Multiple-locus variable-number tandem-repeat analysis (MLVA) of macrolide-susceptible and -resistant Mycoplasma pneumoniae in children in Taiwan.

BACKGROUND/PURPOSE: To date, molecular typing studies on Mycoplasma pneumoniae are limited. We evaluated the molecular types of Mycoplasma pneumoniae in pediatric patients in Taiwan in 2016. METHODS: We used real-time quantitative PCR on respiratory specimens to identify M. pneumoniae in children with community-acquired pneumonia. The domain V of their 23S rRNA were sequenced for detection of macrolide-resistant point mutations. Molecular typing with multiple locus variable-number tandem repeat analysis (MLVA) was done for both macrolide-susceptible and -resistance M. pneumoniae samples. RESULTS: M. pneumoniae was detected in 22% (180/826) respiratory samples during the study period. Among all M. pneumoniae-positive samples, 24% (43/180) had harbored macrolide-resistant genotypes, and 86% (37/43) of them were A2063G mutation. Forty-two macrolide-resistant strains and 20 randomly selected macrolide-susceptible strains underwent MLVA profiling. MLVA 4-5-7-2 was the most frequent type (32/62, 52%), followed by 4-5-7-3 (17/62, 27%) and 1-5-6-2 (9/62, 15%). There was a strong association between MLVA 4-5-7-2 and macrolide resistance (p < 0.001). In contrast, M 4-5-7-3 and 1-5-6-2 were related to macrolide susceptibility (p < 0.001, and p = 0.025, respectively). CONCLUSION: Macrolide resistance was relatively low (24%) in this age group in 2016 in Taiwan, and A2063G was the dominant point mutation. MLVA 4-5-7-2 was associated with macrolide resistance.

Intestinal Dysbiosis Featuring Abundance of Ruminococcus gnavus Associates With Allergic Diseases in Infants.

BACKGROUND & AIMS: Dysbiosis of the intestinal microbiota has been associated with development of allergies in infants. However, it is not clear what microbes might contribute to this process. We investigated what microbe(s) might be involved in analyses of infant twins and mice. METHODS: We studied fecal specimens prospectively in a twin cohort (n = 30) and age-matched singletons (n = 14) born at National Taiwan University Children's Hospital, Taipei, Taiwan, from April 2011 to March 2013. Clinical parameters (gestational age, birth body weight, mode of delivery and feeding, immunizations, and medical events) were recorded. Fecal samples were collected beginning immediately after birth and for 1 year; the children were followed until 3 years of age and allergic symptoms (repetitive and continuous for at least 6 months) were noted. A skin prick test was used to ascertain atopy. Bacterial communities in fecal samples were profiled by 16S ribosomal RNA-based polymerase chain reaction-temporal temperature gradient gel electrophoresis and next-generation sequencing. BALB/c mice without and with ovalbumin sensitization/challenge were infected with candidate bacteria by oral gauge intragastric intubation. Fecal, serum, lung, and colon tissue samples were collected from mice and analyzed for mechanisms of allergy development. RESULTS: During the investigation period, 20 children (45.5%) developed allergic diseases, including respiratory (allergic rhinitis and asthma) and skin (atopic dermatitis and eczema) allergies. Lachnospiraceae were detected at significantly higher frequency in allergic infants than nonallergic infants (P < .004); the high fecal count of Lachnospiraceae in allergic subjects appeared at 2 months of age and persisted until 12 months of age. The enrichment of Lachnospiraceae in allergic infants was attributed to the overgrowth of Ruminococcus gnavus, which tended to have a low frequency in nonallergic subjects (P = .0004). Increased R gnavus was observed before the onset of allergic manifestations, and was associated with respiratory allergies (P < .002) or respiratory allergies coexistent with atopic eczema (P < .001). In mice, endogenous R gnavus grew rapidly after sensitization and challenge with ovalbumin. Mice gavaged with purified R gnavus developed airway hyper-responsiveness and had histologic evidence of airway inflammation (asthma). Expansion of R gnavus in mice stimulated secretion of cytokines (interleukin [IL] 25, IL33, and thymic stromal lymphopoietin) by colon tissues, which activated type 2 innate lymphoid cells and dendritic cells to promote differentiation of T-helper 2 cells and production of their cytokines (IL4, IL5, and IL13). This led to infiltration of the colon and lung parenchyma by eosinophils and mast cells. CONCLUSIONS: In a study of a twin cohort (some infants with, some without allergies), we associated development of allergies, particularly respiratory allergies, with increased fecal abundance of R gnavus. Mice fed R gnavus developed airway inflammation, characterized by expansion of T-helper 2 cells in the colon and lung, and infiltration of colon and lung parenchyma by eosinophils and mast cells.

An integrative genomic analysis identifies Bhmt2 as a diet-dependent genetic factor protecting against acetaminophen-induced liver toxicity.

Acetaminophen-induced liver toxicity is the most frequent precipitating cause of acute liver failure and liver transplant, but contemporary medical practice has mainly focused on patient management after a liver injury has been induced. An integrative genetic, transcriptional, and two-dimensional NMR-based metabolomic analysis performed using multiple inbred mouse strains, along with knowledge-based filtering of these data, identified betaine-homocysteine methyltransferase 2 (Bhmt2) as a diet-dependent genetic factor that affected susceptibility to acetaminophen-induced liver toxicity in mice. Through an effect on methionine and glutathione biosynthesis, Bhmt2 could utilize its substrate (S-methylmethionine [SMM]) to confer protection against acetaminophen-induced injury in vivo. Since SMM is only synthesized in plants, Bhmt2 exerts its beneficial effect in a diet-dependent manner. Identification of Bhmt2 and the affected biosynthetic pathway demonstrates how a novel method of integrative genomic analysis in mice can provide a unique and clinically applicable approach to a major public health problem.

One-pot making of sequence-restricted DNA dumbbells.

A method to build sequence-restricted DNA dumbbells was developed. 5'-exonuclease converts end sequences of DNA targets into sticky ends. Self-looping oligonucleotides with complementary 3'-overhangs are ligated to form dumbbells by DNA polymerase and ligase in a sequence-restricted manner. These reactions proceed in one pot at one temperature. We demonstrated one use of this method to 'tunnel' sequencing libraries into dumbbells for the Pacific Biosciences (PacBio) platform. Readouts of an Illumina P5/P7-ended 16S library from a standard microbial community confirmed successful tunneling. Twelve fecal samples additionally showed significant correlations between standard and tunneled 16S sequence variants on the PacBio platform. We further extended the method at a genomic scale to build a ∼0.45 Mbp giant dumbbell on chromosome 6. Sequences inside the dumbbells were successfully protected from a cocktail of exonucleases. Roughly 11-fold enrichment was achieved for the dumbbell-guarded region compared to the vicinity.

Cross-protective humoral immunity to coronaviruses from SARS coronavirus 2-naïve sera of children with Kawasaki disease.

OBJECTIVES: SARS coronavirus 2 (SARS-CoV-2)-associated multi-system inflammatory syndrome in children indicates that viruses can trigger a Kawasaki disease (KD)-like hyperinflammation. A plausible hypothesis was that coronavirus-specific 'holes' in humoral immunity could cause both diseases. METHODS: To determine whether SARS-CoV-2-naïve patients with KD have inferior humoral immunity for the novel coronavirus, sera of children with KD and control children from year 2015 to 2021 were subjected to ELISA, microwestern, and neutralization assays to evaluate the capabilities in recognizing the receptor-binding domain of SARS-CoV-2, spotting spike proteins of three respiratory syndromic coronaviruses, and blocking SARS-CoV-2 from binding to angiotensin-converting enzyme 2 receptors in vitro, respectively. RESULTS: 29 patients with KD before 2019, 74 patients with KD in 2019 or 2020, 54 non-febrile controls, and 24 febrile controls were included in the study. SARS-CoV-2 was recognized on ELISA for both patients with KD in 2016 and those with KD in 2020. Microwestern demonstrated cross-reactive IgG in an all-or-none manner towards three spike proteins of syndromic coronaviruses regardless of sample year or KD status. The ratio between the sera that recognized all spike proteins and those that recognized none (51 vs. 47) was significantly higher from patients with KD than from non-febrile controls (17 vs. 32; p 0.047) but not from febrile controls (13 vs. 11; p 0.85). Most positive sera (12 of 17 controls, 5 of 8 patients with KD before 2019, and 28 of 33 patients with KD in 2019 or 2020) offered protection comparable to low-titre sera from the WHO reference panel. DISCUSSION: Humoral immunity of SARS-CoV-2-naïve children with KD was not inferior to that of controls in offering cross-protection against the novel coronavirus.

Next-generation computational genetic analysis: multiple complement alleles control survival after Candida albicans infection.

Candida albicans is a fungal pathogen that causes severe disseminated infections that can be lethal in immunocompromised patients. Genetic factors are known to alter the initial susceptibility to and severity of C. albicans infection. We developed a next-generation computational genetic mapping program with advanced features to identify genetic factors affecting survival in a murine genetic model of hematogenous C. albicans infection. This computational tool was used to analyze the median survival data after inbred mouse strains were infected with C. albicans, which provides a useful experimental model for identification of host susceptibility factors. The computational analysis indicated that genetic variation within early classical complement pathway components (C1q, C1r, and C1s) could affect survival. Consistent with the computational results, serum C1 binding to this pathogen was strongly affected by C1rs alleles, as was survival of chromosome substitution strains. These results led to a combinatorial, conditional genetic model, involving an interaction between C5 and C1r/s alleles, which accurately predicted survival after infection. Beyond applicability to infectious disease, this information could increase our understanding of the genetic factors affecting susceptibility to autoimmune and neurodegenerative diseases.

A critical role of TAK1 in B-cell receptor-mediated nuclear factor kappaB activation.

The kinase TAK1 is essential for T-cell receptor (TCR)-mediated nuclear factor kappaB (NF-kappaB) activation and T-cell development. However, the role of TAK1 in B-cell receptor (BCR)-mediated NF-kappaB activation and B-cell development is not clear. Here we show that B-cell-specific deletion of TAK1 impaired the transition from transitional type 2 to mature follicular (FO) B cells and caused a marked decrease of marginal zone (MZ) B cells. TAK1-deficient B cells exhibited an increase of BCR-induced apoptosis and impaired proliferation in response to BCR ligation. Importantly, TAK1-deficient B cells failed to activate NF-kappaB after BCR stimulation. Thus, TAK1 is critical for B-cell maturation and BCR-induced NF-kappaB activation.

The role of interleukin-1 in wound biology. Part I: Murine in silico and in vitro experimental analysis.

BACKGROUND: Wound healing is a multistep, complex process that involves the coordinated action of multiple cell types. Conflicting results have been obtained when conventional methods have been used to study wound biology. Therefore, we analyzed the wound response in a mouse genetic model. METHODS: We analyzed inflammatory mediators produced within incisional wounds induced in 16 inbred mouse strains. Computational haplotype-based genetic analysis of inter-strain differences in the level of production of 2 chemokines in wounds was performed. An in vitro experimental analysis system was developed to investigate whether interleukin (IL)-1 could affect chemokine production by 2 different types of cells that are present within wounds. RESULTS: The level of 2 chemokines, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein 1α, exhibited very large (75- and 463-fold, respectively) interstrain differences within wound tissue across this inbred strain panel. Genetic variation within Nalp1, an inflammasome component that regulates IL-1 production, correlated with the interstrain differences in KC and macrophage inhibitory protein 1α production. Consistent with the genetic correlation, IL-1ß was shown to stimulate KC production by murine keratinocyte and fibroblast cell lines in vitro. CONCLUSIONS: Genetic variation within Nalp1 could contribute to interstrain differences in wound chemokine production by altering the amount of IL-1 produced.

The role of interleukin-1 in wound biology. Part II: In vivo and human translational studies.

BACKGROUND: In the accompanying paper, we demonstrate that genetic variation within Nalp1 could contribute to interstrain differences in wound chemokine production through altering the amount of interleukin (IL)-1 produced. We further investigate the role of IL-1 in incisional wound biology and its effect on wound chemokine production in vivo and whether this mechanism could be active in human subjects. METHODS: A well-characterized murine model of incisional wounding was used to assess the in vivo role of IL-1 in wound biology. The amount of 7 different cytokines/chemokines produced within an experimentally induced skin incision on a mouse paw and the nociceptive response was analyzed in mice treated with an IL-1 inhibitor. We also investigated whether human IL-1ß or IL-1α stimulated the production of chemokines by primary human keratinocytes in vitro, and whether there was a correlation between IL-1ß and chemokine levels in 2 experimental human wound paradigms. RESULTS: Administration of an IL-1 receptor antagonist to mice decreased the nociceptive response to an incisional wound, and reduced the production of multiple inflammatory mediators, including keratinocyte-derived chemokine (KC) and macrophage inhibitory protein (MIP)-1α, within the wounds. IL-1α and IL-1ß stimulated IL-8 and GRO-α (human homologues of murine keratinocyte-derived chemokine) production by primary human keratinocytes in vitro. IL-1ß levels were highly correlated with IL-8 in human surgical wounds, and at cutaneous sites of human ultraviolet B-induced sunburn injury. CONCLUSIONS: IL-1 plays a major role in regulating inflammatory mediator production in wounds through a novel mechanism; by stimulating the production of multiple cytokines and chemokines, it impacts clinically important aspects of wound biology. These data suggest that administration of an IL-1 receptor antagonist within the perioperative period could decrease postsurgical wound pain.

Integrated Counts of Carbohydrate-Active Protein Domains as Metabolic Readouts to Distinguish Probiotic Biology and Human Fecal Metagenomes.

Bowel microbiota is a "metaorgan" of metabolisms on which quantitative readouts must be performed before interventions can be introduced and evaluated. The study of the effects of probiotic Clostridium butyricum MIYAIRI 588 (CBM588) on intestine transplantees indicated an increased percentage of the "other glycan degradation" pathway in 16S-rRNA-inferred metagenomes. To verify the prediction, a scoring system of carbohydrate metabolisms derived from shotgun metagenomes was developed using hidden Markov models. A significant correlation (R = 0.9, p < 0.015) between both modalities was demonstrated. An independent validation revealed a strong complementarity (R = -0.97, p < 0.002) between the scores and the abundance of "glycogen degradation" in bacteria communities. On applying the system to bacteria genomes, CBM588 had only 1 match and ranked higher than the other 8 bacteria evaluated. The gram-stain properties were significantly correlated to the scores (p < 5 × 10-4). The distributions of the scored protein domains indicated that CBM588 had a considerably higher (p < 10-5) proportion of carbohydrate-binding modules than other bacteria, which suggested the superior ability of CBM588 to access carbohydrates as a metabolic driver to the bowel microbiome. These results demonstrated the use of integrated counts of protein domains as a feasible readout for metabolic potential within bacteria genomes and human metagenomes.

The BTB-kelch protein KLHL6 is involved in B-lymphocyte antigen receptor signaling and germinal center formation.

BTB-kelch proteins can elicit diverse biological functions but very little is known about the physiological role of these proteins in vivo. Kelch-like protein 6 (KLHL6) is a BTB-kelch protein with a lymphoid tissue-restricted expression pattern. In the B-lymphocyte lineage, KLHL6 is expressed throughout ontogeny, and KLHL6 expression is strongly upregulated in germinal center (GC) B cells. To analyze the role of KLHL6 in vivo, we have generated mouse mutants of KLHL6. Development of pro- and pre-B cells was normal but numbers of subsequent stages, transitional 1 and 2, and mature B cells were reduced in KLHL6-deficient mice. The antigen-dependent GC reaction was blunted (smaller GCs, reduced B-cell expansion, and reduced memory antibody response) in the absence of KLHL6. Comparison of mutants with global loss of KLHL6 to mutants lacking KLHL6 specifically in B cells demonstrated a B-cell-intrinsic requirement for KLHL6 expression. Finally, B-cell antigen receptor (BCR) cross-linking was less sensitive in KLHL6-deficient B cells compared to wild-type B cells as measured by proliferation, Ca2+ response, and activation of phospholipase Cgamma2. Our results strongly point to a role for KLHL6 in BCR signal transduction and formation of the full germinal center response.

Shared IgG Infection Signatures vs. Hemorrhage-Restricted IgA Clusters in Human Dengue: A Phenotype of Differential Class-Switch via TGFß1.

Phenotypic manifestations of infectious diseases are closely related to individual immune responses. Methods to extract information from patients' own immune reactions would be of great use for both diagnosis and treatment. Dengue fever is one of the diseases that clinical aggravations could occur paradoxically after humoral immunity appears. This property makes dengue fever an excellent disease model to explore. A principal component analyses (PCAs)-based framework derived from a prior vaccination study was developed. The framework was verified by successful demonstrations of known IgG signatures from a Mexico Dengue data set. Afterward the pipeline was tested upon de novo IgG and IgA libraries of Dengue patients from southern Taiwan. We discovered four infection signatures within IgG repertoires, two of which were identical to previous reports. However, it was IgA but not IgG that could differentiate hemorrhagic from non-hemorrhagic patients. IgA repertoires were found more diversified among bleeders, from whom seven signature clusters were characterized. The expressions of transforming growth factor beta 1 (TGFß1) and accordingly mediated class-switch activity of IgA were distinct only among the PCA-segregated bleeding group. In sum, intercontinental sharing of IgG signatures in dengue fever was demonstrated via a unified working flow. Differential regulation of IgA class-switch with associated diversity expansion plus existences of hemorrhage-restricted clusters were shown. The ability of the framework to find common IgG signatures would implicate applications to infections even from unknown pathogens. The clusters within IgA repertoires could offer perspectives to other IgA-related bleeding disorders such as Henoch-Schönlein purpura or IgA nephropathy. Substantiated grounds for IgA-specific effector function via TGFß1-mediated class-switch would be a new factor to consider for infectious diseases.

Network Signatures of IgG Immune Repertoires in Hepatitis B Associated Chronic Infection and Vaccination Responses.

The repertoire of IgG antibody responses to infection and vaccination varies depending on the characteristics of the immunogen and the ability of the host to mount a protective immune response. Chronic hepatitis B virus (HBV) infections are marked by persistent infection and immune tolerance to vaccination. This disease offers a unique opportunity to discover key repertoire signatures during infection and in response to vaccination. Complementarity determining region 3 of an antibody heavy chain (CDR-H3) has a major impact on the antigenic specificity of an antibody. We used next-generation sequencing to characterize the CDR-H3 sequences in paired siblings of 4 families in which only one member of each pair had chronic HBV infection. Blood samples were obtained before and 2 weeks after HBV vaccination. The analysis revealed a huge network of sequence-related CDR-H3 clones found almost exclusively among carriers. In contrast, vaccination induced significant increases of CDR-H3 cluster diversities among siblings without hepatitis B. Several vaccination-associated clone clusters were identified. Similar findings of vaccination-associated clone networks were observed in healthy adults receiving HBV boosters. These strategies can be used to identify signatures of other infectious diseases and accelerate discoveries of antibody sequences with important biomedical implications.

Postnatal outcome of fetal cardiac echogenic foci.

BACKGROUND AND PURPOSE: Cardiac echogenic foci are found frequently during fetal echocardiographic investigations and may be related to increased mineralization of the papillary muscles. However, data from postnatal follow-up are limited. This study investigated the clinical characteristics and postnatal echocardiographic findings in infants with cardiac echogenic foci identified prenatally. METHODS: Between March 1995 and April 1998, 43 fetuses were noted to have cardiac echogenic foci during the second trimester. Postnatal evaluation was completed for 20 of these 43 fetuses. No other congenital malformations were noted during the fetal stage or after birth. Postnatal echocardiography was performed from 17 months to 4 years and 7 months after birth. RESULTS: Seven (35%) infants had persistent cardiac echogenic foci. However, only one had mild mitral valve prolapse without mitral regurgitation. All fetuses had left ventricular (LV) foci and three also had right ventricular (RV) foci. One infant who had a LV focus prenatally was noted to have a RV focus on postnatal follow-up. Among the three infants with prenatal biventricular involvement, only one had biventricular involvement on postnatal follow-up. Other cardiac echogenic foci had disappeared in all infants. The probability of persistence of foci decreased with age and reached 50% at the age of 4 years and 4 months. Thereafter, cardiac echogenic foci tended to regress and only 11% of infants had persistence at the last follow-up. No significant difference was found in the rate of persistence between children with univentricular foci and those with biventricular foci. CONCLUSIONS: Although some fetal cardiac echogenic foci may persist after birth, fetal echogenic foci were not associated with significant intracardiac or extracardiac anomalies.

Cd14 SNPs regulate the innate immune response.

CD14 is a monocytic differentiation antigen that regulates innate immune responses to pathogens. Here, we show that murine Cd14 SNPs regulate the length of Cd14 mRNA and CD14 protein translation efficiency, and consequently the basal level of soluble CD14 (sCD14) and type I IFN production by murine macrophages. This has substantial downstream consequences for the innate immune response; the level of expression of at least 40 IFN-responsive murine genes was altered by this mechanism. We also observed that there was substantial variation in the length of human CD14 mRNAs and in their translation efficiency. sCD14 increased cytokine production by human dendritic cells (DCs), and sCD14-primed DCs augmented human CD4T cell proliferation. These findings may provide a mechanism for exploring the complex relationship between CD14 SNPs, serum sCD14 levels, and susceptibility to human infectious and allergic diseases.

Computational genetic mapping in mice: the ship has sailed.

Computational haplotype-based genetic mapping can be used to discover new biological mechanisms, disease-related pathways, and unexpected uses for existing drugs. Here we discuss the benefits and limitations of this methodology, its impact on translational medicine, and its future course.

Essential role of TAK1 in thymocyte development and activation.

The protein kinase TAK1 mediates the activation of NF-kappaB in response to stimulation by proinflammatory cytokines and microbial pathogens in the innate immunity pathways. However, the physiological function of TAK1 in the adaptive immunity pathways is unclear. By engineering mice lacking TAK1 in T cells, here, we show that TAK1 is essential for thymocyte development and activation in vivo. Deletion of TAK1 prevented the maturation of single-positive thymocytes displaying CD4 or CD8, leading to reduction of T cells in the peripheral tissues. Thymocytes lacking TAK1 failed to activate NF-kappaB and JNK and were prone to apoptosis upon stimulation. Our results provide the genetic evidence that TAK1 is required for the activation of NF-kappaB in thymocytes and suggest that TAK1 plays a central role in both innate and adaptive immunity.

The specific and essential role of MAVS in antiviral innate immune responses.

The mitochondrial antiviral signaling protein (MAVS) mediates the activation of NFkappaB and IRFs and the induction of interferons in response to viral infection. In vitro studies have also suggested that MAVS is required for interferon induction by cytosolic DNA, but the in vivo evidence is lacking. By generating MAVS-deficient mice, here we show that loss of MAVS abolished viral induction of interferons and prevented the activation of NFkappaB and IRF3 in multiple cell types, except plasmacytoid dendritic cells (pDCs). However, MAVS was not required for interferon induction by cytosolic DNA or by Listeria monocytogenes. Mice lacking MAVS were viable and fertile, but they failed to induce interferons in response to poly(I:C) stimulation and were severely compromised in immune defense against viral infection. These results provide the in vivo evidence that the cytosolic viral signaling pathway through MAVS is specifically required for innate immune responses against viral infection.