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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.

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.

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 A retrospective analysis of the records of all 1,503 infants admitted to our NICU during the past 6 years (from April 2013 to March 2019) was performed 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. Of the infants transferred to other facilities, 81% of infants born at our hospital were born to mothers transported to our obstetric 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, both accepting and transferring infants for convalescent care has become an integral part of NICU practice to bridge the gaps between higher level care facility and home. Establishment of well-defined transfer criteria and appropriate allocation of medical and staff resources among relevant facilities is desirable.

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-1β, 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.

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.

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.

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.

Objective: The aim of this study was to examine the effects of a newly established neonatal intensive care unit (NICU on clinical work practice and educational activity at Nippon Medical School Musashikosugi Hospital. Methods: This retrospective study analyzed the clinical records of all neonates admitted to the NICU from December 2010 through November 2013. Anthropometric data clinical status problems and outcomes of patients and the related obstetrical history were extracted and analyzed. Results: Of the 568 neonatal admissions about half were related to preterm birth (49% and low birth weight (55%. Forty‑eight percent of patients were born via caesarean delivery. Maternal hypertension diabetes and thyroid disease were found in 8% 5% and 2% of cases respectively. Mechanical ventilatory support was provided for 20% of patients. Neonates from multiple pregnancy and with significant congenital anomalies accounted for 17% and 10% of all patients respectively. Five patients died during hospitalization. In addition training was provided in the NICU for an average of 10 residents and 20 medical students per year. Conclusion: Since the NICU was established closer cooperation beyond the framework of a single department has come to be needed. In addition NICUs in teaching hospitals are expected to provide opportunities for medical students and residents to observe and participate in multidisciplinary medical care. (J Nippon Med Sch 2014; 81: 328―332

Peters' plus syndrome is a rare autosomal recessive condition characterized by a combination of typical ocular defects and other systemic abnormalities. We present a case of this uncommon syndrome that we diagnosed during a fetal ultrasonographical examination. Because the patient exhibited microcephaly and anterior staphyloma of the right eye and because impending rupture was feared, we performed ophthalmectomy during the neonatal period. Fetal ophthalmological anomalies are often detected during ultrasonographic examination targeting other systemic abnormalities, with positive family histories providing important diagnostic clues. This case is, to our knowledge, the first to be reported of prenatally diagnosed Peters' plus syndrome in a patient with no known family history in whom total blindness was prevented with an early referral to specialists.

Hypophosphatasia (HPP), caused by mutations in the gene ALPL encoding tissue-nonspecific alkaline phosphatase (TNALP), is an inherited systemic skeletal disease characterized by mineralization defects of bones and teeth. The clinical severity of HPP varies widely, from a lethal perinatal form to mild odontohypophosphatasia showing only dental manifestations. HPP model mice (Akp2(-/-)) phenotypically mimic the severe infantile form of human HPP; they appear normal at birth but die by 2 weeks of age because of growth failure, hypomineralization, and epileptic seizures. In the present study, we investigated the feasibility of fetal gene therapy using the lethal HPP model mice. On day 15 of gestation, the fetuses of HPP model mice underwent transuterine intraperitoneal injection of adeno-associated virus serotype 9 (AAV9) expressing bone-targeted TNALP. Treated and delivered mice showed normal weight gain and seizure-free survival for at least 8 weeks. Vector sequence was detected in systemic organs including bone at 14 days of age. ALP activities in plasma and bone were consistently high. Enhanced mineralization was demonstrated on X-ray images of the chest and forepaw. Our data clearly demonstrate that systemic injection of AAV9 in utero is an effective strategy for the treatment of lethal HPP mice. Fetal gene therapy may be an important choice after prenatal diagnosis of life-threatening HPP.

Hemoglobin H (HbH) disease is the severe nonfatal form of α-thalassemia syndrome. It is usually caused by molecular defects of 3 of 4 α-globin genes (--/-α) which cause α-globin expression to be decreased. HbH disease is rare in Japan. Here, we report on a 6-year-old girl with HbH disease who had profound hypochromatic and microcytic anemia. Analysis of the α-globin genes of the patient's family showed that the father, who was Japanese, had an abnormal gene with a 3.7-kb deletion (-α(3.7)/αα), and the mother, who was Filipino, had a deletion removing both α-globin genes of the Filipino type (--(FIL)/αα). Neither parent had anemia. The patient was found to have HbH disease with a heterozygous genetic abnormality (--(FIL)/-α(3.7)). Recently, the number of marriages of Japanese to natives of areas where thalassemia is epidemic has increased. Therefore, the incidence of HbH disease can be expected to increase in Japan. Long-term follow-up will be needed to evaluate the long-term complications and to improve the quality of life of patients with HbH disease.

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.

We describe an infant with nemaline myopathy accompanied by hypertrophic cardiomyopathy. The patient required long-term, intermittent positive-pressure ventilation after birth owing to muscle weakness, and cardiac failure developed during infancy. We diagnosed hypertrophic cardiomyopathy with outflow tract obstruction, and treated the heart failure with beta-adrenergic and angiotensin II receptor blockers. We discuss the prognosis of nemaline myopathy combined with hypertrophic cardiomyopathy.

Metachromatic leukodystrophy (MLD) is an autosomal recessive disease caused by mutations in the gene encoding the lysosomal enzyme arylsulfatase A (ASA). In MLD, accumulation of the substrate, sulfated glycoprotein, in the central and peripheral nervous systems results in progressive motor and mental deterioration. Neural progenitor cells are thought to be useful for cell replacement therapy and for cell-mediated gene therapy in neurodegenerative diseases. In the present study, we examined the feasibility of ex vivo gene therapy for MLD using neural progenitor cells. Neural progenitor cells (neurospheres) were prepared from the striatum of E14 embryo MLD knockout mice or GFP transgenic mice and were transduced with the VSV pseudotyped HIV vector carrying the ASA gene (HIV-ASA). For in vivo study, neurospheres from GFP mice were transduced with HIV-ASA and inoculated into the brain parenchyma of adult MLD mice. HIV vector-transduced progenitor cells retained the potential for differentiation into neurons, astrocytes and oligodendrocytes in vitro. Expression of ASA in neurospheres transduced with HIV-ASA was confirmed by spectrophotometric enzyme assay and Western blotting. In vivo, GFP-positive cells were detectable 1 month after injection. These cells included GFAP- and MAP2-positive cells. Immunohistochemistry using anti-ASA antibody demonstrated localization of ASA in both GFP-positive and -negative cells. Partial clearance of accumulated sulfatide was confirmed in vivo in MLD knockout mice. The present findings suggest that ASA enzyme is released from migrated neurospheres and is able to digest sulfatide in surrounding cells. Our results suggest the potential of genetically engineered neural progenitor cells (neurospheres) for ex vivo therapy in MLD.

We report on an infant with Beals syndrome (congenital contractural arachnodactyly [CCA], MIM 121050) with transient cardiomyopathy showing ballon-like dilatation of the left ventricle that was similar to noncompaction. The patients father and two of his brothers were also found to have CCA without cardiovascular complications. CCA, which is caused by a mutation of the gene for fibrillin 2 protein is similar to Marfan syndrome (MIM 154700), which is caused by a mutation of fibrillin 1 but produces a life-threatening cardiovascular complications. This is the first report of CCA with transient cardiomyopathy. We discuss the mechanism of the spontaneous improvement of cardiomyopathy in this case on the basis of expression of the responsible gene.

We report an asymptomatic female with Fabry disease immunohistochemically diagnosed by renal biopsy. She was initially diagnosed as having nephrotic syndrome, and renal biopsy was performed for pathological diagnosis. The renal specimen revealed non-specific findings (minor glomerular abnormalities) for nephrotic syndrome. Numerous laminated bodies in glomerular epithelial cells in electron microscopic findings and accumulations of ceramidetrihexoside immunohistochemically were observed and she was diagnosed with Fabry disease. However, no other laboratory data or clinical findings supported the diagnosis of Fabry disease. Since the efficacy of recombinant human alpha-galactosidase replacement therapy in this disease has been reported, whether enzyme replacement therapy for subclinical Fabry female patients is indicated or not is an important issue.

The prognosis and clinical and biological characteristics of infant leukemia differ from those of leukemia in children 1 year or older. We reviewed the charts of patients younger than 1 year in whom leukemia was diagnosed from January 1981 through December 2003 at our institution. Fourteen infants had leukemia 6 had acute lymphoblastic leukemia（ALL） and 8 had acute myeloid leukemia（AML） . The age of patients at diagnosis ranged from 2 to 11 months. Five of 8 AML patients presented with cutaneous manifestations such as erythema and nodules at diagnosis. Central nervous system（CNS）involvement was seen in 1 AML patient at diagnosis. Hyperleukocytosis of more than 50×10 9 L was seen in 4 of 6 ALL patients and in 4 of 8 AML patients at diagnosis. All ALL patients showed a morphological diagnosis of L1 using the French‑America‑British classification system. For patients with AML the morphological diagnoses were M0 for 1 patient M2 for 1 patient M4 for 2 patients（1 with eosinophilia） M5b for 2 patients and M7 for 2 patients. One patient showing M7 morphology had Down syndrome. Surface markers were examined in 5 of 6 ALL patients and all AML patients. Five ALL patients showed a B‑cell precursor immunophenotype. Two of 5 patients with ALL had CD10‑positive leukemic cells and 3 of 5 patients with ALL had CD10‑negative leukemic cells. All AML patients were positive for CD13 or CD33 or both. Three of 5 patients with ALL showed abnormal chromosomes related to 11q. Six of 7 patients with AML showed abnormal karyotypes. MLL gene rearrangements were seen in 3（2 ALL 1 AML）of 5（2 ALL 3 AML）patients. Serum immunoglobulin M levels were increased in 9 of 14 patients. Complete remission（CR）was achieved in all infants with ALL. Three patients relaps ed and then died of the original disease. One of these 3 patients died after cord blood transplantation. Three ALL patients are alive without leukemia. CR was achieved in 6 of 8 AML patients. Four of 6 patients are alive without leukemia. Infant leukemia patients in our institution had some special features. CNS involvement at diagnosis was seen in only 1 patient and serum IgM levels were higher than those in children whose leukemia was diagnosed at 1 to 10 years of age. （J Nippon Med Sch 2005; 72: 355―363）

Group B Streptococcus (GBS) is an important pathogen that causes neonatal sepsis and meningitis, which have high mortality and morbidity. Most cases of infection are early onset, with late onset infections being less common. Moreover, many cases of infection are caused by type III GBS, while type Ib GBS infections are rare. We report a case of late-onset infection by type Ib GBS. A female neonate weighing 574 g was delivered at 27 weeks' gestation. An endotracheal tube was inserted shortly after birth because of respiratory distress syndrome, and ampicillin was administered by the age of 3 days. At the age of 54 days after cardiopulmonary adaptation had been achieved, the patient presented with tachycardia following refractory apnea and bradycardia, and her skin became pale. She was suspected of having sepsis, and intensive treatment, including intubation and administration of catecholamines, was started. Despite these measures, the patient died after 5 hours after the onset of sepsis. Type Ib GBS infection may be more frequent in Japanese infants because of the low concentration of IgG antibodies against type Ib in pregnant Japanese women.

We examined whether non-hematopoietic BM cells can migrate into the intestinal graft after fetal small intestinal transplantation (FSITx). Fetal small intestine from donor C57BL/6 mice was transplanted into the rectus abdominis of recipient C57BL/6 mice with only green fluorescent protein (GFP) BM cells (syngeneic FSITx). Intestinal grafts were harvested on days 5, 10, and 30 after FSITx and stained immunohistochemically using anti-CD45 antibody (a marker for hematopoietic BM cells). Although there were no GFP-positive cells identified in the epithelium of the graft intestinal villi, there were a few cells positive for both GFP and CD45 in the lamina propria on day 5 after FSITx, and many present on days 10 and 30. In some grafts there were only cells that were GFP positive/CD45 negative (i.e., non-hematopoietic BM cells) found in the lamina propria on days 10 and 30. These data indicate that non-hematopoietic BM cells as well as hematopoietic BM cells can migrate from the recipient's bone marrow, suggesting that recipient mesenchymal stem cells may be strongly implicated in graft regeneration and development after FSITx.

Bone marrow cells contain at least two distinct types of stem cells which are haematopoietic stem cells and mesenchymal stem cells. Both cells have the ability to differentiate into a variety of cell types derived from all three germ layers. Thus, bone marrow stem cells could possibly be used to generate new pancreatic beta cells for the treatment of diabetes. In this study, we investigated the feasibility of bone marrow-derived cells to differentiate into beta cells in pancreas.Using green fluorescent protein transgenic mice as donors, the distribution of haematogenous cells in the pancreas was studied after bone marrow transplantation.In the pancreas of green fluorescent protein chimeric mice, green fluorescent protein-positive cells were found in the islets, but none of these cells expressed insulin. Previous data has suggested that tissue injury can recruit haematopoietic stem cells or their progeny to a non-haematopietic cell fate. Therefore, low-dose streptozotocin (30 or 50 mg/kg on five consecutive days) was injected into the mice 5 weeks after bone marrow transplantation, but no green fluorescent protein-positive cells expressing insulin were seen in the islets or around the ducts of the pancreas.Our data suggests that bone marrow-derived cells are a distinct cell population from islet cells and that transdifferentiation from bone marrow-derived cells to pancreatic beta cells is rarely observed.

We investigated the role of circulating bone marrow cells (BMC) in cardiomyocyte regeneration. BMC, isolated from transgenic mice expressing enhanced green fluorescent protein (GFP), were transplanted into lethally irradiated C57BL6 mice. Five weeks after bone marrow transplantation (BMT), flow cytometric analysis for GFP-positive cells confirmed reconstitution of transplanted bone marrow. Bone marrow transplant mice subsequently underwent left coronary artery ligation (myocardial infarction) or sham-operation, and were killed at 1 mo or 3 mo after operation. Infarct size was similar in bone marrow transplant mice at 1 mo (47.1 +/- 5.9%) and at 3 mo (45.3 +/- 7.8%), and echocardiography at 2 and 8 wk revealed decreasing left ventricular function. In infarcted heart, GFP-positive cells that expressed desmin and troponin T-C were identified by confocal microscopy. GFP and troponin T-C double-positive cells were predominantly in the peri-infarcted region (1 mo, 365 +/- 45 cells/50 sections; 3 mo: 458 +/- 100 cells/50 sections; p0.05 versus noninfarct, infarct, and sham-operated regions). Furthermore, BMC mobilization and differentiation into cardiomyocytes was found to be complete within 1 mo after myocardial infarction. These results demonstrate that circulating BMC undergo mobilization and differentiation in cardiac cells after myocardial infarction. Future studies are required to determine the molecular signaling mechanisms responsible for this phenomenon.

Studies have indicated that bone marrow contains both hematopoietic stem cells and mesenchymal stem cells that can differentiate into a variety of mesenchymal tissues, such as bone, cartilage, muscle, and adipose tissue. Therefore, bone marrow cells are thought to be very useful for cell and gene therapy for various diseases. However, the multipotentiality of these cells remains unclear. To address this issue, we established a chimeric model mouse stably reconstituted with green fluorescent protein (GFP)-marked bone marrow cells. We injected bone marrow cells from GFP-transgenic C57BL/6 mice into the tail veins of recipient wild-type C57BL/6 mice that had been irradiated with a lethal dose of 10 Gy from a cesium source. Microscopic examination and fluorescence-assisted cell sorter (FACS) analysis showed that bone marrow cells, including mesenchymal cells, were almost completely reconstituted with GFP+ cells 5 weeks after transplantation. FACS analysis with lineage-specific antibodies confirmed that the GFP+ cells could differentiate into all types of blood cells. To confirm the usefulness of this mouse model, we studied the role of circulating bone marrow-derived cells in healing of damaged intestine. We performed amputation and anastomosis of the jejunum 10 cm from the pyloric region of the stomach. On the third day after operation, a large number of GFP+ cells were infiltrated in the area of anastomosis, and these cells were positive for CD45 and F4/80 antigens. In 7 days, several cells became negative for CD45 and F4/80 and positive for alpha smooth muscle actin antigen, which is specific for smooth muscle. This finding suggested that bone marrow-derived cells had differentiated into smooth muscle. Because reconstituted bone marrow cells as opposed to injected bone marrow cells, behave naturally, this model is ideal for studying the multipotentiality of bone marrow cells in vivo.

Fabry disease is a systemic disease caused by genetic deficiency of a lysosomal enzyme, alpha-galactosidase A (alpha-gal A), and is thought to be an important target for enzyme replacement therapy. We studied the feasibility of gene-mediated enzyme replacement for Fabry disease. The adeno-associated virus (AAV) vector containing the alpha-gal A gene was injected into the right quadriceps muscles of Fabry knockout mice. A time course study showed that alpha-gal A activity in plasma was increased to approximately 25% of normal mice and that this elevated activity persisted for up to at least 30 weeks without development of anti-alpha-gal A antibodies. The alpha-gal A activity in various organs of treated Fabry mice remained 5-20% of those observed in normal mice. Accumulated globotriaosylceramide in these organs was completely cleared by 25 weeks after vector injection. Reduction of globotriaosylceramide levels was also confirmed by immunohistochemical and electronmicroscopic analyses. Echocardiographic examination of treated mice demonstrated structural improvement of cardiac hypertrophy 25 weeks after the treatment. AAV vector-mediated muscle-directed gene transfer provides an efficient and practical therapeutic approach for Fabry disease.

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.

Gaucher disease is an autosomal recessively inherited lysosomal storage disease in which a deficiency of glucocerebrosidase is associated with the accumulation of glucocerebroside in reticuloendothelial cells. Clinically, 3 types of Gaucher disease have been defined on the basis of the presence or absence of neurological symptoms. The frequency of gallbladder involvement is reportedly greater in patients with type 1 Gaucher disease than in healthy persons. We report a case of recurrent cholelithiasis and liver failure in a patient with type 2 Gaucher disease who showed severe progressive neurological involvement.

A case of herpes simplex virus (HSV encephalitis in a neonate after delivery from a woman whose genital HSV infection had been treated with acyclovir is reported. The main approach to prevent genital HSV infection in the neonate is interruption of transmission at the time of delivery. Guidelines for prophylactic therapy with acyclovir have been established but the risk of neonatal infection remains. A fever began to develop in a male neonate delivered vaginally from a 35‑year‑old woman. Treatment with intravenous acyclovir was started on the basis of a diagnosis of HSV encephalitis because polymerase chain reaction was positive for HSV in the cerebrospinal fluid. The mother had had a first genital HSV infection during the second trimester but treatment with injected acyclovir had caused the blisters and erosion to resolve by the time of delivery. Important steps for preventing neonatal HSV infection are the appropriate treatment of mothers with a history of genital HSV infection the assessment of delivery methods and the appropriate treatment of neonates. (J Nippon Med Sch 2013; 80: 456―459

Background. Bone marrow transplantation is reportedly effective in preventing the progression of neurological deterioration in lysosomal storage disorders, although the mechanism underlying the therapeutic effects remains to be elucidated. Recent research on stem cell biology suggests that bone marrow cells contain nonhematopoietic stem cells, including brain precursor cells. To evaluate the contribution of bone marrow cells as carriers for cell and gene therapy of neurological disorders, we studied the fate of transplanted bone marrow cells in the adult mouse brain. Methods. Bone marrow cells were genetically marked with a retroviral vector containing the green fluorescence protein gene and then transplanted into irradiated mice by either systemic infusion or direct injection. To identify cell types, brain sections were stained with specific antibodies against neuronal cell markers—neuron specific enolase for neurons, glial fibrillary acidic protein (GFAP) for astrocytes, carbonic anhydrase II (CAII) for oligodendrocytes, and ionized calcium binding adaptor molecule 1 (Iba1) for microglia—and then examined under a confocal microscope. Results. Twenty-four weeks after systemic infusion, transplanted cells expressed Iba1 but none of the other brain cell markers. Conversely, 12 weeks after direct injection, transplanted cells were stained with antibodies against GFAP, CAII, and Iba1. Conclusions. Bone marrow contains cells capable of differentiating into oligodendrocytes, astrocytes, and microglia when exposed to the brain microenvironment. Autologous bone marrow cells may be useful as carriers for ex vivo gene therapy for lysosomal disorders with neurological symptoms.

An 11‑year‑old girl with Kostmann syndrome developed refractory pneumonia. Culture of oral discharge throat‑swab specimens and blood could not identity the causative organism and systemic antimicrobial therapy failed to achieve improvement. We then performed diagnostic bronchoalveolar lavage（BAL）and culture of BAL fluid（BALF）yielded Pseudomonas aeruginosa. Therapeutic BAL using gentamicin produced a striking improvement of her pneumonia. Conclusion: In immunocompromised children with pneumonia BAL helps to identify the causative organism. If the patient is unresponsive to systemic antimicrobial therapy BAL using antimicrobial agents is also worth trying.（J Nippon Med Sch 2001; 68: 340―343）

Background: Simple and efficient method for the selection of transduced cells would greatly facilitate the clinical utilization of retrovirus vectors. We developed a therapeutic bicistronic retrovirus vector for Gaucher disease, MFG-GC-GFP, which contains the human glucocerebrosidase (GC) gene and the green fluorescent protein (GFP) gene of the jellyfish Aequorea victoria as a vital selection marker, and investigated its applicability as gene therapy for Gaucher disease. Methods and Results: A packaging cell line, GP + envAM12, was transfected with MFG-GC-GFP and, thus, produced a high titer recombinant virus (1.0 × 106 c.f.u./mL) in the culture supernatant. The expression level of GFP was correlated with the virus production in cells. The recombinant virus infected skin fibroblasts from a Gaucher patient and a sorted fraction of the cells expressing GFP by flow cytometry exhibited almost a six-fold higher activity of GC than normal fibroblasts. Conclusions: These data indicate that MFG-GC-GFP enables the one-step purification of a transduced fraction of target cells and is, therefore, considered to be a useful therapeutic vector for the experimental gene therapy of Gaucher disease.

Group B Streptococcus (GBS) is an important pathogen that causes neonatal sepsis and meningitis, which have high mortality and morbidity. Most cases of infection are early onset, with late onset infections being less common. Moreover, many cases of infection are caused by type III GBS, while type Ib GBS infections are rare. We report a case of late-onset infection by type Ib GBS. A female neonate weighing 574 g was delivered at 27 weeks' gestation. An endotracheal tube was inserted shortly after birth because of respiratory distress syndrome, and ampicillin was administered by the age of 3 days. At the age of 54 days after cardiopulmonary adaptation had been achieved, the patient presented with tachycardia following refractory apnea and bradycardia, and her skin became pale. She was suspected of having sepsis, and intensive treatment, including intubation and administration of catecholamines, was started. Despite these measures, the patient died after 5 hours after the onset of sepsis. Type Ib GBS infection may be more frequent in Japanese infants because of the low concentration of IgG antibodies against type Ib in pregnant Japanese women.

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.

The aim of the study was to determine factors that affect adverse long-term pulmonary outcome in premature infants.This retrospective analysis was done using 306 clinical records of preterm singleton neonates at32 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.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.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.

An 8 years old Japanese boy born in non-sanguineous parents has been in good health until he was found to have macrothrombocytic thrombocytopenia when he developed purpura after common cold and ingestion of aspirin. His hearing ability and renal function were normal. He had no cataract. Hematological studies revealed normal hemoglobin, normal white cell counts with normal differentials and mild thrombocytopenia (94×103/μl). Numerous giant platelets larger than red cells were identified on blood films. Dohle bodies and other inclusion bodies were not found in his neutrophils. The patient's platelet in platelet-rich plasma did not aggregate spontaneously and caused normal aggregation/agglutination in response to epinephrine, ADP, collagen and ristocetin, respectively. Major platelet membrane glycoproteins such as GPIa/IIa, Ib/IX, IIIa, and so on were indistinguishable from those of normal platelets. Clot retraction and coagulation studies were normal. However, calpain activity in the patient's platelets solubilized with 1% Triton X-100 was found to be 218.3% of that in platelets from normal control donors (n=6) when calculated per platelet and 135.7% of the controls when calculated per protein concentration. The patient's mother also showed moderate numbers of giant platelets and moderately increased activity of platelet calpain. Thus, the patient's platelets can not be classified into any congenital platelet disorder so far known and seem to be a new congenital disease in which giant platelets are associated with increased platelet calpain acitvity.

C1q deficiency is a rare complement deficiency in the early part of the complement cascade. Patients with C1q deficiency have severe recurring life-threatening infections and systemic lupus erythematosus (SLE)-like symptoms. We report on a boy with recurrent life-threatening infections and SLE-like recurrent skin conditions before 2 years of age. Immunological studies revealed an undetectable level of C1q. No abnormality was observed in the urine, but renal biopsy showed segmental granulonephritis. However, the changes observed were atypical for SLE nephritis. This case of C1q deficiency was unusual because the SLE-like symptoms appeared earlier than that normally seen in complement deficiency. Therefore, this case provides insights into the development of autoimmune disease, particularly in the early phase of component deficiency, and in managing renal disease that may develop in the future.

Zinc protoporphyrin (ZPP) is generated in the erythrocytes of patients with lead toxication and iron deficiency anemia by the incorporation of zinc instead of ferrous iron into protoporphyrin. Although the measurement of ZPP has been thought to be a potential screening test for evaluating iron deficiency, no simple and reliable ZPP assay method has been available until recently. A newly developed hematofluorometer allows us to quickly determine the level of ZPP in a drop of unprocessed whole blood. In this study, we measured the concentration of ZPP in 732 children including 22 patients with iron deficiency anemia using this hematofluorometer. The cut off value of whole blood ZPP in children was determined to be 38.2 micrograms ZPP/dl whole blood. The level of ZPP showed a good negative correlation with the concentration of hemoglobin. The specificity and the sensitivity of this assay were 97.2% and 81.8%, respectively. These results indicate that this ZPP assay using the newly-developed hematofluorometer is a practical way to screen for iron deficiency anemia.

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.