Escherichia coli A0 34/86 (EcO83) is a probiotic strain used in newborns to prevent nosocomial infections and diarrhoea. This bacterium stimulates both pro- and anti-inflammatory cytokine production and its intranasal administration reduces allergic airway inflammation in mice. Despite its benefits, there are concerns about the use of live probiotic bacteria due to potential systemic infections and gene transfer. Extracellular vesicles (EVs) derived from EcO83 (EcO83-EVs) might offer a safer alternative to live bacteria. This study characterizes EcO83-EVs and investigates their interaction with host cells, highlighting their potential as postbiotic therapeutics. EcO83-EVs were isolated, purified, and characterised following the Minimal Information of Studies of Extracellular Vesicles (MISEV) guidelines. Ex vivo studies conducted in human nasal epithelial cells showed that EcO83-EVs increased the expression of proteins linked to oxidative stress and inflammation, indicating an effective interaction between EVs and the host cells. Further in vivo studies in mice demonstrated that EcO83-EVs interact with nasal-associated lymphoid tissue, are internalised by airway macrophages, and stimulate neutrophil recruitment in the lung. Mechanistically, EcO83-EVs activate the NF-κΒ signalling pathway, resulting in the nitric oxide production. EcO83-EVs demonstrate significant potential as a postbiotic alternative to live bacteria, offering a safer option for therapeutic applications. Further research is required to explore their clinical use, particularly in mucosal vaccination and targeted immunotherapy strategies.

• Keywords
• EVs, Ec083, NF‐κΒ signalling, bacterial extracellular vesicles, macrophage, nitric oxide, postbiotics, probiotic,
• MeSH
• Administration, Intranasal * MeSH
• Epithelial Cells metabolism   MeSH
• Escherichia coli * metabolism   MeSH
• Extracellular Vesicles * metabolism   MeSH
• Humans MeSH
• Lymphoid Tissue metabolism   MeSH
• Macrophages metabolism   MeSH
• Mice MeSH
• NF-kappa B metabolism   MeSH
• Oxidative Stress MeSH
• Lung microbiology   metabolism   MeSH
• Probiotics * administration & dosage   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• NF-kappa B MeSH

INTRODUCTION: The immune systems of both the mother and the newborn face significant challenges during birth. Proper immune regulation after birth is essential for the survival of neonates. Numerous studies have demonstrated that the neonatal immune system is relatively immature, particularly in its adaptive arm, placing the primary responsibility for immune surveillance on innate immunity. METHODS: Given the significant role of neutrophils in protecting the neonate after birth, we conducted a study investigating the properties of neutrophils in newborn cord blood using various methodological approaches. RESULTS: Our findings demonstrate the presence of immature low-density neutrophils in the cord blood, which are likely responsible for the observed elevated expression of genes coding for proteins essential to antimicrobial response, including myeloperoxidase, neutrophils elastase, and defensins. DISCUSSION: We propose that these cells function normally and support the protection of newborns early after birth. Furthermore, our results suggest that the mode of delivery might significantly influence the programming of neutrophil function. The presented findings emphasize the importance of distinct neutrophil subpopulations in neonatal immunity and their potential impact on early postnatal health.

• Keywords
• cord blood, defensins, myeloperoxidase, neutrophils, oxidative burst,
• MeSH
• Anti-Infective Agents * metabolism   MeSH
• Fetal Blood MeSH
• Humans MeSH
• Neutrophils * MeSH
• Infant, Newborn MeSH
• Immunity, Innate MeSH
• Proteins metabolism   MeSH
• Check Tag
• Humans MeSH
• Infant, Newborn MeSH
• Publication type
• Journal Article MeSH
• Comment MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Anti-Infective Agents * MeSH
• Proteins MeSH

Early postnatal events are important for the development of the neonatal immune system. Harboring the pioneering microorganisms forming the microbiota of the neonatal gastrointestinal tract is important for priming the immune system, as well as inducing appropriate tolerance to the relatively innocuous environmental antigens and compounds of normal healthy microbiota. Early postnatal supplementation of suitable, safe probiotics could accelerate this process. In the current study, the immunomodulatory capacity of the probiotic strain of Escherichia coli O83:K24:H31 (EcO83) was characterized in vitro and in vivo. We compared the capacity of EcO83 with and without hemolytic activity on selected immune characteristics in vitro as determined by flow cytometry and quantitative real-time PCR. Both strains with and without hemolytic activity exerted comparable capacity on the maturation of dendritic cells while preserving the induction of interleukin 10 (Il10) expression in dendritic cells and T cells cocultured with EcO83 primed dendritic cells. Early postnatal supplementation with EcO83 led to massive but transient colonization of the neonatal gastrointestinal tract, as detected by in vivo bioimaging. Early postnatal EcO83 administration promoted gut barrier function by increasing the expression of claudin and occludin and the expression of Il10. Early postnatal EcO83 application promotes maturation of the neonatal immune system and promotes immunoregulatory and gut barrier functions.

• Keywords
• E. coli O83:K24:H31, IL-10, dendritic cell, early postnatal probiotic administration, indol amine 2,3 dioxygenase, luciferase, probiotic,
• MeSH
• Dendritic Cells MeSH
• Escherichia coli MeSH
• Interleukin-10 MeSH
• Humans MeSH
• Microbiota * MeSH
• Infant, Newborn MeSH
• Probiotics * pharmacology   MeSH
• Check Tag
• Humans MeSH
• Infant, Newborn MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Interleukin-10 MeSH

INTRODUCTION: Probiotic administration seems to be a rational approach to promote maturation of the neonatal immune system. Mutual interaction of the microbiota with the host immune system is critical for the setting of appropriate immune responses including a tolerogenic one and thevmaintenance of homeostasis. On the other hand, our knowledge on the modes of actions of probiotics is still scarce. METHODS: In our study, probiotic strain Escherichia coli O83:K24:H31 (EcO83) was administered to neonates of allergic mothers (AMs; neonates with increased risk for allergy development) within 48 h after the delivery, and the impact of this early postnatal supplementation on allergy incidence and selected immune markers has been analyzed 10 years after the primary EcO83 administration. RESULTS: We have observed decreased allergy incidence in 10-year-old children supplemented with EcO83 (13 of 52 children were allergic) in comparison with non-supplemented children of AMs (16 of 42 children were allergic). The early postnatal EcO83 supplementation appeared to limit the allergy in the high-risk group (children of AMs) compared to that in the low-risk group (children of healthy mothers). Dendritic cells (DCs) in the peripheral blood of EcO83-supplemented children do not differ significantly in cell surface presence of CD83. The immunomodulatory capacity of EcO83 on DCs was tested in vitro as well. Both directly isolated myeloid and in vitro monocyte-derived DCs from cord blood increased CD83 expression together with interleukin (IL)-10 secretion after EcO83 stimulation. The effect of early postnatal EcO83 supplementation on the microbiota composition of 10-year-old children was characterized by next-generation sequencing, and we have not observed significant changes in the microbiota composition of EcO83-supplemented and non-supplemented children at the age of 10 years. CONCLUSIONS: Early postnatal EcO83 supplementation appears to lower allergy incidence in children of AMs. It seems that the beneficial effect of EcO83 is mediated via modulation of DC functional capacities without impacting the microbiota composition. Larger-scale studies will be necessary to confirm these preliminary findings.

• Keywords
• CD83, Escherichia coli O83:K24:H31, IL-10, allergy, cord blood, dendritic cell, flow cytometry, probiotic,
• MeSH
• Hypersensitivity * epidemiology   prevention & control   MeSH
• Dendritic Cells MeSH
• Child MeSH
• Escherichia coli physiology   MeSH
• Incidence MeSH
• Humans MeSH
• Microbiota * MeSH
• Monocytes MeSH
• Infant, Newborn MeSH
• Probiotics * MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Infant, Newborn MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Continuous increasing incidence of allergic diseases is calling for identifying early prognostic markers pointing to increased risk of allergy development and establishing protocols for preventive strategies limiting allergy development in predisposed individuals. It is important to better understand the critical events occurring in early postnatal life, especially the interaction of a newborn with microbial compounds important for the maturation of the neonatal immune system and setting immunoregulatory responses as well. Dendritic cells (DC) together with the cytokine microenvironment play an important role in priming of immune responses. The capacity of monocyte-derived DC (moDC) from cord blood of children of healthy and allergic mothers to respond to microbial antigens (Escherichia coli O86 (EcO86) and delipidated Bacillus firmus (DBF)) was tested by flow cytometry and quantitative real-time PCR. Both EcO86 and DBF were able to promote maturation of moDC, but moDC of children of allergic mothers expressed higher levels of activation markers CD80 and CD83. Increased gene expression of IL-6 and lower expression of indol-amine 2,3 dioxygenase were observed in moDC of neonates of allergic mothers, in comparison to healthy ones. A higher gene expression and an increased presence of activation markers on moDC of newborns of allergic mothers indicate a generally higher reactivity of these cells, possibly enabling easier development of inappropriate immune response after an allergen encounter.

• MeSH
• Hypersensitivity blood   diagnosis   MeSH
• Antigens, Bacterial immunology   MeSH
• Antigens, Surface genetics   metabolism   MeSH
• Biomarkers metabolism   MeSH
• Cytokines genetics   metabolism   MeSH
• Dendritic Cells immunology   MeSH
• Fetal Blood MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Mothers MeSH
• Monocytes immunology   MeSH
• Infant, Newborn MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Infant, Newborn MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antigens, Bacterial MeSH
• Antigens, Surface MeSH
• Biomarkers MeSH
• Cytokines MeSH

Allergic diseases represent a major issue in clinical and experimental immunology due to their high and increasing incidence worldwide. Allergy status of the mother remains the best predictor of an individual's increased risk of allergy development. Dysregulation of the balance between different branches of immune response, chiefly excessive polarization towards Th2, is the underlying cause of allergic diseases. Regulatory T cells (Tregs) play a pivotal role in the timely establishment of physiological immune polarization and are crucial for control of allergy. In our study we used flow cytometry to assess Tregs in cord blood of newborns of healthy (n = 121) and allergic (n = 108) mothers. We observed a higher percentage of Tregs (CD4+CD25+CD127lowFoxP3+) in cord blood of children of allergic mothers. However, the percentage of cells expressing extracellular (PD-1, CTLA-4, GITR) and intracellular (IL-10, TGF-β) markers of function was lower (significantly for PD-1 and IL-10) within Tregs of these children. Furthermore, Helios- induced Tregs in the cord blood of children of allergic mothers were decreased. These results were supported by a decrease in plasma levels of IL-10 and TGF-β in cord blood of newborns of allergic mothers, implying lower tolerogenic capacity on the systemic level. Taken together, these findings reflect deficient function of Tregs in the group with higher risk of allergy development. This may be caused by a lower maturation status of the immune system, specifically Tregs, at birth. Such immaturity may represent an important mechanism involved in the increased risk of allergy in children of allergic mothers.

• MeSH
• Hypersensitivity blood   immunology   MeSH
• Cytokines blood   MeSH
• Fetal Blood immunology   MeSH
• Immunoglobulin E blood   MeSH
• Humans MeSH
• Mothers MeSH
• Disease Susceptibility blood   immunology   MeSH
• Infant, Newborn MeSH
• T-Lymphocytes, Regulatory immunology   MeSH
• Risk MeSH
• Check Tag
• Humans MeSH
• Infant, Newborn MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytokines MeSH
• Immunoglobulin E MeSH