HLA-G 5'URR regulatory polymorphisms are associated with the risk of developing gliomas.

BACKGROUND: Human leukocyte antigen G (HLA-G) belongs to non-classical MHC class I molecules that is involved in the suppression of immune response. As HLA-G plays important role in the maintenance of fetal tolerance, its overexpression has been associated with tumor progression. For the regulation of HLA-G levels, genetic variants within the 5' upstream regulatory region (5'URR) are of crucial importance. Our study aimed to analyze the association between 16 HLA-G 5'URR variants, sHLA-G level and clinical variables in glioma patients. METHODS: We investigated 59 patients with gliomas (mean age 54.70 ± 15.10 years) and 131 healthy controls (mean age 41.45 ± 9.75 years). Patient's blood was obtained on the day of surgical treatment. The HLA-G 5'URR polymorphisms were typed by direct sequencing and the plasma level of sHLA-G assessed by ELISA. RESULTS: Haploblock within HLA-G 5'URR consisting of -762T, -716G, -689G, -666T, -633A, followed by -486C and -201A alleles were significantly more frequent in patients with gliomas than in the controls (p < 0.05). No correlation of HLA-G 5'URR variants with sHLA-G plasma level was found. Analysis of HLA-G 5'URR variants with main clinical variables in patients with grade IV gliomas revealed that haploblock carriers of -762CT, -716TG, -689AG, -666GT, -633GA, -486AC, -477GC, -201GA followed by -369AC carriers tend to have lower age at onset as compared to other genotype carriers (p = 0.04). CONCLUSION: Our results suggest genetic association of HLA-G 5'URR variants with risk of developing gliomas and possible contribution of HLA-G to disease pathology.

Characterisation of collagen type I matrices for pathophysiologically relevant spatial cancer cell cultures.

Specific cues provided to cells by the extracellular matrix (ECM) are determined by its composition. Except of collagens other naturally occurring ECM components should be considered in designing 3D models of diseases. We used spectrophotometric and rheological measurements and confocal imaging to characterise collagen matrices of human origin that can be modified by clinically relevant ECM components. pH of the neutralising solution, but not incubation of solidified collagen matrices in serum-free culture medium with pH 5.0-9.0 affected distribution of collagen fibres. Admixture of fibronectin or tenascin-C influenced assembly kinetics and resulted in slight increase in the Young's moduli of the matrices, indicating their incorporation into the collagen matrices. Co-localization of fibronectin with collagen fibres was confirmed by fluorescence imaging. Various cell types relevant for tumour tissue were able to proliferate within the matrices suggesting that they can be used to study role of ECM components in cancer in spatial models.

PIXE analysis of iron in rabbit cerebellum after exposure to radiofrequency electromagnetic fields.

PURPOSE: We investigated iron accumulation and the possible mechanisms in the rabbit cerebellum after the exposure to the real GSM and generated radiofrequency electromagnetic fields (RF EMF) using inductively coupled plasma mass spectrometry (ICP MS) and particles induced X-ray emission (PIXE). MATERIALS AND METHODS: Four groups of rabbits were exposed to the real EMF, generated EMF, combination of both the real and generated signals and the control group with no exposition. For determination of iron concentration in the four groups of cerebellum samples ICP MS was used. Iron accumulation in samples by PIXE analysis using the 3 MeV proton beam was carried out. RESULTS: Iron concentration measured by ICP MS revealed no significant differences for all the groups. PIXE results showed a focal accumulation of iron with the size up to 3 mm. Highest concentration of iron after exposure to real signal was observed. CONCLUSION: We suggest that the iron accumulation after the exposure to RF ELF is not the result of higher permeability of blood-brain barrier and leaking out of iron from the bloodstream into the brain cells and tissues. It could be the result of an iron actuation and its redistribution in the tissue (Fig. 2, Ref. 86).

Phenotypical modifications of immune cells are enhanced by extracellular matrix.

Immune cells not only constitute tumour microenvironment but they may even affect disease prognosis as a result of dual functional roles that they may play in tumour tissues. Two frequently used established immune cell lines (lymphocytic Jurkat and monocytic THP-1) were used to test whether microenvironmental factors, especially molecular components of extracellular matrix, can shape the phenotype of immune cells. Proliferation, morphological and phenotypical analyses were applied to compare behaviour of the immune cells, typically cultured as suspensions in culture medium, with their behaviour in collagen type I-based and Matrigel-based 3D cultures. Density of both immune cell types in routine suspension cultures affected their subsequent proliferation in extracellular matrices. THP-1 cells appeared to be more sensitive to their surrounding microenvironment as judged from extracellular matrix type-dependent changes in their cell doubling times and from slight increase in their diameters in both extracellular matrix-containing cell cultures. Moreover, even chemically uninduced monocytic THP-1 cells were present in a minor fraction as CD68 positive cell population in collagen type I matrix indicating their partial differentiation to macrophages. Observed modifications of immune cells by microenvironmental factors may have profound implications for their roles in healthy and pathological tissues.

Social Noise Exposure in a Sample of Slovak University Students.

PURPOSE: Social noise exposure is currently an emerging problem in adolescents and young adults. Various leisure time activities may be responsible for hearing impairment (temporary or permanent hearing threshold shift or hearing loss). The study aimed to quantify environmental noise from various sources-voluntary (social) noise (personal music players (PMPs), high-intensity noise exposure events), and road traffic noise and to detect hearing disorders in relation to individual listening to PMPs in the sample of young adults living and studying in Bratislava, the capital city of Slovakia. METHODS: The study included 1003 university students (306 men and 697 women, average age 23.1 ± 2) living in Bratislava for 4 or more years; 347 lived in the student housing facility exposed to road traffic noise (LAeq = 67.6 dB) and 656 in the control one (LAeq = 53.4 dB). Respondents completed a validated ICBEN 5-grade scale "noise annoyance questionnaire". In the exposed group a significant source of annoyance was road traffic noise (p < 0.001), noise from entertainment facilities (p < 0.001), industrial noise (p < 0.001), and noise from neighboring flats (p = 0.003). The exposure to PMPs was objectified by the conversion of the subjective evaluation of the volume setting and duration. With the cooperation of the Ear, Nose and Throat (ENT)specialist, we arranged audiometric examinations on the pilot sample of 41 volunteers. RESULTS: From the total sample of respondents, 79.2% reported the use of a PMP in the course of the last week, and the average time was 285 minutes. There was a significant difference in PMP use between the road traffic noise-exposed (85.6%) and the control group (75.8%) (p = 0.01). Among PMP users 30.7% exceeded the lower action value (LAV) for industry (LAeq,8h = 80 dB). On a pilot sample of volunteers (n = 41), audiometry testing was performed indicating a hearing threshold shift at higher frequencies in 22% of subjects. CONCLUSIONS: The results of the study on a sample of young healthy individuals showed the importance of exposure to social noise as well as to road traffic noise and the need for prevention and intervention.

Neuronal morphology alterations in autism and possible role of oxytocin.

Current understanding of the neuroanatomical abnormalities in autism includes gross anatomical changes in several brain areas and microstructural alterations in neuronal cells as well. There are many controversies in the interpretation of the imaging data, evaluation of volume and size of particular brain areas, and their functional translation into a broad autism phenotype. Critical questions of neuronal pathology in autism include the concept of the reversible plasticity of morphological changes, volume alterations of brain areas, and both short- and long-term consequences of adverse events present during the brain development. At the cellular level, remodeling of the actin cytoskeleton is considered as one of the critical factors associated with the autism spectrum disorders. Alterations in the composition of the neuronal cytoskeleton, in particular abnormalities in the polymerization of actin filaments and their associated proteins underlie the functional consequences in behavior resulting in symptoms and clinical correlates of autism spectrum disorder. In the present review, a special attention is devoted to the role of oxytocin in experimental models of neurodevelopmental disorders manifesting alterations in neuronal morphology.

Extracellular matrix affects different aspects of cell behaviour potentially involved in response to aminolevulinic acid-based photoinactivation.

Two-dimensional cell cultures do not seem to be reliable models for anticancer drug discovery and validation. Numerous in vitro tumour models of different complexity have been evaluated with the aim to enhance anticancer drug development, but whether all these models could be considered as physiologically relevant is a question. Even type of the extracellular matrix may markedly influence experimental results and supposedly also clinical treatment outcome. By using three human oesophageal cell lines and three-dimensional cultures based on collagen type I, abundant component of stromal tissue, and Matrigel, a surrogate of basement membrane, we tested the impact of extracellular matrix on different aspects of cell behaviour. We applied live cell fluorescence confocal microscopy in combination with image analysis and supplemented it with immunohistochemical analysis of differentiation markers in fixed samples. We found that cell morphogenesis, differentiation, extracellular vesicle formation, protoporphyrin IX production from aminolevulinic acid and response to subsequent photodynamic intervention induced by red light may be affected by the type of extracellular matrix and these modifications occur in a cell-type dependent manner. Our results demonstrate that the choice of the correct extracellular matrix for in vitro tumour models is crucial for gathering clinically relevant information from in vitro experimental studies.

Merkel-like cell distribution in the epithelium of the human vagina. An immunohistochemical and TEM study.

Human Merkel cells (MCs) were first described by Friedrich S. Merkel in 1875 and named "Tastzellen" (touch cells). Merkel cells are primarily localized in the basal layer of the epidermis and concentrated in touch-sensitive areas. In our previous work, we reported on the distribution of MCs in the human esophagus, so therefore we chose other parts of the human body to study them. We selected the human vagina, because it has a similar epithelium as the esophagus and plays very important roles in reproduction and sexual pleasure. Due to the fact that there are very few research studies focusing on the innervation of this region, we decided to investigate the occurrence of MCs in the anterior wall of the vagina. The aim of our research was to identify MCs in the stratified squamous non-keratinized epithelium of the human vagina in 20 patients. For the identification of Merkel cells by light microscopy, we used antibodies against simple-epithelial cytokeratins (especially anti-cytokeratin 20). We also tried to identify them using transmission electron microscopy. Our investigation confirmed that 10 (50 %) of 20 patients had increased number of predominantly intraepithelial CK20 positive "Merkel-like" cells (MLCs) in the human vaginal epithelium. Subepithelial CK20 positive MLCs were observed in only one patient (5%). We tried to identify them also using transmission electron microscopy. Our investigation detected some unique cells that may be MCs. The purpose of vaginal innervation is still unclear. There are no data available concerning the distribution of MCs in the human vagina, so it would be interesting to study the role of MCs in the vaginal epithelium, in the context of innervation and epithelial biology.

Effects of fractionated whole-brain irradiation on cellular composition and cognitive function in the rat brain.

PURPOSE: The aim of this study was investigate whether histopathological changes in the neurogenic region correlate with appropriate cognitive impairment in the experimental model of radiation-induced brain injury. MATERIALS AND METHODS: Adult male Wistar rats randomized into sham (0 Gy) and two experimental groups (survived 30 and 100 days after treatment) received fractionated whole-brain irradiation (one 5 Gy fraction/week for four weeks) with a total dose of 20 Gy of gamma rays. Morris water maze cognitive testing, histochemistry, immunohistochemistry and confocal microscopy were used to determine whether the cognitive changes are associated with the alteration of neurogenesis, astrocytic response and activation of microglia along and/or adjacent to well-defined pathway, subventricular zone-olfactory bulb axis (SVZ-OB axis). RESULTS: Irradiation revealed altered cognitive functions usually at 100 days after treatment. Neurodegenerative changes were characterized by a significant increase of Fluoro-Jade-positive cells 30 days after irradiation accompanied by a steep decline of neurogenesis 100 days after treatment. A strong astrocytic response and upregulation of the activated microglia were seen in both of experimental groups. CONCLUSIONS: Results shows that fractionated irradiation led to cognitive impairment closely associated with accerelation of neuronal cell death, inhibition of neurogenesis, activation of astrocytes and microglia indicate early delayed radiation-induced changes.

Effects of testosterone and estradiol on anxiety and depressive-like behavior via a non-genomic pathway.

Besides their known slow genomic effects, testosterone and estradiol have rapid effects in the brain. However, their impact on mood-related behavior is not clear. The aim of this study was to investigate the non-genomic pathway of testosterone and estradiol in the amygdala in relation to anxiety and depressive-like behavior. Sham-operated and gonadectomized male rats (GDX) supplemented with testosterone propionate, estradiol, or olive oil were used. Five minutes after administration, anxiety and depression-like behavior were tested. Estradiol increased anxiolytic behavior in the open-field test compared to the GDX group, but administration of testosterone had no significant effect. Besides, c-Fos expression in the medial nucleus of the amygdala significantly increased after testosterone treatment compared to the GDX group, while no significant difference was observed in the central and the basolateral nuclei of the amygdala in the testosterone-treated group compared to the GDX group. In conclusion, estradiol had an anxiolytic effect via a rapid pathway, but no rapid effect of testosterone on anxiety was found. Further studies elucidating whether the rapid effect is mediated by a non-genomic pathway are needed.

Does the non-genomic effect of testosterone on social anxiety require the presence of a classical steroid receptor?

Steroid hormones may act through a rapid mechanism that does not require an intracellular steroid receptor and its effects on gene expression. In this study we have analysed this so-called non-genomic effect of testosterone on social anxiety in rats of both sexes using androgen and oestrogen receptor blockers. Male rats were divided into four groups: SHAM-CTRL (a sham operated group treated with oil as vehicle, n=10), SHAM-TST (a sham operated group treated with testosterone at a dose of 1 mg/kg, n=10), GDX-CTRL (a castrated group treated with oil, n=10) and GDX-TST (a castrated group treated with testosterone at a dose of 1 mg/kg, n=10). Female rats were divided into two groups: OVX-CTRL (an ovariectomized group treated with oil, n=10) and OVX-TST (an ovariectomized group treated with testosterone, n=10). The intracellular androgen receptor was blocked with flutamide and both intracellular oestrogen receptors were blocked with tamoxifen (a selective oestrogen receptor modulator). Rats were tested one hour after oil or testosterone administration in the social interaction test. Although the concentration of testosterone was higher in testosterone groups, no significant difference in social interaction was observed between the groups. In summary, in this first study focusing on the non-genomic effects of testosterone on social interaction no rapid effects of testosterone in adult rats were found. Further studies should analyse potential non-genomic effects of testosterone on other forms of social behaviour.

Chronic renal insufficiency does not induce behavioral and cognitive alteration in rats.

In humans, chronic kidney disease (CKD) is associated with cognitive decline, increase in anxiety, or depression. The underlying mechanisms of these changes remain unclear. The aim of this study was to elucidate whether and how experimentally induced long-term CKD affects cognitive functions in rats. Thirty male Wistar rats underwent 5/6 nephrectomy (5/6 Nx), an established model of CKD, or sham surgery. Development of CKD was monitored using biochemical analyses and confirmed by renal histology. Behavioral tests of anxiety, depression and spatial behavior were performed before, and at 3 and 9 months after the surgery. CKD in 5/6 Nx rats was characterized by significant decrease of renal function, e.g., glomerular filtration rate, and progressive glomerulosclerosis, tubular atrophy, and interstitial fibrosis; and increased plasma uremic toxins. Mortality was higher in 5/6 Nx rats in comparison with controls. Compared to control group, the surviving 5/6 Nx rats presented similar general locomotor activity, depression traits, and spatial abilities (p=0.43, p=0.84, p=0.71, respectively). At 9 months, lower anxiety in the light-dark box test was observed in 5/6 Nx rats if compared with the control group (p=0.02). Despite the development of progressive CKD in 5/6 Nx rats, no expected behavioral changes were observed. Further experimental studies associating behavioral responses to severity of CKD are definitely needed to confirm the solely psychosocial aspect background of CKD-associated cognitive impairment in humans.

The effect of testosterone on the formation of brain structures.

It has been confirmed in several studies that testosterone can significantly affect brain development. Following metabolism of this hormone by 5α-reductase to dihydrotestosterone, testosterone may act via androgen receptors, or after conversion by aromatase to estradiol, it may act via estrogen receptors. The parts of the brain which are changed under the influence of sex hormones are known as sexually dimorphic nuclei, especially in the preoptic area of the hypothalamus. Nevertheless, evidence suggests that testosterone also influences the structure of the hippocampus, specifically CA1 and CA3 areas of the hippocampus, as well as the amygdala. These brain areas are designed to convert information from short-term into long-term memory. In this review, we summarize the effects of testosterone on the organization of brain structures with respect to spatial cognitive abilities in small rodents.

The anxiolytic effect of testosterone in the rat is mediated via the androgen receptor.

Endogenous and exogenous testosterone affects several behavioural traits as shown in human and animal studies. The effects of testosterone can be mediated via androgen or oestrogen receptors, but also via rapid non-genomic effects. The aim of this study was to evaluate whether a single testosterone injection has effects, mediated via the androgen receptor, on anxiety in intact male rats. We hypothesised that administration of testosterone will have an anxiolytic effect, mediated by the androgen receptor. Intact adult male Wistar rats were divided into groups: control, flutamide, testosterone and testosterone with flutamide. Testosterone and flutamide (as an androgen receptor blocker) were applied once, intramuscularly, at a dose of 5mg/kg. Twenty four hours later, rats underwent the following behavioural tests to analyse anxiety: open field test, elevated plus maze and light-dark box. Testosterone was measured in plasma to confirm elevated levels in groups that received testosterone. The levels of testosterone were 2.5-3 fold higher amongst rats administered with testosterone compared to controls. Flutamide did not affect plasma testosterone concentrations. Testosterone administration had no effect on anxiety in the open field and elevated plus maze. In the light-dark transition task, testosterone increased the time spent in the light part of the maze by 80%, an effect which was blocked by flutamide, and which was in support of our hypothesis. Flutamide-treated rats spent more time in the central square of the open field. Using the light-dark box we have shown that a single injection of testosterone decreases anxiety in adult male rats. This effect of increased testosterone was mediated via the androgen receptor as flutamide blocked the anxiolytic effect of exogenous testosterone. Treatment with flutamide blocked the effects of endogenous testosterone and had anxiolytic effects in the open field, suggesting a non-linear relationship between genomic effects of T and anxiety.