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Citrullinated fibrinogen (C-Fbg) has been detected in rheumatoid arthritis; however, few studies have reported the role of C-Fbg in other inflammatory diseases. This study aimed to clarify the changes in serum C-Fbg associated with the bacteremia phase.We measured serum C-Fbg concentration in bacteremia patients. C-Fbg levels at each phase of bacteremia, classified by white blood cell (WBC) count and neutrophil left shift change, were compared with those of healthy control (HC). The correlation between C-Fbg concentration and certain inflammatory markers, or citrullinated histone H3 concentration was assessed. Multiple linear regression (MLR) analysis was used to examine the association of log C-Fbg with certain inflammatory markers.Serum C-Fbg levels were significantly higher in bacteremia patients than in HC (p 0.001) and positively correlated with WBC and neutrophil count. Further, C-Fbg levels were significantly higher in phases III and IV of bacteremia than in HC (p 0.001). MLR analysis indicated that log C-Fbg had a stronger relationship with log neutrophil counts than other certain inflammatory markers (p 0.01).Serum C-Fbg levels increased in bacteremia patients, and this was consistent with an influx of neutrophils into the blood stream in accordance with the bacteremia phase.

Backgrounds Eosinophilic gastrointestinal disorders (EGID) are a group of conditions with increased eosinophilic infiltration in any part of the gastrointestinal tract. Although an allergic reaction to certain foods is considered the main cause of EGID, their detailed pathomechanism has not yet been elucidated, nor have proper management strategies been fully established. Moreover, some patients with intractable EGID are resistant to such therapies as an empirical elimination diet and corticosteroids. Methods We retrospectively analyzed the medical records of 4 children with intractable eosinophilic gastroenteritis (EGE) managed with elemental diet therapy (EDT) using an amino acid-based formula. Results All patients displayed resolution of their symptoms after 2 weeks of EDT. Three patients successfully completed food reintroduction and could return to their normal life. No adverse events related to EDT were recorded. Conclusions EDT appears effective and safe for treating pediatric intractable EGE symptoms. Afterwards, the patient's dietary habits should be considered during carefully monitored food reintroduction. With the relatively small number of published case reports and no randomized trials, more study is needed on EDT for EGE.

Although the effectiveness of subarachnoid continuous drug infusion has been established in cancer pain management, its clinical use in children is rare. A 14-year-old girl with neurofibromatosis type I complained of right leg pain stemming from a growing tumor on her right buttock. Continuous and breakthrough right leg pain were unbearable, even at high doses of systemic opioids that caused severe constipation and deep sedation. Subsequent continuous infusion of bupivacaine and morphine through a subarachnoid catheter effectively relieved the girl's pain. The corresponding decrease in systemic opioid also improved her activities of daily living. The patient eventually died of cachexia due to the rapidly growing buttock lesion that was pathologically confirmed post-mortem as a malignant peripheral nerve sheath tumor. Subarachnoid continuous drug infusion may be very useful in controlling severe pain with few side-effects, even in the field of pediatric palliative care.

In the spring of 2015, we experienced a cluster of 4 sporadic cases of yersiniosis in children in Nagano prefecture, a rural area of Japan. Two patients developed appendicitis-like episodes; one had acute gastroenteritis, and the other had bacteremia associated with liver abscess. The causative agent of these infections was Yersinia enterocolitica serogroup O:8. None of the patients had an underlying illness, and all have recovered completely. The patients were neither socially nor geographically related to each other. These 4 consecutive cases suggest that Y. enterocolitica O:8 has spread substantially in the middle part of Japan, and that this virulent strain might be more common than previously reported in our country.

Rubinstein-Taybi syndrome (RSTS) is a multisystem developmental disorder characterized by facial dysmorphisms, broad thumbs and halluces, growth retardation, and intellectual disability. In about 8% of RSTS cases, mutations are found in EP300. Previously, the EP300 mutation has been shown to cause the highly variable RSTS phenotype. Using exome sequencing, we identified a de novo EP300 frameshift mutation in a proband with coloboma, facial asymmetry and imperforate anus with minimal RSTS features. Previous molecular studies have demonstrated the importance of EP300 in oculogenesis, supporting the possibility that EP300 mutation may cause ocular coloboma. Since a wide phenotypic spectrum is well known in EP300-associated RSTS cases, the atypical phenotype identified in our proband may be an example of rare manifestations of RSTS.

To report the characteristics of Japanese shaken baby syndrome (SBS) in a regional Japanese children's hospital and verify previously reported idiosyncratic features that differ from those of SBS in Western countries: (1) a considerably higher frequency of mothers as perpetrators, (2) older ages of abusive parents, and (3) a higher rate of premature infant birth.We reviewed medical charts obtained between 2002 and 2012 at Nagano Children's Hospital, Japan.Thirty-seven SBS cases with abusive head trauma were found, among which 11 (30 %) of the perpetrators were mothers, 9 (24 %) fathers, 3 (8 %) brothers, and 14 (37 %) involved both parents. A history of premature birth was present in 2 (5 %) of the cases. The mean age of the mother was 32.4 years and that of the father was 31.3 years.The clinical characteristics of Japanese SBS in this study are largely comparable to those seen in Western countries. Compared with the previously reported idiosyncratic features, there was a decreased predominance of mothers as perpetrators of SBS, suggestive of an increasing culpability of fathers. We also noticed that the ratio of premature births was more similar to those seen in Western surveys, while the mean ages of abusive Japanese parents remained older. Such varying results warrant a further nationwide survey.

In the spring of 2015, we experienced a cluster of 4 sporadic cases of yersiniosis in children in Nagano prefecture, a rural area of Japan. Two patients developed appendicitis-like episodes; one had acute gastroenteritis, and the other had bacteremia associated with liver abscess. The causative agent of these infections was Yersinia enterocolitica serogroup O:8. None of the patients had an underlying illness, and all have recovered completely. The patients were neither socially nor geographically related to each other. These 4 consecutive cases suggest that Y. enterocolitica O:8 has spread substantially in the middle part of Japan, and that this virulent strain might be more common than previously reported in our country.

We report a six-year-old girl with seizures induced by both micturition and defecation. Several days after unprovoked generalised tonic-clonic seizures, she developed reflex seizures characterised by the extension of both arms and rhythmic jerking of her upper body. No abnormal findings were noted on brain magnetic resonance imaging. Interictal electroencephalography (EEG) showed spike-and-wave activity on central electrode recording, and rhythmic fast activity was recorded by central electrodes during the ictal EEG upon micturition. The combination of clobazam and phenytoin was effective for both unprovoked and reflex seizures. Although some previous reports have described reflex seizures triggered by either micturition or defecation, this is the first case report of reflex seizures induced by both micturition and defecation in the same patient. Based on a comparison with previous cases of reflex seizures induced either by micturition or defecation, the neuronal pathway from the pelvic base musculature to the supplementary motor area may be responsible for the condition in our patient.

Summary Purpose: De novo STXBP1 mutations have been found in individuals with early infantile epileptic encephalopathy with suppression-burst pattern (EIEE). Our aim was to delineate the clinical spectrum of subjects with STXBP1 mutations, and to examine their biologic aspects. Methods: STXBP1 was analyzed in 29 and 54 cases of cryptogenic EIEE and West syndrome, respectively, as a second cohort. RNA splicing was analyzed in lymphoblastoid cells from a subject harboring a c.663 + 5G>A mutation. Expression of STXBP1 protein with missense mutations was examined in neuroblastoma2A cells. Results: A total of seven novel STXBP1 mutations were found in nine EIEE cases, but not in West syndrome. The mutations include two frameshift mutations, three nonsense mutations, a splicing mutation, and a recurrent missense mutation in three unrelated cases. Including our previous data, 10 of 14 individuals (71%) with STXBP1 aberrations had the onset of spasms after 1 month, suggesting relatively later onset of epileptic spasms. Nonsense-mediated mRNA decay associated with abnormal splicing was demonstrated. Transient expression revealed that STXBP1 proteins with missense mutations resulted in degradation in neuroblastoma2A cells. Discussion: Collectively, STXBP1 aberrations can account for about one-third individuals with EIEE (14 of 43). These genetic and biologic data clearly showed that haploinsufficiency of STXBP1 is the important cause for cryptogenic EIEE.

Fukuyama T, Tanaka M, Nakazawa Y, Motoki N, Inaba Y, Higuchi T, Koike K. Prophylactic treatment for hypertension and seizure in a case of allogeneic hematopoietic stem cell transplantation after posterior reversible encephalopathy syndrome. Pediatr Transplantation 2011: 15: E169–E173. © 2010 John Wiley & Sons A/S. Abstract: A six-yr-old boy developed PRES after induction chemotherapy for the relapse of acute lymphoblastic leukemia. Two months after PRES, he underwent BMT from an unrelated HLA-mismatched donor. There were many risk factors for PRES in the BMT including the long-term use of FK506 and methylprednisolone, grade III graft-versus-host disease, thrombotic microangiopathy, and sepsis. Prophylactic treatment for hypertension with nicardipine in conjunction with close monitoring of the magnesium level and the use of valproic acid might be an effective management approach to prevent post-transplant PRES.

Ets1 is a member of the Ets winged helix-turn-helix transcription factor family. Ets transcription factors are activators and repressors of transcription, important in the development of organisms throughout the Metazoa (9, 10, 22, 38, 48, 50). Previous Ets1 knockout mice show partial perinatal lethality. Surviving mice and chimeras derived from Ets1-targeted cells exhibit pan-lymphoid defects. These defects include reduced numbers of thymocytes, lymphoid cells in the spleen, and NK and NKT cells; loss of detectable NK and NKT cell activity; decreased proliferation and elevated apoptosis in mature T cells (4, 6, 40, 58); increased differentiation of B cells to plasma cells with elevated serum immunoglobulin M; B-cell receptor signaling defects (12); abnormalities in T-cell receptor (TCR) β-selection in thymopoiesis; and an ineffective Th1 response (11, 19). Complicating the assessment of Ets1 function is the fact that Ets1-null mice have been studied not only on a mixed genetic background (C57BL/6 × 129Sv) predisposed to autoimmunity (53) but also with a line of Ets1-null mice that expresses a very low level of a neomorphic protein (∼1 to 5%) lacking the pointed domain of Ets1 (7, 59). These Ets1-null mice present with a deficiency in CD8+ thymocyte development and an elevated proportion of single-positive effector memory (CD62L− CD44+) cells in both CD4+ and CD8+ peripheral T-cell populations (7). The mammalian Ets1 gene produces two major protein isoforms, full-length p51-Ets1 and p42-Ets1, which arise from alternative splicing of exon VII of Ets1 hnRNA with no disruption in the reading frame (5, 24, 29). The p51-Ets1 and p42-Ets1 isoforms have common and distinctive physical properties that allow for both functional redundancy and isoform-specific activities. Shared domains include the Pointed (PNT), transactivation, and ETS DNA binding domains. The domain encoded by exon VII has been identified as a negative regulator and modulator of DNA binding, a mediator of homo- and heterotypic protein-protein interactions, and a target of calcium-mediated phosphorylation (2, 8, 14, 28, 46). Collectively, these distinctions contribute to the Ets1 isoform-specific modulation of particular Ets1 target genes. Exon VII-mediated homotypic and AML1 (Runx1)-Ets1 heterotypic protein-protein interactions augment transcription of MMP-3 and granulocyte-macrophage colony-stimulating factor, respectively (2, 35, 36). Conversely, other target genes, such as the VE-cadherin gene, are transcriptionally activated more strongly by the p42-Ets1 variant in a cell context-specific manner (33). These distinctions are physiologically relevant; ectopic expression of the p42-Ets1 isoform but not the full-length p51-Ets1 isoform in DLD-1 colon cancer cells restores Fas-mediated apoptosis (32). A similar proapoptotic role for the p42-Ets1 isoform has been suggested for MDA-MB-231 invasive breast cancer cells (3). While much work characterizing the functions of Ets1 has been done, relatively little is known about the interplay between the two protein isoforms in mediating these functions in vivo. To extend the functional assessment of Ets1 protein isoforms in vivo, we have generated Ets1ΔVII gene-targeted mice, which express only the p42-Ets1 isoform (lacking the full-length p51-Ets1 isoform). Analyses of Ets1ΔVII mice have demonstrated a requirement for the full-length p51-Ets1 isoform in the regulation of lymphopoiesis and homeostasis. Genes important in these processes and misexpressed in Ets1ΔVII homozygotes include those for p16Ink4a, p27Kip1, and CD44 and provide possible mechanisms for the phenotypes observed.

Targeted disruption of the Friend leukemia integration 1 (Fli-1) proto-oncogene results in severe dysmegakaryopoiesis and embryonic lethality. We used morula-stage aggregation as a strategy to further clarify the hematopoietic defects of the Fli-1 gene-targeted mice. Analyses of lineage expression of Fli-1+/- and Fli-1-/- cells in the peripheral blood and bone marrow of chimeric mice consistently demonstrated reduced numbers of neutrophilic granulocytes and monocytes and increased numbers of natural killer (NK) cells. Transplantation studies using sorted Fli-1 mutant cells produced similar findings. Clonal culture studies of bone marrow cells revealed increased numbers of granulocytic and early erythroid progenitors in the Fli-1+/- cells. The sorted Fli-1-/- bone marrow cells revealed specific down-regulation of CCAAT/enhancer binding protein-α (C/EBPα) and C/EBPϵ, and the receptors for granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF), consistent with their critical roles in granulopoiesis. Collectively, these observations suggest previously unknown physiologic roles for Fli-1 in granulocytic, erythroid, and NK cell proliferation and differentiation. Production of chimeras by morula-stage embryo aggregation is an effective way to unravel cell-autonomous hematopoietic defects in gene-targeted mice.

We encountered a pediatric case of bacteremia and possible cholecystitis due to Moraxella osloensis that was treated successfully. We confirmed the diagnosis with the presence of a high serum titer of the antibody to the organism. Furthermore, 16S rRNA sequencing was performed to identify the bacteria.

The Ets family of transcription factors is one of a growing number of master regulators of development. This family was originally defined by the presence of a conserved DNA binding domain, the Ets domain. To date, nearly 30 members of this family have been identified and implicated in a wide range of physiological and pathological processes. Despite the likely importance of Ets-family members, each of their precise roles has not been delineated. Herein, we describe the elucidation of essential functions of a few of these family members in vivo using knockout mouse models. Of the knockouts generated to date, the majority shows important functions in hematopoiesis, ranging from PU.1, a principle regulator of myelo-lymphopoiesis, to Spi-B which regulates the proper function of terminally differentiated cells. Ets1 was shown to be of intermediate importance as a regulator of pan-lymphoid development. Other Ets family members such as Fli1 and TEL1 display distinct and/or overlapping functions in vasculo/angiogenesis, hemostasis and hematopoiesis. The remaining knockouts generated, Ets2 and Er81, show non-hematopoietic defects related to extraembryonic development and neurogenesis, respectively. The pioneering group of knockout models described reveals only the most distinct functions of each of these Ets family members. A better understanding of the roles and hierarchies of Ets family members in cellular differentiation will come with the generation of new null alleles in previously untargeted family members, more mutant alleles in members already disrupted, double knockouts, ES cell differentiation and chimera rescue experiments, and tissue-specific inducible knockouts.

We examined the effects of retinoids on the human mast cell development using a serum-deprived culture system. When 10-week cultured mast cells derived from CD34+ cord blood cells were used as target cells, both all-trans retinoic acid (ATRA) and 9-cis RA inhibited the progeny generation under stimulation with stem cell factor (SCF) in a dose-dependent manner (the number of progeny grown by SCF plus RA at 10−7 mol/L was one tenth of the value obtained by SCF alone). The early steps in mast cell development appear to be less sensitive to RA according to the single CD34+c-kit+ cord blood cell culture study. The optimal concentration of RAs also reduced the histamine concentration in the cultured mast cells (3.00 ± 0.47 pg per cell in SCF alone, 1.44 ± 0.18 pg per cell in SCF+ATRA, and 1.41 ± 0.10 pg per cell in SCF+9-cis RA). RT-PCR analyses showed the expression of RAR, RARβ, RXR, and RXRβ messenger ribonucleic acid (mRNA) in 10-week cultured mast cells. The addition of an RAR-selective agonist at 10−10 mol/L to 10−7 mol/L decreased the number of mast cells grown in SCF, whereas an RXR-selective agonist at up to 10−8 mol/L was inactive. Among RAR subtype selective retinoids used at 10−9 mol/L to 10−7 mol/L, only the RAR agonist was equivalent to ATRA at 10−7 mol/L in its ability to inhibit mast cell growth. Conversely, the addition of excess concentrations of a RAR antagonist profoundly counteracted the retinoid-mediated suppressive effects. These results suggest that RA inhibits SCF-dependent differentiation of human mast cell progenitors through a specific receptor.

Background, To assess the clinical application of thrombopoietin (TPO) for thrombocytopenia of patients receiving cord blood (CB) or bone marrow (BM) transplants, we examined whether various types of hematopoietic progenitors including megakaryocyte (MK) progenitors from CB and BM exerted different proliferative and differentiative potential in the presence of TPO. Methods. The development of MK, granulocyte-macrophage, and erythroid/mixed erythroid (E/Mix) progenitors in a serum-deprived liquid culture medium supplemented with TPO was compared between CD34 + CB and BM cells. Results. The CD34 + CB cells generated 30-fold more MKs than the CD34 + BM cells, but the CB-derived MKs were more immature. A single-cell culture study showed that CB CD34 + CD38 - cells as well as CD34 + CD38 + cells proliferated in response to TPO, whereas the two subpopulations of CD34 + BM cells showed little multiplication. In short-term liquid cultures containing CD34 + CB or BM cells, TPO significantly increased the absolute numbers of various types of colony-forming cells, compared with the input values. In particular, MK progenitors and E/Mix progenitors in CB were amplified to a substantially greater extent than in BM. The superior response of CD34 + CB cells to TPO observed in this study may be due in part to the use of cryopreserved cells. Conclusions. Our results suggest that TPO alone cannot only stimulate megakaryocytopoiesis but also increase the numbers of various types of hematopoietic progenitors, and that quantitative and qualitative differences in TPO-dependent hematopoietic progenitor development exist between CB and BM.

The cytoskeletal and/or nuclear matrix molecules responsible for morphological changes associated with apoptosis were identified using monoclonal antibodies (mAbs). We developed mAbs against Triton X-100-insoluble components of HL-60 cells pretreated with all-trans retinoic acid. In particular, one mAb recognized a 22-kDa protein that exhibited intriguing behavior by forming an aggregate and appearing as a speck during apoptosis induced by retinoic acid and other anti-tumor drugs. Cloning and sequencing of its cDNA revealed that this protein comprises 195 amino acids and that its C-terminal half has a caspase recruitment domain (CARD) motif, characteristic of numerous proteins involved in apoptotic signaling. We referred to this protein as ASC (apoptosis-associated speck-like protein containing a CARD). The ASC gene was mapped on chromosome 16p11.2-12. The antisense oligonucleotides of ASC were found to reduce the expression of ASC, and consequently, etoposide-mediated apoptosis of HL-60 cells was suppressed. Our results indicate that ASC is a novel member of the CARD-containing adaptor protein family.

To clarify the roles of megakaryocytes and platelets in the responses associated with infection and inflammation, we examined the effects of interleukin (IL) 1, the common mediator of the inflammatory process, on the development and secretory functions of megakaryocytes generated from CD34(+)cord blood cells under stimulation with thrombopoietin (TPO). The addition of IL-1alpha did not influence the generation, endomitosis or expression of surface makers of megakaryocytes, compared with TPO alone. However, IL-1alphaenhanced the ability of megakaryocytes to produce IL-8 and growth-regulating oncogene-alpha(GRO-alpha) in the presence of TPO. In contrast, the production of regulated on activation with normal T cell expressed and secreted (RANTES), platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG) were not potentiated. A flow cytometric analysis and a reverse transcription-polymerase chain reaction analysis revealed IL-1 receptor type I (IL-1RI) expression of megakaryocytes generated by TPO. Moreover, the addition of an anti-IL-1RI monoclonal antibody significantly decreased the TPO plus IL-1alpha-induced secretion of IL-8 by the cultured megakaryocytes to the level attained by TPO alone. These results suggest that the production of IL-8 and GRO-alpha (but not RANTES), PF4 and beta-TG, by megakaryocytes is potentiated by signalling through IL-1RI with the aid of TPO. Thus, megakaryocytes and platelets may play an important role in the development of inflammation via chemokine release.

We previously reported the aberrant growth of granulocyte-macrophage (GM) progenitors induced by a combination of stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in juvenile chronic myelogenous leukemia (JCML). We examined here the effects of thrombopoietin (TPO) on the proliferation and differentiation of hematopoietic progenitors in JCML. In serum-deprived single-cell cultures of normal bone marrow (BM) CD34+CD38high cells, the addition of TPO to the culture containing SCF + GM-CSF resulted in an increase in the number and size of GM colonies. In the JCML cultures, in contrast, the number of SCF + GM-CSF–dependent GM colonies was not increased by the addition of TPO. However, the TPO addition caused an enlargement of GM colonies in cultures from the JCML patients to a significantly greater extent compared with the normal controls. There was no difference in the type of the constituent cells of GM colonies with or without TPO grown by JCML BM cells. A flow cytometric analysis showed that the c-Mpl expression was found on CD13+ myeloid cells generated by CD34+CD38high BM cells from JCML patients, but was at an undetectable level in normal controls. The addition of TPO to the culture containing SCF or SCF + GM-CSF caused a significant increase in the production of GM colony-forming cells by JCML CD34+CD38neg/lowpopulation, indicating the stimulatory effects of TPO on JCML primitive hematopoietic progenitors. Normal BM cells yielded a significant number of megakaryocytes as well as myeloid cells in response to a combination of SCF, GM-CSF, and/or TPO. In contrast, megakaryocytic cells were barely produced by the JCML progenitors. Our results may provide a fundamental insight that the administration of TPO enhances the aberrant growth of GM progenitors rather than the recovery of megakaryocytopoiesis.

We recently established a human granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent cell line (HML) from colony-constituent cells grown by peripheral blood cells of a patient with acute megakaryoblastic leukaemia. The HML cells possessed megakaryocytic features, as determined by cytochemical, electron microscopic and flow cytometric analysis. In the present study we examined the effects of retinoic acid (RA) on the development of HML cells. All-trans-RA, 13-cis-RA and 9-cis-RA at 10−8 mol/l to 10−5 mol/l inhibited the GM-CSF-dependent cell growth. Some of the RA-treated cells contained prominent azurophilic granules and were positive for peroxidase. They also reacted with Biebrich scarlet, Luxol fast blue and a monoclonal antibody against eosinophil peroxidase. In addition, exposure to RA increased the frequency and the intensity of major basic protein-positive cells. However, eosinophil-derived neurotoxin and eosinophil cationic protein were not detected or were only detected at a low level in the lysates of the HML cells treated with RA. Although IL-5 alone could not stimulate cell growth, the addition of IL-5 to the cultures containing stem cell factor + all-trans-RA was required for the expression of the eosinophilic phenotype. These results suggest that the HML cell line is a megakaryoblastic cell line with the potential to differentiate into the eosinophilic lineage. HML cells may be a useful model for elucidating the eosinophilic differentiation programme.

In normal hematopoiesis, stem cell factor (SCF) stimulates survival, proliferation and differentiation of hematopoietic progenitors. Although SCF acts synergistically with a variety of cytokines, the mechanism of growth factor-cooperation remains to be determined. To analyze the synergism between SCF and granulocyte-macrophage colony-stimulating factor (GM-CSF), we established a new megakaryoblastic cell line, HML-2, by culture in the presence of both SCF and GM-CSF. While SCF alone or GM-CSF alone supported modest cell growth, SCF and GM-CSF together induced substantial growth of this cell line. SCF alone tyrosine-phosphorylated several bands including the 145 kDa subunit of c-kit. GM-CSF alone did not cause the tyrosine phosphorylation of the 145 kDa subunit, but markedly up-regulated the expression of the 145 kDa subunit of c-kit. The combination of SCF and GM-CSF resulted in a synergistic increase in tyrosine phosphorylation of the 145 kDa subunit of c-kit. Several proliferation inhibitors which removed the two-factor interaction on the growth of the HML-2 cells down-regulated the 145 kDa subunit of c-kit. Thus, a synergistic increase in tyrosine phosphorylation of the 145 kDa subunit of c-kit may be one possible mechanism underlying the cooperation of SCF and GM-CSF on the HML-2 cell growth.

Health examination programs for five-year-old children are aimed at effectively detecting developmental disorders, such as attention deficit/hyperactivity disorders (AD/HD), learning disorders (LD), higher functioning autistic spectrum disorders (HFASD), and other abnormalities. Tests usually include a questionnaire and observation of group playing, verbal communication, and soft neurological signs; however, it is often difficult to detect children who have LD with visual cognitive dysfunctions through such conventional examination techniques. Here, we analyzed the efficacy of using a battery of visual cognitive function tests to identify such cases.We employed four simple tests to evaluate visual cognitive function in addition to a standard health examination for five-year-old between April 2008 and March 2010. To analyze visual cognitive function tests, the results were scored and the applicability of these tests was verified by comparisons with established tests.A total of 653 five-year-old children underwent health examinations, and 48 children were referred to the hospital for further examinations. As a result, 34 children were newly diagnosed with developmental disorders, including HFASD, AD/HD, LD, and mild intellectual disturbances. Strong correlations were seen between the scores of these four examinations and those of other established tests, such as the performance intelligence score, the perceptual organization index of WISC-III, and the Frostig visual development test score. An additional benefit of our method was that parents could easily recognize developmental disorders in their children through direct observation of these examinations.We concluded that the battery of visual cognitive function tests was simple and useful for detecting developmental disorders in the health examinations of five-year-old children.

Background: Myoclonic astatic epilepsy (MAE) is an idiopathic and generalized childhood epileptic syndrome. Although several studies have reported on cognitive function and intellectual outcome in MAE, little is known about the behavioral problems associated with this disease. The aim of this study was to clarify behavior and cognitive function in children with MAE. Methods: Four children who were diagnosed as having MAE using the proposed criteria of the International Classification of Epilepsies were retrospectively analyzed using patient records with regard to clinical and neuropsychological findings such as age of seizure onset, semiology and severity of seizures, treatment course, behavioral problems, EEG findings, and WISC-III and Pervasive Developmental Disorders Autism Society Japan Rating Scale (PARS) scores that indicate the tendency of autistic behaviors. Results: Disease onset in our cohort ranged from 9 to 35 months of age. Seizures were controlled within 3-8 months over a follow-up period of 4-12 years. All patients had borderline normal intelligence (mean IQ: 75.8 at 5-7 years of age) and exhibited impaired coordination, clumsiness, hyperactivity or impulsivity, and impaired social interaction after improvement of seizures. Though these autistic social problems manifested gradually, the children were able to adapt to school life with appropriate support. Interestingly, the behaviors of impaired social interaction and hyperactivity or impulsivity had already been observed before the onset of MAE in most patients, whose PARS scores were all higher than reference values (

In the treatment of Kawasaki disease, resistance to high-dose immunoglobulin intravenous (IGIV) can occur. The neutrophil morphology analyses in 17 patients revealed that transient pseudo-Pelger-Huët anomaly was more frequently detected in the IGIV-resistant group. This finding may aid the prediction of IGIV resistance.

Acute encephalitis with refractory, repetitive partial seizures (AERRPS) is a peculiar form of encephalitis mainly affecting children. Although not usually lethal, we report a case of sudden unexpected death in epilepsy (SUDEP) 3 years after the onset of AERRPS. A 6-year-old boy was admitted to our hospital because of fever and extremely refractory partial and secondary generalized seizures with delirium and psychiatric change. The seizures were highly resistant to anticonvulsants and suppressed only by large dose intravenous administration of midazolam. Seven months after the onset, the seizures were ameliorated by treatment with potassium bromide and clorazepate. After the acute phase, the patient developed complex partial seizures that tended to present with cyanosis. At the age of 10, he was found lying prone in respiratory arrest with facial pallor. Although he regained cardiac function after being taken to our emergency room, the patient died 12 days later. Six SUDEP cases after the onset of AERRPS, including this one, have been reported to date. Since epilepsy following AERRPS is one of the risk factors of SUDEP, clinicians should consider SUDEP to be a rare but high risk syndrome in AERRPS-afflicted children.

We report two cases of symptomatic West syndrome with severe respiratory syncytial virus (RSV)-induced bronchiolitis: one was a 9-month-old boy who was hospitalized for shock, and the other was a 15-month-old boy in pre-shock condition. Both cases needed mechanical ventilation for approximately 2 weeks. Seizures from the primary disease worsened in both patients during the infection, and both needed long periods of hospitalization, which resulted in a considerable reduction in their quality of life and that of their families. According to a one-year epidemiological survey of RSV infection conducted in 2004-2005 in Nagano prefecture, 7 of 238 hospitalized RSV cases were found to have basic neuromuscular disorders. Compared to patients with chronic lung disease or other primary diseases, they were older, had higher incidence of mechanical ventilation, and required longer hospitalization. Neuromuscular disorders may thus be an important risk factor for severe forms of RSV infection. Although children with such disorders should be protected from RSV, they are currently excluded from the indication for palivizumab administration as passive immunization against RSV in Japan.

De novo STXBP1 mutations have been found in individuals with early infantile epileptic encephalopathy with suppression-burst pattern (EIEE). Our aim was to delineate the clinical spectrum of subjects with STXBP1 mutations, and to examine their biologic aspects.STXBP1 was analyzed in 29 and 54 cases of cryptogenic EIEE and West syndrome, respectively, as a second cohort. RNA splicing was analyzed in lymphoblastoid cells from a subject harboring a c.663 + 5GA mutation. Expression of STXBP1 protein with missense mutations was examined in neuroblastoma2A cells.A total of seven novel STXBP1 mutations were found in nine EIEE cases, but not in West syndrome. The mutations include two frameshift mutations, three nonsense mutations, a splicing mutation, and a recurrent missense mutation in three unrelated cases. Including our previous data, 10 of 14 individuals (71%) with STXBP1 aberrations had the onset of spasms after 1 month, suggesting relatively later onset of epileptic spasms. Nonsense-mediated mRNA decay associated with abnormal splicing was demonstrated. Transient expression revealed that STXBP1 proteins with missense mutations resulted in degradation in neuroblastoma2A cells.Collectively, STXBP1 aberrations can account for about one-third individuals with EIEE (14 of 43). These genetic and biologic data clearly showed that haploinsufficiency of STXBP1 is the important cause for cryptogenic EIEE.

A six-yr-old boy developed PRES after induction chemotherapy for the relapse of acute lymphoblastic leukemia. Two months after PRES, he underwent BMT from an unrelated HLA-mismatched donor. There were many risk factors for PRES in the BMT including the long-term use of FK506 and methylprednisolone, grade III graft-versus-host disease, thrombotic microangiopathy, and sepsis. Prophylactic treatment for hypertension with nicardipine in conjunction with close monitoring of the magnesium level and the use of valproic acid might be an effective management approach to prevent post-transplant PRES.

Chimerism analysis by polymerase chain reaction amplification of short tandem repeats (PCR-STR) has become a routine diagnostic procedure for evaluating grafts and assessing the likeliness of original disease recurrence after allogeneic stem cell transplantation. Following a sex-mismatched hematopoietic stem cell transplantation (HSCT), we monitored the clinical course of a 61-year old male AML M6 patient with trisomy 8 using PCR-STR with a TH01 locus on 11p15 and fluorescence in situ hybridization (FISH) analysis specific for alpha satellite DNA on chromosome 8. Ten months after HSCT, FISH analysis showed 24.8% recipient cells, but PCR-STR demonstrated 100% donor type chimerism. Further XY FISH analysis of May-Grünwald-Giemsa-stained bone marrow samples clearly demonstrated relapse of the original disease and G-banding analysis of bone marrow samples at relapse showed that an additional chromosomal abnormality, del(11) (p10), had deleted the PCR-STR detection site in all recipient type cells. As such, clinicians should consider the possibility that unexpected karyotype changes may invalidate PCR-STR analysis findings, especially when conflicting results appear among chimerism analyses.

It is thought that the nuclear nonchromatin structures, such as the nuclear matrix and lamina, play regulatory roles in gene expression. In this study, we identified an insoluble protein that was associated with the chromatin-depleted nuclear structure of proliferating human leukemia HL-60 cells. Preparation of the chromatin-depleted nuclear structure, referred to as the nuclear matrix-intermediate filament scaffold (Fey, E., Krochmalnic, G., and Penman, S. (1986) J. Cell. Biol. 102, 1654-1665), involved cell extraction using a series of buffers containing Triton X-100, DNase I, and 2 M NaCl. A yeast two-hybrid assay revealed that this protein bound to the catalytic subunit of protein phosphatase-1 (PP1). Furthermore, it inhibited PP1 activity in vitro. We therefore named it scapinin (scaffold-associated PP1 inhibiting protein). cDNA cloning revealed that scapinin had two splicing variants of 448 amino acids (scapinin-S) and 518 amino acids (scapinin-L). Scapinin was down-regulated by differentiation in HL-60 cells. These results suggest that scapinin is a putative regulatory subunit of PP1 and is involved in transformed or immature phenotypes of HL-60 cells. We also describe the presence of scapinin family proteins from worm to human.

Forward and reverse genetic approaches facilitate the molecular dissection of individual gene functions and the integration of individual gene functions into multi-gene processes in the context of the whole organism. Variations in mutant phenotypes due to genetic background differences have been well documented through the analysis of mouse mutants. Nevertheless, recommendations concerning the assessment of genetic background as it impacts on phenotype, and utilization of genetic background differences to identify and integrate gene functions have been largely overlooked. Genetic background as it relates to immunological mutants will be discussed utilizing an Ets1-targeted allele to exemplify phenotypic variation due to background. Marker-assisted strategies for the identification of genetic modifiers, especially those linked to the targeted locus, will also be considered.

The regulation mechanism of circulating thrombopoietin (TPO) level in human newborns remains unknown. In the present study, we examined whether the TPO concentrations in cord blood were influenced by the difference in the delivery method and the presence or absence of maternal/fetal complications. Cortisol levels were simultaneously measured to assess the adrenal response of fetuses. Both the TPO level and the cortisol level were substantially greater in the neonates delivered vaginally with and without the complications than in those delivered by cesarean section without the complications. The binding assay showed that the incubation of mpl+/BaF3 cells with cortisol gave rise to a significant decrease in the binding sites of TPO. These results suggest that the stress to the fetuses near the time of delivery affects the cord blood TPO levels, which may be mediated in part by the action of cortisol on the TPO-mpl binding system.

We examined the effects of granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), and thrombopoietin (TPO), alone or in combination, on the generation of neutrophils by bone marrow (BM) cells from three patients with severe congenital neutropenia (SCN) through the use of a serum-deprived liquid culture system. Synergistic effects of G-CSF and SCF on the neutrophil production by BM CD34+CD38+c-kit+ cells were observed in SCN patients as well as in normal controls. The addition of TPO to the culture containing G-CSF and SCF further augmented the growth of neutrophils in the two groups. Single-cell culture experiments revealed that the three-factor combination caused increases in both the number and size of neutrophil colonies compared with G-CSF + SCF in normal BM cells, whereas only a significant increment in the colony size was observed in SCN patients. Even in the presence of SCF or SCF + TPO, the concentrations of G-CSF necessary for the substantial production of neutrophils by CD34+CD38+c-kit+ cells were higher in two patients compared with the levels obtained by normal control cells. In addition, TPO did not accelerate the maturation of neutrophilic cells supported by G-CSF + SCF. When BM CD34+CD38−c-kit+ cells were targeted, the addition of TPO to the culture containing G-CSF and SCF was required for significant neutrophil colony growth in the two groups. These results suggest that TPO enhances the G-CSF-dependent neutrophil production with the aid of SCF in this disorder.

In the present study, we investigated the effects of stem cell factor (SCF) and/or thrombopoietin (TPO) on the cell production by cord blood CD34+ cells using a serum-deprived liquid culture system. Although SCF alone supported a modest production of neutrophilic cells and a remarkable generation of mast cells, the addition of TPO to the culture containing SCF caused an apparent generation of neutrophilic cells, identified by immunocytochemical staining and flow cytometric analysis. The significant production of neutrophilic cells by SCF and TPO was persistently observed from 2 weeks to 2 to 3 months of culture. The interaction between SCF and TPO on the neutrophilic cell generation was greater than the combined effects of SCF with granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). The addition of neutralizing antibody against G-CSF or GM-CSF did not influence the SCF + TPO-dependent neutrophilic cell production. A single-cell culture study showed that not only CD34+CD38+ c-kit+ cells but also CD34+CD38−c-kit+ cells were responsible for the neutrophilic cell generation. In clonal cell cultures, GM progenitors as well as erythroid progenitors and multipotential progenitors expanded in the cultures supplemented with SCF and TPO. The neutrophilic cells grown by SCF + TPO were at myeloblast to band cell stages, and scarcely matured to segmented neutrophils. In addition, the cells generated by SCF + TPO were stained with monoclonal antibodies against myeloperoxidase, elastase, lactoferrin, and CD11b, but they had negligible levels of alkaline phosphatase (ALP) and CD35. The replating of the CD34−c-kit−/low CD15+ cells grown by SCF + TPO into a culture containing SCF + G-CSF permitted both the terminal maturation into segmented cells and the appearance of ALP and CD35. These results indicate the existence of a G-CSF/GM-CSF–independent system of neutrophilic cell production.

In a serum-free liquid culture, thrombopoietin (TPO) selectively stimulated the growth of megakaryocytic cells from CD34-positive cord blood cells. Using these cultured cells, we investigated cytokine production by human megakaryocytes. Day 10 megakaryocytes (2 x 10(5)) secreted > 1000 pg/ml of interleukin (IL)-8, in contrast to small amounts of IL-1beta and IL-6. A time-course study showed that the IL-8 production of megakaryocytes occurred at the late phase of the culture period. The megakaryocyte-conditioned medium had the chemotactic potential of polymorphonuclear leucocytes, which was abrogated by the addition of anti-IL-8 antibody, suggesting the secretion of biologically active IL-8. The combination of TPO and IL-1alpha was required for a significant augmentation of the IL-8 secretion. Direct evidence for IL-8 synthesis in megakaryocytes was provided by reverse transcription-polymerase chain reaction on purified CD41b+ cells and by the detection of intracellular IL-8 in CD41b+ cells. These results suggest that TPO stimulates not only the proliferation and differentiation of the progenitors capable of megakaryocytic lineage expression but also IL-8 release by the megakaryocytic cells with the aid of IL-1.

At 6 months of age, the patient was diagnosed as having neuroblastoma stage IV and was given the chemotherapy, local irradiation, and operation. The treatment was completed in September 1989. In 1992, at 6 years of age, her height was -3 SD and growth hormone secretion was depressed. She had been supplemented with recombinant human growth hormone (rhGH). Because the white blood cell counts began to decrease gradually in 1993, the rhGH therapy was interrupted on January 19, 1994. The rhGH supplement was resumed after a 3-month interval because of the parent's desire. Pancytopenia soon became apparent. She was diagnosed as having myelodysplastic syndrome (MDS) as a refractory anemia with an excess of blasts in transformation with monosomy 7. The rhGH therapy was interrupted again, without any improvement of the MDS. In culture studies, neither rhGH nor insulin-like growth factor-1 stimulated proliferation of her bone marrow cells. These data suggested that the treatment with rhGH after the chemotherapy played some role in the promotion, but not acceleration, of the MDS.