Josu de la Fuente

Microchimerism in female bone marrow and bone decades after fetal mesenchymal stem-cell trafficking in pregnancy

Fetal cells enter maternal blood during pregnancy and persist in women with autoimmune disease. The frequency of subsequent fetomaternal microchimerism in healthy women and its cell type is unknown. To test the hypothesis that fetal mesenchymal stem cells persist in maternal organs, we studied female bone marrow and ribs. Male cells were identified by XY fluorescence in-situ hybridisation in marrow-derived mesenchymal stem cells and in rib sections from all women with male pregnancies, but not in controls (9/9 vs 0/5, p=0.0005). We conclude that fetal stem cells transferred into maternal blood engraft in marrow, where they remain throughout life. This finding has implications for normal pregnancy, for obstetric complications that increase fetomaternal trafficking, and for graft survival after transplantation.

Human Fetal Mesenchymal Stem Cells as Vehicles for Gene Delivery

First‐trimester fetal blood contains a readily expandable population of stem cells, human fetal mesenchymal stem cells (hfMSCs), which might be exploited for autologous intrauterine gene therapy. We investigated the self‐renewal and differentiation of hfMSCs after transduction with onco‐retroviral and lentiviral vectors. After transduction with either a MoMuLV retrovirus or an HIV‐1‐based lentiviral vector carrying the β‐galactosidase and green fluorescent reporter gene, respectively, transgene expression, self‐renewal, and differentiation capabilities were assessed 2 and 14 weeks later. Transduction with the lentiviral vector resulted in higher efficiencies than with the MoMuLV‐based vector (mean, 97.7 ± 1.4% versus 80.2 ± 5.4%; p = .02). Transgene expression was maintained with lentiviral‐transduced cells (94.6 ± 2.6%) but decreased over 14 weeks in culture with onco‐retroviral‐transduced cells (48.3 ± 3.9%). The self‐renewal capability of these cells and their ability to undergo osteogenic, adipogenic, and myogenic differentiation was unimpaired after transduction with either vector. Finally, clonal expansion of lentivirally modified cells was expanded over 20 population doublings with maintenance of multiline age differentiation capacity. These results suggest that hfMSCs may be suitable targets for ex vivo genetic manipulation with onco‐retroviral or lentiviral vectors without affecting their stem cell properties.

Abnormalities in the myeloid progenitor compartment in Down syndrome fetal liver precede acquisition of GATA1 mutations

Down syndrome (DS) children have a high frequency of acute megakaryoblastic leukemia (AMKL) in early childhood. At least 2 in utero genetic events are required, although not sufficient, for DS-AMKL: trisomy 21 (T21) and N-terminal-truncating GATA1 mutations. To investigate the role of T21 in DS-AMKL, we compared second trimester hemopoiesis in DS without GATA1 mutations to gestation-matched normal controls. In all DS fetal livers (FLs), but not marrows, megakaryocyte-erythroid progenitor frequency was increased (55.9% +/- 4% vs 17.1% +/- 3%, CD34(+)CD38(+) cells; P < .001) with common myeloid progenitors (19.6% +/- 2% vs 44.0% +/- 7%) and granulocyte-monocyte (GM) progenitors (15.8% +/- 4% vs 34.5% +/- 9%) commensurately reduced. Clonogenicity of DS-FL versus normal FL CD34(+) cells was markedly increased (78% +/- 7% vs 15% +/- 3%) affecting megakaryocyte-erythroid ( approximately 7-fold higher) and GM and colony-forming unit-granulocyte, erythrocyte macrophage, megakaryocyte (CFU-GEMM) progenitors. Replating efficiency of CFU-GEMM was also markedly increased. These data indicate that T21 itself profoundly disturbs FL hemopoiesis and they provide a testable hypothesis to explain the increased susceptibility to GATA1 mutations in DS-AMKL and DS-associated transient myeloproliferative disorder.

Dyskeratosis congenita: Advances in the understanding of the telomerase defect and the role of stem cell transplantation

Abstract:  DC is a multisystem bone marrow failure syndrome exhibiting marked clinical and genetic heterogeneity. X‐linked, autosomal dominant and autosomal recessive subtypes are recognized. The gene mutated in X‐linked DC (DKC1) encodes a highly conserved nucleolar protein called dyskerin. Dyskerin associates with the H/ACA motif class of small nucleolar RNAs in small nucleolar ribonucleoprotein particles that are important in guiding the conversion of uracil to pseudouracil during the maturation of ribosomal RNA. Dyskerin also associates with the TERC, which is important in the maintenance of telomeres. Mutations in TERC have been identified in patients with autosomal dominant DC and in a subset of patients with aplastic anemia and myelodysplasia. Recently, heterozygous mutations in TERT have been found in some patients with autosomal dominant DC and aplastic anemia. Additionally, patients with the severe multisystem disorder, Hoyeraal–Hreidarsson syndrome, have been found to have DKC1 mutations. Collectively, these observations have demonstrated that classical DC, Hoyeraal–Hreidarsson syndrome and a subset of aplastic anemia are due to a primary defect in telomerase. The critical role of telomeres and telomerase in humans is seen in the multisystem abnormalities found in these patients, including the increased incidence of malignancy. As bone marrow failure is the principal cause of death, conventional allografts have been attempted with limited success due to the high rate of pulmonary and endothelial complications. However, outcomes have improved with the use of non‐myeloablative protocols, although the follow up is too short to evaluate long term toxicity and the natural course of the disease and it may be that correction of the telomerase defect is essential for the treatment of these patients.

Tartrate-resistant acid phosphatase isoform 5b: a novel serum marker for monitoring bone disease in multiple myeloma.

Tartrate‐resistant acid phosphatase isoform‐5b (TRACP‐5b), a new marker reflecting osteoclast activity, and osteoprotegerin (OPG) were measured in 121 patients with multiple myeloma (MM) at diagnosis, and in 63 of them during pamidronate administration, to define their correlation with the extent of bone disease and disease activity in MM. Radiographic evaluation of the skeleton, measurement of other markers of bone remodelling, including N‐terminal cross‐linking telopeptide of type‐I collagen (NTX), bone alkaline phosphatase and osteocalcin and of markers of disease activity (beta2‐microglobulin, paraprotein, interleukin‐6 (IL‐6), were also performed. Levels of TRACP‐5b were increased (p < .0001), while OPG was decreased in MM patients compared to controls (p < .01). TRACP‐5b levels were associated with the radiographically assessed severity of bone disease (p < .0001) as well as with levels of NTX, IL‐6 and beta2‐microglobulin (p < .001, for each biochemical parameter, respectively). The combination of pamidronate with VAD‐chemotherapy produced a reduction in TRACP‐5b, NTX, IL‐6, paraprotein and beta2‐microglobulin levels from the 2nd month of treatment, with no effect on bone formation and OPG. A strong correlation was observed between changes in TRACP‐5b and changes in NTX, IL‐6 and beta2‐microglobulin, while TRACP‐5b predicted the disease progression in 5 patients. These findings suggest that TRACP‐5b is increased in MM, reflects the extent of myeloma bone disease and may have a predictive value. TRACP‐5b has also proved to be very useful for monitoring antimyeloma treatment, which had no effect on OPG levels.

Sickle cell disease: An international survey of results of HLA-identical sibling hematopoietic stem cell transplantation

Despite advances in supportive therapy to prevent complications of sickle cell disease (SCD), access to care is not universal. Hematopoietic cell transplantation is, to date, the only curative therapy for SCD, but its application is limited by availability of a suitable HLA-matched donor and lack of awareness of the benefits of transplant. Included in this study are 1000 recipients of HLA-identical sibling transplants performed between 1986 and 2013 and reported to the European Society for Blood and Marrow Transplantation, Eurocord, and the Center for International Blood and Marrow Transplant Research. The primary endpoint was event-free survival, defined as being alive without graft failure; risk factors were studied using a Cox regression models. The median age at transplantation was 9 years, and the median follow-up was longer than 5 years. Most patients received a myeloablative conditioning regimen (n = 873; 87%); the remainder received reduced-intensity conditioning regimens (n = 125; 13%). Bone marrow was the predominant stem cell source (n = 839; 84%); peripheral blood and cord blood progenitors were used in 73 (7%) and 88 (9%) patients, respectively. The 5-year event-free survival and overall survival were 91.4% (95% confidence interval, 89.6%-93.3%) and 92.9% (95% confidence interval, 91.1%-94.6%), respectively. Event-free survival was lower with increasing age at transplantation (hazard ratio [HR], 1.09; P < .001) and higher for transplantations performed after 2006 (HR, 0.95; P = .013). Twenty-three patients experienced graft failure, and 70 patients (7%) died, with the most common cause of death being infection. The excellent outcome of a cohort transplanted over the course of 3 decades confirms the role of HLA-identical sibling transplantation for children and adults with SCD.

Hyperhemolysis syndrome in sickle cell disease: case report (recurrent episode) and literature review

BACKGROUND: Hyperhemolysis syndrome (HS) has been well described both in sickle cell disease (SCD) and non‐SCD patients. The pathogenesis remains unclear. The possible mechanisms include bystander hemolysis, suppression of erythropoiesis, and destruction of red cells (RBCs) due to contact lysis via activated macrophages.

Poly(A)-specific ribonuclease deficiency impacts telomere biology and causes dyskeratosis congenita

Dyskeratosis congenita (DC) and related syndromes are inherited, life-threatening bone marrow (BM) failure disorders, and approximately 40% of cases are currently uncharacterized at the genetic level. Here, using whole exome sequencing (WES), we have identified biallelic mutations in the gene encoding poly(A)-specific ribonuclease (PARN) in 3 families with individuals exhibiting severe DC. PARN is an extensively characterized exonuclease with deadenylation activity that controls mRNA stability in part and therefore regulates expression of a large number of genes. The DC-associated mutations identified affect key domains within the protein, and evaluation of patient cells revealed reduced deadenylation activity. This deadenylation deficiency caused an early DNA damage response in terms of nuclear p53 regulation, cell-cycle arrest, and reduced cell viability upon UV treatment. Individuals with biallelic PARN mutations and PARN-depleted cells exhibited reduced RNA levels for several key genes that are associated with telomere biology, specifically TERC, DKC1, RTEL1, and TERF1. Moreover, PARN-deficient cells also possessed critically short telomeres. Collectively, these results identify a role for PARN in telomere maintenance and demonstrate that it is a disease-causing gene in a subset of patients with severe DC.

Pamidronate is superior to ibandronate in decreasing bone resorption, interleukin‐6 and β2‐microglobulin in multiple myeloma

Abstract: Objectives: Bisphosphonates have been found to reduce skeletal events in patients with multiple myeloma (MM). This is the first randomised trial to compare the efficacy of pamidronate and ibandronate, a third‐generation aminobisphosphonate, in bone turnover and disease activity in MM patients. Methods: Patients with MM, stage II or III, were randomly assigned to receive either pamidronate 90 mg (group I: 23 patients) or ibandronate 4 mg (group II: 21 patients) as a monthly intravenous infusion in addition to conventional chemotherapy. Skeletal events, such as pathologic fractures, hypercalcaemia, and bone radiotherapy were analysed. Bone resorption markers [N‐terminal cross‐linking telopeptide of type‐I collagen (NTX) and tartrate‐resistant acid phosphatase type 5b (TRACP‐5b)], bone formation markers (bone alkaline phosphatase and osteocalcin), markers of disease activity (paraprotein, CRP, β2‐microglobulin), and interleukin‐6 (IL‐6) were also studied. Results: In both groups, the combination of chemotherapy with either pamidronate or ibandronate produced a reduction in bone resorption and tumour burden as measured by NTX, IL‐6, paraprotein, CRP, and β2‐microglobulin from the second month of treatment, having no effect on bone formation. TRACP‐5b also had a significant reduction in the pamidronate group from the second month of treatment and in the ibandronate group from the sixth month. However, there was a greater reduction of NTX, IL‐6, and β2‐microglobulin in group I than in group II, starting at the second month of treatment (P = 0.002, 0.001, and 0.004, respectively) and of TRACP‐5b, starting at the fourth month (P = 0.014), that being continued throughout the 10‐month follow‐up of this study. There was no difference in skeletal events during this period. A significant correlation was observed between changes of NTX and changes of TRACP‐5b, IL‐6, and β2‐microglobulin from the second month for patients of both groups. Conclusions: These results suggest that a monthly dose of 90 mg of pamidronate is more effective than 4 mg of ibandronate in reducing osteoclast activity, bone resorption, IL‐6, and possibly tumour burden in MM. TRACP‐5b has also proved to be a useful new marker for monitoring bisphosphonates treatment in MM.

Similar outcome of upfront-unrelated and matched sibling stem cell transplantation in idiopathic paediatric aplastic anaemia. A study on behalf of the UK Paediatric BMT Working Party, Paediatric Diseases Working Party and Severe Aplastic Anaemia Working Party of EBMT

We explored the feasibility of unrelated donor haematopoietic stem cell transplant (HSCT) upfront without prior immunosuppressive therapy (IST) in paediatric idiopathic severe aplastic anaemia (SAA). This cohort was then compared to matched historical controls who had undergone first‐line therapy with a matched sibling/family donor (MSD) HSCT (n = 87) or IST with horse antithymocyte globulin and ciclosporin (n = 58) or second‐line therapy with unrelated donor HSCT post‐failed IST (n = 24). The 2‐year overall survival in the upfront cohort was 96 ± 4% compared to 91 ± 3% in the MSD controls (P = 0·30) and 94 ± 3% in the IST controls (P = 0·68) and 74 ± 9% in the unrelated donor HSCT post‐IST failure controls (P = 0·02).The 2‐year event‐free survival in the upfront cohort was 92 ± 5% compared to 87 ± 4% in MSD controls (P = 0·37), 40 ± 7% in IST controls (P = 0·0001) and 74 ± 9% in the unrelated donor HSCT post‐IST failure controls (n = 24) (P = 0·02). Outcomes for upfront‐unrelated donor HSCT in paediatric idiopathic SAA were similar to MSD HSCT and superior to IST and unrelated donor HSCT post‐IST failure. Front‐line therapy with matched unrelated donor HSCT is a novel treatment approach and could be considered as first‐line therapy in selected paediatric patients who lack a MSD.