Gonadotropin levels in urine during early postnatal period in small for gestational age preterm male infants with fetal growth restriction.

OBJECTIVE: The objective of this study was to estimate gonadotropin concentrations in small for gestational age (SGA) male infants with the reactivation of the hypothalamic-pituitary-gonadal axis during the first few months of life that is important for genital development. STUDY DESIGN: We prospectively examined 15 SGA and 15 appropriate for gestational age (AGA) preterm male infants between 2013 and 2014 at Kyoto University Hospital. Gonadotropin concentrations (luteinizing hormone (LH) and follicle-stimulating hormone (FSH)) were measured in serial urine samples from the postnatal days 7 to 168 and compared between SGA and AGA infants using the Mann-Whitney test. RESULTS: A longitudinal analysis showed that SGA infants had higher LH and lower FSH concentrations (P=0.004 and P=0.006, respectively) than AGA infants. CONCLUSION: Male infants who are SGA at birth because of fetal growth restriction have gonadotropin secretion abnormalities in the first few months of life.

CD146 is a novel marker for highly tumorigenic cells and a potential therapeutic target in malignant rhabdoid tumor.

Malignant rhabdoid tumor (MRT) is a rare, highly aggressive pediatric malignancy that primarily develops during infancy and early childhood. Despite the existing standard of intensive multimodal therapy, the prognosis of patients with MRT is dismal; therefore, a greater understanding of the biology of this disease is required to establish novel therapies. In this study, we identified a highly tumorigenic sub-population in MRT, based on the expression of CD146 (also known as melanoma cell adhesion molecule), a cell adhesion molecule expressed by neural crest cells and various derivatives. CD146+ cells isolated from four MRT cell lines by cell sorting exhibited enhanced self-renewal and invasive potential in vitro. In a xenograft model using immunodeficient NOD/Shi-scid IL-2Rγ-null mice, purified CD146+ cells obtained from MRT cell lines or a primary tumor exhibited the exclusive ability to form tumors in vivo. Blocking of CD146-related mechanisms, either by short hairpin RNA knockdown or treatment with a polyclonal antibody against CD146, effectively suppressed tumor growth of MRT cells both in vitro and in vivo via induction of apoptosis by inactivating Akt. Furthermore, CD146 positivity in immunohistological analysis of 11 MRT patient samples was associated with poor patient outcomes. These results suggest that CD146 defines a distinct sub-population in MRT with high tumorigenic capacity and that this marker represents a promising therapeutic target.

Response to thyrotropin-releasing hormone stimulation tests in preterm infants with transient hypothyroxinemia of prematurity.

OBJECTIVE: Whether hormone supplementation is necessary for infants with transient hypothyroxinemia of prematurity (THOP) remains controversial, and further analysis of the hypothalamus-pituitary-thyroid axis of infants with THOP is necessary. STUDY DESIGN: Thyrotropin-releasing hormone (TRH) stimulation tests were performed at 2 weeks of age in 50 infants with a gestational age of 30 weeks or less, and the data were analyzed retrospectively. RESULT: Subjects were divided into three groups; group A consisted of euthyroid infants, group B consisted of infants with THOP and group C consisted of hypothyroid infants. The basal and peak thyroid-stimulating hormone level of group C in response to TRH stimulation tests was significantly higher than the others, but no differences were observed between groups A and B. CONCLUSION: The response of infants with THOP to the TRH stimulation test was not different from that of euthyroid infants, which suggested that their hypothalamic-pituitary-thyroid axis was appropriately regulated in infants with THOP.

In vitro expansion of CD34(+)CD38(-) cells under stimulation with hematopoietic growth factors on AGM-S3 cells in juvenile myelomonocytic leukemia.

Using serum-containing culture, we examined whether AGM-S3 stromal cells, alone or in combination with hematopoietic growth factor(s), stimulated the proliferation of CD34(+) cells from patients with juvenile myelomonocytic leukemia (JMML). AGM-S3 cells in concert with stem cell factor plus thrombopoietin increased the numbers of peripheral blood CD34(+) cells to approximately 20-fold of the input value after 2 weeks in nine JMML patients with either PTPN11 mutations or RAS mutations, who received allogeneic hematopoietic transplantation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) also augmented the proliferation of JMML CD34(+) cells on AGM-S3 cells. The expansion potential of CD34(+) cells was markedly low in four patients who achieved spontaneous hematological improvement. A large proportion of day-14-cultured CD34(+) cells were negative for CD38 and cryopreservable. Cultured JMML CD34(+)CD38(-) cells expressed CD117, CD116, c-mpl, CD123, CD90, but not CXCR4, and formed GM and erythroid colonies. Day-7-cultured CD34(+) cells from two of three JMML patients injected intrafemorally into immunodeficient mice stimulated with human GM-CSF after transplantation displayed significant hematopoietic reconstitution. The abilities of OP9 cells and MS-5 cells were one-third and one-tenth, respectively, of the value obtained with AGM-S3 cells. Our culture system may provide a useful tool for elucidating leukemogenesis and for therapeutic approaches in JMML.

Subcutaneous fat accumulation in early infancy is more strongly associated with motor development and delay than muscle growth.

AIM: Physical growth in neurologically healthy preterm infants affects motor development. This study investigated the separate relationships between muscle and fat in infancy and later motor development and physical growth. METHODS: Muscle thickness and subcutaneous fat thickness of the anterior thigh were measured using ultrasound images obtained from neurologically healthy preterm infants at birth, 3, 6, 12 and 18 months' corrected age. We also obtained the Pediatric Evaluation of Disability Inventory and Alberta Infant Motor Scale scores at 18 months' corrected age to assess motor ability and motor delay. RESULTS: Thirty preterm infants completed the study protocol. There was a significant positive correlation between motor ability and increments in subcutaneous fat thickness during the first 3 and 6 months' corrected age (r = 0.48 and 0.40, p < 0.05, respectively), but not between motor ability and muscle thickness growth in any of the periods. A secondary, logistic regression analysis showed that increments in subcutaneous fat thickness during the first 3 months were a protective factor for motor delay. CONCLUSION: Subcutaneous fat accumulation in early infancy is more strongly associated with motor development and delay than muscle growth.

[Stem cell and self-renewal].

Stem cells are defined as cells with the ability for self-renewal and differentiation. Hematopoietic stem cells are well known, and their application is useful for the treatment of various kinds of diseases. Recently, neural stem cells have been identified even in the adult brain, which has up to now been considered to be a tissue with no regenerative capacity. In addition, it has emerged that tissue stem cells can differentiate into various kinds of cells beyond their original characteristics. Here, we discuss the self-renewal mechanisms of embryonic stem (ES) cells, hematopoietic stem cells and neural stem cells.

Characterization of the gammac chain among 27 unrelated Japanese patients with X-linked severe combined immunodeficiency (X-SCID).

X-linked severe combined immunodeficiency (X-SCID) is a rare fatal disease that is caused by mutations in the gene encoding the gammac chain. In this study, 27 unrelated Japanese patients with X-SCID were examined in terms of their genetic mutations and surface expression of the gammac chain. Among 25 patients examined, excluding two patients with large deletions, 23 different mutations were identified in the IL2RG gene, including 10 novel mutations. One patient bearing an extracellular mutation and all three of the patients bearing intracellular mutations after exon 7 expressed the gammac chain on the cell surface. Overall, 84% of patients lacked surface expression of the gammac chain leading to a diagnosis of X-SCID.

Chimeric cytokine receptor can transduce expansion signals in interleukin 6 receptor alpha (IL-6Ralpha)-, IL-11Ralpha-, and gp130-low to -negative primitive hematopoietic progenitors.

We generated transgenic mice expressing chimeric receptors, which comprise extracellular domains of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor and transmembrane and cytoplasmic domains of the mouse leukemia inhibitory factor receptor. In suspension cultures of lineage-negative (Lin(-)), 5-fluorouracil-resistant bone marrow cells of the transgenic mice, a combination of hGM-CSF and stem cell factor (SCF) induced exponential expansions of mixed colony-forming unit. The combination of hGM-CSF and SCF was effective on enriched, Lin(-)Sca-1(+)c-kit(+) progenitors and increased either mixed colony-forming unit or cobblestone area-forming cells. In case of stimulation with hGM-CSF and SCF, interleukin-6 (IL-6) and SCF, or IL-11 and SCF, the most efficient expansion was achieved with hGM-CSF and SCF. When Lin(-)Sca-1(+)c-kit(+)CD34(-) further enriched progenitors were clone sorted and individually incubated in the presence of SCF, hGM-CSF stimulated a larger number of cells than did IL-6, IL-6 and soluble IL-6 receptor (IL-6R), or IL-11. These data suggest the presence of IL-6Ralpha-, IL-11Ralpha-, and gp130-low to -negative primitive hematopoietic progenitors. Such primitive progenitors are equipped with signal transduction molecules and can expand when these chimeric receptors are genetically introduced into the cells and stimulated with hGM-CSF in the presence of SCF.

Hypomethylation of the proximal and intronic regulatory regions of the IFN-gamma gene is not essential for its transcription by naive CD4+ T cells cultured with IL-4.

Recently, long-term preculture with IL-4 or IL-7 has been reported to induce IFN-gamma-producing ability in naive CD4+ T cells without stimulation via TCR. The mechanism of IFN-gamma-transcription in naive CD4+ T cells precultured with IL-4 was analyzed and compared with that in typical Th1 cells by focusing on the TATA proximal and first intronic regulatory regions of the IFN-gamma gene. Both regulatory regions in these IL-4-primed naive CD4+ T cells, which produce a large amount of IFN-gamma upon stimulation with PMA and ionomycin, were completely methylated in contrast to the same hypomethylated regions in Th1 cells. DNase I hypersensitive site analysis suggested that both regulatory regions in IL-4-primed naive CD4+ T cells were not active for IFN-gamma-expression. Moreover, we demonstrated that the composition of transcriptional factors that can bind to the proximal regulatory region is different between IL-4-primed naive CD4+ T cells and Th1 cells. These results indicated that the transcriptional machinery involved in the expression of the IFN-gamma gene by CD4+ T cells varied depending on their modes of differentiation in both the responsive regulatory regions and the specific nuclear factors.

STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells.

Embryonic stem (ES) cells can be maintained in an undifferentiated state in the presence of leukemia inhibitory factor (LIF). LIF acts through a receptor complex composed of a low affinity LIF receptor (LIFRbeta) and gp130. We reported that the intracellular domain of gp130 plays an important role in self-renewal of ES cells. In the present study, we examined the signaling pathway through which gp130 contributes to the self-renewal of ES cells. Mutational analysis of the cytoplasmic domain of gp130 revealed that the tyrosine residue of gp130 responsible for STAT3 activation is necessary for self-renewal of ES cells, while that required for SHP2 and MAP kinase activation was dispensable. Next, we constructed a fusion protein composed of the entire coding region of STAT3 and the ligand binding domain of the estrogen receptor. This construction (STAT3ER) induced expression of junB (one of the targets of STAT3) in ES cells in the presence of the synthetic ligand 4-hydroxytamoxifen (4HT), thereby indicating that STAT3ER is a conditionally active form. ES cells transfected with STAT3ER cultured in the presence of 4HT maintained an undifferentiated state. Taken together, these results strongly suggest that STAT3 activation is required and sufficient to maintain the undifferentiated state of ES cells.

Cytoplasmic domains of the leukemia inhibitory factor receptor required for STAT3 activation, differentiation, and growth arrest of myeloid leukemic cells.

Leukemia inhibitory factor (LIF) induces growth arrest and macrophage differentiation of mouse myeloid leukemic cells through the functional LIF receptor (LIFR), which comprises a heterodimeric complex of the LIFR subunit and gp130. To identify the regions within the cytoplasmic domain of LIFR that generate the signals for growth arrest, macrophage differentiation, and STAT3 activation independently of gp130, we constructed chimeric receptors by linking the transmembrane and intracellular regions of mouse LIFR to the extracellular domains of the human granulocyte macrophage colony-stimulating factor receptor (hGM-CSFR) alpha and betac chains. Using the full-length cytoplasmic domain and mutants with progressive C-terminal truncations or point mutations, we show that the two membrane-distal tyrosines with the YXXQ motif of LIFR are critical not only for STAT3 activation, but also for growth arrest and differentiation of WEHI-3B D+ cells. A truncated STAT3, which acts in a dominant negative manner was introduced into WEHI-3B D+ cells expressing GM-CSFRalpha-LIFR and GM-CSFRbetac-LIFR. These cells were not induced to differentiate by hGM-CSF. The results indicate that STAT3 plays essential roles in the signals for growth arrest and differentiation mediated through LIFR.

Activation of Stat3 by cytokine receptor gp130 ventralizes Xenopus embryos independent of BMP-4.

Stat3 is one of the main signaling components of cytokine receptors, including gp130. Here we show that activation of cytokine receptor gp130 resulted in a dramatic ventralization of Xenopus embryos and that the ventralization correlated well with Stat3 activation potential of the receptor. This finding led to identification of Xenopus Stat3 (Xstat3), which showed a 95% homology to its murine and human counterparts, at the amino acid level, and was expressed from the one-cell stage throughout development. The mechanism of gp130/XStat3-mediated ventralization proved to be independent of BMP-4. gp130/Xstat3 stimulation inhibited Smad2-induced ectopic axis formation in embryos and Smad2-dependent luciferase activity. A dominant-negative Stat3, in contrast, dorsalized Xenopus embryos, resulting in ectopic axis formation. We propose that Stat3-mediated signaling has the capacity to modify dorsoventral patterning in the early development of Xenopus.

IL-4 and prostaglandin E2 inhibit hypomethylation of the 5' regulatory region of IFN-gamma gene during differentiation of naive CD4+ T cells.

We have previously shown that prostaglandin E2 (PGE2) and IL-4 inhibit the priming of IFN-gamma-production during the differentiation of naive CD4+ T cells from human cord blood by different signal-transducing mechanisms. To compare and analyse the molecular mechanisms by which PGE2 and IL-4 inhibit the priming of IFN-gamma production, we investigated the effects of PGE2 and IL-4 on the methylation of the IFN-gamma gene during the in vitro differentiation of naive CD4+ T cells. In human naive CD4+ T cells, which produce primarily IL-2 and a little amount of IFN-gamma, the IFN-gamma gene was methylated. After stimulation via TCR, CD4+ T cells produced IFN-gamma and the CpG dinucleotide contained within the TATA proximal regulatory element of the IFN-gamma gene was partially hypomethylated. Both IL-4 and PGE2 inhibited the hypomethylation of this site and the acquisition of IFN-gamma-producing ability. In contrast to the SnaBI site in the TATA proximal regulatory element, the HpalI site in the first intron of the IFN-gamma gene of the CD4+ T cells from cord blood was completely methylated even after stimulation via TCR. 5-azacytidine restored the IFN-gamma-producing ability of these cells treated with IL-4 and PGE2. These findings suggest that, although the signal transduction that inhibits the priming of IFN-gamma-production is different for each reagent, the protection from hypomethylation of the regulatory region of the IFN-gamma gene is involved in the molecular mechanisms by which these reagents inhibit the priming of IFN-gamma-production during the differentiation of human naive CD4+ T cells.

A selective switch-on system for self-renewal of embryonic stem cells using chimeric cytokine receptors.

Propagation of embryonic stem (ES) cells with an undifferentiated pluripotential phenotype depends on leukemia inhibitory factor (LIF). The LIF receptor complex is composed of a heterodimer of LIF receptor alpha (LIFR alpha) and gp130. To activate LIFR signaling pathways independently from endogenous ones, we constructed chimeric receptors by linking the extracellular domain of human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha or beta (hGMR alpha or beta) to the transmembrane and cytoplasmic regions of either mouse LIFR alpha or gp130. hGMR alpha-mLIFR/hGMR beta-mgp130 or hGMR alpha-mgp130/hGMR beta-mgp130, but not hGMR alpha-mLIFR/hGMR beta-mLIFR, preserved the self-renewal activity in A3 ES cells. All of these chimeric receptors were phosphorylated after hGM-CSF stimulation without phosphorylation of endogenous gp130. Phosphorylation of the signal transducer and activator of transcription 3 through chimeric receptors correlated with the undifferentiated phenotype. Therefore, these chimeric receptors prove useful to analyze mechanisms of the self-renewal of ES cells.

X-linked severe combined immunodeficiency with gamma delta T cells.

A patient with X-linked severe combined immunodeficiency (X-SCID) was found to have a deletion mutation of a four base pair in the transmembrane domain of the IL-2 receptor gamma chain gene, a subunit shared by the receptors for IL-4, IL-7, IL-9, and IL-15 (common gamma chain; gamma c). He had very few alpha beta T cells but had a considerable number of gamma delta T cells in his peripheral blood. Fluorescence in situ hybridization (FISH) analysis showed that the gamma delta T cells in his peripheral blood were not of maternal origin. He had received a Bacillus Calmette-Guerin (BCG) vaccination before recognition of the disease, and the BCG infection remained quiescent with no reaction for 19 months. After successful bone marrow transplantation, the site of the BCG vaccination showed a reaction, and live BCG were detected. It is useful to consider the relationship between the existence of gamma delta T cells and BCG in this case, and it is suggested that gamma delta T cells may be, in a given situation, less dependent on the gamma c chain than are alpha beta T cells.

Negative signaling in B cells by surface immunoglobulins.

Cross-linking of surface immunoglobulins generates negative signals that cause B-cell death unless appropriate rescue signals are provided. Surface IgM is the main transducer of the negative signaling, but surface IgD and IgG may also transduce negative signaling when cross-linked intensively. In the surface IgM+, IgD+ human malignant B lymphoma cell lines B104 and DND-39, cross-linking of surface IgM by anti-IgM antibodies induced cell death. Anti-IgM antibody-induced B104 cell death was inhibited by stimulation with alpha- and beta-interferons but not stimulation with anti-CD40 antibody or IL-4, whereas anti-IgM antibody-induced DND-39 cell death was inhibited by stimulation with anti-CD40 antibody but not stimulation with alpha- and beta-interferons. Anti-IgM antibody-stimulated B104 cells had morphologic features compatible with necrosis, whereas anti-IgM antibody-stimulated DND-39 cells showed morphologic features of apoptosis. CD11a/CD54-dependent cell adhesion induced by stimulation with anti-CD40 antibody was involved in anti-CD40 antibody-mediated inhibition of anti-IgM antibody-induced DND-39 cells. In normal human mature B cells, cross-linking of surface IgM induced different signaling consequences, including DNA synthesis or cell division (positive signaling) or cell cycle arrest or death (negative signaling). In this system, too, CD40-transduced signal inhibited anti-IgM antibody-induced negative signaling, and CD11a/CD54-dependent cell adhesion played a role in the rescue process. It is suggested that quantitatively different intensities of surface IgM cross-linking induce qualitatively different signaling consequences; relatively weak cross-linking may induce DNA synthesis; moderate cross-linking may induce DNA synthesis with cell cycle arrest at the G2/M interphase; and intense cross-linking may induce apoptotic cell death. The reasons for this difference are not yet known. Further elucidation of the molecular mechanisms responsible for surface IgM-mediated negative signaling and its rescue signaling may contribute toward development of therapy for allergic disorders by artificial modulation of specific immunoglobulin production.

Signal transduction pathway of interleukin-4 and interleukin-13 in human B cells derived from X-linked severe combined immunodeficiency patients.

Interleukin-4 (IL-4) and IL-13 are functionally similar cytokines. The functional IL-4 receptor (IL-4R) consists of the IL-4R alpha chain (IL-4R alpha) and the IL-2R gamma chain (gamma c), which is shared by the IL-2, IL-7, IL-9, and IL-15 receptors. The functional IL-13R is thought to involve the IL-4R alpha but not gamma c. In this study, we have analyzed activation of members of the Janus tyrosine kinase (Jak) family and signal transducers and activators of transcription (STAT) 6 induced by IL-4 and IL-13 in Epstein-Barr virus-transformed B cells derived from two patients of X-linked severe combined immunodeficiency, who have mutations of the gamma c gene in the extracellular and intracellular domains. In these B cells, IL-4 failed to induce tyrosine phosphorylation of Jak3 and activation of STAT6, or activation of these molecules was significantly decreased compared with Epstein-Barr virus-transformed normal B cells. In contrast, IL-13 activated STAT6 in these cells as well as normal B cells. However, Jak3 was not activated by IL-13, even in normal B cells. These results clearly indicated that gamma c is essential for activation of Jak3 and STAT6 in the signal transduction pathway of IL-4 in human B cells and that IL-13 does not utilize gamma c but activates STAT6 through an alternative pathway, which is not impaired in B cells of X-linked severe combined immunodeficiency patients.