Co-Expression Analysis of Airway Epithelial Transcriptome in Asthma Patients with Eosinophilic vs. Non-Eosinophilic Airway Infiltration.

Asthma heterogeneity complicates the search for targeted treatment against airway inflammation and remodeling. We sought to investigate relations between eosinophilic inflammation, a phenotypic feature frequent in severe asthma, bronchial epithelial transcriptome, and functional and structural measures of airway remodeling. We compared epithelial gene expression, spirometry, airway cross-sectional geometry (computed tomography), reticular basement membrane thickness (histology), and blood and bronchoalveolar lavage (BAL) cytokines of n = 40 moderate to severe eosinophilic (EA) and non-eosinophilic asthma (NEA) patients distinguished by BAL eosinophilia. EA patients showed a similar extent of airway remodeling as NEA but had an increased expression of genes involved in the immune response and inflammation (e.g., KIR3DS1), reactive oxygen species generation (GYS2, ATPIF1), cell activation and proliferation (ANK3), cargo transporting (RAB4B, CPLX2), and tissue remodeling (FBLN1, SOX14, GSN), and a lower expression of genes involved in epithelial integrity (e.g., GJB1) and histone acetylation (SIN3A). Genes co-expressed in EA were involved in antiviral responses (e.g., ATP1B1), cell migration (EPS8L1, STOML3), cell adhesion (RAPH1), epithelial-mesenchymal transition (ASB3), and airway hyperreactivity and remodeling (FBN3, RECK), and several were linked to asthma in genome- (e.g., MRPL14, ASB3) or epigenome-wide association studies (CLC, GPI, SSCRB4, STRN4). Signaling pathways inferred from the co-expression pattern were associated with airway remodeling (e.g., TGF-ß/Smad2/3, E2F/Rb, and Wnt/ß-catenin).

Molecular and Immunological Identification of Low Allergenic Fruits among Old and New Apple Varieties.

About 50-70% of patients allergic to birch pollen suffer from sensitization after apple ingestion. Apple allergenicity was established in only few varieties. Studies were performed on apple fruits of 21 traditional and nine modern varieties organically, intensively, or integratively produced. The aim of the study was to assess whether the factors like cultivation method, maturity stage, genotype, or type of tissue place an impact on the allergenic potential of apples. To answer these questions, we used semiquantitative real-time PCR, ELISA, and immunoblotting. Apple allergen genes present divergent expression across apple cultivars. Expression of the Mal d 1.06A correlates with the Mal d 1 level and is affected by the cultivation method and maturity of the fruit. The content of the main allergen Mal d 1 varied widely across cultivars. Interestingly, in our study, the Gala variety presented a low Mal d 1 concentration regardless of the cultivation method. Based on the Mal d 1.06A expression, the Mal d 1 protein content, and the immunoreactivity assay, the Kandil Sinap, Kosztela, Rumianka from Alma-Ata, Kantówka Gdanska, Reinette Coulon, and Gala cultivars emerged as potentially hypoallergenic apple cultivars. Our study allowed distinguishing between potentially low, medium, and highly allergenic varieties.

Reticular Basement Membrane Thickness Is Associated with Growth- and Fibrosis-Promoting Airway Transcriptome Profile-Study in Asthma Patients.

Airway remodeling in asthma is characterized by reticular basement membrane (RBM) thickening, likely related to epithelial structural and functional changes. Gene expression profiling of the airway epithelium might identify genes involved in bronchial structural alterations. We analyzed bronchial wall geometry (computed tomography (CT)), RBM thickness (histology), and the bronchial epithelium transcriptome profile (gene expression array) in moderate to severe persistent (n = 21) vs. no persistent (n = 19) airflow limitation asthmatics. RBM thickness was similar in the two studied subgroups. Among the genes associated with increased RBM thickness, the most essential were those engaged in cell activation, proliferation, and growth (e.g., CDK20, TACC2, ORC5, and NEK5) and inhibiting apoptosis (e.g., higher mRNA expression of RFN34, BIRC3, NAA16, and lower of RNF13, MRPL37, CACNA1G). Additionally, RBM thickness correlated with the expression of genes encoding extracellular matrix (ECM) components (LAMA3, USH2A), involved in ECM remodeling (LTBP1), neovascularization (FGD5, HPRT1), nerve functioning (TPH1, PCDHGC4), oxidative stress adaptation (RIT1, HSP90AB1), epigenetic modifications (OLMALINC, DNMT3A), and the innate immune response (STAP1, OAS2). Cluster analysis revealed that genes linked with RBM thickness were also related to thicker bronchial walls in CT. Our study suggests that the pro-fibrotic profile in the airway epithelial cell transcriptome is associated with a thicker RBM, and thus, may contribute to asthma airway remodeling.

Analysing the effectiveness of topical bleeding care following tooth extraction in patients receiving dual antiplatelet therapy-retrospective observational study.

BACKGROUND: Patients using antiplatelet drugs following infarctions, acute coronary syndrome or stroke pose a significant clinical problem if it is necessary to perform surgery, including dental surgery, since they are at risk of prolonged or secondary post-extraction bleeding. Discontinuation of this therapy is associated with a high risk of serious thromboembolic complications. The purpose of this study was to assess the effectiveness of TachoSil fibrin-collagen patches in stopping and preventing of secondary post-extraction bleeding in patients undergoing chronic antiplatelet therapy. METHODS: The study was conducted through retrospective examination of the medical records of 153 patients using chronic antiplatelet therapy and those qualified for tooth extraction. The largest group comprised 74 patients using aspirin and clopidogrel as dual platelet antiaggregation therapy; in this group 75 tooth extractions were carried out. In all of the patients TachoSil fibrin-collagen patches and stiches were applied to the wounds resulting from tooth removal. RESULTS: Following tooth extraction, primary bleeding was stopped in all the patients and their wounds closed via coagulation within 20-30 min. In eight cases, accounting for 4.9% of the patients, secondary bleeding occurred and was successfully stopped only by applying a pressure dressing soaked in tranexamic acid. Secondary bleeding occurred in three patients on the second day and in five patients on the third day following tooth removal. CONCLUSION: Topical application of TachoSil patches following tooth removal in patients using single or dual antiplatelet therapy effectively stopped bleeding and prevented secondary bleeding after tooth extraction.

Genetic testing in Poland and Ukraine: should comprehensive germline testing of BRCA1 and BRCA2 be recommended for women with breast and ovarian cancer?

PURPOSE: To characterize the spectrum of BRCA1 and BRCA2 pathogenic germline variants in women from south-west Poland and west Ukraine affected with breast or ovarian cancer. Testing in women at high risk of breast and ovarian cancer in these regions is currently mainly limited to founder mutations. METHODS: Unrelated women affected with breast and/or ovarian cancer from Poland (n = 337) and Ukraine (n = 123) were screened by targeted sequencing. Excluded from targeted sequencing were 34 Polish women who had previously been identified as carrying a founder mutation in BRCA1. No prior testing had been conducted among the Ukrainian women. Thus, this study screened BRCA1 and BRCA2 in the germline DNA of 426 women in total. RESULTS: We identified 31 and 18 women as carriers of pathogenic/likely pathogenic (P/LP) genetic variants in BRCA1 and BRCA2, respectively. We observed five BRCA1 and eight BRCA2 P/LP variants (13/337, 3.9%) in the Polish women. Combined with the 34/337 (10.1%) founder variants identified prior to this study, the overall P/LP variant frequency in the Polish women was thus 14% (47/337). Among the Ukrainian women, 16/123 (13%) women were identified as carrying a founder mutation and 20/123 (16.3%) were found to carry non-founder P/LP variants (10 in BRCA1 and 10 in BRCA2). CONCLUSIONS: These results indicate that genetic testing in women at high risk of breast and ovarian cancer in Poland and Ukraine should not be limited to founder mutations. Extended testing will enhance risk stratification and management for these women and their families.

FANCM and RECQL genetic variants and breast cancer susceptibility: relevance to South Poland and West Ukraine.

BACKGROUND: FANCM and RECQL have recently been reported as breast cancer susceptibility genes and it has been suggested that they should be included on gene panel tests for breast cancer predisposition. However, the clinical value of testing for mutations in RECQL and FANCM remains to be determined. In this study, we have characterised the spectrum of FANCM and RECQL mutations in women affected with breast or ovarian cancer from South-West Poland and West Ukraine. METHODS: We applied Hi-Plex, an amplicon-based enrichment method for targeted massively parallel sequencing, to screen the coding exons and proximal intron-exon junctions of FANCM and RECQL in germline DNA from unrelated women affected with breast cancer (n = 338) and ovarian cancer (n = 89) from Poland (n = 304) and Ukraine (n = 123). These women were at high-risk of carrying a genetic predisposition to breast and/or ovarian cancer due to a family history and/or early-onset disease. RESULTS: Among 427 women screened, we identified one carrier of the FANCM:c.1972C > T nonsense mutation (0.23%), and two carriers of the frameshift insertion FANCM:c.1491dup (0.47%). None of the variants we observed in RECQL were predicted to be loss-of-function mutations by standard variant effect prediction tools. CONCLUSIONS: Our study of the Polish and Ukrainian populations has identified a carrier frequency of truncating mutations in FANCM consistent with previous reports. Although initial reports suggesting that mutations in RECQL could be associated with increased breast cancer risk included women from Poland and identified the RECQL:c.1667_1667 + 3delAGTA mutation in 0.23-0.35% of breast cancer cases, we did not observe any carriers in our study cohort. Continued screening, both in research and diagnostic settings, will enable the accumulation of data that is needed to establish the clinical utility of including RECQL and FANCM on gene panel tests.

A comparison of replicative senescence and doxorubicin-induced premature senescence of vascular smooth muscle cells isolated from human aorta.

Senescence of vascular smooth muscle cells (VSMCs) contributes to aging as well as age-related diseases of the cardiovascular system. Senescent VSMCs have been shown to be present in atherosclerotic plaques. Both replicative (RS) and stress-induced premature senescence (SIPS) accompany cardiovascular diseases. We aimed to establish the signature of RS and SIPS of VSMCs, induced by a common anticancer drug, doxorubicin, and to discover the so far undisclosed features of senescent cells that are potentially harmful to the organism. Most of the senescence hallmarks were common for both RS and SIPS; however, some differences were observed. 32 % of doxorubicin-treated cells were arrested in the G2/M phase of the cell cycle, while 73 % of replicatively senescing cells were arrested in the G1 phase. Moreover, on the basis of alkaline phosphatase activity measurements, we show that a 7-day treatment with doxorubicin (dox), does not cause precocious cell calcification, which is a characteristic feature of RS. We did not observe calcification even though after 7 days of dox-treatment many other markers characteristic for senescent cells were present. It can suggest that dox-induced SIPS does not accelerate the mineralization of vessels. We consider that detailed characterization of the two types of cellular senescence can be useful in in vitro studies of potential anti-aging factors.

Status of Oxidative Stress during Low-Risk Labour: Preliminary Data.

Pregnancy and childbirth are associated with the forming of reactive oxygen species that generate oxidative stress. Oxidative stress is a factor that may adversely affect the development of the fetus and the course of labour. Monitoring the parameters of oxidative stress can be used to assess the risk of health issues in the course of pregnancy and the condition of the newborn. Therefore, the analysis of oxidative stress in the physiological course of labour is the basis for understanding the role of oxidative stress in the pathogenesis of miscarriages and neonatal health circumstances. The study aimed to assess oxidative stress of mother-child pairs in the venous blood and umbilical cord blood at the time of physiological labour. One hundred and sixty-eight mother-child pairs were recruited to donate the mother's venous blood in the first stage of labour and the venous umbilical cord blood after the newborn's birth. Total antioxidant status (TAS), the activity of superoxide dismutase (SOD) with cofactors (Zn, Cu, Mn) and the activity of glutathione peroxidase (GPx) were analysed in venous blood plasma and umbilical cord blood. TAS value (p = 0.034), GPx activity (p < 0.001) and Zn concentration (p = 0.007) were significantly lower in maternal blood plasma as compared to neonatal umbilical cord blood. However, the activity of SOD (p = 0.013) and the concentration of Cu (p < 0.001) were significantly higher in the blood of mothers than of new-borns. The concentration of Mn in the plasma of the mother's blood and the umbilical cord blood of the newborns was similar. Our research indicates higher levels of antioxidant enzyme (GPx) and total antioxidant potential (TAS) in umbilical cord blood compared to maternal blood, which may suggest depletion of redox reserves in women's blood during labour.

Detection of viral DNA sequences in sporadic colorectal cancers in relation to CpG island methylation and methylator phenotype.

There is evidence that insertion of viral DNA into a mammalian genome can lead to alterations of methylation patterns. The aim of the present study was to examine the presence of DNA sequences of five human DNA viruses (assessed by PCR): JC polyoma virus (JCV), human adenovirus (AdV), Epstein-Barr virus (EBV), Kaposi sarcoma-associated herpesvirus (KSHV/HHV8) and human papillomavirus (HPV) in a cohort of 186 sporadic colorectal cancers (CRCs) and related these data with the methylation status of six CpG island methylator phenotype (CIMP)-specific genes (MLH1, CACNA1G, NEUROG1, IGF2, SOCS1, RUNX3) and seven cancer-related genes markers (p16, MINT1, MINT2, MINT31, EN1, SCTR and INHBB) assessed by methylation-specific PCR in 186 and 134 CRC cases, respectively. The AdV, KSHV and HPV were detected in four (2%), two (1%) and zero CRC cases, respectively, and thus were excluded from further analyses. Although 19% and 9% of the CRCs were positive for EBV and JCV, respectively, no associations between virus presence and CpG island methylation were found after correction for multiple testing. Our results demonstrate that the presence of DNA sequences of JCV and EBV in CRC is unrelated to the methylation of the 13 cancer-related CpG islands and CIMP.

Apple allergy: Causes and factors influencing fruits allergenic properties-Review.

BACKGROUND: Apple tree fruits (Malus × domestica Borkh.) are a rich source of nutrients and nutraceuticals and are recommended as a part of the healthy, staple diet. However, apples could be also the cause of allergies including severe reactions. Allergies to fruits like apples are predominantly associated with pollinosis. In North and Central Europe, sensitisation to apples is caused mainly by cross-reactive birch pollen aeroallergen, whereas in the Mediterranean area of Europe, apple allergy is mostly associated with allergies to peach. The allergenicity of apples differ across cultivars but only a few varieties were studied. Some factors changing apples allergenicity were identified, including unmodifiable and potentially modifiable factors for example cultivation method, ripening stage and storage conditions. AIM: This review presents current knowledge about the molecular basis of apple allergenicity and factors influencing its level. CONCLUSIONS: Selecting cultivars with low potential of allergenicity, removing apple peel and heat treatment could reduce the risk of severe allergy reaction incidence and presumably can be used in birch pollen immunotherapy.

Helicobacter pylori cagA gene polymorphism affects the total antioxidant capacity of human saliva.

BACKGROUND: We aimed to evaluate the total antioxidant capacity (TAC) of saliva in healthy Helicobacter pylori-positive and negative saliva individuals. MATERIALS AND METHODS: A total of 102 human saliva samples were checked for the presence of H. pylori DNA (ureA and cagA gene fragments). TAC of saliva was estimated by ABTS radical cation (ABTS( +)) decolorization assay. RESULTS: PCR analysis revealed that 36 subjects were ureA-/cagA-, 24 were ureA+/cagA- and 42 were ureA+/cagA+. Smoking habits had no evident effect on H. pylori infection. We found that TAC of the ureA-/cagA- material, after 10 seconds reaction reflecting fast-reacting antioxidants, was significantly higher than of ureA+/cagA- and ureA+/cagA+ samples (p < .01 and p < .001, respectively). Similar results were obtained for reaction time of 3 minutes measuring slow-reacting antioxidants (p < .001). We also estimated ureA+/cagA- and ureA+/cagA+ samples alone and reported a statistically significant decrease in the TAC(3 min) value of ureA+/cagA+ compared with ureA+/cagA- samples (p < .05). CONCLUSIONS: Our data demonstrated that altered redox equilibrium may be associated with more frequent occurrence of H. pylori in the saliva samples.

The Spectrum of FANCM Protein Truncating Variants in European Breast Cancer Cases.

Germline protein truncating variants (PTVs) in the FANCM gene have been associated with a 2-4-fold increased breast cancer risk in case-control studies conducted in different European populations. However, the distribution and the frequency of FANCM PTVs in Europe have never been investigated. In the present study, we collected the data of 114 European female breast cancer cases with FANCM PTVs ascertained in 20 centers from 13 European countries. We identified 27 different FANCM PTVs. The p.Gln1701* PTV is the most common PTV in Northern Europe with a maximum frequency in Finland and a lower relative frequency in Southern Europe. On the contrary, p.Arg1931* seems to be the most common PTV in Southern Europe. We also showed that p.Arg658*, the third most common PTV, is more frequent in Central Europe, and p.Gln498Thrfs*7 is probably a founder variant from Lithuania. Of the 23 rare or unique FANCM PTVs, 15 have not been previously reported. We provide here the initial spectrum of FANCM PTVs in European breast cancer cases.

Targeted massively parallel sequencing characterises the mutation spectrum of PALB2 in breast and ovarian cancer cases from Poland and Ukraine.

Loss-of-function germline mutations in the PALB2 gene are associated with an increase of breast cancer risk. The purpose of this study was to characterise the spectrum of PALB2 mutations in women affected with breast or ovarian cancer from South-West Poland and West Ukraine. We applied Hi-Plex, an amplicon-based enrichment method for targeted massively parallel sequencing, to screen the coding exons and proximal intron-exon junctions of PALB2 in germline DNA from unrelated women affected with breast cancer (n = 338) and ovarian cancer (n = 89) from Poland (n = 304) and Ukraine (n = 123). These women were at high-risk of carrying a genetic predisposition to breast and/or ovarian cancer due to a family history and/or early-onset disease. Targeted-sequencing identified two frameshift deletions: PALB2:c.509_510del; p.R170Ifs in three women affected with breast cancer and PALB2:c.172_175del;p.Q60Rfs in one woman affected with ovarian cancer. A number of other previously described missense (some predicted to be damaging by PolyPhen-2 and CADD) and synonymous mutations were also identified in this population. This study is consistent with previous reports that PALB2:c.509_510del and PALB2:c.172_175del are recurrent mutations associated with breast cancer predisposition in Polish women with a family history of the disease. Our study contributes to the accumulating evidence indicating that PALB2 should be included in genetic testing for breast cancer susceptibility in these populations to enhance risk assessment and management of women at high-risk of developing breast cancer. This data could also contribute to ongoing work that is assessing the possible association between ovarian cancer risk and PALB2 mutations for which there is currently no evidence.

Irrelevance of CHEK2 variants to diagnosis of breast/ovarian cancer predisposition in Polish cohort.

CHEK2 gen encodes cell cycle checkpoint kinase 2 that participates in the DNA repair pathway, cell cycle regulation and apoptosis. Mutations in CHEK2 gene may result in kinase inactivation or reduce both catalytic activity and capability of binding other proteins. Some studies indicate that alterations in CHEK2 gene confers increase the risk of breast cancer and some other malignancies, while the results of other studies are inconclusive. Thus the significance of CHEK2 mutations in aetiology of breast cancer is still debatable. The aim of our study was to evaluate the relationship between the breast/ovarian cancer and CHEK2 variants by: i) the analysis of the frequency of selected CHEK2 variants in breast and ovarian cancer patients compared to the controls; ii) evaluation of relationships between the certain CHEK2 variants and clinico-histopathological and pedigree data. The study was performed on 284 breast cancer patients, 113 ovarian cancer patients and 287 healthy women. We revealed the presence of 430T > C, del5395 and IVS2 + 1G > A variants but not 1100delC in individuals from both study and control groups. We did not observe significant differences between cancer patients and controls neither in regard to the frequency nor to the type of CHEK2 variants. We discussed the potential application of CHEK2 variants in the evaluation of breast and ovarian cancer predisposition.

Polymorphisms in methyl-group metabolism genes and risk of sporadic colorectal cancer with relation to the CpG island methylator phenotype.

BACKGROUND: The CpG island methylator phenotype (CIMP), together with extensive promoter methylation, is regarded as one of the mechanisms involved in colorectal carcinogenesis. The mechanisms underlying CIMP in sporadic colorectal cancer are poorly understood. Genes involved in methyl-group metabolism are likely to affect DNA methylation and thereby influence an individual's risk of CIMP. The aim of the present study was to evaluate whether polymorphisms in the genes encoding methyl-group metabolism pathway predispose to CIMP+ and/or CIMP- CRC. METHODS: We examined the potential association between the polymorphisms of MTHFR 677C>T, TS 5'UTR 2R/3R, TS 3'UTR 1494del6, DeltaDNMT3B -149C>T and DNMT3B -283T>C in a group of 46 CIMP+ CRC cases, 140 CIMP- CRC cases and 140 healthy controls. The CIMP status of the CRC cases was determined by MS-PCR in tumor tissue by a panel of five markers (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1), which was also followed by analyzing hMLH1 methylation and BRAF V600E mutation. RESULTS: The variant allele homozygote genotype for the DeltaDNMT3B -283T>C polymorphism was associated with a decreased risk for CIMP+ CRC (OR: 0.31, 95%CI: 0.09-0.73, p=0.009). Individuals with TS 3R/3R had an increased risk of CIMP- CRC (OR: 2.21, 95%CI: 1.23-4.91, p=0.01). Moreover, the carriers of 3R allele had an increased risk of CIMP- CRC (OR: 1.45, 95%CI: 1.10-2.13, p=0.01). CONCLUSION: This study provides support to the hypothesis that methyl-group metabolism plays a role in the etiology of both CIMP+ and CIMP- colorectal cancers but has a different impact on a distinct molecular subgroups of colorectal cancer.