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1.
J Phys Chem Lett ; 10(22): 7230-7236, 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31689111

RESUMO

The unique properties of liquid water mainly arise from its hydrogen bond network. The geometry and dynamics of this network play a key role in shaping the characteristics of soft matter, from simple solutions to biosystems. Here we report an anomalous intrinsic fluorescence of HCl and NaOH aqueous solutions at room temperature that shows important differences in the excitation and emission bands between the two solutes. From ab initio time-dependent density functional theory modeling we propose that fluorescence emission could originate from hydrated ion species contained in transient cavities of the bulk solvent. These cavities, which are characterized by a stiff surface, could provide an environment that, upon trapping the excited state, suppresses the fast nonradiative decay and allows the slower radiative channel to become a possible decay pathway.

2.
JCI Insight ; 2019 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-31689241

RESUMO

γδ T cells account for a large fraction of human intestinal intraepithelial lymphocytes (IELs) endowed with potent anti-tumor activities. However, little is known about their origin, phenotype and clinical relevance in colorectal cancer (CRC). To determine γδ IEL gut-specificity, homing and functions, γδ T cells were purified from human healthy blood, lymph nodes, liver, skin, intestine either disease-free or affected by CRC or generated from thymic precursors. The constitutive expression of NKp46 specifically identifies a new subset of cytotoxic Vδ1 T cells representing the largest fraction of gut-resident IELs. The ontogeny and gut-tropism of NKp46pos/Vδ1 IELs depends both on distinctive features of Vδ1 thymic precursors and gut-environmental factors. Either the constitutive presence of NKp46 on tissue-resident Vδ1 intestinal IELs or its induced-expression on IL-2/IL-15 activated Vδ1 thymocytes are associated with anti-tumor functions. Higher frequencies of NKp46pos/Vδ1 IELs in tumor-free specimens from CRC patients correlate with a lower risk of developing metastatic III/IV disease stages. Additionally, our in vitro settings reproducing CRC tumor-microenvironment inhibited the expansion of NKp46pos/Vδ1 cells from activated thymic precursors. These results parallel the very low frequencies of NKp46pos/Vδ1 IELs able to infiltrate CRC, thus providing new insights to either follow-up cancer progression or develop novel adoptive cellular therapies.

3.
Pharm Pat Anal ; 8(5): 193-202, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31612798

RESUMO

The worldwide active patent portfolio has nearly doubled in numbers and strength since 2000. The number of active pharmaceutical patent families has tripled in the same time period. The quantitative growth results mostly from a surge of patents from China, half of them classified in A61K36 ('medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants'). High-quality patents exhibit a slower growth curve, and cluster within the three areas biologicals; heterocyclic compounds, and cancer drugs. However, the highest concentration of high-quality patents was found when selecting patents listing inventors from at least two out of the five most important countries of origin for pharmaceutical patents: China, EP countries, Japan, South Korea and the USA.

4.
J Bone Miner Res ; 34(11): 2133-2148, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31295380

RESUMO

Controlling oxidative stress through the activation of antioxidant pathways is crucial in bone homeostasis, and impairments of the cellular defense systems involved contribute to the pathogenesis of common skeletal diseases. In this work we focused on the dipeptidyl peptidase 3 (DPP3), a poorly investigated ubiquitous zinc-dependent exopeptidase activating the Keap1-Nrf2 antioxidant pathway. We showed Dpp3 expression in bone and, to understand its role in this compartment, we generated a Dpp3 knockout (KO) mouse model and specifically investigated the skeletal phenotype. Adult Dpp3 KO mice showed a mild growth defect, a significant increase in bone marrow cellularity, and bone loss mainly caused by increased osteoclast activity. Overall, in the mouse model, lack of DPP3 resulted in sustained oxidative stress and in alterations of bone microenvironment favoring the osteoclast compared to the osteoblast lineage. Accordingly, in vitro studies revealed that Dpp3 KO osteoclasts had an inherent increased resorptive activity and ROS production, which on the other hand made them prone to apoptosis. Moreover, absence of DPP3 augmented bone loss after estrogen withdrawal in female mice, further supporting its relevance in the framework of bone pathophysiology. Overall, we show a nonredundant role for DPP3 in the maintenance of bone homeostasis and propose that DPP3 might represent a possible new osteoimmunological player and a marker of human bone loss pathology. © 2019 American Society for Bone and Mineral Research.

6.
Stem Cells Transl Med ; 8(10): 1107-1122, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31140762

RESUMO

Defective functionality of thymic epithelial cells (TECs), due to genetic mutations or injuring causes, results in altered T-cell development, leading to immunodeficiency or autoimmunity. These defects cannot be corrected by hematopoietic stem cell transplantation (HSCT), and thymus transplantation has not yet been demonstrated to be fully curative. Here, we provide proof of principle of a novel approach toward thymic regeneration, involving the generation of thymic organoids obtained by seeding gene-modified postnatal murine TECs into three-dimensional (3D) collagen type I scaffolds mimicking the thymic ultrastructure. To this end, freshly isolated TECs were transduced with a lentiviral vector system, allowing for doxycycline-induced Oct4 expression. Transient Oct4 expression promoted TECs expansion without drastically changing the cell lineage identity of adult TECs, which retain the expression of important molecules for thymus functionality such as Foxn1, Dll4, Dll1, and AIRE. Oct4-expressing TECs (iOCT4 TEC) were able to grow into 3D collagen type I scaffolds both in vitro and in vivo, demonstrating that the collagen structure reproduced a 3D environment similar to the thymic extracellular matrix, perfectly recognized by TECs. In vivo results showed that thymic organoids transplanted subcutaneously in athymic nude mice were vascularized but failed to support thymopoiesis because of their limited in vivo persistence. These findings provide evidence that gene modification, in combination with the usage of 3D biomimetic scaffolds, may represent a novel approach allowing the use of postnatal TECs for thymic regeneration. Stem Cells Translational Medicine 2019;8:1107-1122.

8.
Lancet Haematol ; 6(5): e239-e253, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30981783

RESUMO

BACKGROUND: Wiskott-Aldrich syndrome is a rare, life-threatening, X-linked primary immunodeficiency characterised by microthrombocytopenia, infections, eczema, autoimmunity, and malignant disease. Lentiviral vector-mediated haemopoietic stem/progenitor cell (HSPC) gene therapy is a potentially curative treatment that represents an alternative to allogeneic HSPC transplantation. Here, we report safety and efficacy data from an interim analysis of patients with severe Wiskott-Aldrich syndrome who received lentiviral vector-derived gene therapy. METHODS: We did a non-randomised, open-label, phase 1/2 clinical study in paediatric patients with severe Wiskott-Aldrich syndrome, defined by either WAS gene mutation or absent Wiskott-Aldrich syndrome protein (WASP) expression or a Zhu clinical score of 3 or higher. We included patients who had no HLA-identical sibling donor available or, for children younger than 5 years of age, no suitable 10/10 matched unrelated donor or 6/6 unrelated cord blood donor. After treatment with rituximab and a reduced-intensity conditioning regimen of busulfan and fludarabine, patients received one intravenous infusion of autologous CD34+ cells genetically modified with a lentiviral vector encoding for human WAS cDNA. The primary safety endpoints were safety of the conditioning regimen and safety of lentiviral gene transfer into HSPCs. The primary efficacy endpoints were overall survival, sustained engraftment of genetically corrected HSPCs, expression of vector-derived WASP, improved T-cell function, antigen-specific responses to vaccinations, and improved platelet count and mean platelet volume normalisation. This interim analysis was done when the first six patients treated had completed at least 3 years of follow-up. The planned analyses are presented for the intention-to-treat population. This trial is registered with ClinicalTrials.gov (number NCT01515462) and EudraCT (number 2009-017346-32). FINDINGS: Between April 20, 2010, and Feb 26, 2015, nine patients (all male) were enrolled of whom one was excluded after screening; the age range of the eight treated children was 1·1-12·4 years. At the time of the interim analysis (data cutoff April 29, 2016), median follow-up was 3·6 years (range 0·5-5·6). Overall survival was 100%. Engraftment of genetically corrected HSPCs was successful and sustained in all patients. The fraction of WASP-positive lymphocytes increased from a median of 3·9% (range 1·8-35·6) before gene therapy to 66·7% (55·7-98·6) at 12 months after gene therapy, whereas WASP-positive platelets increased from 19·1% (range 4·1-31·0) to 76·6% (53·1-98·4). Improvement of immune function was shown by normalisation of in-vitro T-cell function and successful discontinuation of immunoglobulin supplementation in seven patients with follow-up longer than 1 year, followed by positive antigen-specific response to vaccination. Severe infections fell from 2·38 (95% CI 1·44-3·72) per patient-year of observation (PYO) in the year before gene therapy to 0·31 (0·04-1·11) per PYO in the second year after gene therapy and 0·17 (0·00-0·93) per PYO in the third year after gene therapy. Before gene therapy, platelet counts were lower than 20 × 109 per L in seven of eight patients. At the last follow-up visit, the platelet count had increased to 20-50 × 109 per L in one patient, 50-100 × 109 per L in five patients, and more than 100 × 109 per L in two patients, which resulted in independence from platelet transfusions and absence of severe bleeding events. 27 serious adverse events in six patients occurred after gene therapy, 23 (85%) of which were infectious (pyrexia [five events in three patients], device-related infections, including one case of sepsis [four events in three patients], and gastroenteritis, including one case due to rotavirus [three events in two patients]); these occurred mainly in the first 6 months of follow-up. No adverse reactions to the investigational drug product and no abnormal clonal proliferation or leukaemia were reported after gene therapy. INTERPRETATION: Data from this study show that gene therapy provides a valuable treatment option for patients with severe Wiskott-Aldrich syndrome, particularly for those who do not have a suitable HSPC donor available. FUNDING: Italian Telethon Foundation, GlaxoSmithKline, and Orchard Therapeutics.


Assuntos
Terapia Genética , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/metabolismo , Lentivirus/genética , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Criança , Pré-Escolar , Feminino , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Lactente , Itália , Masculino , Mutação , Linfócitos T/imunologia , Linfócitos T/metabolismo , Condicionamento Pré-Transplante/métodos , Resultado do Tratamento , Síndrome de Wiskott-Aldrich/sangue , Síndrome de Wiskott-Aldrich/diagnóstico , Proteína da Síndrome de Wiskott-Aldrich/genética
9.
Front Immunol ; 10: 447, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30949166

RESUMO

The thymus plays a fundamental role in establishing and maintaining central and peripheral tolerance and defects in thymic architecture or AIRE expression result in the development of autoreactive lymphocytes. Patients with partial DiGeorge Syndrome (pDGS) and Down Syndrome (DS) present alterations in size and architecture of the thymus and higher risk to develop autoimmunity. We sought to evaluate thymic architecture and thymocyte development in DGS and DS patients and to determine the extent to which thymic defects result in immune dysregulation and T cell homeostasis perturbation in these patients. Thymi from pediatric patients and age-matched controls were obtained to evaluate cortex and medullary compartments, AIRE expression and thymocyte development. In the same patients we also characterized immunophenotype of peripheral T cells. Phenotypic and functional characterization of thymic and peripheral regulatory T (Treg) cells was finally assessed. Histologic analysis revealed peculiar alterations in thymic medulla size and maturation in DGS and DS patients. Perturbed distribution of thymocytes and altered thymic output was also observed. DGS patients showed lower mature CD4+ and CD8+ T cell frequency, associated with reduced proportion and function of Tregs both in thymus and peripheral blood. DS patients showed increased frequency of single positive (SP) thymocytes and thymic Treg cells. However, Tregs isolated both from thymus and peripheral blood of DS patients showed reduced suppressive ability. Our results provide novel insights on thymic defects associated with DGS and DS and their impact on peripheral immune dysregulation. Indeed, thymic abnormalities and defect in thymocyte development, in particular in Treg cell number and function could contribute in the pathogenesis of the immunodysregulation present in pDGS and in DS patients.

10.
Front Immunol ; 10: 629, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30984193

RESUMO

The identification of Receptor activator of nuclear factor kappa B ligand (RANKL) and its cognate receptor Receptor activator of nuclear factor kappa B (RANK) during a search for novel tumor necrosis factor receptor (TNFR) superfamily members has dramatically changed the scenario of bone biology by providing the functional and biochemical proof that RANKL signaling via RANK is the master factor for osteoclastogenesis. In parallel, two independent studies reported the identification of mouse RANKL on activated T cells and of a ligand for osteoprotegerin on a murine bone marrow-derived stromal cell line. After these seminal findings, accumulating data indicated RANKL and RANK not only as essential players for the development and activation of osteoclasts, but also for the correct differentiation of medullary thymic epithelial cells (mTECs) that act as mediators of the central tolerance process by which self-reactive T cells are eliminated while regulatory T cells are generated. In light of the RANKL-RANK multi-task function, an antibody targeting this pathway, denosumab, is now commonly used in the therapy of bone loss diseases including chronic inflammatory bone disorders and osteolytic bone metastases; furthermore, preclinical data support the therapeutic application of denosumab in the framework of a broader spectrum of tumors. Here, we discuss advances in cellular and molecular mechanisms elicited by RANKL-RANK pathway in the bone and thymus, and the extent to which its inhibition or augmentation can be translated in the clinical arena.

11.
Front Immunol ; 10: 316, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31031743

RESUMO

Background: Primary Immunodeficiencies (PIDs) are a heterogeneous group of genetic immune disorders. While some PIDs can manifest with more than one phenotype, signs, and symptoms of various PIDs overlap considerably. Recently, novel defects in immune-related genes and additional variants in previously reported genes responsible for PIDs have been successfully identified by Next Generation Sequencing (NGS), allowing the recognition of a broad spectrum of disorders. Objective: To evaluate the strength and weakness of targeted NGS sequencing using custom-made Ion Torrent and Haloplex (Agilent) panels for diagnostics and research purposes. Methods: Five different panels including known and candidate genes were used to screen 105 patients with distinct PID features divided in three main PID categories: T cell defects, Humoral defects and Other PIDs. The Ion Torrent sequencing platform was used in 73 patients. Among these, 18 selected patients without a molecular diagnosis and 32 additional patients were analyzed by Haloplex enrichment technology. Results: The complementary use of the two custom-made targeted sequencing approaches allowed the identification of causative variants in 28.6% (n = 30) of patients. Twenty-two out of 73 (34.6%) patients were diagnosed by Ion Torrent. In this group 20 were included in the SCID/CID category. Eight out of 50 (16%) patients were diagnosed by Haloplex workflow. Ion Torrent method was highly successful for those cases with well-defined phenotypes for immunological and clinical presentation. The Haloplex approach was able to diagnose 4 SCID/CID patients and 4 additional patients with complex and extended phenotypes, embracing all three PID categories in which this approach was more efficient. Both technologies showed good gene coverage. Conclusions: NGS technology represents a powerful approach in the complex field of rare disorders but its different application should be weighted. A relatively small NGS target panel can be successfully applied for a robust diagnostic suspicion, while when the spectrum of clinical phenotypes overlaps more than one PID an in-depth NGS analysis is required, including also whole exome/genome sequencing to identify the causative gene.

12.
J Allergy Clin Immunol ; 144(3): 825-838, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30926529

RESUMO

BACKGROUND: Thrombocytopenia is a serious issue for all patients with classical Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) because it causes severe and life-threatening bleeding. Lentiviral gene therapy (GT) for WAS has shown promising results in terms of immune reconstitution. However, despite the reduced severity and frequency of bleeding events, platelet counts remain low in GT-treated patients. OBJECTIVE: We carefully investigated platelet defects in terms of phenotype and function in untreated patients with WAS and assessed the effect of GT treatment on platelet dysfunction. METHODS: We analyzed a cohort of 20 patients with WAS/XLT, 15 of them receiving GT. Platelet phenotype and function were analyzed by using electron microscopy, flow cytometry, and an aggregation assay. Platelet protein composition was assessed before and after GT by means of proteomic profile analysis. RESULTS: We show that platelets from untreated patients with WAS have reduced size, abnormal ultrastructure, and a hyperactivated phenotype at steady state, whereas activation and aggregation responses to agonists are decreased. GT restores platelet size and function early after treatment and reduces the hyperactivated phenotype proportionally to WAS protein expression and length of follow-up. CONCLUSIONS: Our study highlights the coexistence of morphologic and multiple functional defects in platelets lacking WAS protein and demonstrates that GT normalizes the platelet proteomic profile with consequent restoration of platelet ultrastructure and phenotype, which might explain the observed reduction of bleeding episodes after GT. These results are instrumental also from the perspective of a future clinical trial in patients with XLT only presenting with microthrombocytopenia.

13.
Stem Cells ; 37(7): 876-887, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30895693

RESUMO

In spite of the progress in gene editing achieved in recent years, a subset of genetic diseases involving structural chromosome abnormalities, including aneuploidies, large deletions and complex rearrangements, cannot be treated with conventional gene therapy approaches. We have previously devised a strategy, dubbed chromosome transplantation (CT), to replace an endogenous mutated chromosome with an exogenous normal one. To establish a proof of principle for our approach, we chose as disease model the chronic granulomatous disease (CGD), an X-linked severe immunodeficiency due to abnormalities in CYBB (GP91) gene, including large genomic deletions. We corrected the gene defect by CT in induced pluripotent stem cells (iPSCs) from a CGD male mouse model. The Hprt gene of the endogenous X chromosome was inactivated by CRISPR/Cas9 technology thus allowing the exploitation of the hypoxanthine-aminopterin-thymidine selection system to introduce a normal donor X chromosome by microcell-mediated chromosome transfer. X-transplanted clones were obtained, and diploid XY clones which spontaneously lost the endogenous X chromosome were isolated. These cells were differentiated toward the myeloid lineage, and functional granulocytes producing GP91 protein were obtained. We propose the CT approach to correct iPSCs from patients affected by other X-linked diseases with large deletions, whose treatment is still unsatisfactory. Stem Cells 2019;37:876-887.

14.
Haematologica ; 104(9): 1744-1755, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30792210

RESUMO

The balance between self-renewal and differentiation is crucial to ensure the homeostasis of the hematopoietic system, and is a hallmark of hematopoietic stem cells. However, the underlying molecular pathways, including the role of micro-RNA, are not completely understood. To assess the contribution of micro-RNA, we performed micro-RNA profiling of hematopoietic stem cells and their immediate downstream progeny multi-potent progenitors from wild-type control and Pbx1-conditional knockout mice, whose stem cells display a profound self-renewal defect. Unsupervised hierarchical cluster analysis separated stem cells from multi-potent progenitors, suggesting that micro-RNA might regulate the first transition step in the adult hematopoietic development. Notably, Pbx1-deficient and wild-type cells clustered separately, linking micro-RNAs to self-renewal impairment. Differential expression analysis of micro-RNA in the physiological stem cell-to-multi-potent progenitor transition and in Pbx1-deficient stem cells compared to control stem cells revealed miR-127-3p as the most differentially expressed. Furthermore, miR-127-3p was strongly stem cell-specific, being quickly down-regulated upon differentiation and not re-expressed further downstream in the bone marrow hematopoietic hierarchy. Inhibition of miR-127-3p function in Lineage-negative cells, achieved through a lentiviral-sponge vector, led to severe stem cell depletion, as assessed with serial transplantation assays. miR-127-3p-sponged stem cells displayed accelerated differentiation, which was uncoupled from proliferation, accounting for the observed stem cell reduction. miR-127-3p overexpression in Lineage-negative cells did not alter stem cell pool size, but gave rise to lymphopenia, likely due to lack of miR-127-3p physiological downregulation beyond the stem cell stage. Thus, tight regulation of miR-127-3p is crucial to preserve the self-renewing stem cell pool and homeostasis of the hematopoietic system.

15.
J Autoimmun ; 98: 132-148, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30704941

RESUMO

Primary immunodeficiencies (PIDs) are inherited disorders of the immune system, associated with a considerable increase in susceptibility to infections. PIDs can also predispose to malignancy, inflammation and autoimmunity. There is increasing awareness that some aspects of the immune dysregulation in PIDs may be linked to intestinal microbiota. Indeed, the gut microbiota and its metabolites have been shown to influence immune functions and immune homeostasis both locally and systemically. Recent studies have indicated that genetic defects causing PIDs lead to perturbations in the conventional mechanisms underlying homeostasis in the gut, resulting in poor immune surveillance at the intestinal barrier, which associates with altered intestinal permeability and bacterial translocation. Consistently, a substantial proportion of PID patients presents with clinically challenging IBD-like pathology. Here, we describe the current body of literature reporting on dysbiosis of the gut microbiota in different PIDs and how this can be either the result or cause of immune dysregulation. Further, we report how infections in PIDs enhance pathobionts colonization and speculate how, in turn, pathobionts may be responsible for increased disease susceptibility and secondary infections in these patients. The potential relationship between the microbial composition in the intestine and other sites, such as the oral cavity and skin, is also highlighted. Finally, we provide evidence, in preclinical models of PIDs, for the efficacy of microbiota manipulation to ameliorate disease complications, and suggest that the potential use of dietary intervention to correct dysbiotic flora in PID patients may hold promise.

16.
Immunol Rev ; 287(1): 73-90, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30565244

RESUMO

Mutations of the recombinase activating genes (RAG) in humans underlie a broad spectrum of clinical and immunological phenotypes that reflect different degrees of impairment of T- and B-cell development and alterations of mechanisms of central and peripheral tolerance. Recent studies have shown that this phenotypic heterogeneity correlates, albeit imperfectly, with different levels of recombination activity of the mutant RAG proteins. Furthermore, studies in patients and in newly developed animal models carrying hypomorphic RAG mutations have disclosed various mechanisms underlying immune dysregulation in this condition. Careful annotation of clinical outcome and immune reconstitution in RAG-deficient patients who have received hematopoietic stem cell transplantation has shown that progress has been made in the treatment of this disease, but new approaches remain to be tested to improve stem cell engraftment and durable immune reconstitution. Finally, initial attempts have been made to treat RAG deficiency with gene therapy.

18.
Int J Mol Sci ; 19(10)2018 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-30322134

RESUMO

Mesenchymal stem cells (MSCs) are recognized as an attractive tool owing to their self-renewal and differentiation capacity, and their ability to secrete bioactive molecules and to regulate the behavior of neighboring cells within different tissues. Accumulating evidence demonstrates that cells prefer three-dimensional (3D) to 2D culture conditions, at least because the former are closer to their natural environment. Thus, for in vitro studies and in vivo utilization, great effort is being dedicated to the optimization of MSC 3D culture systems in view of achieving the intended performance. This implies understanding cell⁻biomaterial interactions and manipulating the physicochemical characteristics of biomimetic scaffolds to elicit a specific cell behavior. In the bone field, biomimetic scaffolds can be used as 3D structures, where MSCs can be seeded, expanded, and then implanted in vivo for bone repair or bioactive molecules release. Actually, the union of MSCs and biomaterial has been greatly improving the field of tissue regeneration. Here, we will provide some examples of recent advances in basic as well as translational research about MSC-seeded scaffold systems. Overall, the proliferation of tools for a range of applications witnesses a fruitful collaboration among different branches of the scientific community.

19.
Blood ; 132(22): 2362-2374, 2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30254128

RESUMO

ARPC1B is a key factor for the assembly and maintenance of the ARP2/3 complex that is involved in actin branching from an existing filament. Germline biallelic mutations in ARPC1B have been recently described in 6 patients with clinical features of combined immunodeficiency (CID), whose neutrophils and platelets but not T lymphocytes were studied. We hypothesized that ARPC1B deficiency may also lead to cytoskeleton and functional defects in T cells. We have identified biallelic mutations in ARPC1B in 6 unrelated patients with early onset disease characterized by severe infections, autoimmune manifestations, and thrombocytopenia. Immunological features included T-cell lymphopenia, low numbers of naïve T cells, and hyper-immunoglobulin E. Alteration in ARPC1B protein structure led to absent/low expression by flow cytometry and confocal microscopy. This molecular defect was associated with the inability of patient-derived T cells to extend an actin-rich lamellipodia upon T-cell receptor (TCR) stimulation and to assemble an immunological synapse. ARPC1B-deficient T cells additionally displayed impaired TCR-mediated proliferation and SDF1-α-directed migration. Gene transfer of ARPC1B in patients' T cells using a lentiviral vector restored both ARPC1B expression and T-cell proliferation in vitro. In 2 of the patients, in vivo somatic reversion restored ARPC1B expression in a fraction of lymphocytes and was associated with a skewed TCR repertoire. In 1 revertant patient, memory CD8+ T cells expressing normal levels of ARPC1B displayed improved T-cell migration. Inherited ARPC1B deficiency therefore alters T-cell cytoskeletal dynamics and functions, contributing to the clinical features of CID.

20.
Artigo em Inglês | MEDLINE | ID: mdl-30184340

RESUMO

Biomimetic scaffolds are extremely versatile in terms of chemical composition and physical properties, which can be defined to accomplish specific applications. One property that can be added is the production/release of bioactive soluble factors, either directly from the biomaterial, or from cells embedded within the biomaterial. We reasoned that pursuing this strategy would be appropriate to setup a cell-based therapy for RANKL-deficient Autosomal Recessive Osteopetrosis, a very rare skeletal genetic disease in which lack of the essential osteoclastogenic factor RANKL impedes osteoclast formation. The exogenously administered RANKL cytokine is effective in achieving osteoclast formation and function in vitro and in vivo, thus, we produced murine Rankl-/- MSCs overexpressing human soluble RANKL (hsRL) following lentiviral transduction (LVhsRL). Here, we described a three-dimensional (3D) culture system based on a Magnesium-doped hydroxyapatite/collagen I (MgHA/Col) biocompatible scaffold closely reproducing bone physicochemical properties. MgHA/Col-seeded murine MSCs showed improved properties, as compared to two-dimensional (2D) culture, in terms of proliferation and hsRL production, with respect to LVhsRL-transduced cells. When implanted subcutaneously in Rankl-/- mice, these cell constructs were well tolerated, colonized by host cells, and intensely vascularized. Of note, in the bone of Rankl-/- mice that carried scaffolds with either WT or LVhsRL-transduced Rankl-/- MSCs, we specifically observed formation of TRAP+ cells, likely due to sRL released from the scaffolds into circulation. Thus, our strategy proved to have the potential to elicit an effect on the bone; further work is required to maximize these benefits and achieve improvements of the skeletal pathology in the treated Rankl-/- mice. Stem Cells Translational Medicine 2018.

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