The Role of Apelin and Apelin Receptor Expression in Migration and Invasiveness of Colon Cancer Cells.

BACKGROUND/AIM: Colon cancer is the second deadliest malignancy for human. Its correlation with obesity has led to an increasing number of studies focusing on the role of adipokines in colon cancer development. Apelin, which belongs to the family of adipokines, affects several pathological processes, including heart diseases, obesity and carcinogenesis. In this study, we examined the importance of apelin and apelin receptor (APJ) during motility regulation of colon cancer cells. MATERIALS AND METHODS: Colon cancer cells with overexpression of apelin receptor, as well as cells with down-regulation of apelin were used in this study. Migration and invasion ability was tested using Transwell® filters. The proteolytic activity was analyzed with fluorescent-substrate degradation assay and gelatin zymography. We also used confocal microscopy to examine migratory protrusion formation and the localization of MT1-MMP. The levels of AKT and ERK kinases were evaluated using Western blotting assay. RESULTS: Overexpression of APJ receptor resulted in increased migration and invasion abilities through stimulation of migratory protrusion formation and proteolytic activity. These processes were mediated by PI3K/AKT and MAPK signaling pathways. Opposite effect was obtained when the level of apelin was down-regulated. CONCLUSION: The level of apelin and its receptor is strictly connected with regulation of migration and invasion of colon cancer cells. Therefore, apelinergic system seems to be a promising target for anti-cancer therapy.

Combination of Selected MET and EGFR Inhibitors Decreases Melanoma Cells' Invasive Abilities.

We have previously shown that combination of foretinib, an inhibitor of MET (hepatocyte growth factor receptor), with gefitinib or lapatinib, inhibitors of EGFR (epidermal growth factor receptor), has a synergistic cytotoxic effect on melanoma cells. However, there are cancer cells resistant to drugs' treatment which are still able to invade. Thus, in this study, we examined the influence of these drugs on invasive abilities of melanoma cells. To investigate cell migration and invasion, Transwell inserts and wound healing assay were used. Cell viability was evaluated by XTT method, while invadopodia formation by immunocytochemistry. Level of phosphorylated Src kinase (pSrc) was verified by Western blot. Proteolytic activity of cells was analyzed using gelatin conjugated with fluorescein degradation assay and gelatin zymography. Combination of used inhibitors diminished cell movement, resulting in smaller distances covered by cells, and decreased the ratio of cells with ability to cross the Transwell inserts. These inhibitors induced changes in formation of invadopodia and actin cytoskeleton organization. Their application also decreased the level of pSrc kinase. Furthermore, used drugs led to reduction of proteolytic activity of examined cells. Our data support the idea that simultaneous targeting of EGFR and MET could be a promising therapeutic strategy inhibiting not only tumor cell growth but also its metastasis.

Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells.

Epidermal and hepatocyte growth factors can stimulate invasive abilities of melanoma cells, while treatment with combination of their receptors' (EGFR and MET, respectively) inhibitors reduces viability of these cells, as we have previously shown. Proposed therapy has potential; however, used drugs block more than one goal effectively, what raises the question about the real target of analysed inhibitors. For this reason, we analysed direct involvement of these receptors in the invasion of melanoma cells inducing EGFR and MET up- and down-regulations in examined cells. Results were acquired with assays evaluating cell migration and invasion (scratch wound assay, Transwell filter-based method and single-cell tracking). We revealed that cells' motile abilities are increased after EGFR overexpression and decreased following EGFR and MET silencing. This outcome correlates with elevated (EGFR up-regulation) or reduced (EGFR/MET down-regulation) number of formed invadopodia, visualized with immunofluorescence, and their rate of proteolytic abilities, evaluated by fluorescent gelatin degradation assay, and gelatin zymography, compared to control cells. Above-mentioned data indicate that both-EGFR and MET signalling is directly connected with melanoma cells invasion, what establishes these receptors as promising targets for anti-cancer treatment.

Evaluation of Apelin and Apelin Receptor Level in the Primary Tumor and Serum of Colorectal Cancer Patients.

Colorectal cancer is the second deadliest tumor, which has a positive correlation with obesity which led to increasing interest in the relationship between adipokines and cancer progression. Apelin is a secreted peptide involved in regulation of tumor progression and invasiveness. In this study, we examined apelin and apelin receptor expression level in colorectal cancer. Apelin, and its receptor mRNA, and protein expression levels were measured in tumor tissue of 56 surgically treated colorectal adenocarcinoma (CRC) patients. We also analyzed apelin and apelin receptor protein levels in sera of 56 CRC patients and 27 healthy controls. The mRNA and protein level of this peptide and its receptor was higher in tumors than that in control tissue. Serum levels of apelin and apelin receptor were increased in CRC patients in comparison to controls. The concentration of serum apelin level significantly increased in individuals with lymph node and distant metastasis. Obtained results suggest that apelin could be an important factor in progression of colorectal carcinoma.

Apelin Effects Migration and Invasion Abilities of Colon Cancer Cells.

Colon cancer is one of the most common cancer types. Its positive correlation with general obesity has led to increasing amounts of research focusing on the role of adipokines in colon cancer development. Apelin is a peptide released by adipose tissue that could affect many cellular processes connected with carcinogenesis. In this study, we examined the role of apelin in the motility regulation of colon cancer cells. We showed that the effect of four different apelin peptides increased the ability of cancer cells to migrate and invade examined cells trough influencing migratory protrusions formation and actin cytoskeleton rearrangement. Additionally, using confocal microscopy, we noticed that apelin stimulated the proteolytic activity of cancer cells, especially increasing the level of membrane-type 1 matrix metalloprotease. Taken together, apelin increased the movement of colon cancer cells through several possible mechanisms. Moreover, better understanding the process through which apelin regulates cancer development is still necessary to the creation of novel anti-cancer therapy.

Chronic Inflammatory Microenvironment in Epidermodysplasia Verruciformis Skin Lesions: Role of the Synergism Between HPV8 E2 and C/EBPß to Induce Pro-Inflammatory S100A8/A9 Proteins.

Persistent genus ß-HPV (human papillomavirus) infection is a major co-factor for non-melanoma skin cancer in patients suffering from the inherited skin disease epidermodysplasia verruciformis (EV). Malignant EV lesions are particularly associated with HPV type 5 or 8. There is clinical and molecular evidence that HPV8 actively suppresses epithelial immunosurveillance by interfering with the recruitment of Langerhans cells, which may favor viral persistence. Mechanisms how persistent HPV8 infection promotes the carcinogenic process are, however, less well understood. In various tumor types chronic inflammation has a central role in tumor progression. The calprotectin complex consisting of S100A8 and S100A9 proteins has recently been identified as key driver of chronic and tumor promoting inflammation in skin carcinogenesis. It induces chemotaxis of neutrophil granulocytes and modulates inflammatory as well as immune responses. In this study, we demonstrate that skin lesions of EV-patients are massively infiltrated by inflammatory cells, including CD15+ granulocytes. At the same time we observed a very strong expression of S100A8 and S100A9 proteins in lesional keratinocytes, which was mostly confined to the suprabasal layers of the epidermis. Both proteins were hardly detected in non-lesional skin. Further experiments revealed that the HPV8 oncoproteins E6 and E7 were not involved in S100A8/A9 up-regulation. They rather suppressed differentiation-induced S100A8/A9 expression. In contrast, the viral transcription factor E2 strongly enhanced PMA-mediated S100A8/A9 up-regulation in primary human keratinocytes. Similarly, a tremendous up-regulation of both S100 proteins was observed, when minute amounts of the PMA-inducible CCAAT/enhancer binding protein ß (C/EBPß), which is expressed at low levels in the suprabasal layers of the epidermis, were co-expressed together with HPV8 E2. This confirmed our previous observation that C/EBPß interacts and functionally synergizes with the HPV8 E2 protein in differentiation-dependent gene expression. Potent synergistic up-regulation of S100A8/A9 was seen at transcriptional and protein levels. S100A8/A9 containing supernatants from keratinocytes co-expressing HPV8 E2 and C/EBPß significantly induced chemotaxis of granulocytes in migration assays supporting the relevance of our finding. In conclusion, our data suggest that the HPV8 E2 protein actively contributes to the recruitment of myeloid cells into EV skin lesions, which may support chronic inflammation and progression to skin cancer.

Identification of C/EBPα as a novel target of the HPV8 E6 protein regulating miR-203 in human keratinocytes.

Patients suffering from Epidermodysplasia verruciformis (EV), a rare inherited skin disease, display a particular susceptibility to persistent infection with cutaneous genus beta-human papillomavirus (beta-HPV), such as HPV type 8. They have a high risk to develop non-melanoma skin cancer at sun-exposed sites. In various models evidence is emerging that cutaneous HPV E6 proteins disturb epidermal homeostasis and support carcinogenesis, however, the underlying mechanisms are not fully understood as yet. In this study we demonstrate that microRNA-203 (miR-203), a key regulator of epidermal proliferation and differentiation, is strongly down-regulated in HPV8-positive EV-lesions. We provide evidence that CCAAT/enhancer-binding protein α (C/EBPα), a differentiation-regulating transcription factor and suppressor of UV-induced skin carcinogenesis, directly binds the miR-203 gene within its hairpin region and thereby induces miR-203 transcription. Our data further demonstrate that the HPV8 E6 protein significantly suppresses this novel C/EBPα/mir-203-pathway. As a consequence, the miR-203 target ΔNp63α, a proliferation-inducing transcription factor, is up-regulated, while the differentiation factor involucrin is suppressed. HPV8 E6 specifically down-regulates C/EBPα but not C/EBPß expression at the transcriptional level. As shown in knock-down experiments, C/EBPα is regulated by the acetyltransferase p300, a well-described target of cutaneous E6 proteins. Notably, p300 bound significantly less to the C/EBPα regulatory region in HPV8 E6 expressing keratinocytes than in control cells as demonstrated by chromatin immunoprecipitation. In situ analysis confirmed congruent suprabasal expression patterns of C/EBPα and miR-203 in non-lesional skin of EV-patients. In HPV8-positive EV-lesions both factors are potently down-regulated in vivo further supporting our in vitro data. In conclusion our study has unraveled a novel p300/C/EBPα/mir-203-dependent mechanism, by which the cutaneous HPV8 E6 protein may expand p63-positive cells in the epidermis of EV-patients and disturbs fundamental keratinocyte functions. This may drive HPV-mediated pathogenesis and may potentially also pave the way for skin carcinogenesis in EV-patients.

Strontium-doped organic-inorganic hybrids towards three-dimensional scaffolds for osteogenic cells.

Biomimetic organic-inorganic hybrid bioscaffolds are developed to complement or replace damaged fragments in bone tissue surgery. The aim of this work was to develop a simple and fast method to prepare composite material for bone engineering, avoiding time consuming and complex methodologies. The resulting materials (also called in this work as hybrid composites or hybrid scaffolds) have a three-dimensional macroporous polymer-like network derived from triethoxyvinylsilane (TEVS) and 2-hydroxyethylmethacrylate (HEMA) monomers, with incorporated calcium, strontium, and phosphate ions. The materials were fully characterized using FT-IR, biomineralization studies, scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy, scratch tests, Young's modulus and compressive strength tests, and gas physisorption. We report a comprehensive study on the in vitro effect of novel strontium doped materials on human bone cells. In vitro investigations were conducted using a normal human osteoblast cell line that mimics the cellular events of the in vivo intramembranous bone formation process. The materials do not have a negative impact on the survival of the normal human osteoblasts; moreover, materials doped with strontium show that not only are cells able to survive, but they also attach to and grow on a bioscaffolds surface. For this reason, they may be used in future in vivo experiments.

Recognition of bacterial signal peptides by mammalian formyl peptide receptors: a new mechanism for sensing pathogens.

Formyl peptide receptors (FPRs) are G-protein-coupled receptors that function as chemoattractant receptors in innate immune responses. Here we perform systematic structure-function analyses of FPRs from six mammalian species using structurally diverse FPR peptide agonists and identify a common set of conserved agonist properties with typical features of pathogen-associated molecular patterns. Guided by these results, we discover that bacterial signal peptides, normally used to translocate proteins across cytoplasmic membranes, are a vast family of natural FPR agonists. N-terminally formylated signal peptide fragments with variable sequence and length activate human and mouse FPR1 and FPR2 at low nanomolar concentrations, thus establishing FPR1 and FPR2 as sensitive and broad signal peptide receptors. The vomeronasal receptor mFpr-rs1 and its sequence orthologue hFPR3 also react to signal peptides but are much more narrowly tuned in signal peptide recognition. Furthermore, all signal peptides examined here function as potent activators of the innate immune system. They elicit robust, FPR-dependent calcium mobilization in human and mouse leukocytes and trigger a range of classical innate defense mechanisms, such as the production of reactive oxygen species, metalloprotease release, and chemotaxis. Thus, bacterial signal peptides constitute a novel class of immune activators that are likely to contribute to mammalian immune defense against bacteria. This evolutionarily conserved detection mechanism combines structural promiscuity with high specificity and enables discrimination between bacterial and eukaryotic signal sequences. With at least 175,542 predicted sequences, bacterial signal peptides represent the largest and structurally most heterogeneous class of G-protein-coupled receptor agonists currently known for the innate immune system.

Differential regulation of human papillomavirus type 8 by interferon regulatory factors 3 and 7.

The genus ß human papillomavirus (HPV) type 8 is associated with nonmelanoma skin cancer in patients with epidermodysplasia verruciformis, and evidence for its protumorigenic potential in the general population increases. To date, strategies to suppress genus ß HPV infections are limited. Interferon regulatory factors IRF-3 and IRF-7 play key roles in the activation of the innate immune response to viral infections. In this study, we show for the first time that both IRF-3 and IRF-7 regulate transcription of a papillomavirus, but with opposing effects. IRF-7, expressed in the suprabasal layers of human epidermis, increased HPV8 late promoter activity via direct binding to viral DNA. UV-B light-induced activation of the HPV8 promoter involved IRF-7 as a downstream effector. In contrast, IRF-3, expressed in all layers of human epidermis, induced strong HPV8 suppression in primary keratinocytes. IRF-3-mediated suppression prevailed over IRF-7-induced HPV8 transcription. Unlike the E6 oncoprotein of the mucosal high-risk HPV16, the HPV8 E6 protein did not bind to IRF-3 and only weakly antagonized its activity. Strong antiviral activity was also observed, when keratinocytes were treated with potent IRF-3 activators, poly(I:C) or RNA bearing 5' phosphates. In conclusion, we show that IRF-3 activation induces a state of cell-autonomous immunity against HPV in primary human keratinocytes. Our study suggests that local application of IRF-3-activating compounds might constitute an attractive novel therapeutic strategy against HPV8-associated diseases, particularly in epidermodysplasia verruciformis patients.

Association of UDP-glucuronosyltransferase 1A9 (UGT1A9) gene polymorphism with kidney allograft function.

BACKGROUND: UDP-glucuronosyltransferases (UGTs) are a group of enzymes involved in the detoxification and excretion of xeno- and endobiotics. Polymorphic variants of the UGT1A9 gene were shown to influence exposition to mycophenolate mophetil (MMF), a common immunosuppressive drug used in kidney allograft recipients. Therefore, the aim of this study was to evaluate an association between key clinical features of kidney post-transplant course in patients receiving MMF therapy and UGT1A9-2152C>T and -275 T>A SNPs, known to induce UGT1A9 gene expression and UGT1A9 98T>C, resulting in reduced enzyme activity. MATERIAL/METHODS: DNA was isolated from peripheral blood of kidney allograft recipients (n=103) and a control group representing the background population of Poland (n=450). Presence of the analyzed SNP was detected using the PCR restriction fragment length polymorphism (RFLP) method. Accuracy of the applied method was confirmed by DNA sequencing. RESULTS: In patients carrying the UGT1A9-2152T and -275A minor alleles we observed a trend of increased risk of acute allograft rejection within 3 months after transplantation, but this difference was at the border of significance. However, the UGT1A9 98C allele was found to be associated with diminished estimated glomerular filtration rate (eGFR) during the first year after engraftment and transient proteinuria in the first and second month post-transplantation. This association was not observed for UGT1A9-2152C>T and -275 T>A. Our data show that transplanted kidney function may be affected in patients carrying UGT1A9 98C allele and receiving MMF. CONCLUSIONS: Genotyping of the functional UGT1A9 SNP may be of practical use in kidney transplant recipients.

[Microbiological assesssment of efficiency chemothermal disinfection of blood contaminated hospital textiles]. / Ocena mikrobiologiczna skutecznosci dezynfekcji chemiczno-termicznej bielizny szpitalnej zanieczyszczonej krwia.

Thermal disinfection should be applied to laundering procedures of hospital textiles contaminated with blood. Currently, there is an increasing number of hospital textiles composed of cotton-polyester blends that cannot endure high temperatures of thermal disinfection. Besides, decreasing the temperature of chemothermal disinfection enhances the possibility of micro-organisms to survive the laundering procedure. The aim of this study was to prepare a new method for the microbiological evaluation of disinfecting laundering procedures for hospital textiles contaminated with blood. The bactericidal activity of chemical disinfectants for chemothermal disinfection was determined by simulating a laundering procedure for hospital textiles in the laboratory according to procedure of National Institute of Hygiene - DF/05/03. Bioindicators cotton carriers inoculated with Enterococcus faecium were used for determinating the antibacterial effects for hospital textiles contaminated with blood. High concentrations of bovine albumin and/or sheep erythrocytes were used as substrate for simulating human blood. The results showed that the bactericidal activity of chemical disinfectants for chemothermal disinfection hospital textiles in the event of massive organic contamination--heavily soiled with blood, shall be evaluated using carrier test in following conditions: test organism- Enterococcus faecium, interfering substances--6 g/l bovine albumin solution added to preparation.

[The effect of antibacterial substances on spread resistance of bacteria]. / Wplyw substancji antybakteryjnych na rozprzestrzenianie sie opornosci bakterii.

The evaluation of influence biocides on phenomenon of spread resistance bacteria is wide discussed particularly in the medical area. Current issue is examinated mechanisms of spread bacterial resistance in the areas using antibiotics and disinfectants and in natural environment. Selection of resistance bacteria is connected with using biocides against the rules in medical care and disinfection. Biocides using in static concentrations do not act as bacteriocidal substances and contribute to survival rate of resistance bacteria. Disinfectants use correctly to the areas and in right using concentrations prevent spread of resistance bacteria.

[The wastewater treatment significance in the control sanitarian and epidemiological state of environment]. / Znaczenie oczyszczania biologicznego w kontroli stanu sanitarno-epidemiologicznego srodowiska.

The municipal wastewater consist of organic, inorganic and biological contaminations. The most of human and animals pathogens are found in municipal wastewater responsible for water-borne and waterwashed diseases. Wastewater biological treatment is effective methods to reduce the transmission route of this pathogens. Different kind of methods (microfiltration/coagulation) and technology (aerobic/anaerobic stabilization) treated municipal wastewater, secondary effluent, primary and excess sludge are used to inactivation viruses, bacteria and protozoan. Chemical disinfection with CaO significantly affects inactivation of helminthes eggs during the hygienization of sludge. However the efficiency of pathogens disinfection particularly depend on contact time and concentration of disinfectants.

[Assessment of disinfection and sterilization processes in dental practice as an important factors in prevention of infections]. / Ocena prawidlowonci procesów dezynfekcji i sterylizacji w wybranych gabinetach stomatologicznych jako istotnych elementów zapobiegania zakazeniom.

The dental health-care settings is an environment where disease transmission occurs easily. Prevention of cross infection is therefore a crucial aspect of dental practice and dental clinic stuffmust adopt certain basic routines while practicing. Infections may be transmitted in the dental operatory through direct contact with blood, oral fluids or other secretions; via indirect contact with contaminated instruments, equipment or environmental surfaces; or by contact with airborne contaminants present in either droplet splatter or aerosols of oral and respiratory fluids. Strategies to prevent dental patient infections have focused on disinfection and sterilization. This study evaluates basic routines in prevention of cross-infection in the dentistry. The sample comprised 100 dentists, who completed questionnaires. Based on inquires the conditions for disinfection and sterilization of medical devices were assessed. The following issues were taken into consideration: the way of disinfection and preparation of the disinfectants, the localization of disinfection, preparing to disinfection, washing and packing of dental devices, the frequency of disinfection, methods of sterilization and the monitoring system, type of sterilizers and the available cycles. The dental practices are well equiped to proceed the steam sterilization, but 33% of dentists don't know the available cycles in their autoclaves. Only 35% of them made sterilization process protocols. Very common are three failures of instruments disinfections: multiple use of disinfectant, adding of disinfectant, adding new instruments. There is still need for improvement in disinfection and sterilization in dental practice, especially including: monitoring and documentation of sterilization process, proper use of disinfectants according to manufactures instructions, frequent disinfection of surfaces which contact with patients. Dental stuff should take part in advanced training courses about disinfection and sterilization.

[The principle of registration, evaluation and testing of disinfecting preparations]. / Zasady rejestrowania, opiniowania i badania preparatów dezynfekcyjnych.

Disinfectants are used to produce a state in which the number of living micro-organisms has been reduced to a level which is appropriate to the practical situation. For any products which are included in the Biocidal Directive 98/8/EC, for which specific activity is claimed, test data has to be approved by the regulatory authority and a product license obtained before the product can be offered for sale. Disinfectants can be recorded as biocidal products or medical devices. Presently, it is possible to measure the activity of a product on defined micro-organisms in specified experimental conditions. Efficacy is the result of the use of a product according to a defined application. To allow different requirements in different areas of application, separate tests and pass criteria have been or will be prepared for each of following three areas of application: medical, veterinary and group comprising food, industrial, domestic and institutional areas. The laboratory methods to be used for testing the activity of chemical disinfectants meets the European standards. The tests are categorised on a modular basis as follows: phase 1 tests, phase 2 step 1 tests, phase 2 step 2 tests and phase 3 tests. In order to claim that a product has disinfectant properties, suitable for use in the medical area, the product shall be tested according to European standards: phase 2 step 1 tests, phase 2 step 2 tests. Phase 1 tests are not required to support claims for chemical disinfectants used in human medicine. Only phase 1 tests are required to support claims for active substances for which no particular area of application is specified. Medical devices are subjects to the European Directive 93/42/EEC which requires that a product must carry a CE mark. Disinfectants which are intended specifically by its manufacturer to be used on medical devices are themselves medical devices and so these products, as well as conforming to the instrument disinfection European standards as specified in EN 14885, are also required to carry a CE mark.