Middle East respiratory syndrome coronavirus vaccine based on a propagation-defective RNA replicon elicited sterilizing immunity in mice.

Self-amplifying RNA replicons are promising platforms for vaccine generation. Their defects in one or more essential functions for viral replication, particle assembly, or dissemination make them highly safe as vaccines. We previously showed that the deletion of the envelope (E) gene from the Middle East respiratory syndrome coronavirus (MERS-CoV) produces a replication-competent propagation-defective RNA replicon (MERS-CoV-ΔE). Evaluation of this replicon in mice expressing human dipeptidyl peptidase 4, the virus receptor, showed that the single deletion of the E gene generated an attenuated mutant. The combined deletion of the E gene with accessory open reading frames (ORFs) 3, 4a, 4b, and 5 resulted in a highly attenuated propagation-defective RNA replicon (MERS-CoV-Δ[3,4a,4b,5,E]). This RNA replicon induced sterilizing immunity in mice after challenge with a lethal dose of a virulent MERS-CoV, as no histopathological damage or infectious virus was detected in the lungs of challenged mice. The four mutants lacking the E gene were genetically stable, did not recombine with the E gene provided in trans during their passage in cell culture, and showed a propagation-defective phenotype in vivo. In addition, immunization with MERS-CoV-Δ[3,4a,4b,5,E] induced significant levels of neutralizing antibodies, indicating that MERS-CoV RNA replicons are highly safe and promising vaccine candidates.

Childhood and adolescent lymphoma in Spain: incidence and survival trends over 20 years.

BACKGROUND: Lymphoma is the third most common malignancy in children (0-14 years) and the first in adolescents (15-19 years). This population-based study-the largest ever done in Spain-analyses incidence and survival of lymphomas among Spanish children and adolescents. PATIENTS AND METHODS: 1664 lymphoma cases (1983-2007) for incidence and 1030 for survival (1991-2005) followed until 31/12/2010, were provided by 11 cancer registries. Age-adjusted incidence rates (ASRw) to the world standard population were obtained; incidence trends were modelled using the Joinpoint programme, observed survival (OS) was estimated with Kaplan-Meier and trends tested with a log-rank test. Results are presented according to the International Classification of Childhood Cancer-3. RESULTS: In Spain, the ASRw0-14 for lymphomas was 17.5 per 1.000.000 child-years and 50.0 the specific rate for adolescents. Overall incidence increased significantly during 1983-1997 with no increases thereafter. Patients over 9 years old showed significant rising trends for all subtypes, except for Burkitt lymphoma (BL) in adolescents. During 2001-2005 (age 0-19 years), 5-year OS was 94 (90-98), 73 (64-83) and 86 (78-94) for Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL) and BL, respectively. No improvement in survival was found. The incidence in Spain was higher than overall European rates, but within the range of that in Southern Europe. Comparing OS in Spain 1991-1995 and 2001-2005 with results for Europe of the Automated Childhood Cancer Information System (ACCIS) (1988-1997) and the European cancer registry-based study on survival and care of cancer patients (EUROCARE) (2000-2007), it was similar for HL and lower for NHL and BL. CONCLUSIONS: Systematic monitoring and analysis of lymphoma paediatric data would provide clinical and epidemiological information to improve the health care of these patients and the outcomes for these malignancies in Spain.

[Update on L-asparaginase treatment in paediatrics]. / Actualización del tratamiento con L-asparraginasa en Pediatría.

L-asparaginase (L-ASP) is one of the cornerstones of the treatment of acute lymphoblastic leukemia and non-Hodgkin lymphoma. It is an enzyme of bacterial origin capable of transforming L-asparagine to aspartic acid. The extracellular depletion of L-asparagine inhibits protein synthesis in lymphoblasts, inducing their apoptosis. Numerous studies have demonstrated that treatment with L-ASP improves survival of patients, but there are clear differences in the characteristics of the three currently available formulations. This article reviews the dosage, activity and side effects of the two L-ASP derived from Escherichia coli (native and pegylated), and the one derived from Erwinia chrysanthemi (Erwinia ASP). Despite its indisputable indication over the past50 years, there are still many points of contention, and its use is still marked by the side effects of the inhibition of protein synthesis. The short half-life of native forms, and the most frequently used parenteral administration by intramuscular injections, affects the quality of life of the patients. Therefore, recent studies claim to evaluate alternatives, such as the formulation of longer half-life pegylated L-ASP, and the use of intravenous formulations. There are encouraging results to date with both preparations. Still, further studies are needed to establish which should be the formulation and frontline indicated route of administration, optimal dosing, and management of adverse effects.

[Protocol for the study and treatment of immune thrombocytopenic purpura (ITP). ITP-2010]. / Protocolo de estudio y tratamiento de la trombocitopenia inmune primaria (PTI-2010).

Primary immune thrombocytopenia (ITP), formerly known as immune thrombocytopenic purpura, is a disease in which clinical and therapeutic management has always been controversial. The ITP working group of the Spanish Society of Paediatric Haematology and Oncology has updated its guidelines for diagnosis and treatment of ITP in children based on current guidelines, literature review, clinical trials and member consensus. The primary objective was to lessen clinical variability in diagnostic and therapeutic procedures in order to obtain best clinical results with minimal adverse events and good quality of life.

Allogeneic hemopoietic stem cell transplantation for childhood acute lymphoblastic leukemia in second complete remission-similar outcomes after matched related and unrelated donor transplant: a study of the Spanish Working Party for Blood and Marrow Transplantation in Children (Getmon).

The authors report the results of 58 children with ALL in 2CR after related (n = 31) or unrelated (n = 27) AHSCT. Characteristics at diagnosis and initial and after relapse antileukemic treatment were similar in the related donor (RD) and the unrelated donor (UD) groups. Conditioning consisted of TBI/CY +/- VP-16 for patients > or = 3 years old (n = 43) and Bu/CY for the rest. Median recipient age was 8 years (range 1-17) in the RD and 9 years (range 3-14) in the UD group. Median follow-up was 54 months (range 24-80) and 52 months (range 22-85) in the RD and the UD groups repectively. The 5-year EFS probability was 43 +/- 9% for the RD group and 36 +/- 9% in the UD group (p = .25). The transplant-related mortality was 16% in the RD and 37% in the UD group (p = .016). In the RD group 36.7% of patients relapsed versus 18.6% in the UD group (p = .05). GvHD associated with organ failure or infection caused most of the transplant-related deaths in both groups. Survivor quality of life for both groups was good (Lansky score < or = 90).

Long-term outcome of allogeneic or autologous haemopoietic cell transplantation for acute lymphoblastic leukaemia in second remission in children. GETMON experience 1983-1998.

We present a retrospective study of long-term outcome and predictive factors of survival and relapse in 219 paediatric patients with acute lymphoblastic leukaemia (ALL) in second remission. They received allogeneic (allo) or autologous (auto) haemopoietic cell transplantation (HCT) depending on the availability of a matched sibling donor. The probability of event-free survival (EFS) for the total patient group was 0.35+0.03 at 14 years. No significant differences were observed for EFS between allo- and auto-HCT: 0.39+0.05 vs 0.32+0.04 (P=0.43). A better EFS was seen in patients with a late relapse (LR) (P=0.06 and 0.02, for allogeneic and autologous respectively). Significantly better EFS was observed in allo-HCT patients under 10 years of age and in auto-HCT patients with leukocytes at diagnosis below 25 x 109/l and late relapse. Predictive factors of failure in both groups were early relapse (ER), medullary relapse and age over 10 years. The probability of relapse (RP) for the total group of patients was 0.57+0.03, and it was significantly higher in auto-HCT patients: 0.65+0.04 vs 0.42+0.06 (P=0.002). Factors predictive for relapse were medullary and early relapse, auto-HCT and WBC >25 x 109/l at diagnosis.

Autologous stem cell transplantation for advanced Hodgkin's disease in children. Spanish group for BMT in children (GETMON), Spain.

This study evaluates the outcome of myeloablative chemo-radiotherapy and autologous stem cell transplantation (ASCT) in children with Hodgkin's disease (HD). Twenty children aged 5 to 18 years (median 10.8 years) at diagnosis, with relapsed, refractory or very poor prognosis HD, underwent ASCT in eight hospitals of our country. Status at transplant was: second complete remission (CR2): n = 12; further CR (CR >2): n = 3, partial remission (PR): n = 2, relapse: n = 2 and first CR (CR1): n = 1. Eighteen patients received chemotherapy-based conditioning regimens: cyclophosphamide, carmustine and etoposide (CBV): 11 (55%), carmustine, etoposide, cytarabine and melphalan (BEAM): 5, other: 2; and two patients were conditioned with TBI/Cy. Peripheral blood (PB) was the source of progenitor cells in 12 patients, BM in seven, and BM plus PB, in one. All patients engrafted. One patient died of sepsis and multiorgan failure at day 28 after transplantation. All four patients with measurable disease (PR or relapse) at transplantation attained complete remission. Five patients relapsed 5-34 months after transplant (median: 11 months). Eighteen children remain alive with a median survival time of 40 months. The projected 5-year overall survival and event-free survival (EFS) rates were 0.95 and 0.62. High-dose therapy with stem cell rescue can lead to durable remissions in children with advanced HD. Bone Marrow Transplantation (2000) 25, 31-34.

Serum malondialdehyde in HIV-seropositive children negatively correlates with CD4+ lymphocytes count.

Human immunodeficiency virus (HIV) infection is associated with oxidative stress as it has been demonstrated in adult-seropositive individuals. We show in this study that serum malondialdehyde (MDA) concentration of HIV-infected children was significantly higher than in control children. A negative correlation (r = -0.515) was found in HIV-infected children between their CD4+ lymphocyte count, and MDA concentration but not with serum antioxidant status. The increase of MDA concentration in HIV-seropositive children confirms the involvement of oxidative stress in the pathophysiology of this infection also in childhood. Because of the importance of oxidative stress and antioxidants for HIV viral replication, the adequacy of an adjuvant therapy with antioxidants should be considered; an adequate candidate for it could be N-acetylcysteine.

Serum malondialdehyde in HIV seropositive children.

Human immunodeficiency virus (HIV) infection is associated with oxidative stress as it has been demonstrated in adult seropositive individuals. We show in this study that serum malondialdehyde (MDA) concentration of HIV infected children was significantly higher than in control children. Moreover, a statistically significant decreased serum antioxidant status was detected in HIV infected children when compared with controls. No correlation was found in HIV infected children between their clinical or immunological categories, CD4+ lymphocyte count or CD4+/CD8+ ratio, and MDA concentration or serum antioxidant status. Newborn from HIV seropositive mothers had also a higher MDA concentration in cord blood serum than their corresponding controls from HIV seronegative mothers, whereas no difference could be established in the serum antioxidant status between both groups. No apparent correlation could be established between birth weight, gestational age or APGAR test values, and MDA in any of these groups. The results presented, (i.e., the increase of MDA concentration in HIV seropositive infants and children, and the decrease in serum total antioxidants in HIV seropositive children) confirm the involvement of oxidative stress in the pathophysiology of this infection also in childhood. Because of the importance of oxidative stress and antioxidants for HIV viral replication, the adequacy of an adjuvant therapy with antioxidants should be considered; an adequate candidate for it could be N-acetyl-cysteine.

Factors regulating megakaryocyte progenitor commitment to polyploidization.

Recognizable megakaryocytes are polyploid cells generated by a clonogenic, diploid progenitor, termed CFU-MKC (colony forming unit, megakaryocyte). In order to quantify polyploidization, ploidy histograms of megakaryocytes determined by microphotometric or flow cytometric measurements of megakaryocyte DNA have generally been used. However these techniques provide no information on the rate of commitment of CFU-MKC to polyploidy. Using a technique of clonal analysis determining the distributions of the number of doublings (NbD) undergone by CFU-MKC before committing to polyploidization, the polyploidization probability of CFU-MKC could be derived. This probability was found to be a constant independent from CFU-MKC mitotic history, since NbD distributions are exponential functions characterized by a constant rate of decay per doubling. By studying the effects of growth factors on polyploidization probability, it was also shown that: (1) this parameter is negatively regulated by growth factors contained in poke-weed or WEHI conditioned media, as well as by erythropoietin; (2) commitment to polyploidization does not require prior CFU-MKC division; (3) bipotent erythroid-megakaryocyte progenitors have a lower polyploidization probability than CFU-MKC; (4) determination of polyploidization probability reflects the activity of growth factors with greater accuracy than megakaryocyte colony count.

Factors regulating megakaryocyte progenitor commitment to polyploidization.

Recognizable megakaryocytes are polyploid cells generated by a clonogenic, diploid progenitor, termed CFU-MKC (colony forming unit, megakaryocyte). In order to quantify polyploidization, ploidy histograms of megakaryocytes determined by microphotometric or flow cytometric measurements of megakaryocyte DNA have generally been used. However these techniques provide no information on the rate of commitment of CFU-MKC to polyploidy. Using a technique of clonal analysis determining the distributions of the number of doublings (NbD) undergone by CFU-MKC before committing to polyploidization, the polyploidization probability of CFU-MKC could be derived. This probability was found to be a constant independent from CFU-MKC mitotic history, since NbD distributions are exponential functions characterized by a constant rate of decay per doubling. By studying the effects of growth factors on polyploidization probability, it was also shown that: (1) this parameter is negatively regulated by growth factors contained in poke-weed or WEHI conditioned media, as well as by erythropoietin; (2) commitment to polyploidization does not require prior CFU-MKC division; (3) bipotent erythroid-megakaryocyte progenitors have a lower polyploidization probability than CFU-MKC; (4) determination of polyploidization probability reflects the activity of growth factors with greater accuracy than megakaryocyte colony count.

Anaemia during pregnancy as a risk factor for infant iron deficiency: report from the Valencia Infant Anaemia Cohort (VIAC) study.

A prospective cohort study with a 1-year follow-up of 156 neonates was carried out specifically designed to test the hypothesis that there is a positive relationship between iron deficiency during pregnancy and the development of the same disease in newborn infants. Exposure was defined as being born of a mother with ferropenic anaemia at delivery, and cases as the infants who developed iron deficiency during their first year of life. A statistically significant positive association was detected with an odds ratio of 6.57 (95% confidence limits 1.81-25.97). A stratified analysis was also performed to control the effect of potential confounders such as socio-economic variables, feeding practices and other factors linked with the iron status of infants. This second analytical procedure showed no alteration in the association detected in the simple analysis but that there was a statistically significant strong interaction between the quantity of cow's milk intake and the ferropenic status of the mother. These results show a relationship between iron deficiency of the mother at delivery and the development of iron deficiency in the infants. These new findings could be important in the development of new prevention programmes applied to pregnant women.

Abnormal splenic megakaryopoiesis in MPSV-induced myeloproliferative disease.

The myeloproliferative sarcoma virus (MPSV) induces a murine myeloproliferative syndrome characterized by an erythromyelemia, an anemia, a thrombocytopenia associated with a myeloproliferation in the spleen and a splenic and medullar fibrosis. We have used the in-vitro plasma clot technique to measure megakaryocytic precursors in the spleen and bone-marrow of MPSV-infected mice. We report that megakaryocytic colonies are increased, in number (X75), in concentration (X9) and in size, in the spleen but not in the bone-marrow of neoplastic mice. Furthermore, these splenic precursors are hypersensitive to growth factors present in the anemic mouse serum used in the culture system. These data show that the thrombocytopenia observed in the MPSV-induced neoplasia does not result from a lack of megakaryocyte precursors, but rather from an excess of megakaryocyte destruction. This ineffective splenic megakaryopoiesis associated with the presence of a massive splenic fibrosis make the MPS-induced neoplasia a suitable model for studying the perturbation of megakaryopoiesis in myeloproliferative syndrome associated with fibrosis.

Organization of haematopoietic stem cells and their relationship to mastocytopoiesis.

Haematopoiesis is a dynamic process in which differentiated blood cells are regularly replaced by the proliferation and differentiation of stem cells. Two types of haematopoietic stem cells (HSC) can be distinguished: totiopotent HSC capable of giving rise to cells of all haematopoietic lineages, and pluripotent HSC capable of giving rise only to the myeloid lineages. HSC are characterized by their capacity for self-renewal and differentiation in committed stem cells determined towards one of the haematopoietic lineages. These committed stem cells, also named progenitor cells or colony-forming units (CFU), have the capacity to give rise, in semi-solid medium, to colonies composed of differentiated cells. It has recently been shown that mast cells originate from a pluripotent HSC and that CFU Baso/Masto can be detected from murine and human bone marrow and blood cells. The in vivo regulation of the proliferation and differentiation of HSC is not well known. Several studies have emphasized the role of the haematopoietic microenvironment and of long- and short-range factors in such regulation. The in vitro growth of HSC is strictly dependent on growth factors, also named CSF (colony-stimulating factors) and interleukin (IL), in particular IL3. IL3 is a glycoprotein which induces the proliferation and differentiation of pluripotent and committed HSC and which seems to have a preferential role in mast cell regulation.

[Histiocytosis X: localized and disseminated forms. Apropos of 13 cases]. / Histiocitosis X: formas localizadas y diseminadas. A propósito de 13 casos.

The authors present a study of 13 patients, between 7 months and 4 years of age diagnosed, of histiocytosis X, nine of them with a disseminated form and 4 with a localized form. The clinical and histopathologic types of presentation are analyzed, applying the usual scores of prognostic evaluation, with emphasis on the low mortality rate (one death), considering that there are several patients with bad prognostic scores. This is explained by the positive effect of chemotherapy used in these cases.

[Granulocyte-macrophage precursor cells in umbilical cord blood]. / Progenitores granulocito-macrofágicos en sangre de cordón umbilical.

Authors study growth patterns of granulocytic-macrophagic progenitors in umbilical cord blood of 21 full-term newborns, using collagen gel culture method. Predominantly they obtain pure macrophagic colonies or mixed granulocytic colonies. After 7 days of incubation mean and SD of clusters in 103.22 +/- 75.88, and mean and SD of colonies is 34.26 +/- 23.1. After 14 days clusters falls down until 47.2 +/- 30.48, but colonies goes up to 74.2 +/- 32.78. Lineal regression analysis shows a correlation coefficient of 0.809 between 7 days and 14 days colonies. These results demonstrate the different behaviour between myelopoietic progenitor from umbilical cord blood and same progenitor from adult blood and are probably related to different nature of both progenitors.