| BackgroundThe incidence of sepsis is more than 200 cases per 100,000 people, and 50-95 cases per 100,000 people for severe sepsis. The incidence of sepsis has been increasing by 8.7% each year over the past two decades. Despite a long history of research into the prevention and treatment of sepsis, the mortality rate of severe sepsis still ranges from 20% to 50%, severe sepsis has become the main causes of death in intensive care units. Initially, the pro-inflammatory response was considered to be the main cause of death in sepsis patients. However, many therapeutic approaches that block the early pro-inflammatory mediators, such as TNF-α antibody and interleukin-1 receptor antagonist, have not proved to be of benefit in improving outcomes of septic patients. More recently, Lymphocyte apoptosis and immunosuppression are increasingly considered as vital processes in the pathogenesis of sepsis. It has been shown that preventing lymphocyte apoptosis and strengthening immune function can improve prognosis in animal models and clinical trials.Tumor necrosis factor-alpha has been considered as an essential pro-inflammatory cytokine in patients with sepsis. However, Docke et al pointed out that up-regulation of tumor necrosis factor-alpha levels indicated restoration of monocyte function and recovery of an antimicrobial response. Tumor necrosis factor-alpha secretion in vitro induced by lipopolysaccharide was a valuable parameter for assessing immune status of sepsis patients. Hall et al. also showed that whole blood ex vivo tumor necrosis factor-alpha response may be used as a biomarker to monitor immune function of sepsis patients.Thymosin alpha 1 is a hormone secreted from the thymus gland, first described and characterized by Goldstein et al. Thymosin alpha 1 is considered as one of the immunomodulatory peptides, which has been clinically used as immunotherapy in the treatment of chronic hepatitis B and C viral infection. Thymosin alpha 1 has many biological activities in the immune system, such as activating natural killer cells, stimulating T cell proliferation, differentiation, maturation, and blocking lymphocyte apoptosis. Therefore, as an immunoregulator, thymosin alpha 1 may be appropriate to treat sepsis. Some clinical studies showed a beneficial effect of thymosin alpha 1 for treating severe sepsis. However, due to heterogeneity in patients with severe sepsis, thymosin alpha 1 may show significant efficacy in some patients, but not in others. However, these studies failed to point out the candidates most appropriate for thymosin alpha 1 treatment. The aim of the present study was to identify the candidates most appropriate for thymosin alpha 1 treatment in patients with severe sepsis, and the effects of thymosin alpha 1 on APACHEII score, SOFA score and lymphocyte count of severe sepsis patients. In order to observe the effects of thymosin alpha 1 on immune function in patients with severe sepsis, we evaluated tumor necrosis factor-alpha levels in peripheral blood mononuclear cells stimulated in vitro by lipopolysaccharide.Objectives1. To investigate the effects of thymosin alpha 1 on 28-day prognosis and survival time of patients with severe sepsis;2. To investigate the effects of thymosin alpha 1 on APACHEII score, SOFA score and lymphocyte count of patients with severe sepsis;3. To investigate the effects of thymosin alpha 1 on immune function of patients with severe sepsis.Methods1. The ethicsWe retrospectively collected severe sepsis patient information between January 2013 and December 2014 at our department. For the retrospective analysis, informed consent was not obtained because the data were analyzed anonymously. To observe the effects of thymosin alpha 1 on immune function in patients with severe sepsis, tumor necrosis factor-alpha levels were assessed in vitro in peripheral blood mononuclear cells stimulated by lipopolysaccharide. For the in vitro studies, informed consent was obtained from patients or their relatives if patients were unable to sign. The study protocol was approved by the ethics committee of Zhujiang Hospital of Southern Medical University, and the approval number was 2014-ZZYXK-003.2. PatientsSevere sepsis patient information between January 2013 and December 2014 was retrospectively collected in the present study. The criteria for severe sepsis were in accord with the 2001 International Standard of Sepsis Definition. Exclusion criteria were pregnancy, patients with malignant tumors, autoimmune diseases, or blood system diseases (such as aplastic anemia or leukemia), and patients who received immune stimulating agents other than thymosin alpha 1, immune inhibitors, or hormone therapy within the previous 1 months. Patients aged less than 18 years or for whom prognostic information was not available (due to loss at follow-up) were also excluded from the study. Finally,244 severe sepsis patients were included in this study. Among the 244 patients, male patients were 169 cases and female 75 cases. Mean age was (63.0±16.4) years. Primary sites of infection causing severe sepsis were as follows:Pulmonary infection in 183 cases, abdominal infection in 32 cases, urinary tract infection in 11 cases, other parts of infection in 18 cases. For the in vitro experiments,12 cases of patients who were diagnosed with severe sepsis and did not meet the exclusion criteria were selected in January 2015.3. Data collectionData for gender, age, site of infection causing severe sepsis, APACHEII score, SOFA score, lymphocyte count, and prognosis were retrospectively obtained from patients’medical records (or by contacting patients’ relatives to obtain prognostic information). (All of the above detection indicators were completed in the inspection department and intensive care unit of our hospital).4. Grouping(1) Among the 244 patients, according to thymosin alpha 1 treatment, severe sepsis patients were divided into thymosin alpha 1 (Tα1) group and non-Tα1 group. 117 patients in non-Tα1 group received conventional therapy based on international guidelines for management of severe sepsis and septic shock in 2008. The other 127 patients in Tα1 group received conventional therapy plus thymosin alpha 1, administered as subcutaneous injections (1.6 mg once every 12 h, for 7 consecutive days). (2) Patients in both groups were stratified according to lymphocyte count on Day 1 (the day when patients were diagnosed with severe sepsis) into the following subgroups:≥1.00×109lymphocytes/L,0.50-1.00×109 lymphocytes/L, and≤ 0.50×109 lymphocytes/L.5. In vitro experiments(1) Peripheral blood mononuclear cells separationIn each of 12 patients with severe sepsis,4 ml of whole blood was collected and stored in vacuum tubes with lithium heparin anticoagulant. Each 4-ml blood sample was mixed with 4 ml of phosphate-buffered saline, and then carefully layered over 3 ml of Ficoll-Paque in a centrifuge tube. After centrifugation in a horizontal centrifuge at 2000 revolutions per min for 30 min, the mixed liquid was divided into three layers. Ficoll-Paque containing peripheral blood mononuclear cells in the middle layer was carefully removed by pipette, placed in another centrifuge tube, and then mixed with phosphate-buffered saline. This preparation was centrifuged at 1000 revolutions per min for 10 min to remove the upper layer of liquid that contained platelets. The remaining sediment was re-suspended in phosphate-buffered saline to obtain the final peripheral blood mononuclear cell fraction.(2) The effects of thymosin alpha 1 on tumor necrosis factor-alpha levels in peripheral blood mononuclear cells stimulated in vitro by lipopolysaccharide.RPMI 1640 medium containing 10% fetal calf serum was divided into three groups:control, lipopolysaccharide and lipopolysaccharide +Tα1. For the lipopolysaccharide +Tα1 group, thymosin alpha 1 was added to the medium to obtain a concentration of 200 μg/ml. For the control group and lipopolysaccharide group, the same volume of phosphate-buffered saline was added to the medium. For each group, Peripheral blood mononuclear cells were cultured at a concentration of 106 cells/ml medium, for 8 h at 37℃ in a 5% CO2 incubator. Subsequently, for the lipopolysaccharide and lipopolysaccharide+Tα1 groups, phosphate-buffered saline containing lipopolysaccharide was added to the medium to obtain a concentration of 10μg lipopolysaccharide /ml (with the same volume of phosphate-buffered saline as for the control group). Peripheral blood mononuclear cells were then cultured for another 6 h. The subsequent supernatants were collected from each group medium. The concentration of tumor necrosis factor-alpha in supernatants was detected using a human tumor necrosis factor-alpha ELISA kit.6. Statistical analysisStatistical analyses were carried out using SPSS version 19.0 software. The data were presented as frequency for categorical variables, mean±standard deviation (SD) for normal distributions or median with interquartile range (IQR) for non-normal distributions. Categorical data were compared using a chi-square test. Continuous variables were tested by t test for normal distributions or Mann-Whitney U test for non-normal distributions. The survival time was estimated by Kaplan-Meier analysis, and a log-rank test was used for comparisons. Tumor necrosis factor-alpha concentrations were tested by one-way analysis of variance (ANOVA), followed by a least significant difference (LSD) test for multiple comparisons. A two-sided P-value less than 0.05 was considered statistically significant in all tests.Results1. Patients and baseline characteristicsTwo hundred and forty-four patients were included in the present study.127 cases were included in Tα1 group,117 cases in non-Tα1 group. At baseline, both groups were similar with regard to sex, age, APACHE Ⅱ score, SOFA score, and lymphocyte count (P> 0.05). Although APACHE II and SOFA scores were higher on the day when patients were diagnosed with severe sepsis in Tα1 group, and lymphocyte counts lower, there were no significant differences between groups (P> 0.05). After patients were stratified according to lymphocyte count, each subgroup was also similar with regard to age, sex, APACHE II score, SOFA score, and lymphocyte counts in the two groups (P> 0.05).2. Comparison of the prognosis of patientsFifty-four of 127 patients in Tα1 group and 59 of 117 patients in non-Tα1 group died within 28 days after being diagnosed with severe sepsis; the difference between groups was not statistically significant (P> 0.05). When stratified according to lymphocyte count, there was no difference between both groups for all-cause mortality at 28 days for patients with≥1.00×109 lymphocytes/L, furthermore, in the subgroup with 0.50-1.00×109lymphocytes/L, all-cause mortality at 28 days was not also significantly different between the two groups (P> 0.05). However, in the subgroup with lymphocyte count<0.50×109/L, all-cause mortality at 28 days was significantly lower for Tα1 group than for non-Tα1 group (P< 0.05).3. Comparison of the survival timeThe survival time of patients was not significantly different between Ta 1 group and non-Tα1 group, and survival analysis curves for both groups were similar (log-rank test, P> 0.05). When stratified according to lymphocyte count, there were no also differences between the two groups for the survival analysis curves for patients with≥1.00×109 lymphocytes/L and 0.50-1.00×109lymphocytes/L (log-rank test, P> 0.05). But in the subgroup with lymphocyte count≤0.50×109/L, the survival time of patients in Tα1 group was significantly longer than those in non-Tα1 group (log-rank test, P< 0.05).4. Dynamic changes in APACHE II score, SOFA scores and lymphocyte countAPACHE II scores were significantly lower in Tα1 group and non-Tα1 group on Day 7 than on Day 1 (P< 0.05). The decrease in APACHE II score on Day 7 was more apparent in Tα1 group, and the difference between groups was statistically significant (P< 0.05). Decreases in SOFA scores on Day 7 followed a similar tendency as for APACHE II scores in the two groups (however, there was no significant difference in non-Tα1 group on Day 7 and on Day 1), but without a statistically significant difference in dynamic changes between the groups (P> 0.05). Compared with Day 1, lymphocyte count was significantly increased on Day 7 in both groups (P< 0.05). Compared with non-Tα1 group, changes in lymphocyte count were more significant in Tα1 group (P< 0.05).5. Outcomes of in vitro experimentsThere was a significant difference in tumor necrosis factor-alpha concentration in control group, lipopolysaccharide group and lipopolysaccharide+Tα1 group (P< 0.05). The tumor necrosis factor-alpha levels in the lipopolysaccharide +Tα1 group were markedly higher than in the control and lipopolysaccharide groups (P< 0.05). The tumor necrosis factor-alpha concentration in the lipopolysaccharide group was also statistically significantly higher than in the control group (P< 0.05).Conclusions1. Thymosin alpha 1 can significantly improve 28-day prognosis and prolong the survival time of severe sepsis patients with lymphocyte count≤0.50×109/L;2. Thymosin alpha 1 can significantly reduce APACHE Ⅱ score and SOFA score and increase lymphocyte count of severe sepsis patients, and improve the patient’s condition;3.Thymosin alpha 1 can significantly improve immune function in patients with severe sepsis. |
PDF Full Text Request