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Nutrition has broad impacts on all physiological processes. However, how nutrition affects human immunity remains largely unknown. Here we explored the impact of a dietary intervention on both immunity and the microbiota by performing a post hoc analysis of a clinical trial in which each of the 20 participants sequentially consumed vegan or ketogenic diets for 2 weeks ( NCT03878108 ). Using a multiomics approach including multidimensional flow cytometry, transcriptomic, proteomic, metabolomic and metagenomic datasets, we assessed the impact of each diet, and dietary switch, on host immunity and the microbiota. Our data revealed that overall, a ketogenic diet was associated with a significant upregulation of pathways and enrichment in cells associated with the adaptive immune system. In contrast, a vegan diet had a significant impact on the innate immune system, including upregulation of pathways associated with antiviral immunity. Both diets significantly and differentially impacted the microbiome and host-associated amino acid metabolism, with a strong downregulation of most microbial pathways following ketogenic diet compared with baseline and vegan diet. Despite the diversity of participants, we also observed a tightly connected network between datasets driven by compounds associated with amino acids, lipids and the immune system. Collectively, this work demonstrates that in diverse participants 2 weeks of controlled dietary intervention is sufficient to significantly and divergently impact host immunity, which could have implications for precision nutritional interventions. ClinicalTrials.gov registration: NCT03878108 .
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Dieta Cetogênica , Dieta Vegana , Humanos , Proteômica , Ensaios Clínicos como AssuntoRESUMO
Background: Both sex and prior exposure to pathogens are known to influence responses to immune challenges, but their combined effects are not well established in humans, particularly in early innate responses critical for shaping subsequent outcomes. Methods: We employed systems immunology approaches to study responses to a replication-defective, herpes simplex virus (HSV) 2 vaccine in men and women either naive or previously exposed to HSV. Results: Blood transcriptomic and cell population profiling showed substantial changes on day 1 after vaccination, but the responses depended on sex and whether the vaccinee was naive or previously exposed to HSV. The magnitude of early transcriptional responses was greatest in HSV naive women where type I interferon (IFN) signatures were prominent and associated negatively with vaccine-induced neutralizing antibody titers, suggesting that a strong early antiviral response reduced the uptake of this replication-defective virus vaccine. While HSV seronegative vaccine recipients had upregulation of gene sets in type I IFN (IFN-α/ß) responses, HSV2 seropositive vaccine recipients tended to have responses focused more on type II IFN (IFN-γ) genes. Conclusions: These results together show that prior exposure and sex interact to shape early innate responses that then impact subsequent adaptive immune phenotypes. Funding: Intramural Research Program of the NIH, the National Institute of Allergy and Infectious Diseases, and other institutes supporting the Trans-NIH Center for Human Immunology, Autoimmunity, and Inflammation. The vaccine trial was supported through a clinical trial agreement between the National Institute of Allergy and Infectious Diseases and Sanofi Pasteur. Clinical trial number: NCT01915212.
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Vacinas contra Herpesvirus , Imunidade Inata , Fatores Sexuais , Feminino , Humanos , Masculino , Anticorpos Neutralizantes , Herpesvirus Humano 2 , Vacinas contra Herpesvirus/imunologia , Vacinas Atenuadas , Herpes Simples/prevenção & controleRESUMO
Acute viral infections can have durable functional impacts on the immune system long after recovery, but how they affect homeostatic immune states and responses to future perturbations remain poorly understood1-4. Here we use systems immunology approaches, including longitudinal multimodal single-cell analysis (surface proteins, transcriptome and V(D)J sequences) to comparatively assess baseline immune statuses and responses to influenza vaccination in 33 healthy individuals after recovery from mild, non-hospitalized COVID-19 (mean, 151 days after diagnosis) and 40 age- and sex-matched control individuals who had never had COVID-19. At the baseline and independent of time after COVID-19, recoverees had elevated T cell activation signatures and lower expression of innate immune genes including Toll-like receptors in monocytes. Male individuals who had recovered from COVID-19 had coordinately higher innate, influenza-specific plasmablast, and antibody responses after vaccination compared with healthy male individuals and female individuals who had recovered from COVID-19, in part because male recoverees had monocytes with higher IL-15 responses early after vaccination coupled with elevated prevaccination frequencies of 'virtual memory'-like CD8+ T cells poised to produce more IFNγ after IL-15 stimulation. Moreover, the expression of the repressed innate immune genes in monocytes increased by day 1 to day 28 after vaccination in recoverees, therefore moving towards the prevaccination baseline of the healthy control individuals. By contrast, these genes decreased on day 1 and returned to the baseline by day 28 in the control individuals. Our study reveals sex-dimorphic effects of previous mild COVID-19 and suggests that viral infections in humans can establish new immunological set-points that affect future immune responses in an antigen-agnostic manner.
Assuntos
COVID-19 , Imunidade Inata , Memória Imunológica , Vacinas contra Influenza , Caracteres Sexuais , Linfócitos T , Vacinação , Feminino , Humanos , Masculino , Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Interleucina-15/imunologia , Receptores Toll-Like/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Monócitos , Imunidade Inata/genética , Imunidade Inata/imunologia , Análise de Célula Única , Voluntários SaudáveisRESUMO
OBJECTIVES: Premature cardiovascular events in systemic lupus erythematosus (SLE) contribute to morbidity and mortality, with no effective preventive strategies described to date. Immune dysregulation and metabolic disturbances appear to play prominent roles in the induction of vascular disease in SLE. The peroxisome proliferator activated receptor-gamma agonist pioglitazone (PGZ suppresses vascular damage and immune dysregulation in murine lupus and improves endothelial dysfunction in other inflammatory diseases. We hypothesised that PGZ could improve vascular dysfunction and cardiometabolic parameters in SLE. METHODS: Eighty SLE subjects with mild to severe disease activity were randomised to a sequence of PGZ followed by placebo for 3 months, or vice versa, in a double-blind, cross-over design with a 2-month wash-out period. Primary endpoints were parameters of endothelial function and arterial inflammation, measured by multimodal assessments. Additional outcome measures of disease activity, neutrophil dysregulation, metabolic disturbances and gene expression studies were performed. RESULTS: Seventy-two subjects completed the study. PGZ was associated with a significant reduction in Cardio-Ankle Vascular Index (a measure of arterial stiffness) compared with placebo. Various metabolic parameters improved with PGZ, including insulin resistance and lipoprotein profiles. Circulating neutrophil extracellular trap levels also significantly decreased with PGZ compared with placebo. Most adverse events experienced while on PGZ were mild and resolved with reduction in PGZ dose. CONCLUSION: PGZ was well tolerated and induced significant improvement in vascular stiffness and cardiometabolic parameters in SLE. The results suggest that PGZ should be further explored as a modulator of cardiovascular disease risk in SLE. TRIAL REGISTRATION NUMBER: NCT02338999.
RESUMO
Viral infections can have profound and durable functional impacts on the immune system. There is an urgent need to characterize the long-term immune effects of SARS-CoV-2 infection given the persistence of symptoms in some individuals and the continued threat of novel variants. Here we use systems immunology, including longitudinal multimodal single cell analysis (surface proteins, transcriptome, and V(D)J sequences) from 33 previously healthy individuals after recovery from mild, non-hospitalized COVID-19 and 40 age- and sex-matched healthy controls with no history of COVID-19 to comparatively assess the post-infection immune status (mean: 151 days after diagnosis) and subsequent innate and adaptive responses to seasonal influenza vaccination. Identification of both sex-specific and -independent temporally stable changes, including signatures of T-cell activation and repression of innate defense/immune receptor genes (e.g., Toll-like receptors) in monocytes, suggest that mild COVID-19 can establish new post-recovery immunological set-points. COVID-19-recovered males had higher innate, influenza-specific plasmablast, and antibody responses after vaccination compared to healthy males and COVID-19-recovered females, partly attributable to elevated pre-vaccination frequencies of a GPR56 expressing CD8+ T-cell subset in male recoverees that are "poised" to produce higher levels of IFNγ upon inflammatory stimulation. Intriguingly, by day 1 post-vaccination in COVID-19-recovered subjects, the expression of the repressed genes in monocytes increased and moved towards the pre-vaccination baseline of healthy controls, suggesting that the acute inflammation induced by vaccination could partly reset the immune states established by mild COVID-19. Our study reveals sex-dimorphic immune imprints and in vivo functional impacts of mild COVID-19 in humans, suggesting that prior COVID-19, and possibly respiratory viral infections in general, could change future responses to vaccination and in turn, vaccines could help reset the immune system after COVID-19, both in an antigen-agnostic manner.
RESUMO
COVID-19 exhibits extensive patient-to-patient heterogeneity. To link immune response variation to disease severity and outcome over time, we longitudinally assessed circulating proteins as well as 188 surface protein markers, transcriptome, and T cell receptor sequence simultaneously in single peripheral immune cells from COVID-19 patients. Conditional-independence network analysis revealed primary correlates of disease severity, including gene expression signatures of apoptosis in plasmacytoid dendritic cells and attenuated inflammation but increased fatty acid metabolism in CD56dimCD16hi NK cells linked positively to circulating interleukin (IL)-15. CD8+ T cell activation was apparent without signs of exhaustion. Although cellular inflammation was depressed in severe patients early after hospitalization, it became elevated by days 17-23 post symptom onset, suggestive of a late wave of inflammatory responses. Furthermore, circulating protein trajectories at this time were divergent between and predictive of recovery versus fatal outcomes. Our findings stress the importance of timing in the analysis, clinical monitoring, and therapeutic intervention of COVID-19.
Assuntos
COVID-19/imunologia , Citocinas/metabolismo , Células Dendríticas/metabolismo , Expressão Gênica/imunologia , Células Matadoras Naturais/metabolismo , Índice de Gravidade de Doença , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , COVID-19/mortalidade , Estudos de Casos e Controles , Células Dendríticas/citologia , Feminino , Humanos , Células Matadoras Naturais/citologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Transcriptoma/imunologia , Adulto JovemRESUMO
Changes in maternal immunity during pregnancy can result in an altered immune state, and as a natural perturbation, this provides an opportunity to understand functional interactions of the immune system in vivo. We report characterization of maternal peripheral immune phenotypes for 33 longitudinally sampled normal pregnancies, using clinical measurements of complete blood counts and major immune cell populations, as well as high parameter flow cytometry for 30 leukocyte antigens characterizing 79 cell populations, and monitoring of 1305 serum proteins using the SomaLogic platform. Cellular analyses characterized transient changes in T cell polarization and more persistent alterations in T and B cell subset frequencies and activation. Serum proteomic analysis identified a potentially novel set of 7 proteins that are predictive of gestational age: DDR1, PLAU, MRC1, ACP5, ROBO2, IGF2R, and GNS. We further show that gestational age can be predicted from the parameters obtained by complete blood count tests and clinical flow cytometry characterizing 5 major immune cell populations. Inferring gestational age from this routine clinical phenotyping data could be useful in resource-limited settings that lack obstetric ultrasound. Overall, both the cellular and proteomic analyses validate previously reported phenotypic immunological changes of pregnancy and uncover potentially new alterations and predictive markers.
Assuntos
Idade Gestacional , Leucócitos/imunologia , Primeiro Trimestre da Gravidez/imunologia , Gravidez/imunologia , Adolescente , Adulto , Biomarcadores/sangue , Feminino , Humanos , Imunofenotipagem , Leucócitos/metabolismo , Pessoa de Meia-Idade , Gravidez/sangue , Primeiro Trimestre da Gravidez/sangueRESUMO
One of the mechanisms by which malignancies can induce immune suppression is through the production of cytokines that affect the maturation and differentiation of inflammatory cells in the tumor microenvironment. Semaphorin 4D (Sema4D) is a proangiogenic cytokine produced by several malignancies, which has been described in the regulation of the immune system. In the present study, we examined the role of human head and neck squamous cell carcinoma (HNSCC)-secreted Sema4D on myeloid cell differentiation. CD33(+) cells cultured in HNSCC cell line-derived conditioned medium differentiated into myeloid derived suppressor cells (MDSC) (CD33(+)CD11b(+)HLA-DR(-/low)). The addition of anti-Sema4D Ab to HNSCC conditioned medium significantly reduced the expansion of the MDSC population. Similarly, knockdown of Sema4D in an HNSCC cell line resulted in a loss of MDSC function as shown by a decrease in the production of the immune-suppressive cytokines arginase-1, TGF-ß, and IL-10 by MDSC, concomitant with recovery of T cell proliferation and IFN-γ production following stimulation of CD3/CD28. Importantly, CD33(+) myeloid and T cells cultured in conditioned medium of HNSCC cells in which Sema4D was knocked down promoted antitumor inflammatory profile, through recovery of the effector T cells (CD4(+)T-bet(+) and CD8(+)T-bet(+)), as well as a decrease in regulatory T cells (CD4(+)CD25(+)FOXP3(+)). We also showed that Sema4D was comparable to GM-CSF in its induction of MDSC. Collectively, this study describes a novel immunosuppressive role for Sema4D in HNSCC through induction of MDSC, and it highlights Sema4D as a therapeutic target for future studies to enhance the antitumorigenic inflammatory response in HNSCC and other epithelial malignancies.
Assuntos
Antígenos CD/imunologia , Carcinoma de Células Escamosas/imunologia , Neoplasias de Cabeça e Pescoço/imunologia , Tolerância Imunológica/imunologia , Células Mieloides/imunologia , Semaforinas/imunologia , Evasão Tumoral/imunologia , Diferenciação Celular/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Immunoblotting , Ativação Linfocitária/imunologia , Células Mieloides/citologia , RNA Interferente Pequeno , Carcinoma de Células Escamosas de Cabeça e Pescoço , Linfócitos T Reguladores/imunologia , TransfecçãoRESUMO
Graft-versus-host disease (GVHD) remains a significant complication of allogeneic transplantation. We previously reported that the adenosine A(2A) receptor (A(2A)R) specific agonist, ATL146e, decreases the incidence and severity of GVHD in a mouse transplant model. There is increasing interest in treatments that increase CD4(+)CD25(high)Foxp3(+) regulatory T cells (Tregs) to suppress GVHD. Our current study found in vitro that A(2A)R selective agonists enhanced TGF-ß-induced generation of mouse Tregs 2.3- to 3-fold. We demonstrated in vivo suppression of GVHD with specific A(2A)R agonists in two different murine GVHD transplant models associated with profound increases in both circulating and target tissue Tregs of donor origin. Three different A(2A)R agonists of differing potency, ATL146e, ATL370, and ATL1223, all significantly inhibited GVHD-associated weight loss and mortality. At the same time, Tregs shown to be of donor origin increased 5.1- to 7.4-fold in spleen, 2.7- to 4.6-fold in peripheral blood, 2.3- to 4.7-fold in colon, and 3.8- to 4.6-fold in skin. We conclude that specific activation of A(2A)R inhibits acute GVHD through an increase of donor-derived Tregs. Furthermore, the increased presence of Tregs in target tissues (colon and skin) of A(2A)R-specific agonist-treated mice is likely the mechanistic basis for the anti-inflammatory effect preventing acute GVHD.
Assuntos
Agonistas do Receptor A2 de Adenosina/farmacologia , Ácidos Cicloexanocarboxílicos/farmacologia , Regulação para Baixo/imunologia , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Purinas/farmacologia , Linfócitos T Reguladores/imunologia , Regulação para Cima/imunologia , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Ácidos Cicloexanocarboxílicos/administração & dosagem , Feminino , Doença Enxerto-Hospedeiro/metabolismo , Tolerância Imunológica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Purinas/administração & dosagem , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/transplante , Fator de Crescimento Transformador beta/fisiologiaRESUMO
Adiponectin is expressed in adipose tissue by adipogenic transcription factors including PPARgamma, C/EBPalpha, and ADD1/SREBP1c. Because cAMP-response element binding protein (CREB) is also a central transcriptional activator of adipocyte differentiation, we evaluated CREB to determine if it stimulates adiponectin gene expression. To accomplish this, we evaluated the effects of activated CREB on the promoter activity of the mouse adiponectin gene, and identified the cAMP-response element (CRE) in the promoter. The constitutively active form of CREB increased the promoter activity of the mouse adiponectin gene. In addition, transfection studies using 5' serial deleted promoters revealed the presence of a putative CRE located between the -1250 and -1000bp region. Furthermore, an electrophoresis mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis demonstrated that CREB bound to the region between -1022 and -995 in the adiponectin promoter. Insulin-like growth factor (IGF-1), which activate CREB, increased the adiponectin promoter activity. However, this stimulation was prevented by the dominant negative form of CREB (ACREB) and pretreatment with PD098059, indicating that IGF-1 stimulate adiponectin expression through CREB phosphorylation via the ERK pathway. Importantly, the transactivation of adiponectin expression by CREB was inhibited by ATF3. Coimmunoprecipitation and GST pull-down assay revealed that ATF3 bound to CREB and prevented CREB phosphorylation induced during differentiation of 3T3-L1 adipocytes. Collectively, these findings demonstrate that CREB is a positive regulator of mouse adiponectin gene expression in adipocytes, which play an important role in the regulation of adiponectin expression in response to growth factor.
Assuntos
Adipócitos/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ativação Transcricional , Células 3T3-L1 , Fator 3 Ativador da Transcrição/metabolismo , Adiponectina/genética , Animais , Sequência de Bases , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Regiões Promotoras GenéticasRESUMO
Cisplatin is one of the most effective antineoplastic drugs, but it has undesirable side effects such as hepatotoxicity at high doses. This study investigated the protective effect of macelignan, isolated from Myristica fragrans HOUTT. (nutmeg), against cisplatin-induced hepatotoxicity and the possible mechanisms involved in these effects in mice. Pretreatment with macelignan for 4 d significantly prevented the increased serum enzymatic activities of alanine and aspartate aminotransferase in a dose-dependent manner. The results also showed that the protective effects of macelignan on cisplatin-induced hepatotoxicity may be associated with the mitogen activated protein kinase (MAPK) signaling pathway. Cisplatin-induced phosphorylation of c-Jun N-terminal kinase1/2 (JNK1/2) and extracellular signal-regulated kinase1/2 (ERK1/2) was abrogated by pretreatment with macelignan, however, that of p38 was not significantly affected. It was also found that macelignan attenuated the expression of phosphorylated c-Jun in cisplatin-treated mice. Accordingly, it is suggested that the hepatoprotective effects of macelignan could be related to activation of the MAPK signaling pathway, especially JNK and c-Jun, its substrate. The present findings suggest that co-treatment of cisplatin with macelignan may provide more advantage than cisplatin treatment alone in cancer therapy.
Assuntos
Antineoplásicos/antagonistas & inibidores , Antineoplásicos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Cisplatino/antagonistas & inibidores , Cisplatino/toxicidade , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Lignanas/farmacologia , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Western Blotting , Doença Hepática Induzida por Substâncias e Drogas/patologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Testes de Função Hepática , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Myristica/química , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Transcrição/efeitos dos fármacosRESUMO
Neuronatin (Nnat) was initially identified as a selectively-expressed gene in neonatal brains, but its expression has been also identified in pancreatic beta-cells. Therefore, to investigate the possible functions that Nnat may serve in pancreatic beta-cells, two Nnat isotypes (alpha and beta) were expressed using adenoviruses in murine MIN6N8 pancreatic beta-cells, and the cellular fates and the effects of Nnat on insulin secretion, high glucose-induced apoptosis, and functional impairment were examined. Nnatalpha and Nnatbeta were primarily localized in the endoplasmic reticulum (ER), and their expressions increased insulin secretion by increasing intracellular calcium levels. However, under chronic high glucose conditions, the Nnatbeta to Nnatalpha ratio gradually increased in proportion to the length of exposure to high glucose levels. Moreover, adenovirally-expressed Nnatbeta was inclined to form aggresome-like structures, and we found that Nnatbeta aggregation inhibited the function of the proteasome. Therefore, when glucose is elevated, the expression of Nnatbeta sensitizes MIN6N8 cells to high glucose stress, which in turn, causes ER stress. As a result, expression of Nnatbeta increased hyperglycemia-induced apoptosis. In addition, the expression of Nnatbeta under high glucose conditions decreased the expression of genes important for beta-cell function, such as glucokinase (GCK), pancreas duodenum homeobox-1 (PDX-1), and insulin. Collectively, Nnat may play a critical factor in normal beta-cell function, as well as in the pathogenesis of type 2 diabetes.
Assuntos
Apoptose/efeitos dos fármacos , Glucose/farmacologia , Células Secretoras de Insulina/fisiologia , Insulina/metabolismo , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Animais , Apoptose/fisiologia , Sequência de Bases , Cálcio/metabolismo , Linhagem Celular , Primers do DNA/genética , Retículo Endoplasmático/metabolismo , Expressão Gênica , Secreção de Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Obesos , Proteínas do Tecido Nervoso/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
OBJECTIVE: Peroxisome proliferator-activated receptor (PPAR) alpha/gamma dual agonists have the potential to be used as therapeutic agents for the treatment of type 2 diabetes. This study evaluated the function of macelignan, a natural compound isolated from Myristica fragrans, as a dual agonist for PPARalpha/gamma and investigated its antidiabetes effects in animal models. RESEARCH DESIGN AND METHODS: GAL4/PPAR chimera transactivation was performed and the expression of PPARalpha/gamma target genes was monitored to examine the ability of macelignan to activate PPARalpha/gamma. Additionally, macelignan was administrated to obese diabetic (db/db) mice to investigate antidiabetes effects and elucidate its molecular mechanisms. RESULTS: Macelignan reduced serum glucose, insulin, triglycerides, free fatty acid levels, and triglycerides levels in the skeletal muscle and liver of db/db mice. Furthermore, macelignan significantly improved glucose and insulin tolerance in these mice, and without altering food intake, their body weights were slightly reduced while weights of troglitazone-treated mice increased. Macelignan increased adiponectin expression in adipose tissue and serum, whereas the expression and serum levels of tumor necrosis factor-alpha and interleukin-6 decreased. Macelignan downregulated inflammatory gene expression in the liver and increased AMP-activated protein kinase activation in the skeletal muscle of db/db mice. Strikingly, macelignan reduced endoplasmic reticulum (ER) stress and c-Jun NH(2)-terminal kinase activation in the liver and adipose tissue of db/db mice and subsequently increased insulin signaling. CONCLUSIONS: Macelignan enhanced insulin sensitivity and improved lipid metabolic disorders by activating PPARalpha/gamma and attenuating ER stress, suggesting that it has potential as an antidiabetes agent for the treatment of type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Retículo Endoplasmático/efeitos dos fármacos , Lignanas/farmacologia , Lignanas/uso terapêutico , PPAR alfa/agonistas , PPAR gama/agonistas , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/metabolismo , Animais , Linhagem Celular , Proteínas Substratos do Receptor de Insulina , Lignanas/química , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Obesos , Estrutura Molecular , Myristica/química , Estresse Fisiológico , TapsigarginaRESUMO
In this study, we investigated the protective effect of macelignan, isolated from Myristica fragrans Houtt. (nutmeg) against tert-butylhydroperoxide (t-BHP)-induced cytotoxicity in a human hepatoma cell line, HepG2. The tetrazolium dye colorimetric test (MTT test) and lactate dehydrogenase (LDH) assay were used to monitor cell viability and necrosis, respectively. Lipid peroxidation [malondialdehyde (MDA) formation] was estimated by the fluorometric method. Intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA), and DNA damage was detected using single cell gel electrophoresis (comet assay). The results showed that macelignan significantly reduced the cell growth inhibition and necrosis caused by t-BHP. Furthermore, macelignan ameliorated lipid peroxidation as demonstrated by a reduction in MDA formation in a dose-dependent manner. It was also found that macelignan reduced intracellular ROS formation and DNA damaging effect caused by t-BHP. These results strongly suggest that macelignan has significant protective ability against oxidative damage caused by reactive intermediates.
Assuntos
Dano ao DNA/efeitos dos fármacos , Lignanas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , terc-Butil Hidroperóxido/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Relação Dose-Resposta a Droga , Humanos , L-Lactato Desidrogenase/metabolismo , Lignanas/química , Peroxidação de Lipídeos/efeitos dos fármacos , Malondialdeído/metabolismo , Estrutura Molecular , Myristica , Oxirredução/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Fatores de TempoRESUMO
Vibrio vulnificus is a pathogenic bacterium that causes gastroenteritis and primary septicemia. To identify factors involved in microbial adherence to the host cells, we investigated bacterial proteins capable of binding to fibronectin, one of the main components comprised of the extracellular matrix of mammalian cells. A protein of approximately 35 kDa was purified from the extracts of V. vulnificus by its property to bind to immobilized fibronectin. This protein was identified as OmpU, one of the major outer membrane proteins of V. vulnificus. In binding assays using immobilized fibronectin, the number of ompU mutant cells bound to fibronectin was only 4% of that of wild-type cells bound to fibronectin. In addition, the exogenous addition of antibodies against OmpU resulted in a decreased ability of wild-type V. vulnificus to adhere to fibronectin. The ompU mutant was also defective in its adherence to RGD tripeptide (5% of the adherence of the wild type to RGD), cytoadherence to HEp-2 cells (7% of the adherence of the wild type to HEp-2), cytotoxicity to cell cultures (39% of the cytotoxicity of the wild type), and mortality in mice (10-fold increase in the 50% lethal dose). The ompU mutant complemented with the intact ompU gene restored its abilities for adherence to fibronectin, RGD tripeptide, and HEp-2 cells; cytotoxicity to HEp-2 cells; and mouse lethality. This study indicates that OmpU is an important virulence factor involved in the adherence of V. vulnificus to the host cells.
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Adesinas Bacterianas/fisiologia , Aderência Bacteriana , Vibrioses/microbiologia , Vibrio vulnificus/patogenicidade , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Animais , Aderência Bacteriana/genética , Modelos Animais de Doenças , Matriz Extracelular/microbiologia , Fibronectinas/química , Humanos , Camundongos , Mutação , Oligopeptídeos/química , Vibrio vulnificus/genéticaRESUMO
Free-living amoebae of the genus Acanthamoeba are causative agents of granulomatous amebic encephalitis and amebic keratitis. Because the virulence of Acanthamoeba culbertsoni cultured in the laboratory is restored by consecutive brain passages, we examined the genes induced in mouse brain-passaged A. culbertsoni by differential display reverse transcriptase polymerase chain reaction (DDRT-PCR). Enhanced A. culbertsoni virulence was observed during the second mouse brain passage, i.e., infected mouse mortality increased from 5% to 70%. Ten cDNAs induced during mouse brain passage were identified by DDRT-PCR and this was confirmed by northern blot analysis. BlastX searches of these cDNAs indicated the upregulations of genes encoding predictive NADH-dehydrogenase, proteasomal ATPase, and GDP-mannose pyrophosphorylase B, which have previously been reported to be associated with A. culbertsoni virulence factors.
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Acanthamoeba/genética , Acanthamoeba/patogenicidade , Amebíase/parasitologia , DNA de Protozoário/fisiologia , Regulação da Expressão Gênica , Amebíase/mortalidade , Animais , Northern Blotting/métodos , Encéfalo/parasitologia , Clonagem Molecular/métodos , DNA Complementar/biossíntese , DNA de Protozoário/biossíntese , Perfilação da Expressão Gênica/métodos , Genes de Protozoários/genética , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Inoculações Seriadas , Regulação para Cima , Virulência/genéticaRESUMO
Peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear receptor of ligand-activated transcription factors, regulates the expression of key genes involved in lipid and glucose metabolism or adipocyte differentiation. Ligands for this receptor have emerged as potent insulin sensitizers used in the treatment of Type2 diabetes. Ginseng saponins or ginsenosides are reported to provide anti-diabetic activity as well as to modulate glucose metabolism, although the mechanism remains unclear. In this study, we examined the effect of ginsenosides on activation of PPARgamma and adipogenes in 3T3-L1. Using a GAL-4/PPARgamma transactivation assay, 20(S)-protopanaxatriol (PPT), one of the ginsenoside metabolites, was found to increase PPARgamma-transactivation activity dose-dependently with similar activity as troglitazone, a well-known PPARgamma agonist. PPT enhanced adipogenesis by increasing the expression of PPARgamma target genes such as aP2, LPL and PEPCK. Furthermore, PPT significantly increased expression of glucose transporter 4 (GLUT4). These results indicate that PPT can be developed as a PPARgamma agonist for the improvement of insulin resistance associated with diabetes.
Assuntos
Adipócitos/metabolismo , Hipoglicemiantes , PPAR gama/agonistas , Pirazóis/farmacologia , Células 3T3 , Adipócitos/efeitos dos fármacos , Animais , Western Blotting , Células COS , Diferenciação Celular/efeitos dos fármacos , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Ginsenosídeos/farmacologia , Transportador de Glucose Tipo 4/biossíntese , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Fenóis , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saponinas/farmacologia , Ativação TranscricionalRESUMO
The protective effect of panduratin A, isolated from Kaempferia pandurata ROXB. (Zingiberaceae), against tert-butylhydroperoxide (t-BHP)-induced cytotoxicity was investigated in a human hepatoma cell line, HepG2. The tetrazolium dye colorimetric test (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay) was used to monitor cytotoxicity. Lipid peroxidation [malondialdehyde (MDA) formation] and intracellular glutathione level were estimated by fluorometric methods. Intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA). Panduratin A significantly reduced the cell growth inhibition caused by t-BHP. Furthermore, panduratin A ameliorated lipid peroxidation as demonstrated by a reduction in MDA formation, and attenuated glutathione (GSH) depletion in a dose-dependent manner. It was also found that panduratin A reduced intracellular ROS formation caused by t-BHP. These results strongly suggest that panduratin A has significant protective ability against oxidative damage caused by reactive intermediates.
Assuntos
Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , terc-Butil Hidroperóxido/antagonistas & inibidores , terc-Butil Hidroperóxido/toxicidade , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Chalconas , Humanos , Estresse Oxidativo/fisiologia , Extratos Vegetais/isolamento & purificação , Rizoma , ZingiberaceaeRESUMO
Adherence of a pathogen to the host cell is one of the critical steps in microbial infections. Naegleria fowleri, a causative agent of primary amoebic meningoencephalitis in humans, is expected to interact with extracellular components of the host, such as fibronectin, in a receptor-mediated mode. In this study, we investigated the interaction between N. fowleri and fibronectin to understand its cytopathology. In binding assays using immobilized fibronectin, the number of amoebae bound to fibronectin was increased compared to the controls, and was dependent on the amount of coated fibronectin present. A fibronectin binding protein of 60 kDa was found in extracts of N. fowleri. Western blot and immunolocalization assays using integrin alpha(5)/FnR antibodies showed that a 60 kDa protein reacted with the antibodies in extracts of N. fowleri, which was localized on the surface of N. fowleri. Preincubation of N. fowleri with the integrin antibodies significantly inhibited amoebic binding to fibronectin and cytotoxicity to the CHO cells. Additionally, protein kinase C activity was detected in the extract of N. fowleri. When N. fowleri was pretreated with protein kinase C activator or inhibitor, the abilities of amoebic adhesion to fibronectin and cytotoxicity to the host cells were markedly affected compared to untreated amoebae. These results suggest that an amoebic integrin-like receptor and protein kinase C play important roles in amoebic cellular processes in response to fibronectin.