Inclusion criteria: patients with SAP, and infected necrosis, with APACHE II > 8, and follow-up carried out on hospital admittance and discharge [16]. While the reported limit of its usefulness is up to 48 h after admittance, it was considered appropriate to measure until hospital discharge. Amylase, lipase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, total and direct bilirubin, hemoglobin, hematocrit, leukocytes, glucose, calcium, potassium, sodium, creatinine, partial thromboplastin time and prothrombin time, were measured. Surgical interventions, length of ICU and total hospital stay, complications, and death when applicable were also determined.

Blood samples were obtained by venipuncture immediately before the onset of anaesthesia for the first necrosectomy, with a sample taken every 48 h; and the last was taken just prior to discharge, regardless of their destination (due to the large quantity of samples, those considered for the study were the initial baseline and final). Two vials were collected (10 mL each), one containing 0.1% of ethylene-diamine-tetra-acetic acid (EDTA), and other dry. Plasma and serum were separated by centrifugation at 3000 rpm for 10 min at room temperature and stored at −80˚C until use.

The SAP is associated with systemic inflammatory response syndrome, and concentrations of tumor necrosis factor-alpha (TNF-α), interleukin-32 (IL-32), interleukin- 6 (IL-6); transforming grow factor beta1 (TGF-β1), and interleukin-2 (IL-2) were measured. These cytokines were evaluated in order to discern between the possibilities of survival/death throughout the course of the disease. Measurement of immunoreactive cytokines in the plasma was carried out by solid phase immune-absorbent assay with the multiple sandwich principle (ELISA). An eight-point pattern curve was prepared and optical density was read at 450 nm. Results are expressed in pg/mL and ng/L.

Kits were used following the manufacturer’s specifications (R&D Systems^® USA). Monoclonal antibodies specific for TNF-α, IL-6, TGF-β1 and IL-2 were precoated onto microplates. Standards and samples were pipetted into the wells and any TNF-α, IL-6, TGF-β1 and IL-2 present were bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for TNF-α, IL-6, TGF-β1 and IL-2 was added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells and color developed in proportion to the amount of TNF-α, IL-6, TGF-β1 and IL-2 bound in the initial step. The color development was stopped and the intensity of the color was measured.

The IL-32 was established using ELISA kits (My Biosource^® USA), following the manufacturers protocol. IL-32 ELISA is based on the biotin double antibody sandwich technique to assay human IL-32; which isadded to the wells, pre-coated with IL-32 monoclonal antibody, and then incubated. Afterwards, anti-IL-32 antibodies labeled with biotin, to unite with streptavidinHRP formingan immune complex. Unbound enzymes are removed after incubation and washing; and substrate A and B are added. The solution will turn blue and change to yellow with the effects of the acid. The intensity of the color is measured at 450 nm optical density.

The local Research and Ethics Committee with registration number R-2009-1301-86 approved the study. Encrypted identification codes were assigned to assure patient confidentiality; and the study was performed in accordance with international and national laws, and taking into consideration all principles described in the Declaration of Helsinki, updated in the year 2000 in Edinburg, Scotland. Patients or relatives signed informed consents prior to participation.

The statistical analysis was performed with SPSS. Quantitative data were expressed as mean ± SD, and qualitative variables were expressed as frequencies and percentages. Data did not follow a normal distribution (Shapiro-Wilk test), and comparisons were analyzed using the Mann-Whitney U test between groups, for continuous variables. A two tailed value of p < 0.05 was considered significant.

Levels considered normal in healthy volunteers were 47.00 ± 7.00 ng/L. All of the measurements, in all patients included were significantly incremented throughout the study, suggesting the important inflammatory state of those included (p < 0.0001). Survivors managed with necrosectomy at the onset had 147.44 ± 85.06 ng/L and increased to 215 ± 124 ng/L at the end. In non-survivors levels rose even more and persisted until the end without change: 296.29 ± 111.99 ng/L and 295.43 ± 111.66 ng/L, respectively. The higher level of IL-32 was found in patients subjected to necrosectomy plus VAC plus Drotrecogin alpha. Survivors, at onset and end, had similar levels with 455.60 ± 203 ng/L and 434.8 ± 194 ng/L, respectively. The maximum peak was observed in deceased patient 725 ng/L at the onset and 693.77 ng/L at the end. We found positive correlation between TNF-α and the baseline IL-32 (p < 0.016) (Figures 1(a) and 2(c)).

TNF-α was not detected in healthy individuals. Baseline values in survivors of necrosectomy were 10.75 ± 3.16 pg/mL, and the final result 13.37 ± 2.85 pg/mL. The highest values were reported in non-survivors with 33.7 ± 5.96 pg/mL at study onset, and 30.96 ± 5.91 pg/mL at the end (p < 0.0001). Survivors who underwent necrosectomy plus VAC had 17.90 ± 4.84 pg/mL at onset, and 14.48 ± 2.45 pg/mL at the end; vs. baseline values in non-survivors 20.21 ± 5.59 pg/mL, and an important increase 34.30 ± 9.90 pg/mL at the end (p < 0.007). The initial values in the survivors of necrosectomy plus VAC plus Drotrecogin alpha decreased from 26.30 ± 9.29 to 17.15 ± 4.57 pg/mL at the end; vs. the initial value in the deceased patient 31 pg/mL, and 8.31 pg/mL at the end. These results suggest that all patients with SAP were found a continuous inflammatory state throughout the study, which tended to diminish in survivors when the SAP improved. In contrast, the expression tended to increase in non-survivors. The over expression of TNF-α in SAP was predictable from the onset, persisting throughout the study and predominating in those who experienced fatal conclusions; which, can be translated as the importance of the involvement of the Th1 cytokines in SAP (Figure 1(b)).

The IL-6 is a pro-inflammatory cytokine produced in response to stimuli such as the over-expression of TNF-α. The normal value of IL-6 was 3.03 ± 0.81 pg/mL with over-expression in the study groups from the onset until the end of follow-up. Levels of IL-6 in survivors tended to decrease at the end of the study; however, the initial serum levels in non-survivors were significantly increased, mostly in patients who underwent necrosectomy and those subjected to necrosectomy plus VAC plus Drotrecogin alpha (p < 0.0001); although the difference between them was not significant in the final results, as shown in Figure 1(c), despite the maximum increase that was expected within the first 24 - 48 h.

The TGF-β1 is a potent, pro-inflammatory and pro-fibrogenic cytokine with an important role in cellular differentiation, growth and apoptosis. The normal value was 3778 ± 1222 pg/mL. The initial serum levels in survivors was significantly increased: 45928.58 ± 16399.10 pg/mL (necrosectomy), 22150.58 ± 8152.34 pg/mL (necrosectomy plus VAC), and 37153.45 ± 18626.70 pg/mL (necrosectomy plus VAC and Drotrecogin alpha); and, at the end diminished significantly: 11773.88 ± 4003.15 pg/mL (necrosectomy, p < 0.007), 12805.22 ± 2849.30 pg/mL (necrosectomy plus VAC, p < 0.0001), and 6885.34 ± 2492.79 pg/mL (necrosectomy plus VAC plus Drotrecogin alpha, p < 0.02). Interestingly, the initial values in non-survivors were significantly lower compared to survivors: 8975.23 ± 3183.15 (necrosectomy), 8873.25 ± 1982.81 (necrosectomy plus VAC) and 330.50 pg/mL (necrosectomy plus VAC plus Drotrecogin alpha). There was atendency for increased expression at the end of the study, prior to death: 10,079.12 ± 2878.72 pg/mL (necrosectomy), 15857.66 ± 4545.36 pg/mL (necrosectomy plus VAC, p < 0.003) and 19290.50 pg/mL (necrosectomy plus VAC plus Drotrecogin alpha). The over-expression of TGF-β1 could be considered characteristic of SAP; however, were the expression significantly diminished it could have negative repercussions on recovery; preventing pancreas regeneration and affecting multiple organs and systems (Figure 1(d)). There was negative correlation between the TNF-α and baseline TGF-β (p < 0.039) (Figure 2(d)).

Figure 1(e) shows IL-2 results. The IL-2 is considered a predominant anti-inflammatory. Normal serum levels were 28.36 ± 4.95 pg/mL, with over-expression at the onset in survivors of necrosectomy 40.67 ± 7.33 pg/mL, and 52.69 ± 6.46 pg/mL at the end (p < 0.013); necrosectomy plus VAC had 41.3 ± 8.2 pg/mL at the onset, and 31.7 ± 3.6 pg/mL at the end (p < 0.004); however, in the group with necrosectomy plus VAC plus Drotrecogin alpha, the initial and final expressions were not significantly different, 30.21 ± 8.40 pg/mL vs. 35.98 ± 6.98 pg/mL, respectively. On the other hand, there was a significant increase in baseline serum levels in non-survivors of the necrosectomy group vs. normal levels; possibly in an attempt to compensate for the severe inflammatory state in those patients: 92.63 ± 26.91 pg/mL (p < 0.0004) that remained increased until the end 74.87 ± 24.82. This cytokine behaved more evenly in the other managed groups, without significant differences between survivors and non-survivors. There was a tendency for negative correlation between the IL-2 and baseline TGF- β (p < 0.052) (Figure 2(e)).