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1.
Emerg Infect Dis ; 27(10): 2718-2720, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34545803

RESUMO

Genomic surveillance can provide early insights into new circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. While conducting genomic surveillance (1,663 cases) from December 2020-April 2021 in Arizona, USA, we detected an emergent E484K-harboring variant, B.1.243.1. This finding demonstrates the importance of real-time SARS-CoV-2 surveillance to better inform public health responses.


Assuntos
COVID-19 , SARS-CoV-2 , Arizona/epidemiologia , Genômica , Humanos , Saúde Pública
2.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445351

RESUMO

Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell-cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other.


Assuntos
Diagnóstico por Imagem/métodos , Proteínas/metabolismo , Análise de Célula Única/métodos , Anticorpos/metabolismo , Comunicação Celular , Imunofluorescência/métodos , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacocinética , Formaldeído/química , Peroxidase do Rábano Silvestre/análise , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Técnicas Imunoenzimáticas/métodos , Tonsila Palatina/química , Tonsila Palatina/citologia , Tonsila Palatina/metabolismo , Inclusão em Parafina , Proteínas/análise , Sensibilidade e Especificidade , Coloração e Rotulagem/métodos
3.
Cells ; 10(6)2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34063986

RESUMO

Understanding the composition, regulation, and function of complex biological systems requires tools that quantify multiple transcripts at their native cellular locations. However, the current multiplexed RNA imaging technologies are limited by their relatively low sensitivity or specificity, which hinders their applications in studying highly autofluorescent tissues, such as formalin-fixed paraffin-embedded (FFPE) tissues. To address this issue, here we develop a multiplexed in situ RNA profiling approach with a high sensitivity and specificity. In this approach, transcripts are first hybridized by target-specific oligonucleotide probes in pairs. Only when these two independent probes hybridize to the target in tandem will the subsequent signal amplification by oligonucleotide hybridization occur. Afterwards, horseradish peroxidase (HRP) is applied to further amplify the signal and stain the target with cleavable fluorescent tyramide (CFT). After imaging, the fluorophores are chemically cleaved and the hybridized probes are stripped by DNase and formamide. Through cycles of RNA staining, fluorescence imaging, signal cleavage, and probe stripping, many different RNA species can be profiled at the optical resolution. In applying this approach, we demonstrated that multiplexed in situ RNA analysis can be successfully achieved in both fixed, frozen, and FFPE tissues.

4.
Methods Mol Biol ; 2344: 3-6, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34115348

RESUMO

As we approach the twentieth anniversary of completing the international Human Genome Project, the next (and arguably most significant) frontier in biology consists of functionally understanding the proteins, which are encoded by the genome and play a crucial role in all of biology and medicine. To accomplish this challenge, different proteomics strategies must be devised to examine the activities of gene products (proteins) at scale. Among them, protein microarrays have been used to accomplish a wide variety of investigations such as examining the binding of proteins and proteoforms to DNA, small molecules, and other proteins; characterizing humoral immune responses in health and disease; evaluating allergenic proteins; and profiling protein patterns as candidate disease-specific biomarkers. In Protein Microarray for Disease Analysis: Methods and Protocols, expert researchers involved in the field of protein microarrays provide concise descriptions of the methodologies that they currently use to fabricate microarrays and how they apply them to analyze protein interactions and responses of proteins to dissect human disease.


Assuntos
Doenças Transmissíveis/diagnóstico , Análise Serial de Proteínas , Proteínas/análise , Proteômica , Humanos
5.
Methods Mol Biol ; 2344: 47-64, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34115351

RESUMO

A novel protein microarray technology, called high-density nucleic acid programmable protein array (HD-NAPPA), enables the serological screening of thousands of proteins at one time. HD-NAPPA extends the capabilities of NAPPA, which produces protein microarrays on a conventional glass microscope slide. By comparison, HD-NAPPA displays proteins in over 10,000 nanowells etched in a silicon slide. Proteins on HD-NAPPA are expressed in the individual isolated nanowells, via in vitro transcription and translation (IVTT), without any diffusion during incubation. Here we describe the method for antibody biomarker identification using HD-NAPPA, including four main steps: (1) HD-NAPPA array protein expression, (2) primary antibodies (serum/plasma) probing, (3) secondary antibody visualization, and (4) image scanning and data processing.


Assuntos
Anticorpos/química , Análise Serial de Proteínas , Biomarcadores/análise , Humanos
6.
Molecules ; 26(8)2021 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-33921211

RESUMO

Understanding the composition, function and regulation of complex cellular systems requires tools that quantify the expression of multiple proteins at their native cellular context. Here, we report a highly sensitive and accurate protein in situ profiling approach using off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this method, protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and CFT. Subsequently, the fluorophores are efficiently cleaved by mild chemical reagents, which simultaneously deactivate HRP. Through reiterative cycles of protein staining, fluorescence imaging, fluorophore cleavage, and HRP deactivation, multiplexed protein quantification in single cells in situ can be achieved. We designed and synthesized the high-performance CFT, and demonstrated that over 95% of the staining signals can be erased by mild chemical reagents while preserving the integrity of the epitopes on protein targets. Applying this method, we explored the protein expression heterogeneity and correlation in a group of genetically identical cells. With the high signal removal efficiency, this approach also enables us to accurately profile proteins in formalin-fixed paraffin-embedded (FFPE) tissues in the order of low to high and also high to low expression levels.


Assuntos
Amidas/metabolismo , Corantes Fluorescentes/metabolismo , Proteômica , Epitopos/metabolismo , Células HeLa , Peroxidase do Rábano Silvestre , Humanos , Proteínas do Fator Nuclear 90/metabolismo , Tonsila Palatina/metabolismo , Inclusão em Parafina , Análise de Célula Única , Fixação de Tecidos
7.
Gastric Cancer ; 24(4): 858-867, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33661412

RESUMO

BACKGROUND: Around 10% of gastric carcinomas (GC) contain Epstein-Barr virus (EBV) DNA. We characterized the GC-specific antibody response to this common infection, which may provide a noninvasive method to detect EBV-positive GC and elucidate its contribution to carcinogenesis. METHODS: Plasma samples from EBV-positive (n = 28) and EBV-negative (n = 34) Latvian GC patients were immune-profiled against 85 EBV proteins on a multi-microbial Nucleic Acid Programmable Protein Array (EBV-NAPPA). Antibody responses were normalized for each sample as ratios to the median signal intensity (MNI) across all antigens, with seropositivity defined as MNI ≥ 2. Antibodies with ≥ 20% sensitivity at 95% specificity for tumor EBV status were verified by enzyme-linked immunosorbent assay (ELISA) and validated in independent samples from Korea and Poland (n = 24 EBV-positive, n = 65 EBV-negative). RESULTS: Forty anti-EBV IgG and eight IgA antibodies were detected by EBV-NAPPA in ≥ 10% of EBV-positive or EBV-negative GC patients, of which nine IgG antibodies were discriminative for tumor EBV status. Eight of these nine were verified and seven were validated by ELISA: anti-LF2 (odds ratio = 110.0), anti-BORF2 (54.2), anti-BALF2 (44.1), anti-BaRF1 (26.7), anti-BXLF1 (12.8), anti-BRLF1 (8.3), and anti-BLLF3 (5.4). The top three had areas under receiver operating characteristics curves of 0.81-0.85 for distinguishing tumor EBV status. CONCLUSIONS: The EBV-associated GC-specific humoral response was exclusively directed against lytic cycle immediate-early and early antigens, unlike other EBV-associated malignancies such as nasopharyngeal carcinoma and lymphoma where humoral response is primarily directed against late lytic antigens. Specific anti-EBV antibodies could have utility for clinical diagnosis, epidemiologic studies, and immune-based precision treatment of EBV-positive GC.

8.
J Proteome Res ; 20(1): 409-419, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33108201

RESUMO

Chronic Helicobacter pylori infection is the major risk factor for gastric cancer (GC). However, only some infected individuals develop this neoplasia. Previous H. pylori serology studies have been limited by investigating small numbers of candidate antigens. Therefore, we evaluated humoral responses to a nearly complete H. pylori immunoproteome (1527 proteins) among 50 GC cases and 50 controls using Nucleic Acid Programmable Protein Array (NAPPA). Seropositivity was defined as median normalized intensity ≥2 on NAPPA, and 53 anti-H. pylori antibodies had >10% seroprevalence. Anti-GroEL exhibited the greatest seroprevalence (77% overall), which agreed well with ELISA using whole-cell lysates of H. pylori cells. After an initial screen by H. pylori-NAPPA, we discovered and verified that 12 antibodies by ELISA in controls had ≥15% of samples with an optical reading value exceeding the 95th percentile of the GC group. ELISA-verified antibodies were validated blindly in an independent set of 100 case-control pairs. As expected, anti-CagA seropositivity was positively associated with GC (odds ratio, OR = 5.5; p < 0.05). After validation, six anti-H. pylori antibodies showed lower seropositivity in GC, with ORs ranging from 0.44 to 0.12 (p < 0.05): anti-HP1118/Ggt, anti-HP0516/HsIU, anti-HP0243/NapA, anti-HP1293/RpoA, anti-HP0371/FabE, and anti-HP0875/KatA. Among all combinations, a model with anti-Ggt, anti-HslU, anti-NapA, and anti-CagA had an area under the curve of 0.73 for discriminating GC vs. controls. This study represents the first comprehensive assessment of anti-H. pylori humoral profiles in GC. Decreased responses to multiple proteins in GC may reflect mucosal damage and decreased bacterial burden. The higher prevalence of specific anti-H. pylori antibodies in controls may suggest immune protection against GC development.


Assuntos
Infecções por Helicobacter , Helicobacter pylori , Neoplasias Gástricas , Anticorpos Antibacterianos , Antígenos de Bactérias , Proteínas de Bactérias , Infecções por Helicobacter/diagnóstico , Infecções por Helicobacter/epidemiologia , Humanos , Estudos Soroepidemiológicos
9.
Nat Commun ; 11(1): 5301, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067450

RESUMO

The Human Proteome Organization (HUPO) launched the Human Proteome Project (HPP) in 2010, creating an international framework for global collaboration, data sharing, quality assurance and enhancing accurate annotation of the genome-encoded proteome. During the subsequent decade, the HPP established collaborations, developed guidelines and metrics, and undertook reanalysis of previously deposited community data, continuously increasing the coverage of the human proteome. On the occasion of the HPP's tenth anniversary, we here report a 90.4% complete high-stringency human proteome blueprint. This knowledge is essential for discerning molecular processes in health and disease, as we demonstrate by highlighting potential roles the human proteome plays in our understanding, diagnosis and treatment of cancers, cardiovascular and infectious diseases.


Assuntos
Doença/genética , Proteoma/genética , Projeto Genoma Humano , Humanos , Proteoma/química , Proteoma/metabolismo , Proteômica
10.
Cancer Epidemiol Biomarkers Prev ; 29(12): 2475-2485, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32994341

RESUMO

In spite of the progress made in treatment and early diagnosis, breast cancer remains a major public health issue worldwide. Although modern image-based screening modalities have significantly improved early diagnosis, around 15% to 20% of breast cancers still go undetected. In underdeveloped countries, lack of resources and cost concerns prevent implementing mammography for routine screening. Noninvasive, low-cost, blood-based markers for early breast cancer diagnosis would be an invaluable alternative that would complement mammography screening. Tumor-specific autoantibodies are excellent biosensors that could be exploited to monitor disease-specific changes years before disease onset. Although clinically informative autoantibody markers for early breast cancer screening have yet to emerge, progress has been made in the development of tools to discover and validate promising autoantibody signatures. This review focuses on the current progress toward the development of autoantibody-based early screening markers for breast cancer.See all articles in this CEBP Focus section, "NCI Early Detection Research Network: Making Cancer Detection Possible."

11.
Sci Rep ; 10(1): 13323, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770037

RESUMO

Baculovirus mediated-insect cell expression systems have been widely used for producing heterogeneous proteins. However, to date, there is still the lack of an easy-to-manipulate system that enables the high-throughput protein characterization in insect cells by taking advantage of large existing Gateway clone libraries. To resolve this limitation, we have constructed a suite of Gateway-compatible pIEx-derived baculovirus expression vectors that allow the rapid and cost-effective construction of expression clones for mass parallel protein expression in insect cells. This vector collection also supports the attachment of a variety of fusion tags to target proteins to meet the needs for different research applications. We first demonstrated the utility of these vectors for protein expression and purification using a set of 40 target proteins of various sizes, cellular localizations and host organisms. We then established a scalable pipeline coupled with the SONICC and TEM techniques to screen for microcrystal formation within living insect cells. Using this pipeline, we successfully identified microcrystals for ~ 16% of the tested protein set, which can be potentially used for structure elucidation by X-ray crystallography. In summary, we have established a versatile pipeline enabling parallel gene cloning, protein expression and purification, and in vivo microcrystal screening for structural studies.


Assuntos
Baculoviridae , Expressão Gênica , Vetores Genéticos , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Cristalografia por Raios X , Microscopia Eletrônica de Transmissão , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Células Sf9 , Spodoptera
12.
Sci Rep ; 10(1): 12318, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32703985

RESUMO

To further understand the molecular pathogenesis of desmoplastic small round cell tumor (DSRCT), a fatal malignancy occurring primarily in adolescent/young adult males, we used next-generation RNA sequencing to investigate the gene expression profiles intrinsic to this disease. RNA from DSRCT specimens obtained from the Children's Oncology Group was sequenced using the Illumina HiSeq 2000 system and subjected to bioinformatic analyses. Validation and functional studies included WT1 ChIP-seq, EWS-WT1 knockdown using JN-DSRCT-1 cells and immunohistochemistry. A panel of immune signature genes was also evaluated to identify possible immune therapeutic targets. Twelve of 14 tumor samples demonstrated presence of the diagnostic EWSR1-WT1 translocation and these 12 samples were used for the remainder of the analysis. RNA sequencing confirmed the lack of full-length WT1 in all fusion positive samples as well as the JN-DSRCT-1 cell line. ChIP-seq for WT1 showed significant overlap with genes found to be highly expressed, including IGF2 and FGFR4, which were both highly expressed and targets of the EWS-WT1 fusion protein. In addition, we identified CD200 and CD276 as potentially targetable immune checkpoints whose expression is independent of the EWS-WT1 fusion gene in cultured DSCRT cells. In conclusion, we identified IGF2, FGFR4, CD200, and CD276 as potential therapeutic targets with clinical relevance for patients with DSRCT.


Assuntos
Tumor Desmoplásico de Pequenas Células Redondas/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Oncologia , Terapia de Alvo Molecular , Adolescente , Linhagem Celular Tumoral , Criança , Pré-Escolar , Análise por Conglomerados , Feminino , Humanos , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Masculino , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica
13.
J Proteome Res ; 19(7): 2525-2528, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32610914
14.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 6): 278-289, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32510469

RESUMO

µNS is a 70 kDa major nonstructural protein of avian reoviruses, which cause significant economic losses in the poultry industry. They replicate inside viral factories in host cells, and the µNS protein has been suggested to be the minimal viral factor required for factory formation. Thus, determining the structure of µNS is of great importance for understanding its role in viral infection. In the study presented here, a fragment consisting of residues 448-605 of µNS was expressed as an EGFP fusion protein in Sf9 insect cells. EGFP-µNS(448-605) crystallization in Sf9 cells was monitored and verified by several imaging techniques. Cells infected with the EGFP-µNS(448-605) baculovirus formed rod-shaped microcrystals (5-15 µm in length) which were reconstituted in high-viscosity media (LCP and agarose) and investigated by serial femtosecond X-ray diffraction using viscous jets at an X-ray free-electron laser (XFEL). The crystals diffracted to 4.5 Šresolution. A total of 4227 diffraction snapshots were successfully indexed into a hexagonal lattice with unit-cell parameters a = 109.29, b = 110.29, c = 324.97 Å. The final data set was merged and refined to 7.0 Šresolution. Preliminary electron-density maps were obtained. While more diffraction data are required to solve the structure of µNS(448-605), the current experimental strategy, which couples high-viscosity crystal delivery at an XFEL with in cellulo crystallization, paves the way towards structure determination of the µNS protein.


Assuntos
Elétrons , Lasers , Proteínas Recombinantes de Fusão/química , Reoviridae/metabolismo , Proteínas não Estruturais Virais/química , Difração de Raios X/métodos , Animais , Cristalização , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Células Sf9 , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Viscosidade , Raios X
15.
Cells ; 9(4)2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32244728

RESUMO

The ability to perform highly sensitive and multiplexed in-situ protein analysis is crucial to advance our understanding of normal physiology and disease pathogenesis. To achieve this goal, we here develop an approach using cleavable biotin-conjugated antibodies and cleavable fluorescent streptavidin (CFS). In this approach, protein targets are first recognized by the cleavable biotin-labeled antibodies. Subsequently, CFS is applied to stain the protein targets. Though layer-by-layer signal amplification using cleavable biotin-conjugated orthogonal antibodies and CSF, the protein detection sensitivity can be enhanced at least 10-fold, compared with the current in-situ proteomics methods. After imaging, the fluorophore and the biotin unbound to streptavidin are removed by chemical cleavage. The leftover streptavidin is blocked by biotin. Upon reiterative analysis cycles, a large number of different proteins with a wide range of expression levels can be profiled in individual cells at the optical resolution. Applying this approach, we have demonstrated that multiple proteins are unambiguously detected in the same set of cells, regardless of the protein analysis order. We have also shown that this method can be successfully applied to quantify proteins in formalin-fixed paraffin-embedded (FFPE) tissues.


Assuntos
Proteínas/análise , Estreptavidina/química , Anticorpos/metabolismo , Biotina/química , Fluorescência , Corantes Fluorescentes/química , Formaldeído/química , Células HeLa , Histonas/metabolismo , Humanos , Antígeno Ki-67/metabolismo , Lisina/metabolismo , Metilação , Inclusão em Parafina , Fixação de Tecidos
16.
Biomaterials ; 247: 119975, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32278213

RESUMO

The tumor microenvironment has been demonstrated to play a crucial role in modulating cancer progression. Amongst various cell types within the tumor microenvironment, cancer associated fibroblasts (CAFs) are in abundance, serving to modulate the biophysical properties of the stromal matrix, through excessive deposition of extracellular matrix (ECM) proteins that leads to enhanced tumor progression. There is still a critical need to develop a fundamental framework on the role of tumor-stromal cell interactions on desmoplasia and tumorigenicity. Herein, we developed a 3D microengineered organotypic tumor-stroma model incorporated with breast cancer cells surrounded by CAF-embedded collagen matrix. We further integrated our platform with atomic force microscopy (AFM) to study the dynamic changes in stromal stiffness during active tumor invasion. Our findings primarily demonstrated enhanced tumor progression in the presence of CAFs. Furthermore, we highlighted the crucial role of crosstalk between tumor cells and CAFs on stromal desmoplasia, where we identified the role of tumor-secreted PDGF-AA/-BB on elevated matrix stiffness. Inhibition of the activity of PDGFRs in CAFs led to attenuation of stromal stiffness. Overall, our work presents a well-controlled tumor microenvironment model capable of dissecting specific biophysical and biochemical signaling cues which lead to stromal desmoplasia and tumor progression.


Assuntos
Fibroblastos Associados a Câncer , Neoplasias , Fibroblastos , Humanos , Transdução de Sinais , Células Estromais , Microambiente Tumoral
17.
J Vis Exp ; (152)2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31710025

RESUMO

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.


Assuntos
Análise Serial de Proteínas/métodos , Inibidores de Proteínas Quinases/farmacologia , Humanos , Proteínas Quinases
18.
Ann Rheum Dis ; 78(12): 1699-1705, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31471297

RESUMO

OBJECTIVE: To find autoantibodies (AAbs) in serum that could be useful to predict incidence of radiographic knee osteoarthritis (KOA). DESIGN: A Nucleic-acid Programmable Protein Arrays (NAPPA) platform was used to screen AAbs against 2125 human proteins in sera at baseline from participants free of radiographic KOA belonging to the incidence and non-exposed subcohorts of the Osteoarthritis Initiative (OAI) who developed or not, radiographic KOA during a follow-up period of 96 months. NAPPA-ELISA were performed to analyse reactivity against methionine adenosyltransferase two beta (MAT2ß) and verify the results in 327 participants from the same subcohorts. The association of MAT2ß-AAb levels with KOA incidence was assessed by combining several robust biostatistics analysis (logistic regression, Receiver Operating Characteristic and Kaplan-Meier curves). The proposed prognostic model was replicated in samples from the progression subcohort of the OAI. RESULTS: In the screening phase, six AAbs were found significantly different at baseline in samples from incident compared with non-incident participants. In the verification phase, high levels of MAT2ß-AAb were significantly associated with the future incidence of KOA and with an earlier development of the disease. The incorporation of this AAb in a clinical model for the prognosis of incident radiographic KOA significantly improved the identification/classification of patients who will develop the disorder. The usefulness of the model to predict radiographic KOA was confirmed on a different OAI subcohort. CONCLUSIONS: The measurement of AAbs against MAT2ß in serum might be highly useful to improve the prediction of OA development, and also to estimate the time to incidence.


Assuntos
Autoanticorpos/sangue , Diagnóstico Precoce , Articulação do Joelho/diagnóstico por imagem , Osteoartrite do Joelho/diagnóstico , Autoanticorpos/imunologia , Biomarcadores/sangue , Estudos de Casos e Controles , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Seguimentos , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/sangue , Osteoartrite do Joelho/epidemiologia , Valor Preditivo dos Testes , Curva ROC , Radiografia , Espanha/epidemiologia
19.
Artigo em Inglês | MEDLINE | ID: mdl-31245298

RESUMO

The identification of microbial biomarkers is critical for the diagnosis of a disease early during infection. However, the identification of reliable biomarkers is often hampered by a low concentration of microbes or biomarkers within host fluids or tissues. We have outlined a multi-platform strategy to assess microbial biomarkers that can be consistently detected in host samples, using Borrelia burgdorferi, the causative agent of Lyme disease, as an example. Key aspects of the strategy include the selection of a macaque model of human disease, in vivo Microbial Antigen Discovery (InMAD), and proteomic methods that include microbial biomarker enrichment within samples to identify secreted proteins circulating during infection. Using the described strategy, we have identified 6 biomarkers from multiple samples. In addition, the temporal antibody response to select bacterial antigens was mapped. By integrating biomarkers identified from early infection with temporal patterns of expression, the described platform allows for the data driven selection of diagnostic targets.


Assuntos
Biomarcadores , Borrelia burgdorferi/isolamento & purificação , Doença de Lyme/diagnóstico , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Técnicas Bacteriológicas , Biomarcadores/sangue , Biomarcadores/urina , Borrelia burgdorferi/imunologia , Diagnóstico Precoce , Humanos , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Macaca mulatta , Proteômica , Soro/química , Urina/química
20.
Cancer Res ; 79(12): 3139-3151, 2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-30992322

RESUMO

Tumor-stroma interactions significantly influence cancer cell metastasis and disease progression. These interactions are partly comprised of the cross-talk between tumor and stromal fibroblasts, but the key molecular mechanisms within the cross-talk that govern cancer invasion are still unclear. Here, we adapted our previously developed microfluidic device as a 3D in vitro organotypic model to mechanistically study tumor-stroma interactions by mimicking the spatial organization of the tumor microenvironment on a chip. We cocultured breast cancer and patient-derived fibroblast cells in 3D tumor and stroma regions, respectively, and combined functional assessments, including cancer cell migration, with transcriptome profiling to unveil the molecular influence of tumor-stroma cross-talk on invasion. This led to the observation that cancer-associated fibroblasts (CAF) enhanced invasion in 3D by inducing expression of a novel gene of interest, glycoprotein nonmetastatic B (GPNMB), in breast cancer cells, resulting in increased migration speed. Importantly, knockdown of GPNMB blunted the influence of CAF on enhanced cancer invasion. Overall, these results demonstrate the ability of our model to recapitulate patient-specific tumor microenvironments to investigate the cellular and molecular consequences of tumor-stroma interactions. SIGNIFICANCE: An organotypic model of tumor-stroma interactions on a microfluidic chip reveals that CAFs promote invasion by enhancing expression of GPNMB in breast cancer cells.


Assuntos
Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/patologia , Fibroblastos/patologia , Glicoproteínas de Membrana/metabolismo , Técnicas Analíticas Microfluídicas/métodos , Organoides/patologia , Microambiente Tumoral , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Movimento Celular , Técnicas de Cocultura , Feminino , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Humanos , Glicoproteínas de Membrana/genética , Modelos Biológicos , Invasividade Neoplásica , Organoides/metabolismo
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