Assuntos
Plaquetas/ultraestrutura , Diabetes Mellitus Tipo 2/sangue , Integrina beta3/efeitos dos fármacos , Substâncias Macromoleculares/química , Pravastatina/farmacologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Colesterol/sangue , Estudos Cross-Over , Feminino , Humanos , Integrina beta3/análise , Substâncias Macromoleculares/sangue , Masculino , Pessoa de Meia-Idade , Ativação Plaquetária , Pravastatina/uso terapêuticoRESUMO
AIM: We investigated the occurrence and thrombin generating mechanisms of circulating microparticles (MP) in patients with multiple organ dysfunction syndrome (MODS) and sepsis. METHODS: MP, isolated from blood of patients (n = 9) and healthy controls (n = 14), were stained with cell-specific monoclonal antibodies (MoAbs) or anti-tissue factor (anti-TF) MoAb and annexin V, and analyzed by flow cytometry. To assess their thrombin-generating capacity, MP were reconstituted in normal plasma. The coagulation activation status in vivo was quantified by plasma prothrombin fragment F1+2- and thrombin-antithrombin (TAT) measurements. RESULTS: Annexin V-positive MP in the patients originated predominantly from platelets (PMP), and to a lesser extent from erythrocytes, endothelial cells (EMP) and granulocytes (GMP). Compared to healthy controls, the numbers of annexin V-positive PMP and TF-exposing MP were decreased (p = <0.001 for both), EMP were decreased (E-selectin, p = 0.003) or found equal (CD144, p = 0.063), erythrocyte-derived MP were equal (p = 0.726), and GMP were increased (p = 0.008). GMP numbers correlated with plasma concentrations of elastase (r = 0.70, p = 0.036), but not with C-reactive-protein or interleukin-6 concentrations. Patient samples also contained reduced numbers of annexin V-negative PMP, and increased numbers of erythrocyte-derived MP and GMP (p = 0.005, p = 0.021 and p <0.001, respectively). Patient MP triggered thrombin formation, which was reduced compared to the healthy controls (p = 0.008) and strongly inhibited by an anti-factor XII MoAb (two patients), by anti-factor XI MoAb (eight patients) or by anti-TF MoAb (four patients). Concentrations of F1+2 and TAT were elevated (p = 0.005 and p = 0.001, respectively) and correlated inversely with the number of circulating MP (and r = -0.51, p = 0.013, and r = -0.65, p = 0.001, respectively) and their thrombin generation capacity (F1+2: r= -0.62, p = 0.013). CONCLUSIONS: In patients with MODS and sepsis relatively low numbers of MP are present that differ from controls in their cellular origin, numbers and coagulation activation mechanisms.
Assuntos
Células Sanguíneas/metabolismo , Membrana Celular/metabolismo , Insuficiência de Múltiplos Órgãos/sangue , Sepse/sangue , Trombofilia/etiologia , Proteínas de Fase Aguda/metabolismo , Adulto , Idoso , Células Sanguíneas/fisiologia , Células Sanguíneas/ultraestrutura , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/metabolismo , Plaquetas/fisiologia , Plaquetas/ultraestrutura , Proteína C-Reativa/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Elastase Pancreática/sangue , Tamanho da Partícula , Fosfolipídeos/efeitos adversos , Fosfolipídeos/sangue , Fosfolipídeos/metabolismo , Trombina/biossíntese , Trombina/efeitos dos fármacos , Trombofilia/sangueRESUMO
A study was conducted to investigate the relationship between intra-uterine bacterial contamination, endotoxin levels and the development of endometritis in cows that experienced a dystocia or retained their placenta. Fifteen healthy cows, 31 cows with retained placenta (RP) and 13 cows that had dystocia were clinically examined 1 or 2 days after parturition when a uterine swab for bacteriological examination was taken. In addition, plasma and uterine lochia samples were collected to determine lipopolysaccharide (LPS) and the plasma IgG anti-LPS concentrations. Subsequently, 15 RP and 6 dystocia cows were initially left untreated and another uterine swab was collected at 2 and 4 wk postpartum. Immediately after calving, RP cows had significantly higher LPS levels in uterine lochia (average of 2.24 x 10(4) Endotoxin Units (EU)/mL) as compared to dystocia and healthy postpartum cows (average of 0.10 and 0.26 EU/mL, respectively). However, plasma LPS levels were below the detection limit (<0.036 EU/mL platelet-rich plasma) in all groups of cows. IgG anti-LPS levels in plasma were not significantly different between the 3 groups immediately postpartum (average of 26, 16 and 44 Median Units (MU)/mL) for healthy, dystocia and RP cows, respectively), but they were significantly lower when compared to plasma IgG anti-LPS levels of healthy cows at more than 2 months postpartum (mean 83 MU/mL). High LPS levels in lochia at 1 or 2 days postpartum were significantly related to abnormal cervical discharge, the presence of Escherichia coli, black pigmented gram-negative anaerobes and Clostridium spp. shortly after calving, and Arcanobacterium pyogenes and gram-negative anaerobes in the uterus at 14 days postpartum. These results suggest that the presence of E. coli and LPS (endotoxins) in lochia early postpartum favor the development of uterine infections by A. pyogenes and gram-negative anaerobes later postpartum. LPS were not observed in plasma, suggesting that either they are not absorbed into the blood, or they are efficiently detoxified by IgG anti-LPS or other detoxification mechanisms.
Assuntos
Doenças dos Bovinos/microbiologia , Distocia/veterinária , Endometrite/microbiologia , Endotoxinas/análise , Placenta Retida/veterinária , Útero/microbiologia , Animais , Bovinos , Doenças dos Bovinos/metabolismo , Clostridium/isolamento & purificação , Distocia/complicações , Endometrite/metabolismo , Escherichia coli/isolamento & purificação , Feminino , Bactérias Gram-Positivas/isolamento & purificação , Imunoglobulina G/sangue , Lipopolissacarídeos/análise , Lipopolissacarídeos/imunologia , Placenta Retida/complicações , Gravidez , Transtornos Puerperais/microbiologia , Transtornos Puerperais/veterinária , Útero/metabolismoRESUMO
We prospectively examined 464 febrile patients (median age, 61 years) for predictors of in-hospital death, by use of univariate and multivariate logistic regression using clinical data (age, underlying disease, duration of fever, chills, and shock on admission) and plasma endotoxin, TNF-alpha, IL-6, IL-10, and procalcitonin levels. The mortality rate was 4.6-fold higher (95% confidence interval [CI], 1.8-12) in 31 patients with shock on admission, 7 of whom died; the strongest association with mortality was the endotoxin concentration (relative risk, 13.7; 95% CI, 1. 4-136), which predicted 5 of the deaths with a 5% false-positive rate. For 433 patients without shock on admission, mortality (26 deaths) was associated with age and underlying disease: clinical data predicted 30% of the deaths, whereas IL-6 and procalcitonin levels identified an extra 10% with a 5% false-positive rate. When febrile patients are screened on hospital admission to identify those with a high risk for mortality, clinical judgment on the basis of age, underlying disease, and recent history outweighs the predictive value of endotoxin, cytokine, and procalcitonin levels. Only in patients who present with shock will measurement of endotoxin levels help predict those who will likely die at the cost of few false-positive results.
Assuntos
Calcitonina/sangue , Infecções Comunitárias Adquiridas/mortalidade , Citocinas/sangue , Endotoxinas/sangue , Febre/etiologia , Precursores de Proteínas/sangue , Sepse/mortalidade , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Bacteriemia/microbiologia , Bactérias/classificação , Bactérias/isolamento & purificação , Peptídeo Relacionado com Gene de Calcitonina , Infecções Comunitárias Adquiridas/diagnóstico , Endotoxemia/diagnóstico , Feminino , Hospitalização , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Valor Preditivo dos Testes , Estudos Prospectivos , Fatores de Risco , Sepse/diagnóstico , Choque/diagnósticoRESUMO
The aim of the present study was to establish whether gastro-intestinal (GI) complaints observed during and after ultra-endurance exercise are related to gut ischaemia-associated leakage of endotoxins [lipopolysaccharide (LPS)] into the circulation and associated cytokine production. Therefore we collected blood samples from 29 athletes before, immediately after, and 1, 2 and 16 h after a long-distance triathlon for measurement of LPS, tumour necrosis factor-alpha and interleukin-6 (IL-6). As the cytokine response would trigger an acute-phase response, characteristic variables of these responses were also measured, along with creatine kinase (CK) to obtain an indicator of muscle damage. There was a high incidence (93% of all participants) of GI symptoms; 45% reported severe complaints and 7% of the participants abandoned the race because of severe GI distress. Mild endotoxaemia (5-15 pg/ml) was evident in 68% of the athletes immediately after the race, as also indicated by a reduction in IgG anti-LPS levels. In addition, we observed production of IL-6 (27-fold increase immediately after the race), leading to an acute-phase response (20-fold increase in C-reactive protein and 12% decrease in pre-albumin 16 h after the race). The extent of endotoxaemia was not correlated with the GI complaints or the IL-6 response, but did show a correlation with the elevation in C-reactive protein (r(s) 0.389; P=0.037). Creatine kinase levels were increased significantly immediately post-race, and increased further in the follow-up period. Creatine kinase levels did not correlate with those of either IL-6 or C-reactive protein. It is therefore concluded that LPS does enter the circulation after ultra-endurance exercise and may, together with muscle damage, be responsible for the increased cytokine response and hence GI complaints in these athletes.
Assuntos
Reação de Fase Aguda , Citocinas/metabolismo , Endotoxemia/metabolismo , Gastroenteropatias/metabolismo , Resistência Física/fisiologia , Corrida/fisiologia , Adulto , Análise de Variância , Anticorpos Antibacterianos/sangue , Peso Corporal , Proteína C-Reativa/análise , Creatina Quinase/sangue , Feminino , Humanos , Imunoglobulina G/imunologia , Interleucina-6/sangue , Lipopolissacarídeos/sangue , Lipopolissacarídeos/imunologia , Masculino , Pré-Albumina/análise , Fator de Necrose Tumoral alfa/análise , Equilíbrio HidroeletrolíticoRESUMO
A chromogenic assay is presented for the determination of endotoxin (LPS) in blood. The assay is based upon the LPS-dependent activation of Limulus amebocyte lysate (LAL), and the subsequent measurement of the activated enzyme with a chromogenic substrate. Handling and stability of the reagents, details of the assay method in tubes or microtiter-plates, recovery of LPS from spiked blood in platelet-rich plasma (PRP), platelet-poor plasma (PPP) and serum, as well as the possibility to store plasma samples will be discussed. A clinical evaluation of the assay is provided by S. van Deventer et al. in this volume.
Assuntos
Endotoxinas/sangue , Compostos Cromogênicos , Estabilidade de Medicamentos , Humanos , Indicadores e Reagentes , Teste do Limulus , Lipopolissacarídeos/sangue , Compostos OrgânicosRESUMO
Early detection of Gram-negative septicemia or endotoxemia may become feasible with sensitive and reliable endotoxin (LPS) measurements. We recently published an assay for LPS in blood, based upon the LPS dependent activation of Limulus amebocyte lysate (LAL) and the subsequent chromogenic measurement of the activated enzyme(s). Inhibitors and activated clotting factors potentially interfering in the assay were removed by dilution and heating. In the present study we describe the further improvement of the assay. Optimal conditions include: blood anticoagulated with 30 I.U./ml heparin; dilution of the platelet-rich plasma (PRP) in water; 5 min. heating at 75 degrees C; 15 mM Mg2+, 1.5 mM Ca2+, 125 mM Na+, 50 mM TRIS, pH 8.5 in the LAL activation step; substrate step without extra addition of Ca2+, Mg2+, or Na+, but in the presence of 50 mM TRIS at pH = 9.5. Under those optimal conditions less than 10 pg LPS per ml blood (PRP) can easily be detected. Prospective clinical trials are presently envisaged to investigate the clinical usefulness of this extremely sensitive LPS assay.