Mirella Pasino

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.

Familial nonrandom inactivation linked to the X inactivation centre in heterozygotes manifesting haemophilia A

A basic tenet of the Lyon hypothesis is that X inactivation occurs randomly with respect to parental origin of the X chromosome. Yet, nonrandom patterns of X inactivation are common – often ascertained in women who manifest recessive X-linked disorders despite being heterozygous for the mutation. Usually, the cause of skewing is cell selection disfavouring one of the cell lineages created by random X inactivation. We have identified a three generation kindred, with three females who have haemophilia A because of extreme skewing of X inactivation. Although they have both normal and mutant factor VIII (FVIII) alleles, only the mutant one is transcribed; and, they share an XIST allele that is never transcribed. The skewing in this case seems to result from an abnormality in the initial choice process, which prevents the chromosome bearing the mutant FVIII allele from being an inactive X.

Analysis of 18 novel mutations in the factor VIII gene.

Summary. We describe 18 novel mutations, unreported in the Haemophilia A mutation Databases, that have been identified in a cohort of unrelated, Italian patients affected with haemophilia A (HA). Screening of the factor VIII gene (FVIII) was performed using denaturing high‐performance liquid chromatography (DHPLC) and direct sequencing. Eight mutations were characterized as non‐missense alterations, and the remaining 10 were missense mutations. Heterozygosity for the identified mutations was observed in the female relatives of patients belonging to eight families with sporadic cases. In an attempt to understand better the causative effect of the mutations and the clinical variability of the patients, missense mutation consequences were investigated for: (1) the nature of the new amino acid; (2) the location of the substituted amino acid within crystallographic and theoretical models; and (3) the degree of conservation of the native residue in factor VIII (FVIII) protein and FVIII‐related protein family aligned sequences. These research tools have provided evidence that the mutations we describe involve residues that were conserved, at least in FVIII proteins, in all the species we compared.

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.

Germ-line origin of intron 1 inversion in two haemophilia A families.

Summary.  Factor VIII gene inversion of intron 1 has recently been reported to be the mutation responsible for haemophilia A in about 5% of severe cases. In our series of patients, which is made up of 77 Italian cases negative for intron 22 inversion, the mutation was found in three sporadic and in one familial patients, with an overall frequency of 5.2%. The carrier status of the patients’ female relatives was assessed by mutation analysis and showed that only two‐thirds of cases could be considered truly sporadic. The germ‐line origin of the mutation was investigated in the two sporadic families by haplotype analysis on genomic DNA of the patients’ maternal grandparents. These studies indicated that both mutation events had occurred in the germ cell lines of the patients’ healthy grandfather, suggesting that, as already demonstrated for the inversion of intron 22, the male germ cell line is more susceptible to the intrachromosome recombination which leads to the inversion of intron 1.

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.

Cyto-morphologic evaluation of bone marrow in infants with disseminated neuroblastoma

We studied the prevalence and degree of tumor cell infiltration (TCI) in bone marrow (BM) aspirates of 89 infants with stage 4/4 S neuroblastoma and correlated them with MYCN gene status and patient outcome. TCI was scored 0, +, ++, and +++, the last corresponding to an infiltration greater than 10%. TCI 0 was more frequent in stage 4 than in stage 4 S. TCI + and ++ were equally represented. TCI +++ was found only in 9 patients, all with typical stage 4 features (bone or lung involvement). Overall survival was not significantly influenced by the presence and degree of TCI.

Treatment of Poor-Risk Neuroblastoma with Intensive Chemotherapy and Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor

Advanced neuroblastoma is one of the most lethal pediatric malignancies. Several antitumor compounds are able to induce tumor regression, and a dose-response relationship has been demonstrated for some of them. A significant improvement in both response rate and duration of survival has thus been obtained by treatment intensification [1–4]. In the Italian experience, the median survival time of children treated with aggressive chemotherapy has been doubled compared with historical controls [5]. However, the higher initial response rate and the prolonged remission time has resulted in only a marginal improvement in cure rate, presently not exceeding 25% [6, 7]. Hematopoietic growth factors (colony-stimulating factors, CSFs), by mitigating the myelotoxic effect of chemotherapy, may reduce the treatment-related morbidity and thus permit the delivery of higher dosages and the shortening of intervals between chemotherapy courses [8, 9].

EFFICIENCY OF RETROVIRAL TRANSDUCTION OF CD 34+ CELLS FROM UMBILICAL CORD BLOOD DEPENDS ON GROWTH FACTOR STIMULATION. 134

The feasibility of gene marking or gene therapy protocols making use of purified CD34+ cells greatly depends on the efficiency of stable transduction. The great potentiality of umbilical cord blood CD34+ cells prompted us to evaluate which factor(s) play a major role(s) in influencing the transduction efficiency of retroviral vectors. CD34+ cells, isolated with an immunomagnetic device (MiniMacs, Miltenyi Biotech, Germany), 90-95% pure as assessed by cytofluorimetric analysis, were transduced with the LXSN retroviral vector carrying the neomicine resistance gene. Infection was made with virus-containing supernatant of Am12 env+ packaging cell line, having a titer of 2×106 PFU/ml. All the infections were performed using a multiplicity of infection of 10 PFU/cell. Multiple or single infections were performed with the following schedules: i) up to 6 infection lasting one hour over 3 days; ii) a 72 hr infections substituting fresh viral supernatant and cytokines every 24 hrs; iii) a single 72 hr infection. In most experiments, CD34+ cells were preincubated with different cytokine cocktails for 24 or 48 hrs before infection. Efficiency of transduction was evaluated both by clonogenic assays and semiquantitative PCR analysis performed either immediately or after 7 day expansion in the presence of cytokines and G-418. G-418-resistant CFU-E, BFU-E and CFU-GM colonies were scored after 7 and 14 days. We provided evidence that the efficiency of transduction was mainly dependent on cell preincubation with growth factors rather than on the number of infections. Precisely, infection performed on freshly isolated CD34+ was almost ineffective (0.4%) while 48 hr preincubation in the presence of SCF*+IL-6+IL-3 allowed to reach a level of transduction up to 40% regardeless of the number of infections. In this respect it is noteworthy that a single infection, lasting as long as a multiple infection course gave identical results. In conclusion the results obtained suggest that high efficiency of transduction of umbilical cord blood CD34+ cells can be achieved in liquid, stroma free, cultures.

HUMAN UMBILICAL CORD BLOOD CD34+ CELL EXPANSION IN THE PRESENCE OF FLT-LIGAND(FL). 133

Human umbilical cord blood (HUCB) is an important source of progenitor cells which are able to reconstitute hematopoiesis. The in vitro expansion of isolated CD34 positive cells should provide a sufficient number of hematopoietic cells for transplants in large reciplents and would increase vector-mediated gene transfer efficiency. In vitro expansion of progenitor cells requires the identification of the cytokine combinations which result in the greatest increase of committed cells, without exhausting the early progenitor pool. In this study we tested the effect of three colony stimulating-factor (CSF) combinations [SCF*+IL-6(CSF2); SCF*+IL-6+FL (CSF3); SCF*+IL-6+FL+IL-3 (CSF4)] on CD34+ cells isolated(MiniMACS, Miltenyi Biotec, Germany) from HUCB. CD34+ cells, ≥97% pure in all samples, were ≥97% CD33- (range 97-99%) and ≥97% CD38+ (range 97-99.8%). The absolute number of total cells and cell subsets, evaluated after 7 and 11 days of liquid cultures, showed the following median fold increase:Table