Epithelial cells remove precancerous cells by cell competition via MHC class I-LILRB3 interaction.

Epithelial cells have an ability termed 'cell competition', which is an immune surveillance-like function that extrudes precancerous cells from the epithelial layer, leading to apoptosis and clearance. However, it remains unclear how epithelial cells recognize and extrude transformed cells. Here, we discovered that a PirB family protein, leukocyte immunoglobulin-like receptor B3 (LILRB3), which is expressed on non-transformed epithelial cells, recognizes major histocompatibility complex class I (MHC class I) that is highly expressed on transformed cells. MHC class I interaction with LILRB3 expressed on normal epithelial cells triggers an SHP2-ROCK2 pathway that generates a mechanical force to extrude transformed cells. Removal of transformed cells occurs independently of natural killer (NK) cell or CD8+ cytotoxic T cell-mediated activity. This is a new mechanism in that the immunological ligand-receptor system generates a mechanical force in non-immune epithelial cells to extrude precancerous cells in the same epithelial layer.

Accumulation of annexin A2 and S100A10 prevents apoptosis of apically delaminated, transformed epithelial cells.

In various epithelial tissues, the epithelial monolayer acts as a barrier. To fulfill its function, the structural integrity of the epithelium is tightly controlled. When normal epithelial cells detach from the basal substratum and delaminate into the apical lumen, the apically extruded cells undergo apoptosis, which is termed anoikis. In contrast, transformed cells often become resistant to anoikis and able to survive and grow in the apical luminal space, leading to the formation of multilayered structures, which can be observed at the early stage of carcinogenesis. However, the underlying molecular mechanisms still remain elusive. In this study, we first demonstrate that S100A10 and ANXA2 (Annexin A2) accumulate in apically extruded, transformed cells in both various cell culture systems and murine epithelial tissues in vivo. ANXA2 acts upstream of S100A10 accumulation. Knockdown of ANXA2 promotes apoptosis of apically extruded RasV12-transformed cells and suppresses the formation of multilayered epithelia. In addition, the intracellular reactive oxygen species (ROS) are elevated in apically extruded RasV12 cells. Treatment with ROS scavenger Trolox reduces the occurrence of apoptosis of apically extruded ANXA2-knockdown RasV12 cells and restores the formation of multilayered epithelia. Furthermore, ROS-mediated p38MAPK activation is observed in apically delaminated RasV12 cells, and ANXA2 knockdown further enhances the p38MAPK activity. Moreover, the p38MAPK inhibitor promotes the formation of multilayered epithelia of ANXA2-knockdown RasV12 cells. These results indicate that accumulated ANXA2 diminishes the ROS-mediated p38MAPK activation in apically extruded transformed cells, thereby blocking the induction of apoptosis. Hence, ANXA2 can be a potential therapeutic target to prevent multilayered, precancerous lesions.

Analysis of Cytokine Profiles in Pediatric Myocarditis Multicenter Study.

Acute myocarditis (AM) is an inflammatory disease of the heart muscle that can progress to fulminant myocarditis (FM), a severe and life-threatening condition. The cytokine profile of myocarditis in children, especially in relation to fulminant myocarditis, is not well understood. This study aims to evaluate the cytokine profiles of acute and fulminant myocarditis in children. Pediatric patients diagnosed with myocarditis were included in the study. Cytokine levels were measured using a multiplexed fluorescent bead-based immunoassay. Statistical analysis was performed to compare patient characteristics and cytokine levels between FM, AM, and healthy control (HC) groups. Principal component analysis (PCA) was applied to cytokine groups that were independent among the FM, AM, and HC groups. The study included 22 patients with FM and 14 with AM patients. We identified four cytokines that were significantly higher in the FM group compared to the AM group: IL1-RA (p = 0.002), IL-8 (p = 0.005), IL-10 (p = 0.011), and IL-15 (p = 0.005). IL-4 was significantly higher in the AM group compared to FM and HC groups (p = 0.006 and 0.0015). PDGF-AA, and VEGF-A were significantly lower in the FM group than in the AM group (p = 0.013 and <0.001). Similar results were obtained in PCA. Cytokine profiles might be used to differentiate pediatric FM from AM, stratify severity, and predict prognosis. The targeted therapy that works individual cytokines might provide a potential treatment for reducing the onset of the FM and calming the condition, and further studies are needed.

Changes in trends of pediatric ß- and γ-herpesvirus infections during the COVID-19 pandemic: A single-center observational study.

Nonpharmaceutical interventions (NPIs) to control COVID-19 have decreased the incidence of many pediatric infectious diseases. The epidemiology of ß- and γ-herpesvirus infections might have been affected by NPIs. The aim of this study was to elucidate changes in trends in ß- and γ-herpesvirus infections and complex febrile seizures (cFS) of viral etiology before and during the COVID-19 pandemic. Between April 2017 and March 2021, febrile children aged ≤5 years were enrolled. Detection of EBV, CMV, HHV-6B, and HHV-7 DNA in serum was performed using real-time PCR. The epidemiology of viral infections and cFS were compared between the prepandemic and pandemic periods. During the observation period, 1432 serum samples were collected. The mean number of febrile children decreased during the pandemic period, but the number of patients with HHV-6B infection increased from 35 (9.3% of all febrile children) per year before the pandemic to 43 (15.5%) during the pandemic. The change in the proportion of patients with primary HHV-6B infection was 6.50% (95% confidence interval [CI], 2.05%-11.3%; p = 0.0047). The mean number of patients with cFS decreased during the pandemic period, but the number of patients with HHV-6B-associated cFS was stable throughout the observation period. Therefore, the change in proportion of patients with cFS caused by primary HHV-6B infection was 49.5% (95% CI, 12.2%-60.5%; p = 0.0048). The disease burden of primary HHV-6B infection among patients in the emergency room remained unchanged, with a significant increase in the relative proportion after the COVID-19 pandemic began.

Serological analysis of severe acute respiratory syndrome coronavirus 2 infection in children with Kawasaki disease.

Multisystem inflammatory syndrome in children (MIS-C) was reported as a severe complication of coronavirus disease 2019; an infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and was suggested to be associated with Kawasaki disease (KD) in terms of severe systemic inflammation and mucocutaneous symptoms. Because severe gastrointestinal symptoms and systemic shock are more frequently observed with MIS-C, patients with mild MIS-C might have been diagnosed with KD. In this study, titers of IgG antibodies against the SARS-CoV-2 S (S-IgG) and N proteins (N-IgG) were measured in 99 serum samples collected from patients with KD treated between January 2020 and December 2021 to evaluate the relationship between KD and SARS-CoV-2 infection. S-IgG were detected in only one patient out of 99 patients. This patient had coronavirus disease 2019 (COVID-19) 10 months before KD onset, and was unlikely MIS-C. According to characters of S-IgG and N-IgG, the patients was unlikely infected with SARS-CoV-2 just before the onset of KD. In addition to this study, the 26th Nationwide Survey and previous studies showed an association between KD and SARS-CoV-2 to be unlikely. In conclusion, SARS-CoV-2 infection was not observed in patients with KD until Delta predominance in Japan by the method of detecting SARS-CoV-2 IgG.

Analysis of the reliability of rapid diagnostic tests for varicella, including breakthrough cases.

In the era of universal varicella vaccination, diagnosis of varicella is challenging, especially for breakthrough cases. We sought to clarify the reliability of direct varicella-zoster virus (VZV) loop-mediated isothermal amplification (LAMP) and DermaQuick® VZV using the immunochromatography technique as rapid diagnostic tests for varicella. In addition, the usefulness of saliva as a sample type for direct LAMP was investigated. Among the 46 enrolled patients with suspected VZV infection, 31 patients (67.3%) were positive for the nucleic acid test based on real-time PCR from skin swab samples. Direct LAMP of skin swabs was positive in 29 (63.0%) of 46 patients. DermaQuick® VZV was positive in 25 (54.3%) of 46 patients. VZV DNA was detected in only 48.4% of oral swabs with the direct LAMP method. With real-time polymerase chain reaction (PCR) as the standard for diagnosing varicella, the sensitivity and specificity of DermaQuick® VZV were 80.7% and 100%, respectively. The sensitivity and specificity of direct LAMP from skin swabs were 93.6% and 100%, respectively. The sensitivity and specificity of real-time PCR for DNA extracted from oral swabs were 74.2% and 93.3%, respectively. Thus, oral swab samples are not suitable for breakthrough varicella diagnosis. Although DermaQuick® VZV is considered the most convenient point-of-care test for varicella, its sensitivity and specificity were lower than those of direct VZV LAMP.

Systemic herpes simplex virus infection in a child with eczema herpeticum.

Eczema herpeticum (EH) is a disseminated cutaneous infection with herpes simplex virus (HSV) that develops in patients with atopic dermatitis. The kinetics and clinical significance of HSV viremia in EH are poorly understood. Herein, we report HSV DNAemia in a child with EH 12 months after the completion of chemotherapy for Hodgkin lymphoma.

Basal extrusion of single-oncogenic mutant cells induces dome-like structures with altered microenvironments.

At the initial stage of carcinogenesis, oncogenic transformation occurs in single cells within epithelial layers. However, the behavior and fate of the newly emerging transformed cells remain enigmatic. Here, using originally established mouse models, we investigate the fate of RasV12-transformed cells that appear in a mosaic manner within epithelial tissues. In the lung bronchial epithelium, most majority of RasV12-transformed cells are apically extruded, whereas noneliminated RasV12 cells are often basally delaminated leading to various noncell-autonomous changes in surrounding environments; macrophages and activated fibroblasts are accumulated, and normal epithelial cells overlying RasV12 cells overproliferate and form a convex multilayer, which is termed a 'dome-like structure'. In addition, basally extruded RasV12 cells acquire certain features of epithelial-mesenchymal transition (EMT). Furthermore, the expression of COX-2 is profoundly elevated in RasV12 cells in dome-like structures, and treatment with the COX inhibitor ibuprofen suppresses the recruitment of activated fibroblasts and moderately diminishes the formation of dome-like structures. Therefore, basal extrusion of single-oncogenic mutant cells can induce a tumor microenvironment and EMT and generate characteristic precancerous lesions, providing molecular insights into the earlier steps of cancer development.

Inherited Chromosomally Integrated Human Herpesvirus 6 Is a Risk Factor for Spontaneous Abortion.

BACKGROUND: Human herpesvirus 6 (HHV-6) can be genetically transmitted from parent to child as inherited chromosomally integrated HHV-6 (iciHHV-6). HHV-6 reactivation occurs in pregnant women with iciHHV-6. We found no sex differences in the frequency of index cases with iciHHV-6 but inheritance from the father was more common. We evaluated the association between iciHHV-6 status and spontaneous abortion. METHODS: iciHHV-6 was confirmed by high viral DNA copy numbers in whole blood and somatic cells. The origin of integrated viral genome, paternal or maternal, was examined using the same method. The pregnancy history of 23 mothers in families with iciHHV-6 and 285 mothers in families without iciHHV-6 was abstracted. RESULTS: Of 23 iciHHV-6 index cases, 8 mothers and 15 fathers had iciHHV-6. Spontaneous abortion rates in mothers with and mothers without/fathers with iciHHV-6 and mothers in families without iciHHV-6 were 27.6%, 10.3%, and 14.8%, respectively (P = .012). Mothers with iciHHV-6 (odds ratio [OR], 6.41; 95% confidence interval [CI], 1.10-37.4) and maternal age at the most recent pregnancy ≥40 years (OR, 3.91; 95% CI, 1.30-11.8) were associated with 2 or more spontaneous abortions. CONCLUSIONS: Mothers with iciHHV-6 is a risk factor for spontaneous abortion.

Immune response against SARS-CoV-2 in pediatric patients including young infants.

Pediatric cases of the coronavirus disease 2019 (COVID-19) are generally mild or asymptomatic, and are usually detected by virological examination following close contact with COVID-19 patients, often the children's parents. The detailed clinical features and virological data of pediatric patients with COVID-19, particularly young infants, remain unclear. Here, the clinical and virological characteristics of four children with COVID-19 including two young infants were investigated. One- and 4-month-old boys with COVID-19 were both asymptomatic, and seroconversion was demonstrated. These findings suggest that even young infants can mount an immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), despite having weaker immune defenses than adolescents and adults. Three-year-old boy, who was SARS-CoV-2-negative, was admitted to the same room as his SARS-CoV-2-positive father due to the lack of caregivers. Although he was asymptomatic, he had seroconverted to SARS-CoV-2. Eleven-year-old boy, who was sibling of the 3-year-old boy, was also SARS-CoV-2-negative. He was isolated in his own room and did not seroconvert. If young children are SARS-CoV-2 negative, they should be isolated from their SARS-CoV-2-positive parents. This may be difficult in practice, if parents with COVID-19 are the only available caregivers. In such situations, the most appropriate measures should be taken for each patient.

Frequency of subclinical herpes zoster in pediatric hematology-oncology patients receiving chemotherapy: A retrospective cohort analysis.

Varicella-zoster virus (VZV) reactivation from the enteric nervous system can cause ileus (Ogilvie's syndrome) in adult patients. Since no pediatric cases have been described, we sought to retrospectively analyze VZV reactivation in pediatric hematology-oncology patients to determine whether VZV infection including subclinical VZV reactivation can induce gastrointestinal complications such as Ogilvie's syndrome. Thirty-five patients who received chemotherapy at our institution between September 2013 and June 2018 were included. Serum samples were collected weekly during hospitalization and every 3 months during outpatient maintenance chemotherapy. A real-time polymerase chain reaction assay was used to measure VZV DNA load in serum. The clinical features of patients with VZV infection were retrospectively analyzed. Of 1165 serum samples, 7 (0.6%) were positive for VZV DNA. VZV DNA was detected in 3 of 35 patients. In patient A, VZV DNA was detected during two episodes. The first episode involved varicella-like eruptions caused by the Oka VZV vaccine strain. The second episode involved herpes zoster (HZ) caused by the same strain. Patients B and C had a clinical course that was typical for HZ caused by wild-type VZV. No gastrointestinal symptoms were observed at the time of VZV infection in these three patients. VZV DNA was not detected in any other samples. No pediatric cases with Ogilvie's syndrome caused by VZV reactivation were demonstrated in this cohort. Additionally, no subclinical VZV reactivation was found in this cohort. Further study is needed to elucidate the precise incidence of pediatric Ogilvie's syndrome caused by VZV reactivation.

Chromosomally integrated human herpesvirus 6 in the Japanese population.

The objectives of the work are to elucidate the incidence and virological findings of chromosomally integrated human herpesvirus 6 (ciHHV-6) in Japanese population and to analyze an association between ciHHV-6 and the clinical manifestation of exanthema subitum (ES). Real-time polymerase chain reaction was performed to determine HHV-6 DNA loads in 2347 cord blood samples from healthy neonates (cohort A), febrile children less than 5 years old (cohort B), and hematopoietic cell transplant recipients (cohort C). CiHHV-6 was confirmed by detection of high copy numbers of viral DNA in somatic cells. The integration site was determined by fluorescent in situ hybridization analysis. In the ciHHV-6 subjects of cohorts A and B, HHV-6 antibody titers were measured, the history of ES was obtained, and the incidence of ES was compared with non-ciHHV-6 children without primary HHV-6B infection in the cohort B. CiHHV-6 was detected in 14 (0.60%) of the 2347 samples: A (6/1006, 0.60%), B (6/790, 0.76%), and C (2/551, 0.36%). The integration sites were on chromosome 22q in seven cases, Yp in two cases, and 17q and Xp in one case. No past history of ES was observed in 11 of the 12 subjects. Nine children with ciHHV-6 underwent serological analysis and were found to be positive for HHV-6 IgG antibodies. Incidence of ES was statistically higher in the control subjects than the ciHHV-6 subjects (P = 0.0039). In Japan, the frequency of ciHHV-6 was 0.60%. A high incidence of ciHHV-6A, specifically in chromosome 22, is a characteristic finding among the Japanese. CiHHV-6 may interfere with the clinical symptoms of primary HHV-6B infection.

A GIRL WITH ADENOVIRUS TYPE 2 INDUCED INTERSTITIAL PNEUMONITIS ACUTELY EXACERBATED WITH PRIMARY HUMAN HERPESVIRUS 7 INFECTION.

We encountered a previously healthy 3-year-old girl with interstitial pneumonitis that initially developed due to human adenovirus type 2 infection and exacerbated by primary human herpesvirus 7 infection. A comprehensive serum biomarker analysis showed patterns that differed by viral infection, suggesting that respiratory and lymphotropic viral infections might have different pathophysiology in interstitial pneumonitis.

Future Perspective for ALK-Positive Anaplastic Large Cell Lymphoma with Initial Central Nervous System (CNS) Involvement: Could Next-Generation ALK Inhibitors Replace Brain Radiotherapy for the Prevention of Further CNS Relapse?

Central nervous system (CNS) involvement in anaplastic large cell lymphoma (ALCL) at diagnosis is rare and leads to poor prognosis with the use of the standard ALCL99 protocol alone. CNS-directed intensive chemotherapy, such as an increased dose of intravenous MTX, increased dose of dexamethasone, intensified intrathecal therapy, and high-dose cytarabine, followed by cranial irradiation, has been shown to improve survival in this population. In this paper, the authors describe a 14-year-old male with an intracranial ALCL mass at onset who received CNS-directed chemotherapy followed by 23.4 Gy of whole-brain irradiation. After the first systemic relapse, the CNS-penetrating ALK inhibitor, alectinib, was applied; it has successfully maintained remission for 18 months without any adverse events. CNS-penetrating ALK inhibitor therapy might prevent CNS relapse in pediatric ALK-positive ALCL. Next-generation ALK inhibitors could be introduced as a promising treatment option, even for primary ALCL with CNS involvement, which could lead to the omission of cranial irradiation and avoid radiation-induced sequalae. Further evidence of CNS-penetrating ALK inhibitor combined therapy for primary ALK-positive ALCL is warranted to reduce radiation-induced sequalae in future treatments.

Similarities in rotavirus vaccine viral shedding and immune responses in pairs of twins.

Objectives: Intestinal rotavirus (RV) vaccine replication and host immune response are suggested to be affected by several factors, including maternal antibodies, breastfeeding history, and gut microbiome, which are thought to be similar in pairs of twins. The aim of this study was to determine whether viral shedding from the fecal RV vaccine strain Rotarix® (RV1) and IgG and IgA responses to RV show similarity in pairs of twins. Methods: Quantitative reverse transcription polymerase chain reaction specific to RV vaccine strain RV1 was used to monitor fecal RV1 viral shedding. RV IgG and IgA titers were measured using an in-house enzyme-linked immunosorbent assay. Fecal RV1 viral shedding and immune responses were compared between twins and singletons with mixed effects and fixed effects models. Results: A total of 347 stool and 54 blood samples were collected from four pairs of twins and twelve singletons during the observation period. Although the kinetics of fecal RV1 viral shedding and immune responses differed among vaccinated individuals, they appeared to be similar within twin pairs. RV shedding after the first dose (P=0.049) and RV IgG titers during the entire observation period (P=0.015) had a significantly better fit in the fixed effect model that assumed that twins have the same response versus the model that assumed that twins have a different response. Conclusions: The similarity of RV vaccine viral replication in intestine and host immune responses in twin pairs was demonstrated using statistical analysis.

Identification of the Kinase-Substrate Recognition Interface between MYPT1 and Rho-Kinase.

Protein kinases exert physiological functions through phosphorylating their specific substrates; however, the mode of kinase-substrate recognition is not fully understood. Rho-kinase is a Ser/Thr protein kinase that regulates cytoskeletal reorganization through phosphorylating myosin light chain (MLC) and the myosin phosphatase targeting subunit 1 (MYPT1) of MLC phosphatase (MLCP) and is involved in various diseases, due to its aberrant cellular contraction, morphology, and movement. Despite the importance of the prediction and identification of substrates and phosphorylation sites, understanding of the precise regularity in phosphorylation preference of Rho-kinase remains far from satisfactory. Here we analyzed the Rho-kinase-MYPT1 interaction, to understand the mode of Rho-kinase substrate recognition and found that the three short regions of MYPT1 close to phosphorylation sites (referred to as docking motifs (DMs); DM1 (DLQEAEKTIGRS), DM2 (KSQPKSIRERRRPR), and DM3 (RKARSRQAR)) are important for interactions with Rho-kinase. The phosphorylation levels of MYPT1 without DMs were reduced, and the effects were limited to the neighboring phosphorylation sites. We further demonstrated that the combination of pseudosubstrate (PS) and DM of MYPT1 (PS1 + DM3 and PS2 + DM2) serves as a potent inhibitor of Rho-kinase. The present information will be useful in identifying new substrates and developing selective Rho-kinase inhibitors.

A Case of Aseptic Meningitis Without Skin Rash Caused by Oka Varicella Vaccine.

Here, we present a previously healthy adolescent with aseptic meningitis without skin rash caused by varicella vaccine derived from the Oka/Biken strain; the patient received a single dose of varicella vaccine at 1 year of age. Pediatricians should be aware of the potential for reactivation of varicella vaccine derived from the Oka/Biken strain, which can cause aseptic meningitis in vaccinated children even in the absence of a skin rash.

Rotavirus genotypes and clinical outcome of natural infection based on vaccination status in the post-vaccine era.

Rotavirus (RV) is a leading cause of gastroenteritis in children. In Japan, Rotarix (RV1; GlaxoSmithKline), which is a monovalent vaccine derived from human RV (G1P[8]), has been introduced since November 2011, and RotaTeq (RV5; MSD) which is an pentavalent, human-bovine mono-reassortant vaccine (G1, G2, G3, G4, and P1A[8]), has been introduced since July 2012. Long-term follow-up on vaccine efficacy and RV genotypical change should be carried out in order to control RV infection. The RV gastroenteritis (RVGE) outbreak occurred during the 2018/2019 season in Aichi prefecture, Japan. Therefore, the molecular epidemiology of RV among three different groups of RVGE, which were outpatients who received RV1, those who received RV5, and those without vaccination, was explored. Clinical features of RVGE patients were compared among the three patient groups. Children less than 15 years of age with gastroenteritis who visited any of seven pediatric practices between January and June 2019 were enrolled in the study. G, P, and E genotypes were determined by direct sequencing of reverse transcription-polymerase chain reaction products amplified from stool samples. Among 110 patients, there were 27, 28, and 55 in the RV1-vaccinated, RV5-vaccinated, and unvaccinated groups, respectively. The most frequent genotype was G8P[8] (92/110 patients, 83.6%). Genotype distributions did not significantly differ among the three patient groups (P = .125). Mean Vesikari score was significantly lower among RV1-vaccinated (7.1) and RV5-vaccinated patients (6.4) than among unvaccinated patients (10.2) (P < .001). Even in RVGE patients treated in an outpatient clinic, RV vaccine reduced the severity of the disease in this cohort.

Correlation between anti-S IgG and neutralizing antibody titers against three live SARS-CoV-2 variants in BNT162b2 vaccine recipients.

We analyzed serially collected serum samples from healthy adults who underwent BNT162b2 vaccination to elucidate the association between spike (S)-IgG antibody titers determined by ELISA using the WHO international standard (NIBSC code 20/136) and neutralizing antibody titers against three live SARS-CoV-2 variants. This study included 53 health care workers who received two doses of the BNT162b2 vaccine. S-IgG and nucleocapsid (N)-IgG antibody titers were measured by ELISA. Neutralizing (NT) antibody responses against three variants (Wuhan D614 G: KUH003, Alpha, and Delta) were evaluated before and after the first and second vaccination. N-IgG were not detected in any serum samples. S-IgG antibody titers remarkably increased after two BNT162b2 vaccine doses in all participants. S-IgG antibody titers were strongly correlated with NT titers against three variants of live viruses: KUH003 (r = 0.86), Alpha (r = 0.72), and Delta (r = 0.84). Serum samples from participants after one dose of BNT162b2 neutralized Alpha efficiently (median titer, 113.0), but median NT titers against KUH003 and Delta variants were lower, 57.0 and 28.0, respectively (p < .01). Two doses of the BNT162b2 vaccine elicited a strong immune response in this study. The second dose was required for induction of a strong booster effect. Serum collected from BNT162b2 vaccine recipients contained significantly lower neutralizing activity against Delta than that of against KUH003 (p < .0001) and Alpha (p < .0001). If a new variant emerges, live virus-based NT titers should be examined in serum obtained from vaccine recipients to evaluate vaccine efficacy for protection against infection.