RESUMEN
BACKGROUND: The immune response of critically ill patients, such as those with sepsis, severe trauma, or major surgery, is heterogeneous and dynamic, but its characterization and impact on outcomes are poorly understood. Until now, the primary challenge in advancing our understanding of the disease has been to concurrently address both multiparametric and temporal aspects. METHODS: We used a clustering method to identify distinct groups of patients, based on various immune marker trajectories during the first week after admission to ICU. In 339 severely injured patients, we initially longitudinally clustered common biomarkers (both soluble and cellular parameters), whose variations are well-established during the immunosuppressive phase of sepsis. We then applied this multi-trajectory clustering using markers composed of whole blood immune-related mRNA. RESULTS: We found that both sets of markers revealed two immunotypes, one of which was associated with worse outcomes, such as increased risk of hospital-acquired infection and mortality, and prolonged hospital stays. This immunotype showed signs of both hyperinflammation and immunosuppression, which persisted over time. CONCLUSION: Our study suggest that the immune system of critically ill patients can be characterized by two distinct longitudinal immunotypes, one of which included patients with a persistently dysregulated and impaired immune response. This work confirms the relevance of such methodology to stratify patients and pave the way for further studies using markers indicative of potential immunomodulatory drug targets.
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Biomarcadores , Heridas y Lesiones , Humanos , Masculino , Femenino , Biomarcadores/sangre , Biomarcadores/análisis , Persona de Mediana Edad , Adulto , Heridas y Lesiones/inmunología , Heridas y Lesiones/sangre , Análisis por Conglomerados , Enfermedad Crítica , Unidades de Cuidados Intensivos/estadística & datos numéricos , Unidades de Cuidados Intensivos/organización & administración , Anciano , Sepsis/sangre , Sepsis/inmunología , Estudios LongitudinalesRESUMEN
Sepsis induces intense, dynamic and heterogeneous host response modulations. Despite improvement of patient management, the risk of mortality and healthcare-associated infections remains high. Treatments to counterbalance immune response are under evaluation, but effective biomarkers are still lacking to perform patient stratification. The design of the present study was defined to alleviate the limitations of existing literature: we selected patients who survived the initial hyperinflammatory response and are still hospitalized at day 5-7 after ICU admission. Using the Immune Profiling Panel (IPP), a fully automated RT-qPCR multiplex prototype, we optimized a machine learning model combining the IPP gene expression levels for the identification of patients at high risk of worsening, a composite endpoint defined as death or secondary infection, within one week after sampling. This was done on 332 sepsis patients selected from two retrospective studies. The IPP model identified a high-risk group comprising 30% of patients, with a significant increased proportion of worsening events at day 28 compared to the low-risk group (49% vs. 28%, respectively). These preliminary results underline the potential clinical application of IPP for sepsis patient stratification in a personalized medicine perspective, that will be confirmed in a larger prospective multicenter study.
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Biomarcadores , Sepsis , Humanos , Sepsis/inmunología , Masculino , Femenino , Anciano , Persona de Mediana Edad , Aprendizaje Automático , Estudios Retrospectivos , PronósticoRESUMEN
Guided biomarker-personalized immunotherapy is advancing rapidly as a means to rejuvenate immune function in injured patients who are the most immunosuppressed. A recent study introduced a fully automated interferon-γ release assay (IGRA) for monitoring the functionality of T lymphocytes in patients with septic shock. While a significant decrease in IFN-γ release capacity was observed, a significant correlation with CD8 lymphocyte absolute count was also reported, raising the question of whether ex-vivo IFN-γ production would be only a surrogate marker for lymphocyte count or if these two parameters conveyed distinct and complementary information. In a large cohort of more than 353 critically ill patients following various injuries (sepsis, trauma, major surgery), the primary objective of the present study was to simultaneously evaluate the association between ex vivo IFN-γ release and CD8 cell count with regard to adverse outcome. Our findings provide a clear-cut result, as they distinctly demonstrate that IGRA offers higher-quality information than CD8 count in terms of an independent association with the occurrence of an adverse outcome. These results strengthen the case for incorporating IGRA into the array of biomarkers of interest for defining endotypes in sepsis. This holds especially true given that fully automated tests are now readily available and could be used in routine clinical practice.
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Ensayos de Liberación de Interferón gamma , Sepsis , Humanos , Ensayos de Liberación de Interferón gamma/métodos , Interferón gamma , Enfermedad Crítica , Terapia de Inmunosupresión , Recuento de Linfocitos , Linfocitos T CD8-positivos , BiomarcadoresRESUMEN
BACKGROUND: The development of stratification tools based on the assessment of circulating mRNA of genes involved in the immune response is constrained by the heterogeneity of septic patients. The aim of this study is to develop a transcriptomic score based on a pragmatic combination of immune-related genes detected with a prototype multiplex PCR tool. METHODS: As training cohort, we used the gene expression dataset obtained from 176 critically ill patients enrolled in the REALISM study (NCT02638779) with various etiologies and still hospitalized in intensive care unit (ICU) at day 5-7. Based on the performances of each gene taken independently to identify patients developing ICU-acquired infections (ICU-AI) after day 5-7, we built an unweighted score assuming the independence of each gene. We then determined the performances of this score to identify a subgroup of patients at high risk to develop ICU-AI, and both longer ICU length of stay and mortality of this high-risk group were assessed. Finally, we validated the effectiveness of this score in a retrospective cohort of 257 septic patients. RESULTS: This transcriptomic score (TScore) enabled the identification of a high-risk group of patients (49%) with an increased rate of ICU-AI when compared to the low-risk group (49% vs. 4%, respectively), with longer ICU length of stay (13 days [95% CI 8-30] vs. 7 days [95% CI 6-9], p < 0.001) and higher ICU mortality (15% vs. 2%). High-risk patients exhibited biological features of immune suppression with low monocytic HLA-DR levels, higher immature neutrophils rates and higher IL10 concentrations. Using the TScore, we identified 160 high-risk patients (62%) in the validation cohort, with 30% of ICU-AI (vs. 18% in the low-risk group, p = 0.06), and significantly higher mortality and longer ICU length of stay. CONCLUSIONS: The transcriptomic score provides a useful and reliable companion diagnostic tool to further develop immune modulating drugs in sepsis in the context of personalized medicine.
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Sepsis , Transcriptoma , Humanos , Estudios Retrospectivos , Enfermedad Crítica , Sepsis/diagnóstico , Sepsis/genética , Unidades de Cuidados Intensivos , Progresión de la EnfermedadRESUMEN
OBJECTIVES: There is a crucial unmet need for biomarker-guided diagnostic and prognostic enrichment in clinical trials evaluating immune modulating therapies in critically ill patients. Low monocyte expression of human leukocyte antigen-DR (mHLA-DR), considered as a reference surrogate to identify immunosuppressed patients, has been proposed for patient stratification in immunostimulation approaches. However, its widespread use in clinic has been somewhat hampered by technical constraints inherent to flow cytometry technology. The objective of the present study was to evaluate the ability of a prototype multiplex polymerase chain reaction tool (immune profiling panel [IPP]) to identify immunosuppressed ICU patients characterized by a low mHLA-DR expression. DESIGN: Retrospective observational cohort study. SETTING: Adult ICU in a University Hospital, Lyon, France. PATIENTS: Critically ill patients with various etiologies enrolled in the REAnimation Low Immune Status Marker study (NCT02638779). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: mHLA-DR and IPP data were obtained from 1,731 blood samples collected from critically ill patients with various etiologies and healthy volunteers. A partial least square regression model combining the expression levels of IPP markers was trained and used for the identification of samples from patients presenting with evidence of immunosuppression, defined here as mHLADR less than 8,000 antibodies bound per cell (AB/C). The IPP gene set had an area under the receiver operating characteristic curve (AUC) of 0.86 (95% CI 0.83-0.89) for the identification of immunosuppressed patients. In addition, when applied to the 123 patients still in the ICU at days 5-7 after admission, IPP similarly enriched the number of patients with ICU-acquired infections in the immunosuppressed group (26%), in comparison with low mHLA-DR (22%). CONCLUSIONS: This study reports on the potential of the IPP gene set to identify ICU patients presenting with mHLA-DR less than 8,000 AB/C. Upon further optimization and validation, this molecular tool may help in the stratification of patients that could benefit from immunostimulation in the context of personalized medicine.
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Enfermedad Crítica , Monocitos , Adulto , Humanos , Estudios Retrospectivos , Antígenos HLA-DR/genética , Biomarcadores , AnticuerposRESUMEN
Immune responses affiliated with COVID-19 severity have been characterized and associated with deleterious outcomes. These approaches were mainly based on research tools not usable in routine clinical practice at the bedside. We observed that a multiplex transcriptomic panel prototype termed Immune Profiling Panel (IPP) could capture the dysregulation of immune responses of ICU COVID-19 patients at admission. Nine transcripts were associated with mortality in univariate analysis and this 9-mRNA signature remained significantly associated with mortality in a multivariate analysis that included age, SOFA and Charlson scores. Using a machine learning model with these 9 mRNA, we could predict the 28-day survival status with an Area Under the Receiver Operating Curve (AUROC) of 0.764. Interestingly, adding patients' age to the model resulted in increased performance to predict the 28-day mortality (AUROC reaching 0.839). This prototype IPP demonstrated that such a tool, upon clinical/analytical validation and clearance by regulatory agencies could be used in clinical routine settings to quickly identify patients with higher risk of death requiring thus early aggressive intensive care.
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COVID-19 , Enfermedad Crítica , Humanos , ARN Mensajero , Hospitalización , Reacción en Cadena de la PolimerasaRESUMEN
Background: Novel biomarkers are needed to progress toward individualized patient care in sepsis. The immune profiling panel (IPP) prototype has been designed as a fully-automated multiplex tool measuring expression levels of 26 genes in sepsis patients to explore immune functions, determine sepsis endotypes and guide personalized clinical management. The performance of the IPP gene set to predict 30-day mortality has not been extensively characterized in heterogeneous cohorts of sepsis patients. Methods: Publicly available microarray data of sepsis patients with widely variable demographics, clinical characteristics and ethnical background were co-normalized, and the performance of the IPP gene set to predict 30-day mortality was assessed using a combination of machine learning algorithms. Results: We collected data from 1,801 arrays sampled on sepsis patients and 598 sampled on controls in 17 studies. When gene expression was assayed at day 1 following admission (1,437 arrays sampled on sepsis patients, of whom 1,161 were alive and 276 (19.2%) were dead at day 30), the IPP gene set showed good performance to predict 30-day mortality, with an area under the receiving operating characteristics curve (AUROC) of 0.710 (CI 0.652-0.768). Importantly, there was no statistically significant improvement in predictive performance when training the same models with all genes common to the 17 microarray studies (n = 7,122 genes), with an AUROC = 0.755 (CI 0.697-0.813, p = 0.286). In patients with gene expression data sampled at day 3 following admission or later, the IPP gene set had higher performance, with an AUROC = 0.804 (CI 0.643-0.964), while the total gene pool had an AUROC = 0.787 (CI 0.610-0.965, p = 0.811). Conclusion: Using pooled publicly-available gene expression data from multiple cohorts, we showed that the IPP gene set, an immune-related transcriptomics signature conveys relevant information to predict 30-day mortality when sampled at day 1 following admission. Our data also suggests that higher predictive performance could be obtained when assaying gene expression at later time points during the course of sepsis. Prospective studies are needed to confirm these findings using the IPP gene set on its dedicated measurement platform.
RESUMEN
Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. The immune system plays a key role in sepsis onset and remains dysregulated over time in a heterogeneous manner. Here, we decipher the heterogeneity of the first week evolution of the monocyte HLA-DR (mHLA-DR) surface protein expression in septic patients, a key molecule for adaptive immunity onset. We found and verified four distinctive trajectories endotypes in a discovery (n = 276) and a verification cohort (n = 102). We highlight that 59% of septic patients exhibit low or decreasing mHLA-DR expression while in others mHLA-DR expression increased. This study depicts the first week behavior of mHLA-DR over time after sepsis onset and shows that initial and third day mHLA-DR expression measurements is sufficient for an early risk stratification of sepsis patients. These patients might benefit from immunomodulatory treatment to improve outcomes. Going further, our study introduces a way of deciphering heterogeneity of immune system after sepsis onset which is a first step to reach a more comprehensive landscape of sepsis.
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Antígenos HLA-DR/metabolismo , Monocitos/inmunología , Sepsis/inmunología , Anciano , Biomarcadores , Diferenciación Celular , Linaje de la Célula , Estudios de Cohortes , Progresión de la Enfermedad , Femenino , Humanos , Inmunomodulación , Masculino , Monitorización Inmunológica , Fenotipo , Pronóstico , Sepsis/diagnóstico , Regulación hacia ArribaRESUMEN
BACKGROUND: Sepsis is the leading cause of mortality for critically ill patients worldwide. Patients develop T lymphocyte dysfunctions leading to T-cell exhaustion associated with increased risk of death. As interleukin-7 (IL-7) is currently tested in clinical trials to reverse these dysfunctions, it is important to evaluate the expression of its specific CD127 receptor on the T-cell surface of patients with septic shock. Moreover, the CD127lowPD-1high phenotype has been proposed as a T-cell exhaustion marker in chronic viral infections but has never been evaluated in sepsis. The objective of this study was first to evaluate CD127 and CD127lowPD-1high phenotype in septic shock in parallel with functional T-cell alterations. Second, we aimed to reproduce septic shock-induced T-cell alterations in an ex vivo model. METHODS: CD127 expression was followed at the protein and mRNA levels in patients with septic shock and healthy volunteers. CD127lowPD-1high phenotype was also evaluated in parallel with T-cell functional alterations after ex vivo activation. To reproduce T-cell alterations observed in patients, purified T cells from healthy volunteers were activated ex vivo and their phenotype and function were evaluated. RESULTS: In patients, neither CD127 expression nor its corresponding mRNA transcript level was modified compared with normal values. However, the percentage of CD127lowPD-1high T cells was increased while T cells also presented functional alterations. CD127lowPD-1high T cells co-expressed HLA-DR, an activation marker, suggesting a role for T-cell activation in the development of this phenotype. Indeed, T-cell receptor (TCR) activation of normal T lymphocytes ex vivo reproduced the increase of CD127lowPD-1high T cells and functional alterations following a second stimulation, as observed in patients. Finally, in this model, as observed in patients, IL-7 could improve T-cell proliferation. CONCLUSIONS: The proportion of CD127lowPD-1high T cells in patients was increased compared with healthy volunteers, although no global CD127 regulation was observed. Our results suggest that TCR activation participates in the occurrence of this T-cell population and in the development of T-cell alterations in septic shock. Furthermore, we provide an ex vivo model for the investigation of the pathophysiology of sepsis-induced T-cell immunosuppression and the testing of innovative immunostimulant treatments.
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Choque Séptico/sangre , Linfocitos T/fisiología , Anciano , Femenino , Francia , Humanos , Interleucina-7/análisis , Interleucina-7/sangre , Interleucina-7/fisiología , Subunidad alfa del Receptor de Interleucina-7/análisis , Subunidad alfa del Receptor de Interleucina-7/sangre , Recuento de Linfocitos/métodos , Masculino , Persona de Mediana Edad , Fenotipo , Receptor de Muerte Celular Programada 1/análisis , Receptor de Muerte Celular Programada 1/sangre , Choque Séptico/fisiopatologíaRESUMEN
OBJECTIVES: Septic shock is the primary cause of death in ICUs. A better comprehension of its pathophysiology, in particular, the immune alteration mechanisms, opened new therapeutic perspectives such as the recombinant interleukin-7. The use of biomarkers could improve the identification of eligible patients for this therapy. The soluble form of the interleukin-7 appears as a promising candidate in this regard since an association between its high plasmatic level and mortality in critically ill patients has been demonstrated. Because there are no data available on the transcriptional regulation of the interleukin-7 receptor in such patients, this study aimed to explore the expression level of different interleukin-7 receptor transcripts after septic shock and evaluate their association with mortality. DESIGN: Retrospective discovery cohort (30 patients) and validation cohort (177 patients). SETTING: Two French ICUs (discovery study) and six French ICUs (validation study). PATIENTS: Adult septic shock patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The quantification of several interleukin-7 receptor transcripts using specific reverse transcription quantitative polymerase chain reaction designs allowed for global evaluation of interleukin-7 receptor gene expression in whole blood. In the discovery cohort, all interleukin-7 receptor transcripts studied were expressed at lower levels in septic shock patients than in healthy volunteers. Interleukin-7 receptor gene expression at day 3 after septic shock diagnosis was associated with day 28 mortality. Patients at a lower risk of death showed higher expression levels. These results were confirmed in the independent validation cohort. Interestingly, using a threshold obtained on the discovery cohort, we observed in the validation cohort a high negative predictive value for day 28 mortality for the transcript encoding the membrane form of interleukin-7 receptor (0.86; 95% CI, 0.79-0.93). CONCLUSIONS: Interleukin-7 receptor transcripts appear as biomarkers of impaired adaptive immune response in septic shock patients and as a promising tool for patient stratification in clinical trials evaluating immunoadjuvant therapies.
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Enfermedad Crítica/mortalidad , Subunidad alfa del Receptor de Interleucina-7/sangre , Choque Séptico/sangre , Choque Séptico/mortalidad , Adulto , Anciano , Biomarcadores/sangre , Estudios de Cohortes , Cuidados Críticos/métodos , Femenino , Francia , Mortalidad Hospitalaria , Humanos , Unidades de Cuidados Intensivos , Masculino , Persona de Mediana Edad , Pronóstico , Medición de RiesgoRESUMEN
PURPOSE: Intensive care unit (ICU)-acquired infections (IAI) result in increased hospital and ICU stay, costs and mortality. To date, no biomarker has shown sufficient evidence and ease of application in clinical routine for the identification of patients at risk of IAI. We evaluated the association of the systemic mRNA expression of two host response biomarkers, CD74 and IL10, with IAI occurrence in a large cohort of ICU patients. METHODS: ICU patients were prospectively enrolled in a multicenter cohort study. Whole blood was collected on the day of admission (D1) and on day 3 (D3) and day 6 (D6) after admission. Patients were screened daily for IAI occurrence and data were censored after IAI diagnosis. mRNA expression levels of biomarkers were measured using RT-qPCR. Fine and Gray competing risk models were used to assess the association between gene expression and IAI occurrence. RESULTS: A total of 725 patients were analyzed. At least one IAI episode occurred in 137 patients (19%). After adjustment for shock and sepsis status at admission, CD74 and IL10 levels were found to be significantly associated with IAI occurrence [subdistribution hazard ratio (95% confidence interval) 0.67 (0.46-0.97) for CD74 D3/D1 expression ratio and 2.21 (1.63-3.00) for IL10 at D3]. IAI cumulative incidence was significantly different between groups stratified according to CD74 or IL10 expression (Gray tests p < 0.001). CONCLUSION: Our results suggest that two immune biomarkers, CD74 and IL10, could be relevant tools for the identification of IAI risk in ICU patients.
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Antígenos de Diferenciación de Linfocitos B/sangre , Infección Hospitalaria/epidemiología , Antígenos de Histocompatibilidad Clase II/sangre , Unidades de Cuidados Intensivos , Interleucina-10/sangre , ARN Mensajero/metabolismo , Adulto , Antígenos de Diferenciación de Linfocitos B/genética , Biomarcadores/sangre , Infección Hospitalaria/diagnóstico , Femenino , Regulación de la Expresión Génica , Antígenos de Histocompatibilidad Clase II/genética , Hospitalización , Humanos , Incidencia , Unidades de Cuidados Intensivos/estadística & datos numéricos , Interleucina-10/genética , Masculino , Estudios Prospectivos , ARN Mensajero/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Medición de RiesgoAsunto(s)
Enfermedad Crítica/mortalidad , Subunidad alfa del Receptor de Interleucina-7/sangre , Biomarcadores/sangre , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Humanos , Unidades de Cuidados Intensivos , Receptores de Interleucina-7/uso terapéutico , Estadísticas no ParamétricasRESUMEN
OBJECTIVES: Hospital-acquired infections (HAI) are associated with significant mortality and morbidity and prolongation of hospital stay, adding strain on limited hospital resources. Despite stringent infection control practices some children remain at high risk of developing HAI. The development of biomarkers which could identify these patients would be useful. In this study our objective was to evaluate mRNA candidate biomarkers for HAI prediction in a pediatric intensive care unit. DESIGN: Serial blood samples were collected from patients admitted to pediatric intensive care unit between March and June 2012. Candidate gene expression (IL1B, TNF, IL10, CD3D, BCL2, BID) was quantified using RT-qPCR. Comparisons of relative gene expression between those that did not develop HAI versus those that did were performed using Mann Whitney U-test. PATIENTS: Exclusion criteria were: age <28 days or ≥16 years, expected length of stay < 24 hours, expected survival < 28 days, end-stage renal disease and end-stage liver disease. Finally, 45 children were included in this study. MAIN RESULTS: The overall HAI rate was 30% of which 62% were respiratory infections. Children who developed HAI had a three-fold increase in hospital stay compared to those who did not (27 days versus 9 days, p<0.001). An increased expression of cytokine genes (IL1B and IL10) was observed in patients who developed HAI, as well as a pro-apoptosis pattern (higher expression of BID and lower expression of BCL2). CD3D, a key TCR co-factor was also significantly down-modulated in patients who developed HAI. CONCLUSIONS: To our knowledge, this is the first study of mRNA biomarkers of HAI in the paediatric population. Increased mRNA expressions of anti-inflammatory cytokine and modulation of apoptotic genes suggest the development of immunosuppression in critically ill children. Immune monitoring using a panel of genes may offer a novel stratification tool to identify HAI risk.
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Biomarcadores/metabolismo , Infección Hospitalaria/diagnóstico , Infección Hospitalaria/microbiología , ARN Mensajero/metabolismo , Adolescente , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Complejo CD3/metabolismo , Niño , Preescolar , Aberraciones Cromosómicas , Control de Enfermedades Transmisibles , Citocinas/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Lactante , Recién Nacido , Inflamación , Unidades de Cuidado Intensivo Pediátrico , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Tiempo de Internación , Masculino , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Control de Calidad , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcripción GenéticaRESUMEN
OBJECTIVES: Septic syndromes are the leading cause of death in intensive care units. They are characterized by the development of immune dysfunctions such as endotoxin tolerance (ET), whose intensity and duration are associated with increased risk of nosocomial infections and mortality. Alarmins S100A8 and S100A9 have been shown to be increased after septic shock. Importantly, a delayed S100A9 mRNA increase predicts hospital-acquired infection in patients. The aim of this study was to investigate the regulation of S100A8 and S100A9 mRNA expression in an ex vivo model of ET. SUBJECTS AND MEASUREMENTS: ET was reproduced ex vivo by priming healthy peripheral blood mononuclear cells (number of donors â=â9 to 10) with low-dose endotoxin (2 ng/ml) before stimulation with high dose endotoxin (100 ng/ml). S100A8 and S100A9 mRNA levels were measured by quantitative real-time polymerase chain reactions. MAIN RESULTS: ET was established by observing decreased TNFα and increased IL-10 transcriptomic responses to two subsequent endotoxin challenges. Interestingly, ET was associated with increased S100A8 and S100A9 mRNA expression ex vivo. We showed that IL-10 played a role in this process, since S100A8 and S100A9 mRNA increases were significantly abrogated by IL-10 blockade in the model. Conversely, treatment with rIFN-γ, a pro-inflammatory and immunostimulating molecule known to block ET induction, was able to restore normal S100A8 and S100A9 mRNA in this model. CONCLUSIONS: In this ex vivo model, we observed that S100A8 and S100A9 mRNA expression was significantly increased during ET. This reproduced ex vivo the observations we had previously made in septic shock patients. Interestingly, IL-10 blockade and rIFN-γ treatment partially abrogated S100A8/A9 mRNA increases in this model. Pending confirmation in larger, independent clinical studies, these preliminary results suggest that S100A8 and S100A9 mRNA levels might be used as surrogate markers of ET and as stratification tools for personalized immunotherapy in septic shock patients.
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Calgranulina A/genética , Calgranulina B/genética , Tolerancia Inmunológica/efectos de los fármacos , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Lipopolisacáridos/farmacología , Modelos Biológicos , Adulto , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-10/genética , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Cardiac troponin I (cTnI) is a specific marker of myocardial injury. In blood of patients with cardiovascular diseases, cTnI is released as a mixture of free, complexed and post-translationally modified forms. METHODS: The cTnI forms present in the plasma from 8 patients with acute myocardial infarction (AMI) have been analysed by two-dimensional gel electrophoresis (2-DE) and Western Blot using anti-cTnI mAb 19C7 and anti-phosphorylated cTnI (Serines 22-23) mAb 5E6. RESULTS: After immunoextraction of cTnI in plasma samples by 19C7 and 2-DE separation, 4 different forms were detected by 19C7 in 7 out the 8 AMI plasma samples. Two 29 kDa spots corresponding to intact free cTnI forms were detected at pIs 5.2 and 5.4. However, spot with pI 5.4 was also recognized by mAb 5E6, and should be bis-phosphorylated cTnI. Two 55 kDa spots with pIs 6.6 and 6.7 could be IC complexes. CTnI forms with pIs lower than the theoretical pI were also found in free cTnI and phosphorylated cTnI purified materials. CONCLUSIONS: 2-DE analysis of AMI plasma showed the presence of acidic cTnI forms, one of them being phosphorylated. The clinical significance of these forms has to be further investigated.
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Infarto del Miocardio/sangre , Troponina I/sangre , Troponina I/química , Western Blotting , Electroforesis en Gel Bidimensional , Humanos , Punto Isoeléctrico , Fosforilación , Isoformas de Proteínas/sangre , Isoformas de Proteínas/química , Troponina I/aislamiento & purificaciónRESUMEN
The troponin (Tn) complex is composed of troponin T, troponin C and troponin I. The cardiac isoform of TnI (cTnI) is modified and released in blood of patients with cardiovascular diseases as a heterogeneous mixture of free, complexed and posttranslationally modified forms. With the aim to determine later, whether specific forms of cTnI could be associated with the different pathologies leading to cTnI release, the cTnI forms present in the plasma from 64 patients with acute myocardial infarction (AMI) have been analysed by SELDI-TOF MS using anti-TnI mAbs coupled to PS20 ProteinChips arrays. Upfront immunoaffinity enrichment using anti-cTnI 19C7 mAb allowed us to detect cTnI and bis-phosphorylated cTnI in 11/12 and 9/12 analyses respectively, as well as truncated cTnI in plasma with concentration of cTnI as low as 8 ng/mL. Cardiac troponin C (cTnC) and covalent TnIC complex were also found in pools of plasma with higher concentrations of cTnI. MAb 19C7-affinity SELDI-TOF MS analysis performed after immunopurification of one pool of AMI plasma with anti-free cTnI, anti-cTnC, and anti-phosphorylated cTnI mAbs indicated that intact and bis-phosphorylated cTnI were mostly under the free form. Besides, a 18 718 m/z peak could correspond to a truncated phosphorylated form initially complexed with cTnC.
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Infarto del Miocardio/sangre , Isoformas de Proteínas/análisis , Troponina I/análisis , Western Blotting , Humanos , Fosforilación , Análisis por Matrices de Proteínas , Isoformas de Proteínas/inmunología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Troponina I/metabolismoRESUMEN
Monoclonal antibody D32.10 produced by immunizing mice with a hepatitis C virus (HCV)-enriched pellet obtained from plasmapheresis of a chronically HCV1b-infected patient binds HCV particles derived from serum of different HCV1a- and HCV1b-infected patients. Moreover, this monoclonal has been shown to recognize both HCV envelope proteins E1 and E2. In an attempt to provide novel insight into the membrane topology of HCV envelope glycoproteins E1 and E2, we localized the epitope recognized by D32.10 on the E1 and/or E2 sequence using Ph.D.-12 phage display peptide library technology. Mimotopes selected from the phage display dodecapeptide library by D32.10 shared partial similarities with 297RHWTTQGCNC306 of the HCV E1 glycoprotein and with both 613YRLWHYPCT621 and 480PDQRPYCWHYPPKPC494 of the HCV E2 glycoprotein. Immunoreactivity of D32.10 with overlapping peptides corresponding to these three HCV regions confirmed these localizations and suggested that the three regions identified are likely closely juxtaposed on the surface of serum-derived particles as predicted by the secondary model structure of HCV E2 derived from the tick-borne encephalitis virus E protein. This assertion was supported by the detection of specific antibodies directed against these three E1E2 regions in sera from HCV-infected patients.