Search Results (118)

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

Biotechnological research increasingly focuses on developing new drugs to counter the rise of antibiotic-resistant strains in hospitals. This study aimed to create bacterial lysates from antibiotic-resistant pathogens isolated from patients and medical instruments across hospital departments. Identification was performed based on morphological, cultural, and biochemical characteristics, as well as 16S rRNA gene sequencing using the BLAST algorithm. Strain viability was assessed using the Miles and Misra method, while sensitivity to eight antibacterial drug groups and biosafety between cultures were evaluated using agar diffusion. From 15 clinical sources, 25 pure isolates were obtained, and their phenotypic and genotypic properties were studied. Carbohydrate fermentation testing confirmed that the isolates belonged to the genera Escherichia, Citrobacter, Klebsiella, Acinetobacter, Pseudomonas, Staphylococcus, Haemophilus, and Streptococcus. The cultures exhibited good viability (10⁹–10¹⁰ CFU/mL) and compatibility with each other. Based on prevalence and clinical significance, three predominant hospital pathogens (Klebsiella pneumoniae 12 BL, Pseudomonas aeruginosa 3 BL, and Acinetobacter baumannii 24 BL) were selected to develop a bacterial lysate consortium. Lysates were prepared with physical disruption using a French press homogenizer. The resulting product holds industrial value and may stimulate the immune system to combat respiratory pathogens prevalent in Kazakhstan’s healthcare settings. Full article

Background: Multidrug-resistant (MDR) Escherichia coli (E. coli) strains pose a significant public health challenge, which has led to the exploration of alternative therapeutic strategies. Due to their antibacterial and immunomodulatory properties, bacteriophages have emerged as promising therapeutic agents. Methods: This study investigates the effects of GADS24, a novel lytic bacteriophage of E. coli, on human-monocyte-derived dendritic cells (DCs). DCs are exposed to purified GADS24 phage, bacterial lysate, or a combination of both. Flow cytometry was used to assess the expression of surface markers (HLA-DR, CD80, CD83, and CD86), and ELISA was used to measure cytokine production (IL-10 and IL-12p70). Results: Following treatment with bacterial lysate, a significant increase in DC maturation markers was observed. The GADS24 phage alone induced a moderate upregulation of these markers, decreased IL-10 secretion, and increased IL-12p70. Combining bacterial lysate and phage tempered the maturation response compared to the lysate treatment alone. Conclusion: These findings suggest that GADS24 exerts antibacterial activity and modulates host immunity by influencing DC maturation and cytokine production. Due to its dual antimicrobial and immunomodulatory functions, GADS24 is likely to be a valuable adjunctive therapy for multidrug-resistant (MDR) bacterial infections. Furthermore, in vivo studies are necessary to confirm these promising in vitro results. Full article

Background: Bacterial lysates are known to modulate the immune response against respiratory infections. However, the effects of the commercial bacterial lysate Pulmonarom^® on dendritic cells—particularly human monocyte-derived dendritic cells (moDCs)—have not been studied. Additionally, limited data are available on the expression of Toll-like receptors (TLRs) and cytokines following stimulation with bacterial lysates. Methods: Human monocytes were isolated from buffy coats and differentiated into moDCs. Pulmonarom^® was lyophilized, quantified, and used to stimulate moDCs. Ultrastructural changes were evaluated using transmission electron microscopy. The expression of TLRs and selected cytokines was analyzed by flow cytometry. Results: Pulmonarom^® stimulation induced morphological changes in moDCs, including an increased number of dendrites and lysosomes. It also led to the upregulation of MHC class II molecules and TLRs 2, 3, 6, and 7. Additionally, the production of IL-4, IL-6, IL-8, and MCP-1 was significantly increased. Conclusions: Pulmonarom^® promotes moDC maturation, characterized by enhanced antigen presentation capabilities and lysosomal activity, along with increased expression of specific TLRs and cytokines. These features suggest a trained immunity phenotype in moDCs, potentially improving their ability to initiate adaptive immune responses against respiratory pathogens. To our knowledge, this is the first study to investigate the immunomodulatory effects of Pulmonarom^® on human moDCs, providing novel insights into its potential as an immunotherapeutic adjuvant. Full article

Mycobacterium indicus pranii (MIP), an atypical mycobacterium originally developed as an anti-leprosy vaccine, has emerged as a potent immunomodulator with diverse therapeutic applications. Despite its clinical significance, molecular mechanisms underlying MIP’s immunomodulatory properties remain largely unexplored. Bacterial phosphatases are recognized as crucial virulence factors that enable pathogens to evade host defenses by modulating host immune signaling pathways, including phosphoinositide metabolism. MIP_07528 was identified as a putative protein tyrosine phosphatase B (PtpB) ortholog through in silico analysis, with significant sequence conservation observed within catalytic domains of pathogenic mycobacterial PtpB proteins. Phosphatase activity was detected in both cell lysate and culture filtrate fractions, revealing differential expression patterns between MIP and M. tuberculosis. Upregulation of MIP_07528 was demonstrated under oxidative stress, suggesting involvement in stress adaptation. The recombinant protein exhibited distinctive kinetic properties, characterized by higher substrate affinity yet increased susceptibility to oxidative inactivation compared to its M. tuberculosis counterpart. In macrophages, MIP_07528 suppressed pro-inflammatory cytokines while enhancing anti-inflammatory IL-10 production. These findings establish MIP_07528 as a functional phosphatase that may contribute to MIP’s immunomodulatory properties. This work advances understanding of phosphatase function in non-pathogenic mycobacteria while providing insights into virulence factor evolution and establishing a foundation for novel antimicrobial strategies. Full article

Purpose: To evaluate the sterility and biological functionality of platelet lysate eye drops stored in BTI ophthalmic devices for PRGF delivery under different storage conditions and simulated use scenarios. Methods: Eye drops were prepared using platelet lysate and stored in BTI tubes under three different conditions: ≤−15 °C, 2–8 °C, and room temperature (RT) for 72 h. Simulated use was performed for 72 h with controlled drop dispensing. Bacterial contamination was assessed according to European Pharmacopoeia sterility testing principles. The biological activity of the eye drops was assessed using in vitro proliferation assays with primary human keratocytes (HKs) and human corneal epithelial cells (HCEs). Statistical analyses were performed to compare the effects of different storage conditions and application scenarios. Results: No bacterial contamination was detected in platelet lysate eye drops stored under any of the conditions tested, regardless of simulated use. Proliferation assays showed that eye drops enhanced the growth of HK and HCE cells compared to the control medium. No significant differences in proliferation were observed between storage conditions. Conclusions: Platelet lysate eye drops maintain sterility and biological functionality when stored in BTI ophthalmic devices at ≤−15 °C, 2–8 °C and RT for up to 72 h of simulated use. These results support the feasibility of using BTI eye drop devices in clinical settings while ensuring safety and efficacy. Full article

Growing evidence supports the use of nitrate-reducing bacterial strains as probiotics to enhance the benefits of nitrate metabolism for both oral and systemic health. This study aimed to test the nitrate reductase activity of Levilactobacillus brevis CD2 (DSM-27961/CNCM I-5566), a strain widely used as a starter culture in fermented foods and recognized for its multifaceted health-promoting probiotic properties. We also sought to determine whether the probiotic lysate enhances nitrate reduction ex vivo using six salivary samples from healthy subjects while evaluating its potential influence on pH and buffering capacity. Considering the established link between lactate metabolism and nitrite production, we assessed the salivary levels of D-lactate after a 3-hour incubation with or without Lv. brevis. The results indicate that Lv. brevis CD2 exhibits significant intrinsic and concentration-dependent nitrate reductase activity. Additionally, treatment with Lv. brevis for 3 h significantly increased nitrite generation across all saliva samples, with further enhancement observed after the addition of exogenous nitrates. Lv. brevis also significantly improved salivary pH and buffering capacity, particularly when combined with nitrate. Furthermore, the probiotic treatment resulted in reduced levels of salivary D-lactate. To further support and validate our in vitro and ex vivo findings, we evaluated the oral nitrate-reducing activity in saliva samples from healthy individuals treated for four weeks with Lv. brevis CD2 lozenges. Of note, the results indicated that the probiotic group showed a significant increase in oral nitrate-reducing capacity compared to baseline and placebo after four weeks of treatment. Overall, our study suggests that Lv. brevis CD2 acts as a nitrate-reducing probiotic, providing new insights into its health benefits and complementing findings from previous studies. Full article

Helicobacter pylori is a pathogenic bacterium that causes gastric and extragastric diseases. We have previously demonstrated that one of the mechanisms of H. pylori-associated chronic immune thrombocytopenia involves immune complexes of platelets, a H. pylori protein Lpp20 and an anti-Lpp20 antibody. However, it remains unclear how Lpp20 enters the body. We hypothesize that bacterial extracellular vesicles (bEVs) transport Lpp20. Thus, this study assessed Lpp20 in the bEVs released from seven clinical H. pylori isolates, using immunoprecipitation (IP), immunoblotting (IB), and surface plasmon resonance imaging (SPRi), with anti-GroEL (a marker of bEVs) and anti-Lpp20 antibodies. Lpp20 and bEVs were each detected in lysates of all seven strains. IP–IB experiments demonstrated that bEVs containing Lpp20 were produced by five of the strains (J99, SS1, HPK5, JSHR3, and JSHR31). SPRi using an anti-Lpp20 antibody demonstrated significantly higher reflectance from the strain HPK5 than from its lpp20-disrupted strains (p < 0.01), indicating localization of Lpp20 on the bEVs’ surface; Lpp20 may also be contained within bEVs. The bEVs containing Lpp20 were not detected from two clinical H. pylori strains (26695 and JSHR6) or from two lpp20-disrupted strains (26695ΔLpp20 and HPK5ΔLpp20). Differences in Lpp20 detection in bEVs are likely due to variations in bEV production resulting from strain diversity. Full article