Therapy-related myelodysplastic syndrome in childhood: a retrospective study of 36 patients in Japan.

We report here a retrospective analysis of 36 children with therapy-related myelodysplastic syndrome (t-MDS) diagnosed between 1990 and 1999 in Japan. Their median age was 7.7 years and the median latency period for the development of t-MDS was 38.5 months. The primary tumors were hematologic in 15 of the cases and nonhematologic in 21. Chromosomal abnormalities were detected in 32/34(94%) patients: abnormalities of chromosomes 5and/or 7 in 41% and notably, 11q23 abnormalities in 31%. The prognosis of children with t-MDS was very poor as compared to children with primary MDS (5 year survival: 16% versus 54%, p<0.0001).

Thrombopoietin enhances neutrophil production by bone marrow hematopoietic progenitors with the aid of stem cell factor in congenital neutropenia.

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.

Proliferative and differentiative potential of thrombopoietin-responsive precursors: expression of megakaryocytic and erythroid lineages.

We investigated changes in proliferative potential and surface markers during human megakaryocytic differentiation, using megakaryocytic cells grown by thrombopoietin (TPO). Cells grown in response to TPO from CD34+ cord blood cells in a liquid culture system expressed CD41b at a frequency of 92% and CD42b at a frequency of 80% on day 10, whereas cells expressing other lineage markers constituted less than 2.5% of this population. The cultured cells were divided into CD41b-/CD42b-, CD41b+/CD42b-, and CD41b+/CD42b+ cells. Comparison of their respective proliferative potentials showed that CD41b-/CD42b- cells generated megakaryocytic progeny in response to TPO to a lesser extent, but responded to the combination of growth factors (GFs) more intensely than CD41b+/CD42b- cells. Almost all CD41b+/CD42b+ cells failed to undergo cell division. In the culture containing GFs, some CD41b-/CD42b- cells and CD41b+/CD42b- cells gave rise to erythroid as well as megakaryocytic progeny. The potential of these cells to yield erythroid progeny in response to GFs correlated well with their expression of CD34. These results suggest that TPO generates precursors with a potential to differentiate into megakaryocytic and erythroid lineages.

Aberrant growth of granulocyte-macrophage progenitors in juvenile chronic myelogenous leukemia in serum-free culture.

We investigated the properties of granulocyte-macrophage (GM) progenitors obtained from patients with juvenile chronic myelogenous leukemia (JCML). CD34+ bone marrow cells from a patient with JCML, unlike normal bone marrow cells, generated a large number of cells in serum-containing liquid culture without additional hematopoietic factors. In serum-deprived culture, only granulocyte colony-stimulating factor (G-CSF) had a modest stimulatory effect on GM colony growth in normal controls. In contrast, stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3), as well as G-CSF, when tested individually, generated significant numbers of GM colonies in some JCML patients. All two-factor combinations generated significantly more GM colonies in JCML compared with normal controls. In particular, GM-CSF plus SCF exerted an interaction equivalent to the all-factor combination in most patients. Significant differences in the size and constituent cells of GM colonies stimulated by GM-CSF plus SCF were also observed. These results suggest that one possible mechanism for the excessive cell production in JCML is the strong proliferation of GM progenitors induced by hematopoietic factors, especially SCF. According to immunofluorescent analysis, however, it is unlikely that this multiplication is due to an increase in the cell surface expression of c-kit receptors on JCML progenitors.

Terminal morphology of two branches arising from a single stem-axon of pretectal (PSm) neurons in the common carp.

The induction of postsynaptic structures by presynaptic terminals is suggested in a teleost brain. Neurons in the nucleus pretectalis superficialis pars magnocellularis (PSm) in the common carp are known to send fibers to the corpus mamillare (CM) and the nucleus lateralis valvulae (NLV). Individual axons of PSm neurons bifurcate (or give off an axon collateral), both of which reach the target areas in the CM and NLV. The morphology of horseradish peroxidase-labeled terminals in the CM and NLV appears quite different in light microscopy. Terminals in the CM appear as a fine network of beaded (2-4 microns in diameter) fibers, while those in the NLV are larger (8-12 microns in transverse diameter) and cup-shaped, partially enveloping the soma of individual NLV neurons. In electron microscopy, however, these synapses in the CM and NLV share several ultrastructural similarities. Small (0.2 to 0.4-micron thick, 0.4 to 0.7-micron long) spine-like protrusions arising from dendrites in the CM, and from cell bodies in the NLV, invaginate into the axon terminals, and the synaptic junctions are always formed at the base of the protrusion in both areas. Development of this unusual morphology is inferred to be directed from the presynaptic side. The morphological similarity of the spine-like protrusions to the "spinule," which is thought to be formed in response to synaptic activation, is discussed.

Telencephalic ascending gustatory system in a cichlid fish, Oreochromis (Tilapia) niloticus.

Central fiber connections of the gustatory system were examined in a percomorph fish Oreochromis (Tilapia) niloticus by means of the horseradish peroxidase (HRP), biocytin, and carbocyanine dye tracing methods. The primary gustatory areas in tilapia are the facial, glossopharyngeal, and vagal lobes of the medulla. The secondary gustatory nucleus (SGN) is a dumb-bell-shaped structure located in the isthmic region. In the SGN, there are two or three layers of neurons lining the ventromedial periphery of the nucleus and a molecular layer constituting of the major part of the nucleus. The SGN receives bilateral projections from the facial lobes and ipsilateral projections from the glossopharyngeal and vagal lobes. Ascending fibers originating from the SGN form the ipsilateral tertiary gustatory tract. A major part of the tract courses rostrally and terminates ipsilaterally in several diencephalic nuclei: the preglomerular tertiary gustatory nucleus (pTGN), the posterior thalamic nucleus, the nucleus diffusus lobi inferioris, the nucleus centralis of inferior lobe, and the nucleus recessus lateralis. The remaining small fiber bundle enters the medial and lateral forebrain bundles and terminates directly in two telencephalic regions; the area ventralis pars intermedia (Vi) and the area dorsalis pars posterior (Dp). Ascending fibers from the pTGN pass through the lateral forebrain bundle and terminate ipsilaterally in the dorsal region of area dorsalis pars medialis (dDm) of the telencephalon. Following biocytin injections into the dDm, small, round cells were labeled in the pTGN. After biocytin injections into the Vi and Dp of the telencephalon, retrogradely labeled cells were found in the ipsilateral SGN. The results show that the ascending fiber connections of the central gustatory system in the percomorph teleost tilapia are essentially similar to those of mammals. That is, the pathway from the primary gustatory areas (facial, glossopharyngeal, and vagal lobes) through the SGN and pTGN to the dDm in tilapia corresponds with the mammalian gustatory pathway from the solitary nucleus through the pontine taste areas (nucleus parabrachialis) and the thalamic relay nucleus (ventral posteromedial nucleus) to gustatory neocortices. In addition, the pathway from the primary gustatory areas through the SGN to the Vi and Dp in tilapia corresponds with the pathway from the solitary nucleus through the pontine taste areas to the amygdala in mammals.

Quantitative and qualitative differences in thrombopoietin-dependent hematopoietic progenitor development between cord blood and bone marrow.

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.

Analysis of synergism between stem cell factor and granulocyte-macrophage colony-stimulating factor on human megakaryoblastic cells: an increase in tyrosine phosphorylation of 145 kDa subunit of c-kit in two-factor combination.

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.

Cytogenetic clonality analysis in monosomy 7 associated with juvenile myelomonocytic leukemia: clonality in B and NK cells, but not in T cells.

It remains unclear which lymphoid lineages are involved in juvenile myelomonocytic leukemia (JMML). We report a JMML patient who acquired monosomy 7 after intensive chemotherapy. In this case, the expression of monosomy 7 was analyzed in T, B and natural killer (NK) cells highly purified from peripheral blood mononuclear cells of the patient. The fluorescence in situ hybridization method revealed the expression of monosomy 7 in B cells, but not T cells. Half of the NK cells expressed monosomy 7; when NK cells were divided into CD2- and CD2+ populations, this abnormality was positive in 91.1% of CD2- NK cells but in only 14.7% of CD2+ NK cells. These results suggest that, in this JMML patient who acquired monosomy 7 after intensive chemotherapy, B cells and half of NK cells, but not T cells, have monosomy 7.

Preoptic gonadotropin-releasing hormone (GnRH) neurons innervate the pituitary in teleosts.

In most teleosts, there are three groups of gonadotropin-releasing hormone (GnRH) neurons. In this study we addressed the question of GnRH neuronal innervation of the pituitary in the dwarf gourami and the tilapia using immunocytochemistry combined with biocytin tract tracing. Biocytin was applied to the pituitary attached to the brain in vitro. Similar results were obtained in both species. GnRH neurons retrogradely labeled with biocytin were observed only in the preoptic area. These results indicate that preoptic GnRH neurons innervate the pituitary. Negative labeling of biocytin in the terminal-nerve and midbrain GnRH neurons suggests that these two GnRH neuronal populations do not project to the pituitary. Biocytin-positive but GnRH-negative neurons were also observed in the preoptic area and the ventromedial parts of the hypothalamus, suggesting neuropeptidergic and aminergic innervation of the pituitary besides GnRH.

Frequent natural killer cell abnormality in children in an area highly contaminated by the Chernobyl accident.

Since January 1991, we have been performing thyroid surveys and hematologic and immunologic screening on children in Chechersk, Belarus, a city situated in one of the areas most seriously contaminated with high levels of radionuclides after the Chernobyl accident. Ten children selected from 713 children because of goiter did not show a decrease in humoral immunity or in the number and function of T cells. By contrast, natural killer (NK) cell activity against K562 cells was depressed in 4 of these 10 children. The clinical and laboratory findings indicated that previously reported diseases with NK cell dysfunction could be excluded. A comparative analysis of NK cell activity in children from areas with and without high 137Cs levels revealed a high frequency of abnormal NK cell activity only in children from the area contaminated by radioactive fallout. In addition, there was no correlation between NK cell activity and NK cell number as percentage in the children from the area with high 137Cs levels. Neither activity nor number of NK cells was correlated with the body content of 137Cs. Thus, the frequent abnormality of NK cell function may not have been due to actual internal exposure to the long-lived radionuclide.

Gonadotropin-releasing hormone neurons in the gourami midbrain: a double labeling study by immunocytochemistry and tracer injection.

There are three groups of gonadotropin-releasing hormone (GnRH) neurons in the teleost brain. Midbrain GnRH neurons in the dwarf gourami send axons to various areas of the central nervous system. However, it is not clear whether midbrain GnRH neurons form a cell cluster separate from the nucleus of the medial longitudinal fasciculus (nMLF), which has been reported to project to the spinal cord. Thus, we performed a double labeling study. GnRH neurons were immunostained but were very faintly labeled with biocytin injected into the spinal cord. In contrast, nMLF neurons were strongly labeled with biocytin but were GnRH-immunonegative. GnRH neurons are distributed at almost the same rostrocaudal levels as nMLF neurons, but they constitute a separate cell group dorsocaudal to nMLF neurons.

Regression of atypical lymphoid hyperplasia after eradication of Helicobacter pylori.

A rare case of endoscopic and histological regression of a gastric lymphoid mucosal lesion after eradication of Helicobacter pylori is reported. A 72-year-old man was suspected of having a low-grade B-cell gastric mucosa-associated lymphoid tissue (MALT) lymphoma by endoscopic and histological findings. Histology of biopsy specimens showed massive infiltration of atypical lymphocytes and lymphoepithelial lesions. Immunohistochemical staining revealed kappa light chain expression in the infiltrated atypical lymphocytes to be twofold that of lambda light chain. The above diagnosis was thus highly suspected but not confirmed. Antibiotic therapy was given on the basis of evidence of H. pylori infection. Successful eradication of H. pylori resulted in remarkable improvement of endoscopic and histological findings. Follow-up studies were carried out 8 months after eradication, with no evidence of relapse. The eradication of H. pylori appears to be an effective alternative therapy for B-cell lymphoproliferative disease, although longer follow-up and further studies are needed before this treatment can be established.

Cholinergic mechanisms responsible for blood pressure regulation on sympathoexcitatory neurons in the rostral ventrolateral medulla of the rat.

We examined whether reticulospinal sympathoexcitatory neurons in the rostral ventrolateral medulla (RVLM) have muscarinic receptors and ACh inputs, and whether these cholinergic mechanisms on RVLM neurons are involved in the pressor response induced by peripheral administration of physostigmine. Microiontophoretic application of ACh and carbachol enhanced the firing rate of RVLM sympathoexcitatory neurons and the enhancement of RVLM neurons by these cholinoceptor agonists was abolished by the nonselective muscarinic receptor antagonist scopolamine and/or by the M2 muscarinic receptor antagonist methoctramine. Physostigmine and the ACh releaser 3,4-diaminopyridine also enhanced the firing rate of RVLM neurons. Intravenous administration of physostigmine enhanced RVLM sympathoexcitatory neuronal activity and the physostigmine-induced response was reversed by iontophoretic application of scopolamine onto the neurons. These results are consistent with the hypothesis that M2 muscarinic receptors responsible for blood pressure regulation are present on RVLM sympathoexcitatory neurons and these receptors receive ACh inputs. Physostigmine injected systemically may exert a portion of its hypertensive effect through a direct enhancement of cholinergic mechanisms on RVLM sympathoexcitatory neurons.

Lupus nephritis in juvenile myelomonocytic leukemia.

A 13-year-old girl developed lupus nephritis and Hashimoto thyroiditis in the chronic phase of juvenile myelomonocytic leukemia (JMML). At age 7 months, she was diagnosed as having JMML based on the hepatosplenomegaly, leukocytosis, thrombocytopenia, increased levels of fetal hemoglobin, and spontaneous in vitro growth of granulocyte-macrophage progenitors. At the onset of JMML, she had hypergammaglobulinemia, antinuclear antibodies, rheumatoid factors and anti-smooth muscle antibody. She had been placed on oral 6-mercaptopurine for about 12 years, with clinical improvement. At age 13 years, she was found to have hematuria and proteinuria. She also developed arthritis and Raynaud's phenomenon as well. She had antinuclear antibodies, rheumatoid factors, LE phenomenon, beta-1C (C3) nephritic factor (C3NeF), antithyroid antibodies, and hypocomplementemia. The renal biopsy specimens revealed a diffuse increase in the mesangial cells and matrix by light microscopy, and intense staining of IgG, Clq and C3 by immunofluorescence microscopy. The hormonal study ultimately showed decreased thyroid functions. So she was diagnosed as lupus nephritis and Hashimoto thyroiditis. The patient is the first example to show close relationship between stem cell abnormalities in JMML and development of overt autoimmune disorders.

Antitumour activity and adverse reactions of combined treatment with chitosan and doxorubicin in tumour-bearing mice.

We examined the antitumour activity and adverse reactions, such as myelotoxicity, gastrointestinal toxicity and body-weight loss,of the cancer chemotherapy drug doxorubicin when given with chitosan in sarcoma 180-bearing mice. Intraperitoneally administered doxorubicin (5 mg kg(-1)) given on days 1 and 8 with or without orally administered chitosan (200, 400 and 800 mg kg(-1) twice daily) inhibited tumour growth. The orally administered chitosan (400 and 800 mg kg(-1) twice daily) prevented doxorubicin-induced body-weight loss and small-intestinal mucosal injury. Similarly, the reduction of leucocyte number induced by the intraperitoneally administered doxorubicin was restored to normal by the oral administration of chitosan (400 and 800 mg kg(-1) twice daily). It seems likely that the mechanisms by which the orally administered chitosan protects against doxorubicin-induced gastrointestinal toxicity may be due to the formation of doxorubicin-chitosan complex in the small-intestinal mucosa through the diffusion of chitosan into the small-intestinal villi. In conclusion, our data suggest that the oral administration of chitosan prevents the gastrointestinal mucositis associated with doxorubicin therapy.

[Lymphocytic infundibuloneurohypophysitis during the first remission in acute lymphoblastic leukemia].

A 15-year-old girl developed acute lymphoblastic leukemia (ALL). The patient was treated according to the 13th protocol of the Tokyo Children's Cancer Study Group, and thereafter remained free of disease. However, at the age of 20, she complained of polyuria, polydipsia and amenorrhea. Hematological or meningeal relapse was ruled out on the basis of clinical and laboratory findings. The plasma concentrations of GH, TSH, LH, FSH, ACTH and ADH were low or below the detectable limits. There was no increase in urine osmolarity after water deprivation. Arginine, LH-RH, TRH and CRH tolerance tests revealed no or low responses of GH, LH/FSH, TSH, and ACTH/cortisol, respectively. Magnetic resonance imaging demonstrated thickening of the pituitary stalk, which was homogeneously enhanced by gadolinium administration. A biopsy specimen showed fibrosis and infiltration of CD8-positive T lymphocytes in a portion of the pituitary stalk, whereas the adenohypophysis was normal. In addition, no leukemic cells were observed in the samples. Thus, a diagnosis of lymphocytic infundibuloneurohyophysitis (LIN) was established. All the symptoms were improved by treatment with hydrocortisone, L-thyroxine, desamino-8-d-arginine vasopressin, estrogen and gestagen. This is the first reported case of ALL complicated by LIN.

[Abnormalities in regulation system of granulopoiesis].

We studied the effects of several cytokines on the development of granulocyte-macrophage (GM) progenitors using the serum-deprived culture. SCF plays an important role in the GM-CSF- or IL-3-dependent production of neutrophils and macrophages. In vitro colony assay also suggests an increase in sensitivity of GM progenitors to cytokines (GM-CSF, IL-3, G-CSF and/or SCF) in a patient with juvenile chronic myelogenous leukemia. A high level of serum IFN-gamma was associated with leukopenia and thrombocytopenia in a patient with hemophagocytic syndrome. Based on the evidence that IFN-gamma significantly inhibited the proliferation of GM progenitors, IFN-gamma-mediated suppression was suggested as one of the mechanisms causing cytopenia. In patients with aplastic anemia and neutropenia, an increase of serum G-CSF levels was observed when neutrophils decreased remarkably in number. However, the serum SCF levels were constant in these patients. A failure of SCF to enhance colony growth in some patients with aplastic anemia implies qualitative abnormalities of hematopoietic progenitors.

[An analysis of sclerodermatous graft-versus-host-disease after allogeneic bone marrow transplantation: CD8+CD57+T-cell proliferation and increased production of TGF-beta].

A 19-year-old woman with acute lymphoblastic leukemia received an allogeneic bone marrow transplantation (BMT) from an HLA-identical sibling during the second remission, on September 28, 1993. The conditioning regimen consisted of total body irradiation and cyclophosphamide. Short term methotrexate and cyclosporin A were given for prophylaxis of graft-versus-host disease (GVHD). On day 771 after BMT, she complained of bilateral forearm pain, and developed sclerotic lesions on the skin of the abdominal wall, forearms and legs. The diagnosis of sclerodermatous GVHD was established by skin biopsy on day 834. The values of CRP and IgG were elevated, and both antinuclear antibody and anti-DNA antibody became positive. Flow cytometric analysis showed a significant increase in the number of CD57+ cells after appearance of sclerotic change. In addition, 65% of CD8+ cells were positive for CD57. Circulating level of transforming growth factor (TGF)-beta 1 was high. These results suggest that overproduction of CD8+ CD57+ T cells and high level of circulating TGF-beta are related to the development of sclerodermatous GVHD.

[Hemophagocytic syndrome with high level of interferon-gamma in the advanced stage].

Serum cytokines, lymphocyte subsets of peripheral blood and natural killer cell activity were serially assayed in a 3-year-old girl with hemophagocytic syndrome. Laboratory findings showed pancytopenia, increased levels of transaminases and hyperferritinemia and proliferation of histiocytes in bone marrow and pleural effusion. The administration of prednisolone resulted in a temporary improvement followed by a further exacerbation. The patient died in spite of the treatment with VP-16 and THP-adriamycin. Serum interferon-gamma (IFN-gamma) markedly increased in active phases. TNF-alpha, IL-1 beta and GM-CSF concentrations were normal or slightly elevated. The CD4/8 ratio of peripheral lymphocytes and natural killer cell activity also fluctuated according to clinical stage. These results suggested that IFN-gamma was the most important cytokine to activate histiocytes in this case.