RESUMO
The inflammatory response after traumatic brain injury (TBI) can lead to significant secondary brain injury and chronic inflammation within the central nervous system. Cell therapies, including mesenchymal stromal cells (MSC), have led to improvements in animal models of TBI and are under investigation in human trials. One potential mechanism for the therapeutic potential of MSC is their ability to augment the endogenous response of immune suppressive regulatory T cells (Treg). We have recently shown that infusion of human cord blood Treg decreased chronic microgliosis after TBI and altered the systemic immune response in a rodent model. These cells likely use both overlapping and distinct mechanisms to modulate the immune system; therefore, combining Treg and MSC as a combination therapy may confer therapeutic benefit over either monotherapy. However, investigation of Treg + MSC combination therapy in TBI is lacking. In this study, we compared the ability MSC + Treg combination therapy, as well as MSC and Treg monotherapies, to inhibit the neuroinflammatory response to TBI in vivo and in vitro. Treg + MSC combination therapy demonstrated increased potency to reduce the neuro- and peripheral inflammatory response compared to monotherapy; furthermore, the timing of infusion proved to be a significant variable in the efficacy of both MSC monotherapy and Treg + MSC combination therapy in vivo and in vitro.
Assuntos
Lesões Encefálicas Traumáticas/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Linfócitos T Reguladores/imunologia , Animais , Lesões Encefálicas Traumáticas/imunologia , Terapia Combinada/métodos , Modelos Animais de Doenças , Imunidade , Inflamação/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Ratos Sprague-DawleyRESUMO
Traumatic brain injury (TBI) effects both the brain and the immune system. Circulating monocytes/macrophages (Mo /Ma ) after a TBI may play an important role in preserving the blood-brain barrier (BBB), reducing brain edema, and interacting with resident microglia. To elucidate the role of circulating Mo /Ma , we utilized a monocyte/macrophage depletion model in response to TBI in male rats. Clodronate liposomes (CL) were used to deplete circulating Mo /Ma . A controlled cortical impact (CCI) injury model was used to create a TBI. All animals received either CL or PBS liposomes (PL), 48 and 24 hr prior to the procedure, and were sacrificed 72 hr post-injury for analysis of BBB permeability, brain edema, whole blood (Mo /Ma and granulocytes), and/or microglial analysis. Animals undergoing Mo /Ma depletion with CL prior to CCI (CCI-CL) were found to have increased BBB permeability when compared to non-depleted CCI (CCI-PL) animals. At 72 hr following injury, Sham-CL maintained on average an 82% reduction in the whole blood monocytes when compared to Sham-PL (p < 0.001). Monocytes in the whole blood remained significantly lower in CCI-CL animals when compared to CCI-PL (p < 0.001). The number of granulocytes in the whole blood of CCI-CL animals was higher at 3 days when compared to CCI-PL (p < 0.022). Surprisingly, the depletion of Mo /Ma did not affect brain edema. However, the depletion of Mo /Ma did result in a significant decrease in microglia (CCI-CL vs. CCI-PL, p < 0.012). In conclusion, an intact Mo /Ma population is required to repair BBB integrity and microglial response following injury.
Assuntos
Barreira Hematoencefálica/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Animais , Permeabilidade Capilar , Modelos Animais de Doenças , Masculino , Microglia/metabolismo , Ratos Sprague-DawleyRESUMO
Extracellular vesicles (EVs) secreted by mesenchymal stromal cells (MSCs) have been proposed to be a key mechanistic link in the therapeutic efficacy of cells in response to cellular injuries through paracrine effects. We hypothesize that inflammatory stimulation of MSCs results in the release of EVs that have greater anti-inflammatory effects. The present study evaluates the immunomodulatory abilities of EVs derived from inflammation-stimulated and naive MSCs (MSCEv+ and MSCEv, respectively) isolated using a current Good Manufacturing Practice-compliant tangential flow filtration system. Detailed characterization of both EVs revealed differences in protein composition, cytokine profiles, and RNA content, despite similarities in size and expression of common surface markers. MSCEv+ further attenuated release of pro-inflammatory cytokines in vitro when compared to MSCEv, with a distinctly different pattern of EV-uptake by activated primary leukocyte subpopulations. The efficacy of EVs was partially attributed to COX2/PGE2 expression. The present study demonstrates that inflammatory stimulation of MSCs renders release of EVs that have enhanced anti-inflammatory properties partially due to COX2/PGE2 pathway alteration. Stem Cells 2018;36:79-90.
Assuntos
Vesículas Extracelulares/metabolismo , Inflamação/metabolismo , Células-Tronco Mesenquimais/metabolismo , Microscopia Eletrônica de Transmissão/métodos , HumanosRESUMO
Despite intensive research and clinical trials with numerous therapeutic treatments, traumatic brain injury (TBI) is a serious public health problem in the United States. There is no effective FDA-approved treatment to reduce morbidity and mortality associated with TBI. Inflammation plays a pivotal role in the pathogenesis of TBI. We looked to re-purpose existing drugs that reduce immune activation without broad immunosuppression. Teriflunomide, an FDA-approved drug, has been shown to modulate immunological responses outside of its ability to inhibit pyrimidine synthesis in rapidly proliferating cells. In this study, we tested the efficacy of teriflunomide to treat two different injury intensities in rat models of TBI. Our results show that teriflunomide restores blood-brain barrier integrity, decreases inflammation, and increases neurogenesis in the subgranular zone of the hippocampus. While we were unable to detect neurocognitive effects of treatment on memory and special learning abilities after treatment, a 2-week treatment following injury was sufficient to reduce neuroinflammation up to 120 days later.
Assuntos
Lesões Encefálicas Traumáticas/tratamento farmacológico , Lesões Encefálicas Traumáticas/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Crotonatos/uso terapêutico , Microglia/efeitos dos fármacos , Microglia/metabolismo , Toluidinas/uso terapêutico , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Modelos Animais de Doenças , Hidroxibutiratos , Imuno-Histoquímica , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Masculino , Neurogênese/efeitos dos fármacos , Nitrilas , Ratos , Ratos Sprague-Dawley , Tálamo/efeitos dos fármacos , Tálamo/metabolismoRESUMO
BACKGROUND: Traumatic brain injury (TBI) is a major cause of death and disability. TBI results in a prolonged secondary central neuro-inflammatory response. Previously, we have demonstrated that multiple doses (2 and 24 h after TBI) of multipotent adult progenitor cells (MAPC) delivered intravenously preserve the blood-brain barrier (BBB), improve spatial learning, and decrease activated microglia/macrophages in the dentate gyrus of the hippocampus. In order to determine if there is an optimum treatment window to preserve the BBB, improve cognitive behavior, and attenuate the activated microglia/macrophages, we administered MAPC at various clinically relevant intervals. METHODS: We administered two injections intravenously of MAPC treatment at hours 2 and 24 (2/24), 6 and 24 (6/24), 12 and 36 (12/36), or 36 and 72 (36/72) post cortical contusion injury (CCI) at a concentration of 10 million/kg. For BBB experiments, animals that received MAPC at 2/24, 6/24, and 12/36 were euthanized 72 h post injury. The 36/72 treated group was harvested at 96 h post injury. RESULTS: Administration of MAPC resulted in a significant decrease in BBB permeability when administered at 2/24 h after TBI only. For behavior experiments, animals were harvested post behavior paradigm. There was a significant improvement in spatial learning (120 days post injury) when compared to cortical contusion injury (CCI) in groups when MAPC was administered at or before 24 h. In addition, there was a significant decrease in activated microglia/macrophages in the dentate gyrus of hippocampus of the treated group (2/24) only when compared to CCI. CONCLUSIONS: Intravenous injections of MAPC at or before 24 h after CCI resulted in improvement of the BBB, improved cognitive behavior, and attenuated activated microglia/macrophages in the dentate gyrus.
Assuntos
Lesões Encefálicas Traumáticas/cirurgia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células-Tronco Multipotentes/fisiologia , Animais , Barreira Hematoencefálica/fisiopatologia , Proteínas de Ligação ao Cálcio/metabolismo , Permeabilidade Capilar/fisiologia , Citocinas/metabolismo , Modelos Animais de Doenças , Proteínas do Domínio Duplacortina , Injeções Intraventriculares , Masculino , Aprendizagem em Labirinto , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Multipotentes/transplante , Neuropeptídeos/metabolismo , Ratos , Tempo de Reação , Fatores de TempoRESUMO
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.
Assuntos
Lesões Encefálicas Traumáticas/terapia , Dinoprostona/farmacologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Líquido Amniótico/citologia , Animais , Encéfalo/patologia , Lesões Encefálicas Traumáticas/patologia , Contagem de Células , Doença Crônica , Constrição Patológica , Ciclo-Oxigenase 2/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Imunomodulação , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Inflamação/patologia , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Permeabilidade , Ratos Sprague-DawleyRESUMO
Peroxidative damage to human spermatozoa has been shown to be the primary cause of male infertility. The possible role of nitric oxide (NO) in affecting sperm motility, capacitation, and acrosome reaction has been reported, too. The overproduction of NO by the enzyme inducible nitric oxide synthase (iNOS) could be responsible as it has been implicated in the pathogenesis of many diseases. There have been many studies on regulating iNOS function in various tissues, especially by protein-protein interaction; however, no study has looked for iNOS-interacting proteins in the human testis. Here, we have reported the identification of two proteins that interact with iNOS. We initially undertook a popular yeast two-hybrid assay to screen a human testis cDNA library in yeast using an iNOS-peptide fragment (amino acids 181-335) as bait. We verified our data using the mammalian chemiluminescent co-IP method; first, employing the same peptide and, then, a full-length protein co-expressed in HEK293 cells in addition to the candidate protein. In both cases, these two protein partners of iNOS were revealed: (a) sperm acrosome-associated 7 protein and (b) retinoblastoma tumor-suppressor binding protein.
Assuntos
Óxido Nítrico Sintase Tipo II , Testículo , Técnicas do Sistema de Duplo-Híbrido , Humanos , Masculino , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Testículo/metabolismo , Células HEK293 , Ligação ProteicaRESUMO
Mesenchymal stromal cells (MSC) undergo functional maturation upon their migration from bone marrow and introduction to a site of injury. This inflammatory licensing leads to heightened immune regulation via cell-to-cell interaction and the secretion of immunomodulatory molecules, such as anti-inflammatory mediators and antioxidants. Pro-inflammatory cytokines are a recognized catalyst of inflammatory licensing; however, biomechanical forces, such as fluid shear stress, are a second, distinct class of stimuli that incite functional maturation. Here we show mechanotransduction, achieved by exposing MSC to various grades of wall shear stress (WSS) within a scalable conditioning platform, enhances the immunomodulatory potential of MSC independent of classical pro-inflammatory cytokines. A dose-dependent effect of WSS on potency is evidenced by production of prostaglandin E2 (PGE2) and indoleamine 2,3 dioxygenase 1 (IDO1), as well as suppression of tumor necrosis factor-α (TNF- α) and interferon-γ (IFN-γ) production by activated immune cells. Consistent, reproducible licensing is demonstrated in adipose tissue and bone marrow human derived MSC without significant impact on cell viability, cellular yield, or identity. Transcriptome analysis of WSS-conditioned BM-MSC elucidates the broader phenotypic implications on the differential expression of immunomodulatory factors. These results suggest mechanotransduction as a viable, scalable pre-conditioning alternative to pro-inflammatory cytokines. Enhancing the immunomodulatory capacity of MSC via biomechanical conditioning represents a novel cell therapy manufacturing approach.
Assuntos
Mecanotransdução Celular , Células-Tronco Mesenquimais , Citocinas/metabolismo , Dinoprostona/metabolismo , Humanos , Imunomodulação , Células-Tronco Mesenquimais/metabolismoRESUMO
Introduction: Traumatic brain injury is a leading cause of injury-related death and morbidity. Multiple clinical and pre-clinical studies have reported various results regarding sex-based differences in TBI. Our accepted rodent model of traumatic brain injury was used to identify sex-based differences in the pathological features of TBI. Methods: Male and female Sprague-Dawley rats were subjected to either controlled-cortical impact (CCI) or sham injury; brain tissue was harvested at different time intervals depending on the specific study. Blood-brain barrier (BBB) analysis was performed using infrared imaging to measure fluorescence dye extravasation. Microglia and splenocytes were characterized with traditional flow cytometry; microglia markers such as CD45, P2Y12, CD32, and CD163 were analyzed with t-distributed stochastic neighbor embedding (t-SNE). Flow cytometry was used to study tissue cytokine levels, and supplemented with ELISAs of TNF-âº, IL-17, and IL-1ß of the ipsilateral hemisphere tissue. Results: CCI groups of both sexes recorded a higher BBB permeability at 72 hours post-injury than their respective sham groups. There was significant difference in the integrated density value of BBB permeability between the male CCI group and the female CCI group (female CCI mean = 3.08 x 108 ± 2.83 x 107, male CCI mean = 2.20 x 108 ± 4.05 x 106, p = 0.0210), but otherwise no differences were observed. Traditional flow cytometry did not distinguish any sex-based difference in regards to splenocyte cell population after CCI. t-SNE did not reveal any significant difference between the male and female injury groups in the activation of microglia. Cytokine analysis after injury by flow cytometry and ELISA was limited in differences at the time point of 6 hours post-injury. Conclusion: In our rodent model of traumatic brain injury, sex-based differences in pathology and neuroinflammation at specified time points are limited, and only noted in one specific analysis of BBB permeability.
Assuntos
Lesões Encefálicas Traumáticas , Animais , Barreira Hematoencefálica/metabolismo , Lesões Encefálicas Traumáticas/patologia , Citocinas/metabolismo , Feminino , Masculino , Microglia/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Traumatic brain injury (TBI) results in a cascade of cellular responses, which produce neuroinflammation, partly due to the activation of microglia. Accurate identification of microglial populations is key to understanding therapeutic approaches that modify microglial responses to TBI and improve long-term outcome measures. Notably, previous studies often utilized an outdated convention to describe microglial phenotypes. We conducted a temporal analysis of the response to controlled cortical impact (CCI) in rat microglia between ipsilateral and contralateral hemispheres across seven time points, identified microglia through expression of activation markers including CD45, CD11b/c, and p2y12 receptor and evaluated their activation state using additional markers of CD32, CD86, RT1B, CD200R, and CD163. We identified unique sub-populations of microglial cells that express individual or combination of activation markers across time points. We further portrayed how the size of these sub-populations changes through time, corresponding to stages in TBI response. We described longitudinal changes in microglial population after CCI in two different locations using activation markers, showing clear separation into cellular sub-populations that feature different temporal patterns of markers after injury. These changes may aid in understanding the symptomatic progression following TBI and help define microglial subpopulations beyond the outdated M1/M2 paradigm.
Assuntos
Lesões Encefálicas Traumáticas , Microglia , Animais , Biomarcadores/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , RatosRESUMO
Traumatic brain injury (TBI) causes both physical disruption of the blood brain barrier (BBB) and altered immune responses that can lead to significant secondary brain injury and chronic inflammation within the central nervous system (CNS). Cell therapies, including mesenchymal stromal cells (MSC), have been shown to restore BBB integrity and augment endogenous splenic regulatory T cells (Treg), a subset of CD4+ T cells that function to regulate immune responses and prevent autoimmunity. We have recently shown that infusion of human cord blood-derived Treg decreased neuroinflammation after TBI in vivo and in vitro. However, while both cells have demonstrated anti-inflammatory and regenerative potential, they likely utilize differing, although potentially overlapping, mechanisms. Furthermore, studies investigating these two cell types together, as a combination therapy, are lacking. In this study, we compared the ability of Treg+MSC combination therapy, as well as MSC and Treg monotherapies, to improve BBB permeability in vivo and suppress inflammation in vitro. While Treg+MSC combination did not significantly augment potency in vivo, our in vitro data demonstrates that combination therapy may augment therapeutic potency and immunosuppressive potential compared to Treg or MSC monotherapy.
Assuntos
Barreira Hematoencefálica/imunologia , Lesões Encefálicas Traumáticas , Tolerância Imunológica , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Linfócitos T Reguladores , Animais , Lesões Encefálicas Traumáticas/imunologia , Lesões Encefálicas Traumáticas/terapia , Modelos Animais de Doenças , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/transplanteRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0233263.].
RESUMO
Traumatic brain injury (TBI) causes a profound inflammatory response within the central nervous system and peripheral immune system, which contributes to secondary brain injury and further morbidity and mortality. Preclinical investigations have demonstrated that treatments that downregulate microglia activation and polarize them toward a reparative/anti-inflammatory phenotype have improved outcomes in preclinical models. However, no therapy to date has translated into proven benefits in human patients. Regulatory T cells (Treg) have been shown to downregulate pathologic immune responses of the innate and adaptive immune system across a variety of pathologies. Furthermore, cellular therapy has been shown to augment host Treg responses in preclinical models; yet, studies investigating the use of Treg as a therapeutic for TBI are lacking. In a rodent TBI model, we demonstrate that human umbilical cord blood Treg modulate the central and peripheral immune response after injury in vitro and in vivo.
Assuntos
Lesões Encefálicas Traumáticas/imunologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Imunidade/imunologia , Imunofenotipagem/métodos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Animais , Lesões Encefálicas Traumáticas/patologia , Modelos Animais de Doenças , Humanos , Ratos , Ratos Sprague-DawleyRESUMO
In the U.S., approximately 1.7 million people suffer traumatic brain injury each year, with many enduring long-term consequences and significant medical and rehabilitation costs. The primary injury causes physical damage to neurons, glia, fiber tracts and microvasculature, which is then followed by secondary injury, consisting of pathophysiological mechanisms including an immune response, inflammation, edema, excitotoxicity, oxidative damage, and cell death. Most attempts at intervention focus on protection, repair or regeneration, with regenerative medicine becoming an intensively studied area over the past decade. The use of stem cells has been studied in many disease and injury models, using stem cells from a variety of sources and applications. In this study, human adipose-derived mesenchymal stromal cells (MSCs) were administered at early (3 days) and delayed (14 days) time points after controlled cortical impact (CCI) injury in rats. Animals were routinely assessed for neurological and vestibulomotor deficits, and at 32 days post-injury, brain tissue was processed by flow cytometry and immunohistochemistry to analyze neuroinflammation. Treatment with HB-adMSC at either 3d or 14d after injury resulted in significant improvements in neurocognitive outcome and a change in neuroinflammation one month after injury.
Assuntos
Lesões Encefálicas Traumáticas/terapia , Transplante de Células-Tronco Mesenquimais , Tecido Adiposo/citologia , Animais , Encéfalo/patologia , Lesões Encefálicas Traumáticas/patologia , Lesões Encefálicas Traumáticas/psicologia , Modelos Animais de Doenças , Humanos , Inflamação/patologia , Masculino , Aprendizagem em Labirinto , Células-Tronco Mesenquimais/citologia , Destreza Motora , Neurogênese , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Clinical trials in trauma populations are exploring the use of clinical cellular therapeutics (CCTs) like human mesenchymal stromal cells (MSC) and mononuclear cells (MNC). Recent studies demonstrate a procoagulant effect of these CCTs related to their expression of tissue factor (TF). We sought to examine this relationship in blood from severely injured trauma patients and identify methods to reverse this procoagulant effect. Human MSCs from bone marrow, adipose, and amniotic tissues and freshly isolated bone marrow MNC samples were tested. TF expression and phenotype were quantified using flow cytometry. CCTs were mixed individually with trauma patients' whole blood, assayed with thromboelastography (TEG), and compared with healthy subjects mixed with the same cell sources. Heparin was added to samples at increasing concentrations until TEG parameters normalized. Clotting time or R time in TEG decreased relative to the TF expression of the CCT treatment in a logarithmic fashion for trauma patients and healthy subjects. Nonlinear regression curves were significantly different with healthy subjects demonstrating greater relative decreases in TEG clotting time. In vitro coadministration of heparin normalized the procoagulant effect and required dose escalation based on TF expression. TF expression in human MSC and MNC has a procoagulant effect in blood from trauma patients and healthy subjects. The procoagulant effect is lower in trauma patients possibly because their clotting time is already accelerated. The procoagulant effect due to MSC/MNC TF expression could be useful in the bleeding trauma patient; however, it may emerge as a safety release criterion due to thrombotic risk. The TF procoagulant effect is reversible with heparin.
Assuntos
Coagulação Sanguínea , Ferimentos e Lesões/sangue , Adulto , Biomarcadores/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Estudos de Casos e Controles , Feminino , Heparina/farmacologia , Humanos , Masculino , TromboelastografiaRESUMO
Traumatic brain injury (TBI) results in a cascade of cellular responses, which produce neuroinflammation, partly due to microglial activation. Transforming from surveying to primed phenotypes, microglia undergo considerable molecular changes. However, specific microglial profiles in rat remain elusive due to tedious methodology and limited availability of reagents. Here, we present a flow cytometry-based analysis of rat microglia 24 h after TBI using the controlled cortical impact model, validated with a bioinformatics approach. Isolated microglia are analyzed for morphological changes and their expression of activation markers using flow cytometry, traditional gating-based analysis methods and support the data by employing bioinformatics statistical tools. We use CD45, CD11b/c, and p2y12 receptor to identify microglia and evaluate their activation state using CD32, CD86, RT1B, CD200R, and CD163. The results from logic-gated flow cytometry analysis was validated with bioinformatics-based analysis and machine learning algorithms to detect quantitative changes in morphology and marker expression in microglia due to activation following TBI.
Assuntos
Biomarcadores/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Biologia Computacional , Citometria de Fluxo , Microglia/metabolismo , Animais , Lesões Encefálicas Traumáticas/patologia , Polaridade Celular , Tamanho Celular , Microglia/patologia , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Lesões Encefálicas/terapia , Encéfalo/irrigação sanguínea , Sangue Fetal/transplante , Inflamação/terapia , Cordão Umbilical/citologia , Animais , Barreira Hematoencefálica/patologia , Encéfalo/patologia , Lesões Encefálicas/complicações , Lesões Encefálicas/patologia , Extravasamento de Materiais Terapêuticos e Diagnósticos/patologia , Humanos , Imunomodulação , Inflamação/complicações , Inflamação/patologia , Masculino , Ratos Sprague-Dawley , Baço/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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.
Assuntos
Diferenciação Celular , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Animais , Modelos Animais de Doenças , HumanosRESUMO
BACKGROUND: Human multipotent adult progenitor cells (MAPC®) are an emerging therapy for traumatic brain injury (TBI); however, clinically translating a therapy involves overcoming many factors in vivo which are not present in pre-clinical testing. In this study we examined clinical parameters in vitro that may impact cell therapy efficacy. METHODS: MAPC were infused through varying gauged needles and catheters with and without chlorhexidine, and their viability tested with trypan blue exclusion. MAPC were co-cultured with phenytoin and celecoxib at relevant clinical concentrations for 1 h and 24 h. Anti-inflammatory potency was tested using a stimulated rat splenocyte co-culture and ELISA for TNF-α production. MAPC were cultured under different osmolar concentrations and stained with propidium iodide for viability. Anti-inflammatory potency was tested by co-culture of MAPC with naïve lymphocytes activated by CD3/CD28 beads, and Click-iT® Plus EdU was used to quantify proliferation by flow cytometry. RESULTS: The mean viability of the MAPC infused via needles was 95 ± 1%; no difference was seen with varying flow rate, but viability was notably reduced by chlorhexidine. MAPC function was not impaired by co-culture with phenytoin, celecoxib, or combination with both. Co-culture with phenytoin showed a decrease in TNF-α production as compared to the MAPC control. MAPC cultured at varying osmolar concentrations all had viabilities greater than 90% with no statistical difference between them. Co-culture of MAPC with CD3/CD28 activated PBMCs showed a significant reduction in proliferation as measured by EdU uptake. DISCUSSION: Needle diameter, phenytoin, celecoxib, and a relevant range of osmolarities do not impair MAPC viability or anti-inflammatory potency in vitro.