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Article in Chinese | WPRIM | ID: wpr-936370


OBJECTIVE@#To study the cytokine patterns in patients with rheumatoid arthritis (RA) and healthy individuals and identify candidate serum biomarkers for clinical diagnosis of RA.@*METHODS@#This study was conducted among 59 patients diagnosed with RA in our hospital from 2015 to 2019 with 46 age- and gender-matched healthy subjects who received regular physical examinations in our hospital as the control group. Serological autoimmune profiles of 5 RA patients and 5 healthy control subjects were obtained from human cytokine microarrays. We selected 4 differentially expressed cytokines (LIMPII, ROBO3, Periostin and IGFBP-4) and 2 soluble cytokine receptors of interest (2B4 and Tie-2) and examined their serum levels using enzyme-linked immunosorbent assay in 54 RA patients and 41 healthy control subjects. Spearman correlation test was performed to assess the correlation of serum cytokine and soluble receptor expression levels with the clinical features including rheumatoid factor (RF), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), disease activity score (DAS28) and health assessment questionnaire (HAQ). Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic capability of these cytokines.@*RESULTS@#We identified 6 dysregulated cytokines and soluble receptors (2B4, LIMPII, Tie-2, ROBO3, periostin and IGFBP-4) in RA patients (P < 0.01). The serum levels of LIMPII, ROBO3 and periostin were significantly correlated with the disease activity indicators including RF (P < 0.001), CRP (P < 0.001), DAS28 (P < 0.001) and HAQ (P < 0.001) in RA patients. Among the 6 candidate cytokines, 2B4 showed the largest area under the curve (AUC) of 0.861 for RA diagnosis (P < 0.001), followed then by LIMPII, ROBO3, periostin, Tie-2 and IGFBP-4.@*CONCLUSION@#Serum levels of LIMPII, ROBO3 and periostin can be indicative of the disease activity of RA, and serum 2B4, LIMPII, periostin, ROBO3, IGFBP-4 and Tie-2 levels may serve as biomarkers for the diagnosis of RA.

Humans , Arthritis, Rheumatoid/diagnosis , Biomarkers , C-Reactive Protein , Cytokines , Insulin-Like Growth Factor Binding Protein 4 , Protein Array Analysis , Receptors, Cell Surface
Chinese Journal of Biotechnology ; (12): 4075-4082, 2021.
Article in Chinese | WPRIM | ID: wpr-921488


The rapid screening of tumor markers is a challenging task for early diagnosis of cancer. This study aims to use highly sensitive chemiluminescent protein microarray technology to efficiently screen a variety of low abundance tumor related markers. A new material, termed integrated polydimethylsiloxane modified silica gel (iPDMS), was obtained by adding a surface polymerization initiator with olefin end to the conventional polydimethylsiloxane, and fixing into the three-dimensional structure of polydimethylsiloxane by thermal crosslinking through silicon hydrogen bonding. In order to make the iPDMS material resistant to non-specific protein adsorption, a poly(OEGMA) polymer brush was synthesized by surface-initiated atom transfer radical polymerization at the active initiation site. Finally, 20 tumor-related antigens were printed into the specific areas of the microarray by high-throughput spray printing technology, and assembled into 48-well detection microtiterplates of the iPDMS microarray. It was found the VEGFR and VEGF121 autoantibodies that obtained from 8 common tumors (breast cancer, lung cancer, colon cancer, gastric cancer, liver cancer, leukemia, lymphoma and ovarian cancer) can be used as potential tumor markers. The chemiluminescence labeled iPDMS protein microarray can be used for the screening of tumor autoantibodies at early stage.

Adsorption , Autoantibodies , Dimethylpolysiloxanes , Protein Array Analysis , Silica Gel , Surface Properties
Chinese Journal of Biotechnology ; (12): 1360-1367, 2021.
Article in Chinese | WPRIM | ID: wpr-878637


Imported malaria has become a major risk factor for malaria prevention and control in China. How to screen malaria quickly for people entering China is an urgent problem to be solved. Protein microarrays are widely used in high-throughput screening and diagnosis. In this study, surface plasmon resonance (SPR) technique for malaria detection was established by using the specific adsorption surface treated by polyethylene glycol polymer, and the malaria specific antigen HRP2 was used as capture probe. The optimal concentration of antigen, sensitivity and specificity of detection, as well as anti-interference ability of the chip were analyzed. The SPR protein chip was applied to detect specific antibodies of malignant malaria in serum with the advantage of label-free, instant and fast. Compared with fluorescence quantitative PCR, there were no significant difference in sensitivity and specificity between the two methods. This study lays a foundation for further development of protein microarray for malaria typing identification, and it is conducive to the rapid screening of malaria for people entering.

Humans , Antibodies , China , Malaria/diagnosis , Protein Array Analysis , Surface Plasmon Resonance
Article in English | WPRIM | ID: wpr-816620


BACKGROUND: Adrenal cortical carcinoma (ACC) is a rare cancer with a variable prognosis. Several prognostic factors of ACC have been previously reported, but a proteomic analysis has not yet been performed. This study aimed to investigate prognostic biomarkers for ACC using a proteomic approach.METHODS: We used reverse-phase protein array data from The Cancer Proteome Atlas, and identified differentially expressed proteins in metastatic ACCs. Multivariate Cox regression analysis adjusted by age and staging was used for survival analysis, and the C-index and category-free net reclassification improvement (cfNRI) were utilized to evaluate additive prognostic value.RESULTS: In 46 patients with ACC, cyclin B1, transferrin receptor (TfR1), and fibronectin were significantly overexpressed in patients with distant metastasis. In multivariate models, high expression of cyclin B1 and TfR1 was significantly associated with mortality (hazard ratio [HR], 6.13; 95% confidence interval [CI], 1.02 to 36.7; and HR, 6.59; 95% CI, 1.14 to 38.2; respectively), whereas high fibronectin expression was not (HR, 3.92; 95% CI, 0.75 to 20.4). Combinations of high cyclin B1/high TfR1, high cyclin B1/high fibronectin, and high TfR1/high fibronectin were strongly associated with mortality ([HR, 13.72; 95% CI, 1.89 to 99.66], [HR, 9.22; 95% CI, 1.34 to 63.55], and [HR, 18.59; 95% CI, 2.54 to 135.88], respectively). In reclassification analyses, cyclin B1, TfR1, fibronectin, and combinations thereof improved the prognostic performance (C-index, 0.78 to 0.82–0.86; cfNRI, all P values <0.05).CONCLUSION: In ACC patients, the overexpression of cyclin B1, TfR1, and fibronectin and combinations thereof were associated with poor prognosis.

Humans , Adrenocortical Carcinoma , Biomarkers , Cyclin B1 , Cyclins , Fibronectins , Mortality , Neoplasm Metastasis , Prognosis , Protein Array Analysis , Proteome , Proteomics , Receptors, Transferrin , Transferrin
Article in Chinese | WPRIM | ID: wpr-879936


OBJECTIVE@#To detect the differentially expressed inflammatory proteins in acute gouty arthritis (AGA) with protein chip.@*METHODS@#The Raybiotech cytokine antibody chip was used to screen the proteomic expression in serum samples of 10 AGA patients and 10 healthy individuals. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were applied to determine the biological function annotation of differentially expressed proteins and the enrichment of signal pathways. ELISA method was used to verify the differential protein expression in 60 AGA patients and 60 healthy subjects. The ROC curve was employed to evaluate the diagnostic value of differential proteins in AGA patients.@*RESULTS@#According to|log@*CONCLUSIONS@#Proteomics can be applied to identify the biomarkers of AGA, which may be used for risk prediction and diagnosis of AGA patients.

Humans , Arthritis, Gouty/diagnosis , Cytokines/genetics , Gene Expression Profiling , Gene Expression Regulation , Inflammation , Protein Array Analysis , Proteomics
Biol. Res ; 52: 8, 2019. tab, graf
Article in English | LILACS | ID: biblio-1011404


BACKGROUND: Cervical cancer (CC) ranks third in the morbidity and mortality of female cancer around the world. Derlin1 has been found to be overexpressed in several human cancers. However, it is still unclear about its roles in CC. The research aims to explore the relationship between Derlin1 and CC. METHODS: We purchased a human CC tissues microarray, which contained CC tissues and corresponding para-cancerous tissues from 93 patients with primary cervical squamous cell carcinoma. Immunohistochemical staining was used to confirm the expression of Derlin1 in these tissues. And we detected the differential expression of Derlin1 in cervical cancer cell lines and normal cervical epithelial cells (H8). Further, the cervical cancer cell lines SiHa and C33A were used as an in vitro model, which was down-regulated the expression of Derlin1 using siRNA interference technology. The effects of Derlin1 down-regulating in CC cell lines on cell proliferation and migration were detected by CCK8 assay and transwell assay, respectively. The effect of Derlin1 down-regulating on apoptosis was analyzed by flow cytometry, and apoptosis-related proteins were detected using western blotting. In-depth mechanisms were studied using western blotting. In addition, the effects of Derlin1 up-regulating in normal cervical epithelial cells also were exposed. RESULTS: Derlin1 was significantly elevated in CC tissues (81.7%, 76/93), and the expression of Derlin 1 was positively correlated with the tumor size, pathological grade, and lymph node metastasis in CC patients. And Derlin 1 was high expressed in cervical cancer cell lines compared to H8 cells. Knockdown of Derlin 1 in cervical cancer cell lines inhibited cell proliferation and migration. Moreover, knockdown of Derlin 1 induced apoptosis and affected the expression of apoptosis-related proteins, including Bcl-2, Bax, Bim, caspase3 and caspase9. Further experiments showed that AKT/mTOR signal pathway might be involve in this processes that knockdown of Derlin 1 inhibited the expression of p-AKT and p-mTOR. Over-expression of Derlin 1 in H8 cells promoted cell proliferation and migration via up-regulated the expression of p-AKT and p-mTOR. CONCLUSION: Derlin 1 is an oncogene in CC via AKT/mTOR pathway. It might be a potential therapeutic target for CC.

Humans , Female , Carcinoma, Squamous Cell/metabolism , Signal Transduction/physiology , Uterine Cervical Neoplasms/metabolism , Proto-Oncogene Proteins c-akt/metabolism , TOR Serine-Threonine Kinases/metabolism , Membrane Proteins/metabolism , Immunohistochemistry , Carcinoma, Squamous Cell/pathology , Uterine Cervical Neoplasms/pathology , Apoptosis , Protein Array Analysis , Cell Line, Tumor , Cell Proliferation , Proto-Oncogene Proteins c-akt/physiology
Article in English | WPRIM | ID: wpr-773003


Characterization of the colon cancer immunome and its autoantibody signature from differentially-reactive antigens (DIRAGs) could provide insights into aberrant cellular mechanisms or enriched networks associated with diseases. The purpose of this study was to characterize the antibody profile of plasma samples from 32 colorectal cancer (CRC) patients and 32 controls using proteins isolated from 15,417 human cDNA expression clones on microarrays. 671 unique DIRAGs were identified and 632 were more highly reactive in CRC samples. Bioinformatics analyses reveal that compared to control samples, the immunoproteomic IgG profiling of CRC samples is mainly associated with cell death, survival, and proliferation pathways, especially proteins involved in EIF2 and mTOR signaling. Ribosomal proteins (e.g., RPL7, RPL22, and RPL27A) and CRC-related genes such as APC, AXIN1, E2F4, MSH2, PMS2, and TP53 were highly enriched. In addition, differential pathways were observed between the CRC and control samples. Furthermore, 103 DIRAGs were reported in the SEREX antigen database, demonstrating our ability to identify known and new reactive antigens. We also found an overlap of 7 antigens with 48 "CRC genes." These data indicate that immunomics profiling on protein microarrays is able to reveal the complexity of immune responses in cancerous diseases and faithfully reflects the underlying pathology.

Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Biomarkers, Tumor , Genetics , Allergy and Immunology , Metabolism , Case-Control Studies , Colonic Neoplasms , Allergy and Immunology , Metabolism , Computational Biology , Methods , Computer Simulation , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Immunoglobulin G , Allergy and Immunology , Protein Array Analysis , Methods
Article in English | WPRIM | ID: wpr-713693


Triple-negative breast cancer (TNBC) has a higher risk of death within 5 years of being diagnosed than the other forms of breast cancer. It is the second leading cause of death due to cancer among women. Currently, however, no diagnostic blood-based biomarker exists to identify the early stages of TNBC. To address this point, we utilized a human protein microarray system to identify serum autoantibodies that showed different expression patterns between TNBC and normal serum samples, and identified five autoantibodies showing TNBC-specific expression. Among them, we selected the thioredoxin-like 2 (TXNL2) autoantibody and evaluated its diagnostic relevance by dot blot analysis with the recombinant TXNL2 protein. We demonstrated that the TXNL2 autoantibody showed 2- to 6-fold higher expression in TNBC samples than in normal samples suggesting that serum TXNL2 autoantibodies are potential biomarkers for TNBC.

Female , Humans , Autoantibodies , Biomarkers , Breast Neoplasms , Cause of Death , Protein Array Analysis , Triple Negative Breast Neoplasms
Article in English | WPRIM | ID: wpr-715839


PURPOSE: We investigated B-cell lymphoma 2 (BCL2) regulation across DNA, RNA, protein, and methylation status according to molecular subtype of breast cancer using The Cancer Genome Atlas (TCGA) database. MATERIALS AND METHODS: We analyzed clinical and biological data on 1,096 breast cancers from the TCGA database. Biological data included reverse phase protein array (RPPA), mRNA sequencing (mRNA-seq), mRNA microarray, methylation, copy number alteration linear, copy number alteration nonlinear, and mutation data. RESULTS: The luminal A and luminal B subtypes showed upregulated expression of RPPA and mRNAseq and hypomethylation compared to the human epidermal growth factor receptor 2 (HER2) and triple-negative subtypes (all p < 0.001). No mutations were found in any subjects. High mRNA-seq and high RPPA were strongly associated with positive estrogen receptor, positive progesterone receptor (all p < 0.001), and negative HER2 (p < 0.001 and p=0.002, respectively). Correlation analysis revealed a strong positive correlation between protein and mRNA levels and a strong negative correlation between methylation and protein and mRNA levels (all p < 0.001). The high BCL2 group showed superior overall survival compared to the low BCL2 group (p=0.006). CONCLUSION: The regulation of BCL2 was mainly associated with methylation across the molecular subtypes of breast cancer, and luminal A and luminal B subtypes showed upregulated expression of BCL2 protein, mRNA, and hypomethylation. Although copy number alteration may have played a minor role, mutation status was not related to BCL2 regulation. Upregulation of BCL2 was associated with superior prognosis than downregulation of BCL2.

Humans , Breast Neoplasms , Breast , DNA , Down-Regulation , Estrogens , Gene Expression Regulation , Genome , Lymphoma, B-Cell , Methylation , Phenobarbital , Prognosis , Protein Array Analysis , ErbB Receptors , Receptors, Progesterone , RNA , RNA, Messenger , Up-Regulation
Annals of Dermatology ; : 322-330, 2018.
Article in English | WPRIM | ID: wpr-715492


BACKGROUND: Empirical evidences for efficacy of hot spring (HS) water in inflammatory skin disorders have not been substantiated with sufficient, immunological “hard evidence”. Mageumsan HS water, characterized by its weakly-alkaline properties and low total dissolved solids content, has been known to alleviate various immune-inflammatory skin diseases, including atopic dermatitis (AD). OBJECTIVE: The trial attempted to quantitatively analyze in vitro expression levels of chemical mediators in cutaneous inflammation from HaCaT cell line treated with Mageumsan HS, and suggest the likely mode of action through which it exerts the apparent anti-inflammatory effects in AD. METHODS: Using membrane-based human antibody array kit, customized to include 30 different, keratinocyte-derived mediator proteins, their expression levels (including interleukin [IL]-1, IL-6, IL-8, thymic stromal lymphopoietin, thymus and activation-regulated chemokine, and granulocyte macrophage colony-stimulating factor) were assessed in vitro. Selected key proteins were further quantified with enzyme-linked immunosorbent assay. RESULTS: There was a clear pattern of overall suppression of the mediators, especially those noted for their pro-inflammatory role in AD (monocyte chemoattractant protein [MCP]-1, regulated on activation, normal T cell expressed and secreted, cutaneous T-cell-attracting chemokine, Eotaxin, and macrophage inflammatory protein-1α, etc.). Also, reduced expression of involucrin and cytokeratin 1 was also reduced in the HS-treated group. CONCLUSION: The present study has shown that Mageumsan HS water may exert its effects on inflammatory skin disorders through regulation of proinflammatory cytokines. These evidences are to be supported with further future investigations to elucidate immunological mechanism behind these beneficial effects of HS water in the chronically inflamed skin of AD.

Humans , Cell Line , Chemokine CCL17 , Chemokine CCL27 , Cytokines , Dermatitis, Atopic , Enzyme-Linked Immunosorbent Assay , Granulocytes , Hot Springs , In Vitro Techniques , Inflammation , Interleukin-6 , Interleukin-8 , Interleukins , Keratins , Macrophages , Protein Array Analysis , Skin , Skin Diseases , Water
Article in English | WPRIM | ID: wpr-690626


<p><b>OBJECTIVE</b>To identify potential serum biomarkers for distinguishing between latent tuberculosis infection (LTBI) and active tuberculosis (TB).</p><p><b>METHODS</b>A proteome microarray containing 4,262 antigens was used for screening serum biomarkers of 40 serum samples from patients with LTBI and active TB at the systems level. The interaction network and functional classification of differentially expressed antigens were analyzed using STRING 10.0 and the TB database, respectively. Enzyme-linked immunosorbent assays (ELISA) were used to validate candidate antigens further using 279 samples. The diagnostic performances of candidate antigens were evaluated by receiver operating characteristic curve (ROC) analysis. Both antigen combination and logistic regression analysis were used to improve diagnostic ability.</p><p><b>RESULTS</b>Microarray results showed that levels of 152 Mycobacterium tuberculosis (Mtb)-antigen- specific IgG were significantly higher in active TB patients than in LTBI patients (P < 0.05), and these differentially expressed antigens showed stronger associations with each other and were involved in various biological processes. Eleven candidate antigens were further validated using ELISA and showed consistent results in microarray analysis. ROC analysis showed that antigens Rv2031c, Rv1408, and Rv2421c had higher areas under the curve (AUCs) of 0.8520, 0.8152, and 0.7970, respectively. In addition, both antigen combination and logistic regression analysis improved the diagnostic ability.</p><p><b>CONCLUSION</b>Several antigens have the potential to serve as serum biomarkers for discrimination between LTBI and active TB.</p>

Adolescent , Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , Antibodies, Bacterial , Antibody Specificity , Antigens, Bacterial , Biomarkers , Blood , Latent Tuberculosis , Blood , Diagnosis , Logistic Models , Mycobacterium tuberculosis , Protein Array Analysis , Methods , Proteome , Genetics , Proteomics , Methods , ROC Curve
Braz. j. med. biol. res ; 50(5): e5981, 2017. tab, graf
Article in English | LILACS | ID: biblio-839288


Different pathways act synergistically to participate in many biological processes. Thus, the purpose of our study was to extract dysregulated pathways to investigate the pathogenesis of colorectal cancer (CRC) based on the functional dependency among pathways. Protein-protein interaction (PPI) information and pathway data were retrieved from STRING and Reactome databases, respectively. After genes were aligned to the pathways, each pathway activity was calculated using the principal component analysis (PCA) method, and the seed pathway was discovered. Subsequently, we constructed the pathway interaction network (PIN), where each node represented a biological pathway based on gene expression profile, PPI data, as well as pathways. Dysregulated pathways were then selected from the PIN according to classification performance and seed pathway. A PIN including 11,960 interactions was constructed to identify dysregulated pathways. Interestingly, the interaction of mRNA splicing and mRNA splicing-major pathway had the highest score of 719.8167. Maximum change of the activity score between CRC and normal samples appeared in the pathway of DNA replication, which was selected as the seed pathway. Starting with this seed pathway, a pathway set containing 30 dysregulated pathways was obtained with an area under the curve score of 0.8598. The pathway of mRNA splicing, mRNA splicing-major pathway, and RNA polymerase I had the maximum genes of 107. Moreover, we found that these 30 pathways had crosstalks with each other. The results suggest that these dysregulated pathways might be used as biomarkers to diagnose CRC.

Humans , Adenoma/genetics , Adenoma/metabolism , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Protein Interaction Maps/genetics , Area Under Curve , Biomarkers, Tumor/genetics , Case-Control Studies , Gene Expression Profiling/methods , Gene Expression Regulation , Principal Component Analysis , Protein Array Analysis , Reference Values , RNA Splicing , Signal Transduction , Transcriptome
Assiut University Bulletin for Environmental Researches. 2017; 20 (1): 11-22
in English | IMEMR | ID: emr-188416


The present study aimed to assess the levels of some heavy metals [Cu, Fe, Pb, Zn and Cd] in muscles of wild and farmed Oreochromis niloticus as well as to evaluate the human hazard index associated with fish consumption

In addition, total protein, molecular weights and band counts of sarcoplasmic proteins were investigated with sodium dodecyl sulfate polyacrylamide gel electrophoresis [SDS-PAGE] method. The obtained results revealed that the accumulation of Cu has the highest value in farmed Oreochromis niloticus compared to wild Oreochromis niloticus while the highest Fe, Pb, Zn and Cd concentrations were recorded in wild Oreochromis niloticus compared to farmed Oreochromis niloticus

The calculated hazard index [HI] indicated that all metals had low HI except Pb and Cd levels in both wild and farmed fish were higher than their permissible limits for fish as a human food

There was no significant [P> 0.05] difference between wild and farmed Oreochromis niloticus in total protein

Wild fish predominant farmed fish in the number of separated proteins

Wild fish muscle protein showed 12 protein bands, while farmed fish muscle proteins showed 11 protein bands. Also, these bands were differed in quantitative parameters. Wild fish had unique bands [MW. 198.13, 97.92, 56.77 and 29.75] while farmed fish had unique bands [MW. 121.62, 79.05 and 26,16]

The current data found that there are differences in electrophoretic pattern and heavy metals accumulation between wild and farmed Oreochromis niloticus

Humans , Heavy Metal Poisoning , Protein Array Analysis , Muscles/chemistry , Fishes , Electrophoresis, Polyacrylamide Gel , Food Analysis , Fisheries
Article in English | WPRIM | ID: wpr-72761


Plasmodium vivax produces numerous caveola-vesicle complex (CVC) structures beneath the membrane of infected erythrocytes. Recently, a member helical interspersed subtelomeric (PHIST) superfamily protein, PcyPHIST/CVC-81₉₅, was identified as CVCs-associated protein in Plasmodium cynomolgi and essential for survival of this parasite. Very little information has been documented to date about PHIST/CVC-81₉₅ protein in P. vivax. In this study, the recombinant PvPHIST/CVC-81₉₅ N and C termini were expressed, and immunoreactivity was assessed using confirmed vivax malaria patients sera by protein microarray. The subcellular localization of PvPHIST/CVC-81₉₅ N and C termini in blood stage parasites was also determined. The antigenicity of recombinant PvPHIST/CVC-81₉₅ N and C terminal proteins were analyzed by using serum samples from the Republic of Korea. The results showed that immunoreactivities to these proteins had 61% and 43% sensitivity and 96.9% and 93.8% specificity, respectively. The N terminal of PvPHIST/CVC-81₉₅ which contains transmembrane domain and export motif (PEXEL; RxLxE/Q/D) produced CVCs location throughout the erythrocytic-stage parasites. However, no fluorescence was detected with antibodies against C terminal fragment of PvPHIST/CVC-81₉₅. These results suggest that the PvPHIST/CVC-81₉₅ is localized on the CVCs and may be immunogenic in natural infection of P. vivax.

Humans , Antibodies , Erythrocytes , Fluorescence , Malaria, Vivax , Membranes , Parasites , Plasmodium cynomolgi , Plasmodium vivax , Plasmodium , Protein Array Analysis , Republic of Korea , Sensitivity and Specificity
Article in English | WPRIM | ID: wpr-13392


The discovery and understanding of antigenic proteins are essential for development of a vaccine against malaria. In Plasmodium falciparum, Pf92 have been characterized as a merozoite surface protein, and this protein is expressed at the late schizont stage, but no study of Pv92, the orthologue of Pf92 in P. vivax, has been reported. Thus, the protein structure of Pv92 was analyzed, and the gene sequence was aligned with that of other Plasmodium spp. using bioinformatics tools. The recombinant Pv92 protein was expressed and purified using bacterial expression system and used for immunization of mice to gain the polyclonal antibody and for evaluation of antigenicity by protein array. Also, the antibody against Pv92 was used for subcellular analysis by immunofluorescence assay. The Pv92 protein has a signal peptide and a sexual stage s48/45 domain, and the cysteine residues at the N-terminal of Pv92 were completely conserved. The N-terminal of Pv92 was successfully expressed as soluble form using a bacterial expression system. The antibody raised against Pv92 recognized the parasites and completely merged with PvMSP1-19, indicating that Pv92 was localized on the merozoite surface. Evaluation of the human humoral immune response to Pv92 indicated moderate antigenicity, with 65% sensitivity and 95% specificity by protein array. Taken together, the merozoite surface localization and antigenicity of Pv92 implicate that it might be involved in attachment and invasion of a merozoite to a new host cell or immune evasion during invasion process.

Animals , Humans , Mice , Computational Biology , Cysteine , Fluorescent Antibody Technique , Immune Evasion , Immunity, Humoral , Immunization , Malaria , Merozoites , Parasites , Plasmodium falciparum , Plasmodium vivax , Plasmodium , Protein Array Analysis , Protein Sorting Signals , Schizonts , Sensitivity and Specificity
Rio de Janeiro; s.n; 2015. xiii,55 p. ilus, tab, graf.
Thesis in Portuguese | LILACS | ID: lil-774205


A leptospirose é uma zoonose cosmopolita grave causada por bactérias patogênicas do gêneroLeptospira sp. Existem mais de 260 sorovares divididos em 13 espécies patogênicas cujaprevalência varia em diferentes regiões geográficas. O teste de aglutinação microscópica(MAT), considerado o padrão-ouro, é realizado apenas em poucos laboratórios de referência etem baixa sensibilidade no início da doença. Assim, o desenvolvimento de um teste dediagnóstico precoce sensível e universalmente eficaz permitirá o tratamento adequado erefletirá a verdadeira incidência da doença. Portanto, foi desenvolvido anteriormente ummicroarranjo de proteínas que compreendeu o genoma de L. interrogans Copenhageni cepaFiocruz L1-130. A resposta imune por IgG foi investigada utilizando soros de pacientes emfase aguda e convalescente da doença, infectados pela referida espécie, obtidos emSalvador/BA. O estudo permitiu a identificação de antígenos para os quais foram detectadosanticorpos nos pacientes estudados, mas não em indivíduos saudáveis. O objetivo do presentetrabalho foi identificar, dentro desse grupo de antígenos imunodominantes anteriormentedefinido, aqueles que podem ser utilizados em um ensaio para o diagnóstico de pacientesinfectados por diferentes espécies e sorovares de Leptospira. As respostas por anticorpos IgGde 308 indivíduos diagnosticados com leptospirose em áreas de transmissão endêmicas e nãoendêmicas de diferentes locais em todo o mundo e 32 indivíduos saudáveis de uma área nãoendêmica dos Estados Unidos foram analisadas. Nove antígenos foram identificados comosororeativos em até cinco das áreas avaliadas localizadas em diferentes continentes (África,Europa, Ásia, América do Norte, América do Sul e Oceania). Entre estes, os segmentos dasproteínas Ligs mostraram ser diferencialmente reativos em todos os locais...

Leptospirosis is a serious cosmopolitan zoonosis caused by pathogenic bacteria of thegenus Leptospira sp. There are over 260 serovars divided into 13 pathogenic species whoseprevalence varies in different geographical regions. The microscopic agglutination test(MAT), considered the gold standard, is performed only in a few reference laboratories andhas low sensitivity in the early course of the disease. Thus, the development of an earlydiagnostic test, sensitive and universally efficient allows the right treatment and reflects thetrue incidence of the disease. Therefore, a protein microarray comprising the genome of L.interrogans serovar Copenhageni Fiocruz L1-130 strain was previously developed. The IgGimmune response was investigated using sera from patients in acute and convalescent phaseof the disease, infected by that species, obtained in Salvador/BA. The study allowed theidentification of antigens to which antibodies were detected in the studied patients but not inhealthy individuals. The aim of this project was to identify within that immunodominantantigens group previously defined, those that can be used in a test for the diagnosis of patientsinfected with different species and serovars of Leptospira. We investigated the response byIgG antibodies of 308 individuals diagnosed with leptospirosis in endemic and non-endemictransmission areas of different locations worldwide and 32 healthy subjects from a nonendemicarea of the United States. Nine antigens were identified as seroreactive up to five ofthe evaluated areas located in different continents (Africa, Europe, Asia, North and SouthAmerica and Oceania). Among these, the segments of Lig proteins were shown to bedifferentially reactive in all locations...

Humans , Leptospirosis/diagnosis , Leptospirosis/epidemiology , Protein Array Analysis , Sensitivity and Specificity , Enzyme-Linked Immunosorbent Assay
Gut and Liver ; : 665-671, 2015.
Article in English | WPRIM | ID: wpr-216102


BACKGROUND/AIMS: To screen for serum protein/peptide biomarkers of hepatitis B virus (HBV)-associated chronic hepatic lesions in an attempt to profile the progression of HBV-associated chronic hepatic lesions using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) techniques. METHODS: Using SELDI-TOF MS, serum protein/peptide profiles on the CM10 ProteinChip arrays were obtained from a training group including 26 HBV-associated hepatocellular carcinoma patients with liver cirrhosis (LC), 30 HBV-associated LC patients, 85 patients at different stages of liver fibrosis, and 30 asymptomatic HBV carriers. The most valuable SELDI peak for predicting the progression to LC in HBV-infected patients was identified. RESULTS: A SELDI peak of M/Z 5805 with value for predicting LC in HBV-infected patients was found and was identified as a peptide of the C-terminal fraction of the fibrinogen alpha-chain precursor, isoform 1. CONCLUSIONS: The peptide of the C-terminal fraction of the fibrinogen alpha-chain precursor, isoform 1 with M/Z 5805, may be a serological biomarker for progression to LC in HBV-infected patients.

Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , Biomarkers/blood , Carcinoma, Hepatocellular/virology , Disease Progression , Hepatitis B virus , Hepatitis B, Chronic/blood , Liver Cirrhosis/blood , Liver Neoplasms/virology , Protein Array Analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Immune Network ; : 66-72, 2015.
Article in English | WPRIM | ID: wpr-70038


Currently, detecting biochemical differences before and after allogeneic stem cell transplantation (SCT) for improved prediction of acute graft-versus-host disease (aGVHD) is a major clinical challenge. In this pilot study, we analyzed the kinetics of circulating adipokine levels in patients with or without aGVHD before and after allogeneic SCT. Serum samples were obtained and stored at -80degrees C within 3 hours after collection, prior to conditioning and at engraftment after transplantation. A protein array system was used to measure the levels of 7 adipokines of patients with aGVHD (n=20) and without aGVHD (n=20). The resistin level at engraftment was significantly increased (p<0.001) after transplantation, regardless of aGVHD occurrence. In the non-aGVHD group, the concentrations of the hepatocyte growth factor (HGF) (mean values+/-SD; 206.6+/-34.3 vs. 432.3+/-108.9 pg/ml, p=0.040) and angiopoietin-2 (ANG-2) (mean values+/-SD; 3,197.2+/-328.3 vs. 4,471.8+/-568.4 pg/ml, p=0.037) at engraftment were significantly higher than those of the pre-transplant period, whereas in the aGVHD group, the levels of adipokines did not change after transplantation. Our study suggests that changes in serum HGF and ANG-2 levels could be considered helpful markers for the subsequent occurrence of aGVHD.

Humans , Adipokines , Angiopoietin-2 , Graft vs Host Disease , Hepatocyte Growth Factor , Kinetics , Pilot Projects , Protein Array Analysis , Resistin , Stem Cell Transplantation
Braz. j. med. biol. res ; 47(10): 834-841, 10/2014. tab, graf
Article in English | LILACS | ID: lil-722173


In this study, biomarkers and transcriptional factor motifs were identified in order to investigate the etiology and phenotypic severity of Down syndrome. GSE 1281, GSE 1611, and GSE 5390 were downloaded from the gene expression ominibus (GEO). A robust multiarray analysis (RMA) algorithm was applied to detect differentially expressed genes (DEGs). In order to screen for biological pathways and to interrogate the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, the database for annotation, visualization, and integrated discovery (DAVID) was used to carry out a gene ontology (GO) function enrichment for DEGs. Finally, a transcriptional regulatory network was constructed, and a hypergeometric distribution test was applied to select for significantly enriched transcriptional factor motifs. CBR1, DYRK1A, HMGN1, ITSN1, RCAN1, SON, TMEM50B, and TTC3 were each up-regulated two-fold in Down syndrome samples compared to normal samples; of these, SON and TTC3 were newly reported. CBR1, DYRK1A, HMGN1, ITSN1, RCAN1, SON, TMEM50B, and TTC3 were located on human chromosome 21 (mouse chromosome 16). The DEGs were significantly enriched in macromolecular complex subunit organization and focal adhesion pathways. Eleven significantly enriched transcription factor motifs (PAX5, EGR1, XBP1, SREBP1, OLF1, MZF1, NFY, NFKAPPAB, MYCMAX, NFE2, and RP58) were identified. The DEGs and transcription factor motifs identified in our study provide biomarkers for the understanding of Down syndrome pathogenesis and progression.

Animals , Humans , Mice , Rats , Amino Acid Motifs/genetics , Computational Biology/methods , Down Syndrome/genetics , Gene Regulatory Networks/genetics , Transcription Factors/analysis , Algorithms , Biomarkers/analysis , Databases, Genetic , Down Syndrome/etiology , Gene Expression , Gene Ontology , Molecular Sequence Annotation/methods , Phenotype , Protein Array Analysis/methods , Up-Regulation/genetics
Article in English | WPRIM | ID: wpr-232680


We developed a novel simple microwell plate that allows researchers to culture cells on chips easily without the use of tubes and pumps for studies of cell proliferation, apoptosis and pharmacology. Unlike any other previous design, this device can be put into an incubator directly and maintains cells in an environment with stable temperature, humidity, air pressure, oxygen and CO2 concentration. We cultured PC-12 cells in these microwell plates and measured the cell proliferation and apoptosis. The microwell plate is recommended for use in all research including those involving rare samples and expensive reagents.

Animals , Rats , Apoptosis , Cell Culture Techniques , Cell Proliferation , PC12 Cells , Protein Array Analysis