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PURPOSE: The importance of the DNA damage response in mediating effects of radiotherapy (RT) has galvanized efforts to target this pathway with radiosensitizers. Yet early clinical trials of this approach have failed to yield a benefit in unselected populations. We hypothesized that ataxia-telangiectasia mutated (Atm)-null tumors would demonstrate genotype-specific synergy between RT and an inhibitor of the DNA damage response protein ataxia-telangiectasia and Rad3-related (ATR) kinase. EXPERIMENTAL DESIGN: We investigated the synergistic potential of the ATR inhibitor (ATRi) RP-3500 and RT in two Atm-null and isogenic murine models, both in vitro and in vivo. Staining of γ-H2AX foci, characterization of the immune response via flow cytometry, and tumor rechallenge experiments were performed to elucidate the mechanism of interaction. To examine genotype specificity, we tested the interaction of ATRi and RT in a Brca1-null model. Finally, patients with advanced cancer with ATM alterations were enrolled in a phase I/II clinical trial to validate preclinical findings. RESULTS: Synergy between RP-3500 and RT was confirmed in Atm-null lines in vitro, characterized by an accumulation of DNA double-strand breaks. In vivo, Atm-null tumor models had higher rates of durable control with RT and ATRi than controls. In contrast, there was no synergy in tumors lacking Brca1. Analysis of the immunologic response indicated that efficacy is largely mediated by cell-intrinsic mechanisms. Lastly, early results from our clinical trial showed complete responses in patients. CONCLUSIONS: Genotype-directed radiosensitization with ATRi and RT can unleash significant therapeutic benefit and could represent a novel approach to develop more effective combinatorial synthetic cytotoxic RT-based treatments.
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Chlamydia muridarum (Cm) has reemerged as a moderately prevalent infectious agent in research mouse colonies. Despite its' experimental use, there are limited studies evaluating Cm's effects on immunocompetent mice following its natural route of infection. A Cm field isolate was administered (orogastric gavage) to 8-week-old female BALB/cJ (C) mice. After confirming shedding (through 95d), these mice were cohoused with naïve C57BL/6J (B6), C, and Swiss (J:ARC[S]) mice (n=28/strain) for 30 days. Cohoused mice (n=3-6 exposed and 1-6 control/strain) were evaluated 7, 14, 21, 63, 120, and 180 days post-cohousing (DPC) via hemograms, serum biochemistry analysis, fecal qPCR, histopathology, and Cm MOMP immunohistochemistry. Immunophenotyping was performed on spleen (B6, C, S; n=6/strain) and intestines (B6; n=6) at 14 and 63 DPC. Serum cytokine concentrations were measured (B6; n=6 exposed and 2 control) at 14 and 63 DPC. All B6 mice were shedding Cm by 3 through 180 DPI. One of 3 C and 1 of 6 S mice began shedding Cm at 3 and 14 DPC, respectively, with the remaining shedding thereafter. Clinical pathology was nonremarkable. Minimal-to-moderate enterotyphlocolitis and gastrointestinal associated lymphoid tissue (GALT) hyperplasia was found in numerous infected mice. Cm antigen was frequently detected in GALT-associated surface intestinal epithelial cells. Splenic immunophenotyping revealed increased monocytes and shifts in T cell population subsets in all strains/timepoints. Gastrointestinal immunophenotyping (B6) revealed sustained increases in total inflammatory cells and elevated cytokine production in innate lymphoid cells and effector T cells (large intestine). Elevated concentrations of pro-inflammatory cytokines were detected in the serum (B6). These results demonstrate that while clinical disease was not appreciated, 3 commonly utilized strains of mice are susceptible to chronic enteric infections with Cm which may alter various immune responses. Considering the widespread use of mice to model GI disease, institutions should consider excluding Cm from their colonies.
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Introduction: In vivo studies of cancer biology and assessment of therapeutic efficacy are critical to advancing cancer research and ultimately improving patient outcomes. Murine cancer models have proven to be an invaluable tool in pre-clinical studies. In this context, multi-parameter flow cytometry is a powerful method for elucidating the profile of immune cells within the tumor microenvironment and/or play a role in hematological diseases. However, designing an appropriate multi-parameter panel to comprehensively profile the increasing diversity of immune cells across different murine tissues can be extremely challenging. Methods: To address this issue, we designed a panel with 13 fixed markers that define the major immune populations -referred to as the backbone panel- that can be profiled in different tissues but with the option to incorporate up to seven additional fluorochromes, including any marker specific to the study in question. Results: This backbone panel maintains its resolution across different spectral flow cytometers and organs, both hematopoietic and non-hematopoietic, as well as tumors with complex immune microenvironments. Discussion: Having a robust backbone that can be easily customized with pre-validated drop-in fluorochromes saves time and resources and brings consistency and standardization, making it a versatile solution for immuno-oncology researchers. In addition, the approach presented here can serve as a guide to develop similar types of customizable backbone panels for different research questions requiring high-parameter flow cytometry panels.
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Corantes Fluorescentes , Neoplasias , Animais , Camundongos , Citometria de Fluxo/métodos , Neoplasias/metabolismo , Microambiente TumoralRESUMO
Cell-intrinsic mechanisms of immunogenicity in ovarian cancer (OC) are not well understood. The presence of damaging mutations in the SWI/SNF chromatin remodeling complex, such as the SMARCA4 (BRG1) catalytic subunit, has been associated with improved response to ICB, however the mechanism by which this occurs is unclear. The aim of this current study was to examine the alterations in tumor cell-intrinsic and extrinsic immune signaling caused by SMARCA4 loss. Using OC models with loss-of-function mutations in SMARCA4 , we found that SMARCA4 loss resulted in increased cancer cell-intrinsic immunogenicity, characterized by upregulation of long-terminal RNA repeats such as endogenous retroviruses, increased expression of interferon-stimulated genes, and upregulation of antigen presentation machinery. Notably, this response was dependent on IRF3 signaling, but was independent of the type I interferon receptor. Mice inoculated with cancer cells bearing SMARCA4 loss demonstrated increased activation of cytotoxic T cells and NK cells in the tumor microenvironment as well as increased infiltration with activated dendritic cells. These results were recapitulated when animals bearing SMARCA4- proficient tumors were treated with a BRG1 inhibitor, suggesting that modulation of chromatin remodeling through targeting SMARCA4 may serve as a strategy to reverse immune evasion in OC.
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Human induced pluripotent stem cells (hiPSCs) were differentiated into a specific mesoderm subset characterized by KDR+CD56+APLNR+ (KNA+) expression. KNA+ cells had high clonal proliferative potential and specification into endothelial colony-forming cell (ECFCs) phenotype. KNA+ cells differentiated into perfused blood vessels when implanted subcutaneously into the flank of nonobese diabetic/severe combined immunodeficient mice and when injected into the vitreous of type 2 diabetic mice (db/db mice). Transcriptomic analysis showed that differentiation of hiPSCs derived from diabetics into KNA+ cells was sufficient to change baseline differences in gene expression caused by the diabetic status and reprogram diabetic cells to a pattern similar to KNA+ cells derived from nondiabetic hiPSCs. Proteomic array studies performed on retinas of db/db mice injected with either control or diabetic donor-derived KNA+ cells showed correction of aberrant signaling in db/db retinas toward normal healthy retina. These data provide "proof of principle" that KNA+ cells restore perfusion and correct vascular dysfunction in db/db mice.
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Background: Emerging evidence of antibody-independent functions, as well as the clinical efficacy of anti-CD20 depleting therapies, helped to reassess the contribution of B cells during multiple sclerosis (MS) pathogenesis. Objective: To investigate whether CD19+ B cells may share expression of the serine-protease granzyme-B (GzmB), resembling classical cytotoxic CD8+ T lymphocytes, in the peripheral blood from relapsing-remitting MS (RRMS) patients. Methods: In this study, 104 RRMS patients during different treatments and 58 healthy donors were included. CD8, CD19, Runx3, and GzmB expression was assessed by flow cytometry analyses. Results: RRMS patients during fingolimod (FTY) and natalizumab (NTZ) treatment showed increased percentage of circulating CD8+GzmB+ T lymphocytes when compared to healthy volunteers. An increase in circulating CD19+GzmB+ B cells was observed in RRMS patients during FTY and NTZ therapies when compared to glatiramer (GA), untreated RRMS patients, and healthy donors but not when compared to interferon-ß (IFN). Moreover, regarding Runx3, the transcriptional factor classically associated with cytotoxicity in CD8+ T lymphocytes, the expression of GzmB was significantly higher in CD19+Runx3+-expressing B cells when compared to CD19+Runx3- counterparts in RRMS patients. Conclusions: CD19+ B cells may exhibit cytotoxic behavior resembling CD8+ T lymphocytes in MS patients during different treatments. In the future, monitoring "cytotoxic" subsets might become an accessible marker for investigating MS pathophysiology and even for the development of new therapeutic interventions.
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Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Adulto , Antígenos CD19/uso terapêutico , Antígenos CD20 , Linfócitos B/metabolismo , Feminino , Cloridrato de Fingolimode/uso terapêutico , Acetato de Glatiramer/uso terapêutico , Humanos , Interferon beta/uso terapêutico , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/tratamento farmacológico , Natalizumab/uso terapêutico , Peptídeos , Linfócitos TRESUMO
BACKGROUND: Neurofilament Light (NfL) chain levels in both cerebrospinal fluid (CSF) and serum have been correlated with the reduction of axonal damage in multiple sclerosis (MS) patients treated with Natalizumab (NTZ). However, little is known about the function of plasmacytoid cells in NTZ-treated MS patients. OBJECTIVE: To evaluate CSF NfL, serum levels of soluble-HLA-G (sHLA-G), and eventual tolerogenic behavior of plasmacytoid dendritic cells (pDCs) in MS patients during NTZ treatment. METHODS: CSF NfL and serum sHLA-G levels were measured using an ELISA assay, while pDCs (BDCA-2+) were accessed through flow cytometry analyses. RESULTS: CSF levels of NfL were significantly reduced during NTZ treatment, while the serum levels of sHLA-G were increased. Moreover, NTZ treatment enhanced tolerogenic (HLA-G+, CD274+, and HLA-DR+) molecules and migratory (CCR7+) functions of pDCs in the peripheral blood. CONCLUSION: These findings suggest that NTZ stimulates the production of molecules with immunoregulatory function such as HLA-G and CD274 programmed death-ligand 1 (PD-L1) which may contribute to the reduction of axonal damage represented by the decrease of NfL levels in patients with MS.
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AIMS/HYPOTHESIS: Hypothalamic inflammation and sympathetic nervous system hyperactivity are hallmark features of the metabolic syndrome and type 2 diabetes. Hypothalamic inflammation may aggravate metabolic and immunological pathologies due to extensive sympathetic activation of peripheral tissues. Loss of somatostatinergic (SST) neurons may contribute to enhanced hypothalamic inflammation. METHODS: The present data show that leptin receptor-deficient (db/db) mice exhibit reduced hypothalamic SST neurons, particularly in the periventricular nucleus. We model this finding, using adeno-associated virus delivery of diphtheria toxin subunit A (DTA) driven by an SST-cre system to deplete these neurons in Sstcre/gfp mice (SST-DTA). RESULTS: SST-DTA mice exhibit enhanced hypothalamic c-Fos expression and brain inflammation as demonstrated by microglial and astrocytic activation. Bone marrow from SST-DTA mice undergoes skewed haematopoiesis, generating excess granulocyte-monocyte progenitors and increased proinflammatory (C-C chemokine receptor type 2; CCR2hi) monocytes. SST-DTA mice exhibited a 'diabetic retinopathy-like' phenotype: reduced visual function by optokinetic response (0.4 vs 0.25 cycles/degree; SST-DTA vs control mice); delayed electroretinogram oscillatory potentials; and increased percentages of retinal monocytes. Finally, mesenteric visceral adipose tissue from SST-DTA mice was resistant to catecholamine-induced lipolysis, displaying 50% reduction in isoprenaline (isoproterenol)-induced lipolysis compared with control littermates. Importantly, hyperglycaemia was not observed in SST-DTA mice. CONCLUSIONS/INTERPRETATION: The isolated reduction in hypothalamic SST neurons was able to recapitulate several hallmark features of type 2 diabetes in disease-relevant tissues.
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Tecido Adiposo/metabolismo , Medula Óssea/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hipotálamo/metabolismo , Neurônios/metabolismo , Retina/metabolismo , Somatostatina/metabolismo , Animais , Encéfalo/metabolismo , Diabetes Mellitus Tipo 2/diagnóstico , Toxina Diftérica/toxicidade , Eletrorretinografia , Citometria de Fluxo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo RealRESUMO
OBJECTIVE: Characterization of partial remission using the insulin dose-adjusted HbA1c (IDAA1c) ≤ 9 definition in a multiethnic Brazilian population of children and adolescents with type 1 diabetes (T1D), in addition with the determination of both Class II HLA genotype and autoantibodies. METHODS: We analyzed the prevalence of partial remission in 51 new-onset T1D patients with a median time follow-up of 13 months from diagnosis. For this study, anti-GAD65, anti-IA2 and HLA class II genotyping were considered. RESULTS: Partial remission occurred in 41.2% of T1D patients until 3 months after diagnosis, mainly in those aged 5-15 years. We have demonstrated a significant increase in the haplotypes of class II HLA DRB1*0301-DQB1*0201 in children and adolescents with a partial remission phase of the disease (42.9% vs 21.7% in non-remitters, P = .0291). This haplotype was also associated with the reduction of anti-IA2 antibodies production. Homozygote DRB1*03-DQB1*0201/DRB1*03-DQB1*0201 children had the lowest prevalence of IA-2A antibodies (P = .0402). However, this association does not correlate with the time of the remission phase. CONCLUSION: Although the number of patients studied was reduced, our data suggested that the association between genetics and decrease in antibody production to certain islet auto-antigen may contribute, at least in part, to the remission phase of T1D.
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Autoanticorpos/biossíntese , Diabetes Mellitus Tipo 1 , Antígenos de Histocompatibilidade Classe II/genética , Adolescente , Adulto , Autoanticorpos/genética , Brasil/epidemiologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/epidemiologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Antígenos HLA-DQ/genética , Cadeias HLA-DRB1/genética , Haplótipos , Humanos , Lactente , Masculino , Remissão Espontânea , Adulto JovemRESUMO
RATIONALE: There is incomplete knowledge of the impact of bone marrow cells on the gut microbiome and gut barrier function. OBJECTIVE: We postulated that diabetes mellitus and systemic ACE2 (angiotensin-converting enzyme 2) deficiency would synergize to adversely impact both the microbiome and gut barrier function. METHODS AND RESULTS: Bacterial 16S rRNA sequencing and metatranscriptomic analysis were performed on fecal samples from wild-type, ACE2-/y, Akita (type 1 diabetes mellitus), and ACE2-/y-Akita mice. Gut barrier integrity was assessed by immunofluorescence, and bone marrow cell extravasation into the small intestine was evaluated by flow cytometry. In the ACE2-/y-Akita or Akita mice, the disrupted barrier was associated with reduced levels of myeloid angiogenic cells, but no increase in inflammatory monocytes was observed within the gut parenchyma. Genomic and metatranscriptomic analysis of the microbiome of ACE2-/y-Akita mice demonstrated a marked increase in peptidoglycan-producing bacteria. When compared with control cohorts treated with saline, intraperitoneal administration of myeloid angiogenic cells significantly decreased the microbiome gene expression associated with peptidoglycan biosynthesis and restored epithelial and endothelial gut barrier integrity. Also indicative of diabetic gut barrier dysfunction, increased levels of peptidoglycan and FABP-2 (intestinal fatty acid-binding protein 2) were observed in plasma of human subjects with type 1 diabetes mellitus (n=21) and type 2 diabetes mellitus (n=23) compared with nondiabetic controls (n=23). Using human retinal endothelial cells, we determined that peptidoglycan activates a noncanonical TLR-2 (Toll-like receptor 2) associated MyD88 (myeloid differentiation primary response protein 88)-ARNO (ADP-ribosylation factor nucleotide-binding site opener)-ARF6 (ADP-ribosylation factor 6) signaling cascade, resulting in destabilization of p120-catenin and internalization of VE-cadherin as a mechanism of deleterious impact of peptidoglycan on the endothelium. CONCLUSIONS: We demonstrate for the first time that the defect in gut barrier function and dysbiosis in ACE2-/y-Akita mice can be favorably impacted by exogenous administration of myeloid angiogenic cells.
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Bactérias/metabolismo , Transplante de Medula Óssea , Permeabilidade Capilar , Diabetes Mellitus Tipo 2/cirurgia , Microbioma Gastrointestinal , Mucosa Intestinal/irrigação sanguínea , Mucosa Intestinal/microbiologia , Intestino Delgado/irrigação sanguínea , Intestino Delgado/microbiologia , Neovascularização Fisiológica , Peptidil Dipeptidase A/deficiência , Fator 6 de Ribosilação do ADP , Junções Aderentes/metabolismo , Enzima de Conversão de Angiotensina 2 , Animais , Células Cultivadas , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/microbiologia , Diabetes Mellitus Tipo 2/fisiopatologia , Modelos Animais de Doenças , Disbiose , Humanos , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Intestino Delgado/enzimologia , Intestino Delgado/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptidoglicano/metabolismo , Peptidil Dipeptidase A/genética , Recuperação de Função FisiológicaRESUMO
Macrophages are important in mounting an innate immune response to injury as well as in repair of injury. Gene expression of Rho proteins is known to be increased in fibrotic models; however, the role of these proteins in idiopathic pulmonary fibrosis (IPF) is not known. Here, we show that BAL cells from patients with IPF have a profibrotic phenotype secondary to increased activation of the small GTPase Rac1. Rac1 activation requires a posttranslational modification, geranylgeranylation, of the C-terminal cysteine residue. We found that by supplying more substrate for geranylgeranylation, Rac1 activation was substantially increased, resulting in profibrotic polarization by increasing flux through the mevalonate pathway. The increased flux was secondary to greater levels of acetyl-CoA from metabolic reprogramming to ß oxidation. The polarization mediated fibrotic repair in the absence of injury by enhancing macrophage/fibroblast signaling. These observations suggest that targeting the mevalonate pathway may abrogate the role of macrophages in dysregulated fibrotic repair.
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Fibrose Pulmonar Idiopática/metabolismo , Macrófagos/metabolismo , Ácido Mevalônico/metabolismo , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Adolescente , Adulto , Idoso , Animais , Feminino , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Oxirredução , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Macrophage activation is implicated in the development of pulmonary fibrosis by generation of profibrotic molecules. Although NADPH oxidase 4 (NOX4) is known to contribute to pulmonary fibrosis, its effects on macrophage activation and mitochondrial redox signaling are unclear. Here, we show that NOX4 is crucial for lung macrophage profibrotic polarization and fibrotic repair after asbestos exposure. NOX4 was elevated in lung macrophages from subjects with asbestosis, and mice harboring a deletion of NOX4 in lung macrophages were protected from asbestos-induced fibrosis. NOX4 promoted lung macrophage profibrotic polarization and increased production of profibrotic molecules that induce collagen deposition. Mechanistically, NOX4 further augmented mitochondrial ROS production and induced mitochondrial biogenesis. Targeting redox signaling and mitochondrial biogenesis prevented the profibrotic polarization of lung macrophages by reducing the production of profibrotic molecules. These observations provide evidence that macrophage NOX4 is a potentially novel therapeutic target to halt the development of asbestos-induced pulmonary fibrosis.
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Asbestose/metabolismo , Macrófagos Alveolares/fisiologia , Macrófagos/fisiologia , NADPH Oxidase 4/metabolismo , Biogênese de Organelas , Adulto , Idoso , Animais , Linhagem Celular , Polaridade Celular , Feminino , Fibrose , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Fenótipo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Previously, we showed that mesenchymal stem cells (MSC) can be mobilized into peripheral blood using electroacupuncture (EA) at acupoints, LI-4, LI-11, GV-14, and GV-20. The purpose of this study was to determine whether EA-mobilized MSC could be harvested and expanded in vitro to be used as an autologous cell therapy in horses. Peripheral blood mononuclear cells (PBMC) isolated from young and aged lame horses (n = 29) showed a marked enrichment for MSCs. MSC were expanded in vitro (n = 25) and administered intravenously at a dose of 50 x 106 (n = 24). Treatment resulted in significant improvement in lameness as assessed by the American Association of Equine Practitioners (AAEP) lameness scale (n = 23). MSCs exhibited immunomodulatory function by inhibition of lymphocyte proliferation and induction of IL-10. Intradermal testing showed no immediate or delayed immune reactions to MSC (1 x 106 to 1 x 104). In this study, we demonstrated an efficient, safe and reproducible method to mobilize and expand, in vitro, MSCs in sufficiently high concentrations for therapeutic administration. We confirm the immunomodulatory function of these cells in vitro. This non-pharmacological and non-surgical strategy for stem cell harvest has a broad range of biomedical applications and represents an improved clinically translatable and economical cell source for humans.
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Mobilização de Células-Tronco Hematopoéticas , Imunomodulação , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Animais , Proliferação de Células , Separação Celular , Cavalos , Linfócitos/citologia , Linfócitos/imunologia , Células-Tronco Mesenquimais/citologia , Transplante AutólogoRESUMO
The present study investigates the effects of xenotransplantation of Adipose Tissue Mesenchymal Stem Cells (AT-MSCs) in animals after ventral root avulsion. AT-MSC has similar characteristics to bone marrow mesenchymal stem cells (BM-MSCs), such as immunomodulatory properties and expression of neurotrophic factors. In this study, Lewis rats were submitted to surgery for unilateral avulsion of the lumbar ventral roots and received 5 × 10(5) AT-MSCs via the lateral funiculus. Two weeks after cell administration, the animals were sacrificed and the moto neurons, T lymphocytes and cell defense nervous system were analyzed. An increased neuronal survival and partial preservation of synaptophysin-positive nerve terminals, related to GDNF and BDNF expression of AT-MSCs, and reduction of pro-inflammatory reaction were observed. In conclusion, AT-MSCs prevent second phase neuronal injury, since they suppressed lymphocyte, astroglia and microglia effects, which finally contributed to rat motor-neuron survival and synaptic stability of the lesioned motor-neuron. Moreover, the survival of the injected AT- MSCs lasted for at least 14 days. These results indicate that neuronal survival after lesion, followed by mesenchymal stem cell (MSC) administration, might occur through cytokine release and immunomodulation, thus suggesting that AT-MSCs are promising cells for the therapy of neuronal lesions.
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Transplante de Células-Tronco Mesenquimais , Neurônios Motores/patologia , Radiculopatia/terapia , Medula Espinal/transplante , Tecido Adiposo/citologia , Tecido Adiposo/transplante , Animais , Xenoenxertos , Humanos , Imunomodulação , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Regeneração Nervosa , Neuroproteção , Radiculopatia/imunologia , Radiculopatia/metabolismo , Radiculopatia/patologia , Ratos , Medula Espinal/fisiopatologia , Raízes Nervosas Espinhais/fisiopatologia , Sinapses/imunologia , Sinapses/metabolismo , Sinapses/patologia , Sinaptofisina/metabolismo , Linfócitos T/imunologiaAssuntos
Linfócitos B/fisiologia , Células Dendríticas/fisiologia , Fosfatos de Dinucleosídeos/uso terapêutico , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Receptor Toll-Like 9/agonistas , Transferência Adotiva/métodos , Animais , Linfócitos B/efeitos dos fármacos , Complexo CD3/metabolismo , Células Dendríticas/efeitos dos fármacos , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito/toxicidade , Fragmentos de Peptídeos/toxicidadeRESUMO
Myelodysplastic syndromes (MDS) are clonal disorders involving hematopoietic stem cells (HSC) characterized by ineffective hematopoiesis. In addition to HSC defects, a defective hematopoiesis supporting capacity of mesenchymal stromal cells (MSCs) in the microenvironment niche has been implicated in MDS pathophysiology. The interaction between the dysfunctional MSCs MDS and HSC regulates diverse adhesion-related processes, such as progenitor cell survival, proliferation, differentiation, and self-renewal. As previously reported, a microarray analysis identified serine protease inhibitor kunitz-type 2 (SPINT2), an inhibitor of hepatocyte growth factor (HGF) activation, to be downregulated in MSCs from MDS patients. To define the role of SPINT2 in MDS hematopoietic microenvironment, an analysis of the effect of SPINT2 silencing in MSCs was carried out. We herein reported significantly lower levels of SPINT2 whereas HGF was expressed at higher levels in MSCs from MDS patients compared with healthy controls. SPINT2 underexpression results in an increased expression, production, and secretion of HGF and stromal cell-derived factor 1 (SDF-1) by MSCs. An increased adhesion of normal HSC or malignant cells onto MSCs silenced for SPINT2 was also observed. The altered MSCs adhesion in SPINT2-knockdown cells was correlated with increased CD49b and CD49d expression and with a decrease in CD49e expression. Our results suggest that the SPINT2 underexpression in the MSC from MDS patients is probably involved in the adhesion of progenitors to the bone marrow niche, through an increased HGF and SDF-1 signaling pathway.
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Regulação para Baixo , Células-Tronco Hematopoéticas/metabolismo , Glicoproteínas de Membrana/biossíntese , Células-Tronco Mesenquimais/metabolismo , Síndromes Mielodisplásicas/metabolismo , Adolescente , Adulto , Adesão Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Quimiocina CXCL12/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Células-Tronco Hematopoéticas/patologia , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Integrina alfa5/metabolismo , Masculino , Células-Tronco Mesenquimais/patologia , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/patologiaAssuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Fatores de Transcrição Forkhead/genética , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Inflamação/tratamento farmacológico , Neurite Autoimune Experimental/tratamento farmacológico , Animais , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Citocinas/metabolismo , Bainha de Mielina/fisiologia , Neuritos/fisiologia , Neurite Autoimune Experimental/patologia , Ratos , Ratos Endogâmicos Lew , Células Th1/efeitos dos fármacos , Células Th1/metabolismo , Células Th17/efeitos dos fármacos , Células Th17/metabolismoRESUMO
BACKGROUND: A growing body of evidence supports the hypothesis that vitamin D is an important environmental factor in the etiology of T-cell-mediated autoimmune diseases such as multiple sclerosis (MS). AIM: The purpose of this study was exploring the mechanisms underlying the beneficial effect of vitamin D3 in encephalomyelitis (EAE). METHODS: We treated monophasic experimental autoimmune EAE, induced in Lewis rat, with vitamin D3 and adoptively transfer tolerogenic bone marrow-derived DCs generated in the presence of vitamin D3. RESULTS: This study provides evidence that the in vivo administration of vitamin D3, as well as the adoptive transfer of vitamin D3 -induced IDO(+) immature/tolerogenic dendritic cells, leads to a significant increase in the percentage of CD4(+) CD25(+) Foxp3(+) regulatory T cells in the lymph nodes in a rat model of MS, experimental autoimmune EAE. Concomitant with the increase in this cell population, there is a significant decrease in the number of autoreactive T cells in the central nervous system. Bone marrow-derived DCs cultivated in the presence of vitamin D3 present a tolerogenic profile with high IL-10, TNFα, and IDO expression and decreased MHC-II and CD80 expression. The adoptive transfer of IDO (+) DCs induces a significant increase in the percentage of CD4(+) CD25(+) Foxp3(+) T cells in the lymph nodes, comparable with vitamin D3 treatment. CONCLUSION: These mechanisms contribute actively to the generation of a microenvironment in the lymph nodes that suppresses the activation of encephalitogenic T cells, resulting in the downregulation of the inflammatory response in the central nervous system.
Assuntos
Colecalciferol/farmacologia , Colecalciferol/uso terapêutico , Células Dendríticas/efeitos dos fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Índice de Gravidade de Doença , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Células Dendríticas/imunologia , Células Dendríticas/patologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Ratos , Ratos Endogâmicos Lew , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologiaRESUMO
INTRODUCTION: The seroprevalence of human T-cell leukemia virus type 1 (HTLV-1) is very high among Brazilians (1:200). HTLV-1 associated myelopathy or tropical spastic paraparesis (HAM/TSP) is the most common neurological complication of HTLV-1 infection. HAM/TSP can present with an acute/subacute form of longitudinally extensive myelitis, which can be confused with lesions seen in aquaporin-4 antibody (AQP4-Ab) positive neuromyelitis optica spectrum disorders (NMOSD) on MRI. Moreover, clinical attacks in patients with NMOSD have been shown to be preceded by viral infections in around 30% of cases. OBJECTIVE: To evaluate the frequency of AQP4-Ab in patients with HAM/TSP. To evaluate the frequency of HTLV-1 infection in patients with NMOSD. PATIENTS AND METHODS: 23 Brazilian patients with HAM/TSP, 20 asymptomatic HTLV-1+ serostatus patients, and 34 with NMOSD were tested for AQP4-Ab using a standardized recombinant cell based assay. In addition, all patients were tested for HTLV-1 by ELISA and Western blotting. RESULTS: 20/34 NMOSD patients were positive for AQP4-Ab but none of the HAM/TSP patients and none of the asymptomatic HTLV-1 infected individuals. Conversely, all AQP4-Ab-positive NMOSD patients were negative for HTLV-1 antibodies. One patient with HAM/TSP developed optic neuritis in addition to subacute LETM; this patient was AQP4-Ab negative as well. Patients were found to be predominantly female and of African descent both in the NMOSD and in the HAM/TSP group; Osame scale and expanded disability status scale scores did not differ significantly between the two groups. CONCLUSIONS: Our results argue both against a role of antibodies to AQP4 in the pathogenesis of HAM/TSP and against an association between HTLV-1 infection and the development of AQP4-Ab. Moreover, the absence of HTLV-1 in all patients with NMOSD suggests that HTLV-1 is not a common trigger of acute attacks in patients with AQP4-Ab positive NMOSD in populations with high HTLV-1 seroprevalence.