Pier Giorgio Mori

Flow cytometric and functional characterization of AC133+ cells from human umbilical cord blood

AC133+ cells may represent an alternative source of transplantable haemopoietic progenitor cells to CD34+ cells. Here, we have addressed the characterization of umbilical cord blood (UCB) AC133+ cells and compared their immunophenotypic and functional features with those of UCB CD34+ cells. UCB AC133+ and CD34+ cell fractions were purified by magnetic cell sorting, analysed by flow cytometry, tested for their content in blast cell colony‐forming units (CFU‐Bl), erythroid and granulocyte–macrophage colony‐forming units before and after expansion in the presence of various haemopoietic growth factor combinations. Median AC133+ cell yield was 62·3%, and median AC133+ population purity was 97·9%. AC133+ cells were found to contain significantly more CFU‐Bl than CD34+ cells; furthermore, the replating efficiency, i.e. the number of CFU‐Bl capable of generating secondary colonies, was higher in the former than in the latter cells. Both AC133+ and CD34+ cells displayed an increased ability to give rise to committed progenitors after 7‐day expansion in liquid cultures. These data suggest that the AC133+ cell subset is a heterogeneous pool of immature and more differentiated cells that can be maintained and expanded in well‐defined culture conditions. In comparison with CD34+ cells, UCB AC133+ cells appear to contain a higher number of early haemopoietic progenitors.

Pentasomy 21 in leukemia complicating Diamond-Blackfan anemia

We present the cytogenetic pattern of a leukemic infant with Diamond-Blackfan anemia (DBA). The karyotype was characterized by clonal evolution involving consecutive gains of chromosome 21 up to pentasomy. No chromosomal changes were present in normal lymphocytes. Such a karyotype evolution has been described in some cases of acute leukemia associated with Down Syndrome, but rarely in non-Down cases.

Phase II Trial of Methylprednisolone Pulse Therapy in Childhood Chronic Thrombocytopenia

Nineteen children with chronic idiopathic thrombocytopenia (ITP) were treated with a single intravenous injection of methylprednisolone (MP), 15 mg/kg/day, for 3 consecutive days. The 3-day pulses gave rise to a positive and fast therapeutic response with increase of the platelet count in about three quarters of the patients. The platelet count remained above 50 X 10(9)/1 for more than 1 month in 10 children. Eight out of them still presented a safe platelet count (greater than 50 X 10(9)/1) 4 months after the onset of the therapy. The MP therapy improved the platelet count more in the older children and possibly in the females. No severe side effects were observed. Our results suggest that this therapeutic approach could be useful in the management of acute bleeding episodes in children with chronic ITP.

von Willebrand factor: biological function and molecular defects.

The human von Willebrand factor (vWF) plays a pivotal role in the mechanisms of blood clotting and platelet thrombus formation; it also binds and stabilizes factor VIII procoagulant protein. The biological functions of vWF are dependent on distinct molecular domains responsible for the specificity and affinity for ligands. The multimeric structure of vWF provides an array of binding sites that allow multivalent interactions, thus supporting the formation of stable platelet aggregates at the site of vascular injury, particularly under flow conditions characterized by high shear stress. Quantitative and qualitative abnormalities of vWF cause the most common congenital bleeding disorder in humans, the von Willebrand disease (vWD). This review will provide an update on the recent advances toward the elucidation of structure-function relationships and the detection of molecular defects leading to vWD and will highlight the revised classification of vWD.

Treatment of Acute Idiopathic Thrombocytopenic Purpura (AITP): Cooperative Italian Study Group Results

A cooperative Italian study group on acute idiopathic thrombocytopenic purpura (AITP) has been designed to evaluate efficacy and safety of no treatment at the onset of the disease and sequential treatment with immunoglobulin and high dose steroid. One hundred thirty-eight patients with AITP entered in the trial. Eleven patients were treated before the end of the waiting period because of bleeding. One hundred twenty-seven (92%) received no treatment for the first 10 days of the disease, 65 patients (51.18%) recovered spontaneously, 62 patients were treated with immunoglobulin, and 52 (83.8%) of them responded positively but only 36 (58.06%) permanently. There was no statistical difference between the results obtained with 400 mg/kg for 5 days versus 200 mg/kg. Twenty-four patients were treated with high doses of steroids, 20 (83.3%) with positive response, and 10 (41.66%) were permanently cured. Four (3.14%) of the patients enrolled in the protocol still had active disease at the end of treatment, and 10 relapsed within 4 months after the end of the treatment.

Mutation analysis impact on the genetic counseling of sporadic hemophilia B families

Previous studies have shown that hemophilia B (HB) is the result of several different mutations, mostly single nucleotide substitutions, in the factor IX (FIX) gene. In order to evaluate the impact of mutation analysis on genetic counseling in sporadic and uninformative HB familial pedigrees, we re‐analyzed by the conformation sensitive gel electrophoresis (CSGE) technique 14 patients, previously studied by restriction fragment length polymorphisms (RFLPs). A single mutation was present within the FIX gene of each patient: 12 mutations were single base substitutions, 1 was a base insertion, and 1 was a four nucleotide deletion; 4/12 mutations have not been described so far. By identifying the detrimental mutations in affected males, carrier status was correctly diagnosed in all the women we studied; 3/12 de novo events were found in maternal meioses with a 25% mutation rate. Identification of the genetic defect was also successfully applied to three prenatal diagnoses.

A homozygosity state for 20210A prothrombin variant in a young womanas cause of a deep venous thrombosisduring pregnancy

To the Editor: Venous thromboembolism represents one of the main causes of morbidity and, less frequently, of mortality among the adult population. It is the third most common cardiovascular disease besides acute ischemic heart disease and stroke (1). Congenital de®ciency of the hemostasis main natural inhibitors (antithrombin III, protein C or protein S) is associated with an increased risk of venous thrombosis, even though these genetic defects are found in only 5±10% (2) of all patients with thrombosis. Recently, several studies have demonstrated that even other genetic factors such as the G1621A mutation in Factor V, the G20210A mutation in Factor II (prothrombin), and the C677T mutation in methylenetetrahydrofolate reductase (MTHFR) genes may increase the risk of thromboembolic events, and they may be responsible for thrombosis in young subjects in absence of other risk factors (3±5). Factor V and Factor II are signi®cant in heterozygosity, while MTHFR is signi®cant only in homozygosity and when associated with the increased homocysteine and low folate plasma levels. In particular the mutation in the 3k untranslated region of the prothrombin gene (G20210A) was found to be associated with increased prothrombin plasma levels. Pregnancy is assumed to be a triggering factor of thrombotic events (6), and this risk may increase when these genetic conditions are present. Here we report a thrombotic event during the ®rst pregnancy of a 22-yr-old woman. D.P.M. had painful swelling of the upper left leg at the 35th week of an uneventful pregnancy. Thrombosis of the left femoral and iliac veins was demonstrated by doppler ultrasonography. Calcium heparin treatment was given (5000 I.U. twice a day) and continued for 10 d after cesarean section; then oral anticoagulant with 4-hydroxycumarin was started. The International Normalized Ratio (INR) was maintained between 2 and 3. Anticoagulant therapy is still ongoing. Natural inhibitor dosage in the proband performed before starting anticoagulant treatment showed that antithrombin III, protein C, and free and total protein S were normal. Lupus anticoagulant (LA) was negative. Prothrombin plasma level was 1.5 U/ml, higher than in the control group (n.v. 0.50±1.1 U/ ml). To evaluate the impact of genetic defects in thrombosis, DNA was extracted from 1 ml of peripheral blood, and the most common genetic factors involved in deep vein thrombosis (DVT), mutations for FV Leiden, C677T MTHFR, and G20210A prothrombin genes were investigated by the aim of polymerase chain reaction (PCR). Density gradient gel electrophoresis (DGGE) was used to detect FV Leiden (7), while the fragments obtained from MTHFR and FII ampli®cation were digested by restriction enzimes Hinf I and Hind III, respectively, as described (4, 5). In our patient, no mutation of the FV or MTHFR genes was detected, while a homozygous state for the prothrombin gene 20210A allele was found. An association between the prothrombin gene variant in the heterozygous state, and a threefold risk of developing venous thrombosis has been shown (5). Among the Caucasian population the 20210A allele frequency in healthy controls is 1±2%, while it is 5±8% (8) in patients with episodes of DVT. Literature reports that homozygous Factor II point mutation is quite rare among patients with venous thrombosis, and an accurate risk assessment has not yet been identi®ed. An accurate anamnesis of this family was performed in order to identify possible hemostatic disorders or other factors predisposing to thrombosis. The probands father had a heart infarction when he was 50 yr old. The paternal grandfather had a stroke when he was 52, while the 49-yr-old mother was asymptomatic. The father was found heterozygous for prothrombin variant and normal for Factor V and MTHFR; the mother was heterozygous for both Factor II and Factor V mutation and normal for the MTHFR gene. Prothrombin plasma level was 1.20 in the father and 1.10 U/ml in the mother. No mutations were found in the Eur J Haematol 2000: 65: 80±81 Printed in UK. All rights reserved Copyright # Munksgaard 2000

Transforming growth factor beta-1 (TGF-β1) released by an Epstein-Barr virus (EBV) positive spontaneous lymphoblastoid cell line from a patient with Kostmann's congenital neutropenia inhibits the growth of normal committed haemopoietic progenitors in vitro

Summary This study reports the characterization of a spontaneous lymphoblastoid cell line (LCL) raised from the peripheral blood of a patient with Kostmanns congenital neutropenia. The LCL was composed of EBV‐infected polyclonal B cells and displayed surface markers and pattern of growth in vitro typical of normal LCLs. The supernatant of the LCL contained a colony inhibiting activity (CIA) that decreased the cloning efficiency of normal committed haemopoietic progenitors and was identified as immunoreactive transforming growth factor β1 (TGF‐β1) by neutralization experiments with a specific antiserum. Control studies with a panel of LCLs spontaneously derived from the peripheral blood of patients seropositive for Epstein‐Barr virus (EBV) infections showed that 5/30 LCLs produced a CIA. This CIA was not identifiable as TGF‐β1 but rather was due to the combined effects of tumour necrosis factor α (TNFα). tumour necrosis factor β (TNFβ) and interferon α (IFNα), that were present in the LCL supernatants. The hypothesis that the B cells latently infected by EBV in vivo and possibly expanded as a consequence of the infection may have contributed to the inhibition of the patient granulopoiesis by releasing TGF‐β1 will be discussed.

Chronic Childhood Neutropenia: Studies on the in vitro Clonogenicity of Bone Marrow Myeloid Progenitors

Bone marrow mononuclear cells (MNC) from 6 pediatric patients with chronic neutropenia were tested for myeloid colony formation upon stimulation with the supernatant of the 5637 cell line or with recombinant granulocyte-macrophage colony-stimulating factor or interleukin 3. Heterogeneous patterns of response of myeloid progenitors were observed in the individual patients, with no colony growth in 2 cases and abnormalities of colony formation or composition in two additional cases. Morphologic and surface marker analyses showed that the bone marrow of some patients contained an excess of lymphocytes with an altered subset distribution. In order to investigate whether or not there was a relationship between the latter abnormality and the observed clonogenic defects, marrow MNC were tested for myeloid colony formation before and after lymphocyte depletion. No evidence for a cell-mediated suppression of colony growth was obtained; likewise, patient sera failed to inhibit colony formation by normal bone marrow myeloid progenitors. Taken together, these data make it unlikely that, in our cases, immunologic mechanisms were responsible for the pathogenesis of chronic neutropenia.

Platelet Antibody Detection in Pediatric Immune Thrombocytopenic Purpura: Evaluation of Three Screening Methods

Background and objectives: Immune thrombocytopenic purpura (ITP) is a common hematologic disorder, two forms of which occur in children. The detection of circulating platelet antibodies is helpful in diagnosis. Materials and methods: We evaluated three different immunological methods for detecting platelet antibodies in the serum of children with ITP. These were: a solid‐phase red‐cell adherence test (SPRCA), an enzyme immunoassay (EIA), and an immunofluorescence test (PSIF). Results: The sensitivity of the methods in detecting IgG antibodies ranged from 28.1 (EIA) to 39.4% (SPRCA). We also looked for IgM antibodies by PSIF, thus raising the sensitivity of this test from 32.0 to 40.0%. A combination of two tests (SPRCA and EIA) allowed us to detect 61.8% positive samples. By doing all three tests, we obtained 71.3% positive samples. Finally, we reached 73.5% by adding PSIF for IgM. We found a higher frequency of circulating antibodies in both acute and chronic ITP at onset than in clinical remission. There were a few positive sera in chronic ITP, but not in the acute form in remission. Conclusion: The individual tests each have a relatively low sensitivity, but the combination of all three increases the diagnostic effectiveness. The finding of platelet antibodies during remission may predict evoluation toward a systemic autoimmune state.