Microglial diversity along the hippocampal longitudinal axis impacts synaptic plasticity in adult male mice under homeostatic conditions.

The hippocampus is a plastic brain area that shows functional segregation along its longitudinal axis, reflected by a higher level of long-term potentiation (LTP) in the CA1 region of the dorsal hippocampus (DH) compared to the ventral hippocampus (VH), but the mechanisms underlying this difference remain elusive. Numerous studies have highlighted the importance of microglia-neuronal communication in modulating synaptic transmission and hippocampal plasticity, although its role in physiological contexts is still largely unknown. We characterized in depth the features of microglia in the two hippocampal poles and investigated their contribution to CA1 plasticity under physiological conditions. We unveiled the influence of microglia in differentially modulating the amplitude of LTP in the DH and VH, showing that minocycline or PLX5622 treatment reduced LTP amplitude in the DH, while increasing it in the VH. This was recapitulated in Cx3cr1 knockout mice, indicating that microglia have a key role in setting the conditions for plasticity processes in a region-specific manner, and that the CX3CL1-CX3CR1 pathway is a key element in determining the basal level of CA1 LTP in the two regions. The observed LTP differences at the two poles were associated with transcriptional changes in the expression of genes encoding for Il-1, Tnf-α, Il-6, and Bdnf, essential players of neuronal plasticity. Furthermore, microglia in the CA1 SR region showed an increase in soma and a more extensive arborization, an increased prevalence of immature lysosomes accompanied by an elevation in mRNA expression of phagocytic markers Mertk and Cd68 and a surge in the expression of microglial outward K+ currents in the VH compared to DH, suggesting a distinct basal phenotypic state of microglia across the two hippocampal poles. Overall, we characterized the molecular, morphological, ultrastructural, and functional profile of microglia at the two poles, suggesting that modifications in hippocampal subregions related to different microglial statuses can contribute to dissect the phenotypical aspects of many diseases in which microglia are known to be involved.

Indoleamine 2,3-dioxygenase and regulatory t cells in intestinal mucosa in children with inflammatory bowel disease.

Impaired immune regulation has been suggested as an underlying mechanism of inflammatory bowel disease. Indoleamine 2,3-dioxygenase (IDO) and regulatory T cells expressing FOXP3 are crucial elements of immune regulation. Conversion of FOXP3- lymphocytes to Tregs is one of the functions of IDO. The aim of this study was to evaluate the number of cells expressing FOXP3 and IDO in the lamina propria of intestinal mucosa and to evaluate correlations between these parameters and disease activity. Sixty-six children newly diagnosed with inflammatory bowel disease (41 patients with ulcerative colitis and 25 patients with Crohn’s disease) were included in the study. Clinical activity of the disease was assessed by the Pediatric Ulcerative Colitis Activity Index and the Pediatric Crohn’s Disease Activity Index. Histopathological activity was scored according to the system described by Geboes. The infiltration of FOXP3+ and IDO+ cells was evaluated by immunohistochemistry. Sixteen patients with a diagnosis of irritable bowel syndrome (IBS) served as a control group. Lamina propria demonstrated a significantly higher infiltration of FOXP3+ and IDO+ cells in inflammatory bowel disease compared to the control group (p=0.001, p=0.004, respectively). The number of IDO+ and FOXP3+ cells correlated with clinical and histopathologic activity of Crohn’s disease. A positive correlation between the number of IDO+ and FOXP3+ cells was found in both types of inflammatory disease but not in patients with IBS. We conclude that indoleamine dioxygenase and FOXP3+ cells are upregulated in the intestinal mucosa of children with inflammatory bowel disease. IDO mediated conversion of FOXP3 -T cells to Tregs predominantly occurs in inflammation.

Structural colour printing from a reusable generic nanosubstrate masked for the target image.

Structural colour printing has advantages over traditional pigment-based colour printing. However, the high fabrication cost has hindered its applications in printing large-area images because each image requires patterning structural pixels in nanoscale resolution. In this work, we present a novel strategy to print structural colour images from a pixelated substrate which is called a nanosubstrate. The nanosubstrate is fabricated only once using nanofabrication tools and can be reused for printing a large quantity of structural colour images. It contains closely packed arrays of nanostructures from which red, green, blue and infrared structural pixels can be imprinted. To print a target colour image, the nanosubstrate is first covered with a mask layer to block all the structural pixels. The mask layer is subsequently patterned according to the target colour image to make apertures of controllable sizes on top of the wanted primary colour pixels. The masked nanosubstrate is then used as a stamp to imprint the colour image onto a separate substrate surface using nanoimprint lithography. Different visual colours are achieved by properly mixing the red, green and blue primary colours into appropriate ratios controlled by the aperture sizes on the patterned mask layer. Such a strategy significantly reduces the cost and complexity of printing a structural colour image from lengthy nanoscale patterning into high throughput micro-patterning and makes it possible to apply structural colour printing in personalized security features and data storage. In this paper, nanocone array grating pixels were used as the structural pixels and the nanosubstrate contains structures to imprint the nanocone arrays. Laser lithography was implemented to pattern the mask layer with submicron resolution. The optical properties of the nanocone array gratings are studied in detail. Multiple printed structural colour images with embedded covert information are demonstrated.

Expression of the androgen receptor in the testes and the concentrations of gonadotropins and sex steroid hormones in male turkeys (Meleagris gallopavo) during growth and development.

Androgens, including testosterone (T) and androstenedione (A4), are essential for puberty, fertility and sexual functions. The biological activity of those hormones is mediated via the androgen receptor (AR). The regulation of androgen action in birds is poorly understood. Therefore, the present study analysed mRNA and protein expression of AR in the testes, plasma concentrations of the luteinizing hormone (LH), follicle-stimulating hormone (FSH), T, A4 and oestradiol (E2), as well as the levels of T, A4 and E2 in testicular homogenates of male turkeys (Meleagris gallopavo) at the age of 4, 8, 12, 16, 20, 24 and 28weeks. Plasma concentrations of LH and FSH, as well as plasma and testicular levels of T and A4 began to increase at 20weeks of age. The lowest plasma levels of E2 were noted at 20weeks relative to other growth stages. The 20th week of life seems to be the key phase in the development of the reproductive system of turkeys. The AR protein was found in the nuclei of testicular cells in all examined growth stages. Higher expression of AR protein in the testes beginning at 20weeks of age was accompanied by high plasma concentrations of LH and high plasma and testicular levels of androgens. This relationship seems to be necessary to regulate male sexual function.

Concentrations of the adrenocorticotropic hormone, corticosterone and sex steroid hormones and the expression of the androgen receptor in the pituitary and adrenal glands of male turkeys (Meleagris gallopavo) during growth and development.

Androgens take part in the regulation of puberty and promote growth and development. They play their biological role by binding to a specific androgen receptor (AR). The aim of this study was to evaluate the expression of AR mRNA and protein in the pituitary and adrenal glands, to localize AR protein in luteinizing hormone (LH)-producing pituitary and adrenocortical cells, to determine plasma concentrations of adrenocorticotropic hormone (ACTH) and corticosterone and the concentrations of corticosterone, testosterone (T), androstenedione (A4) and oestradiol (E2) in the adrenal glands of male turkeys at the age of 4, 8, 12, 16, 20, 24 and 28weeks. The concentrations of hormones and the expression of AR varied during development. The expression of AR mRNA and protein in pituitary increased during the growth. The increase of AR mRNA levels in pituitary occurred earlier than increase of AR protein. The percentage of pituitary cells expressing ARs in the population of LH-secreting cells increased in week 20. It suggests that AR expression in LH-producing pituitary cells is determined by the phase of development. The drop in adrenal AR mRNA and protein expression was accompanied by an increase in the concentrations of adrenal androgens. Those results could point to the presence of a compensatory mechanism that enables turkeys to avoid the potentially detrimental effects of high androgen concentrations. Our results will expand our knowledge of the role of steroids in the development of the reproductive system of turkeys from the first month of age until maturity.

Immunohistochemical evidence of the co-localisation of cocaine and amphetamine regulatory peptide with neuronal isoform of nitric oxide synthase, vasoactive intestinal peptide and galanin within the circular muscle layer of the human caecum.

The enteric nervous system consists of about one hundred million of neurons. In big mammals (including humans) intestinal enteric neuronal cells are grouped into three types of intramural ganglia located within myenteric, as well as outer and inner submucosal plexuses, which are connected by numerous nerve fibres. Both nerve fibres and cell bodies located in the gastrointestinal tract utilise a broad spectrum of active substances. One of them is cocaine- and amphetamine-regulated transcript peptide (CART). The goal of the current study was to determinate the distribution and degree of co-localisation of CART with substances taking part in intestinal motor activity by double labelling immunofluorescence technique. During the study CART-, neuronal isoform of nitric oxide synthase (nNOS)-, vasoactive intestinal peptide (VIP)- and/or galanin (GAL) - like immunoreactive (LI) nerve fibres in the circular muscle layer of the human caecum were observed in all patients studied. The degree of co-localisation of particular substances with CART depended on their type. The majority of CART-LI fibres contained simultaneously nNOS, slightly lower degree of co-localisation was observed in the case of the VIP, while simultaneously CART- and GAL-positive nerve fibres were observed less often.

Deep Learning Glioma Grading with the Tumor Microenvironment Analysis Protocol for Comprehensive Learning, Discovering, and Quantifying Microenvironmental Features.

Gliomas are primary brain tumors that arise from neural stem cells, or glial precursors. Diagnosis of glioma is based on histological evaluation of pathological cell features and molecular markers. Gliomas are infiltrated by myeloid cells that accumulate preferentially in malignant tumors, and their abundance inversely correlates with survival, which is of interest for cancer immunotherapies. To avoid time-consuming and laborious manual examination of images, a deep learning approach for automatic multiclass classification of tumor grades was proposed. As an alternative way of investigating characteristics of brain tumor grades, we implemented a protocol for learning, discovering, and quantifying tumor microenvironment elements on our glioma dataset. Using only single-stained biopsies we derived characteristic differentiating tumor microenvironment phenotypic neighborhoods. The study was complicated by the small size of the available human leukocyte antigen stained on glioma tissue microarray dataset - 206 images of 5 classes - as well as imbalanced data distribution. This challenge was addressed by image augmentation for underrepresented classes. In practice, we considered two scenarios, a whole slide supervised learning classification, and an unsupervised cell-to-cell analysis looking for patterns of the microenvironment. In the supervised learning investigation, we evaluated 6 distinct model architectures. Experiments revealed that a DenseNet121 architecture surpasses the baseline's accuracy by a significant margin of 9% for the test set, achieving a score of 69%, increasing accuracy in discerning challenging WHO grade 2 and 3 cases. All experiments have been carried out in a cross-validation manner. The tumor microenvironment analysis suggested an important role for myeloid cells and their accumulation in the context of characterizing glioma grades. Those promising approaches can be used as an additional diagnostic tool to improve assessment during intraoperative examination or subtyping tissues for treatment selection, potentially easing the workflow of pathologists and oncologists.

Lung impairment in scleroderma.

Scleroderma typically manifests as fibrosis of the skin, but may also involve other organs, particularly the lungs. Interstitial lung disease and functional abnormalities are observed in the majority of patients. The aim of this study was to evaluate radiological changes in the lungs and their correlation with functional disorders in scleroderma patients. The study was conducted in 37 scleroderma patients (F/M-31/6). High resolution computed tomography (HRCT), Warrick score system and spirometry, body plethysmography, and lung diffusion examinations (DLco) were performed. The HRCT showed septal and subpleural lines in 70%, ground-glass opacities in 51%, and honeycomb lungs in 30% of the cases. The DLco values were decreased in 92% of the patients. Total lung capacity (TLC) showed a restrictive pattern in 24% of the patients, and only in 11% of them obstruction predominated. The Warrick score correlated inversely with both DLco (r=0.36; p>0.05). Interstitial lung disease often coexists with scleroderma and is accompanied by functional lung abnormalities.

Seasonal changes in the expression of the androgen receptor in the testes of the domestic goose (Anser anser f. domestica).

It is generally acknowledged that seasonal fluctuations in the morphology and function of bird testes are primarily regulated by seasonal changes in circulating concentrations of testosterone (T) which mediates its action via the androgen receptor (AR). However, it has not yet been elucidated whether gonadal sensitivity to androgens also varies across the bird reproductive cycle. In order to answer the above question, this study makes the first ever attempt to account for the gonadal expression of the AR gene and protein in relation to circulating and testicular T concentrations in the gonads of male birds during the reproductive cycle. The experimental model used in this study was the domestic goose, Anser anser f. domestica, a species with three distinct phases of the annual reproductive cycle: the breeding season in March, the non-breeding season in July and the sexual reactivation phase in November. The plasma and testicular T concentrations were highest in the breeding season, followed by a dramatic decline in the non-breeding season with a successive rise in the sexual reactivation phase. Interestingly, we observed the divergent effect of season on AR mRNA and protein expression. Whereas the AR gene expression showed a nearly inverse relationship with T levels, the seasonal variations in AR protein levels primarily reflected the differences in T concentrations. The results of our study also indicated that regardless of the examined phase of the season, an abundance of AR protein was found only in the nuclei of Leydig and Sertoli cells and myoid cells. The above supports the observation that somatic cells are the targets for androgen action in bird testes. Summarizing, this study revealed that seasonal variations in sensitivity to androgens in the gonads of male birds are reflected in variations in the availability of their cognate receptors. Furthermore, a different pattern of seasonal expression of the AR gene and protein suggests that the AR system is subject to complex regulation that includes both steroid-dependent and steroid-independent factors.

Gliomas induce and exploit microglial MT1-MMP expression for tumor expansion.

Diffuse infiltration of glioma cells into normal brain tissue is considered to be a main reason for the unfavorable outcomes of patients with malignant gliomas. Invasion of glioma cells into the brain parenchyma is facilitated by metalloprotease-mediated degradation of the extracellular matrix. Metalloproteases are released as inactive pro-forms and get activated upon cleavage by membrane bound metalloproteases. Here, we show that membrane type 1 metalloprotease (MT1-MMP) is up-regulated in glioma-associated microglia, but not in the glioma cells. Overexpression of MT1-MMP is even lethal for glioma cells. Glioma-released factors trigger the expression and activity of MT1-MMP via microglial toll-like receptors and the p38 MAPK pathway, as deletion of the toll-like receptor adapter protein MyD88 or p38 inhibition prevented MT1-MMP expression and activity in cultured microglial cells. Microglial MT1-MMP in turn activates glioma-derived pro-MMP-2 and promotes glioma expansion, as shown in an ex vivo model using MT1-MMP-deficient brain tissue and a microglia depletion paradigm. Finally, MyD88 deficiency or microglia depletion largely attenuated glioma expansion in 2 independent in vivo models.

Adiponectin affects uterine steroidogenesis during early pregnancy and the oestrous cycle: An in vitro study.

Reproduction in females is an energetically demanding process. We assumed that adiponectin (ADPN), known for its role in energy balance maintenance, is also engaged in the regulation of uterine steroidogenesis in the pig. We determined the impact of ADPN alone or in combination with insulin (INS) on testosterone (T), estrone (E1) and estradiol (E2) secretion by porcine endometrium and myometrium, uterine expression of CYP17A1 and CYP19A3 genes, and endometrial abundance of P450C17 and P450AROM proteins during the peri-implantation period and the oestrous cycle, using radioimmunoassay, qPCR, and Western Blot, respectively. During pregnancy, in the endometrial explants from days 10-11, ADPN decreased CYP17A1 gene expression, P450C17 protein abundance and T secretion, whereas increased E1 secretion. On days 12-13 of pregnancy, ADPN decreased CYP17A1 and CYP19A3 expression, P450C17 and P450AROM protein abundance and E1 secretion, but stimulated T secretion. On days 15-16 of pregnancy, ADPN decreased P450C17 protein accumulation but enhanced CYP19A3 expression and E1 secretion. On days 27-28 of pregnancy, ADPN increased CYP17A1 and CYP19A3 mRNA content and T secretion in this tissue and decreased P450C17 content. ADPN effect on myometrial explants was dependent on stage of gestation or oestrous cycle. Moreover, INS treatment modulated basal and ADPN-affected steroidogenic enzymes gene and protein expression and steroids secretion. The results obtained indicate that ADPN may affect processes required for successful implantation such as steroidogenesis. ADPN and INS were also shown to modulate each other action, which indicates that the proper course of uterine steroidogenesis may be dependent on both hormones' interaction.

The expression of chemerin and its receptors (CMKLR1, GPR1, CCRL2) in the porcine uterus during the oestrous cycle and early pregnancy and in trophoblasts and conceptuses.

Recent research has demonstrated that chemerin may take part in the regulation of reproduction. The aim of this study was to determine the expression of chemerin system - chemerin and its receptors, chemokine-like receptor 1 (CMKLR1), G protein-coupled receptor 1 (GPR1) and C-C chemokine receptor-like 2 (CCRL2) - in the porcine uterus during the oestrous cycle and early pregnancy, and in trophoblasts and conceptuses by real-time PCR and western blotting. Chemerin concentrations in uterine luminal flushings (ULF) were determined using ELISA test. In the endometrium, the highest expression of chemerin and GPR1 proteins was observed during the mid-luteal phase; CMKLR1, during the late luteal phase; and CCRL2, during the follicular phase of the cycle. In the myometrium, chemerin protein expression was enhanced during the early luteal phase, and chemerin receptor proteins were highly expressed during the follicular phase. In the endometrium of pregnant pigs, the highest expression of chemerin and CCRL2 protein was observed during implantation; CMKLR1, during placentation; and GPR1, during embryo migration. In the myometrium, chemerin and CCRL2 protein expression increased at the end of implantation, and the expression of CMKLR1 and GPR1 protein was enhanced during implantation. In the conceptuses and trophoblasts, the highest expression of chemerin system proteins was observed during placentation, with the exception of GPR1 protein in the trophoblasts. The highest concentrations of the analysed adipokine were observed in ULF during the luteal phase of the cycle and during maternal recognition of pregnancy. This is the first study to demonstrate that the expression of the chemerin system in the porcine uterus, conceptuses and trophoblasts, and chemerin concentrations in ULF are influenced by the hormonal milieu in different stages of the oestrous cycle and in early pregnancy. The present results also suggest that chemerin is implicated in the regulation of reproductive functions in pigs.

Molecular mechanisms leading to neuroprotection/ischemic tolerance: effect of preconditioning on the stress reaction of endoplasmic reticulum.

Ischemic tolerance can be developed by prior ischemic non-injurious stimulus preconditioning. The molecular mechanisms underlying ischemic tolerance are not yet fully understood. The purpose of this study is to evaluate the effect of preconditioning/preischemia on ischemic brain injury. We examined the endoplasmic reticulum stress response (unfolded protein response (UPR)) by measuring the mRNA and protein levels of specific genes such as ATF6, GRP78, and XBP1 after 15 min 4-VO ischemia and different times of reperfusion (1, 3, and 24 h). The data from the group of naïve ischemic rats were compared with data from the group of preconditioned animals. The results of the experiments showed significant changes in the gene expression at the mRNA level in the all ischemic/reperfusion phases. The influence of preischemia on protein level of XBP was significant in later ischemic times and at 3 h, the reperfusion reached 230% of the controls. The protein levels of GRP78 in preischemic animals showed a significant increase in ischemic and reperfusion times. They exceeded to 50% levels of corresponding naïve ischemic/reperfusion groups. Preconditioning also induced remarkable changes in the levels of ATF6 protein in the ischemic phase (about 170%). The levels of ATF6 remained elevated in earlier reperfusion times (37 and 62%, respectively) and persisted significantly elevated after 24 h of reperfusion. This data suggest that preconditioning paradigm (preischemia) underlies its neuroprotective effect by the attenuation of ER stress response after acute ischemic/reperfusion insult.

Molecular analysis of endoplasmic reticulum stress response after global forebrain ischemia/reperfusion in rats: effect of neuroprotectant simvastatin.

Simvastatin is a cholesterol-lowering agent whose functional significance and neuroprotective mechanism in ischemic brain injury is not yet solved. The purpose of this study is to evaluate the effect of simvastatin on ischemic brain injury. We examined the endoplasmic reticulum stress response (UPR/unfolded protein response), by measuring the mRNA and protein levels of specific genes such as ATF6, GRP78, and XBP1 after 15 min 4-VO ischemia and different times of reperfusion (1, 3, and 24 h). The results from the group of naïve ischemic rats were compared with results from the group of pre-treated animals with simvastatin. The results of the experiments showed significant increase in all genes at the mRNA level in ischemic phase (about 43% for XBP1, 58% for GRP78, and 39% for ATF6 more than control). The protein level of XBP1 was decreased in pre-treated animals at ischemic phase and first hour of reperfusion (about 15% less), and did not reach control levels. The protein levels of GRP78 were maximal at third hour of reperfusion in statin group with a small decrease at 24 h of reperfusion in both groups. The levels of ATF6 mRNA in statin-treated animals was higher in comparison to non-statin animals at the ischemic phase and the third hour of reperfusion (about 35% higher), which was also translated into the higher protein level. This could indicate that one of the main proteins targeted to enhance neuroprotective effect to ER during the first two hours of reperfusion was ATF6 protein, the levels of which were 60% higher than in non-treated animals. These data suggest that simvastatin, in addition to the proposed neuroprotective effect, exerts a neuroprotective role in the attenuation of ER stress response after acute ischemic/reperfusion insult.

Diagnostic value of plasma expression of microRNAs complementary to Drosha and Dicer in lung cancer patients.

OBJECTIVE: Lung cancer (LC) is diagnosed mostly in advanced, non-operable stage, with poor prognosis. The analysis of microRNAs may be a useful tool for early and non-invasive detection of cancer. Dicer and Drosha are enzymes with an essential role for microRNA biogenesis. The aim of our study was to analyze the expression of miRNA-27a-3p, miRNA-31, miRNA-182, miRNA-195 with the ability to reciprocal regulation of Dicer and Drosha expression in lung cancer patients. PATIENTS AND METHODS: The relative expression of microRNAs was detected by qPCR in plasma of 160 LC patients. The U-Mann Whitney test was used to compare the relative expression between particular groups of lung cancer patients and healthy individuals. The diagnostic value of microRNAs examination was analyzed using a receiver operating curve. RESULTS: We demonstrated that the plasma levels of miRNA-27, miRNA-31 and miRNA-182 were significantly higher and miRNA-195 significantly lower in the whole group of LC patients and in patients with early stages of NSCLC, in comparison with healthy donors. ROC analysis showed that four studied microRNAs have a potential diagnostic value for early stages of NSCLC with AUC=0.95 for miRNA-27a (94% sensitivity and 81% specificity, p=0.0001), 0.71 for miRNA-31 (73% sensitivity and 61% specificity, p=0.001) 0.77 for miRNA-182 (70% sensitivity and 79% specificity, p=0.0001) and 0.82 for miRNA-195 (74% sensitivity and 80% specificity, p=0.0001). CONCLUSIONS: We have proved that the expression of miRNA-27a-3p, miRNA-31, miRNA-182, and miRNA-195 in patients with LC is different from the expression of these molecules in healthy people. The examination of these microRNAs in plasma could be used in non-invasive lung cancer diagnosis.

Serum gastrin concentration and changes in G and D cell densities in gastric antrum in children with chronic gastritis.

BACKGROUND: Elevated gastrin concentration leading to gastritis is explained as the effect of change in the density of D and G cells. The aim of the study was to determine and compare fasting serum gastrin concentrations, G and D cell densities in gastric antrum mucosa in children with chronic gastritis and in children with no gastritis or Helicobacter pylori infection. MATERIAL AND METHODS: A total of 184 patients aged 6-18 years, with chronic abdominal pain underwent endoscopic examination. We created three groups: I--patients with chronic gastritis and H. pylori infection; II--patients with chronic gastritis but no H. pylori infection; III--patients with neither gastric mucosal abnormalities nor H. pylori infection. G and D cell densities were determined in the biopsy specimens (using Rbalpha H Gastrin & Somatostatin antibodies). Fasting serum gastrin concentrations were measured using a Beckmann gamma-counter and a GASK-PR kit. RESULTS: The mean serum gastrin concentration in group I was higher when compared with group II (p = 0.04) and group III (p = 0.019). No statistically significant differences were found between groups II and III (p = 0.91). There were no statistically significant differences in G and D cell densities between groups. CONCLUSION: The mean G/D cell ratios in groups I and III were almost identical. The mean fasting serum gastrin concentration was higher in children with both chronic gastritis and H. pylori infection compared with patients without infection or without antral inflammation. No difference in the G cell density or D cell density in children was found, regardless of the presence or absence of gastritis or H. pylori infection.

Degradation kinetics and mechanism of pentoxifylline by ultraviolet activated peroxydisulfate.

Degradation of pentoxifylline (PTX) by sodium peroxydisulfate (SPDS) assisted by UV irradiation has been investigated in deionized water. The treatment was more favorable over direct photolysis or peroxydisulfate oxidation alone. The effects of various parameters, including different dosage of oxidant agent, PTX concentration, initial solution pH levels, and the presence of inorganic ions like chloride, nitrate and carbonate have been evaluated. The rate of PTX decomposition depends on the oxidant agent dose. The highest degradation was determined at pH 10.5, which can be explained by the generation of additional hydroxyl radicals (HO˙) in the reaction between sulfate radicals and hydroxide ions. The presence of inorganic ions, especially the carbonate ions quench valuable sulfate radicals and have successfully retarded the PTX decomposition. Six PTX oxidation products were identified using UPLC-QTOF-MS for trials in a basic environment. The main degradation product (3,7-dimethyl-6-(5-oxohexyloxy)-3,7-dihydro-2H-purin-2-one) was isolated by column chromatography and identified by 1HNMR and LC MS analyzes.

Some chemotherapeutics-treated colon cancer cells display a specific phenotype being a combination of stem-like and senescent cell features.

Colorectal cancer (CRC) is the second leading cause of death among cancer patients in the Northern countries. CRC can reappear a long time after treatment. Recent clinical studies demonstrated that, in response to chemotherapy, cancer cells may undergo stress-induced premature senescence (SIPS), which typically results in growth arrest. Nonetheless, these senescent cells were reported to divide in an atypical manner and thus contribute to cancer re-growth. Therefore, we examined if SIPS escape may follow treatment with chemotherapeutics used clinically: 5-fluorouracil (5-FU), oxaliplatin (OXA) and irinotecan (IRINO). To mimic the therapeutic regimes we exposed human colon cancer HCT116 and SW480 cells to repeated cycles of drug treatment. The cells treated with 5-FU or IRINO exhibited several hallmarks of SIPS: growth arrest, increased size and granularity, polyploidization, augmented activity of the SA-ß-galactosidase, accumulation of P21 and CYCLIN D1 proteins, and the senescence-associated secretory phenotype. Moreover, re-population of the cancer cell cultures was delayed upon treatment with the senescence-inducing agents. At the same time, we detected a subpopulation of senescent colon cancer cells with features of stemness: elevated NANOG expression, exclusion of Hoechst 33342 (typical for side population) and increased CD24 expression. Additionally, rare, polyploid cells exhibited blastocyst-like morphology and produced progeny. In parallel, majority of chemotherapeutics-treated cells underwent mesenchymal to epithelial transition, as the percentage of CD44-positve cells was reduced, and levels of E-cadherin (epithelial marker) were elevated. Our study demonstrates that a subpopulation of chemotherapeutics-treated colon cancer cells display a specific phenotype being a combination of stem-like and senescent cell features. This may contribute to their resistance to chemotherapy and their ability to re-grow cancer after completion of therapeutic intervention.

Serum from patients with chronic obstructive pulmonary disease promotes proangiogenic behavior of the vascular endothelium.

OBJECTIVE: It has been documented that COPD is a risk factor for lung cancer. In COPD patients, changes in lung angiogenesis - a critical process in the development of lung cancer - have been poorly investigated. We aimed to determine whether serum from COPD patients could promote the proangiogenic capabilities of endothelial cells in vitro. PATIENTS AND METHODS: The research was carried out using sera from COPD patients and healthy volunteers, endothelial cells EA.hy926, and bronchial epithelial cells. The concentration of angiogenic molecules was quantified using ELISA tests. The proliferation and migration of EA.hy926 were tested using fluorescence-based methods. Tube formation was analyzed with a commercially available assay. RESULTS: Sera from COPD patients and conditioned media generated by epithelial cells exposed to these sera stimulate proliferation, but not migration, of EA.hy926. This coincided with increased tube formation in both experimental regimens. The sera from COPD patients contained increased levels of CCL2, CCL21, and HGF, whereas the conditioned media generated by epithelial cells treated with these sera exhibited increased levels of CCL2, CCL21, CXCL8, FGF, and sICAM-1. The concentration of angiogenic markers in the sera and conditioned media, and their effect on the behavior of the endothelium were independent of smoking status (COPD and controls), stage of obstruction, and disease group (COPD). CONCLUSIONS: The increased incidence of lung malignancy in COPD patients may be associated, at least to some extent, with the direct and indirect proangiogenic activity of their sera (via alterations in the secretome of epithelial cells).