Lubomír Janda

Dietary cereal prolamins differ in composition and gastrointestinal processing, but their shared and protein-source-specific effects on intestinal homeostasis remain incompletely defined. BALB/c mice were fed for five weeks with diets in which 70% of total protein was supplied by vital wheat gluten, purified gliadin, or purified avenin, alongside casein-fed controls. Intestinal morphology, antigen-specific plasma immunoglobulin G and A (IgG, IgA), mucosal gene expression, and small-intestinal proteomics were analysed. No overt histopathological injury or generalised inflammatory response was detected in any group. Gliadin and avenin both increased ileal villus length; avenin additionally elicited the broadest and most frequent prolamin-specific IgG response, associated with reduced intestinal interleukin-10 expression in responding animals. Proteomic profiling revealed a shared structural-metabolic signature alongside protein-specific changes in lipid handling, junctional regulation, and epithelial immune signalling. These findings indicate that wheat-and oat-derived prolamins differentially influence intestinal homeostasis and antigen recognition even at matched dietary doses, informing their evaluation as functional food ingredients. Graphical

Oats are widely included in gluten-free diets, yet the biological effects of oat prolamins (avenins) on intestinal homeostasis remain incompletely understood. Here, we compared purified wheat gliadins and oat avenins administered at equivalent concentrations in a BALB/c murine model. After five weeks of dietary exposure, no overt intestinal pathology was observed, as villous architecture and inflammatory cytokine expression remained largely unchanged. Nevertheless, both gliadin and avenin induced antigen-specific humoral responses, with avenin eliciting broader and more frequent IgG reactivity. Proteomic analysis of the small intestine revealed coordinated alterations in pathways related to epithelial homeostasis, antigen handling, and immune regulation. Reduced expression of caspase-8 and interleukin-18 suggested disrupted epithelial regulatory balance, while increased markers of antigen transport indicated enhanced mucosal antigen sampling. In parallel, antibody-responsive animals exhibited reduced IL-10 expression, consistent with impaired regulatory control. Together, these findings support a model in which dietary prolamins actively modulate mucosal regulatory networks and induce a primed mucosal state characterised by immune activation without overt inflammation. Given the substantially lower natural abundance of avenins compared with gliadins, these effects likely reflect dose-dependent responses rather than intrinsic pathogenicity. This model could additionally serve as a useful experimental platform for monitoring the combined effects of dietary additives and prolamins on intestinal barrier integrity and mucosal immune responses.

Background: In view of the ever‐increasing representation of Staphylococcus spp. strains resistant to various antibiotics, the development of in vivo models for evaluation of novel antimicrobials is of utmost importance.Methods: In this article, we describe the development of a fully immunocompetent porcine model of extensive skin and soft tissue damage suitable for testing topical antimicrobial agents that matches the real clinical situation. The model was developed in three consecutive stages with protocols for each stage amended based on the results of the previous one.Results: In the final model, 10 excisions of the skin and underlying soft tissue were created in each pig under general anesthesia, with additional incisions to the fascia performed at the base of the defects and immediately inoculated with Staphylococcus aureus suspension. One pig was not inoculated and used as the negative control. Subsequently, the bandages were changed on Days 4, 8, 11, and 15. At these time points, a filter paper imprint technique (FPIT) was made from each wound for semi‐quantitative microbiological evaluation. Tissue samples from the base of the wound together with the adjacent intact tissue of three randomly selected defects of each pig were taken for microbiological, histopathological, and molecular‐biological examination. The infection with the inoculated S. aureus strains was sufficient during the whole experiment as confirmed by both FPIT and from tissue samples. The dynamics of the inflammatory markers and clinical signs of infection are also described.Conclusions: A successfully developed porcine model is suitable for in vivo testing of novel short‐acting topical antimicrobial agents.

Neural tissue from any source is an excellent material for tubulin isolation, as the dendrites and axons of neurons are rich in microtubules. Here, we present a procedure for extracting tubulin that can be employed, with minor modifications, for neural tissue from multiple sources. In the presented protocol, a new clarification step of the crude lysate has been introduced, which led to a significant reduction in the initial amount of insoluble debris before the first polymerization step occurred. This additional step allowed the processing of additional tissue while using the same instrumentation and, thus, increasing the relative volume of the processed homogenate. The newly introduced step has no significant effect on the quality of purified tubulin, as confirmed by in vitro activity assays and transmission electron microscopy. The described procedure contains all crucial steps, including tissue collection, transport, tissue homogenization, tubulin isolation cycles, and final polishing by ion exchange chromatography using FPLC and subsequent activity measurement assays. The homogeneity of purified tubulin was more than 97%, as confirmed by MS/MS analysis utilizing electrospray ionization and MALDI-TOF.

Neural tissue from any source is an excellent material for tubulin isolation, as the dendrites and axons of neurons are rich in microtubules. Here, we present a procedure for extracting tubulin that can be employed, with minor modifications, for neural tissue from multiple sources. In the presented protocol, a new clarification step of the crude lysate has been introduced, which led to a significant reduction in the initial amount of insoluble debris before the first polymerization step occurred. This additional step allowed the processing of additional tissue while using the same instrumentation and, thus, increasing the relative volume of the processed homogenate. The newly introduced step has no significant effect on the quality of purified tubulin, as confirmed by in vitro activity assays and transmission electron microscopy. The described procedure contains all crucial steps, including tissue collection, transport, tissue homogenization, tubulin isolation cycles, and final polishing by ion exchange chromatography using FPLC and subsequent activity measurement assays. The homogeneity of purified tubulin was more than 97%, as confirmed by MS/MS analysis utilizing electrospray ionization and MALDI-TOF.

Staphylococcus aureus is a gram-positive, facultatively anaerobic coccus capable of causing infectious diseases in animals and humans. Especially dangerous are multidrug-resistant forms with poor or even no response to available treatments. The study aimed to verify the effect of enzybiotics on the healing of S. aureus-infected skin wounds in an experimental pig model. Two pigs were included in the experiment and wounds (10/pig) of 5 × 5 cm in size with 2 cm spacing were made by incision on their backs. The wounds were infected with a methicillin (oxacillin) and amoxicillin-resistant strain of S. aureus (MRSA). The experimental groups consisted of individual wounds that were infected with one sequence type of S. aureus at a concentration of 2 × 109 CFU/mL. Two wounds were left untreated (N), four wounds were using hydrogel with added lysostaphin, and four wounds were treated using hydrogel with added lysostaphin and endolysin. Subsequently, samples were taken from each wound on days 4, 7, 11, and 14. The material was processed using a standard histological technique of paraffin blocks and the sections were stained with hematoxylineosin. The results show that these defects present a full spectrum of reparative changes with re-epithelialization with alternating sections of necrosis and newly formed granulation tissue with an accompanying round cell inflammatory infiltrate in edematous tissue and surface scabs. On the surface of the wounds and also in smaller groups in the newly formed granulation tissue, coccoid formations corresponding to S. aureus are visible. Compared to untreated wounds, hydrogel dressings with added lysostaphin or lysostaphin and endolysin trapped greater numbers of S. aureus cocci colonies, which subsequently died off to a large extent. Enzybiotics may have interesting potential in the topical therapy of MRSA-infected skin wounds.

Celiac disease is a common gastroenterological illness. Current diagnostics of the disease are based on serological markers and histology of duodenal biopsies. Hitherto, a strict gluten-free diet is the only effective treatment and is necessary for good control of the disease. Serological tests in current use have very high specificity and sensitivity for diagnostics, but in follow-up they have some limitations. Their levels do not accurately reflect mucosal healing, and they are unable to detect minimal transgressions in the diet. This problem is significant in patients with IgA deficiency, and there exist no robust follow-up tools for monitoring these patients’ adherence to treatment. For their follow-up, we currently use IgG-based tests, and these antibodies persist for a long time even when a patient has stopped consuming gluten. More accurate and specific biomarkers are definitely needed. Adherence to a gluten-free diet is essential not only for intestinal mucosa healing and alleviation of symptoms but also for preventing complications associated with celiac disease. Here, we summarize current evidence regarding noninvasive biomarkers potentially useful for follow-up not only of patients with IgA deficiency but for all patients with celiac disease. We describe several very promising biomarkers with potential to be part of clinical practice in the near future.

Antimicrobial peptides, including the human cathelicidin LL-37, offer a possible solution to the global problem of bacterial resistance to antibiotics. LL-37 peptide has potent antimicrobial effects against current multi-drug resistant bacterial strains. The peptide itself is also characterized by a very diverse range of immunomodulatory effects. The aim of this study was to produce antimicrobially active peptide LL-37 in E. coli in high yields using an own expression system pUbEx100 with the fusion protein ubiquitin. The results showed that the peptide GLL-37 could be produced in high amounts, but this peptide did not have antimicrobial activity compared to synthetically produced LL-37. CD spectroscopy results showed that the produced peptide GLL-37 is in α-helix form in contrast to the sLL-37 (random-coil form). The recombinant peptide GLL-37 can not bind to the membrane in the α-helix form, it would have to be in the form of a random-coil. This study confirms by CD spectroscopy the previously observed mechanism of access of LL-37 peptide to the bacterial membrane obtained by NMR.

Antimicrobial peptides, including the human cathelicidin LL-37, offer a possible solution to the global problem of bacterial resistance to antibiotics. LL-37 peptide has potent antimicrobial effects against current multidrug-resistant bacterial strains. The peptide itself is also characterized by a very diverse range of immunomodulatory effects. The aim of this study was to produce antimicrobially active peptide LL-37 in E. coli in high yields using an own expression system pUbEx100 with the fusion protein ubiquitin. The results showed that the peptide GLL-37 could be produced in high amounts, but this peptide did not have antimicrobial activity compared to synthetically produced LL-37. The synthetic peptide LL-37 (sLL-37) alone showed potent activity against multidrug-resistant strains Acinetobacter baumannii and Pseudomonas aeruginosa. CD spectroscopy results showed that the produced peptide GLL-37 is in α-helix form in contrast to the sLL-37 (random-coil form). The recombinant peptide GLL-37 can not bind to the membrane in the α-helix form, it would have to be in the form of a random-coil. This study confirms by CD spectroscopy the previously observed mechanism of access of LL-37 peptide to the bacterial membrane obtained by NMR.