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Background/Aim. Colorectal cancer (CRC) is one of the most common cancers in the population, often leading to lethal outcomes. Myeloid-derived suppressor cells (MDSCs) belong to a heterogeneous group of immature cells thought to have an immunosuppressive effect that may aid in tumor development and spreading. The aim of this study was to analyze the frequency and significance of MDSC-like cells at different stages in patients with CRC. Methods. Peripheral blood (PB) samples of 83 patients at different stages of the disease and 12 healthy subjects (control group) were analyzed. MDSC-like cells were identified and enumerated in the PB samples of the participants based on the immuno-phenotypic characteristics of the cells. Results. A statistically significant increase in the absolute and relative number of polymorphonuclear (PMN) MDSC (PMN-MDSC)-like cells was observed in the PB of all the patients with CRC, com-pared to the healthy control group (p < 0.0001). No significant increase was observed in monocytic MDSC (M-MDSC)-like cells when they were analyzed without CRC stage stratification (p > 0.05). When the relative and absolute numbers of PMN-MDSC-like cells were analyzed in relation to the stages of CRC disease (TNM classification), a statistically significant difference was observed between the control group and patients in stages III and IV of the dis-ease (p = 0.0005 vs. p = 0.0003 and p < 0.0001 vs. p < 0.0001, respectively). There was, as well, a significant difference when the numbers of PMN-MDSC-like cells in patients in stages I and II were compared to numbers in patients in stage IV of the CRC (p = 0.0161 vs. p < 0.0001 and p = 0.0065 vs. p < 0.0001, respectively). A statistically significant difference in the relative and absolute number of M-MDSC-like cells was observed only between patients in stages II and IV of the disease (p = 0.0014 and p = 0.0002, respectively). The highest number of MDSC-like cells was observed in stage IV of the disease according to the TNM classification. A positive correlation between the presence of these cells and the number of organs affected by metastatic changes was observed (p < 0.0001 for the relative and absolute number of PMN-MDSC-like cells and p = 0.003 and p = 0.0004 for the relative and absolute number of M-MDSC-like cells). Conclusion. CRC patients had a statistically significant increase in PMN-MDSC-like cells compared to healthy controls. The increase in absolute and relative numbers of these cells mostly follows the growth and progression of CRC, while a statistically significant difference in the number of M-MDSC-like cells is observed only between stages II and IV of the disease. The absolute and relative numbers of both subtypes of MDSC-like cells significantly correlate with the number of organs affected by CRC metastases.

Background/Aim. The injuries of the facial nerve lead to paralysis of the mimic musculature, which is conditioned by functional disorders accompanied by deformity of varying degrees depending on the intensity and location of the injury. Surgical treatment is a method of choice to treat an injured nerve. Injuries in the parotid lodge area are repaired by direct neurosuture in combination with platelet-rich plasma (PRP). Methods. The experimental study was carried out on 48 chinchilla male rabbits (Oryctolagus cuniculus), of about the same weight (2,500–3,000 gr), aged between 3 and 4 months in two surgical stages, in two different periods – six and ten weeks after the first surgical procedure. The animals were divided into four groups: Group I (suture); Group II [suture and fibrin glue (FG)]; Group III (suture and PRP); Group IV (sutures, FG, and PRP). Each group had two subgroups based on the duration of the experiment (six and ten weeks). A part of the dissected nerve in the length of 5 mm was subjected to histologic verification, where the number of axons and Schwann cells was determined and expressed numerically based on the histological sample of the tissue of the observed nerve. The extent of the presence of connective tissue and the degree of neo-vascularisation is shown by the description of histological samples by grades (connective tissue 1-4, neovascularisation 1-3). Results. Our results showed that all parameters of re-generation of damaged nerve showed a significantly higher regeneration efficiency after six and ten weeks of intervention in groups treated with PRP therapy with or without using FG. Conclusion. The use of PRP and the stimulating effect of activated growth factors results in the regeneration of the facial nerve in the sense of replication of the Schwann cells and the number of axons, with a high degree of neovascularization and minimal proliferation of connective tissue, which histologically corresponds to a healthy nerve.

Background/Aim. Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) 2019 infection represents a global problem. At this moment, in October 2020, there is no vaccine or efficient treatment for infected patients. Treatment with blood plasma rich with anti-SARS-CoV-2 specific antibodies might be a safe, and effective therapy for COVID-19 patients. Methods. A total of 768 patients were analyzed in this study, whose samples were collected in a time interval from May 1, 2020, till August 15, 2020. Patients were enrolled in the study from COVID-19 hospitals and out-clinics. In-house ELISA tests were developed to measure the concentration of anti-S1S2 spike and anti-nucleoprotein (np) (IgG, IgA, IgM) SARS-CoV-2 antibodies. Blood convalescent plasma was selectively collected from recovered patients according to specific antibodies concentration. Results. The highest concentrations of anti-S1S2 spike or anti-np specific IgG antibodies were detected in patients with the moderate/heavy clinical form of the infection. An extremely high concentration of anti-S1S2 spike IgG and anti-np IgG was demonstrated in 3% and 6% of patients who recovered from severe COVID-19, respectively. Of tested hospitalized patients, 63% and 51% had modest levels of anti-S1S2 spike and anti-np, respectively. After 60 days, in our selected donors, concentrations of anti- S1S2 spike IgG and anti-np IgG antibodies increased in 67% and 58% of donors, respectively. Conclusion. In-house developed ELISA tests enable a novel protocol for selecting convalescent blood plasma donors recovered from SARS-CoV-2 infection.

Background/Aim. In the spirit of personalized medicine, determining caries biomarkers in the saliva and gingival crevicular fluid (GCF) attracts great attention in the current dental re-search. The concentration of GCF cytokines is illustrative in depicting the processes in tooth structures. Their relevance must be inspected with aspects of tooth position and caries lesion level. Different impacts of dental restoration materials on GCF IL-1β and TNF-α could be used as a parameter for estimating local inflammation. This paper aimed to estimate the concentrations of the proinflammatory cytokines (IL-1β and TNF-α) in the GCF and to correlate them with caries extension, tooth position, and different restorative biomaterials. Methods. GCF samples were collected from 90 periodontally healthy patients demonstrating at least one tooth with proximal caries and one intact tooth, at the baseline, 7 and 30-days post-treatment. The biomarkers’ profile was investigated in relation to different levels of caries extension (superficial, pulpitis, gangrenous, root affection), defect size, and restorative biomaterial. Results. Before therapy, caries level was significantly associated with GCF IL-1β concentration, demonstrating the lowest level in gangrenous (C4) and superficial caries (C2). Thirty days after therapy, root affection (C5) was characterized by the highest IL-1β concentration. Different dental fillings showed various GCF cytokine changes. CPC induced a significant IL-1β increase in more than 70% of treated patients. Caries lesion size was insignificantly associated with GCF levels of these proinflammatory cytokines, where larger defects were followed by an average cytokine increase. Considering the tooth position be-fore therapy, IL-1β had the highest level in GCF samples from caries-affected canines and second molars, while TNF-α showed the highest levels from canines GCF. Dental restoration induced cytokine increase in canines (IL-1β and TNF-α), 1st and 2nd molars GCF (IL-1β). Conclusion. Inflammation intensity of tooth structures was directly reflected in IL-1β and TNF-α concentrations. Dental restoration significantly affects IL-1β and TNF-α levels, depending on the used dental filling-type material. The profile of these cytokines varied in GCF samples of the tooth with different anatomical positions, where canines and molars demonstrated the highest level. An increase of these proinflammatory cytokines in the absence of any symptomatic manifestation of the inflammatory response can be considered as a possible tooth reparation parameter.

Background/Aim. Recent studies indicate that adipokines have an important role in bone physiology and pathology. Recent data indicate that adipokine leptin functions as a regulator of bone growth at multiple levels, systemically and locally. So far, it has been shown that leptin influences bone volume and bone mineral density in a population with metabolic and/or hormonal abnormality. Data concerning leptin values in non-obese children with fractures are scarce. Methods. This study included 93 non-obese children with long bone fractures (LBF), 14 children with short bone fractures (SBF), and 19 healthy children. Leptin concentration was determined in two blood samples (day 0 and day 21) and analyzed according to gender, fracture type, anatomical localization of the fracture, fracture topography, callus formation, and the healing outcome. Results. Children with LBF demonstrated significantly increased leptin levels compared to the control group (both day 0/day 21). In the control group, girls had significantly more leptin than boys. Leptin value was significantly influenced by anatomical localization since boys and girls with humerus fracture and girls with femur fracture had the highest average leptin concentration in the initial sample. Boys with incomplete callus formation had the highest leptin concentration (both day 0 /day 21), significantly elevated compared to boys’ samples in the control group, boys’ samples with an intermediary and well-formed callus, and also increased compared to the initial samples of girls with incomplete callus. Better callus formation in girls was associated with an increment of leptin concentrations in the second over the initial sample. Girls with partially and satisfactorily formed callus had significantly increased leptin concentration in the second sample (day 21) compared to the boys’ group. Conclusion. Leptin concentration was significantly increased (both samples) in children with LBF compared to children with SBF and corresponding controls. Leptin concentration was highly influenced by gender. High blood leptin concentrations in boys or low leptin concentrations in girls immediately upon fracture could be used to identify groups of children with incomplete callus formation.

Background/Aim. The inflammatory response is of utmost importance in bone healing, but the precise role of cells and cytokines remains unclear. In our study, we examined the as-sociation between interleukin-1β (IL-1β), tumor necrosis fac-tors alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) concentrations, fracture configuration, and callus formation. Methods. Serum cyto-kine concentrations were determined in 78 non-obese chil-dren with long bone fractures (group 1), 10 children with fin-ger fractures (group 2), and 10 healthy controls (group 3). Blood samples were taken immediately after fracture upon hospital admission for groups 1 and 2. Differences in cyto-kine concentrations were analyzed among groups and catego-rized according to fracture configuration and callus for-mation. Results. IL-1β and TNF-α levels were lower in pa-tients that went on to produce incomplete callus compared with patients that formed complete callus. Surprisingly, the average IL-1β concentration was highest in the healthy con-trol group. The only significant correlation between IL-1β and TNF-α was in the group with moderate callus formation. MCP-1 level was slightly increased in all patient groups com-pared to controls, with no mutual difference. An average IL-8 level showed a clear decrease tendency from the group with incompletely formed callus toward the group with completely formed callus compared to controls, without significant dif-ference. Children with epiphysiolysis had the lowest concen-trations of cytokines compared with all other fracture types including transverse, oblique, and spiral. There were signifi-cantly lower concentrations of IL-1β and MCP-1 in patients with less fragment displacement compared with patients with greater fragment displacement. Conclusion. The systemic in-flammatory response is important in physiological bone heal-ing. High early production of IL-1β, TNF-α, and MCP-1 is associated with greater callus formation and better healing outcome, while increased IL-8 level is associated with poor callus formation and worse healing outcome. Our results in-dicate that epiphysiolysis and larger fragment displacement are associated with delayed fracture healing.

Background/Aim. Myeloid-derived suppressor cells (MDSCs) suppress immune responses via a series of inhibitory mechanisms, which ultimately could lead to tumor growth. B7-H4 expression is significantly associated with poor outcome and promotion of tumor cell proliferation, invasion and migration in patients with various cancers. Data concerning B7-H4 expression in lung cancers (LC), either on tumor or immunological cells, are still sporadic. The aim was to estimate and correlate the number of CD14+B7-H4+MDSCs in blood and lung tumor microcirculation with clinical stage, histology type of tumor, tumor node metastasis (TNM) stadium, nodal status and disease outspread. Methods. The study included 44 lung cancer patients (III and IV clinical stage) and 30 healthy controls. CD14+B7-H4+ MDSC number was estimated by flow cytometry in blood and tumor microcirculation samples of each patient. Results. CD14+B7-H4+ MDSCs number was significantly higher in patients’ samples compared to controls. CD14+B7-H4+ MDSC number was significantly increased in tumor compared to blood sample of the same patient. Clinical stage III patients had the increased number of the CD14+B7-H4+ MDSC compared to stage IV, in both types of samples. According to histology, small cell lung cancer (SCLC) patients had the highest average CD14+B7-H4+ MDSCs number, significantly increased compared to patients with squamous and large cell LC histology type. Tumor size was directly associated with the number of the CD14+B7-H4+ MDSC, both in blood and tumor samples. Furthermore, nodal involvement was associated with the gradual increase of the CD14+B7-H4+ MDSC number, being the highest in the N3 group, again both in blood and tumor samples. Finally, we detected higher CD14+B7-H4+ MDSCs number in the samples of patients without metastases. Conclusion. CD14+B7-H4+ MDSCs n umber in L C patients is significantly associated with tumor histology type, lymph node involvement, disease extent degree and tumor size. Concerning their large number in LC tumor microenvironment together with immunosuppressive capacities, CD14+B7-H4+ MDSCs could represent important tumor promoting factor in LC pathophysiology.

Background/Aim. Recent data imply the significance of certain cytokines in the appearance and development of prostate cancer (PC), as well as their association with pathohistological and/or clinical characteristics of PC. The aim of this study was to examine the relationship between the IL-10 concentration with histopathological and clinical characteristics of PC patients. Methods. IL-10 concentration was determined in serum, urine and prostate massage secret (pms) samples of 88 CP patients (initially and after therapy), 20 benign prostatic hyperplasia (BPH) patients and 15 healthy controls. Results. Compared to BPH and control groups, PC patients had the highest average serum IL-10 concentration. Interestingly, BPH patients demonstrated the highest concentration of IL-6 in urine and pms samples. Also, patients with G3 gradus and with the highest Gleason score (4 + 4) demonstrated the highest IL-10 serum level. PC patients without any histopathological sign of tumor invasion had a significantly increased serum IL-10, either before or after the therapy, compared to the patient group with evident invasion of tumor cells. The therapy induced different IL-10 profile in serum and urine samples. After the therapy, there was a clear significant IL-10 increase in serum of patients with unfavorable Gleason score (4 + 4), with present infiltration of tumor cells in peritumoral tissue (lymphatic, vascular and combined) and in patients with high tumor volume. Conclusion. PC patients without any histopathological signs of tumor invasion before the therapy have significantly increased serum IL-10 concentration compared to those with the signs of tumor invasion. There is a clear dissociation of IL-10 values between a serum sample and local, urine and pms samples from a particular patient. After the therapy, high IL-10 serum concentration is present only in patients with high Gleason score, pre-sent infiltration of peritumoral tissue and high tumor volume.

Vitamin D receptor (VDR) gene polymorphisms could play a significant role in the susceptibility and pathogenesis of osteoarthritis (OA), the most common degenerative joint disorder in humans. The current study involved 94 OA patients and 100 healthy, asymptomatic controls. VDR variants FokI (rs2228570), TaqI (rs731236), ApaI (rs7975232) and EcoRV (rs4516035) were genotyped using TaqMan-based real-time PCR. Adjusted odds ratio (OR) analysis showed that VDR TaqI and FokI polymorphisms are significantly associated with susceptibility to OA (OR=1.986, P=0.001 and OR=1.561, P=0.017, respectively). Joint-specific analysis showed that the VDR TaqI polymorphism was associated with risk of hip OA (OR=1.930, P=0.005) and knee OA (OR=1.916, P=0.028), while the VDR FokI polymorphism was associated with higher risk of knee OA (OR=2.117, P=0.012). VDR TaqI and FokI polymorphisms are associated with the occurrence of persistent pain (P=0.005 and P=0.027, respectively), while ApaI was associated with a family history of OA (p=0.004). The VDR FokI and TaqI genetic variants significantly contribute to osteoarthritis susceptibility, the occurrence of persistent pain, and potentially to joint-specific OA risk.

Background/Aim. Exposure of living organisms to γ-radiation results in the overproduction of free radicals. The aim of the study was to test if the subacute administration of micronized zeolite (MZC) accomplishes radioprotective role based on the evaluation of the status of oxidative stress (OS) in the brain and 8-hydroxyguanosine (8-OH-dG) in the plasma of the rats exposed to the single γ-ray irradiation of 2 and/or 10 Gray (Gy). Methods. Wistar rats were on a four-week normal or 5% MZC supplemented diet and afterward exposed to the single γ-ray irradiation of 2 and 10 Gy. Groups of rats were: a) on a normal diet (the control group, and 2Gy and 10Gy groups); b) on 5% MZC supplemented diet (the control group – MZC, MZC + 2Gy, and MZC + 10Gy groups). We measured malondialdehyde (MDA), glutathione (GSH) total, and activity of total super-oxide dismutase (tSOD) and manganese superoxide dismutase (MnSOD) in vulnerable brain regions (cerebellum, hippocampus, and cerebral cortex) and 8-OH-dG in plasma. Results. Lower MDA was found in the MZC+2Gy and MZC+10Gy groups compared to the 2Gy and 10Gy groups. Activity od total SOD was higher in the MZC+10Gy group than in the 10Gy one. GSH was the highest in the 10Gy group. Compared to the control group, 8-OH-dG was extremely higher in groups radiated with 10 Gy regardless of a diet, but slightly lower in the MZC+2Gy and 2Gy groups. Conclusion. Subacute MZC pretreatment accomplished partial radioprotective effect in irradiated rats compared to non-irradiated rats, based on suppressed SOD activity at 2 Gy, and reduced brain MDA when exposed to 2 Gy and 10 Gy.

Background/Aim. Sepsis represents a significant global burden, with an estimated 48.9 million cases and 11.0 million sepsis-related deaths recently recorded worldwide. The aim of this observational study was to assess a prognostic value of some readily available routine biomarkers: presepsin, procalcitonin, C-reactive protein (CRP), white blood cell (WBC) count, platelet count, mean platelet volume (MPV), and lactate, as well as their combination regarding the outcome in a cohort of critically ill adult patients with secondary sepsis. Methods. A total of 86 critically ill patients with secondary sepsis due to peritonitis, pancreatitis, and severe trauma, admitted to the surgical intensive care unit, were enrolled in this prospective study. Blood samples for biomarker analysis were collected in three time points: on admission (the 1st day) and on the 3rd, and 5th day after admission. The Sequential Organ Failure Assessment (SOFA) score, the Simplified Acute Physiology Score (SAPS) II, and the Acute Physiology and Chronic Health Evaluation (APACHE) II score were calculated and recorded within the first 24 hours after admission (1st day). SOFA and SAPS II scores were recorded daily. The primary end-point was hospital mortality. Results. Values of each applied score were expectedly significantly higher in non-survivors in all time points. Regarding investigated parameters, only presepsin levels were significantly higher in non-survivors in all time points; MPV levels on the 3rd and 5th day; serum lactate levels on the 3rd day; CRP levels and WBC count on the 5th day. Clinical accuracy of parameters in predicting lethal outcomes was investigated in all time points. On the 1st day, apart from all three scores, only presepsin demonstrated statistically significant discriminative power regarding outcome (AUC of 0.670). Apart from SAPS II and SOFA score, on the 3rd day presepsin, MPV, and lactate (AUCs of 0.716, 0.667, and 0.642, respectively) and on the 5th day presepsin, MPV, CRP, and WBC count (AUCs of 0.790, 0.681, 0.643 and 0.654, respectively) were good predictors of the lethal outcome. Composite bioscore (presepsin, MVP, and lactate) on the 3rd day had the highest AUC of 0.820 in comparison with individual scores and parameters. The independent predictor of the lethal outcome on the 1st day was presepsin (p < 0.05) and on the 3rd day MPV (p < 0.01). Conclusion. Composite bioscore is superior to routine biomarkers and established scoring systems in predicting mortality in adult critically ill patients with secondary sepsis.

Background/Aim. Lung cancer (LC) is one of the leading causes of mortality. Disease progression and advanced disease are characterized by the unprotective immune response due to M2 macrophage polarization, myeloid-derived suppressor cells (MDSC) activity, cytokine imbalance, and regulatory T lymphocyte activity. The aim of this study was to investigate the association between Th1/Th2 cytokines and CD14+B7H4+ monocyte (Mo) number in LC patients. Methods. We investigated principal Th1/Th2 cytokines and CD14+B7H4+ Mo number in blood and tumor microcirculation samples of 41 LC patients (III and IV clinical stage) and 30 healthy participants (control group). Results. The serum concentrations of investigated cytokines in all patients vs. heathy controls did not differ significantly. Stratification in groups according to tumor histology, disease extent, and tumor size revealed significant differences. LC patients with different histology types demonstrated significant differences, both in serum and tumor microcirculation samples. The presence of metastasis was associated with increased IFN-γ/IL-4 in blood and increased IL-13 in tumor microcirculation samples. Tumor microcirculation samples of the largest tumors were characterized by the Th2 cytokine profile. Investigation of CD14+B7H4+ Mo in blood samples demonstrated a significant association of extreme value of this cell population with elevated IL-2/IL-13. Patients with the highest CD14+B7H4+ Mo number in tumor microcirculation samples demonstrated significant increments of IL-4, IL-13, IL-10, and TGF-β1. Conclusion. LC patients demonstrated polarization of cytokine response associated with microenvironment origin, tumor histology type, tumor size, and disease extent. The highest number of CD14+ B7H4+ monocytes is significantly associated with the Th2 cytokine profile.

Introduction/Objective Epidemiological studies have established cigarette smoking as one of the most significant risk factors in pathogenesis of laryngeal squamous cell carcinoma (LSCC). One of the possible underlying mechanism is chronic inflammation, but published data regarding the effect of tobacco on systemic immune response is inconsistent. The goal of this study was to evaluate concentrations of serum proinflammatory cytokines [interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α] in patients with LSCC and in healthy subjects according to cigarette smoking. Methods Fifty-nine LSCC patients and 44 healthy controls were enrolled in the study. Samples of peripheral blood and details of tobacco use were gathered from the examinees. Flow cytometry was performed to analyze serum concentrations of IL-6, IL-1β, and TNF-α. The results were compared according to active smoking status. Results Statistical analysis revealed no significant difference between smoking LSCC patients and smoking healthy subjects. Additionally, investigated cytokines were not significantly different in healthy subjects according to smoking status. In non-smoking participants with LSCC, concentrations of serum IL-1β and TNF-α were higher (p < 0.05) in comparison with smoking LSCC patients. Conclusion Findings of our study may indicate that smoking leads to the suppression of proinflammatory response in LSCC patients, whilst proinflammatory response is unaffected by cigarettes in healthy subjects.

Background/Aim. Gram-positive and Gram-negative bacteria may induce different inflammatory patterns. The aim of this study was to examine the association of the myeloid-derived suppressor cells (MDSCs) with the type of infecting microorganisms (Gram positive, Gram negative, polymicrobial) and underlying cause of secondary sepsis (peritonitis, pancreatitis, trauma). Methods. Totally, 40 critically ill patients with secondary sepsis were enrolled in the prospective study. Two patients without documented positive blood culture were excluded. We detected and enumerated both main subsets of MDSCs: granulocytic (G)-MDSCs and monocytic (M)-MDSCs on the Days 1 and 5. Blood was simultaneously drawn for a blood culture. The patients with different underlying causes of sepsis (peritonitis, pancreatitis, trauma) were perceived as separated groups and the frequencies and absolute numbers of their G-MDSCs and M-MDSCs were compared. Results. Both main MDSC subpopulations were accumulated significantly in Grampositive sepsis. Univariate logistic regression analyses of investigated variables regarding Gram-positive sepsis on the Day 5 revealed that G-MDSCs absolute number along with both MMDSCs frequency and absolute number had statistically significant power for predicting Gram-positive sepsis. Stepwise multivariate logistic regression analyses of the variables on the Day 5 determined that M-MDSCs absolute number was independent predictor of Gram-positive sepsis [odds ratio (OR) 1.012; p < 0.05]. Clinical accuracy of neutrophil (Ne)/GMDSCs (Ne/G-MDSCs) and monocyte (Mo)/M-MDSCs (Mo/M-MDSCs) ratios in predicting nature of bacteremia and outcome were investigated. Discriminative power of both Ne/G-MDSCs and Mo/M-MDSCs ratios in predicting Grampositive blood culture was statistically significant both on the Day 1 and Day 5 [areas under curve (AUCs): 0.684 and 0.692, and 0.707 and 0.793, respectively). Ne/G-MDSCs both on the Day 1 and Day 5 were statistically significant predictors of lethal outcome (AUCs: 0.694 and 0.678, respectively). There were no statistically significant differences in G-MDSCs and M-MDSCs among different three groups of patients regarding peritonitis, pancreatitis and trauma as causes of sepsis neither on the Day 1 nor on the Day 5. Conclusion. Gram-positive infectious agents were powerful inducers of MDSCs generation in sepsis. Also, underlying causes of secondary sepsis might not seem to influence the MDSCs accumulation.

Background/Aim. Role of myeloid-derived suppressor cells (MDSCs) in human host response to sepsis still needs to be clarified. The aim of our study was to determine whether frequency and/or absolute numbers of the MDSCs were associated with outcome in critically ill patients with secondary sepsis and/or septic shock. Methods. Total of 40 critically ill patients with secondary sepsis were enrolled in a prospective study. We detected and enumerated both main subsets of MDSCs: granulocytic (G)-MDSCs and monocytic (M)- MDSCs on the Day 1 (the day of hospital admission) and the Day 5 after the. The primary end-point was hospital mortality. Results. Increased frequencies and absolute numbers of subpopulations corresponding to MDSCs were associated with poor outcome. As far as relative kinetics was concerned, in both survivors and non-survivors, sepsis duration from 1th to 5th day was accompanied by an increase in MDSCs values of both investigated subpopulations. In contrast to findings of stepwise multivariate logistic regression analysis of the variables on the Day 1, on the Day 5 it was determined that the Sequential Organ Failure Assessment (SOFA) score (OR 2.350; p < 0.05) and G-MDSCs frequencies (OR 3.575; p < 0.05) were independent predictors of lethal outcome. Conclusion. These findings suggest harmful role of MDSCs in secondary sepsis.

Objective: To investigate epidermal growth factor, transforming growth factor-α and interleukin-8 production in nasal mucosa irrigated with hypertonic 2.3 per cent solution with algae extracts, in comparison to 0.9 per cent NaCl during the first two weeks after surgery for nasal polyposis, in relation to symptoms and local findings. Methods: This prospective study included 20 nasal polyposis patients postoperatively irrigated with hypertonic solution and 20 nasal polyposis patients postoperatively irrigated with isotonic solution. We evaluated nasal symptom score, endoscopic score and mediator levels in nasal secretions before and after irrigation. Results: Following treatment, nasal symptom score and endoscopic score were significantly lower in the hypertonic solution group (p = 0.023; p < 0.001, respectively). The increase in the epidermal growth factor and the decrease in the transforming growth factor-α and interleukin-8 concentration were higher in the hypertonic group (p < 0.001 for all mediators). Conclusion: Irrigation with a hypertonic solution was found to be more effective than an isotonic solution in nasal mucosa reparation. [ABSTRACT FROM AUTHOR]

Background/Aim. Colorectal cancer (CRC) represents one of the most common cancers worldwide. CRC is frequently diagnosed at advanced stages with poor prognosis, indicating the need for new diagnostic and prognostic markers. The aim of this study was to determine systemic and fecal values of galectin- 1 (gal-1) and ratios between gal-1 and proinflammatory cytokines: tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and interferon gamma (IFN-γ), in the patients with CRC and the relationship with clinicopathological aspects of the disease. Methods. The blood samples and feces liquid fraction of 58 patients with CRC were analyzed. The serum and fecal levels of TNF-α, IL-1β and IFN-γ and gal-1 were measured using sensitive enzyme-linked immunosorbent assay (ELISA) kits. Results. The fecal level of gal-1 was increased in the CRC patients with higher nuclear grade and poor tumor tissue differentiation. The gal-1/TNF-α ratio in the serum and feces had a higher trend in the patients with the advanced tumor-nodemetastasis (TNM) stage as well as the detectable lymphatic and blood vessel invasion. The gal-1/TNF-α and gal-1/IFN-γ ratios were increased in the serum of patients with presence of lung/liver metastasis or peritoneal carcinomatosis, while the enhanced gal-1/IL-1 ratio was detected only in the serum of patients with lung metastasis. A positive correlation between the gal-1 value in feces and histological differentiation of tumor and biomarkers alpha-fetoprotein (AFP) and cancer antigen- 19-9 (CA 19-9), respectively, was also observed. The fecal values of gal-1 higher than 13,708.29 pg/g presented a highly sensitive and specific marker for histological differentiation of tumor tissue. Conclusion. We believe that the predomination of gal-1 over pro-inflammatory cytokines TNF-α, IL-1β and IFN- γ in the patients with advanced and progressive CRC may implicate on an immunomodulatory role of gal-1 in the limiting ongoing proinflammatory processes. The fecal values of gal-1 can be used as a valuable marker for the severity of CRC. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 175071, Grant no. 175069 and Grant no. 175103]

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 superoxide dismutase was in stage III, while the highest activity of manganese superoxide dismutase was in stage IV Catalase activity was increasing with the disease progression achieving the maximum in stage III. Conclusions: Results of our study suggest that melanoma is oxidative stress associated disease, as well as deteriorated cell functioning at mitochondrial level.

Background/Aim. Stem cells (SCs) guarantee complete/longterm bone marrow (BM) repopulation after SC-transplants. The aim of the study was to evaluate absolute count of total SCs (determined by ISHAGE-sequential-gating protocol – SCish) and relative frequency of immature CD34+/CD90+ (CD90+SCish) subset in peripheral blood (PB) as predictive factors of mobilization and apheresis product (AP) quality. Methods. Mobilization included chemotherapy and granulocytegrowth- factor (G-CSF). Harvesting was performed by Spectra- Optia-IDL-system. The SCsish were determined as a constitutional part of CD34+ cells in the “stem-cell-region” using FC- 500 flow-cytometer. In this study, the original ISHAGEsequential- gating protocol was modified by introduction of anti-CD90-PE monoclonal-antibody into the analysis of CD90 expression on SCish (CD90+SCish). The results were presented as a percentage of SCish per nucleated-cell count, absolute SCish count in μL of the PB or the AP, percentage of the CD90+SCish expressed to SCish and absolute CD90+SCish count in μL of the PB or the AP. Results. The absolute count of total SCish and CD90+SCish was significantly higher (p = 0.0007 and p = 0.0266, respectively) in the AP than in the PB samples. The CD90+SCish/total SCish indexes from PB were higher than indexes from the AP (p = 0.039). The relative frequency of CD90+SCish showed a highly significant inverse correlation with the absolute count of total SCish in both, the PB and AP (p = 0.0003 and p = 0.0013 respectively). The relative frequency of CD90+SCish from the PB also showed a significant (p = 0.0002) inverse relationship with total SCish count in the AP. Patients with less than 10% CD90+SCish in the PB had evidently higher (p = 0.0025) total SCish count in the AP. Conclusion. We speculate that lower CD90+SCish yield in the AP is not a consequence of an inferior collection efficacy, but most likely a result of several still not fully resolved immature SC cytomorphological/ biophysical features. Therefore, following the mobilization by chemotherapy G-CSF, some logical questions appear – whether we should follow the absolute count of total SCish, or, whether we should test for relative frequency of CD90+SCish prior to harvesting. To reach the final conclusions, it is essential to conduct further controlled and larger investigations concerning the correlation of circulating and harvested SCs with patients' hematopoietic recovery. [Project of the Ministry of Defence of the RS, Project MF/VMA 9/17-19 and Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 41031]

Background/Aim. Survivin is a multifunctional protein abundantly expressed in tumors of various types, including melanoma. There are still sparse data regarding relationship of melanoma cell survivin expression with accepted histopathological characteristics as well as serum concentration. The aim of this study was to investigate the association of local tumor survivin expression (primary tumor and metastatic lesions) and serum concentration with clinical and histopathological parameters in melanoma patients. Methods. The level of survivin expression was determined immunocytochemically in tumor tissue and with ELISA test in the serum of 84 melanoma patients diagnosed from 2009 to 2013 at the Institute for Pathology and Forensic Medicine and Institute for Medical Research at Military Medical Academy, Belgrade, Serbia. Results. The intensity of survivin expression was significantly higher in the patients whose tumor had ulceration, higher mitotic index, higher Clark and Breslow stage, that made vascular invasion or spread through lymphatic vessels in primary tumor, and was significantly higher in the patients with metastatic disease. Survivin expression and the number of survivin positive cells in metastatic lesions were significantly associated with the duration of disease free interval (DFI). The patients with high expression score had almost double shorter DFI comparing to those with weak local survivin expression and a small number of survivin+cells (9 ± 7 vs 19 ± 13 months, respectively). The degree of tumor infiltrating lymphocytes presence in tumor tissue was significantly associated with serum survivin concentration, with lowest average level detected in samples of patients with the highest degree of infiltration. Serum survivin concentrations were highest in samples of melanoma patients with IA American Joint Commission on Cancer (AJCC) clinical stage, pT1a histological stage, patients whose tumors were still in horizontal growth phase, without signs of lympho-hematological disease spreading, with the highest number of mitoses and the smallest Clark index. Conclusion. Survivin expression in tumor tissue and its serum concetration significantly correlate with clinical and histopathological parameters. Serum levels could be important in initial follow-up as indicators of those patients that would have aggressive local tumor growth and spreading. Survivin determination in tumor tissue is of great significance in estimation of DFI.

Background/Aim. Kidney injury molecule-1 (KIM-1) and aquaporin-1 (AQP-1) are potential early urinary biomarkers of clear renal cell carcinoma (cRCC). The aim of this study was to ascertain relationship between the urine concentrations KIM-1 and AQP-1 with tumor size, grade, pT stage and type of operation (radical or partial nephrectomy) in patients with cRCC. Methods. Urinary concentrations of urinary KIM-1 (uKIM-1) and urinary AQP-1 (uAQP-1) were determined by commercially available ELISA kits. The analysis included 40 patients undergoing partial or radical nephrectomy for cRCC and 40 age- and sex-matched healthy adult volunteers. Results. The median preoperative concentrations of KIM-1 in the cRCC group [0.724 ± 1.120 ng/mg urinary creatinine (Ucr)] were significantly greater compared with controls (healthy volunteers) (0.210 ± 0.082 ng/mgUcr) (p = 0.0227). Postoperatively, uKIM-1 concentration decreased significantly to control values (0.177 ± 0.099 ng/mgUcr vs 0.210 ± 0.082 ng/mgUcr, respectively). The size, grade and stage of tumor were correlated positively with preoperative uKIM-1 concentrations. Contrary to these results, concentrations of uAQP-1 in the cRCC group were significantly lower (0.111 ± 0.092 ng/mgUcr) compared with the control group (0.202 ± 0.078 ng/mgUcr) (p = 0.0014). Postoperatively, the concentrations of uAQP-1 increased progressively up to control values, approximately. We find no significant correlation between preoperative uAQP-1 concentrations and tumor size, grade and stage. Conclusion. uKIM-1 was found to be a reliable diagnostic marker of cRCC, based on its significantly increased values before and decreased values after the nephrectomy. [Projekat Ministarstva nauke Republike Srbije, br. III41018]

Background: The immune response in patients with me I anoma 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.

Background/Aim. Melanoma is the most aggresive malignant tumor of the skin. Contradictory data was published on vascular endothelial growth factor (VGEF) in tumor samples and its role in skin melanoma progression and prognosis. The aim of this study was to investigate the significance of VEGF expression as a prognostic parameter in melanoma. Methods. The experimental group included 81 patients with primary skin melanomas treated from 2009 to 2013 at the Military Medical Academy, Belgrade. The control group included 20 patients with dysplastic and 20 with benign naevi. Stratification was done according to gender, age, clinical and patological stage, localization, histologic type, Clark’s, Breslow, mitotic count, regression and ulceration, tumor infiltrating lymphocytes and metastatic spread. Immunohistochemical staining was performed on skin biopsies using DAKO anti-VEGF antibodies (Ab), LSABTM +HRP, Dand microvawe antigen (Ag) retrieval in DAKO pH 9.0 solution. For statistical data analysis was done with ANOVA, Bonferroni, Mann Whitney and Wilcoxon test. Results. The mean intensity of VEGF staining was statistically significantly higher in melanomas than in benign or dysplastic naevi. Furthermore, the highest recorded values were in Ia and IV clinical stages. The majority of melanomas with high intensity of VEGF staining were in pT1a pathological stage. Melanomas with the highest mitotic count (> 6) had a significantly higher intensity of VEGF staining than those with < 2 mitoses. The higest intensity of staining was in melanomas without significant lymphocytic infiltrate and the lowest was in those with brisk lymphocytic infiltrate, thus a statistical difference was siginifant. The mean intensity of VEGF staining was highest in melanomas with lymphovascular invasion. There was no statistically significant difference between VEGF and any other parameter. Conclusion. VEGF in primary skin melanomas plays an important role in tumor progression and is linked to the absence if tumor infiltrating lymphocytes and the presence of lymphovascular invasion. More detailed studies have to be done on VEGF prognostic value in melanoma on a larger number of patients.

Background/Aim. Several cytokines and lymphokines (IL1β, ENA78, IL6, TNFα, IL8 and S100A8) are expressed during dental pulp inflammation. Analysis of gingival crevicu-lar fluid (GCF) offers a non-invasive means of studying gen-eral host response in oral cavity. Although GCF levels of various mediators could reflect the state of inflammation both in dental pulp and gingiva adjacent to a tooth, GCF samples of those without significant gingivitis could be inter-preted as reflection of pulpal process. The aim of this study was to investigate IL9 GCF values in patients with dental car-ies and to assess possible influence of various dental fillings materials on local IL9 production. Methods. The study group included 90 patients, aged 18–70, with inclusion and exclusion criteria in the prospective clinical study. Of the 6 types of material used for the restoration of prepared cavities, 3 were intended for temporary and 3 for definitive restora-tion. According to dental fillings weight, all the participants were divided into 3 groups: those with fillings lighter than 0.50 g, those with 0.50–1.00 g, and those with fillings heavier than 1.00 g. Samples were taken from gingival sulcus using the filter paper technique. Clinical parameters were deter-mined by bleeding index, plaque index (Silness-Lou, 0–3), gingival index (0–3), and gingival sulcus depth. Cytokine con-centrations were assessed using commercially available cy-tomix. Results. According to the weight of dental fillings, there was a clear decreament trend of IL9 values meaning that dental defects greater than 1.00 g of dental filling were associated with lower GCF IL9 concentration. The IL9 val-ues correlated with the degree of gingival index and depth of gingival sulcus, being higher with more advanced gingivitis and more pronounced anatomical changes in the tooth edge. Different filling materials exerted various local IL9 responses. Zink polycarbonate cement and amalgam fillings induced a significant and long-lasting local IL9 decrement, while the use of Tetric EvoCeram and GMA-BISK significantly increased IL9 levels. Conclusion. The obtained results indicate that IL9 GCF could be regarded as a measure of odontoblasts’ re-sponse to the extensity of dental caries. The type of material used for dental fillings could profoundly alter biological func-tion of gingival and pulpal cells. Also, the results obtained in this study suggest that some materials could even enhance wound repair by modulating macrophage activation.

Background/Aim. Interaction between tumor cells and host’s immunoregulatory cells in creation of microenvironment that supports tumor progression is the focus of numerous investigations in recent years. Myeloid-derived suppressor cells (MDSCs) are heterogeneous population of immature dendritic cells, macrophages and granulocytes. In cancer patients, these cells accumulate in tumor microenvironment, tumor-draining lymph nodes, peripheral blood and the liver and their numbers correlate with the stage of the disease and the metastatic disease. The aim of the study was to investigate the effect of interferon alpha on MDSCs percentage in peripheral blood of melanoma patients. Methods. The interferon treated melanoma patients were given subcutaneously interferon alpha, in optimal dose, for a period of at least 6 months before the analysis. Blood samples were collected from the melanoma patients (n = 91) and the age/sex matched healthy controls (n = 8). The following anti-human monoclonal antibodies were used for immunostaining: anti-CD15-FITC, anti-CD33-PE, anti-CD45-ECD, anti-HLA-DR PE/Cy5, anti-CD14-FITC, anti-CD16-PE and anti-CD11b-PE. Results. Comparison of myeloid-derived suppressor cells values in the stage 2 melanoma patients with and without interferon alpha therapy did not show a significant difference. When we compared the MDSCs values in the patients within stage 3 melanoma, we found a significant difference in granulocytic subset values between the interferon alpha-treated and the untreated group. Comparison of values of all suppressor cells populations between the interferon alpha-treated patients and healthy controls showed a significant increase in suppressor cells percentage in the melanoma patients. The granulocytic and total MDSCs values were significantly lower in the interferon alpha treated melanoma patients with progression in comparison with untreated patients with stable disease. Conclusion. We confirmed that interferon alpha effect in stage 3 melanoma patients was reduction in MDSCs percentage. We also found an unexpected bounce back of these suppressor cells levels, many months after the discontinuation of interferon alpha therapy.

Background/Aim. Immune response and consequent inflammatory process which originate on ocular surface after a trauma are mediated by cytokines. Photoablation of corneal stroma performed by excimer laser causes surgically induced trauma. Interleukin-6 (IL-6) is mostly known as a proinflammatory cytokine. However, it also has regenerative and anti-inflammatory effects. It is supposed that this cytokine is likely to play a significant role in the process of corneal wound healing response after photoablation of stroma carried out by laser in situ keratomileusis (LASIK) or photorefractive keratectomy (PRK) methods. The aim of this study was to determine and compare the levels of IL-6 in tears before and after treatment with LASIK and PRK methods. Methods. The study included 68 shortsighted eyes up to -3.0 diopter sphere, i.e. 198 samples of tears (per three samples taken from each of the eyes), divided into two groups according to the kind of excimer laser intervention performed: the group 1 - eyes treated by LASIK method (n = 31), and the group 2 - eyes treated by the PRK method (n = 37). The samples of tears were taken from each eye at the following time points: before excimer laser treatment (0 h, the control group), 1 h after the treatment (1 h) and 24 h after the treatment (24 h). The patients did not use anti-inflammatory therapy 24 h after the intervention. Tear samples were collected using microsurgical sponge. Level of IL-6 in tear fluid was determined by the flow cytometry method, applying a commercial test kit which allowed cytokine detection from a small sample volume. Results. The values of IL-6 were detectable in 16% of samples before LASIK treatment and in 30% of samples before PRK treatment. One h after the treatment IL-6 was detectable in 29% of samples for the LASIK group and 43% of samples for the PRK group, and 24 h after the treatment it was detectable in 19% of samples for the LASIK group and in 57% of samples for the PRK group. When we analyzed the dynamics of IL-6 production in particular groups, we noticed that both in the LASIK and PRK group the number of samples with increased values of IL-6 after 1 h, and after 24 h, was considerably larger than the number of samples with decreased values of IL-6 after the intervention. Analyzing the dynamics of IL-6 concentration changes in the 1 h samples vs 24 h samples there was a statistically significant increase in the number of samples with IL-6 concentration decline in the LASIK group, while at the same time no considerable changes occurred in the PRK group. Comparing average IL-6 values between the two treatment groups in all tear samples at 0 h, 1 h and 24 h after intervention a significantly higher level in the PRK group 24 h after procedure (p = 0.0031) was detected. Conclusion. IL-6 level in tears increases 1 h and 24 h after LASIK and PRK treatments. This increment is significantly larger 24 h after the treatment with the PRK method than with the LASIK method. Changes of IL-6 production levels in tears after excimer laser treatment indicate that this cytokine takes part in the corneal recovery process after stromal photoablation.

Nasal polyposis is a chronic inflammatory disease of the nasal and paranasal sinuses mucosa, characterized by prolaps of edematous mucosa, most commonly from the area of anterior ethmoid. The mean histological characteristics are proliferation of pseudostratified respiratory epithelium, thickening of the basement membrane, focal fibrosis and eosinophilic and lymphocytic infiltration of the lamina propria. Although etiology is unknown, two hypotheses are dominant among the scientists: ”hypothesis of staphylococcal superantigens“ and ”hypothesis of immune barrier dysfunction“. Although we have not yet achieved a full understanding of the precise mechanisms underlying the pathogenesis of this disease, it is known that nasal polyposis is associated with intensive chronic inflammation, followed by dysregulation of chemotaxis, migration, activation and function of eosinophils. A great number of cytokines, chemokines, and adhesion molecules are involved in the regulation of these complex mechanisms. After activation, eosinophils produce and release enzymes, which can lead to the damage of mucosa and tissue remodeling. Hyperactive eosinophils release a new amount of chemokines and cytokines, attracting new eosinophils into the site of inflammation, and may cause the persistence of chronic inflammation.

Bacgraund/Aim. The use of new therapeutic methods to prevent development of fibrothorax as the final complication of the human pleural infections requires research with experimental animals. The aim of this study was to standardize the procedures for the establishment of our own experimental model of empyema in rabbits, since it should be able to offer similar conditions found in human pleural infections. Methods. This experiment included 15 chinchilla rabbits, weighing from 2.3 to 2.8 kg. There were 12 rabbits in the experimental group, while 3 rabbits formed the control group. On the first day, we administered 0.4-0.5 mL of turpentine in the right pleural space of the rabbits from the experimental group in order to provoke sterile exudative pleurisy. After 24 h we injected 1 mL of Staphylococcus aureus and 1 mL of Escherichia coli bacteria in the same concentration of 4.5 x 108 bacteria/mL. Thoracocentesis for the pleural fluid analysis was performed 24, 48, 72, and 96 h after bacteria instillation. In these pleural samples we estimated the number of leucocytes and the values of lactate dehydrogenase (LDH), glucose and pH in pleural fluid, as well as the presence of bacteria. We did not protect the animals with antibiotics, and on the day 7 of the experiment they were sacrificed with the lethal dose of barbiturate (iv). The lung from the empyemic side of all experimental animals and the lung of one control animal were histopathologically examined. Results. A total of 4 animals had a small amount of clear pleural fluids or there was no fluid obtained with thoracocentesis 24 and 48 h after the bacteria instillation. after the bacteria instillation. In the remaining 8 rabbits 24 h after bacteria administration the mean values (± SD) of the parameters monitored were as follows: Le 34.75 ± 6.13 x 109/L, LDH 17,000 ± 4,69 U/L, glucose 1.23 ± 0.45 mmol/L, and pH 6.975 ± 0.15. The obtained values met the criteria for the evaluation of effusion as pleural empyema or complex and complicated pleural effusion (LDH > 1000 U/L, glucose < 2.31 mmol/L and pH < 7.20). Bacterial cultures were positive in 5 out of 8 first pleural samples and in only 2 samples after 48 h of bacteria administration. There was a positive correlation between the number of leukocytes and the LDH value (r = 0.071, p < 0.001), and a negative correlation between the number of leukocytes and the glucose level (r = 0.864, p < 0.001), and the leukocytes number and pH of the pleural fluid (r = 0.894, p < 0.001). The mean glucose value increased after 48 h (3.23 ± 0.44 mmol/L), and the pH value rose after 72 h (7.22 ± 0.03) which was beyond the empyema level. Conclusion. The creation of the experimental empyema model is a very delicate work with uncertain success. Its value and importance are crucial for pleural pathology research. With the intention to obtain a more empyemic pleural reaction we created a model with two different human pathogen bacteria. We generated the satisfactory results, but not as good as those contained in some of the reference literature data.

Background/Aim. Peri-implantitis presents inflammatory process that affects soft and hard supporting tissues of osseointegrated implant based on inflammatory osteoclastogenesis. The aim of this study was to investigate whether receptor activator of nuclear factor kappa B (RANK) concentrations in peri-implant crevicular fluid could be associated with clinical parameters that reflect inflammatory nature of peri-implantitis. Methods. The study included 67 patients, 22 with diagnosed peri-implantitis, 22 persons with healthy peri-implant tissues and 23 patients with periodontitis. Clinical parameters from each patient were recorded and samples of peri-implant/gingival crevicular fluid were collected for the enzyme-linked immunosorbent assay (ELISA) analysis. Results. RANK concentration was significantly increased in samples from the patients with periimplantitis when compared to healthy implants (p < 0.0001), where the average levels were 9 times higher. At the same time RANK concentration was significantly higher in periimplantitis than in periodontitis sites (p < 0.0001). In implant patients pocket depths and bleeding on probing values were positively associated with high RANK concentrations (p < 0.0001). Conclusion. These results revealed association of increased RANK concentration in samples of periimplant/ gingival crevicular fluid with peri-implant inflammation and suggests that RANK could be a pathologic determinant of peri-implantitis, thereby a potential parameter in assessment of peri-implant tissue inflammation and a potential target in designing treatment strategies.

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.

Background/Aim. The function of dental implants depends on their stability in bone tissue over extended period of time, i.e. on osseointegration. The process through which osseointegration is achieved depends on several factors, surgical insertion method being one of them. The aim of this study was to histopathologically compare the impact of the surgical method of implant insertion on the peri-implant bone tissue. Methods. The experiment was performed on 9 dogs. Eight weeks following the extraction of lower premolars implants were inserted using the one-stage method on the right mandibular side and two-stage method on the left side. Three months after implantation the animals were sacrificed. Three distinct regions of bone tissue were histopathologically analyzed, the results were scored and compared. Results. In the specimens of one-stage implants increased amount of collagen fibers was found in 5 specimens where tissue necrosis was also observed. Only moderate osteoblastic activity was found in 3 sections. The analysis of bone-to-implant contact region revealed statistically significantly better results regarding the amount of collagen tissue fibers for the implants inserted in the two-stage method (Wa = 59 < 66,5, α = 0.05), but necrosis was found in all specimens, and no osteoblastic activity. Histopathological analysis of bone-implant interface of one-stage implants revealed increased amount of collagen fibers in all specimens, moderate osteoblastic activity and neovascularization in 2 specimens. No inflammation was observed. The analysis of two-stage implants revealed a marked increase of collagen fibers in 5 specimens, inflammation and bone necrosis were found in only one specimen. There were no statistically significant differences between the two methods regarding bone-implant interface region. Histopathological analysis of bone tissue adjacent to the one-stage implant revealed moderate increase of collagen tissue in only 1 specimen, moderate increase of osteoblasts and osteocytes in 3 specimens. No necrotic tissue was found. The analyzed specimens of bone adjacent to two-stage implants revealed a moderate increase in the number of osteocytes in 3 and a marked increase in 6 specimens respectively. This difference was statistically significant (Wb = 106.5 > 105, α = 0.05). No necrosis and osteoblastic activity were observed. Conclusion. Better results were achieved by the two-stage method in bone-to-implant contact region regarding the amount of collagen tissue, while the results were identical regarding the osteoblastic activity and bone tissue necrosis. There was no difference between the methods in the bone-implant interface region. In the bone tissue adjacent to the implant the results were identical regarding the amount of collagen tissue, osteoblastic reaction and bone tissue necrosis, while better results were achieved by the two-stage method regarding the number of osteocytes.

nema

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 between 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 larager 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 periodas compared to the OR and therefore less possibility of the development of systemic inflammatory respons syndrome in patients treated.

Immunomodulatory treatment of chronic rhinosinusitis with nasal polyposis (CRSwNP) by macrolide antibiotics represents a challenging alternative to conventional therapy and surgery, still being at the very beginning. Immune and inflammatory processes in nasal and paranasal sinus mucosa, crucial in the etiopathogenesis of nasal polyps (NPs) are reflected in levels of various local mediators, found both in mucosa and nasal fluid. In this prospective study, we assessed the immunomodulatory and clinical effects of longterm low-dose oral macrolide treatment in the management of CRSwNP. Twenty-two (n = 22) nonasthmatic, nonallergic patients with CRSwNP were administered clarithromycin (CAM) 500 mg/day single oral dose for eight weeks. We measured the levels of proinflammatory cytokines TNF-a, TNF-b, and IL-1b, Th1 cytokines IL-2, IL-12, and IFN-g, Th2 cytokines IL-4, IL-5, IL-6, and IL-10, and chemokine IL-8 in the nasal fluid samples, before and after treatment, using a flow cytometric method. We also scored each of the 22 patients before and after therapy according to Tsicopoulos' global nasal symptom score and Malm's endoscopic score. Following treatment, we found significantly reduced levels of IL-8 (p