RESUMO
BACKGROUND: Despite recent advances in therapeutic regimens, the prognosis of acute myeloid leukemia (AML) remains poor. Following our previous finding that interleukin-33 (IL-33) promotes cell survival along with activated NF-κB in AML, we further investigated the role of NF-κB during leukemia development. METHODS: Flow cytometry was performed to value the apoptosis and proliferation. qRT-PCR and western blot were performed to detect the expression of IL-6, active caspase 3, BIRC2, Bcl-2, and Bax, as well as activated NF-κB p65 and AKT. Finally, xenograft mouse models and AML patient samples were used to verify the findings observed in AML cell lines. RESULTS: IL-33-mediated NF-κB activation in AML cell lines contributes to a reduction in apoptosis, an increase in proliferation rate as well as a decrease in drug sensitivity, which were reversed by NF-κB inhibitor, Bay-117085. Moreover, IL-33 decreased the expression of active caspase-3 while increasing the levels of BIRC2, Bcl-2, and Bax, and these effects were blocked by Bay-117085. Additionally, NF-κB activation induced by IL-33 increases the production of IL-6 and autocrine activation of AKT. Co-culture of bone marrow stroma with AML cells resulted in increased IL-33 expression by leukemia cells, along with decreased apoptosis level and reduced drug sensitivity. Finally, we confirmed the in vivo pro-tumor effect mediated by IL-33/ NF-κB axis using a xenograft model of AML. CONCLUSION: Our data indicate that IL-33/IL1RL1-dependent signaling contributes to AML cell activation of NF-κB, which in turn causes autocrine IL-6-induced activation of pAKT, supporting IL-33/NF-κB/pAKT as a potential target for AML therapy.
Assuntos
Apoptose , Resistencia a Medicamentos Antineoplásicos , Interleucina-33 , Leucemia Mieloide Aguda , NF-kappa B , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/tratamento farmacológico , Apoptose/efeitos dos fármacos , NF-kappa B/metabolismo , Animais , Interleucina-33/metabolismo , Camundongos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Transdução de Sinais/efeitos dos fármacos , Masculino , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
BACKGROUND: Talaromyces marneffei (T. Marneffei) infection is considered as an indicator of immunosuppression in immunocompromised individuals, leading to multiple organ damage. Our study aimed to evaluate both the clinical characteristics and immunological features of pediatric patients infected with T. marneffei from our institute, providing novel insights into diagnosis and treatment for this life-threatening disease. METHOD: Thirteen pediatric patients with T. marneffei infection were enrolled in Guangzhou Women and Children's Medical Center during 2012 to 2020. Clinical data and laboratory findings were collected and further analyzed. Pearson correlation coefficient was calculated to determine the relationship between serum immunoglobulins (Igs) levels and white blood cell count, or the absolute lymphocyte count. RESULTS: Patients were diagnosed as having T. Marneffei infection mainly based on the results of fungal culture and Gram stain of specimens. The most common presentations were fever (69%), pneumonia (38%) and immunodeficiency (38%). The total levels of Igs (IgE, IgA, and IgM) were positively correlated with both white blood cell count and absolute lymphocyte count. CONCLUSION: Serum Ig expression Pattern in patients diagnosed with T. marneffei infection might serve as an effective prognostic marker which would help with the development of early interventions for children with this fatal disease.
Assuntos
Micoses , Talaromyces , Humanos , Criança , Feminino , Estudos Retrospectivos , Micoses/microbiologia , Hospedeiro Imunocomprometido , Terapia de ImunossupressãoRESUMO
ß-Thalassemia major (ß-TM) is an inherited disorder of hemoglobin (Hb) production, which can cause severe anemia. A compromised immune system has been observed in patients with ß-TM, whereas cytokines have a major role in immune modulation. Interleukin-4 (IL-4), IL-8, IL-13 and transforming growth factor-ß (TGF-ß) are critical in initiating pro-inflammatory responses, and the serum levels of those cytokines may be involved in the pathophysiology of ß-thalassemia (ß-thal). To assess this hypothesis, we studied 23 pediatric patients with ß-TM by measuring serum levels of IL-4, IL-8, IL-13 and TGF-ß, as well as evaluating infection frequency per year, total number of transfusions and serum ferritin (SF) levels, together with age-matched healthy controls. We found that patients with ß-thal had higher IL-8, IL-13 and TGF-ß concentrations than normal controls, whereas markedly decreased serum IL-4 level was documented in patients with ß-TM. Serum IL-4 level of ß-thal patients showed a negative significant correlation with infection frequency, total number of transfusions and SF levels. On the contrary, serum levels of IL-8, IL-13 and TGF-ß exerted a positive relationship with those clinical parameters. Taken together, our study implies that dysregulated cytokine profile might contribute to iron overloads and impair immune cell functions, thus serving as useful biomarkers for diagnosis and evaluation of ß-TM in the future. Our study sheds new light on the pathogenesis of ß-TM.
Assuntos
Talassemia beta , Criança , Humanos , Interleucina-13 , Interleucina-4 , Interleucina-8 , Citocinas , Fator de Crescimento Transformador betaRESUMO
Toxins, encoding by virulence factors, are significant cause of food-borne illnesses and death in the worldwide. Loop-mediated isothermal amplification (LAMP) is one of the widely used methodologies because of the high sensitivity, specificity and rapidity. Nowadays, LAMP has been regarded as an innovative gene amplification technology and emerged as an alternative to PCR-based methodologies in identification of the pathogenic virulent and toxic genetics. The high sensitivity of LAMP enables detection of the pathogens in sample materials even without time consuming and sample preparation. Therefore, we review the typical characteristics of LAMP assay, recent advance in detection of virulence factors and the application of LAMP assay on detection of four commonly virulence factors. As concluded, with the advantages of rapidity, simplicity, sensitivity, specificity and robustness, LAMP is capable of identification the virulence factors. Moreover, the main purpose of this review is to provide theory support for the application of LAMP assay on the virulence factors identification.
Assuntos
Análise de Alimentos/métodos , Contaminação de Alimentos , Técnicas de Amplificação de Ácido Nucleico/métodos , Toxinas Biológicas/análise , Fatores de Virulência/análise , Animais , Humanos , Toxinas Biológicas/genética , Fatores de Virulência/genéticaRESUMO
Regarded as a common genetic element responsible for horizontal gene transfer and wide spread of antimicrobial resistance among a large variety of bacteria, integrons are commonly distributed and considered as a determinant in the acquisition and evolution of virulence and antibiotic resistance. To date, the surveillances of integrons have been widely conducted in clinic, community even husbandry. For exact and accurate integron screening, as well as resistant cassettes, reliable monitoring methods is need. Current methods applied on integron screening are mainly conducted by the screening of integrases, followed by the detection of various gene cassettes inserted into integrons. PCR and PCR-related methods (such as RFLP) are mainly employed under such circumstances. Matured LAMP and Sequencing technology have lowered cost and dramatically increased throughput in integron screening and possessed the advantages in similarity analysis of mutated resistant cassettes. This review focused on the classification and characterization of integrons, antimicrobial resistance of integron and genotyping methods for integrons. In methodology, PCR, LAMP and Sequencing technology were mainly introduced for the screening of various classes' integrons and the detection of resistant gene cassettes. Staphylococcus, Pseudomonas and Enterococcus were selected as typical integron-positive clinical and environmental pathogens screened with three methods mentioned above. With the surveillance of the occurrence of integron and resistance gene cassettes conducted in South China, the review also summarized the occurrence, pathogenicity and virulence mediated by integrons.
Assuntos
Enterococcus/genética , Técnicas de Genotipagem/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Integrons , Sequências Repetitivas Dispersas , Pseudomonas/genética , Staphylococcus/genética , Infecções Bacterianas/microbiologia , China , Farmacorresistência Bacteriana , Enterococcus/isolamento & purificação , Transferência Genética Horizontal , Humanos , Pseudomonas/isolamento & purificação , Staphylococcus/isolamento & purificação , VirulênciaRESUMO
BACKGROUND: Recognized as a resistance mechanism responsible for the emergence and prevalence of antimicrobial resistance, integron is widely distributed and spread among clinical microorganisms and play a key role in the dissemination of such antimicrobial resistance, which may eventually contribute to the unleashing of "Super Bugs" In this study, detection assays based on loop-mediated isothermal amplification (LAMP) methodologies targeting on class 1 to class 3 integrase genes was developed and evaluated. METHODS: LAMP methodology was employed to develop novel detection assays on class 1, 2 and 3 integrons. Firstly, this protocol was specifically designed to detect such integrons by targeting integrase genes intI1, intI2 and intI3. Development, evaluation and optimization of such LAMP assays was studied, including the reaction temperature, volumn, time, sensitivity and specificity of both primers and targets. A total of 1082 strains, including 397 integron positive and 685 integron negative microorganisms, were included for the application verification of the established LAMP assays. RESULTS: The indispensability of each primer was confirmed, and the optimal amplification was obtained under 63 °C for 45 min, with 25 µl reaction found to be the most cost-efficient volume. As application was concerned, all of the 397 integron-positive isolates yielded positive amplicons and other 685 integron-negative bacteria were negative for the integron-LAMP assays, revealing totaling 100% detection rate and specificity. CONCLUSIONS: The established integron-LAMP assays was demonstrated to be a valid and rapid detection method for integrons screening, which may aid in both the laboratory and clinical integron screening for microorganisms.
Assuntos
Farmacorresistência Bacteriana/genética , Integrons/genética , Sequências Repetitivas Dispersas/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Antibacterianos , Bactérias/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Temperatura Alta , Integrases/classificação , Integrases/genética , Sensibilidade e Especificidade , Virulência/genéticaRESUMO
In this work, loop-mediated isothermal amplification based detection assay using bacterial culture and bacterial colony for various common pathogens direct detection had been established, evaluated and further applied. A total of five species of common pathogens and nine detection targets (tlh, tdh and trh for V. Parahaemolyticus, rfbE, stx1 and stx2 for E. coli, oprI for P. aeruginosa, invA for Salmonella and hylA for L. monocytogenes) were performed on bacterial culture and bacterial colony LAMP. To evaluate and optimize this assay, a total of 116 standard strains were included. Then, for each detected targets, 20 random selected strains were applied. Results were determined through both visual observation of the changed color by naked eye and electrophoresis, which increased the accuracy of survey. The minimum adding quantity of each primer had been confirmed, and the optimal amplification was obtained under 65 °C for 45 min with 25 µl reaction volume. The detection limit of bacterial culture LAMP and PCR assay were determined to be 102 and 104 or 105 CFU/reaction, respectively. No false positive amplification was observed when subjecting the bacterial -LAMP assay to 116 reference strains. This was the first report of colony-LAMP and culture-LAMP assay, which had been demonstrated to be a fast, reliable, cost-effective and simple method on detection of various common pathogens.
Assuntos
Técnicas Bacteriológicas , Técnicas de Amplificação de Ácido Nucleico/métodos , Carga Bacteriana/métodos , Genes Bacterianos , Genoma Bacteriano , Humanos , Reação em Cadeia da Polimerase , Reprodutibilidade dos TestesRESUMO
Gram-positive microorganisms are one of leading pathogenic microorganisms in public health, including several typical "Super Bugs" as methicillin-resistant Staphylococcus aureus, Klebsiella pneumoniae carbapenemase and vancomycin-resistant enterococci, which caused a increasement of infections, clinical failures and expenses. Regarded as a common genetic element responsible for horizontal gene transfer, integrons are widely distributed in various pathogens considered as a determinant in the acquisition and evolution of antibiotic resistance. Current investigations mainly focus on the distribution of integrons in Gram-negative microorganisms, while the role of integron in antibiotic resistance among Gram-positive microorganisms remains unclear and need investigation. To date, the surveillances of integrons in Gram-positive microorganism have been widely conducted in clinic, community even husbandry. China remains one of the worst country in antibiotics abuse worldwide and considered as a potential area for the prevalence of antimicrobial microorganisms and the occurrence of various 'Super Bugs'. Recently, the surveillance of the occurrence of integron and resistance gene cassettes was conducted in South China during the first 10 years of the 21st century. Referred to the surveillance in South China and other investigation in Asian countries, this review aims to summarize the occurrence, pathogenicity and virulence mediated by integrons in typical Gram-positive microorganisms (Staphylococcus, Enterococcus, Corynebacterium and Streptococcus) and the role of integrons in antibiotic resistance.
Assuntos
Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/patogenicidade , Infecções por Bactérias Gram-Positivas/microbiologia , Integrons , Animais , Transferência Genética Horizontal , Bactérias Gram-Positivas/metabolismo , Humanos , VirulênciaRESUMO
As a self-protection mechanism, the viable but non-culturable (VBNC) state provides the ability against conventional detection methods among various foodborne pathogens. The ability of forming colonies is lost while metabolism is still maintaining in VBNC state cells. Recently, ethidium monoazide (EMA) and propidium monoazide (PMA) have been widely applied on the detection of foodborne pathogens in VBNC state. Combined with loop-mediated isothermal amplification (LAMP), the PMA/EMA-LAMP showed a significant priority in high sensitivity, specificity and rapidity over conventional PCR based assays. Particularly, PMA/EMA-LAMP has been proved as an effective method in the detection of Escherichia coli, Vibrio parahaemolyticus and Staphylococcus in VBNC state. Based on the current investigations, the VBNC mechanism and current detection method for VBNC-state foodborne pathogens were introduced and discussed in this review.
Assuntos
Bactérias/isolamento & purificação , Técnicas Bacteriológicas/métodos , Microbiologia de Alimentos/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Azidas , Bactérias/genética , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Viabilidade Microbiana , Propídio/análogos & derivados , Sensibilidade e Especificidade , Staphylococcus/genética , Staphylococcus/isolamento & purificação , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/isolamento & purificaçãoRESUMO
In exposure to outer pressure, microorganisms are capable of entry into the Viable But Non-Culturable (VBNC) state, and thus survive under various elimination processing. The survival microorganisms may yield negative results on culturing, and cause false negative for this golden standard methodology. In this study, a novel PMA-LAMP assay on the detection of Enterohemorrhage E. coli and shiga toxins has been developed and evaluated, with further application on a number of food borne E. coli strains. LAMP primers were designed on the target of rfbe for Enterohemorrhage E. coli and stx1with stx2 for shiga toxins. Via specific penetration through the damaged cell membrane of dead cells and intercalating into DNA, PMA could prevent DNA amplification of dead bacteria from LAMP, which enabled the differentiation of bacteria between VBNC state and dead state. The established PMA-LAMP showed significant advantage in rapidity, sensitivity and specificity, compared with regular PCR assay. The applicability had also been verified, demonstrating the PMA-LAMP was capable of detection on Enterohemorrhage E. coli and shiga toxins.
Assuntos
Técnicas Bacteriológicas/métodos , Escherichia coli/isolamento & purificação , Microbiologia de Alimentos/métodos , Toxinas Shiga/isolamento & purificação , Carboidratos Epimerases/genética , Primers do DNA , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli O157/genética , Escherichia coli O157/isolamento & purificação , Inocuidade dos Alimentos/métodos , Doenças Transmitidas por Alimentos/microbiologia , Testes de Sensibilidade Microbiana , Viabilidade Microbiana , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase , Toxina Shiga I/genética , Toxina Shiga II/genética , Toxinas Shiga/genética , Transaminases/genéticaRESUMO
In the Viable but Non-Culturable (VBNC) state, microorganisms may survive under severe external environment. In this study, the specificity and sensitivity of PMA-LAMP assay on the detection of Vibrio Parahemolyticus (V. parahemolyticus) has been developed and evaluated, with further application on a number of food-borne V. parahemolyticus strains. Six primers were designed for recognizing 8 distinct targeting on tlh, tdh and trh gene. Through specific penetration through the damaged cell membrane of dead cells and intercalating into DNA, PMA could prevent DNA amplification of dead bacteria from LAMP, which enabled the differentiation of bacteria between VBNC state and dead state. The established PMA-LAMP showed significant advantage in rapidity, sensitivity and specificity, compared with regular PCR assay. The applicability had also been verified, demonstrating the PMA-LAMP was capable of detection on V. parahaemolyticus.
Assuntos
Toxinas Bacterianas/metabolismo , Gastroenterite/microbiologia , Proteínas Hemolisinas/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , Vibrioses/microbiologia , Vibrio parahaemolyticus/isolamento & purificação , Vibrio parahaemolyticus/patogenicidade , Toxinas Bacterianas/genética , Proteínas Hemolisinas/genética , Humanos , Viabilidade Microbiana , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/crescimento & desenvolvimento , VirulênciaRESUMO
Rho-associated coiled-coil kinase 2 (ROCK2) is classified as a member of the serine/threonine protein kinase family and has been identified as a key driver of the development of various forms of cancer. The cause of ROCK2's impact on acute myeloid leukemia (AML) is still unknown. We found that ROCK2 expression was higher in AML patients, leading to lower complete response rates and worse overall survival. Additionally, ROCK2 expression was elevated in the doxorubicin-resistant leukemia cell line HL-60/ADM when compared to their individual parent cells. Moreover, the suppression or inhibition of ROCK2 leads to enhanced drug sensitivity in both AML cell lines and primary AML specimens, along with a notable decrease in downstream signaling pathways. Furthermore, the suppression of ROCK2 caused disruption of cellular energy production pathways by directly affecting the functionality of proteins within the mitochondrial electron transport chain. Finally, we discovered that TRIM26, a specific E3 ligase, is capable of ubiquitylating ROCK2, and the upregulation of TRIM26 within HL-60/ADM cells resulted in heightened sensitivity to the drug and reduced resistance. Thus, our study presents a new strategy for overcoming drug resistance in AML through targeting ROCK2/AKT/MAPK signaling pathway.
RESUMO
This study aimed to in the management of Kasabach-Merritt phenomenon (KMP), a severe thrombocytopenic coagulopathy that occurs in the presence of an enlarging vascular tumor. Here, we retrospectively evaluated 12 patients with KMP in Guangzhou Women and Children's Medical Center, Guangzhou Medical University, from 2017 to 2021. 12 patients, including 7 females and 5 males, were identified. Tumors were located in the leg (n = 4), neck (n = 1), face (n = 3), chest wall (n = 1), back (n = 2), and retroperitoneum (n = 1). A plaque-like lesion with ecchymosis was the most common cutaneous manifestation. All the patients underwent embolization therapy. Nine patients received steroid treatment and 7 patients were administered with sirolimus. The mean duration of treatment was 1.6 months. All the patients reported in this study were alive when discharged. Embolization combined with steroid and sirolimus appears effective in patients with KMP, as well as in those who experienced disease recurrence. However, a long-term follow-up of the children cured of KMP will be necessary to monitor its recurrence and improve the outcome.
Assuntos
Hemangioendotelioma , Síndrome de Kasabach-Merritt , Sarcoma de Kaposi , Criança , Terapia Combinada , Feminino , Humanos , Lactente , Síndrome de Kasabach-Merritt/tratamento farmacológico , Síndrome de Kasabach-Merritt/patologia , Masculino , Recidiva Local de Neoplasia , Estudos Retrospectivos , SirolimoRESUMO
Through the advancements in recent decades, childhood acute lymphoblastic leukemia (ALL) is gradually becoming a highly curable disease. However, the truth is there remaining relapse in â¼15% of ALL cases with dismal outcomes. RAS mutations, in particular NRAS mutations, were predominant mutations affecting relapse susceptibility. KRAS mutations targeting has been successfully exploited, while NRAS mutation targeting remains to be explored due to its complicated and compensatory mechanisms. Using targeted sequencing, we profiled RAS mutations in 333 primary and 18 relapsed ALL patients and examined their impact on ALL leukemogenesis, therapeutic potential, and treatment outcome. Cumulative analysis showed that RAS mutations were associated with a higher relapse incidence in children with ALL. In vitro cellular assays revealed that about one-third of the NRAS mutations significantly transformed Ba/F3 cells as measured by IL3-independent growth. Meanwhile, we applied a high-throughput drug screening method to characterize variable mutation-related candidate targeted agents and uncovered that leukemogenic-NRAS mutations might respond to MEK, autophagy, Akt, EGFR signaling, Polo-like Kinase, Src signaling, and TGF-ß receptor inhibition depending on the mutation profile.
RESUMO
Rhabdomyosarcoma (RMS) originates from a differentiation block in muscle progenitors. Leptomeningeal metastasis is a rare but devastating complication of RMS which can be caused by dissemination of cancer cells in cerebrospinal fluid (CSF). Here, we present a 4-year-old female with RMS originating from the upper nasal wall. The following histologic and immunohistochemistry analyses combined with molecular testing analysis supported the diagnosis of embryonal rhabdomyosarcoma (ERMS). Results from CSF routine test, magnetic resonance imaging scans and CSF cytology indicated metastatic meningitis, thus confirming the diagnosis of metastatic ERMS in CSF. This is the first report to describe the clinical features of ERMS in CSF.
Assuntos
Líquido Cefalorraquidiano/citologia , Metástase Neoplásica/patologia , Rabdomiossarcoma Embrionário , Biomarcadores Tumorais , Pré-Escolar , Citodiagnóstico , Feminino , Humanos , Imuno-Histoquímica , Imageamento por Ressonância Magnética , Neoplasias Nasais/patologia , Rabdomiossarcoma Embrionário/diagnóstico , Rabdomiossarcoma Embrionário/patologiaRESUMO
OBJECTIVE: To investigate the relationship between single nucleotide polymorphisms (SNPs) of IKAROS family Zinc finger 3 (IKZF3) gene and the risk of acute lymphoblastic leukemia (ALL) in children. METHODS: The peripheral blood samples from 286 children with ALL and 382 healthy children were collected and divided into ALL group and control group, respectively. The genotypes of IKZF3 gene at rs62066988 C > T and rs12946510 C > T were detected by quantitative PCR with TaqMan detection system, and their correlation with ALL was analyzed. RESULTS: The distribution frequencies of CC, CT and TT genotypes at rs62066988 in ALL group were 58.39%, 37.06% and 4.55%, respectively, while those in control group were 69.19%, 27.68% and 3.13%, respectively. The distribution frequencies of CC, CT and TT genotypes at rs12946510 in ALL group were 58.16%, 34.75% and 7.09%, respectively, while those in control group were 55.76%, 37.43% and 6.81%, respectively. Compared with the control group, the distribution frequency of CT/TT genotype at rs62066988 was significantly increased in the ALL group (OR=1.59, 95%CI: 1.16-2.19, P=0.004). However, there was no significant difference in the distribution of rs12946510 C > T polymorphism between ALL group and control group. CONCLUSION: The CT/TT genotype of IKZF3 at the site of rs62066988 is associated with the increased risk of ALL in children.
Assuntos
Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células Precursoras , Alelos , Estudos de Casos e Controles , Criança , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Fator de Transcrição Ikaros/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMO
Food safety and foodborne infections and diseases have been a leading hotspot in public health, and methicillin-resistant Staphylococcus aureus (MRSA) has been recently documented to be an important foodborne pathogen, in addition to its recognition to be a leading clinical pathogen for some decades. Standard identification for MRSA has been commonly performed in both clinical settings and food routine detection; however, most of such so-called "standards," "guidelines," or "gold standards" are incapable of detecting viable but non-culturable (VBNC) cells. In this study, two major types of staphylococcal food poisoning (SFP), staphylococcal enterotoxins A (sea) and staphylococcal enterotoxins B (seb), as well as the panton-valentine leucocidin (pvl) genes, were selected to develop a cross-priming amplification (CPA) method. Limit of detection (LOD) of CPA for sea, seb, and pvl was 75, 107.5, and 85 ng/µl, indicating that the analytical sensitivity of CPA is significantly higher than that of conventional PCR. In addition, a rapid VBNC cells detection method, designated as PMA-CPA, was developed and further applied. PMA-CPA showed significant advantages when compared with PCR assays, in terms of rapidity, sensitivity, specificity, and accuracy. Compared with conventional VBNC confirmation methods, the PMA-CPA showed 100% accordance, which had demonstrated that the PMA-CPA assays were capable of detecting different toxins in MRSA in VBNC state. In conclusion, three CPA assays were developed on three important toxins for MRSA, and in combination with PMA, the PMA-CPA assay was capable of detecting virulent gene expression in MRSA in the VBNC state. Also, the above assays were further applied to real samples. As concluded, the PMA-CPA assay developed in this study was capable of detecting MRSA toxins in the VBNC state, representing first time the detection of toxins in the VBNC state.
RESUMO
Acute myeloid leukemia (AML) is a fatal disease characterized by the accumulation of immature myeloid blasts in the bone marrow (BM). Cytokine provide signals for leukemia cells to improve their survival in the BM microenvironment. Previously, we identified interleukin-33 (IL-33) as a promoter of cell survival in a human AML cell line and primary mouse leukemia cells. In this study, we report that the cell surface expression of IL-33-specific receptor, Interleukin 1 Receptor Like 1 (IL1RL1), is elevated in BM cells from AML patients at diagnosis, and the serum level of IL-33 in AML patients is higher than that of healthy donor controls. Moreover, IL-33 levels are found to be positively associated with IL-6 levels in pediatric patients with AML. In vitro, IL-33 treatment increased IL-6 mRNA expression and protein level in BM and peripheral blood (PB) cells from AML patients. Evidence was also provided that IL-33 inhibits cell apoptosis by activating p38 mitogen-activated protein kinase (MAPK) pathway using human AML cell line and AML patient samples. Finally, we confirmed that IL-33 activated IL-6 expression in a manner that required p38 MAPK pathway using clinical AML samples. Taken together, we identified a potential mechanism of IL-33-mediated survival involving p38 MAPK in pediatric AML patients that would facilitate future drug development.
Assuntos
Regulação Leucêmica da Expressão Gênica/imunologia , Interleucina-33/imunologia , Interleucina-6/imunologia , Leucemia Mieloide Aguda/imunologia , Proteínas de Neoplasias/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Sobrevivência Celular/imunologia , Criança , Feminino , Humanos , Leucemia Mieloide Aguda/patologia , MasculinoRESUMO
OBJECTIVE: To investigate whether the down-regulation of miR-125b can reverse the drug-resistence of doxorubicine-resistant leukemia cell lines or not, so as to explore a new method for treatment of drug-resistant leukemia patients. METHODS: The expression levels of miR125b in doxorubicine drug-sensitive and doxorubicine drug-resistant leukemia cell lines.HL-60, K562 and HL-60/Dox, the K562/Dox were detected by using RT-qPCR; the up-regulation or inhibition of miR-1256 expression in HL-60/Dox were performed by electroporation transfection, then the viability of cells treated with doxorubicine of different concentration was detected by CCK-8 method, the proliferation inhibition curve of cells was drawed, and the IC50 was calculated. RESULTS: The miR-125b expression was obviously up-regulated in drug-resistant cell lines HL-60/DOX and K562/DOX, as compared with HL-60 and K562 cell lines. The miR-125b expression level in HL-60/DOX and K562/DOX cells was 15 times and 5 times higher than that in HL-60 and K562 cells, respectively. The up-regulating or inhibiting expression of miR-125b in HL-60/DOX cells found that the proliferation inhibition rate in cells transfected with miR-125b mimic significantly decreased, compared with control group (P<0.01), while the proliferation inhibition rate in cells transfected with miR-125b inhibitor significantly increased, compared with control group(P<0.01). CONCLUSION: The miR-125b expression in HL-60/Dox and K562/Dox cells has been up-regulated, down-regulation of miR-125b expression can reverse the drug resistance of leukemia cells to doxorubicine.
Assuntos
Regulação para Baixo , Leucemia , Doxorrubicina , Resistencia a Medicamentos Antineoplásicos , Humanos , Células K562 , MicroRNAsRESUMO
OBJECTIVE: To investigate the expression of miR-181a in AML cell lines and explore its effect on cell proliferation. METHODS: The expression of miR-181a in AML cell lines (NB4,HL-60,K562 and MV-4-11) was detected by quantiative polymerase chain reation(qPCR). Moreover, the cell proliferation and cell cycle were evaluated in several cell lines (HL60, NB4 and K562) by using CCK-8 and flow cytometry after the imitative transfection with miR-181a. RESULTS: The miR-181a expression was significantly increased in most AML cell lines, including NB4,HL-60 and MV-4-11, but decreased in a few AML cell lines(K562), as compared with that in control(P<0.05). Overexpressed miR-181a in the cell lines significantly enhanced the cell proliferation, as well as the cell ratio of S-to and G2-phase by miR-181a imitative transfection in vitro. CONCLUSION: Overexpression of miR-181a can promote AML cell proliferation. MiR-181a may play an oncogene role in AML, studying the MiR-181a may provide a new method for treatment of AML.