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1.
Cell Mol Life Sci ; 81(1): 163, 2024 Apr 04.
Article in English | MEDLINE | ID: mdl-38570362

ABSTRACT

Proteolytic release of transmembrane proteins from the cell surface, the so called ectodomain shedding, is a key process in inflammation. Inactive rhomboid 2 (iRhom2) plays a crucial role in this context, in that it guides maturation and function of the sheddase ADAM17 (a disintegrin and metalloproteinase 17) in immune cells, and, ultimately, its ability to release inflammatory mediators such as tumor necrosis factor α (TNFα). Yet, the macrophage sheddome of iRhom2/ADAM17, which is the collection of substrates that are released by the proteolytic complex, is only partly known. In this study, we applied high-resolution proteomics to murine and human iRhom2-deficient macrophages for a systematic identification of substrates, and therefore functions, of the iRhom2/ADAM17 proteolytic complex. We found that iRhom2 loss suppressed the release of a group of transmembrane proteins, including known (e.g. CSF1R) and putative novel ADAM17 substrates. In the latter group, shedding of major histocompatibility complex class I molecules (MHC-I) was consistently reduced in both murine and human macrophages when iRhom2 was ablated. Intriguingly, it emerged that in addition to its shedding, iRhom2 could also control surface expression of MHC-I by an undefined mechanism. We have demonstrated the biological significance of this process by using an in vitro model of CD8+ T-cell (CTL) activation. In this model, iRhom2 loss and consequent reduction of MHC-I expression on the cell surface of an Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line dampened activation of autologous CTLs and their cell-mediated cytotoxicity. Taken together, this study uncovers a new role for iRhom2 in controlling cell surface levels of MHC-I by a dual mechanism that involves regulation of their surface expression and ectodomain shedding.


Subject(s)
Carrier Proteins , Epstein-Barr Virus Infections , Animals , Humans , Mice , ADAM17 Protein/genetics , ADAM17 Protein/metabolism , Carrier Proteins/metabolism , Herpesvirus 4, Human , Major Histocompatibility Complex , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice, Knockout
2.
Int J Mol Sci ; 25(5)2024 Feb 28.
Article in English | MEDLINE | ID: mdl-38474048

ABSTRACT

Spontaneous bacterial peritonitis (SBP) is a severe complication in patients with decompensated liver cirrhosis and is commonly treated with broad spectrum antibiotics. However, the rise of antibiotic resistance requires alternative therapeutic strategies. As recently shown, human amnion-derived mesenchymal stem cells (hA-MSCs) are able, in vitro, to promote bacterial clearance and modulate the immune and inflammatory response in SBP. Our results highlight the upregulation of FOXO1, CXCL5, CXCL6, CCL20, and MAPK13 in hA-MSCs as well as the promotion of bacterial clearance, prompting a shift in the immune response toward a Th17 lymphocyte phenotype after 72 h treatment. In this study, we used an in vitro SBP model and employed omics techniques (next-generation sequencing) to investigate the mechanisms by which hA-MSCs modify the crosstalk between immune cells in LPS-stimulated ascitic fluid. We also validated the data obtained via qRT-PCR, cytofluorimetric analysis, and Luminex assay. These findings provide further support to the hope of using hA-MSCs for the prevention and treatment of infective diseases, such as SBP, offering a viable alternative to antibiotic therapy.


Subject(s)
Bacterial Infections , Peritonitis , Humans , Ascites , Lipopolysaccharides , Amnion , Liver Cirrhosis/complications , Ascitic Fluid/microbiology , Anti-Bacterial Agents/therapeutic use , Peritonitis/drug therapy , Bacterial Infections/microbiology , Forkhead Box Protein O1
3.
Langmuir ; 38(11): 3403-3411, 2022 03 22.
Article in English | MEDLINE | ID: mdl-35262354

ABSTRACT

Biomolecule-targeted imaging represents one of the most difficult challenges in medicine. Nanoerythrosomes (NERs) are nanovesicles obtained after lysis of red blood cells, and they are promising tools for drug delivery and imaging. In this work, a formulation based on NERs functionalized with 7-amino-3-methylcoumarin via cross-linking was tested on rat INS-1E and mouse MIN6 ß-cells and endothelial MSI cell lines. First, the morphology, size, ζ-potentials, and spectroscopic properties of the aggregates were investigated, highlighting that the functionalization did not significantly affect the nanoparticles' physicochemical features. In vitro, the nanoparticles did not significantly affect the proliferation and function of INS-1E and MIN6 ß-cells at different concentrations. Only at the highest concentration tested on the MSI cell line, the formulation inhibited proliferation. Furthermore, NER aggregates were not internalized in both INS-1E and MIN6 cell lines, while a diffuse fluorescence was noticed in the cytosol of the MSI cell line at the highest concentrations. These findings proved that NER formulations might represent a new nanotool for ß-cell imaging as a part of a strategy aimed to prevent any intracellular accumulation, thus reducing/avoiding side effects.


Subject(s)
Endothelial Cells , Insulin-Secreting Cells , Animals , Biological Transport , Cell Line , Endothelial Cells/metabolism , Insulin/metabolism , Insulin-Secreting Cells/metabolism , Mice , Rats
4.
Int J Mol Sci ; 23(2)2022 Jan 13.
Article in English | MEDLINE | ID: mdl-35055040

ABSTRACT

BACKGROUND: Spontaneous bacterial peritonitis (SBP) is a severe and often fatal infection in patients with decompensated cirrhosis and ascites. The only cure for SBP is antibiotic therapy, but the emerging problem of bacterial resistance requires novel therapeutic strategies. Human amniotic mesenchymal stromal cells (hA-MSCs) possess immunomodulatory and anti-inflammatory properties that can be harnessed as a therapy in such a context. METHODS: An in vitro applications of hA-MSCs in ascitic fluid (AF) of cirrhotic patients, subsequently infected with carbapenem-resistant Enterobacterales, was performed. We evaluated the effects of hA-MSCs on bacterial load, innate immunity factors, and macrophage phenotypic expression. RESULTS: hA-MSCs added to AF significantly reduce the proliferation of both bacterial strains at 24 h and diversely affect M1 and M2 polarization, C3a complement protein, and ficolin 3 concentrations during the course of infection, in a bacterial strain-dependent fashion. CONCLUSION: This study shows the potential usefulness of hA-MSC in treating ascites infected with carbapenem-resistant bacteria and lays the foundation to further investigate antibacterial and anti-inflammatory roles of hA-MSC in in vivo models.


Subject(s)
Amnion/cytology , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/metabolism , Peritoneal Fibrosis/etiology , Peritoneal Fibrosis/therapy , Bacterial Load , Biomarkers , Carbapenems/pharmacology , Complement Activation/immunology , Complement System Proteins/immunology , Complement System Proteins/metabolism , Disease Susceptibility , Enterobacter/drug effects , Enterobacter/genetics , Enterobacteriaceae Infections/complications , Enterobacteriaceae Infections/microbiology , Humans , Immunomodulation , Inflammation Mediators , Macrophages , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/cytology , Peritoneal Fibrosis/metabolism , Peritonitis/complications , Peritonitis/microbiology , Phagocytosis , Receptors, Pattern Recognition/metabolism , Treatment Outcome , beta-Lactam Resistance
5.
Int J Mol Sci ; 23(23)2022 Nov 30.
Article in English | MEDLINE | ID: mdl-36499373

ABSTRACT

At present, there is a lack of clinical evidence about the impact and long-term durability of the immune response induced by the third dose of mRNA vaccines. In this study, we followed up the B cell compartment behavior in a cohort of immunocompetent individuals three and six months after the third dose of vaccine. During this period, some subjects contracted the virus. In uninfected vaccinated subjects, we did not report any changes in serum spike-specific IgG levels, with a significant reduction in IgA. Instead, subjects recovered from natural infection showed a significant increase in both specific IgG and IgA. Moreover, we showed a time-related decrease in IgG neutralizing potential to all SARS-CoV-2 variants of concern (VOC) in uninfected compared to recovered subjects, who displayed an increased neutralizing ability, particularly against the omicron variant. Finally, we underlined the presence of a pool of SARS-CoV-2-specific B cells in both groups that are prone to respond to restimulation, as demonstrated by their ability to differentiate into plasma cells and to produce anti-SARS-CoV-2-specific immunoglobulins. These data lead us to assert the long-term effectiveness of the BNT162b2 vaccine in contrasting the severe form of the pathology and prevent COVID-19-associated hospitalization.


Subject(s)
COVID-19 , Memory B Cells , Humans , SARS-CoV-2 , BNT162 Vaccine , COVID-19/prevention & control , RNA, Messenger/genetics , Immunoglobulin G , Antibodies, Viral
6.
Int J Mol Sci ; 23(2)2022 Jan 13.
Article in English | MEDLINE | ID: mdl-35055049

ABSTRACT

Mesenchymal stromal/stem cells (MSCs) are believed to function in vivo as a homeostatic tool that shows therapeutic properties for tissue repair/regeneration. Conventionally, these cells are expanded in two-dimensional (2D) cultures, and, in that case, MSCs undergo genotypic/phenotypic changes resulting in a loss of their therapeutic capabilities. Moreover, several clinical trials using MSCs have shown controversial results with moderate/insufficient therapeutic responses. Different priming methods were tested to improve MSC effects, and three-dimensional (3D) culturing techniques were also examined. MSC spheroids display increased therapeutic properties, and, in this context, it is crucial to understand molecular changes underlying spheroid generation. To address these limitations, we performed RNA-seq on human amnion-derived MSCs (hAMSCs) cultured in both 2D and 3D conditions and examined the transcriptome changes associated with hAMSC spheroid formation. We found a large number of 3D culture-sensitive genes and identified selected genes related to 3D hAMSC therapeutic effects. In particular, we observed that these genes can regulate proliferation/differentiation, as well as immunomodulatory and angiogenic processes. We validated RNA-seq results by qRT-PCR and methylome analysis and investigation of secreted factors. Overall, our results showed that hAMSC spheroid culture represents a promising approach to cell-based therapy that could significantly impact hAMSC application in the field of regenerative medicine.


Subject(s)
Amnion/cytology , Mesenchymal Stem Cells/metabolism , Transcriptome , Biomarkers , Cell Culture Techniques , Cell Differentiation , Cell Separation , Cells, Cultured , Computational Biology/methods , Epigenesis, Genetic , Gene Expression Profiling , Gene Expression Regulation , Gene Ontology , Humans , Immunophenotyping , Mesenchymal Stem Cells/cytology , Molecular Sequence Annotation , Regenerative Medicine
7.
Int J Mol Sci ; 22(2)2021 Jan 06.
Article in English | MEDLINE | ID: mdl-33419219

ABSTRACT

The clinical results of lung transplantation (LTx) are still less favorable than other solid organ transplants in both the early and long term. The fragility of the lungs limits the procurement rate and can favor the occurrence of ischemia-reperfusion injury (IRI). Ex vivo lung perfusion (EVLP) with Steen SolutionTM (SS) aims to address problems, and the implementation of EVLP to alleviate the activation of IRI-mediated processes has been achieved using mesenchymal stromal/stem cell (MSC)-based treatments. In this study, we investigated the paracrine effects of human amnion-derived MSCs (hAMSCs) in an in vitro model of lung IRI that includes cold ischemia and normothermic EVLP. We found that SS enriched by a hAMSC-conditioned medium (hAMSC-CM) preserved the viability and delayed the apoptosis of alveolar epithelial cells (A549) through the downregulation of inflammatory factors and the upregulation of antiapoptotic factors. These effects were more evident using the CM of 3D hAMSC cultures, which contained an increased amount of immunosuppressive and growth factors compared to both 2D cultures and encapsulated-hAMSCs. To conclude, we demonstrated an in vitro model of lung IRI and provided evidence that a hAMSC-CM attenuated IRI effects by improving the efficacy of EVLP, leading to strategies for a potential implementation of this technique.


Subject(s)
Alveolar Epithelial Cells/drug effects , Cold Ischemia/methods , Culture Media, Conditioned/pharmacology , Mesenchymal Stem Cells/drug effects , Reperfusion Injury/drug therapy , A549 Cells , Alveolar Epithelial Cells/metabolism , Amnion/cytology , Apoptosis/drug effects , Apoptosis/genetics , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Survival/drug effects , Cell Survival/genetics , Cells, Cultured , Cytokines/genetics , Gene Expression Regulation/drug effects , Humans , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , NF-kappa B/genetics , Reperfusion Injury/physiopathology
8.
Int J Mol Sci ; 22(4)2021 Feb 19.
Article in English | MEDLINE | ID: mdl-33669517

ABSTRACT

Despite low levels of vascular endothelial growth factor (VEGF)-A, the secretome of human Wharton's jelly (WJ) mesenchymal stromal cells (MSCs) effectively promoted proangiogenic responses in vitro, which were impaired upon the depletion of small (~140 nm) extracellular vesicles (EVs). The isolated EVs shared the low VEGF-A profile of the secretome and expressed five microRNAs, which were upregulated compared to fetal dermal MSC-derived EVs. These upregulated microRNAs exclusively targeted the VEGF-A gene within 54 Gene Ontology (GO) biological processes, 18 of which are associated with angiogenesis. Moreover, 15 microRNAs of WJ-MSC-derived EVs were highly expressed (Ct value ≤ 26) and exclusively targeted the thrombospondin 1 (THBS1) gene within 75 GO biological processes, 30 of which are associated with the regulation of tissue repair. The relationship between predicted microRNA target genes and WJ-MSC-derived EVs was shown by treating human umbilical-vein endothelial cells (HUVECs) with appropriate doses of EVs. The exposure of HUVECs to EVs for 72 h significantly enhanced the release of VEGF-A and THBS1 protein expression compared to untreated control cells. Finally, WJ-MSC-derived EVs stimulated in vitro tube formation along with the migration and proliferation of HUVECs. Our findings can contribute to a better understanding of the molecular mechanisms underlying the proangiogenic responses induced by human umbilical cord-derived MSCs, suggesting a key regulatory role for microRNAs delivered by EVs.


Subject(s)
Extracellular Vesicles/metabolism , Mesenchymal Stem Cells/metabolism , MicroRNAs/metabolism , Neovascularization, Physiologic , Vascular Endothelial Growth Factor A/metabolism , Wharton Jelly/cytology , Cell Movement , Cell Proliferation , Cell Separation , Fetus/cytology , Fluoresceins/metabolism , Gene Ontology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Immunophenotyping , MicroRNAs/genetics , Nanoparticles/chemistry , Reproducibility of Results , Skin/cytology , Succinimides/metabolism , Thrombospondin 1/metabolism , Umbilical Cord/cytology
9.
Biochem Biophys Res Commun ; 522(1): 171-176, 2020 01 29.
Article in English | MEDLINE | ID: mdl-31757423

ABSTRACT

Different approaches have been studied in both preclinical and clinical settings to develop cell-based therapies and/or engineered cell-based therapies to better integrate grafts with the host. In these techniques, much attention is addressed to the use of adult stem cells such as mesenchymal stem cells (MSCs), but identifying and obtaining sufficient numbers of therapeutic cells, and the right route of administration, is often a challenge. In this study, we tested the feasibility of encapsulating human amnion-derived MSCs (hAMSCs) in a semipermeable and biocompatible fiber as a new approach for regenerative medicine. Our data showed that hAMSCs aggregated in the device constitutes an effective system for enhancing, or at least for maintaining, the paracrine activity of these cells in order to better promote tissue regeneration in an immune isolated state. In our new experimental approach, the hAMSCs retained their therapeutic potential, as shown by both the production of specific immunomodulatory/angiogenic factors and immunomodulatory and angiogenic ability observed in vitro. Unlike cell infusion methods, the use of encapsulated-cells leads to minimally invasive approaches, avoiding a direct interaction with the host. Therefore, the potentiality of an allograft or xenograft without the need for immunosuppression, and the lack of tumorigenesis is very intriguing.


Subject(s)
Amnion/cytology , Cell Culture Techniques/instrumentation , Mesenchymal Stem Cells/cytology , Placenta/cytology , Angiogenesis Inducing Agents/metabolism , Biocompatible Materials/chemistry , Cell Adhesion , Cell Aggregation , Cell Culture Techniques/methods , Cell Movement , Cell Survival , Cells, Immobilized/cytology , Cells, Immobilized/metabolism , Female , Human Umbilical Vein Endothelial Cells , Humans , Immunologic Factors/metabolism , Mesenchymal Stem Cells/metabolism , Pregnancy
10.
Differentiation ; 105: 14-26, 2019.
Article in English | MEDLINE | ID: mdl-30553176

ABSTRACT

We isolated a population of proliferating cells from cultured human fetal hepatocytes of 16-22 weeks gestational age. The cells shared a similar phenotype to that of mesenchymal stromal cells (MSCs) according to the International Society for Cellular Therapy (ISCT), including plastic adherence, antigen expression profile, and in vitro multilineage differentiation potential. Fetal liver (FL)-MSCs expressed the albumin gene, and harbored a subpopulation of CK18+ cells (20-40%), which defined their hepatic origin. However, when subjected to in vitro hepatic differentiation, FL-MSCs did not acquire significant liver functions. Quantitative analysis of conditioned medium (CM) collected from cultured cells revealed the presence of growth factors and chemokines with potential liver regenerative properties, the most relevant of which (concentration ≥3000 pg/ml) were SDF-1 alpha, IL-6, MCP-1, IL-8, MIP-1 beta, VEGF-A, Gro-alpha, and HGF. Culturing of FL-MSCs as spheroids significantly enhanced the secretion of HGF and bFGF (approximately 5-fold) compared with culture monolayers. Moreover, CM assessed in vitro induced capillary-like organization and migration of human umbilical vein endothelial cells (HUVECs) and fibroblasts as target cells. Interestingly, exosomes isolated from CM induced similar cellular responses in vitro with high efficiency and in a dose-dependent manner. FL-MSCs underwent several in vitro subcultivations, and did not stimulate allogenic T-cell proliferation thus suggesting a low immunogenicity. Furthermore, 5-year cryopreservation did not affect cell viability (approximately 90% of viable post-thawed FL-MSCs). These observations support the feasibility of a cell bank establishment for allogenic transplantation. We concluded that FL-MSCs or they secreted factors may be a valid alternative to hepatocyte transplantation in liver cell-based therapies.


Subject(s)
Human Embryonic Stem Cells/metabolism , Liver Regeneration , Liver/cytology , Mesenchymal Stem Cells/metabolism , Cells, Cultured , Chemokines/metabolism , Culture Media, Conditioned/pharmacology , Human Umbilical Vein Endothelial Cells/drug effects , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Liver/embryology , Spheroids, Cellular/drug effects
11.
Biochem Biophys Res Commun ; 490(2): 472-479, 2017 08 19.
Article in English | MEDLINE | ID: mdl-28624456

ABSTRACT

Culturing fetal hepatocytes in high cell-density allowed stabilization of the hepatocyte phenotype up to 8 weeks, including the maintenance of liver-specific functions. On the other hand, when cultured at low cell-density, fetal hepatocytes underwent morphological modifications and acquired fibroblastic morphology. Since a switch from E-cadherin to vimentin expression accompanied these changes, we hypothesized the occurrence of epithelial-to-mesenchymal transition when fetal hepatocytes were cultured at low cell-density. Changes in gene expressionsuch as up-regulation of fibrosis-related geneswere also observed, suggesting that the low cell-density culture system promoted the acquisition of a profibrotic phenotype in cultured hepatocytes. The origin of fibrogenic cells in the liver is not well known, and the role of hepatocytes as a source of fibrogenic cells is controversial. Therefore, we hypothesized that hepatocytes undergoing epithelial-to-mesenchymal transition could have a central role in liver fibrosis as a source of fibrogenic cells. To conclude, the high cell-density culture system could be a useful model for in vitro studies requiring long-term cultures of hepatocytes, such as the development of pharmaceutical drugs and mechanisms of viral infections. The low cell-density culture system may provide additional insights into the origin of fibrogenic cells in the liver, thus contributing to the development of novel therapeutic approaches.


Subject(s)
Epithelial-Mesenchymal Transition , Hepatocytes/cytology , Hepatocytes/pathology , Liver Cirrhosis/pathology , Liver/pathology , Cell Count , Cell Culture Techniques/methods , Cells, Cultured , Humans , Liver/embryology
12.
Int J Pharm ; 653: 123904, 2024 Mar 25.
Article in English | MEDLINE | ID: mdl-38355074

ABSTRACT

An amine derivative of hyaluronic acid (HA) was crosslinked to obtain a 3D dried sponge. The sponge was subsequently rehydrated using secretome from human mesenchymal stromal cells (MSCs), resulting in the formation of a hydrogel. The release kinetics analysis demonstrated that the hydrogel effectively sustained secretome release, with 70% of the initially loaded wound-healing-associated cytokines being released over a 12-day period. Tuning the hydrogel properties through heparin crosslinking resulted in a biomaterial with a distinct mechanism of action. Specifically, the presence of heparin enhanced water uptake capacity of the hydrogel and increased its sensitivity to enzymatic degradation. Notably, the heparin crosslinking also led to a significant retention of cytokines within the hydrogel matrix. Overall, the secretome-rehydrated HA hydrogel holds promise as a versatile device for regenerative medicine applications: the non-heparinized hydrogel may function as a biomaterial with low reabsorption rates, sustaining the release of bioactive molecules contained in MSC secretome. In contrast, the heparinized hydrogel may serve as a depot of bioactive molecules with faster reabsorption rates. Given its patch-like characteristic, the HA-based hydrogel appears suitable as topical treatment for external organs, such as the skin.


Subject(s)
Hydrogels , Mesenchymal Stem Cells , Humans , Hydrogels/pharmacology , Hyaluronic Acid/pharmacology , Secretome , Mesenchymal Stem Cells/metabolism , Heparin , Biocompatible Materials/metabolism , Cytokines/metabolism
13.
Front Cell Dev Biol ; 12: 1385712, 2024.
Article in English | MEDLINE | ID: mdl-38882056

ABSTRACT

Mesenchymal stromal/stem cells (MSCs) are a heterogeneous population of multipotent cells that can be obtained from various tissues, such as dental pulp, adipose tissue, bone marrow and placenta. MSCs have gained importance in the field of regenerative medicine because of their promising role in cell therapy and their regulatory abilities in tissue repair and regeneration. However, a better characterization of these cells and their products is necessary to further potentiate their clinical application. In this study, we used unbiased high-resolution mass spectrometry-based proteomic analysis to investigate the impact of distinct priming strategies, such as hypoxia and IFN-γ treatment, on the composition and therapeutic functionality of the secretome produced by MSCs derived from the amniotic membrane of the human placenta (hAMSCs). Our investigation revealed that both types of priming improved the therapeutic efficacy of hAMSCs, and these improvements were related to the secretion of functional factors present in the conditioned medium (CM) and exosomes (EXOs), which play crucial roles in mediating the paracrine effects of MSCs. In particular, hypoxia was able to induce a pro-angiogenic, innate immune response-activating, and tissue-regenerative hAMSC phenotype, as highlighted by the elevated production of regulatory factors such as VEGFA, PDGFRB, ANGPTL4, ENG, GRO-γ, IL8, and GRO-α. IFN-γ priming, instead, led to an immunosuppressive profile in hAMSCs, as indicated by increased levels of TGFB1, ANXA1, THBS1, HOMER2, GRN, TOLLIP and MCP-1. Functional assays validated the increased angiogenic properties of hypoxic hAMSCs and the enhanced immunosuppressive activity of IFN-γ-treated hAMSCs. This study extends beyond the direct priming effects on hAMSCs, demonstrating that hypoxia and IFN-γ can influence the functional characteristics of hAMSC-derived secretomes, which, in turn, orchestrate the production of functional factors by peripheral blood cells. This research provides valuable insights into the optimization of MSC-based therapies by systematically assessing and comparing the priming type-specific functional features of hAMSCs. These findings highlight new strategies for enhancing the therapeutic efficacy of MSCs, particularly in the context of multifactorial diseases, paving the way for the use of hAMSC-derived products in clinical practice.

14.
J Clin Med ; 12(14)2023 Jul 20.
Article in English | MEDLINE | ID: mdl-37510901

ABSTRACT

BACKGROUND: Ischemia/reperfusion injury (IRI), acute rejection (AR), and delayed graft function (DGF) might occur as major complications following kidney transplantation. Thus, the identification of biomarkers for the IRI, AR, and/or DGF development becomes crucial as it may help to guide post-transplant management. Natural killer (NK) cells, hepatic interstitial T-lymphocytes (T-Li), and NK-T cells are crucial in both innate and adaptive immunity after abdominal solid organ transplantation. Hence, the aim of this study was to evaluate the impact of the immune system after graft reperfusion during KT in adults in order to identify predictive biomarkers. METHODS: The NK, T-Li, and NK-T phenotypes and concentrations were retrospectively analyzed in a consecutive series of liver perfusates obtained after organ procurement flushing the abdominal cavity recovered from deceased brain donors (DBDs). Their percentage was compared with the renal transplant recipients' characteristics with kidneys taken from the same DCDs. The hepatic perfusate cells were purified by density gradient centrifugation. Flow cytometric investigation was used to determine their phenotype with the following immunological markers in order to determine the relative percentage of T-Li, NK-T, and NK cells: CD3, CD4, CD8, and CD56. RESULTS: 42 DBDs' liver perfusates were analyzed. The related clinical outcomes of kidney transplant recipients from 2010 to 2020 performed at our Institute were evaluated. Time in days of delayed functional recovery of transplanted kidneys (DGF) (p = 0.02) and the onset of secondary infection from a cytomegalovirus (p = 0.03) were significantly associated with the T-Li percentage. An increased relative risk (HR) of organ survival was significantly associated with the percent cell concentration of T-Li and time to DGF, on COX analysis, were (HR = 1.038, p = 0.04; and HR = 1.029, p = 0.01, respectively). None relevant clinical outcomes in kidney transplant patients were associated with the specificity of the NK and NK-T cell proportions. CONCLUSIONS: A new potential role of T-Li cells was detected in the context of hepatic perfusate from DBDs. It could detect potential impacts in organ allocation, surgical procuring techniques, and in the analysis of IRI pathophysiological events.

15.
Liver Transpl ; 18(2): 226-37, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22034152

ABSTRACT

Although hepatic cell transplantation (CT) holds the promise of bridging patients with end-stage chronic liver failure to whole liver transplantation, suitable cell populations are under debate. In addition to hepatic cells, mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) are being considered as alternative cell sources for initial clinical cell work. Fetal liver (FL) tissue contains potential progenitors for all these cell lineages. Based on the collagenase incubation of tissue fragments, traditional isolation techniques yield only a fraction of the number of available cells. We report a 5-step method in which a portal vein in situ perfusion technique is used for tissue from the late second trimester. This method results in the high viabilities known for adult liver vascular perfusion, addresses the low cell yields of conventional digestion methods, and reduces the exposure of the tissue to collagenase 4-fold. We used donated tissue from gestational weeks 18 to 22, which yielded 1.8 ± 0.7 × 10(9) cells with an average viability of 78%. Because HSC transplantation and MSC transplantation are of interest for the treatment of hepatic failure, we phenotypically confirmed that in addition to hepatic progenitors, the resulting cell preparation contained cells expressing typical MSC and HSC markers. The percentage of FL cells expressing proliferation markers was 45 times greater than the percentage of adult hepatocytes expressing these markers and was comparable to the percentage of immortalized HepG2 liver hepatocellular carcinoma cells; this indicated the strong proliferative capacity of fetal cells. We report a case of human FL CT with the described liver cell population for clinical end-stage chronic liver failure. The patient's Model for End-Stage Liver Disease (MELD) score improved from 15 to 10 within the first 18 months of observation. In conclusion, this human FL cell isolation protocol may be of interest for further clinical translation work on the development of liver cell-based therapies.


Subject(s)
Cell Separation/methods , End Stage Liver Disease/surgery , Fetal Stem Cells/transplantation , Hematopoietic Stem Cell Transplantation , Hepatitis C/complications , Liver Cirrhosis/surgery , Liver/embryology , Mesenchymal Stem Cell Transplantation , Perfusion , Biomarkers/metabolism , Cell Culture Techniques , Cell Survival , Collagenases/metabolism , End Stage Liver Disease/virology , Fetal Stem Cells/metabolism , Gestational Age , Hep G2 Cells , Humans , Immunosuppressive Agents/therapeutic use , Liver/blood supply , Liver/metabolism , Liver Cirrhosis/virology , Male , Middle Aged , Phenotype , Portal Vein/embryology , Treatment Outcome
16.
Front Immunol ; 13: 856657, 2022.
Article in English | MEDLINE | ID: mdl-35401503

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), is modifying human activity all over the world with significant health and economic burden. The advent of the SARS-CoV-2 pandemic prompted the scientific community to learn the virus dynamics concerning transmissibility, epidemiology, and usefulness of vaccines in fighting emerging health hazards. Pieces of evidence suggest that the first and second doses of mRNA vaccines induce a significant antibody response in vaccinated subjects or patients who recovered from SARS-CoV-2 infection, demonstrating the importance of the previously formed memory. The aim of this work has been to investigate the effects of BNT162b2 Pfizer-BioNTech mRNA-based vaccine booster dose in a cohort of 11 uninfected immunocompetent (ICs), evaluating the humoral and cellular responses, with more carefulness on memory B and T cells. Our findings underscore the potential benefit of the third dose of mRNA vaccine on the lifespan of memory B and T cells, suggesting that booster doses could increase protection against SARS-CoV-2 infection.


Subject(s)
COVID-19 , SARS-CoV-2 , BNT162 Vaccine , COVID-19/prevention & control , COVID-19 Vaccines , Humans , Immunity, Cellular , RNA, Messenger/genetics , T-Lymphocytes , Vaccines, Synthetic , mRNA Vaccines
17.
Stem Cell Rev Rep ; 17(3): 981-998, 2021 06.
Article in English | MEDLINE | ID: mdl-33389680

ABSTRACT

Cirrhosis is associated with dysregulated immune cell activation and immune dysfunction. These conditions modify gut flora, facilitate bacterial translocation, and increase susceptibility to bacterial peritonitis and consequent systemic infections by dramatically affecting long-term patient survival. Human amnion-derived mesenchymal stromal cells (hA-MSCs) exert immunomodulatory potential benefit, and have the ability to modulate their actions, especially in situations requiring immune activation through mechanisms not fully understood. In this study, we aimed to investigate, in vitro, the immunostimulant or immunosuppressive effects of hA-MSCs on cellular components of ascitic fluid obtained from cirrhotic patients with refractory ascites. We found that hA-MSCs viability is not affected by ascitic fluid and, interestingly, hA-MSCs diminished the pro-inflammatory cytokine production, and promoted anti-inflammatory M2 macrophage polarization. Moreover, we found that there was no simultaneous significant decrease in the M1-like component, allowing a continual phagocytosis activity of macrophages and NK cells to restore a physiological condition. These data highlight the plasticity of hA-MSCs' immunomodulatory capacity, and pave the way to further understanding their role in conditions such as spontaneous bacterial peritonitis.


Subject(s)
Bacterial Infections , Mesenchymal Stem Cells , Peritonitis , Amnion , Anti-Inflammatory Agents/therapeutic use , Ascites/drug therapy , Bacterial Infections/therapy , Humans , Liver Cirrhosis/therapy , Peritonitis/drug therapy
18.
Stem Cells Int ; 2020: 8889379, 2020.
Article in English | MEDLINE | ID: mdl-32855639

ABSTRACT

The use of cell secreted factors in clinical settings could be an alternative to conventional cell therapy, with the advantage of limiting concerns generally associated with traditional cell transplantation, such as tumorigenicity, immunoreactivity, and carrying of infections. Based on our published data, we predict a potential role for extracellular vesicles (EVs) in contributing to the proangiogenic activity of human fetal dermal cell secretome. Depletion of nanosized EVs from secretome significantly impaired its ability to induce formation of mesh-like structures in vitro. The isolated EVs were characterized for size and concentration by nanoparticle tracking analysis, and for protein markers (Rab5+, Alix+, CD63+, and calnexin-). The microRNA profile of EVs revealed 87 microRNAs significantly upregulated (≥15-fold increase) in fetal compared to adult dermal cell-derived EVs. Interestingly, these upregulated microRNAs included microRNAs with a validated role in angiogenesis according to literature. Moreover, the DIANA-TarBase v7.0 analysis confirmed enrichment in the KEGG signaling pathways associated with angiogenesis and wound healing, with the identification of putative target genes including thrombospondin 1. To validate the in silico data, EVs were also characterized for total protein contents. When tested in in vitro angiogenesis, fetal dermal cell-derived EVs were more effective than their adult counterpart in inducing formation of complete mesh-like structures. Furthermore, treatment of fibroblasts with fetal dermal-derived EVs determined a 4-fold increase of thrombospondin 1 protein amounts compared with the untreated fibroblasts. Finally, visualization of CSFE-labeled EVs in the cytosol of target cells suggested a successful uptake of these particles at 4-8 hours of incubation. We conclude that EVs are important contributors of the proangiogenic effect of fetal dermal cell secretome. Hence, EVs could also serve as vehicle for a successful delivery of microRNAs or other molecules of therapeutic interest to target cells.

19.
Front Immunol ; 11: 54, 2020.
Article in English | MEDLINE | ID: mdl-32117234

ABSTRACT

Human mesenchymal stromal/stem cells (MSCs), being immunoprivileged and having immunomodulatory ability, represent a promising tool to be applied in the field of regenerative medicine. Based on numerous in vitro evidences, the immunological effects of MSCs on immune cells could depend on different mechanisms as cell-to-cell contact and paracrine signals. Furthermore, recent studies have shown that the immunomodulatory activity of MSCs is initiated by activated immune cells; thus, their interaction represents a potential homeostatic mechanism by which MSCs regulate the immune response. MSCs also release exosomes able to give different effects, in a paracrine manner, by influencing inflammatory processes. In this study, we aimed to establish the potential role of human amnion-derived MSCs (hAMSCs), in immunomodulation. We found that the immunosuppressive properties of hAMSCs are not constitutive, but require "supportive signals" capable of promoting these properties. Indeed, we observed that hAMSCs alone are not able to produce an adequate amount of soluble immunomodulatory factors. Here, we studied, in depth, the strong immunomodulatory licensing signal deriving from the direct interaction between hAMSCs and stimulated peripheral blood mononuclear cells. We found that the immunomodulatory effect of hAMSCs also depends on cell-to-cell contact through the contribution of the PDL-1/PD-1 axis. We then investigated the IFN-γ priming of hAMSCs (γ-hAMSCs), which induce the increase of PDL-1 expression, high production of IDO, and upregulation of different immunomodulatory exosome-derived miRNAs. Our miRNA-target network analysis revealed that nine of the deregulated miRNAs are involved in the regulation of key proteins that control both T cell activation/anergy and monocyte differentiation pathways. Finally, we observed that γ-hAMSCs induce in monocytes both M2-like phenotype and the increase of IL-10 production. The extensive implications of MSCs in modulating different aspects of the immune system make these cells attractive candidates to be employed in therapeutic application in immune-based diseases. For these reasons, we aimed, with this study, to shed light on the potential of hAMSCs, and how they could become a useful tool for treating different inflammatory diseases, including end-stage pathologies or adverse effects in transplanted patients.


Subject(s)
Amnion/immunology , Cell Communication/immunology , Immunomodulation/immunology , Lymphocyte Activation/immunology , Mesenchymal Stem Cells/immunology , Amnion/cytology , B7-H1 Antigen/metabolism , Cell Differentiation , Cell Proliferation , Coculture Techniques , Cytokines/metabolism , Exosomes/immunology , Healthy Volunteers , Humans , I-kappa B Kinase/metabolism , Immunity , Immunologic Factors/metabolism , Immunosuppression Therapy , Interferon Regulatory Factor-1/metabolism , Interferon-gamma/pharmacology , Leukocytes, Mononuclear/immunology , Mesenchymal Stem Cells/metabolism , MicroRNAs , Monocytes , Primary Cell Culture , Programmed Cell Death 1 Receptor/metabolism
20.
Stem Cells Int ; 2019: 7486279, 2019.
Article in English | MEDLINE | ID: mdl-30911299

ABSTRACT

The secretion of potential therapeutic factors by mesenchymal stem cells (MSCs) has aroused much interest given the benefits that it can bring in the field of regenerative medicine. Indeed, the in vitro multipotency of these cells and the secretive capacity of both angiogenic and immunomodulatory factors suggest a role in tissue repair and regeneration. However, during culture, MSCs rapidly lose the expression of key transcription factors associated with multipotency and self-renewal, as well as the ability to produce functional paracrine factors. In our study, we show that a three-dimensional (3D) culture method is effective to induce MSC spheroid formation, to maintain the multipotency and to improve the paracrine activity of a specific population of human amnion-derived MSCs (hAMSCs). The regenerative potential of both 3D culture-derived conditioned medium (3D CM) and their exosomes (EXO) was assessed against 2D culture products. In particular, tubulogenesis assays revealed increased capillary maturation in the presence of 3D CM compared with both 2D CM and 2D EXO. Furthermore, 3D CM had a greater effect on inhibition of PBMC proliferation than both 2D CM and 2D EXO. To support this data, hAMSC spheroids kept in our 3D culture system remained viable and multipotent and secreted considerable amounts of both angiogenic and immunosuppressive factors, which were detected at lower levels in 2D cultures. This work reveals the placenta as an important source of MSCs that can be used for eventual clinical applications as cell-free therapies.

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