RESUMEN
Toxoplasma gondii, which causes toxoplasmosis, is prevalent in warm-blooded animals, such as cats, dogs, and humans. T. gondii causes economic losses to livestock production and represents a potential risk to public health. Dogs and cats are common hosts in the epidemiology of toxoplasmosis. The current molecular diagnostic tools for T. gondii infection require high technical skills, a laboratory environment, and complex instruments. Herein, we developed a recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) assay to detect T. gondii. The lowest limit of detection of the assay was 31 copies/µL for the T. gondii B1 gene. In addition, we established a visual RPA-CRISPR/Cas12a lateral flow band assay (RPA-CRISPR/Cas12a-LFA) combined with a digital visualization instrument, which minimized the problem of false-negative results for weakly positive samples and avoided misinterpretation of the results by the naked eye, making the LFA assay results more accurate. The assay established in this study could identify T. gondii within 55 min with high accuracy and sensitivity, without cross-reaction with other tested parasites. The developed assay was validated by establishing a mouse model of toxoplasmosis. Finally, the developed assay was used to investigate the prevalence of T. gondii in stray cats and dogs in Zhejiang province, Eastern China. The positive rates of T. gondii infection in stray cats and dogs were 8.0% and 4.0%, respectively. In conclusion, the RPA-CRISPR/Cas12a-LFA is rapid, sensitive, and accurate for the early diagnosis of T. gondii, showing promise for on-site surveillance. IMPORTANCE: Toxoplasma gondii is a virulent pathogen that puts millions of infected people at risk of chronic disease reactivation. Hosts of T. gondii are distributed worldwide, and cats and dogs are common hosts of T. gondii. Therefore, rapid diagnosis of early T. gondii infection and investigation of its prevalence in stray dogs and cats are essential. Here, we established a visual recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a-assay combined with a lateral flow band assay and a digital visualization instrument. Detailed analyses found that the assay could be used for the early diagnosis of T. gondii without false-negative results. Moreover, we detected the prevalence of T. gondii in stray cats and dogs in Zhejiang province, China. Our developed assay provides technical support for the early diagnosis of T. gondii and could be applied in prevalence surveys of T. gondii in stray dogs and cats.
Asunto(s)
Sistemas CRISPR-Cas , Enfermedades de los Gatos , Enfermedades de los Perros , Toxoplasma , Toxoplasmosis Animal , Gatos , Animales , Perros , Toxoplasma/genética , Toxoplasma/aislamiento & purificación , Enfermedades de los Perros/parasitología , Enfermedades de los Perros/epidemiología , Enfermedades de los Perros/diagnóstico , Enfermedades de los Gatos/parasitología , Enfermedades de los Gatos/epidemiología , Enfermedades de los Gatos/diagnóstico , China/epidemiología , Toxoplasmosis Animal/parasitología , Toxoplasmosis Animal/epidemiología , Toxoplasmosis Animal/diagnóstico , Ratones , Sensibilidad y Especificidad , Proteínas Asociadas a CRISPR/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Técnicas de Diagnóstico Molecular/métodos , Proteínas Bacterianas , EndodesoxirribonucleasasRESUMEN
BACKGROUND: Timely diagnosis of Toxoplasma gondii infection is necessary to prevent and control toxoplasmosis transmission. The gold immunochromatographic assay (GICA) is a means of rapidly detecting pathogen in samples. GICA-based diagnostic methods have been developed to accurately detect pathogens with high sensitivity and specificity, and their application in T. gondii diagnosis is expected to yield good results. METHODS: Colloidal gold test strips were produced using T. gondii C-terminal truncated apical membrane antigen 1 (AMA1C). Colloidal gold-AMA1C and colloidal gold-murine protein conjugate were synthesized under optimal conditions. A nitrocellulose membrane was treated with AMA1C and goat anti-mouse antibody as the test line and control line, respectively. In total, 90 cat serum samples were tested using AMA1C-GICA and a commercial enzyme linked immunosorbent assay (ELISA) kit. The GICA results were digitally displayed using a portable colloidal gold immunochromatographic test strip analyzer (HMREADER). The sensitivity, specificity, and stability of AMA1C-GICA were assessed, and this was then used to examine clinical samples, including 203 human sera, 266 cat sera, and 81 dog sera. RESULTS: AMA1C-GICA had a detection threshold of 1:32 for T. gondii-positive serum. The GICA strips specifically detected T. gondii antibodies and exhibited no reactivity with Plasmodium vivax, Paragonimus kellicotti, Schistosoma japonicum, Clonorchis sinensis, and Schistosoma mansoni. Consequently, 15 (16.7%) positive samples were detected using the AMA1C-GICA and commercial ELISA kits for each of the assays. The receiver-operating characteristic curve showed that GICA had a relative sensitivity of 85.3% and specificity of 92%, with an area under the curve of 98%. After analyzing clinical samples using HMREADER, 1.2%-23.4% of these samples were found to be positive for T. gondii. CONCLUSIONS: This study presents a novel assay that enables timely and efficient detection of serum antibodies against T. gondii, thereby allowing for its early clinical diagnosis. Furthermore, the integration of digital detection using HMREADER can enhance the implementation of GICA.
Asunto(s)
Toxoplasma , Toxoplasmosis , Ratones , Animales , Perros , Humanos , Cromatografía de Afinidad/métodos , Sensibilidad y Especificidad , Inmunoensayo/métodos , Toxoplasmosis/diagnóstico , Ensayo de Inmunoadsorción Enzimática/métodos , Anticuerpos Antihelmínticos , Oro Coloide/análisis , Oro Coloide/químicaRESUMEN
BACKGROUND: Diseases caused by Toxoplasma gondii (T. gondii) have introduced serious threats to public health. There is an urgent need to develop a rapid detection method for T. gondii infection in cats, which are definitive hosts. Recombinant apical membrane antigen 1 (rAMA1) was produced in a prokaryotic expression system and used as the detection antigen. The aim of this study was to evaluate and optimize a reliable indirect enzyme-linked immunosorbent assay (iELISA) method based on rAMA1 for the detection of antibodies against T. gondii in cats. RESULTS: The rAMA1-iELISA method was developed and optimized by the chessboard titration method. There were no cross-reactions between T. gondii-positive cat serum and positive serum for other pathogens, indicating that rAMA1-iELISA could only detect T. gondii in most cases. The lowest detection limit of rAMA1-iELISA was 1:3200 (dilution of positive serum), and the CV of repeated tests within batches and between batches were confirmed to be less than 10%. The results of 247 cat serum samples detected by rAMA1-iELISA (kappa value = 0.622, p < 0.001) were in substantial agreement with commercial ELISA. The ROC curve analysis revealed the higher overall check accuracy of rAMA1-iELISA (sensitivity = 91.7%, specificity = 93.6%, AUC = 0.956, 95% CI 0.905 to 1.000) than GRA7-based iELISA (sensitivity = 91.7%, specificity = 85.5%, AUC = 0.936, 95% CI 0.892 to 0.980). Moreover, the positive rate of rAMA1-iELISA (6.5%, 16/247) was higher than that of GRA7-based iELISA (3.6%, 9/247) and that of commercial ELISA kit (4.9%, 12/247). CONCLUSION: The iELISA method with good specificity, sensitivity, and reproducibility was established and can be used for large-scale detection of T. gondii infection in clinical cat samples.
Asunto(s)
Enfermedades de los Gatos , Toxoplasma , Toxoplasmosis Animal , Gatos , Animales , Antígenos de Protozoos , Sensibilidad y Especificidad , Reproducibilidad de los Resultados , Anticuerpos Antiprotozoarios , Ensayo de Inmunoadsorción Enzimática/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodos , Toxoplasmosis Animal/diagnóstico , Enfermedades de los Gatos/diagnósticoRESUMEN
BACKGROUND: Toxoplasma gondii is an obligate intracellular apicomplexan parasite and is responsible for zoonotic toxoplasmosis. It is essential to develop an effective anti-T. gondii vaccine for the control of toxoplasmosis, and this study is to explore the immunoprotective effects of a live attenuated vaccine in mice and cats. METHODS: First, the ompdc and uprt genes of T. gondii were deleted through the CRISPR-Cas9 system. Then, the intracellular proliferation and virulence of this mutant strain were evaluated. Subsequently, the immune responses induced by this mutant in mice and cats were detected, including antibody titers, cytokine levels, and subsets of T lymphocytes. Finally, the immunoprotective effects were evaluated by challenge with tachyzoites of different strains in mice or cysts of the ME49 strain in cats. Furthermore, to discover the effective immune element against toxoplasmosis, passive immunizations were carried out. GraphPad Prism software was used to conduct the log-rank (Mantel-Cox) test, Student's t test and one-way ANOVA. RESULTS: The RHΔompdcΔuprt were constructed by the CRISPR-Cas9 system. Compared with the wild-type strain, the mutant notably reduced proliferation (P < 0.05). In addition, the mutant exhibited virulence attenuation in both murine (BALB/c and BALB/c-nu) and cat models. Notably, limited pathological changes were found in tissues from RHΔompdcΔuprt-injected mice. Furthermore, compared with nonimmunized group, high levels of IgG (IgG1 and IgG2a) antibodies and cytokines (IFN-γ, IL-4, IL-10, IL-2 and IL-12) in mice were detected by the mutant (P < 0.05). Remarkably, all RHΔompdcΔuprt-vaccinated mice survived a lethal challenge with RHΔku80 and ME49 and WH6 strains. The immunized sera and splenocytes, especially CD8+ T cells, could significantly extend (P < 0.05) the survival time of mice challenged with the RHΔku80 strain compared with naïve mice. In addition, compared with nonimmunized cats, cats immunized with the mutant produced high levels of antibodies and cytokines (P < 0.05), and notably decreased the shedding numbers of oocysts in feces (95.3%). CONCLUSIONS: The avirulent RHΔompdcΔuprt strain can provide strong anti-T. gondii immune responses, and is a promising candidate for developing a safe and effective live attenuated vaccine.
Asunto(s)
Toxoplasma , Toxoplasmosis Animal , Toxoplasmosis , Animales , Gatos , Ratones , Toxoplasma/genética , Linfocitos T CD8-positivos , Vacunas Atenuadas , Proteínas Protozoarias/genética , Citocinas , Ratones Endogámicos BALB C , Anticuerpos Antiprotozoarios , Toxoplasmosis Animal/prevención & controlRESUMEN
BACKGROUND: KIF18A is associated with a variety of tumours; however, the specific mechanism of action of KIF18A in hepatocellular carcinoma (HCC) remains unclear. In this study, in vitro and in vivo experiments were performed with the aim of exploring the potential function and molecular mechanism of kinesin KIF18A in the occurrence and development of HCC. METHODS: We detected the expression of KIF18A in tumour and adjacent tissues as well as cell proliferation, cell invasion and migration in hepatoma cells after silencing KIF18A. KIF18A-silenced hepatoma cells were subcutaneously injected into nude mice to verify the tumorigenicity of KIF18A. We also detected the expression of signal pathway-related proteins in hepatoma cells after KIF18A knockdown with the aim of exploring the association between KIF18A and related signalling pathways. RESULTS: The level of KIF18A protein was higher in liver cancer tissues than adjacent tissues. After silencing KIF18A in SMMC-7721 and HepG2 cells, cell growth was obviously inhibited; the migration and invasion abilities were significantly decreased and the in vivo tumour weight was decreased compared to the control group (0.201 ± 0.088 g vs 0.476 ± 0.126 g, p = 0.009). The expression of cell cycle-related protein (cyclin B1), invasion and metastasis-related proteins (MMP-7 and MMP-9) and Akt-related proteins in hepatoma cells was also decreased after knocking down KIF18A. CONCLUSIONS: KIF18A may promote proliferation, invasion and metastasis of HCC cells by promoting the cell cycle signalling pathway as well as the Akt and MMP-7/MMP-9-related signalling pathways and may serve as a new target for the diagnosis and treatment of HCC.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/secundario , Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Cinesinas/metabolismo , Neoplasias Hepáticas/patología , Animales , Apoptosis , Carcinoma Hepatocelular/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Ratones , Ratones Desnudos , Invasividad Neoplásica , Metástasis de la Neoplasia , Pronóstico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hepatocellular carcinoma (HCC) has a high predilection with portal vein tumor thrombosis (PVTT). However, part of the PVTT type can be found only under the microscopy, which was namely as type I0. The objective of this study was to establish a simple and inexpensive non-invasive model to predict the presentation of PVTT at HCC patients. A total of 815 HCC patients were retrospectively evaluated and randomly assigned into 2 groups: the training group (n = 408) and validation group (n = 407). A new index model, namely WγAL, was built to predict the presence of PVTT in the training subjects and was further validated in the validation subjects. At the optimal cutoff of 8.90, WγAL identified patients with a hazard ratio (HR) of 7.139 for the presence of PVTT. The area under receiver operating characteristic (AUROC) of WγAL was 0.795 (sensitivity: 71.9%; specificity: 78.6%) for differentiation between PVTT and non-PVTT patients in the training group. The AUROC of WγAL in differentiating patients with PVTT type I0 from non-PVTT patients was 0.748 (sensitivity: 72.1%; specificity: 68.4%) with an HR of 5.355. In addition, WγAL > 8.90 was significantly associated with large tumors, multiple tumor numbers, TNM stage III-IV, metastasis, and overall survival in HCC patients. The novel predictive model represents a simple and inexpensive model that can identify the presence of PVTT in HCC patients with a high degree of accuracy, with important clinical significance in the future therapeutic management of HCC patients.
RESUMEN
OBJECTIVE: To evaluate the clinical efficacy and tolerability of pioglitazone monotherapy or in combination with sulfonylurea therapy in the treatment of chinese patients with type 2 diabetes. METHODS: This 12-week study involved 117 patients with type 2 diabetes whose blood glucose was uncontrolled on a stable regimen of diet or oral sulfonylureas for at least 3 months (fasting blood glucose (FBG) 7.8-15.0 mmol/L; postprandial blood glucose (PBG) >11.1 mmol/L)). The patients received once daily pioglitazone 30 mg plus diet (Group PIO-mono) or sulfonylurea (Group PIO-SU). The fasting and postprandial plasma glucose, serum insulin, C-peptid, lipid profile, free fatty acids (FFAs) levels were measured at baseline and every 4 weeks after treatment. Results All patients showed significant improvement in glycemic control over baseline. There were 1.64%, 2.1 mmol/L, 5.2 mmol/L decrease from baseline HbA1c, FBG and PBG levels in the Group PIO-mono and 1.06%, 2.3 mmol/L, 5.4 mmol/L decrease in the Group PIO-SU after 12 weeks treatment, respectivley. Both groups experienced slight decreases in C-peptide and insulin concentrations during 12 weeks period. Both groups showed mean percent decreases in triglyceride and FFA levels(21.8% and 20.7% for PIO-mono, 22.2% and 14.5 for PIO-SU) and increases in HDL-C (21.4% for PIO-mono and 30.8% for PIO-SU), compared with baseline. pioglitazone was well tolerated. CONCLUSION: In patients with type 2 diabetes, pioglitazone monotherapy or in combination with sulfonylurea therapy significantly improves HbA1c, FBG and PBG levels and reserves the beta-cell function with beneficial effects on serum triglyceride and HDL-C levels.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/administración & dosificación , Compuestos de Sulfonilurea/administración & dosificación , Tiazolidinedionas/administración & dosificación , Adulto , Anciano , Quimioterapia Combinada , Femenino , Hemoglobina Glucada/metabolismo , Índice Glucémico/efectos de los fármacos , Humanos , Resistencia a la Insulina , Masculino , Persona de Mediana Edad , Pioglitazona , Resultado del TratamientoRESUMEN
OBJECTIVE: To investigate the effectiveness of alendronate Chinese national product in the prevention and treatment of postmenopausal osteoporosis. METHODS: The 56 postmenopausal women with osteopenia or osteoporosis were randomly divided into two groups: treated with alendronate 10 mg/d (28 cases) orally and placebo (28 cases), for 6 months. All subjects received 600 mg/d of calcium carbonate and vitamin D 1,000 U/d. Bone mineral density (BMD) of the lumbar spine, femoral neck, trochanter and Ward's triangle were measured by dual energy X-ray absorptiometry as well as the markers of bone turnover were analysed at the beginning and the end of the study. RESULTS: The results showed that lumbar spine BMD increased by 5% in the alendronate group (P < 0.01), but decreased in BMD of the lumbar spine and femur in the placebo group (P < 0.05) after 6 months of treatment. In the alendronate group the marker of bone resorption and bone formation were significantly decreased after alendronate therapy. There were no change in placebo group. CONCLUSIONS: Alendronate (Chinese national product) is effective in reducing bone turnover and promoting bone mass of postmenopausal osteoporosis.