Botulinum and Tetanus Neurotoxins.

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) are the most potent toxins known and cause botulism and tetanus, respectively. BoNTs are also widely utilized as therapeutic toxins. They contain three functional domains responsible for receptor-binding, membrane translocation, and proteolytic cleavage of host proteins required for synaptic vesicle exocytosis. These toxins also have distinct features: BoNTs exist within a progenitor toxin complex (PTC), which protects the toxin and facilitates its absorption in the gastrointestinal tract, whereas TeNT is uniquely transported retrogradely within motor neurons. Our increasing knowledge of these toxins has allowed the development of engineered toxins for medical uses. The discovery of new BoNTs and BoNT-like proteins provides additional tools to understand the evolution of the toxins and to engineer toxin-based therapeutics. This review summarizes the progress on our understanding of BoNTs and TeNT, focusing on the PTC, receptor recognition, new BoNT-like toxins, and therapeutic toxin engineering.

Blocking Neuronal Signaling to Immune Cells Treats Streptococcal Invasive Infection.

The nervous system, the immune system, and microbial pathogens interact closely at barrier tissues. Here, we find that a bacterial pathogen, Streptococcus pyogenes, hijacks pain and neuronal regulation of the immune response to promote bacterial survival. Necrotizing fasciitis is a life-threatening soft tissue infection in which "pain is out of proportion" to early physical manifestations. We find that S. pyogenes, the leading cause of necrotizing fasciitis, secretes streptolysin S (SLS) to directly activate nociceptor neurons and produce pain during infection. Nociceptors, in turn, release the neuropeptide calcitonin gene-related peptide (CGRP) into infected tissues, which inhibits the recruitment of neutrophils and opsonophagocytic killing of S. pyogenes. Botulinum neurotoxin A and CGRP antagonism block neuron-mediated suppression of host defense, thereby preventing and treating S. pyogenes necrotizing infection. We conclude that targeting the peripheral nervous system and blocking neuro-immune communication is a promising strategy to treat highly invasive bacterial infections. VIDEO ABSTRACT.

Astrocyte-immune cell interactions in physiology and pathology.

Astrocytes play both physiological and pathological roles in maintaining central nervous system (CNS) function. Here, we review the varied functions of astrocytes and how these might change in subsets of reactive astrocytes. We review the current understanding of astrocyte interactions with microglia and the vasculature and protective barriers in the central nervous system as well as highlight recent insights into physiologic and reactive astrocyte sub-states identified by transcriptional profiling. Our goal is to stimulate inquiry into how these molecular identifiers link to specific functional changes in astrocytes and to define the implications of these heterogeneous molecular and functional changes in brain function and pathology. Defining these complex interactions has the potential to yield new therapies in CNS injury, infection, and disease.

Comprehensive analysis of the global impact and distribution of tick paralysis, a deadly neurological yet fully reversible condition.

SUMMARYTick paralysis is a potentially fatal condition caused by neurotoxins secreted by the salivary glands of certain ticks. Documented cases have been reported worldwide, predominantly in the United States, Canada, and Australia, with additional reports from Europe and Africa. This condition also affects animals, leading to significant economic losses and adverse impacts on animal health and welfare. To date, 75 tick species, mostly hard ticks, have been identified as capable of causing this life-threatening condition. Due to symptom overlap with other conditions, accurate diagnosis of tick paralysis is crucial to avoid misdiagnosis, which could result in adverse patient outcomes. This review provides a comprehensive analysis of the current literature on tick paralysis, including the implicated tick species, global distribution, tick toxins, molecular pathogenesis, clinical manifestations, diagnosis, treatment, control, and prevention. Enhancing awareness among medical and veterinary professionals is critical for improving the management of tick paralysis and its health impacts on both humans and animals.

A venom peptide-induced NaV channel modulation mechanism involving the interplay between fixed channel charges and ionic gradients.

Venoms are used by arthropods either to immobilize prey or as defense against predators. Our study focuses on the venom peptide, Ta3a, from the African ant species, Tetramorium africanum and its effects on voltage-gated sodium (NaV) channels, which are ion channels responsible for the generation of electrical signals in electrically excitable cells, such as neurons. Using the NaV1.7 isoform as our model NaV channel we show that Ta3a prolongs single channel active periods with increased open probability and induces non-inactivating whole-cell currents. Ta3a-affected NaV1.7 channels exhibit a leftward (hyperpolarizing) shift in activation threshold, constitutive activity even in the absence of an activating voltage stimulus, and at cell membrane voltages where channels are normally silent. Current-voltage experiments show that Ta3a shifts the voltage at which NaV current changes direction (reversal potential) by altering the local ionic concentration of permeant ions (Na+) rather than changing the channel's preference for ionic species. We propose a model where Ta3a maintains the positively charged voltage-sensing (S4) domains of the channel in the activated configuration where their electric field is exposed to the extracellular membrane surface to create an ionic bilayer comprising S4 domains and mobile anions (Cl-). This bilayer has a depolarizing effect on the cell membrane, thus reducing the amount of externally applied voltage required for channel activation.

Ancestral sequence reconstruction dissects structural and functional differences among eosinophil ribonucleases.

Evolutionarily conserved structural folds can give rise to diverse biological functions, yet predicting atomic-scale interactions that contribute to the emergence of novel activities within such folds remains challenging. Pancreatic-type ribonucleases illustrate this complexity, sharing a core structure that has evolved to accommodate varied functions. In this study, we used ancestral sequence reconstruction to probe evolutionary and molecular determinants that distinguish biological activities within eosinophil members of the RNase 2/3 subfamily. Our investigation unveils functional, structural, and dynamical behaviors that differentiate the evolved ancestral ribonuclease (AncRNase) from its contemporary eosinophil RNase orthologs. Leveraging the potential of ancestral reconstruction for protein engineering, we used AncRNase predictions to design a minimal 4-residue variant that transforms human RNase 2 into a chimeric enzyme endowed with the antimicrobial and cytotoxic activities of RNase 3 members. This work provides unique insights into mutational and evolutionary pathways governing structure, function, and conformational states within the eosinophil RNase subfamily, offering potential for targeted modulation of RNase-associated functions.

Domoic acid biosynthesis in the red alga Chondria armata suggests a complex evolutionary history for toxin production.

Domoic acid (DA), the causative agent of amnesic shellfish poisoning, is produced by select organisms within two distantly related algal clades: planktonic diatoms and red macroalgae. The biosynthetic pathway to isodomoic acid A was recently solved in the harmful algal bloom-forming diatom Pseudonitzschia multiseries, establishing the genetic basis for the global production of this potent neurotoxin. Herein, we sequenced the 507-Mb genome of Chondria armata, the red macroalgal seaweed from which DA was first isolated in the 1950s, identifying several copies of the red algal DA (rad) biosynthetic gene cluster. The rad genes are organized similarly to the diatom DA biosynthesis cluster in terms of gene synteny, including a cytochrome P450 (CYP450) enzyme critical to DA production that is notably absent in red algae that produce the simpler kainoid neurochemical, kainic acid. The biochemical characterization of the N-prenyltransferase (RadA) and kainoid synthase (RadC) enzymes support a slightly altered DA biosynthetic model in C. armata via the congener isodomoic acid B, with RadC behaving more like the homologous diatom enzyme despite higher amino acid similarity to red algal kainic acid synthesis enzymes. A phylogenetic analysis of the rad genes suggests unique origins for the red macroalgal and diatom genes in their respective hosts, with native eukaryotic CYP450 neofunctionalization combining with the horizontal gene transfer of N-prenyltransferases and kainoid synthases to establish DA production within the algal lineages.

Dual-Modal Cellular Nanoparticles for Continuous Neurotoxin Detoxification.

Neurotoxins are known for their extreme lethality. However, due to their enormous diversity, effective and broad-spectrum countermeasures are lacking. This study presents a dual-modal cellular nanoparticle (CNP) formulation engineered for continuous neurotoxin neutralization. The formulation involves encapsulating the metabolic enzyme N-sulfotransferase (SxtN) into metal-organic framework (MOF) nanoparticle cores and coating them with a natural neuronal membrane, termed "Neuron-MOF/SxtN-NPs". The resulting nanoparticles combine membrane-enabled broad-spectrum neurotoxin neutralization with enzyme payload-enabled continuous neurotoxin neutralization. The studies confirm the protection of the enzyme payload by the MOF core and validate the continuous neutralization of saxitoxin (STX). In vivo studies conducted using a mouse model of STX intoxication reveal markedly improved survival rates compared with control groups. Furthermore, acute toxicity assessments show no adverse effects associated with the administration of Neuron-MOF/SxtN-NPs in healthy mice. Overall, Neuron-MOF/SxtN-NPs represent a unique biomimetic nanomedicine platform poised to effectively neutralize neurotoxins, marking an important advancement in the field of countermeasure nanomedicine.

Mammalian neurotoxins, Blarina paralytic peptides, cause hyperpolarization of human T-type Ca channel hCav3.2 activation.

Among the rare venomous mammals, the short-tailed shrew Blarina brevicauda has been suggested to produce potent neurotoxins in its saliva to effectively capture prey. Several kallikrein-like lethal proteases have been identified, but the active substances of B. brevicauda remained unclear. Here, we report Blarina paralytic peptides (BPPs) 1 and 2 isolated from its submaxillary glands. Synthetic BPP2 showed mealworm paralysis and a hyperpolarization shift (-11 mV) of a human T-type Ca2+ channel (hCav3.2) activation. The amino acid sequences of BPPs were similar to those of synenkephalins, which are precursors of brain opioid peptide hormones that are highly conserved among mammals. However, BPPs rather resembled centipede neurotoxic peptides SLPTXs in terms of disulfide bond connectivity and stereostructure. Our results suggested that the neurotoxin BPPs were the result of convergent evolution as homologs of nontoxic endogenous peptides that are widely conserved in mammals. This finding is of great interest from the viewpoint of the chemical evolution of vertebrate venoms.

New Engineered-Chimeric Botulinum Neurotoxin Mutant Acts as an Effective Bivalent Vaccine Against Botulinum Neurotoxin Serotype A and E.

Botulinum neurotoxins (BoNTs), including serotypes A and E, are potent biotoxins known to cause human poisoning. In addition to the critical protective antigen found in the full BoNT molecule, the receptor binding domain (Hc domain), BoNTs also harbour another essential protective antigen-the light chain-translocation domain (L-HN domain). Leveraging these pivotal protective antigens, we genetically engineered a series of inactivated chimeric molecules incorporating L-HN and Hc domains of BoNT/A and E. The structure of these chimeric molecules, mirror BoNT/A and E, but are devoid of enzyme activity. Experimental findings demonstrated that a lead candidate mEL-HN-mAHc harnessing the inactivated protease LCHN/E with the mutated gangliosides binding site Hc/A (mE-mA) elicited robust immune protection against BoNT/A and E simultaneously in a mouse model, requiring low immune dosages and minimal immunisations. Moreover, mE-mA exhibited high protective efficacy against BoNT/A and E in guinea pigs and New Zealand white rabbits, resulting in elevated neutralising antibody titres. Furthermore, mE-mA proved to be a more stable and safer vaccine compared to formaldehyde-inactivated toxoid. Our data underscore the genetically engineered mE-mA as a highly effective bivalent vaccine against BoNT/A and E, paving the way for the development of polyvalent vaccines against biotoxins.

The analgesic effects of botulinum neurotoxin by modulating pain-related receptors; A literature review.

Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, have been used for the treatment of various central and peripheral neurological conditions. Recent studies have suggested that BoNTs may also have a beneficial effect on pain conditions. It has been hypothesized that one of the mechanisms underlying BoNTs' analgesic effects is the inhibition of pain-related receptors' transmission to the neuronal cell membrane. BoNT application disrupts the integration of synaptic vesicles with the cellular membrane, which is responsible for transporting various receptors, including pain receptors such as TRP channels, calcium channels, sodium channels, purinergic receptors, neurokinin-1 receptors, and glutamate receptors. BoNT also modulates the opioidergic system and the GABAergic system, both of which are involved in the pain process. Understanding the cellular and molecular mechanisms underlying these effects can provide valuable insights for the development of novel therapeutic approaches for pain management. This review aims to summarize the experimental evidence of the analgesic functions of BoNTs and discuss the cellular and molecular mechanisms by which they can act on pain conditions by inhibiting the transmission of pain-related receptors.

Botulinum neurotoxin serotype A inhibited ocular angiogenesis through modulating glial activation via SOCS3.

BACKGROUND: Pathological angiogenesis causes significant vision loss in neovascular age-related macular degeneration and other retinopathies with neovascularization (NV). Neuronal/glial-vascular interactions influence the release of angiogenic and neurotrophic factors. We hypothesized that botulinum neurotoxin serotype A (BoNT/A) modulates pathological endothelial cell proliferation through glial cell activation and growth factor release. METHODS: A laser-induced choroidal NV (CNV) was employed to investigate the anti-angiogenic effects of BoNT/A. Fundus fluorescence angiography, immunohistochemistry, and real-time PCR were used to assess BoNT/A efficacy in inhibiting CNV and the molecular mechanisms underlying this inhibition. Neuronal and glial suppressor of cytokine signaling 3 (SOCS3) deficient mice were used to investigate the molecular mechanisms of BoNT/A in inhibiting CNV via SOCS3. FINDINGS: In laser-induced CNV mice with intravitreal BoNT/A treatment, CNV lesions decreased > 30%; vascular leakage and retinal glial activation were suppressed; and Socs3 mRNA expression was induced while vascular endothelial growth factor A (Vegfa) mRNA expression was suppressed. The protective effects of BoNT/A on CNV development were diminished in mice lacking neuronal/glial SOCS3. CONCLUSION: BoNT/A suppressed laser-induced CNV and glial cell activation, in part through SOCS3 induction in neuronal/glial cells. BoNT/A treatment led to a decrease of pro-angiogenic factors, including VEGFA, highlighting the potential of BoNT/A as a therapeutic intervention for pathological angiogenesis in retinopathies.

A consensus recombinant elapid long-chain α-neurotoxin and how protein folding matters for antibody recognition and neutralization of elapid venoms.

Antivenoms are essential in the treatment of the neurotoxicity caused by elapid snakebites. However, there are elapid neurotoxins, e.g., long-chain α-neurotoxins (also known as long-chain three-finger toxins) that are barely neutralized by commercial elapid antivenoms; so, recombinant elapid neurotoxins could be an alternative or complements for improving antibody production against the lethal long-chain α-neurotoxins from elapid venoms. This work communicates the expression of a recombinant long-chain α-neurotoxin, named HisrLcNTx or rLcNTx, which based on the most lethal long-chain α-neurotoxins reported, was constructed de novo. The gene of rLcNTx was synthesized and introduced into the expression vector pQE30, which contains a proteolytic cleavage region for exscinding the mature protein, and His residues in tandem for affinity purification. The cloned pQE30/rLcNTx was transfected into Escherichia coli Origami cells to express rLcNTx. After expression, it was found in inclusion bodies, and folded in multiple Cys-Cys structural isoforms. To observe the capability of those isoforms to generate antibodies against native long-chain α-neurotoxins, groups of rabbits were immunized with different cocktails of Cys-Cys rLcNTx isoforms. In vitro, and in vivo analyses revealed that rabbit antibodies raised against different rLcNTx Cys-Cys isoforms were able to recognize pure native long-chain α-neurotoxins and their elapid venoms, but they were unable to neutralize bungarotoxin, a classical long-chain α-neurotoxin, and other elapid venoms. The rLcNTx Cys-Cys isoform 2 was the immunogen that produced the best neutralizing antibodies in rabbits. Yet to neutralize the elapid venoms from the black mamba Dendroaspis polylepis, and the coral shield cobra Aspidelaps lubricus, it was required to use two types of antibodies, the ones produced using rLcNTx Cys-Cys isoform 2 and antibodies produced using short-chain α-neurotoxins. Expression of recombinant elapid neurotoxins as immunogens could be an alternative to improve elapid antivenoms; nevertheless, recombinant elapid neurotoxins must be well-folded to be used as immunogens for obtaining neutralizing antibodies.

Protein-Loaded Cellular Nanosponges for Dual-Biomimicry Neurotoxin Countermeasure.

Neurotoxins present a substantial threat to human health and security as they disrupt and damage the nervous system. Their potent and structurally diverse nature poses challenges in developing effective countermeasures. In this study, a unique nanoparticle design that combines dual-biomimicry mechanisms to enhance the detoxification efficacy of neurotoxins is introduced. Using saxitoxin (STX), one of the deadliest neurotoxins, and its natural binding protein saxiphilin (Sxph) as a model system, human neuronal membrane-coated and Sxph-loaded metal-organic framework (MOF) nanosponges (denoted "Neuron-MOF/Sxph-NS") are successfully developed. The resulting Neuron-MOF/Sxph-NS exhibit a biomimetic design that not only emulates host neurons for function-based detoxification through the neuronal membrane coating, but also mimics toxin-resistant organisms by encapsulating the Sxph protein within the nanoparticle core. The comprehensive in vitro assays, including cell osmotic swelling, calcium flux, and cytotoxicity assays, demonstrate the improved detoxification efficacy of Neuron-MOF/Sxph-NS. Furthermore, in mouse models of STX intoxication, the application of Neuron-MOF/Sxph-NS shows significant survival benefits in both therapeutic and prophylactic regimens, without any apparent acute toxicity. Overall, the development of Neuron-MOF/Sxph-NS represents an important advancement in neurotoxin detoxification, offering promising potential for treating injuries and diseases caused by neurotoxins and addressing the current limitations in neurotoxin countermeasures.

Validation of a clinical assay for botulinum neurotoxins through mass spectrometric detection.

Botulism is a paralytic disease due to the inhibition of acetylcholine exocytosis at the neuromuscular junction, which can be lethal if left untreated. Botulinum neurotoxins (BoNTs) are produced by some spore-forming Clostridium bacteria. The current confirmatory assay to test for BoNTs in clinical specimens is the gold-standard mouse bioassay. However, an Endopep-MS assay method has been developed to detect BoNTs in clinical samples using benchtop mass spectrometric detection. This work demonstrates the validation of the Endopep-MS method for clinical specimens with the intent of method distribution in public health laboratories. The Endopep-MS assay was validated by assessing the sensitivity, robustness, selectivity, specificity, and reproducibility. The limit of detection was found to be equivalent to or more sensitive than the mouse bioassay. Specificity studies determined no cross-reactivity between the different serotypes and no false positives from an exclusivity panel of culture supernatants of enteric disease organisms and non-toxigenic strains of Clostridium. Inter-serotype specificity testing with 19 BoNT subtypes was 100% concordant with the expected results, accurately determining the presence of the correct serotype and the absence of incorrect serotypes. Additionally, a panel of potential interfering substances was used to test selectivity. Finally, clinical studies included clinical specimen stability and reproducibility, which was found to be 99.9% from a multicenter evaluation study. The multicenter validation study also included a clinical validation study, which yielded a 99.4% correct determination rate. Use of the Endopep-MS method will improve the capacity and response time for laboratory confirmation of botulism in public health laboratories.

Outpatient Infant Botulism in the United States, 1976-2021.

OBJECTIVE: To characterize cases of outpatients with infant botulism (IB) in the United States (US) identified by the Infant Botulism Treatment and Prevention Program (IBTPP) at the California Department of Public Health (CDPH) from 1976 through 2021. STUDY DESIGN: Outpatient IB cases were defined as patients presenting with an illness consistent with the known paralyzing action of botulinum neurotoxin (BoNT) and with laboratory confirmation. Outpatient cases were distinguished from the majority of patients with IB by the atypical fact that they did not require hospitalization throughout the course of their illness. RESULTS: Of the 4372 cases of IB identified by the IBTPP over a 45-year period (1976-2021), 17 (0.4%) were outpatient cases. Most (11/17; 65%) cases occurred in California. The median age at disease onset was 20 weeks (range = 6 to 55 weeks). The most common symptom among cases was constipation (16/17; 94%). Most patients (16/17; 94%) had at least one cranial nerve palsy, manifested as decreased head control, ptosis, weak cry, or poor suck. CONCLUSIONS: Outpatient IB occurs nationwide, although clinical diagnosis may be difficult because the severity of symptoms do not necessitate hospitalization or more comprehensive clinical intervention. Identification of outpatient cases requires an astute clinician and a capable, willing diagnostic testing laboratory. It is likely that more outpatient cases of IB are occurring than are presently recognized in infants mildly affected by this disease. Healthcare providers should consider the possibility of IB when presented with a previously well infant with failure to thrive, poor feeding, constipation, mild hypotonia, or cranial nerve palsy.

Microcystin-LR sensitizes the Oncorhynchus mykiss intestinal epithelium and interacts with paralytic shellfish toxins to alter oxidative balance.

In the context of harmful algal blooms, fish can be exposed to the combined effects of more than one toxin. We studied the effects of consecutive exposure to Microcystin-LR (MCLR) in vivo and paralytic shellfish toxins (PST) ex vivo/in vitro (MCLR+PST) in the rainbow trout Oncorhynchus mykiss's middle intestine. We fed juvenile fish with MCLR incorporated in the feed every 12 h and euthanized them 48 h after the first feeding. Immediately, we removed the middle intestine to make ex vivo and in vitro preparations and exposed them to PST for one hour. We analyzed glutathione (GSH) and glutathione disulfide (GSSG) contents, glutathione S-transferase (GST), glutathione reductase (GR), catalase (CAT), and protein phosphatase 1 (PP1) activities in ex vivo intestinal strips; apical and basolateral ATP-biding cassette subfamily C (Abcc)-mediated transport in ex vivo everted and non- everted sacs; and reactive oxygen species (ROS) production in isolated enterocytes in vitro. MCLR+PST treatment decreased the GSH content, GSH/GSSG ratio, GST activity, and increased ROS production. GR activity remained unchanged, while CAT activity only increased in response to PST. MCLR inhibited PP1 activity and activated Abcc-mediated transport only at the basolateral side of the intestine. Our results show a combined effect of MCLR+PST on the oxidative balance in the O. mykiss middle intestine, which is not affected by the two toxins groups when applied individually. Basolateral Abcc transporters activation by MCLR treatment could lead to an increase in the absorption of toxicants (including MCLR) into the organism. Therefore, MCLR makes the O. mykiss middle intestine more sensitive to possibly co-occurring cyanotoxins like PST.

Botulinum toxin and conservative treatment strategies in people with cervical dystonia: an online survey.

Isolated cervical dystonia is a focal, idiopathic dystonia affecting the neck muscles. Treatment usually consists of botulinum neurotoxin (BoNT) injections into the dystonic muscles. Our aim is to investigate the use of BoNT treatment and conservative treatments by people living with cervical dystonia. An online survey in English was conducted between June and August 2022. Participants were eligible to participate if they were living with cervical dystonia, were over 18 years old and could read and understand English. The survey consisted of demographic questions, characteristics of dystonia, questions relating to BoNT use and the perceived utility of conservative treatments. The data were analysed descriptively, and open-ended questions were grouped into similar topics represented by direct quotes. We received 128 responses from people with cervical dystonia, with an average age of 59 years and 77% women. Most participants (52%) described their cervical dystonia as mild to moderate with an average pain score of 5/10. Eighty-two (64%) participants were having regular BoNT injections, with overall positive perceived effects. Common activities reported to improve the symptoms were the use of heat packs, massage, relaxation, physiotherapy and participation in general exercise. Common coping strategies reported were getting sufficient rest, having the support of friends and family, and remaining engaged in enjoyable hobbies. We found that most participants received regular BoNT injections and that heat packs, exercise, massage, physiotherapy and relaxation were mostly perceived as effective in reducing the symptoms of cervical dystonia.

Therapy with botulinum neurotoxin for Parkinson's disease.

Botulinum neurotoxin (BoNT) has been in use since the 1970's. Its effect is reached mainly by inhibiting the release of acetylcholine in the synaptic gap of motor neurons or at the motor end plate and the parasympathetic ganglia. In the case of Parkinson's disease, it is used to treat several motor and non-motor symptoms. Within recent years increasingly numerous possible fields of application of BoNT have been found for the treatment of Parkinson's disease, and for some specific symptoms it has in fact become the therapy of choice, while for others it is but one of the therapeutic options that come into consideration when others are not sufficiently effective. In the following, we intend to outline the indications, the possible side effects and also the approvals for therapies with botulinum toxin in the primary and secondary symptoms of Parkinson's disease.

Urinary eosinophil-derived neurotoxin is associated with reduced lung function in pediatric asthma.

INTRODUCTION: Eosinophil-derived neurotoxin (EDN) is a biomarker for eosinophilic activation. Urinary (u) EDN may allow non-invasive monitoring of asthma, but clinical recommendations are lacking. We assessed the potential of uEDN as a marker of disease activity in pediatric asthma. METHODS: We assessed urine samples of 371 children from the German ALLIANCE study cohort, from which we had: 169 preschool wheezers (<6 years), 80 asthmatics (≥6 years), and 122 healthy controls using the ImmunoCAP™ EDN Assay. Creatinine (Cr)-adjusted uEDN values were analyzed using correlations, association tests, (non) parametric statistics, multiple linear, and multivariable regression. RESULTS: uEDN/uCr values were higher in atopic versus non-atopic preschool-aged subjects (p = .035) and associated with the sum of allergen-specific IgE in younger (r = 0.24, p = .003), and older subjects (r = 0.23, p = .043). uEDN/uCr was marginally a good determinant for atopy (p = .078, for subjects aged <6 years, and p = .058 for subjects ≥6 years). Children with the T2-high phenotype had higher uEDN/uCr (p < .001) versus T2-low-irrespective of using uEDN/uCr or blood eosinophils in combination to allergen sIgE for disease phenotyping. uEDN/uCr significantly correlated with reduced lung function among asthmatics (FEV1 z-scores: r = -0.30, p = .007, and FEV1/FVC z-scores: r = -0.24, p = .038). Using multivariable modeling, uEDN/uCr was an independent determinant of FEV1 (p = .038), and to a lesser extent, FEV1/FVC (p = .080). CONCLUSIONS: uEDN/uCr may serve as a non-invasive biomarker for clinical features such as lung function in pediatric asthma. We highlight the utility of uEDN/uCr as a biomarker that can be easily assessed using widely available robust diagnostic immunoassays.