The Study Of The Expression And Clinical Relationship Of Procalcitonin And Th17/Regulatory T Cell In Ventilator-associated Pneumonia After Cardiac Surgery

Part Ⅰ Assessment of the accuracy of serum procalcitonin for the diagnosis and prognosis of ventilator-associated pneumonia after cardiac surgeryObjectiveIn order to provide reference to accurately diagnose ventilator-associated pneumonia(VAP) after cardiac surgery in clinics, we investigated the kinetics alteration of serum Procalcitonin (PCT)、 Pancreatic stone protein (PSP)、Interleukin-6(IL-6) and C-reactive protein (CRP) from early onset VAP patients after cardiac surgery with cardiopulmonary bypass(CPB) and assessed these biomarkers’value for diagnosis and prognosis of VAP after cardiac surgery.MethodsThis study included51consecutive patients aged≥18years who accepted48hours or more of mechanical ventilation at our intensive care unit(ICU) of cardiac surgery (Second Xiang-Ya Hospital,Changsha,China) between February2012and December2012.All of them undergone elective cardiac surgery with the use of CPB.33patients were diagnosed with early onset VAP (mechanical ventilation time less than4days) and the remaining18patients formed a non-VAP group. Follow-up were for28days or until death. In VAP group, patients who died within28days after VAP onset were classified as non-survivors (7patients),others (26patients) were classified as survivors. Blood samples for biologic measurements were drawn before cardiac surgery(baseline). In VAP group,Dayl was defined as the day when the VAP diagnosis was confirmed while Day1was defined as the day when the patients removed the tracheal intubation in the non-VAP group. Serum PCT、PSP、IL-6and CRP level were measured every two days(Day1、Day3、Day5、Day7) in VAP group and non-VAP group.Serum PCT and IL-6were measured by electrochemiluminescence immunoassay. Serum CRP was measured by immune turbidimetry while serum PSP was measured by Enzyme-linked immuno sorbent assay (ELISA).Date collected included preoperative and postoperative characteristics and non-infectious complications. Sequential Organ Failure Assessment (SOFA) were evalued in all the patients on Day1and Day7. Date of positive lower respiratory tract secretions from VAP patients was also collected. Comparsion between two groups were performed using the Student t test、the Mann-Whitney test or the Fisher exact method,when appropriate.Receiver operating characteristic (ROC) curves was used to determine the best threshold values for sensitivity and specificit.Correlations were calculated by Spearman rank correlation. ResultsDuration of mechanical ventilation、Length of stay in the ICU and SOFA on Day1and Day7were significantly higher in VAP group than non-VAP group. Non-survivors group’s SOFA on Dayl and Day7were also significantly higher than those of survivors group.There were no differences in pre-operation characteristics and non-infectious complications between the non-VAP group and VAP group. Serum PCT、 PSP、IL-6and CRP level from all the patients were significantly high compared with their pre-operation baseline level. On Day1,Serum PCT level in patients who were diagnosed with VAP after cardiac surgery were markly higher than those of the non-VAP group. On Dayl, the best PCT cutoff value for VAP diagnosis was5.0ng/ml,with a sensitivity of93.9%and a specificity of83.3%(area under the curve:0.886; P<0.01). None of serum PSP、IL-6、CRP had significant predictive value for VAP diagnosis. Among the patients who were diagnosed with VAP,serum PCT level on Day5and Day7、serum PSP and CRP level on Day7, SOFA on Dayl and Day7all had some predictive value in detecting non-survivors. On Day7,the area under the ROC curve that used PCT to detect non-survivors was0.869(p=0.01),which provided the best predictive value. The best PCT cutoff value for VAP prognosis was6.4ng/ml, with a sensitivity of80.0%and a specificity of96.2%.Used SOFA to detect non-survivors was considerable:the area under the ROC curve of SOFA on Dayl and Day7were0.805and0.865, respectively. For detection of non-survivors by SOFA, a value of14on Day1showed57.1%sensitivity,92.3%specificity and a value of9on Day7showed80.0%sensitivity,84.6%specificity.The area under the ROC curve that used serum PSP and CRP on Day7to detect non-survivors were0.831and0.808. Serum PSP≥494ng/ml and CRP≥87ng/ml on Day7for VAP prognosis provided80.0%.80.0%sensitivity and80.8%、73.1%sensitivity, respectively. There was a postive linear correlation between serum PCT level and SOFA (the Pearson’s r between PCT level and SOFA on Day1and Day7were0.54and0.66respectively, P<0.01).37strains gram-negative bacilli were separated from the lower respiratory tract secretions from33patients with VAP,with a proportion of82%.5strains gram-postive bacteria made up11%and3strans fungus accounted for7%.Conclutions1. Cardiac surgery with CPB has an influence on serum PCT、PSP IL-6and CRP level and they are markly higher in patipents with postoperation complications.2. PCT is a useful marker for diagnosing VAP after cardiac surgery and none of PSP、IL-6、CRP have significant predictive value for VAP diagnosis.3. Continuous monitoring kinetics alteration of serum PCT、PSP、 IL-6and CRP in VAP patipents may contribute to predict the prognosis of VAP. At different phases of VAP, PCT、SOFA、PSP and CRP all have some predictive value in detecting non-survivors.PCT which provides the best sensitivity and specificity may be a good marker for VAP prognosis.4. Majority of pathogenic bacteria in VAP are Gram-negative bacilli. Serum PCT、PSP、IL-6and CRP does not differ between Gram-negative bacilli and Gram-negative bacilli. Part II Research on the expression and effects of Th17/Regulatory T cell in ventilator-associated pneumonia after cardiac surgeryObjectiveTo investigate the imbalance between T helper17cell(Th17cell) and CD4+CD25+Foxp3+regulatory T (Treg) cell, the expression of Thl7/Treg cell key transcription factors Retinoid-related orphan nuclear receptor-yt (ROR-yt)、Forkhead transcription factor3(Foxp3) and related cytokines IL-17、IL-10and IL-6mRNA and protein level、the relationship with serum procalcitonin (PCT) and the possible mechanism during systemic inflammatory response syndrome (SIRS) and early stage of ventilator-associated pneumonia (VAP) after cardiac surgery.Try to discover the relationship between the imbalance of Th17/Treg cell and the ventilator-associated pneumonia and afford the theoretical and experimental proof for the onset and treatment of VAP after cardiac surgery.MethodsThis study included8patients who were diagnosed with VAP and8patients with SIRS who accepted48hours or more of mechanical ventilation but not developed VAP at our intensive care unit(ICU) of cardiac surgery (Second Xiang-Ya Hospital,Changsha,China) between February2012and December2012.Meanwhile,8healthy volunteers formed a normal control group. Peripheral blood mononuclear cell (PBMC) were isolated by Ficoll-Hyaque density gradient centrifugation.Total RNA were extracted using a RNA extraction kit. Total RNA concentrations were determined by spectrophotometer. Total RNA quality were assessed by agarose gel electrophoresis. The expression of Th17/Treg cell key transcription factors ROR-yt. Foxp3and the related cytokines IL-17、IL-10and IL-6mRNA were measured by real time quantitative Reverse transcription polymerase chain reaction (RT-PCR). Serum IL-17、IL-10were measured by Enzyme-linked immunosorbent assay (ELISA) while Serum IL-6、PCT were measured by electrochemiluminescence immunoassay. mRNA calculations were used-△CT relative quantitative method.Comparsion between the two groups were performed using the Student t test or the Mann-Whitney test,when appropriate. Correlations between these index and serum PCT were calculated by Spearman rank correlation.ResultsThe ratio of OD260nm/OD280nm of the total RNA were all between1.8and2.0.28s、18s and5s pattern appeared upon agarose gel electrophoresis.The RNA fluorescence intensity ratio of18s to28s was almost1:2. There were only a peak in all the realtime quantitative PCR melted curves.IL-17、IL-10、IL-6. Foxp3and ROR-γt mRNA in SIRS group and VAP group were higher than those in the normal control group.The difference was significant. IL-10and Foxp3mRNA in VAP group were also significantly higher than those in SIRS group. IL-17、I1-6and ROR-yt mRNA did not differ between SIRS group and VAP group.Serum IL-17、IL-10and IL-6level in SIRS group and VAP group were higher than those in the normal control group.The difference was significant. Serum IL-10level in VAP group were also significantly higher than SIRS group.No differences in the level of serum IL-17and I1-6were found between SIRS group and VAP group. There was a postive linear correlation between serum PCT level and Foxp3、serum IL-10(the Pearson’s r were0.53and0.64respectively, P<0.05)Conclution1.Th17cell involves in the development of SIRS after cardiac surgery.The role of Th17cell in the early stage of ventilator-associated pneumonia needs further discussed.2. Treg cell is activated after cardiac surgery and fights against SIRS-induced inflammatory reaction. Treg cell-mediated anti-inflammation response has already occurred during early stage of ventilator-associated pneumonia and play a role in immunity disorder of VAP patients.3. Serum PCT level has a postive linear correlation with Treg cell key transcription factors Foxp3and serum IL-10level.PCT is parallel with the degree of anti-inflammation activation response to a certain extent.