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Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor (EGFR/ErbB1) and ErbB4. HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Thus, monoclonal antibodies (mAbs) for HB-EGF detection could be an important tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases. However, few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we generated a clone of hybridoma-derived mAb 2-108 by immunizing mice with recombinant human HB-EGF protein expressed by human cells. The mAb 2-108 specifically bound to human HB-EGF but not to mouse HB-EGF and was successful in immunoblotting, even under reducing conditions, immunoprecipitation, and immunofluorescence for unfixed as well as paraformaldehyde-fixed cells. Notably, this mAb was effective in IHC of paraffin-embedded tumor specimens. Epitope mapping analysis showed that mAb 2-108 recognized the N-terminal prodomain in HB-EGF. These results indicate that this new anti-HB-EGF mAb 2-108 would be useful in the diagnosis of HB-EGF-related cancers and would be a strong tool in both basic and clinical research on HB-EGF.

A practice guideline aimed at standardizing the treatment for childhood idiopathic thrombocytopenic purpura (ITP) is presented. This consensus guideline is based on a survey carried out via a questionnaire prepared by the ITP Committee of the Japanese Society of Pediatric Hematology and sent to society members. The survey questionnaire included questions on the diagnosis of ITP submitted for the purpose of revising the ITP diagnostic guideline prepared in 1990 by the Research Group for Intractable Hematopoietic Disorders; a revised diagnostic guideline also is presented.

The improved outcome of acquired aplastic anemia (AA) has revealed later complications, such as myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). We retrospectively analyzed 167 children with severe acquired AA. Eleven of 50 children treated with cyclosporin (CSA) and recombinant human granulocyte colony-stimulating factor (rhG-CSF ) developed MDS/AML; 8 of these were within 36 months of the diagnosis of AA, much earlier than previous reports. Six of the 11 children received rhG-CSF exceeding 10 μg/kg/d, and 9 received rhG-CSF therapy for over 1 year. Ten children showed monosomy 7 at diagnosis of MDS. All of the 11 children were administered both CSA and rhG-CSF. There was no development of MDS/AML among 41 children treated with either CSA or rhG-CSF or among 48 children who underwent bone marrow transplantation. A well-controlled clinical trial is warranted to determine whether therapeutic modalities affect the development of MDS/AML in children with severe acquired AA.

Morphological changes in the testis induced by chemotherapy given according to the Tokyo Children's Cancer Study Group (TCCSG) regimens were studied in children with acute lymphoblastic leukemia (ALL). After informed consent, testicular biopsies were performed 14 times in 12 patients at the end of treatment. The testicular morphology in all cases had sustained a degree of damage. The tubular fertility index (TFI), calculated as the percentage of seminiferous tubules containing identifiable spermatogonia, was from 0 to 42.8% (mean 33.4%) below the normal value. Infiltration of leukemic cells was the most significant factor contributing to the decrease in TFI. There were no differences in the TFI among the TCCSG protocols. Formation of sperm was recognized in six cases, whose ages were 7, 8, 9, 10, 15 and 19 years. In two children, testicular biopsy was performed twice. In the second biopsy, TFI was elevated and sperm formation with the maturation of Leydig cells was observed. A number of other pathological changes were observed: modification of spermatogonia, Sertoli cells and inclusion bodies in spermatogonia, abnormal maturation of Leydig cells, evidence of interstitial fibrosis and thickening of the basement membrane. These results suggest that recent strong chemotherapy for the treatment of ALL might cause severe but not fatal damage to children's testicular tissue. As chemotherapy escalates, more investigation of testicular function will be necessary.

TJ-137 administered to patients with chronic ITP increased the platelet count "slightly" or more in 31.7% of the patients and showed a clinical effect in 40.9% with a rating of "modestly effective" or better. With a low incidence of adverse reactions, TJ-137 is ex pected to be a new drug for the treatment of ITP.

The efficacy of recombinant human interferon alpha-2b (rh IFN alpha-2b) in the treatment of steroid resistant idiopathic thrombocytopenic purpura (ITP) was studied in 50 cases. Forty-one patients treated with rh IFN alpha-2b three times a week, six of 18 (33.3%) in the low dose group (150 x 10(4)IU: 3 MIU) and four of 20 (20.0%) in the high dose group (300 x 10(4)IU: 3 MIU) responded with platelet counts increasing to above 50 x 10(9)/L. Because of the exacerbation of thrombocytopenia and nasal bleeding, treatment was discontinued within 2 weeks in three patients out of 41 cases. On the other hand, six of nine patients (66.7%) treated with 3 MIU of IFN alpha-2b once a week for 8 weeks showed satisfactory response. Treatment with either administration schedule did not result in sustaining platelet counts above 50 x 10(9)/L for a long time after treatment. The results indicate that once a week administration schedule of rh IFN alpha-2b is more efficacious for platelet counts increasing for short period in patients who failed to respond to steroid and other medications than other schedules. The maintenance of this treatment schedule will allow sustained increased platelet levels, resulting in relief of bleeding tendency, while also being cost effective in comparison with other IFN treatment schedules and achieving better patient compliance without flu-like symptoms.

Encouraging results are reported with high-dose chemotherapy and total body irradiation followed by autologous bone marrow transplantation in the treatment of advanced neuroblastoma. However, relapse remains a significant problem. We used high-dose chemotherapy, surgery, intraoperative radiation and an autologous bone marrow transplant treated in vitro to remove tumor cells followed by 13-cis-retinoic acid to treat 36 children with advanced neuroblastoma. This comprehensive treatment appears to improve the survival rate of patients with advanced neuroblastoma, including those with N-myc amplification and bony involvement. The disease-free survival rate was 66% (95% confidence interval, 49-84%) at 3 years. All patients who received 13-cis-retinoic acid developed cheilitis, but no bone marrow depression occurred in these patients. Five patients developed hemolytic uremic syndrome (HUS) post-transplant. This may have been related to the procedure used for total body irradiation. Patients who had their kidneys shielded during this procedure did not develop this syndrome. Patients who received local irradiation at the primary site showed no evidence of relapse in this region, indicating that such therapy may help to prevent a relapse. These data suggest a high rate of 3 year disease-free survival with this treatment strategy. The nonrandomized nature of the study and use of multiple modalities precludes analysis of the specific contribution of each.

Ex vivo expansion of hematopoietic progenitor cells in the umbilical cord blood mononuclear cells (CB-MNC) was investigated in liquid culture system with various combinations of cytokines (stem cell factor [SCF], interleukin [IL]-3, IL-6, granulocyte-colony stimulating factor [G-CSF], erythropoietin [EPO], and interferon [INF]-gamma). Non-lineage-committed hematopoietic progenitor cells and lineage committed hematopoietic progenitor cells were represented as CD34+CD38- and CD34+CD38+ subpopulations, respectively. Although absolute CD34+CD38- cell numbers decreased even in the presence of multicytokines, the combinations of SCF plus IL-6 and SCF plus IL-3 plus IL-6 plus IFN-gamma were significantly effective in maintaining CD34+CD38- cells than the other combinations (P < 0.05). After 4 weeks of culture. CD34+CD38- cells disappeared in all combinations of cytokines. Absolute CD34+CD38+ cell numbers increased in the presence of cytokines. Maximal expansion of CD34+CD38+ cells were observed in the combinations of SCF plus IL-3 plus IL-6 plus EPO (19.8 +/- 3.3-fold) and SCF plus IL-3 plus IL-6 plus G-CSF (18.3 +/- 2.6). The combination of SCF plus IL-3 plus IL-6 was also effective to expand CD34+CD38- cells (15.8 +/- 3.9). However, the expansion was transient and they decreased to zero within 3 weeks. In the combinations of SCF plus IL-6 and SCF plus IL-3 plus IL-6 plus INF-gamma, maximal expansion was inferior to the others but CD34+CD38+ cells were maintained more than 4 weeks. These results suggested that the indication of CBT can be expanded into older children by ex vivo augmentation of CB hematopoietic progenitor cells using multi-cytokines.

We compared the effects of various combinations of cytokines (stem cell factor [SCF], interleukin [IL]-3, IL-6, granulocyte-colony stimulating factor [G-CSF], erythropoietin [EPO]) among the growth of human hematopoietic progenitor cells from cord blood (CB), bone marrow (BM), and peripheral blood mononuclear cells (MNC) mobilized by chemotherapy and G-CSF (PB) in a semi-solid medium. Macroscopic colonies, that were visible to the naked eye, were formed from PB-MNC within 1 week even without cytokines. They consisted of blasts containing macrophage-like cells with immature nuclei on Wright stain, and were strongly accelerated by IL-3. Macroscopic colonies were also formed from CB-MNC. However, they appeared after 1-3 weeks and synergistic effects of SCF with other cytokines, especially EPO, were prominent. Macroscopic colonies were not formed from BM-MNC. Granulocyte-colony stimulating factor was effective in increasing colony forming units of granulocyte macrophage from BM-MNC and they appeared between 1 and 2 weeks. These results suggested that the quality of hematopoietic progenitor cells was different among blood sources. This might lead to different bone marrow recovery patterns after transplantation of each blood source. The appropriate cytokines should be added to evaluate their exact potential.

Since January 1987, chemoprophylaxis and empiric therapy with antifungal drugs have been used routinely in neutropenic children with neoplasms. The incidence of terminal invasive fungal infection (IFI) diagnosed by autopsy findings is compared in two groups: group A, consisting of 25 patients autopsied between January 1982 and December 1986, and group B, consisting of 14 patients autopsied between January 1987 and December 1991. There was no difference in the incidence of IFI between the two groups, but the characteristics of IFI appeared to differ. With the recent intensification of chemotherapy, the patients in group B had more risk factors for IFI. It might therefore have been anticipated that group B patients may have shown an increase in IFI. No such increase was observed, suggesting that the prophylaxis was effective. Autopsy findings also showed that the target organs of IFI were localized and that systemic candidiasis was decreased in group B. It is thus concluded that the present antifungal countermeasures are effective, although some problems still remain.

A human pro-B cell line, named JKB-1, was established from the bone marrow of a 16-year-old girl with acute lymphoblastic leukemia (ALL) in relapse. The origin of the JKB-1 cell line was indicated by its chromosomal and immunologic similarity to the patient's fresh leukemic cells. This cell line has been growing for more than 14 months in suspension culture medium and had a doubling time of about 24 hours. JKB-1 expressed terminal deoxynucleotide transferase (TdT) and early antigens (HLA-DR, CD19, CD24) of B cells, with heavy chain gene rearrangement. However, it did not express late antigens (CD10, CD20, CD21, CD22, CD23) of B cells, light chain gene rearrangement, and cytoplasmic μ-chain. These results suggested that JKB-1 is at the stage of “pro-B” cell or early B-cell precursors. This cell line was induced to differentiate after 7 days of co-incubation with irradiated bone marrow stromal cells because of the expression of pre-B cell antigens (CD10, CD20), cytoplasmic μ-chain, light chain gene rearrangement, and disappearance of TdT. JKB-1 cells adhered to a preestablished bone marrow stromal cell layer with string-like processes under scanning electron microscope. When JKB-1 cells were separated from the stromal layer by a cyclopore membrane with 0.45 μ pore size, they did not differentiate. Bone marrow stromal cell conditioned medium could not induce differentiation either. Thus it was suspected that direct contact between JKB-1 cells and stromal cells was required for differentiation. In methylcellulose semisolid medium, the colony size and number of JKB-1 cells were increased by stem cell factor (SCF), or interleukin (IL)-3, or IL-7, but they were decreased by IL-6. Moreover, SCF synergized with IL-3 or IL-7 to stimulate the proliferation of JKB-1 cells. Because there are very few reproducible models for examing early stages of human B-cell differentiation, the JKB-1 cell line would be useful for studying the relationship between human B-cell differentiation and bone marrow microenvironment, as well as leukemogenesis. © 1994 Wiley-Liss, Inc.

小児の慢性特発性血小板減少性紫斑病(c-ITP) 38例の液性免疫ならびに細胞性免疫能について検討した.非脾摘群ではCD 3, CD 8比率がcontrolに比し有意に低く,脾摘群ではCD 4比率, CD 4/8比がcontrolに比べて有意に低かった. T-cellの百分率はcontrol群に比べ,有意差はなかった.しかし, B-cellの百分率は非脾摘群でcontrolに比べ有意に上昇していた. T-cellのblastogenesisをstimulation indexでみるとPHA刺激では脾摘群も非脾摘群もcontrol群と有意差はないが, Con-A刺激では非脾摘群においてcontrolに比べ,有意に低下していた.自己抗体では,抗DNA抗体は検索した34例全例で陰性であったが,抗核抗体は33例中8例が陽性, 2例が偽陽性であった.これらは,症例数もまだ少なく,絶対的なc-ITPの免疫学的変化を示すものではないと思われるが,従来の成人c-ITPの報告と異なり, B-cellに異常がある可能性も示唆された.

The relationships between transfused cell number of CD34+ cell subpopulations divided by HLA-DR and CD33 antibodies and hematopoietic recovery patterns after peripheral blood progenitor cell transplantation (PBPCT) subsequent to myeloablative chemoradiotherapy were investigated in 14 children with cancer. Both logarithm of transfused CD34+ cell number/10(6)/kg and logarithm of transfused cell number/10(6)/kg of the CD34+HLA-DR+CD33+ subpopulation, which is supposed to be myeloid-committed cells, were correlated with myeloid recovery after PBSCT, though they were not correlated with erythroid or platelets recovery. On the other hand, logarithm of transfused cell number/10(6)/kg of CD34 + HLA-DR-CD33-subpopulation, which is supposed to be immature progenitor cells, was not correlated with myeloid recovery but correlated with erythroid recovery and platelet recovery. These results suggested that rapid myelopoiesis after PBPCT occurs following transfusion of sufficient numbers of myeloid-committed cells and complete hematopoietic reconstitution occurs after transfusion of sufficient numbers of immature hematopoietic progenitor cells.

Background. Patients with neurofibromatosis type 1 (NF1) are prone to develop malignancy, particularly malignant schwannoma and glioma in adults. Methods. To assess the risk for childhood malignancy in NF1, 26,084 patients with cancer younger than 15 years of age registered from 1969 to 1989 in the Japan Children's Cancer Registry were reviewed. The incidence of NF1 in each type of cancer was compared with that in the Japanese population. Results. Fifty-six children with cancer had NF1 in the national registry. The incidence of NF1 (0.21%) was 6.45 times that of the expected estimated rate of 1 per 3000 in the Japanese population. These tumors tended to be type and site specific. The NF1 incidence was extremely high in optical nerve glioma (12.5%), other central nervous system gliomas (0.9%), and malignant schwannoma (31.4%). For nonneural tumors, NF1 incidence was increased in rhabdomyosarcoma (1.36%), particularly those in urogenital organs, and in myelogenous leukemia (0.27%). Epithelial carcinomas were not observed in the group of patients with NF1. Conclusion. The risk for glioma and malignant schwannoma increases in children and adults with NF1. Moreover, the risk for two childhood malignancies, myelogenous leukemia and rhabdomyosarcoma, increases in children with NF1. The NF1 gene seems to increase the risk not only for neural tumors but also for some non-neural tumors in an age-specific, organ-dependent pattern of carcinogenesis.

A total of 91 peripheral blood stem cell collections were performed in 26 children with various malignant tumors and peripheral blood stem cell transplantations (PBSCT) were performed in 15 of the children. There was a positive correlation between logarithm of total CD34+ cells/kg and logarithm of colony-forming unit-granulocyte macrophage (CFU-GM)/kg (r = 0.86). The time elapsed until the white blood cells (WBC) exceeded 1000/microL was related to both CFU-GM (r = 0.67) and CD34+ cell count (r = 0.60). The number of days elapsed until platelet count exceeded 5 x 10(4)/microL was not related to the logarithm of CFU-GM count/10(5) per kg transfused (r = 0.47), but was related to the logarithm of CD34+ cell counts/10(6) per kg transfused (r = 0.73). The number of days elapsed until the reticulocytes exceeded 10% was not related to the logarithm of CFU-GM count/10(5) per kg (r = 0.52), but was related to the logarithm of CD34+ cell counts/10(6) per kg transfused (r = 0.91). Although CD34+ cell counts correlated with the number of CFU-GM, bone marrow regeneration rates in three lineages were predicted more accurately by the number of CD34+ cells transfused than by the number of CFU-GM. These results suggest that measurement of the CD34+ cell count may be useful in predicting bone marrow regeneration rate after PBSCT.

PATIENTS AND METHODS We report patients who were treated for non-Hodgkin's lymphoma (NHL) or Ki-1 antigen-positive (Ki-1) lymphoma with a T-8801 protocol that included etoposide (VP-16) and behenoylcytosine arabinoside. RESULTS Secondary acute myeloid leukemia (AML) developed in 5 of 38 NHL and Ki-1 lymphoma patients, and the cumulative risk at 4 years was 18.4%. The median time from the initiation of the chemotherapy to the development of AML was 21 months (range, 13-30). Four patients had a FAB M5 morphology, and one had FAB M2. In four of five examined cases, chromosomal alterations involving the long arm of chromosome 11 were demonstrated at the time of development of AML. None of the 46 NHL patients who we treated with another protocol (B-8801), using significantly higher cumulative doses of VP-16 than in the case of the patients with T-8801 and a different schedule of VP-16 administration, developed secondary AML. CONCLUSIONS The risk of secondary AML possibly related to the use of VP-16 given twice weekly.

A human leukemia cell line, JK-T1, was established from the bone marrow of a 10-year-old boy with T-cell acute lymphoblastic leukemia. The origin of the leukemic cell line, JK-T1, was demonstrated by its chromosomal and immunologic similarity to the patient's fresh leukemic cells. Karyotypic analysis revealed 46,XY,del(6)(q?),t(8;14)(q24;q13),der(9)t(9;?)(q34;?). In JK-T1, neither rearrangement nor amplification of the c-myc gene was observed apparently because the breakpoint of chromosome 14 was not q11 but q13. JK-T1 was independent of interleukin 2 (IL-2) because of little production of IL-2, little IL-2 receptor (CD25) on the surface, and no response to exogenous IL-2. JK-T1 had lymphocyte function associated antigen-1 (LFA-1) (CD11a, CD18) on its surface and could adhere to the hematologic stromal layer. These characteristics of JK-T1 cell line are considered to be be useful not only for evaluating the role of t(8;14) but also in studying the adhesion molecules of leukemia.

A 2-year-old boy with B-cell lymphoma (stage IV) in the first complete remission received myeloablative treatment followed by peripheral blood stem cell autotransplantation and is surviving event-free for 18 months. Reconstitution of all cell lines occurred very rapidly, ie, a level of 1000 leukocytes per microliter was reached after 9 days, that of 500 granulocytes per microliter after 10 days, and platelets were substituted only once. This result suggests that PBSCT can be safely employed during consolidation therapy for malignant disorders because of its short duration of bone marrow suppression.

Late effects of childhood acute leukemia and its treatment were studied in 776 patients (684 ALL, 73 ANLL, and 9 others) in Japan who had remained in remission for more than 1 year after their first complete remission. Delayed adverse sequelae involve a wide variety of organs and their functions. Short stature was present in 2.61%, obesity in 3.79%, abnormalities of growth hormone secretion in 1.5%, delayed secondary sex characteristics in 1.5% of males and 0.6% of females, motor disturbances in 1.17%, sensory disturbances in 0.91%, intellectual and learning disabilities in 2.48%, abnormal findings in routine neurologic examinations in 1.31%, EEG abnormalities in 4.30%, brain CT abnormalities in 5.09% and cardiac dysfunction in 1.07%. Various other disorders were seen in 20 patients. Many of these delayed adverse sequelae are caused by or related to central nervous system prophylaxis and systemic combination chemotherapy. The results suggest that it is needed to improve therapeutic methods through the stratification of patients by risk factors and detailed analysis of prognostic factors. Moreover it is important to render medical and psychosocial support to long-term survivors of childhood leukemia through interactions between the patient, parents and medical staff.

Since children with acute leukemia have prolonged periods of severe neutropenia due to the disease itself or its intensive chemotherapy, they often develop life-threatening infectious complications. A protected environment and prophylactic antibiotics for patients with severe neutropenia have been shown to decrease the incidence of infectious morbidity. In spite of the high mortality rate of infections, on the other hand, their diagnosis is not well established because the percentage of microbiological detectability by blood culture is low. The differentiation of infections may be possible by using some supplementary measures and particular laboratory parameters. Febrile neutropenic patients with leukemia should promptly receive empiric treatment with combinations of two or three broad-spectrum antibiotics that are bactericidal and synergistic toward the presumed pathogens, or with antifungal drugs if patients are resistant to antibiotics, as well as supportive measures such as granulocyte transfusion, intravenous gamma-globulin and granulocyte colony-stimulating factor.

The Tokyo Children's Cancer Study Group (TCCSG) has performed prospective randomized controlled studies since 1984 for childhood acute lymphoblastic leukemia (ALL). Four hundred and ninety-eight newly diagnosed patients were treated with 5 different regimens designated as S1, S2 for a standard risk group (SRG), H1 and H2 for a high risk group (HRG) and HEX for an extremely high risk group of patients. We added intermediate-dose methotrexate as early intensification therapy in the S2 and H2 groups to reduce extramedullary and medullary relapses. Event-free survival of all patients at 4 years 6 months from the start of this regimen (median follow-up period 32 months) was 67.5%. The CNS relapse rate was significantly decreased to 2.2% compared to previously reported studies and our own experience. There were some unexpected complications of the CNS such as myelopathy and/or leukoencephalopathy. For the SRG early ID-MTX seemed to be useful to improve the prognosis, but triple intrathecal injections (TIT) should be limited in number. The 24Gy cranial irradiation (CRX) was effective but possibly excessive for the HRG. As evidenced by the success of the HEX group regimen, more intensive multi-drug consolidation in the early post-remission phase might be necessary to improve further the prognosis and to make it possible to shorten the treatment periods.

Three antiphospholipid antibodies (aPLs), namely, antiphosphatidylinositol antibody (antiinositol antibody), antiphosphatidylserine antibody (antiserine antibody), and anticardiolipin. beta 2-glycoprotein I complex antibody (antiCL. beta 2-GPI antibody), were determined in 49 children with idiopathic thrombocytopenic purpura (ITP) consisting of 14 newly-diagnosed cases and 35 chronic cases. Determination of aPL was performed twice in the newly-diagnosed patients, once each during the acute and convalescent phases, and once in the chronic patients. The positive rates in the acute and convalescent phases of the newly-diagnosed group and in the chronic group were, respectively, 14.3%, 28.6%, and 18.8% for the antiinositol antibody, 14.3%, 14.3%, and 15.6% for the antiserine antibody, and 21.4%, 28.6%, and 25.0% for either of these 2 antibodies. Thus, antiinositol and antiserine aPLs were present at high incidences; however, all patients were negative for the antiCL. beta 2-GPI antibody. No correlation was noted between either the antiinositol or the antiserine antibody and peripheral platelet count, anti-GP IIb/IIIa antibody or PAIgG. Thus, although some aPLs are present in both acute and chronic pediatric ITP, the aPLs seems to be of an infectious disease type. No results that suggest possible involvement of aPLs in ITP pathology were obtained.

Idiopathic thrombocytopenic purpura (ITP) occurs more commonly in young women during the reproductive years. To obtain information for management of ITP in pregnancy, we performed a nationwide retrospective survey. Findings from a total of 284 pregnant women with ITP and their 286 newborn infants were available for analysis. The bleeding tendency at delivery was managed chiefly with corticosteroid, intravenous high-dose gamma-globulin, and platelet transfusion. Maternal complications occurred in 77 cases (27.1%) and were frequently seen in cases with poor control of ITP. Neonatal abnormalities, which were not influenced by the clinical state of the mother, occurred at a frequency of 17.8%. Thrombocytopenia in neonates occurred in 48 cases (22.4%), and bleeding tendency was found in 16 cases (6.3%) without severe bleeding. Prediction of thrombocytopenia in neonates was difficult. However, infants from splenectomized mothers with well-controlled ITP showed thrombocytopenia more frequently than those from nonsplenectomized mothers. Mothers treated with steroids at doses greater than 15 mg/day showed a high frequency of maternal complications and fetal abnormal body weight. These observations will be useful in the management of pregnant women with ITP and their infants.

We conducted a survey by questionnaire to clarify the actual conditions of neonates born to mothers with autoimmune thrombocytopenic purpura (ATP) in Japan. We investigated 93 pregnancies (1 resulting in twins) in 31 hospitals between 1985 and 1994. Forty-nine of the neonates (52%) had thrombocytopenia (below 150 x 10(9)/L). Nineteen neonates (20%) showed a bleeding tendency, but this was generally mild. In only one neonate (1%) (a case of asymptomatic intracranial hemorrhage, ICH), deep bleeding occurred due to thrombocytopenia. The lowest platelet count of neonates after birth occurred on day 4, not on day 0. There was no correlation between maternal and neonatal platelet counts. However, there was an apparent correlation between the neonatal platelet count on day 0 and the lowest platelet count after birth. Treatment of the mothers with intravenous high-dose gamma-globulin and prednisolone did not prevent risk of neonatal thrombocytopenia significantly.

We evaluated platelet associated immunoglobulin (PIag) G, PaIgM, platelet associated autoantibodies to platelet glycoprotein IIb/IIIa (Pa-GPIIb/IIIa), the percentage of CD5+ B cells and the amount of platelet-bound anti-GPIIb/IIIa monoclonal antibody (mAb) in the peripheral blood of 29 patients with childhood onset chronic immune thrombocytopenic purpura (c-ITP). The percentage of CD5+ B cells ranged from 2 to 8% (4.7 +/- 2.0) in control patients and 1 to 18% (6.2 +/- 4.2) in the ITP patients. There was no overall significant difference between the two groups, but the percentage of CD5+B cells in six of the ITP patients was higher than the mean + 2 s.d. of the controls. There was a significant correlation between the percentage of CD5+ B cells and PaIgM (y = 1.73x + 13.4, r=0.40, P < 0.05). This finding is the basis for the speculation that CD5+ B cells may play an important role in the production of PaIgM in vivo. There was no correlation between the amounts of PaIgG and Pa-GPIIb/IIIa). This suggests that the amount of PaIgG does not accurately reflect of the amount of Pa-GPIIb/IIIa. Furthermore, we have demonstrated that autoantibodies to GPIIb/IIIa are directed to more that one epitope.

To investigate immaturity of hematopoietic progenitor cells in umbilical cord blood mononuclear cells (CB-MNC), the formation of macroscopic colonies and mixed-cell colonies was assayed by methylcellulose culture with various combinations of cytokines (stem cell factor [SCF], interleukin [IL]-3, IL-6, granulocyte-colony stimulating factor [G-CSF], erythropoietin [EPO]) and compared with bone marrow (BM)-MNC. Moreover, distribution of the subpopulations divided by CD34, CD38, HLA-DR and CD33 was compared by flow-cytometry. Colonies derived from CB-MNC were so large that they could be observed with the naked eye and consisted of a variety of types of hematopoietic cells. Mixed-cell colonies were formed to a much greater extent in CB-MNC than in BM-MNC. Addition of EPO, IL-3, and SCF had rapid effects on the growth of mixed-cell colonies. The subpopulations of immature hematopoietic progenitor cells (CD34+, CD38-, HLA-DR-), which are supposed to be able to differentiate into hematopoietic precursors and stromal cells, were significantly higher in CB-MNC (8.7 +/- 6.6%) than in BM-MNC (0.0 +/- 0.1%; P < 0.001). These results suggest that CB is a rich source of immature hematopoietic progenitor cells compared to BM.

This paper reports on a patient with lymphoma syndrome leukemia (LSL) who showed interesting findings on brain computed tomography (CT) and ultrasound scans of the abdomen at the initial presentation. The patient was a 5 year old girl. When she was admitted to our hospital, there were many lymph nodes palpable. The abdomen was distended and the liver and spleen were palpable below the umbilicus. Hematologic examinations revealed a leukocyte count of 275,800/microL with 98% lymphoblasts. Chest X-ray film revealed a mediastinal mass. The diagnosis of LSL was made. Her brain CT scan showed a low density area in the right thalamic region without contrast enhancement; infarction was suspected. Furthermore, her abdominal ultrasound scan showed hepatosplenomegaly, kidney swelling with increasing echogenicity and hydronephrosis and stones in the renal pelvis and bladder. These findings are unprecedentedly rare in cases of childhood acute lymphoblastic leukemia (ALL), much less in LSL.

This retrospective study attempts to clarify the optimal timing for peripheral blood stem cell (PBSC) collection after conventional chemotherapy followed by granulocyte-colony stimulating factor (G-CSF) administration. Leukapheresis was performed 32 times in nine children with various cancers during bone marrow recovery phase following transient pancytopenia after chemotherapy. (On two occasions, leukapheresis was excluded because many leukemic blasts were included). When the number of white blood cells (WBC) exceeded 1.8 x 10(10)/L after administration of G-CSF (200 micrograms/m2, continuous infusion), many more CD34+ cells were contained in the collected peripheral mononuclear cells (P > 0.02) and a sufficient number of PBSC for transplantation (> or = 10 x 10(8) CD34+ cells/kg) was obtained after one run in 15 of 17 leukapheresis sessions. In contrast, sufficient PBSC were obtained only in one of 13 runs of leukapheresis when the number of WBC was < 1.8 x 10(10)/L. The number of WBC on the day when PBSC were collected correlated with collected nuclear cell number (r = 0.60), but not with the CD34+ cell ratio. The ratio was higher only when both platelets and reticulocytes increased in parallel with WBC. We conclude that sufficient PBSC collection is possible after conventional chemotherapy using G-CSF, when hematopoietic recovery is parallel, without the use of high-dose chemotherapy.

Determining the site and severity of blood loss is important in the management of children with gastrointestinal (GI) bleeding. Blood urea nitrogen (BUN) and serum creatinine (Cr) were measured on the day of hospitalization and the ratio of BUN/Cr was calculated in 11 children with 16 episodes of upper GI bleeding and 49 with lower GI bleeding. There was a significant difference between the two GI bleeding groups with regard to BUN/Cr ratio (p less than 0.001). When the ratio was 30 or above, the specificity of upper GI bleeding was 98% with a sensitivity of 68.8%. A linear relationship was found between the BUN/Cr ratio and delta Hb (delta Hb = 0.08 x BUN/Cr +/- 0.8 g/dl) for bleeding originating from the upper GI tract. This study confirms that measurement of the BUN/Cr ratio is useful for localizing the source of bleeding to the upper GI tract and also demonstrates its usefulness as an estimation of the severity of blood loss from the upper GI tract.

We investigated 332 children with malignancy who were admitted to our University Hospital from 1966 to 1985 and examined the annual changes in the main cause of death in 64 autopsied children with leukemia. It was found that, recently, infection has been the most important factor and the main cause, but the increase in fungal infection is also drastic. Considering this situation, we introduced an oral combined administration of high-dose amphotericin B syrup and trimethoprim-sulfa-methoxazole for the prophylaxis of infections at the time of remission induction, and this regimen was found to be effective for protection against bacterial and mycotic infection in children whose neutropenia was in the range of 1000/μl to 100/μ1. We also studied the effects of the nutritional condition on the immunological function of children with leukemia undergoing intensive chemotherapy. We found that the concentration of total serum protein tends to decrease after the induction therapy, and there were decreases of albumin, prealbumin, and transferrin, in particular. The changes were in accordance with the decrease in immunoglobulin. Lentinan, the immunomodulator, was intravenously administered every 2 weeks to the children to whom intensification chemotherapy was repeatedly given, and changes in the subpopulation of peripheral lymphocytes were followed. Restorations of PHA responsiveness and a population of killer T cells were found in children receiving Lentinan.

This report describes a modified ELISA-plaque assay that we have succeeded in developing and utilizing for the detection of platelet antibody-secreting cells (PASC). We investigated the effects of various culture conditions on the formation and specificity of plaques obtained from spleen cells and peripheral blood leukocytes (PBL) of mice immunized with human platelets. The values of PASC using this plaque assay were compared with the corresponding serum platelet-binding IgG (PBIgG) levels. The results revealed that plaque formation from spleen cells was well correlated with the number of inoculations, whereas that from PBL was not. The rise of PBIgG levels in sera was shown to be delayed, as compared with the plaque formation from spleen cells.

The effect of fresh frozen plasma infusions was reported for the female child who suffered from hemolytic uremic syndrome at the age of 1 2/12 years and repeated episodes of relapse and recurrence during the following several years.Her thrombocytopenia in relapse period responded significantly to high-dose intravenous immunoglobulin therapy using Fc intact immunoglobulin.The latter effects may represent the important suggestion in the interpretation of effectiveness of plasma infusion therapy for the patient with hemolytic uremic syndrome.The studies were also described on the platelet function, the values of anti thrombin III and complement component of her sera and the metabolism of prostaglandin.

We report an 8-year-old boy with Ph-positive chronic myelogenous leukemia (CML) who showed a testicular tumor as the first manifestation of blastic crisis. Pathological examination of the excised testis revealed marked infiltration of immature cells, whereas bone marrow remained in the chronic phase. Immunohistochemical and molecular examinations disclosed the immature cells to have B-lymphocyte surface determinants and immunoglobulin JH rearrangement. DNA obtained from the testis and peripheral blood cells showed similarly rearranged bcr configurations, indicating that infiltrating cells in the testis originated from the CML clone. Bone marrow showed B-lymphoid blastic crisis 4 months after testicular involvement.

Infection is the major complication occurring in children with cancer and is often fatal. Since the clinical manifestations of infection are slight, except for fever, a definitive diagnosis is difficult in the early phase of infection, but using laboratory parameters, the differentiation of bacterial infections may be possible to some extent. The infection cannot be treated only by antimicrobial chemotherapy, but supportive measures such as neutrophil transfusion, and other biological medications should be given in consideration of the deficiency of the immune defense mechanisms of the patient. Anticancer treatment is also needed, along with antimicrobial treatment. The prophylactic use of antimicrobial drugs is still controversial but it is effective, depending on the type of infection for which it is used, and on the status of the defective defense mechanism in the patient who is on cancer chemotherapy.

Platelet-associated IgG (PAIgG) levels were measured in 60 children with ITP (46-chronic, 14-acute) using Fab-anti Fab radioimmunoassay method described by McMillan et al. In some patients platelet binding IgG in serum (PBIgG) was also determined at the same time. Patients with ITP had significantly greater PAIgG levels than 30 normal subjects and 13 non-immune thrombocytopenic controls. Elevated PAIgG values did not correlate with parameters of platelet size (mean platelet volume; MPV and percentage of large platelet; PLP) and so these data indicated that high levels of PAIgG in ITP were not due to nonspecific adhesion of serum IgG to megathrombocytes usually increased in this disorder, but due to specific immunological reaction. PBIgG IgG values were also elevated in patients with pretreated chronic ITP, but high levels remained even after successful splenectomy. Furthermore, serial determination of PAIgG values were obtained in some patients with chronic ITP who underwent splenectomy and with acute ITP who achieved spontaneous remission. PAIgG returned to normal levels when thrombocytopenia disappeared. PAIgG seems to be the most reproducible indicator which reflects transition of the clinical picture in this disorder.

This work was supported in part by a grant-in-aid from the Japan Medical Research Foundation and by Scientific Research Grants (project No. 57770632) from the Ministry of Education, Science and Culture of Japan.

A total of 126 children with chronic idiopathic thrombocytopenic purpura, including 35 splenectomized cases, were investigated in a long-term follow-up study, with regard to residual hematologic and immunologic abnormalities, complications and physical growth. Such hemorrhagic symptoms as petechiae, ecchymosis and epistaxis were still observed in about 22%-28% of the patients with a period of morbidity ranging from 3 to 15 years after onset. Residual thrombocytopenia below 150,000/microliters was found in 62% of patients within 5 years, 59% within 5 to 9 years and 57% within 10-14 years after onset. Other abnormalities were mild anemia, low serum level of IgA or IgM, positive antinuclear antibody, rheumatoid factor, and positive Coombs test in a small number of patients. Increased platelet-associated IgG was still obtained in patients with subnormal platelet counts whose morbid periods were 6 to 27 years after onset. Investigation of the patients by questionnaire revealed such complications as obesity, striae atrophicae, abdominal pain, headache, cataract, Perthes' disease, and cardiac complication in some patients. No apparent disturbances except for obesity were observed in their physical growth.

Platelet volume was measured in 20 healthy children, 18 patients with acute idiopathic thrombocytopenic purpura (ITP), 24 patients with chronic ITP, 22 patients with aplastic anemia, and 17 patients with acute leukemia in childhood. The authors compared the platelet volume of these patients by use of peak platelet volume (PPV or mode of the platelet volume), mean platelet volume (MPV) and the percent of large platelets (PLP) as parameters of the platelet volume, and then studied the relationship between the platelet volume and the specific gravity of platelets from these patients. The following was the platelet volume of normal children: PPV was 6.7 +/- 1.1 micrometer 3, MPV was 9.9 +/- 1.6 micrometer 3, PLP was 26.0 +/- 7.7%. All parameters of the platelet volume were remarkably large in chronic ITP, but only PPV was small in acute ITP. On the other hand, all parameters were small in both aplastic anemia and acute leukemia. In acute ITP and chronic ITP, the platelet volume returned to normal value after these thrombocytopenias disappeared. The platelet volume, however, still remained small even after remission in acute leukemia. There was better correlation between MPV and PLP than PPV and PLP. The density analysis of the platelets obtained from platelet-rich plasma (PRP) revealed that PLP in each medium (1.010 approximately 1.052) of normal PRP increased slowly in proportion to the increase of specific density of the media. On the contrary, most of large platelets obtained from the patients with acute ITP and chronic ITP were especially contained in the medium of high specific density (1.052). Large platelets of aplastic anemia and acute leukemia, however, were found in both low and high density media. The authors believe that analysis of the platelet volume using above mentioned three parameters would provide good weapon in early differential diagnosos of acute ITP, chronic ITP, and aplastic anemia. And these findings also may suggest the difference of thrombopoiesis between acute ITP and chronic ITP.

The pathogenesis of the anemia of chronic renal failure is multifactorial. Anabolic steroid has been reported to be of therapeutic value, but it is difficult to use because of its side effect especially in children. We used an analog of activated vitamin D (1α-OH-D3) in 6 haemodialysis patients with anemia. After lα-OH-D3 treatment, hemoglobin and hematocrit values didn't change in these patients, but frequency of blood transfusion has been decreased. We examined serum erythropoietin by in vitro bioassay and haemoaggulutination inhibition assay. And we also examined the effect of various vitamin D metabolites on in vitro growth of granulopoietic and erythropoietic colonies. lα-OH-D3 is suspected to have anabolic steroid like effect.