Publications by authors named "Beatriz Martín-Antonio"

31 Publications

Defining an Ultra-Low Risk Group in Asymptomatic IgM Monoclonal Gammopathy.

Cancers (Basel) 2021 Apr 23;13(9). Epub 2021 Apr 23.

Amyloidosis and Myeloma Unit, Department of Hematology, Hospital Clínic of Barcelona, 08036 Barcelona, Spain.

We analyzed 171 patients with asymptomatic IgM monoclonal gammopathies (64 with IgM monoclonal gammopathy of undetermined significance-MGUS and 107 with smoldering Waldenström macroglobulinemia - SWM) who had a bone marrow (BM) evaluation performed at diagnosis. Abnormal free-light chain ratio (53% vs. 31%) and mutation prevalence (66% vs. 30%) were higher in patients with SWM. No other differences were found among groups. With a median follow-up of 4.3 years, 14 patients progressed to Waldenström macroglobulinemia, 1 to amyloidosis, and 28 died without progression. The mutation was found in 53% of patients (available in 160 patients). Multivariate analysis showed that immunoparesis (subhazard ratio-SHR 10.2, 95% confidence interval-CI: 4.2-24.8; < 0.001) and BM lymphoplasmacytic infiltration ≥ 20% (SHR: 6, 95% CI: 1.6-22.1; = 0.007) were associated with higher risk of progression. We developed a risk model based on these two risk factors. In the absence of both variables, an ultra-low risk group was identified (SHR 0.1, 95% CI 0.02-0.5; = 0.004), with 3% and 6% of cumulative incidence of progression at 10 and 20 years, respectively. Bootstrap analysis confirmed the reproducibility of these results. This study finds immunoparesis and BM infiltration as biomarkers of progression as well as a low-risk group of progression in asymptomatic IgM monoclonal gammopathies.
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http://dx.doi.org/10.3390/cancers13092055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8122982PMC
April 2021

Editorial: Understanding the Cytokine Release Syndrome: Toward Improving Cancer Immunotherapy.

Front Immunol 2021 19;12:666703. Epub 2021 Mar 19.

Department of Experimental Hematology, Instituto de Investigación Sanitaria-Fundación Jiménez Diaz, Madrid, Spain.

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http://dx.doi.org/10.3389/fimmu.2021.666703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8017175PMC
March 2021

Natural Killer Cells in Immunotherapy: Are We Nearly There?

Cancers (Basel) 2020 Oct 27;12(11). Epub 2020 Oct 27.

Department of Hematology, Hospital Clinic, IDIBAPS, 08036 Barcelona, Spain.

Natural killer (NK) cells are potent anti-tumor and anti-microbial cells of our innate immune system. They are equipped with a vast array of receptors that recognize tumor cells and other pathogens. The innate immune activity of NK cells develops faster than the adaptive one performed by T cells, and studies suggest an important immunoregulatory role for each population against the other. The association, observed in acute myeloid leukemia patients receiving haploidentical killer-immunoglobulin-like-receptor-mismatched NK cells, with induction of complete remission was the determinant to begin an increasing number of clinical studies administering NK cells for the treatment of cancer patients. Unfortunately, even though transfused NK cells demonstrated safety, their observed efficacy was poor. In recent years, novel studies have emerged, combining NK cells with other immunotherapeutic agents, such as monoclonal antibodies, which might improve clinical efficacy. Moreover, genetically-modified NK cells aimed at arming NK cells with better efficacy and persistence have appeared as another option. Here, we review novel pre-clinical and clinical studies published in the last five years administering NK cells as a monotherapy and combined with other agents, and we also review chimeric antigen receptor-modified NK cells for the treatment of cancer patients. We then describe studies regarding the role of NK cells as anti-microbial effectors, as lessons that we could learn and apply in immunotherapy applications of NK cells; these studies highlight an important immunoregulatory role performed between T cells and NK cells that should be considered when designing immunotherapeutic strategies. Lastly, we highlight novel strategies that could be combined with NK cell immunotherapy to improve their targeting, activity, and persistence.
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http://dx.doi.org/10.3390/cancers12113139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694052PMC
October 2020

CAR T cells targeting options in the fight against multiple myeloma.

Panminerva Med 2021 Mar 21;63(1):37-45. Epub 2020 Sep 21.

IRCCS Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST), Meldola, Forlì-Cesena, Italy.

Introduction: Multiple myeloma (MM) is a hematological malignancy in which patients present with bone marrow infiltration of clonal terminally-differentiated plasma cells. Monoclonal protein in the serum and/or urine is frequently detected. Over the past decade, important progress has been made in the comprehension of disease biology and treatment personalization. Much work has been put into the development of chimeric antigen receptor (CAR) gene-modified T-cell therapy thought to be a promising therapeutic option for pluritreated patients with refractory MM.

Evidence Acquisition: We performed an analysis of clinical trials registered at the international repository clinicaltrials.gov using "CAR" OR "CAR T" AND "multiple myeloma" as search terms to understand what were the antigens targeted by CAR T strategies and what was the trade-off of their exploitation. The search retrieved a list of 103 trials that was manually filtered to eliminate follow-up and observational or not-pertinent trials.

Evidence Synthesis: Most studies employed anti-BCMA targeting either alone (62/94; 66%), or in combination with a second target (12/94; 13%). The second target most studied was SLAMF7 (CD319) explored by 4/94 (4%) clinical trials. Other antigens investigated and described here include: CD44v6, CD38, CD138, MUC1, CD56, CD19, Igk light chain, Lewis Y, CD229 and GPRC5D.

Conclusions: Targeting an appropriate antigen(s) is the key to both safety and efficacy of CAR T approaches in MM as there is dearth of tumor-specific antigens. Most antigens tested are merely enriched on MM cells. Working with tumor-enriched antigens requires careful assessment of the balance between harm (toxicity) and benefit (disease eradication) to the patient. This review provides an up-to-date overview of the avenues that are being explored.
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http://dx.doi.org/10.23736/S0031-0808.20.04146-4DOI Listing
March 2021

Senescence in the Development and Response to Cancer with Immunotherapy: A Double-Edged Sword.

Int J Mol Sci 2020 Jun 18;21(12). Epub 2020 Jun 18.

Department of Hematology, Hospital Clinic, IDIBAPS, 08036 Barcelona, Spain.

Cellular senescence was first described as a physiological tumor cell suppressor mechanism that leads to cell growth arrest with production of the senescence-associated secretory phenotype known as SASP. The main role of SASP in physiological conditions is to attract immune cells to clear senescent cells avoiding tumor development. However, senescence can be damage-associated and, depending on the nature of these stimuli, additional types of senescence have been described. In the context of cancer, damage-associated senescence has been described as a consequence of chemotherapy treatments that were initially thought of as a tumor suppressor mechanism. However, in certain contexts, senescence after chemotherapy can promote cancer progression, especially when immune cells become senescent and cannot clear senescent tumor cells. Moreover, aging itself leads to continuous inflammaging and immunosenescence which are responsible for rewiring immune cells to become defective in their functionality. Here, we define different types of senescence, pathways that activate them, and functions of SASP in these events. Additionally, we describe the role of senescence in cancer and its treatments, including how aging and chemotherapy contribute to senescence in tumor cells, before focusing on immune cell senescence and its role in cancer. Finally, we discuss potential therapeutic interventions to reverse cell senescence.
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http://dx.doi.org/10.3390/ijms21124346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352478PMC
June 2020

Point-Of-Care CAR T-Cell Production (ARI-0001) Using a Closed Semi-automatic Bioreactor: Experience From an Academic Phase I Clinical Trial.

Front Immunol 2020 20;11:482. Epub 2020 Mar 20.

Institut D'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain.

Development of semi-automated devices that can reduce the hands-on time and standardize the production of clinical-grade CAR T-cells, such as CliniMACS Prodigy from Miltenyi, is key to facilitate the development of CAR T-cell therapies, especially in academic institutions. However, the feasibility of manufacturing CAR T-cell products from heavily pre-treated patients with this system has not been demonstrated yet. Here we report and characterize the production of 28 CAR T-cell products in the context of a phase I clinical trial for CD19+ B-cell malignancies (NCT03144583). The system includes CD4-CD8 cell selection, lentiviral transduction and T-cell expansion using IL-7/IL-15. Twenty-seven out of 28 CAR T-cell products manufactured met the full list of specifications and were considered valid products. cell expansion lasted an average of 8.5 days and had a mean transduction rate of 30.6 ± 13.44%. All products obtained presented cytotoxic activity against CD19+ cells and were proficient in the secretion of pro-inflammatory cytokines. Expansion kinetics was slower in patient's cells compared to healthy donor's cells. However, product potency was comparable. CAR T-cell subset phenotype was highly variable among patients and largely determined by the initial product. T and T were the predominant T-cell phenotypes obtained. 38.7% of CAR T-cells obtained presented a T or T phenotype, in average, which are the subsets capable of establishing a long-lasting T-cell memory in patients. An in-depth analysis to identify individual factors contributing to the optimal T-cell phenotype revealed that cell expansion leads to reduced numbers of T, T, and T cells, while T cells increase, both due to cell expansion and CAR-expression. Overall, our results show for the first time that clinical-grade production of CAR T-cells for heavily pre-treated patients using CliniMACS Prodigy system is feasible, and that the obtained products meet the current quality standards of the field. Reduced expansion may yield CAR T-cell products with increased persistence .
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http://dx.doi.org/10.3389/fimmu.2020.00482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259426PMC
March 2021

Nectin-2 Expression on Malignant Plasma Cells Is Associated with Better Response to TIGIT Blockade in Multiple Myeloma.

Clin Cancer Res 2020 09 8;26(17):4688-4698. Epub 2020 Jun 8.

Department of Hematology, Hospital Clínic de Barcelona, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain.

Purpose: T-cell immunoreceptor with Ig and ITIM domain (TIGIT) blockade could represent an alternative therapeutic option to release the immune response in patients with multiple myeloma. Here we analyzed the expression of TIGIT and its ligands poliovirus receptor (PVR) and nectin-2 in the bone marrow (BM) of patients with monoclonal gammopathies and the efficacy of TIGIT blockade activating antimyeloma immunity.

Experimental Design: Expression levels of TIGIT and its ligands were characterized by flow cytometry and ELISA. TIGIT blockade was analyzed in functional assays with peripheral T cells. BM cells were studied with NanoString technology, real-time PCR, and patient BM cell models.

Results: TIGIT and its ligands are highly expressed in the BM of patients with multiple myeloma, suggesting that may play a role in restraining immune activation. TIGIT blockade depleted FoxP3 Tregs while increasing proliferation of IFNγ-producing CD4 T cells from patients with multiple myeloma. PVR ligation inhibited CD8 T-cell signaling and cell proliferation which could be overcome with anti-TIGIT mAb. However, BM cells showed a remarkable heterogeneity in immune signature. Accordingly, functional BM assays revealed that only some patients respond to checkpoint blockade. Thus, response to TIGIT blockade correlated with low frequency of TIGIT cells and high nectin-2 expression on malignant plasma cells.

Conclusions: TIGIT blockade efficiently reinvigorated peripheral T cells from patients with multiple myeloma. However, in the BM, the efficacy of blocking anti-TIGIT mAb to achieve tumor cell death may depend on the expression of TIGIT and nectin-2, becoming potential predictive biomarkers for identifying patients who may benefit from TIGIT blockade.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3673DOI Listing
September 2020

potential of human mesenchymal stem cells for corneal epithelial regeneration.

Regen Med 2020 03 30;15(3):1409-1426. Epub 2020 Apr 30.

Barcelona Tissue Bank, Banc de Sang I Teixits (BST), Barcelona, Spain.

To determine the potential of mesenchymal stem cells (MSC) for corneal epithelial regeneration . Bone marrow MSC (BM-MSC) and adipose tissue MSC were analyzed for corneal epithelial and mesenchymal markers, using limbal stem cells and corneal cells as controls. MSC with better potential were cultured with specific mediums for epithelial induction. Transepithelial electric resistance and wound healing assay with human corneal epithelial cells were performed. BM-MSC showed better potential, increased corneal markers, and higher transepithelial electric resistance values when induced with limbal epithelial culture medium. Induced BM-MSC promoted better wound healing of human corneal epithelial cells by paracrine secretion. BM-MSC has potential for corneal epithelial induction in a protocol compatible with human application.
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http://dx.doi.org/10.2217/rme-2019-0067DOI Listing
March 2020

Preclinical development of a humanized chimeric antigen receptor against B cell maturation antigen for multiple myeloma.

Haematologica 2021 01 1;106(1):173-184. Epub 2021 Jan 1.

Hospital Clinic, IDIBAPS, Josep Carreras Leukaemia Research Institute, Barcelona, Spain.

Multiple myeloma is a prevalent and incurable disease, despite the development of new and effective drugs. The recent development of chimeric antigen receptor (CAR)-T cell therapy has shown impressive results in the treatment of patients with relapsed or refractory hematological B cell malignancies. In the recent years, B-cell maturation antigen (BCMA) has appeared as a promising antigen to target using a variety of immuno-therapy treatments including CART cells, for MM patients. To this end, we generated clinical-grade murine CART cells directed against BCMA, named ARI2m cells. Having demonstrated its efficacy, and in an attempt to avoid the immune rejection of CART cells by the patient, the single chain variable fragment was humanized, creating ARI2h cells. ARI2h cells demonstrated comparable in vitro and in vivo efficacy to ARI2m cells, and superiority in cases of high tumor burden disease. In terms of inflammatory response, ARI2h cells showed a lower TNFα production and lower in vivo toxicity profile. Large-scale expansion of both ARI2m and ARI2h cells was efficiently conducted following Good Manufacturing Practice guidelines, obtaining the target CART cell dose required for treatment of multiple myeloma patients. Moreover, we demonstrate that soluble BCMA and BCMA released in vesicles impacts on CAR-BCMA activity. In summary, this study sets the bases for the implementation of a clinical trial (EudraCT code: 2019-001472-11) to study the efficacy of ARI2h cell treatment for multiple myeloma patients.
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http://dx.doi.org/10.3324/haematol.2019.228577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7776337PMC
January 2021

Development of a Novel Anti-CD19 Chimeric Antigen Receptor: A Paradigm for an Affordable CAR T Cell Production at Academic Institutions.

Mol Ther Methods Clin Dev 2019 Mar 6;12:134-144. Epub 2018 Dec 6.

Institut d'Investigacions Biomèdiques August Pi i Sunyer - IDIBAPS, Rosselló 153, 08036 Barcelona, Spain.

Genetically modifying autologous T cells to express an anti-CD19 chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19+ B cell malignancies in several clinical trials (CTs). Making this treatment available to our patients prompted us to develop a novel CART19 based on our own anti-CD19 antibody (A3B1), followed by CD8 hinge and transmembrane region, 4-1BB- and CD3z-signaling domains. We show that A3B1 CAR T cells are highly cytotoxic and specific against CD19+ cells , inducing secretion of pro-inflammatory cytokines and CAR T cell proliferation. , A3B1 CAR T cells are able to fully control disease progression in an NOD.Cg- /SzJ (NSG) xenograph B-ALL mouse model. Based on the pre-clinical data, we conclude that our CART19 is clearly functional against CD19+ cells, to a level similar to other CAR19s currently being used in the clinic. Concurrently, we describe the implementation of our CAR T cell production system, using lentiviral vector and CliniMACS Prodigy, within a medium-sized academic institution. The results of the validation phase show our system is robust and reproducible, while maintaining a low cost that is affordable for academic institutions. Our model can serve as a paradigm for similar institutions, and it may help to make CAR T cell treatment available to all patients.
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http://dx.doi.org/10.1016/j.omtm.2018.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6319086PMC
March 2019

Immunotherapy: A Novel Era of Promising Treatments for Multiple Myeloma.

Int J Mol Sci 2018 Nov 15;19(11). Epub 2018 Nov 15.

Department of Hematology, Hospital Clinic, IDIBAPS, 08036 Barcelona, Spain.

Multiple myeloma (MM) remains an incurable hematological malignancy characterized by clonal proliferation of malignant plasma cells in bone marrow. In the last 20 years, the introduction of autologous stem cell transplantation, followed by proteasome inhibitors and immunomodulatory agents, increased the survival of MM patients by 50%. However, still a high proportion of patients relapse and become refractory, especially, high-risk patients with adverse cytogenetics where these treatment combinations have shown limited benefit. Therefore, novel strategies, such as immunotherapy, have been developed in the last few years to help improve the survival of these patients. Immunotherapy treatments include a high number of different strategies used to attack the tumor cells by using the immune system. Here, we will review the most successful immunotherapy strategies published up to date in patients with relapsed or refractory (R/R) MM, including monoclonal antibodies targeting specific antigens on the tumor cells, antibodies combined with cytotoxic drugs or Antibodies Drug Conjugates, immune checkpoint inhibitors which eliminate the barriers that damper immune cells and prevent them from attacking tumor cells, bi-specific T-cell engagers antibodies (BiTEs), bi-specific antibodies and the infusion of chimeric antigen receptor-modified T cells. We overview the results of clinical studies that have been presented up to date and also review pre-clinical studies describing potential novel treatments for MM.
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http://dx.doi.org/10.3390/ijms19113613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6274949PMC
November 2018

CAR-T Cell Therapy: A Door Is Open to Find Innumerable Possibilities of Treatments for Cancer Patients

Turk J Haematol 2018 11 6;35(4):217-228. Epub 2018 Sep 6.

Institut d’Investigacions Biomèdiques August Pi i Sunyer Hospital, Clinic of Hematology, Barcelona, Spain

Seven years ago a chronic lymphocytic leukemia patient was for the first time successfully treated with chimeric antigen receptor (CAR)-modified T cells (CAR-T cells) to target CD19 overexpression in tumor cells. This was the beginning of the development of a new type of immunotherapy treatment in cancer patients. Since then, identification of novel antigens expressed in tumor cells and optimization of both CAR constructs and protocols of administration have opened up new avenues for the successful treatment of other hematological malignancies. However, research still continues to avoid some problems such as toxicities associated with the treatment and to find strategies to avoid tumor cell immune evasion mechanisms. On the other hand, for solid tumors, CAR-T therapy results are still in an early phase. In contrast to hematological malignancies, the complex tumor heterogeneity of solid tumors has led to the research of novel and challenging strategies to improve CAR-T cell activity. Here, we will review the main clinical results obtained with CAR-T cells in hematological malignancies, specifically focusing on CAR-T-19 and CAR-T against B-cell maturation antigen (CAR-T-BCMA). Moreover, we will mention the main problems that decrease CAR-T cell activity in solid tumors and the strategies to overcome them. Finally, we will present some of the first clinical results obtained for solid tumors.
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http://dx.doi.org/10.4274/tjh.2018.0196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6256819PMC
November 2018

A novel predictive approach for GVHD after allogeneic SCT based on clinical variables and cytokine gene polymorphisms.

Blood Adv 2018 07;2(14):1719-1737

Department of Statistics, Universidad Carlos III de Madrid, Madrid, Spain.

Despite considerable advances in our understanding of the pathophysiology of graft-versus-host disease (GVHD), its prediction remains unresolved and depends mainly on clinical data. The aim of this study is to build a predictive model based on clinical variables and cytokine gene polymorphism for predicting acute GVHD (aGVHD) and chronic GVHD (cGVHD) from the analysis of a large cohort of HLA-identical sibling donor allogeneic stem cell transplant (allo-SCT) patients. A total of 25 SNPs in 12 cytokine genes were evaluated in 509 patients. Data were analyzed using a linear regression model and the least absolute shrinkage and selection operator (LASSO). The statistical model was constructed by randomly selecting 85% of cases (training set), and the predictive ability was confirmed based on the remaining 15% of cases (test set). Models including clinical and genetic variables (CG-M) predicted severe aGVHD significantly better than models including only clinical variables (C-M) or only genetic variables (G-M). For grades 3-4 aGVHD, the correct classification rates (CCR1) were: 100% for CG-M, 88% for G-M, and 50% for C-M. On the other hand, CG-M and G-M predicted extensive cGVHD better than C-M (CCR1: 80% vs. 66.7%, respectively). A risk score was calculated based on LASSO multivariate analyses. It was able to correctly stratify patients who developed grades 3-4 aGVHD ( < .001) and extensive cGVHD ( < .001). The novel predictive models proposed here improve the prediction of severe GVHD after allo-SCT. This approach could facilitate personalized risk-adapted clinical management of patients undergoing allo-SCT.
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http://dx.doi.org/10.1182/bloodadvances.2017011502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058238PMC
July 2018

Deleterious Effect of Steroids on Cytomegalovirus Infection Rate after Allogeneic Stem Cell Transplantation Depends on Pretransplant Cytomegalovirus Serostatus of Donors and Recipients.

Biol Blood Marrow Transplant 2018 10 9;24(10):2088-2093. Epub 2018 May 9.

Hematology Department, Hospital Clínic Barcelona, Barcelona, Spain; Institut d'Investigació Biomèdica August Pi I Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain; Institut Josep Carreras, Barcelona, Spain.

This study examined the impact of prednisone (PDN) on cytomegalovirus (CMV) infection after allogeneic stem cell transplantation (allo-SCT) according to donor and recipient CMV serostatus. Seventy-five patients underwent allo-SCT from June 2010 to July 2012. The risk of CMV infection according to donor and recipient serostatus was defined as follows: high risk (HR; D-/R+), intermediate risk (IR; D+/R+ and D+/R-), and low risk (D-/R-). Forty-five patients (60%) developed CMV infection, and 46 patients (61%) received steroids (PDN ≥ 1 mg/kg/day) to treat acute graft-versus-host disease. CMV infection was more common in those treated with steroids than in those not treated with steroids (70% versus 44%, respectively, P < .05). Overall, 40% of patients had recurrent CMV infection (50% PDN versus 24% no PDN, P < .05). Steroids had no impact on the incidence of CMV infection or its recurrence in HR patients; however, steroids did prolong the need for antiviral treatment. The incidence of CMV infection in IR patients was higher in those receiving PDN (80% PDN versus 41% no PDN, P = .01); recurrence rates were also higher (55% PDN versus 18% no PDN, P = .02). We analyzed CMV-specific immune reconstitution in the first 22 patients of the series and observed that patients on steroids had lower levels of CMV-specific lymphocytes TCD8 (P < .05 on days +60, +100, and +180) and that CMV-specific immune reconstitution (defined as lymphocytes CD8/IFN ≥ 1 cell/µL) was achieved later (after day +100 post-SCT) in the steroid group.
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http://dx.doi.org/10.1016/j.bbmt.2018.05.001DOI Listing
October 2018

Loss of the Immune Checkpoint CD85j/LILRB1 on Malignant Plasma Cells Contributes to Immune Escape in Multiple Myeloma.

J Immunol 2018 04 12;200(8):2581-2591. Epub 2018 Mar 12.

Amyloidosis and Myeloma Unit, Department of Hematology, Hospital Clínic, August Pi i Sunyer Biomedical Research Institute, University of Barcelona, 08036 Barcelona, Spain;

Mechanisms of immune regulation may control proliferation of aberrant plasma cells (PCs) in patients with monoclonal gammopathy of undetermined significance (MGUS) preventing progression to active multiple myeloma (MM). We hypothesized that CD85j (), an inhibitory immune checkpoint for B cell function, may play a role in MM pathogenesis. In this study, we report that patients with active MM had significantly lower levels of CD85j and its ligand S100A9. Decreased CD85j expression could also be detected in the premalignant condition MGUS, suggesting that loss of CD85j may be an early event promoting tumor immune escape. To gain insight into the molecular mechanisms underlying CD85j functions, we next enforced expression of CD85j in human myeloma cell lines by lentiviral transduction. Interestingly, gene expression profiling of CD85j-overexpressing cells revealed a set of downregulated genes with crucial functions in MM pathogenesis. Furthermore, in vitro functional assays demonstrated that CD85j overexpression increased susceptibility to T cell- and NK-mediated killing. Consistently, ligation of CD85j decreased the number of PCs from individuals with MGUS but not from patients with MM. In conclusion, downregulation of inhibitory immune checkpoints on malignant PCs may provide a novel mechanism of immune escape associated with myeloma pathogenesis.
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http://dx.doi.org/10.4049/jimmunol.1701622DOI Listing
April 2018

Natural Killer Cells: Angels and Devils for Immunotherapy.

Int J Mol Sci 2017 Aug 29;18(9). Epub 2017 Aug 29.

Department of Hematology, Hospital Clinic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain.

In recent years, the relevance of the immune system to fight cancer has led to the development of immunotherapy, including the adoptive cell transfer of immune cells, such as natural killer (NK) cells and chimeric antigen receptors (CAR)-modified T cells. The discovery of donor NK cells' anti-tumor activity in acute myeloid leukemia patients receiving allogeneic stem cell transplantation (allo-SCT) was the trigger to conduct many clinical trials infusing NK cells. Surprisingly, many of these studies did not obtain optimal results, suggesting that many different NK cell parameters combined with the best clinical protocol need to be optimized. Various parameters including the high array of activating receptors that NK cells have, the source of NK cells selected to treat patients, different cytotoxic mechanisms that NK cells activate depending on the target cell and tumor cell survival mechanisms need to be considered before choosing the best immunotherapeutic strategy using NK cells. In this review, we will discuss these parameters to help improve current strategies using NK cells in cancer therapy. Moreover, the chimeric antigen receptor (CAR) modification, which has revolutionized the concept of immunotherapy, will be discussed in the context of NK cells. Lastly, the dark side of NK cells and their involvement in inflammation will also be discussed.
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http://dx.doi.org/10.3390/ijms18091868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5618517PMC
August 2017

The Genotype of the Donor for the (GT)n Polymorphism in the Promoter/Enhancer of FOXP3 Is Associated with the Development of Severe Acute GVHD but Does Not Affect the GVL Effect after Myeloablative HLA-Identical Allogeneic Stem Cell Transplantation.

PLoS One 2015 16;10(10):e0140454. Epub 2015 Oct 16.

Department of Hematology, Hospital General Universitario Gregorio Marañón, Madrid, Spain; Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), Madrid, Spain.

The FOXP3 gene encodes for a protein (Foxp3) involved in the development and functional activity of regulatory T cells (CD4+/CD25+/Foxp3+), which exert regulatory and suppressive roles over the immune system. After allogeneic stem cell transplantation, regulatory T cells are known to mitigate graft versus host disease while probably maintaining a graft versus leukemia effect. Short alleles (≤(GT)15) for the (GT)n polymorphism in the promoter/enhancer of FOXP3 are associated with a higher expression of FOXP3, and hypothetically with an increase of regulatory T cell activity. This polymorphism has been related to the development of auto- or alloimmune conditions including type 1 diabetes or graft rejection in renal transplant recipients. However, its impact in the allo-transplant setting has not been analyzed. In the present study, which includes 252 myeloablative HLA-identical allo-transplants, multivariate analysis revealed a lower incidence of grade III-IV acute graft versus host disease (GVHD) in patients transplanted from donors harboring short alleles (OR = 0.26, CI 0.08-0.82, p = 0.021); without affecting chronic GVHD or graft versus leukemia effect, since cumulative incidence of relapse, event free survival and overall survival rates are similar in both groups of patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0140454PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608671PMC
June 2016

Overexpression of GYS1, MIF, and MYC is associated with adverse outcome and poor response to azacitidine in myelodysplastic syndromes and acute myeloid leukemia.

Clin Lymphoma Myeloma Leuk 2015 Apr 23;15(4):236-44. Epub 2014 Oct 23.

Department of Haematology, University Hospital Virgen del Rocío, Instituto de Biomedicina de Sevilla (IBIS)/CSIC/Universidad de Sevilla, Sevilla, Spain.

Background: The prognosis of myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML) is very heterogeneous.

Patients And Methods: We analyzed the prognostic value of several genes in a cohort of 85 MDS and AML patients.

Results: Overexpression of glycogen synthase 1 and macrophage migration inhibitory factor genes had an adverse outcome in multivariate analysis (P = .003 and P < .001, respectively). Furthermore, the higher expression of myelocytomatosis oncogene was associated with a lower response to azacitidine (P = .03).

Conclusion: In the current study we identified a specific gene expression profile as prognostic factors for response to azacitidine and survival in MDS and AML.
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http://dx.doi.org/10.1016/j.clml.2014.10.003DOI Listing
April 2015

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.

Haematologica 2014 Jul 11;99(7):1168-75. Epub 2014 Apr 11.

Josep Carreras Leukemia Research Institute, Cell Therapy Program of the University of Barcelona, Faculty of Medicine, Barcelona, Spain Instituciò Catalana de Reserca i Estudis Avançats (ICREA), Barcellona, Spain

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.
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http://dx.doi.org/10.3324/haematol.2014.103580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4077077PMC
July 2014

Gene and miRNA expression profiles of hematopoietic progenitor cells vary depending on their origin.

Biol Blood Marrow Transplant 2014 May 23;20(5):630-9. Epub 2014 Jan 23.

Department of Hematology/Hospital Clinic/IDIBAPS and Institute of Research Josep Carreras/University of Barcelona.

Hematopoietic progenitor cells (HPCs) from granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (G-PB), bone marrow (BM), or umbilical cord blood (CB) have differing biological properties and differing kinetics of engraftment post-transplantation, which might be explained, at least in part, by differing gene and miRNA expression patterns. To assess the differences in gene and miRNA expression, we analyzed whole genome expression profiles as well as the expression of 384 miRNAs in CD34(+) cells isolated from 18 healthy individuals (6 individuals per subtype of HPC source). We identified 43 genes and 36 miRNAs differentially expressed in the various CD34(+) cell sources. We observed that CD34(+) cells from CB and BM showed similar gene and miRNA expression profiles, whereas CD34(+) cells from G-PB had a very different expression pattern. Remarkably, 20 of the differentially expressed genes are targets of the differentially expressed miRNAs. Of note, the majority of genes differentially expressed in CD34(+) cells from G-PB are involved in cell cycle regulation, promoting the process of proliferation, survival, hematopoiesis, and cell signaling, and are targets of overexpressed and underexpressed miRNAs in CD34(+) cells from the same source. These data suggest significant differences in gene and miRNA expression among the various HPC sources used in transplantation. We hypothesize that the differentially expressed genes and miRNAs involved in cell cycle and proliferation might explain the differing kinetics of engraftment observed after transplantation of hematopoietic stem cells obtained from these different sources.
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http://dx.doi.org/10.1016/j.bbmt.2014.01.022DOI Listing
May 2014

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

PLoS One 2013 18;8(10):e76781. Epub 2013 Oct 18.

Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076781PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3800010PMC
August 2014

Fucosylation with fucosyltransferase VI or fucosyltransferase VII improves cord blood engraftment.

Cytotherapy 2014 Jan 1;16(1):84-9. Epub 2013 Oct 1.

Department of Stem Cell Transplantation and Cellular Therapy, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.

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.
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http://dx.doi.org/10.1016/j.jcyt.2013.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3883688PMC
January 2014

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

Haematologica 2014 Feb 20;99(2):243-51. Epub 2013 Sep 20.

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.
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http://dx.doi.org/10.3324/haematol.2013.086959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3912953PMC
February 2014

Self-renewing human bone marrow mesenspheres promote hematopoietic stem cell expansion.

Cell Rep 2013 May 25;3(5):1714-24. Epub 2013 Apr 25.

Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid 28029, Spain.

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.
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http://dx.doi.org/10.1016/j.celrep.2013.03.041DOI Listing
May 2013

Impact of global and gene-specific DNA methylation pattern in relapsed multiple myeloma patients treated with bortezomib.

Leuk Res 2013 Jun 8;37(6):641-6. Epub 2013 Feb 8.

Amyloidosis and Myeloma Unit, Department of Hematology, Hospital Clínic, Barcelona, Institut d'Investigacions Biomèdiques August Pi I Sunyer, José Carreras Leukaemia Research Institute, University of Barcelona, Barcelona, Spain.

We studied seventy-five patients with relapsed MM treated with bortezomib-based regimens. DNA was isolated from bone marrow samples at the time of relapse. Global methylation was determined by ELISA, and CpG island DNA methylation profile of 30 genes by a DNA methylation PCR system. Patients with more than 3.95% of total DNA methylated achieved better overall survival (OS) (p=0.004). A relatively low methylation percentage (<1.07%) of NFKB1 was also associated with longer OS after bortezomib treatment (p=0.015). The combination of highly methylated global genome with low NFKB1 methylation status defined a specific subset of patients with better prognosis.
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http://dx.doi.org/10.1016/j.leukres.2013.01.013DOI Listing
June 2013

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

Expert Rev Hematol 2010 Aug;3(4):411-27

Hematology Department/Hospital Clínic of Barcelona, Institut d'Investigacions Biomediques August Pi i Sunyer, University of Barcelona, Avenida Villarroel 170, 08036-Barcelona, Spain.

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.
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http://dx.doi.org/10.1586/ehm.10.40DOI Listing
August 2010

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

Haematologica 2011 Jan 17;96(1):102-9. Epub 2010 Sep 17.

Department of Hematology, Hospital Clinic University of Barcelona, Barcelona, Spain.

Unlabelled: 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.
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http://dx.doi.org/10.3324/haematol.2010.026401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3012773PMC
January 2011

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

Fish Shellfish Immunol 2010 Feb 10;28(2):296-302. Epub 2009 Nov 10.

IFAPA Centro El Toruño, Consejería de Agricultura y Pesca, Junta de Andalucía. Apartado 16, 11500 El Puerto de Santa María, Cádiz, Spain.

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.
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http://dx.doi.org/10.1016/j.fsi.2009.11.006DOI Listing
February 2010

Genomic characterization and gene expression analysis of four hepcidin genes in the redbanded seabream (Pagrus auriga).

Fish Shellfish Immunol 2009 Mar;26(3):483-91

IFAPA Centro El Toruño, CICE, Junta de Andalucía, Camino Tiro de Pichón s/n, 11500 El Puerto de Santa María, Cádiz, Spain.

Hepcidin antimicrobial peptides (HAMPs) are key molecules of the innate immune system against bacterial infections and in iron metabolism. In this study we report the molecular cloning and genomic characterization of four HAMP genes (referred to as HAMP1, HAMP2, HAMP3 and HAMP4) in the redbanded seabream (Pagrus auriga). All these genes possessed the eight characteristic cysteine residues involved in protein folding. No canonical sequence for convertase-mediated processing of the HAMP3 propeptide was identified. At the genomic level, all four HAMP genes consisted of two introns and three exons. Phylogenetic analysis revealed that HAMPs could group in two main clusters with HAMP2, HAMP3 and HAMP4 belonging to the more complex and diversified HAMP2-like group of acanthopterygians. Quantitation of mRNA levels in adult tissues showed that HAMP1 was ubiquitously expressed, HAMP2 mainly in kidney, spleen and intestine, whereas HAMP3 and HAMP4 in liver. During development, HAMP2 and HAMP3 were expressed at a high level in embryos. Moreover, the expression levels of the four HAMP genes increased between 5 and 15 days after hatching when larvae started external feeding. Induction experiments with lipopolysaccharide revealed significant changes in gene expression of the four HAMP genes in kidney, liver and spleen. However, expression profiles differed in magnitude and time course response. HAMP1 mRNAs increased rapidly in kidney at 1 h p.i. whereas HAMP2 did later at 24 h. Moreover, HAMP4 transcripts increased more than 5000-fold in liver whereas HAMP2 mRNAs dropped significantly in spleen at 3 h p.i. All these data suggest that HAMPs are involved in the response against bacterial infections although additional functions in iron regulation and embryogenesis in fish should be considered.
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http://dx.doi.org/10.1016/j.fsi.2009.01.012DOI Listing
March 2009

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

Fish Shellfish Immunol 2008 Jul 24;25(1-2):57-65. Epub 2007 Dec 24.

IFAPA Centro El Toruño, Consejería de Innovación Ciencia y Empresa, Junta de Andalucía, Molecular Biology Laboratory, Camino Tiro de pichón s/n, 11500 El Puerto de Santa María, Cádiz, Spain.

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.
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http://dx.doi.org/10.1016/j.fsi.2007.12.009DOI Listing
July 2008