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1.
Biol Chem ; 401(2): 249-262, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-31299006

RESUMO

Rickettsial species have independently lost several genes owing to reductive evolution while retaining those predominantly implicated in virulence, survival, and biosynthetic pathways. In this study, we have identified a previously uncharacterized Rickettsia conorii gene RC0497 as an N-acetylmuramoyl-L-alanine amidase constitutively expressed during infection of cultured human microvascular endothelial cells at the levels of both mRNA transcript and encoded protein. A homology-based search of rickettsial genomes reveals that RC0497 homologs, containing amidase_2 family and peptidoglycan binding domains, are highly conserved among the spotted fever group (SFG) rickettsiae. The recombinant RC0497 protein exhibits α-helix secondary structure, undergoes a conformational change in the presence of zinc, and exists as a dimer at higher concentrations. We have further ascertained the enzymatic activity of RC0497 via demonstration of its ability to hydrolyze Escherichia coli peptidoglycan. Confocal microscopy on E. coli expressing RC0497 and transmission immunoelectron microscopy of R. conorii revealed its localization predominantly to the cell wall, septal regions of replicating bacteria, and the membrane of vesicles pinching off the cell wall. In summary, we have identified and functionally characterized RC0497 as a peptidoglycan hydrolase unique to spotted fever rickettsiae, which may potentially serve as a novel moonlighting protein capable of performing multiple functions during host-pathogen interactions.

2.
Front Immunol ; 11: 595039, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33414785

RESUMO

A parasitic protozoan Trypanosoma cruzi (T. cruzi) is the etiologic agent of Chagas disease. Previously, we have identified T. cruzi antigens TcG2 and TcG4 as potential vaccine candidates, cloned in eukaryotic expression vector pCDNA3.1 (referred as p2/4) and tested their ability to elicit protection from T. cruzi infection. In the present study, we subcloned the two antigens in a nanoplasmid that is optimized for delivery, antigen expression, and regulatory compliance standards, and evaluated the nanovaccine (referred as nano2/4) for prophylactic protection against repeat T. cruzi infections. For this, C57BL/6 mice were immunized with two doses of p2/4 or nano2/4 at 21 days interval, challenged with T. cruzi 21 days after 2nd immunization, and euthanized at 10- and 21-days post-infection (pi) corresponding to parasite dissemination and replication phase, respectively. Some mice were re-challenged 21 days pi and monitored at 7 days after re-infection. Without the help of a vaccine, T. cruzi elicited delayed and sub-par T cell activation and low levels of effector molecules that failed to control tissue dissemination and replication of the parasite and provided no protection against repeat challenge infection. The nano2/4 was most effective in eliciting an early activation and production of IFN-γ by CD4+T effector/effector memory (TEM) cells and cytolytic perforin (PFN) and granzyme B (GZB) molecules by CD4+ and CD8+ TEM subsets at 10 days pi that was followed by robust expansion of CD4+ and CD8+ TEM and TCM cells with further increase in IFN-γ production at 21 days pi. Consequently, nano2/4-immunized mice exhibited potent control of parasite dissemination at 10 days pi, and tissue parasite burden and tissue inflammatory infiltrate and necrosis were barely detectable at 21 days pi. Furthermore, nano2/4-immunized mice responded to re-challenge infection with high levels of effector molecules production by CD4+ and CD8+ TEM subpopulations that offered even better control of tissue parasite burden than was observed after 1st infection. In comparison, non-vaccinated/infected mice exhibited clinical features of sickness and 59% mortality within 7 days after re-infection. In conclusion, we show that delivery of TcG2 and TcG4 in nanoplasmid offers excellent, protective T cell immunity against repeat T. cruzi infections.

3.
Mediators Inflamm ; 2019: 3481430, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31182931

RESUMO

Macrophages (Mφ) play a central role in coordinating host response to pathogens, cellular injury, and environmental stimuli. Herein, we report multidimensional, nuclear proteomic analyses of protein expression and posttranslational modifications (PTMs) that control biological processes during Mφ activation. For this, Mφ were incubated with IFN-γ/LPS and IL-4, and their differentiation to proinflammatory (M1) and anti-inflammatory (M2a, referred as M2 for simplicity throughtout the manuscript) phenotypes was confirmed by detection of CD64 and CD206 surface markers and TNF-α, arginase I, and iNOS-dependent nitrite levels. We used a sequential method of organellar enrichment and labeling of nuclear fractions with BODIPY FL-maleimide fluorescence dye followed by two-dimensional electrophoresis (2DE) to capture quantitative changes in abundance and S-nitrosylated (SNO) proteome signatures. Exact same gels were then labeled with Pro-Q Diamond to detect protein phosphorylation. MALDI-TOF/TOF MS analysis of the protein spots with fold change of ≥|1.5| in any of the groups yielded 229 identifications. We found that 145, 78, and 173 protein spots in M1 Mφ and 105, 81, and 164 protein spots in M2 Mφ were changed in abundance, S-nitrosylation, and phosphorylation, respectively, with respect to M0 controls (fold change: ≥|1.5|, p ≤ 0.05). Targeted analysis by immunoprecipitation and Western blotting was performed to verify the differential abundance and phosphorylation levels of two of the proteins in M1 and M2 (vs. M0) Mφ. Ingenuity Pathway Analysis of the nuclear proteome datasets showed that the abundance and posttranslational (SNO and Phosphor) modifications of the proteins predicted to be involved in cytoskeletal organization/cell movement, phagocytosis/endocytosis, and cell proliferation/cell death were differentially regulated with proinflammatory and anti-inflammatory activation of Mφ.


Assuntos
Macrófagos/metabolismo , Animais , Western Blotting , Eletroforese em Gel Bidimensional , Citometria de Fluxo , Imunoprecipitação , Espectrometria de Massas , Camundongos , Óxido Nítrico/metabolismo , Fosforilação , Análise de Componente Principal , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
4.
JAMA Oncol ; 5(2): 243-247, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30419088

RESUMO

Importance: Brain metastases are a common source of morbidity for patients with cancer, and limited data exist to support the local therapeutic choice between surgical resection and stereotactic radiosurgery (SRS). Objective: To evaluate local control of brain metastases among patients treated with SRS vs surgical resection within the European Organization for the Research and Treatment of Cancer (EORTC) 22952-26001 phase 3 trial. Design, Setting, and Participants: This unplanned, exploratory analysis of the international, multi-institutional randomized clinical trial EORTC 22952-26001 (conducted from 1996-2007) was performed from February 9, 2017, through July 25, 2018. The EORTC 22952-26001 trial randomized patients with 1 to 3 brain metastases to whole-brain radiotherapy vs observation after complete surgical resection or before SRS. Patients in the present analysis were stratified but not randomized according to local modality (SRS or surgical resection) and treated per protocol with 1 to 2 brain metastases and tumors with a diameter of no greater than 4 cm. Interventions: Surgical resection or SRS. Main Outcomes and Measures: The primary end point was local recurrence of treated lesions. Cumulative incidence of local recurrence was calculated according to modality (surgical resection vs SRS) with competing risk regression to adjust for prognostic factors and competing risk of death. Results: A total of 268 patients were included in the analysis (66.4% men; median age, 60.7 years [range, 26.9-81.1 years]); 154 (57.5%) underwent SRS and 114 (42.5%) underwent surgical resection. Median follow-up time was 39.9 months (range, 26.0-1982.0 months). Compared with the SRS group, patients undergoing surgical resection had larger metastases (median 28 mm [range, 10-40 mm] vs 20 mm [range, 4-40 mm]; P < .001), more frequently had 1 brain metastasis (112 [98.2%] vs 114 [74.0%]; P < .001), and differed in location (parietal, 21 [18.4%] vs 61 [39.6%]; posterior fossa, 30 [26.3%] vs 12 [7.8%]; P < .001). In adjusted models, local recurrence was similar between the SRS and surgical resection groups (hazard ratio [HR], 1.15; 95% CI, 0.72-1.83). However, when stratified by interval, patients with surgical resection had a much higher risk of early (0-3 months) local recurrence compared with those undergoing SRS (HR, 5.94; 95% CI, 1.72-20.45), but their risk decreased with time (HR for 3-6 months, 1.37 [95% CI, 0.64-2.90]; HR for 6-9 months, 0.75 [95% CI, 0.28-2.00]). At 9 months or longer, the surgical resection group had a lower risk of local recurrence (HR, 0.36; 95% CI, 0.14-0.93). Conclusions and Relevance: In this exploratory analysis, local control of brain metastases was similar between SRS and surgical resection groups. Stereotactic radiosurgery was associated with improved early local control of treated lesions compared with surgical resection, although the relative benefit decreased with time. Trial Registration: ClinicalTrials.gov Identifier: NCT00002899.


Assuntos
Neoplasias Encefálicas/terapia , Procedimentos Neurocirúrgicos , Radiocirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/secundário , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Procedimentos Neurocirúrgicos/efeitos adversos , Procedimentos Neurocirúrgicos/mortalidade , Radiocirurgia/efeitos adversos , Radiocirurgia/mortalidade , Medição de Risco , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Carga Tumoral
5.
Front Immunol ; 9: 3014, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30687302

RESUMO

Recent discovery that much of the mammalian genome does not encode protein-coding genes (PCGs) has brought widespread attention to long noncoding RNAs (lncRNAs) as a novel layer of biological regulation. Enhancer lnc (elnc) RNAs from the enhancer regions of the genome carry the capacity to regulate PCGs in cis or in trans. Spotted fever rickettsioses represent the consequence of host infection with Gram-negative, obligate intracellular bacteria in the Genus Rickettsia. Despite being implicated in the pathways of infection and inflammation, the roles of lncRNAs in host response to Rickettsia species have remained a mystery. We have profiled the expression of host lncRNAs during infection of susceptible mice with R. conorii as a model closely mimicking the pathogenesis of human spotted fever rickettsioses. RNA sequencing on the lungs of infected hosts yielded reads mapping to 74,964 non-coding RNAs, 206 and 277 of which were determined to be significantly up- and down-regulated, respectively, in comparison to uninfected controls. Following removal of short non-coding RNAs and ambiguous transcripts, remaining transcripts underwent in-depth analysis of mouse lung epigenetic signatures H3K4Me1 and H3K4Me3, active transcript markers (POLR2A, p300, CTCF), and DNaseI hypersensitivity sites to identify two potentially active and highly up-regulated elncRNAs NONMMUT013718 and NONMMUT024103. Using Hi-3C sequencing resource, we further determined that genomic loci of NONMMUT013718 and NONMMUT024103 might interact with and regulate the expression of nearby PCGs, namely Id2 (inhibitor of DNA binding 2) and Apol10b (apolipoprotein 10b), respectively. Heterologous reporter assays confirmed the activity of elncRNAs as the inducers of their predicted PCGs. In the lungs of infected mice, expression of both elncRNAs and their targets was significantly higher than mock-infected controls. Induced expression of NONMMUT013718/Id2 in murine macrophages and NONMMUT024103/Apol10b in endothelial cells was also clearly evident during R. conorii infection in vitro. Finally, shRNA mediated knock-down of NONMMUT013718 and NONMMUT024103 elncRNAs resulted in reduced expression of endogenous Id2 and Apl10b, demonstrating the regulatory roles of these elncRNAs on their target PCGs. Our results provide very first experimental evidence suggesting altered expression of pulmonary lncRNAs and elncRNA-mediated regulation of PCGs involved in immunity and during host interactions with pathogenic rickettsiae.


Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , RNA Longo não Codificante/genética , Infecções por Rickettsia/genética , Transcrição Genética , Animais , Linhagem Celular , Mapeamento Cromossômico , Biologia Computacional/métodos , Modelos Animais de Doenças , Epigenômica , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno/imunologia , Camundongos , Rickettsia/imunologia , Infecções por Rickettsia/imunologia , Infecções por Rickettsia/microbiologia , Sítio de Iniciação de Transcrição
6.
Mediators Inflamm ; 2017: 3427461, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29147069

RESUMO

Endothelial cell interactions with lipopolysaccharide (LPS) involve both activating and repressing signals resulting in pronounced alterations in their transcriptome and proteome. Noncoding RNAs are now appreciated as posttranscriptional and translational regulators of cellular signaling and responses, but their expression status and roles during endothelial interactions with LPS are not well understood. We report on the expression profile of long noncoding (lnc) RNAs of human microvascular endothelial cells in response to LPS. We have identified a total of 10,781 and 8310 lncRNA transcripts displaying either positive or negative regulation of expression, respectively, at 3 and 24 h posttreatment. A majority of LPS-induced lncRNAs are multiexonic and distributed across the genome as evidenced by their presence on all chromosomes. Present among these are a total of 44 lncRNAs with known regulatory functions, of which 41 multiexonic lncRNAs have multiple splice variants. We have further validated splice variant-specific expression of EGO (NONHSAT087634) and HOTAIRM1 (NONHSAT119666) at 3 h and significant upregulation of lnc-IL7R at 24 h. This study illustrates the genome-wide regulation of endothelial lncRNA splice variants in response to LPS and provides a foundation for further investigations of differentially expressed lncRNA transcripts in endothelial responses to LPS and pathophysiology of sepsis/septic shock.


Assuntos
Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Lipopolissacarídeos/farmacologia , RNA Longo não Codificante/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos
7.
J Innate Immun ; 9(2): 203-216, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27902980

RESUMO

BACKGROUND: Chronic inflammation and oxidative stress are hallmarks of chagasic cardiomyopathy (CCM). In this study, we determined if microparticles (MPs) generated during Trypanosoma cruzi (Tc) infection carry the host's signature of the inflammatory/oxidative state and provide information regarding the progression of clinical disease. METHODS: MPs were harvested from supernatants of human peripheral blood mononuclear cells in vitro incubated with Tc (control: LPS treated), plasma of seropositive humans with a clinically asymptomatic (CA) or symptomatic (CS) disease state (vs. normal/healthy [NH] controls), and plasma of mice immunized with a protective vaccine before challenge infection (control: unvaccinated/infected). Macrophages (mφs) were incubated with MPs, and we probed the gene expression profile using the inflammatory signaling cascade and cytokine/chemokine arrays, phenotypic markers of mφ activation by flow cytometry, cytokine profile by means of an ELISA and Bioplex assay, and oxidative/nitrosative stress and mitotoxicity by means of colorimetric and fluorometric assays. RESULTS: Tc- and LPS-induced MPs stimulated proliferation, inflammatory gene expression profile, and nitric oxide (∙NO) release in human THP-1 mφs. LPS-MPs were more immunostimulatory than Tc-MPs. Endothelial cells, T lymphocytes, and mφs were the major source of MPs shed in the plasma of chagasic humans and experimentally infected mice. The CS and CA (vs. NH) MPs elicited >2-fold increase in NO and mitochondrial oxidative stress in THP-1 mφs; however, CS (vs. CA) MPs elicited a more pronounced and disease-state-specific inflammatory gene expression profile (IKBKB, NR3C1, and TIRAP vs. CCR4, EGR2, and CCL3), cytokine release (IL-2 + IFN-γ > GCSF), and surface markers of mφ activation (CD14 and CD16). The circulatory MPs of nonvaccinated/infected mice induced 7.5-fold and 40% increases in ∙NO and IFN-γ production, respectively, while these responses were abolished when RAW264.7 mφs were incubated with circulatory MPs of vaccinated/infected mice. CONCLUSION: Circulating MPs reflect in vivo levels of an oxidative, nitrosative, and inflammatory state, and have potential utility in evaluating disease severity and the efficacy of vaccines and drug therapies against CCM.


Assuntos
Micropartículas Derivadas de Células/metabolismo , Doença de Chagas/imunologia , Inflamação/imunologia , Leucócitos Mononucleares/imunologia , Macrófagos/imunologia , Trypanosoma cruzi/imunologia , Vacinas/imunologia , Animais , Doenças Assintomáticas , Linhagem Celular , Micropartículas Derivadas de Células/imunologia , Citocinas/metabolismo , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Inflamação/genética , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Estresse Oxidativo , Vacinação
8.
Infect Immun ; 84(12): 3527-3541, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27698021

RESUMO

Trypanosoma cruzi is the causative agent of chronic chagasic cardiomyopathy. Why macrophages (mφs), the early responders to infection, fail to achieve parasite clearance is not known. Mouse (RAW 264.7) and human (THP-1 and primary) mφs were infected for 3 h and 18 h with T. cruzi TcI isolates, SylvioX10/4 (SYL, virulent) and TCC (nonpathogenic), which represent mφ stimulation and infection states, respectively. Mφs incubated with lipopolysaccharide and gamma interferon (LPS/IFN-γ) and with interleukin-4 (IL-4) were used as controls. We monitored the cytokine profile (using enzyme-linked immunosorbent assay [ELISA]), reactive oxygen species (ROS; fluorescent probes), nitric oxide (·NO; Griess assay), and metabolic state using a custom-designed mitoxosome array and Seahorse XF24 Analyzer. LPS/IFN-γ treatment of mφs elicited a potent increase in production of tumor necrosis alpha (TNF-α) at 3 h and of ROS and ·NO by 18 h. Upon SYL infection, murine mφs elicited an inflammatory cytokine profile (TNF-α ≫ TGF-ß + IL-10) and low levels of ·NO and ROS production. LPS/IFN-γ treatment resulted in the inhibition of oxidative metabolism at the gene expression and functional levels and a switch to the glycolytic pathway in mφs, while IL-4-treated mφs utilized oxidative metabolism to meet energy demands. SYL infection resulted in an intermediate functional metabolic state with increased mitoxosome gene expression and glycolysis, and IFN-γ addition shut down the oxidative metabolism in SYL-infected mφs. Further, TCC- and SYL-stimulated mφs exhibited similar levels of cell proliferation and production of TNF-α and ROS, while TCC-stimulated mφs exhibited up to 2-fold-higher levels of oxidative metabolism and ·NO production than SYL-infected mφs. Inhibiting ATP-coupled O2 consumption suppressed the ·NO generation in SYL-infected mφs. Mitochondrial oxygen consumption constitutes a mechanism for stimulating ·NO production in mφs during T. cruzi infection. Enhancing the oxidative metabolism provides an opportunity for increased ·NO production and pathogen clearance by mφs to limit disease progression.


Assuntos
Macrófagos/metabolismo , Macrófagos/parasitologia , Óxido Nítrico/biossíntese , Trypanosoma cruzi/fisiologia , Animais , Linhagem Celular , Sobrevivência Celular , Regulação da Expressão Gênica/fisiologia , Genes Mitocondriais/fisiologia , Camundongos , Espécies Reativas de Oxigênio
9.
Radiat Oncol ; 10: 248, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26626714

RESUMO

PURPOSE: Stereotactic radiosurgery (SRS) alone is an increasingly common treatment strategy for brain metastases. However, existing prognostic tools for overall survival (OS) were developed using cohorts of patients treated predominantly with approaches other than SRS alone. Therefore, we devised novel risk scores for OS and distant brain failure (DF) for melanoma brain metastases (MBM) treated with SRS alone. METHODS AND MATERIALS: We retrospectively reviewed 86 patients treated with SRS alone for MBM from 2009-2014. OS and DF were estimated using the Kaplan-Meier method. Cox proportional hazards modeling identified clinical risk factors. Risk scores were created based on weighted regression coefficients. OS scores range from 0-10 (0 representing best OS), and DF risk scores range from 0-5 (0 representing lowest risk of DF). Predictive power was evaluated using c-index statistics. Bootstrapping with 200 resamples tested model stability. RESULTS: The median OS was 8.1 months from SRS, and 54 (70.1 %) patients had DF at a median of 3.3 months. Risk scores for OS were predicated on performance status, extracranial disease (ED) status, number of lesions, and gender. Median OS for the low-risk group (0-3 points) was not reached. For the moderate-risk (4-6 points) and high-risk (6.5-10) groups, median OS was 7.6 months and 2.4 months, respectively (p < .0001). Scores for DF were predicated on performance status, ED status, and number of lesions. Median time to DF for the low-risk group (0 points) was not reached. For the moderate-risk (1-2 points) and high-risk (3-5 points) groups, time to DF was 4.8 and 2.0 months, respectively (p < .0001). The novel scores were more predictive (c-index = 0.72) than melanoma-specific graded prognostic assessment or RTOG recursive partitioning analysis tools (c-index = 0.66 and 0.57, respectively). CONCLUSIONS: We devised novel risk scores for MBM treated with SRS alone. These scores have implications for prognosis and treatment strategy selection (SRS versus whole-brain radiotherapy).


Assuntos
Neoplasias Encefálicas/secundário , Neoplasias Encefálicas/cirurgia , Melanoma/secundário , Melanoma/cirurgia , Neoplasias Cutâneas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/mortalidade , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Melanoma/mortalidade , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Radiocirurgia , Estudos Retrospectivos , Fatores de Risco , Adulto Jovem
10.
Mol Cell Biochem ; 405(1-2): 265-79, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25956512

RESUMO

The present study was aimed to investigate the relation between nuclear factor kappa beta (NFκB) activation and downstream up-regulation of vascular endothelial growth factor (VEGF) in diabetic retinopathy (DR). Moreover the study was intended to evaluate the role of VEGF gene single nucleotide polymorphisms (SNPs) in DR occurrence and to investigate the functional relevance of VEGF gene SNPs in terms of VEGF expression in DR. Serum level of VEGF, VEGF R1 (receptor 1), VEGF R 2 (receptor 2) and NFκB (p50/65) activity was measured by enzyme linked immune sorbent assay. Genotyping and allelic composition of different SNPs i.e., rs2010963, rs3025039, rs1570360 and rs 2071559 were investigated by Taqman SNP genotyping assay. VEGF, NFκB p50/p65, and VEGF R1 & R2 gene expressions were quantified by real time quantitative polymerase chain reaction. Increased NFκB p50/p65 activity and expressions were observed in non proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR) subjects compared to type 2 diabetes mellitus without retinopathy (DNR) group. Significantly elevated levels of serum VEGF and highest VEGF expression were found among PDR subjects compared to DNR or NPDR subjects. CC genotype and C allele of rs2010963 and TT genotype and T allele of rs3025039 were significantly over represented among PDR subjects compared to DNR group. Increased activation of NFκß in NPDR and PDR subjects might involve increased up regulation of VEGF. VEGF SNPs i.e., rs2010963 C allele and rs3025039 T allele might be associated with PDR occurrence and in turn regulates VEGF expression among PDR subjects.


Assuntos
Retinopatia Diabética/genética , NF-kappa B/genética , Polimorfismo de Nucleotídeo Único/genética , Regulação para Cima/genética , Fator A de Crescimento do Endotélio Vascular/genética , Alelos , Diabetes Mellitus Tipo 2/genética , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Receptores de Fatores de Crescimento do Endotélio Vascular/genética
11.
Can J Diabetes ; 37(6): 401-7, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24321721

RESUMO

OBJECTIVE: The present study was intended to investigate whether oxidative stress is the key regulator to alter neuroretinal biochemical homeostasis and in turn aggravate the process of diabetic retinopathy by inducing nitrosative stress in the retinal neurovascular unit. METHODS: Peripheral blood mononuclear cell reactive oxygen species level was measured by flow cytometry along with spectrophotometric detection of malondialdehyde (MDA) and glutamate from serum or plasma and a vitreous sample of study groups (i.e. subjects with proliferative diabetic retinopathy [PDR], type 2 diabetes without retinopathy [DNR] and healthy controls [HCs]). Further, nitrosative stress assessment was performed by spectrophotometric and enzyme-linked immunosorbent assay-based detection of serum and vitreous nitrite and nitrotyrosine concentrations, respectively. RESULTS: The plasma glutamate level remains insignificant between subjects with PDR and DNR (p=0.505) or in HC (p=0.1344) individuals. However, serum MDA (p=0.0004), nitrite (p=0.0147) and nitrotyrosine (p=0.0129) were found to be strikingly higher among PDR subjects compared with the DNR group. Significantly increased levels of peripheral blood mononuclear cell reactive oxygen species (p<0.0001), vitreous glutamate (p=0.0009, p<0.0001), MDA (p=0.0058, p=0.0003), nitrite (p=0.0014, p<0.0001) and nitrotyrosine (p=0.0008, p<0.0001) were found in PDR subjects compared with DNR and HC subjects, respectively. CONCLUSIONS: Our observation suggests that oxidative stress is associated with impairment in neuroretinal biochemical homeostasis among PDR subjects, which further augments retinal nitrosative stress and thus worsens the pathogenic process of retinopathy among PDR subjects.


Assuntos
Retinopatia Diabética/metabolismo , Ácido Glutâmico/sangue , Estresse Oxidativo , Retina/metabolismo , Corpo Vítreo/metabolismo , Adulto , Estudos de Casos e Controles , Retinopatia Diabética/fisiopatologia , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Malondialdeído/metabolismo , Pessoa de Meia-Idade , Nitritos/sangue , Espécies Reativas de Oxigênio/metabolismo , Retina/fisiopatologia , Tirosina/análogos & derivados , Tirosina/sangue
13.
Diabetes Res Clin Pract ; 100(3): 376-84, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23602454

RESUMO

AIM: This study aimed to evaluate whether hyperglycemia mediated increased formation of advanced glycation end products (AGEs) was associated with erythrocyte antioxidant enzyme activity in subjects with different stages of diabetic retinopathy (DR). METHODS: Serum level of AGEs was determined by enzyme linked immunosorbent assay. Erythrocyte superoxide dismutase (SOD), glutathione reductase (GR) and catalase activity were estimated by enzymatic reaction based spectrophotometric assay in patients with type 2 diabetes with proliferative diabetic retinopathy (PDR), non-proliferative diabetic retinopathy (NPDR) and no retinopathy (DNR) and also in healthy non-diabetic controls (HC). RESULT: Erythrocyte SOD and GR activity was significantly lower among NPDR (p=0.024, 0.0017, respectively) and PDR (p=0.0003, 0.0001, respectively) subjects compared with DNR individuals. A significant inverse correlation was observed between serum AGEs and erythrocyte SOD or GR activity in DNR (p=0.0019; r=-0.3033, p=0.0021; r=-0.3015, respectively), NPDR (p=0.0001; r=-0.4602, p=0.0003; r=-0.4161, respectively), and PDR (p<0.0001; r=-0.6753, p<0.0001; r=-0.5854, respectively) individuals. CONCLUSION: Poor glycemia may be the key factor enhancing AGE formation, which may be associated with lower erythrocyte SOD and GR activity along with increased catalase activity in DR.


Assuntos
Antioxidantes/metabolismo , Retinopatia Diabética/metabolismo , Eritrócitos/metabolismo , Hiperglicemia/metabolismo , Catalase/metabolismo , Feminino , Hemoglobina A Glicada/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Oxirredução , Superóxido Dismutase/metabolismo
14.
Retina ; 33(1): 207-16, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22653543

RESUMO

PURPOSE: To evaluate erythrocyte redox state and its surrogates in patients with different stages of diabetic retinopathy and their association with cellular metabolic derangement developed in retinal microvascular cells. METHODS: Sixty type 2 diabetic patients with nonproliferative diabetic retinopathy (NPDR), 85 patients with proliferative diabetic retinopathy (PDR), and 70 patients with diabetes but without retinopathy were considered as diabetic control (DC) for the study. In addition, 65 normal individuals without diabetes were enrolled as healthy control in this study. Erythrocyte oxidized nicotinamide adenine dinucleotide phosphate / reduced nicotinamide adenine dinucleotide phosphate (NADP / NADPH), oxidized nicotinamide adenine dinucleotide / reduced nicotinamide adenine dinucleotide (NAD / NADH) glutathione, plasma and vitreous lactate, and pyruvate levels were determined by enzymatic reaction-based spectrophotometric assay for the patients and individuals. RESULT: Erythrocyte NADP+ to NADPH ratio to NADPH ratio was found to be significantly higher among NPDR and PDR patients compared with DC subjects (P < 0.0001). Erythrocyte-reduced glutathione was significantly decreased in patients of NPDR (P = 0.0004) and patients of PDR (P = 0.0157) compared to DC. Erythrocyte NAD to NADH ratio was also significantly decreased in patients of NPDR (P < 0.0001) and PDR (P < 0.0001) compared to DC subjects. Lactate to pyruvate ratio of plasma was elevated significantly in patients with NPDR compared with DC (P < 0.0001) and those having PDR (P = 0.0046). In the vitreous fluid, the lactate to pyruvate ratios were found to be significantly lower in normal individuals without diabetes compared with patients having PDR (P < 0.0001). CONCLUSION: Hyperglycemia-mediated erythrocyte redox state alterations might be a potential risk factor for the development of NPDR in poorly controlled diabetic subjects.


Assuntos
Diabetes Mellitus Tipo 2/sangue , Retinopatia Diabética/sangue , Eritrócitos/metabolismo , Hiperglicemia/sangue , NADP/metabolismo , NAD/metabolismo , Glicemia/metabolismo , Pressão Sanguínea , Estudos Transversais , Membrana Eritrocítica/metabolismo , Feminino , Angiofluoresceinografia , Teste de Tolerância a Glucose , Glutationa/metabolismo , Hemoglobina A Glicada/metabolismo , Humanos , Ácido Láctico/sangue , Masculino , Pessoa de Meia-Idade , Oxirredução , Ácido Pirúvico/sangue
15.
Retina ; 32(6): 1197-203, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22105495

RESUMO

PURPOSE: New blood vessel formation in the retina because of prolonged hypoxia is believed to be directly associated with increased expression of several growth factors and angiogenic cytokines. In the present study, we made an attempt to investigate the possible association of the promoter polymorphisms of interleukin 6, tumor necrosis factor α, and interleukin 10 for the pathogenesis of proliferative diabetic retinopathy (PDR). METHODS: This case-control study comprised 493 volunteers (253 PDR cases and 240 diabetic controls). Cases and controls were ascertained such that age, sex, nutrition, and glycemic status were matched. Genotypes were determined by polymerase chain reaction-based methods. RESULTS: Interleukin 10-1082GG (P = 0.0037; odds ratio [OR] = 2.232), tumor necrosis factor α-238AA (P = 0.0001; OR = 5.791), and GA (P = 0.0015; OR = 1.909) genotypes were significantly associated with PDR occurrence. The interleukin 10-1082G allele (P = 0.0048, OR = 1.4442) and the tumor necrosis factor α-238A allele (P = 0.0001; OR = 2.2897) were significantly increased among PDR cases. CONCLUSION: From our study, it may be concluded that the genetic variation, that is, tumor necrosis factor α-238A and interleukin 10-1082G alleles are the potent risk factors for the pathogenesis of PDR.


Assuntos
Retinopatia Diabética/genética , Interleucina-10/genética , Interleucina-6/genética , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Fator de Necrose Tumoral alfa/genética , Adulto , Idoso , Alelos , Estudos de Casos e Controles , Estudos Transversais , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase
16.
Eur J Med Chem ; 46(11): 5283-92, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21907466

RESUMO

Three new series of 4-hydroxy-8-trifluoromethyl-quinoline derivatives were synthesized through multi step reactions. All the newly synthesized compounds were characterized by spectral and elemental analyses. The structure of 5j was evidenced by X-ray crystallographic study. The newly synthesized title compounds were evaluated for their antimicrobial activities including antimycobacterial activity. Amongst the tested compounds, 5b, 5e, 5h, 5j, 6c and 7c displayed promising antimicrobial activity. The mode of action of these active compounds was carried out by docking of receptor enoyl-ACP reductase with newly synthesized candidate ligands, 5b, 5e, 5h, 5j and 6c.


Assuntos
Antituberculosos/síntese química , Antituberculosos/farmacologia , Desenho de Fármacos , Hidrazinas/síntese química , Hidrazinas/farmacologia , Modelos Moleculares , Quinolinas/química , Antituberculosos/química , Bactérias/efeitos dos fármacos , Hidrazinas/química , Testes de Sensibilidade Microbiana , Conformação Molecular
17.
Eur J Med Chem ; 46(10): 4834-45, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21880400

RESUMO

New series of quinoline-oxazolidinone hybrid molecules were synthesized based on the preliminary docking studies. All the newly synthesized compounds were characterized by spectral analyses. The newly synthesized compounds were screened for their antimycobacterial properties based on the promising preliminary antibacterial screening results. Amongst tested compounds, compounds 8a, 8j and 13a were active at 0.65 µg/mL against Mycobacterium tuberculosis H(37)Rv strain. The mode of action of these active compounds was carried out by docking of receptor enoyl-ACP reductase with newly synthesized candidate ligands 8a, 8j and 13a. These compounds exhibited well established bonds with one or more amino acids in the receptor active pocket. From the docking studies, compound 8j was considered to be the best inhibitor.


Assuntos
Antituberculosos/química , Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Oxazolidinonas/química , Oxazolidinonas/farmacologia , Quinolinas/química , Quinolinas/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Antituberculosos/síntese química , Bactérias/efeitos dos fármacos , Domínio Catalítico , Desenho de Fármacos , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/química , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Humanos , Modelos Moleculares , Mycobacterium tuberculosis/enzimologia , Oxazolidinonas/síntese química , Ligação Proteica , Quinolinas/síntese química , Tuberculose/tratamento farmacológico
18.
Eur J Med Chem ; 46(6): 2503-12, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21489660

RESUMO

Three new series of quinoline-4-yl-1,2,3-triazoles carrying amides, sulphonamides and amidopiperazines were synthesized through multi-step reactions. The required intermediate, [1-(6-methoxy-2-methylquinolin-4-yl)-1H-1,2,3-triazol-4-yl]methanol (2) was prepared by treating 4-azido-6-methoxy-2-methylquinoline (1) with propargyl alcohol. Three different series of compounds were synthesized from this intermediate. All the newly synthesized compounds were characterized by spectral and elemental analyses. The structure of 2 was confirmed by X-ray crystallographic study. Further, the title compounds were evaluated for their in vitro anti-bacterial activity against five different bacterial strains and antimycobacterial activity against Mycobacterium tuberculosis H37Rv, Mycobacterium smegmatis (ATCC 19420) and Mycobacterium fortuitum (ATCC 19542). Title compounds, 6a, 6d, 6i, 6j, 7e, 10a and 10i were found to be active against Mycobacterium tuberculosis H37Rv strain and could be lead molecules of interest.


Assuntos
Amidas/química , Antituberculosos/farmacologia , Escherichia coli/efeitos dos fármacos , Piperazinas/química , Pseudomonas/efeitos dos fármacos , Quinolinas/farmacologia , Streptococcus/efeitos dos fármacos , Triazóis/farmacologia , Antituberculosos/síntese química , Antituberculosos/química , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Escherichia coli/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Pseudomonas/crescimento & desenvolvimento , Quinolinas/síntese química , Quinolinas/química , Estereoisomerismo , Streptococcus/crescimento & desenvolvimento , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/química
19.
Retina ; 31(7): 1412-20, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21394064

RESUMO

BACKGROUND: Eales disease (ED) is an idiopathic, inflammatory, venoocclusive disorder of peripheral retina resulting in retinal angiogenesis and vitreous hemorrhage. The objective of the present study is to investigate the expression and activation of gelatinase associated with the retinal neovascularization in ED and the relation between the levels of gelatinase and the cytokine tumor necrosis factor-α, known to upregulate matrix metalloproteinase (MMP) expression on various cells. METHODS: Vitreous and serum samples from 19 patients with ED who underwent retinal surgery were estimated for levels of MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1, tissue inhibitor of metalloproteinase-2, and tumor necrosis factor-α by enzyme-linked immunosorbent assay method. Matrix metalloproteinase-2 and MMP-9 activities in serum and vitreous samples were evaluated by gelatin zymography method. Vitreous samples from 16 patients with macular hole undergoing vitrectomy were used as controls. RESULTS: Among the 2 gelatinase examined in vitreous and serum samples, only level and activity of MMP-9 were significantly higher in serum (P = 0.0001) and vitreous (P = 0.0002) samples of patients with ED than those of control subjects. Simultaneously, a positive correlation was found between intraocular tumor necrosis factor-α and MMP-9 concentration (Spearman correlation coefficient, r = 0.7040, P = 0.0023) in patients with ED. CONCLUSION: Increase in MMP-9 activity and its concentration in serum and vitreous of patients with ED compared with that of control subjects and correlation between intraocular levels of MMP-9 and tumor necrosis factor-α in patients with ED seem to provide a plausible explanation for inflammation-mediated angiogenesis during the development of this condition.


Assuntos
Metaloproteinase 2 da Matriz/sangue , Metaloproteinase 9 da Matriz/sangue , Neovascularização Patológica/sangue , Neovascularização Retiniana/sangue , Vasculite Retiniana/sangue , Fator de Necrose Tumoral alfa/sangue , Corpo Vítreo/metabolismo , Adulto , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/cirurgia , Vasculite Retiniana/cirurgia , Inibidor Tecidual de Metaloproteinase-1/sangue , Inibidor Tecidual de Metaloproteinase-2/sangue , Vitrectomia
20.
Eur J Med Chem ; 45(8): 3374-83, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20537437

RESUMO

Four new series of quinoline derivatives were synthesized starting from 2-trifluoromethyl aniline through multi-step reactions. In the reaction sequence, substituted aniline was cyclized to 4-hydroxy quinoline 1, which was then transformed to 4-chloro-2,8-bis(trifluoromethyl)quinoline 2. The key scaffold 4-hydrazinyl-2,8-bis(trifluoromethyl)quinoline 3, obtained from the compound 2, was successfully converted to target quinoline derivatives, viz. hydrazones 4a-t, ureas 5a-e, thioureas 6a-c and pyrazoles 7a-d, in good yields. The newly synthesized title compounds were evaluated for their in vitro antibacterial activity against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae (recultured) and antituberculosis activity against Mycobacterium tuberculosis H(37)Rv and MDR-TB. Preliminary results indicated that most of the hydrazone derivatives demonstrated very good antibacterial and antituberculosis activities while other derivatives showed moderate activity.


Assuntos
Antituberculosos/síntese química , Antituberculosos/farmacologia , Bactérias/efeitos dos fármacos , Quinolinas/síntese química , Quinolinas/farmacologia , Antituberculosos/química , Cristalografia por Raios X , Testes de Sensibilidade Microbiana , Quinolinas/química
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