Doctor of Philosophy, Universidad De Barcelona (2012)
Master of Science, Universidad De Barcelona (2009)
Master of Science, University of Havana (2007)
Bachelor of Science, University of Havana (2003)
Research in xenotransplantation implies a high experimental complexity comprising molecular, cellular, and in vivo studies to investigate the mechanisms of xenograft immune rejection and functional failure, as well as the strategies to counteract them. After major advances associated with the identification of the carbohydrate xenoantigens and their elimination through genomic edition of the source pigs, the study of the cellular immune response against the xenograft is gaining particular attention. Xenogeneic cell-based assays that put together pig cells and human leukocytes such as monocytes, NK cells, and T cells are relevant to address this hurdle. Thus, we describe here coculture, co-stimulatory, and cytotoxicity assays for investigating the cellular and molecular mechanisms of xenograft rejection. These techniques allow elucidating the key pathways that take place during the xenogeneic immune response in a simplified setting. Treatment with either pro-inflammatory or anti-inflammatory cytokines can be used for studying the regulation of adhesion, co-stimulatory molecules, and receptors involved in triggering the immune response under various conditions. Furthermore, these assays can be used for the follow-up of the immune response of in vivo studies as well as for the development of tolerogenic approaches that promote xenograft survival.
View details for DOI 10.1007/978-1-0716-0255-3_7
View details for PubMedID 32002904
Current treatment options are not providing an adequate solution for cartilage defects. Articular cartilage lesions in particular are not able to repair spontaneously and progressively degenerate with an arthrosic pattern. Aiming to solve this pressing medical need, xenotransplantation of porcine chondrocytes could be developed as a new therapeutic approach. Xenotransplantation is gaining much attention, thanks to the advances in animal genetic engineering and progress in the characterization of the rejection mechanisms that prevent long-term graft survival. In this regard, our team has identified various targets for intervention that should be tested in a meaningful animal model to prove their relevance in rejection of xenogeneic cartilage. To this end, we have recently established a discordant xenotransplantation model by injecting three million porcine articular chondrocytes (PAC) into the femorotibial joint of Lewis rats. This chapter describes this new model, which can be used to assess the immunoregulatory effect of a variety of strategies designed to inhibit rejection of xenogeneic PAC both at the humoral and cellular levels.
View details for DOI 10.1007/978-1-0716-0255-3_17
View details for PubMedID 32002914
Acute graft-versus-host disease (GVHD) is a leading cause of mortality after allogeneic hematopoietic cell transplantation (HCT) mediated by dysregulated T-cell immune reconstitution. Given the role of the T-cell immunoglobulin and mucin 1 (TIM-1) surface protein in many immune processes, including organ transplantation tolerance, we asked if TIM-1 might drive post-transplant inflammation and acute GVHD. TIM-1 binds to phosphatidylserine (PtdSer), and agonism of TIM1 on immune cells is proinflammatory. HCT conditioning results in a significant supply of PtdSer from apoptosis and cellular debris. Using murine models, treatment with an antagonistic anti-TIM-1 monoclonal antibody (mAb) protects against acute GVHD while maintaining graft-versus-tumor effects. In contrast, the addition of exogenous free PtdSer worsened GVHD in a TIM-1-dependent manner. Importantly, TIM-1 blockade did not alter the expansion of donor T cells in vitro or in vivo. Instead, TIM-1 blockade reduces proinflammatory cytokines and promotes anti-inflammatory factors like carbonic anhydrase 1 and serum amyloid A1 in the gut tissue. This is mediated by TIM-1 on donor cells, as HCT of wild-type (WT) bone marrow (BM) and conventional T (Tcon) cells into TIM-1-/- knockout (KO) recipient mice showed little survival advantage compared with WT recipients, whereas WT recipients of TIM-1-/- KO Tcon cells or TIM1-/- KO BM had improved survival, in part due to the expression of TIM-1 on donor invariant natural killer T cells, which drives inflammation. Finally, in a humanized mouse xenograft GVHD model, treatment with anti-human TIM-1 antagonist mAb reduced GVHD disease burden and mortality. This supports TIM-1 as important for GVHD pathogenesis and as a target for the prevention of GVHD.
View details for DOI 10.1182/bloodadvances.2019000286
View details for PubMedID 31714958
Mesenchymal stem cells (MSCs) have been used as a therapy to modulate diverse biological processes. To fulfill the requirements for different MSC therapies, safe and effective gene transfer methods for MSCs are critical. Calcium phosphate transfection is an inexpensive and well-described method without discernible biosafety issues; however, an optimal protocol has not been developed for MSCs. In this report, we optimized the protocol of calcium phosphate transfection for murine MSCs, and compared this protocol with other gene transfer methods in different strains of mice and in human cells. We found that transfection efficiency and cell viability showed an inverse relationship depending on serum concentration during the process of calcium phosphate transfection, in which 2% serum was chosen in the optimized protocol. The optimized protocol of calcium phosphate transfection showed a fine balance between efficiency (about 70-80%) and viability (doubling original cell number) compared to other methods. Human MSCs were more resistant to this protocol (about 30% efficiency) compared with murine MSCs. Moreover, MSC potential for osteogenesis, adipogenesis, and chondrogenesis was not affected by calcium phosphate transfection. Finally, MSCs transfected with the luciferase gene were injected into the murine distal femoral bone marrow cavity to monitor gene expression overtime in vivo. MSCs in the bone marrow environment showed extended expression of the luciferase that was transfected by calcium phosphate. This report provides an optimized protocol for calcium phosphate transfection for murine MSCs and characterizes gene over-expression in MSCs in the in vitro and in vivo environments.
View details for DOI 10.1089/ten.TEC.2019.0147
View details for PubMedID 31441373
Selective depletion of natural anti-Galα1-3Galβ1-4GlcNAc (so-called anti-αGal) antibodies is achieved in α1,3-galactosyltransferase knockout (Gal-KO) mice by administration of the soluble glycoconjugate of αGal GAS914. This molecule removed up to 90% of natural circulating anti-αGal antibodies without causing unspecific production of cytokines in wild-type (CBA) and Gal-KO mice. However, the removal of anti-αGal antibodies in Gal-KO mice with GAS914 in the context of sepsis after cecal ligation and puncture (CLP) was associated with a significant increase in the production of leptin, CXLC1, CXLC13, and TIMP-1 cytokines compared to vehicle (PBS)-treated controls. Despite the current lack of understanding of the underlying mechanism, our data suggest a putative role of natural anti-αGal antibodies in the regulation of some cytokines during sepsis.
View details for DOI 10.1134/S0006297917020122
View details for Web of Science ID 000395066100012
View details for PubMedID 28320304
Chronic obstructive pulmonary disease is a major health problem becoming a leading cause of morbidity and mortality worldwide. A large part of these disorders is associated with acute exacerbations resulting from infection by bacteria, such as non-typeable Haemophilus influenzae (NTHi). Our understanding of the pathogenesis of these exacerbations is still elusive. We demonstrate herein that NTHi infection of mice chronically exposed to cigarette smoke (CS), an experimental model of chronic obstructive pulmonary disease (COPD), not only causes acute pulmonary inflammation but also impairs the production of interleukin (IL)-22, a cytokine with potential anti-bacterial activities. We also report that mice lacking IL-22, as well as mice exposed to CS, have a delayed clearance of NTHi bacteria and display enhanced alveolar wall thickening and airway remodeling compared with controls. Supplementation with IL-22 not only boosted bacterial clearance and the production of anti-microbial peptides but also limited lung damages induced by infection both in IL-22(-/-) and CS-exposed mice. In vitro exposure to CS extract altered the NTHi-induced IL-22 production by spleen cells. This study shows for the first time that a defect in IL-22 is involved in the acute exacerbation induced by NTHi infection during experimental COPD and opens the way to innovative therapeutic strategies.
View details for DOI 10.1038/mi.2016.40
View details for Web of Science ID 000395807400014
View details for PubMedID 27143304
Xenogeneic chondrocytes and allogeneic mesenchymal stem cells (MSC) are considered a potential source of cells for articular cartilage repair. We here assessed the immune response triggered by xenogeneic chondrocytes when injected intraarticularly, as well as the immunoregulatory effect of allogeneic bone marrow-derived MSC after systemic administration. To this end, a discordant xenotransplantation model was established by injecting three million porcine articular chondrocytes (PAC) into the femorotibial joint of Lewis rats and monitoring the immune response. First, the fate of MSC injected using various routes was monitored in an in vivo imaging system. The biodistribution revealed a dependency on the injection route with MSC injected intravenously (i.v.) succumbing early after 24 h and MSC injected intraperitoneally (i.p.) lasting locally for at least 5 days. Importantly, no migration of MSC to the joint was detected in rats previously injected with PAC. MSC were then administered either i.v. 1 week before PAC injection or i.p. 3 weeks after to assess their immunomodulatory function on humoral and adaptive immune parameters. Anti-PAC IgM and IgG responses were detected in all PAC-injected rats with a peak at week 2 postinjection and reactivity remaining above baseline levels by week 18. IgG2a and IgG2b were the predominant and long-lasting IgG subtypes. By contrast, no anti-MSC antibody response was detected in the cohort injected with MSC only, but infusion of MSC before PAC injection temporarily augmented the anti-PAC antibody response. Consistent with a cellular immune response to PAC in PAC-injected rats, cytokine/chemokine profiling in serum by antibody array revealed a distinct pattern relative to controls characterized by elevation of multiple markers at week 2, as well as increases in proliferation in draining lymph nodes. Notably, systemic administration of allogeneic MSC under the described conditions did not diminish the immune response. IL-2 measurements in cocultures of rat peripheral blood lymphocytes with PAC indicated that PAC injection induced some T-cell hyporesponsiveness that was not enhanced in the cohorts additionally receiving MSC. Thus, PAC injected intraarticularly in Lewis rats induced a cellular and humoral immune response that was not counteracted by the systemic administration of allogeneic MSC under the described conditions.
View details for DOI 10.3389/fimmu.2017.01465
View details for PubMedID 29163532
View details for PubMedCentralID PMC5681521
Cellular therapies based on permanent genetic modification of conventional T cells have emerged as a promising strategy for cancer. However, it remains unknown if modification of T cell subsets, such as Tregs, could be useful in other settings, such as allograft transplantation. Here, we use a modular system based on a chimeric antigen receptor (CAR) that binds covalently modified mAbs to control Treg activation in vivo. Transient expression of this mAb-directed CAR (mAbCAR) in Tregs permitted Treg targeting to specific tissue sites and mitigated allograft responses, such as graft-versus-host disease. mAbCAR Tregs targeted to MHC class I proteins on allografts prolonged islet allograft survival and also prolonged the survival of secondary skin grafts specifically matched to the original islet allograft. Thus, transient genetic modification to produce mAbCAR T cells led to durable immune modulation, suggesting therapeutic targeting strategies for controlling alloreactivity in settings such as organ or tissue transplantation.
View details for PubMedID 29046484
The IL-22 signaling pathway is critical for regulating mucosal defense and limiting bacterial dissemination. IL-22 is unusual among interleukins because it does not directly regulate the function of conventional immune cells, but instead targets cells at outer body barriers, such as respiratory epithelial cells. Consequently, IL-22 signaling participates in the maintenance of the lung mucosal barrier by controlling cell proliferation and tissue repair, and enhancing the production of specific chemokines and anti-microbial peptides. Pseudomonas aeruginosa is a major pathogen of ventilator-associated pneumonia and causes considerable lung tissue damage. A feature underlying the pathogenicity of this bacterium is its capacity to persist and develop in the host, particularly in the clinical context of nosocomial lung infections. We aimed to investigate the ability of P. auruginosa to disrupt immune-epithelial cells cross-talk. We found that P. aeruginosa escapes the host mucosal defenses by degrading IL-22, leading to severe inhibition of IL-22-mediated immune responses. We demonstrated in vitro that, protease IV, a type 2 secretion system-dependent serine protease, is responsible for the degradation of IL-22 by P. aeruginosa. Moreover, the major anti-proteases molecules present in the lungs were unable to inhibit protease IV enzymatic activity. In addition, tracheal aspirates of patients infected by P. aeruginosa contain protease IV activity which further results in IL-22 degradation. This so far undescribed cleavage of IL-22 by a bacterial protease is likely to be an immune-evasion strategy that contributes to P. aeruginosa-triggered respiratory infections.
View details for DOI 10.1080/21505594.2016.1253658
View details for PubMedID 27792459
View details for PubMedCentralID PMC5626239
Progression of chronic obstructive pulmonary disease (COPD) is linked to episodes of exacerbations caused by bacterial infections due to Streptococcus pneumoniae. Our objective was to identify during COPD, factors of susceptibility to bacterial infections among cytokine network and their role in COPD exacerbations. S. pneumoniae was used to sub-lethally challenge mice chronically exposed to air or cigarette smoke (CS) and to stimulate peripheral blood mononuclear cells (PBMC) from non-smokers, smokers and COPD patients. The immune response and the cytokine production were evaluated. Delayed clearance of the bacteria and stronger lung inflammation observed in infected CS-exposed mice were associated with an altered production of IL-17 and IL-22 by innate immune cells. This defect was related to a reduced production of IL-1β and IL-23 by antigen presenting cells. Importantly, supplementation with recombinant IL-22 restored bacterial clearance in CS-exposed mice and limited lung alteration. In contrast with non-smokers, blood NK and NKT cells from COPD patients failed to increase IL-17 and IL-22 levels in response to S. pneumoniae, in association with a defect in IL-1β and IL-23 secretion. This study identified IL-17 and IL-22 as susceptibility factors in COPD exacerbation. Therefore targeting such cytokines could represent a potent strategy to control COPD exacerbation.
View details for DOI 10.1016/j.ebiom.2015.09.040
View details for PubMedID 26870795
View details for PubMedCentralID PMC4740310
Vector-borne diseases (VBD) challenge our understanding of emerging diseases. Recently, arthropod vectors have been involved in emerging anaphylactic diseases. In particular, the immunoglobulin E (IgE) antibody response to the carbohydrate Galα1-3Galβ1-(3)4GlcNAc-R (α-gal) following a tick bite was associated with allergies to red meat, cetuximab, and gelatin. By contrast, an anti-α-gal IgM antibody response was shown to protect against mosquito-borne malaria. Herein, we highlight the interplay between the gut microbiota, vectors, transmitted pathogens, and the regulation of the immune response as a model to understand the protective or allergic effect of α-gal. Establishing the source of α-gal in arthropod vectors and the immune response to vector bites and transmitted pathogens will be essential for diagnosing, treating, and ultimately preventing these emerging anaphylactic and other vector-borne diseases.
View details for DOI 10.1016/j.pt.2015.06.016
View details for PubMedID 26433250
Advances in animal transgenesis may allow using xenogeneic chondrocytes in tissue-engineering applications for clinical cartilage repair. Porcine cartilage is rejected by humoral and cellular mechanisms that could be overcome by identifying key molecules triggering rejection and developing effective genetic-engineering strategies. Accordingly, high expression of α1,2-fucosyltransferase (HT) in xenogeneic cartilage protects from galactose α1,3-galactose (Gal)-mediated antibody responses. Now, we studied whether expression of a complement inhibitor provides further protection. First, porcine articular chondrocytes (PAC) were isolated from non-transgenic, single and double transgenic pigs expressing HT and moderate levels of human CD59 (hCD59) and their response to human serum was assessed. High recombinant expression of human complement regulatory molecules hCD59 and hDAF was also attained by retroviral transduction of PAC for further analyses. Complement activation on PAC after exposure to 20 % human serum for 24 hours mainly triggered the release of pro-inflammatory cytokines IL-6 and IL-8. Transgenic expression of HT and hCD59 did not suffice to fully counteract this effect. Nevertheless, the combination of blocking anti-Gal antibodies (or C5a) and high hCD59 levels conferred very high protection. On the contrary, high hDAF expression attained the most dramatic reduction in IL-6/IL-8 secretion by a single strategy, but the additional inhibition of anti-Gal antibodies or C5a did not provide further improvement. Notably, we demonstrate that both hCD59 and hDAF inhibit anaphylatoxin release in this setting. In conclusion, our study identifies genetic-engineering approaches to prevent humoral rejection of xenogeneic chondrocytes for use in cartilage repair.
View details for Web of Science ID 000366141600018
View details for PubMedID 26579969
We have previously described that boosted natural xenoantibodies in rats cross-react to bacteria by targeting carbohydrate antigens. This type of immunization is associated with reduced survival after cecal ligation and puncture (CLP). In the present study, we investigated further this phenomenon by immunizing Lewis rats with three intraperitoneal injections, every other day, of hamster blood compared to saline-injected control animals. One day after the last injection, CLP was performed to produce a low-grade sepsis. Induction of xenoantibodies was associated with a reduction in animal survival after CLP relative to controls (45% vs. 90%, p<0.01). No bacterial blood load was observed after CLP in this model either with or without xenoantibody enhancement, indicating that the augmented mortality was not mediated by a direct effect of boosted xenoantibodies over blood bacteria. Nevertheless, the xenoimmunization produced a systemic inflammatory response in all rats. Additionally, a lack of weight gain at the time of CLP was present in animals that died after the procedure, which was not observed in surviving rats and controls. The cytokine profile at the time of CLP in animals that died after the procedure was characterized by an increase in the serum level of several cytokines, particularly adipokines. In contrast, the cytokine profile at CLP of xenoimmunized rats that survived the procedure was characterized by a reduction in the level of cytokines. In conclusion, this study failed to show a direct effect of boosted xenoantibodies over blood bacterial isolates as cause for the decreased survival after CLP. However, it evidenced that non-infectious systemic inflammation may lead to a pattern of augmented cytokines, particularly adipokines, which impairs survival after subsequent CLP. Therefore, the profile of cytokines existing before the infectious insult appears more crucial than that resulting from the condition for the outcome of sepsis.
View details for DOI 10.1371/journal.pone.0125472
View details for Web of Science ID 000354917300017
View details for PubMedID 25984763
View details for PubMedCentralID PMC4436005
Natural antibodies include a subset described as xenoantibodies considered to be directed at microorganisms and also cross-react with antigens of unrelated species. In this study, we generated T-cell-independent (TI) and T-cell-dependent (TD) xenoantibodies in Lewis rats with hamster and pig blood injections. TI anti-hamster and anti-pig IgM and IgG xenoantibodies cross-reacted with Enterococcus faecalis but not with Escherichia coli isolated from the blood of Lewis rats after cecal ligation and puncture (CLP). TI anti-pig IgM xenoantibodies also showed some reactivity with two human blood isolates of E. faecalis. In contrast, TD xenoantibodies did not show any reactivity with rat or human bacteria. TI and TD anti-hamster and anti-pig IgM and IgG xenoantibodies showed cross-reactivity with lymphocytes and endothelial cells from species distinct to that used for immunization. Glycan array analysis and inhibition assays identified antibodies against melibiose and L-rhamnose as mediators of anti-hamster and anti-porcine xenoantibody cross-reactivity with E. faecalis. A rise in TI anti-hamster and anti-pig xenoantibodies was accompanied by decreased survival of Lewis rats in a low-severity sepsis model of CLP. Therefore, TI xenoantibodies in the rat include anti-carbohydrate antibodies reactive to bacteria of endogenous flora. Enhancement of these antibodies may result in more severe infectious diseases caused by these microorganisms.
View details for DOI 10.1159/000355305
View details for Web of Science ID 000331774900004
View details for PubMedID 24246417
Tissue-based xenografts such as cartilage are rejected within weeks by humoral and cellular mechanisms that preclude its clinical application in regenerative medicine. The problem could be overcome by identifying key molecules triggering rejection and the development of genetic-engineering strategies to counteract them. Accordingly, high expression of α1,2-fucosyltransferase (HT) in xenogeneic cartilage reduces the galactose α1,3-galactose (Gal) antigen and delays rejection. Yet, the role of complement activation in this setting is unknown.To determine its contribution, we assessed the effect of inhibiting C5 complement component in α1,3-galactosyltransferase-knockout (Gal KO) mice transplanted with porcine cartilage and studied the effect of human complement on porcine articular chondrocytes (PAC).Treatment with an anti-mouse C5 blocking antibody for 5 weeks enhanced graft survival by reducing cellular rejection. Moreover, PAC were highly resistant to complement-mediated lysis and primarily responded to human complement by releasing IL-6 and IL-8. This occurred even in the absence of anti-Gal antibody and was mediated by both C5a and C5b-9. Indeed, C5a directly triggered IL-6 and IL-8 secretion and up-regulated expression of swine leukocyte antigen I (SLA-I) and adhesion molecules on chondrocytes, all processes that enhance cellular rejection. Finally, the use of anti-human C5/C5a antibodies and/or recombinant expression of human complement regulatory molecule CD59 (hCD59) conferred protection in correspondence with their specific functions.Our study demonstrates that complement activation contributes to rejection of xenogeneic cartilage and provides valuable information for selecting approaches for complement inhibition.
View details for DOI 10.1016/j.joca.2013.09.002
View details for Web of Science ID 000329266700017
View details for PubMedID 24041966
Cellular studies are essential in the xenotransplantation field in order to investigate the cellular immune responses triggered by xenogeneic cells and identify the key molecules involved. A series of functional studies can be conducted with this purpose that include treatment with proinflammatory cytokines and xenogeneic cell-based assays that put together pig cells and human leukocytes such as monocytes, NK cells, and T cells. The choice of the pig cell type is critical to appropriately model the transplant setting of interest. Thus, pig endothelial cells are commonly used for studying the rejection process of vascularized organs. Treatment with cytokines allows studying the regulation of adhesion, costimulatory molecules, and receptors involved in triggering the immune response in an attempt to reproduce the more complex in vivo situation. The adhesion assays are used to determine the capacity of human leukocytes to adhere to porcine cells under various conditions. Furthermore, we describe coculture, costimulatory, and cytotoxicity assays for investigating the cellular and molecular mechanisms that take place during the xenogeneic immune response.
View details for DOI 10.1007/978-1-61779-845-0_7
View details for PubMedID 22565992
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