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Chronic inflammation creates tumor microenvironment (TME) that facilitates colorectal cancer (CRC) cell proliferation, migration, metastasis, and tumor progression. Interleukin-6 (IL-6) is a proinflammatory cytokine with a pleiotropic effect on CRC development. We aimed to evaluate IL-6 expression in tumor cells and in immune cells in TME, to assess the serum level and IL6 -174 G/C genotype distribution and to correlate the results with selected morphologic and clinical parameters that may add useful information in understanding the mechanisms of human CRC progression. A total of 153 patients with CRC were recruited in the current study. We assessed the IL-6 serum concentration through the ELISA method, the expression of IL-6 in tumor and in immune cells by immunohistochemical and double immunofluorescence staining, the MSI status by immunоhistochemistry for 4 mismatch repair (MMR) proteins, and the genotype distributions for IL6 -174G/C (rs1800795) single-nucleotide polymorphism through PCR-RFLP method. Our results showed that serum IL-6 level were increased in CRC patients as compared with healthy controls (P<0.0001), and in patients with cancers with advanced histologic type (type IV). However, the higher concentration (above the median of 55.71 pg/mL) was with borderline association with longer survival of the patients after surgical therapy (P=0.055, Log rank test). We also found that IL-6+ immune cells prevailed in the invasive front (IF) of tumors compared with the tumor stroma (TS) (P<0.0001). More IL-6+ cells were recruited in the tumors with less advanced histologic type (I+II), with stronger inflammatory infiltrate in the IF, in early pTNM stages (I+II), without lymph node and distant metastases and the higher levels of IL-6+ cells, especially in the IF, were associated with longer survival (P=0.012). The results of our study suggest that although the serum levels of IL-6 are higher in CRC, the increased IL-6+ cells in tumor microenvironment, both in the invasive front and in tumor stroma, as well as the higher serum levels are associated with good prognostic variables and longer survival of the patients mainly in the early stages of CRC.

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Introduction: Pancreatic ß-cells and α-cells have been found in the murine extrahepatic biliary ducts but not in the gallbladder. However, there has been no information reported in the specialized literature about the presence of glucagon- and insulin-expressing endocrine cells in porcine bile ducts and gallbladder. Aim: We aimed to perform an immunohistochemical study to identify glucagon- and insulin-positive cells and their distribution in the porcine extrahepatic biliary ducts and gallbladder. Method: The immunohistochemical method was used to detect the presence and distribution of glucagon- and insulin-positive endocrine cells in the common hepatic duct (ductus hepaticus communis), common bile duct (ductus choledochus), cystic duct (ductus cysticus), and gallbladder (vesica fellea) of male pigs. Chromogranin A was used as a typical marker for endocrine cells. Results: The density of chromogranin A-, glucagon- and insulin-positive cells per field was the largest in the common bile duct, followed by the common hepatic duct, cystic duct, and gallbladder. The three types of endocrine cells showed specific localization in the superficial and deep glands of the studied organs. Conclusion and clinical importance: The distribution of glucagon- and insulin-immunopositive endocrine cells in the porcine extrahepatic biliary tract was established for the first time as a new source of these hormones. The presence of α- and ß-cells in the epithelium of extrahepatic bile ducts can be applied in treatment of diabetes, taking into account the possibility to reprogram the biliary epithelium to mentioned pancreatic endocrine cell types.

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CONTEXT: There is data about the existence of some endocrine cells in the epithelial layer of the bile duct in humans and rats. OBJECTIVE: We evaluated Ghrelin-, Insulin-, Glucagon- and Somatostatin-positive cells in peribiliary glands, mast cells, and nerve fibres. MATERIALS AND METHODS: Wistar rats were used for dietary manipulation with a 15% fructose solution for 12 weeks. Tissue samples were elaborated with immunohistochemistry for Insulin, Glucagon, Ghrelin, and Somatostatin. Glucose and lipid parameters were studied. RESULTS: In treated animals, Ghrelin+ and Insulin+ cells in perybiliary glands (PBGs) were significantly increased. In the male fructose group there was a significant increase of the homeostasis model assessment insulin resistance (HOMA-IR). CONCLUSIONS: Stem/progenitor cells in extrahepatic bile tree (EHBT) could be a source of Insulin-producing cells in metabolic syndrome. Fructose treatment induces the increase of Ghrelin+ and Insulin+ cells in PBGs and the elevation of Insulin and Ghrelin plasma concentration.

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Background: Interleukin (IL)-17A and IL-17F, expressed mainly by a novel subset of CD-positive (+) T-helper (Th) cells of the immune system, has been closely related to inflammatory conditions underlying colorectal cancer pathogenesis. Accordingly, we conducted a case-control study to investigate the association of common single nucleotide polymorphisms (SNP) in the IL17A and IL17F genes (rs2275913 and rs763780, respectively) with the susceptibility and severity of CRC patients from the Bulgarian population. Methods and Materials: 136 patients with histologically confirmed CRC diagnosis and 116 healthy individuals were recruited in the present study. Genotypes were determined by the restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) technique. Results: The IL17A heterozygous A/G-genotype was overrepresented among the control group (p = 0.003). Additionally, the carriers of the heterozygous A/G-genotype had a 2.39-fold lower risk for CRC compared to the G/G-genotype (OR = 0.418, p = 0.006). Our results also indicated that in the advanced CRC stages (III + IV) the heterozygous genotype (A/G) appeared to be less frequent (p = 0.024, χ2-test). Among the patients with detected distant metastases, the A/G-carriers were the smallest part (14.3%) compared to the homozygous genotypes A/A (42.9%) and G/G (42.8%), p = 0.006. There was no association of the studied IL17F rs763780 SNP with susceptibility and severity of CRC among the studied subjects, although the heterozygous C/T-carriers had shorter median survival compared to the T/T-carriers (p = 0.129). Conclusions: Our study finds a protective role of heterozygosity for the IL17A-197A/G SNP and negative effects of the A-allele on CRC progression.

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Context: Gastric ghrelin-positive endocrine cells (GHR + EC) were most dense in the oxyntic mucosa.Objective: We evaluated ECs and contractile activity in rat stomach with metabolic disorders.Materials and methods: Male Wistar rats were divided into two groups: Control (n = 9) received tap water and Fructose (n = 9) drank 15% fructose solution for 12 weeks. Streptozotocin was applied in a dose of 20 mg/kg b.w. two weeks after the beginning of the experiment on Fructose group. Smooth-muscle strips from the stomach were influenced by Angiotensin II for analysis of parameters of contractions. Stomach samples were elaborated with immunohistochemistry for ghrelin, somatostatin, gastrin antibodies and with double immunofluorescence.Results: In treated animals, GHR + EC were significantly increased in the corpus where somatostatin-positive cells were decreased. Contractile activity was decreased.Conclusions: The increase number of GHR + EC was discussed in the context of Somatostatin and Gastrin-positive ECs variations and correlated with the decrease of smooth muscle contraction.

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The predominant role of IL-6 in cancer is its key promotion of tumour growth. IL-6 binds IL-6 receptor (IL-6R) and the membrane-bound glycoprotein gp130. The complex I-6/IL-6R/gp130 starts the Janus kinases (JAKs) and signal transducer and activator of transcription 3 (STAT3) or JAK/STAT3 pathway. IL-6 R exits in two forms: a membrane-bound IL-6Rα subunit (mIL-6R) that participates in classic signalling pathway and soluble IL-6R subunit (sIL-6R) engaged in trans-signalling. The pro-tumour functions of IL-6 are associated with STAT3, a major oncogenic transcription factor that triggers up-regulation of target genes responsible for tumour cell survival. IL-6 combined with TGF-ß induces proliferation of pathogenic Th17 cells. The anti-tumour function of IL-6 is the promotion of anti-tumour immunity. IL-6 trans-signaling contributed to transmigration of lymphocytes in high endothelial venules (HEV). Dendritic cell (DC) secreted IL-6 in the lymph node influences the activation, distribution and polarisation of the immune response. Elevated serum levels of IL-6 and increased expression of IL-6 in tumour tissue are negative prognostic marker for patients' survival.

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Our aim was to analyzed the significance of CD11c and CD123 positive DCs and their relations with some clinical and pathologic parameters of patients with non-small cell lung cancer (NSCLC). The immunohistochemical expression of CD11c and CD123, was evaluated in 40 patients with NSCLC. After analysis we found that 35.3% of the patients in the T3-4 tumour stage had a high CD11c infiltration in the tumour stroma, while 100% of the patients in the T1-2 tumour stage had low infiltration (p = 0.03). We also found that 71.4% of patients in the M1 stage had a high infiltration with CD123 in the tumour stroma, whereas only 15.6% of patients without metastases had high infiltration, analogous data are also found in comparing the distribution of CD123 in the tumour border (p = 0.002 or p = 0.002). Comparing the density of CD123 in the border of lymph node involvement, we found that only 7.14% of patients without metastases had low infiltration with dendritic cells, whereas in patients with metastatic lymph nodes that percentage was 41.7% (p = 0.008). In conclusion results suggest that CD11c- and CD123-positive DCs play an important role in antitumour immunity and can be predictive factor for tumour development in patients with NSCLC.

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The present study was directed to the development of EPR methodology for distinguishing cells with different proliferative activities, using "redox imaging." Three nitroxide radicals were used as redox sensors: (a) mito-TEMPO-cell-penetrating and localized mainly in the mitochondria; (b) methoxy-TEMPO-cell-penetrating and randomly distributed between the cytoplasm and the intracellular organelles; and (c) carboxy-PROXYL-nonpenetrating in living cells and evenly distributed in the extracellular environment. The experiments were conducted on eleven cell lines with different proliferative activities and oxidative capacities, confirmed by conventional analytical tests. The data suggest that cancer cells and noncancer cells are characterized by a completely different redox status. This can be analyzed by EPR spectroscopy using mito-TEMPO and methoxy-TEMPO, but not carboxy-PROXYL. The correlation analysis shows that the EPR signal intensity of mito-TEMPO in cell suspensions is closely related to the superoxide level. The described methodology allows the detection of overproduction of superoxide in living cells and their identification based on the intracellular redox status. The experimental data provide evidences about the role of superoxide and hydroperoxides in cell proliferation and malignancy.

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Dendritic cells (DCs) use effective mechanisms to combat antigens and to bring about adaptive immune responses through their ability to stimulate näive T cells. At present, four major cell types are categorised as DCs: Classical or conventional (cDCs), Plasmacytoid (pDCs), Langerhans cells (LCs), and monocyte-derived DCs (Mo-DCs). It was suggested that pDCs, CD1c+ DCs and CD141+ DCs in humans are equivalent to mouse pDCs, CD11b+ DCs and CD8α+ DCs, respectively. Human CD141+ DCs compared to mouse CD8α+ DCs have remarkable functional and transcriptomic similarities. Characteristic markers, transcription factors, toll-like receptors, T helpers (Th) polarisation, cytokines, etc. of DCs are discussed in this review. Major histocompatibility complex (MHC) I and II antigen presentation, cross-presentation and Th polarisation are defined, and the dual role of DCs in the tumour is discussed. Human DCs are the main immune cells that orchestrate the immune response in the tumour microenvironment.

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BACKGROUND: Inflammation that occurred in the tumor microenvironment was characterized by abundant macrophage infiltration, playing role in innate immunity. Multinucleate giant cells (MGCs) occur in a variety of inflammatory, hyperplastic, and neoplastic thyroid disorders. They also have been recognized as a feature of papillary thyroid carcinoma (PTC). AIM: The aim of this study was to evaluate cases of PTC for the presence of macrophages, and estimate CD68+ TAMs density in tumor stroma, margin and the surrounding tissue. We assessed also MGCs. METHODS: Macrophages and MGCs densities were correlated with clinicopathologic parameters to assess the possible prognostic significance. We investigated 56 patients immunohistochemically and immunofluorescence with antibodies against CD68 and IL-17. RESULTS: A statistically significant correlation was established between PTC patients in III stage, containing many MGCs, and PTC in I and II stage, with many MGCs. Eighty Percent of patients in III stage showed many MGCs in comparison with patients in I and II stage, where many MGCs were found only in 21,1% (χ2 = 6.189, p = 0.013). CONCLUSION: Our study demonstrates that the increased density of MGCs is associated with advanced stage of PTC, and therefore with tumor progression and that cases of PTC should be carefully screened for their presence.

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BACKGROUND: Non-small cell lung cancer (NSCLC) is an insidious metastasis condition of the lungs often presenting no symptoms at the onset. Defining markers for quick determination of prognosis is essential for building up a treatment strategy. AIM: The aim of this study is to define the role of the Neutrophils-to-Lymphocytes ratio (NLR) and Platelets-to- Lymphocytes ratio (PLR) as biomarkers in patients with NSCLC, according to the stage and prognosis of the disease. METHODS: We investigated 20 patients with NSCLC. NLR and PLR are calculated and are evaluated according to the presence or absence of metastasis, stage of the disease, histological type and survival rate. RESULTS: We found that thirteen of the patients had low NLR, while the rest 7 had high NLR (mean 3.15). By analysing PLR we found that 11 patients have low and 9 have high level of PLR (mean 1.42). After the correlations have been made we discovered that in 90.1% of the patients with low PLR no lymph metastasises were detected, while in 50% of the patients with high PLR lymph metastasises were observed (χ2 = 3.99; P = 0.046). We also discovered that in 84.6% of the patients with low NLR lymph metastases were absent, while in 42.9% with high NLR lymph metastasises were present (χ 2 = 1.83; P = 0.176). CONCLUSION: In conclusion, NLR and PLR were discovered as prominent biomarkers which provide relatively fast determination for prognosis in patients with NSCLC.

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BACKGROUND: Papillary thyroid carcinoma (PTC) accounts for 80% of the thyroid malignancies that are characterised by slow growth and an excellent prognosis. Over-expression of SMAD4 protein restores TGF-ß signalling, determines a strong increase in anti-proliferative effect and reduces invasive potential of tumour cells expressing it. AIM: The study aimed to analyse the immunohistochemical expression of TGF-ß1 and its downstream phosphorylated SMAD4, element and of the inhibitory SMAD7 PTC variants and their association with the localisation of TAMs within the tumour microenvironment. METHODS: For this retrospective study we investigated 69 patients immunohistochemistry with antibodies against TGF-ß, TGF - ß-RII, SMAD4, SMAD7, CD68+ macrophages. RESULTS: Patients with low infiltration with CD68+ cells in tumour stroma has significantly shorter survival (median of 129.267 months) compared to those with high CD68+ cells infiltration (p = 0.034). From the analysis of CD68+ cells in tumour border and tumour stroma correlated with expression of TGF-ß1 / SMAD proteins, we observed that the positive expression of TGF-ß1 in tumour cytoplasm, significantly correlated with increased number of CD68+ cells in tumour border (X2 = 5,945; p = 0.015). CONCLUSION: TGF-ß enhances motility and stimulates recruitment of monocytes, macrophages and other immune cells while directly inhibiting their anti-tumour effector functions.

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BACKGROUND: Anti-inflammatory cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-ß1 have a complex role in the development of colorectal cancer (CRC). Dendritic cells (DCs) are the cellular component of the inflammatory microenvironment in the tumor and infiltration of tumors by DCs is associated with better prognosis and fewer metastases. METHODS: In the present study, we explored the role of two single nucleotide polymorphisms (SNPs) in the promoter regions of TGFB1 and IL10 genes and their associations with infiltrating DCs in CRC.A case-control study was designed. Genotyping was performed via the polymerase chain reaction-restriction fragment length polymorphism method and DC infiltration was determined immunohistochemically. RESULTS: For the TGFΒ1 -509C/T SNP, we found that the T allele was less frequent in patients than in controls (p = 0.031) and the TT-genotype had a 2.74-fold lower risk for CRC than the CC-genotype (odds ratio = 0.365, 95% confidence interval = 0.15-0.88, p = 0.015). Additionally, the TT carriers had the shortest median survival (14.4 months) (p = 0.045). The C-allele genotypes had a significantly longer survival compared to TT carriers (p = 0.018). The CC genotype was associated with a lower cellular density of CD11c in the invasive margin of the tumor (p = 0.033), whereas there was an opposite finding for CD83+ DCs (p = 0.037). Carriers of A-allele genotypes of the IL10 -1080A/G SNP had significantly lower CD83+ cells (p = 0.046) in the tumor invasive margin. CONCLUSIONS: The T-allele of the TGFB1 -509C/T SNP might be a protective factor for development of CRC, although, in the course of the disease, this variant allele might be associated with more unfavorable prognosis of the patients.

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A characteristic feature of inflamed lungs in bronchial asthma (BA) is airway remodeling. Due to limited information on this topic in the literature, we aimed to explore the possible role of polymorphisms in the promoter region of the macrophage elastase gene MMP12 82A>G (rs2276109) as a predisposing factor for BA in an ethnic Bulgarian population. Using restriction fragment length polymorphism analysis of polymerase chain reaction-amplified fragments (PCR-RFLP), we performed genotype analysis of 58 patients and 119 control individuals. We found statistically significant differences in the distribution of genotypes (P = .008) and alleles (P = .004) between patients and nonaffected controls. In the dominant model, carriers of the G allele genotypes had 3.6-fold lower risk for BA, compared with those with the AA genotype, after adjustment for age and sex (odds ratio [OR], -0.277; 95% confidence interval [CI], .12-.65; P = .003). The results of our study suggest that the variant G allele of the MMP12 -82 A>G promoter polymorphism might be considered protective for development of BA in ethnic Bulgarian adults residing in central Bulgaria.

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PURPOSE: Chronic inflammation is a key component in the development and progression of colorectal cancer (CRC). A notable hallmark of the inflammation process is the release of pro-inflammatory cytokines by infiltrating cells of the immune system. Defects in dendritic cells (DCs) recruitment, maturation and cytokine release are a hallmark of the CRC strategy to escape immune surveillance.The purpose of our study was to evaluate the possible role of IL-12B polymorphism in the promoter region of the IL-12B gene (rs17860508) as a genetic factor contributing to the risk for CRC development. Additionally, we aimed to evaluate the influence of this polymorphism on DCs infiltration in tumor microenvironment. METHODS: IL-12Bpro polymorphism was genotyped by Amplification Refractory Mutation System- Polymerase Chain Reaction (ARMS-PCR). Immunohistochemistry was performed for DCs infiltration. RESULTS: Statistically significant correlation between the expression of S100 and CD1a DCs and the 11- genotype of the studied polymorphism was found. No statistically significant difference in genotype distribution between cases and controls was observed (p=0.163). Analysis of the overall survival (OS) of genotyped patients revealed a tendency among the carriers of the 22-genotype to have shorter survival of 36 months versus the 11- and 12-cariers- 61 months (log rank, p=0.117). CONCLUSIONS: The IL-12Bpro polymorphism does not constitute a risk factor for CRC development. However, genotype-11 might have a complex role in the recruitment and maturation of DCs in tumor microenvironment.

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The intracellular redox balance (redox status) is a dynamic system that may change via many factors. Mitochondria are one of the most important among them. These organelles are the main intracellular source of energy. They are essential for maintaining cellular homeostasis due to regulation of many biochemical processes. The mitochondrial dynamics change during cellular activities and in some cases, can cause an overproduction of reactive oxygen species (ROS), which encourages the induction of oxidative DNA damage and up- or down-regulation of phosphatases, proliferative/anti-proliferative factors, apoptotic/anti-apoptotic factors, etc. Moreover, mitochondrial dysfunction and redox imbalance can continuously support and contribute to a wide range of pathologies, termed as "free radical diseases" (e.g., cancer, neurodegeneration, atherosclerosis, inflammation, etc.). This review article is focused on the mitochondrial dysfunction and cellular redox status as a hallmark of cell homeostasis and diagnostic marker of cancer. It is intended to broad readership - from students to specialists in the field.

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Chronic inflammation and remodelling of the small airways are features related to chronic obstructive pulmonary disease (COPD). In the current study, we aimed to explore the possible role of MMP12 -82 A > G (rs2276109) promoter polymorphism in the development of COPD in a population from Bulgaria (167 patients with COPD and 119 control individuals). The genotype and allele distributions differed significantly between COPD patients and controls (p = .010 and p = .043, respectively, χ2 test). The genotypes containing at least one variant G allele (AA + GG) were more frequent in the control group than in patients (36.1% vs. 22.2%) determining 2.96-fold lower risk for COPD after adjustment for age, sex and smoking habits (OR = 0.338, 95%CI: 0.168-0.682, p = .002). Our results suggest that carriers of genotypes with at least one copy of minor G allele of rs2276109 might have lower risk for COPD development, with no marked effect on the lung function and severity of the disease.

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BACKGROUND: The aberrant activation of Wnt signalling pathway may be a common denominator for the development of thyroid tumorigenesis. It was announced that the loss of E-cadherin rather than ß-catenin mutation represents a crucial event in determining the degree of differentiation of thyroid carcinomas. AIM: The aim of the study was to evaluate the expression of E-cadherin and ß-catenin in the thyroid cancer tissue and to correlate these data with some histological and clinical parameters of the tumours. MATERIAL AND METHODS: We investigated 112 patients, having thyroid tumours - papillary, follicular, anaplastic and oncocytic carcinomas immunohistochemically with antibodies against E-cadherin and ß-catenin. Survival analyses were done. RESULTS: E-cadherin expression was focally retained in the tumour cell membranes and the tumour cell cytoplasm of the papillary, follicular and oncocytic thyroid cancers, weather in anaplastic cancers it was almost lost (p = 0.0042, and p = 0.019, respectively, Fisher's Exact Test). The expression of ß-catenin in tumour cytoplasm and membrane in papillary cancers was higher as compared to that in the other tumours (p = 0.111, and p = 0.0104, respectively). CONCLUSION: Not surprisingly, the presence of aberrant expression of E-cadherin and ß-catenin in thyroid cancer has been associated with better patients' prognosis and better differentiated tumour histology.

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The aim of this study was to examine the expression of TGF-ß1 and TGF-ß receptor type II (RII) and the impact of the -509C>T single nucleotide polymorphism (SNP) in the gene in relation to clinicopathological factors in gastric cancer (GC). Using immunohistochemistry we investigated 43 patients with GC for expression of TGF-ß1 and TGF-ß-RII. Consequently, RFLP-PCR was performed to analyze the presence of -509C>T polymorphism in the TGF-ß1 gene. We found that 72.1% of GCs had cytoplasmic TGF-ß1 expression and 27.9% were negative. The TGF-ß1 receptor type II was expressed on tumor cell membranes in 58.1%. TGF-ß1 positivity in tumor cytoplasm correlated positively with TGF-ß1-RII expression in tumor cytoplasm in 67.4% of cases (2 = 8.02; p = 0.005). Also, the results showed that patients with low and moderate tumor differentiation had TGF-ß1-RII positivity in 53.3% and 81.8% resp. (2 = 6.58; p = 0,037). The analysis of genotype distribution of the -509C>T SNP in the promoter region of TGF-ß1 gene and clinical stage distribution revealed that among the 32 patients in III-IV clinical stage 53.1% were heterozygous (TC), 34.4% were homozygous for the C-allele and 12.5% were homozygous for the variant T-allele (2 = 3.31; p = 0.069). In conclusion the expression of TGF-ß1 was related to shorter survival time and rapid progression for the GC patients. Additionally, the variant T-allele of the studied polymorphism was associated with worse prognosis for GC patients.

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