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Autologous bone marrow mononuclear cells (BMMNCs) infused after severe traumatic brain injury have shown promise for treating the injury. We evaluated their impact in children, particularly their hypothesized ability to preserve the blood-brain barrier and diminish neuroinflammation, leading to structural CNS preservation with improved outcomes. We performed a randomized, double-blind, placebo-sham-controlled Bayesian dose-escalation clinical trial at two children's hospitals in Houston, TX and Phoenix, AZ, USA (NCT01851083). Patients 5-17â years of age with severe traumatic brain injury (Glasgow Coma Scale score ≤ 8) were randomized to BMMNC or placebo (3:2). Bone marrow harvest, cell isolation and infusion were completed by 48 h post-injury. A Bayesian continuous reassessment method was used with cohorts of size 3 in the BMMNC group to choose the safest between two doses. Primary end points were quantitative brain volumes using MRI and microstructural integrity of the corpus callosum (diffusivity and oedema measurements) at 6â months and 12â months. Long-term functional outcomes and ventilator days, intracranial pressure monitoring days, intensive care unit days and therapeutic intensity measures were compared between groups. Forty-seven patients were randomized, with 37 completing 1-year follow-up (23 BMMNC, 14 placebo). BMMNC treatment was associated with an almost 3-day (23%) reduction in ventilator days, 1-day (16%) reduction in intracranial pressure monitoring days and 3-day (14%) reduction in intensive care unit (ICU) days. White matter volume at 1â year in the BMMNC group was significantly preserved compared to placebo [decrease of 19 891 versus 40 491, respectively; mean difference of -20 600, 95% confidence interval (CI): -35 868 to -5332; P = 0.01], and the number of corpus callosum streamlines was reduced more in placebo than BMMNC, supporting evidence of preserved corpus callosum connectivity in the treated groups (-431 streamlines placebo versus -37 streamlines BMMNC; mean difference of -394, 95% CI: -803 to 15; P = 0.055), but this did not reach statistical significance due to high variability. We conclude that autologous BMMNC infusion in children within 48 h after severe traumatic brain injury is safe and feasible. Our data show that BMMNC infusion led to: (i) shorter intensive care duration and decreased ICU intensity; (ii) white matter structural preservation; and (iii) enhanced corpus callosum connectivity and improved microstructural metrics.
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Trasplante de Médula Ósea , Lesiones Traumáticas del Encéfalo , Trasplante Autólogo , Humanos , Niño , Lesiones Traumáticas del Encéfalo/terapia , Masculino , Femenino , Adolescente , Método Doble Ciego , Preescolar , Trasplante de Médula Ósea/métodos , Trasplante Autólogo/métodos , Imagen por Resonancia Magnética , Resultado del Tratamiento , Leucocitos Mononucleares/trasplante , Teorema de BayesRESUMEN
BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are multipotent adult cells that can be isolated from tissues including bone marrow [MSC(BM)], adipose [MSC(AT)] and umbilical cord [MSC(CT)]. Previous studies have linked expression of tissue factor (TF) on MSC surfaces to a procoagulant effect. Venous thromboembolism (VTE), immediate blood-mediated inflammatory reaction (IBMIR) and microvascular thrombosis remain a risk with intravascular MSC therapy. We examined the effect of low molecular weight heparin (LMWH) on clinical-grade MSCs using calibrated automated thrombography (CAT). METHODS: Clinical grade MSC(BM)s, MSC(AT)s and MSC(CT)s harvested at passage 4 were added to normal pooled plasma (NPP) to a final concentration of either 400 000 or 50 000 cells/mL. LMWH was added to plasma in increments of 0.1 U/mL. Thrombin generation (TG) was measured using CAT. Flow cytometry was conducted on the cells to measure MSC phenotype and TF load. RESULTS: Presence of MSCs decreased lag time and increased peak TG. All cell lines demonstrated a dose response to LMWH, with MSC(AT) demonstrating the least thrombogenicity and most sensitivity to LMWH. TG was significantly reduced in all cell lines at doses of 0.2 U/mL LMWH and higher. DISCUSSION: All MSC types and concentrations had a decrease in peak thrombin and TG with increasing amounts of LMWH. While this in vitro study cannot determine optimal dosing, it suggests that LMWH can be effectively used to lower the risk of VTE associated with intravascular administration of MSCs. Future in vivo work can be done to determine optimal dosing and effect on IBMIR and VTE.
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Coagulantes , Trombosis , Tromboembolia Venosa , Adulto , Humanos , Heparina de Bajo-Peso-Molecular/farmacología , Heparina de Bajo-Peso-Molecular/uso terapéutico , Tromboembolia Venosa/tratamiento farmacológico , Coagulantes/uso terapéutico , Trombina/uso terapéutico , Heparina/uso terapéuticoRESUMEN
Traumatic brain injury (TBI) is soon predicted to become the third leading cause of death and disability worldwide. After the primary injury, a complex set of secondary injuries develops hours and days later with prolonged neuroinflammation playing a key role. TBI and other inflammatory conditions are currently being treated in preclinical and clinical trials by a number of cellular therapies. Mesenchymal stem cells (MSC) are of great interest due to their widespread usage, safety, and relative ease to isolate and culture. However, there has been a wide range in efficacy reported using MSC clinically and in preclinical models, likely due to differences in cell preparations and a significant amount of donor variability. In this study, we seek to find a correlation between in vitro activity and in vivo efficacy. We designed assays to explore the responsiveness of MSC to immunological cues to address the immunomodulatory properties of MSC, one of their primary modes of therapeutic activity in TBI. Our results showed intrinsic differences in the immunomodulatory capacity of MSC preparations from different bone marrow and amniotic fluid donors. This difference mirrored the therapeutic capacity of the MSC in an experimental model of TBI, an effect confirmed using siRNA knockdown of COX2 followed by overexpressing COX2. Among the immunomodulatory factors assessed, the therapeutic benefit correlated with the secretion of prostaglandin E2 (PGE2 ) by MSC prior to treatment, suggesting that measurement of PGE2 could be a very useful potency marker to create an index of predicted efficacy for preparations of MSC to treat TBI. Stem Cells 2017;35:1416-1430.
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Lesiones Traumáticas del Encéfalo/terapia , Dinoprostona/farmacología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Líquido Amniótico/citología , Animales , Encéfalo/patología , Lesiones Traumáticas del Encéfalo/patología , Recuento de Células , Enfermedad Crónica , Constricción Patológica , Ciclooxigenasa 2/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Inmunomodulación , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Inflamación/patología , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patología , Permeabilidad , Ratas Sprague-DawleyRESUMEN
Preclinical studies using bone marrow derived cells to treat traumatic brain injury have demonstrated efficacy in terms of blood-brain barrier preservation, neurogenesis, and functional outcomes. Phase 1 clinical trials using bone marrow mononuclear cells infused intravenously in children with severe traumatic brain injury demonstrated safety and potentially a central nervous system structural preservation treatment effect. This study sought to confirm the safety, logistic feasibility, and potential treatment effect size of structural preservation/inflammatory biomarker mitigation in adults to guide Phase 2 clinical trial design. Adults with severe traumatic brain injury (Glasgow Coma Scale 5-8) and without signs of irreversible brain injury were evaluated for entry into the trial. A dose escalation format was performed in 25 patients: 5 controls, followed 5 patients in each dosing cohort (6, 9, 12 ×106 cells/kg body weight), then 5 more controls. Bone marrow harvest, cell processing to isolate the mononuclear fraction, and re-infusion occurred within 48 hours after injury. Patients were monitored for harvest-related hemodynamic changes, infusional toxicity, and adverse events. Outcome measures included magnetic resonance imaging-based measurements of supratentorial and corpus callosal volumes as well as diffusion tensor imaging-based measurements of fractional anisotropy and mean diffusivity of the corpus callosum and the corticospinal tract at the level of the brainstem at 1 month and 6 months postinjury. Functional and neurocognitive outcomes were measured and correlated with imaging data. Inflammatory cytokine arrays were measured in the plasma pretreatment, posttreatment, and at 1 and 6 month follow-up. There were no serious adverse events. There was a mild pulmonary toxicity of the highest dose that was not clinically significant. Despite the treatment group having greater injury severity, there was structural preservation of critical regions of interest that correlated with functional outcomes. Key inflammatory cytokines were downregulated. Treatment of severe, adult traumatic brain injury using an intravenously delivered autologous bone marrow mononuclear cell infusion is safe and logistically feasible. There appears to be a treatment signal as evidenced by central nervous system structural preservation, consistent with previous pediatric trial data. Inflammatory biomarkers are downregulated after cell infusion. Stem Cells 2016 Video Highlight: https://youtu.be/UiCCPIe-IaQ Stem Cells 2017;35:1065-1079.
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Células de la Médula Ósea/citología , Lesiones Traumáticas del Encéfalo/terapia , Leucocitos Mononucleares/trasplante , Adulto , Conducta , Biomarcadores/sangre , Lesiones Traumáticas del Encéfalo/sangre , Lesiones Traumáticas del Encéfalo/patología , Cuerpo Calloso/patología , Citocinas/sangre , Femenino , Sustancia Gris/patología , Humanos , Mediadores de Inflamación/metabolismo , Masculino , Tractos Piramidales/patología , Resultado del TratamientoRESUMEN
Although hepatic cell transplantation (CT) holds the promise of bridging patients with end-stage chronic liver failure to whole liver transplantation, suitable cell populations are under debate. In addition to hepatic cells, mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) are being considered as alternative cell sources for initial clinical cell work. Fetal liver (FL) tissue contains potential progenitors for all these cell lineages. Based on the collagenase incubation of tissue fragments, traditional isolation techniques yield only a fraction of the number of available cells. We report a 5-step method in which a portal vein in situ perfusion technique is used for tissue from the late second trimester. This method results in the high viabilities known for adult liver vascular perfusion, addresses the low cell yields of conventional digestion methods, and reduces the exposure of the tissue to collagenase 4-fold. We used donated tissue from gestational weeks 18 to 22, which yielded 1.8 ± 0.7 × 10(9) cells with an average viability of 78%. Because HSC transplantation and MSC transplantation are of interest for the treatment of hepatic failure, we phenotypically confirmed that in addition to hepatic progenitors, the resulting cell preparation contained cells expressing typical MSC and HSC markers. The percentage of FL cells expressing proliferation markers was 45 times greater than the percentage of adult hepatocytes expressing these markers and was comparable to the percentage of immortalized HepG2 liver hepatocellular carcinoma cells; this indicated the strong proliferative capacity of fetal cells. We report a case of human FL CT with the described liver cell population for clinical end-stage chronic liver failure. The patient's Model for End-Stage Liver Disease (MELD) score improved from 15 to 10 within the first 18 months of observation. In conclusion, this human FL cell isolation protocol may be of interest for further clinical translation work on the development of liver cell-based therapies.
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Separación Celular/métodos , Enfermedad Hepática en Estado Terminal/cirugía , Células Madre Fetales/trasplante , Trasplante de Células Madre Hematopoyéticas , Hepatitis C/complicaciones , Cirrosis Hepática/cirugía , Hígado/embriología , Trasplante de Células Madre Mesenquimatosas , Perfusión , Biomarcadores/metabolismo , Técnicas de Cultivo de Célula , Supervivencia Celular , Colagenasas/metabolismo , Enfermedad Hepática en Estado Terminal/virología , Células Madre Fetales/metabolismo , Edad Gestacional , Células Hep G2 , Humanos , Inmunosupresores/uso terapéutico , Hígado/irrigación sanguínea , Hígado/metabolismo , Cirrosis Hepática/virología , Masculino , Persona de Mediana Edad , Fenotipo , Vena Porta/embriología , Resultado del TratamientoRESUMEN
We examined an autologous mononuclear-cell-therapy-based approach to treat cerebral palsy using autologous umbilical cord blood or bone-marrow-derived mononuclear cells. The primary objective was to determine if autologous cells are safe to administer in children with cerebral palsy. The secondary objectives were to determine if there was improvement in motor function of patients 12 months after infusion using the Gross Motor Function Measure and to evaluate impact of treatment on corticospinal tract microstructure as determined by radial diffusivity measurement. This Phase 1/2a trial was a randomized, blinded, placebo-controlled, crossover study in children aged 2-10 years of age with cerebral palsy enrolled between November 2013 and November 2016. Participants were randomized to 2:1 treatment:placebo. Treatment was either autologous bone-marrow-derived mononuclear cells or autologous umbilical cord blood. All participants who enrolled and completed their baseline visit planned to return for follow-up visits at 6 months, 12 months and 24 months after the baseline visit. At the 12-month post-treatment visit, participants who originally received the placebo received either bone-marrow-derived mononuclear cell or umbilical cord blood treatment. Twenty participants were included; 7 initially randomized to placebo, and 13 randomized to treatment. Five participants randomized to placebo received bone-marrow-derived mononuclear cells, and 2 received umbilical cord blood at the 12-month visit. None of the participants experienced adverse events related to the stem cell infusion. Cell infusion at the doses used in our study did not dramatically alter motor function. We observed concordant bilateral changes in radial diffusivity in 10 of 15 cases where each corticospinal tract could be reconstructed in each hemisphere. In 60% of these cases (6/10), concordant decreases in bilateral corticospinal tract radial diffusivity occurred post-treatment. In addition, 100% of unilateral corticospinal tract cases (3/3) exhibited decreased corticospinal tract radial diffusivity post-treatment. In our discordant cases (n = 5), directionality of changes in corticospinal tract radial diffusivity appeared to coincide with handedness. There was a significant improvement in corticospinal tract radial diffusivity that appears related to handedness. Connectivity strength increased in either or both pathways (corticio-striatal and thalamo-cortical) in each participant at 12 months post-treatment. These data suggest that both stem cell infusions are safe. There may be an improvement in myelination in some groups of patients that correlate with small improvements in the Gross Motor Function Measure scales. A larger autologous cord blood trial is impractical at current rates of blood banking. Either increased private banking or matched units would be required to perform a larger-scale trial.
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BACKGROUND AND OBJECTIVES: In a phase 1 amyotrophic lateral sclerosis (ALS) study, autologous infusions of expanded regulatory T-lymphocytes (Tregs) combined with subcutaneous interleukin (IL)-2 were safe and well tolerated. Treg suppressive function increased and disease progression stabilized during the study. The present study was conducted to confirm the reliability of these results. METHODS: Participants with ALS underwent leukapheresis, and their Tregs were isolated and expanded in a current Good Manufacturing Practice facility. Seven participants were randomly assigned in a 1:1 ratio to receive Treg infusions (1 × 106 cells/kg) IV every 4 weeks and IL-2 (2 × 105 IU/m2) injections 3 times/wk or matching placebo in a 24-week randomized controlled trial (RCT). Six participants proceeded into a 24-week dose-escalation open-label extension (OLE). Two additional participants entered directly into the OLE. The OLE included dose escalation of Treg infusions to 2 × 106 cells/kg and 3 × 106 cells/kg at 4-week intervals. RESULTS: The Treg/IL-2 treatments were safe and well tolerated, and Treg suppressive function was higher in the active group of the RCT. A meaningful evaluation of progression rates in the RCT between the placebo and active groups was not possible due to the limited number of enrolled participants aggravated by the COVID-19 pandemic. In the 24-week OLE, the Treg/IL-2 treatments were also safe and well tolerated in 8 participants who completed the escalating doses. Treg suppressive function and numbers were increased compared with baseline. Six of 8 participants changed by an average of -2.7 points per the ALS Functional Rating Scale-Revised, whereas the other 2 changed by an average of -10.5 points. Elevated levels of 2 markers of peripheral inflammation (IL-17C and IL-17F) and 2 markers of oxidative stress (oxidized low-density lipoprotein receptor 1 and oxidized LDL) were present in the 2 rapidly progressing participants but not in the slower progressing group. DISCUSSION: Treg/IL-2 treatments were safe and well tolerated in the RCT and OLE with higher Treg suppressive function. During the OLE, 6 of 8 participants showed slow to no progression. The 2 of 8 rapid progressors had elevated markers of oxidative stress and inflammation, which may help delineate responsiveness to therapy. Whether Treg/IL-2 treatments can slow disease progression requires a larger clinical study (ClinicalTrials.gov number, NCT04055623). CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that Treg infusions and IL-2 injections are safe and effective for patients with ALS.
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Esclerosis Amiotrófica Lateral , Tratamiento Farmacológico de COVID-19 , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Biomarcadores , Progresión de la Enfermedad , Humanos , Inflamación , Interleucina-2/efectos adversos , Linfocitos T ReguladoresRESUMEN
INTRODUCTION: Resection and reconstruction of the esophagus remains fraught with morbidity and mortality. Recently, data from a porcine reconstruction model revealed that segmental esophageal reconstruction using an autologous mesenchymal stromal cell-seeded polyurethane graft (Cellspan esophageal implant [CEI]) can facilitate esophageal regrowth and regeneration. To this end, a patient requiring a full circumferential esophageal segmental reconstruction after a complex multiorgan tumor resection was approved for an investigational treatment under the Food and Drug Administration Expanded Access Use (Investigational New Drug 17402). METHODS: Autologous adipose-derived mesenchymal stromal cells (Ad-MSCs) were isolated from the Emergency Investigational New Drug patient approximately 4 weeks before surgery from an adipose tissue biopsy specimen. The Ad-MSCs were grown and expanded under current Good Manufacturing Practice manufacturing conditions. The cells were then seeded onto a polyurethane fiber mesh scaffold (Cellspan scaffold) and cultured in a custom bioreactor to manufacture the final CEI graft. The cell-seeded scaffold was then shipped to the surgical site for surgical implantation. After removal of a tumor mass and a full circumferential 4 cm segment of the esophagus that was invaded by the tumor, the CEI was implanted by suturing the tubular CEI graft to both ends of the remaining native esophagus using end-to-end anastomosis. RESULTS: In this case report, we found that a clinical-grade, tissue-engineered esophageal graft can be used for segmental esophageal reconstruction in a human patient. This report reveals that the graft supports regeneration of the esophageal conduit. Histologic analysis of the tissue postmortem, 7.5 months after the implantation procedure, revealed complete luminal epithelialization and partial esophageal tissue regeneration. CONCLUSIONS: Autologous Ad-MSC seeded onto a tubular CEI tissue-engineered graft stimulates tissue regeneration following implantation after a full circumferential esophageal resection.
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International regulatory authorities view risk management as an essential production need for the development of innovative, somatic cell-based therapies in regenerative medicine. The available risk management guidelines, however, provide little guidance on specific risk analysis approaches and procedures applicable in clinical cell therapy manufacturing. This raises a number of problems. Cell manufacturing is a poorly automated process, prone to operator-introduced variations, and affected by heterogeneity of the processed organs/tissues and lot-dependent variability of reagent (e.g., collagenase) efficiency. In this study, the principal challenges faced in a cell-based product manufacturing context (i.e., high dependence on human intervention and absence of reference standards for acceptable risk levels) are identified and addressed, and a risk management model approach applicable to manufacturing of cells for clinical use is described for the first time. The use of the heuristic and pseudo-quantitative failure mode and effect analysis/failure mode and critical effect analysis risk analysis technique associated with direct estimation of severity, occurrence, and detection is, in this specific context, as effective as, but more efficient than, the analytic hierarchy process. Moreover, a severity/occurrence matrix and Pareto analysis can be successfully adopted to identify priority failure modes on which to act to mitigate risks. The application of this approach to clinical cell therapy manufacturing in regenerative medicine is also discussed.
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Tratamiento Basado en Trasplante de Células y Tejidos , Modelos Organizacionales , Garantía de la Calidad de Atención de Salud , Gestión de Riesgos/organización & administración , Gestión de Riesgos/normasRESUMEN
Secondary alveolar bone grafts (ABGs) are the standard treatment for the alveolar defect in patients with cleft lip and palate (CLP), but remain invasive and have several disadvantages such as delayed timing of alveolar repair, donor-site complications, graft resorption, and need for multiple surgeries. Earlier management of the alveolar defect (primary ABG) would be ideal, but is limited by the minimal bony donor sites available in the infant. In this study we used a critical-size alveolar bone defect model in the rat to investigate the use of Wharton's Jelly (WJ), the stem cell-rich connective tissue matrix of the umbilical cord, to generate bone within the alveolar cleft. Human WJ was isolated and implanted into a critical-size alveolar bone defect model representative of secondary cleft ABG surgery in 10-11-week-old male Sprague-Dawley rats. The defects were monitored with CT imaging of living animals to evaluate bone regrowth and healing over 24 weeks, followed by histomorphometric evaluation at 24 weeks, after the last CT scan. CT data confirmed that the defect size was critical and did not lead to the union of the bones in the control animals (n = 12) for the entire duration of the study. New bone growth was stimulated leading to partial-to-full closure of the defect in the animals treated with WJ (n = 12). Twenty four weeks postoperatively, the percent increase in new bone formation in the WJ-treated group (156.58% ± 20.67%) was markedly higher than that in the control group (50.36% ± 21.07%) (p < 0.05). Histomorphometric data also revealed significantly greater new bone formation in WJ-treated versus control animals, confirming CT findings. qPCR analysis of human Alu elements was unable to detect any appreciable long-term persistence of human cells in the new bone, indicating that WJ may enhance bone growth by mediating osteoinduction in the host tissue, rather than through osteogenic differentiation of WJ-embedded cells. Impact statement In this study, Wharton's Jelly enhanced bone growth in a preclinical alveolar defect model, indicating its potential use as a natural adjunct in the repair of the alveolar cleft defect in patients with cleft lip and palate (CLP). The clinical success of this approach would represent a paradigm shift in the treatment of patients with CLP by reducing or eliminating the need for subsequent secondary alveolar bone graft and reducing their number of lifetime surgeries.
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Fisura del Paladar/cirugía , Gelatina de Wharton , Animales , Regeneración Ósea/fisiología , Diferenciación Celular/fisiología , Supervivencia Celular/fisiología , Células Cultivadas , Humanos , Osteogénesis/fisiología , Ratas , Ratas Sprague-Dawley , Microtomografía por Rayos XRESUMEN
BACKGROUND: Tacrolimus is a substrate of cytochrome P-450 (CYP) 3A enzyme and of the drug transporter ABCB1. We have investigated the effects of possible relevant CYP3A5 and ABCB1 single nucleotide polymorphisms (SNPs) present in both donors and recipients on tacrolimus blood levels achieved in a population of 32 Caucasian liver transplant patients. MATERIAL/METHODS: At 1, 3 and 6 months after transplantation, tacrolimus doses (mg/kg/day) and trough blood levels (C(0)) were determined. Polymerase chain reaction followed by restriction fragment length polymorphism analysis was used for genotyping CYP3A5*3 [6986A>G] as well as ABCB1 at exons 21 [2677G>T] and 26 [3435C>T]. RESULTS: 87.5% of the population showed a CYP3A5*3/*3 genotype. For the ABCB1 SNPs, in the case of 3435C>T the total frequency observed for the allelic variant was 50%. For the 2677G>T, the total frequency of the allelic variant was 12.5%, lower than in other Caucasian populations and without any significant linkage with 3435C>T. At 3 and 6 months after transplantation, tacrolimus dose requirements were significantly higher in patients receiving a liver with one copy of the *1 allele compared to those homozygous for the *3 allele (0.111+/-0.057 vs. 0.057+/-0.030 [P<0.05] at 3 month and 0.086+/-0.051 vs. 0.044+/-0.025 [P<0.05] at 6 month). For the recipients' genotypes, the presence of at least one *1 copy tended, though not statistically significantly, to increase tacrolimus doses. With regard to the ABCB1 SNPs, they did not show any influence on tacrolimus dosing requirements. CONCLUSIONS: Pharmacogenetic analysis of CYP3A5 in the donor could contribute to determine the appropriate initial dosage of tacrolimus in liver transplant patients.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Citocromo P-450 CYP3A/genética , Inmunosupresores/administración & dosificación , Trasplante de Hígado/inmunología , Polimorfismo de Nucleótido Simple , Tacrolimus/administración & dosificación , Subfamilia B de Transportador de Casetes de Unión a ATP , Femenino , Genotipo , Homocigoto , Humanos , Inmunosupresores/sangre , Masculino , Farmacogenética , Tacrolimus/sangre , Población Blanca/genéticaRESUMEN
OBJECTIVES: Functional and quantitative alterations and senescence of circulating and expanded endothelial progenitor cells (EPC), as well as systemic and tissue modifications of angiogenetic and inflammatory molecules, were evaluated for predicting age-related vessel wall remodeling, correlating them to intima media thickness (IMT) in the common carotid artery (CCA), a biomarker of early cardiovascular disease and aortic root dilation. POPULATIONS AND METHODS: A homogenous Caucasian population was included in the study, constituted by 160 healthy subjects (80 old subjects, mean age 72⯱â¯6.4, range 66-83â¯years; and 80 younger blood donors, mean age 26.2⯱â¯3.4, range 21-33â¯years), and 60 old subjects (mean age 73⯱â¯1.4 (range 66-83) years) with aortic root dilatation and hypertension, and 60 old people (70⯱â¯2.8 (age range 66-83)) with sporadic ascending aorta aneurysm (AAA). In addition, 20 control individuals (10 men and 10 women, mean age: 65⯱â¯8), were also included in the study for evaluating the gene expression's levels, in aorta tissues. Appropriate techniques, practises, protocols, gating strategies and statistical analyses were performed in our evaluations. RESULTS: Interestingly, old people had a significantly reduced functionality and a high grade of senescence (high SA-ß-Gal activity and high levels of TP53, p21 and p16 genes) of EPC expanded than younger subjects. The values of related parameters progressively augmented from the old subjects, in good healthy shape, to subjects with hypertension and aorta dilation, and AAA. Moreover, they significantly impacted the endothelium than the alterations in EPC number. No changes, but rather increased systemic levels of VEGF and SDF-1 were also assessed in old people vs. younger donors. Old people also showed significantly increased systemic levels of inflammatory cytokines, and a reciprocal significant reduction of systemic s-Notch 1 than younger subjects. These parameters, also including the number EPC alterations, resulted to be significantly sustained in old people bearers of an inflammatory combined genotype. Consistent with these data, a reduced expression of Notch-1 gene, accompanied by a sustained expression of inflammatory genes (i.e. TLR4, IL-1ß, IL-6 and IL-17) were detected in aortic tissues from old control people and AAA cases. Finally, we detected the biological effects induced by all the detected alterations on vessel wall age-related remodeling, by evaluating the IMT in the population studied and correlating it to these alterations. The analysis demonstrated that the unique independent risk predictors for vascular ageing are age, the EPC reduced migratory activity and senescence, high grade of expression of genes inducing EPC senescence and chronic tissue and systemic inflammation. CONCLUSIONS: Thus, we propose these parameters, of easy determination in biological samples (i.e. blood and tissue samples) from alive human population, as optimal biomarkers for vascular ageing.
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Envejecimiento/fisiología , Aorta/fisiología , Células Progenitoras Endoteliales/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/análisis , Arteria Carótida Común/fisiología , Grosor Intima-Media Carotídeo , Quimiocina CXCL12/análisis , Quimiotaxis , Femenino , Humanos , Masculino , Receptor Notch1/genética , Receptor Toll-Like 4/genética , Factor A de Crecimiento Endotelial Vascular/análisis , Adulto JovenRESUMEN
Aim: Traumatic brain injury is a complex condition consisting of a mechanical injury with neurovascular disruption and inflammation with limited clinical interventions available. A growing number of studies report systemic delivery of human umbilical cord blood (HUCB) as a therapy for neural injuries. Materials & methods: HUCB cells from five donors were tested to improve blood-brain barrier integrity in a traumatic brain injury rat model at a dose of 2.5 × 107 cells/kg at 24 or 72 h postinjury and for immunomodulatory activity in vitro. Results & Conclusion: We observed that cells delivered 72 h postinjury significantly restored blood-brain barrier integrity. HUCB cells reduced the amount of TNF-α and IFN-γ released by activated primary rat splenocytes, which correlated with the expression of COX2 and IDO1.
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Lesiones Encefálicas/terapia , Encéfalo/irrigación sanguínea , Sangre Fetal/trasplante , Inflamación/terapia , Cordón Umbilical/citología , Animales , Barrera Hematoencefálica/patología , Encéfalo/patología , Lesiones Encefálicas/complicaciones , Lesiones Encefálicas/patología , Extravasación de Materiales Terapéuticos y Diagnósticos/patología , Humanos , Inmunomodulación , Inflamación/complicaciones , Inflamación/patología , Masculino , Ratas Sprague-Dawley , Bazo/patología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
For several decades, multipotent mesenchymal stromal cells (MSCs) have been extensively studied for their therapeutic potential across a wide range of diseases. In the preclinical setting, MSCs demonstrate consistent ability to promote tissue healing, down-regulate excessive inflammation and improve outcomes in animal models. Several proposed mechanisms of action have been posited and demonstrated across an array of in vitro models. However, translation into clinical practice has proven considerably more difficult. A number of prominent well-funded late-phase clinical trials have failed, thus calling out for new efforts to optimize product delivery in the clinical setting. In this review, we discuss novel topics critical to the successful translation of MSCs from pre-clinical to clinical applications. In particular, we focus on the major routes of cell delivery, aspects related to hemocompatibility, and potential safety concerns associated with MSC therapy in the different settings.
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Diferenciación Celular , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/inmunología , Animales , Modelos Animales de Enfermedad , HumanosRESUMEN
We present a case of posterior ventricular septal rupture associated to left ventricular aneurysm manged, during peri-operative period, by transthoracic and transesophageal echocardiography. Three-dimensional transesophageal echocardiography findings add adjunctive and more accurate information regarding morphological details of the ventricular septal rupture rather than two-dimensional echocardiography, allowing, meanwhile, the detection of the outcome of the surgical repair.
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Background: After long-term intensive training, considerable morphological and functional heart changes occur in professional athletes. Such changes arise progressively and regress upon interruption of the physical activity. Morphological and functional alterations on heart are known as "Athlete's heart" condition. Objective: This study aims to compare echocardiographic parameters in two different groups of professional athletes. Furthermore, a prospective study is performed analyzing the echocardiographic changes occurring in 12 professional players in 3 years of follow-up. Materials and Methods: 78 football players were examined from July 2011 to May 2016 (40 enrolled in Group A and 38 in Group B). Twelve players of GROUP A were followed for 3 consecutive seasons. The general clinical examination, the cardiopulmonary evaluation, the ECG, the ergometer stress test, the spirometric examination and the standard cardiac eco color doppler test were recorded. Results: Left ventricle dimensions, left atrium dimensions, and interventricular septum dimensions were higher in A players than in B players. Moreover, following up 12 players for 3 years, a statistically significant increase of such values was observed. Discussion: In A players, higher dimensions of the left chambers and the interventricular septum were observed, compared to B players. No statistically significant difference was found regarding the ejection fraction. The 3 years follow-up showed a statistically significant increase of both left chambers and interventricular septum dimensions, particularly in the second and third year. Conclusions: These findings demonstrated that A players have higher echocardiographic parameters respect to B players. The results of this study support the scientific theory that long-term intensive training influences heart function, inducing "athlete's heart" with morphological adaptations. No significant echocardiographic variation within the examined sample was observed for different roles (goalkeeper, defender, midfielder, or attacker) or skills of individual players.
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The second half of the 20th century witnessed the birth of organ transplantation, and failing organs can now be replaced with healthy ones procured from living or cadaveric donors, allowing their recipient to start, or return to, an active life. Major milestones in the field were set in the eighties and nineties at the University of Pittsburgh Medical Center (UPMC), an institution that made it a mission to spread its expertise internationally. A successful partnership between UPMC and the Region of Sicily gave rise to the Mediterranean Institute for Transplantation and Highly Specialized Therapies (ISMETT), the only Italian facility entirely dedicated to transplantation of all solid organs and therapies for the treatment of end-stage organ failure. In its first seven years of activity, ISMETT has become a major referral center for patients from the entire Mediterranean Basin and the Middle East. Despite the fact that organ transplantation is the current gold standard for end-stage organ failure, the field is facing a worldwide emergency represented by the chronic shortage of organ donors. Research aimed at understanding the molecular networks involved in organ-specific ageing and their relationship with maintenance networks and organ failure should be actively encouraged and supported as it could ultimately allow to control organ performance and lifespan, increasing the number of organs available for transplant.
Asunto(s)
Regeneración/fisiología , Medicina Regenerativa , Trasplante , Envejecimiento/fisiología , Animales , Humanos , Región MediterráneaRESUMEN
End-stage organ failure is a major cause of death worldwide that can occur in patients of all ages and transplantation is the current standard of care for chronic end-stage disease of many organs. Despite the success of organ transplantation, it is becoming clear that there will never be enough organs made available through donation to meet the increasing demand. The past decade's rapid advancement in stem cell biology and tissue engineering generated an explosive outburst of reports that gave rise to regenerative medicine, a new field that promises to "fix" damaged organs through regeneration provided by transplanted cells, stimulation of endogenous repair mechanisms, or implantation of bioengineered tissue. Whether, and if so when, regenerative medicine will keep its promise is uncertain. As we continue to strive to find new effective solutions, alternative approaches based on the development of targeted, preventive interventions aimed at maintaining normal organ function, instead of repairing organ damage, should also be pursued.
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Insuficiencia Multiorgánica/terapia , Medicina Regenerativa/tendencias , Historia del Siglo XVIII , Historia del Siglo XIX , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Medicina Regenerativa/historiaRESUMEN
Immobilizing hydrated soft tissue specimens for atomic force microscopy (AFM) is a challenge. Here, we describe a simple and very cost-effective immobilization method, based on the use of transglutaminase in an aqueous environment, and successfully apply it to AFM characterization of human native Wharton's Jelly (nWJ), the gelatinous connective tissue matrix of the umbilical cord. A side-by-side comparison with a widely used polyphenolic protein-based tissue adhesive (Corning Cell-Tak), which is known to bind strongly to virtually all inorganic and organic surfaces in aqueous environments, shows that both adhesives successfully immobilize nWJ in its physological hydrated state. The cost of transglutaminase, however, is over 3000-fold lower than that of Cell-Tak, making it a very attractive method for immobilizing soft tissues for AFM characterization.
Asunto(s)
Técnicas de Preparación Histocitológica/métodos , Microscopía de Fuerza Atómica/métodos , Gelatina de Wharton/diagnóstico por imagen , Gelatina de Wharton/fisiología , Materiales Biocompatibles , Fenómenos Biomecánicos , Módulo de Elasticidad , Humanos , Adhesivos TisularesRESUMEN
UNLABELLED: Cell banked epidermal skin progenitor cells have the potential to provide an "off-the-freezer" product. Such cells may provide a skin donor area-independent cell-spray grafting therapy for the treatment of burns. We first characterized fetal skin samples of gestational ages ranging from 6 to 21 weeks. As the results suggest that the phenotypic differentiation occurs after 10 weeks, which may complicate follow-up in vitro studies, we developed and compared different cell isolation techniques for human fetal skin-derived epithelial cells from tissue ages 6 to 9 weeks. We initially screened seven methods of characterization, concluding that two methods warranted further investigation: incubating the epidermal tissue in Petri-dishes with culture medium for spontaneous cell outgrowth, and wiping the epidermal tissue onto a dry Petri-dish culture surface followed by adding culture medium. Non-controllable culture contamination with dermal cells was the reason for excluding the other five methods. The results suggest that epidermal cells can be isolated from tissue exhibiting a single homogeneous layer of CK15(+) basal keratinocytes up to week 9. At later gestational ages, the ongoing skin differentiation results in a multi-layer basal structure and progenitors associated with the hair bulb would have to be considered. Spraying the resulting cells with a clinical spray device was successfully demonstrated in an in vitro model. CONCLUSION: Gestational age 6-9 weeks epidermal human fetal skin cells from the basal layer can be reproducibly isolated and transferred into culture for studies on the development of skin cell transplantation therapies.