Ivan Stanojevic

A subpopulation that may correspond to granulocytic myeloid-derived suppressor cells reflects the clinical stage and progression of cutaneous melanoma

Seventy-eight melanoma patients and 10 healthy individuals were examined. Follow-up examinations of all melanoma patients were performed regularly every three months. Myeloid-derived suppressor cells (MDSC) were defined as lineage negative (CD3(-), CD19(-), CD56(-)), HLA-DR(-/low), CD11b(+) and CD33(+). Classification of granulocytic (GrMDSC) and monocytic (MoMDSC) subsets was based on the CD15 and CD14 expression, respectively. Unlike the MoMDSC, that were present in 60% of healthy controls and 15% of melanoma patients, the GrMDSC were present in all examined participants, and the melanoma patients were found to have statistically higher frequencies compared with healthy controls. Accordingly, we kept focused on GrMDSC frequencies in relation to the melanoma stages and course of the disease. The GrMDSC values are highest in stage IV melanoma patients, with statistical significance compared with stages IA, IB, IIA and IIB. Patients with progression had statistically higher GrMDSC counts comparing with those with stable disease (P = 0.0079). Patients who had progression-free interval (PFI) < 12 months showed significantly higher GrMDSC values compared with those with PFI > 12 months (P = 0.0333). GrMDSC showed significant negative correlation with PFI intervals (P = 0.0095). The GrMDSC subset was predominant in all our patients. We confirmed that GrMDSC do accumulate early in the peripheral blood of melanoma patients and their frequencies correlate narrowly with the clinical stage and the spread of the disease. The increase in GrMDSC frequencies correlates well with a progressive disease and could be considered a potential predictive biomarker of high-risk melanoma cases that are more likely to have a shorter PFI.

Effects of different anesthetic agents on GM-CSF, MCP1, IL1α and TNFα levels in rat sepsis model.

Anesthetic agents could alter the course and outcome of physical trauma, as well as experimentally or naturally occurring severe infections, by regulating several immune response mechanisms. The aim of our study was to investigate the influence of several commercially used anesthetic agents (ketamine, propofole, pentylentetrazole - PTZ) on cytokine concentrations, animal survival and pathohistological changes in the model of rat sepsis. In adult, male Wistar rats after different anesthetic treatment and induction of sepsis by cecal ligation and punction we estimated serum levels of IL1α, TNFα, GM-CSF and MCP-1 at 12h intervals. After 48h of sepsis induction, the largest number of animals survived in the group treated with PTZ (47%), while the lowest survival rate was in the propofole treatment group (24%). Contrary to survival rate, the most abundant pathohistological changes were seen on preparations from PTZ and than in ketamine/PTZ treated groups, without any significant changes in the CNS of propofole treated animals. In the propofole treated group there was a prominent increament of GM-CSF values at 12h and 24h, followed by a significant decreament at 36h. These changes were negatively correlated to the survival rate in this group. This group had the lowest levels of MCP1 at all evaluated time intervals. After high initial levels, IL1α and TNFα levels fell to undetectable concentrations and at 24h increased to a high level. In PTZ as well as ketamine groups, at 12 h interval, GM-CSF levels were lower than in the propofole treated group. Contrary, MCP-1 levels were higher in these groups comparing to propofole group. After a high initial peak, IL1α levels decreased to low but detectable levels, followed by an intensive rise in ketamine treated, but with further decrement in pentazole treated groups. TNFα levels were low through all evaluated intervals in both these groups. Our results indicate that induction of anaesthesia of animals with sepsis with variuos anesthetic agents is connected to different pathohistological CNS changes, distinct serum cytokine profiles and diverse survival rates.

Early inflammatory response following elective abdominal aortic aneurysm repair: A comparison between endovascular procedure and conventional, open surgery

BACKGROUND/AIM Abdominal aorta aneurysm (AAA) represents a pathological enlargment of infrarenal portion of aorta for over 50% of its lumen. The only treatment of AAA is a surgical reconstruction of the affected segment. Until the late XX century, surgical reconstruction implied explicit, open repair (OR) of AAA, which was accompanied by a significant morbidity and mortality of the treated patients. Development of endovascular repair of (EVAR) AAA, especially in the last decade, offered another possibility of surgical reconstruction of AAA. The preliminary results of world studies show that complications of such a procedure, as well as morbidity and mortality of patients, are significantly lower than with OR of AAA. The aim of this paper was to present results of comparative clinical prospective study of early inflammatory response after reconstruction of AAA be tween endovascular and open, conventional surgical technique. METHODS A comparative clinical prospective study included 39 patients, electively operated on for AAA within the period of December 2008 - February 2010, divided into two groups. The group I counted 21 (54%) of the patients, 58-87 years old (mean 74.3 years), who had been submited to EVAR by the use of excluder stent graft. The group II consisted of 18 (46%) of the patients, 49-82 (mean 66.8) years, operated on using OR technique. All of the treated patients in both groups had AAA larger than 50 mm. The study did not include patients who have been treated as urgent cases, due to the rupture or with simptomatic AAA. Clinical, biochemical and inflamatory parameters in early postoperative period were analyzed, in direct postoperative course (number of leucocytes, thrombocytes, serum circulating levels of cytokine--interleukine (IL)-2, IL-4, IL-6 and IL-10). Parameters were monitored on the zero, first, second, third and seventh postoperative days. The study was approved by the Ethics Commitee of the Military Medical Academy. RESULTS The study showed a statistically significantly shorter time of treatment in the EVAR group (average 90 min) compared to the OR group (average 136 min). Also, there was a statistically significantly less blood loss in the patients operated on by the use of EVAR surgery (average 60 mL) as compared to the patients treated with OR techinique (average 495 mL), as well as a shorter postoperative hospitalization of patients in the EVAR group (average 4 days) compared to the OR group (average 8 days). The OR group was detected with a statistically significant increase of leucocytes and statistically significant fall of the number of thrombocytes in comparison with the EVAR group in all the investigated terms. A significant concentration rise of IL-2 in the OR group and concentration rise of IL-6 in the EVAR group was shown 24 hours after the procedure, whereas on the second postoperative day there was detected a significant fall of IL-6 in the EVAR group. IL-4 concentration in the OR group was significantly higher as of the third postoperative day in comparison to the EVAR group. There was no significant difference in IL-10 concentration between the groups. CONCLUSION The EVAR techinique is a safer and less invasive and less traumatic procedure for patients than the OR of AAA. Following the EVAR, there are less inflammatory reactions in the early postoperative period as compared to the OR and therefore less possibility of the development of systemic inflammatory respons syndrome in patients treated.

In vitro antioxidant activity of thiazolidinone derivatives of 1,3-thiazole and 1,3,4-thiadiazole

The initial steps in preclinical drug developing research concern the synthesis of new compounds for specific therapeutic use which needs to be confirmed by in vitro and then in vivo testing. Nine thiazolidinone derivatives (numerically labeled 1-9) classified as follows: 1,3-thiazole-based compounds (1 and 2); 1,3,4-thiadiazole based compounds (3 and 4); substituted 5-benzylideno-2-adamantylthiazol[3,2-b][1,2,4]triazol-6(5H)ones (5-8); and an ethylaminothiazole-based chalcone (9), were tested for antioxidant activity (AOA) by using three in vitro assays: DPPH (1,1-diphenyl-2-picrylhydrazyl scavenging capacity test); FRAP (ferric reducing antioxidant power test); and TBARS (thiobarbituric acid reactive substances test). Compounds 1-4 and 9 in particular are newly synthesized compounds. Also, traditional antioxidants Vitamins E and C and α-lipoic acid (α-LA) were tested. The results of DPPH testing: Vitamin C 94.35%, Vitamin E 2.99% and α-LA 1.57%; compounds: 4 33.98%; 2 18.73%; 1 15.62%; 5 6.59%; 3 4.99%; 6-9 demonstrated almost no AOA. The results of TBARS testing (% of LPO inhibition): Vitamin C 62.32%; Vitamin E 36.29%; α-LA 51.36%; compounds: 1 62.11%; 5 66.71%; 9 60.93%; 4, 6 and 7 demonstrated ∼50%; 3 and 8 displayed ∼38%; 2 23.51%. By FRAP method, Vitamins E and C showed equal AOA, ∼100%, unlike α-LA (no AOA), and AOA of the tested compounds (expressed as a fraction of the AOA of Vitamin C) were: 2 and 4-75%; 8, 3 and 1-45%; 5-7 and 9-27%. Different red-ox reaction principles between these assays dictate different AOA outcomes for a single compound. Vitamin C appeared to be the superior antioxidant out of the traditional antioxidants; and compound 4 was superior to other tested thiazolidinone derivatives. Vitamin C appeared to be the superior antioxidant out of the traditional antioxidants; and compound 4 was superior to other tested thiazolidinone derivatives. Phenyl-functionalized benzylidene, amino-carbonyl functional domains and chelating ligand properties of the thiazolidinone derivatives correlated with AOA.

Disulfiram moderately restores impaired hepatic redox status of rats subchronically exposed to cadmium

Abstract Examination of cadmium (Cd) toxicity and disulfiram (DSF) effect on liver was focused on oxidative stress (OS), bioelements status, morphological and functional changes. Male Wistar rats were intraperitoneally treated with 1 mg CdCl2/kg BW/day; orally with 178.5 mg DSF/kg BW/day for 1, 3, 10 and 21 days; and co-exposed from 22nd to 42nd day. The co-exposure nearly restored previously suppressed total superoxide dismutase (SOD), catalase (CAT) and increased glutathione peroxidase (GPx) activities; increased previously reduced glutathione reductase (GR) and total glutathione-S-transferase (GST) activities; reduced previously increased superoxide anion radical (O2·−) and malondialdehyde (MDA) levels; increased zinc (Zn) and iron (Fe), and decreased copper (Cu) (yet above control value), while magnesium (Mg) was not affected; and decreased serum alanine aminotransferases (ALT) levels. Histopathological examination showed signs of inflammation process as previously demonstrated by exposure to Cd. Overall, we ascertained partial liver redox status improvement, compared with the formerly Cd-induced impact. Graphical Abstract

The simple isocratic HPLC—UV method for the simultaneous determination of reduced and oxidized glutathione in animal tissue

The aim of our work was to optimize and apply simple high-performance liquid chromatography method with ultraviolet detection (HPLC—UV) for simultaneous determination of reduced (GSH) and oxidized (GSSG) glutathione in biological matrix (specifically, the rat liver tissue was used herein), since the ratio between oxidized and reduced glutathione forms (GSSG—GSH) has been recognized as an important biological marker of oxidatively depleted GSH in oxidative stress (OS)-associated diseases and poisonings. An isocratic chromatographic separation of GSH and GSSG (2.8 min and 6.3 min, respectively) was performed with the mobile phase consisted of sodium perchlorate solution (pH adjusted to 2.8) at flow rate of 1 mL min−1, detection set at 215 nm, and column temperature of 40 °C. The method offers short run time, linearity in the range of 0.01—200 μM concentration for both compounds (R2 = 1), low limits of detection and quantification (GSH: 0.18 μM and 0.56 μM, GSSG: 0.52 μM and 1.58 μM, respectively), precision...

CLINICAL CASE REPORT OF A LARGE ANTROCHOANAL POLYP

Antrochoanal polyps are benign lesions originating from the mucosa of the maxillary sinus. Nasal obstruction and rhinorrhea are their main symptoms. Their endoscopical and radiological appearance makes them relatively easy to diagnose. These polyps are usually presented unilaterally, although bilateral presentation is also possible. We described two cases of atypically giant antrochoanal polyps: in a 15-year-old child and in a 38-year-old man. In both cases, the diagnosis was done by nasal endoscopy and computed tomography (CT) of the paranasal sinuses and supported by histopathological analysis. In the first patient, the excised polyp had the histological characteristic of an angiomatous antrochoanal polyp. Because of their unusual dimension, the combined transoral and endonasal endoscopic approach was performed for complete polyp excision. We discussed the clinical, histopathological and immunohistochemical characteristics of choanal polyps in comparison to inflammatory nasal polyps, and the applicable surgical techniques for treatment of these polyps.

High Interleukin 27 Production is Associated with Early Clinical Stage and Localized Disease in Patients with Melanoma

Summary Background: The immune response in patients with melanoma is an important focus of research due to the tumor’s resistance and immunotherapy possibilities. IL-27 is one of the cytokines with antitumor properties. The role of IL-27 in the pathogenesis of melanoma is still unclear. The aim of this study was to examine the association between serum IL-27 levels and the clinical parameters of melanoma patients. Methods: The IL-27 concentration was determined by commercial ELISA in serum samples from melanoma patients (n=72) and healthy control subjects (n=44). Patients were classified according to AJCC clinical stage, TNM stage, the length of progression-free interval (PFI) and the extent of the disease (localized or widespread). Results: Average IL-27 values were increased in patients with early stages of melanoma compared to patients with terminal stages and control values. The highest IL-27 concentration was found in stage IIa. Patients in stages III and IV had significantly lower values of IL-27 compared to control. Patients with localized melanoma and shorter PFI had insignificantly increased IL-27 levels compared to patients with widespread disease and longer PFI. Patients with metastatic disease and stage TNM4 had significantly lower average IL-27 values compared to control. Patients with high production of IL-27 (>1000 pg/mL) were most numerous in IIa AJCC stage, with initial tumor size TNM2 and in the group of patients with localized disease. Conclusions: High levels of IL-27 in patients with melanoma are associated with the initial stages and localized disease.

Neutrophil-to-Lymphocyte Ratio, Monocyte-to-Lymphocyte Ratio, Platelet-to-Lymphocyte Ratio, and Mean Platelet Volume-to-Platelet Count Ratio as Biomarkers in Critically Ill and Injured Patients: Which Ratio to Choose to Predict Outcome and Nature of Bacteremia?

Background Neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), platelet-to-lymphocyte ratio (PLR), and mean platelet volume-to-platelet count (MPV/PC) ratio are readily available parameters that might have discriminative power regarding outcome. The aim of our study was to assess prognostic value of these biomarkers regarding outcome in critically ill patients with secondary sepsis and/or trauma. Methods A total of 392 critically ill and injured patients, admitted to surgical ICU, were enrolled in a prospective observational study. Leukocyte and platelet counts were recorded upon fulfilling Sepsis-3 criteria and for traumatized Injury Severity Score > 25 points. Patients were divided into four subgroups: peritonitis, pancreatitis, trauma with sepsis, and trauma without sepsis. Results NLR and MPV/PC levels were significantly higher in nonsurvivors (AUC/ROC of 0.681 and 0.592, resp., in the peritonitis subgroup; 0.717 and 0.753, resp., in the pancreatitis subgroup); MLR and PLR did not differ significantly. There was no significant difference of investigated biomarkers between survivors and nonsurvivors in trauma patients with and without sepsis except for PLR in the trauma without sepsis subgroup (significantly higher in nonsurvivors, AUC/ROC of 0.719). Independent predictor of lethal outcome was NLR in the whole cohort and in the peritonitis subgroup as well as MPV in the pancreatitis subgroup. Also, there were statistically significant differences in MPV/PC, MLR, and PLR values regarding nature of bacteremia. In general, the lowest levels had been found in patients with Gram-positive blood cultures. Conclusions NLR and MPV were very good independent predictors of lethal outcome. For the first time, we demonstrate that nature of bacteremia influences MPV/PC, MLR, and PLR. In heterogeneous cohort subgroup, analysis is essential.

Association Between Oxidative Stress and Melanoma Progression

Summary Background: Overproduction of free radicals accompanied with their insufficient removal/neutralization by antioxidative defense system impairs redox hemostasis in living organisms. Oxidative stress has been shown to be involved in all the stages of carcinogenesis and malignant melanocyte transformation. The aim of this study was to examine association between oxidative stress development and different stages of melanoma. Methods: The measured oxidative stress parameters included: superoxide anion radical, total and manganese superoxide dismutase, catalase and malondialdehyde. Oxidative stress parameters were measured spectrophotometrically in serum samples from melanoma patients (n=72) and healthy control subjects (n=30). Patients were classified according to AJCC clinical stage. Results: Average superoxide anion and malondialdehyde concentrations were significantly higher in melanoma patients than in control group, with the highest value of superoxide anion in stage III, while malondialdehyde highest value was in stage IV. The activity of total and manganese superoxide dismutase was insignificantly higher in melanoma patients than in control group, while catalase activity was significantly higher. The highest activity of total activity of manganese superoxide dismutase was in stage IV. Catalase activity was increasing with the disease progression achieving the maximum in stage III. Conclusion: Results of our study suggest that melanoma is oxidative stress associated disease, as well as deteriorated cell functioning at mitochondrial level.