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Background: Food protein‐induced allergic proctocolitis (FPIAP) is the leading cause of rectal bleeding in infants. Tolerance is presumed to develop until the first year of age, although natural history studies are scarce, making the determination of the ideal duration for any intervention, challenging. Intestinal microbiota (IM) is crucial in food allergy development; however, data for FPIAP remain limited. This study aimed to assess FPIAP remission after 3 months of milk avoidance and its correlation with IM longitudinal changes. Methods: A prospective observational study of infants aged ≤6 months with a diagnosis of FPIAP. After 3 months of management according to a clinical algorithm, infants were subjected to a milk challenge using either a cow (CM) or a goat (GM) milk formula in a random order. Stool samples were collected longitudinally for microbiome analysis. Results: Out of 61 infants, 57 were challenged (29 with CM, 28 with GM). Of these, 55 (96.5%) achieved tolerance, with no difference in tolerance rates between CM (28/29) and GM (27/28). The average age of tolerance development was 6.3 months. Enterobacteriaceae clusters (Klebsiella‐ and Shigella‐dominated) were most often represented in samples from symptomatic infants. In contrast, Bacteroides and Bifidobacteria clusters emerged later, in apparently healthy infants. Conclusion: A 3‐month intervention was sufficient for almost all infants to achieve tolerance. GM was tolerated equally well to CM. Symptomatic FPIAP is associated with immature enterotypes, while disease remission coincides with microbiome changes in time. [ABSTRACT FROM AUTHOR]

Citrus brown mite, Eutetranychus orientalis (Klein), is one of the most serious pests on fruit, vegetable and ornamental plants in the world. The present study aimed to evaluate the toxicity, and the repellency/attractancy effects of five essential oils (clove, castor, cinnamon, black cumin and anise) on E. orientalis under laboratory conditions. Also, field experiments were performed to investigate the efficacy of two essential oils (castor oil and black cumin 10 %), compared with other acaricides and predacious mites. Two acaricides (abamectin 5% EC [Biomectin™] and fenpyroximate 5 % SC [Ortus ]), in addition to the predacious mites Phytoseiulus persimilis and Euseius scutalis, applied at two levels of release (200 and 300 individuals/tree). The results showed that after 1 h of the treatment, essential oils of black cumin and anise recorded the highest percentage of repellency (80-100 %) followed by castor oil (30-90 %), and clove oil (50 and 70 %), while cinnamon was the lowest. In addition, castor oil showed good repellency effect after 6 h, while black cumin has the same effect after 1 h of application. Field experiments revealed that castor oil gave the best efficacy in suppressing E. orientalis population compared with control, followed by black cumin oil. While applying the acaricides fenpyroximate and abamectin recorded good reduction of E. orientalis with mortality of 88.85 and 87.92 %, respectively. The numbers of E. orientalis were affected by releasing the biological control agent P. persimilis at 200 and 300 individuals/tree, where the numbers of E. orientalis decreased on trees to 1.5, 1.0 indiv./10 leaves after 15 days of release compared with pre-treatment account 3.0, 2.5 indiv./10 leaves, respectively. [ABSTRACT FROM AUTHOR]

Populations of Tetranychus urticae Koch (Acari: Tetranychidae), and its predatory mite, Agistemus exsertus Gonzalez (Acari: Stigmaeidae), were determined on four soybean varieties, namely, Giza 22, 35, 82 and 111, during the 2016 season, and in addition some biological aspects of both species were studied. The results obtained revealed that Giza 35 variety was the most susceptible variety with significant difference between the mean numbers of T. urticae compared with other varieties. The mean rate of infestation was 11.89 individuals/25 cm² of leaves, while the mean number of T. urticae was 7.11 and 3.54 individuals/25 cm² of leaves for Giza 111 and Giza 82 without significant difference. Giza 22 recorded the lowest mean number of T. urticae, 1.53 individuals/25 cm². The population dynamics of T. urticae showed that infestation increased gradually from mid-June reach its maximum at the end of July, and then decreased gradually at the beginning of August, reaching its minimum number at the beginning of September before harvest time. As for A. exsertus, data showed that the mean number of A. exsertus was the highest on Giza 35 (2.46 individuals/25 cm²) followed by Giza 22 (2.13 individuals/25 cm²), but Giza 82 and Giza 111 mean numbers were 1.99, and 1.91 individuals/25 cm². Life cycle of tetranychid males was the shortest when reared on Giza 35 with significant difference with other varieties. As for females the shortest life cycle was when reared on Giza 35 with significant difference with Giza 22 which recorded the longest life cycle for males and females, respectively. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Polyethylene glycols (PEG) or macrogols are polymers of ethylene oxide widely used in drugs either as active substances or, more commonly, as excipients. We report a Caucasian 32-year-old woman with referred anaphylaxis almost instantly after oral intake of a macrogol-containing laxative. Despite an anaphylactic reaction, the patient showed negative results for both the skin test and specific IgE to monomer, while the basophil activation test and oral challenge were positive. The patient was later successfully vaccinated with a polysorbate 80-containing SARS-CoV-2 vaccine following an additional work-up. As a result, the inactive form of PEG cannot be fully diagnosed, and it is considered a "hidden" allergen. Allergens like polysorbates need special consideration due to their possible cross-reactivity by their specific derivatives.

Background: Immunoglobulin-E(IgE)-mediated hypersensitivity to cow's milk allergens is a frequent cause of severe and life-threatening anaphylactic reactions. Besides case histories and controlled food challenges, the detection of the IgE antibodies specific to cow's milk allergens is important for the diagnosis of cow-milk-specific IgE sensitization. Cow´s milk allergen molecules provide useful information for the refined detection of cow-milk-specific IgE sensitization., Methods: A micro-array based on ImmunoCAP ISAC technology was developed and designated milk allergen micro-array (MAMA), containing a complete panel of purified natural and recombinant cow's milk allergens (caseins, α-lactalbumin, β-lactoglobulin, bovine serum albumin-BSA and lactoferrin), recombinant BSA fragments, and α-casein-, α-lactalbumin- and β-lactoglobulin-derived synthetic peptides. Sera from 80 children with confirmed symptoms related to cow's milk intake (without anaphylaxis: n = 39; anaphylaxis with a Sampson grade of 1-3: n = 21; and anaphylaxis with a Sampson grade of 4-5: n = 20) were studied. The alterations in the specific IgE levels were analyzed in a subgroup of eleven patients, i.e., five who did not and six who did acquire natural tolerance., Results: The use of MAMA allowed a component-resolved diagnosis of IgE sensitization in each of the children suffering from cow's-milk-related anaphylaxis according to Sampson grades 1-5 requiring only 20-30 microliters of serum. IgE sensitization to caseins and casein-derived peptides was found in each of the children with Sampson grades of 4-5. Among the grade 1-3 patients, nine patients showed negative reactivity to caseins but showed IgE reactivity to alpha-lactalbumin ( n = 7) or beta-lactoglobulin ( n = 2). For certain children, an IgE sensitization to cryptic peptide epitopes without detectable allergen-specific IgE was found. Twenty-four children with cow-milk-specific anaphylaxis showed additional IgE sensitizations to BSA, but they were all sensitized to either caseins, alpha-lactalbumin, or beta-lactoglobulin. A total of 17 of the 39 children without anaphylaxis lacked specific IgE reactivity to any of the tested components. The children developing tolerance showed a reduction in allergen and/or peptide-specific IgE levels, whereas those remaining sensitive did not., Conclusions: The use of MAMA allows for the detection, using only a few microliters of serum, of IgE sensitization to multiple cow's milk allergens and allergen-derived peptides in cow-milk-allergic children with cow-milk-related anaphylaxis.

Purpose of Review: This review aims to report all the recent studies that are implicated in DNA methylation analysis in the field of allergy and to underline the complexity of the study methodologies and results., Recent Findings: Although the growing number of DNA methylation studies have yet to point to a specific mechanism, herein we provide an overview of the majority of pathways considered to be implicated and highlight particular genes, like KNH2 , ATPAF2 and ZNF385A , for their potential as biomarkers., Summary: The epigenetic profile of respiratory allergic diseases, and particularly DNA methylation, has been investigated in various populations, so as to gain a better understanding of its role in pathogenesis. Through our analysis, multiple links are presented between differential DNA methylation loci and IgE sensitization, lung functionality and severity of the disease. Additionally, associations of this epigenetic change with maternal asthma, age, sex and environmental factors are described, thus uncovering specific gene families that, after further examination could be used as methylation biomarkers in cases of allergic disease., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Four EMA-approved vaccines against SARS-CoV-2 are currently available. Data regarding antibody responses to initial vaccination regimens in patients with inflammatory bowel diseases (IBD) are limited., Methods: We conducted a prospective, controlled, multicenter study in tertiary Greek IBD centers. Participating patients had completed the initial vaccination regimens (1 or 2 doses, depending on the type of COVID-19 vaccine) at least 2 weeks before study enrolment. Anti-S1 IgG antibody levels were measured. Demographic and adverse events data were collected., Results: We tested 403 patients (Crohn's disease, 58.9%; male, 53.4%; median age, 45 years) and 124 healthy controls (HCs). Following full vaccination, 98% of patients seroconverted, with mRNA vaccines inducing higher seroconversion rates than viral vector vaccines (P = .021). In total, IBD patients had lower anti-S1 levels than HCs (P < .001). In the multivariate analysis, viral vector vaccines (P < .001), longer time to antibody testing (P < .001), anti-TNFα treatment (P = .013), and age (P = .016) were independently associated with lower anti-S1 titers. Vedolizumab monotherapy was associated with higher antibody levels than anti-TNFα or anti-interleukin-12/IL-23 monotherapy (P = .023 and P = .032). All anti- SARS-CoV-2 vaccines were safe., Conclusions: Patients with IBD have impaired antibody responses to anti-SARS-CoV-2 vaccination, particularly those receiving viral vector vaccines and those on anti-TNFα treatment. Older age also hampers antibody production after vaccination. For those low-response groups, administration of accelerated or prioritized booster vaccination may be considered., (© The Author(s) 2022. Published by Oxford University Press on behalf of Crohn’s & Colitis Foundation. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The susceptibility of the atopic population to respiratory infections (RI) has not been fully elucidated. This susceptibility is attributed to the immune dysregulation that characterizes atopic diseases. Although, the exact mechanisms involved are not fully understood, there is evidence that shows that the maturation of innate immunity progresses differently in patients with atopy. Objective: The aim of the study was to evaluate the susceptibility to viral RIs (VRI) based on the number and duration of them in different age groups in subjects with atopy and subjects without atopy. Methods: Seventy-eight subjects (39 healthy and 39 with atopy) were included in the study. All the subjects were evaluated by a specialist and defined as being atopic if they had a clinical history and/or symptoms compatible with any allergic diseases and relevant sensitizations. Epidemiologic data were recorded based on a standardized questionnaire, which included recording habits, conditions, and living environment as well as the history of viral infections during the last year. Results: In our population, children with atopy were found to be more susceptible to viral RIs than children without atopy (p = 0.02), whereas there was no difference in susceptibility between healthy adults and adults with atopy (18-45 years old). More specifically, the atopic age group 2-5 years old showed the higher susceptibility to VRIs. Conclusion: This study provided evidence that children with atopy, especially at ages 2-5 years old, had more numerically and prolonged RIs than did the subjects without atopy. These clinical findings support the hypothesis of distracted maturation of innate immunity in subjects with atopy.

Background: Immunoglobulin E (IgE)-mediated cow's milk allergy (CMA) can be life-threatening and affects up to 3% of children. Hypoallergenic infant formulas based on hydrolyzed cow's milk protein are increasingly considered for therapy and prevention of cow's milk allergy. The aim of this study was to investigate the allergenic activity and ability to induce T cell and cytokine responses of an infant formula based on extensively hydrolyzed cow's milk protein (whey) (eHF, extensively hydrolyzed formula) supplemented with Galactooligosaccharides (GOS) and Limosilactobacillus fermentum CECT5716 (LF) to determine its suitability for treatment and prevention of CMA., Methods: eHF and standard protein formula based on intact cow's milk proteins (iPF) with or without Galactooligosaccharide (GOS) and Limosilactobacillus fermentum CECT5716 (LF) were investigated with allergen-specific antibodies and tested for IgE reactivity and allergenic activity in basophil degranulation assays with sera from cow's milk (CM)-allergic infants/children. Their ability to stimulate T cell proliferation and cytokine secretion in cultured peripheral blood mononuclear cells (PBMC) from CM-allergic infants and children was studied with a FACS-based carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assay and xMAP Luminex fluorescent bead-based technology, respectively., Results: An eHF supplemented with GOS and LF exhibiting almost no IgE reactivity and allergenic activity was identified. This eHF induced significantly lower inflammatory cytokine secretion as compared to an intact protein-based infant formula but retained T cell reactivity., Conclusions: Due to strongly reduced allergenic activity and induction of inflammatory cytokine secretion but retained T cell reactivity, the identified eHF may be used for treatment and prevention of CMA by induction of specific T cell tolerance.

Bacteriophages represent the most extensive group of viruses within the human virome and have a significant impact on general health and well-being by regulating bacterial population dynamics. Staphylococcus aureus , found in the anterior nostrils, throat and skin, is an opportunistic pathobiont that can cause a wide range of diseases, from chronic inflammation to severe and acute infections. In this study, we developed a human cell-based homeostasis model between a clinically isolated strain of S. aureus 141 and active phages for this strain (PYO Sa141 ) isolated from the commercial Pyophage cocktail (PYO). The cocktail is produced by Eliava BioPreparations Ltd. (Tbilisi, Georgia) and is used as an add-on therapy for bacterial infections, mainly in Georgia. The triptych interaction model was evaluated by time-dependent analysis of cell death and inflammatory response of the nasal and bronchial epithelial cells. Inflammatory mediators (IL-8, CCL5/RANTES, IL-6 and IL-1β) in the culture supernatants were measured by enzyme-linked immunosorbent assay and cell viability was determined by crystal violet staining. By measuring trans-epithelial electrical resistance, we assessed the epithelial integrity of nasal cells that had differentiated under air-liquid interface conditions. PYO Sa141 was found to have a prophylactic effect on airway epithelial cells exposed to S. aureus 141 by effectively down-regulating bacterial-induced inflammation, cell death and epithelial barrier disruption in a time-dependent manner. Overall, the proposed model represents an advance in the way multi-component biological systems can be simulated in vitro .

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: Acute bronchiolitis is one of the most common respiratory infections in infancy. Although most infants with bronchiolitis do not get hospitalized, infants with hospitalized bronchiolitis are more likely to develop wheeze exacerbations during the first years of life. The objective of this prospective cohort study was to develop machine learning models to predict incidence and persistence of wheeze exacerbations following the first hospitalized episode of acute bronchiolitis. Methods: One hundred thirty-one otherwise healthy term infants hospitalized with the first episode of bronchiolitis at a tertiary pediatric hospital in Athens, Greece, and 73 age-matched controls were recruited. All patients/controls were followed up for 3 years with 6-monthly telephone reviews. Through principal component analysis (PCA), a cluster model was used to describe main outcomes. Associations between virus type and the clusters and between virus type and other clinical characteristics and demographic data were identified. Through random forest classification, a prediction model with smallest classification error was identified. Primary outcomes included the incidence and the number of caregiver-reported wheeze exacerbations. Results: PCA identified 2 clusters of the outcome measures (Cluster 1 and Cluster 2) that were significantly associated with the number of recurrent wheeze episodes over 3-years of follow-up (Chi-Squared, p < 0.001). Cluster 1 included infants who presented higher number of wheeze exacerbations over follow-up time. Rhinovirus (RV) detection was more common in Cluster 1 and was more strongly associated with clinical severity on admission ( p < 0.01). A prediction model based on virus type and clinical severity could predict Cluster 1 with an overall error 0.1145 (sensitivity 75.56% and specificity 91.86%). Conclusion: A prediction model based on virus type and clinical severity of first hospitalized episode of bronchiolitis could predict sensitively the incidence and persistence of wheeze exacerbations during a 3-year follow-up. Virus type (RV) was the strongest predictor., Competing Interests: NP reports personal fees from Novartis, personal fees from Nutricia, personal fees from HAL, personal fees from MENARINI/FAES FARMA, personal fees from SANOFI, personal fees from MYLAN/MEDA, personal fees from BIOMAY, personal fees from AstraZeneca, personal fees from GSK, personal fees from MSD, personal fees from ASIT BIOTECH, personal fees from Boehringer Ingelheim, grants from Gerolymatos International SA, grants from Capricare, outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Makrinioti, Maggina, Lakoumentas, Xepapadaki, Taka, Megremis, Manioudaki, Johnston, Tsolia, Papaevangelou and Papadopoulos.)

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 development of methods and miniaturized systems for fast and reliable quantitative determinations at the Point-of-Care is a top challenge and priority in diagnostics. In this work, a compact bench-top system, based on White Light Reflectance Spectroscopy, is introduced and evaluated in an application with high clinical interest, namely the determination of C-Reactive protein (CRP) in human blood samples. The system encompassed all the necessary electronic and optical components for the performance of the assay, while the dedicated software provided the sequence and duration of assay steps, the reagents flow rate, the real-time monitoring of sensor response, and data processing to deliver in short time and accurately the CPR concentration in the sample. The CRP assay included two steps, the first comprising the binding of sample CRP onto the chip immobilized capture antibody and the second the reaction of the surface immunosorbed CRP molecules with the detection antibody. The assay duration was 12 min and the dynamic range was from 0.05 to 200 μg/mL, covering both normal values and acute inflammation incidents. There was an excellent agreement between CRP values determined in human plasma samples using the developed device with those received for the same samples by a standard diagnostic laboratory method.

L-Dopa decarboxylase (DDC) is the most significantly co-expressed gene with ACE2, which encodes for the SARS-CoV-2 receptor angiotensin-converting enzyme 2 and the interferon-inducible truncated isoform dACE2. Our group previously showed the importance of DDC in viral infections. We hereby aimed to investigate DDC expression in COVID-19 patients and cultured SARS-CoV-2-infected cells, also in association with ACE2 and dACE2. We concurrently evaluated the expression of the viral infection- and interferon-stimulated gene ISG56 and the immune-modulatory, hypoxia-regulated gene EPO. Viral load and mRNA levels of DDC, ACE2, dACE2, ISG56 and EPO were quantified by RT-qPCR in nasopharyngeal swab samples from COVID-19 patients, showing no or mild symptoms, and from non-infected individuals. Samples from influenza-infected patients were analyzed in comparison. SARS-CoV-2-mediated effects in host gene expression were validated in cultured virus-permissive epithelial cells. We found substantially higher gene expression of DDC in COVID-19 patients (7.6-fold; p = 1.2e-13) but not in influenza-infected ones, compared to non-infected subjects. dACE2 was more elevated (2.9-fold; p = 1.02e-16) than ACE2 (1.7-fold; p = 0.0005) in SARS-CoV-2-infected individuals. ISG56 (2.5-fold; p = 3.01e-6) and EPO (2.6-fold; p = 2.1e-13) were also increased. Detected differences were not attributed to enrichment of specific cell populations in nasopharyngeal tissue. While SARS-CoV-2 virus load was positively associated with ACE2 expression (r≥0.8, p<0.001), it negatively correlated with DDC, dACE2 (r≤-0.7, p<0.001) and EPO (r≤-0.5, p<0.05). Moreover, a statistically significant correlation between DDC and dACE2 expression was observed in nasopharyngeal swab and whole blood samples of both COVID-19 and non-infected individuals (r≥0.7). In VeroE6 cells, SARS-CoV-2 negatively affected DDC, ACE2, dACE2 and EPO mRNA levels, and induced cell death, while ISG56 was enhanced at early hours post-infection. Thus, the regulation of DDC, dACE2 and EPO expression in the SARS-CoV-2-infected nasopharyngeal tissue is possibly related with an orchestrated antiviral response of the infected host as the virus suppresses these genes to favor its propagation., Competing Interests: The authors have declared that no competing interests exist.

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.

The airway epithelium is the primary site where inhaled and resident microbiota interacts between themselves and the host, potentially playing an important role on allergic asthma development and pathophysiology. With the advent of culture independent molecular techniques and high throughput technologies, the complex composition and diversity of bacterial communities of the airways has been well-documented and the notion of the lungs' sterility definitively rejected. Recent studies indicate that the microbial composition of the asthmatic airways across the spectrum of disease severity, differ significantly compared with healthy individuals. In parallel, a growing body of evidence suggests that bacterial viruses (bacteriophages or simply phages), regulating bacterial populations, are present in almost every niche of the human body and can also interact directly with the eukaryotic cells. The triptych of airway epithelial cells, bacterial symbionts and resident phages should be considered as a functional and interdependent unit with direct implications on the respiratory and overall homeostasis. While the role of epithelial cells in asthma pathophysiology is well-established, the tripartite interactions between epithelial cells, bacteria and phages should be scrutinized, both to better understand asthma as a system disorder and to explore potential interventions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tzani-Tzanopoulou, Skliros, Megremis, Xepapadaki, Andreakos, Chanishvili, Flemetakis, Kaltsas, Taka, Lebessi, Doudoulakakis and Papadopoulos.)

Aim: The aim of the study was to evaluate computed tomography (CT) artifacts and image recognition of the CyberKnife system. Regarding fiducial markers, VISICOIL of 0.5 mm × 5.0 mm and 0.75 mm × 5.0 mm, ball-shaped Gold Anchor (GA) of 0.28 mm × 10 mm and 0.28 mm × 20 mm, were compared with the standard cylinder marker of 0.9 mm × 3.0 mm (ACCULOC)., Background: Recently, various kinds of commercial fiducial markers have been available in CyberKnife treatment., Materials and Methods: The CT images of a water equivalent gel with each fiducial marker were acquired for the evaluation of CT artifacts. The evaluation was performed using the standard deviation of Hounsfield Unit (HU) value for a rectangle region near the fiducial marker. Then, to evaluate the image recognition, each fiducial marker was located to overlap in the target locating system (TLS) for the two sites; the vertebral bone and the pubic bone., Results: For CT artifacts, the standard deviations of the VISICOIL of 0.5 mm × 5.0 mm was the smallest. The image recognition of four fiducial markers had a value close to the standard cylinder marker and was feasible for common use, but was slightly poorer when using GA of 0.28 mm × 10 mm in the dynamic conditions., Conclusion: Our results indicated that VISICOIL 0.5 × 5.0 mm and the GAs can be used nearly always for CyberKnife treatment in spite of their much thinner needles than those of cylinder types., (© 2019 Greater Poland Cancer Centre. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: There are no financial or other issues that might lead to conflict of interest., (Copyright © 2020 The Korean Academy of Asthma, Allergy and Clinical Immunology · The Korean Academy of Pediatric Allergy and Respiratory Disease.)

Background: Infections with human rhinoviruses (RVs) are responsible for millions of common cold episodes and the majority of asthma exacerbations, especially in childhood. No drugs specifically targeting RVs are available., Objective: We sought to identify specific anti-RV molecules based on DNAzyme technology as candidates to a clinical study., Methods: A total of 226 candidate DNAzymes were designed against 2 regions of RV RNA genome identified to be sufficiently highly conserved between virus strains (ie, the 5'-untranslated region and cis-acting replication element) by using 3 test strains: RVA1, RVA16, and RVA29. All DNAzymes were screened for their cleavage efficiency against in vitro-expressed viral RNA. Those showing any catalytic activity were subjected to bioinformatic analysis of their reverse complementarity to 322 published RV genomic sequences. Further molecular optimization was conducted for the most promising candidates. Cytotoxic and off-target effects were excluded in HEK293 cell-based systems. Antiviral efficiency was analyzed in infected human bronchial BEAS-2B cells and ex vivo-cultured human sinonasal tissue., Results: Screening phase-generated DNAzymes characterized by either good catalytic activity or by high RV strain coverage but no single molecule represented a satisfactory combination of those 2 features. Modifications in length of the binding domains of 2 lead candidates, Dua-01(-L12R9) and Dua-02(-L10R11), improved their cleavage efficiency to an excellent level, with no loss in eminent strain coverage (about 98%). Both DNAzymes showed highly favorable cytotoxic/off-target profiles. Subsequent testing of Dua-01-L12R9 in BEAS-2B cells and sinonasal tissue demonstrated its significant antiviral efficiency., Conclusions: Effective and specific management of RV infections with Dua-01-L12R9 might be useful in preventing asthma exacerbations, which should be verified by clinical trials., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

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<0.05) in relation to other groups. A significant (P<0.05) hypomethylation of the hepatic GR promoter was revealed in males of the R2 group and a tendency towards the same in the R1 group, along with increased (P<0.001) GR gene expression in both restricted groups. A significant increase (P<0.05) in hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene expression was found in male lambs of both undernourished groups, accompanied by increased (P<0.01) protein levels, while no differences were detected for glucose-6-phosphatase (G6Pase) mRNA abundance and protein levels. In female lambs, no differences between groups were observed for any parameter studied. These data represent potential mechanisms by which insults in early life may lead to persistent physiological changes in the offspring.

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.

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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®)

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 20nm 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 11.5ng 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.)

Genetic polymorphisms in the human solute carrier family 11 member 1 (SLC11A1) gene predispose to susceptibility to infectious/inflammatory diseases and cancer. Human susceptibility to these diseases exhibits allelic association with a polymorphic regulatory Z-DNA-forming microsatellite of a (GT/AC)n repeat. The carriage of different alleles may influence chromatin remodeling and accessibility by transcription factors. Of particular importance is the binding site for the Activating Protein 1 (AP-1) elements, (ATF-3 and c-Jun), adjacent to the 5' sequence of the Z-DNA-forming polymorphism. The aim of the study was to characterize the transcriptional mechanisms controlling different alleles of SLC11A1 expression by ATF-3 and c-Jun. Allele 2, [T(GT)5AC(GT)5AC(GT)10GGCAGA(G)6], and Allele 3, [T(GT)5AC(GT)5AC(GT)9GGCAGA(G)6], were subcloned into the PGL2Basic vector. Transient transfections of THP-1 cells with the constructs, in the presence or absence of pATF-3 were preformed. Luciferase expression was determined. To document the recruitment of ATF-3 and c-Jun, to the polymorphic promoter alleles in vivo, we performed ChIP assays with transient transfected THP-1 cells treated with or without lipopolyssacharides. Our data documented that ATF-3 suppresses the transcriptional activation of Allele-3, and this suppression is enhanced in the presence of lipopolyssacharides. Our findings suggest that ATF-3 and c-Jun may influence heritable variation in SLC11A1-dependent innate resistance to infection and inflammation both within and between populations.

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: E-selectin, an intercellular adhesion molecule that plays important roles in metastasis of tumor cells, has a polymorphism in exon 4 that results in the substitution of a serine by an arginine within the extracellular domain of the receptor, which increases its affinity for ligands. No evidence exist on the role of E-selectin polymorphism in pancreatic cancer., Methods: Eighty pancreatic cancer patients and 160 cases of normal healthy control subjects were investigated for genotype and allelic frequencies of S128R polymorphism of E-selectin gene by PCR-RFLPs., Results: The frequencies for "AA," "CA," and "CC" genotypes were 46.25%, 50%, and 3.75% in patients, and 63.75%, 26.9%, and 9.4% in healthy subjects, respectively. The "C" carriers group of patients ("CA + CC" genotype) and the "C" allele were over-represented among the pancreatic cancer cases (P = 0.012 and 0.096, respectively). Advanced T stage, the presence of lymph node and other adverse pathologic characteristics were not significantly correlated with either the "CA + CC" genotype group of patients or the presence of "C" allele., Conclusions: E-selectin S128R "C" allele may confer an increased susceptibility to pancreatic cancer development, while its carriage status does not appear to be related to the 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.

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.

The plasmid profiles of virulent Rhodococcus equi strains isolated on three horse-breeding farms located in different parts of Hungary were investigated. From 49 soil samples collected on the three farms, 490 R. equi isolates (10 from each sample) were obtained and tested for the presence of 15- to 17-kDa antigens (VapA) by immunoblotting and PCR. Ninety-eight VapA-positive isolates were detected from 30 of the 49 culture-positive samples with a prevalence ranging from 13.1% to 23.2%. Of the 98 virulent isolates, 70 contained an 85-kb type I plasmid, 13 contained an 87-kb type I plasmid, and 15 contained an 85-kb type III plasmid which had been uniquely isolated from soil isolates in the United States. This study demonstrates that the virulent form of R. equi is very widespread in the soil environment of these stud farms in Hungary and the plasmid pattern is different from farm to farm.

We have developed a new analytical transmission electron microscope (TEM), called coincidence TEM, which, in principle, enables observation of elemental mapping images at a high signal-to-noise ratio. We have previously reported the successful observation of an elemental mapping image of a specimen, but over a very long period of time (168 h). To solve this inefficiency, we installed a gamma-type imaging energy filter in the coincidence TEM to remove the no-loss electrons, which are mainly transmitted electrons. This has enabled the intensity of the background signals in the coincidence measurement to be markedly reduced. The coincidence TEM with a gamma-type imaging energy filter allows the coincidence image to be observed in 3 h, thus, the measurement time is shortened by two orders of magnitude. Moreover, the use of a silicon drift detector (SDD) will shorten the measurement time.