Juan M. Borreguero-León

Matrix metalloproteinase-9, -10, and tissue inhibitor of matrix metalloproteinases-1 blood levels as biomarkers of severity and mortality in sepsis

IntroductionMatrix metalloproteinases (MMPs) play a role in infectious diseases through extracellular matrix (ECM) degradation, which favors the migration of immune cells from the bloodstream to sites of inflammation. Although higher levels of MMP-9 and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) have been found in small series of patients with sepsis, MMP-10 levels have not been studied in this setting. The objective of this study was to determine the predictive value of MMP-9, MMP-10, and TIMP-1 on clinical severity and mortality in a large series of patients with severe sepsis.MethodsThis was a multicenter, observational, and prospective study carried out in six Spanish Intensive Care Units. We included 192 (125 surviving and 67 nonsurviving) patients with severe sepsis and 50 age- and sex-matched healthy controls in the study. Serum levels of MMP-9, MMP-10, TIMP-1, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-10 were measured in patients with severe sepsis at the time of diagnosis and in healthy controls.ResultsSepsis patients had higher levels of MMP-10 and TIMP-1, higher MMP-10/TIMP-1 ratios, and lower MMP-9/TIMP-1 ratios than did healthy controls (P < 0.001). An association was found between MMP-9, MMP-10, TIMP-1, and MMP-9/TIMP-1 ratios and parameters of sepsis severity, assessed by the SOFA score, the APACHE-II score, lactic acid, platelet count, and markers of coagulopathy. Nonsurviving sepsis patients had lower levels of MMP-9 (P = 0.037), higher levels of TIMP-1 (P < 0.001), lower MMP-9/TIMP-1 ratio (P = 0.003), higher levels of IL-10 (P < 0.001), and lower TNF-α/IL-10 ratio than did surviving patients. An association was found between MMP-9, MMP-10, and TIMP-1 levels, and TNF-α and IL-10 levels. The risk of death in sepsis patients with TIMP-1 values greater than 531 ng/ml was 80% higher than that in patients with lower values (RR = 1.80; 95% CI = 1.13 to 2.87;P = 0.01; sensitivity = 0.73; specificity = 0.45).ConclusionsThe novel findings of our study on patients with severe sepsis (to our knowledge, the largest series reporting data about MMP levels in sepsis) are that reduced MMP-9/TIMP-1 ratios and increased MMP-10 levels may be of great pathophysiologic significance in terms of severity and mortality, and that TIMP-1 levels may represent a biomarker to predict the clinical outcome of patients with sepsis.

Association between serum soluble CD40 ligand levels and mortality in patients with severe sepsis

IntroductionCD40 Ligand (CD40L) and its soluble counterpart (sCD40L) are proteins that exhibit prothrombotic and proinflammatory properties on binding to their cell surface receptor CD40. The results of small clinical studies suggest that sCD40L levels could play a role in sepsis; however, there are no data on the association between sCD40L levels and mortality of septic patients. Thus, the aim of this study was to determine whether circulating sCD40L levels could be a marker of adverse outcome in a large cohort of patients with severe sepsis.MethodsThis was a multicenter, observational and prospective study carried out in six Spanish intensive care units. Serum levels of sCD40L, tumour necrosis factor-alpha and interleukin-10, and plasma levels of tissue factor were measured in 186 patients with severe sepsis at the time of diagnosis. Serum sCD40L was also measured in 50 age- and sex-matched controls. Survival at 30 days was used as the endpoint.ResultsCirculating sCD40L levels were significantly higher in septic patients than in controls (P = 0.01), and in non-survivors (n = 62) compared to survivors (n = 124) (P = 0.04). However, the levels of CD40L were not different regarding sepsis severity. Logistic regression analysis showed that sCD40L levels >3.5 ng/mL were associated with higher mortality at 30 days (odds ratio = 2.89; 95% confidence interval = 1.37 to 6.07; P = 0.005). The area under the curve of sCD40L levels >3.5 ng/mL as predictor of mortality at 30 days was 0.58 (95% CI = 0.51 to 0.65; P = 0.03).ConclusionsIn conclusion, circulating sCD40L levels are increased in septic patients and are independently associated with mortality in these patients; thus, its modulation could represent an attractive therapeutic target.

Association of sepsis-related mortality with early increase of TIMP-1/MMP-9 ratio.

Objective Higher circulating levels of tissue inhibitor of matrix metalloproteinases (TIMP)-1 at the time of severe sepsis diagnosis have been reported in nonsurviving than in surviving patients. However, the following questions remain unanswered: 1) Does TIMP-1/MMP-9 ratio differ throughout the first week of intensive care between surviving and non-surviving patients? 2) Is there an association between TIMP-1/MMP-9 ratio and sepsis severity and mortality during such period? 3) Could TIMP-1/MMP-9 ratio during the first week be used as an early biomarker of sepsis outcome? 4) Is there an association between TIMP-1/MMP-9 ratio and coagulation state and circulating cytokine levels during the first week of intensive care in these patients? The present study sought to answer these questions. Methods Multicenter, observational and prospective study carried out in six Spanish Intensive Care Units (ICUs) of 295 patients with severe sepsis. Were measured circulating levels of TIMP-1, MMP-9, tumour necrosis factor (TNF)-alpha, interleukin (IL)-10 and plasminogen activator inhibitor (PAI)-1 at day 1, 4 and 8. End-point was 30-day mortality. Results We found higher TIMP-1/MMP-9 ratio during the first week in non-surviving (n = 98) than in surviving patients (n = 197) (p<0.01). Logistic regression analyses showed that TIMP-1/MMP-9 ratio at days 1, 4 and 8 was associated with mortality. Receiver operating characteristic (ROC) curves showed that TIMP-1/MMP-9 ratio at days 1, 4 and 8 could predict mortality. There was an association between TIMP-1/MMP-9 ratio and TNF-alpha, IL-10, PAI-1 and lactic acid levels, SOFA score and platelet count at days 1, 4 and 8. Conclusions The novel findings of our study were that non-surviving septic patients showed persistently higher TIMP-1/MMP-9 ratio than survivors ones during the first week, which was associated with severity, coagulation state, circulating cytokine levels and mortality; thus representing a new biomarker of sepsis outcome.

Prognostic Value of Malondialdehyde Serum Levels in Severe Sepsis: A Multicenter Study

Objective The oxidant/antioxidant state in septic patients has only been studied in small series. We wished to determine whether malondialdehyde (MDA) serum levels were associated with severity and 30-day mortality in a large series of patients with sepsis. Methods We performed an observational, prospective, multicenter study in six Spanish Intensive Care Units. Serum levels of MDA were measured in a total of 228 patients (145 survivors and 83 non-survivors) with severe sepsis and 100 healthy controls. Results Serum levels of MDA were higher in severe septic patients than in healthy controls. Non-surviving septic patients had higher MDA values than survivors. MDA serum levels were associated with severity markers (lactic acid, SOFA, APACHE-II) and coagulation indices. Regression analysis showed that MDA serum levels were associated with 30-day survival (Hazard ratio = 1.05; 95% confidence interval = 1.009–1.091; p = 0.016). Receiver operating characteristic analysis showed that the area under curve of MDA serum levels to predict 30-day survival was 0.62 (95% CI = 0.56–0.69; P = 0.002). The risk of death in septic patients with MDA serum levels above 4.11 nmol/mL was higher than in patients with lower values (Hazard Ratio = 2.43; 95% CI = 1.49–3.94; p<0.001). Conclusions The novel findings of our study on severe septic patients, to our knowledge the largest series providing data on the oxidative state, are that elevated MDA serum levels probably represent an unbalanced oxidant state and are related with poor prognosis in patients with severe sepsis.

Red blood cell distribution width during the first week is associated with severity and mortality in septic patients.

Objective Higher values of red blood cell distribution width (RDW) have been found in non-surviving than in surviving septic patients. However, it is unknown whether RDW during the first week of sepsis evolution is associated with sepsis severity and early mortality, oxidative stress and inflammation states, and these were the aims of the study. Methods We performed a prospective, observational, multicenter study in six Spanish Intensive Care Units with 297 severe septic patients. We measured RDW, serum levels of malondialdehyde (MDA) to assess oxidative stress, and tumour necrosis factor (TNF)-α to assess inflammation at days 1, 4, and 8. The end-point was 30-day mortality. Results We found higher RDW in non-surviving (n = 104) than in surviving (n = 193) septic patients at day 1 (p = 0.001), day 4 (p = 0.001), and day 8 (p = 0.002) of ICU admission. Cox regression analyses showed that RDW at day 1 (p<0.001), 4 (p = 0.005) and 8 (p = 0.03) were associated with 30-day mortality. Receiver operating characteristic curves showed that RDW at day 1 (p<0.001), 4 (p<0.001), and 8 (p<0.001) could be used to predict 30-day mortality. RDW showed a positive correlation with serum MDA levels at day 1 and day 4, with serum TNF-α levels at days 4 and 8, and with SOFA score at days 1, 4 and 8. Conclusions The major findings of our study were that non-surviving septic patients showed persistently higher RDW during the first week of ICU stay than survivors, that RDW during the first week was associated with sepsis severity and mortality, that RDW during the first week could be used as biomarker of outcome in septic patients, and that there was an association between RDW, serum MDA levels, and serum TNF-α levels during the first week.

Serum Levels of Caspase-Cleaved Cytokeratin-18 and Mortality Are Associated in Severe Septic Patients: Pilot Study

Objective Apoptosis is increased in sepsis. Cytokeratin 18 (CK-18), a protein of the intermediate filament group present in most epithelial and parenchymal cells, is cleaved by the action of caspases and released into the blood as caspase-cleaved CK (CCCK)-18 during apoptosis. Circulating levels of CCCK-18 have scarcely been explored in septic patients. In one study with 101 severe septic patients, the authors reported higher serum CCCK-18 levels in non-survivors than in survivors; however, the sample size was too small to demonstrate an association between serum CCCK-18 levels and early mortality and whether they could be used as a biomarker to predict outcomes in septic patients. Thus, these were the objectives of this study with a large series of patients. Methods We performed a prospective, multicenter, observational study in six Spanish Intensive Care Units with 224 severe septic patients. Blood samples were collected at the time that severe sepsis was diagnosed to determine serum levels of CCCK-18, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-10. The end point was 30-day mortality. Results Non-surviving patients (n = 80) showed higher serum CCCK-18 levels (P<0.001) than survivors (n = 144). Multiple logistic regression analysis showed that serum CCCK-18 levels>391 u/L were associated with 30-day survival (Odds ratio = 2.687; 95% confidence interval = 1.449–4.983; P = 0.002), controlling for SOFA score, serum lactic acid levels and age. Kaplan-Meier survival analysis showed that the risk of death in septic patients with serum CCCK-18 levels >391 u/L was higher than in patients with lower values (Hazard Ratio = 3.1; 95% CI = 1.96–4.84; P<0.001). Serum CCCK-18 levels were positively associated with serum levels of IL-6 and lactic acid, and with SOFA and APACHE scores. Conclusions The major novel finding of our study, the largest cohort of septic patients providing data on circulating CCCK-18 levels, was that serum CCCK-18 levels are associated with mortality in severe septic patients.

The 372 T/C genetic polymorphism of TIMP-1 is associated with serum levels of TIMP-1 and survival in patients with severe sepsis

IntroductionPrevious studies have found higher circulating levels of tissue inhibitor of matrix metalloproteinase (TIMP)-1 in nonsurviving septic patients than in surviving septic patients, and an association between the 372 T/C genetic polymorphism of TIMP-1 and the risk of developing certain diseases. However, the relationship between genetic polymorphisms of TIMP-1, circulating TIMP-1 levels and survival in patients with severe sepsis has not been examined, and this was the objective of the study.MethodsThis multicentre, prospective, observational study was carried out in six Spanish ICUs. We determined the 372 T/C genetic polymorphism of TIMP-1 (rs4898), serum levels of TIMP-1, matrix metalloproteinase (MMP)-9, MMP-10, TNFα, IL-10 and plasma plasminogen activator inhibitor-1 (PAI-1). Survival at 30 days from ICU admission was the endpoint assessed. The association between continuous variables was carried out using Spearmans rank correlation coefficient or Spearmans rho coefficient. Multivariate logistic regression analysis was applied to determine the association between the 372 T/C genetic polymorphism and survival 30 days from ICU admission.ResultsOf 275 patients with severe sepsis, 80 had genotype CC, 55 had genotype CT and 140 had genotype TT of the 372 T/C genetic polymorphism of TIMP-1. Patients with the T allele showed higher serum levels of TIMP-1 than patients without the T allele (P = 0.004). Multiple logistic regression analysis showed that the T allele was associated with higher mortality at 30 days (odds ratio = 2.08; 95% confidence interval = 1.06 to 4.09; P = 0.03). Survival analysis showed that patients with the T allele presented lower 30-day survival than patients without the T allele (χ2 = 5.77; P = 0.016). We found an association between TIMP-1 levels and levels of MMP-9 (ρ = -0.19; P = 0.002), MMP-10 (ρ = 0.55; P <0.001), TNFα (ρ = 0.56; P <0.001), IL-10 (ρ = 0.48; P <0.001) and PAI-1 (ρ = 0.49; P <0.001).ConclusionThe novel findings of our study are that septic patients with the T allele in the 372 T/C genetic polymorphism of TIMP-1 showed higher serum TIMP-1 levels and lower survival rate. The determination of the 372 T/C genetic polymorphism of TIMP-1 thus has prognostic implications and could help in the selection of patients who may benefit from modulation of the MMP/TIMP balance.

Association between Serum Tissue Inhibitor of Matrix Metalloproteinase-1 Levels and Mortality in Patients with Severe Brain Trauma Injury

Objective Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) play a role in neuroinflammation after brain trauma injury (TBI). Previous studies with small sample size have reported higher circulating MMP-2 and MMP-9 levels in patients with TBI, but no association between those levels and mortality. Thus, the aim of this study was to determine whether serum TIMP-1 and MMP-9 levels are associated with mortality in patients with severe TBI. Methods This was a multicenter, observational and prospective study carried out in six Spanish Intensive Care Units. Patients with severe TBI defined as Glasgow Coma Scale (GCS) lower than 9 were included, while those with Injury Severity Score (ISS) in non-cranial aspects higher than 9 were excluded. Serum levels of TIMP-1, MMP-9 and tumor necrosis factor (TNF)-alpha, and plasma levels of tissue factor (TF) and plasminogen activator inhibitor (PAI)-1 plasma were measured in 100 patients with severe TBI at admission. Endpoint was 30-day mortality. Results Non-surviving TBI patients (n = 27) showed higher serum TIMP-1 levels than survivor ones (n = 73). We did not find differences in MMP-9 serum levels. Logistic regression analysis showed that serum TIMP-1 levels were associated 30-day mortality (OR = 1.01; 95% CI = 1.001–1.013; P = 0.03). Survival analysis showed that patients with serum TIMP-1 higher than 220 ng/mL presented increased 30-day mortality than patients with lower levels (Chi-square = 5.50; P = 0.02). The area under the curve (AUC) for TIMP-1 as predictor of 30-day mortality was 0.73 (95% CI = 0.624–0.844; P<0.001). An association between TIMP-1 levels and APACHE-II score, TNF- alpha and TF was found. Conclusions The most relevant and new findings of our study, the largest series reporting data on TIMP-1 and MMP-9 levels in patients with severe TBI, were that serum TIMP-1 levels were associated with TBI mortality and could be used as a prognostic biomarker of mortality in TBI patients.

Severe Septic Patients with Mitochondrial DNA Haplogroup JT Show Higher Survival Rates: A Prospective, Multicenter, Observational Study

Objective In a previous cohort study (n=96), we found an association between mitochondrial (mt) DNA haplogroup JT and increased survival of severe septic patients, after controlling for age and serum lactic acid levels. The aim of this research was to increase the predictive accuracy and to control for more confounder variables in a larger cohort (n=196) of severe septic patients, to confirm whether mtDNA haplogroup JT influences short and medium-term survival in these patients. Methods We conducted a prospective, multicenter, observational study in six Spanish Intensive Care Units. We determined 30-day and 6-month survival and mtDNA haplogroup in this second cohort of 196 patients and in the global cohort (first and second cohorts combined) with 292 severe septic patients. Multiple logistic regression and Cox regression analyses were used to test for the association of mtDNA haplogroups JT with survival at 30-days and 6-months, controlling for age, sex, serum interleukin-6 levels and SOFA score. Results Logistic and Cox regression analyses showed no differences in 30-day and 6-month survival between patients with mtDNA haplogroup JT and other haplogroups in the first cohort (n=96). In the second cohort (n=196), these analyses showed a trend to higher 30-day and 6-month survival in those with haplogroup JT. In the global cohort (n=292), logistic and Cox regression analyses showed higher 30-day and 6-month survival for haplogroup JT. There were no significant differences between J and T sub-haplogroups in 30-day and 6-month survival. Conclusions The global cohort study (first and second cohorts combined), the largest to date reporting on mtDNA haplogroups in septic patients, confirmed that haplogroup JT patients showed increased 30-day and 6-month survival. This finding may be due to single nucleotide polymorphism defining the whole haplogroup JT and not separately for J or T sub-haplogroups.

Serum levels of caspase-cleaved cytokeratin-18 in patients with severe traumatic brain injury are associated with mortality: a pilot study.

Objective There have been found apoptotic changes in brain tissue samples from animals and humans after a traumatic brain injury (TBI). The protein cytokeratin 18 (CK-18), present in epithelial cells, is cleaved by the action of caspases during apoptosis, and the resulting fragments are released into the blood as caspase-cleaved CK (CCCK)-18. Circulating levels of CCCK-18, as biomarker of apoptosis, have been determined in patients with different processes; however, it has not been explored in TBI patients. Thus, the objective of this study was to determine whether there is an association between serum CCCK-18 levels and mortality and whether such levels could be used as a biomarker to predict outcomes in TBI patients. Methods A prospective, observational, multicenter study carried out in six Spanish Intensive Care Units. We included patients with severe TBI defined as Glasgow Coma Scale (GCS) lower than 9; and were excluded those patients with Injury Severity Score (ISS) in non-cranial aspects higher than 9. We measured serum CCCK-18 levels at admission. The end-point of the study was 30-day mortality. Results Surviving patients (n = 73) showed lower serum CCCK-18 levels (P = 0.003) than non-survivors (n = 27). On ROC analysis, the area under the curve (AUC) for serum CCCK-18 levels as predictor of 30-day mortality was 0.69 (95% CI = 0.59–0.78; P = 0.006). We found in survival analysis that patients with serum CCCK-18 higher than 201 u/L had higher 30-day mortality than patients with lower levels (Hazard ratio = 3.9; 95% CI = 1.81–8.34; P<0.001). Regression analyses showed that serum CCCK-18 levels higher than 201 u/L were associated with 30-day mortality (OR = 8.476; 95% CI = 2.087–34.434; P = 0.003) after controlling for age and GCS. Conclusions The novel finding of our study was that serum CCCK-18 levels are associated with 30-day mortality and could be used as a prognostic biomarker in patients with severe TBI.