Tiziana Lanza

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.

Etanercept as a salvage treatment for refractory aplastic anemia

About 10–15% of patients with acquired aplastic anemia (AAA) have resistant/recurrent disease not eligible for standard treatment like hematopoietic stem cell transplantation and/or combined immunosuppression. We report a 17‐year‐old male with an 11 years history of AAA who, after two courses of immunosuppression, was red cell transfusion‐dependent, severely thrombocytopenic, refractory to platelet transfusion, had iron overload and post‐transfusion HCV infection. This patient achieved transfusion independence from platelets and normalized Hb after treatment with the anti‐TNF agent Etanercept. Over a 12 months follow‐up he experienced only transient increase of liver transaminases. Pediatr Blood Cancer 2009;52:522–525.

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.

Possible role of toll‐like receptor 9 polymorphism in chemotherapy‐related invasive mold infections in children with hematological malignancies

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Insight into molecular changes of the FIX protein in a series of Italian patients with haemophilia B

Summary.  Deficiency or dysfunction of factor IX FIX leads to haemophilia B (HB), an X‐linked, recessive, bleeding disorder. On a molecular basis, HB is due to a heterogeneous spectrum of mutations spread throughout the F9 gene. In several instances, a cause‐effect relation has been elucidated, in others predicted possibilities have been offered by crystallography inspection and by software‐constructed models of the protein. The aim of this study was to contribute to the understanding of HB molecular pathology. The F9 missense mutations we identified in 21 unrelated Italian HB patients by direct sequencing of the whole F9 coding regions were inspected for the causative effect they provoked on the ensuing transcript, and on the protein structure. Each alteration was studied in order to: (i) characterize the defect on the basis of the nature of the mutation; (ii) identify the predicted defect that is induced in the gene and (iii) speculate about the potential, detrimental effects which upset the protein functionality through an idealized FIX model. The resulting data may further contribute to the comprehension of the mechanisms underlying the disease.

Autoimmune neutropenia of infancy: Data from the Italian neutropenia registry

ing these with HFE genetic profiles. The local Ethics Committee approved the protocol. We analyzed data from all the 159 patients admitted in the study period with suspected iron overload based on high TS (above 55% in men and 45% in women) and/or SF (> 322 ng/mL), who had undergone MRI-T2* for heart, liver, spleen, and/or pancreas iron overload and had been screened for the presence of HFE mutations by allele-specific PCR (polymerase chain reaction). The calculations of liver iron concentration (LIC) values were based on liver MRI-T2* measurements, using the Thalassemia-Tools software (Cardiovascular Imaging Solutions, London, UK). Mutations in the HFE gene were identified in 109/159 (68.6%) patients. The most common mutation in our sample was H63D, present in 91 patients (57.2%): 14 (8.8%) were homozygous, 69 (43.4%) heterozygous, and 8 (5%) compound heterozygous for C282Y/H63D. For the C282Y mutation, in contrast, only 5 patients (3.1%) were homozygous and 11 (6.9%) were heterozygous. The S65C mutation was detected in heterozygous state in 2 (2.5%) cases. All 159 patients underwent abdominal MRI-T2* and 126 underwent cardiac MRI-T2* too. Only 3 out of 126 cardiac MRIs had a positive T2* result, mild cardiac overload (T2*: 18.98, 19.14, and 19.8 ms). Of these, two patients had the H63D mutation (1 homozygous and 1 heterozygous) and one patient did not have any of the mutations studied. In the liver, 61 (38.4%) patients had iron overload (T2*:< 11.4 ms and LIC> 2.0 mg/g) of which 57 (35.8%) were light (T2*: 3.83–11.4 ms and LIC: 2.01– 6.86 mg/g), and four (2.5%) moderate (T2*: 2.0–3.8 ms and LIC: 7.06–13.56 mg/g). Of these patients with liver overload, 27.9% were C282Y carriers (8.2% homozygous, 11.5% heterozygous, and 8.2% compound heterozygous C282Y/H63D), and 50.8% carried the H63D mutation (14.8% in homozygosis and 36.1% in heterozygosis). Only 12 (19.7%) patients with liver overload did not have the HFE mutation. The presence of C282Y mutation (in either homo or heterozygosis), compound heterozygous (C282/H63D), and H63D in homozygosis was significantly associated with a higher frequency of iron overload in the liver as measured by T2* (P5 0.001). However, this was not true in patients with H63D in heterozygosis or absence of mutation (P5 0.42), in which overload frequency was 68.4% and 29.1%, respectively. Pancreatic overload was diagnosed in 33 patients (21%), and 56 patients (35.7%) had splenic overload (Table I). The presence of the C282Y was associated with an overall higher frequency of iron overload. There was also a relatively high frequency (37.3%) of abnormal T2* values in H63D mutants both in the liver and in the spleen, and the frequency of splenic iron overload in H63D mutants was similar to that associated with the C282Y mutation. SF results were available for 152 patients. Median SF was 647 ng/mL (72–13,625), and in 138 patients (90.8%) SF was abnormally high. Overall, in 28 patients (18.2%) serum levels were higher than 1,000 ng/mL, in 80 patients (54%) they varied from 501 to 1,000 ng/mL and in 30 (20.3%) they ranged from 324 to 500 ng/mL. Serum TS was obtained from 94 patients, with a median of 42% (31–57) and elevated results in 32 (34%) of the tests. Considering MRI findings as standard, SF was the most sensitive test (sensitivity 94.7%, specificity 11.8%), whereas TS was the most specific (sensitivity 34%, specificity 65.4%), indicating that they might be complementary. Sensitivity and specificity values were similar in patients with and without HFE mutation. Iron overload prevalence was different according to the affected organ or the type of HFE mutation; over 50% of patients with liver iron overload carried the H63D mutation, and two out of three patients who had cardiac iron overload were also H63D carriers. A total of 38% of the H63D carriers presented with iron overload in the liver and spleen and 22% in the pancreas, showing that this mutation alone might correlate with iron overload. It is worth noticing, however, that the absence of HFE mutations does not rule out the presence of other mutations associated with hereditary hemochromatosis. Our study demonstrates, in a large sample of Brazilian patients, that MRI-T2* is a non-invasive, accurate, and sensitive technique for the detection of low levels of iron overload in patients with HH type 1. Excessive iron stores in the liver were detected in 39% of patients, showing that iron accumulation begins in the liver [6]. Given the observation that MRI is an accurate and safe tool to measure iron stores in these organs, we believe that this technology should be incorporated in the investigation of suspected cases of hemochromatosis and contribute to guide therapeutic decisions such as phlebotomy.

Sirolimus for the treatment of multi-resistant autoimmune haemolytic anaemia in children.

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Etanercept treatment in Fanconi anaemia; combined US and Italian experience

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