Search

The study of epigenetics has improved our understanding of mechanisms underpinning gene-environment interactions and is providing new insights in the pathophysiology of respiratory allergic diseases. We reviewed the literature on DNA methylation patterns across different tissues in asthma and/or rhinitis and attempted to elucidate differentially methylated loci that could be used to characterize asthma or rhinitis. Although nasal and bronchial epithelia are similar in their histological structure and cellular composition, genetic and epigenetic regulation may differ across tissues. Advanced methods have enabled comprehensive, high-throughput methylation profiling of different tissues (bronchial or nasal epithelial cells, whole blood or isolated mononuclear cells), in subjects with respiratory conditions, aiming to elucidate gene regulation mechanisms and identify new biomarkers. Several genes and CpGs have been suggested as asthma biomarkers, though research on allergic rhinitis is still lacking. The most common differentially methylated loci presented in both blood and nasal samples are ACOT7, EPX, KCNH2, SIGLEC8, TNIK, FOXP1, ATPAF2, ZNF862, ADORA3, ARID3A, IL5RA, METRNL and ZFPM1. Overall, there is substantial variation among studies, (i.e. sample sizes, age groups and disease phenotype). Greater variability of analysis method detailed phenotypic characterization and age stratification should be taken into account in future studies. © 2022 The Authors. Clinical and Translational Allergy published by John Wiley & Sons Ltd on behalf of European Academy of Allergy and Clinical Immunology.

Introduction: The leukotriene pathway may be implicated in the induction of virus-induced inflammation. Respiratory epithelial cells may express low levels of 5-lipoxygenase (5-LO) and release leukotrienes (LTs) C4, D4, and E4, upon exposure to viruses or other stimuli. Enhanced expression of 5-LO pathway proteins after rhinovirus (RV) infection has previously been described. We hypothesized that anti-leukotriene treatment of epithelial cells, with or without exposure to RV-infected peripheral blood mononuclear cells (PBMCs)-conditioned media, may inhibit RV-induced up-regulation of inflammatory cytokines. Methods: PBMCs from a healthy donor were exposed to RV1B and supernatants were harvested at 48 h post infection. BEAS-2B cells were infected with RV, with or without conditioning with the PBMC supernatant. Treatment with anti-LT agents was performed either on both PBMCs and BEAS-2B or at the bronchial epithelial level only, with varying concentrations of montelukast (CysLT receptor antagonist) or MK-886 [FLAP(5-lipoxygenase-activating-protein) inhibitor]. Evaluation of the inflammatory cytokines IL-8, RANTES, IL-11, IL-6, and IP-10 was performed using ELISA. Results: Our results show that anti-LT treatment of RV-infected bronchial epithelial cells suppresses epithelial RV-mediated cytokine production, independent of conditioning. Conclusions: This observation may represent an indirect mode of action of the anti-leukotrienes in virus-induced asthma. © 2021, The Author(s).

Background: The maturation of innate immune responses in health and atopy is still incompletely understood. Methods: We aimed to evaluate age-related trajectories of the TLR3 and TLR7/8 pathways from birth to adulthood and whether these differ between healthy and atopic individuals. Peripheral blood mononuclear cells (PBMCs) were isolated from 39 otherwise healthy, atopic and 39 non-atopic subjects, aged 0–45 years. Selected cytokines involved in antiviral responses were measured by Luminex in culture supernatants of poly(I:C)- and R848-stimulated PBMCs. The non-parametric correlation between age and cytokine expression and differences in developmental trajectories between healthy and atopic subjects were estimated. Patterns of cytokine development were identified with principal component analysis. Results: Normal innate immune maturation entails significant and progressive age-related changes in the production of IL-1β, TNF-α, MIP-1β, MCP-3, IP-10, IL-10, IL-12p70, and IFN-γ upon TLR3 and/or TLR7/8 stimulation. Individual cytokines made small contributions to the observed variability; chemokines MCP-3 and IP-10 were key contributors. The development of these pathways deviated in atopic subjects with significant differences observed in the trajectories of IL-1β, MIP-1β, and IL-10 syntheses. Conclusion: TLR3 and TLR7/8 pathways mature during childhood, while atopy is associated with an abnormal maturation pattern. Suboptimal responses in Th1, inflammatory cytokine, and chemokine production may be implicated in poor antiviral immunity in atopics. Moreover, the deficient maturation of IL-10 synthesis may be implicated in the breaking of tolerance, characterizing the onset of atopic disease. © 2021 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

The aim of this study was to characterise the effects of maternal undernutrition during gestation on hepatic gluconeogenic enzyme gene expression and to determine whether such effects are mediated through epigenetic changes in the glucocorticoid receptor (GR). Pregnant ewes were fed a 50% nutrient-restricted diet from Day 0 to 30 (R1) or from Day 31 to 100 of gestation (R2) or a 100% diet throughout gestation (Control). After parturition lambs were fed to appetite. At 10 months of age offspring were euthanised and livers were removed. Maternal undernutrition did not affect offspring bodyweight at birth or at 10 months of age. However, liver weight of males of the R2 group was lower (P

The aim of this study was to assess the immune response to parainfluenza virus type 3 (PIV3), rhinovirus 1B (RV1B) and intracellular Toll-like receptors (TLR) agonists in nasal epithelial cells (NECs) from patients with allergic rhinitis and healthy controls. NECs were obtained from eight patients with allergic rhinitis (AR) and 11 non-atopic healthy controls (HC) by nasal scraping, grown to confluence and exposed to PIV3, RV1B infection or TLR-3 and TLR-7/8 agonists. Interferon (IFN)-λ1, IFN-α, IFN-β and regulated on activation, normal T expressed and secreted (RANTES) release into the cell culture supernatants was assessed at 8, 24 and 48 h upon infection or 8 and 24 h after stimulation with poly(I:C) and R848. mRNA levels of IFNs, RANTES, interferon regulatory transcription factor (IRF)3, IRF7 and viral gene copy number were determined using real-time polymerase chain reaction (RT-PCR). PIV3 but not RV1B replication 48 h after infection was significantly lower (P < 0·01) in NECs from AR patients compared to HC. PIV3 infection induced significantly less IFN-λ1 (both protein and mRNA) in NECs from AR compared to HC. IFN-β mRNA expression and RANTES protein release and mRNA expression tended to be smaller in AR compared HC cells in response to both viruses. Stimulation with TLR-3 agonist [poly (I:C)] induced similar IFN-λ1 and RANTES generation in AR and HC subjects. Viral infections in NECs induced IRF7 expression, which correlated with IFN and RANTES expression. These data suggest that virus proliferation rates and the immune response profile are different in nasal epithelial cells from patients with allergic rhinitis compared to healthy individuals. © 2016 British Society for Immunology

The proinflammatory cytokine interleukin-17A (IL-17A) is known to mediate antimicrobial activity, but its role during rhinovirus (RV) infections and in asthma needs further investigation. Therefore, we addressed the role of IL-17A during allergic asthma and antiviral immune response in human and murine immunocompetent cells. In this study we found that asthmatic children with a RV infection in their upper airways have upregulated mRNA levels of the antiviral cytokine interferon type I (IFN)-β and the transcription factor T-box 21 (TBX21) and reduced levels of IL-17A protein in their peripheral blood mononuclear cells (PBMCs). We also found that IL-17A inhibited RV1b replication in infected human lung epithelial cells A549. Furthermore, by using gene array analysis we discovered that targeted deletion of Il17a in murine lung CD4 + T cells impaired Oas1g mRNA downstream of Ifnβ, independently from RV infection. Additionally, in PBMCs of children with a RV infection in their nasalpharyngeal fluid OAS1 gene expression was found downregulated. Finally RV1b inhibited IL-17A production in lung CD4 + T cells in a setting of experimental asthma. These results indicate that the RV1b inhibits IL-17A in T helper type 17 cells and IL-17A clears RV1b infection in epithelial cells. In both cases IL-17A contributes to fend off RV1b infection by inducing genes downstream of interferon type I pathway.

Cadmium selenide quantum dots have been incorporated to a lateral flow assay for the specific and very simple detection of different mycobacterial DNA targets within only a few minutes, bypassing the complexity of conventional DNA hybridization assays. The method extends our previous work on protein detection using an identical procedure. © 2015 Elsevier Ltd.

The study aimed to investigate whether the genetic polymorphisms in the 3. 'UTR of the caprine SLC11A1 gene are functional, and to assess the role of MAP as a regulatory parameter in gene expression. To this goal we constructed plasmids expressing the Luciferase reporter gene in transient transfections of a mouse (Balb/c) macrophage cell line (RAW264.7), incorporating those polymorphisms that our previous work indicated as more prominent in terms of SLC11A1 expression and responsiveness to MAP infection. Gene expression variation was recorded on the average of the respective measurements after exposure to Mycobacterium avium subsp. paratuberculosis (MAP) combined with microbial antigens and cytokines. In silico analysis of the region under study allowed identification of one cis-acting RNA element, five putative transcriptional regulatory elements and 85 3. 'end microRNA binding sites. The two polymorphic regions (regions A and B) of the 3. 'UTR of the caprine SLC11A1 gene were recognized as regulators of its activity, at transcriptional and post-transcriptional level. The GT16 polymorphism at region A, combined with the GT8 polymorphism at region B, results in up-regulation of the SLC11A1 gene. The specific genotype was also found to be more responsive to MAP exposure at a statistically significant level. © 2015 Elsevier B.V.

Background: Nasopharyngeal (NP) bacterial colonization is necessary for subsequent respiratory and/or invasive infection. Our study aimed at comparing NP bacterial colonization rates between children with and without symptoms of an acute viral respiratory tract infection and examining associations between identified microorganisms. Methods: Children 3 months to 6 years of age with and without an acute viral respiratory tract infection were recruited, and a questionnaire was filled. NP samples were examined for Streptococcus pneumoniae (SP), Haemophilus influenzae (HI), Moraxella catarrhalis (MC), Staphylococcus aureus and Streptococcus pyogenes by culture. Viruses were detected with polymerase chain reaction. Results: Median age of the 386 recruited children was 23.4 months, and 127 had no respiratory symptoms. More asymptomatic subjects were found negative for all bacteria tested (P < 0.01). SP (P < 0.01), MC (P = 0.001) and mixed bacterial colonization patterns were more frequent among symptomatic children (P < 0.05). Colonization of symptomatic, virus-positive children with MC was higher than in asymptomatic and/or virus-negative children (P = 0.005). The highest HI and MC colonization rates were recorded in association with influenza virus. A strongly negative association between SP and S. aureus, a higher rate of HI detection among SP colonized children and an increased likelihood of MC detection in the presence of HI were observed. HI colonization was more likely in the presence of respiratory syncytial virus and MC colonization was associated with rhinovirus detection. Conclusions: Viruses are associated with different NP bacterial colonization patterns. Observed pathogens' associations may play a role in disease, and continuous surveillance is required to follow possible effects of interventions such as vaccines. © 2015 Wolters Kluwer Health, Inc.

11th European Congress on Epileptology -- JUN 29-JUL 03, 2014 -- Stockholm, SWEDEN Ankarali, Handan Camdeviren/0000-0002-3613-0523; Ankarali, Seyit/0000-0003-3752-0846 WOS: 000337979300577 …

Leishmaniosis is a zoonose caused by protozoans of the genus Leishmania. The need for accurate diagnostic investigation of cases of leishmaniosis has rendered today the use of molecular biology techniques broadly applicable. However, the reliable application of these methods requires highly-specialised personnel, dedicated equipment and space. The aim of this study was the design and construction of functionalized gold nanoparticles (AuNPs) that would be incorporated into an easily applicable DNA detection methodology for the identification of Leishmania spp. in clinical samples.AuNPs 20. nm in diameter were conjugated with four oligonucleotide probes, targeting kinetoplastid minicircle DNA of Leishmania spp. In the absence of complimentary DNA, AuNPs-probes precipitate under acid environment causing a change of color from red to purple, which can be detected by visual observation. In the presence of target DNA the color of the solution remains red. The specific methodology was applied to positive and negative control samples and whole blood collected from dogs with suspected canine leishmaniosis. The method's minimum detection limit was defined to 11.5. ng of target DNA per μl of sample. Repeatability and reproducibility were 100%. Relative sensitivity and specificity referenced to PCR were calculated to 92% and 100% regarding collectively control and clinical samples.The proposed approach can be considered an appealing diagnostic solution especially for screening purposes in enzootic areas, where detection of very small amounts of the targeted analyte is not top priority. © 2013 Elsevier B.V.

Foodborne illness is a major cause of morbidity and mortality especially for children, even in the developed world. The aim of this study was to assess the microbial safety of food of animal origin intended for consumption by children in Greece. Sampling involved 8 categories of retail products and was completed with a collection of 850 samples. These were tested by PCR and/or culture for Listeria monocytogenes, Campylobacter spp., Escherichia coli O157, Salmonella spp., Cronobacter sakazakii, Brucella spp., and Mycobacterium avium subsp paratuberculosis (MAP). The number of positive results recorded collectively for the pathogens under investigation over the total number of samples tested was 3.52% and 0.12% by PCR and culture, respectively. The most frequently detected pathogen was enterohemorrhagic E. coli (1.29%) followed by Brucella (0.82%) and Listeria (0.82%). DNA belonging to MAP was detected in 0.35% of samples, which was also the percentage of positivity recorded for Campylobacter. The percentage for Salmonella was 0.12%. It can be concluded from the results that there is no indication of noncompliance for the tested food samples. However, detection of DNA belonging to pathogens that are transmissible to humans through food is indicative that constant vigilance regarding food safety is an absolute necessity. © 2014 Institute of Food Technologists®.

Foodborne illness is a major cause of morbidity and mortality especially for children, even in the developed world. The aim of this study was to assess the microbial safety of food of animal origin intended for consumption by children in Greece. Sampling involved 8 categories of retail products and was completed with a collection of 850 samples. These were tested by PCR and/or culture for Listeria monocytogenes, Campylobacter spp., Escherichia coli O-157, Salmonella spp., Cronobacter sakazakii, Brucella spp., and Mycobacterium avium subsp paratuberculosis (MAP). The number of positive results recorded collectively for the pathogens under investigation over the total number of samples tested was 3.52% and 0.12% by PCR and culture, respectively. The most frequently detected pathogen was enterohemorrhagic E. coli (1.29%) followed by Brucella (0.82%) and Listeria (0.82%). DNA belonging to MAP was detected in 0.35% of samples, which was also the percentage of positivity recorded for Campylobacter. The percentage for Salmonella was 0.12%. It can be concluded from the results that there is no indication of noncompliance for the tested food samples. However, detection of DNA belonging to pathogens that are transmissible to humans through food is indicative that constant vigilance regarding food safety is an absolute necessity.

Johne's disease or paratuberculosis is a chronic, progressive intestinal disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). One of the genes that have been targeted with regard to resistance or sensitivity to paratuberculosis is the SLC11A1 (solute carrier family 11 member A1). Here we extend our previous work to the sequence and structure analysis of the caprine SLC11A1 gene and we assess the functional impact of the most frequent polymorphisms of the 3' UTR region of the SLC11A1 gene to its expression in goat macrophages exposed in vitro to MAP. The role of these polymorphisms in primary immune response is also investigated with connection to gene expression of two interleukins (IL), one of which pro (IL-1a), and the other anti-inflammatory (IL-10). In order to assess gene response, quantitative detection of the SLC11A1, IL-10 and IL1a mRNA was performed by real time PCR before, and at 1, 3 and 24. h after exposure of primary cultures of peripheral blood monocyte-derived macrophages to MAP, collected from 54 goats of the Greek native goat breed. Sequence analysis of the 3' UTR end of the caprine SLC11A1 gene determined its full length to be 522 bases. Structure analysis confirmed the presence of two microsatellites consisted of a variable number of guanine-thymine repeats (regions A and B). The homozygous B7 genotype [B(GTn)7/7] was associated at a statistically significant level with increased expression of the SLC11A1 and IL-1α genes indicating increased in vitro responsiveness and therefore resistance of mononuclear derived macrophages to MAP infection. © 2013 Elsevier B.V.

Protozoa of the genus Leishmania are the causative agents of leishmaniosis. Although the polymerase chain reaction (PCR) has proved very effective in the detection of Leishmania DNA, a standardized method does not exist. In this study we attempt a comparative evaluation between one real time PCR (Method D), two in-house (Methods A and C), and a commercially available PCR assay (Method B) for the detection of Leishmania DNA, in order to support reliable diagnostic investigation of leishmaniosis. This evaluation was performed in regard to relative specificity and sensitivity, minimum detection limit (MDL), repeatability and reproducibility using cultured isolates and clinical samples. All the methods under study produced the expected result with the positive and negative controls. However with regard to clinical samples, Method C showed a statistically significant higher level of positivity. Relative sensitivity and specificity of Methods A, B and D in comparison to C was calculated respectively at 50.7%, 43%, 40%, and 90.8%, 93.4% and 89.5%. The MDL for Methods A-D was defined respectively at 30.7, 5, 3.7, and 5 promastigotes/ml. Repeatability and reproducibility were excellent in all cases with only the exception of Method A regarding reproducibility with a different brand of PCR reagents. The results that were recorded indicate that evaluation of PCR assays before their application for research and clinical diagnosis can provide useful evidence for their reliable application. Within this context the use of internal amplification controls and the confirmation of the specificity of the amplification product is recommended. © 2012 Elsevier Inc.

Stromal cell derived factor-1 (SDF-1), a CXC chemokine that plays an important role in the tumor growth, angiogenesis and metastasis of tumor cells, has a polymorphism at position 801 of its 3′-untranslated region, known as SDF1-3′A. The SDF1-3′A polymorphism has been investigated in various types of cancer, but no information is currently available on its role in pancreatic cancer. In this study, 80 pancreatic cancer patients and 160 normal healthy control subjects were investigated for the genotype and allelic frequencies of the SDF-1 gene using PCR-RFLP. The genotype frequencies for GG, GA and AA were 21.25, 77.5 and 1.25% in patients, and 42.5, 55 and 2.5% in healthy subjects, respectively. The A carrier group (GA+AA genotype) and the A allele were overrepresented among the patients with pancreatic cancer (p=0.015 and p=0.031, respectively). The GA+AA genotype was statistically correlated with an advanced T stage and the presence of lymph node metastasis, and displayed a clear trend towards significance in relation to the presence of distant metastatic disease (p=0.061). Only T stage was significantly related to A allele frequency (p=0.004). SDF1-3′ A allele carriers were more prevalent among cancer patients than among normal subjects. SDF1-3′ A allele carrier status may imply a higher risk of pancreatic cancer, while the presence of the A allele in pancreatic cancer patients may be related to aggressive features of this malignancy.

Objectives: This case-control study was performed to evaluate the association between a specific caspase-9 polymorphism as well as the genetic polymorphism -31G/C located in the cycle-dependent elements/cell cycle homology regions repressor element of the human survivin promoter and the risk of pancreatic cancer. Methods: Eighty patients with pancreatic cancer and 160 healthy controls were investigated for genotype and allelic frequencies of caspase-9 1263A/G and survivin -31G/C polymorphisms by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism. Results: The G carrier group of patients and the G allele of caspase-9 1263A/G were overrepresented among the pancreatic cancer cases. With regard to tumor characteristics, a statistically significant association was detected between the survivin C carrier group of patients and the advanced T stage as well as the presence of lymph node metastasis. Conclusions: The caspase-9 G allele confers increased susceptibility to pancreatic cancer development, and the survivin C carriage status may be related to aggressive features of this malignancy. Copyright © 2010 by Lippincott Williams & Wilkins.

MicroRNAs (miRs) are single-stranded RNAs of 18-25 nucleotides. These molecules regulate gene expression at the post-transcriptional level; several of these are differentially expressed in asthma as well as in viral acute respiratory infections (ARIs), the main triggers of acute asthma exacerbations. In recent years, miRs have been studied in order to discover drug targets as well as biomarkers for diagnosis, disease severity and prognosis. We describe recent findings on miR expression and function in asthma and their role in the regulation of viral ARIs, according to cell tissue specificity and asthma severity. By combining the above information, we identify miRs that may be important in virus-induced asthma exacerbations. This is the first attempt to link miR profiles of asthmatic patients and ARI-induced miRs, addressing the question of whether there might be a specific miR deficit in asthmatic subjects that make them more susceptible and/or reactive to infection. Copyright © 2020 The Korean Academy of Asthma, Allergy and Clinical Immunology • The Korean Academy of Pediatric Allergy and Respiratory Disease.