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1.
Diabetologia ; 64(11): 2575-2588, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34430981

RESUMO

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.


Assuntos
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 Real
2.
Diabetologia ; 64(7): 1674-1689, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33770194

RESUMO

AIMS/HYPOTHESIS: Homo sapiens evolved under conditions of intermittent food availability and prolonged fasting between meals. Periods of fasting are important for recovery from meal-induced oxidative and metabolic stress, and tissue repair. Constant high energy-density food availability in present-day society contributes to the pathogenesis of chronic diseases, including diabetes and its complications, with intermittent fasting (IF) and energy restriction shown to improve metabolic health. We have previously demonstrated that IF prevents the development of diabetic retinopathy in a mouse model of type 2 diabetes (db/db); however the mechanisms of fasting-induced health benefits and fasting-induced risks for individuals with diabetes remain largely unknown. Sirtuin 1 (SIRT1), a nutrient-sensing deacetylase, is downregulated in diabetes. In this study, the effect of SIRT1 stimulation by IF, fasting-mimicking cell culture conditions (FMC) or pharmacological treatment using SRT1720 was evaluated on systemic and retinal metabolism, systemic and retinal inflammation and vascular and bone marrow damage. METHODS: The effects of IF were modelled in vivo using db/db mice and in vitro using bovine retinal endothelial cells or rat retinal neuroglial/precursor R28 cell line serum starved for 24 h. mRNA expression was analysed by quantitative PCR (qPCR). SIRT1 activity was measured via histone deacetylase activity assay. NR1H3 (also known as liver X receptor alpha [LXRα]) acetylation was measured via western blot analysis. RESULTS: IF increased Sirt1 mRNA expression in mouse liver and retina when compared with non-fasted animals. IF also increased SIRT1 activity eightfold in mouse retina while FMC increased SIRT1 activity and expression in retinal endothelial cells when compared with control. Sirt1 expression was also increased twofold in neuronal retina progenitor cells (R28) after FMC treatment. Moreover, FMC led to SIRT1-mediated LXRα deacetylation and subsequent 2.4-fold increase in activity, as measured by increased mRNA expression of the genes encoding ATP-binding cassette transporter (Abca1 and Abcg1). These changes were reduced when retinal endothelial cells expressing a constitutively acetylated LXRα mutant were tested. Increased SIRT1/LXR/ABC-mediated cholesterol export resulted in decreased retinal endothelial cell cholesterol levels. Direct activation of SIRT1 by SRT1720 in db/db mice led to a twofold reduction of diabetes-induced inflammation in the retina and improved diabetes-induced visual function impairment, as measured by electroretinogram and optokinetic response. In the bone marrow, there was prevention of diabetes-induced myeloidosis and decreased inflammatory cytokine expression. CONCLUSIONS/INTERPRETATION: Taken together, activation of SIRT1 signalling by IF or through pharmacological activation represents an effective therapeutic strategy that provides a mechanistic link between the advantageous effects associated with fasting regimens and prevention of microvascular and bone marrow dysfunction in diabetes.


Assuntos
Angiopatias Diabéticas/prevenção & controle , Jejum/fisiologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Animais , Bovinos , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Células Cultivadas , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Experimental/terapia , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/metabolismo , Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Hipoglicemiantes/farmacologia , Receptores X do Fígado/genética , Receptores X do Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ratos , Retina/efeitos dos fármacos , Retina/patologia , Neurônios Retinianos/efeitos dos fármacos , Neurônios Retinianos/metabolismo , Neurônios Retinianos/patologia , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sirtuína 1/efeitos dos fármacos , Sirtuína 1/genética , Sirtuína 1/metabolismo
3.
Circ Res ; 125(11): 969-988, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31610731

RESUMO

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.


Assuntos
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 Fisiológica
4.
Pediatr Diabetes ; 21(4): 606-614, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32078220

RESUMO

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.


Assuntos
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 Jovem
5.
J Cell Mol Med ; 23(2): 1562-1571, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30484958

RESUMO

The role of tumour microenvironment in neoplasm initiation and malignant evolution has been increasingly recognized. However, the bone marrow mesenchymal stromal cell (BMMSC) contribution to disease progression remains poorly explored. We previously reported that the expression of serine protease inhibitor kunitz-type2 (SPINT2/HAI-2), an inhibitor of hepatocyte growth factor (HGF) activation, is significantly lower in BMMSC from myelodysplastic syndromes (MDS) patients compared to healthy donors (HD). Thus, to investigate whether this loss of expression was due to SPINT2/HAI-2 methylation, BMMSC from MDS and de novo acute myeloid leukaemia (de novo AML) patients were treated with 5-Azacitidine (Aza), a DNA methyltransferase inhibitor. In MDS- and de novo AML-BMMSC, Aza treatment resulted in a pronounced SPINT2/HAI-2 levels up-regulation. Moreover, Aza treatment of HD-BMMSC did not improve SPINT2/HAI-2 levels. To understand the role of SPINT2/HAI-2 down-regulation in BMMSC physiology, SPINT2/HAI-2 expression was inhibited by lentivirus. SPINT2 underexpression resulted in an increased production of HGF by HS-5 stromal cells and improved survival of CD34+ de novo AML cells. We also observed an increased adhesion of de novo AML hematopoietic cells to SPINT2/HAI-2 silenced cells. Interestingly, BMMSC isolated from MDS and de novo AML patients had increased expression of the integrins CD49b, CD49d, and CD49e. Thus, SPINT2/HAI-2 may contribute to functional and morphological abnormalities of the microenvironment niche and to stem/progenitor cancer cell progression. Hence, down-regulation in SPINT2/HAI-2 gene expression, due to methylation in MDS-BMMSC and de novo AML-BMMSC, provides novel insights into the pathogenic role of the leukemic bone marrow microenvironment.


Assuntos
Azacitidina/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Glicoproteínas de Membrana/genética , Síndromes Mielodisplásicas/tratamento farmacológico , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Integrina alfa2/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos
6.
Ann Hematol ; 98(11): 2507-2521, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31493004

RESUMO

Microparticles are sub-micron vesicles possessing protein and other materials derived from the plasma membrane of their parent cells, and literature suggests that they may have a role in the pathophysiology and downstream manifestations of sickle cell disease (SCD). The contributions of red blood cells microparticles (RMP) to the pathogenic mechanisms and clinical phenotypes of SCD are largely unknown. There is a controversy as to whether the proportions of intravascular hemolysis (approximately ≤ 30% of total hemolysis) would be enough to explain some complications seen in patients with SCD. We investigated RMP among 138 SCD patients and 39 HbAA individuals. Plasma RMPs were quantified by flow cytometry, plasma hemoglobin and heme by colorimetric assays, and haptoglobin and hemopexin by ELISA. The patients had higher RMP, plasma hemoglobin, and heme compared to the controls. On the contrary, haptoglobin and hemopexin were depleted in the patients. The RMP correlated positively with heme, lactate dehydrogenase, plasma hemoglobin, serum bilirubin, reticulocyte counts, and tricuspid regurgitant jet velocity of the patients. Contrarily, it correlated negatively with HbF, hemopexin, red blood cells counts, hemoglobin concentration, and haptoglobin. Although patients treated with hydroxyurea had lower RMP, this did not attain statistical significance. Patients with sickle leg ulcer and elevated tricuspid regurgitant jet velocity had higher levels of RMP. In conclusion, these data suggest that RMPs are associated with hemolysis and may have important roles in the pathophysiology and downstream complications of SCD.


Assuntos
Anemia Falciforme/sangue , Micropartículas Derivadas de Células/metabolismo , Eritrócitos/metabolismo , Hemólise , Adolescente , Adulto , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/patologia , Biomarcadores/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
7.
Biochim Biophys Acta Mol Basis Dis ; 1863(2): 450-461, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27840303

RESUMO

New drug development for neoplasm treatment is nowadays based on molecular targets that participate in the disease pathogenesis and tumor phenotype. Herein, we describe a new specific pharmacological hematopoietic cell kinase (HCK) inhibitor (iHCK-37) that was able to reduce PI3K/AKT and MAPK/ERK pathways activation after erythropoietin induction in cells with high HCK expression: iHCK-37 treatment increased leukemic cells death and, very importantly, did not affect normal hematopoietic stem cells. We also present evidence that HCK, one of Src kinase family (SFK) member, regulates early-stage erythroid cell differentiation by acting as an upstream target of a frequently deregulated pathway in hematologic neoplasms, PI3K/AKT and MAPK/ERK. Notably, HCK levels were highly increased in stem cells from patients with some diseases, as Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML), that are associated with ineffective erythropoiesis These discoveries support the exploration of the new pharmacological iHCK-37 in future preclinical and clinical studies.


Assuntos
Inibidores Enzimáticos/farmacologia , Eritropoetina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-hck/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-hck/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adulto , Idoso , Morte Celular/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Feminino , Fator de Transcrição GATA1/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/metabolismo , Adulto Jovem
8.
Immunology ; 146(3): 486-95, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26302057

RESUMO

Paracoccidioidomycosis is a systemic infection prevalent in Latin American countries. Disease develops after inhalation of Paracoccidioides brasiliensis conidia followed by an improper immune activation by the host leucocytes. Dendritic cells (DCs) are antigen-presenting cells with the unique ability to direct the adaptive immune response by the time of activation of naive T cells. This study was conducted to test whether extracts of P. brasiliensis would induce maturation of DCs. We found that DCs treated with extracts acquired an inflammatory phenotype and upon adoptive transfer conferred protection to infection. Interestingly, interleukin-10 production by CD8(+) T cells was ablated following DC transfer. Further analyses showed that lymphocytes from infected mice were high producers of interleukin-10, with CD8(+) T cells being the main source. Blockage of cross-presentation to CD8(+) T cells by modulated DCs abolished the protective effect of adoptive transfer. Collectively, our data show that adoptive transfer of P. brasiliensis-modulated DCs is an interesting approach for the control of infection in paracoccidioidomycosis.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Interleucina-10/biossíntese , Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Paracoccidioidomicose/prevenção & controle , Transferência Adotiva , Animais , Antígenos de Fungos/farmacologia , Diferenciação Celular/imunologia , Apresentação Cruzada , Citocinas/biossíntese , Células Dendríticas/citologia , Células Dendríticas/microbiologia , Feminino , Vacinas Fúngicas/imunologia , Vacinas Fúngicas/farmacologia , Mediadores da Inflamação/metabolismo , Interleucina-10/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout
9.
Front Immunol ; 15: 1374943, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38605953

RESUMO

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.


Assuntos
Corantes Fluorescentes , Neoplasias , Animais , Camundongos , Citometria de Fluxo/métodos , Neoplasias/metabolismo , Microambiente Tumoral
10.
bioRxiv ; 2024 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-39071441

RESUMO

Chlamydia muridarum (Cm) has reemerged as a moderately prevalent infectious agent in research mouse colonies. Despite its' experimental use, few studies evaluate 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 observed in 15 and 47 of 76 Cm-infected mice, respectively. 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). Results demonstrate that while clinical disease was not appreciated, 3 commonly utilized strains of mice are susceptible to chronic enteric Cm infection which may alter various immune responses. Considering the widespread use of mice to model GI disease, institutions should consider excluding Cm from their colonies.

11.
Clin Cancer Res ; : OF1-OF14, 2024 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-39109923

RESUMO

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.

12.
bioRxiv ; 2023 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-37609261

RESUMO

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.

13.
Mult Scler ; 18(7): 1038-41, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22041091

RESUMO

Intrathecal immunoglobulin synthesis in an oligoclonal pattern is the most common immunologic abnormality detected in MS patients. Various treatments, such as immunomodulators and immunosuppressors, have not been found to modify it. Natalizumab hinders migration of encephalitogenic T-cells into the central nervous system (CNS), reducing inflammatory response. Its impact on CSF oligoclonal bands (OCBs) has not been demonstrated. This report describes its effect in four out of six patients with multiple sclerosis after a mean of 10 infusions: the CSF was negative for OCBs at the second lumbar puncture. In conclusion, natalizumab treatment can reduce CSF OCBs to undetectable levels, although the clinical significance of this observation is not yet known.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Esclerose Múltipla Recidivante-Remitente/líquido cefalorraquidiano , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Bandas Oligoclonais/líquido cefalorraquidiano , Adulto , Linfócitos B/efeitos dos fármacos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Feminino , Humanos , Masculino , Esclerose Múltipla Recidivante-Remitente/imunologia , Natalizumab , Adulto Jovem
14.
Front Immunol ; 13: 750660, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35197967

RESUMO

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.


Assuntos
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 T
15.
Sci Adv ; 8(9): eabm5559, 2022 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-35245116

RESUMO

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.

16.
J Neuroinflammation ; 8(1): 2, 2011 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-21214939

RESUMO

The plasmacytoid dendritic cells (pDCs) express a high level of Toll-like receptor 9 (TLR-9), which recognizes viral DNA. Activated via TLR-9, pDCs also secrete large amounts of type I interferon which are involved either in stimulation or down regulation of immune response in multiple sclerosis (MS). In the present study, we determinate pDCs levels by flow cytometry in Cerebrospinal Fluid (CSF) and Peripheral Blood from MS patients in relapsing and in remitting phases of the disease, comparing with other non-inflammatory diseases (OND). We provide evidence that MS patients in relapse without any treatment have a significantly (p < 0.01) higher percentage of pDCs in CSF than do patients in remission or those with OND. No change in the percentage of pDCs was observed in the peripheral blood of any of these patients. The increase of pDCs in central nervous system during relapse may be explained either by a virus infection or a down regulatory process.


Assuntos
Líquido Cefalorraquidiano/citologia , Células Dendríticas/metabolismo , Esclerose Múltipla Recidivante-Remitente/líquido cefalorraquidiano , Esclerose Múltipla Recidivante-Remitente/prevenção & controle , Adulto , DNA Viral/líquido cefalorraquidiano , DNA Viral/imunologia , Células Dendríticas/citologia , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/sangue , Esclerose Múltipla Recidivante-Remitente/imunologia , Recidiva , Receptor Toll-Like 9/imunologia
17.
Nanotechnology ; 22(26): 265103, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21576788

RESUMO

Our data demonstrate that multi-walled carbon nanotubes (MWCNTs) are internalized by macrophages, subsequently activating them to produce interleukin (IL)-12 (IL-12). This cytokine induced the proliferative response of T lymphocytes to a nonspecific mitogen and to ovalbumin (OVA). This increase in the proliferative response was accompanied by an increase in the expression of pro-inflammatory cytokines, such as interferon-gamma (IFNγ), tumor necrosis factor-alpha (TNFα) and IL-6, in mice inoculated with MWCNTs, whether or not they had been immunized with OVA. A decrease in the expression of transforming growth factor-beta (TGFß) was observed in the mice treated with MWCNTs, whereas the suppression of the expression of both TGFß and IL-10 was observed in mice that had been both treated and immunized. The activation of the T lymphocyte response by the pro-inflammatory cytokines leads to an increase in antibody production to OVA, suggesting the important immunostimulatory effect of carbon nanotubes.


Assuntos
Formação de Anticorpos/imunologia , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Macrófagos/metabolismo , Nanotubos de Carbono/química , Linfócitos T/imunologia , Regulação para Cima/imunologia , Animais , Antígenos/imunologia , Linfócitos B/imunologia , Endocitose , Regulação da Expressão Gênica , Interleucina-12/genética , Interleucina-12/metabolismo , Ativação Linfocitária/imunologia , Ativação de Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Nanotubos de Carbono/ultraestrutura , Ovalbumina/imunologia , Análise Espectral Raman
18.
Front Cell Neurosci ; 15: 705618, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34381335

RESUMO

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.

19.
JCI Insight ; 5(13)2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32641586

RESUMO

In diabetic dyslipidemia, cholesterol accumulates in the plasma membrane, decreasing fluidity and thereby suppressing the ability of cells to transduce ligand-activated signaling pathways. Liver X receptors (LXRs) make up the main cellular mechanism by which intracellular cholesterol is regulated and play important roles in inflammation and disease pathogenesis. N, N-dimethyl-3ß-hydroxy-cholenamide (DMHCA), a selective LXR agonist, specifically activates the cholesterol efflux arm of the LXR pathway without stimulating triglyceride synthesis. In this study, we use a multisystem approach to understand the effects and molecular mechanisms of DMHCA treatment in type 2 diabetic (db/db) mice and human circulating angiogenic cells (CACs), which are hematopoietic progenitor cells with vascular reparative capacity. We found that DMHCA is sufficient to correct retinal and BM dysfunction in diabetes, thereby restoring retinal structure, function, and cholesterol homeostasis; rejuvenating membrane fluidity in CACs; hampering systemic inflammation; and correcting BM pathology. Using single-cell RNA sequencing on lineage-sca1+c-Kit+ (LSK) hematopoietic stem cells (HSCs) from untreated and DMHCA-treated diabetic mice, we provide potentially novel insights into hematopoiesis and reveal DMHCA's mechanism of action in correcting diabetic HSCs by reducing myeloidosis and increasing CACs and erythrocyte progenitors. Taken together, these findings demonstrate the beneficial effects of DMHCA treatment on diabetes-induced retinal and BM pathology.


Assuntos
Medula Óssea/efeitos dos fármacos , Ácidos Cólicos/farmacologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Retina/efeitos dos fármacos , Animais , Medula Óssea/patologia , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Colesterol/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Inflamação/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipogênese/efeitos dos fármacos , Lipogênese/fisiologia , Receptores X do Fígado/metabolismo , Camundongos , Retina/patologia
20.
Vaccine ; 37(11): 1377-1383, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30755368

RESUMO

Exosomes may represent an interesting antigenic pulse for new forms of anti-tumor immunotherapy. We evaluated exosomes from serum of patients with acute myeloid leukemia (AML) as an antigenic source for dendritic cells (DC) and the effects upon antitumor cytotoxicity, assessed by the percentage of specific lysis of K562 leukemic cells in co-cultures. Surprisingly, incubation of exosomes with DCs decreased lysis of K562, which may correspond to a mechanism of tumor evasion in vivo. However, when immature DCs were pulsed with exosomes purified from K562 culture supernatants, the lysis of target cells was notably enhanced, associated with a substantial increase in the expression of the maturation marker CD83. Thus, the development of vaccines using patients' exosomes would probably add no benefits to the treatment of AML; alternately, exosomes from cultured cells may represent an effective way for maturing DCs into a cytotoxic phenotype, without the immunosuppression observed with patients' exosomes.


Assuntos
Células Dendríticas/imunologia , Exossomos/imunologia , Tolerância Imunológica , Leucemia Mieloide Aguda/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Técnicas de Cocultura , Testes Imunológicos de Citotoxicidade , Feminino , Humanos , Imunoterapia/métodos , Células K562 , Leucemia Mieloide Aguda/terapia , Masculino , Pessoa de Meia-Idade
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