RESUMO
BACKGROUND: A growing number of clinical trials have shown that regulatory T (Treg) cell transfer may have a favorable effect on the maintenance of self-tolerance and immune homeostasis in different conditions such as graft-versus-host disease (GvHD), solid organ transplantation, type 1 diabetes, and others. In this context, the availability of a robust manufacturing protocol that is able to produce a sufficient number of functional Treg cells represents a fundamental prerequisite for the success of a cell therapy clinical protocol. However, extended workflow guidelines for nonprofit manufacturers are currently lacking. Despite the fact that different successful manufacturing procedures and cell products with excellent safety profiles have been reported from early clinical trials, the selection and expansion protocols for Treg cells vary a lot. The objective of this study was to validate a Good Manufacturing Practice (GMP)-compliant protocol for the production of Treg cells that approaches the whole process with a risk-management methodology, from process design to completion of final product development. High emphasis was given to the description of the quality control (QC) methodologies used for the in-process and release tests (sterility, endotoxin test, mycoplasma, and immunophenotype). RESULTS: The GMP-compliant protocol defined in this work allows at least 4.11 × 109 Treg cells to be obtained with an average purity of 95.75 ± 4.38% and can be used in different clinical settings to exploit Treg cell immunomodulatory function. CONCLUSIONS: These results could be of great use for facilities implementing GMP-compliant cell therapy protocols of these cells for different conditions aimed at restoring the Treg cell number and function, which may slow the progression of certain diseases.
Assuntos
Doença Enxerto-Hospedeiro , Linfócitos T Reguladores , Terapia Baseada em Transplante de Células e Tecidos , Humanos , Tolerância Imunológica , Estudos ProspectivosRESUMO
BACKGROUND: The COVID-19 pandemic and the lockdown period lasted from March to May 2020, resulted in a highly stressful situation yielding different negative health consequences, including the worsening of smoking habit. METHODS: A web-based cross-sectional study on a convenient sample of 1013 Italian ever smokers aged 18 years or more was conducted. Data were derived from surveys compiled by three different groups of people: subjects belonging to Smoking Cessation Services, Healthcare Providers and Nursing Sciences' students. All institutions were from Northern Italy. The primary outcome self-reported worsening (relapse or increase) or improvement (quit or reduce) of smoking habit during lockdown period. Multiple unconditional (for worsening) and multinomial (for improving) logistic regressions were carried out. RESULTS: Among 962 participants, 56.0% were ex-smokers. Overall, 13.2% of ex-smokers before lockdown reported relapsing and 32.7% of current smokers increasing cigarette intake. Among current smokers before lockdown, 10.1% quit smoking and 13.5% decreased cigarette intake. Out of 7 selected stressors related to COVID-19, four were significantly related to relapse (OR for the highest vs. the lowest tertile ranging between 2.24 and 3.62): fear of being infected and getting sick; fear of dying due to the virus; anxiety in listening to news of the epidemic; sense of powerlessness in protecting oneself from contagion. In addition to these stressors, even the other 3 stressors were related with increasing cigarette intensity (OR ranging between 1.90 and 4.18): sense of powerlessness in protecting loved ones from contagion; fear of losing loved ones due to virus; fear of infecting other. CONCLUSION: The lockdown during the COVID-19 pandemic was associated with both self-reported relapse or increase smoking habit and also quitting or reduction of it.
Assuntos
COVID-19 , Fumantes , COVID-19/epidemiologia , Controle de Doenças Transmissíveis , Estudos Transversais , Humanos , Pandemias , Recidiva , Fumar/epidemiologiaRESUMO
BACKGROUND: Here, we isolated, expanded and functionally characterized regulatory T cells (Tregs) from patients with end stage kidney and liver disease, waiting for kidney/liver transplantation (KT/LT), with the aim to establish a suitable method to obtain large numbers of immunomodulatory cells for adoptive immunotherapy post-transplantation. METHODS: We first established a preclinical protocol for expansion/isolation of Tregs from peripheral blood of LT/KT patients. We then scaled up and optimized such protocol according to good manufacturing practice (GMP) to obtain high numbers of purified Tregs which were phenotypically and functionally characterized in vitro and in vivo in a xenogeneic acute graft-versus-host disease (aGVHD) mouse model. Specifically, immunodepressed mice (NOD-SCID-gamma KO mice) received human effector T cells with or without GMP-produced Tregs to prevent the onset of xenogeneic GVHD. RESULTS: Our small scale Treg isolation/expansion protocol generated functional Tregs. Interestingly, cryopreservation/thawing did not impair phenotype/function and DNA methylation pattern of FOXP3 gene of the expanded Tregs. Fully functional Tregs were also isolated/expanded from KT and LT patients according to GMP. In the mouse model, GMP Tregs from LT or KT patient proved to be safe and show a trend toward reduced lethality of acute GVHD. CONCLUSIONS: These data demonstrate that expanded/thawed GMP-Tregs from patients with end-stage organ disease are fully functional in vitro. Moreover, their infusion is safe and results in a trend toward reduced lethality of acute GVHD in vivo, further supporting Tregs-based adoptive immunotherapy in solid organ transplantation.
Assuntos
Criopreservação/métodos , Falência Renal Crônica/imunologia , Hepatopatias/imunologia , Linfócitos T Reguladores/citologia , Adulto , Idoso , Animais , Transplante de Células , Metilação de DNA , Feminino , Fatores de Transcrição Forkhead/genética , Doença Enxerto-Hospedeiro , Humanos , Imunoterapia , Falência Renal Crônica/cirurgia , Hepatopatias/cirurgia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Pessoa de Meia-Idade , FenótipoRESUMO
Progressive Supranuclear Palsy (PSP) is a rare neurodegenerative disease whose etiopathogenesis remains elusive. The intraneuronal accumulation of hyperphosphorylated Tau, a pivotal protein in regulating microtubules (MT), leads to include PSP into tauopathies. Pathological hallmarks are well known in neural cells but no word yet if PSP-linked dysfunctions occur also in other cell types. We focused on bone marrow mesenchymal stromal cells (MSCs) that have recently gained attention for therapeutic interventions due to their anti-inflammatory, antiapoptotic and trophic properties. Here, we aimed to investigate MSCs biology and to disclose if any disease-linked defect occurs in this non-neuronal compartment. First, we found that cells obtained from patients showed altered morphology and growth. Next, Western blotting analysis unravelled the imbalance in α-tubulin post-translational modifications and in MT stability. Interestingly, MT mass is significantly decreased in patient cells at baseline and differently changes overtime compared to controls, suggesting their inability to efficiently remodel MT cytoskeleton during ageing in culture. Thus, our results provide the first evidence that defects in MT regulation and stability occur and are detectable in a non-neuronal compartment in patients with PSP. We suggest that MSCs could be a novel model system for unravelling cellular processes implicated in this neurodegenerative disorder.
Assuntos
Células-Tronco Mesenquimais/patologia , Microtúbulos/patologia , Paralisia Supranuclear Progressiva/patologia , Acetilação , Idoso , Proliferação de Células , Forma Celular , Células Cultivadas , Feminino , Humanos , Imunofenotipagem , Masculino , Células-Tronco Mesenquimais/metabolismo , Microtúbulos/metabolismo , Pessoa de Meia-Idade , Processamento de Proteína Pós-Traducional , Tubulina (Proteína)/metabolismoRESUMO
BACKGROUND AIMS: Growing evidence supports the therapeutic potential of bone marrow (BM)-derived stem/progenitor cells for end-stage liver disease (ESLD). We recently demonstrated that CD133+ stem/progenitor cell (SPC) reinfusion in patients with ESLD is feasible and safe and improve, albeit transiently, liver function. However, the mechanism(s) through which BM-derived SPCs may improve liver function are not fully elucidated. METHODS: Here, we characterized the circulating SPCs compartment of patients with ESLD undergoing CD133+ cell therapy. Next, we set up an in vitro model mimicking SPCs/liver microenvironment interaction by culturing granulocyte colony-stimulating factor (G-CSF)-mobilized CD133+and LX-2 hepatic stellate cells. RESULTS: We found that patients with ESLD show normal basal levels of circulating hematopoietic and endothelial progenitors with impaired clonogenic ability. After G-CSF treatment, patients with ESLD were capable to mobilize significant numbers of functional multipotent SPCs, and interestingly, this was associated with increased levels of selected cytokines potentially facilitating SPC function. Co-culture experiments showed, at the molecular and functional levels, the bi-directional cross-talk between CD133+ SPCs and human hepatic stellate cells LX-2. Human hepatic stellate cells LX-2 showed reduced activation and fibrotic potential. In turn, hepatic stellate cells enhanced the proliferation and survival of CD133+ SPCs as well as their endothelial and hematopoietic function while promoting an anti-inflammatory profile. DISCUSSION: We demonstrated that the interaction between CD133+ SPCs from patients with ESLD and hepatic stellate cells induces significant functional changes in both cellular types that may be instrumental for the improvement of liver function in cirrhotic patients undergoing cell therapy.
Assuntos
Antígeno AC133/metabolismo , Doença Hepática Terminal/terapia , Células Estreladas do Fígado/citologia , Fígado/citologia , Transplante de Células-Tronco/métodos , Células-Tronco/metabolismo , Proliferação de Células , Técnicas de Cocultura , Doença Hepática Terminal/patologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células Estreladas do Fígado/fisiologia , Humanos , Fígado/metabolismo , Fígado/patologia , Neovascularização Fisiológica , Células-Tronco/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismoRESUMO
BACKGROUND: The trophic, anti-apoptotic and regenerative effects of bone marrow mesenchymal stromal cells (MSC) may reduce neuronal cell loss in neurodegenerative disorders. METHODS: We used MSC as a novel candidate therapeutic tool in a pilot phase-I study for patients affected by progressive supranuclear palsy (PSP), a rare, severe and no-option form of Parkinsonism. Five patients received the cells by infusion into the cerebral arteries. Effects were assessed using the best available motor function rating scales (UPDRS, Hoehn and Yahr, PSP rating scale), as well as neuropsychological assessments, gait analysis and brain imaging before and after cell administration. RESULTS: One year after cell infusion, all treated patients were alive, except one, who died 9 months after the infusion for reasons not related to cell administration or to disease progression (accidental fall). In all treated patients motor function rating scales remained stable for at least six-months during the one-year follow-up. CONCLUSIONS: We have demonstrated for the first time that MSC administration is feasible in subjects with PSP. In these patients, in whom deterioration of motor function is invariably rapid, we recorded clinical stabilization for at least 6 months. These encouraging results pave the way to the next randomized, placebo-controlled phase-II study that will definitively provide information on the efficacy of this innovative approach. Trial registration ClinicalTrials.gov NCT01824121.
Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Transtornos Parkinsonianos/terapia , Paralisia Supranuclear Progressiva/terapia , Idoso , Fenômenos Biomecânicos , Medula Óssea/patologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos Parkinsonianos/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Paralisia Supranuclear Progressiva/diagnóstico por imagem , Paralisia Supranuclear Progressiva/fisiopatologia , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
On November 10, 2014, the representatives of all six certified Good Manufacturing Practices (GMP) cell factories operating in the Lombardy Region of Italy convened a 1-day workshop in Milan titled "Management Models for the Development And Sustainability of Cell Factories: Public-Private Partnership?" The speakers and panelists addressed not only the many scientific, technological and cultural challenges faced by Lombardy Cell Factories, but also the potential impact of advanced therapy medicinal products (ATMPs) on public health and the role played by translational research in this process. Future perspectives for research and development (R&D) and manufacturing processes in the field of regenerative medicine were discussed as well. This report summarizes the most important issues raised by the workshop participants with particular emphasis on strengths and limitations of the R&D and manufacturing processes for innovative therapeutics in Lombardy and what can be improved in this context while maintaining GMP standards. The participants highlighted several strategies to translate patient-specific advanced therapeutics into scaled manufacturing products for clinical application. These included (i) the development of a synergistic interaction between public and private institutions, (ii) better integration with Italian regulatory agencies and (iii) the creation of a network among Lombardy cell factories and other Italian and European institutions.
Assuntos
Técnicas de Cultura de Células , Engenharia Celular , Laboratórios/organização & administração , Modelos Organizacionais , Terapias em Estudo , Pesquisa Biomédica/métodos , Pesquisa Biomédica/organização & administração , Pesquisa Biomédica/normas , Biotecnologia/organização & administração , Biotecnologia/normas , Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células/normas , Engenharia Celular/métodos , Engenharia Celular/normas , Humanos , Itália , Avaliação de Programas e Projetos de Saúde/normas , Melhoria de Qualidade , Terapias em Estudo/métodos , Terapias em Estudo/normasRESUMO
In June 2015, European Medicines Agency/Committee for Advanced Therapies (CAT) released the new version of the reflection paper on classification of advanced therapy medicinal products (ATMPs) established to address questions of borderline cases in which classification of a product based on genes, cells or tissues is unclear. The paper shows CAT's understanding of substantial manipulation and essential function(s) criteria that define the legal scope of cell-based medicinal products. This article aims to define the authors' viewpoint on the reflection paper. ATMP classification has intrinsic weaknesses derived from the lack of clarity of the evolving concepts of substantial manipulation and essential function(s) as stated in the EU Regulation, leading to the risk of differing interpretations and misclassification. This might result in the broadening of ATMP scope at the expense of other products such as cell/tissue transplants and blood products, or even putting some present and future clinical practice at risk of being classified as ATMP. Because of the major organizational, economic and regulatory implications of product classification, we advocate for increased interaction between CAT and competent authorities (CAs) for medicines, blood and blood components and tissues and cells or for the creation of working groups including representatives of all parties as recently suggested by several CAs.
Assuntos
Tomada de Decisões , Transplantes/classificação , HumanosRESUMO
Adipose-derived mesenchymal stem cells (ADMSCs) are an ideal population for regenerative medical application. Both the isolation procedure and the culturing conditions are crucial steps, since low yield can limit further cell therapies, especially when minimal adipose tissue harvests are available for cell expansion. To date, a standardized procedure encompassing both isolation sites and expansion methods is missing, thus making the choice of the most appropriate conditions for the preparation of ADMSCs controversial, especially in view of the different applications needed. In this study, we compared the effects of three different commercial media (DMEM, aMEM, and EGM2), routinely used for ADMSCs expansion, and two supplements, FBS and human platelet lysate, recently proven to be an effective alternative to prevent xenogeneic antibody transfer and immune alloresponse in the host. Notably, all the conditions resulted in being safe for ADMSCs isolation and expansion with platelet lysate supplementation giving the highest isolation and proliferation rates, together with a commitment for osteogenic lineage. Then, we proved that the high ADMSC hematopoietic supportive potential is performed through a constant and abundant secretion of both GCSF and SCF. In conclusion, this study further expands the knowledge on ADMSCs, defining their identity definition and offers potential options for in vitro protocols for clinical production, especially related to HSC expansion without use of exogenous cytokines or genetic modifications.
Assuntos
Tecido Adiposo/citologia , Meios de Cultura/química , Células-Tronco Mesenquimais/citologia , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Voluntários Saudáveis , HumanosRESUMO
BACKGROUND: Progressive Supranuclear Palsy (PSP) is a sporadic and progressive neurodegenerative disease which belongs to the family of tauopathies and involves both cortical and subcortical structures. No effective therapy is to date available. METHODS/DESIGN: Autologous bone marrow (BM) mesenchymal stem cells (MSC) from patients affected by different type of parkinsonisms have shown their ability to improve the dopaminergic function in preclinical and clinical models. It is also possible to isolate and expand MSC from the BM of PSP patients with the same proliferation rate and immuphenotypic profile as MSC from healthy donors. BM MSC can be efficiently delivered to the affected brain regions of PSP patients where they can exert their beneficial effects through different mechanisms including the secretion of neurotrophic factors.Here we propose a randomized, placebo-controlled, double-blind phase I clinical trial in patients affected by PSP with MSC delivered via intra-arterial injection. DISCUSSION: To our knowledge, this is the first clinical trial to be applied in a no-option parkinsonism that aims to test the safety and to exploit the properties of autologous mesenchymal stem cells in reducing disease progression. The study has been designed to test the safety of this "first-in-man" approach and to preliminarily explore its efficacy by excluding the placebo effect. TRIAL REGISTRATION: NCT01824121.
Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Paralisia Supranuclear Progressiva/terapia , Pesquisa Translacional Biomédica , Adulto , Linhagem Celular Tumoral , Humanos , Fatores de Crescimento Neural/metabolismo , Transplante AutólogoRESUMO
Human mesenchymal stem cells (MSCs) are multipotent cells offering valuable hopes for the treatment of degenerative diseases. MSCs can be found among differentiated cells in many tissues and organs but, unfortunately, their phenotypic similarity hinders a robust cell characterization and discrimination from diverse tissue harvests. MicroRNAs (miRNAs) are crucial managers of gene expression with intriguing and still poorly known roles in stem cell maintenance and differentiation. To identify miRNAs that can discriminate among MSCs, we performed a whole-genome comparative miRNA expression profiling analysis on adipose (AD), bone marrow (BM) and cord blood (CB) derived MSCs, all three considered among the most promising in the field of regenerative medicine. miRNA expression patterns were very similar, meeting their extensive phenotypic and functional overlaps. An in-depth comparison of the few most differentially expressed miRNAs allowed the identification of a highly restricted molecular signature consisting of 5 BMMSC, 11 ADMSC and 11 CBMSC specific miRNAs. Functional analysis of their validated targets allowed the identification of an "environmental-niche memory" for BMMSC and an "epithelial" commitment for ADMSC, providing new insights into the molecular mechanisms discriminating between these MSCs, a crucial element to identify the most appropriate stem cell source for clinical application.
Assuntos
Medula Óssea/química , Células-Tronco Hematopoéticas/citologia , Células-Tronco Mesenquimais/citologia , MicroRNAs/genética , Nicho de Células-Tronco , Adipogenia , Tecido Adiposo/química , Tecido Adiposo/citologia , Morte Celular , Proliferação de Células , Forma Celular , Sobrevivência Celular , Condrogênese , Meios de Cultura/química , Sangue Fetal/química , Sangue Fetal/citologia , Citometria de Fluxo , Perfilação da Expressão Gênica/métodos , Genoma Humano , Células-Tronco Hematopoéticas/química , Humanos , Imunofenotipagem , Células-Tronco Mesenquimais/química , OsteogêneseRESUMO
Background Recently, mesenchymal stromal cells (MSCs) have gained recognition for their clinical utility in transplantation to induce tolerance and to improve/replace pharmacological immunosuppression. Cord blood (CB)-derived MSCs are particularly attractive for their immunological naivety and peculiar anti-inflammatory and anti-apoptotic properties. OBJECTIVES: The objective of this study was to obtain an inventory of CB MSCs able to support large-scale advanced therapy medicinal product (ATMP)-based clinical trials. STUDY DESIGN: We isolated MSCs by plastic adherence in a GMP-compliant culture system. We established a well-characterized master cell bank and expanded a working cell bank to generate batches of finished MSC(CB) products certified for clinical use. The MSC(CB) produced by our facility was used in approved clinical trials or for therapeutic use, following single-patient authorization as an immune-suppressant agent. RESULTS: We show the feasibility of a well-defined MSC manufacturing process and describe the main indications for which the MSCs were employed. We delve into a regulatory framework governing advanced therapy medicinal products (ATMPs), emphasizing the need of stringent quality control and safety assessments. From March 2012 to June 2023, 263 of our Good Manufacturing Practice (GMP)-certified MSC(CB) preparations were administered as ATMPs in 40 subjects affected by Graft-vs.-Host Disease, nephrotic syndrome, or bronco-pulmonary dysplasia of the newborn. There was no infusion-related adverse event. No patient experienced any grade toxicity. Encouraging preliminary outcome results were reported. Clinical response was registered in the majority of patients treated under therapeutic use authorization. CONCLUSIONS: Our 10 years of experience with MSC(CB) described here provides valuable insights into the use of this innovative cell product in immune-mediated diseases.
Assuntos
Sangue Fetal , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Controle de Qualidade , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Sangue Fetal/citologia , Feminino , Transplante de Células-Tronco Mesenquimais/métodos , Masculino , Adulto , Pessoa de Meia-Idade , Adolescente , Idoso , Adulto Jovem , CriançaRESUMO
In the last years, mesenchymal stem cells (MSCs) have been identified as an attractive cell population in regenerative medicine. In view of future therapeutic applications, the study of specific differentiation-related gene expression is a pivotal prerequisite to define the most appropriate MSC source for clinical translation. In this context, it is crucial to use stable housekeeping genes (HGs) for normalization of qRT-PCR to obtain validated and comparable results. By our knowledge, an exhaustive validation study of HGs comparing MSCs from different sources under various differentiation conditions is still missing. In this pivotal study, we compared the expression levels of 12 genes (ACTB, Β2M, EF1alpha, GAPDH, GUSB, PPIA, RPL13A, RPLP0, TBP, UBC, YWHAZ and 18S rRNA) to assess their suitability as HGs in MSCs during adipogenic, osteogenic and chondrogenic differentiation. We demonstrated that many of the most popular HGs including 18S rRNA, B2M and ACTB were inadequate for normalization, whereas TBP/YWHAZ/GUSB were frequently identified among the best performers. Moreover, we showed the dramatic effects of suboptimal HGs choice on the quantification of cell differentiation markers, thus interfering with a reliable comparison of the lineage potential properties among various MSCs. Thus, in the emerging field of regenerative medicine, the identification of the most appropriate MSC source and cell line is so crucial for the treatment of patients that being inaccurate in the first step of the stem cell characterization can bring important consequences for the patients and for the promising potential of stem cell therapy.
Assuntos
Adipócitos/metabolismo , Condrócitos/metabolismo , Genes Essenciais , Células-Tronco Mesenquimais/metabolismo , Osteócitos/metabolismo , Proteínas 14-3-3 , Adipócitos/citologia , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Biomarcadores/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular , Células Cultivadas , Condrócitos/citologia , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Glucuronidase/genética , Glucuronidase/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Osteócitos/citologia , Reação em Cadeia da Polimerase em Tempo Real , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismoRESUMO
Focal segmental glomerulosclerosis (FSGS) is the most frequent acquired renal condition resulting in end stage kidney disease in children. We describe a cell therapy treatment with human allogeneic bone marrow mesenchymal stem cells (MSC) in a 13-year-old patient developing recurrent FSGS after renal transplantation, which was not responding to conventional therapy. This treatment relied on the following measurements:clinical and laboratory evaluation of renal function, proteome array, biopsy, short tandem repeat assay. Before MSC treatment, the patient needed weekly plasmapheresis to achieve proteinuria-to-creatininuria ratio below 5. After three MSC infusions without adverse events, the patient has a stable renal function and the proteinuria target was reached without plasmapheresis. In addition, some circulating inflammatory factors decreased and their levels were still low after one year. This is the first report of an MSC treatment in an FSGS patient. Even though different factors may have contributed to the clinical results, after MSC infusion a stable reduction in the serum level of several inflammatory factors has been registered and the patient does not need anymore plasmapheresis to keep proteinuria under control. In addition, this encouraging single case let us identify some putative efficacy biomarkers that could be of clinical interest in chronic kidney diseases.
Assuntos
Glomerulosclerose Segmentar e Focal/cirurgia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Adolescente , Sobrevivência Celular , Células Cultivadas , Citometria de Fluxo , Glomerulosclerose Segmentar e Focal/etiologia , Glomerulosclerose Segmentar e Focal/fisiopatologia , Humanos , Imunofenotipagem , Transplante de Rim/efeitos adversos , Masculino , Células-Tronco Mesenquimais/metabolismo , Transplante Homólogo , Resultado do TratamentoRESUMO
The transplantation of two cord blood (CB) units obtained from unrelated donors (double CBT) is an effective strategy for adult patients with hematologic malignancies. Sustained hematopoiesis after double CBT is usually derived from a single donor, and only a few transplantation recipients displaying a stable mixed donor-donor chimerism have been reported. We investigated the mechanisms underlying single-donor predominance in double CBT by studying in vitro the role of the graft-versus-graft cell-mediated immune effect in two-way mixed-lymphocyte culture, along with the contribution of differential hematopoietic progenitor (HP) potency in HP mixed cultures. Results for the two-way mixed-lymphocyte culture showed that despite the weak and variable alloantigen-specific cytotoxic potential displayed by CB mononuclear cells, an immune-mediated dominance for one of the two CB units was detected in the majority of experiments. Alloantigen-induced cytotoxic activity was directed toward both CB-HP and phytohemagglutinin (PHA)-activated T lymphoblastoid cells. The CB unit with the higher fold expansion of CD34(+) cells in single-expansion culture was prevalent in the HP mixed-expansion culture, as shown by DNA chimerism evaluation. Based on these data, we hypothesize that the dominant CB unit is able to develop prevalent cytotoxic activity toward activated lymphocytes of the other CB unit, thereby preventing them from exerting alloantigen-specific cytotoxic potential against both activated lymphocytes and HPs of the dominant unit. In accordance with this hypothesis, we propose the evaluation of alloantigen-induced cytotoxic activity generated in two-way mixed-lymphocyte culture and directed toward PHA-activated T lymphoblastoid cells as a tool to identify the potentially predominant CB unit before double CBT.
Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical , Sangue Fetal/imunologia , Isoantígenos/imunologia , Antígenos CD34/imunologia , Proliferação de Células , Sangue Fetal/citologia , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/terapia , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , Fito-Hemaglutininas/imunologia , Cultura Primária de Células , Doadores de Tecidos , Quimeras de Transplante , Transplante HomólogoRESUMO
BACKGROUND: Human mesenchymal stem cells (hMSC) have recently raised the attention because of their therapeutic potential in the novel context of regenerative medicine. However, the safety of these new and promising cellular products should be carefully defined before they can be used in the clinical setting, as. The protein expression profile of these cells might reveal potential hazards associated with senescence and tumoral transformation which may occur during culture. Proteomic is a valuable tool for hMSC characterization and identification of possible changes during expansion. RESULTS: We used Surface Enhanced Laser Desorption/Ionization-Time Of Flight-Mass Spectrometry (SELDI-ToF-MS) to evaluate the presence of stable molecular markers in adipose tissue-derived mesenchymal stem cells (AD-MSC) produced under conditions of good manufacturing practices (GMP). Proteomic patterns of cells prepared were consistent, with 4 up-regulated peaks (mass-to-charge ratio (m/z) 8950, 10087, 10345, and 13058) through subculture steps (P0-P7) with similar trend in three donors. Among the differentially expressed proteins found in the cytoplasmic and nuclear fractions, a cytoplasmic 10.1 kDa protein was upregulated during culture passages and was identified as S100A6 (Calcyclin). CONCLUSIONS: This study suggests for the first time that common variation could occur in AD-MSC from different donors, with the identification of S100A6, a protein prevalently related to cell proliferation and cell culture condition. These results support the hypothesis of common proteomic changes during MSCs expansion and could give important insight in the knowledge of molecular mechanisms intervening during MSC expansion.
RESUMO
Cellular therapy is a novel form of medical or surgical treatment using cells in place of or in addition to traditional chemical drugs. The preparation of cellular products - called advanced therapy medicinal products - ATMP in Europe, requires compliance with good manufacturing practices (GMP). Based on long-term experience in blood component manufacturing, product traceability and hemovigilance, selected blood services may represent ideal settings for the development and experimental use of ATMP. International harmonization of the protocols and procedures for the preparation of ATMP is of paramount importance to facilitate the development of multicenter clinical trials with adequate sample size, which are urgently needed to determine the clinical efficacy of ATMP. This article describes European regulations on cellular therapy and summarizes the activities of the 'Franco Calori' Cell Factory, a GMP unit belonging to the department of regenerative medicine of a large public university hospital, which acquired a certification for the GMP production of ATMP in 2007 and developed nine experimental clinical protocols during 2003-2011.
Assuntos
Segurança do Sangue/normas , Terapia Baseada em Transplante de Células e Tecidos/normas , Medicina Regenerativa/normas , Segurança do Sangue/métodos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Ensaios Clínicos como Assunto/métodos , Ensaios Clínicos como Assunto/normas , Europa (Continente) , Humanos , Estudos Multicêntricos como Assunto/métodos , Estudos Multicêntricos como Assunto/normas , Medicina Regenerativa/métodosRESUMO
Cognitive behavioral therapy (CBT) is the most successful protocol in gambling disorder (GD) treatment. However, it presents some weaknesses, especially concerning relapse prevention (RP). RP is one of the most important therapeutic steps, aiming at managing cravings and to avoid future relapse increasing perceived self-efficacy. Encouraging results come from the blending of psychotherapy and virtual reality (VR), containing gambling cues. The goal of Alter Game (approved by the Ethical Commission, Prot. No. 69346) is verifying the efficacy of an innovative psychological treatment for GD based on the integration of traditional CBT therapy and an immersive VR cue exposure therapy using a serious virtual game, which is a game designed for purposes other than entertainment. RP in virtual cue-exposure therapy allows pathological gamblers to manage the urge to gamble and to avoid relapse by becoming aware of which internal and external triggers are related to craving. We hypothesize that the integrated intervention will be more effective than simple CBT with regard to self-efficacy, craving, and gambling-related distortions. Four virtual ecological environments were developed, and a virtual app, Exludo, interfaced with a computerized multiparametric acquisition system for biofeedback, was created. A sample of about 60 patients aged between 18 and 65 with GD referring to the Addiction Medicine Unit of Verona (Rossi Hospital) will be recruited. Patients will be randomly assigned to the CBT group (16 CBT sessions) or the CBT + VR group (8 CBT sessions + 8 VR cue-exposure therapy sessions). The MCMI-III, the BIS-11, and the SOGS will be used to evaluate inclusion and exclusion criteria, while the Gambling Related Cognitions Scale and the Multidimensional Gambling Self-Efficacy Scale will be used to verify changes as a function of the treatment. Craving will be evaluated through VAS, and psychophysiological variables will be assessed through biofeedback. A pre-test/post-test experimental design with a 1-month follow-up will be conducted. This study will examine an innovative psychotherapeutic protocol for GD treatment, and it will help in identifying new virtual tools to increase the efficacy of traditional therapeutic approaches that could also be applied to treat other addictions.
RESUMO
Nicotine addiction is a widespread, worldwide epidemic, causing six million deaths per year. A large variety of treatments for smoking cessation are currently available, including Cytisine, which is a promising drug due to its low cost and high safety levels. Notwithstanding the important amount of research on tobacco addiction treatments, smoking remains one of the most difficult substance use disorders to treat, probably also due to the fact that pharmacological treatment often overlooks other maintaining factors in this addiction, such as sensory impact and cue reactivity. To address this gap in both treatment protocols and scientific literature, we propose a study protocol in which we will compare the effects of combining Cytisine with Nirdosh, a herbal tobacco substitute, to Cytisine only in two groups of patients (C + N and C) who will also undergo exposure to four different virtual reality settings that will assess the importance of environmental cues. We will further assess mood and craving in the two samples, and include a control group taken from the general population. We expect the C + N group to report a more positive mood and a lower sensitivity to tobacco-related environmental cues.