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BACKGROUND: Alanine aminotransferase (ALT) has been used as a sensitive marker for liver injury in people and in preclinical toxicity studies. But measurement of ALT isoenzymes, ALT1 and ALT2, was reported to be of more diagnostic value. The aim of this study is to develop an ideal pair of anti-ALT1 monoclonal antibodies (MAbs) of high specificity and affinity, and subsequently prepare a Immunochromatographic lateral flow device (LFD) for rapid test of ALT1 in human serums. METHODS: The complete coding sequence of ALT1 gene (1500 bp) was cloned from human hepatoma G2 cells (HepG2) and inserted into the expression vector pET-32a(+). ALT1 recombinant protein was routinely prepared by E. coli BL21 (DE3) expression and Ni(2+) affinity purification. Balb/c mice were immunized with purified ALT1 and the splenocytes were fused with Sp2/0 myeloma cells. The positive clones, verified by indirect enzyme-linked immunosorbent assay (ELISA) using purified ALT1, were subcloned to single clones by limiting dilution process. A MAb pair was selected from the obtained MAbs according the sandwich ELISA pairing results and then used for lateral flow device (LFD) production. After evaluation of the sensitivity and specificity, the LFD strips were employed to test human serum samples with known ALT activity levels. RESULTS: ALT1 recombinant protein was expectedly prepared by expression and purification. A total of 8 stable clones that produced antibodies specifically recognizing ALT1 protein were developed. After sandwich ELISA pairing, an ideal pair of anti-ALT1 MAbs, designated as BD7 and DG3, were selected and proved to be of high specificity, titer and affinity. Based on the MAb pair, LFD strips specifically for ALT1 rapid test were subsequently prepared. The detection threshold of the LFD strips was 12 U/L. No cross reaction was found. CONCLUSIONS: The ALT1 LFD with high sensitivity and specificity was successfully developed. It is valuable for testing ALT1 protein in human sera and can be a beneficial complement for traditional ALT test.
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Alanina Transaminase/análise , Anticorpos Monoclonais Murinos/biossíntese , Alanina Transaminase/sangue , Alanina Transaminase/imunologia , Animais , Anticorpos Monoclonais Murinos/química , Cromatografia de Afinidade , Escherichia coli , Feminino , Células Hep G2 , Humanos , Hibridomas , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/análise , Proteínas Recombinantes/sangue , Proteínas Recombinantes/imunologiaRESUMO
Background: Recent studies have revealed changes in microbiota constitution and metabolites associated with tumor progression, however, no causal relation between microbiota or metabolites and diffuse large B-cell lymphoma (DLBCL) has yet been reported. Methods: We download a microbiota dataset from the MiBioGen study, a metabolites dataset from the Canadian Longitudinal Study on Aging (CLSA) study, and a DLBCL dataset from Integrative Epidemiology Unit Open genome-wide association study (GWAS) project. Mendelian randomization (MR) analysis was conducted using the R packages, TwoSampleMR and MR-PRESSO. Five MR methods were used: MR-Egger, inverse variance weighting (IVW), weighted median, simple mode, and weighted mode. Reverse MR analyses were also conducted to explore the causal effects of DLBCL on the microbiome, metabolites, and metabolite ratios. Pleiotropy was evaluated by MR Egger regression and MR-PRESSO global analyses, heterogeneity was assessed by Cochran's Q-test, and stability analyzed using the leave-one-out method. Results: 119 microorganisms, 1,091 plasma metabolite, and 309 metabolite ratios were analyzed. According to IVW analysis, five microorganisms were associated with risk of DLBCL. The genera Terrisporobacter (OR: 3.431, p = 0.049) andgenera Oscillibacter (OR: 2.406, p = 0.029) were associated with higher risk of DLBCL. Further, 27 plasma metabolites were identified as having a significant causal relationships with DLBCL, among which citrate levels had the most significant protective causal effect against DLBCL (p = 0.006), while glycosyl-N-tricosanoyl-sphingadienine levels was related to higher risk of DLBCL (p = 0.003). In addition, we identified 19 metabolite ratios with significant causal relationships to DLBCL, of which taurine/glutamate ratio had the most significant protective causal effect (p = 0.005), while the phosphoethanolamine/choline ratio was related to higher risk of DLBCL (p = 0.009). Reverse MR analysis did not reveal any significant causal influence of DLBCL on the above microbiota, metabolites, and metabolite ratios (p > 0.05). Sensitivity analyses revealed no significant heterogeneity or pleiotropy (p > 0.05). Conclusion: We present the first elucidation of the causal influence of microbiota and metabolites on DLBCL using MR methods, providing novel insights for potential targeting of specific microbiota or metabolites to prevent, assist in diagnosis, and treat DLBCL.
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It is necessary to explore new targets for the treatment of colon adenocarcinoma (COAD) according to the tumor microenvironment. The expression levels of JAML and CXADR were analyzed by bioinformatics analysis and validation of clinical samples. JAML over-expression CD8+ T cell line was constructed, and the proliferation activity was detected by MTT. The production of inflammatory factors was detected by ELISA. The expression of immune checkpoint PD-1 and TIM-3 was detected by Western blot. The apoptosis level was detected by flow cytometry and apoptosis markers. The AOM/DSS mouse model of colorectal cancer was constructed. The expression levels of JAML, CXADR and PD-1 were detected by PCR and Western blot, and the proportion of CD8+ T cells and exhausted T cells were detected by flow cytometry. The expression levels of JAML and CXADR were significantly decreased in colon cancer tissues. Overexpression of JAML can promote the proliferation of T cells, secrete a variety of inflammatory factors. Overexpression of CXADR can reduce the proliferation of colorectal cancer cells, promote apoptosis, and down-regulate the migration and invasion ability of tumor cells. Both JAML agonists and PD-L1 inhibitors can effectively treat colorectal cancer, and the combined use of JAML agonists and PD-L1 inhibitors can enhance the effect. JAML can promote the proliferation and toxicity of CD8+ T cells and down-regulate the expression of immune checkpoints in colon cancer. CXADR can inhibit the proliferation of cancer cells and promote the apoptosis. JAML agonist can effectively treat colorectal cancer by regulating CD8+ T cells.
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Carcinogênese , Neoplasias Colorretais , Microambiente Tumoral , Animais , Humanos , Masculino , Camundongos , Apoptose/efeitos dos fármacos , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Carcinogênese/efeitos dos fármacos , Carcinogênese/imunologia , Carcinogênese/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/patologia , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
Lung adenocarcinoma (LUAD) is the most commonly diagnosed subtype of lung cancer worldwide. Inhibitor of growth 3 (ING3) serves as a tumor suppressor in many cancers. This study aimed to elucidate the role of ING3 in the progression of LUAD and investigate the underlying mechanism related to integrin ß4 (ITGB4) and Src/focal adhesion kinase (FAK) signaling. ING3 expression in LUAD tissues and the correlation between ING3 expression and prognosis were analyzed by bioinformatics databases. After evaluating ING3 expression in LUAD cells, ING3 was overexpressed to assess the proliferation, cell cycle arrest, migration and invasion of LUAD cells. Then, ITGB4 was upregulated to observe the changes of malignant activities in ING3-overexpressed LUAD cells. The transplantation tumor model of NCI-H1975 cells in nude mice was established to analyze the antineoplastic effect of ING3 upregulation in vivo. Downregulated ING3 expression was observed in LUAD tissues and cells and lower ING3 expression predicated the poor prognosis. ING3 upregulation restrained the proliferation, migration, invasion and induced the cell cycle arrest of NCI-H1975 cells. Additionally, ITGB4 expression was negatively correlated with ING3 expression in LUAD tissue. ING3 led to reduced expression of ITGB4, Src and p-FAK. Moreover, ITGB4 overexpression alleviated the effects of ING3 upregulation on the malignant biological properties of LUAD cells. It could be also found that ING3 upregulation limited the tumor volume, decreased the expression of ITGB4, Src and p-FAK, which was restored by ITGB4 overexpression. Collectively, ING3 inhibited the malignant progression of LUAD by negatively regulating ITGB4 expression to inactivate Src/FAK signaling.
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Adenocarcinoma de Pulmão , Adenocarcinoma , Neoplasias Pulmonares , Animais , Camundongos , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica , Integrina beta4/genética , Neoplasias Pulmonares/metabolismo , Camundongos Nus , Quinases da Família src , HumanosRESUMO
CircLRIG1, a newly discovered circRNA, has yet to have its potential function and biological processes reported. This study explored the role of circLRIG1 in the development and progression of bladder carcinoma and its potential molecular mechanisms. Techniques such as qRT-PCR, Western blot, various cellular assays, and in vivo models were used to investigate mRNA and protein levels, cell behavior, molecular interactions, and tumor growth. The results showed that both circLRIG1 and LRIG1 were significantly reduced in bladder carcinoma tissues and cell lines. Low circLRIG1 expression was associated with poor patient prognosis. Overexpressing circLRIG1 inhibited bladder carcinoma cell growth, migration, and invasion, promoted apoptosis, and decreased tumor growth and metastasis in vivo. Importantly, circLRIG1 was found to sponge miR-214-3p, enhancing LRIG1 expression, and its overexpression also modulated protein levels of E-cadherin, N-cadherin, Vimentin, and LRIG1. Similar effects were observed with LRIG1 overexpression. Notably, a positive correlation was found between circLRIG1 and LRIG1 expression in bladder carcinoma tissues. Additionally, the tumor-suppressing effect of circLRIG1 was reversed by overexpressing miR-214-3p or silencing LRIG1. The study concludes that circLRIG1 suppresses bladder carcinoma progression by enhancing LRIG1 expression via sponging miR-214-3p, providing a potential strategy for early diagnosis and treatment of bladder carcinoma.
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Carcinoma , MicroRNAs , Neoplasias da Bexiga Urinária , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Bexiga Urinária/metabolismo , Bexiga Urinária/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Carcinoma/genética , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Glicoproteínas de Membrana/metabolismoRESUMO
Since the surface immunogenic protein (Sip) of group B streptococcus was identified, it's immunogenicity and its potential as a universal vaccine candidate has been evaluated extensively. We developed recombinant Sip protein and used it for monoclonal antibody generation to develop immunochromatographic test kit for GBS detection. The test of bacteria and culture media revealed the correlation between Sip protein expression and diagnosis discrepancy, which has never been reported. Furthermore, not only the surface accessibility of the Sip protein may vary from strains or serotypes; the secretion level of Sip protein may also vary.
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Vibrio parahaemolyticus is widely present in brackish water all over the world, causing infections in certain aquatic animals. It is also a foodborne pathogen that causes diarrhea in humans. The aim of this study is to develop an immunochromatographic lateral flow assay (LFA) for rapid detection of V. parahaemolyticus in both aquatic products and human feces of diarrheal patients. Two monoclonal antibody (MAb) pairs, GA1a-IC9 and IC9-KB4c, were developed and proven to be highly specific and sensitive to V. parahaemolyticus. Based on the two MAb pairs, two types of LFA strips were prepared. Their testing limits for V. parahaemolyticus culture were both 1.2×103CFU/ml. The diagnostic sensitivities and specificities were both 100% for the 32 tested microbial species, including 6 Vibrio species. Subsequently, the LFA strips were used to test Whiteleg shrimps and human feces. The type II strip showed a higher diagnostic sensitivity. Its sensitivity and specificity for hepatopancreas and fecal samples from 13 Whiteleg shrimps and fecal samples from 146 human diarrheal patients were all 100%. In conclusion, our homemade type II LFA is a very promising testing device for rapid and convenient detection of V. parahaemolyticus infection not only in aquatic animals, but also in human diarrheal patients. This sensitive immunochromtographic LFA allows rapid detection of V. parahaemolyticus without requirement of culture enrichment.
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Técnicas de Tipagem Bacteriana/métodos , Cromatografia de Afinidade/métodos , Alimentos Marinhos/microbiologia , Vibrioses/diagnóstico , Vibrioses/microbiologia , Vibrio parahaemolyticus/isolamento & purificação , Animais , Anticorpos Monoclonais , Organismos Aquáticos/microbiologia , Linhagem Celular , Cromatografia de Afinidade/instrumentação , Cromatografia de Afinidade/veterinária , Diarreia/microbiologia , Modelos Animais de Doenças , Fezes/microbiologia , Feminino , Inocuidade dos Alimentos , Coloide de Ouro/química , Humanos , Hibridomas/microbiologia , Camundongos Endogâmicos BALB C , Palaemonidae , Penaeidae/microbiologia , Sensibilidade e Especificidade , Especificidade da Espécie , Fatores de Tempo , Vibrioses/imunologia , Vibrioses/veterinária , Vibrio parahaemolyticus/classificação , Vibrio parahaemolyticus/patogenicidadeRESUMO
OBJECTIVE: Helicobacter pylori stool antigen (HpSA) test is a convenient and reliable non-invasive diagnosis for H. pylori infection. The aim of this study is to develop an immunochromatographic testing device for rapid detection of HpSA among Chinese patients. DESIGN AND METHODS: Monoclonal antibodies (McAb) targeting H. pylori were developed by conventional methods and paired by sandwich ELISA. The lateral flow device (LFD) was prepared using the selected McAb pair. A total of 867 clinically separated bacterial strains, including 56H. pylori strains, were employed to test the sensitivity and specificity. Subsequently, the LFD was used to test 1200 human fecal samples, with a commercial HpSA testing device as comparison. RESULTS: Two McAb pairs targeting H. pylori, DF2a/EE10b and IH10b/EE10b, were developed and proven to be of high specificity and sensitivity. After testing the cultures of 56 clinically separated H. pylori strains, the final LFD product was prepared using the mixture of DF2a and IH10b as capture antibodies and EE10b as the detection antibody. The testing threshold for H. pylori culture was 1.0 × 10(4)cfu/mL. The sensitivity and specificity were both 100% for the 867 tested bacterial cultures. The testing results of 1200 fecal samples showed that the positive and negative agreement rates between the homemade LFD and the commercial testing device were 99.75% and 99.87%, respectively. CONCLUSION: Our homemade HpSA LFD can be a very promising testing device for rapid diagnosis and epidemic screening of H. pylori infection among Chinese patients.
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Antígenos de Bactérias/análise , Cromatografia de Afinidade/instrumentação , Infecções por Helicobacter/diagnóstico , Helicobacter pylori/isolamento & purificação , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Cromatografia de Afinidade/métodos , Ensaio de Imunoadsorção Enzimática , Fezes/química , Infecções por Helicobacter/microbiologia , Helicobacter pylori/imunologia , Humanos , Camundongos , Sensibilidade e Especificidade , Fatores de TempoRESUMO
To prevent epidemic and pandemic cholera disease, an indispensible approach is to develop cholera vaccines based on comprehensive epitope information of this pathogen. This study aimed to utilize our previously raised monoclonal antibody IXiao3G6, which can recognize an epitope in lipopolysaccharide (LPS) sites of Ogawa, to identify mimetic peptides, which may represent Ogawa LPS's epitope information. A phage display library screening using IXiao3G6 antibody resulted in identification of a mimic peptide (MP) with high avidity. A recombinant protein, containing one cholera toxin subunit B (CTB) and two MP repeats (CTB-(MP)2), was subsequently constructed and investigated for its immunological characteristics. The findings collectively demonstrated that the MP presenting phages and CTB-(MP)2 recombinant protein were both capable of inhibiting the interaction between IXiao3G6 and Ogawa/Ogawa LPS specifically in a dose-dependent manner.
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Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Proteínas Recombinantes/imunologia , Anticorpos Antibacterianos/química , Anticorpos Antibacterianos/genética , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Epitopos/química , Epitopos/genética , Escherichia coli/genética , Biblioteca de Peptídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Vibrio cholerae O1/imunologiaRESUMO
BACKGROUND: All the advanced NSCLC patients that received EGFR-TKI therapy will eventually relapse after a period of efficacy. The aim of this study is to investigate the serum biomarkers as potential predictive factors for the efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) targeted therapy in advanced non-small cell lung cancer. METHODS: Twenty self-paired serum samples were collected from 9 advanced NSCLC patients that evaluated as disease control (SD or PR) after gefinitib therapy, at the time points of before and after gefinitib treatment but 2 weeks before being evaluated as disease progress. All samples were pre-separated by WCX microbeads, and then detected on the MALDI-TOF-MS platform of Bruker Autoflex. ClinProTools (Version: 2.1) was used to analyze the differentially expressed proteins. RESULTS: There were 7 protein peaks (m/z), 3 242.09, 8 690.36, 2 952.64, 3 224.04, 1 450.51, 1 887.8 and 3 935.73 found statistically differentially expressed between the self-paired samples. Three proteins (3 242.09, 2 952.64 and 3 224.04) were down-regulated and four proteins (8 690.36, 1 450.51, 1 887.8 and 3 935.73) up-regulated in gefinitib treated sera. CONCLUSIONS: The data here suggest that several specific protein peaks might indicate gefinitib resistance, yet the identities of these proteins and the mechanisms underlying the responsiveness to gefinitib treatment need further investigation.