Insights into Isolation and Purification Strategies of Egg Allergens

Eggs are a great source of protein in the human diet. They are consumed in tens of millions of tons globally per year. In addition, egg proteins, which are known food allergens, are included in many food products due to their excellent techno-functional properties. Hen’s eggs are the most consumed, but other edible avian eggs are occasionally used as gourmet ingredients or delicacies. With a high presence in the food market, the risk of accidental exposure to egg allergens is high. Hen egg allergy ranks among the top three food allergens in infants and young children. The complex structure and similar physicochemical properties of egg proteins limit their separation and purification, making further research challenging. Egg composition is influenced by age, disease, medicine, and environmental stress, and the target protein is often present in negligible amounts or polymorphic forms. To investigate the immunoreactivity of proteins from eggs of different bird species, it is necessary to consistently and quantitatively extract and purify proteins while avoiding harsh conditions. The conformational shape of allergens is impacted by denaturation, which can remove or expose IgE-binding epitopes and change the allergenic potential of proteins. This review presents findings from a literature survey on the isolation and purification strategies utilized for egg allergens from culinary-relevant bird eggs

Probiotic Supplementation Improves Hematological Indices and Morphology of Red Blood Cells and Platelets in Obese Women: A Double-Blind, Controlled Pilot Study

Background/Objectives: The prevalence of obesity worldwide has rapidly increased. Numerous studies showed a beneficial effect of probiotics in obese individuals, and changes in hematological parameters are observed in obesity. Therefore, the aim of this study was to investigate the effect of a novel probiotic approach on the red blood cells (RBCs) and platelets. Methods: Twenty-five obese women participated in a randomized, placebo-controlled study and were divided into the experimental group (one capsule daily containing Lactiplantibacillus plantarum 299v (DSM9843), Saccharomyces cerevisiae var. boulardii, and 40 mg octacosanol; n = 13) and the placebo group (n = 12). Blood samples were collected for light microscopic examination, morphometric analysis, and an automated hematology analyzer. A possible relationship between hematological parameters and body mass index (BMI), a common indicator of obesity, was investigated using Spearman correlation. The plasma concentration of soluble P-selectin and fibrinogen were determined using an ELISA assay. All measurements were performed before (T0) and after 12 weeks of supplementation (T1). Results: The three-month supplementation of probiotics improved hemoglobin levels, chromic status, and red blood cell morphology. The mean platelet volume (MPV), a measure of platelet size, was restored to normal levels, platelet morphology was improved, and the number of activated platelets was significantly reduced (p < 0.05). A strong negative correlation (r = −0.5904, p < 0.05) was found between BMI and platelet distribution width (PDW), a measure of variation in platelet size and shape. Conclusions: The results show that the probiotic approach improves morphology and normalizes the values of disturbed hematological parameters of RBCs and platelets in obese women

Memantine reduces the expression of IFN-gamma and IL-17 by CD4+ t cells in aged EAE rats

The average age of patients with multiple sclerosis (MS) has risen in recent decades. The incidence of late-onset MS has also increased. More than half of people living with an MS diagnosis are over 55. Ageing affects the composition and function of N-methyl-D-aspartate receptors (NMDARs), regardless of whether they are expressed on neuronal or immune cells. The aim of this study was to investigate the effects of memantine, a non-competitive NMDAR antagonist, on CD4 + T lymphocytes in young and aged rats immunized for experimental autoimmune encephalomyelitis (EAE). Memantine was administered by oral gavage to 3- and 24-month-old female Dark Agouti rats for 7 consecutive days from the first day post-immunization (dpi) or from the 7 th dpi. Mononuclear cells were isolated from the lymph nodes draining the site of immunization or the spinal cord and analysed by flow cytometry. A histopathologic analysis of the spinal cord was performed. Memantine administration more effectively reduced the mean neurological score and histological score in aged rats. Memantine reduced the expression of IFN-gamma and IL-17 by CD4 + T lymphocytes derived from draining lymph nodes or the spinal cord to a greater extent in aged rats. On the other hand, there was no difference in apoptosis of CD4 + T lymphocytes between control and memantine-treated young rats, whereas apoptosis of these cells was significantly reduced in memantine-treated aged rats. NMDARs have a greater effect on IFN-gamma and IL-17 synthesis by CD4 + T cells and apoptosis of these cells in aged than in young EAE rats

Modulation of BCG-induced immune response using β-glucan: boosting immune regulation and reducing severe inflammatory reactions

BCG (Bacillus Calmette-Gue´rin) vaccination is well-known for its ability to stimulate immune responses, which lead to trained immunity and various inflammatory responses. The addition of β-glucan, a recognized immunomodulator, may help redirect this response toward a more balanced immune response. The interaction between BCG and β-glucan in modulating monocyte and macrophage populations remains an unexplored topic. Aim of this study was to investigate the effects of BCG and β-glucan on immune cell populations in mice. Sixteen animals were divided into four test groups, with three groups receiving intraperitoneal treatment with BCG, β-glucan, or a combination of both. Flow cytometry analysis of peritoneal cells, spleen, bone marrow, and PBMC showed varying immune responses among the groups. BCG alone triggered a robust inflammatory reaction, increasing the population of M1 macrophages and dendritic cells, along with the expression of MHCII and CD11b. β-glucan also expanded these populations, indicating independent immune activation. However, in the BCG+β-glucan group, a decrease in inflammatory monocytes was observed, suggesting that β-glucan tempers excessive inflammation when combined with BCG. The reduction of MHCII+CD11blowF4/80+CD11c- macrophages in BCG+β-glucan group compared to the β-glucan group alone suggests that β-glucan modulates BCG-induced inflammation by favoring immune regulation over prolonged activation. Furthermore, MHCII+CD19+ B cells were progressively reduced in the BCG and β-glucan group, most significantly in the BCG+β-glucan group, indicating a shift in B cell dynamics toward regulatory or antibody-secreting phenotypes. These findings indicate that β-glucan does not merely enhance BCGinduced activation but modulates it, balancing inflammatory and regulatory immune pathways. The combination of BCG and βglucan leads to controlled immune activation, limiting excessive inflammation while supporting immune adaptation. *The authors marked with an asterisk equally contributed to the work

Amino Acid Substitutions in Bacteriocin Lactolisterin BU Reveal Functional Domains Involved in Biological Activity Against Staphylococcus aureus

The emergence of multidrug-resistant pathogens has driven the development of novel antimicrobial peptides (AMPs) as therapeutic alternatives. Lactolisterin LBU (LBU) is a bacteriocin with promising activity against Gram-positive bacteria, including Staphylococcus aureus. In this study, we designed and evaluated a panel of amino acid variants of LBU to investigate domain–activity relationships and improve activity. Peptides were commercially synthesized, and their effect was evaluated for minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), hemolytic activity, cytotoxicity, in vivo toxicity, and virulence modulation. AlphaFold3 structural prediction of LBU revealed a four-helix topology with amphipathic and hydrophobic segments. Helical wheel projections identified helices I and IV as amphipathic, suggesting their potential involvement in membrane interaction and activity. Glycine-to-alanine substitutions at helix I markedly increased antimicrobial activity but altered toxicity profiles. In contrast, changes at helix junctions and kinks reduced antimicrobial activity. We also showed differential regulation of virulence genes upon sub-MIC treatment. Overall, rational substitution enabled identification of residues critical for activity and toxicity, providing insights into therapeutic tuning of lactolisterin-based peptides

Next-Generation Pertussis Vaccines

Pertusis je respiratorna bolest koja se može sprečiti vakcinacijom, a izaziva je Bordetella pertussis, prisutna u ustima, nosu i grlu zaražene osobe. Kako su ljudi jedini rezervoar ove bakterije, potpuna vakcinacija protiv pertusisa i visoka stopa imunizacije od izuzetnog su značaja, naročito imajući u vidu da su odojčad mlađa od godinu dana najugroženija od teških oblika bolesti i smrtnog ishoda. Vakcinacija celim ćelijama (engl. WCV – whole-cell vaccine) sadrži neaktivne bakterije, dok acelularna vakcina (engl. ACV – acellular pertussis vaccine) sadrži proteinske komponente Bordetella pertussis, kao što su inaktivisani pertusis toksin, filamentni hemaglutinin, pertaktin i fimbrije. Acelularna vakcina razvijena je kao odgovor na izveštaje o neželjenim reakcijama koje su se javljale nakon primene vakcine sa celim ćelijama u određenim zemljama. Uvođenje acelularne vakcine tokom 90-ih godina prošlog veka dovelo je do postepenog porasta broja slučajeva pertusisa. WCV vakcina izaziva snažniji imunološki odgovor, sličniji prirodnoj infekciji, i pruža dugotrajniju zaštitu. U zemljama koje koriste ACV preporučuje se pojačan nadzor, kao i uključivanje dodatnih doza (buster doza). Posebna pažnja se posvećuje vakcinaciji trudnica u cilju zaštite novorođenčadi. Vakcine koje sadrže rekombinantni, genetski detoksifikovani pertusis toksin nedavno su registrovane, a njihova prednost je što omogućavaju manju količinu antigena po dozi za postizanje zaštite. Ipak, postoji potreba za razvojem nove generacije pertusis vakcina koje bi zadržale prednosti vakcina sa celim ćelijama – kao što su indukcija adekvatnijeg imunološkog odgovora i dugotrajna zaštita – o čemu će ovde biti reči.Pertussis is a vaccine-preventable, respiratory disease caused by Bordetella pertussis, present in the mouth, nose, and throat of an infected person. As humans are the sole reservoir, complete vaccination against pertussis and high vaccination coverage is of utmost importance, especially as infants aged <1 year are at greatest risk of serious disease and death. Whole-cell pertussis vaccine (WCV) contains nonviable bacteria and acellular pertussis vaccine (ACV) - contains protein components from B. pertussis such as inactivated pertussis toxin, filamentous hemagglutinin, pertactin, and fimbriae. The acellular vaccine was developed in response to reports of adverse reactions upon administering the whole-cell vaccine in certain countries. The introduction of acellular vaccine in the 90s has led to a lower level gradual increase of the number of pertussis cases. The WCV vaccine stimulates a strong immune response more similar to infection and protection which is more durable. For countries that have adopted ACV increased monitoring is advised as well as the inclusion of booster doses. Special focus is given on the vaccination of pregnant women to protect the newborns. Recombinant, genetically detoxified pertussis toxin containing vaccines have been recently registered the benefit being that this reduces the amount of antigen needed per dose to confer protection. Nevertheless, a need exists for the development of a new generation of pertussis vaccines, which will have the benefits of whole cell vaccine, such as the induction of more adequate immune response and durability of protection, which will be discussed here

Bioinformatic Selection of Mannose-Specific Lectins from Allium genus as SARS-CoV-2 Inhibitors Analysing Protein–Protein Interaction

Mannose-specific lectins are carbohydrate-binding proteins known for their antiviral potential. This study uses a bioinformatic approach to investigate the possibility of lectins from Allium sativum (garlic) and Allium ursinum (wild garlic) as inhibitors of SARS-CoV-2 entry. The information spectrum method (ISM) identified key interaction frequencies between the SARS-CoV-2 spike protein and these lectins, explicitly targeting the receptor-binding domain (RBD) and glycosylated asparagine residues, including N234. Lectins from Allium species showed a high affinity for oligomannose-type glycans on the spike protein, potentially blocking virus entry by preventing the spike-ACE2 receptor interaction. We propose that Allium lectins are promising candidates for further experimental validation as SARS-CoV-2 inhibitors, offering potential therapeutic applications in managing viral infections

A novel type of autoaggregation in lactic acid bacteria promoted by new AggS aggregation factor from Streptococcus thermophilus CC40-4S

Autoaggregation in lactic acid bacteria is considered a beneficial probiotic trait and can be used in the food and medical industries to enhance the properties of utilized microorganisms. Currently, a group of aggregation-promoting factors (APFs) in lactic acid bacteria, known as Snowflake Forming Collagen Binding Aggregation Factors (SFCBAFs), is well described. These are large proteins with a molecular mass of over 170 kDa, containing collagen-binding domains and a repeat region, forming a unique autoaggregation phenotype. Here we describe a new type of autoaggregation in lactic acid bacteria found in Streptococcus thermophilus CC40-4S. The whole genome of the autoaggregation-positive strain S. thermophilus CC40-4S was sequenced, and bioinformatic analysis predicted a putative gene aggS involved in autoaggregation, located on the chromosome and flanked by insertion sequences. The aggS gene disruption by homologous recombination using the temperature-sensitive vector pSC led to the loss of the aggregation phenotype. Cloning and heterologous expression in Lactococcus lactis subsp. cremoris MG1363 confirmed the role of the AggS protein in autoaggregation, given that a strong aggregation phenotype was obtained. Like SFCBAF-type APFs, AggS is a large protein (237 kDa) with a repeat region, but it does not contain collagen-binding domains and forms an autoaggregation phenotype with small aggregates. To our knowledge, this is the first report on a gene coding for an aggregation-promoting factor in S. thermophilus

mRNA-Based Vaccines – Challenges in the Manufacturing Process

Pandemija COVID-19 je poslužila kao katalizator za ubrzani razvoj i primenu iRNK vakcina, ističući njihovu ključnu ulogu u suzbijanju infektivnih bolesti. Brzina razvoja iRNK vakcina, uz istovremeno ispunjavanje strogih bezbednosnih standarda, potvrdila je superiornost ove tehnologije u odgovoru na globalne zdravstvene izazove. U cilju postizanja optimalne efikasnosti, stabilnosti i bezbednosti, primenjuju se napredne tehnike, uključujući upotrebu modifikovanog uridina i/ili samoreplikujuće iRNK, čime se obezbeđuje adekvatan i dugotrajan imunski odgovor, uz usklađenost sa regulatornim i kliničkim zahtevima. Takođe, jedna od ključnih prednosti ove vakcinalne platforme je brzina i fleksibilnost proizvodnje u odnosu na tradicionalne vakcine, pri čemu standarizovani procesi i modularna priroda omogućavaju jednostavnu adaptaciju na nove varijante patogena, bez potrebe za značajnim izmenama proizvodnog postupka. Proizvodni postupak počinje dizajniranjem i proizvodnjom plazmidne DNK, koja se nakon prečišćavanja linearizuje, a zatim koristi kao matrica za sintezu iRNK molekula u reakciji in vitro transkripcije. Sintetisana iRNK mora proći kroz nekoliko faza prečišćavanja kako bi se eliminisali kontaminanti poput DNK, proteina i dr. Sledeći i najznačajni korak za stabilnost je enkapsulaciju iRNK, najčešće pomoću lipidnih nano-čestica (LNP) ili polimernih nanočestica, čime se omogućava efikasna dostava do ciljnih ćelija. Svi koraci proizvodnje su u skladu sa regulatornim standardima i praćeni strogim testovima kontrole kvaliteta, kako bi se osigurala bezbednost, efikasnost i konzistentnost vakcine. Trenutno su u kliničkim ispitivanjima iRNK vakcine protiv različitih virusnih i bakterijskih infekcija. Osim toga, iRNK tehnologija pokazuje veliki potencijal za personalizovanu terapiju, uključujući lečenje retkih genetskih i autoimunih bolesti, kao i razvoj individualizovanih onkoloških terapija.The rapid development of mRNA vaccines, while simultaneously meeting safety standards, has confirmed the superiority of this technology in responding to global health challenges, especially during the COVID-19 pandemic. To achieve optimal efficacy, stability, and safety of mRNA vaccines, advanced techniques such as the use of modified uridine and/or self-amplifying mRNA are employed, ensuring a robust and durable immune response while maintaining compliance with regulatory and clinical requirements. Additionally, one of the key advantages of mRNA vaccine platform is the speed and flexibility of production compared to traditional vaccines. Standardized processes and the modular nature of mRNA technology allow for easy adaptation to new pathogen variants without significant modifications to the manufacturing process. The production process begins with the design and synthesis of plasmid DNA, which is linearized and then used as a template for synthesizing mRNA molecules via in vitro transcription. The synthesized mRNA undergoes multiple purification steps to remove contaminants such as residual DNA, proteins, and other impurities. The next and most crucial step for stability is mRNA encapsulation, typically using lipid nanoparticles (LNPs) or polymeric nanoparticles, enabling efficient delivery to target cells. All manufacturing steps comply with regulatory standards and are accompanied by rigorous quality control testing to ensure the safety, efficacy, and consistency of the vaccine. Currently, mRNA vaccines are undergoing clinical trials for various viral and bacterial infections. Furthermore, mRNA technology holds great potential for personalized therapies, including the treatment of rare genetic and autoimmune diseases, as well as the development of individualized cancer therapies

Herd Immunity to the Measles, Mumps and Rubella Viruses Among the Belgradian Population in May, 2024

Background/Objectives: In the Republic of Serbia, measles vaccination was first introduced in 1971, while combined vaccination (measles, mumps, rubella) was made mandatory in 1996 as part of the national vaccination program. Reported prevalence values for 2023 were <0.75 cases per 100K population for measles, 0.09 cases per 100K for mumps, and no cases of rubella. Methods: This cross-sectional study was performed in May, 2024 as part of the project “Herd Immunity to Vaccine-Preventable and Other Relevant Infections in the Belgradian Population.” It focused on assessing herd immunity to measles, mumps and rubella (MMR) among residents insofar as these remain a public concern despite the availability of vaccines. A total of 2533 subjects were distributed across nine age groups, covering those aged 1–70+ years and various professional groups residing in Belgrade. Participants were stratified by age and activity. Upon obtaining individual information by online questionnaire and receiving a signed statement of informed consent, blood samples were obtained for IgG antibody testing (ELISA) to determine MMR serological status. The results were compared to national and international immunization standards to evaluate herd immunity levels. Results: Our results indicate varying levels of immunity for each virus, with specific demographic groups showing different immunity levels. Total measles seroprevalence during this study was 74.7%, with significant variation across all age groups. While high seropositivity was observed in both children (90.7%) and elder age groups (98.4%), middle-aged individuals in the age group 30–49 years showed significantly lower IgG levels. Between 2021 and 2023, there were no registered cases of rubella detected in Serbia, which indicates a high level of immunity. This was confirmed here with consistently high IgG levels across all age groups, with an average seropositivity of 94.8%. Average mumps seropositivity across all age groups was 85.1%. The lowest value was in the young child (1–5 years) age group (76.1%); the highest was in the elderly group (92.6%). Conclusions: The current findings suggest that the Belgradian population has strong overall immunity to MMR, yet with some concerns regarding measles immunity in middle-aged adults, suggesting a potential need for catch-up vaccinations. While rubella status indicates strong herd immunity and minimal risk of outbreaks, mumps immunity in some groups (children, middle-aged adults) is below the protective threshold. While it is still sufficient to prevent widespread transmission, it should be closely observed. To our knowledge, this study is the first of its kind to provide data about MMR seroprevalence in Belgrade. Findings indicate the need for constant surveillance and revaccination of vulnerable/seronegative groups