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OBJECTIVE: To investigate the cytogenetic characteristics and prognostic risk factors for elderly patients with newly diagnosed elderly acute myeloid leukemia(AML). METHODS: Cytogenetic test results of 76 elderly patients with AML admitted to the First Affiliated Hospital of the University of Science and Technology of China (Anhui Provincial Hospital) from April 2015 to December 2021 were retrospectively analyzed, and analyzed clinical characteristics of patients and risk factors influencing prognosis. RESULTS: According to cytogenetic risk stratification, 76 newly treated elderly AML patients were divided into the favorable, intermediate, and unfavorable groups with 6(7.9%), 58(76.3%), and 12(15.8%) cases, respectively. There was no significant difference in the patient's clinical characteristics and prognosis with the cytogenetics-risk classification groups. Correlation analysis showed that patients' objective response rate (ORR) was related to the age of onset and the mutation status of the CEBPA gene. Logistic regression analysis found that age ≥70 years was an independent risk factor for patients' ORR (OR=0.110, P=0.005). Remission determined the 1-year OS rate (OR=0.049, P=0.005). CONCLUSION: There is no significant difference in clinical characteristics among aged AML patients treated at initial treatment in different cytogenetic risk groups. The age of onset ≥70 years is the determinant of whether patients can obtain ORR, and the rate of ORR is closely related to the 1-year OS rate.
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Although tyrosine kinase inhibitors (TKIs), including imatinib, have greatly improved clinical treatment of patients with chronic myeloid leukemia (CML), drug resistance remains a major obstacle. Studies on the mechanisms underlying imatinib resistance and other alternative drugs are urgently needed. Liquid chromatography tandem mass spectrometry was applied to investigate the differences in proteomics and phosphoproteomics between K562 and K562/G (imatinib resistant K562). Multiple bioinformatics analyses were performed to unveil the differential signal pathways. CCK-8 was used to detect cell proliferation. Flow cytometry was performed to analyze reactive oxygen species (ROS), cell cycle, and cell apoptosis. Western blotting and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) were used to observe the changes of ROS and autophagy associated with imatinib resistance in CML. Our results indicated that ROS-autophagy formed one negative feedback loop and was associated with imatinib resistance. Additionally, the limited-rate enzymes of serine synthesis pathway were escalated in K562/G, which could contribute to the increased cyclin-dependent kinases and cell proliferation index. According to phosphoproteomics data, K562/G cells exhibited abnormal phosphorylation of splicing signals. These results revealed that it could be one useful strategy to correct metabolism shift and oxidative stress, or moderately regulate autophagy. Future research should focus on the discovery of potential targets in ROS-autophagy loop.
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Antineoplásicos , Leucemia Mielógena Crónica BCR-ABL Positiva , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis , Autofagia , Resistencia a Antineoplásicos , Humanos , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Proteómica , Especies Reactivas de OxígenoRESUMEN
This is a retrospective study comparing the effectiveness of umbilical cord blood transplantation (UCBT) and chemotherapy for patients in the first complete remission period for acute myeloid leukemia with KMT2A-MLLT3 rearrangements. A total of 22 patients were included, all of whom achieved first complete remission (CR1) through 1-2 rounds of induction chemotherapy, excluding patients with an early relapse. Twelve patients were treated with UCBT, and 10 patients were treated with chemotherapy after 2 to 4 courses of consolidation therapy. The 3-year overall survival (OS) of the UCBT group was 71.3% (95% CI, 34.4-89.8%), and that of the chemotherapy group was 10% (95% CI, 5.89-37.3%). The OS of the UCBT group was significantly higher than that of the chemotherapy group (P = 0.003). The disease-free survival (DFS) of the UCBT group was 60.8% (95% CI, 25.0-83.6%), which was significantly higher than the 10% (95% CI, 5.72-35.8%) of the chemotherapy group (P = 0.003). The relapse rate of the UCBT group was 23.6% (95% CI, 0-46.8%), and that of the chemotherapy group was 85.4% (95% CI, 35.8-98.4%), which was significantly higher than that of the UCBT group (P < 0.001). The non-relapse mortality (NRM) rate in the UCBT group was 19.8% (95% CI, 0-41.3%), and that in the chemotherapy group was 0.0%. The NRM rate in the UCBT group was higher than that in the chemotherapy group, but there was no significant difference between the two groups (P = 0.272). Two patients in the UCBT group relapsed, two died of acute and chronic GVHD, and one patient developed chronic GVHD 140 days after UCBT and is still alive, so the GVHD-free/relapse-free survival (GRFS) was 50% (95% CI, 17.2-76.1%). AML patients with KMT2A-MLLT3 rearrangements who receive chemotherapy as their consolidation therapy after CR1 have a very poor prognosis. UCBT can overcome the poor prognosis and significantly improve survival, and the GRFS for these patients is very good. We suggest that UCBT is a better choice than chemotherapy for KMT2A-MLLT3 patients.
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Sangre Fetal/trasplante , N-Metiltransferasa de Histona-Lisina/genética , Leucemia Mieloide Aguda/terapia , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas Nucleares/genética , Adolescente , Adulto , Niño , Preescolar , Quimioterapia de Consolidación , Supervivencia sin Enfermedad , Femenino , Reordenamiento Génico , Enfermedad Injerto contra Huésped , Humanos , Quimioterapia de Inducción , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Masculino , Persona de Mediana Edad , Pronóstico , Adulto JovenRESUMEN
Imatinib (IM) is successfully used in the majority of patients with chronic myeloid leukemia (CML), but some patients develop resistance to drug treatment. Insufficient apoptosis results in uncontrolled cell proliferation, which is closely associated with the occurrence of drug resistance. Therefore, it is crucial to identify new biomarkers related to drug resistance. This aim of the present study was to investigate the profile of apoptosis-related proteins in K562 and K562/G (IM-resistant K562 cells) cells, in order to identify new biomarkers. A human apoptosis antibody array was used to screen 46 proteins in the two cells lines, among which 20 proteins were found to be differentially expressed between K562 and K562/G cells. The major proteins included secreted caspase-8, insulin-like growth factor-binding protein (IGFBP)-1, IGFBP-2, IGFBP-3, caspase-3 and p27. IGFBP-1 IGFBP-2 and IGFBP-3 were selected for the follow-up study. Subsequently, reverse transcription-quantitative PCR analysis and western blotting were used to detect the expression levels of the IGFBPs. The results revealed that the expression levels of IGFBP-2 and IGFBP-3 in K562/G cells were significantly decreased compared with those in K562 cells, whereas the IGFBP-1 level was higher. Moreover, no significant correlation was observed between IGFBP-1 or IGFBP-2 and the level of the BCR-ABL fusion protein, whereas decreasing IGFBP-3 levels were associated with increasing BCR-ABL levels. These results suggested that IGFBP-1, IGFBP-2 and IGFBP-3 could be useful novel biomarkers for IM resistance in CML.
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MicroRNAs (miRNAs) are involved in various processes from the development to drug resistance of tumors, including chronic myeloid leukemia (CML). In this study, we examined the STAT5-related miRNA-expression profile in CML cell lines (K562 and imatinib-resistant K562/G) by quantitative real-time reverse-transcriptase polymerase chain reactions. MiR-221 expression was markedly decreased in K562/G cells and peripheral blood mononuclear cells from patients with treatment failure, when compared to imatinib-sensitive CML cells and patients with optimal responses respectively. We also observed the expression of STAT5 inversely correlated with miR-221 expression in K562 and KBM5 cells. Additionally, STAT5 was validated as a direct target of miR-221 in dual-luciferase reporter vector assays. MiR-221 restoration and STAT5 knockdown in K562/G cells increased the sensitivity of CML cells to imatinib by reducing the Bcl2: Bax ratio. Collectively, our data suggested that miR-221-STAT5 axis played crucial roles in controlling the sensitivity of CML cells to imatinib.
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Biomarcadores de Tumor/metabolismo , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Mesilato de Imatinib/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , MicroARNs/genética , Factor de Transcripción STAT5/metabolismo , Adolescente , Adulto , Anciano , Antineoplásicos/farmacología , Apoptosis , Biomarcadores de Tumor/genética , Proliferación Celular , Femenino , Estudios de Seguimiento , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Pronóstico , Factor de Transcripción STAT5/genética , Células Tumorales Cultivadas , Adulto JovenRESUMEN
High expression of the Wilms' tumor gene (WT1) in acute myeloid leukemia (AML) has been considered as a sensitive marker of minimal residual disease (MRD). The present study investigated the significance of quantitative analysis of WT1 mRNA, combined with multiparameter flow cytometry (MFC) regarding its efficacy and prognostic as well as relapse prediction value for leukemia patients with hematopoietic stem cell transplantation. Reverse-transcription quantitative polymerase chain reaction analysis demonstrated that the expression of WT1 in the initial and relapse group was significant higher than that in the complete remission (CR) group (P<0.01). WT1 and the donor chimerism were negatively correlated (r=-0.73, P<0.05). In all AML patients, WT1 was the highest in the M3 subtype and the lowest in the M1 subtype. Follow-up of 12 AML patients demonstrated that WT1 gene expression levels markedly decreased after CR, but obviously increased after relapse, as did the rate of the leukemia cells detected by MFC. The combined usage of MFC and WT1 monitoring contributed to an improved detection rate of relapse (91.7%), and may be used to monitor MRD, assess the treatment efficacy and prognosis, and predict the risk of recurrence in leukemia patients without specific molecular markers after allogeneic hematopoietic stem cell transplantation.
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The persistent activation of signal transducer and activator of transcription 5 (STAT5) may principally be attributed to breakpoint cluster region (BCR)-Abelson murine leukemia viral oncogene homolog 1 (ABL1), and have multi-faceted effects in the development of chronic myeloid leukemia (CML). The p53 protein network regulates important mechanisms in DNA damage repair, cell cycle regulation/checkpoints, and cell senescence and apoptosis, as demonstrated by its ability to positively regulate the expression of various pro-apoptotic genes, including B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax). In the present study, it was observed that the mRNA levels of STAT5A and STAT5B were upregulated in patients with imatinib-resistant CML and in the imatinib-resistant K562/G CML cell line. In addition, increased expression of STAT5 was observed in the BCR-ABL1 mutation group, compared with that in the non-BCR-ABL1 mutation group, regardless of patient imatinib resistance state. Elevated levels of reactive oxygen species (ROS) and DNA double-strand breaks were identified in K562/G cells using flow cytometric and phosphorylated H2AX (γ-H2AX) foci immunofluorescence assays, respectively, compared with the imatinib-sensitive K562 cells. The levels of intracellular ROS and γ-H2AX were decreased by the ROS scavenger (N-acetylcysteine), and ROS levels were also markedly reduced by STAT5 inhibitor (SH-4-54). In addition, imatinib significantly inhibited the proliferation of K562 and K562/G cells, with half maximal inhibitory concentration values of 0.17±0.07 and 14.78±0.43 µM, respectively, and the levels of apoptosis were significantly different between K562 and K562/G cells following treatment with imatinib. The mRNA and protein levels of STAT5 and mouse double minute 2 homolog (MDM2) were upregulated, whereas those of Bax were downregulated in K562/G cells, as determined using western blot analysis. Additionally, although the two cell lines exhibited relatively low protein expression levels of p53, lower levels of p53 and TPp53BP1 transcripts were detected in the K562/G cells. Taken together, these findings suggest that the resistance of CML to the tyrosine kinase inhibitor, imatinib, may be associated with persistent STAT5-mediated ROS production, and the abnormality of the p53 pathway.
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Mesilato de Imatinib/administración & dosificación , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Factor de Transcripción STAT5/genética , Proteína p53 Supresora de Tumor/genética , Roturas del ADN de Doble Cadena/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes abl/genética , Histonas/genética , Humanos , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Proteínas Proto-Oncogénicas c-mdm2/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína 1 de Unión al Supresor Tumoral P53/genéticaRESUMEN
Toxoplasma gondii induces polarization of mouse macrophages, including both classically activated macrophages (M1) and alternatively activated macrophages (M2) in a genotype-related manner. Here we present a novel result that the Wh6 strain with type Chinese 1, which is predominantly prevalent in China, induces Arg1 expression in a STAT6-dependent manner in primary rat peritoneal macrophages as compared to the PRU stain with type II, which elicited a high expression of Arg1 in a C/EBPß-dependent manner. In addition, dexamethasone inhibited Arg1 expression in rat macrophages in both treatments. Our data suggest that Arg1 expression, which is abundant in polarized M2 cells, is associated with strain/genotype differences from different pathways.
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Arginasa/metabolismo , Regulación Enzimológica de la Expresión Génica/genética , Macrófagos Peritoneales/enzimología , Macrófagos Peritoneales/parasitología , Toxoplasma/fisiología , Animales , Arginasa/antagonistas & inhibidores , Arginasa/genética , Western Blotting , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , ADN Complementario/biosíntesis , Dexametasona/farmacología , Regulación hacia Abajo , Femenino , Genotipo , Glucocorticoides/farmacología , Ratones , Óxido Nítrico/metabolismo , ARN Mensajero/metabolismo , ARN Protozoario/genética , ARN Protozoario/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Transcripción STAT6/genética , Factor de Transcripción STAT6/metabolismo , Transducción de Señal , Toxoplasma/clasificación , Regulación hacia ArribaRESUMEN
Congenital toxoplasmosis may result in abortion, severe mental retardation and neurologic damage in the offspring. Placental damage is considered as the key event in this disease. Here we show that maternal infection with Toxoplasma gondii Wh3 isolate of genotype Chinese 1, which is predominantly prevalent in China, induced trophoblast apoptosis of pregnant mouse. PCR array analysis of 84 key genes in the biogenesis and functions of mouse mitochondrion revealed that ten genes were up-regulated at least 2-fold in the Wh3 infection group, compared with those in the control. The elevated levels of reactive oxygen species (ROS), malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG), as well as the decreased glutathione (GSH), were observed in the infected mice. The mRNA levels of NADPH oxidase 1 and glutathione peroxidase 6 (GPx6) were significantly increased. The production of excessive ROS was NADPH oxidase-dependent, which contributed to mitochondrial structural damage and mitochondrial dysfunction in placentas, followed by the cleavage of caspase-9 and caspase-3, and finally resulted in apoptosis of trophoblasts. All the above-mentioned phenomena were inhibited by pretreatment with the antioxidant of N-acetylcysteine (NAC). Taken together, we concluded that Wh3 infection during pregnancy may contribute to trophoblast apoptosis by oxidative stress-induced mitochondrial dysfunction and activation of the downstream signaling pathway.
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Complicaciones Parasitarias del Embarazo/patología , Toxoplasma/fisiología , Toxoplasmosis Animal/patología , Trofoblastos/patología , Animales , Apoptosis , Femenino , Genotipo , Masculino , Potenciales de la Membrana , Ratones , Ratones Endogámicos BALB C , Mitocondrias/patología , Mitocondrias/fisiología , Estrés Oxidativo , Placenta/metabolismo , Placenta/fisiopatología , Embarazo , Complicaciones Parasitarias del Embarazo/metabolismo , Complicaciones Parasitarias del Embarazo/parasitología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiología , Toxoplasma/clasificación , Toxoplasma/genética , Toxoplasmosis Animal/metabolismo , Toxoplasmosis Animal/parasitología , TranscriptomaRESUMEN
Toxoplasma gondii infection is the leading cause of fetal intrauterine growth retardation among the five kinds of pathogens termed as TORCH, including Toxoplasma, Rubella virus, Cytomegalo virus, herpes virus and others during pregnancy. Pathogens infect the fetus through the placenta. T. gondii infection may result in congenital toxoplasmosis, miscarriage, stillbirth, and preemie, and increase pregnancy complications. Adaptive immune response induced by T. gondii infection stimulates T cells and macrophages to produce high levels of cytokines. Physiologically, the microenvironment of pregnancy was Th2-dominant. Here we set up a pregnant Sprague-Dawley rat model, and reported the polarization of macrophages induced by genotype Chinese 1 strain (Wh6) of Toxoplasma, and its adverse impact on pregnancy. The results showed that Wh6 infection pre- or in-gestation both led to abnormal pregnancy outcomes. Peritoneal macrophages in pre-gestation infection were polarized toward classically activated macrophages (M1), while in-gestation infection drove macrophages to polarize toward M2 activation. The Th2-dominant immune response in pregnant rat somewhat inhibits the excessive bias of the macrophages toward M1, and partially, toward M2. Infection of pre- and in-gestation may alter the physiological immune microenvironment in pregnant rats, giving rise to abnormal pregnancy outcomes.
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Activación de Macrófagos/fisiología , Macrófagos Peritoneales/fisiología , Complicaciones Infecciosas del Embarazo/parasitología , Toxoplasma/patogenicidad , Toxoplasmosis Animal/parasitología , Animales , Arginasa/metabolismo , Western Blotting , Técnicas de Cultivo de Célula , Citocinas/metabolismo , ADN Protozoario/genética , Femenino , Masculino , Óxido Nítrico/metabolismo , Embarazo , Complicaciones Infecciosas del Embarazo/inmunología , Complicaciones Infecciosas del Embarazo/patología , Resultado del Embarazo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Células TH1/parasitología , Toxoplasmosis Animal/inmunología , Toxoplasmosis Animal/patologíaRESUMEN
In order to accomplish their life cycles, intracellular pathogens, including the apicomplexan Toxoplasma gondii, subvert the innate apoptotic response of infected host cells. However, the precise mechanisms of parasite interference with the apoptotic pathway remain unclear. MicroRNAs (miRNAs) regulate gene expression at the posttranscriptional level. Using T. gondii strain TgCtwh3, which was isolated from felids and possesses the predominant genotype China 1 (ToxoDB(#)9) in China, we analyzed the miRNA expression profile of human macrophages challenged with TgCtwh3. The results showed that miR-17-92 miRNA expression was significantly increased and Bim was decreased in TgCtwh3-infected cells. Database analysis of miR-17-92 miRNAs revealed the potential binding sites in the 3'UTR of Bim, one of the crucial effectors of pro-apoptosis. Furthermore, we demonstrated that the promoter of the miR-17-92 gene cluster which encodes miRNAs was transactivated through the promoter binding of the STAT3 following TgCtwh3 infection. Taken together, we describe a novel STAT3-miR-17-92-Bim pathway, thus providing a mechanistic explanation for inhibition of apoptosis of host cells following Toxoplasma infection.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Macrófagos/parasitología , Proteínas de la Membrana/metabolismo , MicroARNs/genética , Proteínas Proto-Oncogénicas/metabolismo , Factor de Transcripción STAT3/metabolismo , Toxoplasma/fisiología , Adulto , Secuencia de Aminoácidos , Animales , Proteína 11 Similar a Bcl2 , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Interacciones Huésped-Parásitos , Humanos , Macrófagos/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , ARN Largo no Codificante , Adulto JovenRESUMEN
The obligate intracellular parasite Toxoplasma gondii secretes effector molecules into the host cell to modulate host immunity. Previous studies have shown that T. gondii could interfere with host NF-κB signaling to promote their survival, but the effectors of type I strains remain unclear. The polymorphic rhoptry protein ROP18 is a key serine/threonine kinase that phosphorylates host proteins to modulate acute virulence. Our data demonstrated that the N-terminal portion of ROP18 is associated with the dimerization domain of p65. ROP18 phosphorylates p65 at Ser-468 and targets this protein to the ubiquitin-dependent degradation pathway. The kinase activity of ROP18 is required for p65 degradation and suppresses NF-κB activation. Consistently, compared with wild-type ROP18 strain, ROP18 kinase-deficient type I parasites displayed a severe inability to inhibit NF-κB, culminating in the enhanced production of IL-6, IL-12, and TNF-α in infected macrophages. In addition, studies have shown that transgenic parasites carrying kinase-deficient ROP18 induce M1-biased activation. These results demonstrate for the first time that the virulence factor ROP18 in T. gondii type I strains is responsible for inhibiting the host NF-κB pathway and for suppressing proinflammatory cytokine expression, thus providing a survival advantage to the infectious agent.
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FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Protozoarias/metabolismo , Transducción de Señal , Toxoplasma/metabolismo , Factor de Transcripción ReIA/metabolismo , Animales , Western Blotting , Línea Celular , Femenino , Células HEK293 , Interacciones Huésped-Parásitos , Humanos , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Mutación , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteolisis , Proteínas Protozoarias/genética , Serina/metabolismo , Toxoplasma/genética , Toxoplasma/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
T cells have been demonstrated to exert central roles in the development of type 2 DN (T2DN). To explore whether Th1/Th2/Th17/Treg paradigm plays an important role in the development of T2DN, we investigated the proportions of Th1/Th2/Th17/Treg cells and serum levels of relevant cytokines in T2DM patients with various degrees of nephropathy and controls. Moreover, we analyzed the relationships between the Th1/Th2/Th17/Treg paradigm or relevant cytokines with urine albumin:creatinine ratio (UACR). Our study demonstrated that the Th1/Th2/Th17/Treg paradigm skewed to Th1 and Th17 in T2DN patients. UACR was positively related to the proportions of Th1 and Th17 cells, as well as the ratio of Th17:Treg cells, and negatively related to the proportions of Treg cells. Furthermore, serum levels of IL-6, IL-17, IFN-γ, TNF-α, IL-2 and IL-10 were increased in T2DN patients, and positively related to UACR. These data indicate that the alteration of Th1/Th2/Th17/Treg paradigm exists in T2DN patients, which may contribute to the enhanced immune activation and inflammation, and subsequent development and progression of T2DN. These findings may provide one new approach to the underlying mechanisms of the development of T2DN.
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Diabetes Mellitus Tipo 2/inmunología , Linfocitos T Reguladores/inmunología , Células TH1/inmunología , Células Th17/inmunología , Células Th2/inmunología , Anciano , Albuminuria , Estudios de Casos y Controles , Creatinina/orina , Citocinas/sangre , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Nefropatías Diabéticas/etiología , Femenino , Hemoglobina Glucada/metabolismo , Humanos , Inmunofenotipificación , Masculino , Persona de Mediana Edad , Factores de Riesgo , Linfocitos T Reguladores/metabolismo , Células TH1/metabolismo , Células Th17/metabolismo , Células Th2/metabolismoRESUMEN
As one of food-borne parasitic diseases, toxoplasmosis entails the risk of developing reactivation in immunocompromised patients. The synthetic dipeptide pidotimod is a potent immunostimulating agent that improves the immunodefenses in immunodepression. To investigate the efficacy of pidotimod as a preventive treatment, we used a murine model of reactivated toxoplasmosis with cyclophosphamide (CY)-induced immunosuppression. Pidotimod administration significantly restored the body weight and spleen organ index, increased survival time (from 70 to 90%), and decreased the parasitemia (from 80 to 35%) of CY-induced mice with reactivated toxoplasmosis. Cytokine profiles and CD4(+) T cells subpopulation analyses by Cytometric Bead Array and flow cytometry demonstrated that pidotimod treatment resulted in a significant upregulation of pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-2) and Th1 cells (from 3.73 ± 0.39 to 5.88 ± 0.46%) after CY induction in infected mice. Additionally, histological findings and parasite DNA quantification revealed that mice administered with pidotimod had a remarkable reduction of parasite burden (two-log) and amelioration of histopathology in the brains. The in vitro studies showed that pidotimod significantly restored concanavalin A-induced splenocyte proliferation and pro-inflammatory cytokines in the supernatants of splenocyte culture. It could be concluded that the administration of pidotimod in immunocompromised mice significantly increases the Th1-biased immune response, prolongs survival time, and ameliorates the load of parasites in the blood. This is the first report of the preventive effect of pidotimod on reactivated toxoplasmosis.
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Factores Inmunológicos/uso terapéutico , Ácido Pirrolidona Carboxílico/análogos & derivados , Tiazolidinas/uso terapéutico , Toxoplasmosis Animal/prevención & control , Animales , Ciclofosfamida/farmacología , Citocinas/genética , Citocinas/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Inmunosupresores/farmacología , Ratones , Ratones Endogámicos BALB C , Parasitemia , Ácido Pirrolidona Carboxílico/uso terapéutico , Organismos Libres de Patógenos Específicos , Bazo/citología , Bazo/efectos de los fármacos , Toxoplasmosis Animal/inmunologíaRESUMEN
Toxoplasma gondii is an apicomplexan parasite capable of transplacental transmission to cause spontaneous abortion or significant disease in the surviving neonate. Different from the dominant genotypes of T. gondii strains in European and North American which belong to three distinct clonal lineages, type I, type II, and type III, isolates from China possess the predominant genotype of China 1(ToxoDB#9) with a different virulence. The genotype-associated pathogenesis has been investigated previously. Based on two isolates of T. gondii from Chinese wild cats, a murine model of pregnancy and one transwell system in vitro, here we reported differentially polarized activation of macrophages induced by genotype China 1 strains, TgCtwh3 and TgCtwh6 with different virulence to mice, and its impact on trophoblast apoptosis. The results showed that macrophages were alternatively activated when infected with virulent TgCtwh3 while classically activated when infected with low virulent (cyst-forming) TgCtwh6 both in vitro and in vivo. By the analysis of flow cytometry, the percentage of the Th1 cells in two infection groups decreased significantly, and the Th2 cells from spleen escalated only in the virulent TgCtwh3 group. Interestingly, the high parasite burden was noted in the placenta of TgCtwh3-infected group whereas the inflammatory cells infiltration predominates in the TgCtwh6-infected group. In vivo trophoblast apoptosis in TgCtwh3 group was found to be more obvious when compared with TgCtwh6 although it was present in both. The present observations indicate that polarization of macrophages and modulation of Th subsets induced by the isolates with identical genotype but different virulence could contribute to trophoblast apoptosis through different mechanisms, suggesting a virulence-associated pathogenesis of T. gondii in abnormal pregnant outcome.
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Macrófagos/fisiología , Toxoplasma/patogenicidad , Toxoplasmosis Animal/parasitología , Trofoblastos/fisiología , Animales , Apoptosis , Enfermedades de los Gatos/parasitología , Gatos , China/epidemiología , Técnicas de Cocultivo , Citocinas/genética , Citocinas/metabolismo , Femenino , Regulación de la Expresión Génica , Activación de Macrófagos , Ratones , Ratones Endogámicos ICR , Placenta , Embarazo , Proteínas Protozoarias , Toxoplasma/genética , Toxoplasmosis Animal/epidemiología , VirulenciaRESUMEN
OBJECTIVE: CD4(+)CD25(+) regulatory T (Treg) cells and Th17 cells play important roles in peripheral immunity. Oxidized low-density lipoprotein (ox-LDL) is an instrumental factor in atherogenesis. However, the changes of Th17/Treg cells in patients with acute cerebral Infarction (ACI) and impact on Th17/Treg by ox-LDL are not clear. Here, we examined the balance of Th17/Treg in ACI patients and the effect of ox-LDL on this balance. MATERIALS AND METHODS: The frequencies of Th17 and Treg cells, key transcription factors and relevant cytokines were examined in patients with ACI, Transient ischemic attack (TIA) and controls. The correlations of cytokines, inflammatory biomarkers and ox-LDL in serum to Th17/Treg frequency, and the effects of ox-LDL on Th17/Treg cells in vitro were analyzed. RESULTS: ACI patients have shown a significant increase of Th17 frequency, RORγt expression and Th17 related cytokines (IL-17 and IL-6 ) levels, and a clear decline of Treg frequency, Foxp3 expression, suppressive function and regulatory cytokines (IL-10 and TGF-ß1) levels. Furthermore, TIA patients also have notable variation as compared to control group. Serum ox-LDL and inflammatory biomarkers were positively correlated with the frequency of Th17 cells and negatively correlated with the frequency of Treg cells. Treg and Th17 cells from ACI patients were significantly susceptible to ox-LDL-mediated alterations in vitro. CONCLUSIONS: Th17/Treg cells were imbalanced in ACI patients, and ox-LDL may contribute to this imbalance and lead to the occurrence of ACI suggesting their pathogenetic role in ACI.
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Infarto Cerebral/inmunología , Arteriosclerosis Intracraneal/inmunología , Ataque Isquémico Transitorio/inmunología , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Adulto , Anciano , Biomarcadores/sangre , Recuento de Linfocito CD4 , Estudios de Casos y Controles , Células Cultivadas , Infarto Cerebral/sangre , Infarto Cerebral/patología , Citocinas/sangre , Femenino , Factores de Transcripción Forkhead/sangre , Humanos , Mediadores de Inflamación/sangre , Arteriosclerosis Intracraneal/sangre , Arteriosclerosis Intracraneal/patología , Ataque Isquémico Transitorio/sangre , Ataque Isquémico Transitorio/patología , Lipoproteínas LDL/sangre , Masculino , Persona de Mediana Edad , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/sangre , PronósticoRESUMEN
BACKGROUND: Recent population structure studies of T. gondii revealed that a few major clonal lineages predominated in different geographical regions. T. gondii in South America is genetically and biologically divergent, whereas this parasite is remarkably clonal in North America and Europe with a few major lineages including Types I, II and III. Information on genotypes and mouse virulence of T. gondii isolates from China is scarce and insufficient to investigate its population structure, evolution, and transmission. METHODOLOGY/PRINCIPAL FINDINGS: Genotyping of 23 T. gondii isolates from different hosts using 10 markers for PCR-restriction fragment length polymorphism analyses (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) revealed five genotypes; among them three genotypes were atypical and two were archetypal. Fifteen strains belong to the Chinese 1 lineage, which has been previously reported as a widespread lineage from swine, cats, and humans in China. Two human isolates fall into the type I and II lineages and the remaining isolates belong to two new atypical genotypes (ToxoDB#204 and #205) which has never been reported in China. Our results show that these genotypes of T. gondii isolates are intermediately or highly virulent in mice except for the strain TgCtwh6, which maintained parasitemia in mice for 35 days post infection although it possesses the uniform genotype of Chinese 1. Additionally, phylogenetic network analyses of all isolates of genotype Chinese 1 are identical, and there is no variation based on the sequence data generated for four introns (EF1, HP2, UPRT1 and UPRT7) and two dense granule proteins (GRA6 and GRA7). CONCLUSION/SIGNIFICANCE: A limited genetic diversity was found and genotype Chinese 1 (ToxoDB#9) is dominantly circulating in mainland China. The results will provide a useful profile for deep insight to the population structure, epidemiology and biological characteristics of T. gondii in China.
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ADN Protozoario/genética , Ratones/parasitología , Toxoplasma/genética , Toxoplasma/patogenicidad , Toxoplasmosis Animal/parasitología , Adulto , Anciano , Animales , Gatos , China/epidemiología , ADN Protozoario/clasificación , Marcadores Genéticos , Variación Genética , Genotipo , Técnicas de Genotipaje , Humanos , Persona de Mediana Edad , Filogenia , Filogeografía , Polimorfismo de Longitud del Fragmento de Restricción , Porcinos , Toxoplasma/clasificación , Toxoplasma/aislamiento & purificación , Toxoplasmosis Ocular/epidemiología , Toxoplasmosis Ocular/parasitología , VirulenciaRESUMEN
BACKGROUND: Different from three clonal lineages of Toxoplasma gondii in North America and Europe, the genotype China 1 is predominantly prevalent in China. However, there are different virulent isolates within China 1, such as virulent TgCtwh3 and avirulent TgCtwh6, and little is known about differences in macrophage activation between them. The objective of this study focused on cytokine production, phenotype and markers of activated macrophages, and correlated signaling pathway induced by the two isolates. METHODS: Adherent peritoneal macrophages (termed Wh3-Mφ and Wh6-Mφ, respectively) harvested from infected mice were cultured for detection of Nitric Oxide and arginase activity, and activated markers on Wh3-Mφ/Wh6-Mφ were determined by flow cytometry. In in vitro experiments, the levels of IL-12p40 and TNF-α were measured using ELISA kits, and mRNA expressions of IL-12p40, TNF-α, iNOS, Arg-1 and Ym1 were assayed by real-time PCR. To confirm the activation state of NF-kB p65 in infected cells stained by IF, protein levels of iNOS, Arg-1, Ym1, nuclear NF-κB p65, and phosphorylation of STAT6/STAT3/IκBα were evaluated by Western Blotting. A one-way ANOVA test was used to compare differences among multiple groups. RESULTS: The result revealed that contrary to the virulent TgCtwh3, the less virulent TgCtwh6 isolate induced a significant increase in IL-12p40 and TNF-α. Although both isolates down-regulated CD80, CD86 and MHCII molecule expression on macrophages, TgCtwh3 promoted up-regulation of PD-L2 and CD206. Wh6-Mφ generated a high level of NO whereas Wh3-Mφ up-regulated Ym1 and arginase expression at transcriptional and protein levels. In terms of signaling pathway, TgCtwh3 induced phospho-STAT6, conversely, TgCtWh6 led to NF-κB p65 activation. CONCLUSIONS: The virulent TgCtwh3 isolate induced macrophages to polarize toward alternatively activated cells with STAT6 phosphorylation, whereas the less virulent TgCtwh6 elicited the development of classically activated macrophages with nuclear translocation of NF-κB p65. This discrepancy suggests that it is necessary to thoroughly analyze the genotype of TgCtwh3 and TgCtwh6, and to further study other effector molecules that contribute to the macrophage polarization in T. gondii.
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Activación de Macrófagos , Macrófagos Peritoneales/inmunología , Toxoplasma/inmunología , Toxoplasma/patogenicidad , Toxoplasmosis Animal/inmunología , Animales , Western Blotting , Células Cultivadas , China , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Perfilación de la Expresión Génica , Inmunofenotipificación , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Toxoplasma/genética , Toxoplasma/aislamiento & purificación , VirulenciaRESUMEN
Pork is known as one of the most important sources of Toxoplasma gondii infection in China. In the present study, 416 fresh pork samples were collected from different locations of Anhui province, Eastern China. Tissue fluid ELISA was conducted to detect the antibodies to T. gondii. Real-time PCR and bioassay were performed to identify the presence of T. gondii DNA and viable parasites, respectively. Seventy-five out of 416 samples (18.03%) demonstrated real-time PCR positive reaction and 42 out of 416 samples (10.1%) showed tissue fluid ELISA positive reaction. One isolate (Tgpkfx171) was obtained through bioassay in mice from 14 samples that demonstrated both PCR and ELISA positive reaction. The isolate and seven positive DNA samples were genotyped using 9 PCR-RFLP markers including SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico. Among these, only the isolate and two positive DNA samples were genotyped with complete data for all loci, belonging to ToxoDB#9 (Chinese 1) and ToxoDB#213, respectively. This is the first report of the prevalence and genetic typing of T. gondii from pork in retail meat stores in China. The present results provide an accurate picture of the risk of exposure to T. gondii in retail pork in China.