Nikolaos Memos

Predictive value of procalcitonin for bowel ischemia and necrosis in bowel obstruction.

BACKGROUND To our knowledge, the predictive value of procalcitonin for bowel strangulation has been evaluated in only 2 experimental studies that had conflicting results. The objective of this study was to evaluate the value of procalcitonin for early diagnosis of intestinal ischemia and necrosis in acute bowel obstruction. METHODS We performed a prospective study of 242 patients with small- or large-bowel obstructions in 2005. A total of 100 patients who underwent operation were divided into groups according to the presence of ischemia (reversible and irreversible) and necrosis, respectively, as follows: ischemia (n = 35) and nonischemia groups (n = 65) and necrosis (n = 22) and nonnecrosis groups (n = 78). Data analyzed included age, sex, vital signs, symptoms, clinical findings, white blood cell count, base deficit, metabolic acidosis, procalcitonin levels on presentation, the time between symptom onset and arrival at the emergency department and the time between arrival and operation, and the cause of the obstruction. RESULTS Procalcitonin levels were greater in the ischemia than the nonischemia group (9.62 vs 0.30 ng/mL; P = .0001) and in the necrosis than the non-necrosis group (14.53 vs 0.32 ng/mL; P = .0001). Multivariate analysis identified procalcitonin as an independent predictor of ischemia (P = .009; odds ratio, 2.252; 95% confidence interval, 1.225-4.140) and necrosis (P = .005; odds ratio, 2.762; 95% confidence interval, 1.356-5.627). Using receiver operating characteristic (ROC) curve analysis, the area under the curve (AUC) of procalcitonin for ischemia and necrosis was 0.77 and 0.87, respectively. A high negative predictive value for ischemia and necrosis of procalcitonin levels <0.25 ng/mL (83% and 95%, respectively) and a positive predictive value of procalcitonin >1 ng/mL were identified (95% and 90%, respectively). CONCLUSION Procalcitonin on presentation is very useful for the diagnosis or exclusion of intestinal ischemia and necrosis in acute bowel obstruction and could serve as an additional diagnostic tool to improve clinical decision-making.

Thyroid surgery with the new harmonic scalpel: A prospective randomized study

BACKGROUND Despite their safety and effectiveness in thyroid surgery, the previous harmonic scalpel instruments are considered large and cumbersome by several surgeons. An innovative technical improvement of the device has been made available since 2008. The objective of this study was to compare the results of total thyroidectomy using the new harmonic scalpel (FOCUS) with that with the previously available device (HARMONIC ACE). METHODS A prospective randomized study of all total thyroidectomies between February and July 2008 was conducted. Patients (n = 90) were randomized to undergo total thyroidectomy with FOCUS (group A, n = 45) or HARMONIC ACE (group B, n = 45). RESULTS No significant differences were identified between the 2 groups in terms of demographics, reoperative thyroid surgery, thyroid gland weight and diameter, pathologic diagnosis, preoperative and postoperative serum PTH and calcium levels, postoperative complications, duration of hospital stay, and final outcome. The mean operative time was less in group A than group B (63 ± 7 min vs 76 ± 8 min, P = .009). CONCLUSION The new harmonic scalpel is a useful adjunct to the armamentarium of the thyroid surgeon. It is safe, effective, and hand friendly, offering great capabilities for delicate tissue grasping and dissection. Use of this device decreased operative time compared with the previously available instrument.

Administration of human protein C improves survival in an experimental model of sepsis

Objective:Study the effect of human protein C (PC) concentrate administration on organ damage and survival in septic rats. Design:Animal study. Setting:University laboratory. Subjects:Male Wistar rats. Interventions:Cecal ligation and puncture (CLP) was performed in 210 rats. Rats were randomly assigned to receive either human protein C (PC) IV 1, 7, and 13 hrs after CLP (CLP+PC) or placebo (CLP). Septic animals were again randomized in a survival group (CLP: n = 50 and CLP+PC: n = 40) that was monitored for 60 hrs and time groups (CLP: n = 60 and CLP+PC: n = 60) that were killed at 6, 12, 24, 36, 48, and 60 hrs after CLP. Brain, heart, lung, liver, kidney, gastric, and colon tissue were removed and postfixed in paraffin sections. Measurements and Main Results:PC infusion increased PC serum levels in early sepsis (median 7.25) compared with late sepsis (median 2, p = .001). Activated protein C/a1-antitrypsin complex levels in the CLP+PC group were significantly increased in late sepsis (60 hrs after CLP) compared with early sepsis (6, 12, and 24 hrs after CLP, p = .009, p = .004, and p = .008, respectively) and to late septic CLP and normal rats (p = .005 and p = .007, respectively). Their IL-6 and tumor necrosis factor a plasma levels were decreased (by 27% and 25%, respectively) at 6 hrs compared with placebo (p = .008 and p = .016). Their serum PC levels were also decreased in CLP+PC survivors compared with nonsurvivors of the same group (median = 1.5 vs. median = 7, p = .001). Apoptosis was reduced in brain (10% vs. 77.8%, p < .001), stomach (66.7% vs. 100%, p < .002) and intestine (33.3% vs. 85.2%, p < .001) compared with placebo. Finally, the survival of septic rats treated with human PC was significantly increased compared with placebo (75% vs. 54%, p = .033). Conclusions:Human Protein C administration increased survival in septic rats, decreased plasma inflammatory cytokines levels and tissue injury in vital organs.

Apoptotic death of renal tubular cells in experimental sepsis.

BACKGROUND AND PURPOSE Renal dysfunction attributable to sepsis was long considered a result of hemodynamic instability and subsequent local ischemia. Recent data show that apoptosis may be implicated also. The purpose of this study was to evaluate the role of apoptosis and the expression of the bax, bcl-2, caspase-8, and cytochrome c proteins in the renal parenchymal cells of rats with sepsis. METHODS Sepsis was induced using cecal ligation and puncture (CLP) in 62 male Wistar rats, which were euthanized 6, 12, 24, 36, 48, or 60 h later. Ten sham-treated animals served as a control group. Another group of 50 animals were subjected to CLP and then supervised for 60 h. Renal apoptosis was evaluated using light and transmission electron microscopy, in situ nick-end labeling (TUNEL), and flow cytometry using 7-amino-actinomycin D (7-AAD). Caspase-mediated apoptosis was assessed using M30 antibody. The expression of the apoptosis-regulator proteins B-cell lymphoma 2 (bcl-2), bcl-2-associated x protein (bax), caspase-8, and cytochrome c was detected immunohistochemically. RESULTS Sepsis increased inflammatory infiltration (p < 0.001) and necrosis (p < 0.001) in renal parenchyma. Apoptosis was significantly more common than in the kidneys of control animals (p = 0.02). Nuclei stained by the TUNEL technique were predominant in the tubular cells of non-survivors (p = 0.05). The time distribution of all types of cell death was increased significantly 6 h after the induction of sepsis, and declined subsequently. Caspase-generated cytokeratin 18 (CK18) new epitope (M30) was significantly more abundant in the kidneys of animals with sepsis than in control rats, with peaks at 6 h and 60 h post-procedure (p < 0.001). In addition, cells initiating apoptosis were significantly more common at 6 h than at 48 h post-CLP (p = 0.014). Caspase-8 protein immunodetection followed the same time pattern as cell death, increasing as early as 6 h post-CLP and decreasing thereafter (p = 0.013). Bax protein expression was elevated significantly early in the course of sepsis (p = 0.037), whereas the other members of the mitochondrial-dependent pathway remained constant. Animals dying from sepsis had a significantly greater prevalence of bax- (p = 0.037) and caspase-8- (p = 0.031) immunoreactive renal cells. CONCLUSION Apoptosis in renal tissue was significantly more common in animals with sepsis than in controls. The time distribution of cell death markers showed a consistent pattern, making early sepsis the likely initiator of the apoptotic events.

Administration of Human Protein-C concentrate prevents apoptotic brain cell death after experimental sepsis

Activated Protein C renders anti-apoptotic properties in neurons and endothelial cells. The aim of the present study was to evaluate the in vivo cytoprotective role of Protein C zymogen (PC) administration in septic rat brain. Male Wistar rats (n=60) were subjected to sepsis via Cecal Ligation and Puncture (CLP). Animals were randomly divided either to receive 100 IU/kg human PC concentrate at 1, 7 and 13 h post CLP (CLP+PC group) or placebo treatment (CLP group). At pre-specified time points (6, 12, 24, 36, 48 and 60 h post CLP) five animals from either group were euthanized and the brain tissue was removed. Apoptosis in both neurons (Neu-N+) and astroglia (GFAP+) was assessed by flow cytometry using 7-aminoactinomycin D (7AAD). Immunohistochemical detection of cleaved caspase 3, bax, bcl-2, cytochrome c and caspase 8 was also performed. PC treated animals had significantly reduced apoptosis in neurons at 6 and 24 h post CLP (p=0.04 and p=0.016 respectively) and necrosis at 6, 12 and 60 h post CLP (p=0.008, p=0.012 and p=0.032 respectively). Astrocyte necrosis was also decreased in septic rats receiving PC (6, 12 and 60 h post CLP p=0.008, p=0.016 and p=0.008 respectively). In addition, active caspase 3, bax, cytochrome c and caspase 8 expression was significantly decreased during early sepsis (6-36 h) while bcl-2 expression was increased (24 h p=0.001 and 60 h p=0.001) in the PC treated animals compared to placebo. PC concentrate administration in experimental sepsis produced a time dependent inhibition of apoptosis in rat neurons and astrocytes. The inhibition of sepsis related apoptosis concerned both the mitochondrial and caspase 8 dependent pathways.

Alternations of 14‐3‐3 θ and β protein levels in brain during experimental sepsis

The 14‐3‐3 family members play a crucial role in the determination of cell fate, exerting their antiapoptotic activity through directly interfering with the critical function of the mitochondrial core proapoptotic machinery. Dimerization of 14‐3‐3 is vital for the interaction with many of its client proteins and is regulated by phosphorylation. In a previous study, we observed time‐dependent neuronal apoptosis during sepsis. Therefore, in the present study, we sought to evaluate the expression of 14‐3‐3 θ and β isoforms in septic brain and their association with apoptosis. Sepsis was induced by a CLP model in Wistar rats that were sacrificed at predefined time points. Flow cytometric analysis showed a sepsis‐induced, time‐dependent alteration of 14‐3‐3 θ and β isoforms in both Neun+ and GFAP+ cells. 14‐3‐3 θ was linearly correlated with apoptosis, and stratified analysis for alive and apoptotic neuronal cells demonstrated a gradual down‐regulation of θ isoform in alive neurons and astrocytes. The phospho‐P38 (pP38) MAP kinase levels were altered in a time‐dependent manner during sepsis, presenting a peak at 6 hr post‐CLP. A significant correlation between the two isoforms of 14‐3‐3 was observed in septic rats, with the θ isoform predominant at all time points. The hippocampus, Purkinje cells, and glia‐like cells showed intense immunohistochemical reactivity for 14‐3‐3 θ isoform, whereas the choroid plexus showed constantly increased β isoform expression. Our results showed that sepsis alters the expression of both 14‐3‐3 θ and β isoforms in a time‐, cell‐, and topography‐dependent manner.

EXTENDED CYTOPROTECTIVE EFFECT OF AUTOPHAGY IN THE LATE STAGES OF SEPSIS AND FLUCTUATIONS IN SIGNAL TRANSDUCTION PATHWAYS IN A RAT EXPERIMENTAL MODEL OF KIDNEY INJURY.

ABSTRACT The impact of a potential autophagy (LC3a/b) deregulation in hyper and in hypo stages during sepsis-induced kidney injury and the temporal profile of phosphorylated extracellular signal-related kinase, P38 (pP38), Akt (pAKT), and 13-3-3&bgr; protein were investigated in the current study, using a rat cecal ligation and puncture (CLP) model, by means of flow cytometry and immunohistochemistry. Cell viability was assessed by protein C zymogen concentrate (PC), 7-aminoactinomycin D (7-AAD) staining and inflammation by S100 protein immunostaining. The impact of reduced kidney inflammation in autophagy was assessed by PC administration, an anti-inflammatory and cytoprotective substance. Sepsis induction increased LC3a/b expression, which presented two peaks at 6 and 36 h after CLP, both in the percentage of positive cells (P = 0.024, P = 0.025, respectively) and in fluorescence intensity. At 6 h when inflammation was already apparent, LC3a/b increase was escorted by phosphorylated extracellular signal-related kinase stimulation and high cell viability (65%), designating autophagy as a cytoprotective mechanism against microbial infection. The phosphorylation of P38 was delayed to 12 h after CLP, when autophagy was reduced. pAkt and 14-3-3&bgr; expression was stimulated between 6 and 36 h after CLP, although a slight inhibition of pAkt within each cell was detected (lower MnIX value). During the second peak, inflammation was intensified, necrosis was significantly increased with LC3a/b+/7-AAD + cells to present a 1.5-fold increase. Protein C zymogen concentrate administration declined autophagy at 6 and 36 h after CLP and reduced necrosis, whereas double positive LC3a/b and 7-AAD cells were increased by 1.68 and 2.78-fold, respectively. These data open new prospectives in sepsis treatment, since they further support that autophagy represents a cytoprotective mechanism triggered by stress conditions, rather than an alternative cell death pathway.

Protein C concentrate in adult septic patients

Sir: We read with great interest the article by Barrato et al. [1] regarding the use of protein C (PC) concentrate administration in adult septic patients with acquired PC deficiency and clinical contraindication to recombinant human-activated PC (rhAPC). The authors showed that the drug was safe on the subject of bleeding complications and effective regarding coagulopathy restoration, and speculated whether PC zymogen (Ceprotin , Baxter) could become an adjuvant therapy in patients who cannot receive rhAPC (Xigris , Elli Lilly). Interestingly, the cases they described mirror our own experience. We have treated (unpublished data) three adult patients with gram-negative sepsis and acquired PC deficiency (plasma levels of PC \ 35%) with PC zymogen (Ceprotin , Baxter). Ceprotin was provided at a dose of 100 U/kg per 6 h for the first 24 h and then 50 IU/ kg daily for 3 days. This dosage scheme resulted in supranormal levels of plasma PC and diminished the laboratory values reflecting the severity of disseminated intravascular coagulation (DIC) such as plasminogen activator inhibitor (PAI-1) and thrombin–antithrombin complexes (TAT). All patients had positive outcome despite high APACHE II admission scores. Although this pilot study of Barrato et al. enriches our knowledge on this topic, there are still several unanswered questions regarding the use, the optimal dose and the duration of treatment with PC zymogen in sepsis-acquired deficiency. We believe that the drug could be tested for survival benefit in sepsis unconnectedly to rhAPC. Since restoration of physiological anticoagulant pathways, such as the PC pathway, have been found to be beneficial [2], the use of PC zymogen could be justified in a prospective study given that a number of issues are resolved. A study regarding the use of human PC zymogen in septic animals was performed from our group. Sepsis was induced using the cecal ligation and puncture (CLP) model in 90 Wistar rats to examine whether PC zymogen administration, provided early in the course of sepsis, has any impact on survival (data presented as an abstract form) [3]. Animals were randomly assigned to receive 100 IU/kg human PC zymogen concentrate (Ceprotin , Baxter) at 1.7 and 13 h post-insult (n = 40) or placebo (n = 50) and had 60 h of monitoring for survival. There was a significant increase in survival rate in favor of PC zymogen-treated animals versus control (75 vs. 54%, P = 0.03). Under this view of encouraging data, we do agree with the authors that a randomized, double blind study is warranted to define the role, if any, of PC zymogen concentrate as an adjunct to the standard therapy in sepsis-acquired PC deficiency.

Comment on: “The effects of supra-normal protein C levels on markers of coagulation, fibrinolysis and inflammation in a human model of endotoxemia” by Spiel et al.

Comment on: “The effects of supra-normal protein C levels on markers of coagulation, fibrinolysis and inflammation in a human model of endotoxemia” by Spiel et al. -