Ethics of placental blood collection and storage.
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Bioethics↗
Biomedical subjects
F Locatelli
Publications and source records attributed to F Locatelli.
A subpopulation of murine bone marrow cells fully differentiates along the myogenic pathway and participates in muscle repair in the mdx dystrophic mouse.
Bone marrow (BM) transplantation in mice suggests the existence of pluripotent cells able to differentiate into skeletal muscle tissue, although sustained myofiber reconstitution has not yet been achieved. We investigated the myogenic potential of mouse BM cells and evaluated whether a BM fraction enriched for cells expressing skeletal muscle markers would ameliorate muscle repair, when compared to whole BM, into the dystrophic mdx mouse. We demonstrate that cells expressing striated-muscle-specific proteins are already present in the BM independently from experimentally forced myogenic conversion. We observed the presence of both markers of early myogenic program such as Pax3, Myf5, MyoD, desmin, and late myogenesis such as myosin heavy chain and alpha-sarcomeric actin. These myogenic cells are more represented in the early nonadherent BM fraction, which generates clones able to fully differentiate into myotubes. Transplantation in mdx mice by intravenous injection of whole BM and a tenfold BM myogenic enriched fraction resulted in BM reconstitution and limited dystrophin restoration. Taken together, these data show that a fraction of BM cells have a definite potential for differentiation along the skeletal muscle pathway and can be recruited by muscle repair mechanisms. They also indicate that factors limiting the degree of muscle recruitment and the host stem cell competition should be assessed in order to evaluate the usefulness of BM-derived myogenic cells into the context of cell-mediated gene therapy of inherited muscle diseases.
Animals↗
Modulated generation of neuronal cells from bone marrow by expansion and mobilization of circulating stem cells with in vivo cytokine treatment.
The aim of the present study is to determine whether the expansion and mobilization of circulating bone marrow (BM) stem cells by in vivo treatment with granulocyte-colony stimulating factor (G-CSF) and stem cell factor (SCF) increase the amount of BM-derived neuronal cells in mouse brain. The presence of BM-derived cells in the brain was traced by transplanting into lethally irradiated adults and newborns adult BM from transgenic mice that ubiquitously expressed enhanced green fluorescent protein (GFP). GFP+ and Y-chromosome+ donor-derived cells were present in several brain areas of all treated mice (cortical and subcortical areas, cerebellum, olfactory bulb). The presence of GFP+ cells expressing nuclear neural specific antigen (NeuN), neurofilament, and beta-III tubulin in cortical forebrain and olfactory bulb (OB) was higher in G-CSF-SCF treated groups (P < 0.05, analysis of variance, Fisher post hoc). We observed that overall the amount of double positive cells was higher in animals treated at birth than in adults and in OB than in forebrain areas (P < 0.05). Temporal cortical areas of cytokine-treated adult animals revealed a mean threefold increase in the number of GFP+ cells expressing the nuclear neural specific antigen (211 +/- 86 GFP+NeuN+/mm(3) in G-CSF + SCF treated mice and 66 +/- 33 GFP+NeuN+/mm(3) in control animals). GFP+ cells coexpressing neuronal markers contain only one nucleus and have a DNA index (a measure of DNA ploidy) identical to that of surrounding neurons, thus excluding donor cell fusion with endogenous cells as a relevant phenomenon under these experimental conditions. Our results indicate that G-CSF and SCF administration modulates the availability of GFP+ cells in the brain and enhances their capacity to acquire neuronal characteristics. Cytokine stimulation of autologous stem cells might be seen as a new strategy for neuronal repair in neurodegenerative diseases.
Age Factors↗
Transplant-related toxicity and mortality: an AIEOP prospective study in 636 pediatric patients transplanted for acute leukemia.
Hematopoietic stem cell transplantation can cure high-risk acute leukemia (AL), but the occurrence of non-leukemic death is still high. The AIEOP conducted a prospective study in order to assess incidence and relationships of early toxicity and transplant-related mortality (TRM) in a pediatric population. Between 1990 and 1997 toxicities reported in eight organs (central nervous system, heart, lungs, liver, gut, kidneys, bladder, mucosa) were classified into three grades (mild, moderate, severe) and prospectively registered for 636 consecutive children who underwent autologous (216) or allogeneic (420) transplantation, either from an HLA compatible related (294), or alternative (126) donor in 13 AIEOP transplant centers. Overall, 47% of the patients are alive in CR (3-year EFS: 45.2%, s.e.: 2.1), 19% died in CR at a median of 60 days (90-day TRM: 14.3%, s.e.: 1.4), 34% relapsed. Toxicity of any organ, but mucosa and gut, was positively correlated with early death; moderate and severe toxicity to heart, lungs, liver and kidneys significantly increased early TRM, with estimated relative risks of 9.1, 5.5, 2.7 and 2.8, respectively, as compared to absent or mild toxicity. Patients with grade III-IV aGVHD experienced more than double (56% vs. 19%) TRM than patients with grade 0-II aGVHD. A higher cumulative toxicity score, estimating the impact of toxicity on TRM, was significantly associated with transplantation from an alternative donor. Quantitative assessment allowed us to describe the extent to which 'grade' of toxicity and 'type' of involved organs were related to mortality and pre-transplant characteristics and yielded a prognostic score potentially useful to compare different conditioning regimens and predict probability of early death.
Adolescent↗
Successful treatment of Griscelli syndrome with unrelated donor allogeneic hematopoietic stem cell transplantation.
Griscelli syndrome (GS) is a rare autosomal recessive disorder, characterized by pigmentary dilution of the skin and hair and in most patients by abnormal regulation of the immune system, which results in a syndrome of macrophage hyperactivation, known as hemophagocytic lymophohistiocytosis (HLH). Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment available for genetically induced HLH. Few cases of successful HSCT from a compatible donor have been reported in children with GS. We describe the first patient with GS cured with an allograft from a compatible unrelated bone marrow donor. We used a novel preparative regimen consisting of busulfan, thiotepa and fludarabine. The demonstrated curative effect of HSCT from an unrelated donor in a patient with genetically determined HLH also supports the use of a systematic diagnostic approach in these patients, in order to identify those with a worse prognosis and needing an urgent allograft in a timely manner.
Bone Marrow Transplantation↗
Improvement over time in outcome for children with acute lymphoblastic leukemia in second remission given hematopoietic stem cell transplantation from unrelated donors.
Aims of this study were to verify whether reduction in transplant-related mortality (TRM) of children with acute lymphoblastic leukemia (ALL) in second complete remission (CR) given allogeneic hematopoietic stem cell transplantation (HSCT) from unrelated volunteers has occurred over time and to investigate the role of other variables on the probabilities of relapse, TRM and event-free survival (EFS). We compared results obtained in 26 children given HSCT before January 1998 with those of 37 patients transplanted beyond that date. In all donor-recipient pairs, histocompatibility was determined by serology for HLA-A and -B antigens and by high-resolution DNA typing for DRB1 antigen. High-resolution molecular typing of HLA class I antigens was employed in 20 of the 37 children transplanted more recently. Probability of both acute and chronic GVHD was comparable in the two groups of patients. In multivariate analysis, children transplanted before January 1998, those with T-lineage ALL and those experiencing grade II-IV acute GVHD had a higher relative risk of TRM at 6 months after transplantation. Relapse rate was unfavorably affected by a time interval between diagnosis and relapse <30 months. The 2-year probability of EFS for children transplanted before and after 1 January 1998 was 27% (10-44) and 58% (42-75), respectively (P = 0.02), this difference remaining significant in multivariate analysis. EFS of unrelated donor HSCT in children with ALL in second CR has improved in the last few years, mainly due to a decreased TRM. This information is of value for counseling of patients with relapsed ALL.
Adolescent↗
A rationale for an individualized administration frequency of epoetin beta: a pharmacological perspective.
Several studies have compared the efficacy of once-weekly subcutaneous (s.c.) epoetin treatment with two or three times weekly treatment in renal anaemia. Epoetin administration frequency has attracted a high level of attention in recent years, and numerous small-scale studies have shown comparable efficacy and tolerability of once-weekly vs more frequent administration. The results of two large-scale, randomized, controlled trials of once-weekly administration of epoetin beta became available recently. One of these studies, by Locatelli et al., was the first to be designed specifically to demonstrate therapeutic equivalence between once-weekly and three times weekly epoetin beta treatment, using rigorous statistical methods. This was a large, multicentre, randomized, parallel group, 24-week study in 173 chronic renal failure patients. Treatment regimens were considered equivalent if: (i) the 90% confidence interval (CI) of the difference between treatment groups was within +/-2% for the time-adjusted area under the haematocrit (Hct) curve (AUC); and (ii) for mean weekly epoetin beta dose, the 90% CI of the ratio of the groups was between 0.8 and 1.25. As recommended by current guidelines for statistical analysis of clinical trial data, multiple analysis populations were examined in order to demonstrate robustness of the results with regard to the population chosen for analysis. Findings from the primary analysis, the per-protocol population, were confirmed by both the intent-to-treat analysis and an exploratory analysis that examined the influence of five patients who received dose increases above the mean. In all three analyses, the 90% CIs were within the pre-specified equivalence ranges for both the difference between treatment groups for Hct AUC and the ratio of mean weekly epoetin beta dose. In conclusion, once-weekly and three times weekly s.c. epoetin beta treatment regimens are statistically equivalent in terms of maintaining stable Hct levels and dose requirements in haemodialysis patients. The agreement of the three analysis populations provides a convincing demonstration of the robustness of the results. These results confirm that a once-weekly epoetin beta regimen is an effective option for management of renal anaemia that may improve patient convenience and compliance.
Drug Administration Schedule↗
Unrelated bone marrow transplantation in thalassemia. The experience of the Italian Bone Marrow Transplant Group (GITMO).
BACKGROUND AND OBJECTIVES: Allogeneic bone marrow transplantation (BMT) is a widely accepted therapeutic approach in homozygous beta-thalassemia. However, the majority of patients do not have a genotypically identical donor within the family. This prompted us to conduct a pilot study to investigate the feasibility of matched unrelated bone marrow transplantation in thalassemia. The major drawback was the high risk of immunologic and transplant-related complications, mainly graft-versus-host disease (GvHD) and graft failure. DESIGN AND METHODS: Our aim was to reduce this risk through careful selection of donor/recipient pairs. HLA haplotypes that show a high linkage disequilibrium among their class I, class II and class III alleles are considered extended or ancestral haplotypes. RESULTS: These haplotypes are conserved and can be shared by apparently unrelated individuals. Our study shows that matching for these haplotypes significantly improves the outcome of unrelated bone marrow transplantation in thalassemia. In fact, results were comparable to those obtained in transplants using HLA-identifical family donors. INTERPRETATION AND CONCLUSIONS: Better results were obtained in patients with lesser iron overload and when the donor shared an identity for the DPB1 alleles.
Adolescent↗
Ex vivo priming for long-term maintenance of antileukemia human cytotoxic T cells suggests a general procedure for adoptive immunotherapy.
Adoptive cellular immunotherapy has proven to be a successful approach in preventing and curing cytomegalovirus infection and Epstein-Barr virus-associated lymphomas after bone marrow transplantation. Translation of this approach for preventing leukemia relapse after bone marrow transplantation might require ex vivo priming and long-term maintenance of leukemia blast-specific T cells. To accomplish this goal, procedures were optimized for the in vitro priming of naive CD8 using dendritic cells activated by CD40 ligation, interleukin-12 (IL-12), and IL-7. Using T lymphocytes and dendritic cells obtained from HLA-matched allogeneic bone marrow transplantation donors and leukemia blasts as a source of tumor antigens, anti-acute myeloid leukemia cytotoxic T lymphocytes (CTLs) were induced. In these experiments, it was found that though it is possible to induce CTLs using immature dendritic cells, IL-12, and IL-7, obtaining long-term CTLs requires the presence of CD4 T cells in the priming phase. Using this approach, long-term antileukemia CTL lines could be generated from 4 of 4 bone marrow donors. Because this procedure does not require definition of the target antigen and because it selects responding cells from a virgin T-cell repertoire, its general application is suggested in adoptive immunotherapy and in the definition of tumor rejection antigens.
Bone Marrow Transplantation↗
Second allogeneic bone marrow transplantation in acute leukemia: results of a survey by the European Cooperative Group for Blood and Marrow Transplantation.
PURPOSE: Leukemic relapse is the most frequent cause of treatment failure after allogeneic hematopoietic stem-cell transplantation (HSCT). To identify prognostic factors affecting the outcome of second HSCT, we performed a retrospective study on patients with acute leukemia (AL) undergoing second HSCT who reported to the Acute Leukemia Working Party of the European Cooperative Group for Blood and Marrow Transplantation registry. PATIENTS AND METHODS: One hundred seventy patients who received second HSCTs for AL experienced relapse after first HSCTs were performed from 1978 to 1997. Status at second HSCT, time between first and second HSCT, conditioning regimen, source of stem cells, treatment-related mortality (TRM), acute graft-versus-host disease (aGVHD), leukemia-free survival (LFS), overall survival (OS), and relapse were considered. RESULTS: Engraftment occurred in 97% of patients. Forty-two patients were alive at last follow-up, with a 5-year OS rate of 26%. The 5-year probability for TRM, LFS, and relapse was 46%, 25%, and 59%, respectively. Grade > or = 2 aGVHD occurred in 59% of patients, and chronic GVHD occurred in 32%. In multivariate analysis, diagnosis, interval to relapse after first HSCT > 292 days, aGVHD at first HSCT, complete remission status at second HSCT, use of total-body irradiation at second HSCT, acute GVHD at second HSCT, and use of bone marrow as source of stem cells at second HSCT were associated with better outcome. CONCLUSION: Second HSCT represents an effective therapeutic option for AL patients relapsed after allogeneic HSCT, with a 3-year LFS rate of 52% for the subset of patients who experienced relapse more than 292 days after receiving the first HSCT and who were in remission before receiving the second HSCT.
Adolescent↗
Anti-CD20 monoclonal antibody for the treatment of severe, immune-mediated, pure red cell aplasia and hemolytic anemia.
Immune-mediated, acquired pure red cell aplasia (PRCA) is a rare disorder frequently associated with other autoimmune phenomena. Conventional immunosuppressive treatment is often unsatisfactory. Rituximab is a monoclonal antibody against the CD20 antigen, highly effective for in vivo B-cell depletion. An 18-month-old girl with both severe PRCA and autoimmune hemolytic anemia, refractory to immunosuppressive treatment, received 2 doses of rituximab, 375 mg/m(2) per week. The drug was well tolerated. After anti-CD20 therapy, substitutive treatment with intravenous immunoglobulin was started. The treatment resulted in marked depletion of B cells; a striking rise in reticulocyte count ensued, with increasing hemoglobin levels, finally leading to transfusion independence. The child is now 5 months off-therapy, with normal hemoglobin and reticulocyte levels. This case suggests a role of anti-CD20 monoclonal antibody for treatment of patients with antibody-mediated hematologic disorders. (Blood. 2001;97:3995-3997)
Anemia, Hemolytic, Autoimmune↗
Comparison of outcomes of unrelated bone marrow and umbilical cord blood transplants in children with acute leukemia.
In order to compare the outcomes of unrelated umbilical cord blood transplants (UCBTs) or bone marrow transplants, 541 children with acute leukemia (AL) transplanted with umbilical cord blood (n = 99), T-cell-depleted unrelated bone marrow transplants (T-UBMT) (n = 180), or nonmanipulated (UBMT) (n = 262), were analyzed in a retrospective multicenter study. Comparisons were performed after adjustment for patient, disease, and transplant variables. The major difference between the 3 groups was the higher number in the UCBT group of HLA mismatches (defined by serology for class I and molecular typing for DRB1). The donor was HLA mismatched in 92% of UCBTs, in 18% of UBMTs, and in 43% of T-UBMTs (P <.001). Other significant differences were observed in pretransplant disease characteristics, preparative regimens, graft-versus-host disease (GVHD) prophylaxis, and number of cells infused. Nonadjusted estimates of 2-year survival and event-free survival rates were 49% and 43%, respectively, in the UBMT group, 41% and 37% in the T-UBMT group, and 35% and 31% in the UCBT group. After adjustment, differences in outcomes appeared in the first 100 days after the transplantation. Compared with UBMT recipients, UCBT recipients had delayed hematopoietic recovery (Hazard ratio [HR] = 0.37; 95% confidence interval [95CI]: 0.27-0.52; P <.001), increased 100 day transplant-related mortality (HR = 2.13; 95CI: 1.20-3.76; P <.01) and decreased acute graft-versus-host disease (aGVHD) (HR = 0.50; 95CI: 0.34-0.73; P <.001). T-UBMT recipients had decreased aGVHD (HR = 0.25; 95CI: 0.17-0.36; P <.0001) and increased risk of relapse (HR = 1.96; 95CI: 1.11-3.45; P =.02). After day 100 posttransplant, the 3 groups achieved similar results in terms of relapse. Chronic GVHD was decreased after T-UBMT (HR = 0.21; 95CI: 0.11-0.37; P <.0001) and UCBT (HR = 0.24; 95CI: 0.01-0.66; P =.002), and overall mortality was higher in T-UBMT recipients (HR = 1.39; 95CI: 0.97-1.99; P <.07). In conclusion, the use of UCBT, as a source of hematopoietic stem cells, is a reasonable option for children with AL lacking an acceptably matched unrelated marrow donor.
Analysis of Variance↗
Hydrogenolysis of cyclohexane over Ir/SiO(2) catalyst: a mechanistic study of carbon--carbon bond cleavage on metallic surfaces.
The hydrogenolysis of cyclohexane catalyzed by supported Ir/SiO(2) has been studied to get mechanistic information on the elementary steps of C--C bond cleavage for cyclic saturated hydrocarbons. The reaction was studied under conditions in which no dehydrogenation to benzene occurs. When a mixture of cyclohexane and H(2) flows over a Ir/SiO(2) catalyst at 200 degrees C and for a H(2)/cyclohexane ratio superior to 40, methane, ethane, propane, n-butane, n-pentane, and n-hexane are identified to be primary products. The hydrogenolysis of ethane and n-hexane has also been studied to clarify several mechanistic questions. To account for the primary products in the above reactions, a mechanism is proposed in which the key step of the carbon--carbon bond cleavage occurs via concerted electronic transfer in dimetallacyclopentane intermediate. The comparison of product distributions in the hydrogenolysis of cyclohexane and that observed for n-hexane led to conclusions about the relative ease of carbon--carbon bond cleavage with respect to surface alkyl isomerization.
Journal Article↗
Treatment of aplastic anaemia with granulocyte-colony stimulating factor and risk of malignancy. Italian Aplastic Anaemia Study Group.
Granulocyte-colony stimulating factor (G-CSF) is being increasingly used in healthy volunteers to harvest haemopoietic stem cells. A possible role of G-CSF in the development of clonal disorders or leukaemia has been suggested. We analysed 144 patients with aplastic anaemia treated with immunosuppression protocols with or without G-CSF, with normal cytogenetics at diagnosis or immediately after immunosuppression. Our findings indicated that the risk of developing myelodysplasia or leukaemia was similar in patients with aplastic anaemia on immunosuppressive treatment with or without G-CSF. Therefore, it seems unlikely that G-CSF causes leukaemia in healthy volunteers.
Adolescent↗
A novel splice site mutation (3157+1G>T) in the dystrophin gene causing total exon skipping and DMD phenotype.
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Alternative Splicing↗
Characterisation of cAMP-dependent protein kinase isoforms in the brain of the crab Chasmagnathus.
In the crab Chasmagnathus learning model, systemic administration of cAMP analogues that are specific activators or inhibitors of cAMP-dependent protein kinase (PKA) proved to respectively facilitate or impair long-term retention. The aims of the present work were to analyse PKA activity distribution in the crab brain and to characterise PKA isoforms. The neuropils from the eyestalk showed higher levels of induced PKA activity when compared with other neuropils of the central nervous system. Two PKA isoforms, homologous to mammalian PKA I and PKA II, were detected from central brain protein extracts using DEAE chromatography. Only PKA II was found in lateral protocerebrum extracts, suggesting a role of this isoform in the processing of visual inputs and in the integration of this information with other sensory inputs. PKA I was observed to be ten-fold more sensitive to cAMP than PKA II. cGMP induced a high activation of both PKA isoforms, similar to that obtained with cAMP. PKA I showed a two-fold greater sensitivity for cGMP than PKA II. An autophosphorylation assay was performed and a protein of 55 kDa, corresponding to phosphorylated R II regulatory subunit, was detected. The presence of a PKA I isoform with high sensitivity for cAMP in the central brain suggests a role of this subtype in long-term memory.
Animals↗
Analysis of immune reconstitution in children undergoing cord blood transplantation.
OBJECTIVE: The aim of this study was to investigate and compare immune reconstitution in allogeneic cord blood transplantation (CBT) and bone marrow transplantation (BMT) recipients. MATERIALS AND METHODS: Twenty-three children underwent CBT from either human leukocyte antigen-identical siblings (11 cases) or unrelated donors (12 cases) were enrolled in the study, together with 23 matched children receiving BMT. Patients were analyzed 2-3 and 12-15 months after transplant. Recovery of T-, B-, and NK-lymphocyte subsets, proliferative in vitro response to mitogens, as well as cytotoxic activities, were investigated. RESULTS: CBT recipients showed a marked increase in the number of B lymphocytes as compared with patients who underwent BMT (p < 0.001). The absolute number of CD3(+) and CD8(+) T cells, as well as the proliferative response to T-cell mitogens, recovered with time after transplantation, irrespective of the source of stem cells used. Recipients of unrelated CBT had a better recovery of CD4(+) T lymphocytes (p < 0.01). Among patients experiencing acute graft-versus-host disease (GVHD), children given CBT had a much greater production of CD4(+) CD45RA(+) T cells than BMT recipients (p < 0.005). Recovery of NK cell number and innate cytotoxic activities was fast, irrespective of the source of stem cells used. CONCLUSIONS: Despite the much lower number of lymphocytes transferred with the graft, recovery of lymphocyte number and function toward normal in CBT recipients was rapid and comparable to that observed after transplantation of bone marrow progenitors. This prompt immune recovery possibly was favored by the reduced incidence and severity of GVHD observed in children who underwent CBT.
Acute Disease↗
Bone marrow transplantation in a case of severe, type II congenital dyserythropoietic anaemia (CDA II).
Type II congenital dyserythropoietic anaemia (CDA-II or HEMPAS) is an autosomal recessive disorder, representing the most frequent form of congenital dyserythropoiesis. It is characterised by normocytic anaemia, variable jaundice and hepato-splenomegaly. Gallbladder disease and secondary haemochromatosis are frequent complications. We report a case characterised by severe transfusion-dependent anaemia. The proband inherited CDA-II in association with beta-thalassaemia trait. Splenectomy did not abolish the transfusion dependence and this, in association with poor compliance to iron-chelation therapy, prompted us to consider bone marrow transplantation (BMT) from his HLA-identical sibling. The preparative regimen included busulfan, thiotepa and fludarabine, and graft-versus-host disease prophylaxis consisted of cyclosporin A and short-term methotrexate. Engraftment of donor cells was prompt and the post-transplant course uncomplicated. The patient is alive and transfusion-independent 36 months after allograft. This is the first case of severe CDA-II to undergo BMT. Analysis of this pedigree suggests that interaction with beta-thalassaemia enhanced the clinical severity of CDA-II, making BMT an attractive therapy for patients with transfusion dependence.
Adult↗