Dysbiosis: An Indicator of COVID-19 Severity in Critically Ill Patients.

Here, we examined the dynamics of the gut and respiratory microbiomes in severe COVID-19 patients in need of mechanical ventilation in the intensive care unit (ICU). We recruited 85 critically ill patients (53 with COVID-19 and 32 without COVID-19) and 17 healthy controls (HCs) and monitored them for up to 4 weeks. We analyzed the bacterial and fungal taxonomic profiles and loads of 232 gut and respiratory samples and we measured the blood levels of Interleukin 6, IgG, and IgM in COVID-19 patients. Upon ICU admission, the bacterial composition and load in the gut and respiratory samples were altered in critically ill patients compared with HCs. During their ICU stay, the patients experienced increased bacterial and fungal loads, drastic decreased bacterial richness, and progressive changes in bacterial and fungal taxonomic profiles. In the gut samples, six bacterial taxa could discriminate ICU-COV(+) from ICU-COV(-) cases upon ICU admission and the bacterial taxa were associated according to age, PaO2/FiO2, and CRP levels. In the respiratory samples of the ICU-COV(+) patients, bacterial signatures including Pseudomonas and Streptococcus were found to be correlated with the length of ICU stay. Our findings demonstrated that the gut and respiratory microbiome dysbiosis and bacterial signatures associated with critical illness emerged as biomarkers of COVID-19 severity and could be a potential predictor of ICU length of stay. We propose using a high-throughput sequencing approach as an alternative to traditional isolation techniques to monitor ICU patient infection.

Variable readthrough responsiveness of nonsense mutations in hemophilia A.

Readthrough therapy relies on the use of small molecules that enable premature termination codons in mRNA open reading frames to be misinterpreted by the translation machinery, thus allowing the generation of full-length, potentially functional proteins from mRNA carrying nonsense mutations. In patients with hemophilia A, nonsense mutations potentially sensitive to readthrough agents represent approximately 16% of the point mutations. The aim of this study was to measure the readthrough effect of different compounds and to analyze the influence of premature termination codon context in selected nonsense mutations causing hemophilia A. To this end, primary fibroblasts from three patients with hemophilia A caused by nonsense mutations (p.W1586X, p.Q1636X and p.R1960X) and Chinese hamster ovary (CHO) cells transfected with 12 different plasmids encoding mutated F8 (p.Q462X, p.Q1705X, p.Q1764X, p.W274X, p.W1726X, p.W2015X, p.W2131X, p.R1715X, p.R1822X, p.R1960X, p.R2071X and p.R2228X) were treated with gentamicin, geneticin, PTC124, RTC13 or RTC14. Responses were assessed by analyzing not only F8 mRNA expression and FVIII biosynthesis (FVIII antigen by ELISA, western blot and immunofluorescence) but also the FVIII activity (by chromogenic assay). In the patients' fibroblasts, readthrough agents neither stabilized F8 mRNA nor increased FVIII protein or activity to detectable levels. In CHO cells, only in five of the 12 F8 variants, readthrough treatment increased both FVIII antigen and activity levels, which was associated with a reduction in intracellular accumulation of truncated forms and an increase in full-length proteins. These results provide experimental evidence of genetic context dependence of nonsense suppression by readthrough agents and of factors predicting responsiveness.

Expression of microRNA-155 in inflammatory cells modulates liver injury.

MicroRNA 155 (miR-155) is involved in immune and inflammatory diseases and is associated with liver fibrosis and steatohepatitis. However, the mechanisms involved in miR-155 regulation of liver injury are largely unknown. The role of miR-155 in acute liver injury was assessed in wild-type (WT), miR-155-/- , and miR-155-/- mice transplanted with WT bone marrow. Additionally, miR-155 expression was evaluated in liver tissue and peripheral blood mononuclear cells of patients with autoimmune hepatitis. Concanavalin A, but not acetaminophen, treatment increased the expression of miR-155 in liver tissue of WT mice. Concanavalin A induced increases in cell death, liver aminotransferases, and expression of proinflammatory cytokines (chemokine [C-X-C motif] ligands 1, 5, 9, 10, and 11; chemokine [C-C motif] ligands 2 and 20; and intercellular cell adhesion molecule 1) in miR-155-/- compared to WT mice. Importantly, these animals showed a significant decrease in cluster of differentiation 4-positive/chemokine (C-X-C motif) receptor 3-positive and forkhead box p3-positive cell recruitment but no changes in other inflammatory cell populations. Mechanistically, miR-155-deficient regulatory T cells showed increased SH2 domain-containing inositol 5-phosphatase 1 expression, a known target of miR-155. Inhibition of SH2 domain-containing inositol 5-phosphatase 1 in miR-155-/- mice restored forkhead box p3 recruitment and reduced liver cytokine expression. Transplantation of bone marrow from WT animals into miR-155-/- mice partially reversed the effect of concanavalin A on miR-155-/- mice as assessed by proinflammatory cytokines and cell death protein expression. Patients with autoimmune hepatitis showed a marked increase in miR-155 expression in the liver but reduced expression of miR-155 in peripheral blood mononuclear cells. CONCLUSION: miR-155 expression is altered in both liver tissue and circulating inflammatory cells during liver injury, thus regulating inflammatory cell recruitment and liver damage; these results suggest that maintaining miR-155 expression in inflammatory cells might be a potential strategy to modulate liver injury. (Hepatology 2018).

Myeloid-derived suppressor cells can be efficiently generated from human hematopoietic progenitors and peripheral blood monocytes.

Myeloid-derived suppressor cells (MDSCs) have an important role in controlling inflammation. As such, they are both a therapeutic target and, based on the administration of ex vivo-generated MDSCs, a therapeutic tool. However, there are relatively few reports describing methods to generate human MDSCs, and most of them rely on cells obtained from peripheral blood monocytes. We investigated alternative approaches to the generation of MDSCs from hematopoietic progenitors and monocytes. Purified CD34+ hematopoietic progenitors from apheresis products and CD14+ cells isolated from buffy coats were cultured in the presence of different combinations of cytokines. The resulting myeloid cell populations were then characterized phenotypically and functionally. Progenitor cells cultured in the presence of SCF+TPO+FLT3-L+GM-CSF+IL-6 gave rise to both monocytic (M)- and granulocytic (G)-MDSCs but production of the latter was partially inhibited by IL-3. M-MDSCs but not G-MDSCs were obtained by culturing peripheral blood monocytes with GM-CSF+IL-6 or GM-CSF+TGF-ß1 for 6 days. CD14 expression was downregulated in the cultured cells. PD-L1 expression at baseline was lower in hematopoietic progenitor cell-derived than in monocyte-derived MDSCs, but was markedly increased in response to stimulation with LPS+IFN-γ. The functionality of the two MDSC subtypes was confirmed in studies of the suppression of allogeneic and mitogen-induced proliferation and by cytokine profiling. Here we describe both the culture conditions that allow the generation of MDSCs and the phenotypical and functional characterization of these cell populations.

Gene therapy using a liver-targeted AAV vector restores nucleoside and nucleotide homeostasis in a murine model of MNGIE.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in TYMP, enconding thymidine phosphorylase (TP). TP deficiency results in systemic accumulation of thymidine and deoxyuridine, which interferes with mitochondrial DNA (mtDNA) replication and leads to mitochondrial dysfunction. To date, the only treatment available for MNGIE patients is allogeneic hematopoietic stem cell transplantation, which is associated with high morbidity and mortality. Here, we report that AAV2/8-mediated transfer of the human TYMP coding sequence (hcTYMP) under the control of a liver-specific promoter prevents the biochemical imbalances in a murine model of MNGIE. hcTYMP expression was restricted to liver, and a dose as low as 2 × 10(11) genome copies/kg led to a permanent reduction in systemic nucleoside levels to normal values in about 50% of treated mice. Higher doses resulted in reductions to normal or slightly below normal levels in virtually all mice treated. The nucleoside reduction achieved by this treatment prevented deoxycytidine triphosphate (dCTP) depletion, which is the limiting factor affecting mtDNA replication in this disease. These results demonstrate that the use of AAV to direct TYMP expression in liver is feasible as a potentially safe gene therapy strategy for MNGIE.

Oxidative Stress in Structural Valve Deterioration: A Longitudinal Clinical Study.

The cause of structural valve deterioration (SVD) is unclear. Therefore, we investigated oxidative stress markers in sera from patients with bioprosthetic heart valves (BHVs) and their association with SVD. Blood samples were taken from SVD (Phase A) and BHV patients during the first 24 (Phase B1) and >48 months (Phase B2) after BHV implantation to assess total antioxidant capacity (TAC), malondialdehyde (MDA), and nitrotyrosine (NT). The results show that MDA levels increased significantly 1 month after surgery in all groups but were higher at 6 months only in incipient SVD patients. NT levels increased gradually for the first 24 months after implantation in the BHV group. Patients with transcatheter aortic valve implantation (TAVI) showed even higher levels of stress markers. After >48 months, MDA and NT continued to increase in BHV patients with a further elevation after 60-72 months; however, these levels were significantly lower in the incipient and established SVD groups. In conclusion, oxidative stress may play a significant role in SVD, increasing early after BHV implantation, especially in TAVI cases, and also after 48 months' follow-up, but decreasing when SVD develops. Oxidative stress potentially represents a target of therapeutic intervention and a biomarker of BHV dysfunction.

Preclinical Assessment of a Gene-Editing Approach in a Mouse Model of Mitochondrial Neurogastrointestinal Encephalomyopathy.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare disease caused by recessive mutations in the TYMP gene, which encodes the enzyme thymidine phosphorylase (TP). In this study, the efficient integration of a TYMP transgene into introns of the Tymp and Alb loci of hepatocytes in a murine model of MNGIE was achieved by the coordinated delivery and activity of CRISPR/Cas9 and a TYMP cDNA. CRISPR/Cas9 was delivered either as mRNA using lipid nanoparticle (LNP) or polymeric nanoparticle, respectively, or in an AAV2/8 viral vector; the latter was also used to package the TYMP cDNA. Insertion of the cDNA template downstream of the Tymp and Alb promoters ensured transgene expression. The best in vivo results were obtained using LNP carrying the CRISPR/Cas9 mRNAs. Treated mice showed a consistent long-term (1 year) reduction in plasma nucleoside (thymidine and deoxyuridine) levels that correlated with the presence of TYMP mRNA and functional enzyme in liver cells. In mice with an edited Alb locus, the transgene produced a hybrid Alb-hTP protein that was secreted, with supraphysiological levels of TP activity detected in the plasma. Equivalent results were obtained in mice edited at the Tymp locus. Finally, some degree of gene editing was found in animals treated only with AAV vectors containing the DNA templates, in the absence of nucleases, although there was no impact on plasma nucleoside levels. Overall, these results demonstrate the feasibility of liver-directed genome editing in the long-term correction of MNGIE, with several advantages over other methods.

Identification of multipotent mesenchymal stromal cells in the reactive stroma of a prostate cancer xenograft by side population analysis.

Cancer stem cells are a distinct cellular population that is believed to be responsible for tumor initiation and maintenance. Recent data suggest that solid tumors also contain another type of stem cells, the mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs), which contribute to the formation of tumor-associated stroma. The Hoechst 33342 efflux assay has proved useful to identify a rare cellular fraction, named Side Population (SP), enriched in cells with stem-like properties. Using this assay, we identified SP cells in a prostate cancer xenograft containing human prostate cancer cells and mouse stromal cells. The SP isolation, subculture and sequential sorting allowed the generation of single-cell-derived clones of murine origin that were recognized as MSC by their morphology, plastic adherence, proliferative potential, adipogenic and osteogenic differentiation ability and immunophenotype (CD45(-), CD81(+) and Sca-1(+)). We also demonstrated that SP clonal cells secrete transforming growth factor beta1 (TGF-beta1) and that their inhibition reduces proliferation and accelerates differentiation. These results reveal the existence of SP cells in the stroma of a cancer xenograft, and provide evidence supporting their MSC nature and the role of TGF-beta1 in maintaining their proliferation and undifferentiated status. Our data also reveal the usefulness of the SP assay to identify and isolate MSC cells from carcinomas.

Transgene expression levels determine the immunogenicity of transduced hematopoietic grafts in partially myeloablated mice.

We investigated whether transgene expression levels influence the immunogenicity of transduced hematopoietic grafts upon transplantation into partially myeloablated mice. To this aim, bone marrow cells (BMCs) transduced with retroviral vectors driving green fluorescent protein (GFP) expression either at high (high-EGFP) or low levels (low-EGFP) were transplanted into congenic recipients conditioned with sublethal doses of total body irradiation (TBI) or busulfan. Virtually all recipients showed evidence of donor engraftment 4 weeks after transplantation. However, as opposed to recipients receiving low-EGFP transduced grafts, the risk of rejecting the EGFP(+) cells by 30 days after transplantation was significantly higher in mice conditioned with busulfan and receiving high-EGFP transduced grafts. Anti-EGFP cellular immune responses were demonstrated in high-EGFP-treated mice conditioned with busulfan by interferon-gamma (IFN-gamma), enzyme-linked immunospot assay (ELISPOT), and cytotoxic T lymphocyte (CTL) assays, in contrast to that observed in mice transplanted with low-EGFP BMC. These results show for the first time that transgene expression levels can be critical for the immunogenicity of gene-modified hematopoietic grafts, especially in immunocompetent or in partially immunosuppressed recipients. These results have profound implications in vector choice and in the design of gene therapy (GT) protocols.

Tolerance induction in experimental autoimmune encephalomyelitis using non-myeloablative hematopoietic gene therapy with autoantigen.

Experimental autoimmune encephalomyelitis (EAE) constitutes a paradigm of antigen (Ag)-specific T cell driven autoimmune diseases. In this study, we transferred bone marrow cells (BMCs) expressing an autoantigen (autoAg), the peptide 40-55 of the myelin oligodendrocytic glycoprotein (MOG(40-55)), to induce preventive and therapeutic immune tolerance in a murine EAE model. Transfer of BMC expressing MOG(40-55) (IiMOG-BMC) into partially myeloablated mice resulted in molecular chimerism and in robust protection from the experimental disease. In addition, in mice with established EAE, transfer of transduced BMC with or without partial myeloablation reduced the clinical and histopathological severity of the disease. In these experiments, improvement was observed even in the absence of engraftment of the transduced hematopoietic cells, probably rejected due to the previous immunization with the autoAg. Splenocytes from mice transplanted with IiMOG-BMC produced significantly higher amounts of interleukin (IL)-5 and IL-10 upon autoAg challenge than those of control animals, suggesting the participation of regulatory cells. Altogether, these results suggest that different tolerogenic mechanisms may be mediating the preventive and the therapeutic effects. In conclusion, this study demonstrates that a cell therapy using BMC expressing an autoAg can induce Ag-specific tolerance and ameliorate established EAE even in a nonmyeloablative setting.

Absence of p.R50X Pygm read-through in McArdle disease cellular models.

McArdle disease is an autosomal recessive disorder caused by the absence of muscle glycogen phosphorylase, which leads to blocked muscle glycogen breakdown. We used three different cellular models to evaluate the efficiency of different read-through agents (including amlexanox, Ataluren, RTC13 and G418) in McArdle disease. The first model consisted of HeLa cells transfected with two different GFP-PYGM constructs presenting the Pygm p.R50X mutation (GFP-PYGM p.R50X and PYGM Ex1-GFP p.R50X). The second cellular model was based on the creation of HEK293T cell lines stably expressing the PYGM Ex1-GFP p.R50X construct. As these plasmids encode murine Pygm cDNA without any intron sequence, their transfection in cells would allow for analysis of the efficacy of read-through agents with no concomitant nonsense-mediated decay interference. The third model consisted of skeletal muscle cultures derived from the McArdle mouse model (knock-in for the p.R50X mutation in the Pygm gene). We found no evidence of read-through at detectable levels in any of the models evaluated. We performed a literature search and compared the premature termination codon context sequences with reported positive and negative read-through induction, identifying a potential role for nucleotide positions -9, -8, -3, -2, +13 and +14 (the first nucleotide of the stop codon is assigned as +1). The Pygm p.R50X mutation presents TGA as a stop codon, G nucleotides at positions -1 and -9, and a C nucleotide at -3, which potentially generate a good context for read-through induction, counteracted by the presence of C at -2 and its absence at +4.

Successful engraftment of gene-corrected hematopoietic stem cells in non-conditioned patients with Fanconi anemia.

Fanconi anemia (FA) is a DNA repair syndrome generated by mutations in any of the 22 FA genes discovered to date1,2. Mutations in FANCA account for more than 60% of FA cases worldwide3,4. Clinically, FA is associated with congenital abnormalities and cancer predisposition. However, bone marrow failure is the primary pathological feature of FA that becomes evident in 70-80% of patients with FA during the first decade of life5,6. In this clinical study (ClinicalTrials.gov, NCT03157804 ; European Clinical Trials Database, 2011-006100-12), we demonstrate that lentiviral-mediated hematopoietic gene therapy reproducibly confers engraftment and proliferation advantages of gene-corrected hematopoietic stem cells (HSCs) in non-conditioned patients with FA subtype A. Insertion-site analyses revealed the multipotent nature of corrected HSCs and showed that the repopulation advantage of these cells was not due to genotoxic integrations of the therapeutic provirus. Phenotypic correction of blood and bone marrow cells was shown by the acquired resistance of hematopoietic progenitors and T lymphocytes to DNA cross-linking agents. Additionally, an arrest of bone marrow failure progression was observed in patients with the highest levels of gene marking. The progressive engraftment of corrected HSCs in non-conditioned patients with FA supports that gene therapy should constitute an innovative low-toxicity therapeutic option for this life-threatening disorder.

Estrogen receptor beta displays cell cycle-dependent expression and regulates the G1 phase through a non-genomic mechanism in prostate carcinoma cells.

BACKGROUND: It is well known that estrogens regulate cell cycle progression, but the specific contributions and mechanisms of action of the estrogen receptor beta (ERbeta) remain elusive. METHODS: We have analyzed the levels of ERbeta1 and ERbeta2 throughout the cell cycle, as well as the mechanisms of action and the consequences of the over-expression of ERbeta1 in the human prostate cancer LNCaP cell line. RESULTS: Both ERbeta1 mRNA and protein expression increased from the G1 to the S phase and decreased before entering the G2/M phase, whereas ERbeta2 levels decreased during the S phase and increased in the G2/M phase. ERbeta1 protein was detected in both the nuclear and non-nuclear fractions, and ERbeta2 was found exclusively in the nucleus. Regarding the mechanisms of action, endogenous ERbeta was able to activate transcription via ERE during the S phase in a ligand-dependent manner, whereas no changes in AP1 and NFkappaB transactivation were observed after exposure to estradiol or the specific inhibitor ICI 182,780. Over-expression of either wild type ERbeta1 or ERbeta1 mutated in the DNA-binding domain caused an arrest in early G1. This arrest was accompanied by the interaction of over-expressed ERbeta1 with c-Jun N-terminal protein kinase 1 (JNK1) and a decrease in c-Jun phosphorylation and cyclin D1 expression. The administration of ICI impeded the JNK1-ERbeta1 interaction, increased c-Jun phosphorylation and cyclin D1 expression and allowed the cells to progress to late G1, where they became arrested. CONCLUSIONS: Our results demonstrate that, in LNCaP prostate cancer cells, both ERbeta isoforms are differentially expressed during the cell cycle and that ERbeta regulates the G1 phase by a non-genomic mechanism.

A reproducible method for the isolation and expansion of ovine mesenchymal stromal cells from bone marrow for use in regenerative medicine preclinical studies.

The use of multipotent mesenchymal stromal cells (MSCs) as candidate medicines for treating a variety of pathologies is based on their qualities as either progenitors for the regeneration of damaged tissue or producers of a number of molecules with pharmacological properties. Preclinical product development programmes include the use of well characterized cell populations for proof of efficacy and safety studies before testing in humans. In the field of orthopaedics, an increasing number of translational studies use sheep as an in vivo test system because of the similarities with humans in size and musculoskeletal architecture. However, robust and reproducible methods for the isolation, expansion, manipulation and characterization of ovine MSCs have not yet been standardised. The present study describes a method for isolation and expansion of fibroblastic-like, adherent ovine MSCs that express CD44, CD90, CD140a, CD105 and CD166, and display trilineage differentiation potential. The 3-week bioprocess proposed here typically yielded cell densities of 1.4 × 104 MSCs/cm2 at passage 2, with an expansion factor of 37.8 and approximately eight cumulative population doublings. The osteogenic potential of MSCs derived following this methodology was further evaluated in vivo in a translational model of osteonecrosis of the femoral head, in which the persistence of grafted cells in the host tissue and their lineage commitment into osteoblasts and osteocytes was demonstrated by tracking enhanced green fluorescent protein-labelled cells. Copyright © 2016 John Wiley & Sons, Ltd.

Response of the human myocardium to ischemic injury and preconditioning: The role of cardiac and comorbid conditions, medical treatment, and basal redox status.

BACKGROUND: The diseased human myocardium is highly susceptible to ischemia/reoxygenation (I/R)-induced injury but its response to protective interventions such as ischemic preconditioning (IPreC) is unclear. Cardiac and other pre-existing clinical conditions as well as previous or ongoing medical treatment may influence the myocardial response to I/R injury and protection. This study investigated the effect of both on myocardial susceptibility to I/R-induced injury and the protective effects of IPreC. METHODS AND RESULTS: Atrial myocardium from cardiac surgery patients (n = 300) was assigned to one of three groups: aerobic control, I/R alone, and IPreC. Lactate dehydrogenase leakage, as a marker of cell injury, and cell viability were measured. The basal redox status was determined in samples from 90 patients. The response to I/R varied widely. Myocardium from patients with aortic valve disease was the most susceptible to injury whereas myocardium from dyslipidemia patients was the least susceptible. Tissue from females was better protected than tissue from males. Myocardium from patients with mitral valve disease was the least responsive to IPreC. The basal redox status was altered in the myocardium from patients with mitral and aortic valve disease. CONCLUSIONS: The response of the myocardium to I/R and IPreC is highly variable and influenced by the underlying cardiac pathology, dyslipidemia, sex, and the basal redox status. These results should be taken into account in the design of future clinical studies on the prevention of I/R injury and protection.

Flow cytometry of the Side Population: tips & tricks.

BACKGROUND: The Side Population (SP) has become an important hallmark for the definition of the stem cell compartment, especially in the detection of these cells and in their physical isolation by fluorescence-activated cell sorting (FACS). SP cells are CD34neg and were discovered using ultraviolet excitation based on the efflux of Hoechst 33342 (Ho342). Although the method works as originally described, we believe that this method is difficult for most investigators. First, because the ability to discriminate SP cells is based on the differential retention of Ho342 during a functional assay; second, because of the difficulties in setting the right experimental and acquisition conditions; and third, because the analysis of the acquired data requires an extensive expertise on flow cytometry to accurately detect the SP events. METHODS: First of all and mainly for the SP application, the laser beam paths were exhaustively checked to ensure the lowest coefficients of variation. Blood suspensions were prepared by erythrocyte lysis with ammonium chloride and hematopoietic cells were labeled with Ho342. RESULTS: The Ho342 concentration and the staining procedure are critical for the optimal resolution of the SP cells. Although UV laser alignment is very important to resolve the dim tail that outlines the SP, the problem with Ho342 excitation is not the Hoechst Blue emission, but rather the Hoechst Red's (because of the weak emission). CONCLUSIONS: Each laboratory must establish its own expected ranges based on its instrument and results may vary slightly due to instrument differences such as the narrowness of the band pass filters, laser power, laser emission wavelength, nozzle type, differential of pressure, light collection system (cuvette versus jet-in-air) and beam shaping optics.

Long-Term Restoration of Thymidine Phosphorylase Function and Nucleoside Homeostasis Using Hematopoietic Gene Therapy in a Murine Model of Mitochondrial Neurogastrointestinal Encephalomyopathy.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a metabolic disorder caused by mutations in TYMP, encoding thymidine phosphorylase (TP). In MNGIE patients, TP dysfunction produces systemic thymidine and deoxyuridine accumulation, which ultimately impairs mitochondrial DNA replication and results in mitochondrial dysfunction. To date, only allogeneic hematopoietic stem cell transplantation has demonstrated long-term clinical efficacy, but high morbidity and mortality associated with this procedure necessitate the search for safer alternatives. In a previous study, we demonstrated that hematopoietic stem cell gene therapy using a lentiviral vector containing the coding sequence of TYMP restored the biochemical homeostasis in an animal model of MNGIE. In the present follow-up study, we show that ectopic expression of TP in the hematopoietic system restores normal nucleoside levels in plasma, as well as in tissues affected in MNGIE such as small intestine, skeletal muscle, brain, and liver. Mitochondrial dNTP pool imbalances observed in liver of the animal model were also corrected by the treatment. The biochemical effects were maintained at least 20 months even with low levels of chimerism. No alterations in the blood cell counts or other toxic effects were observed in association with the lentiviral transduction or TP overexpression. These results further support the notion that gene therapy is a feasible treatment option for MNGIE.

Myeloid-derived suppressor cells expressing a self-antigen ameliorate experimental autoimmune encephalomyelitis.

Previous work by our group showed that transferring bone marrow cells transduced with a self-antigen induced immune tolerance and ameliorated experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). We also found that following retroviral transduction of murine bone marrow (BM) cells, the majority of cells generated and transduced were myeloid-derived suppressor cells (MDSCs). Here, we aimed to determine whether purified antigen-expressing MDSCs have similar therapeutic effects than those of unfractionated BM, and to investigate their potential mechanisms. We performed phenotypic and functional analyses in these cells using the same animal model, and we used purified antigen-expressing MDSCs in preventive and therapeutic approaches. These cells exerted therapeutic effects similar to those of BM cells, which depended upon self-antigen expression. The majority of monocytic (M)-MDSCs expressed the immunosuppressive molecule programmed death ligand-1 (PD-L1), CD80, CD86 and MHC class II molecules. Additionally, the animals infused with antigen-expressing cells exhibited lower percentages of activated T cells and higher percentages of B cells with a regulatory phenotype (B220+CD1dhigh CD5+) in the spleen than their respective controls. MDSCs expressing self-antigens, alloantigens or therapeutic transgenes are tolerogenic and can be exploited therapeutically in autoimmune diseases, transplantation and in gene therapy, respectively.

[Therapeutic applications of stem-cells]. / Aplicaciones terapéuticas de las células madre.

In the last years stem cells (SC) have generated huge expectations and have become a new hope for the development of novel cell therapies in the context of regenerative medicine. So far, the hypothetic therapeutic effects of SC, both of embryonic and adult origin, have been demonstrated only in a very few cases. Embryonic SC are pluripotential and have, in theory, more plasticity to differentiate into a wide range of cell or tissue types. However, the society still has to decide on the ethics of its use. Regarding adult SC, they are readily available and are fully matched. However, whether their potential will translate into therapeutic benefits in humans needs to be determined as yet. This article is intended to give a general overview on this field, based on the current scientific knowledge.

Myeloid-derived suppressor cells are generated during retroviral transduction of murine bone marrow.

Previous work by our group showed that transferring bone marrow cells transduced with an autoantigen into nonmyeloablated mice with experimental autoimmune encephalomyelitis induced immune tolerance and improved symptoms of the disease. Because this effect occurred in the absence of molecular chimerism, we hypothesized that the cells responsible did not have repopulating ability and that they were not mediating central but peripheral tolerance mechanisms. In the present study, we analyzed the immunophenotype of the cells that are generated in the transduction cultures and we evaluated the immunosuppressive activity of the main cell subpopulations produced. We show that both granulocytic (CD11b(+) Gr-1(hi)) and monocytic (CD11b(+) Gr-1(lo)) myeloid-derived suppressor cells (G- and M-MDSCs, respectively) are generated during standard 4-day γ-retroviral transduction cultures (representing about 25% and 40% of the total cell output, respectively) and that the effectively transduced cells largely consist of these two cell types. A third cell population representing about 15% of the transduced cells did not express CD45 or hematopoietic lineage markers and expressed mesenchymal stromal cell markers. Transduced total bone marrow cells and sorted M-MDSCs expressed arginase and inducible nitric oxide synthase activities, produced reactive oxygen species, and inhibited antigen-induced T-cell proliferation in vitro. Transgene-expressing MDSCs could be exploited therapeutically to induce tolerance in autoimmune diseases and in gene therapy protocols.