ELLSA based profiling of surface glycosylation in microorganisms reveals that ß-glucan rich yeasts' surfaces are selectively recognized with recombinant banana lectin.

The surface of microorganisms is covered with polysaccharide structures which are in immediate contact with receptor structures on host's cells and antibodies. The interaction between microorganisms and their host is dependent on surface glycosylation and in this study we have tested the interaction of plant lectins with different microorganisms. Enzyme-linked lectin sorbent assay - ELLSA was used to test the binding of recombinant Musa acuminata lectin - BL to 27 selected microorganisms and 7 other lectins were used for comparison: Soy bean agglutinin - SBA, Lens culinaris lectin - LCA, Wheat germ agglutinin - WGA, RCA120 - Ricinus communis agglutinin, Con A - from Canavalia ensiformis, Sambucus nigra agglutinin - SNA I and Maackia amurensis agglutinin - MAA. The goal was to define the microorganisms' surface glycosylation by means of interaction with the selected plant lectins and to make a comparison with BL. Among the tested lectins most selective binding was observed for RCA120 which preferentially bound Lactobacillus casei DG. Recombinant banana lectin showed specific binding to all of the tested fungal species. The binding of BL to Candida albicans was further tested with fluorescence microscopy and flow cytometry and it was concluded that this lectin can differentiate ß-glucan rich surfaces. The binding of BL to S. boulardii could be inhibited with ß-glucan from yeast with IC50 1.81 µg mL-1 and to P. roqueforti with 1.10 µg mL-1. This unique specificity of BL could be exploited for screening purposes and potentially for the detection of ß-glucan in solutions.

Thyroglobulin specific IgE and a possible link to suspected penicillin induced allergic skin manifestations - cross sectional study.

Porcine 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.

Notwithstanding 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.

Electrospun Gelatin Scaffolds with Incorporated Antibiotics for Skin Wound Healing.

Recent advances in regenerative medicine provide encouraging strategies to produce artificial skin substitutes. Gelatin scaffolds are successfully used as wound-dressing materials due to their superior properties, such as biocompatibility and the ability to mimic the extracellular matrix of the surrounding environment. In this study, five gelatin combination solutions were prepared and successfully electrospun using an electrospinning technique. After careful screening, the optimal concentration of the most promising combination was selected for further investigation. The obtained scaffolds were crosslinked with 25% glutaraldehyde vapor and characterized by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and Fourier-transform infrared spectroscopy. The incorporation of antibiotic agents such as ciprofloxacin hydrochloride and gentamicin sulfate into gelatin membranes improved the already existing antibacterial properties of antibiotic-free gelatin scaffolds against Pseudomonas aeruginosa and Staphylococcus aureus. Also, the outcomes from the in vivo model study revealed that skin regeneration was significantly accelerated with gelatin/ciprofloxacin scaffold treatment. Moreover, the gelatin nanofibers were found to strongly promote the neoangiogenic process in the in vivo chick embryo chorioallantoic membrane assay. Finally, the combination of gelatin's extracellular matrix and antibacterial agents in the scaffold suggests its potential for effective wound-healing treatments, emphasizing the importance of gelatin scaffolds in tissue engineering.

AI-Driven Optimization of PCL/PEG Electrospun Scaffolds for Enhanced In Vivo Wound Healing.

Here, an artificial intelligence (AI)-based approach was employed to optimize the production of electrospun scaffolds for in vivo wound healing applications. By combining polycaprolactone (PCL) and poly(ethylene glycol) (PEG) in various concentration ratios, dissolved in chloroform (CHCl3) and dimethylformamide (DMF), 125 different polymer combinations were created. From these polymer combinations, electrospun nanofiber meshes were produced and characterized structurally and mechanically via microscopic techniques, including chemical composition and fiber diameter determination. Subsequently, these data were used to train a neural network, creating an AI model to predict the optimal scaffold production solution. Guided by the predictions and experimental outcomes of the AI model, the most promising scaffold for further in vitro analyses was identified. Moreover, we enriched this selected polymer combination by incorporating antibiotics, aiming to develop electrospun nanofiber scaffolds tailored for in vivo wound healing applications. Our study underscores three noteworthy conclusions: (i) the application of AI is pivotal in the fields of material and biomedical sciences, (ii) our methodology provides an effective blueprint for the initial screening of biomedical materials, and (iii) electrospun PCL/PEG antibiotic-bearing scaffolds exhibit outstanding results in promoting neoangiogenesis and facilitating in vivo wound treatment.

Comparison of Enzyme-Linked Lectin Sorbent Assay and Flow Cytometry for Profiling Microbial Glycans.

The surface of microorganisms is covered with carbohydrates, which makes them unique, self-sustaining glycan probes. Lectins are able to bind to these probes, and this interaction can be exploited for selecting microorganisms or novel lectins. To examine lectin-microorganism interactions, we have previously developed an enzyme-linked lectin sorbent assay (ELLSA) with whole bacterial cells. To further test the validity of this methodology, here we compare it with flow cytometry. For this purpose, we used biotinylated recombinantly produced lectin from Musa acuminata (BanLec), this lectin's recombinantly produced chimera with green fluorescent protein (BanLec-eGFP) and a lectin from Ricinus communis (RCA120), both biotinylated and FITC labeled. Parallel testing showed equivalent results for the two methods, in terms of the presence or absence of binding, with signal intensity yielding high Pearson correlation coefficient of 0.8 for BanLec and 0.95 for RCA120. The ELLSA method demonstrated multiple advantages, such as reliability and convenience for high-throughput analysis; it also required less lectin and yielded more consistent results. As such, ELLSA proved to be a useful tool for profiling microbial glycan structures or testing novel lectins.

Differences in mouse strains determine the outcome of Der p 2 allergy induction protocols.

In vivo animal models can provide worthy information on various aspects of asthma mechanism and pathogenesis. The genetic predisposition and phenotype of mice may affect the immune response itself. Here we compare the early immune response to Der p 2 or HDM allergen extract upon injection and inhalation in BALB/c and C57BL/6 mice. Female C57BL/6 and BALB/c mice were immunized with Der p 2 allergen subcutaneously followed by inhalation of Der p 2 or HDM extract. After challenge, the mice were euthanized; blood, bronchoalveolar lavage (BAL), spleens and lungs were collected. Cells from BAL were identified by May-Grünwald Giemsa staining and lung leukocyte populations were analyzed by flow cytometry. Serum antibody levels of Der p 2 specific IgE, IgG, IgG1 and IgG2a were assessed by ELISA, and cytokine secretion (IL-4, IFN-γ and IL-10) was evaluated upon stimulation with Der p 2 or HDM extract. The Th2 immune response was confirmed by elevated allergen-specific immunoglobulin E (IgE) and the allergic reaction was evidenced by infiltration of eosinophils and/or neutrophils into BAL. We found that BALB/c mice were inefficient in integrating local with systemic immune response, evidenced by almost no IgG or IgE production upon one subcutaneous injection and subsequent inhalation of Der p 2 allergen; also, the bronchoalveolar lavage infiltrate in these mice consisted of neutrophil infiltration, unlike C57BL/6 mice in which eosinophilic infiltrate predominated. The differences between BALB/c and C57BL/6 mice strains could be exploited for generating different types of responses to the Der p 2 allergen.

Age and gender associated changes in immunoglobulin subclass levels specific to S. pneumoniae, serotype 1.

S. pneumoniae is an important human pathogen which has a polysaccharide capsule with virulent properties. This work aims to estimate the titres of S. pneumoniae specific IgG and IgA isotypes, with respect to age and sex. An in-house whole bacterial cell ELISA was used for the determination of relative levels and endpoint titres of IgG subclasses and IgA1 subclass specific for S. pneumoniae serogroup 1, and to quantify specific IgG1 and IgG2 levels. Significantly lower anti-pneumococcus IgG1 titres were found in older individuals, which was more pronounced in men. Lower IgG2 titres were detected in men over 50 years of age, in comparison to women under 50 years of age. The levels of IgG3 and IgG4 did not differ between different sex and age groups. Lower IgA1 levels were detected in male individuals in both age groups in comparison to females under 50 years of age. The levels of IgG1 showed a moderate correlation with IgG4 in younger individuals of both sexes (r = 0.61 in men and 0.63 in women) which was not noted in the older age group. We highlight the deficiency in humoral immunity in older people, especially male and suggest immunization of this population with pneumococcal vaccines.

BanLec-eGFP Chimera as a Tool for Evaluation of Lectin Binding to High-Mannose Glycans on Microorganisms.

Fluorescently labeled lectins are useful tools for in vivo and in vitro studies of the structure and function of tissues and various pathogens such as viruses, bacteria, and fungi. For the evaluation of high-mannose glycans present on various glycoproteins, a three-dimensional (3D) model of the chimera was designed from the crystal structures of recombinant banana lectin (BanLec, Protein Data Bank entry (PDB): 5EXG) and an enhanced green fluorescent protein (eGFP, PDB 4EUL) by applying molecular modeling and molecular mechanics and expressed in Escherichia coli. BanLec-eGFP, produced as a soluble cytosolic protein of about 42 kDa, revealed ß-sheets (41%) as the predominant secondary structures, with the emission peak maximum detected at 509 nm (excitation wavelength 488 nm). More than 65% of the primary structure was confirmed by mass spectrometry. Competitive BanLec-eGFP binding to high mannose glycans of the influenza vaccine (Vaxigrip®) was shown in a fluorescence-linked lectin sorbent assay (FLLSA) with monosaccharides (mannose and glucose) and wild type BanLec and H84T BanLec mutant. BanLec-eGFP exhibited binding to mannose residues on different strains of Salmonella in flow cytometry, with especially pronounced binding to a Salmonella Typhi clinical isolate. BanLec-eGFP can be a useful tool for screening high-mannose glycosylation sites on different microorganisms.

Direct UV Spectrophotometry and HPLC Determination of Triton X-100 in Split Virus Influenza Vaccine.

One of the most commonly used surfactants in the production of split virus influenza vaccine is nonionic surfactant Triton X-100. After splitting of the virus is accomplished, Triton X-100 is removed from the vaccine by subsequent production steps. Because of toxicity of Triton X-100, which remains in the vaccine in residual amounts, a sufficiently sensitive method for its detection and quantification needs to be defined. Two methods for determination of Triton X-100 residuals were developed: the UV-spectrophotometry and HPLC methods. For both methods, preparation of vaccine samples and removal of proteins and virus particles were crucial: samples were treated with methanol (1:1) and then centrifuged at 25 000 × g for 30 min. After such treatment, the majority of vaccine components that interfered in the UV region were removed, and diluted samples could be directly measured. The chromatographic system included C18 column, step methanol gradient, and detection at 225 nm with a single peak of Triton X-100 at 12.6 min. Both methods were validated and gave satisfactory results for accuracy, precision, specificity, linearity, and robustness. LOQ was slightly lower for the HPLC method. Hence, it was shown that both methods are suitable for analysis of residual amounts of Triton X-100, with the advantages of the UV method being its simplicity and availability in most laboratories.