been generated, using the UCB Selected Lymphocyte Antibody Method (UCB SLAM), that block either binding of murine IL-13 (mIL-13) to mIL-13Ralpha1 and mIL-13Ralpha2, or block recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. Monoclonal antibody (mAb) A was shown to bind to mIL-13 with high affinity (K(D) 11 pM) and prevent binding of mIL-13 to mIL-13Ralpha1. MAb B, that also bound mIL-13 with high affinity (K(D) 8 pM), was shown to prevent recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. In vitro, mAbs A and B similarly neutralised mIL-13-stimulated STAT-6 activation and TF-1 cell proliferation. In vivo, mAbs A and B demonstrated equipotent, dose-dependent inhibition of eotaxin generation in mice stimulated by intraperitoneal administration of recombinant mIL-13. In an allergic lung inflammation model in mice, mAbs A and B equipotently inhibited muc5ac mucin mRNA upregulation in lung tissue measured two days after intranasal allergen challenge. These data support the design of therapeutics for the treatment of allergic airway disease that inhibits assembly of the high affinity IL-13 receptor signalling complex, by blocking the binding of IL-13 to IL-13Ralpha1 and IL-13Ralpha2, or the subsequent recruitment of IL-4Ralpha.
Asunto(s)
Anticuerpos Bloqueadores/inmunología , Anticuerpos Monoclonales/inmunología , Subunidad alfa1 del Receptor de Interleucina-13/antagonistas & inhibidores , Subunidad alfa2 del Receptor de Interleucina-13/antagonistas & inhibidores , Interleucina-13/antagonistas & inhibidores , Receptores de Superficie Celular/antagonistas & inhibidores , Animales , Línea Celular , Línea Celular Tumoral , Quimiocina CCL11/análisis , Quimiocina CCL11/inmunología , Modelos Animales de Enfermedad , Epítopos/inmunología , Humanos , Hipersensibilidad/inmunología , Interleucina-13/inmunología , Subunidad alfa1 del Receptor de Interleucina-13/inmunología , Subunidad alfa2 del Receptor de Interleucina-13/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Mucina 5AC/inmunología , Mucina 5AC/metabolismo , Ovalbúmina/inmunología , Neumonía/inmunología , Neumonía/metabolismo , Conejos , Receptores de Superficie Celular/inmunología , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Factor de Transcripción STAT6/inmunología , Factor de Transcripción STAT6/metabolismoRESUMEN
Prevention of Klebsiella infections by passive immunotherapy has received more attention during the last decade. Both K antigen-and O antigen-specific antisera and monoclonal antibodies (mAbs) have been studied with respect to phagocytosis-enhancing and in vivo protective capacities. Our own work has focussed on the generation of O serogroup-specific rabbit antisera and O antigen specific murine antibodies. O-specific rabbit sera were absorbed extensively with heterologous O antigen strains in order to obtain highly specific typing reagents. Using these for typing a collection of 378 clinical strains, we found that 82% of them belonged to one of the four serogroups O1, O2ab, O3 and O5. Phagocytosis experiments using antisera and mAbs showed that O antigen specific antibodies were able to opsonize non-encapsulated strains, while fully encapsulated bacteria were rather resistant against the opsonizing effect. Nevertheless, in vivo experiments demonstrated a prophylactic effect on both Klebsiella septicemia and pneumonia in a mouse model of lethal infection. Given the limited number of O serogroups, O antigen-specific antibodies may be suited to supplement K antigen-specific hyperimmune globulins for passive immunoprophylaxis of Klebsiella infections.
Asunto(s)
Infecciones por Klebsiella/inmunología , Infecciones por Klebsiella/prevención & control , Klebsiella/inmunología , Antígenos O/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Infecciones por Klebsiella/epidemiología , Ratones , Antígenos O/aislamiento & purificación , Proteínas Opsoninas , Conejos , SerotipificaciónRESUMEN
Interleukin-1 (IL-1) plays an important role in host defenses against microbial pathogens. Excessive production of this cytokine, however, may be responsible in part for the lethality observed during sepsis. Our studies show that interferon-gamma (IFN-gamma) downregulates lipopolysaccharide (LPS)-induced interleukin-1beta (IL-1beta) transcription in primary macrophages. This phenomenon does not occur in splenocytes or bone marrow-derived macrophages from signal transducer and activator of transcription (Stat1)-deficient mice, suggesting that Stat1, a transcription factor involved in IFN signaling, plays a critical role in this process. Moreover, nitric oxide (NO) was also involved in the downregulation of LPS-induced IL-1 by IFN, as addition of the inducible nitric oxide synthase (iNOS) inhibitor L-N(6)-(1-iminoethyl)lysine (NIL) negated the effect. Kinetic analysis of IL-1 and IFN levels in LPS-treated mice in vivo suggests that IFN-mediated inhibition of IL-1 might be an important negative feedback mechanism for limiting IL-1 generation in vivo.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Interferón gamma/fisiología , Interleucina-1/antagonistas & inhibidores , Lipopolisacáridos/antagonistas & inhibidores , Macrófagos/inmunología , Transducción de Señal/inmunología , Transactivadores/fisiología , Animales , Línea Celular , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Femenino , Inyecciones Intraperitoneales , Interferón gamma/biosíntesis , Interleucina-1/genética , Cinética , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C3H , Óxido Nítrico/fisiología , ARN Mensajero/antagonistas & inhibidores , Factor de Transcripción STAT1 , Transcripción Genética/inmunologíaRESUMEN
Persistence of intravenous (i.v.) hyperimmune immunoglobulin (100 mg/kg) directed against clinically predominant serotypes of Pseudomonas and Klebsiella in ill, febrile patients was compared to healthy volunteers to determine if ill patients have a decreased Ig half-life resulting in an increased immunoglobulin requirement. Type-specific antibodies were measured by ELISA for 83 days in eight healthy volunteers and for 35 days in eight ill patients with surgical complications or hematologic malignancy. Mean values and fold rises of antibody concentrations for the two groups were above preinfusion values at 35 days. The antibody fold rises in patients and in healthy volunteers were similar. Type-specific antibody levels in some patients increased after illness coincident with elevation of total immunoglobulins. We conclude that the duration of potentially therapeutic levels of infused type-specific hyperimmune immunoglobulin may persist for a longer period of time than what has been measured for total immunoglobulin. While the mechanism of this persistence remains to be characterized, the possibility of type-specific antibody synthesis induced by immunoglobulin administration must be considered.
Asunto(s)
Inmunoglobulinas/administración & dosificación , Inmunoglobulinas/sangre , Klebsiella/inmunología , Pseudomonas/inmunología , Adulto , Anciano , Estudios de Casos y Controles , Femenino , Semivida , Humanos , Infusiones Intravenosas , Klebsiella/clasificación , Masculino , Persona de Mediana Edad , Antígenos O/inmunología , Pseudomonas/clasificación , SerotipificaciónRESUMEN
The passive infusion of antibodies elicited in rabbits with a detoxified J5 lipopolysaccharide (LPS)/group B meningococcal outer membrane protein complex vaccine protected neutropenic rats from heterologous lethal gram-negative bacterial infection. In this study, active immunization was studied in neutropenic rats infected with Pseudomonas aeruginosa, in the presence or absence of ceftazidime therapy, and with Klebsiella pneumoniae. This vaccine elicited a > 200-fold increase in anti-J5 LPS antibody, which remained elevated throughout the duration of cyclophosphamide-induced neutropenia and for < or = 3 months. There was improved survival among immunized versus control animals: 48% (13/28) versus 7% (2/29) in Pseudomonas-challenged rats; 61% (11/18) versus 0% (0/10) in Pseudomonas- and ceftazidime-treated rats; and 64% (9/14) versus 13% (2/15) in Klebsiella-challenged rats (P < 0.01 for each comparison). Immunized animals had lower levels of bacteria in organs and lower levels of circulating endotoxin at the onset of fever. In conclusion, active immunization with an anti-endotoxin vaccine improved survival after infection with > or = 2 heterologous, clinically relevant bacterial species in immunocompromised animals. Active immunization with this vaccine merits further investigation.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas contra Escherichia coli/administración & dosificación , Escherichia coli/inmunología , Lipopolisacáridos/inmunología , Neisseria meningitidis/inmunología , Sepsis/prevención & control , Animales , Anticuerpos Antibacterianos/sangre , Ceftazidima/uso terapéutico , Cefalosporinas/uso terapéutico , Ciclofosfamida , Modelos Animales de Enfermedad , Supervivencia sin Enfermedad , Endotoxinas/sangre , Femenino , Klebsiella pneumoniae , Neutropenia/inducido químicamente , Pseudomonas aeruginosa , Ratas , Ratas Sprague-Dawley , Sepsis/inmunología , Sepsis/microbiología , Vacunas ConjugadasRESUMEN
Fever is an important regulator of inflammation that modifies expression and bioactivity of cytokines, including tumor necrosis factor (TNF)-alpha. Pulmonary vascular endothelium is an important target of TNF-alpha during the systemic inflammatory response. In this study, we analyzed the effect of a febrile range temperature (39.5 degrees C) on TNF-alpha-stimulated changes in endothelial barrier function, capacity for neutrophil binding and transendothelial migration (TEM), and cytokine secretion in human pulmonary artery endothelial cells (EC). Permeability for [(14)C]BSA tracer was increased by treatment with TNF-alpha, and this effect was augmented by incubating EC at 39.5 degrees C. Treating EC with 2. 5 U/ml TNF-alpha stimulated an increase in subsequent neutrophil adherence and TEM. Incubating EC at 39.5 degrees C caused a 30% increase in TEM but did not modify the enhancement of neutrophil adherence or TEM by TNF-alpha treatment. Analysis of cytokine expression in EC cultures exposed to TNF-alpha at either 37 degrees or 39.5 degrees C revealed three patterns of temperature and TNF-alpha responsiveness. Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-8 were not detectable in untreated EC but were increased after TNF-alpha exposure, and this increase was enhanced at 39.5 degrees C. IL-6 expression was also increased with TNF-alpha exposure, but IL-6 expression was lower in 39.5 degrees C EC cultures. Transforming growth factor-beta(1) was constitutively expressed, and its expression was not influenced either by TNF-alpha or exposure to 39.5 degrees C. These data demonstrate that clinically relevant shifts in body temperature might cause important changes in the effects of proinflammatory cytokines on the endothelium.
Asunto(s)
Temperatura Corporal , Endotelio Vascular/fisiopatología , Fiebre/fisiopatología , Factor de Necrosis Tumoral alfa/fisiología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Proteínas del Choque Térmico HSP72 , Proteínas de Choque Térmico/metabolismo , Humanos , Neutrófilos/fisiología , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Temperatura , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The mechanisms by which superantigens, such as staphylococcal enterotoxin B (SEB), contribute to microbial pathogenicity have been poorly defined. The study of such pathogenic processes has been hampered by the lack of an adequate animal model. We utilized a previously described murine chimeric model to determine the cytokines and cell populations that might be involved in SEB toxicity. In the absence of bone marrow transplantation (BMT), all total body irradiated (TBI) mice died, while all transplanted mice survived up to 6 months. Compared with non-TBI and non-BMT mice, chimeric mice had an increased percentage of CD11b (Mac-1)-positive splenocytes (17 vs. 59%, P < 0.05) and decreased CD45R-positive (B) cells (33 vs. 6%, P < 0.05) at 6 weeks after BMT. The relative numbers of splenocyte CD4 and CD8 cells were similar in chimeric and normal mice. Susceptibility of chimeric animals to 10 or 100 microg SEB was time-dependent: no mice challenged at 2 weeks post-BMT died, but 15% of mice challenged at 4 weeks and 50% of those challenged at 6-8 weeks died. Compared with TBI and non-BMT C3H/HeJ mice, SEB-challenged chimeric mice at 6-8 weeks had (1) increased splenocyte mRNA expression for: IFN-gamma (3.5 x optimally at 1 h), TNF-alpha (6.5 x at 2 h), IL-6 (4.8 x at 4 h), IL-1beta (8.4 x at 4 h), IL-2 (4.7 x at 4 h), and IL-10 (3 x at 16 h), and (2) increased and earlier peak serum levels of IFN-gamma, IL-6, IL-1beta and IL-2, but no increase in serum TNF-alpha or IL-4. These data support the hypothesis that the decreased percentage of B cells and increased macrophages in chimeric mice lead to enhanced T cell-macrophage interactions after SEB administration and a lethal burst of T cell and macrophage cytokine release. This model will provide insight into cell populations and mechanisms that mediate superantigen-induced toxicity.
Asunto(s)
Citocinas/metabolismo , Enterotoxinas/inmunología , Staphylococcus aureus/inmunología , Superantígenos/inmunología , Animales , Antígenos CD4/biosíntesis , Antígenos CD8/biosíntesis , Citocinas/sangre , Citocinas/genética , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Enterotoxinas/administración & dosificación , Enterotoxinas/toxicidad , Femenino , Expresión Génica , Cinética , Antígeno de Macrófago-1/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones SCID , Bazo/citología , Bazo/inmunología , Superantígenos/administración & dosificación , Superantígenos/toxicidadRESUMEN
Fever, a nonspecific acute-phase response, has been associated with improved survival and shortened disease duration in infections, but the mechanisms of these beneficial responses are poorly understood. We previously reported that increasing core temperature of bacterial endotoxin (LPS)-challenged mice to the normal febrile range modified expression of tumor necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), and IL-6, three cytokines critical to mounting an initial defense against microbial pathogens, but survival was not improved in the warmer animals. We speculated that our inability to show a survival benefit of optimized cytokine expression in the warmer animals reflected our use of LPS, a nonreplicating agonist, rather than an infection with viable pathogens. The objective of this study was to determine if increasing murine core temperature altered cytokine expression and improved survival in an experimental bacterial peritonitis model. We showed that housing mice at 35.5 degrees C rather than 23 degrees C increased core temperature from 36.5 to 37.5 degrees C to 39.2 to 39.7 degrees C, suppressed plasma TNF-alpha expression for the initial 48 h, delayed gamma interferon expression, improved survival, and reduced the bacterial load in mice infected with Klebsiella pneumoniae peritonitis. We showed that the reduced bacterial load was not caused by a direct effect on bacterial proliferation and probably reflected enhanced host defense. These data suggest that the increase in core temperature that occurs during bacterial infections is essential for optimal antimicrobial host defense.
Asunto(s)
Fiebre/etiología , Infecciones por Klebsiella/fisiopatología , Klebsiella pneumoniae , Peritonitis/fisiopatología , Animales , Temperatura Corporal , Interferón gamma/sangre , Interleucina-6/sangre , Lipopolisacáridos/toxicidad , Masculino , Ratones , Factor de Necrosis Tumoral alfa/análisisRESUMEN
Gram-negative bacillary sepsis is a leading cause of death among patients hospitalized in intensive care units. While initial clinical studies with the passive administration of anti-endotoxin core-glycolipid (CGL) antibodies for the treatment and prophylaxis of sepsis showed promising results, subsequent studies failed to show a consistent benefit. There appears to be a good correlation between anti-CGL antibody levels at the onset of sepsis and maintenance of antibody levels during sepsis with outcome. Previous clinical studies may have failed because insufficient amounts of antibody were administered early in the course of sepsis. Unlike the case with anti-CGL antibodies, polyvalent, hyperimmune type-specific antibody preparations may prevent the development of infections; however, these antibodies also must be provided in adequate amounts and in close proximity to infection in order to provide a beneficial effect. These pharmacokinetic requirements may limit the utility of passive immunotherapy for the prophylaxis of sepsis. Active immunization of acutely traumatized patients or of rats subsequently rendered neutropenic with cyclophosphamide induced high antibody levels for extended periods of time. Since trauma and other conditions are associated with a Th(2) response, these conditions may favor antibody formation following active immunization. Active immunization with both anti-CGL and/or polyvalent-specific vaccines for the prophylaxis of sepsis with passive supplementation at the onset of sepsis is an approach that merits further investigation.
Asunto(s)
Inmunoterapia Activa , Sepsis/terapia , Animales , Anticuerpos Antibacterianos/biosíntesis , Especificidad de Anticuerpos , Endotoxinas/antagonistas & inhibidores , Infecciones por Bacterias Gramnegativas/terapia , Ratas , Resultado del TratamientoRESUMEN
Fever is a phylogenetically ancient response that is associated with improved survival in acute infections. In endothermic animals, fever is induced by a set of pyrogenic cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1, and IL-6] that are also essential for survival in acute infections. We studied the influence of core temperature on cytokine expression using an anesthetized mouse model in which core temperature was adjusted by immersion in water baths. We showed that raising core temperature from basal (36.5-37.5 degrees C) to febrile (39.5-40 degrees C) levels increased peak plasma TNF-alpha and IL-6 levels by 4.1- and 2. 7-fold, respectively, and changed the kinetics of IL-1beta expression in response to lipopolysaccharide challenge. TNF-alpha levels were increased predominantly in liver, IL-1beta levels were higher in lung, and IL-6 levels were widely increased in multiple organs in the warmer mice. This demonstrates that the thermal component of fever may directly contribute to shaping the host response by regulating the timing, magnitude, and tissue distribution of cytokine generation during the acute-phase response.
Asunto(s)
Temperatura Corporal/fisiología , Citocinas/metabolismo , Endotoxinas/farmacología , Fiebre/fisiopatología , Pirógenos/metabolismo , Animales , Citocinas/sangre , Relación Dosis-Respuesta a Droga , Fiebre/sangre , Fiebre/metabolismo , Proteínas del Choque Térmico HSP72 , Proteínas de Choque Térmico/metabolismo , Interleucina-1/metabolismo , Interleucina-6/sangre , Interleucina-6/metabolismo , Macrófagos del Hígado/fisiología , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos , Pirógenos/sangre , Distribución Tisular/fisiología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Therapy with colony-stimulating factors has been extended beyond their use in accelerating myeloid cell recovery to take advantage of their immune function-enhancing properties. Studies in animal models and with human subjects suggest a potential role as adjunctive therapy in infections of non-neutropenic hosts, including those with sepsis. Granulocyte colony-stimulating factor may play a pivotal role in the induction of lipopolysaccharide desensitization by nontoxic lipid A analogues proposed for the prevention of sepsis; granulocyte macrophage colony-stimulating factor may be useful in reversing the immune paralysis described in later stages of sepsis. Significant issues of exogenous colony-stimulating factor therapy must be addressed, however: the optimal timing, dose, and clinical context (e.g., type of immunosuppression, duration of infection-inciting stimulus) as well as tissue-specificity of the activities and net effect of potentially conflicting responses (e.g., immune restorative and procoagulant effects of granulocyte macrophage colony-stimulating factor). Resolution of these issues will require carefully designed clinical studies with meticulous monitoring of immunologic parameters.
Asunto(s)
Infecciones Bacterianas/prevención & control , Factores de Crecimiento de Célula Hematopoyética/fisiología , Sepsis/prevención & control , Animales , Factor Estimulante de Colonias de Granulocitos/fisiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Humanos , Factor Estimulante de Colonias de Macrófagos/fisiología , Neutropenia/prevención & controlRESUMEN
Recent clinical trials with experimental immunotherapeutic agents for severe sepsis and septic shock have been largely unsuccessful despite seemingly convincing preclinical evidence of significant benefit of these antisepsis therapies. This article reviews basic therapeutic rationale, preclinical evaluation, and clinical trial design of past clinical trials of innovative sepsis treatments. Lessons learned from past failures should provide insights into the design and implementation of successful clinical trials for new anti-sepsis agents in the future.
Asunto(s)
Ensayos Clínicos como Asunto/métodos , Sepsis/terapia , Animales , Humanos , Inmunoterapia , Estudios RetrospectivosRESUMEN
Antibodies to various core glycolipid antigens have been shown to correlate with survival from Gram-negative sepsis. Recent preclinical data also support efficacy of the anti-core glycolipid antibodies in the treatment of sepsis. Failure of some of the previous clinical trials with anti-core glycolipid antibody was probably due to inadequate levels of antibody in those preparations. Future clinical trials must ensure that sufficient amounts of anti-core glycolipid antibodies are present in the circulation of patients with sepsis.
Asunto(s)
Anticuerpos Antibacterianos/uso terapéutico , Vacunas Bacterianas/inmunología , Infecciones por Bacterias Gramnegativas/terapia , Sepsis/terapia , Animales , Anticuerpos Monoclonales/uso terapéutico , Vacunas Bacterianas/uso terapéutico , Endotoxinas/química , Endotoxinas/inmunología , Glucolípidos/inmunología , Infecciones por Bacterias Gramnegativas/prevención & control , Humanos , Antígenos O/química , Antígenos O/inmunología , Sepsis/prevención & control , VacunaciónRESUMEN
Fever improves survival in acute infections, but the effects of increased core temperature on host defenses are poorly understood. Tumor necrosis factor alpha (TNF-alpha) is an early activator of host defenses and a major endogenous pyrogen. TNF-alpha expression is essential for survival in bacterial infections but, if disregulated, can cause tissue injury. In this study, we show that passively increasing core temperature in mice from the basal (36.5 to 37.5 degrees C) to the febrile (39.5 to 40 degrees C) range modifies systemic TNF-alpha expression in response to bacterial endotoxin (lipopolysaccharide). The early TNF-alpha secretion rate is enhanced, but the duration of maximal TNF-alpha production is shortened. We identified Kupffer cells as the predominant source of the excess TNF-alpha production in the warmer animals. The enhanced early TNF-alpha production observed at the higher temperature in vivo could not be demonstrated in isolated Kupffer cells or in precision-cut liver slices in vitro, indicating the participation of indirect pathways. Therefore, expression of the endogenous pyrogen TNF-alpha is regulated by increments in core temperature during fever, generating an enhanced early, self-limited TNF-alpha pulse.
Asunto(s)
Regulación de la Temperatura Corporal/inmunología , Fiebre/inmunología , Lipopolisacáridos/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Modelos Animales de Enfermedad , Macrófagos del Hígado/inmunología , Hígado/inmunología , Masculino , Ratones , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
When given in the presence of gamma interferon (IFN-gamma), otherwise nontoxic doses of lipopolysaccharide (LPS or endotoxin) become highly lethal for mice. The mechanisms of this synergistic toxicity are not known. We considered the possibility that an interaction between the LPS-induced NF-kappaB and IFN-gamma-induced JAK-STAT pathways at the pretranscriptional level may enhance the LPS-induced signals. To test this hypothesis, we incubated murine macrophage RAW 264.7 cells with IFN-gamma for 2 h before addition of different doses of LPS. Consistent with the synergistic induction of inducible nitric oxide synthase mRNA and nitric oxide production by a combination of LPS and IFN-gamma, IFN-gamma strongly augmented LPS-induced NF-kappaB activation and accelerated the binding of NF-kappaB to DNA to as early as 5 min. In agreement with this, IFN-gamma pretreatment promoted rapid degradation of IkappaB-alpha but not that of IkappaB-beta. Inhibition of protein synthesis during IFN-gamma treatment suppressed LPS-initiated NF-kappaB binding. A rapidly induced protein appeared to be involved in IFN-gamma priming. Preincubation of cells with antibodies to tumor necrosis factor alpha or the interleukin-1 receptor partially reduced the priming effect of IFN-gamma. In a complementary manner, LPS enhanced the activation of signal-transducing activator of transcription 1 by IFN-gamma. These data suggest novel mechanisms for the synergy between IFN-gamma and LPS by which they cross-regulate the signal-transducing molecules. Through this mechanism, IFN-gamma may transform a given dose of LPS into a lethal stimulus capable of causing sepsis. It may also serve a beneficial purpose by enabling the host to respond quickly to relatively low doses of LPS and thereby activating antibacterial defenses.
Asunto(s)
Adyuvantes Inmunológicos/fisiología , Proteínas I-kappa B , Interferón gamma/fisiología , Interleucina-1/fisiología , Lipopolisacáridos/farmacología , Activación de Macrófagos/inmunología , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Comunicación Autocrina/efectos de los fármacos , Comunicación Autocrina/inmunología , Línea Celular , Cicloheximida/farmacología , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Activación de Macrófagos/efectos de los fármacos , Ratones , Inhibidor NF-kappaB alfa , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Unión Proteica/efectos de los fármacos , Unión Proteica/inmunología , Inhibidores de la Síntesis de la Proteína/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Besides its well-established effects on granulocytopoiesis, granulocyte colony-stimulating factor (G-CSF) has been shown to have direct effects on the recruitment and bactericidal ability of neutrophils, resulting in improved survival of experimentally infected animals. We studied the effect of G-CSF on the course of experimental pneumonia induced by Klebsiella pneumoniae, an important gram-negative bacillary pulmonary pathogen. Using a highly reproducible murine model, we here show the paradoxical finding that mortality from infection was significantly increased when animals received G-CSF before induction of pneumonia. Administration of G-CSF promoted replication of bacteria in the liver and spleen, thus indicating an impairment rather than an enhancement of antibacterial mechanisms. By contrast, a monoclonal antibody against Klebsiella K2 capsule significantly reduced bacterial multiplication in the lung, liver, and spleen, and abrogated the increased mortality caused by G-CSF. In vitro studies showed a direct effect of G-CSF on K pneumoniae resulting in increased capsular polysaccharide (CPS) production. When bacteria were coincubated with therapeutically achievable concentrations of G-CSF, phagocytic uptake and killing by neutrophils was impaired. Western blot analysis showed three binding sites of G-CSF to K pneumoniae. Binding of 125I-G-CSF to K pneumoniae was displaced by an excess of unlabeled G-CSF, whereas an unrelated cytokine, interleukin-1alpha, did not compete with G-CSF binding to the bacteria. Thus, in this model, the direct effect of G-CSF on a bacterial virulence factor, CPS production, outweighed any beneficial effect of G-CSF on recruitment and stimulation of leukocytes.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Infecciones por Klebsiella/fisiopatología , Klebsiella pneumoniae/efectos de los fármacos , Neumonía Bacteriana/fisiopatología , Animales , Femenino , Infecciones por Klebsiella/mortalidad , Klebsiella pneumoniae/metabolismo , Pulmón/microbiología , Pulmón/fisiopatología , Ratones , Ratones Endogámicos BALB C , Neumonía Bacteriana/mortalidad , Polisacáridos Bacterianos/biosíntesis , Análisis de SupervivenciaRESUMEN
The influence of meropenem, a new carbapenem antibiotic, on cell morphology and in-vitro lipopolysaccharide (LPS) release from Escherichia coli was compared with that of imipenem, ceftazidime, tobramycin and ciprofloxacin. Free and cell-associated LPS was quantified by means of a capture ELISA method based on the recognition of E. coli LPS by monoclonal antibodies. Microscopically, meropenem was found to induce spheroplast formation similar to that seen with imipenem, while ceftazidime and ciprofloxacin induced filament formation. Free and cell-associated LPS levels were low in the presence of meropenem, imipenem, ciprofloxacin and tobramycin, but high in the presence of ceftazidime. Reduced endotoxin release appears to be a common property of carbapenem antibiotics. Morphological changes in bacteria in the presence of antibiotics do not predict their LPS-liberating effect since ciprofloxacin induced low levels of LPS despite causing filament formation.
Asunto(s)
Antibacterianos/farmacología , Ensayo de Inmunoadsorción Enzimática/métodos , Escherichia coli/fisiología , Lipopolisacáridos/metabolismo , Tienamicinas/farmacología , Anticuerpos Monoclonales , Ceftazidima/farmacología , Ciprofloxacina/farmacología , Ensayo de Inmunoadsorción Enzimática/normas , Escherichia coli/efectos de los fármacos , Imipenem/farmacología , Lipopolisacáridos/análisis , Lipopolisacáridos/inmunología , Meropenem , Pruebas de Sensibilidad Microbiana , Antígenos O , Tobramicina/farmacologíaRESUMEN
Bacterial lipopolysaccharide induces tyrosine phosphorylation of paxillin, actin reorganization, and opening of the transendothelial paracellular pathway through which macromoles flux. In this study, lipid A was shown to be the bioactive portion of the lipopolysaccharide molecule responsible for changes in endothelial barrier function. We then studied whether endotoxin-neutralizing protein, a recombinant peptide that is derived from Limulus antilipopolysaccharide factor and targets lipid A, could block the effects of lipopolysaccharide on protein tyrosine phosphorylation, actin organization, and movement of 14C-bovine serum albumin across bovine pulmonary artery endothelial cell monolayers. In the presence of serum, a 6-h exposure to lipopolysaccharide (10 ng/ml) increased transendothelial 14C-albumin flux compared to the simultaneous media control. Coadministration of endotoxin-neutralizing protein (> or =10 ng/ml) with lipopolysaccharide (10 ng/ml) protected against lipopolysaccharide-induced barrier dysfunction. This protection was dose dependent, conferring total protection at endotoxin-neutralizing protein/lipopolysaccharide ratios of > or =10:1. Similarly, endotoxin-neutralizing protein was capable of blocking the lipopolysaccharide-induced endothelial cell responses that are prerequisite to barrier dysfunction, including tyrosine phosphorylation of paxillin and actin depolymerization. Finally, endotoxin-neutralizing protein cross-protected against lipopolysaccharide derived from diverse gram-negative bacteria. Thus, endotoxin-neutralizing protein offers a novel therapeutic intervention for the vascular endothelial dysfunction of gram-negative sepsis and its attendant endotoxemia.
Asunto(s)
Antibacterianos/farmacología , Endotelio Vascular/efectos de los fármacos , Hormonas de Invertebrados/farmacología , Lipopolisacáridos/toxicidad , Actinas/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos , Proteínas de Artrópodos , Bovinos , Células Cultivadas , Endotelio Vascular/metabolismo , Lipopolisacáridos/antagonistas & inhibidores , Fosforilación , Albúmina Sérica Bovina/metabolismo , Relación Estructura-Actividad , Tirosina/metabolismoRESUMEN
Polymyxin B (PMB) is a cyclic decapeptide antibiotic which also binds and neutralizes endotoxin. Unfortunately, PMB can be considerably nephrotoxic at clinically utilized doses, thereby limiting its utility as a therapeutic antiendotoxin reagent. We sought to change the pharmacokinetics and toxicity profile of PMB by covalently linking it to a human immunoglobulin G (IgG) carrier. Conjugates of PMB with IgG were prepared by EDAC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide]-mediated amide formation. Analysis by dot enzyme-linked immunosorbent assay with an anti-PMB monoclonal antibody showed that the purified conjugate contained bound PMB. The IgG-PMB conjugate reacted with lipid A and J5 lipopolysaccharide in Western blot assays in a manner comparable to that of whole antiserum with anti-lipid A reactivity; unconjugated IgG had no reactivity. The PMB bound in the conjugate retained its endotoxin-neutralizing activity compared to that of unbound PMB as evidenced by its dose-dependent inhibition of tumor necrosis factor release by endotoxin-stimulated human monocytes in vitro; unconjugated IgG had no activity. By this assay, the PMB-IgG conjugate was determined to have approximately 3.0 microg of bound functional PMB per 100 microg of total protein of conjugate (five molecules of PMB per IgG molecule). The PMB-IgG conjugate was also bactericidal against clinical strains of Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae relative to unconjugated IgG with MBCs of <4 microg of conjugate per ml for each of the tested strains. The conjugate appeared to be nontoxic at the highest doses deliverable and provided statistically significant protection from death to galactosamine-sensitized, lipopolysaccharide-challenged mice in a dose-dependent fashion when administered prophylactically 2 h before challenge. However, neither free PMB nor the PMB-IgG conjugate could protect mice challenged with endotoxin 2 h after administration. This suggests that these reagents can play a role in prophylaxis but not in therapy of sepsis. These experiments demonstrated that the PMB-IgG conjugate retains bound yet functional PMB as evidenced by its endotoxin-neutralizing activity both in vitro and in vivo. Further work is required to define the role that this or related conjugate compounds may play in the prophylaxis of endotoxin-mediated disease.
Asunto(s)
Antibacterianos/farmacología , Inmunoglobulina G , Inmunoglobulinas/farmacología , Monocitos/efectos de los fármacos , Polimixina B/farmacología , Antibacterianos/inmunología , Portadores de Fármacos , Escherichia coli/efectos de los fármacos , Humanos , Klebsiella pneumoniae/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Monocitos/metabolismo , Polimixina B/inmunología , Pseudomonas aeruginosa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The anti-LPS antibody content of commercial intravenous immunoglobulins was examined by quantitative ELISA using LPS preparations from Escherichia coli, Klebsiella and Pseudomonas aeruginosa O serotypes occurring most frequently in gram-negative septicaemia. Three IgG products from different manufacturers and one IgM-enriched product were tested. Mean antibody levels were significantly higher in the IgM fraction of the IgM-enriched product compared with 'pure' IgG products, indicating that natural antibodies against bacterial LPS belong primarily to the IgM class. Immunoblotting studies showed that antibody specificities were directed mainly against O side chain epitopes. Antibodies against rough mutant LPS representing various chemotypes were detected in IgG but not in IgM products. The virtual absence of antibodies against Vibrio cholerae LPS indicated that human anti-LPS antibodies result from continuous environmental exposure to gram-negative pathogens. These data support the further development of IgM-enriched preparations for prophylaxis and treatment of gram-negative nosocomial infections.