RESUMEN
PURPOSE: Sepsis stimulates pro- and anti-inflammatory immune responses. The innate immune response is critical to organ injury repair. We tested for an association between innate immune function and organ function recovery in a prospective cohort of immune-competent adults with sepsis. METHODS: We conducted a prospective observational cohort study enrolling immune-competent adults with sepsis. We tested innate immune function by quantification of lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF) α production capacity in whole blood samples on hospital days 1, 4, and 6. The primary outcome was organ function recovery on day 4 defined as a 4-point decrease in the composite cardiovascular and respiratory Sequential Organ Failure Assessment (SOFA) score components or a SOFA score ≤2. RESULTS: Patients with sepsis who recovered organ function by day 4 (n = 11) had similar baseline characteristics when compared to those with ongoing organ failure (n = 13). Tumor necrosis factor α production capacity was similar between the 2 groups on hospital days 1 and 4 but significantly different on day 6. Patients who regained organ function recovery had significantly higher TNF-α production capacity on day 6 ( P = .01), which persisted after adjustment for age, Acute Physiology and Chronic Health Evaluation III score, and steroid administration ( P = .03). There was no difference in TNF-α production capacity over time in those who survived to hospital discharge versus nonsurvivors. CONCLUSION: Increasing TNF-α production capacity is associated with improved organ failure recovery. Further studies are needed to evaluate a causal association between innate immune suppression and organ failure recovery as well as predictive accuracy for hospital survival. Impaired TNF-α production as a marker of sepsis-associated innate immune dysfunction may be a feasible target for immune stimulation to decrease time to organ failure recovery.
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Cuidados Críticos , Inmunidad Innata/inmunología , Insuficiencia Multiorgánica/inmunología , Sepsis/inmunología , Biomarcadores/sangre , Humanos , Inmunidad Innata/fisiología , Unidades de Cuidados Intensivos , Recuento de Linfocitos , Persona de Mediana Edad , Insuficiencia Multiorgánica/sangre , Insuficiencia Multiorgánica/fisiopatología , Insuficiencia Multiorgánica/terapia , Puntuaciones en la Disfunción de Órganos , Estudios Prospectivos , Sepsis/sangre , Sepsis/fisiopatología , Sepsis/terapiaRESUMEN
RATIONALE: Degradation of the endothelial glycocalyx, a glycosaminoglycan (GAG)-rich layer lining the vascular lumen, is associated with the onset of kidney injury in animal models of critical illness. It is unclear if similar pathogenic degradation occurs in critically ill patients. OBJECTIVES: To determine if urinary indices of GAG fragmentation are associated with outcomes in patients with critical illnesses such as septic shock or acute respiratory distress syndrome (ARDS). METHODS: We prospectively collected urine from 30 patients within 24 hours of admission to the Denver Health Medical Intensive Care Unit (ICU) for septic shock. As a nonseptic ICU control, we collected urine from 25 surgical ICU patients admitted for trauma. As a medical ICU validation cohort, we obtained serially collected urine samples from 70 patients with ARDS. We performed mass spectrometry on urine samples to determine GAG (heparan sulfate, chondroitin sulfate, and hyaluronic acid) concentrations as well as patterns of heparan sulfate/chondroitin sulfate disaccharide sulfation. We compared these indices to measurements obtained using dimethylmethylene blue, an inexpensive, colorimetric urinary assay of sulfated GAGs. MEASUREMENTS AND MAIN RESULTS: In septic shock, indices of GAG fragmentation correlated with both the development of renal dysfunction over the 72 hours after urine collection and with hospital mortality. This association remained after controlling for severity of illness and was similarly observed using the inexpensive dimethylmethylene blue assay. These predictive findings were corroborated using urine samples previously collected at three consecutive time points from patients with ARDS. CONCLUSIONS: Early indices of urinary GAG fragmentation predict acute kidney injury and in-hospital mortality in patients with septic shock or ARDS. Clinical trial registered with www.clinicaltrials.gov (NCT01900275).
Asunto(s)
Lesión Renal Aguda/orina , Glicosaminoglicanos/orina , Mortalidad Hospitalaria , Choque Séptico/orina , Heridas y Lesiones/orina , Lesión Renal Aguda/diagnóstico , Lesión Renal Aguda/etiología , Lesión Renal Aguda/mortalidad , Biomarcadores/orina , Estudios de Casos y Controles , Colorado , Humanos , Unidades de Cuidados Intensivos/estadística & datos numéricos , Espectrometría de Masas/métodos , Valor Predictivo de las Pruebas , Pronóstico , Estudios Prospectivos , Choque Séptico/complicaciones , Choque Séptico/diagnóstico , Choque Séptico/mortalidad , Índices de Gravedad del Trauma , Heridas y Lesiones/clasificación , Heridas y Lesiones/cirugíaRESUMEN
The determination of complex analytes, present at low concentrations, in biological fluids poses a difficult challenge. This study relies on an optimized method of recovery, enzymatic treatment, and disaccharide analysis by liquid chromatography-tandem mass spectrometry to rapidly determine low concentrations of glycosaminoglycans in human urine. The approach utilizes multiple reaction monitoring (MRM) of glycosaminoglycan disaccharides obtained from treating urine samples with recombinant heparin lyases and chondroitin lyase. This rapid and sensitive method allows the analysis of glycosaminoglycan content and disaccharide composition in urine samples having concentrations 10- to 100-fold lower than those typically analyzed from patients with metabolic diseases, such as mucopolysaccharidosis. The current method facilitates the analysis low (ng/mL) levels of urinary glycosaminoglycans present in healthy individuals and in patients with pathological conditions, such as inflammation and cancers, that can subtly alter glycosaminoglycan content and composition.
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Glicosaminoglicanos/orina , Cromatografía Liquida/instrumentación , Creatinina/orina , Humanos , Espectrometría de Masas en Tándem/instrumentaciónRESUMEN
RATIONALE: Diagnosis of ventilator-associated pneumonia (VAP) is imprecise. OBJECTIVES: To (1) determine whether alternate-day surveillance mini-bronchoalveolar lavage (mini-BAL) in ventilated adults could reduce time to initiation of targeted treatment and (2) evaluate the potential for automated microscopy to reduce analysis time. METHODS: Adult intensive care unit patients who were anticipated to require ventilation for at least a further 48 hours were included. Mini-BALs were processed for identification, quantitation, and antibiotic susceptibility, using (1) clinical culture (50 ± 7 h) and (2) automated microscopy (â¼5 h plus offline analysis). MEASUREMENTS AND MAIN RESULTS: Seventy-seven mini-BALs were performed in 33 patients. One patient (3%) was clinically diagnosed with VAP. Of 73 paired samples, culture identified 7 containing pneumonia panel bacteria (>10(4) colony-forming units/ml) from five patients (15%) (4 Staphylococcus aureus [3 methicillin-resistant S. aureus], 2 Stenotrophomonas maltophilia, 1 Klebsiella pneumoniae) and resulted in antimicrobial changes/additions to two of five (40%) of those patients. Microscopy identified 7 of 7 microbiologically positive organisms and 64 of 66 negative samples compared with culture. Antimicrobial responses were concordant in four of five comparisons. Antimicrobial changes/additions would have occurred in three of seven microscopy-positive patients (43%) had those results been clinically available in 5 hours, including one patient diagnosed later with VAP despite negative mini-BAL cultures. CONCLUSIONS: Microbiological surveillance detected infection in patients at risk for VAP independent of clinical signs, resulting in changes to antimicrobial therapy. Automated microscopy was 100% sensitive and 97% specific for high-risk pneumonia organisms compared with clinical culturing. Rapid microscopy-based surveillance may be informative for treatment and antimicrobial stewardship in patients at risk for VAP.
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Líquido del Lavado Bronquioalveolar/microbiología , Neumonía Asociada al Ventilador/diagnóstico , Adulto , Automatización , Técnicas Bacteriológicas/métodos , Lavado Broncoalveolar/métodos , Femenino , Humanos , Masculino , Microscopía/métodos , Persona de Mediana Edad , Neumonía Asociada al Ventilador/microbiología , Sensibilidad y EspecificidadRESUMEN
Systemic inflammatory illnesses (such as sepsis) are marked by degradation of the endothelial glycocalyx, a layer of glycosaminoglycans (including heparan sulfate, chondroitin sulfate, and hyaluronic acid) lining the vascular lumen. We hypothesized that different pathophysiologic insults would produce characteristic patterns of released glycocalyx fragments. We collected plasma from healthy donors as well as from subjects with respiratory failure due to altered mental status (intoxication, ischemic brain injury), indirect lung injury (non-pulmonary sepsis, pancreatitis), or direct lung injury (aspiration, pneumonia). Mass spectrometry was employed to determine the quantity and sulfation patterns of circulating glycosaminoglycans. We found that circulating heparan sulfate fragments were significantly (23-fold) elevated in patients with indirect lung injury, while circulating hyaluronic acid concentrations were elevated (32-fold) in patients with direct lung injury. N-Sulfation and tri-sulfation of heparan disaccharides were significantly increased in patients with indirect lung injury. Chondroitin disaccharide sulfation was suppressed in all groups with respiratory failure. Plasma heparan sulfate concentrations directly correlated with intensive care unit length of stay. Serial plasma measurements performed in select patients revealed that circulating highly sulfated heparan fragments persisted for greater than 3 days after the onset of respiratory failure. Our findings demonstrate that circulating glycosaminoglycans are elevated in patterns characteristic of the etiology of respiratory failure and may serve as diagnostic and/or prognostic biomarkers of critical illness.
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Enfermedad Crítica , Glicosaminoglicanos/sangre , Insuficiencia Respiratoria/sangre , Adulto , Anciano , Sulfatos de Condroitina/sangre , Femenino , Heparitina Sulfato/sangre , Humanos , Pulmón/fisiopatología , Masculino , Persona de Mediana Edad , Insuficiencia Respiratoria/fisiopatologíaRESUMEN
Sepsis, a systemic inflammatory response to infection, commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity. We postulated that sepsis-associated ALI is initiated by degradation of the pulmonary endothelial glycocalyx, leading to neutrophil adherence and inflammation. Using intravital microscopy, we found that endotoxemia in mice rapidly induced pulmonary microvascular glycocalyx degradation via tumor necrosis factor-α (TNF-α)-dependent mechanisms. Glycocalyx degradation involved the specific loss of heparan sulfate and coincided with activation of endothelial heparanase, a TNF-α-responsive, heparan sulfate-specific glucuronidase. Glycocalyx degradation increased the availability of endothelial surface adhesion molecules to circulating microspheres and contributed to neutrophil adhesion. Heparanase inhibition prevented endotoxemia-associated glycocalyx loss and neutrophil adhesion and, accordingly, attenuated sepsis-induced ALI and mortality in mice. These findings are potentially relevant to human disease, as sepsis-associated respiratory failure in humans was associated with higher plasma heparan sulfate degradation activity; moreover, heparanase content was higher in human lung biopsies showing diffuse alveolar damage than in normal human lung tissue.
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Lesión Pulmonar Aguda/fisiopatología , Endotoxemia/complicaciones , Glicocálix/fisiología , Pulmón/fisiopatología , Neutrófilos/fisiología , Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/prevención & control , Traslado Adoptivo , Animales , Adhesión Celular/fisiología , Modelos Animales de Enfermedad , Endotelio/enzimología , Endotelio/fisiología , Endotoxemia/fisiopatología , Activación Enzimática , Regulación de la Expresión Génica/efectos de los fármacos , Glucuronidasa/análisis , Glucuronidasa/deficiencia , Glucuronidasa/fisiología , Heparitina Sulfato/antagonistas & inhibidores , Heparitina Sulfato/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/genética , Perforación Intestinal/complicaciones , Perforación Intestinal/microbiología , Lipopolisacáridos/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Alveolos Pulmonares/enzimología , Alveolos Pulmonares/patología , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Receptores Tipo I de Factores de Necrosis Tumoral/fisiología , Insuficiencia Respiratoria/enzimología , Insuficiencia Respiratoria/patología , Factor de Necrosis Tumoral alfa/fisiología , Lesión Pulmonar Inducida por Ventilación Mecánica/enzimología , Lesión Pulmonar Inducida por Ventilación Mecánica/patologíaRESUMEN
Cystathionine beta-synthase (CBS) catalyzes the condensation of homocysteine (Hcy) and serine to cystathionine, which is then hydrolyzed to cysteine by cystathionine gamma-lyase. Inactivation of CBS results in CBS-deficient homocystinuria more commonly referred to as classical homocystinuria, which, if untreated, results in mental retardation, thromboembolic complications, and a range of connective tissue disorders. The molecular mechanisms that underlie the pathology of this disease are poorly understood. We report here the generation of a new mouse model of classical homocystinuria in which the mouse cbs gene is inactivated and that exhibits low-level expression of the human CBS transgene under the control of the human CBS promoter. This mouse model, designated "human only" (HO), exhibits severe elevations in both plasma and tissue levels of Hcy, methionine, S-adenosylmethionine, and S-adenosylhomocysteine and a concomitant decrease in plasma and hepatic levels of cysteine. HO mice exhibit mild hepatopathy but, in contrast to previous models of classical homocystinuria, do not incur hepatic steatosis, fibrosis, or neonatal death with approximately 90% of HO mice living for at least 6months. Tail bleeding determinations indicate that HO mice are in a hypercoagulative state that is significantly ameliorated by betaine treatment in a manner that recapitulates the disease as it occurs in humans. Our findings indicate that this mouse model will be a valuable tool in the study of pathogenesis in classical homocystinuria and the rational design of novel treatments.
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Betaína/uso terapéutico , Cistationina betasintasa/deficiencia , Homocistinuria/genética , Animales , Trastornos de la Coagulación Sanguínea/tratamiento farmacológico , Trastornos de la Coagulación Sanguínea/etiología , Cistationina/sangre , Modelos Animales de Enfermedad , Hígado Graso/patología , Fibrosis , Homocistinuria/tratamiento farmacológico , Homocistinuria/patología , Ratones , Ratones TransgénicosRESUMEN
Although the accumulation of neutrophils in the lungs and airways is common to many inflammatory lung diseases, including acute lung injury, the alterations that neutrophils undergo as they leave the peripheral circulation and migrate into the lungs have not been well characterized. Human volunteers were exposed to endotoxin by bronchoscopic instillation. The resulting air space neutrophil accumulation and peripheral blood neutrophils were isolated 16 h later, compared with circulating neutrophils isolated before or after to the pulmonary endotoxin exposure, and compared with circulating neutrophils exposed to endotoxin in vitro. Microarray analysis was performed on air space, circulatory, and in vitro endotoxin-stimulated neutrophils. Functional analysis included the determination of neutrophil apoptosis, chemotaxis, release of cytokines and growth factors, and superoxide anion release. Dramatic gene expression differences were apparent between air space and circulating neutrophils: approximately 15% of expressed genes have altered expression levels, including broad increases in inflammatory- and chemotaxis-related genes, as well as antiapoptotic and IKK-activating pathways. Functional analysis of air space compared with circulating neutrophils showed increased superoxide release, diminished apoptosis, decreased IL-8-induced chemotaxis, and a pattern of IL-8, macrophage inflammatory protein-1beta, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha release different from either unstimulated or LPS-stimulated circulating neutrophils. Many of these changes are not elicited by in vitro treatment with endotoxin. Limited differences were detected between circulating neutrophils isolated before and 16 h after pulmonary endotoxin instillation. These results suggest that neutrophils sequestered in the lung become fundamentally different from those resident in the circulation, and this difference is distinct from in vitro activation with endotoxin.
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Citocinas/genética , Genómica , Macrófagos Alveolares/fisiología , Neutrófilos/fisiología , Movimiento Celular , Quimiotaxis de Leucocito , Regulación de la Expresión Génica , Humanos , Cinética , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Increased nuclear accumulation of NF-kappaB in LPS-stimulated peripheral blood neutrophils has been shown to be associated with more severe clinical course in patients with infection associated acute lung injury. Such observations suggest that differences in neutrophil response may contribute to the pulmonary inflammation induced by bacterial infection. To examine this question, we sequentially measured LPS-induced DNA binding of NF-kappaB in neutrophils collected from healthy humans on at least three occasions, each separated by at least 2 wk, and then determined pulmonary inflammatory responses after instillation of LPS into the lungs. Consistent patterns of peripheral blood neutrophil responses, as determined by LPS-induced NF-kappaB DNA binding, were present in volunteers, with a >80-fold difference between individuals in the mean area under the curve for NF-kappaB activation. The number of neutrophils recovered from bronchoalveolar lavage after exposure to pulmonary LPS was significantly correlated with NF-kappaB activation in peripheral blood neutrophils obtained over the pre-LPS exposure period (r = 0.65, p = 0.009). DNA binding of NF-kappaB in pulmonary neutrophils also was associated with the mean NF-kappaB area under the curve for LPS-stimulated peripheral blood neutrophils (r = 0.63, p = 0.01). Bronchoalveolar lavage levels of IL-6 and TNFRII were significantly correlated with peripheral blood neutrophil activation patterns (r = 0.75, p = 0.001 for IL-6; and r = 0.48, p = 0.049 for TNFRII. These results demonstrate that stable patterns in the response of peripheral blood neutrophils to LPS exist in the human population and correlate with inflammatory response following direct exposure to LPS in the lung.