Alpha-interferon therapy induces improvement of platelet counts in children with chronic idiopathic thrombocytopenic purpura.

PURPOSE: To investigate alpha-interferon (IFN) therapy for children with chronic idiopathic thrombocytopenic purpura (ITP). PATIENTS AND METHODS: Patients with refractory ITP lasting more than 12 months from diagnosis were included if they had platelet counts <50 x 10(9)/L and had received no treatment during the past month. Patients received IFN (3 x 10(6) U/m2 per dose), three times per week for 4 weeks; if partial (<150 x 10(9)/L) or no response was obtained, the same dose was continued for another 8 weeks. In patients with favorable response and subsequent decrease to pre-treatment values, an additional 4 weeks of treatment could be administered. RESULTS: Fourteen patients (ages 4-20 y) receiving 17 IFN courses were included. Mean initial platelet count was 29 +/- 15 x 10(9)/L. A significant increase was achieved during 14 of 17 courses (82.4%). All but two responses were transitory, and platelets returned to initial values after IFN discontinuation (mean 44 +/- 26 days). Considering the best response achieved by each patient, we observed: 1) 10 patients who achieved a sustained improvement of platelet count throughout the treatment period, decreasing to initial values after therapy was stopped; 2) one patient who achieved platelet count >150 x 10(9)/L, remaining with normal platelets at 18 months; 3) one patient who achieved platelet count >150 x 10(9)/L, remaining with platelets between 100 and 140 x 10(9)/L at 48 months; 4) one patient who had no response; and 5) one patient in whom therapy worsened the thrombocytopenia. A mild to moderate flu-like syndrome and a moderate decrease of the absolute neutrophil count were the only side effects observed. CONCLUSION: Interferon therapy induces a significant increase of platelet count and seems to be a valid alternative therapy to attempt the achievement of prolonged remission in refractory ITP, to defer splenectomy in younger children, or to improve platelet count before planned splenectomy.

Effect of early versus late administration of human recombinant erythropoietin on transfusion requirements in premature infants: results of a randomized, placebo-controlled, multicenter trial.

OBJECTIVE: The administration of recombinant human erythropoietin (rHuEPO), started after the first 2 weeks of life, reduces the transfusion requirement in premature infants. However, its use throughout the first 2 weeks of life, when anemia results predominantly from phlebotomy losses, remains controversial. We investigated whether early use of rHuEPO would reduce the total transfusion requirement and/or the number of transfusions throughout the first 2 weeks of life. METHODS: We randomized 114 infants with birth weight (BW) <1250 g to receive rHuEPO (1250 units/kg/week; IV; early group: n = 57) or placebo (late group: n = 57) from day 2 to day 14 of life; subsequently, all the patients received rHuEPO (750 units/kg/week, subcutaneously) for 6 additional weeks. All infants were given oral iron (6 mg/kg/day) and folic acid (2 mg/day). RESULTS: The early group showed higher hematocrit and reticulocyte counts than the late group in the first 3 weeks of life, but there was no difference in the total number of transfusions (early: 1.8 +/- 2.3 vs late: 1.8 +/- 2.5 transfusion/patient) or the transfusion requirement throughout the first 2 weeks of life (early:.8 +/- 1.1 vs late:.9 +/- 1.3) could be demonstrated. In infants with BW <800 g and total phlebotomy losses >30 mL/kg (n = 29), a lower number of transfusions was received by infants in the early group, compared with late group, from the second week to the end of the treatment (early: 3.4 +/- 1.1 vs late: 5.4 +/- 3.7 transfusion/patient). No clinical adverse effects were observed. Thrombocytosis was detected during the treatment with rHuEPO in 31% of the infants. CONCLUSIONS: In the whole population, the early administration of rHuEPO induced a rise of reticulocyte counts, but not enough to reduce the transfusion requirement. The most severely ill infants (BW <800 g and phlebotomy losses >30 mL/kg) seemed to benefit from early use of rHuEPO, and this deserves additional study.

Low levels of serum erythropoietin in children with endemic hemolytic uremic syndrome.

Serum erythropoietin (EPO) levels were measured in ten previously non-transfused children with hemolytic uremic syndrome (HUS). Complete blood cell count, serum EPO, and renal function tests were carried out upon admission and weekly thereafter. Blood samples were obtained: (1) prior to the first transfusion; (2) after the first transfusion but before recovery from renal failure; (3) during the recovery stage. All patients required transfusions (mean 1.8+/-0.8 per child). Absolute values of EPO correlated positively with the hematocrit during the three stages (r = 0.53, 0.36, and 0.12, respectively) which is opposite to expected results. The observed EPO logarithm/predicted EPO logarithm upon admission was low (0.70+/-0.08), falling further during stage 2 (0.57+/-0.03), but increasing thereafter (0.78+/-0.07) without reaching normal values. The reticulocyte production rate followed a parallel course (0.74+/-0.14, 0.54+/-0.11, and 0.60+/-0.10, respectively). On comparing the observed serum EPO levels with those expected, 9 of 11 pre-transfusion samples showed low values; in stage 2, all samples were below normal; in the recovery phase most (77.8%) were still low. Our results show an inadequate EPO synthesis in children with HUS, which could play an important pathogenic role, since it aggravates the severity of the existing hemolytic anemia; the secondary inhibitory effect of repeated transfusions exacerbates this inadequate synthesis.

Randomized trial of CVPP for three versus six cycles in favorable-prognosis and CVPP versus AOPE plus radiotherapy in intermediate-prognosis untreated Hodgkin's disease.

PURPOSE: To evaluate in a randomized trial the impact of three versus six cycles of cyclophosphamide, vinblastine, procarbazine, and prednisone (CVPP) chemotherapy in favorable-prognosis and CVPP versus doxorubicin, vincristine, prednisone, and etoposide (AOPE) plus involved-field radiotherapy (RT) in intermediate-prognosis previously untreated Hodgkin's disease. PATIENTS AND METHODS: Of 256 patients evaluated, 80 with a favorable prognosis according to a prognostic index designed by the Grupo Argentina de Tratamiento de Leucemia Aguda (GATLA) were randomized to three versus six cycles of CVPP without RT and 176 with intermediate risk to CVPP versus AOPE, both for six cycles with RT between the third and fourth cycles of 30 Gy to the involved areas at diagnosis. CVPP consisted of intravenous (I.V.) cyclophosphamide and vinblastine on days 1 and 8, and oral procarbazine and prednisone on days 1 to 14, every 28 days. AOPE consisted of I.V. doxorubicin and vincristine on day 1, oral prednisone on days 1 to 5, and I.V. etoposide on days 1 and 3, every 28 days. RESULTS: Complete remission was obtained in 39 of 41 (95%) patients treated with three cycles of CVPP and 36 of 39 (92%) treated with six cycles in the favorable-risk group (difference not significant [NS]). In the intermediate-risk group, 89 of 92 (97%) treated with CVPP plus RT versus 75 of 84 (89%) treated with AOPE plus RT achieved a complete remission (P = .05). At 60 months, the event-free survival (EFS) and overall survival rates in the favorable-risk group were 80% and 91% for CVPP x 3 and 84% and 97% for CVPP x 6, respectively (P = NS). In the intermediate-risk group, 60-month EFS rate for CVPP plus RT was 85%, compared with 66% for AOPE plus RT (P = .009). The overall survival rate was 95% versus 87% respectively (P = .157). CONCLUSION: Three cycles of CVPP without RT are equally effective as six cycles in the favorable-risk group. However, in the intermediate-group, CVPP plus RT is superior to AOPE plus RT, with significantly fewer events before and after induction (P = .009), without a difference in overall survival.

Correction of iron deficiency with an iron-fortified fluid whole cow's milk in children: results of a pilot study.

PURPOSE: This study assesses the efficacy of an iron-fortified (15 mg Fe, as stabilized ferrous sulfate (SFE-171), per liter) fluid whole cow's milk (IFFWCM) for the treatment of mild iron deficiency in children. Previous studies in healthy adult volunteers showed a mean 10.2 +/- 4.7% iron absorption. PATIENTS AND METHODS: Seventeen children (12 to 48 months old) with iron deficiency (serum iron (SI) < 60 micrograms/dl, transferrin saturation (TS) < 15%, serum ferritin (SF) < 15 ng/ml) were included in this study; 11 of them were anemic. As treatment, they received IFFWCM, instead of the customary whole cow's milk, for at least 4 months; medicinal iron was not administered. Hematocrit (Hct), hemoglobin (Hb), SI, TS, and SF were determined monthly. RESULTS: The Hb increased from 10.3 +/- 0.8 to 12.7 +/- 0.6 g/dl in the group with anemia (delta F-B: 2.4 +/- 1.0 g/dl) and from 12.6 +/- 0.7 to 13.5 +/- 0.3 g/dl in the group without anemia (delta F-B: 0.9 +/- 0.5 g/dl); the difference between both groups was significant (p < 0.01); the rate for Hct values showed a similar pattern. In the whole group, the SI increased to 84.8 +/- 37.4 micrograms/dl, with no difference between children with anemia and children without anemia; TS showed a similar pattern (delta F-B: 19.0 +/- 11.0%). The mean SF increased from 12.1 +/- 2.7 ng/ml to 27.9 +/- 25.4 ng/ml. Normal values for Hct, Hb, SI, and TS were reached by 100% of children; the rate for SF was 56.3%. Time required to reach normal Hct in the children with anemia was 59.4 +/- 33.0 days. Acceptance and tolerance were excellent; no treatment had to be discontinued. The group of patients with anemia was compared with an historical group composed of 55 children matched for age, basal Hct, and achieved Hct increase, treated with medicinal FS (4-6 mg/kg/day): time required to reach normal Hct was shorter in the FS-treated group (39.0 +/- 14.5 days) (p = 0.050). CONCLUSION: The use of IFFWCM alone could be an effective, relatively inexpensive, and well-tolerated treatment of iron deficiency in children.

Spectral [corrected] studies on the cadmium-ion-binding properties of bovine brain S-100b protein.

The effect of Cd2+ binding on bovine brain S-100b protein was studied using c.d. u.v. difference spectroscopy and fluorescence measurements. At pH 7.5, S-100b protein binds two Cd2+ ions per monomer with a Kd value of 3 x 10(-5) M. Addition of Cd2+ resulted in perturbing the single tyrosine residue (Tyr17) in the protein as indicated by u.v. difference spectroscopy and aromatic c.d. measurements. In the presence of Cd2+, the tyrosine residue moves to a more non-polar environment, since a red shift was observed in the u.v. difference spectrum. When the protein was excited at 278 nm, the tyrosine fluorescence emission maximum was centred at 306 nm. Cd2+ addition resulted in an increase in intrinsic fluorescence intensity. Fluorescence titration with Cd2+ indicated the protein binds Cd2+ with a Kd value of 3 x 10(-5) M. 2-p-Toluidinylnaphthalene-6-sulphonate-labelled protein, when excited at 345 nm, had a fluorescence emission maximum at 440 nm. Addition of Cd2+ to labelled protein resulted in a 5-fold increase in fluorescence intensity accompanied by a 5 nm blue shift in the emission maximum, suggesting that the probe, in the presence of Cd2+, moves to a hydrophobic domain. U.v. difference spectroscopic studies indicated a unique Cd2(+)-binding site on the protein, since Cd2+ addition yielded a large positive absorption band in the 240 nm region that is not found with either Ca2+ or Zn2- ions. Similar absorption bands have been observed in Cd-protein complexes such as Cd-metallothionein [Vasak, Kagi & Hill (1981) Biochemistry 20, 2852-2856] and also in model complexes of Cd2+ with 2-mercaptoethanol. This absorption band is believed to arise as a result of charge-transfer transitions between the thiolate and Cd2+. Of the two Cd2- -binding sites on the beta-chain, one must be located at the N-terminal end near the single tyrosine residue, since Cd2- and Zn2+ produced similar effects on the intrinsic protein fluorescence. The other Cd2+ site which is unique to Cd2+ must be Cys84, located at the C-terminal end.

Role of methylenetetrahydrofolate depletion in methotrexate-mediated intracellular thymidylate synthesis inhibition in cultured L1210 cells.

The effect of low methotrexate levels on methylenetetrahydrofolate and four other reduced folate pools in cultured L1210 cells has been examined over a 48-h period. Media folate levels and methotrexate were used to alter intracellular levels of reduced folates, and the distribution among individual reduced folates, so that they could be evaluated in terms of their effects on thymidylate synthesis and cell proliferation. Over the media folate concentration range of 0.25-50 microM, growth rate and thymidylate synthesis remained essentially unchanged while total intracellular reduced folates, determined from the summation of the five individual pools measured, increased approximately 25-fold. The 5-methyltetrahydrofolate and 10-formyltetrahydrofolate pools accounted for over 90% of the total reduced folate at the highest media folate level, while low media folate resulted in a much more equal distribution among the five reduced folates examined. Methotrexate, over the concentration range of 0.25-30 nM, caused extensive growth and intracellular thymidylate synthesis inhibition at media folate levels used in RPMI 1640 media (2.5 microM) and lower. However, growth inhibition was much less at the highest media folate level used, and thymidylate synthesis was not inhibited to a statistically significant extent. Intracellular reduced folates also responded differently to methotrexate depending upon the level of media folate. Depletion of the thymidylate synthase substrate, methylenetetrahydrofolate, could not account for diminished growth or thymidylate synthesis inhibition, since at 0.25 and 2.5 microM media folate no depletion occurred in response to methotrexate and only slight depletion was observed at 50 microM media folate. Dihydrofolate showed a tendency to increase at each of the media folate levels used with the least increase at the highest folate level. However, the ratio of dihydrofolate to total reduced folates was quantitatively most consistent with thymidylate synthesis and growth inhibition results.

Enhancement of polyacrylamide gel slice dissolution in hydrogen peroxide by cupric sulfate.

An improved method is described for quantitation of radio-labelled protein by scintillation counting after polyacrylamide gel electrophoresis. The method is based upon copper catalyzed dissolution of gel slices in hydrogen peroxide under ambient conditions. Complete dissolution of gel sections was accomplished by incubation at 25 degrees C in 30% H2O2 that contained 0.9 mM CuSO4. Recovery of tritiated protein was greater than 90% under these conditions while in the absence of CuSO4 recovery was less than 50%.

Tratamiento de la aplasia medular con globulina antilinfocitaria / Treatment of aplastic anemia with antilymphocyte globulin

Diez pacientes con diagnóstico de aplasia medular adquirida (AM) recibieron globulina antilinfocitaria (GAL) como tratamiento. Dos pacientes eran niños de dos yocho años de edad y el resto eran adultos con una media de 32 años (rango 16-56). El intervalo medio desde el diagnóstico hasta el inicio del tratamiento fue de 5,38 meses (rango 1-20. Cinco pacientes tenían antecedentes de contacto con benceno, organofosforado y piroxicam/ampicilina. Otro paciente tenía diagnóstico ce hemoglobinuria paroxística nocturna. En los cuatro restantes no se pudo determinar la etiología. Nueve pacientes habían recibido tratamiento previamente, seis con corticoides y andrógenos y res sólo con corticoides, sin respuesta. La GAL fue administrada bajo internación, en dosis de 10-20 mg/Kg/día durante cuatro días endos pacientes y en el resto durante ocho días. Entre el séptimo y el décimo día de comenzado el tratamiento, todos los pacientes desarrollaron enfermedad del suero, que fue controlada con prednisona. A los tres meses, tres pacientes tuvieron respuesta completa, y tres pacientes respuesta parcial. Cuatro pacientes no respondieron y de ellos tres fallecierom dos por sepsis y uno por hemorragia intracerebral. Nuestros resultados demuestran una respuesta completa o parcial en el 60% de los pacientes tratados con GAL, por lo que concluimos que la GAL es una opción terapéutica eficaz para el tratamiento de la AM severa o moderada en ausencia de posibilidad de transplante de médula ósea

Tratamiento de la aplasia medular con globulina antilinfocitaria / Treatment of aplastic anemia with antilymphocyte globulin

Diez pacientes con diagnóstico de aplasia medular adquirida (AM) recibieron globulina antilinfocitaria (GAL) como tratamiento. Dos pacientes eran niños de dos yocho años de edad y el resto eran adultos con una media de 32 años (rango 16-56). El intervalo medio desde el diagnóstico hasta el inicio del tratamiento fue de 5,38 meses (rango 1-20. Cinco pacientes tenían antecedentes de contacto con benceno, organofosforado y piroxicam/ampicilina. Otro paciente tenía diagnóstico ce hemoglobinuria paroxística nocturna. En los cuatro restantes no se pudo determinar la etiología. Nueve pacientes habían recibido tratamiento previamente, seis con corticoides y andrógenos y res sólo con corticoides, sin respuesta. La GAL fue administrada bajo internación, en dosis de 10-20 mg/Kg/día durante cuatro días endos pacientes y en el resto durante ocho días. Entre el séptimo y el décimo día de comenzado el tratamiento, todos los pacientes desarrollaron enfermedad del suero, que fue controlada con prednisona. A los tres meses, tres pacientes tuvieron respuesta completa, y tres pacientes respuesta parcial. Cuatro pacientes no respondieron y de ellos tres fallecierom dos por sepsis y uno por hemorragia intracerebral. Nuestros resultados demuestran una respuesta completa o parcial en el 60% de los pacientes tratados con GAL, por lo que concluimos que la GAL es una opción terapéutica eficaz para el tratamiento de la AM severa o moderada en ausencia de posibilidad de transplante de médula ósea (AU)

Evidence that H-enriched human placental ferritin is structurally similar to L-enriched ferritins of other tissues.

Ferritin was purified from normal full-term placenta, and the native structure and subunit composition were characterized. Reversed-phase high-performance liquid chromatographic analysis of the placental ferritin subunits suggested the presence of three subunit types. Using acid urea gel electrophoresis and amino acid analysis, these subunits were tentatively identified as two H-type and one L-type. The relative proportions of the subunit types were approx. 23% H-1, 33% H-2 and 44% L. The native structure of placental ferritin as judged by circular dichroism and fluorescence spectroscopy was quite similar to that of ferritin isolated from horse spleen, a source that is composed predominantly of L subunits. These results are consistent with a ferritin tetracosameric structure whose H and L subunits fit into 24 equivalent sites interchangeably because the secondary and tertiary structures of the two subunit types are very similar.

A comparison by HPLC of ferritin subunit types in human tissues.

Ferritin was isolated from human liver and spleen. Reversed phase high performance liquid chromatography of the ferritin subunits from each tissue yielded the same three chromatographic fractions. Physical and chemical characterization of the three fractions indicated that they represented at least two, perhaps three, chemically distinct subunits.