E. Fred Saunders

Central role of tumour necrosis factor, GM-CSF, and interleukin 1 in the pathogenesis of juvenile chronic myelogenous leukaemia

Summary. In previous studies on patients with juvenile chronic myelogenous leukaemia (JCML), we found excessive proliferation of malignant monocyte‐macrophage elements in the absence of exogenous growth factor, and impaired growth of normal haematopoietic progenitors. In the current study, six newly‐diagnosed JCML patients were investigated to characterize the disease further. In co‐cultures, JCML cell culture supernatant as well as patient plasma obtained at diagnosis produced a striking reduction in numbers of control marrow BFU‐E, CFU‐GM, CFU‐Meg and CFU‐GEMM colonies. Monoclonal anti‐tumour necrosis factor alpha neutralizing antibodies (anti‐TNF‐α Ab) abolished these inhibitory properties. In sharp contrast, JCML supernatants exerted a marked growth‐promoting effect on autologous JCML cells cultured in clonogenic assays. Anti‐TNF‐α Ab and anti‐granulocyte‐macrophage colony‐stimulating factor neutralizing antibodies (anti‐GM‐CSF Ab) both reversed the stimulating effect. Recombinant GM‐CSF and recombinant TNF‐α produced a profound increase in JCML colonies when tested individually and anti‐GM‐CSF Ab reversed the TNF‐α effect. Expression studies of TNF‐α and TNF‐α receptor genes of cultured JCML cells demonstrated mRNAs for both. Further, TNF‐α activity was assayed in a wide variety of cell culture supernatants and in normal and patients’plasma, and only the JCML specimens showed increased TNF‐α values. Recombinant interleukin‐1 alpha (IL‐1α) also stimulated JCML colony growth, but polyclonal anti‐IL‐1 neutralizing antibodies did not suppress JCML colony numbers nor did it reverse the effects of TNF‐α or GM‐CSF. The evidence indicated that the JCML monokine which inhibits normal haematopoiesis is TNF‐α and that the endogenously‐produced TNF‐α and GM‐CSF from JCML cells play an important role in the pathogenesis of the disease by acting as autocrine growth factors. IL‐1α also stimulates JCML cell proliferation as an accessory factor and augments the effect of GM‐CSF, TNF‐α or both.

Hypofibrinogenemia and thrombocytopenia in familial hemophagocytic reticulosis

Familial hemophagocytic reticulosis (FHR) is a lethal disorder of early infancy characterized by infiltration of many tissues by histiocytes. A severe bleeding disorder frequently accompanies the disease. Thrombocytopenia associated with an absence of fibrinogen was demonstrated in six patients. The coagulation abnormality was not caused by disseminated intravascular clotting, because plasma levels of all other clotting factors were normal and there was no proof of increased fibrinolysis. If present, this unusual coagulation disturbance is helpful in establishing the diagnosis of FHR during life.

Diamond-Blackfan Syndrome. II. In Vitro Corticosteroid Effect on Erythropoiesis

Summary: To study the action of corticosteroids on erythroid precursors, (burst forming unit-erythroid and colony forming unit-erythroid) in Diamond-Blackfan Syndrome (DBS), marrow from a newly diagnosed untreated infant was studied in vitro with prednisone and dexamethasone. This patient subsequently proved to be steroid responsive. Colony numbers increased linearly in an erythropoietin (EPO) dose response study. There was marked enhancement of colony numbers at all EPO doses after adding either prednisone (10-6 M) or dexamethasone (10-9 M) to the cultures. The data indicate that corticosteroids augment erythropoiesis at both early (BFU-E) and late (CFU-E) stages of development in DBS. In contrast, marrow from a second infant with DBS, clinically steroid resistant, failed to respond to steroids in vitro.Speculation: It seems reasonable that in some patients with Diamond-Blackfan syndrome, erythroid precursors have a relative insensitivity to erythropoietin (EPO). Corticosteroids appear able to modify EPO receptor sites on the progenitor cell membrane in vitro, thus increasing EPO sensitivity and partially correcting the erythropoietic defect.

Efficacy of Amlodipine in Pediatric Bone Marrow Transplant Patients

The calcium antagonist amlodipine may have the potential for expanded use in children owing to its physiochemistry and pharmacokinetic profile that facilitates once-daily dosing in a liquid formulation. Its safety and efficacy have not been previously evaluated in children. A retrospective analysis of 15 pediatric bone marrow transplant patients who had amlodipine incorporated into their antihypertensive drug regimen reveals significantly lower blood pressure as compared with baseline therapy (123.5 ±2.1 mmHg and 117.2 ±2.2 mmHg, systolic blood pressure before and during amlodipine, P<0.05; 81.5 +1.8 mmHg and 75.5 ±2.6 mmHg, diastolic blood pressure before and during amlodipine, P<0.05). Amlodipine provided improved blood pressure control in this cohort and may provide a valuable pharmacologic alternative for treatment of pediatric hypertension.

Diamond-Blackfan Syndrome. I. Erythropoiesis in Prednisone Responsive and Resistant Disease

Summary: We studied the in vitro proliferative characteristics of marrow erythroid progenitors, colony forming unit-erythroid (CFU-E) and burst forming unit-erythroid (BFU-E), in two infants with Diamond-Blackfan syndrome before and during prednisone treatment. Patient 1, had a brisk erythropoietic response to prednisone and the anemia improved; Patient 2 was steroid-unresponsive. Marrow from Patient 1 yielded linear increases in numbers of colonies in erythropoietin dose-response studies, and the magnitude of response was much greater while on prednisone compared to pre-treatment. Cultures of stem cell rich fractions of marrow from Patient 1 separated by unit gravity sedimentation (STA-PUT) revealed moderately reduced CFU-E and BFU-E numbers before prednisone, but normal colony numbers during treatment. Patient 2 differed because CFU-E and BFU-E were almost absent at all erythropoietin doses before and during therapy. Even stem cell rich marrow fractions initially yielded very low colony numbers which did not increase significantly while on prednisone.These findings suggest that in Patient 1 there were adequate numbers of progenitors that were able to differentiate normally only in the presence of prednisone. In Patient 2 the erythropoietic failure in vivo and in vitro was unaffected by prednisone.Speculation: Diamond-Blackfan Syndrome is a heterogeneous disorder with at least two forms demonstrating different patterns of erythropoiesis. In one form erythroid precursors appear to be quantitatively normal but have a relative erythropoietin insensitivity which is correctable with prednisone. In the other form there is either a marked deficiency of numbers of progenitors or an absolute erythropoietin insensitivity.

Lithium therapy of children with chronic neutropenia.

We treated five children with chronic neutropenia using lithium carbonate and studied the effect in vivo on granulopoiesis. Granulocyte precursors (CFU-C) from blood and marrow, and colony-stimulating activity (CSA) from peripheral blood leukocytes, were assayed in a methylcellulose tissue culture system. Three patterns of response to lithium were seen. In patients with aplastic anemia (one acquired and two Fanconis aplastic anemia) despite increased colony-stimulating activity, CFU-C numbers remained very low and the neutropenia persisted. In a patient with Kostmann neutropenia colony-stimulating activity, and blood and marrow CFU-C numbers increased, but the agranulocytosis was unchanged. An impressive therapeutic effect was seen in one patient with idiopathic neutropenia with low colony-stimulating activity who responded to lithium with an increase in colony-stimulating activity and CFU-C resulting in persisting normal neutrophil counts. Lithium appears useful in treating a select group of neutropenic patients in whom colony-stimulating activity production is responsive to lithium, and the granulocytic progenitor compartment is capable of producing mature neutrophils.

Management of intracerebral hemorrhage in idiopathic thrombocytopenic purpura. Report of four cases.

There has been little comment on the specific management of intracerebral bleeding occurring in patients suffering idiopathic thrombocytopenic purpura. The authors present the cases of four children with intracerebral hemorrhage due to this coagulation disturbance. A plan of management is described based on this experience; it includes immediate control of cerebral edema, emergency splenectomy, supportive care with platelet transfusions and corticosteroids, cerebral angiogtaphy, and a definitive neurosurgical procedure. If necessary, the radiological investigation and surgical therapy can be performed with a single general anesthetic. Three of the patients have survived without major neurological sequelae.

Determination of area under the whole blood concentration versus time curve after first intravenous cyclosporine dose in children undergoing hematopoietic stem cell transplant: limited sampling strategies.

Achievement of target trough cyclosporine whole blood concentrations after hematopoietic stem cell transplant (HSCT) reduces the risk of acute graft versus host disease (aGvHD). In solid organ transplant, prevention of acute graft rejection correlates with achievement of target area under the whole blood concentration versus time curve during the 12-hour dosing interval (AUC-12) after oral administration. This study describes a limited sampling strategy for determination of cyclosporine AUC-12 after administration of the first intravenous (IV) dose in children undergoing HSCT and explores the relationships between individual whole blood concentrations during the dosing interval and the AUC. Children undergoing HSCT and receiving cyclosporine prophylaxis were eligible to participate. The first cyclosporine dose was given as a 2 hour infusion, and eight cyclosporine concentrations were determined at 2 (end of the infusion), 2.5, 3, 4, 6, 8, 10, and 12 hours after the start of the IV infusion. The relationship between AUC-12 and whole blood cyclosporine concentrations at individual time points after IV administration was assessed by the Spearman rho correlation coefficient. Limited sampling strategies were developed using three to six time points by way of multiple linear regression analysis. The agreement between the AUC-12 calculated using all eight data points and the limited sampling strategies was assessed by intraclass coefficient and Bland-Altman analysis. Twenty-four children (0.5-16.9 yr) participated. The mean AUC-12 calculated using all eight concentration versus time points was 2793 ± 1165.6 μg/L·hr. Whole blood cyclosporine concentrations obtained within the first 4 hours from the start of the infusion correlated strongly with AUC-12 (Spearman rho coefficient, 0.717-0.868). Limited sampling strategies were developed to estimate AUC-12 with adjusted r2 of 0.955 to 0.998, mean bias of 0% to 0.93%, and precision of 1.6% to 8.1%. The actual AUC-12 and predicted AUC-12 values agreed strongly (intraclass coefficient, 0.981-0.999). Limited sampling strategies using three to six data points have been developed that will estimate cyclosporine AUC-12 after administration of the first IV dose given over 2 hours. Information regarding the possible association between aGvHD and cyclosporine AUC-12 is not available. The limited sampling strategies described here will facilitate the prospective evaluation of the clinical importance of cyclosporine AUC-12 in the prevention of aGvHD.

Haem Synthesis in the Diamond-Blackfan Syndrome

The response of bone marrow to erythropoietin (EPO) from five children with the Diamond‐Blackfan syndrome, also known as congenital hypoplastic anaemia (CHA), was tested in tissue culture by measurement of haem synthesis. Studies of 13 control marrows indicated that the maximum EPO effect occurred at approximately 70 h incubation using an EPO concentration of 0.2–0.3 units/ml and a nucleated cell concentration of 5 × 106 per culture. Under these conditions, haem synthesis was 121% greater in EPO‐stimulated than in unstimulated cultures. Patients with CHA with anaemia and diminished marrow erythroids had reduced or absent haem synthesis. In one patient, haem production became normal after a spontaneous remission, and was not inhibited by autologous plasma drawn at the time of diagnosis. Plasma from three patients did not show inhibitory activity when cultured with control marrow. In contrast, plasma from an adult with acquired pure red cell aplasia produced striking inhibition of haem synthesis when cultured with control marrow. We conclude that, in comparison to some cases of the adult acquired condition, CHA is not due to inhibitors or antibodies. When present, erythroid precursors in children with CHA are capable of responding normally to EPO with increased haem synthesis.

Congenital Lymphoid Hyperplasia with Persistent Hyperlymphocytosis

THE patient described below has had generalized lymphoid hyperplasia since birth, and a white-cell count in the range of 80,000; approximately 90 per cent of the peripheral leukocytes are lymphocyt...