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1.
PLoS One ; 16(1): e0244806, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33417619

RESUMO

OBJECTIVE: To analyze the effect of statins on cytokines levels in gingival crevicular fluid (GCF) and saliva and on clinical periodontal parameters of middle-aged and elderly patients with type 2 diabetes mellitus (T2DM). METHODS: Systemically healthy controls (C group, n = 62), T2DM patients not taking statins (D group, n = 57) and T2DM patients taking statins (S group, n = 24) were recruited. In each group, subjects (40-85 years) were subclassified into the h (periodontal health)group, the g (gingivitis)group or the p (periodontitis) group according to different periodontal conditions. 17 cytokines in gingival crevicular fluid (GCF) and saliva samples of each subject were measured utilizing the Luminex technology kit. Further, HbA1c (glycated hemoglobin), FPG (fasting plasma glucose), PD (probing depth), CAL (clinical attachment level), BOP (bleeding on probing), GI (gingival index) and PI (periodontal index) were recorded. Data distribution was tested through the Shapiro-Wilk test, upon which the Kruskal-Wallis test was applied followed by Mann-Whitney U test and Bonferroni's correction. RESULTS: Levels of IFN-γ, IL-5, IL-10 and IL-13 in the saliva of the Dh group were significantly lower than those in the Ch group, while factor IL-4 was higher (p<0.05). Levels of MIP-3α, IL-7 and IL-2 in GCF of the Dh group were considerably higher than those in the Ch group (p<0.05), while that of IL-23 was considerably lower. Compared with the Cg group, levels of IFN-γ, IL-4, IL-5, IL-6, IL-10 and IL-13 were significantly lower in the saliva of the Dg group (p<0.05). Lower levels of IFN-γ, IL-5 and IL-10 were detected in the Sg group than those in the Cg group (p<0.05). At the same time, levels of IL-1ß, IL-6, IL-7, IL-13, IL-17, IL-21 and MIP-3α in the gingival crevicular fluid of the Sg group were lower in comparison with the Dg group. In addition, lower levels of IL-4 and higher levels of IL-7 in GCF were identified in the Dg group than those in the Cg group, while in the Sg group, lower levels of IL-4, MIP-1αand MIP-3αwere observed than those in the Cg group (p<0.05). Lower levels of IFN-γ, IL-6, IL-10, IL-13 and I-TAC were found in the Sp group compared with those in the Cp group. The IFN-γ, IL-6 and IL-10 levels were lower in the Dp group than those in the Cp group (p<0.05). Meanwhile, in the Sp group, lower levels of pro-inflammatory factors IFN-γ, IL-1ß, IL-2, IL-6, IL-7, IL-21 and TNF-α, in addition to higher levels of anti-inflammatory factors IL-4 and IL-5 in gingival crevicular fluid, were identified than those in the Dp group. Higher levels of IFN-γ,IL-1ß,IL-2,IL-7,IL-21 and TNF-α and a lower level of IL-5 in the Dp group were identified than those in the Cp group (p<0.05). Moreover, statins were able to substantially reduce PD in T2DM patients with periodontitis, indicating an obvious influence on the levels of cytokines secreted by Th1 cells, Th2 cells and Th17 cells, as revealed by PCA (principal component analysis). CONCLUSION: Statins are associated with reduced PD and cytokines levels in the GCF and saliva of T2DM patients with periodontitis.


Assuntos
Citocinas/análise , Diabetes Mellitus Tipo 2/patologia , Líquido do Sulco Gengival/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Saliva/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/complicações , Feminino , Gengivite/patologia , Humanos , Interferon gama/análise , Interleucina-10/análise , Interleucina-13/análise , Masculino , Pessoa de Meia-Idade , Periodontite/complicações , Periodontite/patologia , Análise de Componente Principal
2.
PLoS One ; 15(12): e0243545, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33326443

RESUMO

Downregulation of the T cell system has been proposed as a mechanism to block immunity in colonic cancer (CC). However, little has been studied about circulating αß and γδ T cells and their immunological status in newly diagnosed patients. The aim of this study was to characterize the αß and γδ T cell subsets in peripheral blood of patients with CC matched with healthy volunteers. In this prospective case-control study, blood samples were obtained from 96 patients with newly diagnosed treatment-naïve infiltrating colonic adenocarcinoma and 48 healthy volunteers. Pathological report at surgery was obtained from all CC patients. A significant decrease in CD3+ γδ T cells and CD3+CD8+ γδ T cells (p<0.001) were observed in CC patients. Apoptosis was significantly increased in all conventional and both αß and γδ T cell subsets in patients with CC vs healthy subjects. γδ T cells were decreased in peripheral blood of patients with microscopic infiltration in tissues, history of cancer and synchronous colon cancer (p < 0.05). IFN-γ was significantly reduced in CC patients compared to controls. Cytotoxic effector γδ T cells TEMRA (CD8 and CD56) are the proportionally most abundant T cells in peripheral blood of CC patients. Patients with CC present a deep downregulation in the systemic T-cell immunity. These variations are evident through all tumor stages and suggest that a deficiency in γδ T cell populations could be preventing control of tumor progression. This fact prove the role of immunomodulation on CC carcinogenesis.


Assuntos
Neoplasias do Colo/imunologia , Linfócitos Intraepiteliais/imunologia , Idoso , Biomarcadores/sangue , Linfócitos T CD8-Positivos/imunologia , Estudos de Casos e Controles , Feminino , Humanos , Interferon gama/análise , Interferon gama/sangue , Linfócitos Intraepiteliais/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/sangue
3.
Exp Parasitol ; 216: 107944, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32619431

RESUMO

Evaluation of the murine isotype antibodies is essential in subunit vaccine development because inbred mouse strains with diverse genetic backgrounds respond different to recombinant proteins. In this regard, the main goal of this study was to measuring and comparing the profile of IgG isotype responses in C57BL/6 mice. For this purpose, the extracellular region of plasmodium vivax thrombospondin-related adhesive protein (PvTRAP) gene was expressed in Escherichia coli Rosetta (DE3)-pET23a. Then, the recombinant PvTRAP alone or emulsified with Freund's complete adjuvant were applied for immunization of the C57BL/6 mice. The role of antibodies and cellular immune responses induced by recombinant PvTRAP were evaluated. The results showed the level of anti-rPvTRAP IgG2c was significantly higher than IgG2a in the groups that received rPvTRAP alone (mean OD490 = 0.798 ± 0.12 and 0.39 ± 0.1, respectively) and emulsified with CFA/IFA (mean OD490 = 1.48 ± 0.07 and 0.605 ± 0.13, respectively; P < 0.05, independent sample t-test). Additionally, the immunized mice with rPvTRAP and rPvTRAP + CFA/IFA had an intermediate-avidity IgG2a antibody but high-avidity IgG2c antibody as well as the mean of serum antibody titers results exhibited that in both rPvTRAP and rPvTRAP + CFA/IFA mouse groups, IgG2a end-point titer (1:3200 and 1:25,600, respectively) was noteworthy lower than IgG2c (1:25,600 and 1:102,400, respectively). Moreover, the results revealed the eliciting significant levels of IFN-γ (P < 0.05, independent sample t-test) and no detectable level of IL-4 in the mouse groups received rPvTRAP alone and emulsified with CFA/IFA as compared to the mouse control groups. In general, our results showed that for correctly interpreting of Th1 immune responses in C57BL/6 mouse strain it is critical to measure IgG2c instead of IgG2a along with IFN-γ.


Assuntos
Imunoglobulina G/sangue , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Células Th1/imunologia , Células Th2/imunologia , Animais , Afinidade de Anticorpos , Dicroísmo Circular , Feminino , Imunofluorescência , Imunoglobulina G/classificação , Interferon gama/análise , Interleucina-4/análise , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/imunologia , Vacinas de Subunidades/imunologia , Vacinas Sintéticas/imunologia
4.
PLoS One ; 15(6): e0234700, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32544206

RESUMO

BACKGROUND: We investigated changes in the interferon-γ levels before and after treatment of latent tuberculosis infection (LTBI) using QuantiFERON-TB Gold Plus (QFT-Plus) and QuantiFERON-TB Gold In-Tube (QFT-GIT) assays. The objective was to assess whether QFT-Plus could serve as a biomarker of LTBI treatment response. METHODS: We prospectively enrolled 44 individuals whose baseline QFT-GIT and QFT-Plus showed positive results at a tertiary referral center in South Korea between March 2017 and March 2018. The results of the QFT-Plus assay were defined as positive if either or both of the antigen tubes (TB1 and/or TB2) were positive. After LTBI treatment, both tests were repeated. RESULTS: The mean age of the participants was 47.6 years. The QFT-GIT and QFT-Plus assays revealed positive results in 42/44 (95.5%) and 41/44 (93.2%) participants after LTBI treatment, showing overall agreement of 93.2%, with a Cohen's kappa value of 0.37 (fair agreement). The differences between pre- and post-LTBI treatment interferon-γ levels were measured using the QFT-GIT and QFT-Plus assays. No significant differences were noted among the 3 values: the median difference in interferon-γ value with QFT-GIT, QFT-Plus TB1, and QFT-Plus TB2 was 0.211 IU/mL (IQR, -0.337-3.347), 0.025 IU/mL (IQR, -0.338-1.368), and 0.180 IU/mL (IQR, -0.490-2.278), respectively (P = 0.401). CONCLUSION: The change in interferon-γ levels before and after LTBI treatment measured using the QFT-Plus assay showed a similar trend to that of the QFT-GIT assay. Considering that the QFT-GIT assay is not a useful biomarker of LTBI treatment response, QFT-Plus also appears not to be useful for this purpose.


Assuntos
Interferon gama/análise , Tuberculose Latente/prevenção & controle , Adulto , Biomarcadores , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , República da Coreia , Teste Tuberculínico/métodos
5.
PLoS Negl Trop Dis ; 14(3): e0008093, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32176691

RESUMO

Human leishmaniasis is a public health problem worldwide for which the development of a vaccine remains a challenge. T cell-mediated immune responses are crucial for protection. Peptide vaccines based on the identification of immunodominant T cell epitopes able to induce T cell specific immune responses constitute a promising strategy. Here, we report the identification of human leukocyte antigen class-I (HLA-I) and -II (HLA-II)-restricted multi-epitope peptides from Leishmania proteins that we have previously described as vaccine candidates. Promastigote Surface Antigen (PSA), LmlRAB (L. major large RAB GTPase) and Histone (H2B) were screened, in silico, for T cell epitopes. 6 HLA-I and 5 HLA-II-restricted multi-epitope peptides, able to bind to the most frequent HLA molecules, were designed and used as pools to stimulate PBMCs from individuals with healed cutaneous leishmaniasis. IFN-γ, IL-10, TNF-α and granzyme B (GrB) production was evaluated by ELISA/CBA. The frequency of IFN-γ-producing T cells was quantified by ELISpot. T cells secreting cytokines and memory T cells were analyzed by flow cytometry. 16 of 25 peptide pools containing HLA-I, HLA-II or HLA-I and -II peptides were able to induce specific and significant IFN-γ levels. No IL-10 was detected. 6 peptide pools were selected among those inducing the highest IFN-γ levels for further characterization. 3/6 pools were able to induce a significant increase of the percentages of CD4+IFN-γ+, CD8+IFN-γ+ and CD4+GrB+ T cells. The same pools also induced a significant increase of the percentages of bifunctional IFN-γ+/TNF-α+CD4+ and/or central memory T cells. We identified highly promiscuous HLA-I and -II restricted epitope combinations from H2B, PSA and LmlRAB proteins that stimulate both CD4+ and CD8+ T cell responses in recovered individuals. These multi-epitope peptides could be used as potential components of a polytope vaccine for human leishmaniasis.


Assuntos
Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Leishmania/imunologia , Leishmaniose Cutânea/imunologia , Proteínas Recombinantes de Fusão/imunologia , Adulto , Antígenos de Protozoários/genética , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito T/genética , Feminino , Citometria de Fluxo , Granzimas/análise , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Interferon gama/análise , Interleucina-10/análise , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Necrose Tumoral alfa/análise , Voluntários , Adulto Jovem
6.
J Pediatr Hematol Oncol ; 42(3): 185-192, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32032240

RESUMO

Although aplastic anemia has been extensively investigated, little is known about their circulating cytokine pattern. The present study was done to evaluate the severity of the disease with the 3 major anti-hematopoietic cytokines interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ). This study is ethically cleared. A total of 102 bone marrow plasma and peripheral blood plasma paired samples were collected from the confirmed acquired aplastic anemia (AAA) patients and 10 control cases after taking written consent and analyzed by the quantitative enzyme-linked immunosorbent assay. The Mann-Whitney U test was used for statistical analysis. Considerably increased levels of IL-2, TNF-α, and IFN-γ were found in the peripheral blood plasma and bone marrow plasma of AAA patients as compared with controls, that is, 45.76±20.61 versus 1.99±1.25, P<0.00001; 26.51±15.62 versus 11.7±3.67, P=0.00188; 17.04±11.64 versus 5.27±1.92, P=0.00034 and 70.54± 37.57 versus 3.12±1.82, P<0.00001; 251.82±243.80 versus 15.66±6.35, P<0.00001; 39.35±22.58 versus 11.12±2.41, P=0.00012, respectively. The IL-2, TNF-α, and IFN-γ levels were observed to be extraordinarily elevated in AAA, but were very low in the control cases. The results confirm that IL-2, TNF-α, and IFN-γ may have an imperative association with the disaster in the bone marrow compartment of AAA patients. The levels and ranges of the observed cytokines can also be predicted by the severity basis of this study.


Assuntos
Anemia Aplástica/imunologia , Interferon gama/análise , Interleucina-2/análise , Fator de Necrose Tumoral alfa/análise , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Medula Óssea/imunologia , Criança , Pré-Escolar , Feminino , Humanos , Índia , Masculino , Pessoa de Meia-Idade , Adulto Jovem
7.
Talanta ; 211: 120761, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32070582

RESUMO

A simple, fast and sensitive amperometric immunosensing method for the determination of the clinically relevant cytokine interferon gamma (IFN-γ) in saliva complying the requirements demanded for this kind of sample is reported. The target analyte was sandwiched between a specific capture antibody covalently immobilized on a screen-printed electrode functionalized by the diazonium salt grafting of p-aminobenzoic acid, and a biotinylated detector antibody labeled with a streptavidin-horseradish peroxidase conjugate. The amperometric responses measured at - 0.20 V vs Ag pseudo-reference electrode upon addition of hydrogen peroxide in the presence of hydroquinone as the redox mediator allowed a calibration plot with a linear range between 2.5 and 2000 pg mL-1 and a low limit of detection (1.6 pg mL-1) to be obtained. In addition, a good selectivity against other non-target proteins was achieved. The developed method was validated by analyzing a WHO 1st International Standard for IFN-γ. In addition, the immunosensor was used for the determination of the endogenous IFN-γ in saliva with results in excellent agreement with those obtained by a commercial ELISA kit.


Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Eletrodos , Imunoensaio/métodos , Interferon gama/análise , Saliva/metabolismo , Humanos , Limite de Detecção , Saliva/química
8.
Biosci Biotechnol Biochem ; 84(1): 208-215, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31532348

RESUMO

Dihomo-γ-linolenic acid (DGLA, C20: 3n-6) is known to have an anti-inflammatory activity, but its range of effects was not well studied because of its limited natural sources. We addressed these issues by constructing an yeast Saccharomyces cerevisiae strain having a complete metabolic pathway for DGLA synthesis by introducing two desaturase and one elongase genes to convert endogenous oleic acid to DGLA. Taking advantage of well-known safety of S. cerevisiae, we previously investigated the efficacy of heat-killed whole DGLA-producing yeast cells on irritant contact dermatitis, and showed that oral intake of this yeast significantly suppressed inflammatory reactions, whereas no such suppression was observed by the intake of 25 times the amount of purified DGLA. Since this method is considered to be a simple and efficient way to suppress inflammation, we examined its effectiveness against allergic contact dermatitis (ACD) in this study and showed that this method was also effective against ACD.


Assuntos
Ácido 8,11,14-Eicosatrienoico/farmacologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Dermatite Alérgica de Contato/terapia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Ácido 8,11,14-Eicosatrienoico/administração & dosagem , Ácido 8,11,14-Eicosatrienoico/metabolismo , Acetona/química , Administração Oral , Animais , Quimiocina CCL2/análise , Quimiocinas/análise , Dermatite Alérgica de Contato/etiologia , Dinitrofluorbenzeno/efeitos adversos , Dinitrofluorbenzeno/imunologia , Orelha Externa/patologia , Feminino , Imunização , Inflamação/terapia , Interferon gama/análise , Camundongos , Ácido Oleico/metabolismo , Azeite de Oliva/química
9.
BMC Biotechnol ; 19(1): 102, 2019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31870349

RESUMO

BACKGROUND: Interferon-gamma (IFN-γ) is an important mediator of type I immune response and has antiviral, immunoregulatory and anti-tumor properties, plays a wide range of roles in inflammation and autoimmune diseases. The aim of this study was to obtain monoclonal antibody (mAb) against caprine IFN-γ by immunizing of BALB/c mice with the purified rIFN-γ. RESULTS: Recombinant caprine IFN-γ was expressed in Escherichia coli strain BL21 (DE3) and monoclonal antibodies against caprine IFN-γ were produced by immunizing of BALB/c mice with rIFN-γ. One hybridoma secreting mAb was screened by enzyme-linked immunosorbent assay (ELISA) which was designated as 2C. MAb secreted by this cell line were analyzed through ELISA, western blot and application of the mAb was evaluated by immunofluorescence analysis using goat lip tissues infected with Orf virus. ELISA analysis revealed that mAb 2C can specifically recognize rIFN-γ protein and culture supernatant of goat peripheral blood mononuclear cells (PBMCs) stimulated by concanavalin A (Con A) but cannot recognize the fusion tag protein of pET-32a. Western blot analysis showed that mAb 2C can specifically react with the purified 34.9 kDa rIFN-γ protein but does not react with the fusion tag protein of pET-32a. Immunofluorescence results demonstrated that mAb 2C can detect IFN-γ secreted in histopathological sites of goats infected with Orf virus. CONCLUSIONS: A caprine IFN-γ-specific mAb was successfully developed in this study. Further analyses showed that the mAb can be used to detect IFN-γ expression level during contagious ecthyma in goats.


Assuntos
Anticorpos Monoclonais/análise , Interferon gama/análise , Interferon gama/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Western Blotting , Ectima Contagioso/sangue , Ectima Contagioso/imunologia , Ectima Contagioso/virologia , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Doenças das Cabras/sangue , Doenças das Cabras/imunologia , Doenças das Cabras/virologia , Cabras , Hibridomas/metabolismo , Interferon gama/sangue , Interferon gama/genética , Leucócitos Mononucleares/imunologia , Camundongos Endogâmicos BALB C , Vírus do Orf/fisiologia
10.
Clin Lab ; 65(11)2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31710451

RESUMO

BACKGROUND: Prostate cancer is one of the most common cancers in males worldwide. Recently, it is well characterized that long non-coding RNAs (lncRNA) play critical roles in the initiation, development, and progression of prostate cancer. NeST, an intergenic lncRNA, was found to be a positive regulator of the pro-inflammatory cytokine, IFN-É£, which is responsible for both antitumor immunity properties as well as tumor evasion. FOXCUT, an-other lncRNA, is mainly a regulator of transcription factor, FOXC1 that is believed to be involved in tumor development and progression. METHODS: In a case-control study, 66 paraffin-embedded prostate tissues representing 36 pathologically confirmed cancer and 30 control samples were examined. The cancer samples were classified in a total of three stages based on PSA levels, tumor volume, and Gleason score. RNA extraction was performed for quantitative determination of IFN-É£, lncRNA NeSt, and lncRNA FOXCUT gene expression in both case and control prostate tissues. RESULTS: Our results showed that NeST lncRNA was significantly up-regulated in prostate cancer samples compared to control, while NeST lncRNA and IFN-É£ gene expression was detected mainly in early stages of prostate cancer. The patients with higher NeST and FOXCUT expression had poor clinical features including PSA levels and tumor volume comparing those with lower expression. Moreover, there was a strong correlation between lncRNA FOXCUT and IFN-É£ expression. CONCLUSIONS: Our data suggests that lncRNA NeST and lncRNA FOXCUT may be able to be introduced as novel molecules involved in prostate cancer development and may provide a potential prognostic biomarker and therapeutic target.


Assuntos
Biomarcadores Tumorais/genética , Detecção Precoce de Câncer/métodos , Neoplasias da Próstata/genética , RNA Longo não Codificante/genética , Idoso , Biomarcadores Tumorais/análise , Estudos de Casos e Controles , Regulação Neoplásica da Expressão Gênica , Humanos , Interferon gama/análise , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Regulação para Cima
11.
Vet Parasitol ; 276: 108990, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31775103

RESUMO

Neospora caninum infection is an important cause of neuromuscular disease in dogs and abortion in cattle, leading to significant economic losses in beef and dairy industries. The protective immunity against apicomplexan parasites, specifically Toxoplasma gondii and N. caninum, is typically achieved by inducing an IL-12-driven Th1 immune response. IL-12 stimulates IFN-γ production, which activates Inducible Nitric Oxide Synthase (iNOS) and promotes consequent Nitric Oxide (NO) synthesis, classically described as one of the main effector mechanisms for parasite elimination. Here, we aimed to evaluate the role played by iNOS during N. caninum infection. Our results show that N. caninum infection in C57BL/6 wild type (WT) mice induce NO production in vivo and in vitro. In agreement, iNOS deficient mice, as well as WT mice treated with iNOS inhibitor aminoguanidine, succumbed during acute infection with a dose lethal to 50 % of the WT mice, and presented significant increase in parasite load when submitted to sub-lethal infection protocols. Interestingly, the lack of control of parasite proliferation observed in iNOS-/- mice was associated with notable CNS inflammation and increased production of the main systemic proinflammatory cytokines (IL-12, IFN-γ, IL-6, TNF and IL-17A). Taken together, our findings show that iNOS plays an important role in restricting N. caninum replication, while also modulates the inflammatory process induced by the infection.


Assuntos
Coccidiose/enzimologia , Neospora/imunologia , Óxido Nítrico Sintase Tipo II/fisiologia , Animais , Coccidiose/parasitologia , Coccidiose/patologia , Interferon gama/análise , Subunidade p40 da Interleucina-12/análise , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/deficiência
12.
Eur Surg Res ; 60(5-6): 186-195, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31597147

RESUMO

BACKGROUND: Interferon gamma (IFNγ) and tumor necrosis factor-related weak inducer of apoptosis (TWEAK) molecules seem to have a potential effect on angiogenic factors such as vascular endothelial growth factor (VEGF). The aim of this study was to assess a possible interplay between IFNγ and TWEAK cytokines and VEGF machinery in the different steps of colorectal carcinogenesis. METHODS: A total of 92 subjects with colonic adenoma or cancer who underwent screening colonoscopy or surgery were prospectively enrolled. Polypoid lesion tissue samples were collected and frozen. Real-time reverse transcription polymerase chain reaction for IFNγ, TWEAK, and VEGF-A mRNA expression was performed. Immunoassays for VEGF-A, VEGF-C, VEGFR-1, VEGFR-2, and VEGFR-3 were also performed. Nonparametric statistics, receiver operating characteristic curve analysis, and logistic multiple regression analysis were used. RESULTS: IFNγ and TWEAK mRNA expression was higher in patients with T2 or more advanced colorectal cancer than in those with adenomas or T1 cancer (p < 0.001 and p = 0.01, respectively). IFNγ and TWEAK mRNA expression levels directly correlated with VEGF-A mRNA expression levels (rho = 0.44, p < 0.001 and rho = 0.29, p = 0.004, respectively). On the contrary, IFNγ and TWEAK mRNA expression levels inversely correlated with VEGF-C protein levels (rho = -0.29, p = 0.04 and rho = -0.31, p = 0.03, respectively). Similarly, IFNγ and TWEAK mRNA expression levels inversely correlated with VEGFR2 protein levels (rho = -0.38, p = 0.033 and rho = -0.40, p = 0.025, respectively). CONCLUSION: This study showed that in colorectal polypoid lesions, IFNγ and TWEAK expressions are directly correlated to VEGF-A expression but inversely correlated with VEGFR2 levels, suggesting a possible feedback mechanism in the regulation of VEGF-A expression.


Assuntos
Neoplasias Colorretais/irrigação sanguínea , Citocina TWEAK/genética , Interferon gama/genética , Neovascularização Patológica/etiologia , Fator A de Crescimento do Endotélio Vascular/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/etiologia , Citocina TWEAK/análise , Feminino , Humanos , Interferon gama/análise , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , RNA Mensageiro/análise , Fator A de Crescimento do Endotélio Vascular/análise , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/análise
13.
Nucleic Acids Res ; 47(16): 8362-8374, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31392985

RESUMO

Many nucleic acid aptamers that bind to target molecules have been reported as antibody alternatives. However, while the affinities of aptamers vary widely, little is known about the relationship between the affinities and their applicabilities for practical use. Here, we developed molecular affinity rulers: a series of DNA aptamers with different affinities that bind to the same area of target molecules, to measure the aptamer and its device applicabilities. For the ruler preparation, we used high-affinity DNA aptamers containing a hydrophobic unnatural base (Ds) as the fifth base. By replacing Ds bases with A bases in Ds-DNA aptamers targeting VEGF165 and interferon-γ, we prepared two sets of DNA aptamers with dissociation constants (KD) ranging from 10-12 to 10-8 M. Using these molecular affinity rulers, we evaluated the sensitivity of DNA aptamers in ELISA (enzyme-linked immunosorbent assay), which showed the clear relationship between aptamer affinities and their detection sensitivities. In sandwich-type ELISA using combinations of aptamers and antibodies, aptamers with KD values lower than ∼10-9 M were required for sufficient sensitivities (limit of detection (LOD) < 10 pM) and signal intensities, but optimizations improved the lower-affinity aptamers' applicabilities. These aptamer affinity rulers could be useful for evaluating and improving aptamer applicabilities.


Assuntos
Adenina/química , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Ensaio de Imunoadsorção Enzimática/métodos , Interferon gama/análise , Fator A de Crescimento do Endotélio Vascular/análise , Animais , Anticorpos Monoclonais/química , Aptâmeros de Nucleotídeos/síntese química , Pareamento de Bases , Sequência de Bases , Biotina/química , Ensaio de Imunoadsorção Enzimática/normas , Humanos , Interações Hidrofóbicas e Hidrofílicas , Interferon gama/química , Cinética , Limite de Detecção , Conformação de Ácido Nucleico , Ligação Proteica , Padrões de Referência , Técnica de Seleção de Aptâmeros , Estreptavidina/química , Fator A de Crescimento do Endotélio Vascular/química
14.
Biosens Bioelectron ; 142: 111532, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31377576

RESUMO

In this paper, a novel label-free electrochemical impedance aptasensor for highly sensitive detection of IFN-γ based on target-induced exonuclease inhibition was constructed. For this purpose, we designed a DNA hairpin modified on the gold electrode whose loop was the aptamer of the IFN-γ, and the stem was 5'-thiol-modified. In the absence of IFN-γ, Exonuclease III (Exo III) and Exonuclease I (Exo I) digested the double-stranded and single-stranded strands of the hairpin DNA, respectively, causing smaller impedance value on the surface of the electrode. In the presence of IFN-γ, the function of Exo III was greatly inhibited by the binding of the aptamer with the target, and it stopped after cutting three bases of the hairpin DNA. Forming a major target-bound aptamer digestion product, it could not be digested by Exo I, so there was larger impedance on the electrode surface. The calibration curve for IFN-γ was linear in the range of 1 pM-50 nM with the detection limit (LOD) of 0.7 pM. The proposed aptasensor proved good selectivity and reproducibility, and low cost. In addition, the biosensor was able to detect IFN-γ in serum samples successfully, which is expected to provide an efficient method for TB diagnosis at early stages.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Interferon gama/sangue , Técnicas Biossensoriais/instrumentação , Impedância Elétrica , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Desenho de Equipamento , Exodesoxirribonucleases/química , Humanos , Interferon gama/análise , Limite de Detecção
15.
Nanoscale ; 11(35): 16362-16367, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31435631

RESUMO

The monitoring and detection of molecular biomarkers play crucial roles in disease diagnosis and treatment. In this work, we proposed a target-responsive netlike hybridization chain reaction (nHCR) DNA nanostructure construction method, which can offer an exceptional signal enhancement, for highly sensitive fluorescence detection of cytokine, interferon-gamma (IFN-γ). The presence of the target cytokine can lead to the conformational change of the aptamer recognition hairpin probes and the liberation of the nHCR initiator strands, which further trigger the nHCR process between two dye-labeled and double hairpin-structured probes to form netlike DNA nanostructures. The formation of the DNA nanostructures brings the dyes into close proximity, resulting in significantly amplified fluorescence resonance energy transfer signals for sensitive and enzyme-free detection of IFN-γ. The present method has a detection limit of 1.2 pM and a dynamic linear range of 5 to 1000 pM for IFN-γ detection. Besides, with the high specificity of the aptamer probe and the significant signal amplification of the nHCR, such an IFN-γ detection strategy shows excellent selectivity and high sensitivity, which can be potentially applied to detect IFN-γ in human serums. With such a demonstration of the detection of IFN-γ, this proposed method can be extended for detecting different types of biomolecules.


Assuntos
DNA/química , Transferência Ressonante de Energia de Fluorescência , Interferon gama/análise , Nanoestruturas/química , Humanos , Limite de Detecção , Hibridização de Ácido Nucleico
16.
PLoS Negl Trop Dis ; 13(8): e0007113, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31425525

RESUMO

Buruli Ulcer (BU) is a cutaneous disease caused by Mycobacterium ulcerans. The pathogenesis of this disease is closely related to the secretion of the toxin mycolactone that induces extensive destruction of the skin and soft tissues. Currently, there are no effective measures to prevent the disease and, despite availability of antibiotherapy and surgical treatments, these therapeutic options are often associated with severe side effects. Therefore, it is important to develop alternative strategies for the treatment of BU. Endolysins (lysins) are phage encoded enzymes that degrade peptidoglycan of bacterial cell walls. Over the past years, lysins have been emerging as alternative antimicrobial agents against bacterial infections. However, mycobacteria have an unusual outer membrane composed of mycolylarabinogalactan-peptidoglycan. To overcome this complex barrier, some mycobacteriophages encode a lipolytic enzyme, Lysin B (LysB). In this study, we demonstrate for the first time that recombinant LysB displays lytic activity against M. ulcerans isolates. Moreover, using a mouse model of M. ulcerans footpad infection, we show that subcutaneous treatment with LysB prevented further bacterial proliferation, associated with IFN-γ and TNF production in the draining lymph node. These findings highlight the potential use of lysins as a novel therapeutic approach against this neglected tropical disease.


Assuntos
Úlcera de Buruli/tratamento farmacológico , Endopeptidases/administração & dosagem , Micobacteriófagos/enzimologia , Mycobacterium ulcerans/efeitos dos fármacos , Animais , Bacteriólise , Úlcera de Buruli/patologia , Modelos Animais de Doenças , Endopeptidases/farmacologia , Feminino , Interferon gama/análise , Linfonodos/imunologia , Camundongos Endogâmicos BALB C , Mycobacterium ulcerans/virologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Resultado do Tratamento , Fator de Necrose Tumoral alfa/análise
17.
Acta Trop ; 199: 105148, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31425673

RESUMO

Toxoplasma gondii, a ubiquitous and obligate intracellular pathogen, belonging to the phylum Apicomplexa, is capable of infecting a broad range of warm-blooded hosts including birds and mammals that is nearly worldwide. Preventive measures for toxoplasmosis are currently lacking and as such, development of novel vaccines is of urgent need. The plant-like calcium-dependent protein kinases (CDPKs) expressed by T. gondii, play important roles in cell invasion, gliding motility, egress and some other developmental processes, in which T. gondii CDPK3 (TgCDPK3) has been implicated as an important virulence factor. In this study, the immune protective function of recombinant TgCDPK3 (rTgCDPK3) against experimental toxoplasmosis in BALB/c were evaluated. We divided the mice into different dose groups of vaccines and all immunizations with purified rTgCDPK3 protein were injected by intramuscular at weeks 0, 2, and 4 in BALB/c mice. The rTgCDPK3 vaccine provided protection was correlated with the development of humoral and cellular immune responses demonstrated through the antigen-specific spleen cell proliferation, release of Th1 cytokines IFN-γ, and the production of the high titers of IgG antibody with a predominance of IgG2a over IgG1. Vaccination with rTgCDPK3 conferred partial protection against acute toxoplasmosis, as demonstrated by prolonged survival rate after lethal challenge. Additionally, the amount of brain tissues cysts in vaccinated mice led to 46.5% reduction compared with non-vaccinated ones. These data demonstrated that rTgCDPK3 inoculation prevents or attenuates the harmful influence of T. gondii infection, and it is a potential vaccine candidate against toxoplasmosis.


Assuntos
Proteínas Quinases/imunologia , Vacinas Protozoárias/imunologia , Toxoplasma/imunologia , Toxoplasmose Animal/prevenção & controle , Animais , Feminino , Interferon gama/análise , Camundongos , Camundongos Endogâmicos BALB C , Vacinação , Vacinas Sintéticas/imunologia
18.
Respirology ; 24(10): 962-971, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31418985

RESUMO

Tuberculous effusion is a common disease entity with a spectrum of presentations from a largely benign effusion, which resolves completely, to a complicated effusion with loculations, pleural thickening and even frank empyema, all of which may have a lasting effect on lung function. The pathogenesis is a combination of true pleural infection and an effusive hypersensitivity reaction, compartmentalized within the pleural space. Diagnostic thoracentesis with thorough pleural fluid analysis including biomarkers such as adenosine deaminase and gamma interferon achieves high accuracy in the correct clinical context. Definitive diagnosis may require invasive procedures to demonstrate histological evidence of caseating granulomas or microbiological evidence of the organism on smear or culture. Drug resistance is an emerging problem that requires vigilance and extra effort to acquire a complete drug sensitivity profile for each tuberculous effusion treated. Nucleic acid amplification tests such as Xpert MTB/RIF can be invaluable in this instance; however, the yield is low in pleural fluid. Treatment consists of standard anti-tuberculous therapy or a guideline-based individualized regimen in the case of drug resistance. There is low-quality evidence that suggests possible benefit from corticosteroids; however, they are not currently recommended due to concomitant increased risk of adverse effects. Small studies report some short- and long-term benefit from interventions such as therapeutic thoracentesis, intrapleural fibrinolytics and surgery but many questions remain to be answered.


Assuntos
Antituberculosos/uso terapêutico , Derrame Pleural/tratamento farmacológico , Tuberculose Pleural/diagnóstico , Tuberculose Pleural/terapia , Adenosina Desaminase/análise , Líquidos Corporais/química , Farmacorresistência Bacteriana , Humanos , Interferon gama/análise , Derrame Pleural/microbiologia , Toracentese , Tuberculose Pleural/complicações
19.
J Infect Dis ; 220(11): 1843-1847, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31332447

RESUMO

Nontuberculous mycobacteria (NTM) infect children with increasing frequency worldwide. Using blood and lymph node tissue from children with NTM lymphadenitis, and uninfected lymph node tissue from community controls, we evaluated helper T (TH) cells in functional assays of TH1/TH17 differentiation and measured the concentration of their associated cytokines at the site of infection. Circulating TH cells from infected children were attenuated in their TH1/TH17 differentiation capacity and expressed less interferon γ and interleukin 17 after polyclonal stimulation. Similar differences were observed at the site of infection, where most cytokine concentrations were unchanged relative to controls. Our data are consistent with a model wherein TH1/TH17 differentiation is attenuated in NTM-infected children.


Assuntos
Diferenciação Celular , Infecções por Mycobacterium/patologia , Micobactérias não Tuberculosas/imunologia , Células Th1/imunologia , Células Th17/imunologia , Adolescente , Sangue/imunologia , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Lactente , Recém-Nascido , Interferon gama/análise , Interleucina-17/análise , Linfonodos/imunologia , Masculino , Infecções por Mycobacterium/imunologia
20.
Clin Chim Acta ; 497: 48-53, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31310745

RESUMO

BACKGROUND: In clinical practice, pleural and peritoneal effusions are usual diagnosis. We evaluated the performance of a hybrid panel of biomarkers in the diagnosis of the main diseases affecting pleura and/or peritoneum. METHODS: Samples of pleural/ peritoneal fluid from 120 patients were evaluated for: CEA (carcinoembryonic antigen), VEGF-A (vascular endothelial growth factor A), PD-L1/B7-H1 (programmed death-ligand 1), NGAL (neutrophil gelatinase-associated lipocalin), TREM-1 (triggering receptor expressed in myeloid cells type-1) and IFNγ (gamma-interferon) by Luminex®; CALP (Calprotectin) by ELISA, and ADA (adenosine deaminase) by enzymatic deamination. RESULTS: For malignant effusion (ME) diagnosis, CEA and NGAL presented superior performance than VEGF-A, PD-L1 and CALP. A CEA-NGAL association showed good sensitivity (86.6%) and accuracy (79.2%). For non-tuberculous infectious effusion (NTBIE), NGAL presented the best performance with sensitivity (75.0%), specificity (62.0%) and accuracy (65.0%) higher than TREM-1 and CALP; however, when associated, although with good sensitivity, there was important decrease in specificity. For tuberculous pleural effusion (TPE), IFNy-ADA presented excellent sensitivity (100%), specificity (87.6%), NPV (100%) and accuracies (~90%). CONCLUSIONS: CEA, NGAL, ADA and IFNy were useful in discriminating ME and TPE. However, for NTBIE diagnosis, the hybrid panel did not demonstrate advantages over the classic parameters.


Assuntos
Adenosina Desaminase/análise , Interferon gama/análise , Derrame Pleural Maligno/diagnóstico , Adenosina Desaminase/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/análise , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Interferon gama/metabolismo , Masculino , Pessoa de Meia-Idade , Paracentese , Adulto Jovem
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