The role of analysis of EVER2 haplotypes in actinic keratosis

Introduction . Polymorphisms of EVER genes are related to increased risk of actinic keratosis (AK), squamous skin cancers (SCC) and cervical cancer. Objective . The aim of this study was to analyze the haplotypes of the EVER 2 gene in 65 subjects with ≥ 5 actinic keratoses and in 274 controls. The correlations between EVER 2 haplotype and selected clinical parameters in patients with actinic keratosis were also evaluated. Material and methods . The analysis involved polymorphisms –917A>T (rs7208422), –988-4G>T (rs62079073) and –1107G>A (rs12452890), which obtained statistically significant results in previous published studies of patients with actinic keratosis and squamous skin cancers. The full blood samples of patients and controls were genotyped using real-time polymerase chain reaction with reagents from Applied Biosystems. Results . In a group of patients with AK and the controls the most frequent haplotypes were AGG and TGA. Haplotype TG (–917A>T and –988-4G>T) containing the promoter T allele of 917A>T was more frequent in patients who had AK onset before the age of 70 years (0.6117 vs. 0.417; p = 0.029) and with more extensive skin lesions (0.6085 vs. 0.414; p = 0.029). Haplotype TA (–988-4G>T and –1107G>A), containing the protective G allele of –988-4G>T, was observed only in patients with AK. There was no presence of haplotype TA in patients with coexisting SCC (the result was near statistical significance, p = 0.059). Conclusions . The results of the study suggest the necessity of analysis of haplotypes in evaluation of predisposition to precancerous skin lesions and skin cancers

Original paper Plasmid-mediated resistance to tetracyclines among Neisseria gonorrhoeae strains isolated in Poland between 2012 and 2013

A b s t r a c t Introduction: One of two main mechanisms of resistance in tetracycline-resistant Neisseria gonorrhoeae (TRNG) is associated with the presence of TetM protein responsible for actively blocking of the tetracycline target site in the 30S ribosomal subunit. This mechanism is encoded by conjugative plasmids. The second mechanism is chromosomal in nature and due to mutations in specific genes. Aim: To determine the incidence and type of tetM determinants in TRNG strains isolated from patients presenting with gonorrhea infection to the Dermatology and Venereology Clinic in Warsaw in 2012-2013. Material and methods: Tetracycline and doxycycline susceptibility was determined by E-Tests. The presence and type of the tetM gene were determined by polymerase chain reaction. Results: Tetracycline resistance was detected in 50.8% of the evaluated strains. The TRNG strains containing the tetM plasmid constituted 13.8% of all the evaluated strains. Dutch type tetM constituted 12.3% and American type tetM 1.5% of all the evaluated strains. In the remaining TRNG strains, resistance to tetracyclines was presumably chromosome-encoded. The minimal inhibitory concentration (MIC) of tetracycline ranged from 0.25 to 32.0 mg/l, MIC 50 = 2.0 mg/l, MIC 90 = 32.0 mg/l. The MIC of doxycycline ranged from 0.25 to 32.0 mg/l, MIC 50 = 4.0 mg/l, MIC 90 = 16.0 mg/l. Conclusions: Unlike most of European countries, in 2012-2013 in Poland, the Dutch type tetM was found to be much more common than the American type. Minimal inhibitory concentration values of tetracycline and doxycycline were similar, with doxycycline exhibiting a somewhat lower effectiveness in vitro than tetracycline towards chromosome-mediated tetracycline resistant strains of N. gonorrhoeae

Synthesis of novel, peptidic kinase inhibitors with cytostatic/cytotoxic activity

The utility of a novel, chemoenzymatic procedure for the stereocontrolled synthesis of small peptides is presented in the preparation and structure optimisation of dipeptides with cytostatic/cytotoxic activity. The method uses Passerini multicomponent reaction for the preparation of racemic scaffold which is then enantioselectively hydrolysed by hydrolytic enzymes. Products of these transformations are further functionalised towards title compounds. Both activity and selectivity towards tumor cells is optimised. Final compound is shown to be an inhibitor of the protein kinase signaling pathway. (C) 2014 Elsevier Ltd. All rights reserved

Syphilis in patients of the Department of Dermatology and Venereology at Medical University of Warsaw in 2015 – epidemiological and clinical characteristics, and coexistence of other sexually transmitted diseases

Introduction . Syphilis is a sexually transmitted disease that can be asymptomatic or associated with various symptoms including systemic manifestations. A total of 1,253 cases of syphilis were registered in Poland in 2015. Syphilis frequently coexists with HIV infection and other sexually transmitted diseases. Objective . Epidemiological and clinical characteristics of the population of patients treated for syphilis in the Department of Dermatology and Venereology, Medical University of Warsaw, and to evaluate the coexistence of other sexually transmitted diseases with special reference to HIV infection. Material and methods . The retrospective study involved an analysis of information included in medical files of 411 consecutive patients treated for syphilis in the Department of Dermatology and Venereology, Medical University of Warsaw, in 2015. Results . As many as 92% of the analyzed patients treated for syphilis were males, and 72.5% were men who have sex with men. Eighty-one percent of the subjects were between 21 and 40 years of age. Out of 325 patients with known HIV test results, 26.5% were HIV-positive. Evaluation of coexistence of other sexually transmitted diseases was difficult because of incomplete data, however gonorrhoea (7.8%) and hepatitis C virus infection (2.7%) were diagnosed more often in the studied group of patients with syphilis than in the general population. Hepatitis C virus infection was found in 6% of individuals co-infected with syphilis and HIV. Conclusion . The results confirm the need to screen patients with syphilis for HIV infection, and HIV-positive patients for syphilis

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

Persistent genus β-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 β (C/EBPβ), 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/EBPβ 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/EBPβ 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

Human Papillomavirus Type 8 Interferes with a Novel C/EBPβ-Mediated Mechanism of Keratinocyte CCL20 Chemokine Expression and Langerhans Cell Migration

<div><p>Infection with genus beta human papillomaviruses (HPV) is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and 8 in patients with epidermodysplasia verruciformis (EV), a genetic skin disease. So far, it has been unknown how these viruses overcome cutaneous immune control allowing their persistence in lesional epidermis of these patients. Here we demonstrate that Langerhans cells, essential for skin immunosurveillance, are strongly reduced in HPV8-positive lesional epidermis from EV patients. Interestingly, the same lesions were largely devoid of the important Langerhans cells chemoattractant protein CCL20. Applying bioinformatic tools, chromatin immunoprecipitation assays and functional studies we identified the differentiation-associated transcription factor CCAAT/enhancer binding protein β (C/EBPβ) as a critical regulator of <em>CCL20</em> gene expression in normal human keratinocytes. The physiological relevance of this finding is supported by our in vivo studies showing that the expression patterns of CCL20 and nuclear C/EBPβ converge spatially in the most differentiated layers of human epidermis. Our analyses further identified C/EBPβ as a novel target of the HPV8 E7 oncoprotein, which co-localizes with C/EBPβ in the nucleus, co-precipitates with it and interferes with its binding to the CCL20 promoter in vivo. As a consequence, the HPV8 E7 but not E6 oncoprotein suppressed C/EBPβ-inducible and constitutive <em>CCL20</em> gene expression as well as Langerhans cell migration. In conclusion, our study unraveled a novel molecular mechanism central to cutaneous host defense. Interference of the HPV8 E7 oncoprotein with this regulatory pathway allows the virus to disrupt the immune barrier, a major prerequisite for its epithelial persistence and procarcinogenic activity.</p> </div

C/EBPβ binds to the enhancer region of CCL20 in vivo.

<p>(A) Nucleotide sequence of the human CCL20 promoter region with twelve putative C/EBP binding sites (underlined). Numbers below the underlined C/EBP binding sites mark the sequences, which display C/EBP DNA binding activity in EMSA. In bold is the DNA sequence tested for C/EBP binding in ChIP assay. (B) ³²P-labeled oligonucleotides containing the respective C/EBP binding sites (nt 294–308, nt 574–584, nt 652–667, nt 716–724, nt 734–748) of the CCL20 promoter were incubated with 5 µg GST, GST-C/EBPα or GST-C/EBPβ fusion proteins and analyzed by EMSA. The arrow indicates complexes corresponding to C/EBP DNA binding activity. (C) Chromatin immunoprecipitation assay was performed using RTS3b cells transfected with the C/EBPβ expression vector. For precipitation anti-C/EBPβ (H-7) antibody was used. Genomic DNA was isolated, amplified by real-time PCR with primers specific for the nt 638–677 region of the CCL20 promoter (in bold). The amplicon was quantified (left panel) and visualized on an agarose gel (right panel). The amount of target DNA precipitated with the control antibody was set at 1. Shown are mean values ± SD from four experiments. The asterisk represents statistical significance, p = 0.02.</p

Langerin-positive cells and CCL20 expression are reduced in lesional skin of EV patients.

<p>Sections of normal human skin (A, B, C) and lesional skin from EV patients (D–J) were stained using antibodies against langerin (A, D) or CCL20 (B, C, E, F) and hematoxylin. To verify the specificity of the immunohistochemical reaction, recombinant CCL20 protein was used in a blocking experiment (C). Lesion positive for HPV8 only (D, E, G–J). Lesion coinfected with HPV5 and HPV8 (F). Validation of active infection was visualized by hematoxylin eosin staining to reveal the characteristic E4 cytoplasmic inclusions typically found in EV lesions (G) along with immunofluorescence staining for the HPV8 E4 protein (green (H)) and FISH staining to identify the presence of amplified HPV genomes (red (I)). The merged image showing the onset of genome amplification in E4-positive cells is shown in J. Cell nuclei are visualized in J by counterstaining with DAPI. Selected cells double positive for E4 protein and HPV8 DNA are marked with arrows. Bars correspond to 100 µm in A–E and G–J, and to 200 µm in F.</p

HPV8 E7 directly interacts with C/EBPβ and suppresses C/EBPβ-induced activation of the CCL20 promoter.

<p>(A) NHK were transfected with CCL20 promoter luciferase construct (0.5 µg) and C/EBPβ (0.4 µg) in the presence or absence of HPV8 E6 (0.8 µg) or HPV8 E7 (0.8 µg) pcDNA3.1+ expression vectors. Total amount of DNA was adjusted with empty vector. After 24 h the luciferase activity was measured and normalized to protein concentration of the respective luciferase extract. The normalized luciferase activity of the control transfection was set at 1. Transfections were conducted in triplicates. Shown are mean values from three independent experiments ± SD. Asterisks represent statistical significance, p<0.001. (B) HPV8 E7 co-localizes with C/EBPβ in the keratinocyte nucleus. RTS3b cells seeded on glass coverslips were co-transfected with Flag-HPV8 E7 and ECFP-C/EBPβ expression vectors and stained with DAPI (blue, first panel) as well as anti-Flag antibody (red, second panel). ECFP-C/EBPβ is shown in third panel (green). Cells were analyzed by deconvolution fluorescence microscopy. The overlays and co-localization (yellow) are displayed in the fourth panel. Bars correspond to 20 µm. (C) In pull-down assays GST, GST-p300, GST-C/EBPα or GST-C/EBPβ were incubated with in vitro-translated HPV8 E7 protein (IVT, input) and precipitated (P) by glutathione Sepharose beads. pcDNA3.1+ vector (control vector) was used as a control for HPV8 E7 in vitro translation. Proteins were visualized by SDS-PAGE and autoradiography. (D) C33A cells were transfected with pFlag-CMV2, the Flag-tagged HPV8 E7 construct or deletion mutants thereof and co-transfected with the C/EBPβ expression vector. After 24 h cell lysates were prepared and precipitated with anti-Flag agarose beads (IP). The precipitates (P) and input (I, 10 µl of the lysates) were analyzed with anti-C/EBPβ or anti-Flag antibodies by Western blot (WB). The anti-Flag antibody light chain present in the precipitates is marked with a star (*).</p