Prevalence of Severe Atopic Dermatitis and Comorbid Chronic Systemic Diseases Is Increasing in Korean Children and Adolescents.

Severe atopic dermatitis (AD) is not a localized cutaneous disease, but a systemic disease that often accompanies comorbidities. In this nationwide population-based study, we aimed to analyze the prevalence of severe AD and chronic systemic diseases in Koreans aged ≤ 20 years between 2011 and 2019 using the data from the Korean Health Insurance Review and Assessment Service. Total AD and severe AD were defined according to the International Classification of Diseases-10 code L20. In children aged 6-20 years, the prevalence of severe AD significantly increased from 0.02% in 2011 to 0.04% in 2019 (P for trend < 0.001), with the ratio of severe AD to total AD increasing from 0.76% in 2011 to 1.10% in 2019 (P for trend < 0.001). The prevalence rates of severe AD significantly increased between 2011 and 2019 in children aged 6-12 years (P for trend < 0.05) and 13-18 years (P for trend < 0.001). Severe AD was more frequently found in males than in females each year (all P < 0.001, from 2011 to 2019). During the period from 2011 to 2019, the prevalence rate of chronic systemic diseases was higher in subjects with severe AD than in those without AD (P < 0.001) or with mild-to-moderate AD (P < 0.001). In conclusion, our results suggest that the prevalence of severe AD is increasing in Korean children and adolescents and is higher in males and older age groups. Moreover, severe AD is associated with chronic systemic diseases. Therefore, more attention should be paid to managing severe AD.

Single missense mutations in Vi capsule synthesis genes confer hypervirulence to Salmonella Typhi.

Many bacterial pathogens, including the human exclusive pathogen Salmonella Typhi, express capsular polysaccharides as a crucial virulence factor. Here, through S. Typhi whole genome sequence analyses and functional studies, we found a list of single point mutations that make S. Typhi hypervirulent. We discovered a single point mutation in the Vi biosynthesis enzymes that control Vi polymerization or acetylation is enough to result in different capsule variants of S. Typhi. All variant strains are pathogenic, but the hyper Vi capsule variants are particularly hypervirulent, as demonstrated by the high morbidity and mortality rates observed in infected mice. The hypo Vi capsule variants have primarily been identified in Africa, whereas the hyper Vi capsule variants are distributed worldwide. Collectively, these studies increase awareness about the existence of different capsule variants of S. Typhi, establish a solid foundation for numerous future studies on S. Typhi capsule variants, and offer valuable insights into strategies to combat capsulated bacteria.

Skin biomarkers predict the development of food allergy in early life.

BACKGROUND: Food allergy (FA) often occurs in early childhood with and without atopic dermatitis (AD). FA can be severe and even fatal. For primary prevention, it is important to find early biomarkers to predict the future onset of FA before any clinical manifestations. OBJECTIVE: Our aim was to find early predictors of future onset of FA in the stratum corneum (SC). METHODS: Skin tape strips were collected from the forearm of newborns (n = 129) at age 2 months, before any signs of clinical FA or AD. Children were clinically monitored until they reached age 2 years to confirm the presence or absence of FA and AD. Skin tape strips were subjected to lipidomic analyses by liquid chromatography-tandem mass spectrometry and cytokine determination by Meso Scale Discovery U-Plex assay. RESULTS: Overall, 9 of 129 infants (7.0%) developed FA alone and 9 of 129 infants (7.0%) developed FA concomitantly with AD. In the stratum corneum of children with future FA and concomitant AD and FA, absolute amounts of unsaturated (N24:1)(C18-sphingosine)ceramide and (N26:1)(C18-sphingosine)ceramide and their relative percentages within the molecular group were increased compared with the amounts and percentages in healthy children, with P values ranging from less than .01 to less than .05 according to ANOVA. The children with future AD had normal levels of these molecules. IL-33 level was upregulated in those infants with future FA but not in those with future AD, whereas thymic stromal lymphopoietin was upregulated in those with future AD but not in those with future FA. Logistic regression analysis revealed strong FA predicting power for the combination of dysregulated lipids and cytokines, with an odds ratio reaching 101.4 (95% CI = 5.4-1910.6). CONCLUSION: Noninvasive skin tape strip analysis at age 2 months can identify infants at risk of FA in the future.

Initial updosing phase of oral immunotherapy improves quality of life and psychological burden in parents of children with food allergy.

Background: Oral immunotherapy (OIT) can impose psychological burdens on patients and their parents due to the necessary preparations and repeated adverse reactions. Objective: To investigate changes in quality of life (QoL) and psychological burden in parents of children receiving OIT for food allergy (FA). Methods: Children aged 3-13 years with FA were enrolled. Parents were asked to fill out the Korean versions of the Food Allergy Quality of Life-Parental Burden (FAQL-PB), the Korean versions of the Food Allergy Quality of Life-Parental Form (K-FAQLQ-PF), the Korean versions of the Beck Anxiety Inventory (K-BAI), and the Korean version of the Patient Health Questionnaire-9 (PHQ-9) for depression before OIT (T1), after 2 months of updosing (T2), and after the end of the updosing phase (T3). Results: A total of 111 parents were enrolled. The total FAQL-PB scores were decreased at T2 and T3 compared with those at T1 (all p < 0.001). Greater improvement in the total FAQL-PB score at T2 was noted in parents with a higher parental burden (FAQL-PB score ≥ 74 points) at baseline than in those with a lower parental burden (p = 0.001). Among the K-FAQLQ-PF domains, "food anxiety" scores were decreased at T2 and T3 compared with those at T1 (p = 0.049 and p = 0.030, respectively), whereas there was no change in "social and dietary limitation" and "emotional impact" scores between T1 and T2 and between T1 and T3. However, no differences were observed in K-BAI and PHQ-9 scores between T1 and T2 and between T1 and T3. Conclusion: Our results suggest that OIT improves parental burden and QoL in parents of children with FA.

Single missense mutations in Vi capsule synthesis genes confer hypervirulence to Salmonella Typhi.

Many bacterial pathogens, including the human exclusive pathogen Salmonella Typhi, express capsular polysaccharides as a crucial virulence factor. Here, through S. Typhi whole genome sequence analyses and functional studies, we found a list of single point mutations that make S . Typhi hypervirulent. We discovered a single point mutation in the Vi biosynthesis enzymes that control the length or acetylation of Vi is enough to create different capsule variants of S. Typhi. All variant strains are pathogenic, but the hyper-capsule variants are particularly hypervirulent, as demonstrated by the high morbidity and mortality rates observed in infected mice. The hypo-capsule variants have primarily been identified in Africa, whereas the hyper-capsule variants are distributed worldwide. Collectively, these studies increase awareness about the existence of different capsule variants of S. Typhi, establish a solid foundation for numerous future studies on S. Typhi capsule variants, and offer valuable insights into strategies to combat capsulated bacteria.

Antimicrobial properties of tomato juice and peptides against typhoidal Salmonella.

Tomatoes are readily available and affordable vegetables that offer a range of health benefits due to their bioactive molecules, such as antioxidants and antimicrobials. In contrast to the widely recognized antioxidant properties of tomatoes, their antimicrobial properties remain largely unexplored. Here, we present our findings on the antimicrobial properties of tomato juice and peptides, namely, tomato-derived antimicrobial peptides (tdAMPs), in relation to their effectiveness against typhoidal Salmonella. Our research has revealed that tomato juice demonstrates significant antimicrobial properties against Salmonella Typhi, a pathogen that specifically affects humans and is responsible for causing typhoid fever. By employing computational analysis of the tomato genome sequence, conducting molecular dynamics simulation, and performing functional analyses, we have successfully identified two tdAMPs, namely, tdAMP-1 and tdAMP-2. These tdAMPs have demonstrated potent antimicrobial properties by effectively disrupting bacterial membranes. The efficacy of tdAMP-2 is shown to be more effective than tdAMP-1. The efficacy of tdAMP-1 and tdAMP-2 has been demonstrated against drug-resistant S. Typhi, as well as hyper-capsular S. Typhi variants that possess hypervirulent characteristics, which are presently circulating in countries with endemicity. Tomato juice, along with the two tdAMPs, has demonstrated effectiveness against uropathogenic Escherichia coli as well. This underscores their potential as viable agents in combating certain Gram-negative pathogens. This study provides valuable insights into the development of effective and sustainable public health strategies that utilize tomato and its derivatives as lifestyle interventions.IMPORTANCEIn this study, we investigate the antimicrobial properties of tomato juice, the most widely consumed affordable vegetables, as well as tomato-derived antimicrobial peptides, in relation to their effectiveness against foodborne pathogens with an emphasis on Salmonella Typhi, a deadly human-specific pathogen.

Bacterial AB toxins and host-microbe interactions.

AB toxins are protein virulence factors secreted by many bacterial pathogens, contributing to the pathogenicity of the cognate bacteria. AB toxins consist of two functionally distinct components: the enzymatic "A" component for pathogenicity and the receptor-binding "B" component for toxin delivery. Consistently, unlike other virulence factors such as effectors, AB toxins do not require additional systems to deliver them to the target host cells. Target host cells are located in the infection site and/or located distantly from infected host cells. The first part of this review discusses the structural and functional features of single-peptide and multiprotein AB toxins in the context of host-microbe interactions, using several well-characterized examples. The second part of this review discusses toxin neutralization strategies, as well as applications of AB toxins relevant to developing intervention strategies against diseases.

Malnutrition and maternal vaccination against typhoid toxin.

Children are particularly susceptible to typhoid fever caused by the bacterial pathogen Salmonella Typhi. Typhoid fever is prevalent in developing countries where diets can be less well-balanced. Here, using a murine model, we investigated the role of the macronutrient composition of the diet in maternal vaccination efficacies of two subunit vaccines targeting typhoid toxin: ToxoidVac and PltBVac. We found that maternal vaccinations protected all offspring against a lethal-dose typhoid toxin challenge in a balanced, normal diet (ND) condition, but the declined protection in a malnourished diet (MD) condition was observed in the PltBVac group. Despite the comparable antibody titers in both MD and ND mothers, MD offspring had a significantly lower level of typhoid toxin neutralizing antibodies than their ND counterparts. We observed a lower expression of the neonatal Fc receptor on the yolk sac of MD mothers than in ND mothers, agreeing with the observed lower antibody titers in MD offspring. Protein supplementation to MD diets, but not fat supplementation, increased FcRn expression and protected all MD offspring from the toxin challenge. Similarly, providing additional typhoid toxin-neutralizing antibodies to MD offspring was sufficient to protect all MD offspring from the toxin challenge. These results emphasize the significance of balanced/normal diets for a more effective maternal vaccination transfer to their offspring.

Delivery, structure, and function of bacterial genotoxins.

Bacterial genotoxins are peptide or protein virulence factors produced by several pathogens, which make single-strand breaks (SSBs) and/or double-strand DNA breaks (DSBs) in the target host cells. If host DNA inflictions are not resolved on time, host cell apoptosis, cell senescence, and/or even bacterial pathogen-related cancer may occur. Two multi-protein AB toxins, cytolethal distending toxin (CDT) produced by over 30 bacterial pathogens and typhoid toxin from Salmonella Typhi, as well as small polyketide-peptides named colibactin that causes the DNA interstrand cross-linking and subsequent DSBs is the most well-characterized bacterial genotoxins. Using these three examples, this review discusses the mechanisms by which these toxins deliver themselves into the nucleus of the target host cells and exert their genotoxic functions at the structural and functional levels.

Glycan-mediated molecular interactions in bacterial pathogenesis.

Glycans are expressed on the surface of nearly all host and bacterial cells. Not surprisingly, glycan-mediated molecular interactions play a vital role in bacterial pathogenesis and host responses against pathogens. Glycan-mediated host-pathogen interactions can benefit the pathogen, host, or both. Here, we discuss (i) bacterial glycans that play a critical role in bacterial colonization and/or immune evasion, (ii) host glycans that are utilized by bacteria for pathogenesis, and (iii) bacterial and host glycans involved in immune responses against pathogens. We further discuss (iv) opportunities and challenges for transforming these research findings into more effective antibacterial strategies, and (v) technological advances in glycoscience that have helped to accelerate progress in research. These studies collectively offer valuable insights into new perspectives on antibacterial strategies that may effectively tackle the drug-resistant pathogens that are rapidly spreading globally.

Neutralization of Typhoid Toxin by Alpaca-Derived, Single-Domain Antibodies Targeting the PltB and CdtB Subunits.

Typhoid toxin is secreted by the typhoid fever-causing bacterial pathogen Salmonella enterica serovar Typhi and has tropism for immune cells and brain endothelial cells. Here, we generated a camelid single-domain antibody (VHH) library from typhoid toxoid-immunized alpacas and identified 41 VHHs selected on the glycan receptor-binding PltB and nuclease CdtB. VHHs exhibiting potent in vitro neutralizing activities from each sequence-based family were epitope binned via competition enzyme-linked immunosorbent assays (ELISAs), leading to 6 distinct VHHs, 2 anti-PltBs (T2E7 and T2G9), and 4 anti-CdtB VHHs (T4C4, T4C12, T4E5, and T4E8), whose in vivo neutralizing activities and associated toxin-neutralizing mechanisms were investigated. We found that T2E7, T2G9, and T4E5 effectively neutralized typhoid toxin in vivo, as demonstrated by 100% survival of mice administered a lethal dose of typhoid toxin and with little to no typhoid toxin-mediated upper motor function defect. Cumulatively, these results highlight the potential of the compact antibodies to neutralize typhoid toxin by targeting the glycan-binding and/or nuclease subunits.

Usefulness of Routine Coagulation Tests in Healthy Children Undergoing Elective Minor Surgery: a 12-Year Retrospective Study.

BACKGROUND: Although routine coagulation tests, such as prothrombin time (PT) and activated partial thromboplastin time (aPTT) are performed before surgery to identify the risk of perioperative bleeding, bleeding complications are rare in minor surgeries, and false-positive results are often observed. Therefore, this study aimed to analyze the common causes of abnormal results of preoperative coagulation tests in previously healthy children undergoing elective minor surgery and determine the usefulness of performing these tests. Additionally, it aimed to identify the distribution of factor XII activity in children with prolonged aPTT. METHODS: The medical records of 363 pediatric patients aged 0 - 18 years, who were referred to the pediatric hematology-oncology department due to abnormal preoperative coagulation tests prior to undergoing minor surgery at the Kyung Hee University Medical Center between March 2008 and October 2020, were retrospectively review-ed. RESULTS: The majority of patients (n = 348, 96%) had prolonged aPTT, few (n = 29, 8%) had a prolonged PT international normalized ratio, and a small number (n = 14, 4%) had both prolonged PT and aPTT. On repeating the coagulation tests, 194 children showed persistent abnormal results. Of these, 184 patients underwent mixing tests, and 176 showed correction for factor deficiency (n = 26) and lupus anticoagulant positive (n = 14). Factor deficiencies included factor XII (n = 16), possibility of von Willebrand disease (vWD; n = 4), factor XI (n = 2), factor VIII (n = 1), factors IX and XII (n = 1), factor VII (n = 1), and factor V (n = 1). The severity of factor deficiency was mild (25 - 38%). One patient with factor VII deficiency received preoperative clotting factors but had postoperative bleeding requiring clotting factor replacement. Another patient with possible vWD received fresh frozen plasma after surgery and had mild symptoms. Linear regression showed no significant correlation between factor XII activity and aPTT in patients with prolonged aPTT (R2 = 0.0002, p = 0.84) or factor XII activity according to aPTT results in those with factor XII deficiency (R2 = 0.04749, p = 0.40). CONCLUSIONS: These results suggest that coagulation tests may be selectively performed in previously healthy children undergoing minor surgery with positive bleeding and/or family history. The distribution of factor XII should be investigated further.

Direct IgG epitope mapping on bacterial AB toxins by cryo-EM.

This cryo-EM protocol was used to determine the B cell epitope map on the CdtB subunit of typhoid toxin, an A2B5 toxin secreted by Salmonella Typhi during infection. Immunoglobulin G (IgG) was directly mixed with typhoid toxin in this protocol, different from our previous cryo-EM protocol that uses the Fab fragments in place of IgG. This simple approach requires smaller amounts of materials, supporting the broader use of this protocol for determining antibody recognition sites on various antigens. For complete details on the use and execution of this protocol, please refer to Ahn et al. (2021) and Nguyen et al. (2021).

Complete Genome Sequence of Salmonella enterica Serovar Typhi Strain ISP2825.

Salmonella enterica serovar Typhi ISP2825, isolated in 1983 from a Chilean patient, is one of the major S. Typhi strains used for research, along with strains Ty2, CT18, and H58. The complete genome sequence of ISP2825, consisting of a 4,774,014-bp circular chromosome, will help us understand typhoid pathogenesis and evolution.

The molecular basis of extensively drug-resistant Salmonella Typhi isolates from pediatric septicemia patients.

Sepsis is a syndromic response to infections and is becoming an emerging threat to the public health sector, particularly in developing countries. Salmonella Typhi (S. Typhi), the cause of typhoid fever, is one primary cause of pediatric sepsis in typhoid endemic areas. Extensively drug-resistant (XDR) S. Typhi is more common among pediatric patients, which is responsible for over 90% of the reported XDR typhoid cases, but the majority of antibiotic resistance studies available have been carried out using S. Typhi isolates from adult patients. Here, we characterized antibiotic-resistance profiles of XDR S. Typhi isolates from a medium size cohort of pediatric typhoid patients (n = 45, 68.89% male and 31.11% female) and determined antibiotic-resistance-related gene signatures associated with common treatment options to typhoid fever patients of 18 XDR S. Typhi representing all 45 isolates. Their ages were 1-13 years old: toddlers aging 1-2 years old (n = 9, 20%), pre-schoolers aging 3-5 years old (n = 17, 37.78%), school-age children aging 6-12 years old (n = 17, 37.78%), and adolescents aging 13-18 years old (n = 2, 4.44%). Through analyzing blaTEM1, dhfR7, sul1, and catA1genes for multidrug-resistance, qnrS, gyrA, gyrB, parC, and parE for fluoroquinolone-resistance, blaCTX-M-15 for XDR, and macAB and acrAB efflux pump system-associated genes, we showed the phenotype of the XDR S. Typhi isolates matches with their genotypes featured by the acquisitions of the genes blaTEM1, dhfR7, sul1, catA1, qnrS, and blaCTX-M-15 and a point mutation on gyrA. This study informs the molecular basis of antibiotic-resistance among recent S. Typhi isolates from pediatric septicemia patients, therefore providing insights into the development of molecular detection methods and treatment strategies for XDR S. Typhi.

The structural basis of Salmonella A2B5 toxin neutralization by antibodies targeting the glycan-receptor binding subunits.

Many bacterial pathogens secrete A(2)B5 toxins comprising two functionally distinct yet complementary "A" and "B" subunits to benefit the pathogens during infection. The lectin-like pentameric B subunits recognize specific sets of host glycans to deliver the toxin into target host cells. Here, we offer the molecular mechanism by which neutralizing antibodies, which have the potential to bind to all glycan-receptor binding sites and thus completely inhibit toxin binding to host cells, are inhibited from exerting this action. Cryogenic electron microscopy (cryo-EM)-based analyses indicate that the skewed positioning of the toxin A subunit(s) toward one side of the toxin B pentamer inhibited neutralizing antibody binding to the laterally located epitopes, rendering some glycan-receptor binding sites that remained available for the toxin binding and endocytosis process, which is strikingly different from the counterpart antibodies recognizing the far side-located epitopes. These results highlight additional features of the toxin-antibody interactions and offer important insights into anti-toxin strategies.

Mechanisms of typhoid toxin neutralization by antibodies targeting glycan receptor binding and nuclease subunits.

Nearly all clinical isolates of Salmonella Typhi, the cause of typhoid fever, are antibiotic resistant. All S. Typhi isolates secrete an A2B5 exotoxin called typhoid toxin to benefit the pathogen during infection. Here, we demonstrate that antibiotic-resistant S. Typhi secretes typhoid toxin continuously during infection regardless of antibiotic treatment. We characterize typhoid toxin antibodies targeting glycan-receptor-binding PltB or nuclease CdtB, which neutralize typhoid toxin in vitro and in vivo, as demonstrated by using typhoid toxin secreted by antibiotic-resistant S. Typhi during human cell infection and lethal dose typhoid toxin challenge to mice. TyTx11 generated in this study neutralizes typhoid toxin effectively, comparable to TyTx4 that binds to all PltB subunits available per holotoxin. Cryoelectron microscopy explains that the binding of TyTx11 to CdtB makes this subunit inactive through CdtB catalytic-site conformational change. The identified toxin-neutralizing epitopes are conserved across all S. Typhi clinical isolates, offering critical insights into typhoid toxin-neutralizing strategies.

Enteric Fever Diagnosis: Current Challenges and Future Directions.

Enteric fever is a life-threatening systemic febrile disease caused by Salmonella enterica serovars Typhi and Paratyphi (S. Typhi and S. Paratyphi). Unfortunately, the burden of the disease remains high primarily due to the global spread of various drug-resistant Salmonella strains despite continuous advancement in the field. An accurate diagnosis is critical for effective control of the disease. However, enteric fever diagnosis based on clinical presentations is challenging due to overlapping symptoms with other febrile illnesses that are also prevalent in endemic areas. Current laboratory tests display suboptimal sensitivity and specificity, and no diagnostic methods are available for identifying asymptomatic carriers. Several research programs have employed systemic approaches to identify more specific biomarkers for early detection and asymptomatic carrier detection. This review discusses the pros and cons of currently available diagnostic tests for enteric fever, the advancement of research toward improved diagnostic tests, and the challenges of discovering new ideal biomarkers and tests.

Salmonella Typhoid Toxin PltB Subunit and Its Non-typhoidal Salmonella Ortholog Confer Differential Host Adaptation and Virulence.

Typhoidal and non-typhoidal Salmonelleae (NTS) cause typhoid fever and gastroenteritis, respectively, in humans. Salmonella typhoid toxin contributes to typhoid disease progression and chronic infection, but little is known about the role of its NTS ortholog. We found that typhoid toxin and its NTS ortholog induce different clinical presentations. The PltB subunit of each toxin exhibits different glycan-binding preferences that correlate with glycan expression profiles of host cells targeted by each bacterium at the primary infection or intoxication sites. Through co-crystal structures of PltB subunits bound to specific glycan receptor moieties, we show that they induce markedly different glycan-binding preferences and virulence outcomes. Furthermore, immunization with the NTS S. Javiana or its toxin offers cross-reactive protection against lethal-dose typhoid toxin challenge. Cumulatively, these results offer insights into the evolution of host adaptations in Salmonella AB toxins, their cell and tissue tropisms, and the design for improved typhoid vaccines and therapeutics.

The role of 9-O-acetylated glycan receptor moieties in the typhoid toxin binding and intoxication.

Typhoid toxin is an A2B5 toxin secreted from Salmonella Typhi-infected cells during human infection and is suggested to contribute to typhoid disease progression and the establishment of chronic infection. To deliver the enzymatic 'A' subunits of the toxin to the site of action in host cells, the receptor-binding 'B' subunit PltB binds to the trisaccharide glycan receptor moieties terminated in N-acetylneuraminic acid (Neu5Ac) that is α2-3 or α2-6 linked to the underlying disaccharide, galactose (Gal) and N-acetylglucosamine (GlcNAc). Neu5Ac is present in both unmodified and modified forms, with 9-O-acetylated Neu5Ac being the most common modification in humans. Here we show that host cells associated with typhoid toxin-mediated clinical signs express both unmodified and 9-O-acetylated glycan receptor moieties. We found that PltB binds to 9-O-acetylated α2-3 glycan receptor moieties with a markedly increased affinity, while the binding affinity to 9-O-acetylated α2-6 glycans is only slightly higher, as compared to the affinities of PltB to the unmodified counterparts, respectively. We also present X-ray co-crystal structures of PltB bound to related glycan moieties, which supports the different effects of 9-O-acetylated α2-3 and α2-6 glycan receptor moieties on the toxin binding. Lastly, we demonstrate that the cells exclusively expressing unmodified glycan receptor moieties are less susceptible to typhoid toxin than the cells expressing 9-O-acetylated counterparts, although typhoid toxin intoxicates both cells. These results reveal a fine-tuning mechanism of a bacterial toxin that exploits specific chemical modifications of its glycan receptor moieties for virulence and provide useful insights into the development of therapeutics against typhoid fever.