983 research outputs found
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Thyroglobulin specific IgE and a possible link to suspected penicillin induced allergic skin manifestations – cross sectional study
No full textPorcine thyroglobulin was important in the discovery of alpha-Gal allergy. Here, the linkage of porcine thyroglobulin-specific IgE with IgE positivity to routinely assessed allergens and to the incoming diagnosis within a population of suspected atopic individuals is explored. IgE, IgA, total IgG and IgG subclasses to porcine thyroglobulin, IgE to bovine, human thyroglobulin and meat extract were measured with ELISA. The following correlations were observed in IgE binding to porcine and bovine thyroglobulin (r = 0.910, p = 1x10−17), porcine and human thyroglobulin (r = 0.635, p = 4x10−6), human and bovine thyroglobulin (r = 0.746, p = 6x10−9) and porcine thyroglobulin and meat extract (r = 0.482, p = 0.0009). Only one out of ten samples which showed binding to porcine thyroglobulin in ELISA tested positive with ImmunoCAP alpha-Gal, implying different epitope/s. Increased IgE binding was detected towards a more electronegative fraction of porcine thyroglobulin separated according to charge and the binding could be partially inhibited by galactose. Anti-thyroglobulin IgE was found in 29.7% of the population, in subjects who were significantly younger, p < 0.0001 and it occurred more frequently in patients referred for testing penicillin specific IgE (OR 2.48, p = 0.0059) and were negative. IgE specific to porcine, bovine and possibly human thyroglobulin may be implicated in post-infectious skin manifestation misinterpreted as penicillin allergy
Modulation of T-Cell-Dependent Humoral Immune Response to Influenza Vaccine by Multiple Antioxidant/Immunomodulatory Micronutrient Supplementation
No full textNotwithstanding prevalence gaps in micronutrients supporting immune functions, the significance of their deficits/supplementation for the efficacy of vaccines is underinvestigated. Thus, the influence of supplementation combining vitamins C and D, zinc, selenium, manganese, and N-acetyl cysteine on immune correlates/surrogates of protection conferred by a quadrivalent influenza vaccine (QIV) in mice was investigated. The supplementation starting 5 days before the first of two QIV injections given 28 days apart increased the serum titres of total and neutralizing IgG against each of four influenza strains from QIV. Accordingly, the frequencies of germinal center B cells, follicular CD4+ T helper (Th) cells, and IL-21-producing Th cells increased in secondary lymphoid organs (SLOs). Additionally, the supplementation improved already increased IgG response to the second QIV injection by augmenting not only neutralizing antibody production, but also IgG2a response, which is important for virus clearance, through favoring Th1 differentiation as indicated by Th1 (IFN-γ)/Th2 (IL-4) signature cytokine level ratio upon QIV restimulation in SLO cell cultures. This most likely partly reflected antioxidant action of the supplement as indicated by splenic redox status analyses. Thus, the study provides a solid scientific background for further research aimed at repurposing the use of this safe and inexpensive micronutrient combination to improve response to the influenza vaccine
Insight into the Probiogenomic Potential of Enterococcus faecium BGPAS1-3 and Application of a Potent Thermostable Bacteriocin
No full textThis study aimed to investigate the probiogenomic features of artisanal bacteriocin-producing Enterococcus faecium BGPAS1-3 and the use of the improved pMALc5HisEk expression vector for overexpressing class II bacteriocins and the application of purified bacteriocin 31 in a milk model as a preservative against L. monocytogenes. The BGPAS1-3 strain was isolated from traditional fresh soft cheese manufactured in households on a small scale in rural locations surrounding Pale Mountain City in Bosnia and Herzegovina. The whole-genome sequencing approach and bioinformatics analyses revealed that the strain BGPAS1-3 was non-pathogenic to humans. The presence of bacteriocin operons suggested the ability of the isolate to suppress the growth of pathogens. Coding regions for three maturated bacteriocins (bacteriocin 31, bacteriocin 32, and enterocin P) produced by BGPAS1-3 were amplified and expressed in Escherichia coli ER2523 using the pMALc5HisEk system. All three bacteriocins were successfully overexpressed and purified after enterokinase cleavage but showed different antimicrobial activity. Bacteriocin 31 showed significantly stronger antimicrobial activity compared with bacteriocin 32. It was the only one that proved to be suitable for use as a food preservative against L. monocytogenes in a milk model
Immunomodulatory impact of the BanLec-Bet v 1 chimera and its variants on antigen-presenting cells
No full textEmploying effective adjuvants and hypoallergenic isoforms is essential for improving the efficacy and safety of allergenspecific immunotherapy (AIT). Banana lectin (BanLec) exhibits immunomodulatory potential, and mutation of histidine
at position 84 to threonine (BanLecH84T) reduced its mitogenicity. Our study aims to assess the immunomodulatory
potential on antigen-presenting cells (APCs) of recombinant chimeras combining the major birch pollen allergen Bet v
1a and its hypoallergenic isoform Bet v 1l with BanLec and BanLecH84T. After the production in Escherichia coli and
purification, chimeras were co-incubated with peripheral blood mononuclear cells (PBMC) from birch pollen-allergic
patients. Subsequently, secreted cytokines were analyzed. Antigen uptake was evaluated by co-incubating the treated
monocyte-derived dendritic cells (MoDC) or HLA-DR7+ EBV-transformed B cells (EBV-BCL) with T cell receptor
(TCR) transgenic (tg) Jurkat reporter cells. The tg TCR recognizes the immunodominant epitope of the Bet v 1, Bet v
1142-153 in the context of HLA-DR7 (Jurkattg). MoDC were analyzed for the upregulation of activation marker expression
(CD80, CD86, HLA-DR) and secreted cytokines (IL-6, IL-8, IL-10, IL-12p40, IL-23, TNF- α, IFN-γ). Furthermore,
chimera’s uptake by APCs was modulated by incubation with different uptake pathway inhibitors/enhancers during
antigen loading, and co-culture with Jurkattg. We found that the co-incubation of PBMC with Bet v 1a-BanLec prompted
the secretion of the anti-inflammatory cytokine IL-10 and augmented the IFN-γ/IL-4 ratio. Co-incubation of chimeras
with EBV-BCL or MoDC revealed that all chimeras strongly activated the allergen-specific Jurkattg. Additionally,
activation marker expression and secreted cytokines of MoDC co-incubated with the respective chimeras indicated that
they led to the activation of these cells. Supplementation of media with low concentrations of glucose stimulated antigen
uptake and subsequent Jurkattg activation. In contrast, antigen uptake was inhibited by sucrose, suggesting interference
with the macropinocytotic uptake pathway. Our results highlight the potential of chimeras to modulate allergen-specific
immune responses, enhancing anti-inflammatory cytokine secretion and promoting allergen-specific T-cell activation
thereby demonstrating their potential in AIT
mRNA vaccine manufacturing – challenges in plasmid DNA cloning vector design
No full textIn the post-COVID-19 era, there has been a notable surge in the development of mRNA vaccines. These vaccines are not only targeting various pathogens beyond SARS-CoV-2 but also hold promise in treating cancer and genetic disorders. These type of vaccines are revolutionizing vaccinology through their inherent possibility for rapid pandemic response, high efficacy, minimal side effects, and cost-effectiveness. Achieving these benefits hinges on seamlessly integrating mRNA production steps, from plasmid DNA (pDNA) design and antigen cloning, in vitro transcription to lipid nanoparticle formulation. A critical initial step in mRNA vaccine production is pDNA cloning vector design. The vector should be carefully constructed considering a copy number of plasmid, vector backbone with a promoter, the origin of replication, multiple cloning sites, polyadenylation signal, and markers for selection. However, despite careful design, challenges like poly-A tail deletion may arise, prompting the exploration of stable large-size and low-copy vectors, as well as linear and bacteriophage vectors. Additionally, for large-scale production and regulatory compliance, vector systems must be scalable and well-documented. This overview aims to elaborate upon the intricacies in pDNA cloning vector design. The focus is on achieving accurate insert sequence, especially those encoding the complex antigens and gene expression, highlighting the critical role of pDNA design in ensuring vaccine effectiveness. Although commercial vectors and automated synthesis facilitate gene construction, challenges still exist. This emphasizes that a multifaceted approach, combining molecular biology techniques, computational tools, and collaboration with experts in microbiology, molecular biology, and vaccine development, is required for successful mRNA vaccine development
To BCG or not to BCG – a case against trained immunity?
No full textTrained immunity (TI) has been a hot button issue in current immunology, and we decided to look at the understudied
effect of TI on chemically induced colitis. We used BALB/c male mice, which are more susceptible to TNBS-induced
colitis, and immunized them subcutaneously with BCG (TI-TNBS group) or saline (control-TNBS group). Seven days
later, we induced colitis by intrarectal injection of TNBS in 50% ethanol. We observed the mice over a period of another
week. Most mice in both groups did not survive, but the mice in the control-TNBS group lived significantly longer. We
investigated the mice who survived in an additional study. We immunized the mice with another dose of BCG and paired
them up with adequate controls (mice who didn’t have colitis induced, immunized with BCG or with saline). Prior to the
second immunization we isolated the PBMCs from a blood sample and analyzed them on a flow cytometer. Two days
later mice were euthanized and their bone marrow, peritoneal, and PBMCs were collected and analyzed on a flow
cytometer. The results showed BCG immunization causes an increase in both CD45R+CD19+ B cells and CD4+ T cells in
the circulation, however after colitis induction, the frequency of these cells is reduced in the circulation of the immunized
mice compared to controls. Given that the immunized animals had a shorter lifespan after colitis induction, it is possible
that the infiltration of these lymphocytes from the peripheral circulation could be a causal factor in aggravated
inflammation. In the peritoneal cavity, BCG immunized animals had a greater percentage of F4/80+CD68+ macrophages,
however after colitis, this ratio was reversed, which could indicate a faster depletion of resident macrophages of the
peritoneal cavity, which are known to have a reparative role in visceral tissue damage. Post colitis, a greater percentage
of peritoneal macrophages expressed iNOS, and the BCG vaccination did increase it slightly in the two days after
immunization, compared to the control group. It was shown that TI could aggravate chemically induced colitis in mice,
and our results seem to support this notion, which we will research further
Amyloid-like aggregation influenced by lead(II) and cadmium(II) ions in hen egg white ovalbumin
No full textThe aggregation of proteins into fibrillar, amyloid-like aggregates generally results in an improved, positive effect on various techno-functional properties within food products, such as gelation, emulsification, and foam stabilization. These highly stable structures, characterized by their repetitive, β-sheet rich motifs, may develop as the result of the thermal treatment of protein-rich food products. Heavy metal ions can influence amyloid-like aggregation of food proteins. Lead(II) and cadmium(II) represent some of the most abundant and common environmental water and food pollutants. In this work, the influence of heavy metal ions, lead and cadmium on amyloid-like aggregation of ovalbumin at high temperatures (90 °C) and under acidic conditions (pH 2.0) was investigated. Ovalbumin is used as a general model for how heavy metals can affect amyloid-like aggregation of a food protein. Structural changes were monitored via Thioflavin T and 8-Anilino-1-naphthalenesulfonic acid fluorescence, Fourier-Transform infrared spectroscopy, atomic force microscopy, dynamic light scattering, as well as computational analyses. The obtained results indicate that the added heavy metal ions bind to different sites within ovalbumin prior to thermal treatment. Lead binding sites are closer to the hydrophobic regions of an protein, while cadmium ion binding sites are more exposed. This specific binding of metal ions affects the morphologies of amyloid-like aggregates, resulting in lead-induced branching of amyloid-like fibrils, or cadmium-induced tangling of fibrils into dense amyloid clusters. This additive effect of heavy metal ions is most evident in ovalbumin samples which contain a mixture of both heavy metal ions
Fatty acid profiling in amyotrophic lateral sclerosis
No full textAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, affecting motor neurons, which is characterized by the progressive loss of motor function. Sufficient evidence has been gathered, either in the form of experimental results from animal disease models or from the assessment of affected human subjects, which implicates metabolic changes in the course of ALS. Hypermetabolism, abnormal glucose tolerance and dyslipidaemia were reported to have high incidence in ALS patients. In fact, according to published studies, BMI is significantly and inversely associated with ALS progression. Here, we discuss basic principles of fatty acid metabolism, composition and the existing knowledge of metabolic and fatty acid composition changes in ALS patients. Literature data suggest that ALS patients have unfavorable changes in the fatty acid profile, especially because of the low level of long-chain polyunsaturated fatty acids. Nutritional interventions and supplementation with various foods or food supplements are currently not showing conclusive results, implying there is more to learn about potential ways to modify this illness
Mass spectrometry analysis reveals impact of peanut roasting on post-translational modifications of key allergens and their hinderence of trypsin digestion
No full textA bottom-up proteomic study, using high-resolution tandem mass spectrometry (HRMS) and, PEAKS Studio X+ was performed to investigate the impact of peanut roasting on readily soluble allergens and their post-translational modification (PTM) profiles. Among four major peanut allergen groups, we found that Ara h 3 prevails in raw peanut extract (PE), similar to Ara h 1, while the opposite is true for Ara h 6, which is enriched in roasted PE; Ara h 2 bands are near the same intensity. HRMS detected more than 40 different types of modification in raw and roasted samples. Distinct variations in the types and occurrence of specific amino acid PTMs were identified between allergens present in raw and roasted samples. Roasting affected the most frequent modifications by enrichment of OxM, HyP, carbamoylation (KR), and deamidation. The PTMs could also be mapped to the regions of IgE-binding epitopes of Ara h 1–3 and Ara h 6. As porcine trypsin is used for HRMS sample preparation and is also a digestive protease, hindrance effects to trypsin efficacy regarding PTMs was assessed. Roasting caused dihydroxylation and formylation PTMs with hindrance effects to trypsin efficacy, while methylation on several K/R showed opposite effects. In the case of methylated R342, results suggested facilitation of tryptic performance at modified residue compared to unmodified counterpart, while in the rest of seven genuine sequences containing modified residues, trend was opposite. Further exploration of how different PTMs could affect digestion efficiencies of major gastric and intestinal peptidases is currently in works and a much-needed assessment to better understand the role of PTMs
