Charcoal-dextran treatment of fetal bovine serum removes an inhibitor of human CFU-megakaryocytes.

Serum has been shown to have an inhibitory effect on the growth of human megakaryocyte colony-forming units (CFU-Meg), both in the methylcellulose and plasma clot systems. Charcoal-dextran (CD) treatment of fetal bovine serum (FBS), a method that is known to adsorb free hormones and various other substances from their protein-bound counterparts, has been used for the improvement of human erythroid colony growth. We now report a CD treatment of FBS to improve human CFU-Meg growth, using a modified plasma clot colony assay. We found 22.9 +/- 1.9 (SEM) colonies in cultures containing CD-treated FBS, as compared to 7.1 +/- 0.9 colonies with control FBS (different at p less than or equal to 0.0001). Serum treated with dextran alone produced 5.0 +/- 1.6 colonies, which did not differ from control FBS. Cultures containing CD-treated human AB serum, human AB plasma, and citrated bovine plasma yielded significantly (p less than 0.05) fewer colonies than those with CD-treated FBS. Endotoxin, cortisol, T3, T4, insulin, estradiol, testosterone, and erythropoietin levels were not changed in a consistent direction by the CD treatment. Our treatment of FBS with CD allows improved growth of human CFU-Meg colonies and likely represents the removal of an as-yet-undefined inhibitor(s) of CFU-Meg. This technique provides the ability to assay for stimulators or potentiators less hampered by the influence of an undefined inhibitor(s).

Pattern of response of megakaryocyte colony-stimulating activity in the serum of patients undergoing bone marrow transplantation.

Bone marrow transplantation has become an accepted form of therapy for several malignant, hematologic, and genetic disorders. Platelet recovery is delayed after bone marrow transplantation. To better understand the mechanisms involved in platelet recovery we studied 23 patients undergoing bone marrow transplantation for the presence of megakaryocyte colony-stimulating activity (Mk-CSA) in their serum. Shortly after beginning the pretransplant preparative regimen the Mk-CSA level in the serum of these patients increased. This increase was transient, and the level returned to baseline, to later increase again. The second increase in Mk-CSA level occurred during the second week after bone marrow transplantation at the time of hematopoietic recovery. Most patients who failed to engraft did not show a rise in Mk-CSA during the second week after transplantation. All patients showing engraftment had an Mk-CSA rise during the second week after transplantation. The difference between these two groups was highly significant (p = 0.0007). The biphasic response of Mk-CSA after bone marrow transplantation is similar to the response seen in a rat model after lethal irradiation. We postulate that the first elevation in Mk-CSA is due to tissue injury and nonspecific response whereas the second elevation of Mk-CSA is a physiologic response to marrow aplasia and associated with effective bone marrow engraftment.

Acute megakaryocytic leukemia (M7) in children.

We analyzed the clinical and laboratory features of eight children (median age, 20 months; range, 13 months to 11 years) with acute megakaryocytic leukemia (M7) and compared the findings with those reported in the literature. The diagnosis was supported by ultrastructural examination for platelet peroxidase or immunophenotyping for glycoprotein IIb/IIIa or the von Willebrand factor protein. Two patients had Down's syndrome. Initial findings included anemia (in all patients), thrombocytopenia (in six), myelofibrosis (in three), lytic bone lesions (in two), and pronounced leukocytosis (in one). Stem cell culture studies of peripheral blood specimens revealed an aberrant phenotype of the megakaryocytes in one patient and reversal to a normal pattern after successful therapy. Remission was achieved in seven of the eight patients after aggressive chemotherapy, and four patients remained in remission 27 to 57 months after diagnosis. Three of these four patients underwent allogeneic bone marrow transplantation. M7 leukemia is not infrequent in children younger than 3 years of age, especially in those with Down's syndrome. The availability of monoclonal antibodies specific to restricted antigens of the megakaryocytic lineage has made the diagnosis of M7 leukemia both possible and practical.

Megakaryocytopoiesis in the human fetus.

After immunohistochemical staining the size and maturational stage of fetal megakaryocytes from 20 human fetuses of 12 to 21 weeks' gestation were compared with those from normal adults. The mean diameter of fetal megakaryocytes was 14.0 microns when stained with antiglycoprotein IIb (Tab) and 15.2 microns when stained with antifactor VIII, which was significantly smaller than adult megakaryocytes, which were 18.4 microns when stained with Tab and 20.6 microns when stained with factor VIII. The proportion of immature (stage II) cells was higher--and the proportion of mature (stage IV) cells was lower--in the fetal tissue than in the adult. The smaller size and shift to a less mature population indicated that there were differences at the non-mitotic phase of the development of megakaryocytes in the fetus. Such differences were probably associated with quantitative and qualitative platelet abnormalities in newborn infants. Understanding the physiology and regulation of megakaryocytopoiesis in fetuses and newborn infants will be invaluable in determining the pathophysiology of platelet dysfunction in the newborn.

Treatment of stage I, IIA, IIIA1 pediatric Hodgkin disease with doxorubicin, bleomycin, vincristine and etoposide (DBVE) and radiation: a Pediatric Oncology Group (POG) study.

PURPOSE: The objectives of this study were to evaluate the feasibility of reducing therapy, while maintaining treatment efficacy, in the context of a cooperative group clinical trial that allowed for clinical staging in early stage Hodgkin disease (HD). PATIENTS AND METHODS: Between August 1992 and December 1993, 51 eligible children < or =21 years of age, 31 male and 20 female, were enrolled in this study which was designed for low stage (IA, IIA, IIIA1) HD. Laparotomy and surgical staging was optional. Five postpubertal patients with Stage IA and IIA disease received only involved-field radiation therapy. The other 46 patients, who form the basis of this report, received combined modality therapy consisting of four courses of doxorubicin, bleomycin, vincristine, and etoposide (DBVE) followed by 2,550 cGy involved-field irradiation. RESULTS: With a median follow-up of 8.4 years, the 6-year overall and event-free survival rates for the 46 patients treated with combination therapy were 98 +/- 2% and 91 +/- 5%, respectively. All patients achieved remission after completion of therapy. There have been four recurrences and a remission death due to gunshot wound. Combined modality therapy was well tolerated. Predominant side effects were gastrointestinal and hemopoietic. There have been no clinically significant cardio-pulmonary side effects so far. CONCLUSION: In clinically staged children with early-stage HD, DBVE and low-dose involved-field irradiation was effective therapy with tolerable side effects and reduced potential for long-term adverse events.

Megakaryocyte colony-stimulating factor (Mk-CSF): its physiologic significance.

Megakaryocyte colony-stimulating activity (Mk-CSA) is required for in vitro megakaryocyte colony formation. Its in vivo significance in megakaryocytopoiesis is unknown. We studied 12 patients undergoing bone marrow transplantation (BMT) at our institution. The bone marrow megakaryocyte progenitor cells (CFU-Mk), the serum level of Mk-CSA, and the platelet count on the 28th day after BMT were studied. Patients with elevated Mk-CSA levels had less CFU-Mk in their bone marrow than did patients with a normal or decreased Mk-CSA (p less than 0.01). Animal experiments using murine models have documented that several purified molecules including erythropoietin, multi-CSF and GM-CSF possess Mk-CSA. The in vitro Mk-CSF of WEHI-3-conditioned medium is multi-CSF. The in vivo significance for megakaryocytopoiesis of these factors is not clear. In the human system, Mk-CSA is increased in conditions with decreased bone marrow megakaryocytes. Recombinant human or primate CSFs have in vitro Mk-CSA utilizing both human and murine cells as targets. However, the presence of these activities does not fully explain the Mk-CSA in human serum rich in Mk-CSA. The precise regulation of human blood cell levels and the studies discussed suggest that there is a specific Mk-CSF that responds to in vivo changes in megakaryocyte numbers. Proof of its physiologic role awaits the isolation of a pure factor.

Successful use of ReFacto continuous infusion in two paediatric patients with severe haemophilia A undergoing orthopaedic surgery.

In this report we describe the successful use of B-domain-deleted recombinant factor VIII (ReFacto) administered by continuous infusion during orthopaedic procedures in two children with severe haemophilia A. Both patients underwent ankle synovectomy and in patient 2, a medial patello-femoral ligament repair was performed in the same operative session. Patient 2 developed septic arthritis A in his knee joint and arthroscopic joint irrigation and debridement was performed 2 weeks after the initial procedure. Surgical cover was initiated with a bolus dose of ReFacto 50 IU kg(-1) followed by continuous infusion at 3.3-4.8 IU kg(-1) h(-1) which was maintained for up to 9 days postoperatively. Patient 2 received an additional bolus dose of 15 IU kg(-1) during the infusion period. All procedures were performed without haemostatic complications and long-term orthopaedic outcomes were good in both patients.

Neuroblastoma-associated opsoclonus-myoclonus treated with intravenously administered immune globulin G.

An 18-month-old black girl had progressive truncal ataxia, opsoclonus, and multifocal myoclonus associated with a nonresectable abdominal ganglioneuroblastoma. Before chemotherapy, she received intravenously administered IgG, 1 gm/kg, for 2 days; within 48 hours of the first dose, there was significant improvement of the opsoclonus-myoclonus and ataxia. She required a 1 gm/kg maintenance dose every 4 to 6 weeks for a total of 12 doses, but is now free of symptomatic after 2 years with no therapy.

Analysis of megakaryocyte ploidy in fetal bone marrow biopsies using a new adaptation of the feulgen technique to measure DNA content and estimate megakaryocyte ploidy from biopsy specimens.

Platelet counts in newborns are similar to those of adults and children. However, newborn infants admitted to intensive care nurseries have a high prevalence of thrombocytopenia. The mechanisms responsible for the increased susceptibility to thrombocytopenia are not known. In addition, some studies have documented functional abnormalities in newborn platelets. In an effort to understand differences between platelets in newborns and in adults, we examined megakaryocyte ploidy in bone marrow from fetuses and compared it with bone marrow from adults, using a modified Feulgen stain to measure DNA of individual megakaryocytes. Faced with small fixed tissue samples, we developed a technique for use on bone marrow biopsies to estimate megakaryocyte ploidy and compared the results obtained with this method to those obtained from bone marrow aspirates. This study demonstrated that the overall mean ploidy of fetal megakaryocytes is decreased compared with adults. Additionally, fetal megakaryocyte ploidy increases as megakaryocyte maturation increases, but not to the same extent that adult megakaryocyte ploidy increases with megakaryocyte maturation. Over the gestational period studied, there was no relationship between gestational age and mean ploidy. The small size, shift to a less mature population, and decreased ploidy of fetal megakaryocytes indicate that there are differences in the post mitotic phase of megakaryocyte development in the fetus. Such differences may be related to quantitative and qualitative platelet abnormalities in the newborn. Understanding the physiology and regulation of megakaryocytopoiesis in the fetus and newborn will be valuable in determining the pathophysiologic basis of platelet dysfunction in the newborn.

A framework for querying a database for structural information on 3D images of macromolecules: A web-based query-by-content prototype on the BioImage macromolecular server.

Nowadays we are experiencing a remarkable growth in the number of databases that have become accessible over the Web. However, in a certain number of cases, for example, in the case of BioImage, this information is not of a textual nature, thus posing new challenges in the design of tools to handle these data. In this work, we concentrate on the development of new mechanisms aimed at "querying" these databases of complex data sets by their intrinsic content, rather than by their textual annotations only. We concentrate our efforts on a subset of BioImage containing 3D images (volumes) of biological macromolecules, implementing a first prototype of a "query-by-content" system. In the context of databases of complex data types the term query-by-content makes reference to those data modeling techniques in which user-defined functions aim at "understanding" (to some extent) the informational content of the data sets. In these systems the matching criteria introduced by the user are related to intrinsic features concerning the 3D images themselves, hence, complementing traditional queries by textual key words only. Efficient computational algorithms are required in order to "extract" structural information of the 3D images prior to storing them in the database. Also, easy-to-use interfaces should be implemented in order to obtain feedback from the expert. Our query-by-content prototype is used to construct a concrete query, making use of basic structural features, which are then evaluated over a set of three-dimensional images of biological macromolecules. This experimental implementation can be accessed via the Web at the BioImage server in Madrid, at http://www.bioimage.org/qbc/index.html.

Designing and executing scientific workflows with a programmable integrator.

MOTIVATION: As in many other fields of science, computational methods in molecular biology need to intersperse information access and algorithm execution in a computational workflow. Users often find difficulties when transferring data between data sources and applications. In most cases there is no standard solution for workflow design and execution and tailored scripting mechanisms are implemented in a case by case basis. RESULTS: In this paper, we present a general purpose 'programmable integrator' that can access information from a variety of sources in a coordinated manner. Its usefulness in complex bioinformatics applications is claimed and supported by some application examples. AVAILABILITY: Tools are freely available to non-profit educations and research institutions. Usage by commercial organizations requires a license agreement. Software requirements: Java v1.3 (http://java.sun.com), Xerces XML Parser (http://xml.apache.org/xerces-j) and Kweelt implementation of XQuery (http://kweelt.sourceforge.net/).

Parvovirus B19 infection in patients receiving cancer chemotherapy: the expanding spectrum of disease.

Initial reports have suggested that human parvovirus B19 infection in immune-compromised individuals may cause prolonged or chronic bone marrow suppression. We report four children receiving cancer chemotherapy who developed atypical patterns of cutaneous and hematologic disease associated with parvoviral infection. B19 infection was demonstrated by specific serologies. This report suggests that patients undergoing myelosuppressive or immunosuppressive therapy may have unusual manifestations of infection and be at risk for complications associated with this common and widespread pathogen.

In vitro megakaryocytopoiesis in children with acute idiopathic thrombocytopenic purpura.

Idiopathic thrombocytopenic purpura (ITP) of childhood is a disorder characterized by a history of previous viral illness followed by acute onset of low circulating platelet count with present or increased megakaryocytes in the bone marrow. The majority of children recover a normal platelet count within 6 months to 1 year after onset of the disease. To better understand the regulation of megakaryocytopoiesis in this disorder, we studied nine patients with childhood ITP with the plasma clot colony assay in vitro for megakaryocyte colony forming units (CFU-Mk). Mononuclear bone marrow cells from some of the patients with ITP contained greater numbers of CFU-Mk and greater numbers of cells per colony than mononuclear bone marrow cells from healthy adult volunteers (p less than 0.026) when the cultures contained no added megakaryocyte colony-stimulating activity (Mk-CSA). The serum from patients with ITP did not stimulate in vitro megakaryocytopoiesis from healthy adult volunteers' bone marrow mononuclear cells above baseline values. These findings are consistent with the hypothesis that a decrease in bone marrow megakaryocytes is needed for Mk-CSA production. Alternatively, Mk-CSA is consumed by active megakaryocytopoiesis in the bone marrow.

Infants with Down's syndrome. Use of cytogenetic studies and in vitro colony assay for granulocyte progenitor to distinguish acute nonlymphocytic leukemia from a transient myeloproliferative disorder.

Infants with Down's syndrome have an increased incidence of acute nonlymphocytic leukemia (ANLL). They are also at risk of developing a transient myeloproliferative syndrome indistinguishable from ANLL except by its eventual clinical recovery. The authors studied five infants with Down's syndrome and leukocytosis with circulating blast forms in their peripheral blood with in vitro cultures of bone marrow colony forming units-granulocyte macrophage (CFU-GM), cytogenetics and peripheral blood neutrophil alkaline phosphatase (NAP) score. Three children developed ANLL, the other two had a transient myeloproliferative syndrome. The in vitro assay for CFU-GM showed abnormal results consistent with ANLL in the children who develop this disorder. Serial cytogenetic studies disclosed the appearance of an abnormal clone in one patient. A combination of clinical parameter in vitro colony studies and cytogenetic studies in these children was helpful in distinguishing ANLL from a myeloproliferative disorder. The NAP scores were not helpful.

Long-term neurologic outcome in children with opsoclonus-myoclonus associated with neuroblastoma: a report from the Pediatric Oncology Group.

A retrospective data collection was performed on 29 children diagnosed with neuroblastoma and opsoclonus-myoclonus between 1983-1993 from Pediatric Oncology Group institutions. The aim was to describe neurologic outcome in children with neuroblastoma and opsoclonus-myoclonus. Age at diagnosis ranged from one month to 4 years (median age, 18 months). The duration of opsoclonus-myoclonus symptoms prior to the diagnosis of neuroblastoma ranged from 6 days to 17 months (median duration, 6 weeks). There was a prevalence of low stage disease according to the POG staging system: stage A (n = 18), stage B (n = 3), stage C (n = 7), stage D (n = 1). There was a predominance of paraspinal primary tumors. There was no case of Nmyc amplification (0/17), and 2/8 cases were diploid. Treatment for neuroblastoma consisted of surgery alone in 19/29 (18 stage A, 1 stage C in thorax), and surgery plus chemotherapy in 10/ 29. No patient received radiotherapy. Treatment for opsoclonus-myoclonus ranged varied. Six children received no treatment for opsoclonus-myoclonus. The following agents were used ACTH (n = 14), prednisone (n = 12), IV IgG (n = 6), immuran (n = 2), depakote (n = 1), and inderal (n = 1). Eighteen of 29 children (62%) had resolution of opsoclonus-myoclonus symptoms. The range of time for recovery was a few days to 3 years. However the majority recovered over several months. Twenty of 29 children (69%) had persistent neurologic deficits including speech delay, cognitive deficits, motor delay, and behavioral problems. Of the 9 children who had complete recovery of opsoclonus-myoclonus without neurologic sequelae, age at diagnosis and duration of symptoms were not different from the entire group. Interestingly, 6/9 children with complete recovery received chemotherapy as part of their treatment. In conclusion, persistent neurologic deficits are characteristic for children with neuroblastoma and opsoclonus-myoclonus. Treatment with chemotherapy may improve the neurologic outcome.

Erythroid hypoplasia. An unusual presentation of childhood acute lymphocytic leukemia.

A 3-year-old girl had pancytopenia and bone marrow erythroid hypoplasia. The pancytopenia resolved without therapy, but the erythroid hypoplasia persisted for four months in spite of a five-week course of corticosteroid therapy. She responded briefly when androgens were added to the corticosteroid regimen, but within three weeks of stopping therapy she developed acute lymphocytic leukemia. The differential diagnosis of RBC aplasia in childhood is discussed. To our knowledge, this is the first case reported with erythroid hypoplasia as a prodrome of acute lymphocytic leukemia of childhood.

Chronic neutropenia: diagnostic approach and prognosis.

Chronic neutropenia is a term used to describe a group of disorders characterized by a persistent neutrophil count of less than 1500 cells/microliters. We studied seven children and three sets of parents. We separated patients into a group with good prognosis and a group at higher risk of infection by using a combination of tests, including bone marrow aspiration and biopsy, steroid stimulation of bone marrow reserve, and in vitro CFU-GM and CSA assays. Children with a normal number of myeloid elements in their bone marrow and a normal bone marrow response to steroid stimulation had a benign course. CFU-GM and CSA assays helped to classify these children's neutropenia when their bone marrow had decreased numbers of myeloid elements. Family studies in three children were consistent with an inherited neutropenia, even when their parents were hematologically normal.

Evaluation of bone marrow megakaryocyte ploidy distributions in persons with normal and abnormal platelet counts.

Using bone marrow smears of the type prepared routinely in clinical practice, we determined megakaryocyte ploidy distributions in five normal persons, eight patients with both normal platelet counts and normal bone marrow morphology, and 18 patients with quantitative platelet disorders. To include 2N and 4N megakaryocytes in the ploidy distribution histograms, all megakaryocytes were identified by serial immunologic labelling with rabbit antiserum to human platelet glycoproteins and rhodamine-conjugated goat anti-rabbit igG. Cell nuclei were concurrently Feulgen stained with bis-aminophenyl-oxdiazole, and the nuclear fluorescent signals were quantified photometrically. A modal megakaryocyte ploidy value of 32N was seen in 10 of the 13 persons with normal platelet counts, and geometric mean megakaryocyte ploidy values averaged 24.9N +/- 7.0N (arithmetic mean +/- SD). In these normal control individuals, 2N and 4N megakaryocytes accounted for 11.1% of all megakaryocytes, and 2.6% of the megakaryocytes were 128N. Shifts to a higher mean ploidy were observed in five of seven patients with idiopathic thrombocytopenic purpura, resulting from increased percentages of 64N and 128N megakaryocytes at the expense of 4N, 8N, and 16N cells. Shifts to lower ploidy were demonstrated in two patients with acute myelogenous leukemia and one patient each with thrombotic thrombocytopenic purpura and isoimmune thrombocytopenia. Four of five patients with essential thrombocythemia had strikingly abnormal megakaryocyte ploidy histograms characterized by the presence of unusually high ploidy 256N and 512N megakaryocytes. These 256N and 512N cells were virtually unique to the patients with essential thrombocythemia.(ABSTRACT TRUNCATED AT 250 WORDS)