The Expression of Hypoxia-Related Biomarkers: A Significance of HIF-1α C1772T Polymorphism as Predictor of Laryngeal Carcinoma Relapse.

INTRODUCTION: The association between the expression of HIF-1α in the laryngeal carcinoma and the prognosis of disease is quite well documented, but the significance of HIF-1α C1772T polymorphism and its relation to disease phenotype have to be clarified. The aim of this study was to investigate the influence of C1772T polymorphism on the clinical-pathological characteristics and disease-free survival after initial surgical treatment of patients with laryngeal carcinoma. MATERIALS AND METHODS: The prospective cohort study included 65 patients with laryngeal carcinoma. Two representative tumor tissue specimens were taken in each patient during surgery; 1 specimen was used to asses HIF-1α C1772T polymorphism and the other 1 to determine the immunohistochemical expression of HIF-1α, VEGF, as well as CD 34 proteins. The comparison of polymorphism frequency between study and control population was conducted by collecting a 5 mL of peripheral venous blood samples in each subject. RESULTS: Clinicopathological characteristics of laryngeal carcinoma didn't affect the expression of hypoxia-related biomarkers, such as HIF-1α, VEGF or MVD. The statistically significant association between HIF-1α and VEGF expression was found (P = .034), but not between HIF-1α expression and MVD value (P = .696). The expression of HIF-1α was significantly higher among CT heterozygotes (P = .029). We found a significantly more recurrence among CT heterozygotes compared with patients with CC homozygous alleles (57.10% and 24.30%, respectively; P = .007). Patients with C1772T polymorphic variants had significantly worse disease-free survival compared with patients without polymorphism (Log-rank test, P = .007). CONCLUSION: HIF-1α C1772T polymorphism was significantly associated with worse disease-free survival which nominates it as a predictor of laryngeal carcinoma relapse. The preoperative assessment of hypoxia-related biomarkers should be used in everyday practice in order to determine the treatment modalities for laryngeal carcinoma.

Prenatal Androgenization Induces Anxiety-Like Behavior in Female Rats, Associated with Reduction of Inhibitory Interneurons and Increased BDNF in Hippocampus and Cortex.

Anxiety is one of the most frequent psychiatric disorders. Despite the fact that most studies describe an anxiolytic effect of testosterone, hyperandrogenemia in mothers is assumed to be related to an increased risk of mood disorders in their offspring. An increasing body of scientific evidence suggests that an altered expression of interneuronal markers of the hippocampus may be the cause of anxiety. The aim of this study was to examine the influence of maternal hyperandrogenemia on behavioral parameters of anxiety-like behavior, neuropeptide Y (NPY) and parvalbumin (PV) expression in the hippocampus, and the level of the brain-derived neurotrophic factor (BDNF) in the hippocampus and cerebral cortex. Pregnant female Wistar albino rats were treated with testosterone undecanoate on the 20th day of gestation. Anxiety-like behavior in adult female offspring was evaluated by the elevated plus maze test and the open field. The number of PV and NPY immunoreactive cells in the hippocampus was determined immunohistochemically. The level of BDNF expression in the hippocampus and cerebral cortex was analyzed with the Western blot test. Prenatal hyperandrogenization increased anxiety-like behavior in female offspring and decreased expression of NPY+ and PV+ in the CA1 region of the hippocampus as compared to the control group. BDNF expression in the hippocampus and cerebral cortex of prenatally androgenized female offspring was significantly increased in comparison with the controls. Prenatal hyperandrogenization may be the cause of anxiety-like behavior in female offspring. Decrease in NPY and PV expression in the hippocampus may explain the possible mechanism of hyperandrogenization induced anxiety.

Chronic prostatitis/chronic pelvic pain syndrome increases susceptibility to seizures in rats and alters brain levels of IL-1ß and IL-6.

Chronic Prostatitis/Chronic Pelvic Pain Syndrome (CP/CPPS) is a result of interplay between psychological, immune, neurological and genetic factors, manifested by variety of urological, as well as brain-related symptoms. However, its relation with brain excitability has not been addressed. herefore, our aim was to assess susceptibility to seizures in rats with CP/CPPS. We induced CP/CPPS in adult rats by intraprostatic injection of 3% λ-carrageenan. Sham operated rats served as controls (0.9% NaCl, Sham). On day 7 upon injection, rats were treated with lindane (4 mg/kg) and observed for convulsive behavior (seizure incidence, latency and severity) and EEG manifestations (number and duration of ictal periods). Interleukin levels (IL-1ß and IL-6) were measured in prostate, hippocampus, thalamus and cerebral cortex. Scrotal skin mechanical pain thresholds were determined and prostates were histologically evaluated. Animals with CP/CPPS showed significantly higher incidence, decreased latency time and augmented severity of lindane-induced seizures compared with Sham group. EEG revealed increased number of ictal periods in CP/CPPS rats. Higher levels of IL-1ß and IL-6 were determined in the thalamus and cortex in CP/CPPS animals vs. Sham. IL-1ß level was higher and IL-6 was lower in prostates from CP/CPPS animals comparing to Sham. CP/CPPS development was verified by histological findings of nonbacterial inflammation in the prostates, as well as by significantly decreased scrotal pain threshold in CP/CPPS animals. On the basis of this research, we concluded that CP/CPPS increases susceptibility to lindane-induced seizures in rats associated with increased level of IL-1ß and IL-6 in the cortex and thalamus.

Differences in Chromatin Texture and Nuclear Fractal Dimension Between Hashimoto's and Lymphocytic Thyroiditis Lymphocytes.

Previous evidence suggested that lymphocytic thyroiditis (LT) was a variant of Hashimoto's thyroiditis (HT), thus the aim of the current study is to quantify structural changes in histological specimens taken from HT and LT patients. A total of 600 images containing a single lymphocyte nucleus (300 nuclei per group) were obtained from 20 patients with HT and LT. In order to quantify changes in the nuclear architecture of investigated lymphocytes, the fractal dimension (FD) and some gray-level co-occurrence matrix texture parameters (angular second moment, inverse difference moment, contrast, entropy, and correlation) were calculated for each nucleus. A statistically significant difference in the FD of the "binary-outlined" nucleus and that of the corresponding "black-and-white" nucleus was detected between HT and LT lymphocyte nuclei. In addition, there was also a statistically significant difference in contrast and correlation between HT and LT lymphocyte nuclei. In conclusion, the results of this study suggested that there was a difference in structural complexity between investigated lymphocyte nuclei; additionally, LT lymphocytes possessed probably more complex texture and larger variations as well as more asymmetrical nuclei compared with HT lymphocytes. Accordingly, these findings indicate that LT is probably not a variant of HT; however, more complex studies are necessary to estimate differences between these types of thyroiditis.

SOX3 can promote the malignant behavior of glioblastoma cells.

PURPOSE: Glioblastoma is the most common and lethal adult brain tumor. Despite current therapeutic strategies, including surgery, radiation and chemotherapy, the median survival of glioblastoma patients is 15 months. The development of this tumor depends on a sub-population of glioblastoma stem cells governing tumor propagation and therapy resistance. SOX3 plays a role in both normal neural development and carcinogenesis. However, little is known about its role in glioblastoma. Thus, the aim of this work was to elucidate the role of SOX3 in glioblastoma. METHODS: SOX3 expression was assessed using real-time quantitative PCR (RT-qPCR), Western blotting and immunohistochemistry. MTT, immunocytochemistry and Transwell assays were used to evaluate the effects of exogenous SOX3 overexpression on the viability, proliferation, migration and invasion of glioblastoma cells, respectively. The expression of Hedgehog signaling pathway components and autophagy markers was assessed using RT-qPCR and Western blot analyses, respectively. RESULTS: Higher levels of SOX3 expression were detected in a subset of primary glioblastoma samples compared to those in non-tumoral brain tissues. Exogenous overexpression of this gene was found to increase the proliferation, viability, migration and invasion of glioblastoma cells. We also found that SOX3 up-regulation was accompanied by an enhanced activity of the Hedgehog signaling pathway and by suppression of autophagy in glioblastoma cells. Additionally, we found that SOX3 expression was elevated in patient-derived glioblastoma stem cells, as well as in oncospheres derived from glioblastoma cell lines, compared to their differentiated counterparts, implying that SOX3 expression is associated with the undifferentiated state of glioblastoma cells. CONCLUSION: From our data we conclude that SOX3 can promote the malignant behavior of glioblastoma cells.

Tuberous sclerosis complex protein 1 expression is affected by VHL Gene alterations and HIF-1α production in sporadic clear-cell renal cell carcinoma.

Alterations in von Hippel-Lindau gene (VHL) do not determine deregulation of hypoxia-inducible factors (HIFs) in clear-cell renal carcinoma (ccRCC). Their effects on tuberous sclerosis proteins (TSC1/2) and heat shock protein 90 (Hsp90) expressions in sporadic ccRCC are unknown. Therefore, we analyze the impact of VHL alterations and HIF-α production on the expression of TSC proteins and Hsp90 in these tumors. Alterations in VHL gene region exhibited 37/47 (78.7%) tumors. Monoallelic inactivation (intragenic mutation or LOH) was found in 10 (21.3%) and biallelic inactivation (intragenic mutation plus LOH) in 27 (57.4%) ccRCCs. Tumorous expression of HIF-α mRNAs, HIF-α, Hsp90 and TSC2 were VHL independent; TSC2 was underexpressed in all tumors by immunostaining (P<0.001). Immunoblotting revealed that TSC1 production was lower in tumors with monoallelic VHL inactivation than in control (P=0.01) and tissues with biallelic VHL inactivation (P=0.019), while tumors lacking HIF-1α (16/47) concurrently overexpressed HIF-2α and underexpressed TSC1 in comparison to controls (P=0.01 for both) and HIF-1α positive tumors (P=0.015 and P=0.050). Significant portion of variability (56.4%) in tumor diameter was explained by oscillations in nuclear grade, and TSC1 and HIF-2α expression in VHL altered tumors. In conclusion, while TSC2 is broadly downregulated in sporadic ccRCC, TSC1 expression is reduced in two subsets of these tumors, those with monoallelic VHL gene inactivation and those with concurrent low HIF-1α and high HIF-2α expression. Hence, the involvement of nuclear grade, TSC1 and HIF-2α in the progression of VHL altered tumors, implies the interplay between pVHL and TSC1.

Effects of IL-33/ST2 pathway in acute inflammation on tissue damage, antioxidative parameters, magnesium concentration and cytokines profile.

AIM: The aim of this study was to examine the role of IL-33/ST2 pathway in a pathogenesis of acute inflammation and its effects on tissue damage, antioxidative capacity, magnesium concentration and cytokine profile in acutely inflamed tissue. MATERIAL AND METHODS: Male mice were randomly divided in four groups: wild-type control group (WT-C), ST2 knockout control group (KO-C), wild-type inflammatory group (WT-I), and ST2 knockout inflammatory group (KO-I). Acute inflammation was induced in WT-I and KO-I by intramuscular injection of turpentine oil, while mice in WT-C and KO-C were treated with saline. After 12h, animals were euthanized, and blood was collected for determination of creatine kinase (CK) and aspartate transaminase (AST) activity. The treated tissue was used for histopathological analysis, determination of volume density of inflammatory infiltrate (Vdii) and necrotic fiber (Vdnf), gene expression of interleukin (IL)-33, ST2, tumor necrosis factor alpha (TNF-alpha), IL-6, IL-12p35, and transforming growth factor beta (TGF-beta), concentration of magnesium (Mg), copper (Cu), selenium (Se), manganese (Mn) and reduced glutathione (GSH), and superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity. RESULTS: Presence of inflammatory infiltration and necrosis in the treated tissue was histopathologically confirmed in WT-I and KO-I. Vdii was significantly higher in WT-I when compared to KO-I, whereas Vdnf did not significantly differ between WT-I and KO-I. CK and AST significantly increased in both inflammatory groups when compared to corresponding control groups. However, the values of CK and AST were significantly higher in WT-I than in KO-I. Mg in the treated tissue was significantly lower in WT-I in comparison to WT-C and KO-I, while there was no significant difference between KO-C and KO-I. There was no significant difference in Cu, Se, and Mn in the treated tissue between WT-C, KO-C, WT-I and KO-I. Gene expression of IL-33 in the treated tissue increased in both inflammatory groups when compared to the corresponding control groups, but it was significantly higher in KO-I than in WT-I. Gene expression of ST2 in the treated tissue was significantly higher in WT-I than in WT-C. Gene expression of TNF-alpha, IL-6, and IL-12p35 in the treated tissue was significantly higher in WT-I and KO-I than in the corresponding control groups, and IL-6 was significantly higher in KO-C than in WT-C. TGF-beta gene expression in the treated tissue was significantly higher in KO-I when compared to WT-I, while there was no difference between WT-C and KO-C. SOD activity decreased at the site of acute inflammation in both inflammatory groups, while the GPx activity increased. GSH in the treated tissue was significantly higher in KO-I than in KO-C or WT-I. CONCLUSION: The results of our study have indicated, to our knowledge for the first time, that IL-33/ST2 pathway plays a role in enhancing inflammation and tissue damage at the site of acute inflammation by affecting the concentration of magnesium and GSH, important for antioxidative capacity, as well as gene expression of anti-inflammatory cytokine TGF-beta.

Co-expression of vascular and lymphatic endothelial cell markers on early endothelial cells present in aspirated coronary thrombi from patients with ST-elevation myocardial infarction.

INTRODUCTION: Angiogenesis is the growth of both new vascular and lymphatic blood vessels from the existing vasculature. During this process, blood endothelial cells (BECs) and lymphatic endothelial cells (LECs) express specific markers, which help their discrimination and easier identification. Since the coronary thrombi material aspirated from patients with ST-elevation myocardial infarction (STEMI) proved as good angiogenesis model, we investigated the expression of CD34 and CD31 as BECs markers, and D2-40, LYVE-1 and VEGFR3 as LEC markers in this material. MATERIALS AND METHODS: Aspirated thrombi were stained immunohistochemically for CD34, CD31, D2-40, LYVE-1 and VEGFR3. Organizational patterns of immunopositive cells were graded as single cells, clusters or microvessels. Double immunofluorescence for CD31, D2-40, LYVE-1 and VEGRF3 was done. Thrombi were also graded as fresh (<1day old), lytic (1-5days old) and organized (>5days old). RESULTS: Serial sections of aspirated thrombi showed concordant BEC and LEC markers immunopositivity. Double immunoflorescence proved co-expression of CD31 and LEC markers on the same cells. Cells expressing LEC markers organized in clusters and microvessels were mainly present in lytic and organized thrombi. CONCLUSION: Co-expression of BEC and LEC markers on the same non-tumorous cell during thrombus neovascularization indicates existing in vivo plasticity of endothelial cells under non-tumorous pathological conditions. It also points that CD34 and CD31 on one hand, and D2-40, LYVE-1 and VEGFR3 immunostaining on the other hand, cannot solely be a reliable indicators whether vessel is lymphatic or not.

Effects of Il-33/St2 pathway on alteration of iron and hematological parameters in acute inflammation.

AIM: The aim of this study was to examine the role of the IL-33/ST2 pathway in pathogenesis of acute inflammation by investigating its possible role in alteration of iron and hematological parameters in experimental model of acute inflammation. MATERIAL AND METHODS: Wild-type and ST2 knockout BALB/c mice were divided into four groups: wild-type control group, ST2-/- control group, wild-type inflammatory group, and ST2-/- inflammatory group. Acute inflammation was induced by intramuscular injection of turpentine oil, while control groups were injected with saline. After 12h animals were anesthetized, and the treated tissue, blood and spleen were collected. Iron concentration in the treated tissue, hemoglobin blood concentration, mean corpuscular hemoglobin (MCH), hematocrit, erythrocyte, neutrophil and lymphocyte blood count, and erythrocytes percentage in spleen were determined. RESULTS: Iron concentration in the treated tissue was significantly higher in wild-type inflammatory group (WT-I) when compared to both, the wild-type control group (WT-C) and ST2-/- inflammatory group (KO-I). There was no significant difference in iron concentration between ST2-/- control group (KO-C) and the KO-I. MCH had significantly decreased in WT-I when compared to WT-C, while there was no significant difference between KO-C and KO-I. Hemoglobin blood concentration significantly increased in KO-I in comparison to KO-C, while it did not significantly differ between WT-I and KO-I. Erythrocyte count and hematocrit had significantly increased, while the percentage of erythrocytes in spleen decreased in both inflammatory groups when compared to their controls. Neutrophil count significantly decreased in WT-I, when compared to WT-C. Lymphocyte count decreased in both inflammatory groups when compared to their controls. CONCLUSION: Results of this study indicate that the IL-33/ST2 axis could have a role in the alteration of iron in acute inflammation, namely in an increase of iron concentration at the site of acute inflammation and a decrease of blood mean corpuscular hemoglobin.

The role of gut hormones in appetite regulation (review).

Eating process is an aggregate of complex and different forms of behavior. Its regulation is based on energy homeostasis and appetite control which includes two components: the homeostatic and the hedonistic control. Important signals in appetite regulation are gut-derived hormones. They are produced by enteroendocrine cells in response to nutrient and energy intake, and achieve their effects by influencing brain structures involved in food intake regulation. The key brain structure involved in this process is the hypothalamus. Gut hormones reach the hypothalamus from the circulation or by the vagal nerve via the nucleus of the solitary tract. Among gut peptides, ghrelin is the only orexigenic hormone, leading to an increase in food intake and body weight. All others, such as cholecystokinin, glucagon like peptide-1, oxyntomodulin, peptide tyrosine tyrosine or pancreatic polypeptide, are anorexigenic, leading to decrease in food intake. Also, gut-derived endocannabinoids exert orexigenic effect on appetite. Keeping in mind the growing problem of obesity, the crucial issue when considering gut derived peptides is to understand their mechanisms of acting because of potential role in clinical therapy, and discovering long-lasting gut peptides or their analogues, with no or minimal side effects.

Activation of neurons in the hypothalamic dorsomedial nucleus via hypothalamic projections of the nucleus of the solitary tract following refeeding of fasted rats.

We report that satiation evokes neuronal activity in the ventral subdivision of the hypothalamic dorsomedial nucleus (DMH) as indicated by increased c-fos expression in response to refeeding in fasted rats. The absence of significant Fos activation following food presentation without consumption suggests that satiation but not craving for food elicits the activation of ventral DMH neurons. The distribution pattern of the prolactin-releasing peptide (PrRP)-immunoreactive (ir) network showed remarkable correlations with the distribution of activated neurons within the DMH. The PrRP-ir fibers and terminals were immunolabeled with tyrosine hydroxylase, suggesting their origin in lower brainstem instead of local, hypothalamic PrRP cells. PrRP-ir fibers arising from neurons of the nucleus of the solitary tract could be followed to the hypothalamus. Unilateral transections of these fibers at pontine and caudal hypothalamic levels resulted in a disappearance of the dense PrRP-ir network in the ventral DMH while PrRP immunoreactivity was increased in transected fibers caudal to the knife cuts as well as in perikarya of the nucleus of the solitary tract ipsilateral to the transections. In accord with these changes, the number of Fos-expressing neurons following refeeding declined in the ipsilateral but remained high in the contralateral DMH. However, the Fos response in the ventral DMH was not attenuated following chemical lesion (neonatal monosodium glutamate treatment) of the hypothalamic arcuate nucleus, another possible source of DMH inputs. These findings suggest that PrRP projections from the nucleus of the solitary tract contribute to the activation of ventral DMH neurons during refeeding, possibly by transferring information on cholecystokinin-mediated satiation.

[Characteristics of galanin and vasoactive intestinal peptide immunoreactivity in the rat amygdala complex].

INTRODUCTION: Morphological features and morphometric parameters of galanin (GAL) and vasoactive intestinal peptide (VIP) immunoreactive neurons and neuronal fibres were studied in all nuclei of adult male rat amygdala. MATERIAL AND METHODS: After perfusion and fixation, rat brains were immunohistochemically stained with antibodies against GAL and VIP and then visualized by avidin-biotin-peroxidase complex. RESULTS AND DISCUSSION: The greatest number of galanin-immunoreactive neurons were identified in the medial part of the central nucleus and in the dorsal part of the medial nucleus. In the first case, most neurons were bipolar (37%), and in the second, they were ovoid (45%). GAL-immunoreactive fibers were identified in the medial nucleus, "bed nucleus" of the accessory olfactory tract, fiontal cortical nucleus, amygdalo-hippocampal area and basolateral nucleus. VIP-immunoreactive neurons were diffusely distributed in more nuclei than the previous, mostly in the lateral, basolateral, and basomedial nucleus. They were mostly ovoid (40%). VIP-immunoreactive fibers were observed in the lateral part oJ'the central nucleus, while long and radially oriented fibers were present in the frontal and dorsal cortical nucleus. CONCLUSION: By distribution analysis of GAL and VIP immunoreactive neurons and fibers, and according to literature data, it can be assumed that the medial part of the central nucleus receives VIP fibers from other parts of the amygdaloid body, and then sends GAL fibers to the medial nucleus.

[C-fos protein expression in the parietal cortex and olfactory tubercle in the hypoxic rat brain].

INTRODUCTION: We have attempted to identify which parts of the brain react to ischemic attack using the four-vessel occlusion model in rats. MATERIAL AND METHODS: We have monitored the expression of c-fos protein in the parietal cortex (R3 and T3) and in the olfactory tubercle (R4 and T4), regions which are supplied with blood by different arteries. The four-vessel occlusion was performed using the Pulsinelli's method and rats were divided into two groups: total ischemia (ligation of four blood vessels or coagulation of the vertebral artheries with bilateral ligation of carotid arteries - R group) and transient ischemic attack (ligation of four blood vessels or coagulation of the vertebral arteries with bilateral repeated ligation of carotid arteries - T group of rats, the so-called preconditioned group). Each of these groups had a control group. RESULTS AND CONCLUSION: The results showed pronounced expression of c-fos neurons in T group of rats which can explain longer survival of neurons. We believe that this model can serve as a good starting point to developing new approaches to the therapy of brain ischemia.