Production of BP178, a derivative of the synthetic antibacterial peptide BP100, in the rice seed endosperm

Background: BP178 peptide is a synthetic BP100-magainin derivative possessing strong inhibitory activity against plant pathogenic bacteria, offering a great potential for future applications in plant protection and other fields. Here we report the production and recovery of a bioactive BP178 peptide using rice seeds as biofactories. - Results: a synthetic gene encoding the BP178 peptide was prepared and introduced in rice plants. The gene was efficiently expressed in transgenic rice under the control of an endosperm-specific promoter. Among the three endosperm-specific rice promoters (Glutelin B1, Glutelin B4 or Globulin 1), best results were obtained when using the Globulin 1 promoter. The BP178 peptide accumulated in the seed endosperm and was easily recovered from rice seeds using a simple procedure with a yield of 21 μg/g. The transgene was stably inherited for at least three generations, and peptide accumulation remained stable during long term storage of transgenic seeds. The purified peptide showed in vitro activity against the bacterial plant pathogen Dickeya sp., the causal agent of the dark brown sheath rot of rice. Seedlings of transgenic events showed enhanced resistance to the fungal pathogen Fusarium verticillioides, supporting that the in planta produced peptide was biologically active. -Conclusions: the strategy developed in this work for the sustainable production of BP178 peptide using rice seeds as biofactories represents a promising system for future production of peptides for plant protection and possibly in other fields

Regulatory T-cell Number in Peripheral Blood at 1 Year Posttransplant as Predictor of Long-term Kidney Graft Survival

Background: Regulatory T (Treg) cells play a role in limiting kidney transplant rejection and can potentially promote long-term transplant tolerance. There are no large prospective studies demonstrating the utility of peripheral blood Treg cells as biomarkers for long-term graft outcome in kidney transplantation. The aim of our study was to analyze the influence of the absolute number of peripheral blood Treg cells after transplantation on long-term death-censored graft survival. Methods: We monitored the absolute numbers of Treg cells by flow cytometry in nonfrozen samples of peripheral blood in 133 kidney transplant recipients, who were prospectively followed up to 2 years after transplantation. Death-censored graft survival was determined retrospectively in January 2017. Results: The mean time of clinical follow-up was 7.4 ± 2.9 years and 24.1% patients suffered death-censored graft loss (DCGL). Patients with high Treg cells 1 year after transplantation and above the median value (14.57 cells/mm3), showed better death-censored graft survival (5-year survival, 92.5% vs 81.4%, Log-rank P = .030). One-year Treg cells showed a receiver operating characteristic - area under curve of 63.1% (95% confidence interval, 52.9-73.2%, P = 0.026) for predicting DCGL. After multivariate Cox regression analysis, an increased number of peripheral blood Treg cells was a protective factor for DCGL (hazard ratio, 0.961, 95% confidence interval, 0.924-0.998, P = 0.041), irrespectively of 1-year proteinuria and renal function. Conclusions: Peripheral blood absolute numbers of Treg cells 1 year after kidney transplantation predict a better long-term graft outcome and may be used as prognostic biomarkers

Phenotypic, genomic and functional characterization reveals no differences between CD138++ and CD138low subpopulations in multiple myeloma cell lines

This is an open-access article distributed under the terms of the Creative Commons Attribution License.Despite recent advances in the treatment of multiple myeloma (MM), it remains an incurable disease potentially due to the presence of resistant myeloma cancer stem cells (MM-CSC). Although the presence of clonogenic cells in MM was described three decades ago, the phenotype of MM-CSC is still controversial, especially with respect to the expression of syndecan-1 (CD138). Here, we demonstrate the presence of two subpopulations - CD138++ (95-99%) and CD138low (1-5%) - in eight MM cell lines. To find out possible stem-cell-like features, we have phenotypically, genomic and functionally characterized the two subpopulations. Our results show that the minor CD138low subpopulation is morphologically identical to the CD138++ fraction and does not represent a more immature B-cell compartment (with lack of CD19, CD20 and CD27 expression). Moreover, both subpopulations have similar gene expression and genomic profiles. Importantly, both CD138++ and CD138low subpopulations have similar sensitivity to bortezomib, melphalan and doxorubicin. Finally, serial engraftment in CB17-SCID mice shows that CD138++ as well as CD138low cells have self-renewal potential and they are phenotypically interconvertible. Overall, our results differ from previously published data in MM cell lines which attribute a B-cell phenotype to MM-CSC. Future characterization of clonal plasma cell subpopulations in MM patients' samples will guarantee the discovery of more reliable markers able to discriminate true clonogenic myeloma cells.This work was supported by the Cooperative Research Thematic Network (RTICs; RD06/0020/0006), the “Junta de Castilla y León. Consejería de Sanidad” (GRS 391/B/09), the “Ministerio de Ciencia e Innovación” (PS09/01897), the “Fundación Memoria D. Samuel Solórzano Barruso” (FS/2-2010) and Asociación Española Contra el Cáncer (AECC)(GCB120981SAN).Peer Reviewe

Effect of mTORC1/mTORC2 inhibition on T cell function: potential role in graft-versus-host disease control

Producción CientíficaThe mechanistic target of rapamycin (mTOR) pathway is crucial for the activation and function of T cells, which play an essential role in the development of graft-versus-host disease (GvHD). Despite its partial ability to block mTOR pathway, the mTORC1 inhibitor rapamycin has shown encouraging results in the control of GvHD. Therefore, we considered that simultaneous targeting of both mTORC1 and mTORC2 complexes could exert a more potent inhibition of T cell activation and, thus, could have utility in GvHD control. To assess this assumption, we have used the dual mTORC1/mTORC2 inhibitors CC214-1 and CC214-2. In vitro studies confirmed the superior ability of CC214-1 versus rapamycin to block mTORC1 and mTORC2 activity and to reduce T cell proliferation. Both drugs induced a similar decrease in Th1/Th2 cytokine secretion, but CC214-1 was more efficient in inhibiting na€ıve T cell activation and the expression of Tcell activation markers. In addition, CC214-1 induced specific tolerance against alloantigens, while preserving anti-cytomegalovirus response. Finally, in a mouse model of GvHD, the administration of CC214-2 significantly improved mice survival and decreased GvHD-induced damages. In conclusion, the current study shows, for the first time, the immunosuppressive ability of CC214-1 on T lymphocytes and illustrates the role of CC214-2 in the allogeneic transplantation setting as a possible GvHD prophylaxis agent.Gerencia Regional de Salud de Castilla y León (Proyecto GRS 726/A13

Detection of antibodies to denatured human leucocyte antigen molecules by single antigen Luminex

The anti-HLA antibody detection has been improved in sensitivity and specificity with solid-phase antigen bead (SAB) assays based on Luminex. However, false positive results due to denatured HLA (dHLA) may arise after single antigen test. The aim of this study was to compare the performance of the two Luminex technology-based anti-HLA detection kits available in the market in showing undesired anti-HLA antibody results. A prospective cohort was assessed for anti-HLA antibodies with single antigen A manufacturer (AM) kit and a comparison cohort with single antigen B manufacturer (BM) kit. A total of 11 out of 90 patients in a prospective cohort presented monospecific HLA-I antibodies with AM, and 5 out of 11 confirmed monospecific reaction with BM. Despite the confirmation of monospecific reaction with both manufacturers, 80% were assigned as dHLA reaction by specific crossmatch. Further comparative cohorts detected four out of six monospecific reactions with BM that were confirmed as possible dHLA reactions. A positive SAB test should rule out a reaction against a dHLA molecule, thus avoidance of prolonged waitlist periods or misattribution of anti-HLA reactions after transplantation

The PARP inhibitor olaparib enhances the sensitivity of Ewing sarcoma to trabectedin

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.Recent preclinical evidence has suggested that Ewing Sarcoma (ES) bearing EWSR1-ETS fusions could be particularly sensitive to PARP inhibitors (PARPinh) in combination with DNA damage repair (DDR) agents. Trabectedin is an antitumoral agent that modulates EWSR1-FLI1 transcriptional functions, causing DNA damage. Interestingly, PARP1 is also a transcriptional regulator of EWSR1-FLI1, and PARPinh disrupts the DDR machinery. Thus, given the impact and apparent specificity of both agents with regard to the DNA damage/DDR system and EWSR1-FLI1 activity in ES, we decided to explore the activity of combining PARPinh and Trabectedin in in vitro and in vivo experiments. The combination of Olaparib and Trabectedin was found to be highly synergistic, inhibiting cell proliferation, inducing apoptosis, and the accumulation of G2/M. The drug combination also enhanced γH2AX intranuclear accumulation as a result of DNA damage induction, DNA fragmentation and global DDR deregulation, while EWSR1-FLI1 target expression remained unaffected. The effect of the drug combination was corroborated in a mouse xenograft model of ES and, more importantly, in two ES patient-derived xenograft (PDX) models in which the tumors showed complete regression. In conclusion, the combination of the two agents leads to a biologically significant deregulation of the DDR machinery that elicits relevant antitumor activity in preclinical models and might represent a promising therapeutic tool that should be further explored for translation to the clinical setting.Enrique de Álava’s lab is supported by the AECC (Asociación Española Contra el Cáncer), the Ministry of Economy and Competitiveness of Spain-FEDER (PI081828, RD06/0020/0059 RD12/0036/0017, PT13/0010/0056, PI110018, ISCIII Sara Borrell postdoc grant CD06/00001), the European Project EuroSARC (FP7-HEALTH-2011- two-stage, Project ID 278742 EUROSARC), Fundación Memoria de D. Manuel Solorzano Barruso, Fundación Cris contra el cancer, and Fundación María García Estrada. JLO was sponsored by the CSIC and the European Social Fund (post-doctoral grant JAE DOC) and is at present funded by the AECC. ATA is sponsored by the Fundaçao para a Ciência e Tecnologia, Portugal (fellowship SFRH/BD/69318/2010). OMT is funded by Fondo de Investigaciones Sanitarias-ISCIII (CES12/021) and the AECC. DHM is funded by the AECC. Work supported by the Xarxa de Bancs de Tumors de Catalunya (XBTC) sponsored by Pla Director d’Oncologia de Catalunya. AMC acknowledges funding from the European Union Seventh Framework Programme (FP7/2007-2013) under a Marie Curie International Reintegration Grant (PIRG-08- GA-2010-276998) and ISCIII-FEDER (CP13/00189).Peer Reviewe

Targeting aberrant DNA methylation in mesenchymal stromal cells as a treatment for myeloma bone disease

© The Author(s) 2021.Multiple myeloma (MM) progression and myeloma-associated bone disease (MBD) are highly dependent on bone marrow mesenchymal stromal cells (MSCs). MM-MSCs exhibit abnormal transcriptomes, suggesting the involvement of epigenetic mechanisms governing their tumor-promoting functions and prolonged osteoblast suppression. Here, we identify widespread DNA methylation alterations of bone marrow-isolated MSCs from distinct MM stages, particularly in Homeobox genes involved in osteogenic differentiation that associate with their aberrant expression. Moreover, these DNA methylation changes are recapitulated in vitro by exposing MSCs from healthy individuals to MM cells. Pharmacological targeting of DNMTs and G9a with dual inhibitor CM-272 reverts the expression of hypermethylated osteogenic regulators and promotes osteoblast differentiation of myeloma MSCs. Most importantly, CM-272 treatment prevents tumor-associated bone loss and reduces tumor burden in a murine myeloma model. Our results demonstrate that epigenetic aberrancies mediate the impairment of bone formation in MM, and its targeting by CM-272 is able to reverse MBD.We thank CERCA Program/Generalitat de Catalunya and the Josep Carreras Foundation for institutional support. E.B. was funded by the Spanish Ministry of Science and Innovation (grant numbers SAF2014-55942-R and SAF2017-88086-R), co-funded by FEDER funds/European Regional Development Fund (ERDF)—a way to build Europe, and a Senior Research Award from the Multiple Myeloma Research Foundation (MMRF). C.O.-d.-S. was funded by the Spanish Ministry of Science, Innovation and Universities, under grant RTI2018-094494-B-C22 (MCIU/AEI/FEDER, UE). M.G. received financial support from the Spanish FIS-ISCIII (PI15/02156 and PI19/01384) and FEDER. A.G.G is funded by a postdoctoral contract of the Asociación Española Contra el Cáncer (AECC). F.P. was funded by grants from Instituto de Salud Carlos III (ISCIII), PI17/00701 and PI19/01352, TRASCAN (EPICA and Immunocell), Fundació La Marató de TV3, the Accelerator award CRUK/AIRC/AECC joint funder-partnership, CIBERONC (CB16/12/00489) and co-financed with FEDER funds and Fundación Ramón Areces (PREMAMM)

Protein Translation Inhibition is Involved in the Activity of the Pan-PIM Kinase Inhibitor PIM447 in Combination with Pomalidomide-Dexamethasone in Multiple Myeloma

Background: Proviral Insertion site for Moloney murine leukemia virus (PIM) kinases are overexpressed in hematologic malignancies, including multiple myeloma. Previous preclinical data from our group demonstrated the anti-myeloma effect of the pan-PIM kinase inhibitor PIM447. Methods: Based on those data, we evaluate here, by in vitro and in vivo studies, the activity of the triple combination of PIM447 + pomalidomide + dexamethasone (PIM-Pd) in multiple myeloma. Results: Our results show that the PIM-Pd combination exerts a potent anti-myeloma effect in vitro and in vivo, where it markedly delays tumor growth and prolongs survival of treated mice. Mechanism of action studies performed in vitro and on mice tumor samples suggest that the combination PIM-Pd inhibits protein translation processes through the convergent inhibition of c-Myc and mTORC1, which subsequently disrupts the function of eIF4E. Interestingly the MM pro-survival factor IRF4 is also downregulated after PIM-Pd treatment. As a whole, all these molecular changes would promote cell cycle arrest and deregulation of metabolic pathways, including glycolysis and lipid biosynthesis, leading to inhibition of myeloma cell proliferation. Conclusions: Altogether, our data support the clinical evaluation of the triple combination PIM-Pd for the treatment of patients with multiple myeloma.This work was supported by funding from Spanish FIS (PI15/00067, PI15/02156 and PI18/01600) and FEDER, AECC (GCB120981SAN), Junta de Castilla y León, Consejería de Sanidad (GRS 862/A/13 and BIO/SA05/14), Fundación Memoria de D. Samuel Solórzano Barruso of the University of Salamanca (FS/22-2015), Fundación Ramón Areces (FRA16/003), Sociedad Española de Hematología y Hemoterapia and Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León. E.M.O. was supported by an Inplant grant from IDIVAL. T.P. is supported by a grant from AECC (INVES18043PAÍN)

Synergistic DNA-damaging effect in multiple myeloma with the combination of zalypsis, bor tezomib and dexamethasone

Despite new advances in multiple myeloma treatment and the consequent improvement in overall survival, most patients relapse or become refractory to treatment. This suggests that new molecules and combinations that may further inhibit important survival pathways for these tumor cells are needed. In this context, zalypsis is a novel compound, derived from marine organisms, with a powerful preclinical anti-myeloma effect based on the sensitivity of malignant plasma cells to DNA-damage induction; and it has already been tested in a phase I/II clinical trial in multiple myeloma. We hypothesized that the addition of this compound to the combination of bortezomib plus dexamethasone may improve efficacy with acceptable toxicity. The triple combination demonstrated strong synergy and higher efficacy compared with double combinations; not only in vitro, but also ex vivo and, especially, in in vivo experiments. The triple combination triggers cell death, mainly through a synergistic induction of DNA damage and a decrease in the nuclear localization of nuclear factor kappa B. Our findings support the clinical evaluation of this combination for relapsed and refractory myeloma patients.This work was in part funded by the Spanish ISCIII-FIS (PI 15/0067 and PI15/02156) and FEDER, the Spanish RTICC (RD12/0036/0058), "Asociación Española Contra el Cancer" (AECC, GCB120981SAN), the regional Council from “Castilla y León” (GRS 1175/A/15 and FIC335U14) and a research grant from Pharmamar SAU. MMS were also supported by the Network of Centers for Regenerative Medicine and Cellular Therapy from Castilla y León, Spain. A-A López-Iglesias was supported by a grant from the Spanish Society of Hematology and Hemotherapy.Peer Reviewe

In vivo murine model of acquired resistance in myeloma reveals differential mechanisms for lenalidomide and pomalidomide in combination with dexamethasone

The development of resistance to therapy is unavoidable in the history of multiple myeloma patients. Therefore, the study of its characteristics and mechanisms is critical in the search for novel therapeutic approaches to overcome it. This effort is hampered by the absence of appropriate preclinical models, especially those mimicking acquired resistance. Here we present an in vivo model of acquired resistance based on the continuous treatment of mice bearing subcutaneous MM1S plasmacytomas. Xenografts acquired resistance to two generations of immunomodulatory drugs (IMiDs; lenalidomide and pomalidomide) in combination with dexamethasone, that was reversible after a wash-out period. Furthermore, lenalidomide-dexamethasone (LD) or pomalidomide-dexamethasone (PD) did not display cross-resistance, which could be due to the differential requirements of the key target Cereblon and its substrates Aiolos and Ikaros observed in cells resistant to each combination. Differential gene expression profiles of LD and PD could also explain the absence of cross-resistance. Onset of resistance to both combinations was accompanied by upregulation of the mitogen-activated protein kinaseextracellular signal-regulated kinase (ERK) kinase (MEK)ERK pathway and addition of selumetinib, a small-molecule MEK inhibitor, could resensitize resistant cells. Our results provide insights into the mechanisms of acquired resistance to LD and PD combinations and offer possible therapeutic approaches to addressing IMiD resistance in the clinic.Peer Reviewe