Sepsis is defined as the systemic inflammatory response to infection, with its severe form associated with evidence of organ dysfunction.1,2 Although scientists have for many years researched the prevention and therapy of sepsis, it seems that the incidence of sepsis has not decreased but continues growing. The yearly incidence of sepsis is 50-95 cases per 100 000, and has been increasing by 9% each year.3 Estimates of sepsis indicate that the total of 750 000 cases in the U.S. every year with a mortality as high as 50% make sepsis the disease with the highest mortality of any major disease.4,5
In sepsis, the initiating stimuli are often bacterial components, which induce the secretion of pro-inflammatory cytokines such as interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α) predominantly from cells of the immune system. High circulating concentrations of these cytokines sometimes indicate an increased risk of mortality6 but treatments antagonizing their activities have not improved patients' survival.7 Among the clinical trials, non-specific high-dose steroid treatments8 were tested in a series of disappointing studies.9,10 Specific therapies against TNF-α involving antibodies11,12-17 or soluble TNF-receptors18 were as unsuccessful as trials blocking IL-1 by IL-1-RA.19-22 These agents proved to be ineffective in improving outcome in sepsis. In addition, during sepsis bacterium often lead to a decrease in both number and function of circulating lymphocytes through the mechanism of cell apoptosis with resultant immunosuppression.
Ulinastatin (UTI) is one of the Kunitz-type protease inhibitors found in urine.23 UTI is synthesized from inter-trypsin inhibitor through proteolytic cleavage by neutrophil elastase at the site of inflammation. Various serine proteases such as trypsin, chymotrypsin, neutrophil elastase, and plasmin are inhibited by UTI. It was generally believed that UTI had the ability to control a series of proinflamatory mediators and cytokines. Thymosin α1 (Tα1) is a naturally occurring thymic peptide first described and characterized by Goldstein et al.24 In the form of a synthetic 28-amino acid peptide, the mechanism of action of the synthetic polypeptide is not completely understood, but it is thought to be related to its immunomodulating activities, centered primarily on the augmentation of T-cell function.
In the present study, the potential efficacy of UTI plus Tα1 in patients with severe sepsis was assessed in a randomized, placebo-controlled setting.
Study design and treatment
The study consisted of three phases: screening, treatment, and follow-up. During the screening phase, patient eligibility was determined, informed consent was obtained, and patients were randomly assigned to receive either UTI (Guangdong Techpool Pharmaceutical Co., China) and Tα1 (Chengdu Diao Group, China), defined as Group A, or placebo, defined as Group B. During the treatment phase, all patients received an intravenous loading dose of 200 kU UTI three times a day plus a subcutaneous dose of 1.6 mg Tα1 twice a day for three days followed by a dose of 100 kU UTI thrice a day plus 1.6 mg Tα1 once a day for four continuous days or an equivalent amounts of placebo. Decisions regarding the use of antimicrobial agents, intravenous fluids, cardiovascular and respiratory support, and surgical intervention were made by each patient's attending physician. After completion of the 7-day treatment phase, patients were followed up for 28, 60 and 90 days.
Patients with a diagnosis of severe sepsis were enrolled if they fulfilled the criteria defined by the 2001 International Sepsis Definitions Conference.1 Patients were not eligible if they met any of the following criteria: (1) age under 18 or over 80 years; (2) incurable malignancies with documented metastases; (3) chronic treatment with high-dose immunosuppressive drugs or high dose nonsteroid anti-inflammatory drugs within the previous 2 days; (4) acute myocardial infarction; (5) chronic compensated organ dysfunction, such as chronic liver disease, dialysis-dependent renal failure, moderate to severe chronic heart failure. Informed consent for study participation was obtained from all patients prior to the performance of any study-related procedures.
Patients were followed throughout the 28, 60 and 90 days study period after receipt of study medication or until death, whichever occurred first. During screening, the patient's history was recorded and a physical evaluation was performed. Samples of blood and other body fluids and specimens from suspected sites of infection were obtained for culture. Vital signs were obtained and hematologic and biochemical tests were carried out at screening and on admission and on 3rd, 8th, and 28th day. These laboratory values and organ-specific parameters were also used to calculate the Acute Physiology and Chronic Health Evaluation II (APACHE II),25 multiple organ failure (MOF),26 Glasgow Coma Scores (GCS).27 Organ dysfunction was diagnosed according to the guideline.
Lymphocyte subset and cytokines detection
Lymphocyte subsets were counted by a Guava EasyCyte flow cytometer. Cytokine levels were determined by ELISA, using commercially available kits from Genzyme, Cambridge, MA, USA.
Numeric data on scores is represented as mean±standard deviation (SD). Welch's t test assessed differences in means for APACHE II, MOF and GCS scores. Student's t test was used to assess differences in lymphocyte subsets and inflammatory mediators. Survival rates over time were estimated by the Kaplan-Meier method. A log rank test was used to analyze the length of supportive ventilation using dopamine, the duration of stay in the ICU and length of antimicrobial therapy. Tests were indicated as significant if P was <0.05 with two-sided analysis. Statistical analyses were calculated using SPSS 13.0 software.
Characteristics of patients on admission
At screening, the following data were recorded: mean age, sex (M/F), body weight (kg), underlying diseases, mean APACHE II score, MOF scores and Glasgow score. This distribution, as well as the demographic characteristics and underlying diseases, the pathogens responsible for the septic episode, the severity of sepsis at study entry were well-balanced across the two treatment groups (P >0.05). During study, the mean length of supportive ventilation using dopamine, stay in the ICU and antimicrobial therapy was found significantly different between the two groups (P <0.01). Treatment with UTI plus Tα1 was found to be well-tolerated and safe without adverse events (Table 1).
Effects of UTI plus Tα1 in patients with severe sepsis
Differences in APACHE II, MOF and GCS between groups over time were found (P=0.041, 0.024 and 0.032, respectively). APACHE II and MOF were reduced significantly after therapy on the 3rd, 8th and 28th day in Group A (P=0.042, 0.000, 0.000 and 0.040, 0.000, 0.000 respectively), so did in Group B only after therapy on 28th day when compared with that on admission (P=0.036, 0.021, respectively). Glasgow score increases were found after therapy on the 8th and 28th in Group A (P=0.000), in Group B did the same merely after therapy on 28th day (P=0.039). There were significant differences in APACHE II, MOF, and Glasgow scores after therapy on the 8th day between Group A and Group B (P =0.012, 0.017 and 0.004, respectively) (Table 2).
Resolution of pre-existing organ dysfunction
Acute intestinal dysfunction: 33 patients already showed intestinal dysfunction on admission (Table 3). A reduction in the prevalence of intestinal dysfunction by 56% was recorded on day 8 in Group A. In Group B, the corresponding reduction was 24% (P=0.058).
Acute neurological dysfunction: 21 patients already showed neurological dysfunction on admission. A reduction in the prevalence of central nervous system (CNS) dysfunction by 45% was recorded on day 8 in Group A. In the placebo group, the corresponding reduction was 20% (P =0.221).
Acute respiratory dysfunction: 39 patients were in respiratory failure on admission. A reduction in the prevalence of respiratory dysfunction by at least 53% was noted on day 8 in Group A. In Group B, the corresponding reduction was 20% (P=0.036).
Acute renal dysfunction: 35 patients had renal dysfunction on admission. A reduction in its prevalence by 63% was seen on day 8 in Group A. In Group B, the corresponding reduction was 25% (P=0.027).
Acute hepatic dysfunction: 30 patients had hepatic failure on admission. In Group A the prevalence of hepatic dysfunction was reduced by 62% on day 8. In Group B, the corresponding reduction was 21% (P=0.28).
Coagulation failure: 31 patients had coagulation failure on admission. A reduction in its prevalence by 27% was recorded on day 8 in Group A. In Group B, the corresponding reduction was 12% (P=0.146).
Survival analysis for Group A and Group B
The effect of UTI plus Tα1 treatment on the mortality of patients with sepsis was analyzed on an intention-to-treat basis (Figure 1). The cumulative survival (54.1%, 54.1% and 47.4% of Group A at days 28, 60, and 90) were increased respectively by 18.7%, 25.9%, and 27.4% compared with 35.4%, 28.2% and 20.0% of Group B (P=0.078, 0.045, and 0.033, respectively).
Comparision of lymphocyte subsets and concentration of inflammatory mediators
There were significant differences in the CD4+/CD8+ ratio after therapy on the 8th, and 28th days between Group A and Group B (P=0.013, 0.039, respectively). In group A, TNF-α and IL-6 after therapy on the 8th and 28th days were reduced significantly when compared to those on admission (all P=0.000). In group B TNF-α and IL-6 after therapy on the 28th were decreased (P=0.012, 0.01 respectively) when compared to those on admission. The differences of TNF-α and IL-6 between Group A and Group B on the 8th after therapy were found (P=0.029, 0.039, respectively). The levels of IL-10 were heightened significantly after therapy on the 8th day in Group A when compared to that on admission (P=0.000), but not in the Group B (Figure 2).
Although treatment of sepsis with new therapies, including high dose corticosteroids28 and non-steroidal anti-inflammatory drugs29 and monoclonal antibodies targeting lipopolysaccharides30,31 and drugs for blocking one factor in the inflammatory cascade,32 have failed to improve survival, septic patients treated with UTI plus Tα1 did show a better performance. This study attempted to address the issue from three perspectives. First, we compared the differences in APACHE II, MOF and GCS between groups. Second, we followed the resolution of pre-existing organ dysfunction. Third, we analyzed the survival rate at 28, 60 and 90 days.
The results of this trial demonstrate a better performance of individual organ function in patients treated with the UTI plus Tα1 which was also reflected in overall organ failure scores. The trend for the improvement in APACHE II, MOF, and Glasgow scores started soon after initiation of treatment. APACHE II and MOF scores were reduced significantly after therapy on the 3rd, 8th and 28th day in Group A, but only on the 28th day in Group B. A GCS increase was found after therapy on the 8th and 28th in Group A, however only on the 28th day in Group B. Although both groups showed significant differences in APACHE II, MOF, and Glasgow scores over time, the Group A recovered sooner than the Group B. After therapy on the 8th and 28th days there was a significant difference in APACHE II, MOF, GCS between Group A and Group B.
In sepsis, the majority of patients have lung dysfunction associated with cardiovascular instability and deteriorating renal function and altered intestinal, liver, cerebral and coagulation function.5 In UTI plus Tα1-treated patients, there was a better resolution of these pre-existing organ failures. Especially on day 8 reductions in the prevalence of intestinal dysfunction by 56%, respiratory dysfunction by 45%, CNS dysfunction by 53%, renal dysfunction 63%, hepatic dysfunction by 62% and coagulation failure 27%, were noted in the Group A. In Group B, the corresponding reductions were 24%, 20%, 20%, 25%, 21% and 12%. There was a better clinical trend in Group A with respect to the intestinal, respiratory, renal, and liver systems and, especially, to the patients' cerebral and coagulation function. It remains open, however, as to whether the possible improvement in CNS and coagulation function is a direct benefit of the drug or rather an indirect consequence of the better performance of the patients' respiratory, kidney, and liver systems. However, recently it was reported that the PMNE concentration correlates with the activities of coagulation and fibrinolysis33 and Ulinastatin could inhibit coagulation and fibrinolysis by inhibiting PMNE activity.34
With UTI plus Tα1, the potential life-saving effect was embodied by the increased cumulative survival and the shorter length of supportive ventilation using dopamine, a shorter stay in the ICU and time on antimicrobial therapy. Lin et al35 found the mortality of patients with MODS who received treatment with UTI and Tα1 decreased from 38.32% to 25.14% (P = 0.0088), compared with a control group at 28 days. The survival benefit provided by treatment with UTI plus Tα1 appeared to occur middle-late in the septic process. It started within the second week after initiation of treatment and was most pronounced at the end of the second week. It was speculated that the time was related to reduction of inflammatory mediators by UTI and recovery of lymphocyte function by Tα1.
In the present study, the ratio of CD4+/CD8+ was corrected after treatment with UTI plus Tα1 and there was a significant difference in the CD4+/CD8+ ratio after therapy between the two groups. In Group B patients had higher levels of TNF-α and IL-6 and lower levels of IL-4 and IL-10 than did Group A patients. This difference was especially prominent the day after therapy was completed, Day 8, as were differences in APACHE II, MOF, GCS and resolution of pre-existing organ dysfunction.
It appears to be difficult to definitely unravel the mechanism by which the beneficial effect of UTI and Tα1 was brought about. According to the previous study, UTI is well known to suppress the production of TNF-α36 and IL-6 and IL-8.37 Tα1 can induce expression of various cytokines by peripheral blood lymphocytes, including interferon-γ, IL-2 and IL-7, and can stimulate maturation of thymocyte cells38 and up-regulates expression of Toll-like receptor (TLR) 2, 5, 8 and 9.39,40 In our study, it was found that the percent of different lymphocyte subset were corrected after Tα1 treatment. It was also observed that a proinflammatory response resulted in release of TNF-α and IL-6 which cause organ dysfunction to some extent. In the meanwhile anti-inflammatory cytokines such as IL-4, IL-10 are produced to protect from sepsis. The balance between these responses changes from minute to minute and determines the final outcome of the disease. From our other data, UTI plus Tα1 treatment achieves both control of the inflammatory reaction and activation of immunocompetence, and tends to quicken the balance between inflammatory injury and anti-inflammatory protection.
1. Levy MM, Fink MP, Marshall JC, Abraham E, Angus D, Cook D, et al. 2001 SSCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Crit Care Med 2003; 31: 1250-1256.
2. Balk RA. Severe sepsis and septic shock. Crit Care Clin 2000; 16: 179-192.
3. Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med 2003; 348: 1546-1554.
4. Anderson RN, Smith BL. Deaths: leading causes for 2001. Natl Vital Stat Rep 2003; 52: 1-85.
5. Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Carcillo J, et al. Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated cost of care. Crit Care Med 2001; 29: 1303-1310.
6. Hack CE, Aarden LA, Thijs LG. Role of cytokines in sepsis. Adv Immunol 1997; 66: 95-101.
7. Netea MG, van der Meer JW, van Deuren M, Kullberg BJ. Proinflammatory cytokines and sepsis syndrome: not enough, or too much of a good thing? Trends Immunol 2003; 24: 254-258.
8. Bone RC, Fisher CJ Jr, Clemmer TP, Slotman GJ, Metz CA, Balk RA. A controlled clinical trial of high-dose methylprednisolone in the treatment of severe sepsis and septic shock. N Engl J Med 1987; 317: 653-658.
9. Cronin L, Cook DJ, Carlet J, Heyland DK, King D, Lansang MA, et al. Corticosteroid treatment for sepsis: a critical appraisal and meta-analysis of the literature. Crit Care Med 1995; 23: 1430-1439.
10. Lefering R, Neugebauer EA. Steroid controversy in sepsis and septic shock: a meta-analysis. Crit Care Med 1995; 23: 1294-1439.
11. Fisher CJ Jr, Opal SM, Dhainaut JF, Stephens S, Zimmerman JL, Nightingale P, et al. Influence of an anti-tumor necrosis factor monoclonal antibody on cytokine levels in patients with sepsis. The CB0006 Sepsis Syndrome Study. Crit Care Med 1993; 21: 318-327.
12. Dhainaut JF, Vincent JL, Richard C, Lejeune P, Martin C, Fierobe L, et al. CDP571, a humanized antibody to human tumor necrosis factor-alpha: safety, pharmacokinetics, immune response, and influence of the antibody on cytokine concentrations in patients with septic shock. CPD571 Sepsis Study Group. Crit Care Med 1995; 23: 1461-1469.
13. Abraham E, Wunderink R, Silverman H, Perl TM, Nasraway S, Levy H, et al. Efficacy and safety of monoclonal antibody to human tumor necrosis factor alpha in patients with the sepsis syndrome. A randomized, controlled, double-blind, multicenter clinical trial. TNF-alpha MAb Sepsis Study Group. JAMA 1995; 273: 934-941.
14. Abraham E, Anzueto A, Gutierrez G, Tessler S, San Pedro G, Wunderink R, et al. Double-blind randomised controlled trial of monoclonal antibody to human tumour necrosis factor in treatment of septic shock. NORASEPT II Study Group. Lancet 1998; 351: 929-933.
15. Cohen J, Carlet J. INTERSEPT: an international, multicenter, placebo controlled trial of monoclonal antibody to human tumor necrosis factor-alpha in patients with sepsis. International Sepsis Trial Study Group. Crit Care Med 1996; 24: 1431-1440.
16. Clark MA, Plank LD, Connolly AB, Streat SJ, Hill AA, Gupta R, et al. Effect of a chimeric antibody to tumor necrosis factor-alpha on cytokine and physiologic responses in patients with severe sepsis: a randomized, clinical trial. Crit Care Med 1998; 26: 1650-1659.
17. Reinhart K, Wiegand Lohnert C, Grimminger F, Kaul M, Withington S, Treacher D, et al. Assessment of the safety and efficacy of the monoclonal anti-tumor necrosis factor antibody-fragment, MAK 195F, in patients with sepsis and septic shock: a multicenter, randomized, placebo-controlled, dose-ranging study. Crit Care Med 1996; 24: 733-742.
18. Fisher CJ Jr, Agosti JM, Opal SM, Lowry SF, Balk RA, Sadoff JC, et al. Treatment of septic shock with the tumor necrosis factor receptor: Fc fusion protein. The Soluble TNF Receptor Sepsis Study Group. N Engl J Med 1996; 334: 1697-1702.
19. Opal SM, Fisher CJ Jr, Dhainaut JF, Vincent JL, Brase R, Lowry SF, et al. Confirmatory interleukin-1 receptor antagonist trial in severe sepsis:a phase III, randomized, double-blind, placebo-controlled, multicenter trial.The Interleukin-1 Receptor Antagonist Sepsis Investigator Group. Crit Care Med 1997; 25: 1115-1124.
20. Fisher CJ Jr, Dhainaut JF, Opal SM, Pribble JP, Balk RA, Slotman GJ, et al. Recombinant human interleukin 1 receptor antagonist in the treatment of patients with sepsis syndrome. Results from a randomized, double-blind, placebo-controlled trial. Phase III rhIL-1ra Sepsis Syndrome Study Group. JAMA 1994; 271: 1836-1843.
21. Fisher CJ Jr, Slotman GJ, Opal SM, Pribble JP, Bone RC, Emmanuel G, et al. Initial evaluation of human recombinant interleukin-1 receptor antagonist in the treatment of sepsis syndrome: a randomized, open-label, placebo-controlled multicenter trial. Crit Care Med 1994; 22: 12-21.
22. Astiz ME, Rackow EC. Septic shock. Lancet 1998; 351: 1501-1505.
23. Jonsson BM, Ohlsson K, Rosengren M. Radioimmunological quantitation of the urinary trypsin inhibitor in normal blood and urine. BiolChem 1989; 370: 1157-1161.
24. Goldstein AL, Guha A, Zatz MM, Hardy MA, White A. Purification and biological activity of thymosin,a hormone of the thymus gland. Proc Natl Acad Sci USA 1972; 69: 1800-1803.
25. Osvaldt AB, Viero P, Borges da Costa MS, Wendt LR, Bersch VP, Rohde L. Evaluation of Ranson, Glasgow, APACHE-II, and APACHE-O criteria to predict severity in acute biliary pancreatitis. Int Surg 2001; 86: 158-161.
26. Marshall JC, Cook DJ, Christou NV, Bernard GR, Sprung CL, Sibbald WJ. Multiple organ dysfunction score are reliable descriptor of a complex outcome. Crit Care Med 1995; 23: 1638-1652.
27. Hadak AM, Caesar RR, Frol AB, Krueger K, Harper CR, Temkin NR. Functional outcome scales in traumatic brain injury: a comparison of the Glasgow Outcome Scale (Extended) and the Functional Status Examination. J Neurotrauma 2005; 22: 1319-1326.
28. Annane D, Bellissant E, Bollaert PE, Briegel J, Keh D, Kupfer Y. Corticosteroids for severe sepsis and septic shock: a systematicreview and meta-analysis. BMJ 2004; 329: 480.
29. Bernard GR, Wheeler AP, Russell JA, Schein R, Summer WR, Steinberg KP, et al. The Effects of Ibuprofen on the Physiology and Survival of Patients with Sepsis.The Ibuprofen in Sepsis Study Group. N Engl J Med 1997; 336: 912-918.
30. Ziegler EJ, Fisher CJ, Sprung CL, Straube RC, Sadoff JC, Foulke GE, et al. Treatment of gram-negative bacteremia and septic shock with HA-1A human monoclonal antibody against endotoxin. A randomized, double-blind, placebo-controlled trial. The HA-1A Sepsis Study Group. N Engl J Med 1991; 324: 429-436.
31. Greenberg RN, Wilson KM, Kunz AY, Wedel NI, Gorelick KJ. Randomized, double-blind phase II study of anti-endotoxin antibody (E5) as adjuvant therapy in humans with serious gramnegative infections. Prog Clin Biol Res 1991; 367: 179-186.
32. Marshall JC. Such stuff as dreams are made on: mediator-directed therapy in sepsis. Nat Rev Drug Discov 2003; 2: 391-405.
33. Nishiyama1 T, Yokoyama T, Yamashita K. Effects of a protease inhibitor, ulinastatin, on coagulation and fibrinolysis in abdominal surgery. J Anesth 2006; 20: 179-182.
34. Knutsen AP, Freeman JJ, Mueller KR, Roodman ST, Bouhasin JD. Thymosin-alpha1 stimulates maturation of CD34+
stem cells into CD3+
cells in an in vitro
thymic epithelia organ coculture model. Int J Immunopharmacol 1999; 21: 15-26.
35. The Cooperative Group of Immunomodulatory Therapy
of Sepsis, Lin HY. Clinical trial with a new immunomodulatory strategy: treatment of severe sepsis with Ulinastatin and Maipuxin. Natl Med J China (Chin) 2007; 87: 451-457.
36. Aosasa S, Ono S, Mochizuki H, Tsujimoto H, Ueno C, Matsumoto A. Mechanism of the inhibitory effect of protease inhibitor on tumor necrosis factor-α production of monocytes. Shock 2001; 15: 101-105.
37. Sato Y, Ishikawa S, Otaki A, Takahashi T, Hasegawa Y, Suzuki M, et al. Induction of acute-phase reactive substances during open-heart surgery and efficacy of ulinastatin. Inhibiting cytokines and postoperative organ injury. Jpn J Thorac Cardiovasc Surg 2000; 48: 428-434.
38. Billich A. Thymosin alpha1. SciClone Pharmaceuticals. Curr Opin Investig Drugs 2002; 3: 698-707.
39. Romani L, Bistoni F, Gaziano R, Bozza S, Montagnoli C, Perruccio K, et al. Thymosin alpha 1 activates dendritic cells for antifungal Th1 resistance through toll-like receptor signaling. Blood 2004; 103: 4232-4239.
40. Witthaut R, Werdan K, Schuster HP. Multiple organ dysfunction syndrome and multiple organ failure. Diagnosis, prognosis and therapeutic concepts. Internist (Berl) 1998; 39: 493-501.