Beatriz Martín-Antonio

Effects of stocking density and feed ration on growth and gene expression in the Senegalese sole (Solea senegalensis): Potential effects on the immune response

Stocking density and ration size are two major factors influencing aquaculture production. To evaluate their effects on growth and immune system in Senegalese sole (Solea senegalensis) juveniles, a 2 x 2 experimental design using two rations (1.0% and 0.25% of the total fish biomass) and two different initial stocking densities (7 and 30 kg m(-2)) was performed throughout a 60 days culture period. Soles fed 1.0% showed a higher specific growth rate (SGR) than those fed 0.25% (3.3-fold). No differences in SGR at 60 days were found between densities in spite of reduced values were detected at high density after 20 days (soles fed 0.25%) and 40 days (soles fed 1%) suggesting a compensatory growth. Physiologically, plasma cortisol levels were elevated in soles at high density (45-fold higher than at 7 kg m(-2)) whereas no differences associated to the feeding ration were observed. To assess the effects at a molecular level, the mRNA levels of genes involved in cellular stress (heat shock proteins HSP70 and HSP90), growth (insulin-like growth factors IGF-I, the spliced variants IGF-Ia and IGFI-b, and IGF-II) and innate immune system (g-type lysozyme and hepcidin (HAMP1)) were quantified. No differences in HSP90 expression were detected between densities or rations. In contrast, IGF-I, IGF-Ia and IGF-II showed reduced transcript levels in liver and HSP70 in liver and kidney at high density. Finally, g-type lysozyme and HAMP1 expression was greatly affected by both factors exhibiting an important reduction in the transcript levels at high density and low ration. Overall, our results show that S. senegalensis juveniles might exhibit satisfactory SGR at high density although the high plasma cortisol levels indicate a crowding stress that could negatively affect the expression levels of some of the genes studied.

Molecular characterization, phylogeny, and expression of c-type and g-type lysozymes in brill (Scophthalmus rhombus).

Lysozymes are key proteins of the innate immune system against bacterial infections. In this study we report the molecular cloning and characterization of the c-type and g-type lysozymes in brill (Scophthalmus rhombus). Catalytic and other conserved residues required for functionality were identified. Phylogenetic analysis revealed distinct evolutionary histories for each lysozyme type. Expression profiles of both lysozyme genes were studied in juvenile tissues using a real-time PCR approach. c-Type lysozyme was expressed mainly in stomach and liver, whereas the g-type was detected in all tissues with highest mRNA levels observed in the spleen. Induction experiments revealed that g-type transcripts increased significantly in head kidney after lipopolysaccharide (25- and 23-fold at 12 and 24h, respectively) and Photobacterium damselae subsp. piscicida (17-fold at 24h) treatments. In contrast, no induction was observed for c-type lysozyme. All these data suggest that g-type lysozyme is involved in the response against bacterial infections, whereas c-type lysozyme may also play a role in digestion.

Self-Renewing Human Bone Marrow Mesenspheres Promote Hematopoietic Stem Cell Expansion

Strategies for expanding hematopoietic stem cells (HSCs) include coculture with cells that recapitulate their natural microenvironment, such as bone marrow stromal stem/progenitor cells (BMSCs). Plastic-adherent BMSCs may be insufficient to preserve primitive HSCs. Here, we describe a method of isolating and culturing human BMSCs as nonadherent mesenchymal spheres. Human mesenspheres were derived from CD45- CD31- CD71- CD146+ CD105+ nestin+ cells but could also be simply grown from fetal and adult BM CD45--enriched cells. Human mesenspheres robustly differentiated into mesenchymal lineages. In culture conditions where they displayed a relatively undifferentiated phenotype, with decreased adherence to plastic and increased self-renewal, they promoted enhanced expansion of cord blood CD34+ cells through secreted soluble factors. Expanded HSCs were serially transplantable in immunodeficient mice and significantly increased long-term human hematopoietic engraftment. These results pave the way for culture techniques that preserve the self-renewal of human BMSCs and their ability to support functional HSCs.

Antigen presenting cell-mediated expansion of human umbilical cord blood yields log-scale expansion of natural killer cells with anti-myeloma activity.

Natural killer (NK) cells are important mediators of anti-tumor immunity and are active against several hematologic malignancies, including multiple myeloma (MM). Umbilical cord blood (CB) is a promising source of allogeneic NK cells but large scale ex vivo expansion is required for generation of clinically relevant CB-derived NK (CB-NK) cell doses. Here we describe a novel strategy for expanding NK cells from cryopreserved CB units using artificial antigen presenting feeder cells (aAPC) in a gas permeable culture system. After 14 days, mean fold expansion of CB-NK cells was 1848-fold from fresh and 2389-fold from cryopreserved CB with >95% purity for NK cells (CD56+/CD3−) and less than 1% CD3+ cells. Though surface expression of some cytotoxicity receptors was decreased, aAPC-expanded CB-NK cells exhibited a phenotype similar to CB-NK cells expanded with IL-2 alone with respect to various inhibitory receptors, NKG2C and CD94 and maintained strong expression of transcription factors Eomesodermin and T-bet. Furthermore, CB-NK cells formed functional immune synapses with and demonstrated cytotoxicity against various MM targets. Finally, aAPC-expanded CB-NK cells showed significant in vivo activity against MM in a xenogenic mouse model. Our findings introduce a clinically applicable strategy for the generation of highly functional CB-NK cells which can be used to eradicate MM.

Impact of constitutional polymorphisms in VCAM1 and CD44 on CD34+ cell collection yield after administration of granulocyte colony-stimulating factor to healthy donors

Background The number of CD34+ cells mobilized from bone marrow to peripheral blood after administration of granulocyte colony-stimulating factor varies greatly among healthy donors. This fact might be explained, at least in part, by constitutional differences in genes involved in the interactions tethering CD34+ cells to the bone marrow. Design and Methods We analyzed genetic characteristics associated with CD34+ cell mobilization in 112 healthy individuals receiving granulocyte colony-stimulating factor (filgrastim; 10 μg/kg; 5 days). Results Genetic variants in VCAM1 and in CD44 were associated with the number of CD34+ cells in peripheral blood after granulocyte colony-stimulating factor administration (P=0.02 and P=0.04, respectively), with the quantity of CD34+ cells ×106/kg of donor (4.6 versus 6.3; P<0.001 and 7 versus 5.6; P=0.025, respectively), and with total CD34+ cells ×106 (355 versus 495; P=0.002 and 522 versus 422; P=0.012, respectively) in the first apheresis. Of note, granulocyte colony-stimulating factor administration was associated with complete disappearance of VCAM1 mRNA expression in peripheral blood. Moreover, genetic variants in granulocyte colony-stimulating factor receptor (CSF3R) and in CXCL12 were associated with a lower and higher number of granulocyte colony-stimulating factor-mobilized CD34+ cells/μL in peripheral blood (81 versus 106; P=0.002 and 165 versus 98; P=0.02, respectively) and a genetic variant in CXCR4 was associated with a lower quantity of CD34+ cells ×106/kg of donor and total CD34+ cells ×106 (5.3 versus 6.7; P=0.02 and 399 versus 533; P=0.01, respectively). Conclusions In conclusion, genetic variability in molecules involved in migration and homing of CD34+ cells influences the degree of mobilization of these cells.

Fucosylation with fucosyltransferase VI or fucosyltransferase VII improves cord blood engraftment

BACKGROUND AIMS Advantages associated with the use of cord blood (CB) transplantation include the availability of cryopreserved units, ethnic diversity and lower incidence of graft-versus-host disease compared with bone marrow or mobilized peripheral blood. However, poor engraftment remains a major obstacle. We and others have found that ex vivo fucosylation can enhance engraftment in murine models, and now ex vivo treatment of CB with fucosyltransferase (FT) VI before transplantation is under clinical evaluation (NCT01471067). However, FTVII appears to be more relevant to hematopoietic cells and may alter acceptor substrate diversity. The present study compared the ability of FTVI and FTVII to improve the rapidity, magnitude, multi-lineage and multi-tissue engraftment of human CB hematopoietic stem and progenitor cells (HSPCs) in vivo. METHODS CD34-selected CB HSPCs were treated with recombinant FTVI, FTVII or mock control and then injected into immunodeficient mice and monitored for multi-lineage and multi-tissue engraftment. RESULTS Both FTVI and FTVII fucosylated CB CD34⁺ cells in vitro, and both led to enhanced rates and magnitudes of engraftment compared with untreated CB CD34⁺ cells in vivo. Engraftment after treatment with either FT was robust at multiple time points and in multiple tissues with similar multi-lineage potential. In contrast, only FTVII was able to fucosylate T and B lymphocytes. CONCLUSIONS Although FTVI and FTVII were found to be similarly able to fucosylate and enhance the engraftment of CB CD34⁺ cells, differences in their ability to fucosylate lymphocytes may modulate graft-versus-tumor or graft-versus-host effects and may allow further optimization of CB transplantation.

Bone marrow mesenchymal stem cells from patients with aplastic anemia maintain functional and immune properties and do not contribute to the pathogenesis of the disease

Aplastic anemia is a life-threatening bone marrow failure disorder characterized by peripheral pancytopenia and marrow hypoplasia. The majority of cases of aplastic anemia remain idiopathic, although hematopoietic stem cell deficiency and impaired immune responses are hallmarks underlying the bone marrow failure in this condition. Mesenchymal stem/stromal cells constitute an essential component of the bone marrow hematopoietic microenvironment because of their immunomodulatory properties and their ability to support hematopoiesis, and they have been involved in the pathogenesis of several hematologic malignancies. We investigated whether bone marrow mesenchymal stem cells contribute, directly or indirectly, to the pathogenesis of aplastic anemia. We found that mesenchymal stem cell cultures can be established from the bone marrow of aplastic anemia patients and display the same phenotype and differentiation potential as their counterparts from normal bone marrow. Mesenchymal stem cells from aplastic anemia patients support the in vitro homeostasis and the in vivo repopulating function of CD34+ cells, and maintain their immunosuppressive and anti-inflammatory properties. These data demonstrate that bone marrow mesenchymal stem cells from patients with aplastic anemia do not have impaired functional and immunological properties, suggesting that they do not contribute to the pathogenesis of the disease.

Transmissible cytotoxicity of multiple myeloma cells by cord blood-derived NK cells is mediated by vesicle trafficking

Natural killer cells (NK) are important effectors of anti-tumor immunity, activated either by the downregulation of HLA-I molecules on tumor cells and/or the interaction of NK-activating receptors with ligands that are overexpressed on target cells upon tumor transformation (including NKG2D and NKP30). NK kill target cells by the vesicular delivery of cytolytic molecules such as Granzyme-B and Granulysin activating different cell death pathways, which can be Caspase-3 dependent or Caspase-3 independent. Multiple myeloma (MM) remains an incurable neoplastic plasma-cell disorder. However, we previously reported the encouraging observation that cord blood-derived NK (CB-NK), a new source of NK, showed anti-tumor activity in an in vivo murine model of MM and confirmed a correlation between high levels of NKG2D expression by MM cells and increased efficacy of CB-NK in reducing tumor burden. We aimed to characterize the mechanism of CB-NK-mediated cytotoxicity against MM cells. We show a Caspase-3- and Granzyme-B-independent cell death, and we reveal a mechanism of transmissible cell death between cells, which involves lipid–protein vesicle transfer from CB-NK to MM cells. These vesicles are secondarily transferred from recipient MM cells to neighboring MM cells amplifying the initial CB-NK cytotoxicity achieved. This indirect cytotoxicity involves the transfer of NKG2D and NKP30 and leads to lysosomal cell death and decreased levels of reactive oxygen species in MM cells. These findings suggest a novel and unique mechanism of CB-NK cytotoxicity against MM cells and highlight the importance of lipids and lipid transfer in this process. Further, these data provide a rationale for the development of CB-NK-based cellular therapies in the treatment of MM.

Genomic polymorphisms of the innate immune system and allogeneic stem cell transplantation

Allogeneic stem cell transplantation (allo-HSCT) is largely employed for treating patients affected by many hematological disorders, but despite the considerable improvement in the treatment of its complications, graft-versus-host disease and infections remain important causes of morbidity and mortality. Innate immunity is crucial in the immune defense against infections after allo-HSCT, and in the biological reactions leading to graft-versus-host disease. Thus, the innate immune system plays an important role in allo-HSCT clinical outcome. It is known now that cytokine gene polymorphisms greatly influence the outcome of allo-HSCT. In addition, genetic variability of some pattern-recognition receptors and antimicrobial peptides represent a promising field to be researched for allo-HSCT impact. Furthermore, more recent work suggests the importance of genetic variability between donor and recipient in the killer cell immunoglobulin-like receptors of the natural killer cells on the allo-HSCT outcome. This article discusses the main cytokines and innate immune gene polymorphisms influencing allo-HSCT outcome, presents new innate immune genes with promising expectations and points at the importance of genetic variability in natural killer cells in allo-HSCT outcome.

Granulocyte colony-stimulating factor produces long-term changes in gene and microRNA expression profiles in CD34+ cells from healthy donors

Granulocyte colony-stimulating factor is the most commonly used cytokine for the mobilization of hematopoietic progenitor cells from healthy donors for allogeneic stem cell transplantation. Although the administration of this cytokine is considered safe, knowledge about its long-term effects, especially in hematopoietic progenitor cells, is limited. On this background, the aim of our study was to analyze whether or not granulocyte colony-stimulating factor induces changes in gene and microRNA expression profiles in hematopoietic progenitor cells from healthy donors, and to determine whether or not these changes persist in the long-term. For this purpose, we analyzed the whole genome expression profile and the expression of 384 microRNA in CD34+ cells isolated from peripheral blood of six healthy donors, before mobilization and at 5, 30 and 365 days after mobilization with granulocyte colony-stimulating factor. Six microRNA were differentially expressed at all time points analyzed after mobilization treatment as compared to the expression in samples obtained before exposure to the drug. In addition, 2424 genes were also differentially expressed for at least 1 year after mobilization. Of interest, 109 of these genes are targets of the differentially expressed microRNA also identified in this study. These data strongly suggest that granulocyte colony-stimulating factor modifies gene and microRNA expression profiles in hematopoietic progenitor cells from healthy donors. Remarkably, some changes are present from early time-points and persist for at least 1 year after exposure to the drug. This effect on hematopoietic progenitor cells has not been previously reported.