RESUMEN
The role of complement in the pathogenesis of venous thromboembolism (VTE) is unclear. We wanted to investigate (1) whether plasma complement component C5 (C5) levels are influenced by genetic variants or chronic inflammation and (2) the association between plasma C5 and risk of future VTE in a nested case-control study of 415 patients with VTE and 848 age- and sex-matched controls derived from the Tromsø Study. Plasma C5 levels were measured at inclusion. Odds ratios (ORs) with 95% confidence intervals (95% CIs) for provoked and unprovoked VTE across tertiles of C5 concentrations were estimated by logistic regression. Adjustment for C-reactive protein (CRP) served as a proxy for general inflammation. Whole-exome sequencing and protein quantitative trait loci analyses were performed to assess genetic influence on C5 concentrations. There was no association between genome-wide or C5-related gene variants and C5 levels. The association between plasma C5 levels and VTE risk displayed a threshold effect, where subjects with C5 levels above the lowest tertile had increased risk of VTE. Subjects in tertile 3 (highest C5 levels) had an age- and sex-adjusted OR of 1.45 (95% CI, 1.07-1.96) compared with tertile 1 (lowest). These statistics were more pronounced for unprovoked VTE (OR, 1.70; 95% CI, 1.11-2.60). Adjustments for body mass index and CRP had minor impact on risk estimates. The OR increased substantially with shorter time between blood sampling and VTE event. In conclusion, plasma C5 was associated with risk of future VTE. C5 levels were not genetically regulated and were only slightly influenced by chronic inflammation.
Asunto(s)
Complemento C5/análisis , Tromboembolia Venosa/sangre , Anciano , Estudios de Casos y Controles , Enfermedad Crónica , Complemento C5/genética , Femenino , Variación Genética , Humanos , Inflamación/sangre , Inflamación/genética , Inflamación/patología , Masculino , Persona de Mediana Edad , Recurrencia , Factores de Riesgo , Tromboembolia Venosa/genética , Tromboembolia Venosa/patologíaRESUMEN
Acute coronary syndrome (ACS) results from inadequate supply of blood flow from the coronary arteries to the heart or ischemia. ACS has an extremely high morbidity and mortality. The levels of biomarkers currently used for detection of ACS also increase in response to myocardial necrosis and other diseases and are not elevated immediately after symptoms appear, thus limiting their diagnostic capacity. Therefore, we aimed to discover new ACS diagnostic biomarkers with high sensitivity and specificity that are specifically related to ACS pathogenesis. Sera from 50 patients with ACS and healthy controls (discovery cohort) each were analyzed using mass spectrometry (MS) to identify differentially expressed proteins, and protein candidates were evaluated as ACS biomarkers in 120 people in each group (validation cohort). α-1-acid glycoprotein 1 (AGP1), complement C5 (C5), leucine-rich α-2-glycoprotein (LRG), and vitronectin (VN) were identified as biomarkers whose levels increase and gelsolin (GSN) as a biomarker whose levels decrease in patients with ACS. We concluded that these biomarkers are associated with the pathogenesis of ACS and can predict the onset of ACS prior to the appearance of necrotic biomarkers.
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Síndrome Coronario Agudo/sangre , Proteínas Sanguíneas/análisis , Proteoma , Proteómica , Síndrome Coronario Agudo/diagnóstico , Anciano , Biomarcadores/sangre , Estudios de Casos y Controles , Complemento C5/análisis , Femenino , Gelsolina/sangre , Glicoproteínas/sangre , Humanos , Masculino , Persona de Mediana Edad , Orosomucoide/análisis , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem , Vitronectina/sangreRESUMEN
Unbiased plasma proteomics in a matched case-control study of treated people with human immunodeficiency virus (PWH) revealed the complement cascade as being among the top pathways enriched in PWH. Specific complement components, namely C5, associated significantly with non-AIDS comorbidity prevalence, and did so more strongly than previously established predictive biomarkers.
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Complemento C5/análisis , Infecciones por VIH/epidemiología , Envejecimiento , Biomarcadores/sangre , Estudios de Casos y Controles , Comorbilidad , VIH , Seronegatividad para VIH , Humanos , Factores InmunológicosRESUMEN
SKY59 or RO7112689 is a humanized monoclonal antibody against complement protein C5 with pH-dependent C5-binding and neonatal Fc receptor-mediated recycling capabilities, which result in long-lasting neutralization of C5. We developed and validated a novel total drug assay for quantification of target-binding competent SKY59 in the presence of endogenous C5 in cynomolgus monkey plasma. The target-binding competent SKY59 was determined after complex formation by the addition of recombinant monkey C5 using goat anti-human IgG-heavy chain monkey-adsorbed polyclonal antibody as a capture antibody and rabbit anti-C5 monoclonal antibody (mAb) non-competing with SKY59 for detection. The total SKY59 assay was shown to be accurate and precise over the range of 0.05-3.2 µg/mL as well as be tolerant to more than 400 µg/mL of C5 (~ 3000-fold molar excess of target). We also developed and validated a total C5 assay, confirmed selectivity and parallelism, and verified the utility of recombinant monkey C5 for the total C5 assay as well as the total SKY59 assay. Furthermore, we used these validated methods to measure SKY59 and C5 concentrations in cynomolgus monkey plasma samples in a toxicology study. This total drug assay can be applied not only to other antibody therapeutics against shed/soluble targets when a non-competing reagent mAb is available but also for clinical studies when a reagent mAb specific for engineered Fc region on a therapeutic mAb is available.
Asunto(s)
Anticuerpos Monoclonales Humanizados/sangre , Bioensayo/métodos , Complemento C5/antagonistas & inhibidores , Monitoreo de Drogas/métodos , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/farmacocinética , Complemento C5/análisis , Complemento C5/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Antígenos de Histocompatibilidad Clase I/metabolismo , Inyecciones Intravenosas , Inyecciones Subcutáneas , Límite de Detección , Macaca fascicularis , Masculino , Modelos Animales , Receptores Fc/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND AND AIMS: Given the lack of effective therapies and high mortality in acute alcohol-associated hepatitis (AH), it is important to develop rationally designed biomarkers for effective disease management. Complement, a critical component of the innate immune system, contributes to uncontrolled inflammatory responses leading to liver injury, but is also involved in hepatic regeneration. Here, we investigated whether a panel of complement proteins and activation products would provide useful biomarkers for severity of AH and aid in predicting 90-day mortality. APPROACH AND RESULTS: Plasma samples collected at time of diagnosis from 254 patients with moderate and severe AH recruited from four medical centers and 31 healthy persons were used to quantify complement proteins by enzyme-linked immunosorbent assay and Luminex arrays. Components of the classical and lectin pathways, including complement factors C2, C4b, and C4d, as well as complement factor I (CFI) and C5, were reduced in AH patients compared to healthy persons. In contrast, components of the alternative pathway, including complement factor Ba (CFBa) and factor D (CFD), were increased. Markers of complement activation were also differentially evident, with C5a increased and the soluble terminal complement complex (sC5b9) decreased in AH. Mannose-binding lectin, C4b, CFI, C5, and sC5b9 were negatively correlated with Model for End-Stage Liver Disease score, whereas CFBa and CFD were positively associated with disease severity. Lower CFI and sC5b9 were associated with increased 90-day mortality in AH. CONCLUSIONS: Taken together, these data indicate that AH is associated with a profound disruption of complement. Inclusion of complement, especially CFI and sC5b9, along with other laboratory indicators, could improve diagnostic and prognostic indications of disease severity and risk of mortality for AH patients.
Asunto(s)
Hepatitis Alcohólica/diagnóstico , Adulto , Biomarcadores/sangre , Estudios de Casos y Controles , Complemento C2/análisis , Complemento C3/análisis , Complemento C4/análisis , Complemento C5/análisis , Factor B del Complemento/análisis , Factor D del Complemento/análisis , Proteínas del Sistema Complemento/análisis , Femenino , Hepatitis Alcohólica/inmunología , Humanos , Masculino , Persona de Mediana Edad , PronósticoRESUMEN
SARS-CoV-2 infection causes an abrupt response by the host immune system, which is largely responsible for the outcome of COVID-19. We investigated whether the specific immune responses in the peripheral blood of 276 patients were associated with the severity and progression of COVID-19. At admission, dramatic lymphopenia of T, B, and NK cells is associated with severity. Conversely, the proportion of B cells, plasmablasts, circulating follicular helper T cells (cTfh) and CD56- CD16+ NK-cells increased. Regarding humoral immunity, levels of IgM, IgA, and IgG were unaffected, but when degrees of severity were considered, IgG was lower in severe patients. Compared to healthy donors, complement C3 and C4 protein levels were higher in mild and moderate, but not in severe patients, while the activation peptide of C5 (C5a) increased from the admission in every patient, regardless of their severity. Moreover, total IgG, the IgG1 and IgG3 isotypes, and C4 decreased from day 0 to day 10 in patients who were hospitalized for more than two weeks, but not in patients who were discharged earlier. Our study provides important clues to understand the immune response observed in COVID-19 patients, associating severity with an imbalanced humoral response, and identifying new targets for therapeutic intervention.
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Linfocitos B/inmunología , COVID-19/patología , Inmunoglobulinas/sangre , Células Asesinas Naturales/inmunología , SARS-CoV-2/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Anciano , COVID-19/inmunología , Complemento C3/análisis , Complemento C4/análisis , Complemento C5/análisis , Femenino , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Recuento de Linfocitos , Linfopenia/inmunología , Masculino , Persona de Mediana Edad , Síndrome de Dificultad Respiratoria/inmunología , Síndrome de Dificultad Respiratoria/patologíaRESUMEN
The complement system may be crucial during dengue virus infection and progression to severe dengue. This study investigates the role of MBL2 genetic variants and levels of MBL in serum and complement proteins in Vietnamese dengue patients. MBL2 genotypes (- 550L/H, MBL2 codon 54), MBL2 diplotypes (XA/XO, YA/XO) and MBL2 haplotypes (LXPB, HXPA, XO) were associated with dengue in the study population. The levels of complement factors C2, C5, and C5a were higher in dengue and dengue with warning signs (DWS) patients compared to those in healthy controls, while factor D levels were decreased in dengue and DWS patients compared to the levels determined in healthy controls. C2 and C5a levels were associated with the levels of AST and ALT and with WBC counts. C9 levels were negatively correlated with ALT levels and WBC counts, and factor D levels were associated with AST and ALT levels and with platelet counts. In conclusions, MBL2 polymorphisms are associated with dengue in the Vietnamese study population. The levels of the complement proteins C2, C4b, C5, C5a, C9, factor D and factor I are modulated in dengue patients during the clinical course of dengue.
Asunto(s)
Biomarcadores/análisis , Virus del Dengue/aislamiento & purificación , Factores Inmunológicos/sangre , Lectina de Unión a Manosa/sangre , Lectina de Unión a Manosa/genética , Polimorfismo Genético , Dengue Grave/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Complemento C2/análisis , Complemento C5/análisis , Complemento C5a/análisis , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Regulación de la Expresión Génica , Genotipo , Haplotipos , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Dengue Grave/sangre , Dengue Grave/genética , Dengue Grave/virología , Índice de Severidad de la Enfermedad , Vietnam/epidemiología , Adulto JovenRESUMEN
BACKGROUND: The mechanisms underlying early atherosclerotic plaque formation are not completely understood. Moreover, plasma biomarkers of subclinical atherosclerosis are lacking. OBJECTIVES: The purpose of this study was to analyze the temporal and topologically resolved protein changes taking place in human aortas with early atherosclerosis to find new potential diagnostic and/or therapeutic targets. METHODS: The protein composition of healthy aortas (media layer) or with early atheroma (fatty streak and fibrolipidic, media and intima layers) was analyzed by deep quantitative multiplexed proteomics. Further analysis was performed by Western blot, immunohistochemistry, real-time polymerase chain reaction, and enzyme-linked immunosorbent assay. Plasma levels of complement C5 were analyzed in relation to the presence of generalized (>2 plaques) or incipient (0 to 2 plaques) subclinical atherosclerosis in 2 independent clinical cohorts (PESA [Progression of Early Subclinical Atherosclerosis] [n = 360] and NEFRONA [National Observatory of Atherosclerosis in Nephrology] [n = 394]). RESULTS: Proteins involved in lipid transport, complement system, immunoglobulin superfamily, and hemostasis are increased in early plaques. Components from the complement activation pathway were predominantly increased in the intima of fibrolipidic plaques. Among them, increased C5 protein levels were further confirmed by Western blot, enzyme-linked immunosorbent assay and immunohistochemistry, and associated with in situ complement activation. Plasma C5 was significantly increased in individuals with generalized subclinical atherosclerosis in both PESA and NEFRONA cohorts, independently of risk factors. Moreover, in the PESA study, C5 plasma levels positively correlated with global plaque volume and coronary calcification. CONCLUSIONS: Activation of the complement system is a major alteration in early atherosclerotic plaques and is reflected by increased C5 plasma levels, which have promising value as a novel circulating biomarker of subclinical atherosclerosis.
Asunto(s)
Enfermedades Asintomáticas , Aterosclerosis , Complemento C5/análisis , Placa Aterosclerótica/metabolismo , Aterosclerosis/sangre , Aterosclerosis/diagnóstico , Biomarcadores/análisis , Activación de Complemento , Femenino , Humanos , Inmunohistoquímica , Masculino , Placa Aterosclerótica/patología , Proteómica/métodosRESUMEN
The present study aimed to investigate the association between gene methylation and leukocytopenia from the perspective of gene regulation. A total of 30 patients confirmed as having postinfection leukocytopenia at People's Hospital of Xinjiang Uygur Autonomous Region between January 2016 and June 2017 were successively recruited as the leukocytopenia group; 30 patients with postinfection leukocytosis were enrolled as the leukocytosis group. In addition, 30 healthy volunteers who received a health examination at the hospital during the same period were included as the normal control group. In each group, four individuals were randomly selected for whole genome methylation screening. After selection of key methylation sites, the remaining samples in each group were used for verification using matrixassisted laser desorption/ionizationtime of flight mass spectrometry. The levels of serum complement factors C3 and C5 in the leukocytopenia group were significantly lower than those in the other two groups (P<0.05). According to wholegenome DNA methylation detection, 66 and 27 methylation loci may be associated with leukocytopenia and leukocytosis, respectively. Most of these abnormal loci are located on chromosomes 2, 6, 7, 1, 17 and 11. The rates of WW domain containing E3 ubiquitin protein ligase 2 gene methylation at cytosinephosphateguanine (CpG)_1, CpG_5/6 and CpG_7 in the leukocytopenia group were higher than in the other two groups (P<0.05); the rate of AKT2 CpG_1 methylation was higher in the leukocytopenia group than in the other two groups (P<0.05); the rate of calciumbinding atopyrelated autoantigen 1 gene CpG_2 methylation was higher in the leukocytosis group than in the normal control group (P<0.05); and the rate of NADPH oxidase 5 gene CpG_3 methylation was higher in the leukocytosis group than in the normal control group (P<0.05). Chemotactic factor secretion and cell migration abnormalities, ubiquitination modification disorders and reduced oxidative burst may participate in infectioncomplicated leukocytopenia. The results of this study shed new light on the molecular biological mechanisms of infectioncomplicated leukocytopenia and provide novel avenues for diagnosis and treatment.
Asunto(s)
Metilación de ADN , Leucopenia/patología , Adulto , Anciano , Proteína C-Reactiva/análisis , Estudios de Casos y Controles , Complemento C3/análisis , Complemento C5/análisis , Islas de CpG , Femenino , Ontología de Genes , Humanos , Recuento de Leucocitos , Leucocitosis/genética , Leucocitosis/patología , Leucopenia/genética , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Polipéptido alfa Relacionado con Calcitonina/análisis , Índice de Severidad de la Enfermedad , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Insulin resistance is associated with high circulating level of complement factor C3. Animal studies suggest that improper complement activation mediates high-fat-diet-induced insulin resistance. Individuals born with low birth weight (LBW) are at increased risk of developing insulin resistance. We hypothesized that high-fat overfeeding (HFO) increase circulating C3 and induce complement activation in a birth weight differential manner. Twenty LBW and 26 normal birth weight (NBW) young men were studied using a randomised crossover design. Insulin resistance was measured after a control-diet and after 5-days HFO by a hyperinsulinemic-euglycemic-clamp. Circulating C4, C3, ficolins, mannose-binding-lectin, complement activation products C3bc, terminal complement complex (TCC) and complement activation capacity were determined using turbidimetry and ELISA. HFO induced peripheral insulin resistance in LBW individuals only, while both groups had the same degree of hepatic insulin resistance after HFO. Viewing all individuals circulating levels of C4, C3, C3bc, TCC and complement activation capacity decreased paradoxically along the development of insulin resistance after HFO (P = 0.0015, P < 0.0001, P = 0.01, P < 0.0001, P = 0.0002, P < 0.0001, P = 0.0006). Birth weight did not influence these results. This might reflect a hitherto unrecognized down-regulatory mechanism of the complement system. More human studies are needed to understand the underlying physiology and the potential consequences of these findings.
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Complemento C3/análisis , Complemento C5/análisis , Conducta Alimentaria , Factores Inmunológicos/sangre , Estudios Cruzados , Ensayo de Inmunoadsorción Enzimática , Voluntarios Sanos , Humanos , Resistencia a la Insulina , Masculino , Nefelometría y TurbidimetríaRESUMEN
BACKGROUND: Multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD) are autoimmune inflammatory demyelinating diseases of the central nervous system. Although distinguished by clinicoradiological and demographic features, early manifestations can be similar complicating management. Antibodies against aquaporin-4 support the diagnosis of NMOSD but are negative in some patients. Therefore, there is unmet need for biomarkers that enable early diagnosis and disease-specific intervention. OBJECTIVE: We investigated whether plasma complement proteins are altered in MS and NMOSD and provide biomarkers that distinguish these diseases. METHODS: Plasma from 54 NMOSD, 40 MS and 69 control donors was tested in multiplex assays measuring complement activation products and proteins. Using logistic regression, we tested whether combinations of complement analytes distinguished NMOSD from controls and MS. RESULTS: All activation products were elevated in NMOSD compared to either control or MS. Four complement proteins (C1inh, C1s, C5 and FH) were higher in NMOSD compared to MS or controls. A model comprising C1inh and terminal complement complex (TCC) distinguished NMOSD from MS (area under the curve (AUC): 0.98), while C1inh and C5 distinguished NMOSD from controls (AUC: 0.94). CONCLUSION: NMOSD is distinguished from MS by plasma complement biomarkers. Selected complement analytes enable differential diagnosis. Findings support trials of anti-complement therapies in NMOSD.
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Proteínas del Sistema Complemento/análisis , Esclerosis Múltiple Recurrente-Remitente/diagnóstico , Neuromielitis Óptica/diagnóstico , Adulto , Anciano , Área Bajo la Curva , Biomarcadores/sangre , Estudios de Casos y Controles , Proteína Inhibidora del Complemento C1/análisis , Complemento C5/análisis , Complejo de Ataque a Membrana del Sistema Complemento/análisis , Diagnóstico Diferencial , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Modelos Lineales , Modelos Logísticos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/sangre , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Esclerosis Múltiple Recurrente-Remitente/inmunología , Neuromielitis Óptica/sangre , Neuromielitis Óptica/tratamiento farmacológico , Neuromielitis Óptica/inmunología , Valor Predictivo de las Pruebas , Pronóstico , Curva ROC , Adulto JovenRESUMEN
INTRODUCTION: Severe trauma triggers a systemic inflammatory response that contributes to secondary complications, such as nosocomial infections, sepsis or multi-organ failure. The present study was aimed to identify markers predicting complications and an adverse outcome of severely injured patients by an integrated clinico-transcriptomic approach. METHODS: In a prospective study, RNA samples from circulating leukocytes from severely injured patients (injury severity score ≥ 17 points; n = 104) admitted to a Level I Trauma Center were analyzed for dynamic changes in gene expression over a period of 21 days by quantitative RT-PCR. Transcriptomic candidates were selected based on whole genome screening of a representative discovery set (n = 10 patients) or known mechanisms of the immune response, including mediators of inflammation (IL-8, IL-10, TNF-α, MIF, C5, CD59, SPHK1), danger signaling (HMGB1, TLR2, CD14, IL-33, IL-1RL1), and components of the heme degradation pathway (HP, CD163, HMOX1, BLVRA, BLVRB). Clinical markers comprised standard physiological and laboratory parameters and scoring systems routinely determined in trauma patients. RESULTS: Leukocytes, thrombocytes and the expression of sphingosine kinase-1 (SPHK1), complement C5, and haptoglobin (HP) have been identified as markers with the best performance. Leukocytes showed a biphasic course with peaks on day 0 and day 11 after trauma, and patients with sepsis exhibited significantly higher leukocyte levels. Thrombocyte numbers showed a typical profile with initial thrombopenia and robust thrombocytosis in week 3 after trauma, ranging 2- to 3-fold above the upper normal value. 'Relative thrombocytopenia' was associated with multi-organ dysfunction, the development of sepsis, and mortality, the latter of which could be predicted within 3 days prior to the time point of death. SPHK1 expression at the day of admission indicated mortality with excellent performance. C5-expression on day 1 after trauma correlated with an increased risk for the development of nosocomial infections during the later course, while HP was found to be a marker for the development of sepsis. CONCLUSIONS: The combination of clinical and transcriptomic markers improves the prognostic performance and may represent a useful tool for individual risk stratification in trauma patients.
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Insuficiencia Multiorgánica/diagnóstico , Medición de Riesgo/métodos , Sepsis/diagnóstico , Síndrome de Respuesta Inflamatoria Sistémica/diagnóstico , Biomarcadores/análisis , Biomarcadores/sangre , Complemento C5/análisis , Complemento C5/biosíntesis , Haptoglobinas/análisis , Haptoglobinas/biosíntesis , Humanos , Puntaje de Gravedad del Traumatismo , Insuficiencia Multiorgánica/sangre , Fosfotransferasas (Aceptor de Grupo Alcohol)/análisis , Fosfotransferasas (Aceptor de Grupo Alcohol)/sangre , Estudios Prospectivos , Sepsis/sangre , Síndrome de Respuesta Inflamatoria Sistémica/sangreRESUMEN
BACKGROUND: The aims of our study were to identify serum biomarkers that distinguish pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) patients from benign pancreatic disease patients and healthy subjects, and to assess the effects of jaundice on biomarker performance. METHODS: Isobaric tags for relative and absolute quantification were used to compare pooled serum and pancreatic juice samples from a test set of 59 and 25 subjects, respectively. Validation was undertaken in 113 independent subjects. RESULTS: Candidate proteins Complement C5, inter-α-trypsin inhibitor heavy chain H3, α1-ß glycoprotein and polymeric immunoglobulin receptor were elevated in cancer, as were the reference markers CA19-9 and Reg3A. Biliary obstruction had a significant effect on the performance of the markers, in particular within the PDAC group where the presence of jaundice was associated with a significant increase in the levels of all six proteins (P<0.01). Consequently, in the absence of jaundice, proteins showed reduced sensitivity for PDAC patients over benign subjects and healthy controls (HCs). Similarly, in the presence of jaundice, markers showed reduced specificity for PDAC patients over benign subjects with jaundice. Combining markers enabled improved sensitivity for non-jaundiced PDAC patients over HCs and improved specificity for jaundiced PDAC patients over jaundiced benign disease subjects. CONCLUSIONS: The presence-absence of jaundice in the clinical scenario severely impacts the performance of biomarkers for PDAC diagnosis and has implications for their clinical translation.
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Biomarcadores de Tumor/sangre , Ictericia Obstructiva/sangre , Jugo Pancreático/citología , Neoplasias Pancreáticas/diagnóstico , Anciano , alfa-Globulinas/análisis , Antígenos de Neoplasias/sangre , Antígeno CA-19-9/sangre , Complemento C5/análisis , Femenino , Glicoproteínas/sangre , Humanos , Inmunoglobulinas/sangre , Ictericia Obstructiva/complicaciones , Lectinas Tipo C/sangre , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/sangre , Proteínas Asociadas a Pancreatitis , Receptores de Inmunoglobulina Polimérica/análisisRESUMEN
The serum bactericidal assay is the correlate of protection for meningococcal disease but the use and comparison of functional immunological assays for the assessment of meningococcal vaccines is complicated by the sourcing of human complement. This is due to high levels of immunity in the population acquired through natural meningococcal carriage and means that many individuals must be screened to find donors with suitably low bactericidal titres against the target strain. The use of different donors for each meningococcal strain means that comparisons of assay responses between strains and between laboratories is difficult. We have developed a method for IgG-depletion of 300 ml batches of pooled human lepirudin-derived plasma using Protein G sepharose affinity chromatography that retains complement activity. However, IgG-depletion also removed C1q. This was also eluted from the affinity matrix, concentrated and added to the complement source. The final complement source retained mean alternative pathway activity of 96.8% and total haemolytic activity of 84.2% in four batches. Complement components C3, C5, properdin and factor H were retained following the process and the IgG-depleted complement was shown to be suitable for use in antibody-mediated complement deposition and serum bactericidal activity assays against serogroup B meningococci. The generation of large IgG-depleted batches of pooled human plasma allows for the comparison of immunological responses to diverse meningococcal strain panels in large clinical trials.
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Proteínas del Sistema Complemento/análisis , Neisseria meningitidis/inmunología , Determinación de Anticuerpos Séricos Bactericidas/métodos , Animales , Anticuerpos Antibacterianos/aislamiento & purificación , Cromatografía de Afinidad , Complemento C1q/análisis , Complemento C3/análisis , Complemento C5/análisis , Factor H de Complemento/análisis , Vía Alternativa del Complemento , Ensayo de Inmunoadsorción Enzimática , Hemólisis , Humanos , Inmunoglobulina G/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVE: Otitis media (OM) is one of the most common childhood diseases. The relative contribution of complement activation in protection and pathogenesis during OM remains largely unknown. The purpose of this study was to investigate the beneficial and pathogenic contributions of complement activation in the middle ear of pediatric patients with recurrent acute otitis media (rAOM), and therefore to provide a rational approach to prevent sequelae of OM such as hearing loss. METHODS: Twenty children undergoing pressure equalization tube placement with or without adenoidectomy for rAOM were enrolled in the study. Bacterial cultures, enzyme-linked immunosorbent assay (ELISA) for complement components and cytokines and western blot for complement activation were performed on middle ear effusion (MEE) and serum samples. The levels of complement C3a, C5a and sC5-b9 in MEEs and serum samples were compared. The levels of these factors were also examined in regards to length of episode. Pearson's correlation coefficients were calculated on variables between C5a and IL-6 or IL-8. Complement gene expression in human middle ear epithelial (HMEE) cells induced by otopathogens was evaluated. Data were analyzed with Student's t test or the Mann-Whitney rank sum test. In all cases, a P value of <0.05 was set as the measure of significance. RESULTS: Our data demonstrated that the complement classical/lectin, alternative and terminal pathways were activated in the middle ear of children with rAOM. Increased complement components of C3a, C5a and sC5-b9 in MEEs were detected in patients with the episode lasting more than six weeks. There was a strong correlation between C5a and IL-6 or IL-8 in the MEEs. Additionally, otopathogens induced enhanced gene expression of factor B and C3 in HMEE cells, which is beneficial for host defense against invading pathogens. CONCLUSION: Our studies provided important new insights on how complement activation contributes to inflammatory process during rAOM. Knowledge of the activity of the complement pathway in patients with rAOM may stimulate the development of new strategies to prevent middle ear inflammatory tissue destruction by directing treatment to specific pathways within the complement cascade.
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Activación de Complemento , Proteínas del Sistema Complemento/análisis , Otitis Media con Derrame/diagnóstico , Enfermedad Aguda , Biomarcadores/análisis , Western Blotting , Preescolar , Estudios de Cohortes , Complemento C3/análisis , Complemento C5/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Estudios de Seguimiento , Humanos , Lactante , Interleucina-6/análisis , Interleucina-8/análisis , Masculino , Ventilación del Oído Medio/métodos , Otitis Media con Derrame/cirugía , Reacción en Cadena de la Polimerasa/métodos , Estudios Prospectivos , Recurrencia , Sensibilidad y Especificidad , Índice de Severidad de la Enfermedad , Estadísticas no Paramétricas , Resultado del TratamientoRESUMEN
Robust host innate immune response to staphylococcal enterotoxin B (SEB) and structurally related superantigens causes toxic shock and various autoimmune diseases. While proinflammatory cytokines are known for mediating SEB-induced toxicity, the role of complement C5a in SEB-mediated shock is less well-understood. An ELISA was developed to measure the complement activation product, C5a, in different murine models of toxic shock. This assay provides easy, quantifiable data for complement activation and its role in various SEB-induced toxic shock models.
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Complemento C5/análisis , Enterotoxinas/inmunología , Choque Séptico/sangre , Infecciones Estafilocócicas/sangre , Superantígenos/inmunología , Animales , Anticuerpos/química , Anticuerpos/inmunología , Complemento C5/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática/métodos , Peroxidasa de Rábano Silvestre/química , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Choque Séptico/inmunología , Infecciones Estafilocócicas/inmunologíaRESUMEN
OBJECTIVE: To investigate the inflammatory markers associated with short-term endotracheal intubation in healthy surgical patients. DESIGN: An observational and prospective study of subjects scheduled for same-day surgery procedures. SETTING: Level I trauma center. PATIENTS: Fourteen healthy patients intubated for same-day surgery procedures. The median duration of surgery was 3 hours. INTERVENTIONS: Serial lavages above the tracheal cuff were obtained at the beginning of surgery, at 1 hour, and at the end of surgery; samples were assayed for cellular counts and levels of cytokines and complement 5a (C5a). RESULTS: The total number of polymorphonuclear cells (PMNs) increased almost 10-fold from intubation to extubation (P < .01). The levels of 3 of the cytokines measured in tracheal lavage supernatants were significantly elevated over the time of intubation: tumor necrosis factor (TNF) (P < .01), interleukin 6 (IL-6) (P < .01), and IL-1ß (P < .025). Levels of IL-8 showed an upward trend over time but were not significantly increased; C5a levels were significantly elevated over time (P < .05). CONCLUSIONS: Short-term intubation in healthy patients resulted in significant tracheal inflammation. Involvement of the innate immune system as documented in the present study provides information that may help to better understand the pathophysiologic characteristics of subglottic stenosis and other endotracheal injuries secondary to endotracheal intubation.
Asunto(s)
Líquido del Lavado Bronquioalveolar/citología , Inmunidad Innata/fisiología , Mediadores de Inflamación/metabolismo , Intubación Intratraqueal/métodos , Traqueítis/etiología , Adulto , Anciano , Procedimientos Quirúrgicos Ambulatorios/métodos , Análisis de Varianza , Estudios de Cohortes , Complemento C5/análisis , Complemento C5/metabolismo , Citocinas/análisis , Citocinas/metabolismo , Femenino , Estudios de Seguimiento , Humanos , Mediadores de Inflamación/análisis , Interleucina-6/análisis , Interleucina-6/metabolismo , Interleucina-8/análisis , Interleucina-8/metabolismo , Intubación Intratraqueal/efectos adversos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Estudios Prospectivos , Valores de Referencia , Medición de Riesgo , Estadísticas no Paramétricas , Procedimientos Quirúrgicos Operativos/métodos , Traqueítis/inmunología , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: The aim of this study was to investigate the effects of PRP on SAOS-2 cells in terms of cytokine expression, cell activity and oxidative stress. DESIGN: Cell line SAOS-2 (1×10(5)cells/mL) were grown in culture medium α-MEM with 10% FBS for 24h and stimulated (or not) with PRP at concentrations of 3, 10 and 20%, LPS (E. coli, 10g/mL) and IL-1ß (1mg/mL) for 24h. The supernatant was collected and analyzed for the expression of cytokines in a panel array, ALP using a commercial kit and NO(2)(-) with Griess reaction method. Also, the cells were analyzed using Western blot for RANKL and slot blotting for nitrotyrosine expression. RESULT: There were no significant differences amongst the groups in terms of NO(2)(-), protein nitrotyrosine content and RANKL expression. However, all stimuli increased ALP activity and in case of PRP, it was in a dose-dependent manner (p<0.001). Also, all stimuli induced an increase in cytokines and chemokines expression, but only PRP promoted an increase of component C5, sICAM-1 and RANTES expression. Whilst IL-1 receptor antagonist (IL-1ra) expression was down-regulated by PRP, both LPS and IL-1ß caused up-regulation of this cytokine. CONCLUSIONS: PRP can stimulate osteoblast activity and cytokine/chemokine release, as well as indicate some of the mediators that can (and cannot) be involved in this activation.
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Fosfatasa Alcalina/análisis , Citocinas/análisis , Osteoblastos/metabolismo , Plasma Rico en Plaquetas/fisiología , Línea Celular Tumoral , Quimiocina CCL5/análisis , Quimiocina CXCL1/análisis , Complemento C5/análisis , Relación Dosis-Respuesta a Droga , Escherichia coli , Factor Estimulante de Colonias de Granulocitos y Macrófagos/análisis , Humanos , Molécula 1 de Adhesión Intercelular/análisis , Proteína Antagonista del Receptor de Interleucina 1/análisis , Interleucina-1beta/farmacología , Interleucinas/análisis , Lipopolisacáridos/farmacología , Óxido Nítrico/análisis , Osteoblastos/efectos de los fármacos , Estrés Oxidativo/fisiología , Ligando RANK/análisis , Tirosina/análogos & derivados , Tirosina/análisisAsunto(s)
Activación de Complemento , Complemento C3/análisis , Complemento C5/análisis , Angioedemas Hereditarios/diagnóstico , Biomarcadores/sangre , Complemento C3/deficiencia , Complemento C3/fisiología , Complemento C5/deficiencia , Complemento C5/fisiología , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Inflamación/diagnóstico , Nefelometría y Turbidimetría/métodos , Valores de Referencia , Manejo de Especímenes/métodosRESUMEN
OBJECTIVES: This study assessed the extent and mechanism of complement activation in community-acquired sepsis at presentation to the emergency department (ED) and following 24 hours of quantitative resuscitation. METHODS: A prospective pilot study of patients with severe sepsis and healthy controls was conducted among individuals presenting to a tertiary care ED. Resuscitation, including antibiotics and therapies to normalize central venous and mean arterial pressure (MAP) and central venous oxygenation, was performed on all patients. Serum levels of Factor Bb (alternative pathway), C4d (classical and mannose-binding lectin [MBL] pathway), C3, C3a, and C5a were determined at presentation and 24 hours later among patients. RESULTS: Twenty patients and 10 healthy volunteer controls were enrolled. Compared to volunteers, all proteins measured were abnormally higher among septic patients (C4d 3.5-fold; Factor Bb 6.1-fold; C3 0.8-fold; C3a 11.6-fold; C5a 1.8-fold). Elevations in C5a were most strongly correlated with alternative pathway activation. Surprisingly, a slight but significant inverse relationship between illness severity (by sequential organ failure assessment [SOFA] score) and C5a levels at presentation was noted. Twenty-four hours of structured resuscitation did not, on average, affect any of the mediators studied. CONCLUSIONS: Patients with community-acquired sepsis have extensive complement activation, particularly of the alternative pathway, at the time of presentation that was not significantly reversed by 24 hours of aggressive resuscitation.