Analysis of Plasmablasts From Children With Kawasaki Disease Reveals Evidence of a Convergent Antibody Response to a Specific Protein Epitope.

BACKGROUND: Kawasaki disease (KD) is a febrile illness of young childhood that can result in coronary artery aneurysms and death. Coronavirus disease 2019 (COVID-19) mitigation strategies resulted in a marked decrease in KD cases worldwide, supporting a transmissible respiratory agent as the cause. We previously reported a peptide epitope recognized by monoclonal antibodies (MAbs) derived from clonally expanded peripheral blood plasmablasts from 3 of 11 KD children, suggesting a common disease trigger in a subset of patients with KD. METHODS: We performed amino acid substitution scans to develop modified peptides with improved recognition by KD MAbs. We prepared additional MAbs from KD peripheral blood plasmablasts and assessed MAb characteristics that were associated with binding to the modified peptides. RESULTS: We report a modified peptide epitope that is recognized by 20 MAbs from 11 of 12 KD patients. These MAbs predominantly use heavy chain VH3-74; two-thirds of VH3-74 plasmablasts from these patients recognize the epitope. The MAbs were nonidentical between patients but share a common complementarity-determining region 3 (CDR3) motif. CONCLUSIONS: These results demonstrate a convergent VH3-74 plasmablast response to a specific protein antigen in children with KD, supporting one predominant causative agent in the etiopathogenesis of the illness.

Allograft Inflammatory Factor-1 Links T-Cell Activation, Interferon Response, and Macrophage Activation in Chronic Kawasaki Disease Arteritis.

BACKGROUND: Kawasaki disease (KD) is widely viewed as an acute arteritis. However, our pathologic studies show that chronic coronary arteritis can persist long after disease onset and is closely linked with arterial stenosis. Transcriptome profiling of acute KD arteritis tissues revealed upregulation of T lymphocyte, type I interferon, and allograft inflammatory factor-1 (AIF1) genes. We determined whether these immune responses persist in chronic KD arteritis, and we investigated the role of AIF1 in these responses. METHODS: Gene expression in chronic KD and childhood control arteries was determined by real-time reverse-transcriptase polymerase chain reaction, and arterial protein expression was determined by immunohistochemistry and immunofluorescence. Allograft inflammatory factor-1 small-interfering ribonucleic acid macrophage treatment was performed to investigate the role of AIF1 in macrophage and T lymphocyte activation. RESULTS: Allograft inflammatory factor-1 protein was highly expressed in stenotic KD arteries and colocalized with the macrophage marker CD68. T lymphocyte and interferon pathway genes were significantly upregulated in chronic KD coronary artery tissues. Alpha interferon-induced macrophage expression of CD80 and major histocompatibility complex class II was dependent on AIF1, and macrophage expression of AIF1 was required for antigen-specific T lymphocyte activation. CONCLUSIONS: Allograft inflammatory factor-1, originally identified in posttransplant arterial stenosis, is markedly upregulated in KD stenotic arterial tissues. T lymphocyte and type I interferon responses persist in chronic KD arteritis. Allograft inflammatory factor-1 may play multiple roles linking type I interferon response, macrophage activation, and antigen-specific T lymphocyte activation. These results suggest the likely importance of lymphocyte-myeloid cell cross-talk in the pathogenesis of KD arteritis and can inform selection of new immunotherapies for clinical trials in high-risk KD children.

The transcriptional profile of coronary arteritis in Kawasaki disease.

BACKGROUND: Kawasaki Disease (KD) can cause potentially life-threatening coronary arteritis in young children, and has a likely infectious etiology. Transcriptome profiling is a powerful approach to investigate gene expression in diseased tissues. RNA sequencing of KD coronary arteries could elucidate the etiology and the host response, with the potential to improve KD diagnosis and/or treatment. METHODS: Deep RNA sequencing was performed on KD (n = 8) and childhood control (n = 7) coronary artery tissues, revealing 1074 differentially expressed mRNAs. Non-human RNA sequences were subjected to a microbial discovery bioinformatics platform, and microbial sequences were analyzed by Metastats for association with KD. RESULTS: T lymphocyte activation, antigen presentation, immunoglobulin production, and type I interferon response were significantly upregulated in KD arteritis, while the tumor necrosis factor α pathway was not differentially expressed. Transcripts from known infectious agents were not specifically associated with KD coronary arteritis. CONCLUSIONS: The immune transcriptional profile in KD coronary artery tissues has features of an antiviral immune response such as activated cytotoxic T lymphocyte and type I interferon-induced gene upregulation. These results provide new insights into the pathogenesis of KD arteritis that can guide selection of new immunomodulatory therapies for high-risk KD patients, and provide direction for future etiologic studies.

Ultrastructural, immunofluorescence, and RNA evidence support the hypothesis of a "new" virus associated with Kawasaki disease.

BACKGROUND: Intracytoplasmic inclusion bodies (ICI) have been identified in ciliated bronchial epithelium of Kawasaki disease (KD) patients using a synthetic antibody derived from acute KD arterial IgA plasma cells; ICI may derive from the KD etiologic agent. METHODS: Acute KD bronchial epithelium was subjected to immunofluorescence for ICI and cytokeratin, high-throughput sequencing, and transmission electron microscopy (TEM). Interferon pathway gene expression profiling was performed on KD lung. RESULTS: An intermediate filament cytokeratin "cage" was not observed around KD ICI, making it unlikely that ICI are overproduced or misfolded human protein aggregates. Many interferon-stimulated genes were detected in the bronchial epithelium, and significant modulation of the interferon response pathway was observed in the lung tissue of KD patients. No known virus was identified by sequencing. Aggregates of virus-like particles (VLP) were detected by TEM in all 3 acute KD patients from whom nonembedded formalin-fixed lung tissue was available. CONCLUSIONS: KD ICI are most likely virus induced; bronchial cells with ICI contain VLP that share morphologic features among several different RNA viral families. Expedited autopsies and tissue fixation from acute KD fatalities are urgently needed to more clearly ascertain the VLP. These findings are compatible with the hypothesis that the infectious etiologic agent of KD may be a "new" RNA virus.

Transforming growth factor-beta signaling pathway in patients with Kawasaki disease.

BACKGROUND: Transforming growth factor (TGF)-ß is a multifunctional peptide that is important in T-cell activation and cardiovascular remodeling, both of which are important features of Kawasaki disease (KD). We postulated that variation in TGF-ß signaling might be important in KD susceptibility and disease outcome. METHODS AND RESULTS: We investigated genetic variation in 15 genes belonging to the TGF-ß pathway in a total of 771 KD subjects of mainly European descent from the United States, the United Kingdom, Australia, and the Netherlands. We analyzed transcript abundance patterns using microarray and reverse transcriptase-polymerase chain reaction for these same genes, and measured TGF-ß2 protein levels in plasma. Genetic variants in TGFB2, TGFBR2, and SMAD3 and their haplotypes were consistently and reproducibly associated with KD susceptibility, coronary artery aneurysm formation, aortic root dilatation, and intravenous immunoglobulin treatment response in different cohorts. A SMAD3 haplotype associated with KD susceptibility replicated in 2 independent cohorts and an intronic single nucleotide polymorphism in a separate haplotype block was also strongly associated (A/G, rs4776338) (P=0.000022; odds ratio, 1.50; 95% confidence interval, 1.25 to 1.81). Pathway analysis using all 15 genes further confirmed the importance of the TGF-ß pathway in KD pathogenesis. Whole-blood transcript abundance for these genes and TGF-ß2 plasma protein levels changed dynamically over the course of the illness. CONCLUSIONS: These studies suggest that genetic variation in the TGF-ß pathway influences KD susceptibility, disease outcome, and response to therapy, and that aortic root and coronary artery Z scores can be used for phenotype/genotype analyses. Analysis of transcript abundance and protein levels further support the importance of this pathway in KD pathogenesis.

Human airway epithelial cell culture to identify new respiratory viruses: coronavirus NL63 as a model.

Propagation of new human respiratory virus pathogens in established cell lines is hampered by a lack of predictability regarding cell line permissivity and by availability of suitable antibody reagents to detect infection in cell lines that do not exhibit significant cytopathic effect. Recently, molecular methods have been used to amplify and identify novel nucleic acid sequences directly from clinical samples, but these methods may be hampered by the quantity of virus present in respiratory secretions at different time points following the onset of infection. Human airway epithelial (HAE) cultures, which effectively mimic the human bronchial environment, allow for cultivation of a wide variety of human respiratory viral pathogens. The goal of the experiments described here was to determine if propagation and identification of a human respiratory virus may be achieved through inoculation of HAE cultures followed by whole transcriptome amplification (WTA) and sequence analysis. To establish proof-of-principle human coronavirus NL63 (HCoV-NL63) was evaluated, and the first visualization of HCoV-NL63 virus by transmission electron microscopy (TEM) is reported. Initial propagation of human respiratory secretions onto HAE cultures followed by TEM and WTA of culture supernatant may be a useful approach for visualization and detection of new human respiratory pathogens that have eluded identification by traditional approaches.

Prevention of infective endocarditis: guidelines from the American Heart Association: a guideline from the American Heart Association Rheumatic Fever, Endocarditis and Kawasaki Disease Committee, Council on Cardiovascular Disease in the Young, and the Council on Clinical Cardiology, Council on Cardiovascular Surgery and Anesthesia, and the Quality of Care and Outcomes Research Interdisciplinary Working Group.

BACKGROUND: The purpose of this statement is to update the recommendations by the American Heart Association (AHA) for the prevention of infective endocarditis, which were last published in 1997. METHODS: and RESULTS: A writing group appointed by the AHA for their expertise in prevention and treatment of infective endocarditis (IE) with liaison members representing the American Dental Association, the Infectious Diseases Society of America and the American Academy of Pediatrics. The writing group reviewed input from national and international experts on IE. The recommendations in this document reflect analyses of relevant literature regarding procedure-related bacteremia and IE; in vitro susceptibility data of the most common microorganisms, which cause IE; results of prophylactic studies in animal models of experimental endocarditis; and retrospective and prospective studies of prevention of IE. MEDLINE database searches from 1950 through 2006 were done for English language articles using the following search terms: endocarditis, infective endocarditis, prophylaxis, prevention, antibiotic, antimicrobial, pathogens, organisms, dental, gastrointestinal, genitourinary, streptococcus, enterococcus, staphylococcus, respiratory, dental surgery, pathogenesis, vaccine, immunization and bacteremia. The reference lists of the identified articles were also searched. The writing group also searched the AHA online library. The American College of Cardiology/AHA classification of recommendations and levels of evidence for practice guidelines were used. The article subsequently was reviewed by outside experts not affiliated with the writing group and by the AHA Science Advisory and Coordinating Committee. CONCLUSIONS: The major changes in the updated recommendations include the following. (1) The committee concluded that only an extremely small number of cases of IE might be prevented by antibiotic prophylaxis for dental procedures even if such prophylactic therapy were 100 percent effective. (2) IE prophylaxis for dental procedures should be recommended only for patients with underlying cardiac conditions associated with the highest risk of adverse outcome from IE. (3) For patients with these underlying cardiac conditions, prophylaxis is recommended for all dental procedures that involve manipulation of gingival tissue or the periapical region of teeth or perforation of the oral mucosa. (4) Prophylaxis is not recommended based solely on an increased lifetime risk of acquisition of IE. (5) Administration of antibiotics solely to prevent endocarditis is not recommended for patients who undergo a genitourinary or gastrointestinal tract procedure. These changes are intended to define more clearly when IE prophylaxis is or is not recommended and to provide more uniform and consistent global recommendations.

Prevention of infective endocarditis: guidelines from the American Heart Association: a guideline from the American Heart Association Rheumatic Fever, Endocarditis and Kawasaki Disease Committee, Council on Cardiovascular Disease in the Young, and the Council on Clinical Cardiology, Council on Cardiovascular Surgery and Anesthesia, and the Quality of Care and Outcomes Research Interdisciplinary Working Group.

BACKGROUND: The purpose of this statement is to update the recommendations by the American Heart Association (AHA) for the prevention of infective endocarditis, which were last published in 1997. METHODS AND RESULTS: A writing group appointed by the AHA for their expertise in prevention and treatment of infective endocarditis (IE) with liaison members representing the American Dental Association, the Infectious Diseases Society of America and the American Academy of Pediatrics. The writing group reviewed input from national and international experts on IE. The recommendations in this document reflect analyses of relevant literature regarding procedure-related bacteremia and IE; in vitro susceptibility data of the most common microorganisms, which cause IE; results of prophylactic studies in animal models of experimental endocarditis; and retrospective and prospective studies of prevention of IE. MEDLINE database searches from 1950 through 2006 were done for English language articles using the following search terms: endocarditis, infective endocarditis, prophylaxis, prevention, antibiotic, antimicrobial, pathogens, organisms, dental, gastrointestinal, genitourinary, streptococcus, enterococcus, staphylococcus, respiratory, dental surgery, pathogenesis, vaccine, immunization and bacteremia. The reference lists of the identified articles were also searched. The writing group also searched the AHA online library. The American College of Cardiology/AHA classification of recommendations and levels of evidence for practice guidelines were used. The article subsequently was reviewed by outside experts not affiliated with the writing group and by the AHA Science Advisory and Coordinating Committee. CONCLUSIONS: The major changes in the updated recommendations include the following. (1) The committee concluded that only an extremely small number of cases of IE might be prevented by antibiotic prophylaxis for dental procedures even if such prophylactic therapy were 100 percent effective. (2) IE prophylaxis for dental procedures should be recommended only for patients with underlying cardiac conditions associated with the highest risk of adverse outcome from IE. (3) For patients with these underlying cardiac conditions, prophylaxis is recommended for all dental procedures that involve manipulation of gingival tissue or the periapical region of teeth or perforation of the oral mucosa. (4) Prophylaxis is not recommended based solely on an increased lifetime risk of acquisition of IE. (5) Administration of antibiotics solely to prevent endocarditis is not recommended for patients who undergo a genitourinary or gastrointestinal tract procedure. These changes are intended to define more clearly when IE prophylaxis is or is not recommended and to provide more uniform and consistent global recommendations.

Prevention of infective endocarditis: guidelines from the American Heart Association: a guideline from the American Heart Association Rheumatic Fever, Endocarditis, and Kawasaki Disease Committee, Council on Cardiovascular Disease in the Young, and the Council on Clinical Cardiology, Council on Cardiovascular Surgery and Anesthesia, and the Quality of Care and Outcomes Research Interdisciplinary Working Group.

BACKGROUND: The purpose of this statement is to update the recommendations by the American Heart Association (AHA) for the prevention of infective endocarditis that were last published in 1997. METHODS AND RESULTS: A writing group was appointed by the AHA for their expertise in prevention and treatment of infective endocarditis, with liaison members representing the American Dental Association, the Infectious Diseases Society of America, and the American Academy of Pediatrics. The writing group reviewed input from national and international experts on infective endocarditis. The recommendations in this document reflect analyses of relevant literature regarding procedure-related bacteremia and infective endocarditis, in vitro susceptibility data of the most common microorganisms that cause infective endocarditis, results of prophylactic studies in animal models of experimental endocarditis, and retrospective and prospective studies of prevention of infective endocarditis. MEDLINE database searches from 1950 to 2006 were done for English-language papers using the following search terms: endocarditis, infective endocarditis, prophylaxis, prevention, antibiotic, antimicrobial, pathogens, organisms, dental, gastrointestinal, genitourinary, streptococcus, enterococcus, staphylococcus, respiratory, dental surgery, pathogenesis, vaccine, immunization, and bacteremia. The reference lists of the identified papers were also searched. We also searched the AHA online library. The American College of Cardiology/AHA classification of recommendations and levels of evidence for practice guidelines were used. The paper was subsequently reviewed by outside experts not affiliated with the writing group and by the AHA Science Advisory and Coordinating Committee. CONCLUSIONS: The major changes in the updated recommendations include the following: (1) The Committee concluded that only an extremely small number of cases of infective endocarditis might be prevented by antibiotic prophylaxis for dental procedures even if such prophylactic therapy were 100% effective. (2) Infective endocarditis prophylaxis for dental procedures is reasonable only for patients with underlying cardiac conditions associated with the highest risk of adverse outcome from infective endocarditis. (3) For patients with these underlying cardiac conditions, prophylaxis is reasonable for all dental procedures that involve manipulation of gingival tissue or the periapical region of teeth or perforation of the oral mucosa. (4) Prophylaxis is not recommended based solely on an increased lifetime risk of acquisition of infective endocarditis. (5) Administration of antibiotics solely to prevent endocarditis is not recommended for patients who undergo a genitourinary or gastrointestinal tract procedure. These changes are intended to define more clearly when infective endocarditis prophylaxis is or is not recommended and to provide more uniform and consistent global recommendations.

Detection of antigen in bronchial epithelium and macrophages in acute Kawasaki disease by use of synthetic antibody.

BACKGROUND: Kawasaki disease (KD) is the most common acquired cardiac disease in children in developed nations. The etiology is unknown, but a ubiquitous infectious agent appears to be likely. Immunoglobulin A plasma cells infiltrate inflamed tissues in acute KD, producing oligoclonal, antigen-driven antibodies. METHODS: To identify antigens important in the pathogenesis of KD, oligoclonal KD antibodies were prepared in vitro and tested by immunohistochemistry experiments on tissues from patients with acute KD and from control subjects and were also tested for reactivity with human inflammatory proteins. RESULTS: By use of synthetic antibody A, specific binding to a cytoplasmic antigen in proximal bronchial epithelium was observed in 10 of 13 patients with acute KD but in 0 of 9 control subjects (P=.001). A subset of macrophages was positive in at least 1 inflamed tissue from all 17 patients with acute KD. Antigen was detected in 9 of 12 acute KD coronary artery aneurysms but in 0 of 10 control coronary arteries (P<.001). The antigen is not immunoglobulin or any of 40 common inflammatory proteins. CONCLUSIONS: We report the first demonstration of a KD-associated antigen in the tissues targeted by the disease. Our findings are consistent with the theory that KD is caused by a previously unidentified respiratory infectious agent with tropism for vascular tissue.

Macrophage infiltration of pancreatic acini and islets in acute Kawasaki disease.

A report of 2 patients who developed diabetes after Kawasaki disease (KD) led us to determine whether macrophages and/or T cells infiltrate acute KD pancreas. Three of 10 acute fatal KD cases had diffuse macrophage infiltration of the pancreas; T cells were not prominent. Affected islets were seen in close proximity to normal islets. These findings may explain the rarity of diabetes after KD.

Inflammatory pulmonary nodules in Kawasaki disease.

Symptomatic pulmonary manifestations of Kawasaki disease (KD) are uncommon. However, epidemiologic, radiologic, and histologic studies have indicated that respiratory symptoms and findings occur in KD and suggest that the KD agent may have a respiratory portal of entry. We report on three young infants with KD who developed pulmonary nodules, in addition to coronary artery aneurysms. Two patients had pathologic specimens available, one from biopsy and the other from autopsy. The nodules had predominantly mononuclear cell infiltrates, which were within the lung parenchyma and infiltrating vessel walls. Immunohistochemical studies of the nodules, using antibodies to common leukocyte antigen (LCA) and factor VIII-related antigen, confirmed the inflammatory nature of the lesions and showed capillary proliferation. IgA plasma-cell infiltration was observed in the nodule, consistent with previous KD findings of IgA plasma-cell infiltration in the vessel walls, kidneys, pancreas, and upper respiratory tract. The two patients with nonfatal KD were treated with intravenous immunoglobulin and aspirin, with resolution of the nodules. We propose that when pulmonary involvement occurs in KD, it ranges from subclinical interstitial micronodular infiltrates to larger inflammatory pulmonary nodules. These pulmonary infiltrates and nodules likely reflect the host response to the etiologic agent of KD, and may resolve with the disease process. Recognition of this pulmonary complication of KD may enable cautious observation of such lesions for spontaneous resolution.

Un nuevo modelo patogénico para la enfermedad de Kawasaki / A new pathogenic model for Kawasaki disease

La identificación de antígenos importantes en la patogenia de la enfermedad de Kawasaki permitirá avanzar en el desarrollo de una prueba diagnóstica, mejorar el tratamiento y, finalmente, prevenir esta enfermedad infantil potencialmente fatal.

Systemic production of vascular endothelial growth factor and fms-like tyrosine kinase-1 receptor in acute Kawasaki disease.

BACKGROUND: Increased vascular permeability is an important event during the initial process of Kawasaki disease (KD). One potential responsible candidate for the induction of vascular hyperpermeability is vascular endothelial growth factor (VEGF). METHODS AND RESULTS: We investigated the expression of VEGF and its receptors (flt-1, KDR) in acute KD tissues at 7 days to 5 weeks of illness. Neuropilin-1, which enhances the binding of VEGF(165) to KDR, was also studied. Abundant expression of VEGF and flt-1 was documented immunohistochemically in many organs from acute KD, including heart and lung. VEGF and flt-1 were colocalized in all vessels that showed edema. These molecules resided in endothelium and vascular media and also in migrating smooth muscle cells in neointima and infiltrating macrophages. Compared with controls, coronary vessels of acute KD had upregulation of VEGF and flt-1 but not KDR or neuropilin-1. KDR was expressed by vessels at 7 days of illness but not later in the illness. Plasma proteins were more extensively bound to the extracellular matrix in coronary vessels in acute KD than controls. Furthermore, elevation of serum VEGF levels was correlated with low serum albumin in acute KD (n=220, r=-0.53, P<0.001). CONCLUSIONS: These findings suggest that VEGF and flt-1 are upregulated in blood vessels in many organs of acute KD. Expression of KDR was limited to the early stage of acute KD. The roles of VEGF in acute KD may involve promotion of vascular permeability and macrophage activation. Low serum albumin may indicate overproduction of VEGF in acute KD.