Adipose tissue-derived stem cells suppress coronary arteritis of Kawasaki disease in vivo.

BACKGROUND: Kawasaki disease (KD) is a systemic inflammatory disease resulting in an acute febrile syndrome commonly affecting children younger than 5 years. Coronary arteritis in KD is occasionally non-responsive to several treatments. Recently, adipose tissue-derived stem cells (ADSCs) have been shown to have anti-inflammatory, immunosuppressive, and tissue-repair characteristics and are considered a useful treatment for inflammatory disease. The present study aimed to elucidate whether the administration of ADSCs can suppress KD-associated vasculitis in vivo. METHODS: Candida albicans water-soluble fraction is often used to model KD via the induction of severe coronary arteritis. Kawasaki disease model mice were intravenously administered ADSCs and phosphate-buffered saline (PBS). On day 29, the mice were sacrificed and hearts from mice in each group were dissected. This was followed by serum collection. Cardiac tissue sections were subjected to histopathological examination to evaluate the inflammatory area. The levels of pro-inflammatory cytokines in the serum were analyzed at days 15 and 29. The survival rates of both groups were compared. RESULTS: The mean inflammatory area in coronary arteritis was significantly lower in the ADSC group compared to the PBS group (P < 0.01). Furthermore, the levels of pro-inflammatory cytokines, such as IL-1ß, IL-12, IL-17, RANTES, INF-γ, and TNF-α, in the ADSC group were significantly lower than those in the PBS group. Moreover, the ADSC group had a significantly higher survival rate than the PBS group. CONCLUSIONS: These findings highlight that ADSCs have anti-inflammatory and immune regulatory functions that could provide novel cell-based therapeutic strategies for severe KD.

Comprehensive and quantitative multilocus methylation analysis reveals the susceptibility of specific imprinted differentially methylated regions to aberrant methylation in Beckwith-Wiedemann syndrome with epimutations.

PURPOSE: Expression of imprinted genes is regulated by DNA methylation of differentially methylated regions (DMRs). Beckwith-Wiedemann syndrome is an imprinting disorder caused by epimutations of DMRs at 11p15.5. To date, multiple methylation defects have been reported in Beckwith-Wiedemann syndrome patients with epimutations; however, limited numbers of DMRs have been analyzed. The susceptibility of DMRs to aberrant methylation, alteration of gene expression due to aberrant methylation, and causative factors for multiple methylation defects remain undetermined. METHODS: Comprehensive methylation analysis with two quantitative methods, matrix-assisted laser desorption/ionization mass spectrometry and bisulfite pyrosequencing, was conducted across 29 DMRs in 54 Beckwith-Wiedemann syndrome patients with epimutations. Allelic expressions of three genes with aberrant methylation were analyzed. All DMRs with aberrant methylation were sequenced. RESULTS: Thirty-four percent of KvDMR1-loss of methylation patients and 30% of H19DMR-gain of methylation patients showed multiple methylation defects. Maternally methylated DMRs were susceptible to aberrant hypomethylation in KvDMR1-loss of methylation patients. Biallelic expression of the genes was associated with aberrant methylation. Cis-acting pathological variations were not found in any aberrantly methylated DMR. CONCLUSION: Maternally methylated DMRs may be vulnerable to DNA demethylation during the preimplantation stage, when hypomethylation of KvDMR1 occurs, and aberrant methylation of DMRs affects imprinted gene expression. Cis-acting variations of the DMRs are not involved in the multiple methylation defects.

Adipose-derived stromal cells grown on a hydroxyapatite scaffold can support hematopoiesis in regenerated bone marrow in vivo.

Osteoblastic cells are a key component of the bone marrow (BM) stem cell niche and help regulate hematopoietic stem cells (HSCs). We have previously demonstrated that adipose-derived stromal cells (ADSCs) can differentiate into both osteogenic and chondrogenic cells in vitro. The current study examined whether the anatomical architecture of the BM could be regenerated in vivo by using ADSCs cultured on a hydroxyapatite (HA) scaffold. ADSCs from GFP transgenic mice were cultured in vitro on an HA scaffold. The scaffold with the attached cells was implanted subcutaneously onto the backs of C57/BL6 (Ly5.2) recipient mice. Lineage-negative (Lin-) Ly5.1 BM cells transduced with a lentiviral vector containing the luciferase (Luc) gene were intravenously administered to the recipient mice after lethal irradiation. Eight weeks after BM transplantation, the scaffolds were removed from the first recipient mice and subcutaneously implanted into lethally irradiated second recipient mice. The biodistribution and kinetics of Luc(+) Ly5.1 cells were monitored by bioluminescence imaging and flow cytometry. Luc(+) hematopoietic cells were present in the scaffolds of the secondary implanted mice for at least 8 months. Subcutaneous injection of G-CSF resulted in wide distribution of bioluminescence signals from the original scaffolds to the whole body. Therefore, BM regenerated using ADSCs grown on an HA scaffold can support HSC populations in vivo, suggesting that a functional BM niche is reconstituted. These results may have a significant impact on the development of therapeutic strategies for various hematopoietic diseases.

Use of tocilizumab to treat arthritis associated with mixed connective tissue disease complicated by ovarian teratoma: a case report.

Mixed connective tissue disease (MCTD) is characterized by mixed features of systemic lupus erythematosus, systemic sclerosis, and polymyositis/dermatomyositis and is rare in children. Here, we report a case of MCTD in a 10-year-old girl who presented at our hospital with arthralgia, Raynaud's phenomenon, and fatigue. Blood tests were positive for anti-U1-ribonucleoprotein (RNP) antibodies and for rheumatoid factors (RFs) IgG-RF and anti-galactose-deficient IgG. Levels of myogenic enzymes and hypergammaglobulinemia were elevated. Macrophages were prominent in bone marrow, with scattered phagocytic macrophages. MCTD was diagnosed based on the patient's symptoms and laboratory findings. Methylprednisolone pulse therapy combined with oral tacrolimus was administered, which led to resolution of symptoms. Three months after pulse therapy, arthralgia worsened and methotrexate was administered. Arthralgia improved but did not resolve. Magnetic resonance imaging performed to investigate the hip pain revealed a mature ovarian teratoma, which was surgically removed. Because the pain persisted and interfered with her daily life, she was treated with tocilizumab for joint pain relief, which decreased the pain level. Tocilizumab is a candidate for additional treatment of juvenile idiopathic arthritis-like arthritis associated with childhood-onset MCTD.

Perinatal risk factors for adverse long-term pulmonary outcome in premature infants: comparison of different definitions of bronchopulmonary dysplasia/chronic lung disease.

BACKGROUND: The aim of the study was to determine factors that affect adverse long-term pulmonary outcome in premature infants. METHODS: This retrospective analysis was done using 306 clinical records of preterm singleton neonates at <32 weeks of gestation. Two definitions of adverse pulmonary outcome were used: chronic lung disease (CLD), defined as a need for supplemental oxygen for at least 28 days after birth; and bronchopulmonary dysplasia (BPD), defined as oxygen dependency for at least 28 days after birth plus at 36 weeks postmenstrual age and/or a need for positive-pressure ventilatory support. Selected perinatal variables were compared between these definitions, and factors related to disease development were identified on multivariate analysis. RESULTS: The incidence of CLD and of BPD were 42% and 17%, respectively. Regardless of the definitions, the incidence of patent ductus arteriosus and of neonatal infection were significantly higher in the patients who met the disease criteria, but that of chorioamnionitis and of small for gestational age (SGA) were significantly higher in the patients only when the BPD definition was applied. Multivariate analysis identified SGA as an independent risk factor for the development of BPD after controlling for gestational age. CONCLUSIONS: Among selected perinatal variables, prenatal risk factors, particularly SGA, contributed to prolonged dependency on oxygen and/or positive-pressure ventilatory support, in combination with neonatal risk factors.

Intussusception in an extremely premature infant following bacterial sepsis.

Intussusception occurring in premature infants is exceedingly rare and shows substantially different characteristics from that in the typical age group or non-premature neonates. We present a case of intussusception in an extremely premature infant following bacterial sepsis, in which necrotizing enterocolitis was initially suspected. The correct diagnosis was made at 35 days old using abdominal ultrasonography, but the general condition of the infant had deteriorated to the point where surgery could not be performed. The patient died of multiple organ failure, and autopsy revealed ileo-ileal intussusception without a recognizable anatomical leading point. Possible mechanisms for this rare clinical entity are discussed.

Role of a Fetal Ultrasound Clinic in Promoting Multidisciplinary and Inter-Facility Perinatal Care.

BACKGROUND: With the increasing rate of high-risk pregnancies, there is an increased need for early evaluation of at-risk fetuses. Fetal ultrasound imaging has become a pivotal part of this evaluation. METHODS: To evaluate the role played by a fetal ultrasound clinic in promoting comprehensive perinatal care of patients with high-risk pregnancies, we retrospectively analyzed the indications and findings of fetal scans and the outcomes of the examined fetuses collected over the past 7 years (2014-2020) by our institute, which is reorganized as a perinatal medical center. RESULTS: During the study period, we conducted 345 fetal scans in high-risk pregnancy cases. Of these, 158 cases (46%) were referrals from other institutes. Eighty-nine neonates were admitted to our neonatal intensive care unit (NICU) after being evaluated, of which 10 neonates underwent surgery during their NICU stays. Thirty-nine pregnant women were referred to other tertiary care hospitals mainly due to fetal diagnoses with complex cardiac anomalies. Fourteen cases resulted in intrauterine fetal death or artificial abortion. CONCLUSIONS: Fetal ultrasound clinics have established their role in facilitating sophisticated regional perinatal care via multidisciplinary and inter-facility cooperation for high-risk pregnancy cases. In addition, providing psychological support and counseling for pregnant women whose fetuses are diagnosed with severe congenital anomalies should not be neglected.

Idiopathic severe constriction of the fetal ductus arteriosus: a possible underestimated pathophysiology.

Idiopathic intrauterine constriction/closure of the ductus arteriosus, which is distinct from that secondary to maternal exposure to non-steroidal anti-inflammatory drugs, such as indomethacin, or structural cardiac defect, is an uncommon event that often results in severe fetal-neonatal morbidity and mortality. We reported a case of idiopathic fetal ductal constriction, in which the diagnosis was confirmed by documentation of an abnormal four-chamber view of the fetal heart at 38 weeks of gestation on obstetric ultrasound examination. A female infant weighing 2,816 g was born by Cesarean section, and her postnatal course was mild; transient tachypnea requiring only several days of supplemental oxygen with spontaneous regression of the abnormal echocardiographic findings by 3 months of age. The incidence of idiopathic constriction/closure of the fetal ductus arteriosus may be underestimated, particularly with a negative history of maternal drug exposure and mild postnatal clinical presentation.

Interfacility Neonatal Transport for Convalescent Care: Improving Regionalized Care.

BACKGROUND: Transfer of infants who no longer need intensive or specialized care from tertiary to community hospitals or clinics contributes to efficient bed utilization in neonatal intensive care units (NICUs). METHODS: We retrospectively analyzed the records of all 1,503 infants admitted to our NICU during the 6-year period from April 2013 through March 2019 to evaluate the impact of interfacility neonatal transport for convalescent care. RESULTS: During the study period, our NICU accepted 33 infants from other tertiary NICUs and transferred 103 infants to other hospitals or clinics before their home discharge for convalescent care. Our NICU covered 39% of the total hospital days of infants accepted from other NICUs. Among infants transferred to other facilities, 81% born at our hospital were born to mothers transported to our obstetrics department as imminent high-risk deliveries; 94% of infants born at other hospitals were moved back to the referring facility. CONCLUSIONS: Interfacility neonatal transport for accepting and transferring infants for convalescent care is now an integral part of NICU practice, to bridge gaps between higher-level care facilities and homes. Establishment of well-defined transfer criteria and appropriate allocation of medical and staff resources among relevant facilities are desirable.

Statistical Evaluation of the First Year of a Neonatal Intensive Care Unit Established in a Medical School Hospital.

BACKGROUND: There has been significant progress in reducing perinatal mortality in Japan. However, due to changes in social conditions, the total fertility rate and the number of births are decreasing, whereas the number of low birth weight infants is increasing along with the number of newborn babies that require intensive care. Further, although the number of high-level perinatal medical centers has increased, so has that of infants who need long-term hospitalization. Conversely, the number of regular obstetric facilities has decreased, thus resulting in insufficient beds for neonatal care. To fill this gap, we established a neonatal intensive care unit (NICU) at our hospital. This study aimed to evaluate our new type by comparing the data from ours with that from other facilities. METHODS: The other facilities assessed were two high-level NICU facilities and two regular obstetric facilities. Data, including sex, gestational age, birth weight, Apgar scores at 1 and 5 min, delivery method, and presence of breathing disorders, were extracted from medical records. RESULTS: The birth weight and gestational age distributions were significantly different in the institutions, except in one facility without a NICU. The new NICU saw more infants with low birth weight and respiratory disorders than the regular obstetric facilities. CONCLUSION: The comparison of birth weight and gestational age distributions, cases of respiratory disorders, and delivery methods indicate that our new NICU is positioned as an intermediate facility between a high-level NICU and a regular obstetrics facility.

Hematopoiesis in regenerated bone marrow within hydroxyapatite scaffold.

We examined whether regenerated bone marrow (BM) with BM-derived stromal cells (BMSCs) on hydroxyapatite (HA) scaffolds can reconstitute the functional niche. Ly5.2 BMSCs on HA scaffolds were implanted s.c. onto the backs of Ly5.2 recipient mice. Lineage negative Ly5.1 BM cells expressing luciferase (luc) were i.v. administered into the recipient mice. Eight weeks after primary transplantation, secondary implantation was performed; the scaffolds removed from the first recipient mice were s.c. implanted into secondary recipient mice. Luc+ cells were detected in the scaffolds for 6 mo after secondary implantation. Injection of G-CSF resulted in wide distribution of bioluminescence from the original scaffolds to the whole body. Even after removing the scaffolds from the secondary recipient mice, luc+ cells were emitted by G-CSF stimulation, indicating that regenerated BM is capable of supporting hematopoietic stem cells (HSCs) and delivering HSCs to native BM in vivo. These data suggest that the functional niche is reconstituted at least partly and that regenerated BM on the scaffold may be used as a portable source of HSCs.

An 8-year-old girl with a recurrence of obstructive sleep apnea syndrome caused by hypertrophy of tubal tonsils 4 years after adenotonsillectomy.

Obstructive sleep apnea syndrome affects 1% to 2% of children. It is caused mainly by upper airway obstruction and manifests as snoring and sleep disturbance. Adenotonsillectomy can improve quality of life because airway obstruction occurs when both tonsils and adenoids are enlarged. We describe an 8-year-old girl with a recurrence of obstructive sleep apnea syndrome caused by hypertrophy of the tubal tonsils 4 years after adenotonsillectomy. The findings from this case highlight the importance of 1) identifying hypertrophy of the residual adenoid and compensatory hypertrophy of the tubal tonsils in patients with obstructive sleep apnea syndrome after adenotonsillectomy and 2) determining the optimal timing of adenotonsillectomy with respect to both the severity of obstructive sleep apnea and compensatory hypertrophy of other lymphoid tissue of Waldeyer's ring, as growth of such tissues is most active during the first several years of life.

Changes in Cytokine Profile during Initial Treatment of Pediatric Hemophagocytic Lymphohistiocytosis Associated with Epstein-Barr Virus.

Hemophagocytic lymphohistiocytosis (HLH) associated with Epstein-Barr virus (EBV) infection can be self-limiting, severe/aggressive, or fatal. We report a case of EBV-HLH with persistent fever, severe pancytopenia, hypertriglyceridemia, and hypofibrinogenemia in a 4-year-old boy. Levels of plasma cytokines and chemokines were measured with a Bio-Plex system at 1, 2, 3, 4, 5, and 8 days after hospital admission. Administration of steroid and high-dose intravenous immunoglobulin (1 g/kg) did not alleviate fever or reduce cytokine production; however, after administration of etoposide (an antineoplastic agent), fever decreased immediately, the patient's general condition improved, and levels of IL-6, IL-10, IL-8, MCP-1, IFN-γ, and TNF-α declined after etoposide administration. In particular, IFN-γ production sharply declined, from 1,104.1 pg/mL to 101.5 pg/mL, and IL-6 level decreased from 229.8 pg/mL to 11.0 pg/mL, on the day after initial etoposide administration. There was no later recurrence of symptoms during treatment with dexamethasone, etoposide, and cyclosporine A. This case suggests that early etoposide administration is critical for treatment success and indicates that etoposide promptly inhibits cytokine production.

Hematopoietic capacity of preterm cord blood hematopoietic stem/progenitor cells.

Full-term cord blood (TCB) hematopoietic stem/progenitor cells (HSC/HPCs) are used for stem cell transplantation and are well characterized. However, the properties of preterm cord blood (PCB) HSC/HPCs remain unclear. In the present study, we compared HSC/HPCs from TCB and PCB with respect to their expression of surface markers, homing capacity and ability to repopulate HSCs in the NOD/Shi-scid mice bone marrow. The proportion of CD34+CD38- cells was significantly higher in PCB. On the other hand, the engraftment rate of TCB CD34+ cells into NOD/Shi-scid mice was significantly higher than PCB CD34+ cells. The expression of VLA4 was stronger among TCB CD34+ cells than PCB CD34+ cells. Moreover, there was a positive correlation between the proportion of CD34+CXCR4+ cells and gestational age. These data suggest that the homing ability of HSCs increases during gestation, so that TCB may be a better source of HSCs for transplantation than PCB.

Prenatal diagnosis of isolated congenitally corrected transposition of the great arteries.

Congenitally corrected transposition of the great arteries (ccTGA) is a rare cardiac defect characterized by the atria connecting with anatomically discordant ventricles and the ventricles connecting with discordant and transposed great arteries, which allows hemodynamic compensation. Most patients with ccTGA have associated intracardiac anomalies, which could be a diagnostic clue, whereas isolated forms are infrequently diagnosed during the neonatal period and in utero. We describe a fetus that was diagnosed with ccTGA and without additional cardiac anomalies at 25 weeks of gestation. The parallel course of the great arteries discovered during a routine obstetric scan indicated this rare cardiac anomaly. Further detailed examination of the ventricular morphology helped to confirm the diagnosis. Despite hemodynamic compensation, the long-term prognosis of ccTGA is uncertain because of the possible development of arrhythmias or heart failure later in life. Our findings showed that fetal echocardiography can detect prenatal ccTGA.

Dextran sulfate and stromal cell derived factor-1 promote CXCR4 expression and improve bone marrow homing efficiency of infused hematopoietic stem cells.

Although the homing of hematopoietic stem cells (HSC) to the bone marrow (BM) is a crucial step in hematopoietic development and BM repopulation, the mechanisms underlying these processes have not been fully clarified. Recent studies suggest that interaction between the chemokine receptor CXCR4 and its ligand, stromal cell-derived factor 1 (SDF-1), plays a critical role in these processes. In addition, dextran sulfate increases plasma SDF-1 levels in mice and nonhuman primates. Thus, we examined the effects of preconditioning with SDF-1 and dextran sulfate on the homing efficiency of HSCs following BM transplantation in mice. We found that the preconditioning of donor mice with either SDF-1 or dextran sulfate enhanced the homing efficiency of infused HSCs in vivo. The greatest effects were obtained with dextran sulfate. Moreover, reverse transcriptase polymerase chain reaction analysis demonstrated that SDF-1 and dextran sulfate increased transcription of a variety of homing-related genes, including those for CXCR4, lymphocyte function associated antigen-1, matrix metalloproteinase-9, very late antigen-4/5, and macrophage inflammatory protein-1. We suggest that whereas SDF-1 directly acts to upregulate CXCR4 expression in HSCs, dextran sulfate acts via multiple pathways involved in the induction of various homing-related molecules, in addition to SDF-1. Thus, preconditioning donors with dextran sulfate offers a novel clinical approach for improving the homing and engraftment of HSCs in the BM.

A case of anhidrotic ectodermal dysplasia presenting with pyrexia, atopic eczema, and food allergy.

Anhidrotic ectodermal dysplasia (AED) is a rare hereditary disorder with a triad of sparse hair, dental hypoplasia, and anhidrosis. Here we report a case of AED with food allergy and atopic eczema. The patient was a 11-month-old boy admitted to our hospital with pyrexia for 2 weeks. He presented with a history of dry skin, eczema, and food allergy to egg. On clinical examination, his body temperature was 38.8°C, with dry skin and eczema almost all over the body, sparse eyebrows, and scalp hair. Laboratory investigations and physical examination did not show any evidence of infection. Radioallergosorbent test was positive to egg yolk, egg white, ovomucoid, milk, house dust, and house dust mite. As the child did not sweat despite the high fever, we performed the sweat test which revealed a total lack of sweat glands. Genetic examination revealed a mutation of the EDA gene and he was diagnosed as AED. His pyrexia improved upon cooling with ice and fan. His mother had lost 8 teeth and her sweat test demonstrated low sweating, suggestive of her being a carrier of AED. Atopy and immune deficiencies have been shown to have a higher prevalence in patients with AED. Disruption of the skin barrier in patients with AED make them more prone to allergic diseases such as atopic eczema, bronchial asthma, allergic rhinitis and food allergy. Careful assessment of the familial history is essential to differentiate AED when examining patients with pyrexia of unknown origin and comorbid allergic diseases.

Therapy-related Secondary Malignancy After Treatment of Childhood Malignancy: Cases from a Single Center.

BACKGROUND: Therapeutic outcomes for childhood malignancy have dramatically improved. However, secondary malignancies are a major concern, as they greatly affect the quality of life of survivors. This retrospective study evaluated the cumulative incidence, clinical features, and outcomes of secondary malignancies at Nippon Medical School Hospital. METHODS: We examined data from 275 cases of primary childhood malignancy diagnosed between 1980 and 2014. Information regarding treatment of the primary malignancy, including irradiation dose, site, and cumulative dose of anticancer drugs, was assessed. We also collected data on secondary malignancy, including patient sex, age at diagnosis, malignancy site, time from primary to secondary malignancy, and outcomes. RESULTS: Secondary malignancies developed in 11 patients and included acute myeloid leukemia (AML) (4), meningioma (4), Ewing sarcoma (1), germ cell tumor (1), and malignant parotid gland tumor (1). The primary malignancies included acute lymphoblastic leukemia (ALL) (9), non-Hodgkin lymphoma (1) and brain tumor (1). In 7 of the 9 ALL patients, chemoradiotherapy was the primary treatment. The meningiomas and 1 solid tumor developed within the radiation field. All AMLs and meningiomas developed within 5 years and after 20 years, respectively, of the primary diagnosis. The 10- and 20-year cumulative incidence rates for secondary malignancy in our hospital were 1.9% and 5.8%, respectively. CONCLUSIONS: Our results revealed that the type of secondary malignancy depends on the interval after the end of treatment for primary malignancy. Meningioma, notably, develops many years after completion of primary malignancy treatment. Early detection during long-term follow-up is therefore essential.

AAV1 mediated co-expression of formylglycine-generating enzyme and arylsulfatase a efficiently corrects sulfatide storage in a mouse model of metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by a deficiency of arylsulfatase A (ASA) and is characterized by deposition of sulfatide in all organs, particularly the nervous system. Recently, formylglycine-generating enzyme (FGE) was found to be essential for activation of sulfatases. This study examined the utility of FGE co-expression in AAV type 1 vector (AAV1)-mediated gene therapy of ASA knockout (MLD) mice. AAV1-ASA alone or AAV1-ASA and AAV1-FGE were co-injected into a single site of the hippocampus. Enzyme assay and immunohistochemical analysis showed that ASA was detected not only in the injected hemisphere but also in the non-injected hemisphere by 7 months after injection. Level of ASA activity and extent of ASA distribution were significantly enhanced by co-introduction of AAV1-FGE. Marked reductions in sulfatide levels were observed throughout the entire brain. The unexpectedly widespread distribution of ASA may be due to a combination of diffusion in extracellular spaces, transport through axons, and circulation in cerebrospinal fluid. The rotarod test revealed improvement of neurological functions. These results demonstrate that direct injection of AAV1 vectors expressing ASA and FGE represents a highly promising approach with significant implications for the development of clinical protocols for MLD gene therapy.