Search Results (125)

Streptococcus pneumoniae remains a major global health threat and is listed by the World Health Organization as a pathogen in urgent need of new antimicrobial strategies. While primarily considered an extracellular pathogen, S. pneumoniae can persist within splenic macrophages in severe disease, creating a protected intracellular niche that may contribute to fulminant sepsis. We recently demonstrated the concept of an mRNA-based therapeutic approach in which host cells produce the pneumococcal bacteriophage endolysin Cpl-1. Here, we investigated whether expression of Cpl-1 in macrophages can target S. pneumoniae residing within host cells. Using the human THP-1 macrophage line, we demonstrated successful translation and intracellular accumulation of bioactive Cpl-1 following IVT-mRNA transfection. Lysates from Cpl-1 mRNA-transfected cells exhibited bacteriolytic activity, and Western blotting as well as immunofluorescent staining confirmed cytosolic endolysin production. Phagocytosis assays using an encapsulated and unencapsulated pneumococcal strain showed a reduction in intracellular bacterial burden in Cpl-1 mRNA-transfected macrophages compared with control and inactive-mutant Cpl-1 mRNA groups, and a flow cytometry-based assay further corroborated a decreased intracellular bacterial signal. Together, these findings suggest that mRNA-encoded Cpl-1 enhances intracellular killing of S. pneumoniae and supports the feasibility of mRNA-based endolysin therapies to target intracellular pneumococcal reservoirs. Full article

Background: Caffeine, although widely used in dermatological and cosmetic products, exhibits limited permeability through the stratum corneum, highlighting the need for strategies for optimizing delivery. The aim of this study was in vitro investigation of the effects of probiotic bacterial lysates and submicellar concentrations of bile acids on caffeine permeation, with a particular focus on permeation kinetics. Methods: Caffeine permeability was evaluated using the Skin Parallel Artificial Membrane Permeability Assay (Skin-PAMPA). Donor and acceptor concentrations were quantified by HPLC at predefined time points (1, 2, 4, 6, and 12 h), followed by calculation of apparent permeability coefficients, cumulative permeation profiles, and interval permeation rates in systems containing probiotic lysates and submicellar concentrations of cholic acid (CA) or deoxycholic acid (DCA). Results: Probiotic lysates significantly reduced caffeine permeability (0.98 ± 0.02 × 10⁻⁶ vs. 1.57 ± 0.14 × 10⁻⁶ cm/s in the control group) and modified transport kinetics resulting in lower early-phase interval permeation rates and reduced cumulative permeation. Conversely, bile acids increased the apparent permeability of caffeine, with the highest value observed in the DCA group (2.30 ± 0.08 × 10⁻⁶ cm/s). Conclusions: Overall, probiotic lysates and bile acids modulated caffeine permeation across the Skin-PAMPA membrane primarily by reshaping permeation kinetics rather than simply changing overall permeability. Their combined effects may provide a basis for designing topical formulations with tailored permeation profiles. Full article

This study evaluated the anti-virulence effect of AuraAqua (Aq), a natural mixture of organic acids, against Pseudomonas. fluorescens. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Aq against P. fluorescens were 0.5% and 1% (v/v), respectively. Growth curve analysis confirmed concentration-dependent inhibition of planktonic growth, while sub-inhibitory Aq (0.25%; 1/2 MIC) significantly reduced biofilm formation. Rhodamine-based assays revealed membrane depolarisation at both 0.25% and 0.5% Aq, with decreased intracellular protein levels, indicating impaired membrane integrity under the tested conditions. In fibronectin adherence assays, the presence of Aq during bacterial contact markedly reduced adherence, whereas short pre-treatment alone produced limited, non-persistent effects, suggesting that continuous exposure is required to interfere with host matrix binding. Supernatants and lysates from Aq-exposed P. fluorescens induced lower extracellular protease activity in a concentration-dependent manner and mitigated P. fluorescens-induced cytotoxicity in primary shrimp (SGP) and tilapia (TGP) gut cells, as measured by lactate dehydrogenase (LDH) release after exposure to bacterial supernatants and lysates. Aq treatment was associated with decreased P. fluorescens internalisation into SGP and TGP cells. Collectively, these data show that Aq exerts multi-target inhibitory and virulence-attenuating effects on P. fluorescens, supporting its potential use in aquaculture environments. Full article

Background/Objectives: Respiratory tract infections (RTIs) are a leading cause of morbidity in children under five, with over 75% experiencing recurrent episodes and an increased risk of asthma by school age, particularly following respiratory syncytial virus (RSV) and rhinovirus (RV) infections. While current therapies primarily address acute symptoms, effective preventive strategies remain limited. Bacterial-derived immunomodulators have emerged as promising interventions, but their mechanisms and pediatric clinical evidence remain incompletely characterized. This narrative review examines preclinical mechanisms and clinical findings for four such agents, contextualizing current evidence and identifying key gaps. Methods: A targeted narrative review of PubMed-indexed literature (inception to September 2025) was conducted. Mechanistic studies, pediatric (0–18 years) clinical trials, and meta-analyses evaluating OM-85, polyvalent mechanical bacterial lysates (PMBL/Ismigen), MV130, and Lactobacillus rhamnosus CRL1505 were included. Outcomes of interest comprised immunological mechanisms, RTI incidence, wheezing, and asthma-related outcomes. Results: All four agents share convergent immunomodulatory mechanisms involving epithelial barrier reinforcement, innate immune activation, and adaptive immune modulation. OM-85 has the most extensive preclinical evidence. PMBL enhances epithelial repair via the IL-23/IL-22 axis, MV130 induces trained immunity, and CRL1505 acts through the gut–lung axis. Clinical evidence varies markedly, with OM-85 showing the most comprehensive data (18 RCTs and 7 meta-analyses), followed by PMBL and MV130, while evidence for CRL1505 remains predominantly preclinical. Conclusions: Despite variable evidence maturity, these agents share a coherent mechanistic rationale and favorable safety profiles, with ongoing studies expected to clarify their clinical role in early-life respiratory prevention. Full article

Background/Objectives: Bacteriophage-based display has been utilized for a variety of purposes, such as to assemble protein libraries and conduct biopanning. We have created a modified lambda (λ) bacteriophage platform, ideal for the display and delivery of proteins. Our system utilizes counter-selection recombineering for versatile modification, temperature-sensitive induction for timely lysate production, and an arabinose-inducible mechanism for high-titer, stable yield. Here, we investigated the ability of this specialized λ phage display platform to stimulate highly specific antibodies in mice against the displayed cancer-variant cell-surface receptor EGFRvIII, demonstrating its potential in cancer immunotherapy and broader vaccine development. Methods: λ display immunogenicity was explored by generating fusion proteins between the λ head protein D and a 13-mer peptide from the N terminus of glioblastoma variant cell-surface receptor, EGFRvIII. The 13-mer peptide was fused to either the N or C terminus of the λD protein while λ remained a dormant lysogen in the bacterial host chromosome. Recombinant phage lysates were then generated with ~420 displayed fusion proteins per phage particle. Mice were injected with purified recombinant λ phage without an adjuvant via both intraperitoneal and intramuscular routes, and sera harvested at various timepoints were profiled for immunogenicity. Results: Analysis of serum samples by ELISA and Western blotting demonstrated the ability of the λD~EGFRvIII phage display, especially in the C-terminal fusion construction, to elicit a robust anti-EGFRvIII humoral response by either injection route. Notably, the antibody response was highly specific to EGFRvIII without exhibiting cross-reactivity to wild-type EGFR. Conclusions: The data generated in this study demonstrate the λ system’s immunotherapeutic potential as a high-titer, stable, self-adjuvanting vector for the stimulation of robust antibody titers with defined specificity. Full article

Probiotics hold considerable promise for treating and preventing inflammatory disease; however, their application is often limited by unclear anti-inflammatory mechanisms and reduced viability following lyophilization. In this study, I thoroughly evaluated the anti-inflammatory potential of Lactiplantibacillus plantarum FS4722 (L. plantarum FS4722) and substantially enhanced strain viability through optimization of the lyoprotectant formulation. Functional assays demonstrated that the fermented supernatant, heat-inactivated bacterial suspension, and cell lysate derived from L. plantarum FS4722 effectively suppressed transcription and expression of inflammatory cytokines in LPS-stimulated RAW 264.7 macrophages. The fermented supernatant exhibited the strongest inhibitory effects, surpassing the reference probiotic Lacticaseibacillus rhamnosus GG (LGG). Mechanistic investigations revealed that anti-inflammatory activity is primarily mediated via inhibition of the MAPK and NF-κB signaling pathways. Furthermore, using component screening combined with response surface methodology, the lyoprotectant formulation (10.00% trehalose, 1.00% sodium carboxymethyl cellulose, and 5.00% skim milk) was optimized, resulting in a lyophilization survival rate of 82.32% while maintaining cellular integrity; in this accelerated stability assessment, the strain retained 78.89% of its activity after 28 days of storage at 4 °C. Collectively, this study provides a robust and efficient approach for probiotic formulation while systematically elucidating the underlying anti-inflammatory mechanisms, thereby offering practical guidance for the development and clinical application of high-performance probiotic products. Full article

Objectives: The present study evaluated the efficacy of a preparation based on bacterial lysates of Streptococcus oralis, Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum in the treatment of experimental periodontitis in rats, compared to metronidazole. Methods: Twenty female Wistar rats were used, divided into three groups: control, bacterial lysates and metronidazole, administered for 10 days by oral lavage/gavage. Periodontitis was induced by ligatures contaminated with bacterial suspensions (10⁹ CFU/mL) for 4 weeks. Lysates were obtained by culturing bacterial strains, centrifugation, washing, heat inactivation, ultrasonication and filtration. The evaluations included biocompatibility on HGF-1 fibroblasts, microbiological stability, clinical parameters, hematological, biochemical and histopathological analyses. Results: The lysates demonstrated the absence of cytotoxicity (cell viability 90–100%) and significant antimicrobial effect at the optimal concentration (2 × 10⁹ CFU/mL equivalent). Both treatments significantly reduced periodontal inflammation, with no statistical differences between them. Systemic immunoinflammatory indices (SII, SIRI, AISI) increased comparably, demonstrating controlled immune mobilization, and ALT was maintained within physiological limits. Histopathological examination revealed a reduction in inflammatory infiltrate, connective tissue reorganization and bone regeneration in both treated groups. Conclusions: Bacterial lysates represent a viable therapeutic alternative with comparable efficacy to metronidazole, favorable safety profile and immunomodulatory potential in the treatment of periodontitis. Full article

Background: Urinary tract infections (UTIs) rank among the most prevalent infectious diseases globally, with recurrent UTIs (rUTIs) posing substantial therapeutic challenges due to the lack of durable protective immunity. While trained immunity augments innate immune responses, its induction and functional significance in bladder-resident group 3 innate lymphoid cells (ILC3s) remain unknown. This study investigates whether ILC3s develop trained immunity following uropathogenic Escherichia coli (UPEC) exposure and how they contribute to mucosal defense against rUTIs. Methods: The ILC3 counts were detected in bladder sections from UTI patients and health controls (HC). A recurrent UTI mouse model was established through primary and secondary urethral UPEC inoculation. Bacterial loads in tissues were assessed, and single-cell suspensions were analyzed via flow cytometry. Bladder naïve- and UPEC-trained ILC3s were adoptively transferred, with evaluations of histopathology, epithelial barrier function, inflammation, and antimicrobial peptides. The in vitro ILC3 cell line MNK-3 was detected for IL-17A and IL-22 production following primary and secondary UPEC lysate stimulation. Results: We demonstrate that primary UPEC infection triggers ILC3 expansion in both human and murine bladders. Upon secondary challenge, these ILC3s develop trained immunity, characterized by enhanced proliferation, amplified IL-17A and IL-22 production, and improved pathogen clearance. Mechanistically, trained ILC3s reinforce urothelial barrier integrity through upregulation of antimicrobial peptides (Reg3b/Reg3g) and attenuate inflammatory pathology by suppressing pro-inflammatory cytokines (IL-6, TNF-α). Conclusions: We uncover an endogenous defense mechanism wherein UPEC primes bladder ILC3s via trained immunity, enabling amplified IL-17A- and IL-22-mediated protection against recurrent infections. These findings establish ILC3-trained immunity as a novel conceptual foundation, providing a basis for developing immunotherapies against rUTIs. Full article

Objectives: The aim of this work was to investigate whether Bacteroides vulgatus (BV), Clostridium perfringens (CP), Parabacteroides distasonis (PD), and Ruminococcus albus (RA) lysates modulate the secretion of IL-17, INF-γ, IL-2, and TGF-β 1 by human HT-29 cells, PBMCs, and monocytes (MON). Results: CP lysate significantly lowered IL-17 secretion by HT-29 cells vs. control (p < 0.05), but only at a dose of 100 µg. RA lysate reduced IL-17 secretion by HT-29 cells vs. control (p < 0.05), but only at a dose of 400 µg, whereas PD lysate significantly decreased IL-17 secretion by HT-29 cells vs. control (p < 0.05) at both doses. The secretion of IL-17 by PBMCs was significantly reduced after administration of BV and PD lysates (100 µg). BV and PD lysates (400 µg) also significantly decreased IL-17 secretion by MON vs. control (p < 0.05). The secretion of INF-γ by HT-29 cells was significantly lowered vs. control (p < 0.05) after administration of PD and CP lysates (400 µg). CP lysates (100 µg and 400 µg) also significantly reduced INF-γ secretion by MON compared with control (p < 0.05). The secretion of INF-γ by PBMCs was significantly reduced vs. control (p < 0.05) after administration of BV and CP lysates (400 µg). Conclusions: In PBMCs, HT-29 cells, and MON, INF-γ and IL-17 concentrations were significantly lowered by selected bacterial lysates in a dose-dependent manner. However, the low values detected in this experiment may not have an impact on systemic immune status. Full article

Background: The periodontal pathogen Tannerella forsythia is auxotrophic for muramic acid (MurNAc), a key component of bacterial peptidoglycan, and dependent on an external supply of MurNAc to maintain pure laboratory cultures. The focus of this study was to find a source of muramic acid and peptidoglycan fragments from a Staphylococcus aureus strain. This would facilitate the isolation of T. forsythia by incorporating peptidoglycan into conventional anaerobic media. Methods: The S. aureus strain ATCC 29213 was chosen as the source. The standardization and quantification of the method included verifying concentrations via spectrophotometry and developing a linear regression model with standard curves for muramic acid and lactic acid. The resulting lysate was used to seed Fastidious Anaerobe Agar (FAA) plates, which were inoculated with strain T. forsythia (ATCC 43037) and incubated in an anaerobic chamber for seven days. Results: The resulting lysate had an optical density ranging from 0.061 to 0.083, which corresponds to a muramic acid concentration of approximately 12 µg/mL. Pure cultures of T. forsythia could then be obtained on FAA plates supplemented with muramic acid (MurNAc) (FAA-Mur). The viability of the axenic T. forsythia culture was confirmed using muramic acid/peptidoglycan fragments of microbial origin. Conclusions: The method presented improves the growth of T. forsythia. Consequently, T. forsythia is available for further investigation into the regular performance of sensitivity tests in periodontics and the routine generation of growth curves for quantitative polymerase chain reaction (qPCR) analysis. Full article

Toll-like receptor 9 (TLR9) and Toll-like receptor 3 (TLR3), which are widely expressed in dendritic cells (DCs), function as key pattern recognition receptors (PRRs) in the immune system. Their primary roles involve specifically detecting pathogen-associated molecular patterns (PAMPs): TLR9 recognizes unmethylated CpG motifs predominantly found in bacterial and viral DNA, while TLR3 identifies viral double-stranded RNA (dsRNA), a molecular signature associated with viral replication. Their specific agonists [CpG ODN (a TLR9 agonist) and poly(I:C) (a TLR3 agonist)] can effectively activate DCs and enhance the expression of immune activation-related molecules. In this study, by establishing a mouse primary dendritic cell model and a glioma-bearing mouse model, and employing techniques such as transcriptome sequencing, we found that combined stimulation with CpG ODN and poly(I:C) significantly enhanced the anti-tumor function of DCs: in vitro, DCs subjected to combined stimulation showed upregulation of anti-tumor-related surface markers, enhanced migratory capacity, and a more effective activation of CD8⁺ T cells; in vivo, a DC vaccine loaded with tumor lysate antigen and stimulated with this combined regimen significantly delayed the progression of glioma in tumor-bearing mice. Further investigation revealed that the underlying mechanism for this enhanced effect may involve TLR9 activation promoting TLR3 upregulation through the p38 MAPK-ATF3 signaling axis. Consequently, we designed a sequential stimulation protocol (first CpG ODN then poly(I:C)), which demonstrated a stronger anti-glioma effect compared to simple combined stimulation. This study provides a new strategy for enhancing the immune efficacy of DC vaccines and has potential significance for promoting the clinical translation of DC vaccines. Full article

Bacterial lysates have emerged as promising immunomodulatory agents that can enhance innate immune responses. Given the crucial role of macrophages in recognizing and controlling intracellular pathogens such as Mycobacterium tuberculosis, this study aimed to evaluate the immunological effects of selected bacterial lysates on human monocyte-derived macrophages (MDMs). We examined the ability of commercial bacterial lysates, Pulmonarom, Ismigen, Uro-Vaxom, and a lysate of M. tuberculosis H37 Ra (LMtb) to stimulate the production of key pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-8. In addition, we investigated whether these lysates could modulate the expression of bactericidal/permeability-increasing protein (BPI), a critical antimicrobial effector, and assessed their ability to reduce the intracellular burden of mycobacteria and induce autophagy. The results demonstrate diverse immunostimulatory profiles among the lysates, highlighting differences in both inflammatory and antimicrobial responses that may be relevant for host-directed therapeutic strategies against tuberculosis. Notably, beyond the in vitro antimycobacterial activity observed for BPI, this protein was also found to be elevated in both serum and bronchoalveolar lavage fluid from patients with active TB, reflecting local and systemic immune activation. Furthermore, the reduction in BPI levels after treatment suggests its potential utility for following the dynamics of infection. Full article

The human gut microbiota helps maintain metabolic balance, supports immune function, and defends against opportunistic pathogens that can disrupt the microbiota ecosystem. An imbalance or dysbiosis in microbial composition is linked to various diseases, including inflammatory bowel disease, metabolic syndromes, and neurodegenerative disorders. Using microbiota modulation with prebiotics and postbiotics is a practical approach to address these imbalances. Prebiotic compounds are defined as substrates that promote metabolic activity and restore microbial patterns. Postbiotics include short-chain fatty acids (SCFAs), microbial cell lysates, and extracellular compounds. This research aims to investigate how the gut microbiota can be modulated in vitro using the prebiotic ColonX and a postbiotic derived from Kombucha fermentation within a controlled GIS1 in vitro system. These products demonstrate potential for modulation, as they support selective bacterial growth and enhance microbial diversity. Prebiotics help stabilize gut pH, while postbiotics play a crucial role in biofilm formation. Together, they provide an innovative approach to treating dysbiosis and enhancing overall gut health. The findings highlight the importance of utilizing prebiotics and postbiotics to modulate gut microbiota in chronic diseases characterized by dysbiosis. This paper is especially relevant for elderly populations, as gut dysbiosis is common, and microbiota modulation supports healthy aging. Full article

Bacterial inclusion bodies (IBs) are still generally considered to be waste products of recombinant protein production, despite various studies that have challenged this conventional view in the last two decades, and have been proposed for use as immobilized enzymes in vivo for biocatalysis. Current advances in genetic and molecular biology make it possible to perform multienzymatic reactions or enzymatic cascades to synthesize valuable products. When cascades need cofactor regener tion, it is difficult to use “cheap” whole cells or their lysates, and “expensive” enzyme purification is required. The capture of enzymatic activity into active IBs (aIBs), well-separable protein aggregates from cell lysate, could represent a usable compromise between purified enzymes and cell lysates. It is shown here that the combination of two polyphosphate kinases (PPKs) in the form of aIBs leads to almost 10-fold ATP regeneration and 100% UTP utilization without degradation into adenosine or uridine. PPKs have been combined with N-acetylhexosamine 1-kinase and N-acetylglucosamine-1-phosphate uridyltransferase to produce valuable UDP-N-acetylglucosamine, but the described approach could be used in various multienzymatic syntheses to avoid enzyme purification and ensure nucleotide triphosphate regeneration. Full article

Skin microbiome-friendly preparations are gaining increasing popularity in the cosmetics and pharmaceutical industries. Fermented plants, lysates, and heat-treated products are used as probiotic ingredients in cosmetics. This is due to the presence of Lactobacillus bacteria, such as acid or acid-rennet whey, which are natural probiotics that can positively impact the skin microbiome. However, due to technological difficulties, the direct use of whey as a cosmetic ingredient is limited. An optimized emulsification method was used to obtain alginate microspheres as carriers of whey. The process parameters were optimized using the Design of Experiments (DoEs) methodology. The effect of three key variables, including the type of probiotic raw material (whey from 1—cows, 2—goats, and 3—mixed), the alginate-to-raw material ratio (1–3%), and sonication time (0.5–1.5 min), on parameters such as encapsulation efficiency, bacterial survival, viscosity, and microspheres size was analyzed. The results obtained demonstrated that the optimal process parameters were the sonication time of 0.5 min and the alginate-to-whey mass ratio of 1.5% for all types of whey material studied. However, the most important factor influencing the properties and functionality of the microspheres was sonication time. The optimized whey-loaded microspheres were incorporated into a preservative-containing emulsion system, in which the viability of whey-derived bacteria was monitored over time. The whey encapsulation process effectively maintained the bacteria’s probiotic properties, protecting their viability despite the presence of preservatives (at a level of 4.92 ± 0.9 log CFU/g after 30 days of formulation storage), thus confirming the feasibility of incorporating liquid whey into skincare formulations. Full article