RESUMO
Acute kidney injury (AKI) is a fatal medical episode caused by sudden kidney damage or failure, leading to the death of patients within a few hours or days. Previous studies demonstrated that exosomes derived from various mesenchymal stem/stromal cells (MSC-exosomes) have positive effects on renal injuries in multiple experimental animal models of kidney diseases including AKI. However, the mass production of exosomes is a challenge not only in preclinical studies with large animals but also for successful clinical applications. In this respect, tangential flow filtration (TFF) is suitable for good manufacturing practice (GMP)-compliant large-scale production of high-quality exosomes. Until now, no studies have been reported on the use of TFF, but rather ultracentrifugation has been almost exclusively used, to isolate exosomes for AKI therapeutic application in preclinical studies. Here, we demonstrated the reproducible large-scale production of exosomes derived from adipose tissue-derived MSC (ASC-exosomes) using TFF and the lifesaving effect of the ASC-exosomes in a lethal model of cisplatin-induced rat AKI. Our results suggest the possibility of large-scale stable production of ASC-exosomes without loss of function and their successful application in life-threatening diseases.
Assuntos
Injúria Renal Aguda/terapia , Tecido Adiposo/citologia , Terapia Biológica/métodos , Exossomos , Células-Tronco Mesenquimais , Injúria Renal Aguda/induzido quimicamente , Animais , Células Cultivadas , Cisplatino , Humanos , RatosRESUMO
The coordination of macrophage polarization is essential for the robust regenerative potential of skeletal muscle. Repair begins with a phase mediated by inflammatory monocytes (IM) and proinflammatory macrophages (M1), followed by polarization to a proregenerative macrophage (M2) phenotype. Recently, regulatory T cells (Tregs) were described as necessary for this M1 to M2 transition. We report that chronic infection with the protozoan parasite Toxoplasma gondii causes a nonresolving Th1 myositis with prolonged tissue damage associated with persistent M1 accumulation. Surprisingly, Treg ablation during chronic infection rescues macrophage homeostasis and skeletal muscle fiber regeneration, showing that Tregs can directly contribute to muscle damage. This study provides evidence that the tissue environment established by the parasite could lead to a paradoxical pathogenic role for Tregs. As such, these findings should be considered when tailoring therapies directed at Tregs in inflammatory settings.
Assuntos
Macrófagos/imunologia , Miosite/imunologia , Linfócitos T Reguladores/imunologia , Toxoplasmose/imunologia , Transferência Adotiva , Animais , Diferenciação Celular/imunologia , Feminino , Citometria de Fluxo , Ativação Linfocitária/imunologia , Ativação de Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/imunologia , Músculo Esquelético/microbiologia , Músculo Esquelético/patologia , Miosite/microbiologia , Miosite/patologia , Reação em Cadeia da Polimerase em Tempo Real , Toxoplasmose/patologiaRESUMO
BACKGROUND: Pulmonary hypertension (PH) is driven by diverse pathogenic etiologies. Owing to their pleiotropic actions, microRNA molecules are potential candidates for coordinated regulation of these disease stimuli. METHODS AND RESULTS: Using a network biology approach, we identify microRNA associated with multiple pathogenic pathways central to PH. Specifically, microRNA-21 (miR-21) is predicted as a PH-modifying microRNA, regulating targets integral to bone morphogenetic protein (BMP) and Rho/Rho-kinase signaling as well as functional pathways associated with hypoxia, inflammation, and genetic haploinsufficiency of BMP receptor type 2. To validate these predictions, we have found that hypoxia and BMP receptor type 2 signaling independently upregulate miR-21 in cultured pulmonary arterial endothelial cells. In a reciprocal feedback loop, miR-21 downregulates BMP receptor type 2 expression. Furthermore, miR-21 directly represses RhoB expression and Rho-kinase activity, inducing molecular changes consistent with decreased angiogenesis and vasodilation. In vivo, miR-21 is upregulated in pulmonary tissue from several rodent models of PH and in humans with PH. On induction of disease in miR-21-null mice, RhoB expression and Rho-kinase activity are increased, accompanied by exaggerated manifestations of PH. CONCLUSIONS: A network-based bioinformatic approach coupled with confirmatory in vivo data delineates a central regulatory role for miR-21 in PH. Furthermore, this study highlights the unique utility of network biology for identifying disease-modifying microRNA in PH.
Assuntos
Biologia Computacional/métodos , Redes Reguladoras de Genes/genética , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/genética , MicroRNAs/fisiologia , Transdução de Sinais/genética , Animais , Células Cultivadas , Humanos , Hipertensão Pulmonar/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Ratos , Ratos Sprague-DawleyRESUMO
Introduction: Ets1 is a lymphoid-enriched transcription factor that regulates B- and Tcell functions in development and disease. Mice that lack Ets1 (Ets1 KO) develop spontaneous autoimmune disease with high levels of autoantibodies. Naïve CD4 + T cells isolated from Ets1 KO mice differentiate more readily to Th17 cells that secrete IL-17, a cytokine implicated in autoimmune disease pathogenesis. To determine if increased IL-17 production contributes to the development of autoimmunity in Ets1 KO mice, we crossed Ets1 KO mice to mice lacking the IL-17 receptor A subunit (IL17RA KO) to generate double knockout (DKO) mice. Methods: In this study, the status of the immune system of DKO and control mice was assessed utilizing ELISA, ELISpot, immunofluorescent microscopy, and flow cytometric analysis of the spleen, lymph node, skin. The transcriptome of ventral neck skin was analyzed through RNA sequencing. S. aureus clearance kinetics in in exogenously infected mice was conducted using bioluminescent S. aureus and tracked using an IVIS imaging experimental scheme. Results: We found that the absence of IL17RA signaling did not prevent or ameliorate the autoimmune phenotype of Ets1 KO mice but rather that DKO animals exhibited worse symptoms with striking increases in activated B cells and secreted autoantibodies. This was correlated with a prominent increase in the numbers of T follicular helper (Tfh) cells. In addition to the autoimmune phenotype, DKO mice also showed signs of immunodeficiency and developed spontaneous skin lesions colonized by Staphylococcus xylosus. When DKO mice were experimentally infected with Staphylococcus aureus, they were unable to clear the bacteria, suggesting a general immunodeficiency to staphylococcal species. γδ T cells are important for the control of skin staphylococcal infections. We found that mice lacking Ets1 have a complete deficiency of the γδ T-cell subset dendritic epidermal T cells (DETCs), which are involved in skin woundhealing responses, but normal numbers of other skin γδ T cells. To determine if loss of DETC combined with impaired IL-17 signaling might promote susceptibility to staph infection, we depleted DETC from IL17RA KO mice and found that the combined loss of DETC and impaired IL-17 signaling leads to an impaired clearance of the infection. Conclusions: Our studies suggest that loss of IL-17 signaling can result in enhanced autoimmunity in Ets1 deficient autoimmune-prone mice. In addition, defects in wound healing, such as that caused by loss of DETC, can cooperate with impaired IL-17 responses to lead to increased susceptibility to skin staph infections.
Assuntos
Doenças Autoimunes , Proteína Proto-Oncogênica c-ets-1 , Receptores de Interleucina-17 , Infecções Estafilocócicas , Animais , Camundongos , Autoanticorpos , Doenças Autoimunes/genética , Autoimunidade , Interleucina-17 , Receptores de Interleucina-17/metabolismo , Staphylococcus aureus , Proteína Proto-Oncogênica c-ets-1/metabolismoRESUMO
BACKGROUND: Glutathione peroxidase-3 (GPx-3) is a selenocysteine-containing plasma protein that scavenges reactive oxygen species in the extracellular compartment. A deficiency of this enzyme has been associated with platelet-dependent thrombosis, and a promoter haplotype with reduced function has been associated with stroke risk. METHODS AND RESULTS: We recently developed a genetic mouse model to assess platelet function and thrombosis in the setting of GPx-3 deficiency. The GPx-3((-/-)) mice showed an attenuated bleeding time and an enhanced aggregation response to the agonist ADP compared with wild-type mice. GPx-3((-/-)) mice displayed increased plasma levels of soluble P-selectin and decreased plasma cyclic cGMP compared with wild-type mice. ADP infusion-induced platelet aggregation in the pulmonary vasculature produced a more robust platelet activation response in the GPx-3((-/-)) than wild-type mice; histological sections from the pulmonary vasculature of GPx-3((-/-)) compared with wild-type mice showed increased platelet-rich thrombi and a higher percentage of occluded vessels. Cremaster muscle preparations revealed endothelial dysfunction in the GPx-3((-/-)) compared with wild-type mice. With a no-flow ischemia-reperfusion stroke model, GPx-3((-/-)) mice had significantly larger cerebral infarctions compared with wild-type mice and platelet-dependent strokes. To assess the neuroprotective role of antioxidants in this model, we found that manganese(III) meso-tetrakis(4-benzoic acid)porphyrin treatment reduced stroke size in GPx-3((-/-)) mice compared with vehicle-treated controls. CONCLUSIONS: These findings demonstrate that GPx-3 deficiency results in a prothrombotic state and vascular dysfunction that promotes platelet-dependent arterial thrombosis. These data illustrate the importance of this plasma antioxidant enzyme in regulating platelet activity, endothelial function, platelet-dependent thrombosis, and vascular thrombotic propensity.
Assuntos
Plaquetas/fisiologia , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Trombose/metabolismo , Difosfato de Adenosina/farmacologia , Animais , Antioxidantes/farmacologia , Tempo de Sangramento , Plaquetas/efeitos dos fármacos , GMP Cíclico/sangue , Modelos Animais de Doenças , Endotélio Vascular/patologia , Endotélio Vascular/fisiologia , Genótipo , Glutationa/sangue , Infarto da Artéria Cerebral Média/tratamento farmacológico , Camundongos , Camundongos Knockout , Selectina-P/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Fatores de Risco , Trombose/tratamento farmacológico , Trombose/epidemiologiaRESUMO
Pulmonary arterial hypertension (PAH) is suspected to be a strong mortality determinant of hemolytic disorders. However, direct contribution of acute intravascular hemolysis to fatal PAH has not been investigated. The roles of nitric oxide (NO) insufficiency and platelet activation in hemolysis-associated fatal PAH have been suspected but not been experimentally studied. We recently generated a unique intravascular hemolysis mouse model in which the membrane toxin, intermedilysin (ILY), exclusively lyses the erythrocytes of transgenically expressing human CD59 mice (ThCD59(RBC)), thereby inducing ILY-dose-dependent massive hemolysis. Using this murine hemolysis model, we found that the acute increase in pulmonary arterial pressure leading to right ventricle failure caused sudden death. Reduced NO bioavailability and massive platelet activation/aggregation leading to the formation of massive thrombosis specifically in the pulmonary microvasculature played the critical roles in pathogenesis of acute hemolysis-associated fatal PAH. Therapeutic interventions enhancing NO bioactivity or inhibiting platelet activation prevented sudden death or prolonged survival time via the suppression of the acute increase in pulmonary arterial pressure and improvement of right ventricle function. These findings further highlight the importance of the inhibition of platelet activation and the enhancement of NO bioavailability for the treatment and prevention of hemolysis-associated (fatal) PAH.
Assuntos
Modelos Animais de Doenças , Hemólise , Hipertensão Pulmonar/mortalidade , Hipertensão Pulmonar/patologia , Óxido Nítrico/farmacocinética , Ativação Plaquetária , Artéria Pulmonar/fisiopatologia , Animais , Bacteriocinas/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Antígenos CD59/fisiologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Eritrócitos/patologia , Fibrinolíticos/farmacologia , Humanos , Hipertensão Pulmonar/complicações , Camundongos , Camundongos Transgênicos , Agregação Plaquetária , Taxa de Sobrevida , Distribuição TecidualRESUMO
Toxoplasma gondii infection has proven to be an ideal model to understand the delicate balance between protective immunity and immune-mediated pathology during infection. Lethal infection causes a collapse of T regulatory cells (Tregs) mediated by the loss of IL-2 and conversion of Tregs to IFN-γ-producing cells. Importantly, these Tregs highly express the Th1 transcription factor Tbet. To determine the role of Tbet in Tregs, we infected Tbx21f/f -Foxp3YFPCre and control Foxp3YFPCre mice with the type II strain of T. gondii, ME49. The majority of Tbx21f/f -Foxp3YFPCre mice succumbed to a nonlethal dose. Notably, parasite burden was reduced in Tbx21f/f -Foxp3YFPCre compared with Foxp3YFPCre control mice. We found that Tbx21f/f -Foxp3YFPCre mice have significantly higher serum levels of proinflammatory cytokines IFN-γ and TNF-α, suggestive of a heightened immune response. To test if CD4+ T cells were driving immunopathology, we treated Tbx21f/f -Foxp3YFPCre mice with anti-CD4-depleting Abs and partially rescued these mice. Broad-spectrum antibiotic treatment also improved survival, demonstrating a role for commensal flora in immunopathology in Tbx21f/f -Foxp3YFPCre mice. RNA sequencing analysis reinforced that Tbet regulates several key cellular pathways, including leukocyte activation, regulation of lymphocyte activation, and cell cycle progression, that help to maintain fitness in Tregs during Th1 responses. Taken together, our data show an important role for Tbet in Tregs in preventing lethal immunopathology during T. gondii infection, further highlighting the protective role of Treg plasticity in controlling immune responses to infection and the microbiota.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Proteínas com Domínio T/metabolismo , Linfócitos T Reguladores/imunologia , Toxoplasmose/imunologia , Animais , Feminino , Fatores de Transcrição Forkhead/genética , Interferon gama/metabolismo , Ativação Linfocitária , Masculino , Camundongos , Proteínas com Domínio T/genética , Toxoplasmose/metabolismo , Toxoplasmose/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Toxoplasmic encephalitis can develop in individuals infected with the protozoan parasite Toxoplasma gondii and is typified by parasite replication and inflammation within the brain. Patients often present with seizures, but the parasite genes and host pathways involved in seizure development and/or propagation are unknown. We previously reported that seizure induction in Toxoplasma-infected mice is parasite strain dependent. Using quantitative trait locus mapping, we identify four loci in the Toxoplasma genome that potentially correlate with seizure development. In one locus, we identify the polymorphic virulence factor, GRA15, as a Toxoplasma gene associated with onset of seizures. GRA15 was previously shown to regulate host NF-κB-dependent gene expression during acute infections, and we demonstrate a similar role for GRA15 in brains of toxoplasmic encephalitic mice. GRA15 is important for increased expression of interleukin 1 beta (IL-1ß) and other IL-1 pathway host genes, which is significant since IL-1 signaling is involved in onset of seizures. Inhibiting IL-1 receptor signaling reduced seizure severity in Toxoplasma-infected mice. These data reveal one mechanism by which seizures are induced during toxoplasmic encephalitis. IMPORTANCE Inflammation in the brain caused by infections lead to seizures and other neurological symptoms. But the microbial products that induce seizures as well as the host pathways downstream of these factors are largely unknown. Using a nonbiased genetic screening approach, we identify 4 loci in the Toxoplasma genome that correlate with the induction of seizures in Toxoplasma-infected mice. One of these loci contains the gene, GRA15, which we demonstrate is associated with seizure development in toxoplasmic encephalitic mice. GRA15 accomplishes this in part by activating host pathways that lead to increased IL-1 receptor signaling and that inhibition of this signaling inhibits Toxoplasma-induced seizures.
Assuntos
Encéfalo/imunologia , Interações Hospedeiro-Parasita/imunologia , Interleucina-1beta/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Transdução de Sinais/imunologia , Toxoplasma/genética , Animais , Encéfalo/parasitologia , Encéfalo/patologia , Feminino , Expressão Gênica , Genoma de Protozoário , Humanos , Interleucina-1beta/genética , Camundongos , Camundongos Endogâmicos C57BL , Convulsões/imunologia , Convulsões/parasitologia , Toxoplasma/imunologia , Toxoplasmose Animal/imunologia , Toxoplasmose Animal/parasitologia , Fatores de VirulênciaRESUMO
To extend the retention time of aerosol-delivered growth factors in the lung for stem cell homing/activation purposes, we examined a formulation of vascular endothelial growth factor (VEGF) complexed to dextran sulfate (DS) and chitosan (CS) polyelectrolytes. Optimal incorporation of VEGF was found at a VEGF/DS/CS ratio of 0.12:1:0.33, which resulted in nanoparticle complexes with diameters of 612+/-79 nm and zeta potentials of -31+/-1 mV. The complexes collapsed in physiological solution, and released VEGF in a biphasic time course in vitro. In rat lungs, however, VEGF delivered in the complex was cleared at a constant exponential decay rate, 8-fold slower than that delivered in free form. The extended VEGF retention was likely due to equilibrium binding of VEGF to DS and to endogenous glycosaminoglycans. A similar retention effect is expected with other glycosaminoglycans-binding proteins (including many growth factors) when complexed with these glycans. Owing to its unique application, this type of complex is, perhaps, better described as a nanoglycan complex.
Assuntos
Pulmão/metabolismo , Polissacarídeos/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Animais , Quitosana/química , Quitosana/uso terapêutico , Sulfato de Dextrana/química , Sulfato de Dextrana/uso terapêutico , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Peptídeos e Proteínas de Sinalização Intercelular/farmacocinética , Pulmão/fisiologia , Nanopartículas/química , Nanopartículas/uso terapêutico , Polissacarídeos/farmacocinética , Ratos , Fator A de Crescimento do Endotélio Vascular/farmacocinéticaRESUMO
Objective: Pediatric burns are a major source of injury and in the absence of adequate care can lead to lifelong functional loss and disfigurement. While split thickness skin autografts are the current standard of care for deep partial and full-thickness burns, this approach is associated with considerable morbidity. For this reason, alternative skin substitutes such as allografts have gained interest. Approach: In the present study, we present a case series of 30 children with various types of burns treated with dehydrated human amnion chorion membrane (dHACM). Results: We show that treatment with dHACM is associated with an excellent rate of healing comparable to split thickness skin grafts with less rate of hypertrophic scar and contracture. Innovation: Treatment with dHACM is particularly attractive as it consists of many tissue regenerative factors, such as growth factors and immune modulators, thus it will reduce the risk of scaring. Conclusion: While dHACM is associated with an increased upfront cost, treating patients with small to moderate-sized burns with dHACM in their regional centers works to decrease downstream costs such as management of prolonged pain from donor-site morbidity, revisional surgeries from scar and contractures of split thickness grafts, and avoiding the cost of transfer to higher level centers of care. Our findings challenge the current standard of care, suggesting that dHACM provides an alternative to the current use of split thickness skin grafting and is a safe, feasible, and potentially superior substitute for the management of small to moderate total body surface area partial and full-thickness pediatric burns.
Assuntos
Âmnio , Queimaduras/cirurgia , Córion , Adolescente , Superfície Corporal , Criança , Pré-Escolar , Desidratação , Feminino , Hospitais Pediátricos , Humanos , Lactente , Masculino , New York , Estudos Retrospectivos , Transplante de Pele , Pele Artificial , Transplante Homólogo , Resultado do Tratamento , CicatrizaçãoRESUMO
A common feature shared by systemic fungal pathogens of environmental origin, such as Cryptococcus neoformans, is their ability to adapt to mammalian core body temperature. In C. neoformans, this adaptation is accompanied by Ccr4-mediated decay of ribosomal protein mRNAs. Here we use the related, but thermo-intolerant species Cryptococcus amylolentus to demonstrate that this response contributes to host-temperature adaptation and pathogenicity of cryptococci. In a C. neoformans ccr4Δ mutant, stabilized ribosomal protein mRNAs are retained in the translating pool, and stress-induced transcriptomic changes are reduced in comparison with the wild type strain, likely due to ineffective translation of transcription factors. In addition, the mutant displays increased exposure of cell wall glucans, and recognition by Dectin-1 results in increased phagocytosis by lung macrophages, linking mRNA decay to adaptation and immune evasion.
Assuntos
Cryptococcus neoformans/genética , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/genética , Termotolerância/genética , Animais , Antígenos de Fungos/imunologia , Cryptococcus/genética , Cryptococcus/imunologia , Cryptococcus/metabolismo , Cryptococcus neoformans/imunologia , Cryptococcus neoformans/metabolismo , Regulação Fúngica da Expressão Gênica , Glucanos/imunologia , Evasão da Resposta Imune/imunologia , Lectinas Tipo C/imunologia , Macrófagos Alveolares/imunologia , Camundongos , Fagocitose/imunologia , Ribonucleases/genéticaRESUMO
BACKGROUND AND PURPOSE: Plasma glutathione peroxidase (GPx-3) is a major antioxidant enzyme in plasma and the extracellular space that scavenges reactive oxygen species produced during normal metabolism or after oxidative insult. A deficiency of this enzyme increases extracellular oxidant stress, promotes platelet activation, and may promote oxidative posttranslational modification of fibrinogen. We recently identified a haplotype (H(2)) in the GPx-3 gene promoter that increases the risk of arterial ischemic stroke among children and young adults. METHODS: The aim of this study is to identify possible relationships between promoter haplotypes in the GPx-3 gene and cerebral venous thrombosis (CVT). We studied the GPx-3 gene promoter from 23 patients with CVT and 123 young controls (18 to 45 years) by single-stranded conformational polymorphism and sequencing analysis. RESULTS: Over half of CVT patients (52.1%) were heterozygous (H(1)H(2)) or homozygous (H(2)H(2)) carriers of the H(2) haplotype compared with 12.2% of controls, yielding a more than 10-fold independent increase in the risk of CVT (OR=10.7; 95% CI, 2.70 to 42.36; P<0.0001). Among women, the interaction of the H(2) haplotype with hormonal risk factors increased the OR of CVT to almost 70 (P<0.0001). CONCLUSIONS: These findings show that a novel GPx-3 promoter haplotype is a strong, independent risk factor for CVT. As we have previously shown that this haplotype is associated with a reduction in transcriptional activity, which compromises antioxidant activity and antithrombotic benefits of the enzyme, these results suggest that a deficiency of GPx-3 leads to a cerebral venous thrombophilic state.
Assuntos
Glutationa Peroxidase/genética , Trombose Intracraniana/epidemiologia , Trombose Intracraniana/genética , Trombose Venosa/epidemiologia , Trombose Venosa/genética , Adolescente , Adulto , Predisposição Genética para Doença/epidemiologia , Glutationa Peroxidase/sangue , Haplótipos , Hormônios , Humanos , Trombose Intracraniana/sangue , Modelos Logísticos , Pessoa de Meia-Idade , Polimorfismo Conformacional de Fita Simples , Regiões Promotoras Genéticas/genética , Fatores de Risco , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/epidemiologia , Acidente Vascular Cerebral/genética , Trombose Venosa/sangueRESUMO
Maintenance of tissue integrity in skeletal muscle requires the immunomodulatory and regenerative functions of muscle-resident regulatory T cells (Tregs). Chronic skeletal muscle infections, such as with Toxoplasma gondii disrupt normal immuno-regulatory networks and lead to pathogenic changes in Treg function. Specifically, Tregs during chronic T. gondii infection reinforce an inflammatory macrophage bias that exacerbates injury in skeletal muscle. In this study, we investigated whether the aberrations in skeletal muscle Treg function during chronic infection could be overcome by treatment with Treg-related factors associated with enhanced muscle regeneration during sterile injury. We show treatment of chronically infected mice with the Treg promoting therapies, interleukin-2 complexed with anti-IL-2 antibody or interleukin-33 (IL-33), did not restore macrophage dynamics or muscle function, respectively, in vivo. However supplementation of known Treg-derived factors, interleukin-10 (IL-10) and amphiregulin (Areg) improved muscle function and skewed macrophages toward a restorative phenotype in the presence of chronic infection. These shifts in macrophage phenotype are coupled with enhanced physiologic parameters of regeneration. Together, these data suggest that while Treg-mediated immuno-regulation is compromised during chronic skeletal muscle infection, supplementation of canonical Treg-derived factors such as IL-10 and Areg can restore immunologic balance and enhance muscle repair.
RESUMO
The robust regenerative potential of skeletal muscle is imperative for the maintenance of tissue function across a host of potential insults including exercise, infection, and trauma. The highly coordinated action of multiple immune populations, especially macrophages, plays an indispensable role in guiding this reparative program. However, it remains unclear how skeletal muscle repair proceeds in a chronically inflamed setting, such as infection, where an active immune response is already engaged. To address this question, we used a cardiotoxin injury model to challenge the reparative potential of chronically infected muscle. Compared with regenerating naive skeletal muscle, infected skeletal muscle exhibited multiple indicators of delayed muscle repair including a divergent morphologic response to injury and dysregulated expression of myogenic regulatory factors. Further, using both flow cytometric and single-cell RNA sequencing approaches, we show that reduced macrophage heterogeneity due to delayed emergence of restorative subsets underlies dysfunctional tissue repair during chronic infection. Our findings highlight how the preexisting inflammatory environment within tissue alters reparative immunity and ultimately the quality of tissue regeneration.
Assuntos
Macrófagos/metabolismo , Desenvolvimento Muscular/imunologia , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/metabolismo , Animais , Doença Crônica , Doenças Transmissíveis/imunologia , Biologia Computacional , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular/genética , Músculo Esquelético/lesões , Músculo Esquelético/patologia , Fatores de Regulação Miogênica/genética , Fatores de Regulação Miogênica/metabolismo , Toxoplasma , ToxoplasmoseRESUMO
BACKGROUND AND PURPOSE: Plasma glutathione peroxidase (GPx-3)-deficiency increases extracellular oxidant stress, decreases bioavailable nitric oxide, and promotes platelet activation. The aim of this study is to identify polymorphisms in the GPx-3 gene, examine their relationship to arterial ischemic stroke (AIS) in a large series of children and young adults, and determine their functional molecular consequences. METHODS: We studied the GPx-3 gene promoter from 123 young adults with idiopathic AIS and 123 age- and gender-matched controls by single-stranded conformational polymorphism and sequencing analysis. A second, independent population with childhood stroke was used for a replication study. We identified 8 novel, strongly linked polymorphisms in the GPx-3 gene promoter that formed 2 main haplotypes (H1 and H2). The transcriptional activity of the 2 most prevalent haplotypes was studied with luciferase reporter gene constructs. RESULTS: The H2 haplotype was over-represented in both patient populations and associated with an independent increase in the risk of AIS in young adults (odds ratio=2.07, 95% CI=1.03 to 4.47; P=0.034) and children (odds ratio=2.13, 95% CI=1.23 to 4.90; P=0.027). In adults simultaneously exposed to vascular risk factors, the risk of AIS approximately doubled (odds ratio=5.18, 95% CI=1.82 to 15.03; P<0.001). Transcriptional activity of the H2 haplotype was lower than that of the H1 haplotype, especially after upregulation by hypoxia (normalized relative luminescence: 3.54+/-0.32 versus 2.47+/-0.26; P=0.0083). CONCLUSIONS: These findings indicate that a novel GPx-3 promoter haplotype is an independent risk factor for AIS in children and young adults. This haplotype reduces the gene's transcriptional activity, thereby compromising gene expression and plasma antioxidant and antithrombotic activities.
Assuntos
Isquemia Encefálica/genética , Predisposição Genética para Doença/genética , Glutationa Peroxidase/genética , Polimorfismo Genético/genética , Regiões Promotoras Genéticas/genética , Acidente Vascular Cerebral/genética , Adulto , Antioxidantes/metabolismo , Coagulação Sanguínea/genética , Isquemia Encefálica/sangue , Isquemia Encefálica/enzimologia , Criança , Análise Mutacional de DNA , Ativação Enzimática/genética , Feminino , Regulação Enzimológica da Expressão Gênica/genética , Frequência do Gene , Marcadores Genéticos/genética , Testes Genéticos , Haplótipos , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Masculino , Mutação/genética , Fatores de Risco , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/enzimologia , Ativação Transcricional/genéticaRESUMO
Iron-sulfur (Fe-S) clusters are essential for mitochondrial metabolism, but their regulation in pulmonary hypertension (PH) remains enigmatic. We demonstrate that alterations of the miR-210-ISCU1/2 axis cause Fe-S deficiencies in vivo and promote PH. In pulmonary vascular cells and particularly endothelium, hypoxic induction of miR-210 and repression of the miR-210 targets ISCU1/2 down-regulated Fe-S levels. In mouse and human vascular and endothelial tissue affected by PH, miR-210 was elevated accompanied by decreased ISCU1/2 and Fe-S integrity. In mice, miR-210 repressed ISCU1/2 and promoted PH. Mice deficient in miR-210, via genetic/pharmacologic means or via an endothelial-specific manner, displayed increased ISCU1/2 and were resistant to Fe-S-dependent pathophenotypes and PH. Similar to hypoxia or miR-210 overexpression, ISCU1/2 knockdown also promoted PH. Finally, cardiopulmonary exercise testing of a woman with homozygous ISCU mutations revealed exercise-induced pulmonary vascular dysfunction. Thus, driven by acquired (hypoxia) or genetic causes, the miR-210-ISCU1/2 regulatory axis is a pathogenic lynchpin causing Fe-S deficiency and PH. These findings carry broad translational implications for defining the metabolic origins of PH and potentially other metabolic diseases sharing similar underpinnings.
Assuntos
Predisposição Genética para Doença , Hipertensão Pulmonar/genética , Hipóxia/complicações , Deficiências de Ferro , Proteínas Ferro-Enxofre/genética , MicroRNAs/genética , Enxofre/deficiência , Animais , Células Cultivadas , Células Endoteliais/fisiologia , Feminino , Humanos , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/patologia , CamundongosRESUMO
BACKGROUND: Immunotherapeutic approaches, such as dendritic cell (DC) vaccination, have emerged as promising strategies in the treatment of glioblastoma. Despite their promise, however, the absence of objective biomarkers and/or immunological monitoring techniques to assess the clinical efficacy of immunotherapy still remains a primary limitation. To address this, we sought to identify a functional biomarker for anti-tumor immune responsiveness associated with extended survival in glioblastoma patients undergoing DC vaccination. METHODS: 28 patients were enrolled and treated in two different Phase 1 DC vaccination clinical trials at UCLA. To assess the anti-tumor immune response elicited by therapy, we studied the functional responsiveness of pre- and post-vaccination peripheral blood lymphocytes (PBLs) to the immunostimulatory cytokines interferon-gamma (IFN-γ) and interleukin-2 (IL-2) in 21 of these patients for whom we had adequate material. Immune responsiveness was quantified by measuring downstream phosphorylation events of the transcription factors, STAT-1 and STAT-5, via phospho-specific flow cytometry. RESULTS: DC vaccination induced a significant decrease in the half-maximal concentration (EC-50) of IL-2 required to upregulate pSTAT-5 specifically in CD3(+)CD8(+) T lymphocytes (p < 0.045). Extended survival was also associated with an increased per cell phosphorylation of STAT-5 in cytotoxic T-cells following IL-2 stimulation when the median post/pre pSTAT-5 ratio was used to dichotomize the patients (p = 0.0015, log-rank survival; hazard ratio = 0.1834, p = 0.018). Patients whose survival was longer than two years had a significantly greater pSTAT-5 ratio (p = 0.015), but, contrary to our expectations, a significantly lower pSTAT-1 ratio (p = 0.038). CONCLUSIONS: Our results suggest that monitoring the pSTAT signaling changes in PBL may provide a functional immune monitoring measure predictive of clinical efficacy in DC-vaccinated patients.
RESUMO
Since the first descriptions of their active functions more than ten years ago, small non-coding RNA species termed microRNA (miRNA) have emerged as essential regulators in a broad range of adaptive and maladaptive cellular processes. With an exceptionally rapid pace of discovery in this field, the dysregulation of many individual miRNAs has been implicated in the development and progression of various cardiovascular diseases. MiRNA are also expected to play crucial regulatory roles in the progression of pulmonary vascular diseases such as pulmonary hypertension (PH), yet direct insights in this field are only just emerging. This review will provide an overview of pulmonary hypertension and its molecular mechanisms, tailored for both basic scientists studying pulmonary vascular biology and physicians who manage PH in their clinical practice. We will describe the pathobiology of pulmonary hypertension and mechanisms of action of miRNA relevant to this disease. Moreover, we will summarize the potential roles of miRNA as biomarkers and therapeutic targets as well as future strategies for defining the cooperative actions of these powerful effectors in pulmonary vascular disease.
RESUMO
To compare 123I-metaiodobenzylguanidine (MIBG) and [Fluorine-18]-2-fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET) in 22 patients with phaeochromocytomas and paragangliomas (PGL) retrospectively and to evaluate the correlation between FDG uptake and Ki-67 proliferative index. Fourteen of 17 (82%) patients at initial diagnosis had positive FDG uptake, more intensely in PGL. Eleven of 12 (92%) patients had positive MIBG uptake. PET and MIBG scintigraphy were concordant in 10 patients, discordant in 6. Combined results yielded no false negative findings and are complementary. Neither maximum standardised uptake value nor visual scores on MIBG correlated with Ki-67.
Assuntos
3-Iodobenzilguanidina , Neoplasias das Glândulas Suprarrenais/diagnóstico por imagem , Fluordesoxiglucose F18 , Feocromocitoma/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Neoplasias das Glândulas Suprarrenais/patologia , Adulto , Idoso , Proliferação de Células , Feminino , Humanos , Antígeno Ki-67/metabolismo , Masculino , Pessoa de Meia-Idade , Paraganglioma/diagnóstico por imagem , Paraganglioma/patologia , Feocromocitoma/patologia , Estudos RetrospectivosRESUMO
PURPOSE: Dendritic cell (DC) vaccines have recently emerged as an innovative therapeutic option for glioblastoma patients. To identify novel surrogates of anti-tumor immune responsiveness, we studied the dynamic expression of activation and inhibitory markers on peripheral blood lymphocyte (PBL) subsets in glioblastoma patients treated with DC vaccination at UCLA. EXPERIMENTAL DESIGN: Pre-treatment and post-treatment PBL from 24 patients enrolled in two Phase I clinical trials of dendritic cell immunotherapy were stained and analyzed using flow cytometry. A univariate Cox proportional hazards model was utilized to investigate the association between continuous immune monitoring variables and survival. Finally, the immune monitoring variables were dichotomized and a recursive partitioning survival tree was built to obtain cut-off values predictive of survival. RESULTS: The change in regulatory T cell (CD3(+)CD4(+)CD25(+)CD127(low)) frequency in PBL was significantly associated with survival (p = 0.0228; hazard ratio = 3.623) after DC vaccination. Furthermore, the dynamic expression of the negative co-stimulatory molecule, CTLA-4, was also significantly associated with survival on CD3(+)CD4(+) T cells (p = 0.0191; hazard ratio = 2.840) and CD3(+)CD8(+) T cells (p = 0.0273; hazard ratio = 2.690), while that of activation markers (CD25, CD69) was not. Finally, a recursive partitioning tree algorithm was utilized to dichotomize the post/pre fold change immune monitoring variables. The resultant cut-off values from these immune monitoring variables could effectively segregate these patients into groups with significantly different overall survival curves. CONCLUSIONS: Our results suggest that monitoring the change in regulatory T cell frequencies and dynamic expression of the negative co-stimulatory molecules on peripheral blood T cells, before and after DC vaccination, may predict survival. The cut-off point generated from these data can be utilized in future prospective immunotherapy trials to further evaluate its predictive validity.