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1.
Pain ; 162(5): 1305-1321, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33259456

RESUMO

ABSTRACT: Severe neuropathic pain is a hallmark of Fabry disease, a genetic disorder caused by a deficiency in lysosomal α-galactosidase A. Pain experienced by these patients significantly impacts their quality of life and ability to perform everyday tasks. Patients with Fabry disease suffer from peripheral neuropathy, sensory abnormalities, acute pain crises, and lifelong ongoing pain. Although treatment of pain through medication and enzyme replacement therapy exists, pain persists in many of these patients. Some has been learned in the past decades regarding clinical manifestations of pain in Fabry disease and the pathological effects of α-galactosidase A insufficiency in neurons. Still, it is unclear how pain and sensory abnormalities arise in patients with Fabry disease and how these can be targeted with therapeutics. Our knowledge is limited in part due to the lack of adequate preclinical models to study the disease. This review will detail the types of pain, sensory abnormalities, influence of demographics on pain, and current strategies to treat pain experienced by patients with Fabry disease. In addition, we discuss the current knowledge of Fabry pain pathogenesis and which aspects of the disease preclinical models accurately recapitulate. Understanding the commonalities and divergences between humans and preclinical models can be used to further interrogate mechanisms causing the pain and sensory abnormalities as well as advance development of the next generation of therapeutics to treat pain in patients with Fabry disease.


Assuntos
Doença de Fabry , Neuralgia , Doença de Fabry/complicações , Humanos , Neuralgia/etiologia , Neurônios , Qualidade de Vida , alfa-Galactosidase/genética , alfa-Galactosidase/uso terapêutico
2.
J R Soc Interface ; 17(167): 20190815, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32546114

RESUMO

Human mesenchymal stromal cells (MSCs) are a leading cell therapy candidate for the treatment of immune and inflammatory diseases due to their potent regulation of immune cells. MSC expression of indoleamine-2,3-dioxygenase (IDO) upon interferon γ (IFNγ) exposure has been proposed as both a sentinel marker and key mediator of MSC immunomodulatory potency. Rather than wait for in vivo exposure to cytokines, MSCs can be pre-licensed during manufacturing to enhance IDO expression. In this study, we systematically examine the relative role that the dose of IFNγ, the duration of pre-licensing and the donor of origin play in dictating MSC production of functional IDO. We find that across three human MSC donors, MSCs increase their expression of IDO in response to both increased dose of IFNγ and duration of pre-licensing. However, with extended pre-licensing, the expression of IDO no longer predicts MSCs ability to suppress activated peripheral blood mononuclear cells. In addition, pre-licensing dose and duration are revealed to be minor modifiers of MSCs inherent potency, and thus cannot be manipulated to boost poor donors to the levels of high-performing donors. Thus, the dose and duration of pre-licensing should be tailored to optimize performance of specific donors and an emphasis on donor selection is needed to realize significant benefits of pre-licensing.


Assuntos
Células-Tronco Mesenquimais , Proliferação de Células , Células Cultivadas , Humanos , Imunomodulação , Indolamina-Pirrol 2,3,-Dioxigenase , Interferon gama , Leucócitos Mononucleares
3.
Front Immunol ; 11: 143, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32158443

RESUMO

Mesenchymal stromal cells (MSCs) are administered locally to treat sites of inflammation. Local delivery is known to cause MSCs to aggregate into "spheroids," which alters gene expression and phenotype. While adherent MSCs are highly efficient in their inhibition of T cells, whether or not this property is altered upon MSC aggregation has not been thoroughly determined. In this study, we discovered that aggregation of MSCs into spheroids causes them to lose their T cell-suppressive abilities. Interestingly, adding budesonide, a topical glucocorticoid steroid, alongside spheroids partially restored MSC suppression of T cell proliferation. Through a series of inhibition and add-back studies, we determined budesonide acts synergistically with spheroid MSC-produced PGE2 to suppress T cell proliferation through the PGE2 receptors EP2 and EP4. These findings highlight critical differences between adherent and spheroid MSC interactions with human immune cells that have significant translational consequences. In addition, we uncovered a mechanism through which spheroid MSC suppression of T cells can be partly restored. By understanding the phenotypic changes that occur upon MSC aggregation and the impact of MSC drug interactions, improved immunosuppressive MSC therapies for localized delivery can be designed.


Assuntos
Imunomodulação/imunologia , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , Esferoides Celulares/imunologia , Esferoides Celulares/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Células da Medula Óssea/metabolismo , Budesonida/farmacologia , Agregação Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Dinoprostona/metabolismo , Dinoprostona/farmacologia , Humanos , Fatores Imunológicos/metabolismo , Ativação Linfocitária , Transdução de Sinais/efeitos dos fármacos , Doadores de Tecidos , Cordão Umbilical/citologia
4.
J Vis Exp ; (147)2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31157773

RESUMO

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, the data obtained from rodent islets are often not fully reproduced in or applicable to human islets due to well-known differences in islet structure and function between the species. Currently, techniques that are available to manipulate gene expression of human islets are very limited. Introduction of transgene into intact islets by adenovirus, plasmid, and oligonucleotides often suffers from low efficiency and high toxicity. Low efficiency is especially problematic in gene downregulation studies in intact islets, which require high efficiency. It has been known that enzymatically-dispersed islet cells reaggregate in culture forming spheroids termed pseudoislets. Size-controlled reaggregation of human islet cells creates pseudoislets that maintain dynamic first phase insulin secretion after prolonged culture and provide a window to efficiently introduce lentiviral short hairpin RNA (shRNA) with low toxicity. Here, a detailed protocol for the creation of human pseudoislets after lentiviral transduction using two commercially available multiwell plates is described. The protocol can be easily performed and allows for efficient downregulation of genes and assessment of dynamism of insulin secretion using human islet cells. Thus, human pseudoislets with lentiviral mediated gene modulation provide a powerful and versatile model to assess gene function within human islet cells.


Assuntos
Inativação Gênica , Ilhotas Pancreáticas/metabolismo , Lentivirus/genética , Transfecção/métodos , Células Cultivadas , Expressão Gênica , Humanos , Secreção de Insulina/genética , Ilhotas Pancreáticas/citologia , RNA Interferente Pequeno/genética , RNA Viral/genética , Transgenes
5.
Front Immunol ; 10: 1080, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31134100

RESUMO

As MSC products move from early development to clinical translation, culture conditions shift from xeno- to xeno-free systems. However, the impact of isolation and culture-expansion methods on the long-term resiliency of MSCs within challenging transplant environments is not fully understood. Recent work in our lab has shown that palmitate, a saturated fatty acid elevated in the serum of patients with obesity, causes MSCs to convert from an immunosuppressive to an immunostimulatory state at moderate to high physiological levels. This demonstrated that metabolically-diseased environments, like obesity, alter the immunomodulatory efficacy of healthy donor MSCs. In addition, it highlighted the need to test MSC efficacy not only in ideal conditions, but within challenging metabolic environments. To determine how the choice of xeno- vs. xeno-free media during isolation and expansion would affect future immunosuppressive function, umbilical cord explants from seven donors were subdivided and cultured within xeno- (fetal bovine serum, FBS) or xeno-free (human platelet lysate, PLT) medias, creating 14 distinct MSC preparations. After isolation and primary expansion, umbilical cord MSCs (ucMSC) were evaluated according to the ISCT minimal criteria for MSCs. Following baseline characterization, ucMSC were exposed to physiological doses of palmitate and analyzed for metabolic health, apoptotic induction, and immunomodulatory potency in co-cultures with stimulated human peripheral blood mononuclear cells. The paired experimental design (each ucMSC donor grown in two distinct culture environments) allowed us to delineate the contribution of inherent (nature) vs. environmentally-driven (nurture) donor characteristics to the phenotypic response of ucMSC during palmitate exposure. Culturing MSCs in PLT-media led to more consistent growth characteristics during the isolation and expansion for all donors, resulting in faster doubling times and higher cell yields compared to FBS. Upon palmitate challenge, PLT-ucMSCs showed a higher susceptibility to palmitate-induced metabolic disturbance, but less susceptibility to palmitate-induced apoptosis. Most striking however, was that the PLT-ucMSCs resisted the conversion to an immunostimulatory phenotype better than their FBS counterparts. Interestingly, examining MSC suppression of PBMC proliferation at physiologic doses of palmitate magnified the differences between donors, highlighting the utility of evaluating MSC products in stress-based assays that reflect the challenges MSCs may encounter post-transplantation.


Assuntos
Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Células-Tronco Mesenquimais/citologia , Palmitatos/metabolismo , Cordão Umbilical/citologia , Plaquetas/citologia , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultura/farmacologia , Humanos , Transplante de Células-Tronco Mesenquimais , Obesidade/sangue , Obesidade/patologia , Palmitatos/sangue
6.
Physiol Rep ; 6(20): e13907, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30370689

RESUMO

Rodent islets are widely used to study the pathophysiology of beta cells and islet function, however, structural and functional differences exist between human and rodent islets, highlighting the need for human islet studies. Human islets are highly variable, deteriorate during culture, and are difficult to genetically modify, making mechanistic studies difficult to conduct and reproduce. To overcome these limitations, we tested whether pseudoislets, created by dissociation and reaggregation of islet cell suspensions, allow for assessment of dynamic islet function after genetic modulation. Characterization of pseudoislets cultured for 1 week revealed better preservation of first-phase glucose-stimulated insulin secretion (GSIS) compared with cultured-intact islets and insulin secretion profiles similar to fresh islets when challenged by glibenclamide and KCl. qPCR indicated that pseudoislets are similar to the original islets for the expression of markers for cell types, beta cell function, and cellular stress with the exception of reduced proinflammatory cytokine genes (IL1B, CCL2, CXCL8). The expression of extracellular matrix markers (ASPN, COL1A1, COL4A1) was also altered in pseudoislets compared with intact islets. Compared with intact islets transduced by adenovirus, pseudoislets transduced by lentivirus showed uniform transduction and better first-phase GSIS. Lastly, the lentiviral-mediated delivery of short hairpin RNA targeting glucokinase (GCK) achieved significant reduction of GCK expression in pseudoislets as well as marked reduction of both first and second phase GSIS without affecting the insulin secretion in response to KCl. Thus, pseudoislets are a tool that enables efficient genetic modulation of human islet cells while preserving insulin secretion.


Assuntos
Técnicas de Transferência de Genes , Glucoquinase/genética , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , RNA Interferente Pequeno/genética , Adulto , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Glucoquinase/metabolismo , Humanos , Lentivirus/genética , Masculino , Pessoa de Meia-Idade , RNA Interferente Pequeno/metabolismo
7.
Mol Ther ; 26(3): 860-873, 2018 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-29352647

RESUMO

The use of mesenchymal stromal cell (MSC) therapy for the treatment of type 2 diabetes (T2D) and T2D complications is promising; however, the investigation of MSC function in the setting of T2D has not been thoroughly explored. In our current study, we investigated the phenotype and function of MSCs in a simulated in vitro T2D environment. We show that palmitate, but not glucose, exposure impairs MSC metabolic activity with moderate increases in apoptosis, while drastically affecting proliferation and morphology. In co-culture with peripheral blood mononuclear cells (PBMCs), we found that MSCs not only lose their normal suppressive ability in high levels of palmitate, but actively support and enhance inflammation, resulting in elevated PBMC proliferation and pro-inflammatory cytokine release. The pro-inflammatory effect of MSCs in palmitate was partially reversed via palmitate removal and fully reversed through pre-licensing MSCs with interferon-gamma and tumor necrosis factor alpha. Thus, palmitate, a specific metabolic factor enriched within the T2D environment, is a potent modulator of MSC immunosuppressive function, which may in part explain the depressed potency observed in MSCs isolated from T2D patients. Importantly, we have also identified a robust and durable pre-licensing regimen that protects MSC immunosuppressive function in the setting of T2D.


Assuntos
Interferon gama/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Palmitatos/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Metabolismo Energético/efeitos dos fármacos , Glucose/metabolismo , Glucose/farmacologia , Humanos , Imunomodulação/efeitos dos fármacos , Inflamação/etiologia , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Interferon gama/farmacologia , Janus Quinase 1/metabolismo , Janus Quinase 2/metabolismo , Fenótipo , Substâncias Protetoras/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia
8.
Stem Cells ; 35(5): 1437-1439, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27758056

RESUMO

Tailoring MSCs to fit the disease. Fresh, cryopreserved and, prelicensed cryopreserved MSC are all being explored to treat numerous diseases, but all are not suitable to treat all conditions. injury. "*" denotes preferred therapeutic strategy when both fresh MSC and cryo-MSC have shown utility in treating the disease but one is more efficacious or logistically suitable. ABBREVIATIONS: CLI, critical limb ischemia; GvHD. graft versus host disease; I/R, ischemia reperfusion (I/R); OI, osteogenesis imperfecta.


Assuntos
Criopreservação , Interferon gama/farmacologia , Células-Tronco Mesenquimais/citologia , Animais , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/patologia
9.
Sci Rep ; 6: 26463, 2016 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-27212469

RESUMO

The ability to use mesenchymal stromal cells (MSC) directly out of cryostorage would significantly reduce the logistics of MSC therapy by allowing on-site cryostorage of therapeutic doses of MSC at hospitals and clinics. Such a paradigm would be especially advantageous for the treatment of acute conditions such as stroke and myocardial infarction, which are likely to require treatment within hours after ischemic onset. Recently, several reports have emerged that suggest MSC viability and potency are damaged by cryopreservation. Herein we examine the effect of cryopreservation on human MSC viability, immunomodulatory potency, growth factor secretion, and performance in an ischemia/reperfusion injury model. Using modifications of established cryopreservation methods we developed MSC that retain >95% viability upon thawing, remain responsive to inflammatory signals, and are able to suppress activated human peripheral blood mononuclear cells. Most importantly, when injected into the eyes of mice 3 hours after the onset of ischemia and 2 hours after the onset of reperfusion, cryopreserved performed as well as fresh MSC to rescue retinal ganglion cells. Thus, our data suggests when viability is maintained throughout the cryopreservation process, MSC retain their therapeutic potency in both in vitro potency assays and an in vivo ischemia/reperfusion model.


Assuntos
Criopreservação/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Traumatismo por Reperfusão/terapia , Doenças Retinianas/terapia , Animais , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Humanos , Camundongos , Células Ganglionares da Retina/citologia
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