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1.
Sci Rep ; 11(1): 17473, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471195

RESUMO

As for all newly-emergent pathogens, SARS-CoV-2 presents with a relative paucity of clinical information and experimental models, a situation hampering both the development of new effective treatments and the prediction of future outbreaks. Here, we find that a simple virus-free model, based on publicly available transcriptional data from human cell lines, is surprisingly able to recapitulate several features of the clinically relevant infections. By segregating cell lines (n = 1305) from the CCLE project on the base of their sole angiotensin-converting enzyme 2 (ACE2) mRNA content, we found that overexpressing cells present with molecular features resembling those of at-risk patients, including senescence, impairment of antibody production, epigenetic regulation, DNA repair and apoptosis, neutralization of the interferon response, proneness to an overemphasized innate immune activity, hyperinflammation by IL-1, diabetes, hypercoagulation and hypogonadism. Likewise, several pathways were found to display a differential expression between sexes, with males being in the least advantageous position, thus suggesting that the model could reproduce even the sex-related disparities observed in the clinical outcome of patients with COVID-19. Overall, besides validating a new disease model, our data suggest that, in patients with severe COVID-19, a baseline ground could be already present and, as a consequence, the viral infection might simply exacerbate a variety of latent (or inherent) pre-existing conditions, representing therefore a tipping point at which they become clinically significant.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , COVID-19/genética , Perfilação da Expressão Gênica/métodos , Regulação para Cima , COVID-19/imunologia , Linhagem Celular , Bases de Dados Genéticas , Feminino , Humanos , Imunidade Inata , Masculino , Modelos Biológicos , Modelos Teóricos , Caracteres Sexuais
2.
Nat Commun ; 12(1): 5205, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471128

RESUMO

Molecular mechanisms associated with human germ cell aplasia in infertile men remain undefined. Here we perform single-cell transcriptome profiling to highlight differentially expressed genes and pathways in each somatic cell type in testes of men with idiopathic germ cell aplasia. We identify immaturity of Leydig cells, chronic tissue inflammation, fibrosis, and senescence phenotype of the somatic cells, as well markers of chronic inflammation in the blood. We find that deregulated expression of parentally imprinted genes in myoid and immature Leydig cells, with relevant changes in the ratio of Lamin A/C transcripts and an active DNA damage response in Leydig and peritubular myoid cells are also indicative of senescence of the testicular niche. This study offers molecular insights into the pathogenesis of idiopathic germ cell aplasia.


Assuntos
Envelhecimento/fisiologia , Dano ao DNA , Inflamação , Testículo/metabolismo , Envelhecimento/genética , Comunicação Celular , Quimiocinas , Perfilação da Expressão Gênica , Células Germinativas , Humanos , Inflamação/patologia , Células Intersticiais do Testículo , Masculino , Fenótipo , Alinhamento de Sequência , Espermatogênese/genética , Espermatogênese/fisiologia , Espermatogônias/metabolismo , Transcriptoma
3.
Int J Mol Sci ; 22(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34445143

RESUMO

Dendritic cells (DCs) dictate the outcomes of tissue-specific immune responses. In the context of autoimmune diseases, DCs instruct T cells to respond to antigens (Ags), including self-Ags, leading to organ damage, or to becoming regulatory T cells (Tregs) promoting and perpetuating immune tolerance. DCs can acquire tolerogenic properties in vitro and in vivo in response to several stimuli, a feature that opens the possibility to generate or to target DCs to restore tolerance in autoimmune settings. We present an overview of the different subsets of human DCs and of the regulatory mechanisms associated with tolerogenic (tol)DC functions. We review the role of DCs in the induction of tissue-specific autoimmunity and the current approaches exploiting tolDC-based therapies or targeting DCs in vivo for the treatment of autoimmune diseases. Finally, we discuss limitations and propose future investigations for improving the knowledge on tolDCs for future clinical assessment to revert and prevent autoimmunity. The continuous expansion of tolDC research areas will lead to improving the understanding of the role that DCs play in the development and treatment of autoimmunity.


Assuntos
Doenças Autoimunes/imunologia , Autoimunidade/imunologia , Células Dendríticas/imunologia , Tolerância Imunológica/imunologia , Animais , Humanos , Linfócitos T Reguladores/imunologia
4.
Blood Adv ; 5(16): 3174-3187, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34424322

RESUMO

Adenosine deaminase 2 deficiency (DADA2) is a rare inherited disorder that is caused by autosomal recessive mutations in the ADA2 gene. Clinical manifestations include early-onset lacunar strokes, vasculitis/vasculopathy, systemic inflammation, immunodeficiency, and hematologic defects. Anti-tumor necrosis factor therapy reduces strokes and systemic inflammation. Allogeneic hematopoietic stem/progenitor cell (HSPC) transplantation can ameliorate most disease manifestations, but patients are at risk for complications. Autologous HSPC gene therapy may be an alternative curative option for patients with DADA2. We designed a lentiviral vector encoding ADA2 (LV-ADA2) to genetically correct HSPCs. Lentiviral transduction allowed efficient delivery of the functional ADA2 enzyme into HSPCs from healthy donors. Supranormal ADA2 expression in human and mouse HSPCs did not affect their multipotency and engraftment potential in vivo. The LV-ADA2 induced stable ADA2 expression and corrected the enzymatic defect in HSPCs derived from DADA2 patients. Patients' HSPCs re-expressing ADA2 retained their potential to differentiate into erythroid and myeloid cells. Delivery of ADA2 enzymatic activity in patients' macrophages led to a complete rescue of the exaggerated inflammatory cytokine production. Our data indicate that HSPCs ectopically expressing ADA2 retain their multipotent differentiation ability, leading to functional correction of macrophage defects. Altogether, these findings support the implementation of HSPC gene therapy for DADA2.


Assuntos
Adenosina Desaminase , Vasculite , Adenosina Desaminase/genética , Animais , Humanos , Inflamação , Peptídeos e Proteínas de Sinalização Intercelular , Macrófagos , Camundongos
5.
Andrology ; 2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34409772

RESUMO

BACKGROUND: Circulating testosterone levels have been found to be reduced in men with severe acute respiratory syndrome coronavirus 2 infection, COVID-19, with lower levels being associated with more severe clinical outcomes. OBJECTIVES: We aimed to assess total testosterone levels and the prevalence of total testosterone still suggesting for hypogonadism at 7-month follow-up in a cohort of 121 men who recovered from laboratory-confirmed COVID-19. MATERIALS AND METHODS: Demographic, clinical, and hormonal values were collected for all patients. Hypogonadism was defined as total testosterone ≤9.2 nmol/L. The Charlson Comorbidity Index was used to score health-significant comorbidities. Descriptive statistics and multivariable linear and logistic regression models tested the association between clinical and laboratory variables and total testosterone levels at follow-up assessment. RESULTS: Circulating total testosterone levels increased at 7-month follow-up compared to hospital admittance (p < 0.0001), while luteinizing hormone and 17ß-estradiol levels significantly decreased (all p ≤ 0.02). Overall, total testosterone levels increased in 106 (87.6%) patients, but further decreased in 12 (9.9%) patients at follow-up, where a total testosterone level suggestive for hypogonadism was still observed in 66 (55%) patients. Baseline Charlson Comorbidity Index score (OR 0.36; p = 0.03 [0.14, 0.89]) was independently associated with total testosterone levels at 7-month follow-up, after adjusting for age, BMI, and IL-6 at hospital admittance. CONCLUSIONS: Although total testosterone levels increased over time after COVID-19, more than 50% of men who recovered from the disease still had circulating testosterone levels suggestive for a condition of hypogonadism at 7-month follow-up. In as many as 10% of cases, testosterone levels even further decreased. Of clinical relevance, the higher the burden of comorbid conditions at presentation, the lower the probability of testosterone levels recovery over time.

6.
Front Immunol ; 12: 641596, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33708227

RESUMO

The effective development of innovative surgical applications and immunosuppressive agents have improved remarkable advancements in solid organ transplantation. Despite these improvements led to prevent acute rejection and to promote short-term graft survival, the toxicity of long-term immunosuppression regiments has been associated to organ failure or chronic graft rejection. The graft acceptance is determined by the balance between the regulatory and the alloreactive arm of the immune system. Hence, enhance regulatory cells leading to immune tolerance would be the solution to improve long-term allograft survival which, by reducing the overall immunosuppression, will provide transplanted patients with a better quality of life. Regulatory T cells (Tregs), and regulatory myeloid cells (MRCs), including regulatory macrophages and tolerogenic dendritic cells, are promising cell populations for restoring tolerance. Thus, in the last decade efforts have been dedicated to apply regulatory cell-based therapy to improve the successful rate of organ transplantation and to promote allogeneic tolerance. More recently, this approach has been translated into clinical application. The aim of this review is to summarize and discuss results on regulatory cell-based strategies, focusing on Tregs and MRCs, in terms of safety, feasibility, and efficacy in clinical studies of organ transplantation.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Células Dendríticas , Rejeição de Enxerto , Macrófagos , Transplante de Órgãos , Linfócitos T Reguladores , Animais , Células Dendríticas/imunologia , Células Dendríticas/transplante , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/terapia , Humanos , Macrófagos/imunologia , Macrófagos/transplante , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/transplante
7.
Andrology ; 9(4): 1043-1052, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33635589

RESUMO

BACKGROUND: Circulating androgens could have a relevant pathobiological role in clinical outcomes in men with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (COVID-19). OBJECTIVES: We aimed to assess: (a) circulating sex steroids levels in a cohort of 286 symptomatic men with laboratory-confirmed COVID-19 at hospital admission compared to a cohort of 281 healthy men; and (b) the association between serum testosterone levels (tT), COVID-19, and clinical outcomes. MATERIALS AND METHODS: Demographic, clinical, and hormonal values were collected for all patients. Hypogonadism was defined as tT ≤9.2 nmol/l. The Charlson Comorbidity Index (CCI) was used to score health-significant comorbidities. Severe clinical outcomes were defined as patients either transferred to intensive care unit (ICU) or death. Descriptive statistics and multivariable linear and logistic regression models tested the association between clinical and laboratory variables and tT levels. Univariable and multivariable logistic regression models tested the association between tT and severe clinical outcomes. RESULTS: Overall, a significantly lower levels of LH and tT were found in patients with COVID-19 compared to healthy controls (all p < 0.0001); conversely, healthy controls depicted lower values of circulating E2 (p < 0.001). Testosterone levels suggestive for hypogonadism were observed in 257 (89.8%) patients at hospital admission. In as many as 243 (85%) cases, hypogonadism was secondary. SARS-CoV-2 infection status was independently associated with lower tT levels (p < 0.0001) and greater risk of hypogonadism (p < 0.0001), after accounting for age, BMI, CCI, and IL-6 values. Lower tT levels were associated with higher risk of ICU admission and death outcomes (all p ≤ 0.05), after accounting for clinical and laboratory parameters. CONCLUSIONS: We unveil an independent association between SARS-CoV-2 infection status and secondary hypogonadism already at hospital admission, with lower testosterone levels predicting the most severe clinical outcomes.


Assuntos
COVID-19/sangue , Testosterona/sangue , Adulto , Idoso , Biomarcadores/sangue , COVID-19/complicações , Estudos de Casos e Controles , Estudos de Coortes , Hormônios Esteroides Gonadais/sangue , Humanos , Hipogonadismo/sangue , Hipogonadismo/etiologia , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento
8.
Trends Cancer ; 7(5): 389-392, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33563576

RESUMO

Despite some success, many patients do not benefit from immunotherapy. New strategies to improve clinical efficacy include identification of novel immune-checkpoint (IC) targets or a combination of immunotherapy with antiangiogenic treatments. Here, we propose the therapeutic use of IC, HLA-G/LILRB, and explore its enhanced synergistic antitumor activity when combined with antiangiogenic therapies.

9.
Diabetes ; 70(1): 171-181, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33122392

RESUMO

The induction of antigen (Ag)-specific tolerance represents a therapeutic option for autoimmune diabetes. We demonstrated that administration of a lentiviral vector enabling expression of insulin B chain 9-23 (InsB9-23) (LV.InsB) in hepatocytes arrests ß-cell destruction in prediabetic NOD mice by generating InsB9-23-specific FoxP3+ T regulatory cells (Tregs). LV.InsB in combination with a suboptimal dose of anti-CD3 monoclonal antibody (combined therapy [CT], 1 × 5 µg [CT5]) reverts diabetes and prevents recurrence of autoimmunity after islet transplantation in ∼50% of NOD mice. We investigated whether CT optimization could lead to abrogation of recurrence of autoimmunity. Therefore, alloislets were transplanted after optimized CT tolerogenic conditioning (1 × 25 µg [CT25]). Diabetic NOD mice conditioned with CT25 when glycemia was <500 mg/dL remained normoglycemic for 100 days after alloislet transplantation and displayed reduced insulitis, but independently from the graft. Accordingly, cured mice showed T-cell unresponsiveness to InsB9-23 stimulation and increased Treg frequency in islet infiltration and pancreatic lymph nodes. Additional studies revealed a complex mechanism of Ag-specific immune regulation driven by CT25, in which both Tregs and PDL1 costimulation cooperate to control diabetogenic cells, while transplanted islets play a crucial role, although transient, recruiting diabetogenic cells. Therefore, CT25 before alloislet transplantation represents an Ag-specific immunotherapy to resolve autoimmune diabetes in the presence of residual endogenous ß-cell mass.


Assuntos
Diabetes Mellitus Tipo 1/cirurgia , Hepatócitos/metabolismo , Células Secretoras de Insulina/metabolismo , Transplante das Ilhotas Pancreáticas/métodos , Peptídeos/uso terapêutico , Animais , Autoimunidade/efeitos dos fármacos , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/prevenção & controle , Técnicas de Transferência de Genes , Sobrevivência de Enxerto/imunologia , Hepatócitos/imunologia , Células Secretoras de Insulina/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Camundongos , Camundongos Endogâmicos NOD , Recidiva , Prevenção Secundária , Linfócitos T Reguladores/imunologia
10.
Clin Transl Immunology ; 9(11): e1214, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33304583

RESUMO

Objectives: Genetic or acquired defects in FOXP3+ regulatory T cells (Tregs) play a key role in many immune-mediated diseases including immune dysregulation polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. Previously, we demonstrated CD4+ T cells from healthy donors and IPEX patients can be converted into functional Treg-like cells by lentiviral transfer of FOXP3 (CD4LVFOXP3). These CD4LVFOXP3 cells have potent regulatory function, suggesting their potential as an innovative therapeutic. Here, we present molecular and preclinical in vivo data supporting CD4LVFOXP3 cell clinical progression. Methods: The molecular characterisation of CD4LVFOXP3 cells included flow cytometry, qPCR, RNA-seq and TCR-seq. The in vivo suppressive function of CD4LVFOXP3 cells was assessed in xenograft-versus-host disease (xeno-GvHD) and FOXP3-deficient IPEX-like humanised mouse models. The safety of CD4LVFOXP3 cells was evaluated using peripheral blood (PB) humanised (hu)- mice testing their impact on immune response against pathogens, and immune surveillance against tumor antigens. Results: We demonstrate that the conversion of CD4+ T cells to CD4LVFOXP3 cells leads to specific transcriptional changes as compared to CD4+ T-cell transduction in the absence of FOXP3, including upregulation of Treg-related genes. Furthermore, we observe specific preservation of a polyclonal TCR repertoire during in vitro cell production. Both allogeneic and autologous CD4LVFOXP3 cells protect from xeno-GvHD after two sequential infusions of effector T cells. CD4LVFOXP3 cells prevent hyper-proliferation of CD4+ memory T cells in the FOXP3-deficient IPEX-like hu-mice. CD4LVFOXP3 cells do not impede in vivo expansion of antigen-primed T cells or tumor clearance in the PB hu-mice. Conclusion: These data support the clinical readiness of CD4LVFOXP3 cells to treat IPEX syndrome and other immune-mediated diseases caused by insufficient or dysfunctional FOXP3+ Tregs.

11.
Front Immunol ; 11: 2194, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33133064

RESUMO

The development of novel approaches to control unwanted immune responses represents an ambitious goal in the management of a number of clinical conditions, including autoimmunity, autoinflammatory diseases, allergies and replacement therapies, in which the T cell response to self or non-harmful antigens threatens the physiological function of tissues and organs. Current treatments for these conditions rely on the use of non-specific immunosuppressive agents and supportive therapies, which may efficiently dampen inflammation and compensate for organ dysfunction, but they require lifelong treatments not devoid of side effects. These limitations induced researchers to undertake the development of definitive and specific solutions to these disorders: the underlying principle of the novel approaches relies on the idea that empowering the tolerogenic arm of the immune system would restore the immune homeostasis and control the disease. Researchers effort resulted in the development of cell-free strategies, including gene vaccination, protein-based approaches and nanoparticles, and an increasing number of clinical trials tested the ability of adoptive transfer of regulatory cells, including T and myeloid cells. Here we will provide an overview of the most promising approaches currently under development, and we will discuss their potential advantages and limitations. The field is teaching us that the success of these strategies depends primarily on our ability to dampen antigen-specific responses without impairing protective immunity, and to manipulate directly or indirectly the immunomodulatory properties of antigen presenting cells, the ultimate in vivo mediators of tolerance.


Assuntos
Transferência Adotiva , Autoantígenos/imunologia , Doenças Autoimunes , Dessensibilização Imunológica , Tolerância Imunológica , Células Mieloides , Linfócitos T Reguladores , Doenças Autoimunes/imunologia , Doenças Autoimunes/terapia , Humanos , Células Mieloides/imunologia , Células Mieloides/transplante , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/transplante
12.
Haematologica ; Online ahead of print2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-33054128

RESUMO

Type 1 regulatory (Tr1) T cells induced by enforced expression of IL-10 (LV-10) are being developed as a novel treatment for chemotherapy-resistant myeloid leukemias. In vivo, LV-10 cells do not cause graft vs host disease while mediating graft vs leukemia (GvL) effect against adult acute myeloid leukemia (AML). Since pediatric AML (pAML) and adult AML are different on a genetic and epigenetic level, we investigate herein whether LV-10 cells also efficiently kill pAML cells. We show that the majority of primary pAML are killed by LV-10 cells, with different levels of sensitivity to killing. Transcriptionally, pAML sensitive to LV-10 killing expressed a myeloid maturation signature. Overlaying the signatures of sensitive and resistant pAML onto the public NCI TARGET pAML dataset revealed that sensitive pAML clustered with M5 monocytic pAML and pAML with MLL rearrangement. Resistant pAML clustered with myelomonocytic leukemias and those bearing the core binding factor translocations inv(16) or t(8;21)(RUNX1-RUNX1T1). Furthermore, resistant pAML upregulated the membrane glycoprotein CD200, which binds to the inhibitory receptor CD200R1 on LV-10 cells. To examine if CD200 expression on target cells can impair LV-10 cell function, we overexpressed CD200 in myeloid leukemia cell lines ordinarily sensitive to LV-10 killing. Indeed, LV-10 cells degranulated less and killed fewer CD200-overexpressing cells compared to controls, indicating that pAML can utilize CD200 expression for immune evasion. Altogether, the majority of pAML are killed by LV-10 cells in vitro, supporting further LV-10 cell development as an innovative cell therapy for pAML.


Assuntos
Leucemia Mieloide Aguda , Linfócitos T Reguladores , Adulto , Linfócitos T CD4-Positivos , Criança , Efeito Enxerto vs Leucemia , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Translocação Genética
13.
Front Immunol ; 11: 1178, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32733439

RESUMO

The non-classical HLA-G is a well-known immune-modulatory molecule. In physiological condition, HLA-G surface expression is restricted to the maternal-fetal interface and to immune-privileged adult tissues, whereas soluble forms of HLA-G are detectable in various body fluids. HLA-G can be de novo expressed in pathological conditions including tumors, chronic infections, or after allogeneic transplantation. HLA-G exerts positive effects modulating innate and adaptive immune responses and promoting tolerance, or detrimental effects inducing immune escape mechanisms. HLA-G locus, in contrast to classical HLA class I gene, is highly polymorphic in the non-coding 3' untranslated region (UTR) and in the 5' upstream regulatory region (5' URR). Variability in these regions influences HLA-G expression by modifying mRNA stability or allowing posttranscriptional regulation in the case of 3' UTR or by sensing the microenvironment and responding to specific stimuli in the case of HLA-G promoter regions (5' URR). The influence of genetic variations on the expression of HLA-G makes it an attractive biomarker to monitor disease predisposition and progression, or response to therapy. Here, we summarize the current knowledge, efforts, and obstacles to generate a general consensus on the correlation between HLA-G genetic variability, protein expression, and disease predisposition. Moreover, we discuss perspectives for future investigation on HLA-G genotype/expression in association with disease predisposition and progression.


Assuntos
Predisposição Genética para Doença , Antígenos HLA-G/genética , Antígenos HLA-G/imunologia , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Genótipo , Humanos , Neoplasias/genética , Neoplasias/imunologia
14.
Front Immunol ; 11: 1260, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32695103

RESUMO

The prominent role of dendritic cells (DC) in promoting tolerance and the development of methods to generate clinical grade products allowed the clinical application of tolerogenic DC (tolDC)-based therapies for controlling unwanted immune responses. We established an efficient method to generate tolerogenic human DC, producing supra-physiological levels of IL-10, by genetically engineering monocyte-derived DC with a bidirectional Lentiviral Vector (bdLV) encoding for IL-10 and a marker gene. DCIL-10 are mature DC, modulate T cell responses, promote T regulatory cells, and are phenotypically and functionally stable upon stimulation. Adoptive transfer of human DCIL-10 in a humanized mouse model dampens allogeneic T cell recall responses, while murine DCIL-10 delays acute graft-vs.-host disease in mice. Our report outlines an efficient method to transduce human myeloid cells with large-size LV and shows that stable over-expression of IL-10 generates an effective cell product for future clinical applications in the contest of allogeneic transplantation.


Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Vetores Genéticos/genética , Tolerância Imunológica , Interleucina-10/genética , Lentivirus/genética , Transdução Genética , Animais , Feminino , Expressão Gênica , Humanos , Tolerância Imunológica/genética , Imunofenotipagem , Camundongos , Monócitos/imunologia , Monócitos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo
15.
Mol Genet Metab ; 130(3): 197-208, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32439268

RESUMO

Mucopolysaccharidosis type I (MPS-I), a lysosomal storage disorder caused by a deficiency of alpha-L-iduronidase enzyme, results in the progressive accumulation of glycosaminoglycans and consequent multiorgan dysfunction. Despite the effectiveness of hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT) in correcting clinical manifestations related to visceral organs, complete improvement of musculoskeletal and neurocognitive defects remains an unmet challenge and provides an impact on patients' quality of life. We tested the therapeutic efficacy of combining HSCT and ERT in the neonatal period. Using a mouse model of MPS-I, we demonstrated that the combination therapy improved clinical manifestations in organs usually refractory to current treatment. Moreover, combination with HSCT prevented the production of anti-IDUA antibodies that negatively impact ERT efficacy. The added benefits of combining both treatments also resulted in a reduction of skeletal anomalies and a trend towards decreased neuroinflammation and metabolic abnormalities. As currently there are limited therapeutic options for MPS-I patients, our findings suggest that the combination of HSCT and ERT during the neonatal period may provide a further step forward in the treatment of this rare disease.


Assuntos
Remodelação Óssea , Modelos Animais de Doenças , Terapia de Reposição de Enzimas/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Iduronidase/fisiologia , Mucopolissacaridose I/terapia , Animais , Animais Recém-Nascidos , Terapia Combinada , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucopolissacaridose I/enzimologia , Mucopolissacaridose I/patologia
16.
Elife ; 92020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32207685

RESUMO

Rett syndrome is an incurable neurodevelopmental disorder caused by mutations in the gene encoding for methyl-CpG binding-protein 2 (MeCP2). Gene therapy for this disease presents inherent hurdles since MECP2 is expressed throughout the brain and its duplication leads to severe neurological conditions as well. Herein, we use the AAV-PHP.eB to deliver an instability-prone Mecp2 (iMecp2) transgene cassette which, increasing RNA destabilization and inefficient protein translation of the viral Mecp2 transgene, limits supraphysiological Mecp2 protein levels. Intravenous injections of the PHP.eB-iMecp2 virus in symptomatic Mecp2 mutant mice significantly improved locomotor activity, lifespan and gene expression normalization. Remarkably, PHP.eB-iMecp2 administration was well tolerated in female Mecp2 mutant or in wild-type animals. In contrast, we observed a strong immune response to the transgene in treated male Mecp2 mutant mice that was overcome by immunosuppression. Overall, PHP.eB-mediated delivery of iMecp2 provided widespread and efficient gene transfer maintaining physiological Mecp2 protein levels in the brain.


Assuntos
Encéfalo/metabolismo , Proteína 2 de Ligação a Metil-CpG/genética , Mutação/genética , Síndrome de Rett/genética , Animais , Modelos Animais de Doenças , Expressão Gênica/fisiologia , Regulação da Expressão Gênica , Terapia Genética/métodos , Camundongos Transgênicos , Transgenes/genética
17.
Methods Enzymol ; 632: 155-192, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32000895

RESUMO

Inhibition of T-cell proliferation is the most common approach to assess human myeloid-derived suppressor cell (MDSC) functions. However, diverse methodologies hinder the comparison of results obtained in different laboratories. In this chapter, we present a T-cell proliferation assay procedure based on allogeneic MDSC and T-cells that is potentially suitable to multi-center studies. The T-cells are isolated from non-cancerous donors and frozen for later use in different research groups. We observed that pure thawed T-cells showed poor proliferative capacities. To retain proliferation, T-cell-autologous mature dendritic cells are supplemented after thawing. MDSC are isolated from clinical samples and represent the sole variant between assays. Flow cytometry is used to assess T-cell proliferation by the dilution of a tracking dye.


Assuntos
Proliferação de Células , Técnicas de Cocultura/métodos , Células Supressoras Mieloides/imunologia , Linfócitos T/imunologia , Separação Celular/métodos , Humanos , Ativação Linfocitária , Células Supressoras Mieloides/citologia , Coloração e Rotulagem/métodos , Linfócitos T/citologia
18.
Cell Mol Immunol ; 17(1): 95-107, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-30842629

RESUMO

Tolerogenic dendritic cells (DCs) are key players in maintaining immunological homeostasis, dampening immune responses, and promoting tolerance. DC-10, a tolerogenic population of human IL-10-producing DCs characterized by the expression of HLA-G and ILT4, play a pivotal role in promoting tolerance via T regulatory type 1 (Tr1) cells. Thus far, the absence of markers that uniquely identify DC-10 has limited in vivo studies. By in vitro gene expression profiling of differentiated human DCs, we identified CD141 and CD163 as surface markers for DC-10. The coexpression of CD141 and CD163 in combination with CD14 and CD16 enables the ex vivo isolation of DC-10 from the peripheral blood. CD14+CD16+CD141+CD163+ cells isolated from the peripheral blood of healthy subjects (ex vivo DC-10) produced spontaneously and upon activation of IL-10 and limited levels of IL-12. Moreover, in vitro stimulation of allogeneic naive CD4+ T cells with ex vivo DC-10 induced the differentiation of alloantigen-specific CD49b+LAG-3+ Tr1 cells. Finally, ex vivo DC-10 and in vitro generated DC-10 exhibited a similar transcriptional profile, which are characterized by an anti-inflammatory and pro-tolerogenic signature. These results provide new insights into the phenotype and molecular signature of DC-10 and highlight the tolerogenic properties of circulating DC-10. These findings open the opportunity to track DC-10 in vivo and to define their role in physiological and pathological settings.


Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Interleucina-10/imunologia , Receptores de Superfície Celular/imunologia , Trombomodulina/imunologia , Células Dendríticas/citologia , Humanos , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia
19.
Front Immunol ; 11: 608614, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33505397

RESUMO

Invariant Natural Killer T (iNKT) cells are a small and distinct population of T cells crucial in immunomodulation. After activation by alpha-GalactosylCeramide (αGC), an exogenic glycolipid antigen, iNKT cells can rapidly release cytokines to enhance specific anti-tumor activity. Several human clinical trials on iNKT cell-based anti-cancer are ongoing, however results are not as striking as in murine models. Given that iNKT-based immunotherapies are dependent mainly on antigen-presenting cells (APC), a human tolerogenic molecule with no murine homolog, such as Human Leucocyte Antigen G (HLA-G), could contribute to this discrepancy. HLA-G is a well-known immune checkpoint molecule involved in fetal-maternal tolerance and in tumor immune escape. HLA-G exerts its immunomodulatory functions through the interaction with immune inhibitory receptors such as ILT2, differentially expressed on immune cell subsets. We hypothesized that HLA-G might inhibit iNKT function directly or by inducing tolerogenic APC leading to iNKT cell anergy, which could impact the results of current clinical trials. Using an ILT2-transduced murine iNKT cell line and human iNKT cells, we demonstrate that iNKT cells are sensitive to HLA-G, which inhibits their cytokine secretion. Furthermore, human HLA-G+ dendritic cells, called DC-10, failed at inducing iNKT cell activation compared to their autologous HLA-G‒ DCs counterparts. Our data show for the first time that the HLA-G/ILT2 ICP is involved in iNKT cell function modulation.


Assuntos
Antígenos CD/imunologia , Células Dendríticas/imunologia , Antígenos HLA-G/imunologia , Receptor B1 de Leucócitos Semelhante a Imunoglobulina/imunologia , Células T Matadoras Naturais/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Cultivadas , Citocinas/imunologia , Humanos , Tolerância Imunológica/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL
20.
Front Neurol ; 10: 916, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31507519

RESUMO

Background: Few studies have investigated the experiences of patients around the conversion to secondary progressive multiple sclerosis (SPMS). ManTra is a mixed-method, co-production research project conducted in Italy and Germany to develop an intervention for newly-diagnosed SPMS patients. In previous project actions, we identified the needs and experiences of patients converting to SPMS via literature review and qualitative research which involved key stakeholders. Aims: The online patient survey aimed to assess, on a larger and independent sample of recently-diagnosed SPMS patients: (a) the characteristics associated to patient awareness of SPMS conversion; (b) the experience of conversion; (c) importance and prioritization of the needs previously identified. Methods: Participants were consenting adults with SPMS since ≤5 years. The survey consisted of three sections: on general and clinical characteristics; on experience of SPMS diagnosis disclosure (aware participants only); and on importance and prioritization of 33 pre-specified needs. Results: Of 215 participants, those aware of their SPMS diagnosis were 57% in Italy vs. 77% in Germany (p = 0.004). In both countries, over 80% of aware participants received a SPMS diagnosis from the neurologist; satisfaction with SPMS disclosure was moderate to high. Nevertheless, 28-35% obtained second opinions, and 48-56% reported they did not receive any information on SPMS. Participants actively seeking further information were 63% in Germany vs. 31% in Italy (p < 0.001). Variables independently associated to patient awareness were geographic area (odds ratio, OR 0.32, 95% CI 0.13-0.78 for Central Italy; OR 0.21, 95% CI 0.08-0.58 for Southern Italy [vs. Germany]) and activity limitations (OR 7.80, 95% CI 1.47-41.37 for dependent vs. autonomous patients). All pre-specified needs were scored a lot or extremely important, and two prioritized needs were shared by Italian and German patients: "physiotherapy" and "active patient care involvement." The other two differed across countries: "an individualized health care plan" and "information on social rights and policies" in Italy, and "psychological support" and "cognitive rehabilitation" in Germany. Conclusions: Around 40% of SPMS patients were not aware of their disease form indicating a need to improve patient-physician communication. Physiotherapy and active patient care involvement were prioritized in both countries.

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