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Frontiers in Immunology 2019Determining antigen specificity is vital for understanding B cell biology and for producing human monoclonal antibodies. We describe here a powerful method for...
Determining antigen specificity is vital for understanding B cell biology and for producing human monoclonal antibodies. We describe here a powerful method for identifying B cells that recognize membrane antigens expressed on cells. The technique depends on two characteristics of the interaction between a B cell and an antigen-expressing cell: antigen-receptor-mediated extraction of antigen from the membrane of the target cell, and B cell activation. We developed the method using influenza hemagglutinin as a model viral membrane antigen, and tested it using acetylcholine receptor (AChR) as a model membrane autoantigen. The technique involves co-culturing B cells with adherent, bioorthogonally labeled cells expressing GFP-tagged antigen, and sorting GFP-capturing, newly activated B cells. Hemagglutinin-specific B cells isolated this way from vaccinated human donors expressed elevated CD20, CD27, CD71, and CD11c, and reduced CD21, and their secreted antibodies blocked hemagglutination and neutralized viral infection. Antibodies cloned from AChR-capturing B cells derived from patients with myasthenia gravis bound specifically to the receptor on cell membrane. The approach is sensitive enough to detect antigen-specific B cells at steady state, and can be adapted for any membrane antigen.
Topics: Adult; Aged; Animals; Antigens, Surface; Autoantigens; B-Lymphocyte Subsets; B-Lymphocytes; Cell Line, Tumor; Cell Separation; Clone Cells; Epitopes, B-Lymphocyte; Hemagglutinin Glycoproteins, Influenza Virus; Humans; Immunophenotyping; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Middle Aged; Myasthenia Gravis; Receptors, Cholinergic
PubMed: 31040853
DOI: 10.3389/fimmu.2019.00829 -
Methods in Molecular Biology (Clifton,... 2017Larger extracellular vesicles, microparticles (MPs) or microvesicles (MVs), especially their acquisition and characterization by flow cytometry (FACS), is increasingly...
Larger extracellular vesicles, microparticles (MPs) or microvesicles (MVs), especially their acquisition and characterization by flow cytometry (FACS), is increasingly in focus of clinical/translational research efforts. Several laboratories have shown that MPs/MVs might be suitable for the diagnosis and predicting prognosis in various diseases including cancer. However, FACS staining of larger extracellular vesicles (EVs) can be difficult and results potentially in false positive and inconsistent data interpretation. Despite that FACS equipment is well maintained and the operators have ample experience, a reliable and for larger EVs optimized staining protocol is missing. Here, we aim to close that gap and provide a working multi-antibody staining protocol for larger EVs isolated from human serum samples. We describe in detail the needed steps as currently done in our laboratory. Staining is demonstrated exemplarily for multi-antibody mix including CD147 , a potential cancer marker if applied in combination with other MP/MV surface markers.
Topics: Antigens, Surface; Biomarkers; Extracellular Vesicles; Flow Cytometry; Humans; Staining and Labeling
PubMed: 28828658
DOI: 10.1007/978-1-4939-7253-1_16 -
Journal of Controlled Release :... Oct 2022Hepatitis B virus (HBV) can rapidly replicate in the hepatocytes after transmission, leading to chronic hepatitis, liver cirrhosis and eventually hepatocellular...
Hepatitis B virus (HBV) can rapidly replicate in the hepatocytes after transmission, leading to chronic hepatitis, liver cirrhosis and eventually hepatocellular carcinoma. Interferon-α (IFN-α) is included in the standard treatment for chronic hepatitis B (CHB). However, this therapy causes serious side effects. Delivering IFN-α selectively to the liver may enhance its efficacy and safety. Imiquimod (IMQ), a Toll-Like Receptor (TLR) 7 agonist, stimulates the release of IFN-α that exhibits potent antiviral activity. However, the poor solubility and tissue selectivity of IMQ limits its clinical use. Here, we demonstrated the use of lipid-based nanoparticles (LNPs) to deliver IMQ and increase the production of IFN-α in the liver. We encapsulated IMQ in two liver-targeted LNP formulations: phospholipid-free small unilamellar vesicles (PFSUVs) and DSPG-liposomes targeting the hepatocytes and the Kupffer cells, respectively. In vitro drug release/retention, in vivo pharmacokinetics, intrahepatic distribution, IFN-α production, and suppression of serum HBV surface antigen (HBsAg) were evaluated and compared for these two formulations. PFSUVs provided >95% encapsulation efficiency for IMQ at a drug-to-lipid ratio (D/L) of 1/20 (w/w) and displayed stable drug retention in the presence of serum. DSPG-IMQ showed 79% encapsulation of IMQ at 1/20 (D/L) and exhibited ∼30% burst release when incubated with serum. Within the liver, PFSUVs showed high selectivity for the hepatocytes while DSPG-liposomes targeted the Kupffer cells. Finally, in an experimental HBV mouse model, PFSUVs significantly reduced serum levels of HBsAg by 12-, 6.3- and 2.2-fold compared to the control, IFN-α, and DSPG-IMQ groups, respectively. The results suggest that the hepatocyte-targeted PFSUVs loaded with IMQ exhibit significant potential for enhancing therapy of CHB.
Topics: Adjuvants, Immunologic; Animals; Antigens, Surface; Antiviral Agents; Hepatitis B Surface Antigens; Hepatitis B virus; Hepatocytes; Imiquimod; Interferon-alpha; Liver Neoplasms; Mice; Toll-Like Receptor 7; Unilamellar Liposomes
PubMed: 36058352
DOI: 10.1016/j.jconrel.2022.08.058 -
Breast Cancer Research and Treatment Aug 2022Trophoblast Cell Surface Antigen 2 (TROP2) is a glycoprotein expressed in many cancers. A TROP2 antibody-drug conjugate (ADC) was effective in metastatic triple-negative...
PURPOSE
Trophoblast Cell Surface Antigen 2 (TROP2) is a glycoprotein expressed in many cancers. A TROP2 antibody-drug conjugate (ADC) was effective in metastatic triple-negative breast cancer (TNBC). We studied TROP2 gene (TACSTD2) expression and associations with tumor and clinical characteristics, as well as selected external genes in primary breast cancer.
METHODS
TACSTD2 gene expression was evaluated using microarray data from I-SPY 1 (n = 149), METABRIC (n = 1992), and TCGA (n = 817). Associations with clinical features (Kruskal-Wallis test, all datasets), chemotherapy response (Wilcoxon rank sum test, I-SPY 1), recurrence free survival (Cox proportional hazard model, I-SPY 1 and METABRIC), and selected genes (Pearson correlations, all datasets) were determined.
RESULTS
TACSTD2 gene expression was detectable in all breast cancer subtypes, with a wide range of expression (all datasets). TACSTD2 gene expression was lower in HER2 + than HR + /HER2- and TNBC (METABRIC: p = 0.03, TCGA p = 0.007), and in HER2 + enriched and luminal B breast cancer (METABRIC: p < 0.001, TCGA: p < 0.001). TACSTD2 expression was higher in grade I vs. II/III tumors (METABRIC: p < 0.001). No association with chemotherapy response (I-SPY 1) or recurrence free survival (I-SPY 1 and METABRIC) was seen. TACSTD2 has significant positive correlations with the expression of epithelial/adhesion genes and proliferative genes, but was inversely correlated with immune genes.
CONCLUSION
TACSTD2 gene expression was seen in all breast cancer subtypes particularly luminal A and TNBC, and correlated with the expression of genes involved in cell epithelial transformation, adhesion, and proliferation, which contribute to tumor growth. These results support the investigation of TROP2 ADC in all subtypes of breast cancer.
Topics: Antigens, Neoplasm; Antigens, Surface; Breast Neoplasms; Cell Adhesion Molecules; Female; Humans; Prognosis; Triple Negative Breast Neoplasms; Trophoblasts
PubMed: 35789445
DOI: 10.1007/s10549-022-06660-x -
Parasites & Vectors May 2018Coccidiosis is recognised as a major parasitic disease in chickens. Eimeria maxima is considered as a highly immunoprotective species within the Eimeria spp. family that...
BACKGROUND
Coccidiosis is recognised as a major parasitic disease in chickens. Eimeria maxima is considered as a highly immunoprotective species within the Eimeria spp. family that infects chickens. In the present research, the surface antigen gene of E. maxima (EmSAG) was cloned, and the ability of EmSAG to stimulate protection against E. maxima was evaluated.
METHODS
Prokaryotic and eukaryotic plasmids expressing EmSAG were constructed. The EmSAG transcription and expression in vivo was performed based on the RT-PCR and immunoblot analysis. The expression of EmSAG in sporozoites and merozoites was detected through immunofluorescence analyses. The immune protection was assessed based on challenge experiments. Flow cytometry assays were used to determine the T cell subpopulations. The serum antibody and cytokine levels were evaluated by ELISA.
RESULTS
The open reading frame (ORF) of EmSAG gene contained 645 bp encoding 214 amino acid residues. The immunoblot and RT-PCR analyses indicated that the EmSAG gene were transcribed and expressed in vivo. The EmSAG proteins were expressed in sporozoite and merozoite stages of E. maxima by the immunofluorescence assay. Challenge experiments showed that both pVAX1-SAG and the recombinant EmSAG (rEmSAG) proteins were successful in alleviating jejunal lesions, decreasing loss of body weight and the oocyst ratio. Additionally, these experiments possessed anticoccidial indices (ACI) of more than 170. Higher percentages of CD4 and CD8 T cells were detected in both EmSAG-inoculated birds than those of the negative control groups (P < 0.05). The EmSAG-specific antibody concentrations of both the rEmSAG and pVAX1-EmSAG groups were much higher than those of the negative controls (P < 0.05). Higher concentrations of IL-4, IFN-γ, TGF-β1 and IL-17 were observed more in both the rEmSAG protein and pVAX1-SAG inoculated groups than those of negative controls (P < 0.05).
CONCLUSIONS
Our findings suggest that EmSAG is capable of eliciting a moderate immune protection and could be used as an effective vaccine candidate against E. maxima.
Topics: Animals; Antigens, Surface; Chickens; Coccidiosis; Cytokines; Eimeria; Immunity, Humoral; Merozoites; Oocysts; Open Reading Frames; Poultry Diseases; Protozoan Vaccines; Sporozoites
PubMed: 29848353
DOI: 10.1186/s13071-018-2906-5 -
Journal of Viral Hepatitis Nov 2014In view of a persistently high prevalence of hepatitis B surface antigen (HBsAg) carriage in our obstetric population, we examined the association between HBsAg carriage...
In view of a persistently high prevalence of hepatitis B surface antigen (HBsAg) carriage in our obstetric population, we examined the association between HBsAg carriage with maternal ABO and rhesus (Rh) blood group phenotypes determined at routine antenatal screening. In a retrospective study, the antenatal screening results of women booked for confinement between 1998 and 2011 in our hospital were examined for the relationship between HBsAg carriage with the ABO and rhesus blood groups, taking into account also the effects of advanced maternal age (≥ 35 years) and parity status (nulliparous or multiparous), and year of birth before or following the availability of the hepatitis B vaccine (1984). HBsAg carriage was found in 9.9%, 9.6%, 9.1% and 10.2% (P = 0.037) for group-A (n = 20 581 or 26.1%), -B (n = 20 744 or 26.4%), -AB (n = 5138 or 6.5%) and -O (n = 32 242 or 41.0%) among the 78705 women in the study cohort. Rhesus negativity was found in 0.6%, and HBsAg carriage was 12.3% and 9.8%, respectively, for the Rh-negative and Rh-positive women (P = 0.071). Carriage rate between group-O and non-O was influenced by nulliparity, age ≥ 35 years and Rh-positive status. Regression analysis indicated that group-B (P = 0.044, aOR = 1.062, 95% CI 1.002-1.127) and group-AB (P = 0.016, aOR = 1.134, 95% CI 1.024-1.256) were associated with HBsAg carriage. Blood groups-B and -AB are associated with increased hepatitis B virus (HBV) infection in our population, and further studies are warranted to elucidate the implications of this on the sequelae of HBV infection.
Topics: ABO Blood-Group System; Adult; Cohort Studies; Female; Hepatitis B; Hepatitis B Surface Antigens; Humans; Pregnancy; Retrospective Studies; Rh-Hr Blood-Group System; Seroepidemiologic Studies
PubMed: 24325347
DOI: 10.1111/jvh.12219 -
Journal of Virology Dec 2022Hepatitis B virus (HBV) is a major risk factor for serious liver diseases. The liver plays a unique role in controlling carbohydrate metabolism to maintain the glucose...
Hepatitis B virus (HBV) is a major risk factor for serious liver diseases. The liver plays a unique role in controlling carbohydrate metabolism to maintain the glucose level within the normal range. Chronic HBV infection has been reported to associate with a high prevalence of diabetes. However, the detailed molecular mechanism underlying the potential association remains largely unknown. Here, we report that liver-targeted delivery of small HBV surface antigen (SHBs), the most abundant viral protein of HBV, could elevate blood glucose levels and impair glucose and insulin tolerance in mice by promoting hepatic gluconeogenesis. Hepatocytes with SHB expression also exhibited increased glucose production and expression of gluconeogenic genes () and () in response to glucagon stimulation. Mechanistically, SHBs increased cellular levels of cyclic AMP (cAMP) and consequently activated protein kinase A (PKA) and its downstream effector cAMP-responsive element binding protein (CREB). SHBs-induced activation of CREB enhanced transcripts of gluconeogenic genes, thus promoting hepatic gluconeogenesis. The elevated cAMP level resulted from increased transcription activity and expression of adenylyl cyclase 1 (AC1) by SHBs through a binary E-box factor binding site (BEF). Taken together, we unveiled a novel pathogenic role and mechanism of SHBs in hepatic gluconeogenesis, and these results might highlight a potential target for preventive and therapeutic intervention in the development and progression of HBV-associated diabetes. Chronic HBV infection causes progressive liver damage and is found to be a risk factor for diabetes. However, the mechanism in the regulation of glucose metabolism by HBV remains to be established. In the current study, we demonstrate for the first time that the small hepatitis B virus surface antigen (SHBs) of HBV elevates AC1 transcription and expression to activate cAMP/PKA/CREB signaling and subsequently induces the expression of gluconeogenic genes and promotes hepatic gluconeogenesis both and . This study provides a direct link between HBV infection and diabetes and implicates that SHBs may represent a potential target for the treatment of HBV-induced metabolic disorders.
Topics: Animals; Mice; Antigens, Surface; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Glucagon; Gluconeogenesis; Glucose; Hepatitis B Surface Antigens; Hepatitis B virus; Hepatitis B, Chronic; Hepatocytes; Liver; Mice, Inbred C57BL
PubMed: 36394315
DOI: 10.1128/jvi.01020-22 -
Molecular Pharmaceutics Jun 2019Exosomes are considered to be vehicles of antigen delivery. The localization of antigen proteins, i.e., whether they lie on the outer surface or inner surface of...
Exosomes are considered to be vehicles of antigen delivery. The localization of antigen proteins, i.e., whether they lie on the outer surface or inner surface of exosomes, might affect antigen presentation after exosomes are taken up by antigen-presenting cells; however, little is known about the importance of this phenomenon. In this study, lactadherin (LA) and group-specific antigen (Gag), exosome-tropic proteins that had previously been shown to cause the localization of luciferase to the outer surface and inner surface of exosomes, respectively [ Takahashi , Y. ; J. Biotechnol. 2013 ; Charoenviriyakul , C. ; Mol. Pharm. 2018 ], were used to examine the importance of the localization of antigen proteins in antigen presentation. Human embryonic kidney cells 293 (HEK293) were selected as exosomes producing cells. First, green fluorescent protein (GFP) was used to trace intracellular behavior of antigen proteins after uptake by murine dendritic DC2.4 cells. GFP-derived fluorescence signals were detected in cells only when GFP-inner-loaded (Gag-GFP) exosomes were added to them. Then, ovalbumin (OVA) was used as a model antigen protein, and OVA-loaded exosomes were added to bone marrow-derived dendritic cells. OVA-inner-loaded (Gag-OVA) exosomes showed enhanced class I antigen presentation capacity as compared with OVA-outer-loaded (OVA-LA) exosomes. Using PKH-labeled exosomes, it was found that the localization of OVA had very little effect on the cellular uptake of exosomes. These results indicate that the loading of antigen proteins inside exosomes helps in efficient antigen presentation.
Topics: Animals; Antigen Presentation; Antigens, Surface; Blotting, Western; Bone Marrow Cells; Cell Line; Dendritic Cells; Exosomes; Flow Cytometry; HEK293 Cells; Humans; Male; Mice; Mice, Inbred C57BL; Microscopy, Electron, Transmission; Milk Proteins
PubMed: 30908053
DOI: 10.1021/acs.molpharmaceut.8b01093 -
Parasitology Research Jan 2022Coccidiosis is an intestinal parasitic disease that causes huge economic losses to the poultry industry globally. Eimeria tenella belonging to protozoon is the causative...
Coccidiosis is an intestinal parasitic disease that causes huge economic losses to the poultry industry globally. Eimeria tenella belonging to protozoon is the causative agent of cecal coccidiosis in chicken, and it causes enormous damage to poultry industry. The surface antigens (SAGs) of apicomplexan parasites have functions of attachment and invasion in host-parasite interaction. As a result of parasitic invasion, host immune response is triggered. However, the immunogenicity and potency of E. tenella surface antigen 6 and 15 (EtSAG 6 and 15), as vaccinal candidate antigen, remain largely unknown. Therefore, gene fragments of E. tenella EtSAG 6 and 15 were amplified and transformed to pET28a prokaryotic vector for recombinant protein expression. The pEGFP-N1 eukaryotic vectors with EtSAG 6 and 15 amplification fragments (pEGFP-N1-EtSAG 5 and 16) were transformed into 293 T cell line. The results of reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis revealed successful expressions of EtSAG 6 and 15 in Escherichia coli and 293 T cells. Subsequently, animal experiments of 49 cobb broilers were performed to evaluate immunoprotection of recombinant proteins and DNA vaccines derived from E. tenella EtSAG 5 and 16 with an immunizing dose of 100 μg, respectively. Chickens vaccinated with rEtSAG 6 protein, rEtSAG 15 protein, pEGFP-N1-EtSAG 6 plasmid, or pEGFP-N1-EtSAG 15 plasmid showed no significant increase in IFN-γor interleukin-4 (IL-4) level compared with control groups. Chickens vaccinated with protein rEtSAG 6, protein rEtSAG 15, pEGFP-N1-EtSAG 6 plasmid, or pEGFP-N1-EtSAG 15 exhibited higher weight gains, lower oocyst output, and lower mean lesion scores, compared with infection control group. Among the four immunized groups, plasmid EGFP-N1-EtSAG 6 (100 μg) group exhibited the highest anticoccidial index (ACI) value (150.20). Overall, plasmids EGFP-N1-EtSAG 6 and 15, as DNA vaccines, provided a more effective immunoprotection for chickens against E. tenella than protein rEtSAG 6 and protein rEtSAG 15 as subunit vaccines. EtSAG 6 and 15 are promising candidate antigen genes for developing coccidiosis vaccine.
Topics: Animals; Antigens, Surface; Chickens; Eimeria tenella; Poultry Diseases; Protozoan Vaccines; Recombinant Proteins; Vaccines, DNA
PubMed: 34816300
DOI: 10.1007/s00436-021-07364-9 -
Pediatric Surgery International Nov 2022Immunotherapy may improve the poor prognosis of high-risk neuroblastoma. Programmed cell death-1 (PD-1) is expressed in several cancers. The tyrosine hydroxylase MYCN...
BACKGROUND
Immunotherapy may improve the poor prognosis of high-risk neuroblastoma. Programmed cell death-1 (PD-1) is expressed in several cancers. The tyrosine hydroxylase MYCN (TH-MYCN) transgenic mouse model is widely used in neuroblastoma research, but detailed information on its immunological background is lacking. Therefore, we studied the immunological tumor microenvironment and tumor cell surface antigen expression in homozygote and hemizygote mice and effects of antibody therapy against PD-1.
METHODS
CD4, CD8, CD11b, and CD11c expression in immune cells from retroperitoneal lymph nodes and spleen was analyzed by flow cytometry. Tumor cell surface antigen expression was confirmed, and data from homozygote and hemizygote mice were compared. Effects of anti-PD-1 antibody were evaluated.
RESULTS
CD4-, CD8-, CD11b-, and CD11c-positive cells were not significantly different in homozygote and hemizygote mice, and CD11b- and CD11c-positive cells were identified in the tumor microenvironment in both. Tumor cells expressed PD-1, and anti-PD-1 antibody had anti-tumor effects and significantly reduced the percentage of living tumor cells in cultures after 2 h.
CONCLUSION
The immunological background is similar in homozygote and hemizygote TH-MYCN transgenic mice, and both have PD-1-positive tumor cells. Anti-PD-1 antibody suppresses tumor growth. This mouse model may be a useful for studying immunotherapy of neuroblastoma.
Topics: Animals; Mice; Antigens, Surface; Apoptosis; Disease Models, Animal; Mice, Transgenic; N-Myc Proto-Oncogene Protein; Neuroblastoma; Tumor Microenvironment; Tyrosine 3-Monooxygenase
PubMed: 36441248
DOI: 10.1007/s00383-022-05292-y