RESUMO
Hepatitis B virus (HBV) infection is a leading cause of liver cirrhosis, liver cancer, and liver failure, affecting 350 million people worldwide. Currently available anti-HBV drugs include (PEGylated-) interferon-α and nucleos(t)ide analogs, which can cause significant side effects and drug-resistance in many cases of long-term treatment. The lack of a reliable and robust in vitro infection system is a major barrier for understanding the HBV life cycle and discovering novel therapeutic targets. In the present study, we demonstrate that overexpression of the hepatitis B surface antigen binding protein (SBP) in HepG2 cells (HepG2-SBP) resulted in their susceptibility to HBV infection. HepG2-SBP cells supported the uptake of the viral surface protein (HBsAg-preS), HBV-pseudotyped virus, and live HBV in patient sera. Moreover, SBP-mediated HBsAg-preS uptake, and HBV pseudotyped virus infections were efficiently blocked by preS1- and SBP-specific antibodies. These observations suggest that SBP is involved in HBV entry and that HepG2-SBP cells can serve as a cellular model to study the post-binding steps of HBV infection.
Assuntos
Proteínas de Transporte/metabolismo , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Receptores Virais/metabolismo , Internalização do Vírus , Células Hep G2 , HumanosRESUMO
HER2 is an orphan receptor tyrosine kinase of the EGFR families and is considered to be a key tumor driver gene [1]. Breast cancer and gastric cancer with HER2 amplification can be effectively treated by its neutralizing antibody, Herceptin. In clinic, Immunohistochemistry (IHC) was used as the primary screening method to diagnose HER2 amplification [2]. However, recent evidence suggested that the frequently used rabbit HER2 antibody 4B5 cross reacted with another family member HER4 [3]. IHC staining with 4B5 also indicated that there was strong non-specific cytoplasmic and nuclear signals in normal gastric mucosal cells and some gastric cancer samples. Using a protein lysate array which covers 85% of the human proteome, we have confirmed that the 4B5 bound to HER4 and a nuclear protein ZSCAN18 besides HER2. The non-specific binding accounts for the unexpected cytoplasmic and nuclear staining of 4B5 of normal gastric epithelium. Finally, we have developed a novel mouse HER2 monoclonal antibody UMAB36 with similar sensitivity to 4B5 but only reacted to HER2 across the 17,000 proteins on the protein chip. In 129 breast cancer and 158 gastric cancer samples, UMAB36 showed 100% sensitivity and specificity comparing to the HER2 FISH reference results with no unspecific staining in the gastric mucosa layer. Therefore, UMAB36 could provide as an alternative highly specific IHC reagent for testing HER2 amplification in gastric cancer populations.
Assuntos
Anticorpos Monoclonais/imunologia , Receptor ErbB-2/imunologia , Especificidade de Anticorpos , Linhagem Celular Tumoral , Reações Cruzadas , Humanos , Imuno-Histoquímica , Análise Serial de Proteínas , Neoplasias Gástricas/imunologiaRESUMO
BACKGROUND: An antibody with cross-reactivity can create unexpected side effects or false diagnostic reports if used for clinical purposes. ERCC1 is being explored as a predictive diagnostic biomarker for cisplatin-based chemotherapy. High ERCC1 expression is linked to drug resistance on cisplatin-based chemotherapy. 8F1 is one of the most commonly used monoclonal antibodies for evaluating ERCC1 expression levels in lung cancer patient tissues, but it has been noted that this antibody cross-reacts with an unknown protein. RESULTS: By using a high density protein microarray chip technology, we discovered that 8F1 not only reacts with its authentic target, ERCC1, but also cross-reacts with an unrelated nuclear membrane protein, PCYT1A. The cross-reactivity is due to a common epitope presented on these two unrelated proteins. Similar to the subcellular localization of ERCC1, IHC tests demonstrated that PCYT1A is localized mainly on nuclear membrane. In this study, we also discovered that the PCYT1A gene expression level is significantly higher than the ERCC1 gene expression level in a certain population of lung cancer patient tissue samples. To develop the best monoclonal antibody for ERCC1 IHC analysis, 18 monoclonal antibodies were generated and 6 of them were screened against our protein microarray chip. Two clones showed high mono-specificity on the protein microarray chip test and both worked for the IHC application. CONCLUSION: In summary, the 8F1 clone is not suitable for ERCC1 IHC assay due to its cross-reactivity with PCYT1A protein. Two newly generated monoclonal antibodies, 4F9 and 2E12, demonstrated ultra-specificity against ERCC1 protein and superior performance for IHC analyses.
Assuntos
Anticorpos Monoclonais/química , Biomarcadores Tumorais/imunologia , Proteínas de Ligação a DNA/imunologia , Endonucleases/imunologia , Análise Serial de Proteínas/métodos , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/química , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Colina-Fosfato Citidililtransferase/imunologia , Colina-Fosfato Citidililtransferase/metabolismo , Reações Cruzadas , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Células HEK293 , Humanos , Imuno-Histoquímica/métodos , Neoplasias Pulmonares/química , Neoplasias Pulmonares/metabolismoAssuntos
Diferenciação Celular/genética , Reprogramação Celular/genética , Fibroblastos/metabolismo , Redes Reguladoras de Genes/genética , Células-Tronco Pluripotentes/fisiologia , Adulto , Técnicas de Cultura de Células , Ensaio de Unidades Formadoras de Colônias , Fibroblastos/citologia , Prepúcio do Pênis/citologia , Humanos , Masculino , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Células-Tronco Pluripotentes/citologia , RNA Interferente Pequeno , Transativadores/genética , Transativadores/metabolismo , Transfecção , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Mesenchymal stem cells (MSCs) have already been proved to be multipotent. Our goal was to evaluate the differentiating ability of rat MSCs into insulin-secreting cells in vitro to cure diabetes resulting from abnormal function of pancreatic islets. MSCs were identified by Fluorescence-activated cell sorting (FACS). Pdx1 is a transcription factor involved in the early endocrine development. Betacellulin (BTC) is a growth factor involved in beta-cell maturation. MSCs were transfected with plasmids carrying rat Pdx1 and BTC genes. Coexpression of Pdx1 and BTC significantly increased the number of nestin-positive epithelium-like progenitors and islet-like spheroids which differentiated from MSCs. In Pdx1- and BTC-expressed (Pdx1+ + BTC+) MSCs, insulin and Glut-2 mRNA levels significantly rose. The number of islet-like cells was also evidently augmented. In response to glucose, Pdx1+ + BTC+ MSCs released insulin and C-peptide. It is concluded that genetic manipulation of transcription factor Pdx1 and growth factor BTC in combination with appropriate differentiating culture could induce MSCs into the pancreatic lineage in vitro and produce islet-like spheroids that could secrete increased levels of insulin in response to glucose.
Assuntos
Diferenciação Celular/fisiologia , Proteínas de Homeodomínio/biossíntese , Células Secretoras de Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Proteínas de Filamentos Intermediários/biossíntese , Células-Tronco Mesenquimais/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Esferoides Celulares/metabolismo , Transativadores/biossíntese , Animais , Betacelulina , Células Cultivadas , Epitélio/metabolismo , Expressão Gênica , Proteínas de Homeodomínio/genética , Células Secretoras de Insulina/citologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Filamentos Intermediários/genética , Células-Tronco Mesenquimais/citologia , Proteínas do Tecido Nervoso/genética , Nestina , Ratos , Ratos Sprague-Dawley , Esferoides Celulares/citologia , Transativadores/genéticaRESUMO
Severe acute respiratory syndrome coronavirus (SARS-CoV) is the pathogen of SARS, which caused a global panic in 2003. We describe here the screening of Chinese herbal medicine-based, novel small molecules that bind avidly with the surface spike protein of SARS-CoV and thus can interfere with the entry of the virus to its host cells. We achieved this by using a two-step screening method consisting of frontal affinity chromatography-mass spectrometry coupled with a viral infection assay based on a human immunodeficiency virus (HIV)-luc/SARS pseudotyped virus. Two small molecules, tetra-O-galloyl-beta-D-glucose (TGG) and luteolin, were identified, whose anti-SARS-CoV activities were confirmed by using a wild-type SARS-CoV infection system. TGG exhibits prominent anti-SARS-CoV activity with a 50% effective concentration of 4.5 microM and a selective index of 240.0. The two-step screening method described here yielded several small molecules that can be used for developing new classes of anti-SARS-CoV drugs and is potentially useful for the high-throughput screening of drugs inhibiting the entry of HIV, hepatitis C virus, and other insidious viruses into their host cells.
Assuntos
Antivirais/farmacologia , Flavonoides/farmacologia , Taninos Hidrolisáveis/farmacologia , Plantas Medicinais/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/patogenicidade , Animais , Antivirais/química , Antivirais/metabolismo , Linhagem Celular , China , Chlorocebus aethiops , Cromatografia de Afinidade , HIV-1/genética , HIV-1/metabolismo , Humanos , Taninos Hidrolisáveis/química , Luciferases/genética , Luciferases/metabolismo , Luteolina , Espectrometria de Massas , Glicoproteínas de Membrana/metabolismo , Testes de Sensibilidade Microbiana , Plantas Medicinais/metabolismo , Glicoproteína da Espícula de Coronavírus , Células Vero , Proteínas do Envelope Viral/metabolismoRESUMO
Heptad repeat regions (HR1 and HR2) are highly conserved sequences located in the glycoproteins of enveloped viruses. They form a six-helix bundle structure and are important in the process of virus fusion. Peptides derived from the HR regions of some viruses have been shown to inhibit the entry of these viruses. SARS-CoV was also predicted to have HR1 and HR2 regions in the S2 protein. Based on this prediction, we designed 25 peptides and screened them using a HIV-luc/SARS pseudotyped virus assay. Two peptides, HR1-1 and HR2-18, were identified as potential inhibitors, with EC(50) values of 0.14 and 1.19microM, respectively. The inhibitory effects of these peptides were validated by the wild-type SARS-CoV assay. HR1-1 and HR2-18 can serve as functional probes for dissecting the fusion mechanism of SARS-CoV and also provide the potential of further identifying potent inhibitors for SARS-CoV entry.