Production of prostate-specific antigen by a breast cancer cell line, Sk-Br-3

International Nuclear Information System (INIS)

Kamali Sarvestani, E.; Ghaderi, A.

2002-01-01

Prostate-specific antigen is a 33-KDa serine protease that is produced predominantly by prostate epithelium. However, it has been shown that about 30-40% of female breast tumors produce prostate-specific antigen and its production is associated with the presence of estrogen and progesterone receptors. We have now developed a new tissue culture system to study prostate-specific antigen production in breast cancer and its association with prognostic factors such as progesterone receptor and c-erbB-2. For this purpose we investigated the ability of prostate-specific antigen production in five different cell lines, including two breast cancer cell lines, Sk-Br-3 and MDA-MB-453. The prostate-specific antigen in tissue culture supernatant and cytoplasm of the Sk-Br-3 cell line was detected by western blotting and immunoperoxidase, respectively. Furthermore, we found lower expression of c-erbB-2 in Sk-Br-3 than non-prostate-specific antigen producer breast cancer cell line, MDA-MB-453. Progesterone receptor was expressed by both prostate-specific antigen-positive and -negative cell lines and only the intensity of staining and the number of positive cells in Sk-Br-3 population was higher than MDA-MB-453. According to our findings prostate-specific antigen can be considered as a good prognostic factor in breast cancer and we suggest that these two cell lines are a good in vitro model to study the relationship of different breast cancer prognostic factors and their regulations

Suicide gene reveals the myocardial neovascularization role of mesenchymal stem cells overexpressing CXCR4 (MSC(CXCR4.

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Jialiang Liang

Full Text Available BACKGROUND: Our previous studies indicated that MSC(CXCR4 improved cardiac function after myocardial infarction (MI. This study was aimed to investigate the specific role of MSC(CXCR4 in neovascularization of infarcted myocardium using a suicide gene approach. METHODS: MSCs were transduced with either lentivirus-null vector/GFP (MSC(Null as control or vector encoding for overexpressing CXCR4/GFP. The MSC derived-endothelial cell (EC differentiation was assessed by a tube formation assay, Dil-ac-LDL uptake, EC marker expression, and VE-cadherin promoter activity assay. Gene expression was analyzed by quantitative RT-PCR or Western blot. The suicide gene approach was under the control of VE-cadherin promoter. In vivo studies: Cell patches containing MSC(Null or MSC(CXCR4 were transduced with suicide gene and implanted into the myocardium of MI rat. Rats received either ganciclovir (GCV or vehicle after cell implantation. After one month, the cardiac functional changes and neovascularization were assessed by echocardiography, histological analysis, and micro-CT imaging. RESULTS: The expression of VEGF-A and HIF-1α was significantly higher in MSC(CXCR4 as compared to MSC(Null under hypoxia. Additionally, MSC(CXCR4 enhanced new vessel formation and EC differentiation, as well as STAT3 phosphorylation under hypoxia. STAT3 participated in the transcription of VE-cadherin in MSC(CXCR4 under hypoxia, which was inhibited by WP1066 (a STAT3 inhibitor. In addition, GCV specifically induced death of ECs with suicide gene activation. In vivo studies: MSC(CXCR4 implantation promoted cardiac functional restoration, reduced infarct size, improved cardiac remodeling, and enhanced neovascularization in ischemic heart tissue. New vessels derived from MSC(CXCR4 were observed at the injured heart margins and communicated with native coronary arteries. However, the derived vessel networks were reduced by GCV, reversing improvement of cardiac function. CONCLUSION: The

Upregulation of bacterial-specific Th1 and Th17 responses that are enriched in CXCR5+CD4+ T cells in non-small cell lung cancer.

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Ma, Qin-Yun; Huang, Da-Yu; Zhang, Hui-Jun; Wang, Shaohua; Chen, Xiao-Feng

2017-11-01

The microbial community in the mucosal surfaces is involved in the development of human cancers, including gastric cancer and colorectal cancer. The respiratory tract in the lung also hosts a distinctive microbial community, but the correlation between this community and lung cancer is largely unknown. Here, we examined the Th1 and Th17 responses toward several bacterial antigens, in CD4 + T cells sourced from the peripheral blood (PB), the lung cancer (LC) tissue, and the gastrointestinal (GI) tract of non-small cell lung cancer (NSCLC) patients. Compared to healthy controls, the NSCLC patients presented significantly higher frequencies of Th1 and Th17 cells reacting to Streptococcus salivarius and S. agalactiae, in the PB, LC, and GI tract. Further investigation showed that the upregulation in anti-bacteria response was likely antigen-specific for two reasons. Firstly, the frequencies of Th1 and Th17 cells reacting to Escherichia coli, a typical GI bacterium, were not upregulated in the PB and the LC of NSCLC patients. Secondly, the S. salivarius and S. agalactiae responses could be partially blocked by Tü39, a MHC class II blocking antibody, suggesting that antigen-specific interaction between CD4 + T cells and antigen-presenting cells was required. We also found that S. salivarius and S. agalactiae could potently activate the monocytes to secrete higher levels of interleukin (IL)-6, IL-12, and tumor necrosis factor, which were Th1- and Th17-skewing cytokines. Interestingly, whereas CXCR5 + CD4 + T cells represented <20% of total CD4 + T cells, they represented 17%-82% of bacteria-specific Th1 or Th17 cells. Together, these data demonstrated that NSCLC patients presented a significant upregulation of bacterial-specific Th1 and Th17 responses that were enriched in CXCR5 + CD4 + T cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression of CXCR3 on Adaptive and Innate Immune Cells Contributes Oviduct Pathology throughout Chlamydia muridarum Infection

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Jiang, Janina; Maxion, Heather; Champion, Cheryl I.; Liu, Guangchao; Kelly, Kathleen A.

2017-01-01

CXCR3 is a chemokine receptor expressed on a wide range of leukocytes, and it is involved in leukocyte migration throughout the blood and lymphatics. Specifically, CXCR3 is required for lymphocyte homing to the genital mucosa. When compared to wild type (WT) mice, CXCR3 deficiency (CXCR3−/−) mice infected with Chlamydia muridarum (C. muridarum) did not display impaired clearance and resolution of infection. However, they possessed significantly higher bacterial burden and lower levels of IFN-γ-producing TH1 cells. The knockouts also demonstrated a significant decrease in the level of activated conventional dendritic cells in the GT, ultimately leading to the decrease in activated TH1 cells. In addition, few activated plasmacytoid dendritic cells, which possess an inflammatory phenotype, were found in the lymph node of infected mice. This reduction in pDCs may be responsible for the decrease in neutrophils, which are acute inflammatory cells, in the CXCR3−/− mice. Due to the significantly reduced level of acute inflammation, these mice also possess a decrease in dilation and pathology in the oviduct. This demonstrates that the CXCR3−/− mice possess the ability to clear C. muridarum infections, but they do so without the increased inflammation and pathology in the GT. PMID:29552679

Engineering HIV-resistant human CD4+ T cells with CXCR4-specific zinc-finger nucleases.

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Craig B Wilen

2011-04-01

Full Text Available HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors on host cells. Individuals homozygous for the ccr5Δ32 polymorphism do not express CCR5 and are protected from infection by CCR5-tropic (R5 virus strains. As an approach to inactivating CCR5, we introduced CCR5-specific zinc-finger nucleases into human CD4+ T cells prior to adoptive transfer, but the need to protect cells from virus strains that use CXCR4 (X4 in place of or in addition to CCR5 (R5X4 remains. Here we describe engineering a pair of zinc finger nucleases that, when introduced into human T cells, efficiently disrupt cxcr4 by cleavage and error-prone non-homologous DNA end-joining. The resulting cells proliferated normally and were resistant to infection by X4-tropic HIV-1 strains. CXCR4 could also be inactivated in ccr5Δ32 CD4+ T cells, and we show that such cells were resistant to all strains of HIV-1 tested. Loss of CXCR4 also provided protection from X4 HIV-1 in a humanized mouse model, though this protection was lost over time due to the emergence of R5-tropic viral mutants. These data suggest that CXCR4-specific ZFNs may prove useful in establishing resistance to CXCR4-tropic HIV for autologous transplant in HIV-infected individuals.

Prostate-specific antigen velocity is not better than total prostate-specific antigen in predicting prostate biopsy diagnosis.

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Gorday, William; Sadrzadeh, Hossein; de Koning, Lawrence; Naugler, Christopher T

2015-12-01

1.) Identify whether prostate-specific antigen velocity improves the ability to predict prostate biopsy diagnosis. 2.) Test whether there is an increase in the predictive capability of models when Gleason 7 prostate cancers are separated into a 3+4 and a 4+3 group. Calgary Laboratory Services' Clinical Laboratory Information System was searched for prostate biopsies reported between January 1, 2009 and December 31, 2013. Total prostate-specific antigen tests were recorded for each patient from January 1, 2007 to the most recent test before their recorded prostate biopsy. The data set was divided into the following three groups for comparison; benign, all prostate cancer and Gleason 7-10. The Gleason grade 7-10 group was further divided into 4+3 and 3+4 Gleason 7 prostate cancers. Prostate-specific antigen velocity was calculated using four different methods found in the literature. Receiver operator curves were used to assess operational characteristics of the tests. 4622 men between the ages of 40-89 with a prostate biopsy were included for analysis. Combining prostate-specific antigen velocity with total prostate-specific antigen (AUC=0.570-0.712) resulted in small non-statistically significant changes to the area under the curve compared to the area under the curve of total prostate-specific antigen alone (AUC=0.572-0.699). There were marked increases in the area under curves when 3+4 and 4+3 Gleason 7 cancers were separated. Prostate-specific antigen velocity does not add predictive value for prostate biopsy diagnosis. The clinical significance of the prostate specific antigen test can be improved by separating Gleason 7 prostate cancers into a 3+4 and 4+3 group. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

The chemokine receptor CXCR3 and its splice variant are expressed in human airway epithelial cells.

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Kelsen, Steven G; Aksoy, Mark O; Yang, Yi; Shahabuddin, Syed; Litvin, Judith; Safadi, Fayez; Rogers, Thomas J

2004-09-01

Activation of the chemokine receptor CXCR3 by its cognate ligands induces several differentiated cellular responses important to the growth and migration of a variety of hematopoietic and structural cells. In the human respiratory tract, human airway epithelial cells (HAEC) release the CXCR3 ligands Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11. Simultaneous expression of CXCR3 by HAEC would have important implications for the processes of airway inflammation and repair. Accordingly, in the present study we sought to determine whether HAEC also express the classic CXCR3 chemokine receptor CXCR3-A and its splice variant CXCR3-B and hence may respond in autocrine fashion to its ligands. We found that cultured HAEC (16-HBE and tracheocytes) constitutively expressed CXCR3 mRNA and protein. CXCR3 mRNA levels assessed by expression array were approximately 35% of beta-actin expression. In contrast, CCR3, CCR4, CCR5, CCR8, and CX3CR1 were <5% beta-actin. Both CXCR3-A and -B were expressed. Furthermore, tracheocytes freshly harvested by bronchoscopy stained positively for CXCR3 by immunofluorescence microscopy, and 68% of cytokeratin-positive tracheocytes (i.e., the epithelial cell population) were positive for CXCR3 by flow cytometry. In 16-HBE cells, CXCR3 receptor density was approximately 78,000 receptors/cell when assessed by competitive displacement of 125I-labeled IP-10/CXCL10. Finally, CXCR3 ligands induced chemotactic responses and actin reorganization in 16-HBE cells. These findings indicate constitutive expression by HAEC of a functional CXC chemokine receptor, CXCR3. Our data suggest the possibility that autocrine activation of CXCR3 expressed by HAEC may contribute to airway inflammation and remodeling in obstructive lung disease by regulating HAEC migration.

Direct Lymph Node Vaccination of Lentivector/Prostate-Specific Antigen is Safe and Generates Tissue-Specific Responses in Rhesus Macaques

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Bryan C. Au

2016-02-01

Full Text Available Anti-cancer immunotherapy is emerging from a nadir and demonstrating tangible benefits to patients. A variety of approaches are now employed. We are invoking antigen (Ag-specific responses through direct injections of recombinant lentivectors (LVs that encode sequences for tumor-associated antigens into multiple lymph nodes to optimize immune presentation/stimulation. Here we first demonstrate the effectiveness and antigen-specificity of this approach in mice challenged with prostate-specific antigen (PSA-expressing tumor cells. Next we tested the safety and efficacy of this approach in two cohorts of rhesus macaques as a prelude to a clinical trial application. Our vector encodes the cDNA for rhesus macaque PSA and a rhesus macaque cell surface marker to facilitate vector titering and tracking. We utilized two independent injection schemas demarcated by the timing of LV administration. In both cohorts we observed marked tissue-specific responses as measured by clinical evaluations and magnetic resonance imaging of the prostate gland. Tissue-specific responses were sustained for up to six months—the end-point of the study. Control animals immunized against an irrelevant Ag were unaffected. We did not observe vector spread in test or control animals or perturbations of systemic immune parameters. This approach thus offers an “off-the-shelf” anti-cancer vaccine that could be made at large scale and injected into patients—even on an out-patient basis.

Direct Lymph Node Vaccination of Lentivector/Prostate-Specific Antigen is Safe and Generates Tissue-Specific Responses in Rhesus Macaques.

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Au, Bryan C; Lee, Chyan-Jang; Lopez-Perez, Orlay; Foltz, Warren; Felizardo, Tania C; Wang, James C M; Huang, Ju; Fan, Xin; Madden, Melissa; Goldstein, Alyssa; Jaffray, David A; Moloo, Badru; McCart, J Andrea; Medin, Jeffrey A

2016-02-19

Anti-cancer immunotherapy is emerging from a nadir and demonstrating tangible benefits to patients. A variety of approaches are now employed. We are invoking antigen (Ag)-specific responses through direct injections of recombinant lentivectors (LVs) that encode sequences for tumor-associated antigens into multiple lymph nodes to optimize immune presentation/stimulation. Here we first demonstrate the effectiveness and antigen-specificity of this approach in mice challenged with prostate-specific antigen (PSA)-expressing tumor cells. Next we tested the safety and efficacy of this approach in two cohorts of rhesus macaques as a prelude to a clinical trial application. Our vector encodes the cDNA for rhesus macaque PSA and a rhesus macaque cell surface marker to facilitate vector titering and tracking. We utilized two independent injection schemas demarcated by the timing of LV administration. In both cohorts we observed marked tissue-specific responses as measured by clinical evaluations and magnetic resonance imaging of the prostate gland. Tissue-specific responses were sustained for up to six months-the end-point of the study. Control animals immunized against an irrelevant Ag were unaffected. We did not observe vector spread in test or control animals or perturbations of systemic immune parameters. This approach thus offers an "off-the-shelf" anti-cancer vaccine that could be made at large scale and injected into patients-even on an out-patient basis.

Construction and Quantitative Validation of Chicken CXCR4 Expression Reporter.

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Es-Haghi, Masoumeh; Bassami, Mohammadreza; Dehghani, Hesam

2016-03-01

Site directional migration is an important biological event and an essential behavior for latent migratory cells. A migratory cell maintains its motility, survival, and proliferation abilities by a network of signaling pathways where CXCR4/SDF signaling route plays crucial role for directed homing of a polarized cell. The chicken embryo due to its specific vasculature modality has been used as a valuable model for organogenesis, migration, cancer, and metastasis. In this research, the regulatory regions of chicken CXCR4 gene have been characterized in a chicken hematopoietic lymphoblast cell line (MSB1). A region extending from -2000 bp upstream of CXCR4 gene to +68 after its transcriptional start site, in addition to two other mutant fragments were constructed and cloned in a promoter-less reporter vector. Promoter activity was analyzed by quantitative real-time RT-PCR and flow cytometry techniques. Our findings show that the full sequence from -2000 to +68 bp of CXCR4 regulatory region is required for maximum promoter functionality, while the mutant CXCR4 promoter fragments show a partial promoter activity. The chicken CXCR4 promoter validated in this study could be used for characterization of directed migratory cells in chicken development and disease models.

CXCR4-specific Nanobodies as potential therapeutics for WHIM syndrome

DEFF Research Database (Denmark)

de Wit, Raymond H; Heukers, Raimond; Brink, Hendrik

2017-01-01

WHIM syndrome is a rare congenital immunodeficiency disease, named after its main clinical manifestations: Warts, Hypogammaglobulinemia, Infections and Myelokathexis. The disease is primarily caused by C-terminal truncation mutations of the chemokine receptor CXCR4. Consequently, these CXCR4-WHIM...... as alternative therapeutics for CXCR4-associated diseases like WHIM syndrome....

Human Leukocyte Antigen-G and Regulatory T Cells during Specific Immunotherapy for Pollen Allergy

DEFF Research Database (Denmark)

Sørensen, Anja Elaine; Johnsen, Claus R; Dalgaard, Louise Torp

2013-01-01

of the cytokine profile towards a TH1-polarized immune response. We investigated the effects of SIT on T cells, on immunomodulation of human leukocyte antigen (HLA)-G, which has been associated with allergy, on regulatory cytokine expression, and on serum allergen-specific antibody subclasses (IgE and IgG4......). Methods: Eleven birch and/or grass pollen-allergic patients and 10 healthy nonatopic controls were studied before and during SIT. Tregs, chemokine receptors, soluble HLA-G (sHLA-G), Ig-like transcript (ILT) 2, specific IgE, and IgG4 were studied. Peripheral blood mononuclear cells (PBMCs) were stimulated...... with pollen extract in vitro and immune factors were evaluated. Results: During SIT, the main changes in the peripheral blood were an increase in CXCR3+CD4+CD25+CD127low/- Tregs and a decrease in CCR4+CD4+CD25+CD127low/- Tregs, an increase in allergen-specific IgG4, and a decrease in sHLA-G during the first...

Lymphoid follicle cells in chronic obstructive pulmonary disease overexpress the chemokine receptor CXCR3.

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Kelsen, Steven G; Aksoy, Mark O; Georgy, Mary; Hershman, Richard; Ji, Rong; Li, Xiuxia; Hurford, Matthew; Solomides, Charalambos; Chatila, Wissam; Kim, Victor

2009-05-01

The mechanisms underlying formation of lung lymphoid follicles (LF) in chronic obstructive pulmonary disease (COPD) are unknown. The chemokine receptor CXCR3 regulates immune responses in secondary lymphoid structures elsewhere in the body and is highly expressed by Th1 lymphocytes in the airway in COPD. Because chemokine receptors control inflammatory cell homing to inflamed tissue, we reasoned that CXCR3 may contribute to LF formation in COPD. We assessed the expression of CXCR3 and its ligands (IP-10/CXCL10, Mig/CXCL9, and ITAC/CXCL11) by LF cells in never-smokers, smokers without COPD, and subjects with COPD. CXCR3, IP-10, Mig, and ITAC expression were assessed in lung sections from 46 subjects (never-smokers, smokers without COPD [S], and subjects with COPD in GOLD stages 1-4) by immunohistochemistry. CXCR3-expressing T cells (CD8+ or CD4+) and B cells (CD20+) were topographically distributed at the follicle periphery and center, respectively. The percentage of immunohistochemically identified CXCR3+ cells increased progressively while proceeding from S through GOLD 3-4 (P < 0.01 for GOLD 3-4 vs. S). Moreover, the number of CXCR3+ follicular cells correlated inversely with FEV(1) (r = 0.60). The CXCR3 ligands IP-10 and Mig were expressed by several cell types in and around the follicle, including CD68+ dendritic cells/ macrophages, airway epithelial cells, endothelial cells, and T and B cells. These results suggest that LF form in the COPD lung by recruitment and/or retention of CXCR3-expressing T and B lymphocytes, which are attracted to the region through production of CXCR3 ligands IP-10 and Mig by lung structural and follicular cells.

Glycosaminoglycans Regulate CXCR3 Ligands at Distinct Levels: Protection against Processing by Dipeptidyl Peptidase IV/CD26 and Interference with Receptor Signaling

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Mieke Metzemaekers

2017-07-01

Full Text Available CXC chemokine ligand (CXCL9, CXCL10 and CXCL11 direct chemotaxis of mainly T cells and NK cells through activation of their common CXC chemokine receptor (CXCR3. They are inactivated upon NH2-terminal cleavage by dipeptidyl peptidase IV/CD26. In the present study, we found that different glycosaminoglycans (GAGs protect the CXCR3 ligands against proteolytic processing by CD26 without directly affecting the enzymatic activity of CD26. In addition, GAGs were shown to interfere with chemokine-induced CXCR3 signaling. The observation that heparan sulfate did not, and heparin only moderately, altered CXCL10-induced T cell chemotaxis in vitro may be explained by a combination of protection against proteolytic inactivation and altered receptor interaction as observed in calcium assays. No effect of CD26 inhibition was found on CXCL10-induced chemotaxis in vitro. However, treatment of mice with the CD26 inhibitor sitagliptin resulted in an enhanced CXCL10-induced lymphocyte influx into the joint. This study reveals a dual role for GAGs in modulating the biological activity of CXCR3 ligands. GAGs protect the chemokines from proteolytic cleavage but also directly interfere with chemokine–CXCR3 signaling. These data support the hypothesis that both GAGs and CD26 affect the in vivo chemokine function.

PEA3 activates CXCR4 transcription in MDA-MB-231 and MCF7 breast cancer cells

Institute of Scientific and Technical Information of China (English)

Shengmei Gu; Li Chen; Qi Hong; Tingting Yan; Zhigang Zhuang; Qiaoqiao wang; Wei Jin; Hua Zhu; Jiong Wu

2011-01-01

CXC chemokine receptor 4 (CXCR4) is a cell surface receptor that has been shown to mediate the metastasis of many solid tumors including lung,breast,kidney,and prostate tumors.In this study,we found that overexpression of ets variant gene 4 (PEA3) could elevate CXCR4 mRNA level and CXCR4 promoter activity in human MDA-MB-231 and MCF-7 breast cancer cells.PEA3 promoted CXCR4 expression and breast cancer metastasis.Chromatin immunoprecipitation assay demonstrated that PEA3 could bind to the CXCR4 promoter in the cells transfected with PEA3 expression vector.PEA3 siRNA attenuated CXCR4 promoter activity and the binding of PEA3 to the CXCR4 promoter in MDA-MB-231 and MCF-7 cells.These results indicated that PEA3 could activate CXCR4 promoter transcription and promote breast cancer metastasis.

Autoantibodies in Serum of Systemic Scleroderma Patients: Peptide-Based Epitope Mapping Indicates Increased Binding to Cytoplasmic Domains of CXCR3

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Andreas Recke

2018-03-01

Full Text Available Systemic sclerosis (SSc is a severe chronic autoimmune disease with high morbidity and mortality. Sera of patients with SSc contain a large variety of autoantibody (aab reactivities. Among these are functionally active aab that bind to G protein-coupled receptors (GPCR such as C-X-C motif chemokine receptor 3 (CXCR3 and 4 (CXCR4. Aab binding to the N-terminal portion of these two GPCRs have been shown to be associated with slower disease progression in SSc, especially deterioration of lung function. Aabs binding to GPCRs exhibit functional activities by stimulating or inhibiting GPCR signaling. The specific functional activity of aabs crucially depends on the epitopes they bind to. To identify the location of important epitopes on CXCR3 recognized by aabs from SSc patients, we applied an array of 36 overlapping 18-20mer peptides covering the entire CXCR3 sequence, comparing epitope specificity of SSc patient sera (N = 32, with positive reactivity with CXCR3 to healthy controls (N = 30. Binding of SSc patient and control sera to these peptides was determined by ELISA. Using a Bayesian model approach, we found increased binding of SSc patient sera to peptides corresponding to intracellular epitopes within CXCR3, while the binding signal to extracellular portions of CXCR3 was found to be reduced. Experimentally determined epitopes showed a good correspondence to those predicted by the ABCpred tool. To verify these results and to translate them into a novel diagnostic ELISA, we combined the peptides that represent SSc-associated epitopes into a single ELISA and evaluated its potential to discriminate SSc patients (N = 31 from normal healthy controls (N = 47. This ELISA had a sensitivity of 0.61 and a specificity of 0.85. Our data reveals that SSc sera preferentially bind intracellular epitopes of CXCR3, while an extracellular epitope in the N-terminal domain that appears to be target of aabs in healthy individuals is not bound by SSc

V3-independent competitive resistance of a dual-X4 HIV-1 to the CXCR4 inhibitor AMD3100.

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Yosuke Maeda

Full Text Available A CXCR4 inhibitor-resistant HIV-1 was isolated from a dual-X4 HIV-1 in vitro. The resistant variant displayed competitive resistance to the CXCR4 inhibitor AMD3100, indicating that the resistant variant had a higher affinity for CXCR4 than that of the wild-type HIV-1. Amino acid sequence analyses revealed that the resistant variant harbored amino acid substitutions in the V2, C2, and C4 regions, but no remarkable changes in the V3 loop. Site-directed mutagenesis confirmed that the changes in the C2 and C4 regions were principally involved in the reduced sensitivity to AMD3100. Furthermore, the change in the C4 region was associated with increased sensitivity to soluble CD4, and profoundly enhanced the entry efficiency of the virus. Therefore, it is likely that the resistant variant acquired the higher affinity for CD4/CXCR4 by the changes in non-V3 regions. Taken together, a CXCR4 inhibitor-resistant HIV-1 can evolve using a non-V3 pathway.

Induction of Robust B Cell Responses after Influenza mRNA Vaccination Is Accompanied by Circulating Hemagglutinin-Specific ICOS+ PD-1+ CXCR3+ T Follicular Helper Cells

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Gustaf Lindgren

2017-11-01

Full Text Available Modified mRNA vaccines have developed into an effective and well-tolerated vaccine platform that offers scalable and precise antigen production. Nevertheless, the immunological events leading to strong antibody responses elicited by mRNA vaccines are largely unknown. In this study, we demonstrate that protective levels of antibodies to hemagglutinin were induced after two immunizations of modified non-replicating mRNA encoding influenza H10 encapsulated in lipid nanoparticles (LNP in non-human primates. While both intradermal (ID and intramuscular (IM administration induced protective titers, ID delivery generated this response more rapidly. Circulating H10-specific memory B cells expanded after each immunization, along with a transient appearance of plasmablasts. The memory B cell pool waned over time but remained detectable throughout the 25-week study. Following prime immunization, H10-specific plasma cells were found in the bone marrow and persisted over time. Germinal centers were formed in vaccine-draining lymph nodes along with an increase in circulating H10-specific ICOS+ PD-1+ CXCR3+ T follicular helper cells, a population shown to correlate with high avidity antibody responses after seasonal influenza vaccination in humans. Collectively, this study demonstrates that mRNA/LNP vaccines potently induce an immunological repertoire associated with the generation of high magnitude and quality antibodies.

Specific interaction of CXCR4 with CD4 and CD8α: Functional analysis of the CD4/CXCR4 interaction in the context of HIV-1 envelope glycoprotein-mediated membrane fusion

International Nuclear Information System (INIS)

Basmaciogullari, Stephane; Pacheco, Beatriz; Bour, Stephan; Sodroski, Joseph

2006-01-01

We investigated possible interactions between HIV-1 receptor (CD4) and the main coreceptors CXCR4 and CCR5. We found that CD4 and CXCR4 coexpressed in 293T cells form a complex that can be immunoprecipitated with antibodies directed against the extracellular domain of either protein. Mutagenesis revealed that the CD4/CXCR4 interaction maps to two previously uncharacterized basic motifs in the cytoplasmic domain of CD4. HIV-1 envelope glycoprotein-mediated membrane fusion was found to be independent of the ability of CD4 and CXCR4 to interact, whether fusion was studied in a virus-cell or a cell-cell model. However, this interaction might explain the adaptation of HIV-1 to CXCR4 as an alternative to CCR5. We found that CXCR4 also interacts with the cytoplasmic domain of CD8α in a way that is similar to the CD4/CXCR4 interaction. The CD4/CXCR4 and CD8α/CXCR4 interactions may thus be involved in cellular signaling pathways shared by the CD4 and CD8α molecules

Involvement of the CXCR7/CXCR4/CXCL12 axis in the malignant progression of human neuroblastoma.

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Julie Liberman

Full Text Available Neuroblastoma (NB is a typical childhood and heterogeneous neoplasm for which efficient targeted therapies for high-risk tumors are not yet identified. The chemokine CXCL12, and its receptors CXCR4 and CXCR7 have been involved in tumor progression and dissemination. While CXCR4 expression is associated to undifferentiated tumors and poor prognosis, the role of CXCR7, the recently identified second CXCL12 receptor, has not yet been elucidated in NB. In this report, CXCR7 and CXCL12 expressions were evaluated using a tissue micro-array including 156 primary and 56 metastatic NB tissues. CXCL12 was found to be highly associated to NB vascular and stromal structures. In contrast to CXCR4, CXCR7 expression was low in undifferentiated tumors, while its expression was stronger in matured tissues and specifically associated to differentiated neural tumor cells. As determined by RT-PCR, CXCR7 expression was mainly detected in N-and S-type NB cell lines, and was slightly induced upon NB cell differentiation in vitro. The relative roles of the two CXCL12 receptors were further assessed by overexpressing CXCR7 or CXCR4 receptor alone, or in combination, in the IGR-NB8 and the SH-SY5Y NB cell lines. In vitro functional analyses indicated that, in response to their common ligand, both receptors induced activation of ERK1/2 cascade, but not Akt pathway. CXCR7 strongly reduced in vitro growth, in contrast to CXCR4, and impaired CXCR4/CXCL12-mediated chemotaxis. Subcutaneous implantation of CXCR7-expressing NB cells showed that CXCR7 also significantly reduced in vivo growth. Moreover, CXCR7 affected CXCR4-mediated orthotopic growth in a CXCL12-producing environment. In such model, CXCR7, in association with CXCR4, did not induce NB cell metastatic dissemination. In conclusion, the CXCR7 and CXCR4 receptors revealed specific expression patterns and distinct functional roles in NB. Our data suggest that CXCR7 elicits anti-tumorigenic functions, and may act as a

Simian Immunodeficiency Virus (SIV-Specific Chimeric Antigen Receptor-T Cells Engineered to Target B Cell Follicles and Suppress SIV Replication

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Kumudhini Preethi Haran

2018-03-01

Full Text Available There is a need to develop improved methods to treat and potentially cure HIV infection. During chronic HIV infection, replication is concentrated within T follicular helper cells (Tfh located within B cell follicles, where low levels of virus-specific CTL permit ongoing viral replication. We previously showed that elevated levels of simian immunodeficiency virus (SIV-specific CTL in B cell follicles are linked to both decreased levels of viral replication in follicles and decreased plasma viral loads. These findings provide the rationale to develop a strategy for targeting follicular viral-producing (Tfh cells using antiviral chimeric antigen receptor (CAR T cells co-expressing the follicular homing chemokine receptor CXCR5. We hypothesize that antiviral CAR/CXCR5-expressing T cells, when infused into an SIV-infected animal or an HIV-infected individual, will home to B cell follicles, suppress viral replication, and lead to long-term durable remission of SIV and HIV. To begin to test this hypothesis, we engineered gammaretroviral transduction vectors for co-expression of a bispecific anti-SIV CAR and rhesus macaque CXCR5. Viral suppression by CAR/CXCR5-transduced T cells was measured in vitro, and CXCR5-mediated migration was evaluated using both an in vitro transwell migration assay, as well as a novel ex vivo tissue migration assay. The functionality of the CAR/CXCR5 T cells was demonstrated through their potent suppression of SIVmac239 and SIVE660 replication in in vitro and migration to the ligand CXCL13 in vitro, and concentration in B cell follicles in tissues ex vivo. These novel antiviral immunotherapy products have the potential to provide long-term durable remission (functional cure of HIV and SIV infections.

Intravascular Immune Surveillance by CXCR6+ NKT Cells Patrolling Liver Sinusoids

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Geissmann Frederic

2005-01-01

Full Text Available We examined the in vivo behavior of liver natural killer T cells (NKT cells by intravital fluorescence microscopic imaging of mice in which a green fluorescent protein cDNA was used to replace the gene encoding the chemokine receptor CXCR6. NKT cells, which account for most CXCR6+ cells in liver, were found to crawl within hepatic sinusoids at 10-20 µm/min and to stop upon T cell antigen receptor activation. CXCR6-deficient mice exhibited a selective and severe reduction of CD1d-reactive NKT cells in the liver and decreased susceptibility to T-cell-dependent hepatitis. CXCL16, the cell surface ligand for CXCR6, is expressed on sinusoidal endothelial cells, and CXCR6 deficiency resulted in reduced survival, but not in altered speed or pattern of patrolling of NKT cells. Thus, NKT cells patrol liver sinusoids to provide intravascular immune surveillance, and CXCR6 contributes to liver-based immune responses by regulating their abundance.

Functional, Antigen-Specific Stem Cell Memory (TSCM CD4+ T Cells Are Induced by Human Mycobacterium tuberculosis Infection

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Cheleka A. M. Mpande

2018-03-01

Full Text Available BackgroundMaintenance of long-lasting immunity is thought to depend on stem cell memory T cells (TSCM, which have superior self-renewing capacity, longevity and proliferative potential compared with central memory (TCM or effector (TEFF T cells. Our knowledge of TSCM derives primarily from studies of virus-specific CD8+ TSCM. We aimed to determine if infection with Mycobacterium tuberculosis (M. tb, the etiological agent of tuberculosis, generates antigen-specific CD4+ TSCM and to characterize their functional ontology.MethodsWe studied T cell responses to natural M. tb infection in a longitudinal adolescent cohort of recent QuantiFERON-TB Gold (QFT converters and three cross-sectional QFT+ adult cohorts; and to bacillus Calmette–Guerin (BCG vaccination in infants. M. tb and/or BCG-specific CD4 T cells were detected by flow cytometry using major histocompatibility complex class II tetramers bearing Ag85, CFP-10, or ESAT-6 peptides, or by intracellular cytokine staining. Transcriptomic analyses of M. tb-specific tetramer+ CD4+ TSCM (CD45RA+ CCR7+ CD27+ were performed by microfluidic qRT-PCR, and functional and phenotypic characteristics were confirmed by measuring expression of chemokine receptors, cytotoxic molecules and cytokines using flow cytometry.ResultsM. tb-specific TSCM were not detected in QFT-negative persons. After QFT conversion frequencies of TSCM increased to measurable levels and remained detectable thereafter, suggesting that primary M. tb infection induces TSCM cells. Gene expression (GE profiling of tetramer+ TSCM showed that these cells were distinct from bulk CD4+ naïve T cells (TN and shared features of bulk TSCM and M. tb-specific tetramer+ TCM and TEFF cells. These TSCM were predominantly CD95+ and CXCR3+, markers typical of CD8+ TSCM. Tetramer+ TSCM expressed significantly higher protein levels of CCR5, CCR6, CXCR3, granzyme A, granzyme K, and granulysin than bulk TN and TSCM cells. M. tb-specific TSCM were also

CXCR3-dependent CD4⁺ T cells are required to activate inflammatory monocytes for defense against intestinal infection.

Directory of Open Access Journals (Sweden)

Sara B Cohen

Full Text Available Chemokines and their receptors play a critical role in orchestrating immunity to microbial pathogens, including the orally acquired Th1-inducing protozoan parasite Toxoplasma gondii. Chemokine receptor CXCR3 is associated with Th1 responses, and here we use bicistronic CXCR3-eGFP knock-in reporter mice to demonstrate upregulation of this chemokine receptor on CD4⁺ and CD8⁺ T lymphocytes during Toxoplasma infection. We show a critical role for CXCR3 in resistance to the parasite in the intestinal mucosa. Absence of the receptor in Cxcr3⁻/⁻ mice resulted in selective loss of ability to control T. gondii specifically in the lamina propria compartment. CD4⁺ T cells were impaired both in their recruitment to the intestinal lamina propria and in their ability to secrete IFN-γ upon stimulation. Local recruitment of CD11b⁺Ly6C/G⁺ inflammatory monocytes, recently reported to be major anti-Toxoplasma effectors in the intestine, was not impacted by loss of CXCR3. However, inflammatory monocyte activation status, as measured by dual production of TNF-α and IL-12, was severely impaired in Cxcr3⁻/⁻ mice. Strikingly, adoptive transfer of wild-type but not Ifnγ⁻/⁻ CD4⁺ T lymphocytes into Cxcr3⁻/⁻ animals prior to infection corrected the defect in inflammatory macrophage activation, simultaneously reversing the susceptibility phenotype of the knockout animals. Our results establish a central role for CXCR3 in coordinating innate and adaptive immunity, ensuring generation of Th1 effectors and their trafficking to the frontline of infection to program microbial killing by inflammatory monocytes.

Molecular Pharmacology of CXCR4 inhibition

DEFF Research Database (Denmark)

Steen, Anne; Rosenkilde, Mette Marie

2012-01-01

pharmacology of well-known CXCR4 antagonists in order to augment the potency and affinity and to increase the specificity of future CXCR4-targeting compounds. In this chapter, binding modes of CXCR4 antagonists that have been shown to mobilize stem cells are discussed. In addition, comparisons between results...

BAG3 promotes stem cell-like phenotype in breast cancer by upregulation of CXCR4 via interaction with its transcript.

Science.gov (United States)

Liu, Bao-Qin; Zhang, Song; Li, Si; An, Ming-Xin; Li, Chao; Yan, Jing; Wang, Jia-Mei; Wang, Hua-Qin

2017-07-13

BAG3 is an evolutionarily conserved co-chaperone expressed at high levels and has a prosurvival role in many tumor types. The current study reported that BAG3 was induced under specific floating culture conditions that enrich breast cancer stem cell (BCSC)-like cells in spheres. Ectopic BAG3 overexpression increased CD44 + /CD24 - CSC subpopulations, first-generation and second-generation mammosphere formation, indicating that BAG3 promotes CSC self-renewal and maintenance in breast cancer. We further demonstrated that mechanically, BAG3 upregulated CXCR4 expression at the post-transcriptional level. Further studies showed that BAG3 interacted with CXCR4 mRNA and promoted its expression via its coding and 3'-untranslational regions. BAG3 was also found to be positively correlated with CXCR4 expression and unfavorable prognosis in patients with breast cancer. Taken together, our data demonstrate that BAG3 promotes BCSC-like phenotype through CXCR4 via interaction with its transcript. Therefore, this study establishes BAG3 as a potential adverse prognostic factor and a therapeutic target of breast cancer.

Peripheral Blood CCR4+CCR6+ and CXCR3+CCR6+ CD4+ T Cells Are Highly Permissive to HIV-1 Infection

OpenAIRE

Gosselin, Annie; Monteiro, Patricia; Chomont, Nicolas; Diaz-Griffero, Felipe; Said, Elias A.; Fonseca, Simone; Wacleche, Vanessa; El-Far, Mohamed; Boulassel, Mohamed-Rachid; Routy, Jean-Pierre; Sekaly, Rafick-Pierre; Ancuta, Petronela

2009-01-01

There is limited knowledge on the identity of primary CD4+ T cell subsets selectively targeted by HIV-1 in vivo. In this study, we established a link between HIV permissiveness, phenotype/homing potential, and lineage commitment in primary CD4+ T cells. CCR4+CCR6+, CCR4+CCR6−, CXCR3+CCR6+, and CXCR3+CCR6− T cells expressed cytokines and transcription factors specific for Th17, Th2, Th1Th17, and Th1 lineages, respectively. CCR4+CCR6+ and CXCR3+CCR6+ T cells expressed the HIV coreceptors CCR5 a...

African-American Men with Gleason Score 3+3=6 Prostate Cancer Produce Less Prostate Specific Antigen than Caucasian Men: A Potential Impact on Active Surveillance.

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Kryvenko, Oleksandr N; Balise, Raymond; Soodana Prakash, Nachiketh; Epstein, Jonathan I

2016-02-01

We assess the difference in prostate specific antigen production between African-American and Caucasian men with Gleason score 3+3=6 prostate cancer. We measured tumor volume in 414 consecutive radical prostatectomies from men with National Comprehensive Cancer Network(®) low risk prostate cancer (348 Caucasian, 66 African-American) who had Gleason score 3+3=6 disease at radical prostatectomy. We then compared clinical presentation, pathological findings, prostate specific antigen, prostate specific antigen density and prostate specific antigen mass (an absolute amount of prostate specific antigen in patient's circulation) between African-American and Caucasian men. The t-test and Wilcoxon rank sum were used for comparison of means. African-American and Caucasian men had similar clinical findings based on age, body mass index and prostate specific antigen. There were no statistically significant differences between the dominant tumor nodule volume and total tumor volume (mean 0.712 vs 0.665 cm(3), p=0.695) between African-American and Caucasian men. Prostates were heavier in African-American men (mean 55.4 vs 46.3 gm, p prostate tissue contributing to prostate specific antigen in African-American men, prostate specific antigen mass was not different from that of Caucasian men (mean 0.55 vs 0.558 μg, p=0.95). Prostate specific antigen density was significantly less in African-American men due to larger prostates (mean 0.09 vs 0.105, p prostate cancer produce less prostate specific antigen than Caucasian men. African-American and Caucasian men had equal serum prostate specific antigen and prostate specific antigen mass despite significantly larger prostates in African-American men with all other parameters, particularly total tumor volume, being the same. This finding has practical implications in T1c cases diagnosed with prostate cancer due to prostate specific antigen screening. Lowering the prostate specific antigen density threshold in African-American men may

Prostate-specific membrane antigen-directed nanoparticle targeting for extreme nearfield ablation of prostate cancer cells.

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Lee, Seung S; Roche, Philip Jr; Giannopoulos, Paresa N; Mitmaker, Elliot J; Tamilia, Michael; Paliouras, Miltiadis; Trifiro, Mark A

2017-03-01

Almost all biological therapeutic interventions cannot overcome neoplastic heterogeneity. Physical ablation therapy is immune to tumor heterogeneity, but nearby tissue damage is the limiting factor in delivering lethal doses. Multi-walled carbon nanotubes offer a number of unique properties: chemical stability, photonic properties including efficient light absorption, thermal conductivity, and extensive surface area availability for covalent chemical ligation. When combined together with a targeting moiety such as an antibody or small molecule, one can deliver highly localized temperature increases and cause extensive cellular damage. We have functionalized multi-walled carbon nanotubes by conjugating an antibody against prostate-specific membrane antigen. In our in vitro studies using prostate-specific membrane antigen-positive LNCaP prostate cancer cells, we have effectively demonstrated cell ablation of >80% with a single 30-s exposure to a 2.7-W, 532-nm laser for the first time without bulk heating. We also confirmed the specificity and selectivity of prostate-specific membrane antigen targeting by assessing prostate-specific membrane antigen-null PC3 cell lines under the same conditions (<10% cell ablation). This suggests that we can achieve an extreme nearfield cell ablation effect, thus restricting potential tissue damage when transferred to in vivo clinical applications. Developing this new platform will introduce novel approaches toward current therapeutic modalities and will usher in a new age of effective cancer treatment squarely addressing tumoral heterogeneity.

RANKL downregulates cell surface CXCR6 expression through JAK2/STAT3 signaling pathway during osteoclastogenesis

Energy Technology Data Exchange (ETDEWEB)

Li, Changhong; Zhao, Jinxia; Sun, Lin; Yao, Zhongqiang; Liu, Rui [Department of Rheumatology and Immunology, Peking University Third Hospital, Beijing 100191 (China); Huang, Jiansheng [Department of Pediatrics, Washington University School of Medicine, St. Louis, MO (United States); Liu, Xiangyuan, E-mail: liu-xiangyuan@263.net [Department of Rheumatology and Immunology, Peking University Third Hospital, Beijing 100191 (China)

2012-12-14

Highlights: Black-Right-Pointing-Pointer CXCR6 is down-regulated during RANKL-induced osteoclastogenesis in RAW264.7 cells. Black-Right-Pointing-Pointer CXCR6 reduction was nearly reversed by inhibition of JAK2/STAT3 signaling pathway. Black-Right-Pointing-Pointer CXCL16 alone does not positively regulate osteoclastogenesis. -- Abstract: The receptor activator of nuclear factor-{kappa}B ligand (RANKL), as a member of the tumor necrosis factor (TNF) family, plays an essential role in osteoclast differentiation and function. Chemokines and their receptors have recently been shown to play critical roles in osteoclastogenesis, however, whether CXCL16-CXCR6 plays role in RANKL-mediated osteoclastogenesis is unknown. In this study, we first reported that RANKL decreased CXCR6 in a dose-dependent manner, which may be through deactivation of Akt and STAT3 signaling induced by CXCL16. Interestingly, RANKL-mediated CXCR6 reduction may be associated to the activation of STAT3 by phosphorylation. When STAT3 activation was blocked by JAK2/STAT3 inhibitor AG490, RANKL failed to shut down CXCR6 expression during osteoclastogenesis. However, CXCL16 alone did not augment RANKL-mediated osteoclast differentiation and did not alter RANKL-receptor RANK mRNA expression. These results demonstrate that reduction of CXCL16-CXCR6 is critical in RANKL-mediated osteoclastogenesis, which is mainly through the activation of JAK2/STAT3 signaling. CXCL16-CXCR6 axis may become a novel target for the therapeutic intervention of bone resorbing diseases such as rheumatoid arthritis and osteoporosis.

Effectiveness of the combined evaluation of KLK3 genetics and free-to-total prostate specific antigen ratio for prostate cancer diagnosis.

Science.gov (United States)

Zambon, Carlo-Federico; Prayer-Galetti, Tommaso; Basso, Daniela; Padoan, Andrea; Rossi, Elisa; Secco, Silvia; Pelloso, Michela; Fogar, Paola; Navaglia, Filippo; Moz, Stefania; Zattoni, Filiberto; Plebani, Mario

2012-10-01

Of serum prostate specific antigen variability 40% depends on inherited factors. We ascertained whether the knowledge of KLK3 genetics would enhance prostate specific antigen diagnostic performance in patients with clinical suspicion of prostate cancer. We studied 1,058 men who consecutively underwent prostate biopsy for clinical suspicion of prostate cancer. At histology prostate cancer was present in 401 cases and absent in 657. Serum total prostate specific antigen and the free-to-total prostate specific antigen ratio were determined. Four polymorphisms of the KLK3 gene (rs2569733, rs2739448, rs925013 and rs2735839) and 1 polymorphism of the SRD5A2 gene (rs523349) were studied. The influence of genetics on prostate specific antigen variability was evaluated by multivariate linear regression analysis. The performance of total prostate specific antigen and the free-to-total prostate specific antigen ratio alone or combined with a genetically based patient classification were defined by ROC curve analyses. For prostate cancer diagnosis the free-to-total prostate specific antigen ratio index alone (cutoff 11%) was superior to total prostate specific antigen (cutoff 4 ng/ml) and to free-to-total prostate specific antigen ratio reflex testing (positive predictive value 61%, 43% and 54%, respectively). Prostate specific antigen correlated with KLK3 genetics (rs2735839 polymorphism p = 0.001, and rs2569733, rs2739448 and rs925013 haplotype combination p = 0.003). In patients with different KLK3 genetics 2 optimal free-to-total prostate specific antigen ratio cutoffs (11% and 14.5%) were found. For free-to-total prostate specific antigen ratio values between 11% and 14.5% the prostate cancer probability ranged from 30.0% to 47.4% according to patient genetics. The free-to-total prostate specific antigen ratio is superior to total prostate specific antigen for prostate cancer diagnosis, independent of total prostate specific antigen results. Free-to-total prostate

submitter Emerging importance of chemokine receptor CXCR3 and its ligands in cardiovascular diseases

CERN Document Server

Altara, R; Brandao, R D; Zeidan, A; Booz, G W; Zouein, F A

2016-01-01

The CXC chemokines, CXCL4, -9, -10, -11, CXCL4L1, and the CC chemokine CCL21, activate CXC chemokine receptor 3 (CXCR3), a cell-surface G protein-coupled receptor expressed mainly by Th1 cells, cytotoxic T (Tc) cells and NK cells that have a key role in immunity and inflammation. However, CXCR3 is also expressed by vascular smooth muscle and endothelial cells, and appears to be important in controlling physiological vascular function. In the last decade, evidence from pre-clinical and clinical studies has revealed the participation of CXCR3 and its ligands in multiple cardiovascular diseases (CVDs) of different aetiologies including atherosclerosis, hypertension, cardiac hypertrophy and heart failure, as well as in heart transplant rejection and transplant coronary artery disease (CAD). CXCR3 ligands have also proven to be valid biomarkers for the development of heart failure and left ventricular dysfunction, suggesting an underlining pathophysiological relation between levels of these chemokines and the deve...

Gliadin induces an increase in intestinal permeability and zonulin release by binding to the chemokine receptor CXCR3.

Science.gov (United States)

Lammers, Karen M; Lu, Ruliang; Brownley, Julie; Lu, Bao; Gerard, Craig; Thomas, Karen; Rallabhandi, Prasad; Shea-Donohue, Terez; Tamiz, Amir; Alkan, Sefik; Netzel-Arnett, Sarah; Antalis, Toni; Vogel, Stefanie N; Fasano, Alessio

2008-07-01

Celiac disease is an immune-mediated enteropathy triggered by gliadin, a component of the grain protein gluten. Gliadin induces an MyD88-dependent zonulin release that leads to increased intestinal permeability, a postulated early element in the pathogenesis of celiac disease. We aimed to establish the molecular basis of gliadin interaction with intestinal mucosa leading to intestinal barrier impairment. Alpha-gliadin affinity column was loaded with intestinal mucosal membrane lysates to identify the putative gliadin-binding moiety. In vitro experiments with chemokine receptor CXCR3 transfectants were performed to confirm binding of gliadin and/or 26 overlapping 20mer alpha-gliadin synthetic peptides to the receptor. CXCR3 protein and gene expression were studied in intestinal epithelial cell lines and human biopsy specimens. Gliadin-CXCR3 interaction was further analyzed by immunofluorescence microscopy, laser capture microscopy, real-time reverse-transcription polymerase chain reaction, and immunoprecipitation/Western blot analysis. Ex vivo experiments were performed using C57BL/6 wild-type and CXCR3(-/-) mouse small intestines to measure intestinal permeability and zonulin release. Affinity column and colocalization experiments showed that gliadin binds to CXCR3 and that at least 2 alpha-gliadin 20mer synthetic peptides are involved in this binding. CXCR3 is expressed in mouse and human intestinal epithelia and lamina propria. Mucosal CXCR3 expression was elevated in active celiac disease but returned to baseline levels following implementation of a gluten-free diet. Gliadin induced physical association between CXCR3 and MyD88 in enterocytes. Gliadin increased zonulin release and intestinal permeability in wild-type but not CXCR3(-/-) mouse small intestine. Gliadin binds to CXCR3 and leads to MyD88-dependent zonulin release and increased intestinal permeability.

Antigen-specific murine T cell clones produce soluble interleukin 2 receptor on stimulation with specific antigens

International Nuclear Information System (INIS)

Wagner, D.K.; York-Jolley, J.; Malek, T.R.; Berzofsky, J.A.; Nelson, D.L.

1986-01-01

In this study, monoclonal antibodies were used to the murine IL 2 receptor (IL 2R) termed 3C7 and 7D4, which bind to different epitopes on the murine IL 2R, to develop an ELISA to measure soluble murine IL 2R. Surprisingly, stimulated murine spleen cells not only expressed cell-associated IL 2R, but also produced a considerable level of cellfree IL 2R in the culture supernatant fluid. To assess the fine specificity of this response, myoglobin-immune murine T cell clones were stimulated with appropriate or inappropriate antigen and syngeneic or allogeneic presenting cells. Proliferation, measured by [ 3 H] thymidine incorporation, and levels of soluble IL 2R were determined at day 4. The production of soluble IL2R displayed the same epitope fine specificity, genetic restriction, and antigen dose-response as the proliferative response. Indeed, in some cases there was sharper discrimination of epitope specificity and genetic restriction with the soluble IL 2R levels. There was also reproducible clone-to-clone variation in the amount of soluble receptor produced in response to antigen among 12 T cell clones and lines tested. In time course experiments, proliferation was greatest at day 3, whereas soluble IL 2R levels continued to rise in subsequent days. To the authors' knowledge, this is the first demonstration of release of secretion of soluble IL 2R by murine T cells, and the first demonstration of the fine specificity and genetic restriction of the induction of soluble IL 2R by specific antigen

CXCL10/CXCR3-Dependent Mobilization of Herpes Simplex Virus-Specific CD8+ TEM and CD8+ TRM Cells within Infected Tissues Allows Efficient Protection against Recurrent Herpesvirus Infection and Disease.

Science.gov (United States)

Srivastava, Ruchi; Khan, Arif A; Chilukuri, Sravya; Syed, Sabrina A; Tran, Tien T; Furness, Julie; Bahraoui, Elmostafa; BenMohamed, Lbachir

2017-07-15

Herpes simplex virus 1 (HSV-1) establishes latency within the sensory neurons of the trigeminal ganglia (TG). HSV-specific memory CD8 + T cells play a critical role in preventing HSV-1 reactivation from TG and subsequent virus shedding in tears that trigger recurrent corneal herpetic disease. The CXC chemokine ligand 10 (CXCL10)/CXC chemokine receptor 3 (CXCR3) chemokine pathway promotes T cell immunity to many viral pathogens, but its importance in CD8 + T cell immunity to recurrent herpes has been poorly elucidated. In this study, we determined how the CXCL10/CXCR3 pathway affects TG- and cornea-resident CD8 + T cell responses to recurrent ocular herpesvirus infection and disease using a well-established murine model in which HSV-1 reactivation was induced from latently infected TG by UV-B light. Following UV-B-induced HSV-1 reactivation, a significant increase in both the number and function of HSV-specific CXCR3 + CD8 + T cells was detected in TG and corneas of protected C57BL/6 (B6) mice, but not in TG and corneas of nonprotected CXCL10 -/- or CXCR3 -/- deficient mice. This increase was associated with a significant reduction in both virus shedding and recurrent corneal herpetic disease. Furthermore, delivery of exogenous CXCL10 chemokine in TG of CXCL10 -/- mice, using the neurotropic adeno-associated virus type 8 (AAV8) vector, boosted the number and function of effector memory CD8 + T cells (T EM ) and tissue-resident memory CD8 + T cells (T RM ), but not of central memory CD8 + T cells (T CM ), locally within TG, and improved protection against recurrent herpesvirus infection and disease in CXCL10 -/- deficient mice. These findings demonstrate that the CXCL10/CXCR3 chemokine pathway is critical in shaping CD8 + T cell immunity, locally within latently infected tissues, which protects against recurrent herpesvirus infection and disease. IMPORTANCE We determined how the CXCL10/CXCR3 pathway affects CD8 + T cell responses to recurrent ocular herpesvirus

Llama-derived single variable domains (nanobodies) directed against chemokine receptor CXCR7 reduce head and neck cancer cell growth in vivo.

Science.gov (United States)

Maussang, David; Mujić-Delić, Azra; Descamps, Francis J; Stortelers, Catelijne; Vanlandschoot, Peter; Stigter-van Walsum, Marijke; Vischer, Henry F; van Roy, Maarten; Vosjan, Maria; Gonzalez-Pajuelo, Maria; van Dongen, Guus A M S; Merchiers, Pascal; van Rompaey, Philippe; Smit, Martine J

2013-10-11

The chemokine receptor CXCR7, belonging to the membrane-bound G protein-coupled receptor superfamily, is expressed in several tumor types. Inhibition of CXCR7 with either small molecules or small interference (si)RNA has shown promising therapeutic benefits in several tumor models. With the increased interest and effectiveness of biologicals inhibiting membrane-bound receptors we made use of the "Nanobody platform" to target CXCR7. Previously we showed that Nanobodies, i.e. immunoglobulin single variable domains derived from naturally occurring heavy chain-only camelids antibodies, represent new biological tools to efficiently tackle difficult drug targets such as G protein-coupled receptors. In this study we developed and characterized highly selective and potent Nanobodies against CXCR7. Interestingly, the CXCR7-targeting Nanobodies displayed antagonistic properties in contrast with previously reported CXCR7-targeting agents. Several high affinity CXCR7-specific Nanobodies potently inhibited CXCL12-induced β-arrestin2 recruitment in vitro. A wide variety of tumor biopsies was profiled, showing for the first time high expression of CXCR7 in head and neck cancer. Using a patient-derived CXCR7-expressing head and neck cancer xenograft model in nude mice, tumor growth was inhibited by CXCR7-targeting Nanobody therapy. Mechanistically, CXCR7-targeting Nanobodies did not inhibit cell cycle progression but instead reduced secretion of the angiogenic chemokine CXCL1 from head and neck cancer cells in vitro, thus acting here as inverse agonists, and subsequent angiogenesis in vivo. Hence, with this novel class of CXCR7 inhibitors, we further substantiate the therapeutic relevance of targeting CXCR7 in head and neck cancer.

Downregulation of CXCR4 Expression and Functionality After Zoledronate Exposure in Canine Osteosarcoma.

Science.gov (United States)

Byrum, M L; Pondenis, H C; Fredrickson, R L; Wycislo, K L; Fan, T M

2016-07-01

The establishment and progression of metastases remains the life-limiting factor for dogs diagnosed with osteosarcoma (OS). The pattern of metastases is likely regulated through interactions between chemokine receptors and chemokines, and perturbations in these signaling cascades responsible for cytoskeletal organization and directional migration have the potential to alter metastatic cell trafficking behaviors. Zoledronate will impair directional migration of OS cells through downregulation of chemokine (C-X-C motif) receptor 4 (CXCR4) expression and functionality. Nineteen archived tumor specimens and plasma from 20 dogs with OS. Prospectively, the expressions of CXCR4 were studied in OS cell lines and spontaneous tumor samples. The effect of zoledronate on CXCR4 expression and functionality was investigated by characterizing responses in 3 OS cell lines. In 19 OS specimens and 20 dogs with OS, changes in CXCR4 expression and circulating CXCR4 concentrations were characterized in response to zoledronate therapy respectively. All canine OS cells express CXCR4, and zoledronate reduces CXCR4 expression and functionality by 27.7% (P < .0001), through augmented proteasome degradation and reduced prenylation of heterotrimeric G-proteins in 33% of tumor cell lines evaluated. In OS-bearing dogs, zoledronate reduces CXCR4 expressions by 40% within the primary tumor compared to untreated controls (P = .03) and also decreases the circulating concentrations of CXCR4 in 18 of 20 dogs with OS. Zoledronate can alter CXCR4 expression and functionality in OS cells, and consequent perturbations in CXCR4 intracellular signaling cascades might influence patterns of metastases. Copyright © 2016 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

CXCR7 controls competition for recruitment of β-arrestin 2 in cells expressing both CXCR4 and CXCR7.

Directory of Open Access Journals (Sweden)

Nathaniel L Coggins

Full Text Available Chemokine CXCL12 promotes growth and metastasis of more than 20 different human cancers, as well as pathogenesis of other common diseases. CXCL12 binds two different receptors, CXCR4 and CXCR7, both of which recruit and signal through the cytosolic adapter protein β-arrestin 2. Differences in CXCL12-dependent recruitment of β-arrestin 2 in cells expressing one or both receptors remain poorly defined. To quantitatively investigate parameters controlling association of β-arrestin 2 with CXCR4 or CXCR7 in cells co-expressing both receptors, we used a systems biology approach combining real-time, multi-spectral luciferase complementation imaging with computational modeling. Cells expressing only CXCR4 maintain low basal association with β-arrestin 2, and CXCL12 induces a rapid, transient increase in this interaction. In contrast, cells expressing only CXCR7 have higher basal association with β-arrestin 2 and exhibit more gradual, prolonged recruitment of β-arrestin 2 in response to CXCL12. We developed and fit a data-driven computational model for association of either CXCR4 or CXCR7 with β-arrestin 2 in cells expressing only one type of receptor. We then experimentally validated model predictions that co-expression of CXCR4 and CXCR7 on the same cell substantially decreases both the magnitude and duration of CXCL12-regulated recruitment of β-arrestin 2 to CXCR4. Co-expression of both receptors on the same cell only minimally alters recruitment of β-arrestin 2 to CXCR7. In silico experiments also identified β-arrestin 2 as a limiting factor in cells expressing both receptors, establishing that CXCR7 wins the "competition" with CXCR4 for CXCL12 and recruitment of β-arrestin 2. These results reveal how competition for β-arrestin 2 controls integrated responses to CXCL12 in cells expressing both CXCR4 and CXCR7. These results advance understanding of normal and pathologic functions of CXCL12, which is critical for developing effective

Pharmacological characterization of [3H]VUF11211, a novel radiolabeled small-molecule inverse agonist for the chemokine receptor CXCR3

NARCIS (Netherlands)

Scholten, Danny J; Wijtmans, M.; van Senten, Jeffrey R; Custers, Hans; Stunnenberg, Ailas; de Esch, Iwan J P; Smit, Martine J; Leurs, Rob

Chemokine receptor CXCR3 has attracted much attention, as it is thought to be associated with a wide range of immune-related diseases. As such, several small molecules with different chemical structures targeting CXCR3 have been discovered. Despite limited clinical success so far, these compounds

Chemokine receptors CCR6 and CXCR3 are necessary for CD4(+) T cell mediated ocular surface disease in experimental dry eye disease.

Science.gov (United States)

Coursey, Terry G; Gandhi, Niral B; Volpe, Eugene A; Pflugfelder, Stephen C; de Paiva, Cintia S

2013-01-01

CD4(+) T cells are essential to pathogenesis of ocular surface disease in dry eye. Two subtypes of CD4(+) T cells, Th1 and Th17 cells, function concurrently in dry eye to mediate disease. This occurs in spite of the cross-regulation of IFN-γ and IL-17A, the prototypical cytokines Th1 and Th17 cells, respectively. Essential to an effective immune response are chemokines that direct and summon lymphocytes to specific tissues. T cell trafficking has been extensively studied in other models, but this is the first study to examine the role of chemokine receptors in ocular immune responses. Here, we demonstrate that the chemokine receptors, CCR6 and CXCR3, which are expressed on Th17 and Th1 cells, respectively, are required for the pathogenesis of dry eye disease, as CCR6KO and CXCR3KO mice do not develop disease under desiccating stress. CD4(+) T cells from CCR6KO and CXCR3KO mice exposed to desiccating stress (DS) do not migrate to the ocular surface, but remain in the superficial cervical lymph nodes. In agreement with this, CD4(+) T cells from CCR6 and CXCR3 deficient donors exposed to DS, when adoptively transferred to T cell deficient recipients manifest minimal signs of dry eye disease, including significantly less T cell infiltration, goblet cell loss, and expression of inflammatory cytokine and matrix metalloproteinase expression compared to wild-type donors. These findings highlight the important interaction of chemokine receptors on T cells and chemokine ligand expression on epithelial cells of the cornea and conjunctiva in dry eye pathogenesis and reveal potential new therapeutic targets for dry eye disease.

Chemokine Receptors CCR6 and CXCR3 Are Necessary for CD4+ T Cell Mediated Ocular Surface Disease in Experimental Dry Eye Disease

Science.gov (United States)

Coursey, Terry G.; Gandhi, Niral B.; Volpe, Eugene A.; Pflugfelder, Stephen C.; de Paiva, Cintia S.

2013-01-01

CD4+ T cells are essential to pathogenesis of ocular surface disease in dry eye. Two subtypes of CD4+ T cells, Th1 and Th17 cells, function concurrently in dry eye to mediate disease. This occurs in spite of the cross-regulation of IFN-γ and IL-17A, the prototypical cytokines Th1 and Th17 cells, respectively. Essential to an effective immune response are chemokines that direct and summon lymphocytes to specific tissues. T cell trafficking has been extensively studied in other models, but this is the first study to examine the role of chemokine receptors in ocular immune responses. Here, we demonstrate that the chemokine receptors, CCR6 and CXCR3, which are expressed on Th17 and Th1 cells, respectively, are required for the pathogenesis of dry eye disease, as CCR6KO and CXCR3KO mice do not develop disease under desiccating stress. CD4+ T cells from CCR6KO and CXCR3KO mice exposed to desiccating stress (DS) do not migrate to the ocular surface, but remain in the superficial cervical lymph nodes. In agreement with this, CD4+ T cells from CCR6 and CXCR3 deficient donors exposed to DS, when adoptively transferred to T cell deficient recipients manifest minimal signs of dry eye disease, including significantly less T cell infiltration, goblet cell loss, and expression of inflammatory cytokine and matrix metalloproteinase expression compared to wild-type donors. These findings highlight the important interaction of chemokine receptors on T cells and chemokine ligand expression on epithelial cells of the cornea and conjunctiva in dry eye pathogenesis and reveal potential new therapeutic targets for dry eye disease. PMID:24223818

Pathogen-group specific association between CXCR1 polymorphisms and subclinical mastitis in dairy heifers.

Science.gov (United States)

Verbeke, Joren; Piepers, Sofie; Peelman, Luc; Van Poucke, Mario; De Vliegher, Sarne

2012-08-01

The chemokine (C-X-C motif) receptor 1 (CXCR1) gene encodes the homonymous receptor for interleukin 8 (IL8) on polymorphonuclear neutrophilic leucocytes (PMNL). Binding causes migration from blood to milk, activation and prolonged survival of PMNL, a crucial process in the innate immune defence of the bovine mammary gland against invading mastitis-causing pathogens. The main objective of this study was to screen the entire coding region of the CXCR1 gene for polymorphisms and to analyse their association with udder health of dairy heifers. One-hundred-and-forty Belgian Holstein heifers originating from 20 commercial dairy farms were genotyped by DNA sequencing. Detailed phenotypic data on udder health was available including quarter bacteriological culture results and somatic cell count (SCC) in early lactation and composite milk SCC during first lactation. In total, 16 polymorphisms (including 8 missense mutations) were detected. Polymorphism c.980A>G was associated with pathogen-group specific IMI: heifers with genotype AG were less likely to have an IMI due to major mastitis pathogens compared with heifers with genotype GG but did not have less IMI by coagulase-negative staphylococci, so-called minor pathogens. CXCR1 genotype was neither associated with quarter SCC in early lactation nor with composite SCC during lactation. Although mastitis susceptibility is influenced by many factors, some genetic polymorphisms potentially have major effects on udder health of heifers, as was shown here. These results trigger us to further study the relationship between CXCR1 polymorphisms and mastitis susceptibility in both observational and experimental trials.

COX-2 and Prostaglandin EP3/EP4 Signaling Regulate the Tumor Stromal Proangiogenic Microenvironment via CXCL12-CXCR4 Chemokine Systems

Science.gov (United States)

Katoh, Hiroshi; Hosono, Kanako; Ito, Yoshiya; Suzuki, Tatsunori; Ogawa, Yasufumi; Kubo, Hidefumi; Kamata, Hiroki; Mishima, Toshiaki; Tamaki, Hideaki; Sakagami, Hiroyuki; Sugimoto, Yukihiko; Narumiya, Shuh; Watanabe, Masahiko; Majima, Masataka

2010-01-01

Bone marrow (BM)–derived hematopoietic cells, which are major components of tumor stroma, determine the tumor microenvironment and regulate tumor phenotypes. Cyclooxygenase (COX)−2 and endogenous prostaglandins are important determinants for tumor growth and tumor-associated angiogenesis; however, their contributions to stromal formation and angiogenesis remain unclear. In this study, we observed that Lewis lung carcinoma cells implanted in wild-type mice formed a tumor mass with extensive stromal formation that was markedly suppressed by COX-2 inhibition, which reduced the recruitment of BM cells. Notably, COX-2 inhibition attenuated CXCL12/CXCR4 expression as well as expression of several other chemokines. Indeed, in a Matrigel model, prostaglandin (PG) E2 enhanced stromal formation and CXCL12/CXCR4 expression. In addition, a COX-2 inhibitor suppressed stromal formation and reduced expression of CXCL12/CXCR4 and a fibroblast marker (S100A4) in a micropore chamber model. Moreover, stromal formation after tumor implantation was suppressed in EP3−/− mice and EP4−/− mice, in which stromal expression of CXCL12/CXCR4 and S100A4 was reduced. The EP3 or EP4 knockout suppressed S100A4+ fibroblasts, CXCL12+, and/or CXCR4+ stromal cells as well. Immunofluorescent analyses revealed that CXCL12+CXCR4+S100A4+ fibroblasts mainly comprised stromal cells and most of these were recruited from the BM. Additionally, either EP3- or EP4-specific agonists stimulated CXCL12 expression by fibroblasts in vitro. The present results address the novel activities of COX-2/PGE2-EP3/EP4 signaling that modulate tumor biology and show that CXCL12/CXCR4 axis may play a crucial role in tumor stromal formation and angiogenesis under the control of prostaglandins. PMID:20110411

Antigen presentation by hapten-specific B lymphocytes. II. Specificity and properties of antigen-presenting B lymphocytes, and function of immunoglobulin receptors

International Nuclear Information System (INIS)

Abbas, A.K.; Haber, S.; Rock, K.L.

1985-01-01

Studies were designed to examine the ability of hapten-binding murine B lymphocytes to present hapten-protein conjugates to protein antigen-specific, Ia-restricted T cell hybridomas. BALB/c B cells specific for TNP or FITC presented hapten-modified proteins (TNP-G1 phi, TNP-OVA, or FITC-OVA) to the relevant T cell hybridomas at concentrations below 0.1 microgram/ml. Effective presentation of the same antigens by B lymphocyte-depleted splenocytes, and of unmodified proteins by either hapten-binding B cells or Ig spleen cells, required about 10(3)-to 10(4)-fold higher concentrations of antigen. The use of two different haptens and two carrier proteins showed that this extremely efficient presentation of antigen was highly specific, with hapten specificity being a property of the B cells and carrier specificity of the responding T cells. The presentation of hapten-proteins by hapten-binding B lymphocytes was radiosensitive and was not affected by the depletion of plastic-adherent cells, suggesting that conventional APCs (macrophages or dendritic cells) are not required in this phenomenon. Antigen-pulsing and antibody-blocking experiments showed that this hapten-specific antigen presentation required initial binding of antigen to surface Ig receptors. Moreover, linked recognition of hapten and carrier determinants was required, but these recognition events could be temporally separated. Finally, an antigen-processing step was found to be necessary, and this step was disrupted by ionizing radiation. These data suggest a role for B cell surface Ig in providing a specific high-affinity receptor to allow efficient uptake or focusing of antigen for its subsequent processing and presentation to T lymphocytes

Circulating precursor CCR7(lo)PD-1(hi) CXCR5⁺ CD4⁺ T cells indicate Tfh cell activity and promote antibody responses upon antigen reexposure.

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He, Jing; Tsai, Louis M; Leong, Yew Ann; Hu, Xin; Ma, Cindy S; Chevalier, Nina; Sun, Xiaolin; Vandenberg, Kirsten; Rockman, Steve; Ding, Yan; Zhu, Lei; Wei, Wei; Wang, Changqi; Karnowski, Alexander; Belz, Gabrielle T; Ghali, Joanna R; Cook, Matthew C; Riminton, D Sean; Veillette, André; Schwartzberg, Pamela L; Mackay, Fabienne; Brink, Robert; Tangye, Stuart G; Vinuesa, Carola G; Mackay, Charles R; Li, Zhanguo; Yu, Di

2013-10-17

Follicular B helper T (Tfh) cells support high affinity and long-term antibody responses. Here we found that within circulating CXCR5⁺ CD4⁺ T cells in humans and mice, the CCR7(lo)PD-1(hi) subset has a partial Tfh effector phenotype, whereas CCR7(hi)PD-1(lo) cells have a resting phenotype. The circulating CCR7(lo)PD-1(hi) subset was indicative of active Tfh differentiation in lymphoid organs and correlated with clinical indices in autoimmune diseases. Thus the CCR7(lo)PD-1(hi) subset provides a biomarker to monitor protective antibody responses during infection or vaccination and pathogenic antibody responses in autoimmune diseases. Differentiation of both CCR7(hi)PD-1(lo) and CCR7(lo)PD-1(hi) subsets required ICOS and BCL6, but not SAP, suggesting that circulating CXCR5⁺ helper T cells are primarily generated before germinal centers. Upon antigen reencounter, CCR7(lo)PD-1(hi) CXCR5⁺ precursors rapidly differentiate into mature Tfh cells to promote antibody responses. Therefore, circulating CCR7(lo)PD-1(hi) CXCR5⁺ CD4⁺ T cells are generated during active Tfh differentiation and represent a new mechanism of immunological early memory. Copyright © 2013 Elsevier Inc. All rights reserved.

Chemokine receptors CXCR2 and CX3CR1 differentially regulate functional responses of bone-marrow endothelial progenitors during atherosclerotic plaque regression

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Herlea-Pana, Oana; Yao, Longbiao; Heuser-Baker, Janet; Wang, Qiongxin; Wang, Qilong; Georgescu, Constantin; Zou, Ming-Hui; Barlic-Dicen, Jana

2015-01-01

Aims Atherosclerosis manifests itself as arterial plaques, which lead to heart attacks or stroke. Treatments supporting plaque regression are therefore aggressively pursued. Studies conducted in models in which hypercholesterolaemia is reversible, such as the Reversa mouse model we have employed in the current studies, will be instrumental for the development of such interventions. Using this model, we have shown that advanced atherosclerosis regression occurs when lipid lowering is used in combination with bone-marrow endothelial progenitor cell (EPC) treatment. However, it remains unclear how EPCs home to regressing plaques and how they augment atherosclerosis reversal. Here we identify molecules that support functional responses of EPCs during plaque resolution. Methods and results Chemokines CXCL1 and CX3CL1 were detected in the vascular wall of atheroregressing Reversa mice, and their cognate receptors CXCR2 and CX3CR1 were observed on adoptively transferred EPCs in circulation. We tested whether CXCL1–CXCR2 and CX3CL1–CX3CR1 axes regulate functional responses of EPCs during plaque reversal. We show that pharmacological inhibition of CXCR2 or CX3CR1, or genetic inactivation of these two chemokine receptors interfered with EPC-mediated advanced atherosclerosis regression. We also demonstrate that CXCR2 directs EPCs to regressing plaques while CX3CR1 controls a paracrine function(s) of these cells. Conclusion CXCR2 and CX3CR1 differentially regulate EPC functional responses during atheroregression. Our study improves understanding of how chemokines and chemokine receptors regulate plaque resolution, which could determine the effectiveness of interventions reducing complications of atherosclerosis. PMID:25765938

CELLISA: reporter cell-based immunization and screening of hybridomas specific for cell surface antigens.

Science.gov (United States)

Chen, Peter; Mesci, Aruz; Carlyle, James R

2011-01-01

Monoclonal antibodies (mAbs) specific for cell surface antigens are an invaluable tool to study immune receptor expression and function. Here, we outline a generalized reporter cell-based approach to the generation and high-throughput screening of mAbs specific for cell surface antigens. Termed CELLISA, this technology hinges upon the capture of hybridoma supernatants in mAb arrays that facilitate ligation of an antigen of interest displayed on BWZ reporter cells in the form of a CD3ζ-fusion chimeric antigen receptor (zCAR); in turn, specific mAb-mediated cross-linking of zCAR on BWZ cells results in the production of β-galactosidase enzyme (β-gal), which can be assayed colorimetrically. Importantly, the BWZ reporter cells bearing the zCAR of interest may be used for immunization as well as screening. In addition, serial immunizations employing additional zCAR- or native antigen-bearing cell lines can be used to increase the frequency of the desired antigen-specific hybridomas. Finally, the use of a cohort of epitope-tagged zCAR (e.g., zCAR(FLAG)) variants allows visualization of the cell surface antigen prior to immunization, and coimmunization using these variants can be used to enhance the immunogenicity of the target antigen. Employing the CELLISA strategy, we herein describe the generation of mAb directed against an uncharacterized natural killer cell receptor protein.

Synaptotagmin 3 deficiency in T cells impairs recycling of the chemokine receptor CXCR4 and thereby inhibits CXCL12 chemokine-induced migration.

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Masztalerz, Agnieszka; Zeelenberg, Ingrid S; Wijnands, Yvonne M; de Bruijn, Rosalie; Drager, Angelika M; Janssen, Hans; Roos, Ed

2007-01-15

Synaptotagmins regulate vesicle trafficking and fusion of vesicles with membranes - processes that have been implicated in cell migration. We therefore hypothesized that synaptotagmins play a role in T-cell migration. Amongst synaptotagmins 1-11, we found synaptotagmin 3 (SYT3) to be the only one that is expressed in T cells. CXCR4-triggered migration was inhibited by antisense synaptotagmin 3 mRNA and by the isolated C2B domain, known to impair oligomerization of all synaptotagmins, but not by a C2B mutant that binds Ca(2+) but does not block oligomerization. The C2B domain also blocked CXCR4-triggered actin polymerization and invasion. However, CXCR4-dependent adhesion in flow was not affected. Surprisingly, we found that little or no SYT3 is present near the plasma membrane but that it is mainly localized in multivesicular bodies, which also contained much of the CXCR4. Impaired SYT3 function blocked CXCR4 recycling and thus led to reduced surface levels of CXCR4. Migration was restored by overexpression of CXCR4. We conclude that STT3 is essential for CXCR4 recycling in T cells and thereby for the maintenance of high CXCR4 surface levels required for migration.

Impact of obesity on the predictive accuracy of prostate-specific antigen density and prostate-specific antigen in native Korean men undergoing prostate biopsy.

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Kim, Jae Heon; Doo, Seung Whan; Yang, Won Jae; Lee, Kwang Woo; Lee, Chang Ho; Song, Yun Seob; Jeon, Yoon Su; Kim, Min Eui; Kwon, Soon-Sun

2014-10-01

To evaluate the impact of obesity on the biopsy detection of prostate cancer. We retrospectively reviewed data of 1182 consecutive Korean patients (≥50 years) with serum prostate-specific antigen levels of 3-10 ng/mL who underwent initial extended 12-cores biopsy from September 2009 to March 2013. Patients who took medications that were likely to influence the prostate-specific antigen level were excluded. Receiver operating characteristic curves were plotted for prostate-specific antigen and prostate-specific antigen density predicting cancer status among non-obese and obese men. A total of 1062 patients (mean age 67.1 years) were enrolled in the analysis. A total of 230 men (21.7%) had a positive biopsy. In the overall study sample, the area under the receiver operator characteristic curve of serum prostate-specific antigen for predicting prostate cancer on biopsy were 0.584 and 0.633 for non-obese and obese men, respectively (P = 0.234). However, the area under the curve for prostate-specific antigen density in predicting cancer status showed a significant difference (non-obese 0.696, obese 0.784; P = 0.017). There seems to be a significant difference in the ability of prostate-specific antigen density to predict biopsy results between non-obese and obese men. Obesity positively influenced the overall ability of prostate-specific antigen density to predict prostate cancer. © 2014 The Japanese Urological Association.

Characterization of antigen-specific, Ia-restricted, L3T4+ cytolytic T lymphocytes and assessment of thymic influence on their self specificity

International Nuclear Information System (INIS)

Golding, H.; Munitz, T.I.; Singer, A.

1985-01-01

The goals of the present study were: (a) to generate antigen-specific L3T4+ cytolytic T lymphocytes (CTL), (b) to determine their major histocompatibility complex (MHC) restriction specificity, and (c) to assess the influence of thymic MHC determinants on their self specificity. The authors found that L3T4+ CTL specific for either trinitrophenyl (TNP)-modified self determinants or minor histocompatibility antigens could be generated from Lyt-2- responder T cells provided that the response cultures were supplemented with supernatants rich in helper factors. Such antigen-specific L3T4+ CTL were Ia-restricted by the criteria that they lysed only Ia+ target cells and that their lysis of Ia+ target cells was specifically inhibited by anti-Ia monoclonal antibodies. The relative frequency of L3T4+ pCTL was found to be only 5-10% of the total anti-TNP pCTL present in the spleens of normal mice. Finally, the authors utilized radiation bone marrow chimeras to assess the influence of the thymic haplotype on the self-Ia specificity of L3T4+ CTL. Both bulk culture and limiting dilution experiments revealed that the self-Ia specificity of L3T4+ anti-TNP CTL from F1----parent and A----B allogeneic chimeras was not markedly skewed toward the haplotype of the chimeric thymus. These results contrast with those obtained previously for L3T4+ anti-TNP Th cells and demonstrate that in the radiation bone marrow chimera model of T cell differentiation, the self specificity of Th cells but not pCTL is markedly influenced by the haplotype of the chimeric thymus

Predictive value of different prostate-specific antigen-based markers in men with baseline total prostate-specific antigen <2.0 ng/mL.

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Fujizuka, Yuji; Ito, Kazuto; Oki, Ryo; Suzuki, Rie; Sekine, Yoshitaka; Koike, Hidekazu; Matsui, Hiroshi; Shibata, Yasuhiro; Suzuki, Kazuhiro

2017-08-01

To investigate the predictive value of various molecular forms of prostate-specific antigen in men with baseline prostate-specific antigen baseline prostate-specific antigen level baseline prostate-specific antigen- and age-adjusted men who did not develop prostate cancer. Serum prostate-specific antigen, free prostate-specific antigen, and [-2] proenzyme prostate-specific antigen were measured at baseline and last screening visit. The predictive impact of baseline prostate-specific antigen- and [-2] proenzyme prostate-specific antigen-related indices on developing prostate cancer was investigated. The predictive impact of those indices at last screening visit and velocities from baseline to final screening on tumor aggressiveness were also investigated. The baseline free to total prostate-specific antigen ratio was a significant predictor of prostate cancer development. The odds ratio was 6.08 in the lowest quintile baseline free to total prostate-specific antigen ratio subgroup. No serum indices at diagnosis were associated with tumor aggressiveness. The Prostate Health Index velocity and [-2] proenzyme prostate-specific antigen/free prostate-specific antigen velocity significantly increased in patients with higher risk D'Amico risk groups and higher Gleason scores. Free to total prostate-specific antigen ratio in men with low baseline prostate-specific antigen levels seems to predict the risk of developing prostate cancer, and it could be useful for a more effective individualized screening system. Longitudinal changes in [-2] proenzyme prostate-specific antigen-related indices seem to correlate with tumor aggressiveness, and they could be used as prognostic tool before treatment and during active surveillance. © 2017 The Japanese Urological Association.

Molecular mechanism of AMD3100 antagonism in the CXCR4 receptor: transfer of binding site to the CXCR3 receptor

DEFF Research Database (Denmark)

Rosenkilde, Mette M; Gerlach, Lars-Ole; Jakobsen, Janus S

2004-01-01

, respectively. Metal ion binding in the cyclam rings of AMD3100 increased its dependence on Asp(262) and provided a tighter molecular map of the binding site, where borderline mutational hits became clear hits for the Zn(II)-loaded analog. The proposed binding site for AMD3100 was confirmed by a gradual build......-up in the rather distinct CXCR3 receptor, for which the compound normally had no effect. Introduction of only a Glu at position VII:06 and the removal of a neutralizing Lys residue at position VII:02 resulted in a 1000-fold increase in affinity of AMD3100 to within 10-fold of its affinity in CXCR4. We conclude...

Antigen-specific human NKT cells from tuberculosis patients produce IL-21 to help B cells for the production of immunoglobulins.

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Wu, Changyou; Li, Zitao; Fu, Xiaoying; Yu, Sifei; Lao, Suihua; Yang, Binyan

2015-10-06

Natural killer T (NKT) cells from mouse and human play an important role in the immune responses against Mycobacterium tuberculosis. However, the function of CD3(+)TCRvβ11(+) NKT cells at the local site of M. tuberculosis infection remains poorly defined. In the present study, we found that after stimulation with M. tuberculosis antigens, NKT cells isolated from tuberculosis (TB) pleural fluid mononuclear cells (PFMCs) produced IL-21 and other cytokines including IFN-γ, TNF-α, IL-2 and IL-17. IL-21-expressing NKT cells in PFMCs displayed effector memory phenotype, expressing CD45RO(high)CD62L(low)CCR7(low). Moreover, NKT cells expressed high levels of CXCR5 and all of IL-21-expressing NKT cells co-expressed CXCR5. The frequency of BCL-6-expression was higher in IL-21-expressing but not in non-IL-21-expressing CD3(+)TCRvβ11(+) NKT cells. Sorted CD3(+)TCRvβ11(+) NKT cells from PFMCs produced IFN-γ and IL-21 after stimulation, which expressed CD40L. Importantly, CD3(+)TCRvβ11(+) NKT cells provided help to B cells for the production of IgG and IgA. Taken together, our data demonstrate that CD3(+)TCRvβ11(+) NKT cells from a local site of M. tuberculosis infection produce IL-21, express CXCR5 and CD40L, help B cells to secrete IgG and IgA, and may participate in local immune responses against M. tuberculosis infection.

Screening for epitope specificity directly on culture supernatants in the early phase of monoclonal antibody production by an ELISA with biotin-labeled antigen.

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Andersen, Ditte C; Jensen, Charlotte H; Gregersen, Annemette; Brandt, Jette; Kliem, Anette; Skjødt, Karsten; Koch, Claus; Teisner, Børge

2004-01-01

This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious advantages using this assay, are that it can be performed directly on culture supernatants in the early phase of monoclonal antibody production, and also works for antigens with repetitive epitopes. Moreover, the bonus effect, i.e., a signal in excess of the reference signal when sets of monoclonal antibodies with different epitope specificity are compared, gives a relative measure of affinity.

Augmentation of antigen-specific immune responses using DNA-fusogenic liposome vaccine

International Nuclear Information System (INIS)

Yoshikawa, Tomoaki; Imazu, Susumu; Gao Jianqing; Hayashi, Kazuyuki; Tsuda, Yasuhiro; Shimokawa, Mariko; Sugita, Toshiki; Niwa, Takako; Oda, Atushi; Akashi, Mitsuru; Tsutsumi, Yasuo; Mayumi, Tadanori; Nakagawa, Shinsaku

2004-01-01

In an attempt to enhance the immunological efficacy of genetic immunization, we investigated a new biological means for delivering antigen gene directly to the cytoplasm via membrane fusion. In this context, we investigated fusogenic liposome (FL) encapsulating DNA as a possible genetic immunization vehicle. RT-PCR analysis indicated that a FL could introduce and express encapsulating OVA gene efficiently and rapidly in vitro. Consistent with this observation, an in vitro assay showed that FL-mediated antigen-gene delivery can induce potent presentation of antigen via the MHC class I-dependent pathway. Accordingly, immunization with FL containing the OVA-gene induced potent OVA-specific Th1 and Th2 cytokine production. Additionally, OVA-specific CTL responses and antibody production were also observed in systemic compartments including the spleen, upon immunization with the OVA-gene encapsulating FL. These findings suggest that FL is an effective genetic immunization carrier system for the stimulation of antigen-specific immune responses against its encoding antigen

Response assessment with the CXCR4-directed positron emission tomography tracer [68Ga]Pentixafor in a patient with extranodal marginal zone lymphoma of the orbital cavities.

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Herhaus, Peter; Habringer, Stefan; Vag, Tibor; Steiger, Katja; Slotta-Huspenina, Julia; Gerngroß, Carlos; Wiestler, Benedikt; Wester, Hans-Jürgen; Schwaiger, Markus; Keller, Ulrich

2017-12-01

CXCR4 belongs to the family of chemokine receptors. Together with its sole known ligand CXCL12 (SDF-1alpha), it has a pivotal role during organogenesis and for homing of hematopoietic stem cells. CXCR4 is overexpressed in various malignancies, and this is often associated with poor prognosis. Therefore, molecular imaging of CXCR4 bears a great potential for diagnostics and selecting patients for CXCR4-directed therapies. The CXCR4-directed positron emission tomography (PET) tracer [ 68 Ga]Pentixafor has been shown to visualize CXCR4 expression in various malignancies in vivo. Whereas this tracer has limitations compared to 18 F-Fluorodeoxyglucose ([ 18 F]FDG) in diagnostic PET imaging in peripheral tumour lesions, it might add valuable information in routine diagnostics and response assessment of tumours in close proximity to the central nervous system (CNS) and malignancies within this organ. As a proof-of-concept, we performed [ 68 Ga]Pentixafor PET imaging in a patient with extranodal marginal zone lymphoma (MZL) of the orbital cavities at diagnosis and for post-therapy response assessment. Compared to routinely conducted [ 18 F]FDG PET, the lymphoma lesions determined by magnetic resonance imaging (MRI) showed high tracer accumulation at diagnosis, which decreased upon treatment. We therefore propose that imaging of CXCR4 with [ 68 Ga]Pentixafor is a potential diagnostic tool for tumours close to or within the CNS and suggest this being studied in clinical trials.

Protective Effect of CXCR3+CD4+CD25+Foxp3+ Regulatory T Cells in Renal Ischemia-Reperfusion Injury

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Cao Jun

2015-01-01

Full Text Available Regulatory T cells (Tregs suppress excessive immune responses and are potential therapeutic targets in autoimmune disease and organ transplantation rejection. However, their role in renal ischemia-reperfusion injury (IRI is unclear. Levels of Tregs and expression of CXCR3 in Tregs were analyzed to investigate their function in the early phase of renal IRI. Mice were randomly divided into Sham, IRI, and anti-CD25 (PC61 + IRI groups. The PC61 + IRI group was established by i.p. injection of PC61 monoclonal antibody (mAb to deplete Tregs before renal ischemia. CD4+CD25+Foxp3+ Tregs and CXCR3 on Tregs were analyzed by flow cytometry. Blood urea nitrogen (BUN, serum creatinine (Scr levels, and tubular necrosis scores, all measures of kidney injury, were greater in the IRI group than in the Sham group. Numbers of Tregs were increased at 72 h after reperfusion in kidney. PC61 mAb preconditioning decreased the numbers of Tregs and aggravated kidney injury. There was no expression of CXCR3 on Tregs in normal kidney, while it expanded at 72 h after reperfusion and inversely correlated with BUN, Scr, and kidney histology score. This indicated that recruitment of Tregs into the kidney was related to the recovery of renal function after IRI and CXCR3 might be involved in the migration of Tregs.

Expression of chemokine receptor CXCR3 in female patients with dry eye at perimenopause

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Hong Zhang

2013-09-01

Full Text Available AIM: To explore the expression and clinical significance of chemotactic factor receptor-3(CXCR3of female patients conjunctival with dry eye at perimenopause. METHODS: Thirty dry eye case(60 eyesin the patient at perimenopause of the First Affiliated Hospital of Xinjiang Medical University and thirty dry eye cases(60 eyesin patient at non-perimenopause were selected. The conjunctival epithelial cells at perimenopause and non-perimenopause in dry eye cases were obtained by impression cytology methods, and then immersed into the centrifugal tube with corresponding number respectively and the expression of CXCR3 in conjunctival epithelium of at perimenopause and non-perimenopause in dry eye cases were detected by flow cytometry. RESULTS: The break-up time(BUTand Schirmer Ⅰ test result of perimenopause was significantly lower than those of non-perimenopause(F=4.076, 5.023; PPr=-0.753, r=-0.684; PP>0.05.CONCLUSION: CXCR3 plays an inflammatory mediators role in dry eye mechanism and its expression level reflects the progress of dry eye at perimenopause.

Allopurinol reduces antigen-specific and polyclonal activation of human T cells

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Damián ePérez-Mazliah

2012-09-01

Full Text Available Allopurinol is the most popular commercially available xanthine oxidase inhibitor and it is widely used for treatment of symptomatic hyperuricaemia, or gout. Although, several anti-inflammatory actions of allopurinol have been demonstrated in vivo and in vitro, there have been few studies on the action of allopurinol on T cells. In the current study, we have assessed the effect of allopurinol on antigen-specific and mitogen-driven activation and cytokine production in human T cells. Allopurinol markedly decreased the frequency of IFN-γ and IL-2-producing T cells, either after polyclonal or antigen-specific stimulation with Herpes Simplex virus 1, Influenza virus, tetanus toxoid and Trypanosoma cruzi-derived antigens. Allopurinol attenuated CD69 upregulation after CD3 and CD28 engagement and significantly reduced the levels of spontaneous and mitogen-induced intracellular reactive oxygen species in T cells. The diminished T cell activation and cytokine production in the presence of allopurinol support a direct action of allopurinol on human T cells, offering a potential pharmacological tool for the management of cell-mediated inflammatory diseases.

Prediagnostic prostate-specific antigen kinetics and the risk of biopsy progression in active surveillance patients.

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Iremashvili, Viacheslav; Barney, Shane L; Manoharan, Murugesan; Kava, Bruce R; Parekh, Dipen J; Punnen, Sanoj

2016-04-01

To analyze the association between prediagnostic prostate-specific antigen kinetics and the risk of biopsy progression in prostate cancer patients on active surveillance, and to study the effect of prediagnostic prostate-specific antigen values on the predictive performance of prostate-specific antigen velocity and prostate-specific antigen doubling time. The study included 137 active surveillance patients with two or more prediagnostic prostate-specific antigen levels measured over a period of at least 3 months. Two sets of analyses were carried out. First, the association between prostate-specific antigen kinetics calculated using only the prediagnostic prostate-specific antigen values and the risk of biopsy progression was studied. Second, using the same cohort of patients, the predictive value of prostate-specific antigen kinetics calculated using only post-diagnostic prostate-specific antigens and compared with that of prostate-specific antigen kinetics based on both pre- and post-diagnostic prostate-specific antigen levels was analyzed. Of 137 patients included in the analysis, 37 (27%) had biopsy progression over a median follow-up period of 3.2 years. Prediagnostic prostate-specific antigen velocity of more than 2 ng/mL/year and 3 ng/mL/year was statistically significantly associated with the risk of future biopsy progression. However, after adjustment for baseline prostate-specific antigen density, these associations were no longer significant. None of the tested prostate-specific antigen kinetics based on combined pre- and post-diagnostic prostate-specific antigen values were statistically significantly associated with the risk of biopsy progression. Historical prediagnostic prostate-specific antigens seems to be not clinically useful in patients diagnosed with low-risk prostate cancer on active surveillance. © 2016 The Japanese Urological Association.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

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Isabel Correa

2018-03-01

Full Text Available Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1 specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells.

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Correa, Isabel; Ilieva, Kristina M; Crescioli, Silvia; Lombardi, Sara; Figini, Mariangela; Cheung, Anthony; Spicer, James F; Tutt, Andrew N J; Nestle, Frank O; Karagiannis, Panagiotis; Lacy, Katie E; Karagiannis, Sophia N

2018-01-01

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

Science.gov (United States)

Correa, Isabel; Ilieva, Kristina M.; Crescioli, Silvia; Lombardi, Sara; Figini, Mariangela; Cheung, Anthony; Spicer, James F.; Tutt, Andrew N. J.; Nestle, Frank O.; Karagiannis, Panagiotis; Lacy, Katie E.; Karagiannis, Sophia N.

2018-01-01

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires. PMID:29628923

CXCL10/CXCR3 Signaling in Glia Cells Differentially Affects NMDA-Induced Cell Death in CA and DG Neurons of the Mouse Hippocampus

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van Weering, Hilmar R. J.; Boddeke, Hendrikus W. G. M.; Vinet, Jonathan; Brouwer, Nieske; de Haas, Alexander H.; van Rooijen, Nico; Thomsen, Allan R.; Biber, Knut P. H.

2011-01-01

The chemokine CXCL10 and its receptor CXCR3 are implicated in various CNS pathologies since interference with CXCL10/CXCR3 signaling alters the onset and progression in various CNS disease models. However, the mechanism and cell-types involved in CXCL10/CXCR3 signaling under pathological conditions

CXCR7 functions as a scavenger for CXCL12 and CXCL11.

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Ulrike Naumann

2010-02-01

Full Text Available CXCR7 (RDC1, the recently discovered second receptor for CXCL12, is phylogenetically closely related to chemokine receptors, but fails to couple to G-proteins and to induce typical chemokine receptor mediated cellular responses. The function of CXCR7 is controversial. Some studies suggest a signaling activity in mammalian cells and zebrafish embryos, while others indicate a decoy activity in fish. Here we investigated the two propositions in human tissues.We provide evidence and mechanistic insight that CXCR7 acts as specific scavenger for CXCL12 and CXCL11 mediating effective ligand internalization and targeting of the chemokine cargo for degradation. Consistently, CXCR7 continuously cycles between the plasma membrane and intracellular compartments in the absence and presence of ligand, both in mammalian cells and in zebrafish. In accordance with the proposed activity as a scavenger receptor CXCR7-dependent chemokine degradation does not become saturated with increasing ligand concentrations. Active CXCL12 sequestration by CXCR7 is demonstrated in adult mouse heart valves and human umbilical vein endothelium.The finding that CXCR7 specifically scavenges CXCL12 suggests a critical function of the receptor in modulating the activity of the ubiquitously expressed CXCR4 in development and tumor formation. Scavenger activity of CXCR7 might also be important for the fine tuning of the mobility of hematopoietic cells in the bone marrow and lymphoid organs.

Improved detection of CXCR4-using HIV by V3 genotyping: application of population-based and "deep" sequencing to plasma RNA and proviral DNA.

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Swenson, Luke C; Moores, Andrew; Low, Andrew J; Thielen, Alexander; Dong, Winnie; Woods, Conan; Jensen, Mark A; Wynhoven, Brian; Chan, Dennison; Glascock, Christopher; Harrigan, P Richard

2010-08-01

Tropism testing should rule out CXCR4-using HIV before treatment with CCR5 antagonists. Currently, the recombinant phenotypic Trofile assay (Monogram) is most widely utilized; however, genotypic tests may represent alternative methods. Independent triplicate amplifications of the HIV gp120 V3 region were made from either plasma HIV RNA or proviral DNA. These underwent standard, population-based sequencing with an ABI3730 (RNA n = 63; DNA n = 40), or "deep" sequencing with a Roche/454 Genome Sequencer-FLX (RNA n = 12; DNA n = 12). Position-specific scoring matrices (PSSMX4/R5) (-6.96 cutoff) and geno2pheno[coreceptor] (5% false-positive rate) inferred tropism from V3 sequence. These methods were then independently validated with a separate, blinded dataset (n = 278) of screening samples from the maraviroc MOTIVATE trials. Standard sequencing of HIV RNA with PSSM yielded 69% sensitivity and 91% specificity, relative to Trofile. The validation dataset gave 75% sensitivity and 83% specificity. Proviral DNA plus PSSM gave 77% sensitivity and 71% specificity. "Deep" sequencing of HIV RNA detected >2% inferred-CXCR4-using virus in 8/8 samples called non-R5 by Trofile, and <2% in 4/4 samples called R5. Triplicate analyses of V3 standard sequence data detect greater proportions of CXCR4-using samples than previously achieved. Sequencing proviral DNA and "deep" V3 sequencing may also be useful tools for assessing tropism.

Immunization of rhesus macaques with Echinococcus multilocularis recombinant 14-3-3 antigen leads to specific antibody response.

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Lampe, Karen; Gottstein, B; Becker, T; Stahl-Hennig, C; Kaup, F-J; Mätz-Rensing, K

2017-01-01

E. multilocularis (Em) is the etiologic agent of alveolar echinococcosis (AE), a severe and potentially fatal disease, primarily affecting the liver of and occurring in aberrant intermediate hosts, e.g., humans and non-human primates. Due to increasing numbers of spontaneous cases of AE in the Old World monkey colonies of the German Primate Center, the question arose as to whether vaccination of non-human primates may represent a useful prophylactic approach. In this pilot study, the recombinant antigen Em14-3-3, which has provided a 97 % protection against E. multilocularis challenge infection in rodent models, was used for the first time to immunize rhesus macaques. In order to increase immunogenicity, the antigen was formulated with different adjuvants including Quil A®, aluminum hydroxide (alum), and muramyl dipeptide (MDP). Also, different vaccination regimens were tested. All vaccinated animals developed antigen-specific antibodies. While Quil A® induced a local adverse reaction, alum proved to be the most potent adjuvant in terms of induced antibody levels, longevity as well as tolerability. In conclusion, our pilot study demonstrated that recombinant Em14-3-3 is safe and immunogenic in rhesus monkeys. As a next step, efficacy of the vaccination remains to be explored.

CXC chemokine receptor 7 (CXCR7 regulates CXCR4 protein expression and capillary tuft development in mouse kidney.

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Sammy Haege

Full Text Available BACKGROUND: The CXCL12/CXCR4 axis is involved in kidney development by regulating formation of the glomerular tuft. Recently, a second CXCL12 receptor was identified and designated CXCR7. Although it is established that CXCR7 regulates heart and brain development in conjunction with CXCL12 and CXCR4, little is known about the influence of CXCR7 on CXCL12 dependent kidney development. METHODOLOGY/PRINCIPAL FINDINGS: We provided analysis of CXCR7 expression and function in the developing mouse kidney. Using in situ hybridization, we identified CXCR7 mRNA in epithelial cells including podocytes at all nephron stages up to the mature glomerulus. CXCL12 mRNA showed a striking overlap with CXCR7 mRNA in epithelial structures. In addition, CXCL12 was detected in stromal cells and the glomerular tuft. Expression of CXCR4 was complementary to that of CXCR7 as it occurred in mesenchymal cells, outgrowing ureteric buds and glomerular endothelial cells but not in podocytes. Kidney examination in CXCR7 null mice revealed ballooning of glomerular capillaries as described earlier for CXCR4 null mice. Moreover, we detected a severe reduction of CXCR4 protein but not CXCR4 mRNA within the glomerular tuft and in the condensed mesenchyme. Malformation of the glomerular tuft in CXCR7 null mice was associated with mesangial cell clumping. CONCLUSIONS/SIGNIFICANCE: We established that there is a similar glomerular pathology in CXCR7 and CXCR4 null embryos. Based on the phenotype and the anatomical organization of the CXCL12/CXCR4/CXCR7 system in the forming glomerulus, we propose that CXCR7 fine-tunes CXCL12/CXCR4 mediated signalling between podocytes and glomerular capillaries.

CXC Chemokine Receptor 7 (CXCR7) Regulates CXCR4 Protein Expression and Capillary Tuft Development in Mouse Kidney

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Haege, Sammy; Mueller, Wiebke; Nietzsche, Sandor; Lupp, Amelie; Mackay, Fabienne; Schulz, Stefan; Stumm, Ralf

2012-01-01

Background The CXCL12/CXCR4 axis is involved in kidney development by regulating formation of the glomerular tuft. Recently, a second CXCL12 receptor was identified and designated CXCR7. Although it is established that CXCR7 regulates heart and brain development in conjunction with CXCL12 and CXCR4, little is known about the influence of CXCR7 on CXCL12 dependent kidney development. Methodology/Principal Findings We provided analysis of CXCR7 expression and function in the developing mouse kidney. Using in situ hybridization, we identified CXCR7 mRNA in epithelial cells including podocytes at all nephron stages up to the mature glomerulus. CXCL12 mRNA showed a striking overlap with CXCR7 mRNA in epithelial structures. In addition, CXCL12 was detected in stromal cells and the glomerular tuft. Expression of CXCR4 was complementary to that of CXCR7 as it occurred in mesenchymal cells, outgrowing ureteric buds and glomerular endothelial cells but not in podocytes. Kidney examination in CXCR7 null mice revealed ballooning of glomerular capillaries as described earlier for CXCR4 null mice. Moreover, we detected a severe reduction of CXCR4 protein but not CXCR4 mRNA within the glomerular tuft and in the condensed mesenchyme. Malformation of the glomerular tuft in CXCR7 null mice was associated with mesangial cell clumping. Conclusions/Significance We established that there is a similar glomerular pathology in CXCR7 and CXCR4 null embryos. Based on the phenotype and the anatomical organization of the CXCL12/CXCR4/CXCR7 system in the forming glomerulus, we propose that CXCR7 fine-tunes CXCL12/CXCR4 mediated signalling between podocytes and glomerular capillaries. PMID:22880115

CXCL1 and CXCL2 Regulate NLRP3 Inflammasome Activation via G-Protein-Coupled Receptor CXCR2.

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Boro, Monoranjan; Balaji, Kithiganahalli Narayanaswamy

2017-09-01

Inflammation is an extensively concerted process that confers protection to the host encountering immune insult. The major inflammatory mediators include IL-1 family members, such as IL-1β, and the functional activation of such molecules is arbitrated by their regulated cleavage brought about by components of a multiprotein complex called inflammasome. In this context, NLR family pyrin domain containing 3 (NLRP3) inflammasome activation often acts as a rate-limiting step in regulating critical cell-fate decisions in various inflammatory scenarios. In this study, we identify the G-protein-coupled receptor CXCR2 (recognizing chemokines CXCL1 and CXCL2) as another arm feeding into the regulated activation of NLRP3 inflammasome in macrophages. We demonstrate that in vivo blocking of CXCL1 and CXCL2 can significantly reduce the Mycobacterium tuberculosis -induced bioactive IL-1β production. Further, CXCL1 could amplify the inflammasome activation in in vivo mouse models of carrageenan-induced inflammation in footpads and air pouches. The mechanistic insights revealed CXCR2-driven protein kinase C μ-dependent integrin-linked kinase to be essential for CXCL1-mediated activation of NLRP3 inflammasome. Blocking the activity of integrin-linked kinase or protein kinase C μ either by small interfering RNA-mediated knockdown or pharmacological inhibitor compromised inflammasome activation and subsequent production of bioactive IL-1β. Taken together, our study demonstrates CXCR2-driven activation of NLRP3 inflammasome in macrophages and indicates a potential host-directed therapeutic target to limit the damaging inflammation associated with overt production of proinflammatory IL-1β. Copyright © 2017 by The American Association of Immunologists, Inc.

CXCR6, a newly defined biomarker of tissue-specific stem cell asymmetric self-renewal, identifies more aggressive human melanoma cancer stem cells.

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Rouzbeh Taghizadeh

2010-12-01

Full Text Available A fundamental problem in cancer research is identifying the cell type that is capable of sustaining neoplastic growth and its origin from normal tissue cells. Recent investigations of a variety of tumor types have shown that phenotypically identifiable and isolable subfractions of cells possess the tumor-forming ability. In the present paper, using two lineage-related human melanoma cell lines, primary melanoma line IGR39 and its metastatic derivative line IGR37, two main observations are reported. The first one is the first phenotypic evidence to support the origin of melanoma cancer stem cells (CSCs from mutated tissue-specific stem cells; and the second one is the identification of a more aggressive subpopulation of CSCs in melanoma that are CXCR6+.We defined CXCR6 as a new biomarker for tissue-specific stem cell asymmetric self-renewal. Thus, the relationship between melanoma formation and ABCG2 and CXCR6 expression was investigated. Consistent with their non-metastatic character, unsorted IGR39 cells formed significantly smaller tumors than unsorted IGR37 cells. In addition, ABCG2+ cells produced tumors that had a 2-fold greater mass than tumors produced by unsorted cells or ABCG2- cells. CXCR6+ cells produced more aggressive tumors. CXCR6 identifies a more discrete subpopulation of cultured human melanoma cells with a more aggressive MCSC phenotype than cells selected on the basis of the ABCG2+ phenotype alone.The association of a more aggressive tumor phenotype with asymmetric self-renewal phenotype reveals a previously unrecognized aspect of tumor cell physiology. Namely, the retention of some tissue-specific stem cell attributes, like the ability to asymmetrically self-renew, impacts the natural history of human tumor development. Knowledge of this new aspect of tumor development and progression may provide new targets for cancer prevention and treatment.

Discovery and computer aided potency optimization of a novel class of small molecule CXCR4 antagonists.

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Victoria Vinader

Full Text Available Amongst the chemokine signalling axes involved in cancer, chemokine CXCL12 acting on chemokine receptor CXCR4 is particularly significant since it orchestrates migration of cancer cells in a tissue-specific metastatic process. High CXCR4 tumour expression is associated with poor prognosis of lung, brain, CNS, blood and breast cancers. We have identified a new class of small molecule CXCR4 antagonists based on the use of computational modelling studies in concert with experimental determination of in vitro activity against CXCL12-induced intracellular calcium mobilisation, proliferation and chemotaxis. Molecular modelling proved to be a useful tool in rationalising our observed potencies, as well as informing the direction of the synthetic efforts aimed at producing more potent compounds.

A novel dendritic cell-based direct ex vivo assay for detection and enumeration of circulating antigen-specific human T cells.

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Carrio, Roberto; Zhang, Ge; Drake, Donald R; Schanen, Brian C

2018-05-07

Although a variety of assays have been used to examine T cell responses in vitro, standardized ex vivo detection of antigen-specific CD4 + T cells from human circulatory PBMCs remains constrained by low-dimensional characterization outputs and the need for polyclonal, mitogen-induced expansion methods to generate detectable response signals. To overcome these limitations, we developed a novel methodology utilizing antigen-pulsed autologous human dendritic target cells in a rapid and sensitive assay to accurately enumerate antigen-specific CD4 + T cell precursor frequency by multiparametric flow cytometry. With this approach, we demonstrate the ability to reproducibly quantitate poly-functional T cell responses following both primary and recall antigenic stimulation. Furthermore, this approach enables more comprehensive phenotypic profiling of circulating antigen-specific CD4 + T cells, providing valuable insights into the pre-existing polarization of antigen-specific T cells in humans. Combined, this approach permits sensitive and detailed ex vivo detection of antigen-specific CD4 + T cells delivering an important tool for advancing vaccine, immune-oncology and other therapeutic studies.

Expression of the chemokine receptor CXCR4 on lymphocytes of leprosy patients

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V.A. Mendonça

2011-12-01

Full Text Available Leprosy is caused by Mycobacterium leprae, which induces chronic granulomatous infection of the skin and peripheral nerves. The disease ranges from the tuberculoid to the lepromatous forms, depending on the cellular immune response of the host. Chemokines are thought to be involved in the immunopathogenesis of leprosy, but few studies have investigated the expression of chemokine receptors on leukocytes of leprosy patients. In the present study, we evaluated 21 leprosy patients (M/F: 16/5 with a new diagnosis from the Dermatology Outpatient Clinic of the University Hospital, Federal University of Minas Gerais. The control group was composed of 20 healthy members (M/F: 15/5 of the community recruited by means of announcements. The expression of CCR2, CCR3, CCR5, and CXCR4 was investigated by flow cytometry on the surface of peripheral blood lymphocytes. There was a decrease in percentage of CD3+CXCR4+ and CD4+CXCR4+ lymphocytes in the peripheral blood of leprosy patients (median [range], 17.6 [2.7-41.9] and 65.3 [3.9-91.9], respectively compared to the control group (median [range], 43.0 [3.7-61.3] and 77.2 [43.6-93.5], respectively. The percentage of CD4+CXCR4+ was significantly lower in patients with the tuberculoid form (median [range], 45.7 [0.0-83.1] of the disease, but not in lepromatous patients (median [range], 81.5 [44.9-91.9]. The CXCR4 chemokine receptor may play a role in leprosy immunopathogenesis, probably directing cell migration to tissue lesions in tuberculoid leprosy patients.

Processing of natural and recombinant CXCR3-targeting chemokines and implications for biological activity.

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Hensbergen, P J; van der Raaij-Helmer, E M; Dijkman, R; van der Schors, R C; Werner-Felmayer, G; Boorsma, D M; Scheper, R J; Willemze, R; Tensen, C P

2001-09-01

Chemokines comprise a class of peptides with chemotactic activity towards leukocytes. The potency of different chemokines for the same receptor often varies as a result of differences in primary structure. In addition, post-translational modifications have been shown to affect the effectiveness of chemokines. Although in several studies, natural CXCR3-targeting chemokines have been isolated, detailed information about the proteins and their possible modifications is lacking. Using a combination of liquid chromatography and mass spectrometry we studied the protein profile of CXCR3-targeting chemokines expressed by interferon-gamma-stimulated human keratinocytes. The biological implications of one of the identified modifications was studied in more detail using calcium mobilization and chemotaxis assays. We found that the primary structure of human CXCL10 is different from the generally accepted sequence. In addition we identified a C-terminally truncated CXCL10, lacking the last four amino acids. Native CXCL11 was primarily found in its intact mature form but we also found a mass corresponding to an N-terminally truncated human CXCL11, lacking the first two amino acids FP, indicating that this chemokine is a substrate for dipeptidylpeptidase IV. Interestingly, this same truncation was found when we expressed human CXCL11 in Drosophila S2 cells. The biological activity of this truncated form of CXCL11 was greatly reduced, both in calcium mobilization (using CXCR3 expressing CHO cells) as well as its chemotactic activity for CXCR3-expressing T-cells. It is concluded that detailed information on chemokines at the protein level is important to characterize the exact profile of these chemotactic peptides as modifications can severely alter their biological activity.

Generation of Tumor Antigen-Specific iPSC-Derived Thymic Emigrants Using a 3D Thymic Culture System

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Raul Vizcardo

2018-03-01

Full Text Available Summary: Induced pluripotent stem cell (iPSC-derived T cells may provide future therapies for cancer patients, but those generated by current methods, such as the OP9/DLL1 system, have shown abnormalities that pose major barriers for clinical translation. Our data indicate that these iPSC-derived CD8 single-positive T cells are more like CD4+CD8+ double-positive T cells than mature naive T cells because they display phenotypic markers of developmental arrest and an innate-like phenotype after stimulation. We developed a 3D thymic culture system to avoid these aberrant developmental fates, generating a homogeneous subset of CD8αβ+ antigen-specific T cells, designated iPSC-derived thymic emigrants (iTEs. iTEs exhibit phenotypic and functional similarities to naive T cells both in vitro and in vivo, including the capacity for expansion, memory formation, and tumor suppression. These data illustrate the limitations of current methods and provide a tool to develop the next generation of iPSC-based antigen-specific immunotherapies. : A barrier for clinical application of iPSC-derived CD8 T cells using OP9/DLL1 is their abnormal biology. Vizcardo et al. show that a 3D thymic culture system enables the generation of a homogeneous antigen-specific T cell subset, named iTEs, which closely mimics naive T cells and exhibits potent anti-tumor activity. Keywords: thymopoiesis, T cell differentiation, iPSC differentiation, adoptive cell transfer, naïve T cell, recent rhymic emigrants, fetal thymus organ culture, immunotherapy, 3D culture, tumor antigen specific T cell

CXCL12 MODULATION OF CXCR4 AND CXCR7 ACTIVITY IN HUMAN GLIOBLASTOMA STEM-LIKE CELLS AND REGULATION OF THE TUMOR MICROENVIRONMENT

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Roberto eWurth

2014-05-01

Full Text Available Chemokines are crucial autocrine and paracrine players in tumor development. In particular, CXCL12, through its receptors CXCR4 and CXCR7, affects tumor progression by controlling cancer cell survival, proliferation and migration, and, indirectly, via angiogenesis or recruiting immune cells.Glioblastoma (GBM is the most prevalent primary malignant brain tumor in adults and despite current multimodal therapies it remains almost incurable. The aggressive and recurrent phenotype of GBM is ascribed to high growth rate, invasiveness to normal brain, marked angiogenesis, ability to escape the immune system and resistance to standard of care therapies. Tumor molecular and cellular heterogeneity severely hinders GBM therapeutic improvement. In particular, a subpopulation of chemo- and radio-therapy resistant tumorigenic cancer stem–like cells (CSCs is believed to be the main responsible for tumor cell dissemination to the brain.GBM cells display heterogeneous expression levels of CXCR4 and CXCR7 that are overexpressed in CSCs, representing a molecular correlate for the invasive potential of GBM. The microenvironment contribution in GBM development is increasingly emphasized. An interplay exists between CSCs, differentiated GBM cells, and the microenvironment, mainly through secreted chemokines (e.g. CXCL12 causing recruitment of fibroblasts, endothelial, mesenchymal and inflammatory cells to the tumor, via specific receptors such as CXCR4.This review covers recent developments on the role of CXCL12/CXCR4-CXCR7 networks in GBM progression and the potential translational impact of their targeting. The biological and molecular understanding of the heterogeneous GBM cell behavior, phenotype and signaling is still limited. Progress in the identification of chemokine-dependent mechanisms that affect GBM cell survival, trafficking and chemo-attractive functions, opens new perspectives for development of more specific therapeutic approaches that include

Discovery and Characterization of an Endogenous CXCR4 Antagonist

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Onofrio Zirafi

2015-05-01

Full Text Available CXCL12-CXCR4 signaling controls multiple physiological processes and its dysregulation is associated with cancers and inflammatory diseases. To discover as-yet-unknown endogenous ligands of CXCR4, we screened a blood-derived peptide library for inhibitors of CXCR4-tropic HIV-1 strains. This approach identified a 16 amino acid fragment of serum albumin as an effective and highly specific CXCR4 antagonist. The endogenous peptide, termed EPI-X4, is evolutionarily conserved and generated from the highly abundant albumin precursor by pH-regulated proteases. EPI-X4 forms an unusual lasso-like structure and antagonizes CXCL12-induced tumor cell migration, mobilizes stem cells, and suppresses inflammatory responses in mice. Furthermore, the peptide is abundant in the urine of patients with inflammatory kidney diseases and may serve as a biomarker. Our results identify EPI-X4 as a key regulator of CXCR4 signaling and introduce proteolysis of an abundant precursor protein as an alternative concept for chemokine receptor regulation.

CXCR4/CXCL12/CXCR7 axis is functional in neuroendocrine tumors and signals on mTOR.

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Circelli, Luisa; Sciammarella, Concetta; Guadagno, Elia; Tafuto, Salvatore; del Basso de Caro, Marialaura; Botti, Giovanni; Pezzullo, Luciano; Aria, Massimo; Ramundo, Valeria; Tatangelo, Fabiana; Losito, Nunzia Simona; Ieranò, Caterina; D'Alterio, Crescenzo; Izzo, Francesco; Ciliberto, Gennaro; Colao, Annamaria; Faggiano, Antongiulio; Scala, Stefania

2016-04-05

To evaluate the possible crosstalk between C-X-C chemokine receptor 4 (CXCR4)/C-X-C motif chemokine 12 (CXCL12)/C-X-C chemokine receptor 7 (CXCR7) axis with the mammalian target of rapamycin (mTOR) pathway in neuroendocrine tumors (NETs). Sixty-one human NETs were included into the study. CXCR4/CXCL12/CXCR7 axis and mTOR pathway were assessed by qRT-PCR and immunohistochemistry (IHC). The effect of mTOR inhibitor, RAD001, was evaluated on CXCR4 pathway through proliferation and p-Erk and p-AKT induction. CXCR4/CXCL12/CXCR7 axis and p-mTOR were found to be active and correlated with grading, Ki67 index and tumor stage. mTOR pathway activation significantly correlated with poor prognosis. In human NET cells, CXCL12 induced mTOR signalling while AMD3100 (CXCR4-antagonist) impaired it. The mTOR-antagonist, RAD001, impaired the CXCL12-dependent induction of CXCR4 downstream effectors. Combination of AMD3100 and RAD001 potentiate cell growth inhibition. CXCR4/CXCL12/CXCR7 axis is active in NETs and signals on mTOR. CXCR4 might be considered a prognostic factor in NETs. Combined treatment with AMD3100 and RAD001 may provide clinical benefits in NET patients with drug-resistant.

Synthesis of AMD3100 for antagonist of CXCR4 and labeled with 99Tcm

International Nuclear Information System (INIS)

Gui Yuan; Xu Zhihong; Zhang Xiaojun; Zhang Shuwen; Liu Jian; Tian Jiahe; Zhang Jinming

2013-01-01

Most of human tumors over-express CXCR4. AMD3100, a nonpeptide antagonist for CXCR4 receptor, can be used for therapy of those tumors. It was found that metal ion complex, such as Cu 2+ , with AMD3100 enhanced its binding affinity to the receptor 10-fold higher as compared to AMD3100 alone. AMD3100 was synthesis from 3-aminopropyl ethylene diamine. 99 Tc m -AMD3100 was labeled directly. Biodistribution studies were carried out in NH mice. SPECT imaging was performed in Hep-G2 tumor bearing mouse. The synthetic yield was 5.8% from 3-aminopropyl ethylene diamine to AMD3100. The labeling yield of 99 Tc m -AMD3100 was over 98%. Biodistribution studies showed high accumulation of radio- tracer in liver which had high-expression of CXCR4. SPECT imaging results showed that uptake in Hep-G2 tumor was high. The results showed that 99 Tc m -AMD3100 was an attractive candidate for further development of SPECT radiotracer potentially suitable for CXCR4. (authors)

Noncompetitive antagonism and inverse agonism as mechanism of action of nonpeptidergic antagonists at primate and rodent CXCR3 chemokine receptors

NARCIS (Netherlands)

Verzijl, D.; Storelli, S.; Scholten, D.J.; Bosch, L.; Reinhart, T.A.; Streblow, D.N.; Tensen, C.P.; Fitzsimons, C.P.; Zaman, G.J.; Pease, J.E.; de Esch, I.J.P.; Smit, M.J.; Leurs, R.

2008-01-01

The chemokine receptor CXCR3 is involved in various inflammatory diseases, such as rheumatoid arthritis, multiple sclerosis, psoriasis, and allograft rejection in transplantation patients. The CXCR3 ligands CXCL9, CXCL10, and CXCL11 are expressed at sites of inflammation, and they attract

IQGAP1 is a novel CXCR2-interacting protein and essential component of the "chemosynapse".

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Nicole F Neel

Full Text Available Chemotaxis is essential for a number of physiological processes including leukocyte recruitment. Chemokines initiate intracellular signaling pathways necessary for chemotaxis through binding seven transmembrane G protein-couple receptors. Little is known about the proteins that interact with the intracellular domains of chemokine receptors to initiate cellular signaling upon ligand binding. CXCR2 is a major chemokine receptor expressed on several cell types, including endothelial cells and neutrophils. We hypothesize that multiple proteins interact with the intracellular domains of CXCR2 upon ligand stimulation and these interactions comprise a "chemosynapse", and play important roles in transducing CXCR2 mediated signaling processes.In an effort to define the complex of proteins that assemble upon CXCR2 activation to relay signals from activated chemokine receptors, a proteomics approach was employed to identify proteins that co-associate with CXCR2 with or without ligand stimulation. The components of the CXCR2 "chemosynapse" are involved in processes ranging from intracellular trafficking to cytoskeletal modification. IQ motif containing GTPase activating protein 1 (IQGAP1 was among the novel proteins identified to interact directly with CXCR2. Herein, we demonstrate that CXCR2 co-localizes with IQGAP1 at the leading edge of polarized human neutrophils and CXCR2 expressing differentiated HL-60 cells. Moreover, amino acids 1-160 of IQGAP1 directly interact with the carboxyl-terminal domain of CXCR2 and stimulation with CXCL8 enhances IQGAP1 association with Cdc42.Our studies indicate that IQGAP1 is a novel essential component of the CXCR2 "chemosynapse".

The prognostic importance of CXCR3 chemokine during organizing pneumonia on the risk of chronic lung allograft dysfunction after lung transplantation.

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Michael Y Shino

Full Text Available Since the pathogenesis of chronic lung allograft dysfunction (CLAD remains poorly defined with no known effective therapies, the identification and study of key events which increase CLAD risk is a critical step towards improving outcomes. We hypothesized that bronchoalveolar lavage fluid (BALF CXCR3 ligand concentrations would be augmented during organizing pneumonia (OP and that episodes of OP with marked chemokine elevations would be associated with significantly higher CLAD risk.All transbronchial biopsies (TBBX from patients who received lung transplantation between 2000 to 2010 were reviewed. BALF concentrations of the CXCR3 ligands (CXCL9, CXCL10 and CXCL11 were compared between episodes of OP and "healthy" biopsies using linear mixed-effects models. The association between CXCR3 ligand concentrations during OP and CLAD risk was evaluated using proportional hazards models with time-dependent covariates.There were 1894 bronchoscopies with TBBX evaluated from 441 lung transplant recipients with 169 (9% episodes of OP and 907 (49% non-OP histopathologic injuries. 62 (37% episodes of OP were observed during routine surveillance bronchoscopy. Eight hundred thirty-eight (44% TBBXs had no histopathology and were classified as "healthy" biopsies. There were marked elevations in BALF CXCR3 ligand concentrations during OP compared with "healthy" biopsies. In multivariable models adjusted for other injury patterns, OP did not significantly increase the risk of CLAD when BAL CXCR3 chemokine concentrations were not taken into account. However, OP with elevated CXCR3 ligands markedly increased CLAD risk in a dose-response manner. An episode of OP with CXCR3 concentrations greater than the 25th, 50th and 75th percentiles had HRs for CLAD of 1.5 (95% CI 1.0-2.3, 1.9 (95% CI 1.2-2.8 and 2.2 (95% CI 1.4-3.4, respectively.This study identifies OP, a relatively uncommon histopathologic finding after lung transplantation, as a major risk factor for CLAD

Highly specific expression of luciferase gene in lungs of naive nude mice directed by prostate-specific antigen promoter

International Nuclear Information System (INIS)

Li Hongwei; Li Jinzhong; Helm, Gregory A.; Pan Dongfeng

2005-01-01

PSA promoter has been demonstrated the utility for tissue-specific toxic gene therapy in prostate cancer models. Characterization of foreign gene overexpression in normal animals elicited by PSA promoter should help evaluate therapy safety. Here we constructed an adenovirus vector (AdPSA-Luc), containing firefly luciferase gene under the control of the 5837 bp long prostate-specific antigen promoter. A charge coupled device video camera was used to non-invasively image expression of firefly luciferase in nude mice on days 3, 7, 11 after injection of 2 x 10 9 PFU of AdPSA-Luc virus via tail vein. The result showed highly specific expression of the luciferase gene in lungs of mice from day 7. The finding indicates the potential limitations of the suicide gene therapy of prostate cancer based on selectivity of PSA promoter. By contrary, it has encouraging implications for further development of vectors via PSA promoter to enable gene therapy for pulmonary diseases

Omega-3 Fatty Acids Supplementation Differentially Modulates the SDF-1/CXCR-4 Cell Homing Axis in Hypertensive and Normotensive Rats.

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Halmenschlager, Luiza; Lehnen, Alexandre Machado; Marcadenti, Aline; Markoski, Melissa Medeiros

2017-08-01

We assessed the effect of acute and chronic dietary supplementation of ω-3 on lipid metabolism and cardiac regeneration, through its influence on the Stromal Derived Factor-1 (SDF-1) and its receptor (CXCR4) axis in normotensive and hypertensive rats. Male Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) were allocated in eight groups (of eight animals each), which received daily orogastric administration of ω-3 (1 g) for 24 h, 72 h or 2 weeks. Blood samples were collected for the analysis of the lipid profile and SDF-1 systemic levels (ELISA). At the end of the treatment period, cardiac tissue was collected for CXCR4 expression analysis (Western blot). The use of ω-3 caused a reduction in total cholesterol levels ( p = 0.044), and acutely activated the SDF-1/CXCR4 axis in normotensive animals ( p = 0.037). In the presence of the ω-3, after 72 h, SDF-1 levels decreased in WKY and increased in SHR ( p = 0.017), and tissue expression of the receptor CXCR4 was higher in WKY than in SHR ( p = 0.001). The ω-3 fatty acid supplementation differentially modulates cell homing mediators in normotensive and hypertensive animals. While WKY rats respond acutely to omega-3 supplementation, showing increased release of SDF-1 and CXCR4, SHR exhibit a weaker, delayed response.

Hypoxia increases the metastatic ability of breast cancer cells via upregulation of CXCR4

LENUS (Irish Health Repository)

Cronin, Patricia A

2010-05-21

Abstract Background Chemokine SDF1α and its unique receptor CXCR4 have been implicated in organ-specific metastases of many cancers including breast cancer. Hypoxia is a common feature of solid tumors and is associated with their malignant phenotype. We hypothesized that hypoxia would upregulate CXCR4 expression and lead to increased chemotactic responsiveness to its specific ligand SDF1α. Methods Three breast cancer cell lines MDA-MB-231, MCF7 and 4T1 were subjected to 48 hrs of hypoxia or normoxia. Cell surface receptor expression was evaluated using flow cytometry. An extracellular matrix invasion assay and microporous migration assay was used to assess chemotactic response and metastatic ability. Results CXCR4 surface expression was significantly increased in the two human breast cancer cell lines, MDA-MB-231 and MCF7, following exposure to hypoxia. This upregulation of CXCR4 cell surface expression corresponded to a significant increase in migration and invasion in response to SDF1-α in vitro. The increase in metastatic potential of both the normoxic and the hypoxic treated breast cancer cell lines was attenuated by neutralization of CXCR4 with a CXCR4 neutralizing mAb, MAB172 or a CXCR4 antagonist, AMD3100, showing the relationship between CXCR4 overexpression and increased chemotactic responsiveness. Conclusions CXCR4 expression can be modulated by the tissue microenvironment such as hypoxia. Upregulation of CXCR4 is associated with increased migratory and invasive potential and this effect can be abrogated by CXCR4 inhibition. Chemokine receptor CXCR4 is a potential therapeutic target in the adjuvant treatment of breast cancer.

Splenic B cells and antigen-specific B cells process anti-Ig in a similar manner

International Nuclear Information System (INIS)

Myers, C.D.; Vitetta, E.S.

1989-01-01

B lymphocytes can process and present antigen to T cells. However, the fate of native antigen after its binding to specific B cells, i.e., the intracellular events involved in the processing and recycling of the antigenic fragments to the cell surface for antigen presentation, are not well understood. In the present study, we demonstrate that murine B cells degrade anti-Ig molecules bound to their surface and release acid soluble fragments into the supernatant. We also demonstrate that the kinetics of this process are identical for anti-mu, anti-delta, and anti-light chain antibodies, indicating that both surface IgM and surface IgD are equally effective in binding antigen and directing its processing. We also describe the effects of azide, chloroquine, and irradiation on this process. To extend these studies to the processing of specifically bound antigen, we demonstrate that highly purified trinitrophenyl antigen-binding cells degrade anti-Ig molecules with the same kinetics as unpurified splenic B cells. Thus, this purified population provides a suitable model system for the analysis of antigen degradation by antigen-specific cells

Screening Immunomodulators To Skew the Antigen-Specific Autoimmune Response.

Science.gov (United States)

Northrup, Laura; Sullivan, Bradley P; Hartwell, Brittany L; Garza, Aaron; Berkland, Cory

2017-01-03

Current therapies to treat autoimmune diseases often result in side effects such as nonspecific immunosuppression. Therapies that can induce antigen-specific immune tolerance provide an opportunity to reverse autoimmunity and mitigate the risks associated with global immunosuppression. In an effort to induce antigen-specific immune tolerance, co-administration of immunomodulators with autoantigens has been investigated in an effort to reprogram autoimmunity. To date, identifying immunomodulators that may skew the antigen-specific immune response has been ad hoc at best. To address this need, we utilized splenocytes obtained from mice with experimental autoimmune encephalomyelitis (EAE) in order to determine if certain immunomodulators may induce markers of immune tolerance following antigen rechallenge. Of the immunomodulatory compounds investigated, only dexamethasone modified the antigen-specific immune response by skewing the cytokine response and decreasing T-cell populations at a concentration corresponding to a relevant in vivo dose. Thus, antigen-educated EAE splenocytes provide an ex vivo screen for investigating compounds capable of skewing the antigen-specific immune response, and this approach could be extrapolated to antigen-educated cells from other diseases or human tissues.

[99mTc]O2-AMD3100 as a SPECT tracer for CXCR4 receptor imaging

International Nuclear Information System (INIS)

Hartimath, Siddesh V.; Domanska, Urszula M.; Walenkamp, Annemiek M.E.; Dierckx, Rudi A.J.O.; Vries, Erik F.J. de

2013-01-01

Purpose: CXCR4 plays an important role in HIV infection, tumor progression, neurogenesis, and inflammation. In-vivo imaging of CXCR4 could provide more insight in the role of this receptor in health and disease. The aim of this study was to investigate [ 99m Tc]O 2 -AMD3100 as a potential SPECT tracer for imaging of CXCR4. Method: AMD3100 was labelled with [ 99m Tc]pertechnetate. A cysteine challenge assay was performed to test the tracer stability. Heterologous and homologous receptor binding assay and internalization assay were performed in CXCR4 expressing Jurkat-T cells. Ex vivo biodistribution was studied in healthy mice at 30, 60, and 120 min after tracer injection. Tumor uptake of the tracer was determined by microSPECT imaging in nude mice xenografted with human PC-3 prostate tumor. Specificity of tracer uptake was determined by blocking studies using an excess of unlabelled AMD3100. Results: AMD3100 was labelled with technetium-99 m with a radiochemical yield of > 98%. The tracer was stable in PBS and mouse plasma for at least 6 h at 37 °C. Heterologous and homologous binding assays with AMD3100 showed IC 50 values of 240 ± 10 μM, and 92 ± 5 μM for [ 125 I]SDF-1α and [ 99m Tc]O 2 -AMD3100 respectively, with negligible receptor internalisation. The tracer showed high uptake in liver, lungs, spleen, thymus, intestine and bone. Blocking dose of AMD3100.8HCl (20 mg/kg) decreased the uptake in these organs (p 99m Tc]O 2 -AMD3100 showed specific tumor accumulation in mice bearing PC-3 xenografts model. Time activity curves (TAC) in AMD3100 pre-treated animals tracer showed 1.7 times less tumor uptake as compared to control animals (p 99m Tc]O 2 -AMD3100 is readily labelled, is stable in plasma and displays a favourable binding affinity for the CXCR4 receptors. [ 99m Tc O 2 -AMD3100 shows specific binding in organs with high CXCR4 expression and in CXCR4 positive tumors. These results justify further evaluation of this radiopharmaceutical as a potential

Developing antigen-specific therapies in multiple sclerosis: a tale of Tantalus or Ulysses?

Science.gov (United States)

van Noort JM

1999-10-01

Autoreactive T-cell responses directed to myelin proteins in the central nervous system are widely believed to be crucial in the pathology of multiple sclerosis (MS). However, effective ways of selectively targeting these T-cells in order to alter the clinical course of MS in a predictable manner has yet to be demonstrated. This review discusses two recent developments of crucial importance to the rational development of antigen-specific therapy in MS. The very idea of antigen-specific therapy in MS has long faced the challenge of determinant spreading, i.e., the development of novel autoimmune responses as the consequence of tissue damage. This phenomenon has led many to expect that in ongoing MS, many different pathogenic specificities would accumulate. Obviously, this would render antigen-specific therapy very difficult. Recent data now suggest that determinant spreading is most likely to be a transient phenomenon limited only to the first stages of tissue damage. A second development has changed our perspective on the specificity of individual T-cells and, thus, on the suitability of various ways to implement antigen-specific therapy. Evidence is rapidly accumulating that T-cell receptors are much more cross-reactive than previously assumed. This notion poses unexpected challenges to therapeutic approaches in MS that are based on selective targeting of autoreactive TCR. Vaccination with TCR peptides, administration of anti-TCR antibodies and development of therapeutically altered peptide ligands all depend on a significant level of predictability of pathogenic TCR. With such predictability now turning out to be much lower than was previously hoped, selective TCR-directed strategies for intervention may therefore turn out to be much less effective than anticipated. In the development of antigen-specific therapies, the use of whole protein tolerogens now seems to be the most promising route. Oral, intranasal or iv. administration of antigen remain viable options

Direct ex vivo detection of HLA-DR3-restricted cytomegalovirus- and Mycobacterium tuberculosis-specific CD4+ T cells.

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Bronke, Corine; Palmer, Nanette M; Westerlaken, Geertje H A; Toebes, Mireille; van Schijndel, Gijs M W; Purwaha, Veenu; van Meijgaarden, Krista E; Schumacher, Ton N M; van Baarle, Debbie; Tesselaar, Kiki; Geluk, Annemieke

2005-09-01

In order to detect epitope-specific CD4+ T cells in mycobacterial or viral infections in the context of human class II major histocompatibility complex protein human leukocyte antigen (HLA)-DR3, two HLA-DR3 tetrameric molecules were successfully produced. One contained an immunodominant HLA-DR3-restricted T-cell epitope derived from the 65-kDa heat-shock protein of Mycobacterium tuberculosis, peptide 1-13. For the other tetramer, we used an HLA-DR3-restricted T-cell epitope derived from cytomegalovirus (CMV) pp65 lower matrix protein, peptide 510-522, which induced high levels of interferon (IFN)-gamma-producing CD4+ T cells in three of four HLA-DR3-positive CMV-seropositive individuals up to 0.84% of CD4+ T cells by intracellular cytokine staining. In peripheral blood mononuclear cells from M. tuberculosis-exposed, Mycobacterium bovis bacille Calmette-Guérin (BCG)-vaccinated, or CMV-seropositive individuals, we were able to directly detect with both tetramers epitope-specific T cells up to 0.62% and 0.45% of the CD4+ T-cell population reactive to M. tuberculosis and CMV, respectively. After a 6-day culture with peptide p510-522, the frequency of CMV-specific tetramer-binding T cells was expanded up to 9.90% tetramer+ CFSElow (5,6-carboxyfluorescein diacetate succinimidyl ester) cells within the CD4+ T-cell population, further confirming the specificity of the tetrameric molecules. Thus, HLA-DR3/peptide tetrameric molecules can be used to investigate HLA-DR3-restricted antigen-specific CD4+ T cells in clinical disease or after vaccination.

Phosphatidylcholine-specific phospholipase C inhibition down- regulates CXCR4 expression and interferes with proliferation, invasion and glycolysis in glioma cells.

Directory of Open Access Journals (Sweden)

Laura Mercurio

Full Text Available The chemokine receptor CXCR4 plays a crucial role in tumors, including glioblastoma multiforme (GBM, the most aggressive glioma. Phosphatidylcholine-specific phospholipase C (PC-PLC, a catabolic enzyme of PC metabolism, is involved in several aspects of cancer biology and its inhibition down-modulates the expression of growth factor membrane receptors interfering with their signaling pathways. In the present work we investigated the possible interplay between CXCR4 and PC-PLC in GBM cells.Confocal microscopy, immunoprecipitation, western blot analyses, and the evaluation of migration and invasion potential were performed on U87MG cells after PC-PLC inhibition with the xanthate D609. The intracellular metabolome was investigated by magnetic resonance spectroscopy; lactate levels and lactate dehydrogenase (LDH activity were analyzed by colorimetric assay.Our studies demonstrated that CXCR4 and PC-PLC co-localize and are associated on U87MG cell membrane. D609 reduced CXCR4 expression, cell proliferation and invasion, interfering with AKT and EGFR activation and expression. Metabolic analyses showed a decrease in intracellular lactate concentration together with a decrement in LDH activity.Our data suggest that inhibition of PC-PLC could represent a new molecular approach in glioma biology not only for its ability in modulating cell metabolism, glioma growth and motility, but also for its inhibitory effect on crucial molecules involved in cancer progression.

Downregulation of miR-221-3p contributes to IL-1β-induced cartilage degradation by directly targeting the SDF1/CXCR4 signaling pathway.

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Zheng, Xin; Zhao, Feng-Chao; Pang, Yong; Li, Dong-Ya; Yao, Sheng-Cheng; Sun, Shao-Song; Guo, Kai-Jin

2017-06-01

Osteoarthritis (OA) is characterized by degradation of chondrocyte extracellular matrix (ECM). Accumulating evidence suggests that microRNAs (miRNAs) are associated with OA, but little is known of their function in chondrocyte ECM degradation. The objective of this study was to investigate the expression and function of miRNAs in OA. miRNA expression profile was determined in OA cartilage tissues and controls, employing Solexa sequencing and reverse transcription quantitative PCR (RT-qPCR). According to a modified Mankin scale, cartilage degradation was evaluated. Functional analysis of the miRNAs on chondrocyte ECM degradation was performed after miRNA transfection and IL-1β treatment. Luciferase reporter assays and western blotting were employed to determine miRNA targets. Expression of miR-221-3p was downregulated in OA cartilage tissues, which was significantly correlated with a modified Mankin scale. Through gain-of-function and loss-of-function studies, miR-221-3p was shown to significantly affect matrix synthesis gene expression and chondrocyte proliferation and apoptosis. Using SW1353 and C28I2 cells, SDF1 was identified as a target of miR-221-3p. SDF1 overexpression resulted in increased expression of catabolic genes such as MMP-13 and ADAMTS-5 in response to IL-1β, but these effects were moderated by miR-221-3p. SDF1 treatment antagonized this effect, while knockdown of SDF1 by shSDF1 induced inhibitory effects on the expression of CXCR4 and its main target genes, similar to miR-221-3p. The results indicate that upregulation of miR-221-3p could prevent IL-1β-induced ECM degradation in chondrocytes. Targeting the SDF1/CXCR4 signaling pathway may be used as a therapeutic approach for OA. miR-221-3p is downregulated in human cartilage tissues. miR-221-3p levels are associated with cartilage degeneration grade. miR-221-3p upregulation prevents IL-1β-induced ECM degradation in chondrocytes. Protection of ECM degradation by miR-223-3p occurs via SDF1/CXCR4

Antigenic determinants of prostate-specific antigen (PSA) and development of assays specific for different forms of PSA.

OpenAIRE

Nilsson, O.; Peter, A.; Andersson, I.; Nilsson, K.; Grundstr?m, B.; Karlsson, B.

1997-01-01

Monoclonal antibodies were raised against prostate-specific antigen (PSA) by immunization with purified free PSA, i.e. not in complex with any protease inhibitor (F-PSA) and PSA in complex with alpha1-anti-chymotrypsin (PSA-ACT). Epitope mapping of PSA using the established monoclonal antibody revealed a complex pattern of independent and partly overlapping antigenic domains in the PSA molecule. Four independent antigenic domains and at least three partly overlapping domains were exposed both...

Consequences of ChemR23 heteromerization with the chemokine receptors CXCR4 and CCR7.

Science.gov (United States)

de Poorter, Cédric; Baertsoen, Kevin; Lannoy, Vincent; Parmentier, Marc; Springael, Jean-Yves

2013-01-01

Recent studies have shown that heteromerization of the chemokine receptors CCR2, CCR5 and CXCR4 is associated to negative binding cooperativity. In the present study, we build on these previous results, and investigate the consequences of chemokine receptor heteromerization with ChemR23, the receptor of chemerin, a leukocyte chemoattractant protein structurally unrelated to chemokines. We show, using BRET and HTRF assays, that ChemR23 forms homomers, and provide data suggesting that ChemR23 also forms heteromers with the chemokine receptors CCR7 and CXCR4. As previously described for other chemokine receptor heteromers, negative binding cooperativity was detected between ChemR23 and chemokine receptors, i.e. the ligands of one receptor competed for the binding of a specific tracer of the other. We also showed, using mouse bone marrow-derived dendritic cells prepared from wild-type and ChemR23 knockout mice, that ChemR23-specific ligands cross-inhibited CXCL12 binding on CXCR4 in a ChemR23-dependent manner, supporting the relevance of the ChemR23/CXCR4 interaction in native leukocytes. Finally, and in contrast to the situation encountered for other previously characterized CXCR4 heteromers, we showed that the CXCR4-specific antagonist AMD3100 did not cross-inhibit chemerin binding in cells co-expressing ChemR23 and CXCR4, demonstrating that cross-regulation by AMD3100 depends on the nature of receptor partners with which CXCR4 is co-expressed.

Antigen specific T-cell responses against tumor antigens are controlled by regulatory T cells in patients with prostate cancer.

Science.gov (United States)

Hadaschik, Boris; Su, Yun; Huter, Eva; Ge, Yingzi; Hohenfellner, Markus; Beckhove, Philipp

2012-04-01

Immunotherapy is a promising approach in an effort to control castration resistant prostate cancer. We characterized tumor antigen reactive T cells in patients with prostate cancer and analyzed the suppression of antitumor responses by regulatory T cells. Peripheral blood samples were collected from 57 patients with histologically confirmed prostate cancer, 8 patients with benign prostatic hyperplasia and 16 healthy donors. Peripheral blood mononuclear cells were isolated and antigen specific interferon-γ secretion of isolated T cells was analyzed by enzyme-linked immunospot assay. T cells were functionally characterized and T-cell responses before and after regulatory T-cell depletion were compared. As test tumor antigens, a panel of 11 long synthetic peptides derived from a total of 8 tumor antigens was used, including prostate specific antigen and prostatic acid phosphatase. In patients with prostate cancer we noted a 74.5% effector T-cell response rate compared with only 25% in patients with benign prostatic hyperplasia and 31% in healthy donors. In most patients 2 or 3 tumor antigens were recognized. Comparing various disease stages there was a clear increase in the immune response against prostate specific antigens from intermediate to high risk tumors and castration resistant disease. Regulatory T-cell depletion led to a significant boost in effector T-cell responses against prostate specific antigen and prostatic acid phosphatase. Tumor specific effector T cells were detected in most patients with prostate cancer, especially those with castration resistant prostate cancer. Since effector T-cell responses against prostate specific antigens strongly increased after regulatory T-cell depletion, our results indicate that immunotherapy efficacy could be enhanced by decreasing regulatory T cells. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Prostate-Specific Antigen (PSA) Test: MedlinePlus Lab Test Information

Science.gov (United States)

... medlineplus.gov/labtests/prostatespecificantigenpsatest.html Prostate-Specific Antigen (PSA) Test To use the sharing features on this ... enable JavaScript. What is a prostate-specific antigen (PSA) test? A prostate-specific antigen (PSA) test measures ...

The role of SDF-1-CXCR4/CXCR7 axis in biological behaviors of adipose tissue-derived mesenchymal stem cells in vitro

Energy Technology Data Exchange (ETDEWEB)

Li, Qiang; Zhang, Aijun; Tao, Changbo; Li, Xueyang; Jin, Peisheng, E-mail: jinps2006@163.com

2013-11-22

Highlights: •SDF-1 pretreating increased the levels of CXCR4, CXCR7 in ADSCs. •SDF-1 improved cells paracrine migration and proliferation abilities. •CXCR4 and CXCR7 could function in ADSCs paracrine, migration and proliferation. -- Abstract: Numerous studies have reported that CXCR4 and CXCR7 play an essential, but differential role in stromal cell-derived factor-1 (SDF-1)-inducing cell chemotaxis, viability and paracrine actions of BMSCs. Adipose tissue-derived mesenchymal stem cells (ADSCs) have been suggested to be potential seed cells for clinical application instead of bone marrow derived stroma cell (BMSCs). However, the function of SDF-1/CXCR4 and SDF-1/CXCR7 in ADSCs is not well understood. This study was designed to analyze the effect of SDF-1/CXCR4 and SDF-1/CXCR7 axis on ADSCs biological behaviors in vitro. Using Flow cytometry and Western blot methods, we found for the first time that CXCR4/CXCR7 expression was increased after treatment with SDF-1 in ADSCs. SDF-1 promoted ADSCs paracrine, proliferation and migration abilities. CXCR4 or CXCR7 antibody suppressed ADSCs paracrine action induced by SDF-1. The migration of ADSCs can be abolished by CXCR4 antibody, while the proliferation of ADSCs was only downregulated by CXCR7 antibody. Our study indicated that the angiogenesis of ADSCs is, at least partly, mediated by SDF-1/CXCR4 and SDF-1/CXCR7 axis. However, only binding of SDF-1/CXCR7 was required for proliferation of ADSCs, and CXCR7 was required for migration of ADSCs induced by SDF-1. Our studies provide evidence that the activation of either axis may be helpful to improve the effectiveness of ADSCs-based stem cell therapy.

Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

Directory of Open Access Journals (Sweden)

Unzueta U

2012-08-01

Full Text Available Ugutz Unzueta,1–3 María Virtudes Céspedes,3,4 Neus Ferrer-Miralles,1–3 Isolda Casanova,3,4 Juan Cedano,5 José Luis Corchero,1–3 Joan Domingo-Espín,1–3 Antonio Villaverde,1–3 Ramón Mangues,3,4 Esther Vázquez1–31Institut de Biotecnologia i de Biomedicina, 2Departamento de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, 3CIBER en Bioingeniería, Biomateriales y Nanomedicina, Bellaterra, Barcelona, 4Oncogenesis and Antitumor Drug Group, Biomedical Research Institute Sant Pau, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 5Laboratory of Immunology, Regional Norte, Universidad de la Republica, Salto, UruguayBackground: Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4 is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing.Results: Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer.Conclusion: Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles

Opposing effects of CXCR3 and CCR5 deficiency on CD8+ T cell-mediated inflammation in the central nervous system of virus-infected mice

DEFF Research Database (Denmark)

de Lemos, Carina; Christensen, Jeanette Erbo; Nansen, Anneline

2005-01-01

and therefore protect mice against the otherwise fatal CD8+ T cell-mediated immune attack. Contrary to expectations, the accumulation of mononuclear cells in cerebrospinal fluid was only slightly delayed compared with mice with normal expression of both receptors. Even more surprising, CXCR3/CCR5 double-deficient......T cells play a key role in the control of viral infection in the CNS but may also contribute to immune-mediated cell damage. To study the redundancy of the chemokine receptors CXCR3 and CCR5 in regulating virus-induced CD8+ T cell-mediated inflammation in the brain, CXCR3/CCR5 double-deficient mice...... mice were more susceptible to intracerebral infection than CXCR3-deficient mice. Analysis of effector T cell generation revealed an accelerated antiviral CD8+ T cell response in CXCR3/CCR5 double-deficient mice. Furthermore, while the accumulation of CD8+ T cells in the neural parenchyma...

Expression and function of the SDF-1 chemokine receptors CXCR4 and CXCR7 during mouse limb muscle development and regeneration.

Science.gov (United States)

Hunger, Conny; Ödemis, Veysel; Engele, Jürgen

2012-10-15

The chemokine, SDF-1/CXCL12, and its receptor, CXCR4, have been implied to play major roles during limb myogenesis. This concept was recently challenged by the identification of CXCR7 as an alternative SDF-1 receptor, which can either act as a scavenger receptor, a modulator of CXCR4, or an active chemokine receptor. We have now re-examined this issue by determining whether SDF-1 would signal to C2C12 myoblasts and subsequently influence their differentiation via CXCR4 and/or CXCR7. In addition, we have analyzed CXCR7, CXCR4, and SDF-1 expression in developing and injured mouse limb muscles. We demonstrate that in undifferentiated C2C12 cells, SDF-1-dependent cell signaling and resulting inhibitory effects on myogenic differentiation are entirely mediated by CXCR4. We further demonstrate that CXCR7 expression increases in differentiating C2C12 cells, which in turn abrogates CXCR4 signaling. Moreover, consistent with the view that CXCR4 and CXCR7 control limb myogenesis in vivo by similar mechanisms, we found that CXCR4 expression is the highest in late embryonic hindlimb muscles and drops shortly after birth when secondary muscle growth terminates. Vice versa, CXCR7 expression increased perinatally and persisted into adult life. Finally, underscoring the role of the SDF-1 system in muscle regeneration, we observed that SDF-1 is continuously expressed by endomysial cells of postnatal and adult muscle fibers. Analysis of dystrophin-deficient mdx mice additionally revealed that muscle regeneration is associated with muscular re-expression of CXCR4. The apparent tight control of limb muscle development and regeneration by CXCR4 and CXCR7 points to these chemokine receptors as promising therapeutic targets for certain muscle disorders. Copyright © 2012 Elsevier Inc. All rights reserved.

Prostate specific antigen in the diagnosis and treatment of adenocarcinoma of the prostate. III. Radiation treated patients

International Nuclear Information System (INIS)

Stamey, T.A.; Kabalin, J.N.; Ferrari, M.

1989-01-01

Serum prostate specific antigen was determined (Yang polyclonal radioimmunoassay) in 183 men after radiation therapy for adenocarcinoma of the prostate. A total of 163 men had received 7,000 rad external beam radiotherapy and 20 had been implanted with iodine-125 seeds. Only 11 per cent of these 183 patients had undetectable prostate specific antigen levels at a mean interval of 5 years since completion of radiotherapy. Prostate specific antigen levels after radiotherapy were directly related to initial clinical stage and Gleason score before treatment. Multiple prostate specific antigen determinations were performed with time in 124 of 183 patients. During year 1 after radiotherapy prostate specific antigen levels were decreasing in 82 per cent of the patients but only 8 per cent continued to decrease beyond year 1. Of 80 patients observed greater than 1 year after completion of radiotherapy 51 per cent had increasing values and 41 per cent had stable values. Increasing prostate specific antigen values after radiotherapy were correlated with progression to metastastic disease and residual cancer on prostate biopsy. Total serum acid phosphatase levels were poorly related to prostate specific antigen levels, were less effective in discriminating patients with metastatic disease and provided no additional information beyond that provided by prostate specific antigen

Preclinical chorioamnionitis dysregulates CXCL1/CXCR2 signaling throughout the placental-fetal-brain axis.

Science.gov (United States)

Yellowhair, Tracylyn R; Noor, Shahani; Maxwell, Jessie R; Anstine, Christopher V; Oppong, Akosua Y; Robinson, Shenandoah; Milligan, Erin D; Jantzie, Lauren L

2018-03-01

In the United States, perinatal brain injury (PBI) is a major cause of infant mortality and childhood disability. For a large proportion of infants with PBI, central nervous system (CNS) injury begins in utero with inflammation (chorioamnionitis/CHORIO) and/or hypoxia-ischemia. While studies show CHORIO contributes to preterm CNS injury and is also a common independent risk factor for brain injury in term infants, the molecular mechanisms mediating inflammation in the placental-fetal-brain axis that result in PBI remain a gap in knowledge. The chemokine (C-X-C motif) ligand 1 (CXCL1), and its cognate receptor, CXCR2, have been clinically implicated in CHORIO and in mature CNS injury, although their specific role in PBI pathophysiology is poorly defined. Given CXCL1/CXCR2 signaling is essential to neural cell development and neutrophil recruitment, a key pathological hallmark of CHORIO, we hypothesized CHORIO would upregulate CXCL1/CXCR2 expression in the placenta and fetal circulation, concomitant with increased CXCL1/CXCR2 signaling in the developing brain, immune cell activation, neutrophilia, and microstructural PBI. On embryonic day 18 (E18), a laparotomy was performed in pregnant Sprague Dawley rats to induce CHORIO. Specifically, uterine arteries were occluded for 60min to induce placental transient systemic hypoxia-ischemia (TSHI), followed by intra-amniotic injection of lipopolysaccharide (LPS). Pups were born at E22. Placentae, serum and brain were collected along an extended time course from E19 to postnatal day (P)15 and analyzed using multiplex electrochemiluminescence (MECI), Western blot, qPCR, flow cytometry (FC) and diffusion tensor imaging (DTI). Results demonstrate that compared to sham, CHORIO increases placental CXCL1 and CXCR2 mRNA levels, concomitant with increased CXCR2 + neutrophils. Interestingly, pup serum CXCL1 expression in CHORIO parallels this increase, with sustained elevation through P15. Analyses of CHORIO brains reveal similarly

CXCR6 Expression Is Important for Retention and Circulation of ILC Precursors

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Sylvestre Chea

2015-01-01

Full Text Available Innate lymphoid cells are present at mucosal sites and represent the first immune barrier against infections, but what contributes to their circulation and homing is still unclear. Using Rag2−/−Cxcr6Gfp/+ reporter mice, we assessed the expression and role of CXCR6 in the circulation of ILC precursors and their progeny. We identify CXCR6 expressing ILC precursors in the bone marrow and characterize their significant increase in CXCR6-deficient mice at steady state, indicating their partial retention in the bone marrow after CXCR6 ablation. Circulation was also impaired during embryonic life as fetal liver from CXCR6-deficient embryos displayed decreased numbers of ILC3 precursors. When injected, fetal CXCR6-deficient ILC3 precursors also fail to home and reconstitute ILC compartments in vivo. We show that adult intestinal ILC subsets have heterogeneous expression pattern of CXCR6, integrin α4β7, CD62L, CD69, and CD44, with ILC1 and ILC3 being more likely tissue resident lymphocytes. Intestinal ILC subsets were unchanged in percentages and numbers in both mice. We demonstrate that the ILC frequency is maintained due to a significant increase of ILC peripheral proliferation, as well as an increased proliferation of the in situ ILC precursors to compensate their retention in the bone marrow.

Consequences of ChemR23 heteromerization with the chemokine receptors CXCR4 and CCR7.

Directory of Open Access Journals (Sweden)

Cédric de Poorter

Full Text Available Recent studies have shown that heteromerization of the chemokine receptors CCR2, CCR5 and CXCR4 is associated to negative binding cooperativity. In the present study, we build on these previous results, and investigate the consequences of chemokine receptor heteromerization with ChemR23, the receptor of chemerin, a leukocyte chemoattractant protein structurally unrelated to chemokines. We show, using BRET and HTRF assays, that ChemR23 forms homomers, and provide data suggesting that ChemR23 also forms heteromers with the chemokine receptors CCR7 and CXCR4. As previously described for other chemokine receptor heteromers, negative binding cooperativity was detected between ChemR23 and chemokine receptors, i.e. the ligands of one receptor competed for the binding of a specific tracer of the other. We also showed, using mouse bone marrow-derived dendritic cells prepared from wild-type and ChemR23 knockout mice, that ChemR23-specific ligands cross-inhibited CXCL12 binding on CXCR4 in a ChemR23-dependent manner, supporting the relevance of the ChemR23/CXCR4 interaction in native leukocytes. Finally, and in contrast to the situation encountered for other previously characterized CXCR4 heteromers, we showed that the CXCR4-specific antagonist AMD3100 did not cross-inhibit chemerin binding in cells co-expressing ChemR23 and CXCR4, demonstrating that cross-regulation by AMD3100 depends on the nature of receptor partners with which CXCR4 is co-expressed.

Prostate-specific antigen (PSA/hK3): a further player in the field of breast cancer diagnostics?

OpenAIRE

Mannello, Ferdinando; Gazzanelli, Giancarlo

2001-01-01

Since its identification, much information has been obtained about prostate-specific antigen (PSA, or human glandular kallikrein 3 [hK3]), a kallikrein-like serine protease that is the most valuable tumour marker for the screening, diagnosis and management of human prostate carcinoma. Recently, it has become widely accepted that PSA is also present in many nonprostatic sources, casting doubts about the specificity of its tissue expression. Here we summarize the findings on the biomolecular ex...

CXCR3 chemokine receptor-induced chemotaxis in human airway epithelial cells: role of p38 MAPK and PI3K signaling pathways.

Science.gov (United States)

Shahabuddin, Syed; Ji, Rong; Wang, Ping; Brailoiu, Eugene; Dun, Na; Yang, Yi; Aksoy, Mark O; Kelsen, Steven G

2006-07-01

Human airway epithelial cells (HAEC) constitutively express the CXC chemokine receptor CXCR3, which regulates epithelial cell movement. In diseases such as chronic obstructive pulmonary disease and asthma, characterized by denudation of the epithelial lining, epithelial cell migration may contribute to airway repair and reconstitution. This study compared the potency and efficacy of three CXCR3 ligands, I-TAC/CXCL11, IP-10/CXCL10, and Mig/CXCL9, as inducers of chemotaxis in HAEC and examined the underlying signaling pathways involved. Studies were performed in cultured HAEC from normal subjects and the 16-HBE cell line. In normal HAEC, the efficacy of I-TAC-induced chemotaxis was 349 +/- 88% (mean +/- SE) of the medium control and approximately one-half the response to epidermal growth factor, a highly potent chemoattractant. In normal HAEC, Mig, IP-10, and I-TAC induced chemotaxis with similar potency and a rank order of efficacy of I-TAC = IP-10 > Mig. Preincubation with pertussis toxin completely blocked CXCR3-induced migration. Of interest, intracellular [Ca(2+)] did not rise in response to I-TAC, IP-10, or Mig. I-TAC induced a rapid phosphorylation (5-10 min) of two of the three MAPKs, i.e., p38 and ERK1/2. Pretreatment of HAEC with the p38 inhibitor SB 20358 or the PI3K inhibitor wortmannin dose-dependently inhibited the chemotactic response to I-TAC. In contrast, the ERK1/2 inhibitor U0126 had no effect on chemotaxis. These data indicate that in HAEC, CXCR3-mediated chemotaxis involves a G protein, which activates both the p38 MAPK and PI3K pathways in a calcium-independent fashion.

Characterization of Antigen-Specific B Cells Using Nominal Antigen-Coated Flow-Beads

Science.gov (United States)

Akl, Ahmed; Lepetit, Maud; Crochette, Romain; Giral, Magali; Lepourry, Julie; Pallier, Annaick; Castagnet, Stéphanie; Dugast, Emilie; Guillot-Gueguen, Cécile; Jacq-Foucher, Marylène; Saulquin, Xavier; Cesbron, Anne; Laplaud, David; Nicot, Arnaud; Brouard, Sophie; Soulillou, Jean-Paul

2013-01-01

In order to characterize the reactivity of B cells against nominal antigens, a method based on the coupling of antigens onto the surface of fluorescent core polystyrene beads was developed. We first demonstrate that murine B cells with a human MOG-specific BCR are able to interact with MOG-coated beads and do not recognize beads coated with human albumin or pp65. B cells purified from human healthy volunteer blood or immunized individuals were tested for their ability to interact with various nominal antigens, including viral, vaccine, self and alloantigens, chosen for their usefulness in studying a variety of pathological processes. A substantial amount of B cells binding self-antigen MOG-coated beads can be detected in normal blood. Furthermore, greater frequencies of B cell against anti-Tetanic Toxin or anti-EBNA1 were observed in primed individuals. This method can reveal increased frequencies of anti-HLA committed B cells in patients with circulating anti-HLA antibodies compared to unsensitized patients and normal individuals. Of interest, those specific CD19 cells were preferentially identified within CD27−IgD+ (i-e naïve) subset. These observations suggest that a broad range of medical situations could benefit from a tool that allows the detection, the quantification and the characterization of antigen-specific blood B cells. PMID:24386360

Cationized dextran nanoparticle-encapsulated CXCR4-siRNA enhanced correlation between CXCR4 expression and serum alkaline phosphatase in a mouse model of colorectal cancer

Directory of Open Access Journals (Sweden)

Abedini F

2012-07-01

Full Text Available Fatemeh Abedini,1 Hossein Hosseinkhani,2 Maznah Ismail,1,3 Abraham J Domb,4 Abdul Rahman Omar,1,5 Pei Pei Chong,1,2 Po-Da Hong,3 Dah-Shyong Yu,6 Ira-Yudovin Farber41Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, Selangor, 2Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan, 3Faculty of Medicine & Health Sciences, Universiti Putra Malaysia, Selangor, Malaysia, 4Institute of Drug Research, The Center for Nanoscience and Nanotechnology, School of Pharmacy-Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel, 5Faculty of Veterinary Medicine, Universiti Putra Malaysia, Selangor, Malaysia, 6Nanomedicine Research Center, National Defense Medical Center, Taipei, TaiwanPurpose: The failure of colorectal cancer treatments is partly due to overexpression of CXCR4 by tumor cells, which plays a critical role in cell metastasis. Moreover, serum alkaline phosphatase (ALP levels are frequently elevated in patients with metastatic colorectal cancer. A polysaccharide, dextran, was chosen as the vector of siRNA. Spermine was conjugated to oxidized dextran by reductive amination process to obtain cationized dextran, so-called dextran-spermine, in order to prepare CXCR4-siRNAs/dextran-spermine nanoparticles. The fabricated nanoparticles were used in order to investigate whether downregulation of CXCR4 expression could affect serum ALP in mouse models of colorectal cancer.Methods: Colorectal cancer was established in BALB/C mice following injection of mouse colon carcinoma cells CT.26WT through the tail vein. CXCR4 siRNA for two sites of the target gene was administered following injection of naked siRNA or siRNA encapsulated into nanoparticles.Results: In vivo animal data revealed that CXCR4 silencing by dextran-spermine nanoparticles significantly downregulated CXCR4 expression compared with naked CXCR4 siRNA. Furthermore, there was

Docking of B-cell epitope antigen to specific hepatitis B antibody

Indian Academy of Sciences (India)

The interaction of pres1 region of hepatitis B virus B-cell epitope antigen with specific hepatitis B neutralizing monoclonal antibody was examined by docking study. We modelled the 3D complex structure of B-cell epitope antigen residues CTTPAQGNSMFPSCCCTKPTDGNCY by homology modelling and docked it with the ...

Pathogenesis of Ovarian Serous Carcinoma as the Basis for Immunologic Directed Diagnosis and Treatment. Project 3 - Development of Antigen-Specific Cancer Vaccines for the Control of Ovarian Cancer

National Research Council Canada - National Science Library

Kurman, Robert

2003-01-01

The purpose of this study is to test whether the greater extent of intracellular spreading of encoded antigen will generate a higher degree of antigen-specific immunity and ant-tumor effects in vaccinated mice...

Interleukin-3 enhances the migration of human mesenchymal stem cells by regulating expression of CXCR4.

Science.gov (United States)

Barhanpurkar-Naik, Amruta; Mhaske, Suhas T; Pote, Satish T; Singh, Kanupriya; Wani, Mohan R

2017-07-14

Mesenchymal stem cells (MSCs) represent an important source for cell therapy in regenerative medicine. MSCs have shown promising results for repair of damaged tissues in various degenerative diseases in animal models and also in human clinical trials. However, little is known about the factors that could enhance the migration and tissue-specific engraftment of exogenously infused MSCs for successful regenerative cell therapy. Previously, we have reported that interleukin-3 (IL-3) prevents bone and cartilage damage in animal models of rheumatoid arthritis and osteoarthritis. Also, IL-3 promotes the differentiation of human MSCs into functional osteoblasts and increases their in-vivo bone regenerative potential in immunocompromised mice. However, the role of IL-3 in migration of MSCs is not yet known. In the present study, we investigated the role of IL-3 in migration of human MSCs under both in-vitro and in-vivo conditions. MSCs isolated from human bone marrow, adipose and gingival tissues were used for in-vitro cell migration, motility and wound healing assays in the presence or absence of IL-3. The effect of IL-3 preconditioning on expression of chemokine receptors and integrins was examined by flow cytometry and real-time PCR. The in-vivo migration of IL-3-preconditioned MSCs was investigated using a subcutaneous matrigel-releasing stromal cell-derived factor-1 alpha (SDF-1α) model in immunocompromised mice. We observed that human MSCs isolated from all three sources express IL-3 receptor-α (IL-3Rα) both at gene and protein levels. IL-3 significantly enhances in-vitro migration, motility and wound healing abilities of MSCs. Moreover, IL-3 preconditioning upregulates expression of chemokine (C-X-C motif) receptor 4 (CXCR4) on MSCs, which leads to increased migration of cells towards SDF-1α. Furthermore, CXCR4 antagonist AMD3100 decreases the migration of IL-3-treated MSCs towards SDF-1α. Importantly, IL-3 also induces in-vivo migration of MSCs towards

The CXCR4–STAT3–IL-10 Pathway Controls the Immunoregulatory Function of Chronic Lymphocytic Leukemia and Is Modulated by Lenalidomide

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Hila Shaim

2018-01-01

Full Text Available Chronic lymphocytic leukemia (CLL cells possess regulatory functions comparable to those of normal B10 cells, a regulatory B cell subset that suppresses effector T-cell function through STAT3-mediated IL-10 production. However, the mechanisms governing IL-10 production by CLL cells are not fully understood. Here, we show that the CXC chemokine ligand 12 (CXCL12–CXCR4–STAT3 axis regulates IL-10 production by CLL cells and their ability to suppress T-cell effector function through an IL-10 mediated mechanism. Knockdown of STAT3 significantly impaired the ability of CLL cells to produce IL-10. Furthermore, experiments to assess the role of lenalidomide, an immunomodulatory agent with direct antitumor effect as well as pleiotropic activity on the immune system, showed that this agent prevents a CXCL12-induced increase in p-S727-STAT3 and the IL-10 response by CLL cells. Lenalidomide also suppressed IL-10-induced Y705-STAT3 phosphorylation in healthy T cells, thus reversing CLL-induced T-cell dysfunction. We conclude that the capacity of CLL cells to produce IL-10 is mediated by the CXCL12–CXCR4–STAT3 pathway and likely contributes to immunodeficiency in patients. Lenalidomide appears to be able to reverse CLL-induced immunosuppression through including abrogation of the CXCL12–CXCR4–S727–STAT3-mediated IL-10 response by CLL cells and prevention of IL-10-induced phosphorylation of Y705-STAT3 in T cells.

B700, a murine melanoma-specific antigen, binds Vitamin D3; conservation of binding among albuminoid molecules

International Nuclear Information System (INIS)

Farzaneh, N.K.; Walden, T.L. Jr.; Hearing, V.J.; Gersten, D.M.

1990-01-01

B700, a murine melanoma-specific antigen, is a member of the serum albumin protein family. Other members of this family include serum albumin (SMA), a-fetoprotein (AFP), vitamin D binding protein (DBP), and C700. The primary structure and biochemical functions of B700, as well as its in vivo metabolic fate are largely unknown. The authors examined the functional characteristics of MSA, AFP, and DBP, and for their ability to specifically bind [ 3 H]-1,25-dihydroxy-vitamin D 3 . Scatchard analysis revealed a single binding site for B700 with a Kd of 51,000 M and a Bmax of 4.51 x 10 -7 . There is no significant difference between the Kd and Bmax values among the albuminoid proteins. However, differences in the binding sites could be distinguished by competition of the 1,25-dihydroxy vitamin D 3 with other steroids. 2nM of vitamin D 3 , vitamin D 2 , or estrogen competed for the specific binding of 1,25-dihydroxy vitamin D 3 by B700 but not by DBP. The MSA binding site for 1,25 dihydroxy vitamin D 3 more closely resembles that of DBP than B700. These data indicate that the binding function of the albuminoid proteins has been conserved in the B700 melanoma antigen

Fluorescent imaging of high-grade bladder cancer using a specific antagonist for chemokine receptor CXCR4.

Science.gov (United States)

Nishizawa, Koji; Nishiyama, Hiroyuki; Oishi, Shinya; Tanahara, Noriko; Kotani, Hirokazu; Mikami, Yoshiki; Toda, Yoshinobu; Evans, Barry J; Peiper, Stephen C; Saito, Ryoichi; Watanabe, Jun; Fujii, Nobutaka; Ogawa, Osamu

2010-09-01

We previously reported that the expression of CXC chemokine receptor-4 (CXCR4) was upregulated in invasive bladder cancers and that the small peptide T140 was a highly sensitive antagonist for CXCR4. In this study, we identified that CXCR4 expression was induced in high-grade superficial bladder tumors, including carcinoma in situ and invasive bladder tumors. To visualize the bladder cancer cells using urinary sediments from the patients and chemically induced mouse bladder cancer model, a novel fluorescent CXCR4 antagonist TY14003 was developed, that is a T140 derivative. TY14003 could label bladder cancer cell lines expressing CXCR4, whereas negative-control fluorescent peptides did not label them. When labeling urinary sediments from patients with invasive bladder cancer, positive-stained cells were identified in all patients with bladder cancer and positive urine cytology but not in controls. Although white blood cells in urine were also labeled with TY14003, they could be easily discriminated from urothelial cells by their shape and size. Finally, intravesical instillation of TY14003 into mouse bladder, using N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced bladder cancer model, demonstrated that fluorescent signals were detected in the focal areas of bladder of all mice examined at 12 weeks of BBN drinking by confocal microscopy and fluorescent endoscopy. On the contrary, all the normal bladders were found to be negative for TY14003 staining. In conclusion, these results indicate that TY14003 is a promising diagnostic tool to visualize small or flat high-grade superficial bladder cancer.

An MHC-restricted antibody-based chimeric antigen receptor requires TCR-like affinity to maintain antigen specificity

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Marcela V Maus

2016-01-01

Full Text Available Chimeric antigen receptors (CARs are synthetic receptors that usually redirect T cells to surface antigens independent of human leukocyte antigen (HLA. Here, we investigated a T cell receptor-like CAR based on an antibody that recognizes HLA-A*0201 presenting a peptide epitope derived from the cancer-testis antigen NY-ESO-1. We hypothesized that this CAR would efficiently redirect transduced T cells in an HLA-restricted, antigen-specific manner. However, we found that despite the specificity of the soluble Fab, the same antibody in the form of a CAR caused moderate lysis of HLA-A2 expressing targets independent of antigen owing to T cell avidity. We hypothesized that lowering the affinity of the CAR for HLA-A2 would improve its specificity. We undertook a rational approach of mutating residues that, in the crystal structure, were predicted to stabilize binding to HLA-A2. We found that one mutation (DN lowered the affinity of the Fab to T cell receptor-range and restored the epitope specificity of the CAR. DN CAR T cells lysed native tumor targets in vitro, and, in a xenogeneic mouse model implanted with two human melanoma lines (A2+/NYESO+ and A2+/NYESO−, DN CAR T cells specifically migrated to, and delayed progression of, only the HLA-A2+/NY-ESO-1+ melanoma. Thus, although maintaining MHC-restricted antigen specificity required T cell receptor-like affinity that decreased potency, there is exciting potential for CARs to expand their repertoire to include a broad range of intracellular antigens.

CXCR4 blockade decreases CD4+ T cell exhaustion and improves survival in a murine model of polymicrobial sepsis.

Science.gov (United States)

Ramonell, Kimberly M; Zhang, Wenxiao; Hadley, Annette; Chen, Ching-Wen; Fay, Katherine T; Lyons, John D; Klingensmith, Nathan J; McConnell, Kevin W; Coopersmith, Craig M; Ford, Mandy L

2017-01-01

Sepsis is a dysregulated systemic response to infection involving many inflammatory pathways and the induction of counter-regulatory anti-inflammatory processes that results in a state of immune incompetence and can lead to multi-organ failure. CXCR4 is a chemokine receptor that, following ligation by CXCL12, directs cells to bone marrow niches and also plays an important role in T cell cosignaling and formation of the immunological synapse. Here, we investigated the expression and function of CXCR4 in a murine model of polymicrobial sepsis. Results indicate that CXCR4 is selectively upregulated on naïve CD4+ and CD8+ T cells and CD4+ central memory T cells following the induction of sepsis, and that CXCR4 antagonism resulted in a significant decrease in sepsis-induced mortality. We probed the mechanistic basis for these findings and found that CXCR4 antagonism significantly increased the number of peripheral CD4+ and CD8+ T cells following sepsis. Moreover, mice treated with the CXCR4 antagonist contained fewer PD-1+ LAG-3+ 2B4+ cells, suggesting that blockade of CXCR4 mitigates CD4+ T cell exhaustion during sepsis. Taken together, these results characterize CXCR4 as an important pathway that modulates immune dysfunction and mortality following sepsis, which may hold promise as a target for future therapeutic intervention in septic patients.

CXCR4 blockade decreases CD4+ T cell exhaustion and improves survival in a murine model of polymicrobial sepsis.

Directory of Open Access Journals (Sweden)

Kimberly M Ramonell

Full Text Available Sepsis is a dysregulated systemic response to infection involving many inflammatory pathways and the induction of counter-regulatory anti-inflammatory processes that results in a state of immune incompetence and can lead to multi-organ failure. CXCR4 is a chemokine receptor that, following ligation by CXCL12, directs cells to bone marrow niches and also plays an important role in T cell cosignaling and formation of the immunological synapse. Here, we investigated the expression and function of CXCR4 in a murine model of polymicrobial sepsis. Results indicate that CXCR4 is selectively upregulated on naïve CD4+ and CD8+ T cells and CD4+ central memory T cells following the induction of sepsis, and that CXCR4 antagonism resulted in a significant decrease in sepsis-induced mortality. We probed the mechanistic basis for these findings and found that CXCR4 antagonism significantly increased the number of peripheral CD4+ and CD8+ T cells following sepsis. Moreover, mice treated with the CXCR4 antagonist contained fewer PD-1+ LAG-3+ 2B4+ cells, suggesting that blockade of CXCR4 mitigates CD4+ T cell exhaustion during sepsis. Taken together, these results characterize CXCR4 as an important pathway that modulates immune dysfunction and mortality following sepsis, which may hold promise as a target for future therapeutic intervention in septic patients.

Rac1 plays a role in CXCL12 but not CCL3-induced chemotaxis and Rac1 GEF inhibitor NSC23766 has off target effects on CXCR4.

Science.gov (United States)

Mills, Shirley C; Howell, Lesley; Beekman, Andrew; Stokes, Leanne; Mueller, Anja

2018-01-01

Cell migration towards a chemotactic stimulus relies on the re-arrangement of the cytoskeleton, which is triggered by activation of small G proteins RhoA, Rac1 and Cdc42, and leads to formation of lamellopodia and actin polymerisation amongst other effects. Here we show that Rac1 is important for CXCR4 induced chemotaxis but not for CCR1/CCR5 induced chemotaxis. For CXCL12-induced migration via CXCR4, breast cancer MCF-7 cells are reliant on Rac1, similarly to THP-1 monocytes and Jurkat T-cells. For CCL3-induced migration via CCR1 and/or CCR5, Rac1 signalling does not regulate cell migration in either suspension or adherent cells. We have confirmed the involvement of Rac1 with the use of a specific Rac1 blocking peptide. We also used a Rac1 inhibitor EHT 1864 and a Rac1-GEF inhibitor NSC23766 to probe the importance of Rac1 in chemotaxis. Both inhibitors did not block CCL3-induced chemotaxis, but they were able to block CXCL12-induced chemotaxis. This confirms that Rac1 activation is not essential for CCL3-induced migration, however NSC23766 might have secondary effects on CXCR4. This small molecule exhibits agonistic features in internalisation and cAMP assays, whereas it acts as an antagonist for CXCR4 in migration and calcium release assays. Our findings strongly suggest that Rac1 activation is not necessary for CCL3 signalling, and reveal that NSC23766 could be a novel CXCR4 receptor ligand. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

Prostate-Specific Antigen (PSA) Test

Science.gov (United States)

... Cancer Prostate Cancer Screening Research Prostate-Specific Antigen (PSA) Test On This Page What is the PSA ... parts of the body before being detected. The PSA test may give false-positive or false-negative ...

Synthesis and evaluation of an 18F-labeled pyrimidine-pyridine amine for targeting CXCR4 receptors in gliomas

International Nuclear Information System (INIS)

Demoin, Dustin Wayne; Shindo, Masahiro; Zhang, Hanwen; Edwards, Kimberly J.; Serganova, Inna; Pillarsetty, Naga Vara Kishore; Lewis, Jason S.; Blasberg, Ronald G.

2016-01-01

Introduction: Chemokine receptor-4 (CXCR4, fusin, CD184) is expressed on several tissues involved in immune regulation and is upregulated in many diseases including malignant gliomas. A radiolabeled small molecule that readily crosses the blood–brain barrier can aid in identifying CXCR4-expressing gliomas and monitoring CXCR4-targeted therapy. In the current work, we have synthesized and evaluated an [ 18 F]-labeled small molecule based on a pyrimidine–pyridine amine for its ability to target CXCR4. Experimental: The nonradioactive standards and the nitro precursor used in this study were prepared using established methods. An HPLC method was developed to separate the nitro-precursor from the nonradioactive standard and radioactive product. The nitro-precursor was radiolabeled with 18 F under inert, anhydrous conditions using the [ 18 F]-kryptofix 2.2.2 complex to form the desired N-(4-(((6-[ 18 F]fluoropyridin-2-yl)amino)methyl)benzyl)pyrimidin-2-amine ([ 18 F]-3). The purified radiolabeled compound was used in serum stability, partition coefficient, cellular uptake, and in vivo cancer targeting studies. Results: [ 18 F]-3 was synthesized in 4–10% decay-corrected yield (to start of synthesis). [ 18 F]-3 (t R ≈ 27 min) was separated from the precursor (t R ≈ 30 min) using a pentafluorophenyl column with an isocratic solvent system. [ 18 F]-3 displayed acceptable serum stability over 2 h. The amount of [ 18 F]-3 bound to the plasma proteins was determined to be > 97%. The partition coefficient (LogD 7.4 ) is 1.4 ± 0.5. Competitive in vitro inhibition indicated 3 does not inhibit uptake of 67 Ga-pentixafor. Cell culture media incubation and ex vivo urine analysis indicate rapid metabolism of [ 18 F]-3 into hydrophilic metabolites. Thus, in vitro uptake of [ 18 F]-3 in CXCR4 overexpressing U87 cells (U87 CXCR4) and U87 WT indicated no specific binding. In vivo studies in mice bearing U87 CXCR4 and U87 WT tumors on the left and right shoulders were carried

Induction of antigen-specific immunity by pH-sensitive carbonate apatite as a potent vaccine carrier

International Nuclear Information System (INIS)

Hebishima, Takehisa; Tada, Seiichi; Takeshima, Shin-nosuke; Akaike, Toshihiro; Ito, Yoshihiro; Aida, Yoko

2011-01-01

Highlights: ► To develop effective vaccine, we examined the effects of CO 3 Ap as an antigen carrier. ► OVA contained in CO 3 Ap was taken up by BMDCs more effectively than free OVA. ► OVA-immunized splenocytes was activated by OVA contained in CO 3 Ap effectively. ► OVA contained in CO 3 Ap induced strong OVA-specific immune responses to C57BL/6 mice. ► CO 3 Ap is promising antigen carrier for the achievement of effective vaccine. -- Abstract: The ability of carbonate apatite (CO 3 Ap) to enhance antigen-specific immunity was examined in vitro and in vivo to investigate its utility as a vaccine carrier. Murine bone marrow-derived dendritic cells took up ovalbumin (OVA) containing CO 3 Ap more effectively than free OVA. Interestingly, mice immunized with OVA-containing CO 3 Ap produced OVA-specific antibodies more effectively than mice immunized with free OVA. Furthermore, immunization of C57BL/6 mice with OVA-containing CO 3 Ap induced the proliferation and antigen-specific production of IFN-γ by splenocytes more strongly than immunization with free OVA. Moreover, no significant differences were detected in the induction of delayed-type hypersensitivity responses, an immune reaction involving an antigen-specific, cell-mediated immune response between OVA-containing CO 3 Ap and OVA-containing alumina salt (Alum), suggesting that CO 3 Ap induced cell-mediated immune response to the same degree as Alum, which is commonly used for clinical applications. This study is the first to demonstrate the induction of antigen-specific immune responses in vivo by CO 3 Ap.

Heparin-associated thrombocytopenia: antibody binding specificity to platelet antigens.

Science.gov (United States)

Lynch, D M; Howe, S E

1985-11-01

Sera from four patients with heparin-associated thrombocytopenia (HAT) were evaluated by a quantitative enzyme-linked immunosorbent assay (ELISA) to detect heparin-dependent serum platelet-bindable immunoglobulin (S-PBIg) and by Western blotting and immunoprecipitation to investigate the specificity of the antibody binding. All HAT sera showed mildly increased S-PBIg (mean, 7.8 fg per platelet; normal, less than 6.0 fg per platelet) to intact target platelets in the ELISA, which was markedly increased in the presence of heparin (mean, 20.9 fg per platelet). This increase was 20-fold greater than normal control sera, which showed a mean differential increase of only 0.5 fg per platelet. Immunoglobulin binding specificity to platelet antigens was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of platelet lysate with transfer of the platelet fractions onto nitrocellulose strips (Western blotting) and subsequent immunoassay using HAT and normal sera. In the presence of heparin, the four HAT patients demonstrated increased binding of immunoglobulin to platelet antigens of apparent molecular weights of 180, 124, and 82 kd. Radiolabeled heparin when incubated with HAT sera, normal sera, or albumin blanks bound to platelet proteins of the same apparent molecular weights. These observations are consistent with current hypotheses suggesting that HAT antibody is directed to heparin-platelet complexes or, alternatively, that heparin induces conformational change of antigenic sites on the platelet membrane.

Simultaneous targeting of prostate stem cell antigen and prostate-specific membrane antigen improves the killing of prostate cancer cells using a novel modular T cell-retargeting system.

Science.gov (United States)

Arndt, Claudia; Feldmann, Anja; Koristka, Stefanie; Cartellieri, Marc; Dimmel, Maria; Ehninger, Armin; Ehninger, Gerhard; Bachmann, Michael

2014-09-01

Recently, we described a novel modular platform technology in which T cell-recruitment and tumor-targeting domains of conventional bispecific antibodies are split to independent components, a universal effector module (EM) and replaceable monospecific/monovalent target modules (TMs) that form highly efficient T cell-retargeting complexes. Theoretically, our unique strategy should allow us to simultaneously retarget T cells to different tumor antigens by combining the EM with two or more different monovalent/monospecific TMs or even with bivalent/bispecific TMs, thereby overcoming limitations of a monospecific treatment such as the selection of target-negative tumor escape variants. In order to advance our recently introduced prostate stem cell antigen (PSCA)-specific modular system for a dual-targeting of prostate cancer cells, two additional TMs were constructed: a monovalent/monospecific TM directed against the prostate-specific membrane antigen (PSMA) and a bivalent/bispecific TM (bsTM) with specificity for PSMA and PSCA. The functionality of the novel dual-targeting strategies was analyzed by performing T cell activation and chromium release assays. Similar to the PSCA-specific modular system, the novel PSMA-specific modular system mediates an efficient target-dependent and -specific tumor cell lysis at low E:T ratios and picomolar Ab concentrations. Moreover, by combination of the EM with either the bispecific TM directed to PSMA and PSCA or both monospecifc TMs directed to either PSCA or PSMA, dual-specific targeting complexes were formed which allowed us to kill potential escape variants expressing only one or the other target antigen. Overall, the novel modular system represents a promising tool for multiple tumor targeting. © 2014 Wiley Periodicals, Inc.

Expression of chemokine receptor CXCR4 in esophageal squamous cell and adenocarcinoma

International Nuclear Information System (INIS)

Gockel, Ines; Galle, Peter R; Junginger, Theodor; Moehler, Markus; Schimanski, Carl C; Heinrich, Christian; Wehler, T; Frerichs, K; Drescher, Daniel; Langsdorff, Christian von; Domeyer, Mario; Biesterfeld, Stefan

2006-01-01

Prognosis of esophageal cancer is poor despite curative surgery. The chemokine receptor CXCR4 has been proposed to distinctly contribute to tumor growth, dissemination and local immune escape in a limited number of malignancies. The aim of our study was to evaluate the role of CXCR4 in tumor spread of esophageal cancer with a differentiated view of the two predominant histologic types – squamous cell and adenocarcinoma. Esophageal cancer tissue samples were obtained from 102 consecutive patients undergoing esophageal resection for cancer with curative intent. The LSAB+ System was used to detect the protein CXCR4. Tumor samples were classified into two groups based on the homogeneous staining intensity. A cut-off between CXCR4w (= weak expression) and CXCR4s (= strong expression) was set at 1.5 (grouped 0 – 1.5 versus 2.0 – 3). Long-term survival rates were calculated using life tables and the Kaplan-Meier method. Using the Cox's proportional hazards analysis, a model of survival prediction was established. The overall expression rate for CXCR4 in esophageal squamous cell carcinoma was 94.1%. Subdividing these samples, CXCR4w was found in 54.9% and CXCR4s in 45.1%. In adenocarcinoma, an overall expression rate of 89.1% was detected with a weak intensitiy in 71.7% compared to strong staining in 29.3% (p = 0.066 squamous cell versus adenocarcinoma). The Cox's proportional hazards analysis identified the pM-category with a hazard ratio (HR) of 1.860 (95% CI: 1.014–3.414) (p = 0.045), the histologic tumor type (HR: 0.334; 95% CI: 0.180–0.618) (p = 0.0001) and the operative approach (transthoracic > transhiatal esophageal resection) (HR: 0.546; 95% CI: 0.324–0.920) (p = 0.023) as independent factors with a possible influence on the long-term prognosis in patients with esophageal carcinoma, whereas CXCR4 expression was statistically not significant (>0.05). Expression of the chemokine receptor CXCR4 in esophageal cancer is of major relevance in both

Epstein-Barr virus nuclear antigen 2 specifically induces expression of the B-cell activation antigen CD23

International Nuclear Information System (INIS)

Wang, F.; Gregory, C.D.; Rowe, M.; Rickinson, A.B.; Wang, D.; Birkenbach, M.; Kikutani, H.; Kishimoto, T.; Kieff, E.

1987-01-01

Epstein-Barr virus (EBV) infection of EBV-negative Burkitt lymphoma (BL) cells includes some changes similar to those seen in normal B lymphocytes that have been growth transformed by EBV. The role of individual EBV genes in this process was evaluated by introducing each of the viral genes that are normally expressed in EBV growth-transformed and latently infected lymphoblasts into an EBV-negative BL cell line, using recombinant retrovirus-mediated transfer. Clones of cells were derived that stably express the EBV nuclear antigen 1 (EBNA-1), EBNA-2, EBNA-3, EBNA-leader protein, or EBV latent membrane protein (LMP). These were compared with control clones infected with the retrovirus vector. All 10 clones converted to EBNA-2 expression differed from control clones or clones expressing other EBV proteins by growth in tight clumps and by markedly increased expression of one particular surface marker of B-cell activation, CD23. Other activation antigens were unaffected by EBNA-2 expression, as were markers already expressed on the parent BL cell line. The results indicate that EBNA-2 is a specific direct or indirect trans-activator of CD23. This establishes a link between an EBV gene and cell gene expression. Since CD23 has been implicated in the transduction of B-cell growth signals, its specific induction by EBNA-2 could be important in EBV induction of B-lymphocyte transformation

Characterization of antigen association with accessory cells: specific removal of processed antigens from the cell surface by phospholipases

International Nuclear Information System (INIS)

Falo, L.D. Jr.; Haber, S.I.; Herrmann, S.; Benacerraf, B.; Rock, K.L.

1987-01-01

To characterize the basis for the cell surface association of processed antigen with the antigen-presenting cell (APC) the authors analyzed its sensitivity to enzymatic digestion. Antigen-exposed APC that are treated with phospholipase and then immediately fixed lose their ability to stimulate antigen-plus-Ia-specific T-T hybridomas. This effect is seen with highly purified phospholipase A 2 and phospholipase C. In addition it is observed with three distinct antigens - ovalbumin, bovine insulin, and poly(LGlu 56 LLys 35 LPhe 9 )[(GluLysPhe)/sub n/]. The effect of phospholipases is highly specific. Identically treated APC are equivalent to control in their ability to stimulate alloreactive hybridomas specific for precisely the same Ia molecule that is corecognized by antigen-plus-Ia-specific hybrids. Furthermore, the antigen-presenting function of enzyme-treated, fixed APC can be reconstituted by the addition of exogenous in vitro processed or processing independent antigens. In parallel studies 125 I-labeled avidin was shown to specifically bind to APC that were previously exposed and allowed to process biotin-insulin. Biotin-insulin-exposed APC that are pretreated with phospholipase bind significantly less 125 I-labeled avidin than do untreated, exposed APC. Identical enzyme treatment does not reduce the binding of avidin to a biotinylated antibody already bound to class II major histocompatibility complex molecules of APC. These studies demonstrate that phospholipase effectively removes processed cell surface antigen

Comparison of cell-based assays for the identification and evaluation of competitive CXCR4 inhibitors.

Directory of Open Access Journals (Sweden)

Anneleen Van Hout

Full Text Available The chemokine receptor CXCR4 is activated by its unique chemokine ligand CXCL12 and regulates many physiological and developmental processes such as hematopoietic cell trafficking. CXCR4 is also one of the main co-receptors for human immunodeficiency virus (HIV entry. Dysfunction of the CXCL12/CXCR4 axis contributes to several human pathologies, including cancer and inflammatory diseases. Consequently, inhibition of CXCR4 activation is recognized as an attractive target for therapeutic intervention. In this regard, numerous agents modifying CXCR4 activity have been evaluated in in vitro experimental studies and pre-clinical models. Here, we evaluated a CXCL12 competition binding assay for its potential as a valuable initial screen for functional and competitive CXCR4 inhibitors. In total, 11 structurally diverse compounds were included in a side-by-side comparison of in vitro CXCR4 cell-based assays, such as CXCL12 competition binding, CXCL12-induced calcium signaling, CXCR4 internalization, CXCL12-guided cell migration and CXCR4-specific HIV-1 replication experiments. Our data indicated that agents that inhibit CXCL12 binding, i.e. the anti-CXCR4 peptide analogs T22, T140 and TC14012 and the small molecule antagonists AMD3100, AMD3465, AMD11070 and IT1t showed inhibitory activity with consistent relative potencies in all further applied CXCR4-related assays. Accordingly, agents exerting no or very weak receptor binding (i.e., CTCE-9908, WZ811, Me6TREN and gambogic acid showed no or very poor anti-CXCR4 inhibitory activity. Thus, CXCL12 competition binding studies were proven to be highly valuable as an initial screening assay and indicative for the pharmacological and functional profile of competitive CXCR4 antagonists, which will help the design of new potent CXCR4 inhibitors.

Sensitivity, Specificity, and Positivity Predictors of the Pneumococcal Urinary Antigen Test in Community-Acquired Pneumonia.

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Molinos, Luis; Zalacain, Rafael; Menéndez, Rosario; Reyes, Soledad; Capelastegui, Alberto; Cillóniz, Catia; Rajas, Olga; Borderías, Luis; Martín-Villasclaras, Juan J; Bello, Salvador; Alfageme, Inmaculada; Rodríguez de Castro, Felipe; Rello, Jordi; Ruiz-Manzano, Juan; Gabarrús, Albert; Musher, Daniel M; Torres, Antoni

2015-10-01

Detection of the C-polysaccharide of Streptococcus pneumoniae in urine by an immune-chromatographic test is increasingly used to evaluate patients with community-acquired pneumonia. We assessed the sensitivity and specificity of this test in the largest series of cases to date and used logistic regression models to determine predictors of positivity in patients hospitalized with community-acquired pneumonia. We performed a multicenter, prospective, observational study of 4,374 patients hospitalized with community-acquired pneumonia. The urinary antigen test was done in 3,874 cases. Pneumococcal infection was diagnosed in 916 cases (21%); 653 (71%) of these cases were diagnosed exclusively by the urinary antigen test. Sensitivity and specificity were 60 and 99.7%, respectively. Predictors of urinary antigen positivity were female sex; heart rate≥125 bpm, systolic blood pressureantibiotic treatment; pleuritic chest pain; chills; pleural effusion; and blood urea nitrogen≥30 mg/dl. With at least six of all these predictors present, the probability of positivity was 52%. With only one factor present, the probability was only 12%. The urinary antigen test is a method with good sensitivity and excellent specificity in diagnosing pneumococcal pneumonia, and its use greatly increased the recognition of community-acquired pneumonia due to S. pneumoniae. With a specificity of 99.7%, this test could be used to direct simplified antibiotic therapy, thereby avoiding excess costs and risk for bacterial resistance that result from broad-spectrum antibiotics. We also identified predictors of positivity that could increase suspicion for pneumococcal infection or avoid the unnecessary use of this test.

Antigen-Specific Th17 Cells Are Primed by Distinct and Complementary Dendritic Cell Subsets in Oropharyngeal Candidiasis.

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Kerstin Trautwein-Weidner

2015-10-01

Full Text Available Candida spp. can cause severe and chronic mucocutaneous and systemic infections in immunocompromised individuals. Protection from mucocutaneous candidiasis depends on T helper cells, in particular those secreting IL-17. The events regulating T cell activation and differentiation toward effector fates in response to fungal invasion in different tissues are poorly understood. Here we generated a Candida-specific TCR transgenic mouse reactive to a novel endogenous antigen that is conserved in multiple distant species of Candida, including the clinically highly relevant C. albicans and C. glabrata. Using TCR transgenic T cells in combination with an experimental model of oropharyngeal candidiasis (OPC we investigated antigen presentation and Th17 priming by different subsets of dendritic cells (DCs present in the infected oral mucosa. Candida-derived endogenous antigen accesses the draining lymph nodes and is directly presented by migratory DCs. Tissue-resident Flt3L-dependent DCs and CCR2-dependent monocyte-derived DCs collaborate in antigen presentation and T cell priming during OPC. In contrast, Langerhans cells, which are also present in the oral mucosa and have been shown to prime Th17 cells in the skin, are not required for induction of the Candida-specific T cell response upon oral challenge. This highlights the functional compartmentalization of specific DC subsets in different tissues. These data provide important new insights to our understanding of tissue-specific antifungal immunity.

In vitro characterization and inhibition of the CXCR4/CXCL12 chemokine axis in human uveal melanoma cell lines

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Antecka Emilia

2007-11-01

Full Text Available Abstract Purpose The CXCR4/CXCL12 chemokine axis may play a critical role in guiding CXCR4+ circulating malignant cells to organ specific locations that actively secrete its ligand CXCL12 (SDF-1 such as bone, brain, liver, and lungs. We sought to characterize the presence of the CXCR4/CXCL12 axis in five uveal melanoma (UM cell lines in vitro. The ability of TN14003, a synthetic peptide inhibitor that targets the CXCR4 receptor complex, to inhibit this axis was also assessed. Methods Immunocytochemistry was performed against CXCR4 to confirm expression of this chemokine receptor in all five UM cell lines. Flow cytometry was preformed to evaluate CXCR4 cell surface expression on all five UM cell lines. A proliferation assay was also used to test effects TN14003 would have on cellular proliferation. Inhibition of cellular migration by specifically inhibiting the CXCR4/CXCL12 axis with TN14003 was also investigated. The binding efficacy of TN14003 to the CXCR4 receptor was assessed through flow cytometric methods. Results The CXCR4 receptor was present on all five UM cell lines. All five cell lines expressed different relative levels of surface CXCR4. TN14003 did not affect the proliferation of the five cell lines (p > 0.05. All cell lines migrated towards the chemokine CXCL12 at a level greater than the negative control (p Conclusion Interfering with the CXCR4/CXCL12 axis, using TN14003 was shown to effectively down regulate UM cell migration in vitro. Knowing that UM expresses the CXCR4 receptor, these CXCR4+ cells may be less likely to colonize distant organs that secrete the CXCL12 ligand, if treated with an inhibitor that binds CXCR4. Further studies should be pursued in order to test TN14003 efficacy in vivo.

Radioimmunoassay for a human prostate specific antigen

International Nuclear Information System (INIS)

Machida, T.; Miki, M.; Ohishi, Y.; Kido, A.; Morikawa, J.; Ogawa, Y.

1983-01-01

As a marker for prostatic cancer, a prostate-specific antigen was purified from human prostatic tissues. Double antibody radioimmunoassay utilizing immune reaction was developed on the basis of the purified prostatic antigen (PA). Measurement results have revealed that PA radioimmunoassay is much better than prostatic acid phosphatase (PAP) radioimmunoassay in the diagnosis of prostatic cancer

Activation of nickel-specific CD4+ T lymphocytes in the absence of professional antigen-presenting cells.

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Nasorri, Francesca; Sebastiani, Silvia; Mariani, Valentina; De Pità, Ornella; Puddu, Pietro; Girolomoni, Giampiero; Cavani, Andrea

2002-01-01

Allergic contact dermatitis ensues from exaggerated T cell responses to haptens. Dendritic cells are required for the initiation of hapten sensitization, but they may not be necessary for disease expression. Here we investigated the antigen-presenting cell requirement of nickel-specific CD4+ lymphocytes isolated from the blood of six allergic individuals. A significant proportion (42 out of 121; 35%) of the T cell clones proliferated in vitro to nickel also in the absence of professional antigen-presenting cells, suggesting a direct T-T hapten presentation. Antigen-presenting-cell-independent T cells showed a predominant T helper 1 phenotype. Nickel recognition by these T cells was major histocompatibility complex class II restricted, not influenced by CD28 triggering, independent from their state of activation, and did not require processing. The capacity of this T cell subset to be directly stimulated by nickel was not due to unique antigen-presenting properties, as both antigen-presenting-cell-dependent and antigen-presenting-cell-independent clones displayed comparable levels of HLA-DR, CD80, and CD86, and were equally capable of presenting nickel to antigen-presenting-cell-independent clones. In contrast, neither T cell types activated antigen-presenting-cell-dependent T lymphocytes. T-T presentation induced T cell receptor downregulation, CD25, CD80, CD86, and HLA-DR upregulation, and interferon-gamma release, although to a lesser extent compared to those induced by dendritic cell-T presentation. Following T-T presentation, the clones did not undergo unresponsiveness and maintained the capacity to respond to dendritic cells pulsed with antigen. In aggregate, our data suggest that antigen-presenting-cell-independent T cell activation can effectively amplify hapten- specific immune responses.

Development of a Unique Small Molecule Modulator of CXCR4

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Yoon, Younghyoun; Lin, Songbai; Sasaki, Maiko; Klapproth, Jan-Michael A.; Yang, Hua; Grossniklaus, Hans E.; Xu, Jianguo; Rojas, Mauricio; Voll, Ronald J.; Goodman, Mark M.; Arrendale, Richard F.; Liu, Jin; Yun, C. Chris; Snyder, James P.; Liotta, Dennis C.; Shim, Hyunsuk

2012-01-01

Background Metastasis, the spread and growth of tumor cells to distant organ sites, represents the most devastating attribute and plays a major role in the morbidity and mortality of cancer. Inflammation is crucial for malignant tumor transformation and survival. Thus, blocking inflammation is expected to serve as an effective cancer treatment. Among anti-inflammation therapies, chemokine modulation is now beginning to emerge from the pipeline. CXC chemokine receptor-4 (CXCR4) and its ligand stromal cell-derived factor-1 (CXCL12) interaction and the resulting cell signaling cascade have emerged as highly relevant targets since they play pleiotropic roles in metastatic progression. The unique function of CXCR4 is to promote the homing of tumor cells to their microenvironment at the distant organ sites. Methodology/Principal Findings We describe the actions of N,N′-(1,4-phenylenebis(methylene))dipyrimidin-2-amine (designated MSX-122), a novel small molecule and partial CXCR4 antagonist with properties quite unlike that of any other reported CXCR4 antagonists, which was prepared in a single chemical step using a reductive amination reaction. Its specificity toward CXCR4 was tested in a binding affinity assay and a ligand competition assay using 18F-labeled MSX-122. The potency of the compound was determined in two functional assays, Matrigel invasion assay and cAMP modulation. The therapeutic potential of MSX-122 was evaluated in three different murine models for inflammation including an experimental colitis, carrageenan induced paw edema, and bleomycin induced lung fibrosis and three different animal models for metastasis including breast cancer micrometastasis in lung, head and neck cancer metastasis in lung, and uveal melanoma micrometastasis in liver in which CXCR4 was reported to play crucial roles. Conclusions/Significance We developed a novel small molecule, MSX-122, that is a partial CXCR4 antagonist without mobilizing stem cells, which can be safer for

Development of a unique small molecule modulator of CXCR4.

Directory of Open Access Journals (Sweden)

Zhongxing Liang

Full Text Available Metastasis, the spread and growth of tumor cells to distant organ sites, represents the most devastating attribute and plays a major role in the morbidity and mortality of cancer. Inflammation is crucial for malignant tumor transformation and survival. Thus, blocking inflammation is expected to serve as an effective cancer treatment. Among anti-inflammation therapies, chemokine modulation is now beginning to emerge from the pipeline. CXC chemokine receptor-4 (CXCR4 and its ligand stromal cell-derived factor-1 (CXCL12 interaction and the resulting cell signaling cascade have emerged as highly relevant targets since they play pleiotropic roles in metastatic progression. The unique function of CXCR4 is to promote the homing of tumor cells to their microenvironment at the distant organ sites.We describe the actions of N,N'-(1,4-phenylenebis(methylenedipyrimidin-2-amine (designated MSX-122, a novel small molecule and partial CXCR4 antagonist with properties quite unlike that of any other reported CXCR4 antagonists, which was prepared in a single chemical step using a reductive amination reaction. Its specificity toward CXCR4 was tested in a binding affinity assay and a ligand competition assay using (18F-labeled MSX-122. The potency of the compound was determined in two functional assays, Matrigel invasion assay and cAMP modulation. The therapeutic potential of MSX-122 was evaluated in three different murine models for inflammation including an experimental colitis, carrageenan induced paw edema, and bleomycin induced lung fibrosis and three different animal models for metastasis including breast cancer micrometastasis in lung, head and neck cancer metastasis in lung, and uveal melanoma micrometastasis in liver in which CXCR4 was reported to play crucial roles.We developed a novel small molecule, MSX-122, that is a partial CXCR4 antagonist without mobilizing stem cells, which can be safer for long-term blockade of metastasis than other reported CXCR4

Prostate-specific antigen lowering effect of metabolic syndrome is influenced by prostate volume.

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Choi, Woo Suk; Heo, Nam Ju; Paick, Jae-Seung; Son, Hwancheol

2016-04-01

To investigate the influence of metabolic syndrome on prostate-specific antigen levels by considering prostate volume and plasma volume. We retrospectively analyzed 4111 men who underwent routine check-ups including prostate-specific antigen and transrectal ultrasonography. The definition of metabolic syndrome was based on the modified Adult Treatment Panel III criteria. Prostate-specific antigen mass density (prostate-specific antigen × plasma volume / prostate volume) was calculated for adjusting plasma volume and prostate volume. We compared prostate-specific antigen and prostate-specific antigen mass density levels of participants with metabolic syndrome (metabolic syndrome group, n = 1242) and without metabolic syndrome (non-prostate-specific antigen metabolic syndrome group, n = 2869). To evaluate the impact of metabolic syndrome on prostate-specific antigen, linear regression analysis for the natural logarithm of prostate-specific antigen was used. Patients in the metabolic syndrome group had significantly older age (P prostate volume (P prostate-specific antigen (non-metabolic syndrome group vs metabolic syndrome group; 1.22 ± 0.91 vs 1.15 ± 0.76 ng/mL, P = 0.006). Prostate-specific antigen mass density in the metabolic syndrome group was still significantly lower than that in the metabolic syndrome group (0.124 ± 0.084 vs 0.115 ± 0.071 μg/mL, P = 0.001). After adjusting for age, prostate volume and plasma volume using linear regression model, the presence of metabolic syndrome was a significant independent factor for lower prostate-specific antigen (prostate-specific antigen decrease by 4.1%, P = 0.046). Prostate-specific antigen levels in patients with metabolic syndrome seem to be lower, and this finding might be affected by the prostate volume. © 2016 The Japanese Urological Association.

Replacement of the V3 domain in the surface subunit of the feline immunodeficiency virus envelope glycoprotein with the equivalent region of a T cell-tropic human immunodeficiency virus type 1 results in a chimeric surface protein that efficiently binds to CXCR4.

Science.gov (United States)

González, Silvia A; Falcón, Juan I; Affranchino, José L

2014-03-01

Feline immunodeficiency virus (FIV) and the T cell-tropic strains of human immunodeficiency virus type 1 (HIV-1) share the use of the chemokine receptor CXCR4 for cell entry. To study this process further we developed a cell surface binding assay based on the expression of a soluble version of the FIV SU C-terminally tagged with the influenza virus hemagglutinin epitope (HA). The specificity of the assay was demonstrated by the following evidence: (1) the SU-HA protein bound to HeLa cells that express CXCR4 but not to MDCK cells that lack this chemokine receptor; and (2) binding of the SU-HA to HeLa cells was blocked by incubation with the CXCR4 antagonist AMD3100 as well as with the anti-CXCR4 monoclonal antibody (MAb) 12G5. Deletion of the V3 region from the FIV SU glycoprotein abolished its ability to bind CXCR4-expressing cells. Remarkably, substitution of the V3 domain of the FIV SU by the equivalent region of the HIV-1 NL4-3 isolate resulted in efficient cell surface binding of the chimeric SU protein to CXCR4. Moreover, transfection of MDCK cells with a plasmid encoding human CXCR4 allowed the association of the chimeric SU-HA glycoprotein to the transfected cells. Interestingly, while cell binding of the chimeric FIV-HIV SU was inhibited by an anti-HIV-1 V3 MAb, its association with CXCR4 was found to be resistant to AMD3100. Of note, the chimeric FIV-HIV Env glycoprotein was capable of promoting CXCR4-dependent cell-to-cell fusion.

CCR5 and CXCR3 are dispensable for liver infiltration, but CCR5 protects against virus-induced T-cell-mediated hepatic steatosis

DEFF Research Database (Denmark)

Holst, P J; Orskov, C; Qvortrup, K

2007-01-01

CCR5 and CXCR3 are important molecules in regulating the migration of activated lymphocytes. Thus, the majority of tissue-infiltrating T cells found in the context of autoimmune conditions and viral infections express CCR5 and CXCR3, and the principal chemokine ligands are expressed within inflam...... of CCR5 is associated with the induction of CD8(+) T-cell-mediated immunopathology consisting of marked hepatic microvesicular steatosis....

TCR-CXCR4 signaling stabilizes cytokine mRNA transcripts via a PREX1-Rac1 pathway: implications for CTCL.

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Kremer, Kimberly N; Dinkel, Brittney A; Sterner, Rosalie M; Osborne, Douglas G; Jevremovic, Dragan; Hedin, Karen E

2017-08-24

As with many immunopathologically driven diseases, the malignant T cells of cutaneous T-cell lymphomas (CTCLs), such as Sézary syndrome, display aberrant cytokine secretion patterns that contribute to pathology and disease progression. Targeting this disordered release of cytokines is complicated by the changing cytokine milieu that drives the phenotypic changes of CTCLs. Here, we characterize a novel signaling pathway that can be targeted to inhibit the secretion of cytokines by modulating either CXCR4 or CXCR4-mediated signaling. We demonstrate that upon ligation of the T-cell antigen receptor (TCR), the TCR associates with and transactivates CXCR4 via phosphorylation of S339-CXCR4 in order to activate a PREX1-Rac1-signaling pathway that stabilizes interleukin-2 (IL-2) , IL-4 , and IL-10 messenger RNA (mRNA) transcripts. Pharmacologic inhibition of either TCR-CXCR4 complex formation or PREX1-Rac1 signaling in primary human T cells decreased mRNA stability and inhibited secretion of IL-2, IL-4, and IL-10. Applying this knowledge to Sézary syndrome, we demonstrate that targeting various aspects of this signaling pathway blocks both TCR-dependent and TCR-independent cytokine secretion from a Sézary syndrome-derived cell line and patient isolates. Together, these results identify multiple aspects of a novel TCR-CXCR4-signaling pathway that could be targeted to inhibit the aberrant cytokine secretion that drives the immunopathogenesis of Sézary syndrome and other immunopathological diseases. © 2017 by The American Society of Hematology.

CXCR5-Dependent Entry of CD8 T Cells into Rhesus Macaque B-Cell Follicles Achieved through T-Cell Engineering.

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Ayala, Victor I; Deleage, Claire; Trivett, Matthew T; Jain, Sumiti; Coren, Lori V; Breed, Matthew W; Kramer, Joshua A; Thomas, James A; Estes, Jacob D; Lifson, Jeffrey D; Ott, David E

2017-06-01

Follicular helper CD4 T cells, T FH , residing in B-cell follicles within secondary lymphoid tissues, are readily infected by AIDS viruses and are a major source of persistent virus despite relative control of viral replication. This persistence is due at least in part to a relative exclusion of effective antiviral CD8 T cells from B-cell follicles. To determine whether CD8 T cells could be engineered to enter B-cell follicles, we genetically modified unselected CD8 T cells to express CXC chemokine receptor 5 (CXCR5), the chemokine receptor implicated in cellular entry into B-cell follicles. Engineered CD8 T cells expressing human CXCR5 (CD8 hCXCR5 ) exhibited ligand-specific signaling and chemotaxis in vitro Six infected rhesus macaques were infused with differentially fluorescent dye-labeled autologous CD8 hCXCR5 and untransduced CD8 T cells and necropsied 48 h later. Flow cytometry of both spleen and lymph node samples revealed higher frequencies of CD8 hCXCR5 than untransduced cells, consistent with preferential trafficking to B-cell follicle-containing tissues. Confocal fluorescence microscopy of thin-sectioned lymphoid tissues demonstrated strong preferential localization of CD8 hCXCR5 T cells within B-cell follicles with only rare cells in extrafollicular locations. CD8 hCXCR5 T cells were present throughout the follicles with some observed near infected T FH In contrast, untransduced CD8 T cells were found in the extrafollicular T-cell zone. Our ability to direct localization of unselected CD8 T cells into B-cell follicles using CXCR5 expression provides a strategy to place highly effective virus-specific CD8 T cells into these AIDS virus sanctuaries and potentially suppress residual viral replication. IMPORTANCE AIDS virus persistence in individuals under effective drug therapy or those who spontaneously control viremia remains an obstacle to definitive treatment. Infected follicular helper CD4 T cells, T FH , present inside B-cell follicles represent a

CXCR4 expression in feline mammary carcinoma cells: evidence of a proliferative role for the SDF-1/CXCR4 axis

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Ferrari Angelo

2012-03-01

Full Text Available Abstract Background Mammary tumours frequently develop in female domestic cats being highly malignant in a large percentage of cases. Chemokines regulate many physiological and pathological processes including organogenesis, chemotaxis of inflammatory cells, as well as tumour progression and metastasization. In particular, the chemokine/receptor pair SDF-1/CXCR4 has been involved in the regulation of metastatic potential of neoplastic cells, including breast cancer. The aim of this study was the immunohistochemical defininition of the expression profile of CXCR4 in primary and metastatic feline mammary carcinomas and the evaluation of the role of SDF-1 in feline mammary tumour cell proliferation. Results A total of 45 mammary surgical samples, including 33 primary tumours (31 carcinomas and 2 adenomas, 6 metastases, and 4 normal mammary tissues were anlyzed. Tumor samples were collected from a total number of 26 animals, as in some cases concurrent occurrence of neoplasm in more than one mammary gland was observed. Tissues were processed for standard histological examination, and all lesions were classified according to the World Health Organization criteria. CXCR4 expression in neoplastic cells was evaluated by immunohistochemistry. The level of CXCR4 immunoreactivity was semi-quantitatively estimated as CXCR4 score evaluating both the number of positive cells and the intensity of staining. Six primary, fibroblast-free primary cultures were obtained from fresh feline mammary carcinomas and characterized by immunofluorescence for CXCR4 and malignant mammary cell marker expression. SDF-1-dependent in vitro proliferative effects were also assayed. CXCR4 expression was observed in 29 out of 31 malignant tissues with a higher CXCR4 score observed in 4 out of 6 metastatic lesions than in the respective primary tumours. In 2 benign lesions analyzed, only the single basaloid adenoma showed a mild positive immunostaining against CXCR4. Normal tissue did

Immunoregulatory adherent cells in human tuberculosis: radiation-sensitive antigen-specific suppression by monocytes

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Kleinhenz, M.E.; Ellner, J.J.

1985-07-01

In human tuberculosis, adherent mononuclear cells (AMC) selectively depress in vitro responses to the mycobacterial antigen tuberculin purified protein derivative (PPD). The phenotype of this antigen-specific adherent suppressor cell was characterized by examining the functional activity of adherent cells after selective depletion of sheep erythrocyte-rosetting T cells or OKM1-reactive monocytes. Adherent cell suppression was studied in the (/sup 3/H)thymidine-incorporation microculture assay by using T cells rigorously depleted of T cells with surface receptors for the Fc portion of IgG (T gamma cells) as antigen-responsive cells. PPD-induced (/sup 3/H)thymidine incorporation by these non gamma T cells was uniformly reduced (mean, 42% +/- 10% (SD)) when autologous AMC were added to non gamma T cells at a ratio of 1:2. Antigen-specific suppression by AMC was not altered by depletion of sheep erythrocyte-rosetting T cells or treatment with indomethacin. However, AMC treated with OKM1 and complement or gamma irradiation (1,500 rads) no longer suppressed tuberculin responses in vitro. These studies identify the antigen-specific adherent suppressor cell in tuberculosis as an OKM1-reactive, non-erythrocyte-rosetting monocyte. The radiosensitivity of this monocyte immunoregulatory function may facilitate its further definition.

Immunoregulatory adherent cells in human tuberculosis: radiation-sensitive antigen-specific suppression by monocytes

International Nuclear Information System (INIS)

Kleinhenz, M.E.; Ellner, J.J.

1985-01-01

In human tuberculosis, adherent mononuclear cells (AMC) selectively depress in vitro responses to the mycobacterial antigen tuberculin purified protein derivative (PPD). The phenotype of this antigen-specific adherent suppressor cell was characterized by examining the functional activity of adherent cells after selective depletion of sheep erythrocyte-rosetting T cells or OKM1-reactive monocytes. Adherent cell suppression was studied in the [ 3 H]thymidine-incorporation microculture assay by using T cells rigorously depleted of T cells with surface receptors for the Fc portion of IgG (T gamma cells) as antigen-responsive cells. PPD-induced [ 3 H]thymidine incorporation by these non gamma T cells was uniformly reduced (mean, 42% +/- 10% [SD]) when autologous AMC were added to non gamma T cells at a ratio of 1:2. Antigen-specific suppression by AMC was not altered by depletion of sheep erythrocyte-rosetting T cells or treatment with indomethacin. However, AMC treated with OKM1 and complement or gamma irradiation (1,500 rads) no longer suppressed tuberculin responses in vitro. These studies identify the antigen-specific adherent suppressor cell in tuberculosis as an OKM1-reactive, non-erythrocyte-rosetting monocyte. The radiosensitivity of this monocyte immunoregulatory function may facilitate its further definition

Prostate-specific membrane antigen and its truncated form PSM'

Czech Academy of Sciences Publication Activity Database

Mlčochová, Petra; Bařinka, Cyril; Tykvart, Jan; Šácha, Pavel; Konvalinka, Jan

2009-01-01

Roč. 69, č. 5 (2009), s. 471-479 ISSN 0270-4137 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : prostate specific membrane antigen * glutamate carboxypeptidase II * prostate cancer Subject RIV: CE - Biochemistry Impact factor: 3.081, year: 2009

Plasma membrane associated, virus-specific polypeptides required for the formation of target antigen complexes recognized by virus-specific cytotoxic T lymphocytes

International Nuclear Information System (INIS)

Domber, E.A.

1986-01-01

These studies were undertaken to define some of the poxvirus-specific target antigens which are synthesized in infected cells and recognized by vaccinia virus-specific CTLs (VV-CTLs). Since vaccinia virus infected, unmanipulated target cells express numerous virus-specific antigens on the plasma membrane, attempts were made to manipulate expression of the poxvirus genome after infection so that one or a few defined virus-specified antigens were expressed on the surface of infected cells. In vitro [ 51 Cr]-release assays determined that viral DNA synthesis and expression of late viral proteins were not necessary to form a target cell which was fully competent for lysis by VV-CTLs. Under the conditions employed in these experiments, 90-120 minutes of viral protein synthesis were necessary to produce a competent cell for lysis by VV-CTLs. In order to further inhibit the expression of early viral proteins in infected cells, partially UV-inactivated vaccinia virus was employed to infect target cells. It was determined that L-cells infected with virus preparations which had been UV-irradiated for 90 seconds were fully competent for lysis by VV-CTLs. Cells infected with 90 second UV-irr virus expressed 3 predominant, plasma membrane associated antigens of 36-37K, 27-28K, and 19-17K. These 3 viral antigens represent the predominant membrane-associated viral antigens available for interaction with class I, major histocompatibility antigens and hence are potential target antigens for VV-CTLs

CXCR4 Is Required by a Nonprimate Lentivirus: Heterologous Expression of Feline Immunodeficiency Virus in Human, Rodent, and Feline Cells

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Poeschla, Eric M.; Looney, David J.

1998-01-01

A heterologous feline immunodeficiency virus (FIV) expression system permitted high-level expression of FIV proteins and efficient production of infectious FIV in human cells. These results identify the FIV U3 element as the sole restriction to the productive phase of replication in nonfeline cells. Heterologous FIV expression in a variety of human cell lines resulted in profuse syncytial lysis that was FIV env specific, CD4 independent, and restricted to cells that express CXCR4, the coreceptor for T-cell-line-adapted strains of human immunodeficiency virus. Stable expression of human CXCR4 in CXCR4-negative human and rodent cell lines resulted in extensive FIV Env-mediated, CXCR4-dependent cell fusion and infection. In feline cells, stable overexpression of human CXCR4 resulted in increased FIV infectivity and marked syncytium formation during FIV replication or after infection with FIV Env-expressing vectors. The use of CXCR4 is a fundamental feature of lentivirus biology independent of CD4 and a shared cellular link to infection and cytopathicity for distantly related lentiviruses that cause AIDS. Their conserved use implicates chemokine receptors as primordial lentivirus receptors. PMID:9658135

Characterization of a human antigen specific helper factor

International Nuclear Information System (INIS)

Richardson, B.

1986-01-01

While antigen (Ag) specific helper factors have been characterized in mice, similar molecules have not been identified in humans. To characterize human antigen specific helper molecules, an IL-2 dependent tetanus toxoid (T.T.) reactive T cell line was fused with a 6-thioguanine resistant CEM line, and hybrids selected in medium containing hypoxanthine and azaserine. Hybrids were screened by culturing the cells with 35 S-Met then reacting the supernatants with T.T. or hepatitis vaccine immobilized on nitrocellulose. One hybrid, TT6BA-O, was identified which secreted a Met-containing molecule which bound T.T. but not hepatitis vaccine. Supernatants from TT6BA-O, but not the parent CEM line, when added to autologous peripheral blood mononuclear cells (PBMC's) stimulated secretion of T.T. specific antibodies (Abs). Specificity controls demonstrated that TT6BA-O supernatant did not induce antibodies to diphtheria toxoid, hepatitis vaccine or pneumococcal polysaccharide, and total immunoglobulin (lg) synthesis was minimally increased. In contrast, pokeweed mitogen stimulated significant lg synthesis as well as Ab's to pneumococcal polysaccharide and T.T. TT6BA-O supernatant induced anti-T.T.Ab's in autologous PBMC's but not PBMC's from 3 unrelated donors, suggesting that the activity of the helper factor is restricted, possibly by the MHC. The molecular weight of the helper factor was estimated at 100,000-150,000 by Sephacryl S-300 chromatography. Finally, the helper factor could be demonstrated to bind and elute from sephorose-immobilized T.T. and anti-DR antisera, but not anti-lg antisera or the T40/25 monoclonal antibody, which binds a nonpolymorphic determinant on the human T cell receptor. These results demonstrate that human Ag specific helper factors exist, bind antigen and bear class II MHC determinants

A radioimmunoassay for human antibody specific for microbial antigens

International Nuclear Information System (INIS)

Tew, J.G.; Burmeister, J.; Greene, E.J.; Pflaumer, S.K.; Goldstein, J.

1977-01-01

A simple and sensitive method for detecting and quantitating antibody specific or microbial antigens is described. Bacterial, fungal, parasitic or viral antigens attached to bromoacetyl cellulose or the intact cells themselves were added to a series of two-fold dilutions of human serum. After a short incubation period, which allowed human antibody to attach to the antigens, the complex was thoroughly washed and carbon-14 labeled anti-human light chain antibody was added to each dilution. The resulting complex was washed, collected on a filter pad, placed in a scintillation vial and radioassayed. The relationship between radioactivity bound and -log 2 of the serum dilution was linear. The endpoint for each assay and a confidence interval was calculated by doing inverse prediction from simple linear regression. Results obtained using this assay indicated the presence of antibody in a pool of normal human sera specific for herpes virus and for both cell surface and intracellular antigens of Streptococcus mutans, Naegleria fowleri and Cryptococcus neoformans. In general the dominant response was against the intracellular antigens rather than cell surface antigens

Targeting CXCR4 reverts the suppressive activity of T-regulatory cells in renal cancer.

Science.gov (United States)

Santagata, Sara; Napolitano, Maria; D'Alterio, Crescenzo; Desicato, Sonia; Maro, Salvatore Di; Marinelli, Luciana; Fragale, Alessandra; Buoncervello, Maria; Persico, Francesco; Gabriele, Lucia; Novellino, Ettore; Longo, Nicola; Pignata, Sandro; Perdonà, Sisto; Scala, Stefania

2017-09-29

With the intent to identify biomarkers in renal cell carcinoma (RCC) the functional status of T-regulatory cells (Tregs) was investigated in primary RCC. Tregs were isolated from tumoral-(TT), peritumoral tissue-(PT) and peripheral blood-(PB) of 42 primary RCC patients and function evaluated through effector T cells (Teff) proliferation, cytokines release and demethylation of Treg Specific Region (TSDR). The highest value of Tregs was detected in TT with the uppermost amount of effector-Tregs-(CD4 + CD25 hi FOXP3 hi CD45RA - ). PB-RCC Tregs efficiently suppress Teff proliferation compared to healthy donor (HD)-Tregs and, at the intrapatient evaluation, TT-derived Tregs were the most suppressive. Higher demethylation TSDR was detected in TT- and PB-RCC Tregs vs HD-Tregs ( P <0,001). CXCR4 is highly expressed on Tregs, thus we wished to modulate Tregs function through CXCR4 inhibition. CXCR4 antagonism, elicited by a new peptidic antagonist, Peptide-R29, efficiently reversed Tregs suppression of Teff proliferation. Thus Tregs functional evaluation precisely reflects Tregs status and may be a reliable biomarker of tumoral immune response. In addition, treatment with CXCR4 antagonist, impairing Tregs function, could improve the anticancer immune response, in combination with conventional therapy and/or immunotherapy such as checkpoints inhibitors.

Targeting CXCR4 in HIV Cell-Entry Inhibition

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Steen, Anne; Schwartz, T W; Rosenkilde, M M

2010-01-01

CXCR4 and CCR5 constitute the two major coreceptors for HIV-1 entry into host cells. In the course of an HIV-infection, a coreceptor switch takes place in approximately half of the patients - from R5 HIV-1 (CCR5 utilizing) strains to X4 HIV-1 (CXCR4 utilizing) strains. Treatment of HIV......-infected individuals with CXCR4 antagonists delays the onset of AIDS by preventing the CCR5 to CXCR4 coreceptor switch. In addition to the endogenous CXCR4 and CCR5 ligands, other chemokines, for example the human herpesvirus 8 encoded CC-chemokine, vCCL2, and modifications hereof, have proven efficient HIV-1 cell...... no oral bioavailability. The hunt for orally active small-molecule CXCR4 antagonists led to the development of monocyclam-based compounds, and recently to the non-cyclam antagonist AMD070, which is orally active and currently in Phase II clinical trial as anti-HIV treatment. Current review provides...

Mutations at the CXCR4 interaction sites for AMD3100 influence anti-CXCR4 antibody binding and HIV-1 entry

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Hatse, Sigrid; Princen, Katrien; Vermeire, Kurt

2003-01-01

The interaction of the CXCR4 antagonist AMD3100 with its target is greatly influenced by specific aspartate residues in the receptor protein, including Asp(171) and Asp(262). We have now found that aspartate-to-asparagine substitutions at these positions differentially affect the binding of four...

Vesnarinone downregulates CXCR4 expression via upregulation of Krüppel-like factor 2 in oral cancer cells

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Uchida Daisuke

2009-08-01

Full Text Available Abstract Background We have demonstrated that the stromal cell-derived factor-1 (SDF-1; CXCL12/CXCR4 system is involved in the establishment of lymph node metastasis in oral squamous cell carcinoma (SCC. Chemotherapy is a powerful tool for the treatment of oral cancer, including oral SCC; however, the effects of chemotherapeutic agents on the expression of CXCR4 are unknown. In this study, we examined the expression of CXCR4 associated with the chemotherapeutic agents in oral cancer cells. Results The expression of CXCR4 was examined using 3 different chemotherapeutic agents; 5-fluorouracil, cisplatin, and vesnarinone (3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl-1-piperazinyl]-2-(1H-quinolinone in B88, a line of oral cancer cells that exhibits high levels of CXCR4 and lymph node metastatic potential. Of the 3 chemotherapeutic agents that we examined, only vesnarinone downregulated the expression of CXCR4 at the mRNA as well as the protein level. Vesnarinone significantly inhibited lymph node metastasis in tumor-bearing nude mice. Moreover, vesnarinone markedly inhibited 2.7-kb human CXCR4 promoter activity, and we identified the transcription factor, Krüppel-like factor 2 (KLF2, as a novel vesnarinone-responsive molecule, which was bound to the CXCR4 promoter at positions -300 to -167 relative to the transcription start site. The forced-expression of KLF2 led to the downregulation of CXCR4 mRNA and impaired CXCR4 promoter activity. The use of siRNA against KLF2 led to an upregulation of CXCR4 mRNA. Conclusion These Results indicate that vesnarinone downregulates CXCR4 via the upregulation of KLF2 in oral cancer.

Emerging Targets in Pituitary Adenomas: Role of the CXCL12/CXCR4-R7 System

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Federica Barbieri

2014-01-01

Full Text Available Chemokines are chemotactic regulators of immune surveillance in physiological and pathological conditions such as inflammation, infection, and cancer. Several chemokines and cognate receptors are constitutively expressed in the central nervous system, not only in glial and endothelial cells but also in neurons, controlling neurogenesis, neurite outgrowth, and axonal guidance during development. In particular, the chemokine CXCL12 and its receptors, CXCR4 and CXCR7, form a functional network that controls plasticity in different brain areas, influencing neurotransmission, neuromodulation, and cell migration, and the dysregulation of this chemokinergic axis is involved in several neurodegenerative, neuroinflammatory, and malignant diseases. CXCR4 primarily mediates the transduction of proliferative signals, while CXCR7 seems to be mainly responsible for scavenging CXCL12. Importantly, the multiple intracellular signalling generated by CXCL12 interaction with its receptors influences hypothalamic modulation of neuroendocrine functions, although a direct modulation of pituitary functioning via autocrine/paracrine mechanisms was also reported. Both CXCL12 and CXCR4 are constitutively overexpressed in pituitary adenomas and their signalling induces cell survival and proliferation, as well as hormonal hypersecretion. In this review we focus on the physiological and pathological functions of immune-related cyto- and chemokines, mainly focusing on the CXCL12/CXCR4-7 axis, and their role in pituitary tumorigenesis. Accordingly, we discuss the potential targeting of CXCR4 as novel pharmacological approach for pituitary adenomas.

Gambogic acid inhibits multiple myeloma mediated osteoclastogenesis through suppression of chemokine receptor CXCR4 signaling pathways.

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Pandey, Manoj K; Kale, Vijay P; Song, Chunhua; Sung, Shen-shu; Sharma, Arun K; Talamo, Giampaolo; Dovat, Sinisa; Amin, Shantu G

2014-10-01

Bone disease, characterized by the presence of lytic lesions and osteoporosis is the hallmark of multiple myeloma (MM). Stromal cell-derived factor 1α (SDF-1α) and its receptor, CXC chemokine receptor 4 (CXCR4), has been implicated as a regulator of bone resorption, suggesting that agents that can suppress SDF1α/CXCR4 signaling might inhibit osteoclastogenesis, a process closely linked to bone resorption. We, therefore, investigated whether gambogic acid (GA), a xanthone, could inhibit CXCR4 signaling and suppress osteoclastogenesis induced by MM cells. Through docking studies we predicted that GA directly interacts with CXCR4. This xanthone down-regulates the expression of CXCR4 on MM cells in a dose- and time-dependent manner. The down-regulation of CXCR4 was not due to proteolytic degradation, but rather GA suppresses CXCR4 mRNA expression by inhibiting nuclear factor-kappa B (NF-κB) DNA binding. This was further confirmed by quantitative chromatin immunoprecipitation assay, as GA inhibits p65 binding at the CXCR4 promoter. GA suppressed SDF-1α-induced chemotaxis of MM cells and downstream signaling of CXCR4 by inhibiting phosphorylation of Akt, p38, and Erk1/2 in MM cells. GA abrogated the RANKL-induced differentiation of macrophages to osteoclasts in a dose- and time-dependent manner. In addition, we found that MM cells induced differentiation of macrophages to osteoclasts, and that GA suppressed this process. Importantly, suppression of osteoclastogenesis by GA was mediated through IL-6 inhibition. Overall, our results show that GA is a novel inhibitor of CXCR4 expression and has a strong potential to suppress osteoclastogenesis mediated by MM cells. Published by Elsevier Inc.

Expression of chemokine CXCL12 and its receptor CXCR4 in folliculostellate (FS) cells of the rat anterior pituitary gland: the CXCL12/CXCR4 axis induces interconnection of FS cells.

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Horiguchi, Kotaro; Ilmiawati, Cimi; Fujiwara, Ken; Tsukada, Takehiro; Kikuchi, Motoshi; Yashiro, Takashi

2012-04-01

The anterior pituitary gland is composed of five types of hormone-producing cells plus folliculostellate (FS) cells, which do not produce classical anterior pituitary hormones. FS cells are interconnected by cytoplasmic processes and encircle hormone-producing cells or aggregate homophilically. Using living-cell imaging of primary culture, we recently reported that some FS cells precisely extend their cytoplasmic processes toward other FS cells and form interconnections with them. These phenomena suggest the presence of a chemoattractant factor that facilitates the interconnection. In this study, we attempted to discover the factor that induces interconnection of FS cells and succeeded in identifying chemokine (CXC)-L12 and its receptor CXCR4 as potential candidate molecules. CXCL12 is a chemokine of the CXC subfamily. It exerts its effects via CXCR4, a G protein-coupled receptor. The CXCL12/CXCR4 axis is a potent chemoattractant for many types of neural cells. First, we revealed that CXCL12 and CXCR4 are expressed by FS cells in rat anterior pituitary gland. Next, to clarify the function of the CXCL12/CXCR4 axis in FS cells, we observed living anterior pituitary cells in primary culture with specific CXCL12 inhibitor or CXCR4 antagonist and noted that extension of cytoplasmic processes and interconnection of FS cells were inhibited. Finally, we examined FS cell migration and invasion by using Matrigel matrix assays. CXCL12 treatment resulted in markedly increased FS cell migration and invasion. These data suggest that FS cells express chemokine CXCL12 and its receptor CXCR4 and that the CXCL12/CXCR4 axis evokes interconnection of FS cells.

SENSITIVITY AND SPECIFICITY OF URINARY BLADDER CANCER ANTIGEN FOR DIAGNOSIS OF BLADDER TUMOR;A COMPARATIVE STUDY WITH URINARY CYTOLOGY

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K. Radkhah

2005-06-01

Full Text Available Cystoscopy and urinary cytology are currently the basis for diagnosis and ‎follow-up of bladder tumors. Research to find a sensitive and specific tumor ‎marker for diagnosis of bladder tumor is actively underway, however, due to low sensitivity ‎and high cost of cytology. This cross-sectional study was performed in 65 patients to evaluate whether urinary bladder ‎cancer (UBC antigen level can predict the presence of active bladder tumor. In patients with ‎inactive tumor, UBC antigen level was determined in addition to standard cystoscopy ‎and cytology for follow-up. Patients with active tumor were ‎subjected to standard treatment and UBC antigen level determination. UBC antigen ‎ levels were measured by ELISA, using monoclonal antibodies ‎specific for UBC antigen. As a control group, UBC antigen level ‎was also determined in 65 persons who had been referred for urinalysis for other reasons. ‎UBC antigen level more than 1 μg/L which was regarded as ‎positive was found in 49.4% of the patients. In control group, 96.9% had UBC antigen < 1μg/L‎. Mean UBC antigen level in patients was ‎3.77 μg/L while it was 0.508 μg/L in controls (P < 0.0001. Sensitivity of ‎UBC antigen was 53.3% and its specificity was 40%. Sensitivity and specificity of urinary cytology was 17.3% and 88.2%, respectively. This difference was statistically ‎significant (P < 0.001. UBC antigen is more sensitive than urinary cytology, although cytology still ‎retains its priority in specificity. It is not yet recommended to replace UBC antigen for ‎cytology due to its low specificity and not favorable sensitivity.

Deficiency in adipocyte chemokine receptor CXCR4 exacerbates obesity and compromises thermoregulatory responses of brown adipose tissue in a mouse model of diet-induced obesity

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Yao, Longbiao; Heuser-Baker, Janet; Herlea-Pana, Oana; Zhang, Nan; Szweda, Luke I.; Griffin, Timothy M.; Barlic-Dicen, Jana

2014-01-01

The chemokine receptor CXCR4 is expressed on adipocytes and macrophages in adipose tissue, but its role in this tissue remains unknown. We evaluated whether deficiency in either adipocyte or myeloid leukocyte CXCR4 affects body weight (BW) and adiposity in a mouse model of high-fat-diet (HFD)-induced obesity. We found that ablation of adipocyte, but not myeloid leukocyte, CXCR4 exacerbated obesity. The HFD-fed adipocyte-specific CXCR4-knockout (AdCXCR4ko) mice, compared to wild-type C57BL/6 control mice, had increased BW (average: 52.0 g vs. 35.5 g), adiposity (average: 49.3 vs. 21.0% of total BW), and inflammatory leukocyte content in white adipose tissue (WAT), despite comparable food intake. As previously reported, HFD feeding increased uncoupling protein 1 (UCP1) expression (fold increase: 3.5) in brown adipose tissue (BAT) of the C57BL/6 control mice. However, no HFD-induced increase in UCP1 expression was observed in the AdCXCR4ko mice, which were cold sensitive. Thus, our study suggests that adipocyte CXCR4 limits development of obesity by preventing excessive inflammatory cell recruitment into WAT and by supporting thermogenic activity of BAT. Since CXCR4 is conserved between mouse and human, the newfound role of CXCR4 in mouse adipose tissue may parallel the role of this chemokine receptor in human adipose tissue.—Yao, L., Heuser-Baker, J., Herlea-Pana, O., Zhang, N., Szweda, L. I., Griffin, T. M., Barlic-Dicen, J. Deficiency in adipocyte chemokine receptor CXCR4 exacerbates obesity and compromises thermoregulatory responses of brown adipose tissue in a mouse model of diet-induced obesity. PMID:25016030

Rapid desensitization induces internalization of antigen-specific IgE on mouse mast cells.

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Oka, Tatsuya; Rios, Eon J; Tsai, Mindy; Kalesnikoff, Janet; Galli, Stephen J

2013-10-01

Rapid desensitization transiently prevents severe allergic reactions, allowing administration of life-saving therapies in previously sensitized patients. However, the mechanisms underlying successful rapid desensitization are not fully understood. We sought to investigate whether the mast cell (MC) is an important target of rapid desensitization in mice sensitized to exhibit IgE-dependent passive systemic anaphylaxis in vivo and to investigate the antigen specificity and underlying mechanisms of rapid desensitization in our mouse model. C57BL/6 mice (in vivo) or primary isolated C57BL/6 mouse peritoneal mast cells (PMCs; in vitro) were passively sensitized with antigen-specific anti-2,4-dinitrophenyl IgE, anti-ovalbumin IgE, or both. MCs were exposed over a short period of time to increasing amounts of antigen (2,4-dinitrophenyl-human serum albumin or ovalbumin) in the presence of extracellular calcium in vitro or by means of intravenous administration to sensitized mice in vivo before challenging the mice with or exposing the PMCs to optimal amounts of specific or irrelevant antigen. Rapidly exposing mice or PMCs to progressively increasing amounts of specific antigen inhibited the development of antigen-induced hypothermia in sensitized mice in vivo and inhibited antigen-induced PMC degranulation and prostaglandin D2 synthesis in vitro. Such MC hyporesponsiveness was induced antigen-specifically and was associated with a significant reduction in antigen-specific IgE levels on MC surfaces. Rapidly exposing MCs to progressively increasing amounts of antigen can both enhance the internalization of antigen-specific IgE on the MC surface and also desensitize these cells in an antigen-specific manner in vivo and in vitro. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Stromal cell derived factor-1α (SDF-1α) directed chemoattraction of transiently CXCR4 overexpressing mesenchymal stem cells into functionalized three-dimensional biomimetic scaffolds

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Thieme, S; Ryser, Martin; Gentsch, Marcus

2009-01-01

Three-dimensional (3D) bone substitute material should not only serve as scaffold in large bone defects but also attract mesenchymal stem cells, a subset of bone marrow stromal cells (BMSCs) that are able to form new bone tissue. An additional crucial step is to attract BMSCs from the surface int...... invaded up to 250 mum into SDF-1alpha-releasing 3D scaffolds, whereas CXCR4-overexpressing BMSC invaded up to 500 mum within 5 days. Thus, the SDF-1alpha/CXCR4 chemoattraction system can be used to efficiently recruit BMSCs into SDF-1alpha-releasing 3D scaffolds in vitro and in vivo....

Use of recombinant purified protein derivative (PPD) antigens as specific skin test for tuberculosis.

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Stavri, Henriette; Bucurenci, Nadia; Ulea, Irina; Costache, Adriana; Popa, Loredana; Popa, Mircea Ioan

2012-11-01

Purified protein derivative (PPD) is currently the only available skin test reagent used worldwide for the diagnosis of tuberculosis (TB). The aim of this study was to develop a Mycobacterium tuberculosis specific skin test reagent, without false positive results due to Bacillus Calmette-Guerin (BCG) vaccination using recombinant antigens. Proteins in PPD IC-65 were analyzed by tandem mass spectrometry and compared to proteins in M. tuberculosis culture filtrate; 54 proteins were found in common. Top candidates MPT64, ESAT 6, and CFP 10 were overexpressed in Escherichia coli expression strains and purified as recombinant proteins. To formulate optimal immunodiagnostic PPD cocktails, the antigens were evaluated by skin testing guinea pigs sensitized with M. tuberculosis H37Rv and BCG. For single antigens and a cocktail mixture of these antigens, best results were obtained using 3 μg/0.1 ml, equivalent to 105 TU (tuberculin units). Each animal was simultaneously tested with PPD IC-65, 2 TU/0.1 ml, as reference. Reactivity of the multi-antigen cocktail was greater than that of any single antigen. The skin test results were between 34.3 and 76.6 per cent the level of reactivity compared to that of the reference when single antigens were tested and 124 per cent the level of reactivity compared to the reference for the multi-antigen cocktail. Our results showed that this specific cocktail could represent a potential candidate for a new skin diagnostic test for TB.

Dual-Functional Nanoparticles Targeting CXCR4 and Delivering Antiangiogenic siRNA Ameliorate Liver Fibrosis.

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Liu, Chun-Hung; Chan, Kun-Ming; Chiang, Tsaiyu; Liu, Jia-Yu; Chern, Guann-Gen; Hsu, Fu-Fei; Wu, Yu-Hsuan; Liu, Ya-Chi; Chen, Yunching

2016-07-05

The progression of liver fibrosis, an intrinsic response to chronic liver injury, is associated with hepatic hypoxia, angiogenesis, abnormal inflammation, and significant matrix deposition, leading to the development of cirrhosis and hepatocellular carcinoma (HCC). Due to the complex pathogenesis of liver fibrosis, antifibrotic drug development has faced the challenge of efficiently and specifically targeting multiple pathogenic mechanisms. Therefore, CXCR4-targeted nanoparticles (NPs) were formulated to deliver siRNAs against vascular endothelial growth factor (VEGF) into fibrotic livers to block angiogenesis during the progression of liver fibrosis. AMD3100, a CXCR4 antagonist that was incorporated into the NPs, served dual functions: it acted as a targeting moiety and suppressed the progression of fibrosis by inhibiting the proliferation and activation of hepatic stellate cells (HSCs). We demonstrated that CXCR4-targeted NPs could deliver VEGF siRNAs to fibrotic livers, decrease VEGF expression, suppress angiogenesis and normalize the distorted vessels in the fibrotic livers in the carbon tetrachloride (CCl4) induced mouse model. Moreover, blocking SDF-1α/CXCR4 by CXCR4-targeted NPs in combination with VEGF siRNA significantly prevented the progression of liver fibrosis in CCl4-treated mice. In conclusion, the multifunctional CXCR4-targeted NPs delivering VEGF siRNAs provide an effective antifibrotic therapeutic strategy.

Salvage radiotherapy for prostate-specific antigen relapse after radical prostatectomy for prostate cancer. A single-center experience

International Nuclear Information System (INIS)

Yoshida, Takahiro; Nakayama, Masashi; Suzuki, Osamu

2011-01-01

The aim of this study was to investigate the efficacy and prognostic factors of salvage radiotherapy for prostate-specific antigen relapse after radical prostatectomy for prostate cancer at a single center in Japan. A retrospective review of the medical records of 51 patients who underwent salvage radiotherapy for prostate-specific antigen relapse after radical prostatectomy was carried out. Salvage radiotherapy was undergone for the single indication of at least two consecutive prostate-specific antigen elevations >0.1 ng/ml. Salvage radiotherapy was delivered to the prostatic bed at a total dose of 60 or 64 Gy. Late toxicity was scored according to the Common Terminology Criteria for Adverse Events 3.0. A total dose of 60 and 64 Gy were administered to 26 and 25 patients, respectively. The median prostate-specific antigen level at the initiation of radiotherapy was 0.29 ng/ml (range, 0.11-1.10 ng/ml). With a median follow-up of 57.3 months (range, 9.9-134.0 months), the prostate-specific antigen relapse-free rate at 5 years was 50.7%. Multivariate analysis using Cox's proportional hazards regression model revealed that the Gleason score at radical prostatectomy ≥8 significantly predicted prostate-specific antigen relapse after salvage radiotherapy (hazard ratio 4.531; 95% confidence interval 1.413-14.535; P=0.011). The prostate-specific antigen relapse-free rate at 5 years in the Gleason score at radical prostatectomy ≤7 and at radical prostatectomy ≥8 was 62.7 and 15.4%, respectively. Salvage radiotherapy was effective for prostate-specific antigen relapse after radical prostatectomy with tolerable toxicities in Japanese patients. A high Gleason score seemed to be a poor prognostic factor. (author)

Engineered artificial antigen presenting cells facilitate direct and efficient expansion of tumor infiltrating lymphocytes

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Coukos George

2011-08-01

Full Text Available Abstract Background Development of a standardized platform for the rapid expansion of tumor-infiltrating lymphocytes (TILs with anti-tumor function from patients with limited TIL numbers or tumor tissues challenges their clinical application. Methods To facilitate adoptive immunotherapy, we applied genetically-engineered K562 cell-based artificial antigen presenting cells (aAPCs for the direct and rapid expansion of TILs isolated from primary cancer specimens. Results TILs outgrown in IL-2 undergo rapid, CD28-independent expansion in response to aAPC stimulation that requires provision of exogenous IL-2 cytokine support. aAPCs induce numerical expansion of TILs that is statistically similar to an established rapid expansion method at a 100-fold lower feeder cell to TIL ratio, and greater than those achievable using anti-CD3/CD28 activation beads or extended IL-2 culture. aAPC-expanded TILs undergo numerical expansion of tumor antigen-specific cells, remain amenable to secondary aAPC-based expansion, and have low CD4/CD8 ratios and FOXP3+ CD4+ cell frequencies. TILs can also be expanded directly from fresh enzyme-digested tumor specimens when pulsed with aAPCs. These "young" TILs are tumor-reactive, positively skewed in CD8+ lymphocyte composition, CD28 and CD27 expression, and contain fewer FOXP3+ T cells compared to parallel IL-2 cultures. Conclusion Genetically-enhanced aAPCs represent a standardized, "off-the-shelf" platform for the direct ex vivo expansion of TILs of suitable number, phenotype and function for use in adoptive immunotherapy.

Detection of Avian Antigen-Specific T Cells Induced by Viral Vaccines

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Dalgaard, Tina Sørensen; Norup, Liselotte Rothmann; Juul-Madsen, Helle Risdahl

2016-01-01

Live attenuated viral vaccines are widely used in commercial poultry production, but the development of new effective inactivated/subunit vaccines is needed. Studies of avian antigen-specific T cells are primarily based on analyses ex vivo after activating the cells with recall antigen. There is ......Live attenuated viral vaccines are widely used in commercial poultry production, but the development of new effective inactivated/subunit vaccines is needed. Studies of avian antigen-specific T cells are primarily based on analyses ex vivo after activating the cells with recall antigen...

Inverse expression of somatostatin and CXCR4 chemokine receptors in gastroenteropancreatic neuroendocrine neoplasms of different malignancy

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Kaemmerer, Daniel; Träger, Tina; Hoffmeister, Maike; Sipos, Bence; Hommann, Merten; Sänger, Järg; Schulz, Stefan; Lupp, Amelie

2015-01-01

Introduction Somatostatin receptors (SSTR) are widely distributed in well-differentiated neuroendocrine neoplasms (NEN) and serve as primary targets for diagnostics and treatment. An overexpression of the chemokine receptor CXCR4, in contrast, is considered to be present mainly in highly proliferative and advanced tumors. Comparative data are still lacking, however, for neuroendocrine carcinomas (NEC). Methods SSTR subtype (1, 2A, 3, 5) and CXCR4 expression was evaluated in G1 (n = 31), G2 (n = 47), and low (G3a; Ki-67: 21–49%; n = 21) and highly proliferative (G3b; Ki-67: >50%, n = 22) G3 (total n = 43) gastroenteropancreatic NEN samples by performing immunohistochemistry with monoclonal rabbit anti-human anti-SSTR and anti-CXCR4 antibodies, respectively, and was correlated with clinical data. Results Both CXCR4 and SSTR were widely expressed in all tumors investigated. CXCR4 expression differed significantly between the G1 and G3 specimens and within the G3 group (G3a to G3b), and was positively correlated with Ki-67 expression. SSTR2A, in contrast, exhibited an inverse association with Ki-67. SSTR2A was highly expressed in G1 and G2 tumors, but was significantly less abundant in G3 carcinomas. Additionally, SSTR1 expression was higher in G3a than in G3b tumors. Conclusion We observed an elevation in CXCR4 and a decrease in SSTR2A expression with increasing malignancy. Interestingly, 23% of the G3 specimens had strong SSTR2A expression. Because CXCR4 was strongly expressed in highly proliferative G3 carcinomas, it is an interesting new target and needs to be validated in larger studies. PMID:26259237

Semi-allogeneic dendritic cells can induce antigen-specific T-cell activation, which is not enhanced by concurrent alloreactivity.

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Wells, James W; Cowled, Chris J; Darling, David; Guinn, Barbara-Ann; Farzaneh, Farzin; Noble, Alistair; Galea-Lauri, Joanna

2007-12-01

Alloreactive T-cell responses are known to result in the production of large amounts of proinflammatory cytokines capable of activating and maturing dendritic cells (DC). However, it is unclear whether these allogeneic responses could also act as an adjuvant for concurrent antigen-specific responses. To examine effects of simultaneous alloreactive and antigen-specific T-cell responses induced by semi-allogeneic DC. Semi-allogeneic DC were generated from the F(1) progeny of inbred strains of mice (C57BL/6 and C3H, or C57BL/6 and DBA). We directly primed antigen-specific CD8(+) and CD4(+) T-cells from OT-I and OT-II mice, respectively, in the absence of allogeneic responses, in vitro, and in the presence or absence of alloreactivity in vivo. In vitro, semi-allogeneic DC cross-presented ovalbumin (OVA) to naïve CD8(+) OT-I transgenic T-cells, primed naïve CD4(+) OT-II transgenic T-cells and could stimulate strong alloreactive T-cell proliferation in a primary mixed lymphocyte reaction (MLR). In vivo, semi-allogeneic DC migrated efficiently to regional lymph nodes but did not survive there as long as autologous DC. In addition, they were not able to induce cytotoxic T-lymphocyte (CTL) activity to a target peptide, and only weakly stimulated adoptively transferred OT-II cells. The CD4(+) response was unchanged in allo-tolerized mice, indicating that alloreactive T-cell responses could not provide help for concurrently activated antigen-specific responses. In an EL4 tumour-treatment model, vaccination with semi-allogeneic DC/EL4 fusion hybrids, but not allogeneic DC/EL4 hybrids, significantly increased mouse survival. Expression of self-Major histocompatibility complex (MHC) by semi-allogeneic DC can cause the induction of antigen-specific immunity, however, concurrently activated allogeneic bystander responses do not provide helper or adjuvant effects.

T Lymphocyte-Endothelial Interactions: Emerging Understanding of Trafficking and Antigen-Specific Immunity

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Christopher Vincent Carman

2015-11-01

Full Text Available Antigen-specific immunity requires regulated trafficking of T cells in and out of diverse tissues in order to orchestrate lymphocyte development, immune surveillance, responses and memory. The endothelium serves as a unique barrier, as well as a sentinel, between the blood and the tissues and as such it plays an essential locally tuned role in regulating T cell migration and information exchange. While it is well established that chemoattractants and adhesion molecules are major determinants of T cell trafficking, emerging studies have now enumerated a large number of molecular players as well as a range of discrete cellular remodeling activities (e.g. transmigratory cups and invadosome-like protrusions, IPLs that participate in directed migration and pathfinding by T cells. In addition to providing trafficking cues, intimate cell-cell interaction between lymphocytes and endothelial cells provide instruction to T cells that influence their activation and differentiation states. Perhaps the most intriguing and underappreciated of these ‘sentinel’ roles is the ability of the endothelium to act as a non-hematopoietic ‘semi-professional’ antigen-presenting cell. Close contacts between circulating T cells and antigen-presenting endothelium may play unique non-redundant roles in shaping adaptive immune responses within the periphery. A better understanding of the mechanisms directing T cell trafficking and the antigen-presenting role of the endothelium may not only increase our knowledge of the adaptive immune response but also empower the utility of emerging immunomodulatory therapeutics.

mDia2 and CXCL12/CXCR4 chemokine signaling intersect to drive tumor cell amoeboid morphological transitions.

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Wyse, Meghan M; Goicoechea, Silvia; Garcia-Mata, Rafael; Nestor-Kalinoski, Andrea L; Eisenmann, Kathryn M

2017-03-04

Morphological plasticity in response to environmental cues in migrating cancer cells requires F-actin cytoskeletal rearrangements. Conserved formin family proteins play critical roles in cell shape, tumor cell motility, invasion and metastasis, in part, through assembly of non-branched actin filaments. Diaphanous-related formin-2 (mDia2/Diaph3/Drf3/Dia) regulates mesenchymal-to-amoeboid morphological conversions and non-apoptotic blebbing in tumor cells by interacting with its inhibitor diaphanous-interacting protein (DIP), and disrupting cortical F-actin assembly and bundling. F-actin disruption is initiated by a CXCL12-dependent mechanism. Downstream CXCL12 signaling partners inducing mDia2-dependent amoeboid conversions remain enigmatic. We found in MDA-MB-231 tumor cells CXCL12 induces DIP and mDia2 interaction in blebs, and engages its receptor CXCR4 to induce RhoA-dependent blebbing. mDia2 and CXCR4 associate in blebs upon CXCL12 stimulation. Both CXCR4 and RhoA are required for CXCL12-induced blebbing. Neither CXCR7 nor other Rho GTPases that activate mDia2 are required for CXCL12-induced blebbing. The Rho Guanine Nucleotide Exchange Factor (GEF) Net1 is required for CXCL12-driven RhoA activation and subsequent blebbing. These results reveal CXCL12 signaling, through CXCR4, directs a Net1/RhoA/mDia-dependent signaling hub to drive cytoskeleton rearrangements to regulate morphological plasticity in tumor cells. These signaling hubs may be conserved during normal and cancer cells responding to chemotactic cues. Copyright © 2017 Elsevier Inc. All rights reserved.

Mutations in the prostate specific antigen (PSA/KLK3) correlate with male infertility.

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Gupta, Nishi; Sudhakar, Digumarthi V S; Gangwar, Pravin Kumar; Sankhwar, Satya Narayan; Gupta, Nalini J; Chakraborty, Baidyanath; Thangaraj, Kumarasamy; Gupta, Gopal; Rajender, Singh

2017-09-11

Prostate specific antigen (PSA/KLK3) is known to be the chief executor of the fragmentation of semenogelins, dissolution of semen coagulum, thereby releasing sperm for active motility. Recent research has found that semenogelins also play significant roles in sperm fertility by affecting hyaluronidase activity, capacitation and motility, thereby making PSA important for sperm fertility beyond simple semen liquefaction. PSA level in semen has been shown to correlate with sperm motility, suggesting that PSA level/activity can affect fertility. However, no study investigating the genetic variations in the KLK3/PSA gene in male fertility has been undertaken. We analyzed the complete coding region of the KLK3 gene in ethnically matched 875 infertile and 290 fertile men to find if genetic variations in KLK3 correlate with infertility. Interestingly, this study identified 28 substitutions, of which 8 were novel (not available in public databases). Statistical comparison of the genotype frequencies showed that five SNPs, rs266881 (OR = 2.92, P  C, was more freuqent in the control group, showing protective association. Our findings suggest that polymorphisms in the KLK3 gene correlate with infertility risk.

The simultaneous ex vivo detection of low-frequency antigen-specific CD4+ and CD8+ T-cell responses using overlapping peptide pools.

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Singh, Satwinder Kaur; Meyering, Maaike; Ramwadhdoebe, Tamara H; Stynenbosch, Linda F M; Redeker, Anke; Kuppen, Peter J K; Melief, Cornelis J M; Welters, Marij J P; van der Burg, Sjoerd H

2012-11-01

The ability to measure antigen-specific T cells at the single-cell level by intracellular cytokine staining (ICS) is a promising immunomonitoring tool and is extensively applied in the evaluation of immunotherapy of cancer. The protocols used to detect antigen-specific CD8+ T-cell responses generally work for the detection of antigen-specific T cells in samples that have undergone at least one round of in vitro pre-stimulation. Application of a common protocol but now using long peptides as antigens was not suitable to simultaneously detect antigen-specific CD8+ and CD4+ T cells directly ex vivo in cryopreserved samples. CD8 T-cell reactivity to monocytes pulsed with long peptides as antigens ranged between 5 and 25 % of that observed against monocytes pulsed with a direct HLA class I fitting minimal CTL peptide epitope. Therefore, we adapted our ICS protocol and show that the use of tenfold higher concentration of long peptides to load APC, the use of IFN-α and poly(I:C) to promote antigen processing and improve T-cell stimulation, does allow for the ex vivo detection of low-frequency antigen-specific CD8+ and CD4+ T cells in an HLA-independent setting. While most of the improvements were related to increasing the ability to measure CD8+ T-cell reactivity following stimulation with long peptides to at least 50 % of the response detected when using a minimal peptide epitope, the final analysis of blood samples from vaccinated patients successfully showed that the adapted ICS protocol also increases the ability to ex vivo detect low-frequency p53-specific CD4+ T-cell responses in cryopreserved PBMC samples.

A computational study of the chemokine receptor CXCR1 bound with interleukin-8

Science.gov (United States)

Wang, Yang; Severin Lupala, Cecylia; Wang, Ting; Li, Xuanxuan; Yun, Ji-Hye; Park, Jae-hyun; Jin, Zeyu; Lee, Weontae; Tan, Leihan; Liu, Haiguang

2018-03-01

CXCR1 is a G-protein coupled receptor, transducing signals from chemokines, in particular the interleukin-8 (IL8) molecules. This study combines homology modeling and molecular dynamics simulation methods to study the structure of CXCR1-IL8 complex. By using CXCR4-vMIP-II crystallography structure as the homologous template, CXCR1-IL8 complex structure was constructed, and then refined using all-atom molecular dynamics simulations. Through extensive simulations, CXCR1-IL8 binding poses were investigated in detail. Furthermore, the role of the N-terminal of CXCR1 receptor was studied by comparing four complex models differing in the N-terminal sequences. The results indicate that the receptor N-terminal affects the binding of IL8 significantly. With a shorter N-terminal domain, the binding of IL8 to CXCR1 becomes unstable. The homology modeling and simulations also reveal the key receptor-ligand residues involved in the electrostatic interactions known to be vital for complex formation. Project supported by the National Natural Science Foundation of China (Grant Nos. 11575021, U1530401, and U1430237) and the National Research Foundation of Korea (Grant Nos. NRF-2017R1A2B2008483 and NRF-2016R1A6A3A04010213).

Quantitative studies of the brain specific antigens GFA, 14-3-2, synaptin C1, D1, D2, D3 and D5 in jimpy mouse

DEFF Research Database (Denmark)

Jacque, C M; Baumann, N A; Bock, E

1976-01-01

Seven antigens specific to the nervous tissue were measured in both Jimpy and control mice. The D5 and the GFA protein, both components of the glia, are strongly increased in the mutant while the neuronal components 14-3-2, synaptin C1, D1, D2 and D3 are unchanged....

CCR3, CCR5, CCR8 and CXCR3 expression in memory T helper cells from allergic rhinitis patients, asymptomatically sensitized and healthy individuals

DEFF Research Database (Denmark)

Holse, Mille; Assing, Kristian; Poulsen, Lars K.

2006-01-01

Chemokine receptors have been suggested to be preferentially expressed on CD4+ T cells with CCR3 and CCR8 linked to the T helper (Th) 2 subset and CCR5 and CXCR3 to the Th1 subset, however this remains controversial....

Blood CXCR3+ CD4 T Cells Are Enriched in Inducible Replication Competent HIV in Aviremic Antiretroviral Therapy-Treated Individuals.

Science.gov (United States)

Banga, Riddhima; Procopio, Francesco A; Ruggiero, Alessandra; Noto, Alessandra; Ohmiti, Khalid; Cavassini, Matthias; Corpataux, Jean-Marc; Paxton, William A; Pollakis, Georgios; Perreau, Matthieu

2018-01-01

We recently demonstrated that lymph nodes (LNs) PD-1 + /T follicular helper (Tfh) cells from antiretroviral therapy (ART)-treated HIV-infected individuals were enriched in cells containing replication competent virus. However, the distribution of cells containing inducible replication competent virus has been only partially elucidated in blood memory CD4 T-cell populations including the Tfh cell counterpart circulating in blood (cTfh). In this context, we have investigated the distribution of (1) total HIV-infected cells and (2) cells containing replication competent and infectious virus within various blood and LN memory CD4 T-cell populations of conventional antiretroviral therapy (cART)-treated HIV-infected individuals. In the present study, we show that blood CXCR3-expressing memory CD4 T cells are enriched in cells containing inducible replication competent virus and contributed the most to the total pool of cells containing replication competent and infectious virus in blood. Interestingly, subsequent proviral sequence analysis did not indicate virus compartmentalization between blood and LN CD4 T-cell populations, suggesting dynamic interchanges between the two compartments. We then investigated whether the composition of blood HIV reservoir may reflect the polarization of LN CD4 T cells at the time of reservoir seeding and showed that LN PD-1 + CD4 T cells of viremic untreated HIV-infected individuals expressed significantly higher levels of CXCR3 as compared to CCR4 and/or CCR6, suggesting that blood CXCR3-expressing CD4 T cells may originate from LN PD-1 + CD4 T cells. Taken together, these results indicate that blood CXCR3-expressing CD4 T cells represent the major blood compartment containing inducible replication competent virus in treated aviremic HIV-infected individuals.

Synthesis and preliminary evaluation of a novel 125I-labeled T140 analog for quantitation of CXCR4 expression

International Nuclear Information System (INIS)

Yanjiang Han; Chinese Academy of Sciences, Beijing; Duanzhi Yin; Mingqiang Zheng; Wei Zhou; Lan Zhan; Yufei Ma; Mingxing Wu; Lingli Shi; Ni Wang; ZhenHong Lee

2010-01-01

The aim of this study was to develop a radiopharmaceutical for the imaging of CXCR4-expressing tumors in vivo. For 125 I-labeling, 125 I-SIB was synthesized and conjugated with the ε-NH 2 group of Ac-TZ14011, a specific CXCR4 antagonist. The specific radioactivity of the product was 5 GBq/μmol and the radiochemical purity (RCP) was 96% (n = 3). After 6 h, the RCP of the product in PBS was 93%. The MCF-7 cell uptake of Ac-TZ14011 was rapid and high. Primary biodistribution studies indicated that 125 I-IB-Ac-TZ14011 was mainly excreted via the kidney, and further evaluation in mice with induced tumors was necessary. (author)

The value of prostate specific antigen and prostate specific antigen density in the diagnosis ad treatment of prostate cancer

International Nuclear Information System (INIS)

Hu Guoying; Yu Mingqi; Feng Xinli

2001-01-01

To study the clinical value of prostate specific antigen (PSA) and prostate specific antigen density (PSAD), the PSA levels of pre-and post-treatment were measured in 28 cases with prostate cancer (Pca) and 80 patients with being Prostate hyperplasia (BPH). PASD was measured in 18 cases Pca and 50 cases BPH of them. The results suggest that the sensitivity, specificity and accuracy of diagnosis for Pca were 85.7%, 80.0% and 81.4%, respectively. The false positive rate was 20%. PSAD is superior to PSA in distinguishing prostate cancer from benign prostate hyperplasia. The false positive rate was only 6%. But in the clinical application, the authors should combine PASD with other materials. The regular observation of post therapeutic PSA is of great value to the earlier discovery of local recurrence and metastasis as well as the judgement of curative effect and prognosis

[68Ga]pentixafor for CXCR4 imaging in a PC-3 prostate cancer xenograft model - comparison with [18F]FDG PET/CT, MRI and ex vivo receptor expression.

Science.gov (United States)

Schwarzenböck, Sarah M; Stenzel, Jan; Otto, Thomas; Helldorff, Heike V; Bergner, Carina; Kurth, Jens; Polei, Stefan; Lindner, Tobias; Rauer, Romina; Hohn, Alexander; Hakenberg, Oliver W; Wester, Hans J; Vollmar, Brigitte; Krause, Bernd J

2017-11-10

The aim was to characterize the properties of [ 68 Ga]Pentixafor as tracer for prostate cancer imaging in a PC-3 prostate cancer xenograft mouse model and to investigate its correlation with [ 18 F]FDG PET/CT, magnetic resonance imaging (MRI) and ex vivo analyses. Static [ 68 Ga]Pentixafor and [ 18 F]FDG PET as well as morphological/ diffusion weighted MRI and 1 H MR spectroscopy was performed. Imaging data were correlated with ex vivo biodistribution and CXCR4 expression in PC-3 tumors (immunohistochemistry (IHC), mRNA analysis). Flow cytometry was performed for evaluation of localization of CXCR4 receptors ( in vitro PC-3 cell experiments). Tumor uptake of [ 68 Ga]Pentixafor was significantly lower compared to [ 18 F]FDG. Ex vivo CXCR4 mRNA expression of tumors was shown by PCR. Only faint tumor CXCR4 expression was shown by IHC (immuno reactive score of 3). Accordingly, flow cytometry of PC-3 cells revealed only a faint signal, cell membrane permeabilisation showed a slight signal increase. There was no significant correlation of [ 68 Ga]Pentixafor tumor uptake and ex vivo receptor expression. Spectroscopy showed typical spectra of prostate cancer. PC-3 tumor uptake of [ 68 Ga]Pentixafor was existent but lower compared to [ 18 F]FDG. No significant correlation of ex vivo tumor CXCR4 receptor expression and [ 68 Ga]Pentixafor tumor uptake was shown. CXCR4 receptor expression on the surface of PC-3 cells was existent but rather low possibly explaining the limited [ 68 Ga]Pentixafor tumor uptake; receptor localization in the interior of PC-3 cells is presumable as shown by cell membrane permeabilisation. Further studies are necessary to define the role of [ 68 Ga]Pentixafor in prostate cancer imaging.

Sensitivity and specificity of three Onchocerca volvulus cloned antigens in diagnosis of onchocerciasis

International Nuclear Information System (INIS)

Atti, Miska Elyemen; TR, Unnasch; Mukhtar, M.M.

1999-01-01

Onchcerciasis is endemic in three geographical regions in Sudan, and presented with variable clinical reactions. The current diagnosis of onchocerciasis is based on detection of live microflaraie in skin snips in addition to clinical signs and history of living in endemic regions. Different serological diagnostic trials using crude soluble antigens of O. volvulus have shown variable degrees of cross reaction with other nematodes co-endemic in the same area. We have studied the sensitivity and specificity of RAL-2, calreticulin and PDI O. volvulus cloned antigens using the ELISA techniques. Eighty serum samples of Onchocerciasis patients, 20 non endemic normal controls and 42 samples of patients of other endemic deseas including Leishmanoasis, malaria, tuberculosis and shestosomiasis were tested RAL-2 gave the best Ig G response, with 83.75% sensitivity and 91. 66% specificity. PDI sensitivity was 20% and specificity 91.66%, while calreticulum showed sensitivity of 37.5% and specificity of 73.30%. IgG3 subclasses was not significantly different from controls while IgG4 was significantly higher in patients. The sensitivity of RAL-2 for IgG4 was 90% with a specificity of 100%. For PDI sensitivity was 25% and 100% specificity while calreticulin resulted in sensitivity of 75% and specificity of 100%. Low levels of circulating IgE to RAL-2 antigen were detected. For all antigens there was significant correlation with gender, age, microflarial load or presence of nodules. (Author)

Shape anisotropy enhanced optomagnetic measurement for prostate-specific antigen detection via magnetic chain formation

DEFF Research Database (Denmark)

Tian, Bo; Wetterskog, Erik; Qiu, Zhen

2017-01-01

anisotropy), and directly increasing the optomagnetic signal (via optical shape anisotropy). We achieve a limit of detection (LOD) of 5.5 pM (0.82 ng/mL) for the detection of a model multivalent molecule, biotinylated anti-streptavidin, in PBS. For the measurements of prostate-specific antigen (PSA) in 50...

Development of a new in vivo kit for detection of prostate specific antigen in human serum using immunoradiometric assay method

International Nuclear Information System (INIS)

Babaei, M. H.; Behradkia, P.; Shafii, M.; Movla, M.; Forutan, H.; Najafi, R.

2006-01-01

Prostate is a leading site for the cancer incidence, accounted for 31.0% of new cancer cases in men. Prostate-specific antigen is widely used in the detection and monitoring of the prostate cancer. Currently, immunoassay is used to detect Prostate-specific antigen in human serum. This technique is based on the interaction between antibody and antigen. The varied immunoassay formats and equipment to run the assays allow the users to measure the analytes rapidly, with the flexibility to run a small or a large number of samples. Among different immunoassay methods, immunoradiometric assay is a more sensitive and valuable detection approach. This study has been made in 4 parts: (1) purification of Prostate-specific antigen from seminal fluid; (2) preparation of hybridoma cells which secrete monoclonal antibody (mAb) against Prostate-specific antigen , (3) selection of pair monoclonal antibody among those antibodies, and finally (4) design of an immunoradiometric assay kit and it's quality control . The results of this study were: (1) obtaining a huge amount of Prostate-specific antigen as semi-purified and purified, that is a valuable material for preparation of standard kits; (2) preparation of 8 kinds of monoclonal antibodies; (3) finding 4 pairs of monoclonal antibodies which react with different epitopes on Prostate-specific antigen molecule; and (4) preparation of immunoradiometric assay kit for measuring Prostate-specific antigen concentration in human serum

Sequence similarity between the erythrocyte binding domain 1 of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals binding residues for the Duffy Antigen Receptor for Chemokines

OpenAIRE

Bolton, Michael J; Garry, Robert F

2011-01-01

Abstract Background The surface glycoprotein (SU, gp120) of the human immunodeficiency virus (HIV) must bind to a chemokine receptor, CCR5 or CXCR4, to invade CD4+ cells. Plasmodium vivax uses the Duffy Binding Protein (DBP) to bind the Duffy Antigen Receptor for Chemokines (DARC) and invade reticulocytes. Results Variable loop 3 (V3) of HIV-1 SU and domain 1 of the Plasmodium vivax DBP share a sequence similarity. The site of amino acid sequence similarity was necessary, but not sufficient, ...

Expression and Function of the Chemokine, CXCL13, and Its Receptor, CXCR5, in Aids-Associated Non-Hodgkin's Lymphoma

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Daniel P. Widney

2010-01-01

Full Text Available Background. The homeostatic chemokine, CXCL13 (BLC, BCA-1, helps direct the recirculation of mature, resting B cells, which express its receptor, CXCR5. CXCL13/CXCR5 are expressed, and may play a role, in some non-AIDS-associated B cell tumors. Objective. To determine if CXCL13/CXCR5 are associated with AIDS-related non-Hodgkin's lymphoma (AIDS-NHL. Methods. Serum CXCL13 levels were measured by ELISA in 46 subjects who developed AIDS-NHL in the Multicenter AIDS Cohort Study and in controls. The expression or function of CXCL13 and CXCR5 was examined on primary AIDS-NHL specimens or AIDS-NHL cell lines. Results. Serum CXCL13 levels were significantly elevated in the AIDS-NHL group compared to controls. All primary AIDS-NHL specimens showed CXCR5 expression and most also showed CXCL13 expression. AIDS-NHL cell lines expressed CXCR5 and showed chemotaxis towards CXCL13. Conclusions. CXCL13/CXCR5 are expressed in AIDS-NHL and could potentially be involved in its biology. CXCL13 may have potential as a biomarker for AIDS-NHL.

Use of Digital Rectal Examination as an Adjunct to Prostate Specific Antigen in the Detection of Clinically Significant Prostate Cancer.

Science.gov (United States)

Halpern, Joshua A; Oromendia, Clara; Shoag, Jonathan E; Mittal, Sameer; Cosiano, Michael F; Ballman, Karla V; Vickers, Andrew J; Hu, Jim C

2018-04-01

Guidelines from the NCCN ® (National Comprehensive Cancer Network®) advocate digital rectal examination screening only in men with elevated prostate specific antigen. We investigated the effect of prostate specific antigen on the association of digital rectal examination and clinically significant prostate cancer in a large American cohort. We evaluated the records of the 35,350 men who underwent digital rectal examination in the screening arm of the Prostate, Lung, Colorectal and Ovarian Cancer Screening trial for the development of clinically significant prostate cancer (Gleason 7 or greater). Followup was 343,273 person-years. The primary outcome was the rate of clinically significant prostate cancer among men with vs without suspicious digital rectal examination. We performed competing risks regression to evaluate the interaction between time varying suspicious digital rectal examination and prostate specific antigen. A total of 1,713 clinically significant prostate cancers were detected with a 10-year cumulative incidence of 5.9% (95% CI 5.6-6.2). Higher risk was seen for suspicious vs nonsuspicious digital rectal examination. Increases in absolute risk were small and clinically irrelevant for normal (less than 2 ng/ml) prostate specific antigen (1.5% vs 0.7% risk of clinically significant prostate cancer at 10 years), clinically relevant for elevated (3 ng/ml or greater) prostate specific antigen (23.0% vs 13.7%) and modestly clinically relevant for equivocal (2 to 3 ng/ml) prostate specific antigen (6.5% vs 3.5%). Digital rectal examination demonstrated prognostic usefulness when prostate specific antigen was greater than 3 ng/ml, limited usefulness for less than 2 ng/ml and marginal usefulness for 2 to 3 ng/ml. These findings support the restriction of digital rectal examination to men with higher prostate specific antigen as a reflex test to improve specificity. It should not be used as a primary screening modality to improve sensitivity. Copyright �

Usefulness of transrectal ultrasound in diagnosing prostate cancer: comparison with digital rectal examination, prostate-specific antigen and prostate-specific antigen density

International Nuclear Information System (INIS)

Yoon, Jung Hwan; Kim, Bo Hyun; Choi, Sang Hee; Kim, Seung Hoon; Choi, Han Yong; Chai, Soo Eung; Yoon, Hye Kyung; Lee, Soon Jin; Choo, In Wook; Kim, Bo Kyung

1998-01-01

To determine the usefulness of transrectal ultrasonography (TRUS) in diagnosing prostate cancer by comparing the sensitivity, specificity, accuracy, and positive and negative predictive values of TRUS with those of serum prostate-specific antigen (PSA), prostate-specific antigen density (PSAD) and digital rectal examination (DRE). Two hundred and ten consecutive patients underwent TRUS-guided prostate biopsy due to elevated PSA and/or abnormal findings on TRUS or DRE. The TRUS findings were analyzed and correlated with pathological diagnosis. PSAD was calculated by dividing the serum PSA level by the prostate volume calculated on TRUS. The sensitivity, specificity, accuracy, and positive and negative predictive values of TRUS were compared with those of PSA, PSAD and DRE. Using ROC curve analysis, the combinations of these diagnostic methods were also evaluated for the determination of efficacy in diagnosing prostate cancer. The sensitivity and specificity of serum PSA (cut-off level, 4ng/ml), PSAD (cut-off level, 0.15ng/ml/cm 3 ), DRE, and TRUS were 96%/17%, 96%/37%, 72%/62%, and 89%/68%, respectively. On TRUS, the sensitivity and specificity of low echoic lesions and those of irregular outer margin were 89%/69%, and 60%/90%, respectively. TRUS was statistically more accurate than other diagnostic methods. Of the combinations of diagnostic methods, TRUS and PSAD were most accurate. TRUS demonstrated lower sensitivity but higher specificity than PSA or PSAD. Although it is an accurate modality for the diagnosis of prostate cancer, it cannot be used as a confirmative test due to its relatively low positive predictive value. A combination of diagnostic methods and random biopsy is needed in patients in whom prostate cancer is suspected.=20

Reprogramming Medulloblastoma-Propagating Cells by a Combined Antagonism of Sonic Hedgehog and CXCR4.

Science.gov (United States)

Ward, Stacey A; Warrington, Nicole M; Taylor, Sara; Kfoury, Najla; Luo, Jingqin; Rubin, Joshua B

2017-03-15

The CXCR4 chemokine and Sonic Hedgehog (SHH) morphogen pathways are well-validated therapeutic targets in cancer, including medulloblastoma. However, single-agent treatments with SHH or CXCR4 antagonists have not proven efficacious in clinical trials to date. Here, we discovered that dual inhibition of the SHH and CXCR4 pathways in a murine model of SHH-subtype medulloblastoma exerts potent antitumor effects. This therapeutic synergy resulted in the suppression of tumor-propagating cell function and correlated with increased histone H3 lysine 27 trimethylation within the promoters of stem cell genes, resulting in their decreased expression. These results demonstrate that CXCR4 contributes to the epigenetic regulation of a tumor-propagating cell phenotype. Moreover, they provide a mechanistic rationale to evaluate the combination of SHH and CXCR4 inhibitors in clinical trials for the treatment of medulloblastoma, as well as other cancers driven by SHH that coexpress high levels of CXCR4. Cancer Res; 77(6); 1416-26. ©2016 AACR . ©2016 American Association for Cancer Research.

Prognostic value of CXCL12 and CXCR4 in inoperable head and neck squamous cell carcinoma

Energy Technology Data Exchange (ETDEWEB)

Rave-Fraenk, Margret; Tehrany, Narges; Leu, Martin; Weber, Hanne Elisabeth; Wolff, Hendrik Andreas [University Medical Center Goettingen, Department of Radiotherapy and Radiation Oncology, Goettingen (Germany); Kitz, Julia [University Medical Center Goettingen, Department of Pathology, Goettingen (Germany); Burfeind, Peter [University Medical Center Goettingen, Department of Human Genetics, Goettingen (Germany); Schliephake, Henning [University Medical Center Goettingen, Department of Oral and Maxillofacial Surgery, Goettingen (Germany); Canis, Martin [University Medical Center Goettingen, Department of Otorhinolaryngology, Head and Neck Surgery, Goettingen (Germany); Beissbarth, Tim [University Medical Center Goettingen, Institute of Medical Statistics, Goettingen (Germany); Reichardt, Holger Michael [University Medical Center Goettingen, Institute for Cellular and Molecular Immunology, Goettingen (Germany)

2016-01-15

The chemokine CXCL12 and its receptor CXCR4 can affect tumor growth, recurrence, and metastasis. We tested the hypothesis that the CXCL12 and CXCR4 expression influences the prognosis of patients with inoperable head and neck cancer treated with definite radiotherapy or chemoradiotherapy. Formalin-fixed paraffin-embedded pretreatment tumor tissue from 233 patients with known HPV/p16{sup INK4A} status was analyzed. CXCL12 and CXCR4 expressions were correlated with pretreatment parameters and survival data by univariate and multivariate Cox regression. CXCL12 was expressed in 43.3 % and CXCR4 in 66.1 % of the samples and both were correlated with HPV/p16{sup INK4A} positivity. A high CXCL12 expression was associated with increased overall survival (p = 0.036), while a high CXCR4 expression was associated with decreased metastasis-free survival (p = 0.034). A high CXCR4 expression could be regarded as a negative prognostic factor in head and neck cancer because it may foster metastatic spread. This may recommend CXCR4 as therapeutic target for combating head and neck cancer metastasis. (orig.) [German] Das Chemokin CXCL12 und sein Rezeptor CXCR4 beeinflussen Tumorwachstum, Auftreten von Rezidiven und Metastasierung. Es wurde die Hypothese geprueft, dass ein Zusammenhang der CXCL12- und CXCR4-Expression mit der Prognose von Patienten bestehe, die wegen eines inoperablen Kopf-Hals-Tumors eine primaere Radio- oder Radiochemotherapie erhielten. Dabei wurde auch der HPV-Status der Patienten beruecksichtigt. Formalinfixierte Proben aus unbehandelten Tumoren von 233 Patienten mit bekanntem HPV/p16{sup INK4A}-Status wurden ausgewertet. Die CXCL12- und CXCR4-Expression wurde mit klinischen Parametern und Ueberlebensdaten mittels uni- und multivariater Cox Regression analysiert. CXCL12 wurde von 43,3 %, CXCR4 von 66,1 % der Tumoren exprimiert, und beide Marker korrelierten mit einer HPV/p16{sup INK4A}-Expression. Eine hohe CXCL12-Expression war mit einem verbesserten

Kinetics of antigen specific and non-specific polyclonal B-cell responses during lethal Plasmodium yoelii malaria

Directory of Open Access Journals (Sweden)

Laurence Rolland

1992-06-01

Full Text Available In order to study the kinetics and composition of the polyclonal B-cell activation associated to malaria infection, antigen-specific and non-specific B-cell responses were evaluated in the spleens of mice infected with Plasmodium yoelii 17 XL or injected with lysed erythrocytes or plasma from P. yoelii infected mice or with P. falciparum culture supernatants. Spleen/body weigth ratio, numbers of nucleated spleen cells and Immunoglobulin-containing and Immunoglobulin-secreting cells increased progressively during the course of infection,in parallel to the parasitemia. A different pattern of kinetics was observed when anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell plaque forming cells response were studied: maximum values were observed at early stages of infection, whereas the number of total Immunoglobulin-containing and Immunoglobulin-secreting cells were not yet altered. Conversely, at the end of infection, when these latter values reached their maximum, the anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell specific responses were normal or even infranormal. In mice injected with Plasmodium-derived material, a higher increase in antigen-specific PFC was observed, as compared to the increase of Immunoglobulin-containing and Immunoglobulin-secreting cell numbers. This suggested a "preferential" (antigen-plus mitogen-induced stimulation of antigen-specific cells rather than a generalized non-specific (mitogen-induced triggering of B-lymphocytes. On the basis of these and previous results, it is suggested that polyclonal B-cell activation that takes place during the course of infection appears as a result of successive waves of antigen-specific B-cell activation.

Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

Science.gov (United States)

Unzueta, Ugutz; Céspedes, María Virtudes; Ferrer-Miralles, Neus; Casanova, Isolda; Cedano, Juan; Corchero, José Luis; Domingo-Espín, Joan; Villaverde, Antonio; Mangues, Ramón; Vázquez, Esther

2012-01-01

Background Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4) is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing. Results Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer. Conclusion Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles, and imaging agents. PMID:22923991

Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells.

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Lynette Beattie

2010-03-01

Full Text Available Kupffer cells (KCs represent the major phagocytic population within the liver and provide an intracellular niche for the survival of a number of important human pathogens. Although KCs have been extensively studied in vitro, little is known of their in vivo response to infection and their capacity to directly interact with antigen-specific CD8(+ T cells. Here, using a combination of approaches including whole mount and thin section confocal microscopy, adoptive cell transfer and intra-vital 2-photon microscopy, we demonstrate that KCs represent the only detectable population of mononuclear phagocytes within granulomas induced by Leishmania donovani infection that are capable of presenting parasite-derived peptide to effector CD8(+ T cells. This restriction of antigen presentation to KCs within the Leishmania granuloma has important implications for the identification of new candidate vaccine antigens and for the design of novel immuno-therapeutic interventions.

Cancer Patient T Cells Genetically Targeted to Prostate-Specific Membrane Antigen Specifically Lyse Prostate Cancer Cells and Release Cytokines in Response to Prostate-Specific Membrane Antigen

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Michael C. Gong

1999-06-01

Full Text Available The expression of immunoglobulin-based artificial receptors in normal T lymphocytes provides a means to target lymphocytes to cell surface antigens independently of major histocompatibility complex restriction. Such artificial receptors have been previously shown to confer antigen-specific tumoricidal properties in murine T cells. We constructed a novel ζ chain fusion receptor specific for prostate-specific membrane antigen (PSMA termed Pz-1. PSMA is a cell-surface glycoprotein expressed on prostate cancer cells and the neovascular endothelium of multiple carcinomas. We show that primary T cells harvested from five of five patients with different stages of prostate cancer and transduced with the Pz-1 receptor readily lyse prostate cancer cells. Having established a culture system using fibroblasts that express PSMA, we next show that T cells expressing the Pz-1 receptor release cytokines in response to cell-bound PSMA. Furthermore, we show that the cytokine release is greatly augmented by B7.1-mediated costimulation. Thus, our findings support the feasibility of adoptive cell therapy by using genetically engineered T cells in prostate cancer patients and suggest that both CD4+ and CD8+ T lymphocyte functions can be synergistically targeted against tumor cells.

Screening for epitope specificity directly on culture supernatants in the early phase of monoclonal antibody production by an ELISA with biotin-labeled antigen

DEFF Research Database (Denmark)

Andersen, Ditte C; Jensen, Charlotte H; Gregersen, Annemette

2004-01-01

This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other...... preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious...

CEACAM3-mediated phagocytosis of human-specific bacterial pathogens involves the adaptor molecule Nck

OpenAIRE

Peterson, Lisa

2010-01-01

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are exploited by human-specific pathogens to anchor themselves to or invade host cells. Interestingly, human granulocytes express a specific isoform, CEACAM3, that can direct efficient, opsonin-independent phagocytosis of CEACAM-binding Neisseria, Moraxella and Haemophilus species. As opsonin-independent phagocytosis of CEACAM-binding Neisseria depends on Src-family protein tyrosine kinase (PTK) phosphorylation of the CEACAM3 ...

Expression cloning of camelid nanobodies specific for Xenopus embryonic antigens.

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Keiji Itoh

Full Text Available Developmental biology relies heavily on the use of conventional antibodies, but their production and maintenance involves significant effort. Here we use an expression cloning approach to identify variable regions of llama single domain antibodies (known as nanobodies, which recognize specific embryonic antigens. A nanobody cDNA library was prepared from lymphocytes of a llama immunized with Xenopus embryo lysates. Pools of bacterially expressed cDNAs were sib-selected for the ability to produce specific staining patterns in gastrula embryos. Three different nanobodies were isolated: NbP1 and NbP3 stained yolk granules, while the reactivity of NbP7 was predominantly restricted to the cytoplasm and the cortex. The isolated nanobodies recognized specific protein bands in immunoblot analysis. A reverse proteomic approach identified NbP1 target antigen as EP45/Seryp, a serine protease inhibitor. Given the unique stability of nanobodies and the ease of their expression in diverse systems, we propose that nanobody cDNA libraries represent a promising resource for molecular markers for developmental biology.

Strategy for eliciting antigen-specific CD8+ T cell-mediated immune response against a cryptic CTL epitope of merkel cell polyomavirus large T antigen

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Gomez Bianca P

2012-10-01

Full Text Available Abstract Background Merkel cell carcinoma (MCC is a relatively new addition to the expanding category of oncovirus-induced cancers. Although still comparably rare, the number of cases has risen dramatically in recent years. Further complicating this trend is that MCC is an extremely aggressive neoplasm with poor patient prognosis and limited treatment options for advanced disease. The causative agent of MCC has been identified as the merkel cell polyomavirus (MCPyV. The MCPyV-encoded large T (LT antigen is an oncoprotein that is theorized to be essential for virus-mediated tumorigenesis and is therefore, an excellent MCC antigen for the generation of antitumor immune responses. As a foreign antigen, the LT oncoprotein avoids the obstacle of immune tolerance, which normally impedes the development of antitumor immunity. Ergo, it is an excellent target for anti-MCC immunotherapy. Since tumor-specific CD8+ T cells lead to better prognosis for MCC and numerous other cancers, we have generated a DNA vaccine that is capable of eliciting LT-specific CD8+ T cells. The DNA vaccine (pcDNA3-CRT/LT encodes the LT antigen linked to a damage-associated molecular pattern, calreticulin (CRT, as it has been demonstrated that the linkage of CRT to antigens promotes the induction of antigen-specific CD8+ T cells. Results The present study shows that DNA vaccine-induced generation of LT-specific CD8+ T cells is augmented by linking CRT to the LT antigen. This is relevant since the therapeutic effects of the pcDNA3-CRT/LT DNA vaccine is mediated by LT-specific CD8+ T cells. Mice vaccinated with the DNA vaccine produced demonstrably more LT-specific CD8+ T cells. The DNA vaccine was also able to confer LT-specific CD8+ T cell-mediated protective and therapeutic effects to prolong the survival of mice with LT-expressing tumors. In the interest of determining the LT epitope which most MCC-specific CD8+ T cells recognize, we identified the amino acid sequence of the

TLR4-NOX4-AP-1 signaling mediates lipopolysaccharide-induced CXCR6 expression in human aortic smooth muscle cells

International Nuclear Information System (INIS)

Patel, Devang N.; Bailey, Steven R.; Gresham, John K.; Schuchman, David B.; Shelhamer, James H.; Goldstein, Barry J.; Foxwell, Brian M.; Stemerman, Michael B.; Maranchie, Jodi K.; Valente, Anthony J.; Mummidi, Srinivas; Chandrasekar, Bysani

2006-01-01

CXCL16 is a transmembrane non-ELR CXC chemokine that signals via CXCR6 to induce aortic smooth muscle cell (ASMC) proliferation. While bacterial lipopolysaccharide (LPS) has been shown to stimulate CXCL16 expression in SMC, its effects on CXCR6 are not known. Here, we demonstrate that LPS upregulates CXCR6 mRNA, protein, and surface expression in human ASMC. Inhibition of TLR4 with neutralizing antibodies or specific siRNA interference blocked LPS-mediated CXCR6 expression. LPS stimulated both AP-1 (c-Fos, c-Jun) and NF-κB (p50 and p65) activation, but only inhibition of AP-1 attenuated LPS-induced CXCR6 expression. Using dominant negative expression vectors and siRNA interference, we demonstrate that LPS induces AP-1 activation via MyD88, TRAF6, ERK1/2, and JNK signaling pathways. Furthermore, the flavoprotein inhibitor diphenyleniodonium chloride significantly attenuated LPS-mediated AP-1-dependent CXCR6 expression, as did inhibition of NOX4 NADPH oxidase by siRNA. Finally, CXCR6 knockdown inhibited CXCL16-induced ASMC proliferation. These results demonstrate that LPS-TLR4-NOX4-AP-1 signaling can induce CXCR6 expression in ASMC, and suggest that the CXCL16-CXCR6 axis may be an important proinflammatory pathway in the pathogenesis of atherosclerosis

Comparison of serum prostate specific antigen levels and bone scintigraphy in patients with prostate carcinoma

International Nuclear Information System (INIS)

Bielickaite, J.; Zadeikaite, R.; Jurkiene, N. and others

2003-01-01

The aim of this study was to analyze the levels of serum prostate specific antigen in patients with and without bone metastases detected by means of bone scintigraphy and to determine the highest prostate specific antigen level in patients without bone metastases. The 50 patients consecutively diagnosed of prostate cancer between 1999 and 2001 in our institution made up the study population. Prostate specific antigen plasmatic levels were determined and bone scintigraphy was performed (whole body study after 99mTc-methyl-diphosphonate administration) in all the patients. In patients with positive bone scans (n=23), the mean prostate specific antigen level was 71.4±35.2 ng/ml and was significantly (p<0.00005) higher than in 14 patients with negative bone scans (mean prostate specific antigen level was 10.1±10.5 ng/ml). Suspicious lesions were found in 13 patients and their mean prostate specific antigen level was 8.5±7.7 ng/ml. Regarding prostate specific antigen levels, no statistically significant differences were found between patients with suspicious lessons and normal bone scans. The highest determined prostate specific antigen level in patients without bone metastases was 18 ng/ml. The bone scintigraphy should be performed in all patients with prostate specific antigen level above 18 ng/ml, but it is of limited value in patients with prostate specific antigen level below 18 ng/ml. (author)

Dysregulation of CXCR3 expression on peripheral blood leukocytes in patients with neovascular age-related macular degeneration

DEFF Research Database (Denmark)

Falk, Mads Krüger; Singh, Amardeep; Faber, Carsten

2014-01-01

concentration of the chemokines CXCL9-11. Methods: The study group consisted of patients with AMD attending our department. Patients referred for other reasons than AMD were enrolled as control persons. The expression of CXCR3 on T-cells and the plasma concentration of CXCL9-11 were measured using flow...

Characterization of Specific Immune Responses to Different Aspergillus Antigens during the Course of Invasive Aspergillosis in Hematologic Patients

Science.gov (United States)

Beauvais, Anne; Beau, Remi; Candoni, Anna; Maertens, Johan; Rossi, Giulio; Morselli, Monica; Zanetti, Eleonora; Quadrelli, Chiara; Codeluppi, Mauro; Guaraldi, Giovanni; Pagano, Livio; Caira, Morena; Giovane, Cinzia Del; Maccaferri, Monica; Stefani, Alessandro; Morandi, Uliano; Tazzioli, Giovanni; Girardis, Massimo; Delia, Mario; Specchia, Giorgina; Longo, Giuseppe; Marasca, Roberto; Narni, Franco; Merli, Francesco; Imovilli, Annalisa; Apolone, Giovanni; Carvalho, Agostinho; Comoli, Patrizia; Romani, Luigina; Latgè, Jean Paul; Luppi, Mario

2013-01-01

Several studies in mouse model of invasive aspergillosis (IA) and in healthy donors have shown that different Aspergillus antigens may stimulate different adaptive immune responses. However, the occurrence of Aspergillus-specific T cells have not yet been reported in patients with the disease. In patients with IA, we have investigated during the infection: a) whether and how specific T-cell responses to different Aspergillus antigens occur and develop; b) which antigens elicit the highest frequencies of protective immune responses and, c) whether such protective T cells could be expanded ex-vivo. Forty hematologic patients have been studied, including 22 patients with IA and 18 controls. Specific T cells producing IL-10, IFN-γ, IL-4 and IL-17A have been characterized through enzyme linked immunospot and cytokine secretion assays on 88 peripheral blood (PB) samples, by using the following recombinant antigens: GEL1p, CRF1p, PEP1p, SOD1p, α1–3glucan, β1–3glucan, galactomannan. Specific T cells were expanded through short term culture. Aspergillus-specific T cells producing non-protective interleukin-10 (IL-10) and protective interferon-gamma (IFN-γ) have been detected to all the antigens only in IA patients. Lower numbers of specific T cells producing IL-4 and IL-17A have also been shown. Protective T cells targeted predominantly Aspergillus cell wall antigens, tended to increase during the IA course and to be associated with a better clinical outcome. Aspergillus-specific T cells could be successfully generated from the PB of 8 out of 8 patients with IA and included cytotoxic subsets able to lyse Aspergillus hyphae. Aspergillus specific T-cell responses contribute to the clearance of the pathogen in immunosuppressed patients with IA and Aspergillus cell wall antigens are those mainly targeted by protective immune responses. Cytotoxic specific T cells can be expanded from immunosuppressed patients even during the infection by using the above mentioned

Transfusion of CXCR4-primed endothelial progenitor cells reduces cerebral ischemic damage and promotes repair in db/db diabetic mice.

Directory of Open Access Journals (Sweden)

Ji Chen

Full Text Available This study investigated the role of stromal cell-derived factor-1α (SDF-1α/CXC chemokine receptor 4 (CXCR4 axis in brain and endothelial progenitor cells (EPCs, and explored the efficacy of CXCR4 primed EPCs in treating ischemic stroke in diabetes. The db/db diabetic and db/+ mice were used in this study. Levels of plasma SDF-1α and circulating CD34+CXCR4+ cells were measured. Brain SDF-1α and CXCR4 expression were quantified at basal and after middle cerebral artery occlusion (MCAO. In in vitro study, EPCs were transfected with adenovirus carrying null (Ad-null or CXCR4 (Ad-CXCR4 followed with high glucose (HG treatment for 4 days. For pathway block experiments, cells were pre-incubated with PI3K inhibitor or nitric oxide synthase (NOS inhibitor for two hours. The CXCR4 expression, function and apoptosis of EPCs were determined. The p-Akt/Akt and p-eNOS/eNOS expression in EPCs were also measured. In in vivo study, EPCs transfected with Ad-null or Ad-CXCR4 were infused into mice via tail vein. On day 2 and 7, the cerebral blood flow, neurologic deficit score, infarct volume, cerebral microvascular density, angiogenesis and neurogenesis were determined. We found: 1 The levels of plasma SDF-1α and circulating CD34+CXCR4+ cells were decreased in db/db mice; 2 The basal level of SDF-1α and MCAO-induced up-regulation of SDF-1α/CXCR4 axis were reduced in the brain of db/db mice; 3 Ad-CXCR4 transfection increased CXCR4 expression in EPCs and enhanced EPC colonic forming capacity; 4 Ad-CXCR4 transfection prevented EPCs from HG-induced dysfunction (migration and tube formation and apoptosis via activation of PI3K/Akt/eNOS signal pathway; 4 Ad-CXCR4 transfection enhanced the efficacy of EPC infusion in attenuating infarct volume and promoting angiogenesis and neurogenesis. Our data suggest that Ad-CXCR4 primed EPCs have better therapeutic effects for ischemia stroke in diabetes than unmodified EPCs do.

Profound blockage of CXCR4 signaling at multiple points using the synergy between plerixafor, mirtazapine, and clotrimazole as a new glioblastoma treatment adjunct.

Science.gov (United States)

Kast, Richard E

2010-10-01

CXCL12 signaling at CXCR4 is important in glioblastoma growth promotion as a migration-directing chemokine and as a mitosis-stimulating cytokine system. Recent developments in other areas of medicine may have made it now possible to comprehensively block glioblastoma's use of CXCL12 signaling. CXCL12 signaling at CXCR4 requires an active intermediate conductance Ca2+-activated K+ channel to function. Plerixafor (AMD3100) is a new small molecular weight inhibitor of CXCR4, FDA approved to aid in stem cell mobilization. Inhibition of CXCR4 by plerixafor is expected to inhibit particularly the glioblastoma stem cell population by inhibiting that sub-population's homing to the protective hypoxic niche. Histamine signals through the H1 receptor in glioblastoma cells to activate the intermediate conductance Ca2+-activated K+ channel also, thereby forming a potential bypass for inhibition of CXCR4-initiated signaling. The antidepressant mirtazapine is perhaps the most potent H1 antagonist in common clinical use. By inhibiting H1 stimulation of intermediate conductance Ca2+-activated K+ channels, it could prevent circumvention of CXCR4 inhibition by that path. The anti-fungal clotrimazole directly inhibits the intermediate conductance Ca2+- activated K+ channel at clinically achievable and well-tolerated doses. These three drugs used simultaneously are potential low morbidity paths to deeply inhibit CXCR4/CXCL12 signaling during cytotoxic glioblastoma treatment.

Multiple sclerosis: a study of CXCL10 and CXCR3 co-localization in the inflamed central nervous system

DEFF Research Database (Denmark)

Sørensen, Torben Lykke; Trebst, Corinna; Kivisäkk, Pia

2002-01-01

T-cell accumulation in the central nervous system (CNS) is considered crucial to the pathogenesis of multiple sclerosis (MS). We found that the majority of T cells within the cerebrospinal fluid (CSF) compartment expressed the CXC chemokine receptor 3 (CXCR), independent of CNS inflammation...

The Cytomegalovirus UL146 Gene Product vCXCL1 Targets Both CXCR1 and CXCR2 as an Agonist

DEFF Research Database (Denmark)

Luttichau, H.R.

2010-01-01

Large DNA viruses, such as herpesvirus and poxvirus, encode proteins that target and exploit the chemokine system of their host. UL146 and UL147 in the cytomegalovirus (CMV) genome encode the two CXC chemokines vCXCL1 and vCXCL2. In this study, vCXCL1 was probed against a panel of the 18 classified...... human chemokine receptors. In calcium mobilization assays vCXCL1 acted as an agonist on both CXCR1 and CXCR2 but did not activate or block any of the other 16 chemokine receptors. vCXCL1 was characterized and compared with CXCL1/GRO alpha, CXCL2/GRO beta, CXCL3/GRO gamma, CXCL5/ENA-78, CXCL6/GCP2, CXCL7...

Rapid production of antigen-specific monoclonal antibodies from a variety of animals

Directory of Open Access Journals (Sweden)

Kurosawa Nobuyuki

2012-09-01

Full Text Available Abstract Background Although a variety of animals have been used to produce polyclonal antibodies against antigens, the production of antigen-specific monoclonal antibodies from animals remains challenging. Results We propose a simple and rapid strategy to produce monoclonal antibodies from a variety of animals. By staining lymph node cells with an antibody against immunoglobulin and a fluorescent dye specific for the endoplasmic reticulum, plasma/plasmablast cells were identified without using a series of antibodies against lineage markers. By using a fluorescently labeled antigen as a tag for a complementary cell surface immunoglobulin, antigen-specific plasma/plasmablast cells were sorted from the rest of the cell population by fluorescence-activated cell sorting. Amplification of cognate pairs of immunoglobulin heavy and light chain genes followed by DNA transfection into 293FT cells resulted in the highly efficient production of antigen-specific monoclonal antibodies from a variety of immunized animals. Conclusions Our technology eliminates the need for both cell propagation and screening processes, offering a significant advantage over hybridoma and display strategies.

CdTe QDs-based prostate-specific antigen probe for human prostate cancer cell imaging

International Nuclear Information System (INIS)

Dong Wei; Guo Li; Wang Meng; Xu Shukun

2009-01-01

L-glutathione (GSH) stabilized CdTe quantum dots (QDs) were directly prepared in aqueous solution. The as-prepared QDs were linked to prostate-specific antigen (PSA) for the direct labeling and linked to immunoglobulin G (IgG) for the indirect labeling of fixed prostate cancer cells. The results indicated that QD-based probes were ideal fluorescent markers with excellent spectral properties and photostability and much better than organic dyes making them very suitable in target detection. Meanwhile, the indirect labeling showed much better specificity than the direct labeling. Furthermore, the prepared CdTe QDs did not show detectable effect on cell growth after having cultured for three days, which suggested that the L-glutathione capped CdTe had scarcely cytotoxicity.

3D label-free prostate specific antigen (PSA) immunosensor based on graphene-gold composites.

Science.gov (United States)

Jang, Hee Dong; Kim, Sun Kyung; Chang, Hankwon; Choi, Jeong-Woo

2015-01-15

Highly sensitive and label-free detection of the prostate specific antigen (PSA) remains a challenge in the diagnosis of prostate cancer. Here, a novel three-dimensional (3D) electrochemical immunosensor capable of sensitive and label-free detection of PSA is reported. This unique immunosensor is equipped with a highly conductive graphene (GR)-based gold (Au) composite modified electrode. The GR-based Au composite is prepared using aerosol spray pyrolysis and the morphology of the composite is the shape of a crumpled GR ball decorated with Au nanoparticles. Unlike the previous research, this novel 3D immunosensor functions very well over a broad linear range of 0-10 ng/mL with a low detection limit of 0.59 ng/mL; furthermore, it exhibits a significantly increased electron transfer and high sensitivity toward PSA. The highest rate of current change with respect to the PSA concentration is 5 μA/(ng/mL). Satisfactory selectivity, reproducibility, and stability of the 3D immunosensor are also exhibited. Copyright © 2014 Elsevier B.V. All rights reserved.

I-125 input into antibodies molecules specific to australian antigen

International Nuclear Information System (INIS)

Abdukayumov, A. M.; Chistyakov, P.G.; Garajshina, G. R.

1999-01-01

There are experimental data on I-125 input into antibodies molecules specific to superficial antigen of hepatitis B virus (australian antigen). Three ways of input are submitted: with the help of T chloramine usage, Bolton-Hunter Reagent and with the help of iodogen. There are also comparative characteristics of iodized products obtained: molar radioactivity, radiochemical frequency, immuno - reactivity. The report also discusses advantages and disadvantages of the used methods for inputting I-125 into antibodies to australian antigen in order to study the possibility of creating radio immunological test system for detecting superficial antigen of B hepatitis

Antigen-specific T cell activation independently of the MHC: chimeric antigen receptor (CAR-redirected T cells.

Directory of Open Access Journals (Sweden)

Hinrich eAbken

2013-11-01

Full Text Available Adoptive T cell therapy has recently shown powerful in initiating a lasting anti-tumor response with spectacular therapeutic success in some cases. Specific T cell therapy, however, is limited since a number of cancer cells are not recognized by T cells due to various mechanisms including the limited availability of tumor-specific T cells and deficiencies in antigen processing or major histocompatibility complex (MHC expression of cancer cells. To make adoptive cell therapy applicable for the broad variety of cancer entities, patient's T cells are engineered ex vivo with pre-defined specificity by a recombinant chimeric antigen receptor (CAR which consists in the extracellular part of an antibody-derived domain for binding with a tumor-associated antigen and in the intracellular part of a TCR-derived signaling moiety for T cell activation. The specificity of CAR mediated T cell recognition is defined by the antibody domain, is independent of MHC presentation and can be extended to any target for which an antibody is available. We discuss the advantages and limitations of MHC-independent T cell targeting by an engineered CAR and review most significant progress recently made in early stage clinical trials to treat cancer.

SDF1-CXCR4 Signaling Contributes to the Transition from Acute to Chronic Pain State.

Science.gov (United States)

Yang, Fei; Sun, Wei; Luo, Wen-Jun; Yang, Yan; Yang, Fan; Wang, Xiao-Liang; Chen, Jun

2017-05-01

Emerging evidence has demonstrated the involvement of stromal cell-derived factor 1 (SDF1, also known as CXCL12)-CXCR4 signaling in a variety of pain state. However, the underlying mechanisms of SDF1-CXCR4 signaling leading to the maintenance of chronic pain states are poorly understood. In the present study, we sought to explore the role of SDF1-CXCR4 signaling in the forming of neuroplasticity by applying a model of the transition from acute to chronic pain state, named as hyperalgesic priming. Utilizing intraplantar bee venom (BV) injection, we successfully established hyperalgesic priming state and found that peripheral treating with AMD3100, a CXCR4 antagonist, or knocking down CXCR4 by intraganglionar CXCR4 small interfering RNA (siRNA) injection could prevent BV-induced primary mechanical hyperalgesia and hyperalgesic priming. Moreover, we showed that single intraplantar active SDF1 protein injection is sufficient to induce acute mechanical hyperalgesia and hyperalgesic priming through CXC4. Intraplantar coinjection of ERK inhibitor, U0126, and PI3K inhibitor, LY294002, as well as two protein translation inhibitors, temsirolimus and cordycepin, prevented the development of SDF1-induced acute mechanical hyperalgesia and hyperalgesic priming. Finally, on the models of complete Freund's adjuvant (CFA)-induced chronic inflammatory pain and spared nerve injury (SNI)-induced chronic neuropathic pain, we observed that knock-down of CXCR4 could both prevent the development and reverse the maintenance of chronic pain state. In conclusion, our present data suggested that through regulating ERK and PI3K-AKT pathways-mediated protein translation SDF1-CXCR4 signaling mediates the transition from acute pain to chronic pain state and finally contributes to the development and maintenance of chronic pain.

Fetal- and uterine-specific antigens in human amniotic fluid.

Science.gov (United States)

Sutcliffe, R G; Brock, D J; Nicholson, L V; Dunn, E

1978-09-01

Removal of the major maternal serum proteins from second trimester amniotic fluid by antibody affinity chromatography revealed various soluble tissue antigens, of which two were fetal-specific skin proteins and another, of alpha2-mobility, was specific to the uterus, and was therefore designated alpha-uterine protein (AUP). These proteins could not be detected in maternal serum by antibody-antigen crossed electrophoresis. The concentration of AUP in amniotic fluid reached a maximum between 10 and 20 weeks of gestation, suggesting that there is an influx of uterine protein into the amniotic fluid at this stage of pregnancy.

Genetic engineering of human NK cells to express CXCR2 improves migration to renal cell carcinoma.

Science.gov (United States)

Kremer, Veronika; Ligtenberg, Maarten A; Zendehdel, Rosa; Seitz, Christina; Duivenvoorden, Annet; Wennerberg, Erik; Colón, Eugenia; Scherman-Plogell, Ann-Helén; Lundqvist, Andreas

2017-09-19

Adoptive natural killer (NK) cell transfer is being increasingly used as cancer treatment. However, clinical responses have so far been limited to patients with hematological malignancies. A potential limiting factor in patients with solid tumors is defective homing of the infused NK cells to the tumor site. Chemokines regulate the migration of leukocytes expressing corresponding chemokine receptors. Various solid tumors, including renal cell carcinoma (RCC), readily secrete ligands for the chemokine receptor CXCR2. We hypothesize that infusion of NK cells expressing high levels of the CXCR2 chemokine receptor will result in increased influx of the transferred NK cells into tumors, and improved clinical outcome in patients with cancer. Blood and tumor biopsies from 14 primary RCC patients were assessed by flow cytometry and chemokine analysis. Primary NK cells were transduced with human CXCR2 using a retroviral system. CXCR2 receptor functionality was determined by Calcium flux and NK cell migration was evaluated in transwell assays. We detected higher concentrations of CXCR2 ligands in tumors compared with plasma of RCC patients. In addition, CXCL5 levels correlated with the intratumoral infiltration of CXCR2-positive NK cells. However, tumor-infiltrating NK cells from RCC patients expressed lower CXCR2 compared with peripheral blood NK cells. Moreover, healthy donor NK cells rapidly lost their CXCR2 expression upon in vitro culture and expansion. Genetic modification of human primary NK cells to re-express CXCR2 improved their ability to specifically migrate along a chemokine gradient of recombinant CXCR2 ligands or RCC tumor supernatants compared with controls. The enhanced trafficking resulted in increased killing of target cells. In addition, while their functionality remained unchanged compared with control NK cells, CXCR2-transduced NK cells obtained increased adhesion properties and formed more conjugates with target cells. To increase the success of NK

New developments in the standardization of total prostate-specific antigen.

Science.gov (United States)

Blijenberg, B G; Storm, B N; Van Zelst, B D; Kruger, A E; Schröder, F H

1999-11-01

Analytical evaluation of the calibration of three recently launched assays for the measurement of total prostate-specific antigen, i.e., IMx Total PSA (Abbott), Elecsys PSA (Roche), and IMMULITE 3rd Generation PSA (DPC). For accuracy assessment two reference materials were applied namely, Stanford 90:10 PSA Calibrator and Certified Reference Material 613 Prostate-Specific Antigen. Dilutions of these preparations were analyzed with all assays. In addition, clinical specimens from known prostate cancer or benign prostate hyperplasia patients and samples taken from an ongoing prostate cancer screening study were used for comparison. Application of the Stanford Calibrator revealed results well within 10% of the calculated values for all assays. Regarding the CRM Calibrator only the IMx Total PSA proved to approach the line of identity. The IMMULITE results differed about 40% and the Elecsys about 18% from the calculated values. The comparison with clinical specimens showed statistically different results for the combination IMMULITE-IMx and for IMMULITE-Elecsys. The regression lines for both collections were: y(IMx) = 0.86x(IMMULITE) +0.12 (n = 104, r = 0.970, Sy/x = 0.883 microg/L) and y(Elecsys) = 0.98x(IMMULITE) +0.38 (n = 97, r = 0.976, Sy/x = 0.733 microg/L). In the lower measuring range (PSA differences were less pronounced. In analytical sense a difference was found for both reference preparations in the assays studied. Clinically, despite improvements in methodology, results for total prostate-specific antigen are still not interchangeable. The possible consequences need to be elaborated.

Specific Nongluten Proteins of Wheat Are Novel Target Antigens in Celiac Disease Humoral Response

Science.gov (United States)

2014-01-01

While the antigenic specificity and pathogenic relevance of immunologic reactivity to gluten in celiac disease have been extensively researched, the immune response to nongluten proteins of wheat has not been characterized. We aimed to investigate the level and molecular specificity of antibody response to wheat nongluten proteins in celiac disease. Serum samples from patients and controls were screened for IgG and IgA antibody reactivity to a nongluten protein extract from the wheat cultivar Triticum aestivum Butte 86. Antibodies were further analyzed for reactivity to specific nongluten proteins by two-dimensional gel electrophoresis and immunoblotting. Immunoreactive molecules were identified by tandem mass spectrometry. Compared with healthy controls, patients exhibited significantly higher levels of antibody reactivity to nongluten proteins. The main immunoreactive nongluten antibody target proteins were identified as serpins, purinins, α-amylase/protease inhibitors, globulins, and farinins. Assessment of reactivity toward purified recombinant proteins further confirmed the presence of antibody response to specific antigens. The results demonstrate that, in addition to the well-recognized immune reaction to gluten, celiac disease is associated with a robust humoral response directed at a specific subset of the nongluten proteins of wheat. PMID:25329597

Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

KAUST Repository

Domina, Maria; Lanza Cariccio, Veronica; Benfatto, Salvatore; D'Aliberti, Deborah; Venza, Mario; Borgogni, Erica; Castellino, Flora; Biondo, Carmelo; D'Andrea, Daniel; Grassi, Luigi; Tramontano, Anna; Teti, Giuseppe; Felici, Franco; Beninati, Concetta

2014-01-01

There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER) provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

Directory of Open Access Journals (Sweden)

Maria Domina

Full Text Available There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

KAUST Repository

Domina, Maria

2014-12-04

There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER) provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

Circulating CD4+CXCR5+ T cells contribute to proinflammatory responses in multiple ways in coronary artery disease.

Science.gov (United States)

Ding, Ru; Gao, Wenwu; He, Zhiqing; Wu, Feng; Chu, Yang; Wu, Jie; Ma, Lan; Liang, Chun

2017-11-01

Coronary artery disease (CAD) is a common subtype of cardiovascular disease. The major contributing event is atherosclerosis, which is a progressive inflammatory condition resulting in the thickening of the arterial wall and the formation of atheromatous plaques. Recent evidence suggests that circulating CD4 + CXCR5 + T cells can contribute to inflammatory reactions. In this study, the frequency, phenotype, and function of circulating CD4 + CXCR5 + T cells in CAD patients were examined. Data showed that circulating CD4 + CXCR5 + T cells in CAD patients were enriched with a PD-1 + CCR7 - subset, which was previously identified as the most potent in B cell help. The CD4 + CXCR5 + T cells in CAD patients also secreted significantly higher levels of IFN-γ, IL-17A, and IL-21 than those from healthy controls. Depleting the PD-1 + population significantly reduced the cytokine secretion. Interestingly, the CD4 + CXCR5 + PD-1 - T cells significantly upregulated PD-1 following anti-CD3/CD28 or SEB stimulation. CD4 + CXCR5 + T cells from CAD patients also demonstrated more potent capacity to stimulate B cell inflammation than those from healthy individuals. The phosphorylation of STAT1 and STAT3 were significantly higher in B cells incubated with CD4 + CXCR5 + T cells from CAD than controls. The IL-6 and IFN-γ expression were also significantly higher in B cells incubated with CD4 + CXCR5 + T cells from CAD. Together, this study demonstrated that CAD patients presented a highly activated CD4 + CXCR5 + T cell subset that could contribute to proinflammatory responses in multiple ways. The possibility of using CD4 + CXCR5 + T cells as a therapeutic target should therefore be examined in CAD patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Cytoplasmic CXCR4 expression in breast cancer: induction by nitric oxide and correlation with lymph node metastasis and poor prognosis

International Nuclear Information System (INIS)

Yasuoka, Hironao; Tsujimoto, Masahiko; Yoshidome, Katsuhide; Nakahara, Masaaki; Kodama, Rieko; Sanke, Tokio; Nakamura, Yasushi

2008-01-01

Lymph nodes constitute the first site of metastasis for most malignancies, and the extent of lymph node involvement is a major criterion for evaluating patient prognosis. The CXC chemokine receptor 4 (CXCR4) has been shown to play an important role in lymph node metastasis. Nitric oxide (NO) may also contribute to induction of metastatic ability in human cancers. CXCR4 expression was analyzed in primary human breast carcinoma with long-term follow-up. The relationship between nitrotyrosine levels (a biomarker for peroxynitrate formation from NO in vivo) and lymph node status, CXCR4 immunoreactivity, and other established clinico-pathological parameters, as well as prognosis, was analyzed. Nitrite/nitrate levels and CXCR4 expressions were assessed in MDA-MB-231 and SK-BR-3 breast cancer cell lines after induction and/or inhibition of NO synthesis. CXCR4 staining was predominantly cytoplasmic; this was observed in 50%(56/113) of the tumors. Cytoplasmic CXCR4 expression was significantly correlated with nitrotyrosine levels and lymph node metastasis. Kaplan-Meier survival curves showed that cytoplasmic CXCR4 expression was associated with reduced disease-free and overall survival. In multivariate analysis, cytoplasmic CXCR4 expression emerged as a significant independent predictor for overall and disease-free survival. Cytoplasmic expression of functional CXCR4 in MDA-MB-231 and SK-BR-3 cells was increased by treatment with the NO donor DETA NONOate. This increase was abolished by L-NAME, an inhibitor of NOS. Our data showed a role for NO in stimulating cytoplasmic CXCR4 expression in vitro. Formation of the biomarker nitrotyrosine was also correlated with CXCR4 expression and lymph node metastasis in vivo. In addition, cytoplasmic CXCR4 expression may serve as a significant prognostic factor for long-term survival in breast cancer

Data on human neutrophil activation induced by pepducins with amino acid sequences derived from β2AR and CXCR4

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André Holdfeldt

2016-09-01

Full Text Available The data described here is related to the research article titled (Gabl et al., 2016 [1]. Pepducins with peptide sequence derived from one of the intracellular domains of a given G-protein coupled receptor (GPCR can either activate or inhibit cell functions. Here we include data on human neutrophil function induced by pepducins derived from β2AR (ICL3-8 and CXCR4 (ATI-2341, respectively. ICL3-8 exerts neither direct activating effect on the NADPH-oxidase as measured by superoxide release nor inhibitory effect on FPR signaling. ATI-2341 dose-dependently triggers neutrophil activation and these cells were subsequently desensitized in their response to FPR2 specific agonists F2Pal10 and WKYMVM. Moreover, the ATI-2341 response is inhibited by PBP10 and the peptidomimetic Pam-(Lys-betaNSpe6-NH2 (both are FPR2 specific inhibitors, but not to the FPR1 specific inhibitor cyclosporine H.

Prostate specific antigen velocity does not aid prostate cancer detection in men with prior negative biopsy.

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Vickers, Andrew J; Wolters, Tineke; Savage, Caroline J; Cronin, Angel M; O'Brien, M Frank; Roobol, Monique J; Aus, Gunnar; Scardino, Peter T; Hugosson, Jonas; Schröder, Fritz H; Lilja, Hans

2010-09-01

Prostate specific antigen velocity has been proposed as a marker to aid in prostate cancer detection. We determined whether prostate specific antigen velocity could predict repeat biopsy results in men with persistently increased prostate specific antigen after initial negative biopsy. We identified 1,837 men who participated in the Göteborg or Rotterdam section of the European Randomized Screening study of Prostate Cancer and who underwent 1 or more subsequent prostate biopsies after an initial negative finding. We evaluated whether prostate specific antigen velocity improved predictive accuracy beyond that of prostate specific antigen alone. Of the 2,579 repeat biopsies 363 (14%) were positive for prostate cancer, of which 44 (1.7%) were high grade (Gleason score 7 or greater). Prostate specific antigen velocity was statistically associated with cancer risk but had low predictive accuracy (AUC 0.55, p <0.001). There was some evidence that prostate specific antigen velocity improved AUC compared to prostate specific antigen for high grade cancer. However, the small increase in risk associated with high prostate specific antigen velocity (from 1.7% to 2.8% as velocity increased from 0 to 1 ng/ml per year) had questionable clinical relevance. Men with prior negative biopsy are at lower risk for prostate cancer at subsequent biopsies with high grade disease particularly rare. We found little evidence to support prostate specific antigen velocity to aid in decisions about repeat biopsy for prostate cancer. 2010 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

THE ANTIGEN-SPECIFIC CELL IN VITRO TESTS FOR POST-VACCINATION ANTIPLAGUE IMMUNITY FORMATION

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A. N. Kulichenko

2017-01-01

Full Text Available The possibility of post-vaccination anti-plague immunity evaluation was researched using antigen-stimulated cells tests in vitro and cytometry analysis. The object of study — the blood samples of 17 people immunised by the live plague vaccine (Yersinia pestis EV epicutaneously. Blood taking was carried out before vaccination and after immunisation on 7 and on 21 days, in 3 and in 6 months. Intensity antigen reactivity of lymphocytes was detected by cell tests in vitro, analysing markers of early (CD45+CD3+CD25+ and late (CD45+CD3+HLA-DR+ lymphocyte activation using flow cytometry. The complex of water-soluble Y. pestis antigens and allergen — pestin PP was tested as antigen. The high stimulating potential was defined of the water-soluble antigens Y. pestis complex. It is shown that coefficient of stimulation of relative level T- lymphocytes which express receptors for IL-2 was positive for all observation times after immunisation. The coefficient of stimulation had maximum values at 21 days (56.37% and at 3 (47.41% months. In identifying HLADR-positive lymphocytes before vaccination, the negative coefficient of stimulation was indicated on 7 and 21 days and the positive coefficient of stimulation was indicated at 3 and at 6 months. Analysis of intensity expression of early and late lymphocyte activation markers dynamics showed the possibility and prospect of application of cellular in vitro tests for the laboratory evaluation of specific reactivity of cellular immunity in both the early (7 days and late (6 months periods after vaccination. The results can be the basis for developing a new algorithm for assessment of immunological effectiveness of vaccination people against plague. It is the algorithm based on the identification of lymphocyte activation markers by antigen stimulation in conditions in vitro.

Genetic modification of mesenchymal stem cells to overexpress CXCR4 and CXCR7 does not improve the homing and therapeutic potentials of these cells in experimental acute kidney injury.

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Gheisari, Yousof; Azadmanesh, Kayhan; Ahmadbeigi, Naser; Nassiri, Seyed Mahdi; Golestaneh, Azadeh Fahim; Naderi, Mahmood; Vasei, Mohammad; Arefian, Ehsan; Mirab-Samiee, Siamak; Shafiee, Abbas; Soleimani, Masoud; Zeinali, Sirous

2012-11-01

The therapeutic potential of bone marrow mesenchymal stem cells (MSCs) in kidney failure has been examined in some studies. However, recent findings indicate that after transplantation, these cells home to kidneys at very low levels. Interaction of stromal derived factor-1 (SDF-1) with its receptor, CXCR4, is of pivotal importance in migration and homing. Recently, CXCR7 has also been recognized as another SDF-1 receptor that interacts with CXCR4 and modulates its functions. In this study, CXCR4 and CXCR7 were separately and simultaneously overexpressed in BALB/c bone marrow MSCs by using a lentiviral vector system and the homing and renoprotective potentials of these cells were evaluated in a mouse model of cisplatin-induced acute kidney injury. Using flow cytometry, immunohistochemistry, and real-time PCR methods for detection of GFP-labeled MSCs, we found that although considerably entrapped in lungs, native MSCs home very rarely to kidneys and bone marrow and this rate cannot be significantly affected by CXCR4 and/or CXCR7 upregulation. Transplantation of neither native nor genetically engineered MSCs ameliorated kidney failure. We concluded that overexpression of CXCR4 and CXCR7 receptors in murine MSCs cannot improve the homing and therapeutic potentials of these cells and it can be due to severe chromosomal abnormalities that these cells bear during ex vivo expansion.

Coexpression of EGFR and CXCR4 predicts poor prognosis in resected pancreatic ductal adenocarcinoma.

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Huanwen Wu

Full Text Available Epidermal growth factor receptor (EGFR is highly expressed in pancreatic ductal adenocarcinoma (PDAC and is involved in tumorigenesis and development. However, EGFR expression alone has limited clinical and prognostic significance. Recently, the cross-talk between EGFR and G-protein-coupled chemokine receptor CXCR4 has become increasingly recognized.In the present study, immunohistochemical staining of EGFR and CXCR4 was performed on paraffin-embedded specimens from 131 patients with surgically resected PDAC. Subsequently, the associations between EGFR expression, CXCR4 expression, EGFR/CXCR4 coexpression and clinicopathologic factors were assessed, and survival analyses were performed.In total, 64 (48.9% patients expressed EGFR, 68 (51.9% expressed CXCR4, and 33 (25.2% coexpressed EGFR and CXCR4. No significant association between EGFR and CXCR4 expression was observed (P = 0.938. EGFR expression significantly correlated with tumor differentiation (P = 0.031, whereas CXCR4 expression significantly correlated with lymph node metastasis (P = 0.001. EGFR/CXCR4 coexpression was significantly associated with lymph node metastasis (P = 0.026, TNM stage (P = 0.048, and poor tumor differentiation (P = 0.004. By univariate survival analysis, both CXCR4 expression and EGFR/CXCR4 coexpression were significant prognostic factors for poor disease-free survival (DFS and overall survival (OS. Moreover, EGFR/CXCR4 coexpression significantly increased the hazard ratio for both recurrence and death compared with EGFR or CXCR4 protein expression alone. Multivariate survival analysis demonstrated that EGFR/CXCR4 coexpression was an independent prognostic factor for DFS (HR = 2.33, P<0.001 and OS (HR = 2.48, P = 0.001.In conclusion, our data indicate that although EGFR expression alone has limited clinical and prognostic significance, EGFR/CXCR4 coexpression identified a subset of PDAC patients with more aggressive tumor characteristics and a significantly worse

Targeting the CXCR4 pathway using a novel anti-CXCR4 IgG1 antibody (PF-06747143 in chronic lymphocytic leukemia

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Manoj K. Kashyap

2017-05-01

Full Text Available Abstract Background The CXCR4-CXCL12 axis plays an important role in the chronic lymphocytic leukemia (CLL-microenvironment interaction. Overexpression of CXCR4 has been reported in different hematological malignancies including CLL. Binding of the pro-survival chemokine CXCL12 with its cognate receptor CXCR4 induces cell migration. CXCL12/CXCR4 signaling axis promotes cell survival and proliferation and may contribute to the tropism of leukemia cells towards lymphoid tissues and bone marrow. Therefore, we hypothesized that targeting CXCR4 with an IgG1 antibody, PF-06747143, may constitute an effective therapeutic approach for CLL. Methods Patient-derived primary CLL-B cells were assessed for cytotoxicity in an in vitro model of CLL microenvironment. PF-06747143 was analyzed for cell death induction and for its potential to interfere with the chemokine CXCL12-induced mechanisms, including migration and F-actin polymerization. PF-06747143 in vivo efficacy was determined in a CLL murine xenograft tumor model. Results PF-06747143, a novel-humanized IgG1 CXCR4 antagonist antibody, induced cell death of patient-derived primary CLL-B cells, in presence or absence of stromal cells. Moreover, cell death induction by the antibody was independent of CLL high-risk prognostic markers. The cell death mechanism was dependent on CXCR4 expression, required antibody bivalency, involved reactive oxygen species production, and did not require caspase activation, all characteristics reminiscent of programmed cell death (PCD. PF-06747143 also induced potent B-CLL cytotoxicity via Fc-driven antibody-dependent cell-mediated cytotoxicity (ADCC and complement-dependent cytotoxicity activity (CDC. PF-06747143 had significant combinatorial effect with standard of care (SOC agents in B-CLL treatment, including rituximab, fludarabine (F-ara-A, ibrutinib, and bendamustine. In a CLL xenograft model, PF-06747143 decreased tumor burden and improved survival as a monotherapy

Specific binding of antigen-antibody in physiological environments: Measurement, force characteristics and analysis

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Gu, Xin; Zhou, Jun; Zhou, Lu; Xie, Shusen; Petti, Lucia; Wang, Shaomin; Wang, Fuyan

2018-05-01

The specific recognition of the antigen by the antibody is the crucial step in immunoassays. Measurement and analysis of the specific recognition, including the ways in which it is influenced by external factors are of paramount significance for the quality of the immunoassays. Using prostate-specific antigen (PSA)/anti-PSA antibody and α-fetoprotein (AFP) /anti-AFP antibody as examples, we have proposed a novel solution for measuring the binding forces between the antigens and their corresponding antibodies in different physiological environments by combining laminar flow control technology and optical tweezers technology. On the basis of the experimental results, the different binding forces of PSA/anti-PSA antibody and AFP/anti-AFP antibody in the same phosphate-buffered saline (PBS) environments are analysed by comparing the affinity constant of the two antibodies and the number of antigenic determinants of the two antigens. In different electrolyte environments, the changes of the binding force of antigens-antibodies are explained by the polyelectrolyte effect and hydrophobic interaction. Furthermore, in different pH environments, the changes of binding forces of antigens-antibodies are attributed to the role of the denaturation of protein. The study aims to recognise the antigen-antibody immune mechanism, thus ensuring further understanding of the biological functions of tumour markers, and it promises to be very useful for the clinical diagnosis of early-stage cancer.

The chemokine CXCL16 and its receptor, CXCR6, as markers and promoters of inflammation-associated cancers.

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Merav Darash-Yahana

2009-08-01

Full Text Available Clinical observations and mouse models have suggested that inflammation can be pro-tumorigenic. Since chemokines are critical in leukocyte trafficking, we hypothesized that chemokines play essential roles in inflammation-associated cancers. Screening for 37 chemokines in prostate cancer cell lines and xenografts revealed CXCL16, the ligand for the receptor CXCR6, as the most consistently expressed chemokine. Immunohistochemistry and/or immunofluorescence and confocal imaging of 121 human prostate specimens showed that CXCL16 and CXCR6 were co-expressed, both on prostate cancer cells and adjacent T cells. Expression levels of CXCL16 and CXCR6 on cancer cells correlated with poor prognostic features including high-stage and high-grade, and expression also correlated with post-inflammatory changes in the cancer stroma as revealed by loss of alpha-smooth muscle actin. Moreover, CXCL16 enhanced the growth of CXCR6-expressing cancer and primary CD4 T cells. We studied expression of CXCL16 in an additional 461 specimens covering 12 tumor types, and found that CXCL16 was expressed in multiple human cancers associated with inflammation. Our study is the first to describe the expression of CXCL16/CXCR6 on both cancer cells and adjacent T cells in humans, and to demonstrate correlations between CXCL16 and CXCR6 vs. poor both prognostic features and reactive changes in cancer stoma. Taken together, our data suggest that CXCL16 and CXCR6 may mark cancers arising in an inflammatory milieu and mediate pro-tumorigenic effects of inflammation through direct effects on cancer cell growth and by inducing the migration and proliferation of tumor-associated leukocytes.

Clone-specific MYD88 L265P and CXCR4 mutation status can provide clinical utility in suspected Waldenström macroglobulinemia/lymphoplasmacytic lymphoma.

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Burnworth, Bettina; Wang, Zhixing; Singleton, Timothy P; Bennington, Angela; Fritschle, Wayne; Bennington, Richard; Brodersen, Lisa Eidenschink; Wells, Denise A; Loken, Michael R; Zehentner, Barbara K

2016-12-01

MYD88 L265P, a diagnostic marker for lymphoplasmacytic lymphoma (LPL)/Waldenström macroglobulinemia (WM) can also be detected in other hematopoietic malignancies. We demonstrate a novel approach to increase the specificity of this marker for WM/LPL diagnosis by combining flow cytometric cell sorting with molecular analysis. Clonal B-lymphocyte and co-occurring clonal plasma cell populations of low-grade B-cell lymphomas were sorted by flow cytometry and analyzed for immunoglobulin gene rearrangements (PCR), and for MYD88 and CXCR4 mutations. Identical clonal origin was confirmed by PCR for 21 LPL/WM cases and MYD88 L265P was detected in both B-cell and plasma cell fractions. 9/20 other B-cell lymphomas with identical light chain restriction on B-cells and plasma cells were genotypically identical by PCR and MYD88 L265P was detected in both cell fractions in 7/9 whereas in 11/20 specimens with different clonal origin, MYD88 L265P was absent (5/11), or only found in B-lymphocytes (4/11), or plasma cells (2/11). CXCR4 mutations were detected in 17/39 cases, but missed in 63% of these without cell sorting. Confirming MYD88L265P in both B-cells and plasma cell fractions can provide a novel and powerful discriminator to distinguish LPL/WM from phenotypically similar disorders. Furthermore, this approach significantly increases CXCR4 detection sensitivity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Toward low-cost affinity reagents: lyophilized yeast-scFv probes specific for pathogen antigens.

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Sean A Gray

Full Text Available The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeast-scFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigen-specific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2-3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewable alternatives to monoclonal antibodies for diagnosis and research.

Prostate Specific Membrane Antigen (PSMA) Targeted Bio-orthogonal Therapy for Metastatic Prostate Cancer

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2017-10-01

AWARD NUMBER: W81XWH-16-1-0595 TITLE: Prostate-Specific Membrane Antigen (PSMA) Targeted Bio -orthogonal Therapy for Metastatic Prostate Cancer...Sep 2016 - 14 Sep 2017 4. TITLE AND SUBTITLE Prostate-Specific Membrane Antigen (PSMA) Targeted Bio -orthogonal Therapy for Metastatic Prostate

Prostate-specific antigen-based prostate cancer screening: Past and future.

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Alberts, Arnout R; Schoots, Ivo G; Roobol, Monique J

2015-06-01

Prostate-specific antigen-based prostate cancer screening remains a controversial topic. Up to now, there is worldwide consensus on the statement that the harms of population-based screening, mainly as a result of overdiagnosis (the detection of clinically insignificant tumors that would have never caused any symptoms), outweigh the benefits. However, worldwide opportunistic screening takes place on a wide scale. The European Randomized Study of Screening for Prostate Cancer showed a reduction in prostate cancer mortality through prostate-specific antigen based-screening. These population-based data need to be individualized in order to avoid screening in those who cannot benefit and start screening in those who will. For now, lacking a more optimal screening approach, screening should only be started after the process of shared decision-making. The focus of future research is the reduction of unnecessary testing and overdiagnosis by further research to better biomarkers and the value of the multiparametric magnetic resonance imaging, potentially combined in already existing prostate-specific antigen-based multivariate risk prediction models. © 2015 The Japanese Urological Association.

Immunophenotyping of Waldenstroms macroglobulinemia cell lines reveals distinct patterns of surface antigen expression: potential biological and therapeutic implications.

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Aneel Paulus

Full Text Available Waldenströms macroglobulinemia (WM is a subtype of Non-Hodgkin's lymphoma in which the tumor cell population is markedly heterogeneous, consisting of immunoglobulin-M secreting B-lymphocytes, plasmacytoid lymphocytes and plasma cells. Due to rarity of disease and scarcity of reliable preclinical models, many facets of WM molecular and phenotypic architecture remain incompletely understood. Currently, there are 3 human WM cell lines that are routinely used in experimental studies, namely, BCWM.1, MWCL-1 and RPCI-WM1. During establishment of RPCI-WM1, we observed loss of the CD19 and CD20 antigens, which are typically present on WM cells. Intrigued by this observation and in an effort to better define the immunophenotypic makeup of this cell line, we conducted a more comprehensive analysis for the presence or absence of other cell surface antigens that are present on the RPCI-WM1 model, as well as those on the two other WM cell lines, BCWM.1 and MWCL-1. We examined expression of 65 extracellular and 4 intracellular antigens, comprising B-cell, plasma cell, T-cell, NK-cell, myeloid and hematopoietic stem cell surface markers by flow cytometry analysis. RPCI-WM1 cells demonstrated decreased expression of CD19, CD20, and CD23 with enhanced expression of CD28, CD38 and CD184, antigens that were differentially expressed on BCWM.1 and MWCL-1 cells. Due to increased expression of CD184/CXCR4 and CD38, RPCI-WM1 represents a valuable model in which to study the effects anti-CXCR4 or anti-CD38 targeted therapies that are actively being developed for treatment of hematologic cancers. Overall, differences in surface antigen expression across the 3 cell lines may reflect the tumor clone population predominant in the index patients, from whom the cell lines were developed. Our analysis defines the utility of the most commonly employed WM cell lines as based on their immunophenotype profiles, highlighting unique differences that can be further studied for

Zinc supplementation induces CD4+CD25+Foxp3+ antigen-specific regulatory T cells and suppresses IFN-γ production by upregulation of Foxp3 and KLF-10 and downregulation of IRF-1.

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Maywald, Martina; Rink, Lothar

2017-08-01

The essential trace element zinc plays a fundamental role in immune function and regulation since its deficiency is associated with autoimmunity, allergies, and transplant rejection. Thus, we investigated the influence of zinc supplementation on the Th1-driven alloreaction in mixed lymphocyte cultures (MLC), on generation of antigen-specific T cells, and analyzed underlying molecular mechanisms. Cell proliferation and pro-inflammatory cytokine production were monitored by [ 3 H]-thymidine proliferation assay and ELISA, respectively. Analysis of surface and intracellular T cell marker was performed by flow cytometry. Western blotting and mRNA analysis were used for Foxp3, KLF-10, and IRF-1 expression. Zinc supplementation on antigen-specific T cells in physiological doses (50 µM) provokes a significant amelioration of cell proliferation and pro-inflammatory cytokine production after reactivation compared to untreated controls. Zinc administration on MLC results in an increased induction and stabilization of CD4 + CD25 + Foxp3 + and CD4 + CD25 + CTLA-4 + T cells (p zinc-induced upregulation of Foxp3 and KLF-10 and downregulation of IRF-1. However, in resting lymphocytes zinc increases IRF-1. In summary, zinc is capable of ameliorating the allogeneic immune reaction by enhancement of antigen-specific iTreg cells due to modulation of essential molecular targets: Foxp3, KLF-10, and IRF-1. Thus, zinc can be seen as an auspicious tool for inducing tolerance in adverse immune reactions.

Pathological Outcome following Radical Prostatectomy in Men with Prostate Specific Antigen Greater than 10 ng/ml and Histologically Favorable Risk Prostate Cancer.

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Yu, Jiwoong; Kwon, Young Suk; Kim, Sinae; Han, Christopher Sejong; Farber, Nicholas; Kim, Jongmyung; Byun, Seok Soo; Kim, Wun-Jae; Jeon, Seong Soo; Kim, Isaac Yi

2016-05-01

Active surveillance is now the treatment of choice in men with low risk prostate cancer. Although there is no consensus on which patients are eligible for active surveillance, prostate specific antigen above 10 ng/ml is generally excluded. In an attempt to determine the validity of using a prostate specific antigen cutoff of 10 ng/ml to counsel men considering active surveillance we analyzed a multi-institution database to determine the pathological outcome in men with prostate specific antigen greater than 10 ng/ml but histologically favorable risk prostate cancer. We queried a prospectively maintained database of men with histologically favorable risk prostate cancer who underwent radical prostatectomy between 2003 and 2015. The cohort was categorized into 3 groups based on prostate specific antigen level, including low-less than 10 ng/ml, intermediate-10 or greater to less than 20 and high-20 or greater. Associations of prostate specific antigen group with adverse pathological and oncologic outcomes were analyzed. Of 2,125 patients 1,327 were categorized with histologically favorable risk disease. However on multivariate analyses the rates of up staging and upgrading were similar between the intermediate and low prostate specific antigen groups. In contrast compared to the intermediate prostate specific antigen group the high group had higher incidences of up staging (p = 0.02) and upgrading to 4 + 3 or greater disease (p = 0.046). Biochemical recurrence-free survival rates revealed no pairwise intergroup differences except between the low and high groups. Patients with preoperatively elevated prostate specific antigen between 10 and less than 20 ng/ml who otherwise had histologically favorable risk prostate cancer were not at higher risk for adverse pathological outcomes than men with prostate specific antigen less than 10 ng/ml. Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

miR-539-5p inhibits experimental choroidal neovascularization by targeting CXCR7.

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Feng, Yifan; Wang, Jing; Yuan, Yuanzhi; Zhang, Xi; Shen, Minqian; Yuan, Fei

2018-03-01

Stromal cell-derived factor-1 (SDF-1) has been previously confirmed to participate in the formation of choroidal neovascularization (CNV) via its receptor, CXC chemokine receptor (CXCR) 4; CXCR7 is a recently identified receptor for SDF-1. The molecular mechanisms and therapeutic value of CXCR7 in CNV remain undefined. In this study, experimental CNV was induced by laser photocoagulation in Brown-Norway pigmented rats, and aberrant CXCR7 overexpression was detected in the retinal pigment epithelial/choroid/sclera tissues of laser-injured eyes. Blockade of CXCR7 activation via CXCR7 knockdown or neutralizing Ab administration inhibited SDF-1-induced cell survival and the tubular formation of human retinal microvascular endothelial cells (HRMECs) in vitro and reduced CNV leakage and lesion size in vivo. By using microRNA array screening and bioinformatic analyses, we identified miR-539-5p as a regulator of CXCR7. Transfection of HRMECs and choroid-retinal endothelial (RF/6A) cells with the miR-539-5p mimic inhibited their survival and tube formation, whereas CXCR7 overexpression rescued the suppressive effect of miR-539-5p. The antiangiogenic activities of the miR-539-5p mimic were additionally demonstrated in vivo by intravitreal injection. ERK1/2 and AKT signaling downstream of CXCR7 is involved in the miR-539-5p regulation of endothelial cell behaviors. These findings suggest that the manipulation of miR-539-5p/CXCR7 levels may have important therapeutic implications in CNV-associated diseases.-Feng, Y., Wang, J., Yuan, Y., Zhang, X., Shen, M., Yuan, F. miR-539-5p inhibits experimental choroidal neovascularization by targeting CXCR7.

Chemokine CXCL13 mediates orofacial neuropathic pain via CXCR5/ERK pathway in the trigeminal ganglion of mice.

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Zhang, Qian; Cao, De-Li; Zhang, Zhi-Jun; Jiang, Bao-Chun; Gao, Yong-Jing

2016-07-11

Trigeminal nerve damage-induced neuropathic pain is a severely debilitating chronic orofacial pain syndrome. Spinal chemokine CXCL13 and its receptor CXCR5 were recently demonstrated to play a pivotal role in the pathogenesis of spinal nerve ligation-induced neuropathic pain. Whether and how CXCL13/CXCR5 in the trigeminal ganglion (TG) mediates orofacial pain are unknown. The partial infraorbital nerve ligation (pIONL) was used to induce trigeminal neuropathic pain in mice. The expression of ATF3, CXCL13, CXCR5, and phosphorylated extracellular signal-regulated kinase (pERK) in the TG was detected by immunofluorescence staining and western blot. The effect of shRNA targeting on CXCL13 or CXCR5 on pain hypersensitivity was checked by behavioral testing. pIONL induced persistent mechanical allodynia and increased the expression of ATF3, CXCL13, and CXCR5 in the TG. Inhibition of CXCL13 or CXCR5 by shRNA lentivirus attenuated pIONL-induced mechanical allodynia. Additionally, pIONL-induced neuropathic pain and the activation of ERK in the TG were reduced in Cxcr5 (-/-) mice. Furthermore, MEK inhibitor (PD98059) attenuated mechanical allodynia and reduced TNF-α and IL-1β upregulation induced by pIONL. TNF-α inhibitor (Etanercept) and IL-1β inhibitor (Diacerein) attenuated pIONL-induced orofacial pain. Finally, intra-TG injection of CXCL13 induced mechanical allodynia, increased the activation of ERK and the production of TNF-α and IL-1β in the TG of WT mice, but not in Cxcr5 (-/-) mice. Pretreatment with PD98059, Etanercept, or Diacerein partially blocked CXCL13-induced mechanical allodynia, and PD98059 also reduced CXCL13-induced TNF-α and IL-1β upregulation. CXCL13 and CXCR5 contribute to orofacial pain via ERK-mediated proinflammatory cytokines production. Targeting CXCL13/CXCR5/ERK/TNF-α and IL-1β pathway in the trigeminal ganglion may offer effective treatment for orofacial neuropathic pain.

Enhanced specificity in immunoscreening of expression cDNA clones using radiolabeled antigen overlay

International Nuclear Information System (INIS)

Chao, S.; Chao, L.; Chao, J.

1989-01-01

A highly sensitive and specific method has been developed for immunoscreening clones from an expression cDNA library. The procedures utilize a radiolabeled antigen detection method described originally for the immunoblotting of plasma proteins. Screening of rat alpha 1-antitrypsin clones was used. Comparison between Western blots of alpha 1-antitrypsin using both labeled antigen and protein A detection methods showed that the former yielded lower background and greater sensitivity than the latter. Further, this technique was shown to have a lower detection limit of less than 20 ng through Western blot analysis of varying concentrations of alpha 1-antitrypsin. The procedures are based on the expression of the protein by cDNA clones containing the DNA inserts in the correct reading frame. Following the transfer of phage proteins to nitrocellulose membranes, the bivalent antibodies bind monovalently to both nitrocellulose-bound-antigen in the phage lysates and radiolabeled antigen. The radiolabeled antigen overlay method is superior to the protein A detection method in sensitivity, specificity and reproducibility. This improved method can be applied in general for screening expression cDNA libraries, provided that the specific antiserum and radiolabeled antigen are available

Immunizations with hepatitis B viral antigens and a TLR7/8 agonist adjuvant induce antigen-specific immune responses in HBV-transgenic mice

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Ying Wang

2014-12-01

Conclusions: Immunization with CL097-conjugated HBV-Ag reversed immune tolerance in HBV-Tg mice and induced antigen-specific immune responses. TLR7/8 agonists appear to be potent adjuvants for the induction of antigen-specific Th1 responses in an immune tolerant state.

Use of a solid-phase radioimmunoassay and formalin-fixed whole bacterial antigen in the detection of antigen-specific immunoglobulin in prostatic fluid.

OpenAIRE

Shortliffe, L M; Wehner, N; Stamey, T A

1981-01-01

The prostatic fluid of two patients with Escherichia coli bacterial prostatitis was analyzed for evidence of a local immune response to bacterial infection. A solid-phase radioimmunoassay was modified to measure the immunoglobulin (Ig)A and IgG antigen-specific antibody responses to infecting bacteria in serum and prostatic fluid from patient. Formalin-fixed whole E. coli were used as antigen. In one patient with acute E. coli prostatic infection, measurements of antigen-specific antibody con...

Agonist-induced CXCR4 and CB2 Heterodimerization Inhibits Gα13/RhoA-mediated Migration.

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Scarlett, Kisha A; White, El-Shaddai Z; Coke, Christopher J; Carter, Jada R; Bryant, Latoya K; Hinton, Cimona V

2018-04-01

G-protein-coupled receptor (GPCR) heterodimerization has emerged as a means by which alternative signaling entities can be created; yet, how receptor heterodimers affect receptor pharmacology remains unknown. Previous observations suggested a biochemical antagonism between GPCRs, CXCR4 and CB2 (CNR2), where agonist-bound CXCR4 and agonist-bound CB2 formed a physiologically nonfunctional heterodimer on the membrane of cancer cells, inhibiting their metastatic potential in vitro However, the reduced signaling entities responsible for the observed functional outputs remain elusive. This study now delineates the signaling mechanism whereby heterodimeric association between CXCR4 and CB2, induced by simultaneous agonist treatment, results in decreased CXCR4-mediated cell migration, invasion, and adhesion through inhibition of the Gα13/RhoA signaling axis. Activation of CXCR4 by its cognate ligand, CXCL12, stimulates Gα13 (GNA13), and subsequently, the small GTPase RhoA, which is required for directional cell migration and the metastatic potential of cancer cells. These studies in prostate cancer cells demonstrate decreased protein expression levels of Gα13 and RhoA upon simultaneous CXCR4/CB2 agonist stimulation. Furthermore, the agonist-induced heterodimer abrogated RhoA-mediated cytoskeletal rearrangement resulting in the attenuation of cell migration and invasion of an endothelial cell barrier. Finally, a reduction was observed in the expression of integrin α5 (ITGA5) upon heterodimerization, supported by decreased cell adhesion to extracellular matrices in vitro Taken together, the data identify a novel pharmacologic mechanism for the modulation of tumor cell migration and invasion in the context of metastatic disease. Implications: This study investigates a signaling mechanism by which GPCR heterodimerization inhibits cancer cell migration. Mol Cancer Res; 16(4); 728-39. ©2018 AACR . ©2018 American Association for Cancer Research.

Role of prostate specific antigen and immediate confirmatory biopsy in predicting progression during active surveillance for low risk prostate cancer.

Science.gov (United States)

Adamy, Ari; Yee, David S; Matsushita, Kazuhito; Maschino, Alexandra; Cronin, Angel; Vickers, Andrew; Guillonneau, Bertrand; Scardino, Peter T; Eastham, James A

2011-02-01

We evaluated predictors of progression after starting active surveillance, especially the role of prostate specific antigen and immediate confirmatory prostate biopsy. A total of 238 men with prostate cancer met active surveillance eligibility criteria and were analyzed for progression with time. Cox proportional hazards regression was used to evaluate predictors of progression. Progression was evaluated using 2 definitions, including no longer meeting 1) full and 2) modified criteria, excluding prostate specific antigen greater than 10 ng/ml as a criterion. Using full criteria 61 patients progressed during followup. The 2 and 5-year progression-free probability was 80% and 60%, respectively. With prostate specific antigen included in progression criteria prostate specific antigen at confirmatory biopsy (HR 1.29, 95% CI 1.14-1.46, p <0.0005) and positive confirmatory biopsy (HR 1.75, 95% CI 1.01-3.04, p = 0.047) were independent predictors of progression. Of the 61 cases 34 failed due to increased prostate specific antigen, including only 5 with subsequent progression by biopsy criteria. When prostate specific antigen was excluded from progression criteria, only 32 cases progressed, and 2 and 5-year progression-free probability was 91% and 76%, respectively. Using modified criteria as an end point positive confirmatory biopsy was the only independent predictor of progression (HR 3.16, 95% CI 1.41-7.09, p = 0.005). Active surveillance is feasible in patients with low risk prostate cancer and most patients show little evidence of progression within 5 years. There is no clear justification for treating patients in whom prostate specific antigen increases above 10 ng/ml in the absence of other indications of tumor progression. Patients considering active surveillance should undergo confirmatory biopsy to better assess the risk of progression. Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Antigenic specificity of serum antibodies in mice fed soy protein

DEFF Research Database (Denmark)

Christensen, Hanne Risager; Bruun, S.W.; Frøkiær, Hanne

2003-01-01

Background: Soybean protein is used in a number of food products but unfortunately is also a common cause of food allergy. Upon ingestion of soy protein, healthy mice like other animals and humans generate a soy-specific antibody response in the absence of signs of illness. Not much is known about...... the relationship between the immunogenic proteins involved in this nondeleterious antibody response and the pathological response associated with food allergy. The objective of the present study was to characterize the antigenic specificity of the soy protein-specific antibody response generated in healthy mice...... ingesting soy protein. Methods: Blood from mice fed a soy-containing diet was analyzed using ELISA and immunoblot for antibody reactivity towards various soy protein fractions and pure soy proteins/subunits. Mice bred on a soy-free diet were used as controls. Results: The detectable antigenic specificity...

A radiogallium-DOTA-based bivalent peptidic ligand targeting a chemokine receptor, CXCR4, for tumor imaging.

Science.gov (United States)

Sano, Kohei; Masuda, Ryo; Hisada, Hayato; Oishi, Shinya; Shimokawa, Kenta; Ono, Masahiro; Fujii, Nobutaka; Saji, Hideo; Mukai, Takahiro

2014-03-01

We have developed a novel radiogallium (Ga)-DOTA-based bivalent peptidic ligand targeting a chemokine receptor, CXCR4, for tumor imaging. A CXCR4 imaging probe with two CXCR4 antagonists (Ac-TZ14011) on Ga-DOTA core, Ga-DOTA-TZ2, was synthesized, and the affinity and binding to CXCR4 was evaluated in CXCR4 expressing cells in vitro. The affinity of Ga-DOTA-TZ2 for CXCR4 was 20-fold greater than the corresponding monovalent probe, Ga-DOTA-TZ1. (67)Ga-DOTA-TZ2 showed the significantly higher accumulation in CXCR4-expressing tumor cells compared with (67)Ga-DOTA-TZ1, suggesting the bivalent effect enhances its binding to CXCR4. The incorporation of two CXCR4 antagonists to Ga-DOTA could be effective in detecting CXCR4-expressing tumors. Copyright © 2014 Elsevier Ltd. All rights reserved.

Pretreatment prostate-specific antigen doubling times: clinical utility of this predictor of prostate cancer behavior

International Nuclear Information System (INIS)

Hanks, Gerald E.; Hanlon, Alexandra L.; Lee, W. Robert; Slivjak, Anne; Schultheiss, Timothy E.

1996-01-01

Purpose: The distribution of pretreatment and posttreatment prostate specific antigen (PSA) doubling times (PSADT) varies widely. This report examines the pretreatment PSADT as an independent predictor of biochemical freedom from disease (bNED) and describes the clinical utility of PSADT. Methods and Materials: Ninety-nine patients with T1-3 NX, M-0 prostate cancer treated between February 1989 and November 1993 have pretreatment PSADTs calculated from three or more PSA levels. Biochemical disease-free (bNED) survival (failure is PSA ≥ 1.5 ngm/ml and rising) is evaluated by multivariate analysis of common prognostic indicators and PSADT. Results: Prostate-specific antigen doubling time (PSADT) is a significant predictor of survival along with radiation dose. Patients with a pretreatment PSADT of < 12 months show 50% failure by 18 months, while those with a PSADT that is not increasing show only 3% failure at 3 years. Conclusions: Prostate-specific antigen doubling time (PSADT) is a predictor of bNED outcome in prostate cancer. Patients with PSADT < 12 months have aggressive disease and should be considered for multimodal therapy. Slow PSADT (≥ 5 years) is observed in 57% of patients, and this end point may be considered in the decision to observe rather than to treat. After treatment failure, the PSADT may be used to determine which patients do not need immediate androgen deprivation

Chemokine receptor CXCR7 regulates the invasion, angiogenesis and tumor growth of human hepatocellular carcinoma cells

Directory of Open Access Journals (Sweden)

Li Fan

2010-04-01

Full Text Available Abstract Background In spite of recent advances in diagnostic and therapeutic measures, the prognosis of hepatocellular carcinoma (HCC patients remains poor. Therefore, it is crucial to understand what factors are involved in promoting development of HCC. Evidence is accumulating that members of the chemokine receptor family are viewed as promising therapeutic targets in the fight against cancer. More recent studies have revealed that chemokine receptor CXCR7 plays an important role in cancer development. However, little is known about the effect of CXCR7 on the process of HCC cell invasion and angiogenesis. The aim of this study is to investigate the expression of CXCR7 in hepatocellular carcinoma tissues and cell lines and to evaluate the role of CXCR7 in tumor growth, angiogenesis and invasion of HCC cells. Methods We constructed CXCR7 expressing shRNA, and CXCR7shRNA was subsequently stably transfected into human HCC cells. We evaluated the effect of CXCR7 inhibition on cell invasion, adhesion, VEGF secretion, tube formation and tumor growth. Immunohistochemistry was done to assess the expression of CXCR7 in human hepatocellular carcinoma tissues and CD31 in tumor of mice. We also evaluated the effect of VEGF stimulation on expression of CXCR7. Results CXCR7 was overexpressed in hepatocellular carcinoma tissues. We showed that high invasive potential HCC cell lines express high levels of CXCR7. In vitro, CXCL12 was found to induce invasion, adhesion, tube formation, and VEGF secretion in SMMC-7721 cells. These biological effects were inhibited by silencing of CXCR7 in SMMC-7721 cells. In addition, we also found that VEGF stimulation can up-regulate CXCR7 expression in SMMC-7721 cells and HUVECs. More importantly, enhanced expression of CXCR7 by VEGF was founctional. In vivo, tumor growth and angiogenesis were suppressed by knockdown of CXCR7 in SMMC-7721 cells. However, silencing of CXCR7 did not affect metastasis of tumor in vivo

CXCR1 regulates pulmonary anti-Pseudomonas host defense

Science.gov (United States)

Carevic, M.; Öz, H.; Fuchs, K.; Laval, J.; Schroth, C.; Frey, N.; Hector, A.; Bilich, T.; Haug, M.; Schmidt, A.; Autenrieth, S. E.; Bucher, K.; Beer-Hammer, S.; Gaggar, A.; Kneilling, M.; Benarafa, C.; Gao, J.; Murphy, P.; Schwarz, S.; Moepps, B.; Hartl, D.

2016-01-01

Pseudomonas aeruginosa is a key opportunistic pathogen causing disease in cystic fibrosis (CF) and other lung diseases such as chronic obstructive pulmonary disease (COPD). However, the pulmonary host defense mechanisms regulating anti-Pseudomonas aeruginosa immunity remain incompletely understood. Here we demonstrate, by studying an airway Pseudomonas aeruginosa infection model, in vivo bioluminescence imaging, neutrophil effector responses and human airway samples, that the chemokine receptor CXCR1 regulates pulmonary host defense against Pseudomonas aeruginosa. Mechanistically, CXCR1 regulated anti-Pseudomonas neutrophil responses through modulation of reactive oxygen species and interference with toll-like receptor 5 expression. These studies define CXCR1 as a novel non-canonical chemokine receptor that regulates pulmonary anti-Pseudomonas host defense with broad implications for CF, COPD and other infectious lung diseases. PMID:26950764

Circulating CXCR5+CD4+ T Follicular-Like Helper Cell and Memory B Cell Responses to Human Papillomavirus Vaccines

Science.gov (United States)

Matsui, Ken; Adelsberger, Joseph W.; Kemp, Troy J.; Baseler, Michael W.; Ledgerwood, Julie E.; Pinto, Ligia A.

2015-01-01

Through the interaction of T follicular helper (Tfh) cells and B cells, efficacious vaccines can generate high-affinity, pathogen-neutralizing antibodies, and memory B cells. Using CXCR5, CXCR3, CCR6, CCR7, PD1, and ICOS as markers, Tfh-like cells can be identified in the circulation and be classified into three functionally distinct subsets that are PD1+ICOS+, PD1+ ICOS-, or PD1-ICOS-. We used these markers to identify different subsets of CXCR5+CD4+ Tfh-like cells in response to highly immunogenic and efficacious vaccines for human papillomaviruses (HPV): Cervarix and Gardasil. In this small study, we used PBMC samples from 11 Gardasil recipients, and 8 Cervarix recipients from the Vaccine Research Center 902 Study to examine the induction of circulating Tfh-like cells and IgD-CD38HiCD27+ memory B cells by flow cytometry. PD1+ICOS+ CXCR3+CCR6-CXCR5+CD4+ (Tfh1-like) cells were induced and peaked on Day (D) 7 post-first vaccination, but not as much on D7 post-third vaccination. We also observed a trend toward increase in PD1+ICOS+ CXCR3-CCR6-CXCR5+CD4+ (Tfh2-like) cells for both vaccines, and PD1+ICOS+ CXCR3-CCR6+CXCR5+CD4+ (Tfh17-like) subset was induced by Cervarix post-first vaccination. There were also minimal changes in the other cellular subsets. In addition, Cervarix recipients had more memory B cells post-first vaccination than did Gardasil recipients at D14 and D30. We found frequencies of memory B cells at D30 correlated with anti-HPV16 and 18 antibody titers from D30, and the induction levels of memory B cells at D30 and PD1+ICOS+Tfh1-like cells at D7 post-first vaccination correlated for Cervarix. Our study showed that induction of circulating CXCR5+CD4+ Tfh-like subsets can be detected following immunization with HPV vaccines, and potentially be useful as a marker of immunogenicity of vaccines. However, further investigations should be extended to different cohorts with larger sample size to better understand the functions of these T cells, as well as

Anti-HIV Effect of Liposomes Bearing CXCR4 Receptor Antagonist ...

African Journals Online (AJOL)

Keywords: Antagonist, CXCR4, Liposomes, Receptor, Inflammation, HIV. Tropical Journal of ... receptors and inhibits HIV-1 entry mediated through CCR3, CCR5, and ..... circulation, facilitating HIV-targeted drug delivery. By tissue distribution ...

CXCL10 is critical for the progression and maintenance of depigmentation in a mouse model of vitiligo

Science.gov (United States)

Rashighi, Mehdi; Agarwal, Priti; Richmond, Jillian M; Harris, Tajie H; Dresser, Karen; Su, Mingwan; Zhou, Youwen; Deng, April; Hunter, Chris A; Luster, Andrew D; Harris, John E

2014-01-01

Vitiligo is an autoimmune disease of the skin that results in disfiguring white spots. There are no FDA-approved treatments for vitiligo, and most off-label treatments yield unsatisfactory results. Vitiligo patients have increased numbers of autoreactive, melanocyte-specific CD8+ T cells in the skin and blood, which are directly responsible for melanocyte destruction. Here we report that gene expression in lesional skin from vitiligo patients reveals an IFN-γ-specific signature, including the chemokine CXCL10. CXCL10 is elevated in both vitiligo patient skin and serum and CXCR3, its receptor, is expressed on pathogenic T cells. To address the function of CXCL10 in vitiligo, we employed a mouse model of disease that also exhibits an IFN-γ-specific gene signature, expression of CXCL10 in the skin, and upregulation of CXCR3 on antigen-specific T cells. Mice that receive Cxcr3−/− T cells develop minimal depigmentation, as do mice lacking Cxcl10 or treated with CXCL10 neutralizing antibody. CXCL9 promotes autoreactive T cell global recruitment to the skin but not effector function while, in contrast, CXCL10 is required for effector function and localization within the skin. Surprisingly, CXCL10 neutralization in mice with established, widespread depigmentation induces reversal of disease, evidenced by repigmentation. These data identify a critical role for CXCL10 in both the progression and maintenance of vitiligo, and thereby support inhibiting CXCL10 as a targeted treatment strategy. PMID:24523323

Design, synthesis, and functionalization of dimeric peptides targeting chemokine receptor CXCR4.

NARCIS (Netherlands)

Demmer, O.; Dijkgraaf, I.; Schumacher, U.; Marinelli, L.; Cosconati, S.; Gourni, E.; Wester, H.J.; Kessler, H.

2011-01-01

The chemokine receptor CXCR4 is a critical regulator of inflammation and immune surveillance, and it is specifically implicated in cancer metastasis and HIV-1 infection. On the basis of the observation that several of the known antagonists remarkably share a C(2) symmetry element, we constructed

Bioinformatics analysis of breast cancer bone metastasis related gene-CXCR4

Institute of Scientific and Technical Information of China (English)

Heng-Wei Zhang; Xian-Fu Sun; Ya-Ning He; Jun-Tao Li; Xu-Hui Guo; Hui Liu

2013-01-01

Objective: To analyze breast cancer bone metastasis related gene-CXCR4. Methods: This research screened breast cancer bone metastasis related genes by high-flux gene chip. Results:It was found that the expressions of 396 genes were different including 165 up-regulations and 231 down-regulations. The expression of chemokine receptor CXCR4 was obviously up-regulated in the tissue with breast cancer bone metastasis. Compared with the tissue without bone metastasis, there was significant difference, which indicated that CXCR4 played a vital role in breast cancer bone metastasis. Conclusions: The bioinformatics analysis of CXCR4 can provide a certain basis for the occurrence and diagnosis of breast cancer bone metastasis, target gene therapy and evaluation of prognosis.

Effect of integrin α5β1 inhibition on SDF-l/CXCR4- mediated choroidal neovascularization

Directory of Open Access Journals (Sweden)

Yang Lyu

2018-05-01

Full Text Available AIM: To investigate the roles of integrins in choroidal neovascularization (CNV and their associations with the stromal cell-derived factor-1 (SDF-1/CXCR4 axis. METHODS: CNV lesions were induced in mice using laser photocoagulation. After CNV induction, all animals were randomly assigned to: control, SDF-1, SDF-1+age-related macular degeneration (AMD 3100 (CXCR4 inhibitor, and SDF-1+ATN161 (integrin α5β1 inhibitor groups; their effects on CNV progression were observed using hematoxylin eosin (HE staining, fundus fluorescein angiography (FFA grading and optical coherence tomography (OCT, and their effects on CXCR4/integrin α5 expression were evaluated using Western blot and double immunofluorescence staining. Hypoxia-exposed endothelial cells (ECs were used to simulate CNV in vitro, they were treated with SDF-1, combined with CXCR4 siRNA/AMD3100 or ATN161, and expression of integrin α5, cell migration and tube formation were analyzed. RESULTS: Integrin subunit α5 increased at 3rd and 7th day and decreased at 14th day in CNV mice, with no significant change of β1-integrin. CXCR4 expression in CNV mice had persistent increase within 14d after induction. SDF-1 treatment significantly promoted the CNV progression during 3-14d. The mean CNV length in AMD3100 and ATN161 group at day 7 was 270.13 and 264.23 μm in HE images, significantly lower than the mean length in SDF-1 (345.70 μm group. AMD3100 and ATN161 also significantly reduced thickness and leakage of CNV induced by SDF-1. Mean integrin α5 positive area in SDF-1 group reached 2.31×104 μm2, significantly higher than control (1.25×104 μm2, which decreased to 1.78×104 μm2 after AMD3100 treatment. About 61.36% of ECs in CNV lesions expressed α5 in SDF-1 group, which significantly decreased to 43.12% after AMD3100 treatment. In vitro, integrin α5 peaked by 6 folds after 6h of hypoxia exposure and CXCR4 gradually increased by up to 2.3 folds after 24h of hypoxia. Approximately 25

The chemokine receptor CXCR4 is required for outgrowth of colon carcinoma micrometastases.

Science.gov (United States)

Zeelenberg, Ingrid S; Ruuls-Van Stalle, Lisette; Roos, Ed

2003-07-01

CXCR4, the receptor for the chemokine stromal cell-derived factor (SDF)-1 (CXCL12), is involved in lymphocyte trafficking. We have demonstrated previously that it is required for invasion of lymphoma cells into tissues and therefore essential for lymphoma metastasis. CXCR4 is also expressed by carcinoma cells, and CXCR4 antibodies were recently shown to reduce metastasis of a mammary carcinoma cell line. This was also ascribed to impaired invasion. We have blocked CXCR4 function in CT-26 colon carcinoma cells by transfection of SDF-1, extended with a KDEL sequence. The SDF-KDEL protein is retained in the endoplasmic reticulum by the KDEL-receptor and binds CXCR4, which is thus prevented from reaching the cell surface. We found that metastasis of these cells to liver and lungs was greatly reduced and often completely blocked. Surprisingly, however, our observations indicate that this was not attributable to inhibition of invasion but rather to impairment of outgrowth of micrometastases: (a) in contrast to the lymphoma cells, metastasis was not affected by the transfected S1 subunit of pertussis toxin. S1 completely inhibited Gi protein signaling, which is required for SDF-1-induced invasion; (b) CXCR4 levels were very low in CT-26 cells grown in vitro but strongly up-regulated in vivo. Strong up-regulation was not seen in the lungs until 7 days after tail vein injection. CXCR4 can thus have no role in initial invasion in the lungs; and (c) CXCR4-deficient cells did colonize the lungs to the same extent as control cells and survived. However, they did not expand, whereas control cells proliferated rapidly after a lag period of > or = 7 days. We conclude that CXCR4 is up-regulated by the microenvironment and that isolated metastatic cells are likely to require CXCR4 signals to initiate proliferation. Our results suggest that CXCR4 inhibitors have potential as anticancer agents to suppress outgrowth of micrometastases.

Cytokine-mediated deployment of SDF-1 induces revascularization through recruitment of CXCR4+ hemangiocytes

Science.gov (United States)

Jin, David K; Shido, Koji; Kopp, Hans-Georg; Petit, Isabelle; Shmelkov, Sergey V; Young, Lauren M; Hooper, Andrea T; Amano, Hideki; Avecilla, Scott T; Heissig, Beate; Hattori, Koichi; Zhang, Fan; Hicklin, Daniel J; Wu, Yan; Zhu, Zhenping; Dunn, Ashley; Salari, Hassan; Werb, Zena; Hackett, Neil R; Crystal, Ronald G; Lyden, David; Rafii, Shahin

2009-01-01

The mechanisms through which hematopoietic cytokines accelerate revascularization are unknown. Here, we show that the magnitude of cytokine-mediated release of SDF-1 from platelets and the recruitment of nonendothelial CXCR4+VEGFR1+ hematopoietic progenitors, ‘hemangiocytes,’ constitute the major determinant of revascularization. Soluble Kit-ligand (sKitL), thrombopoietin (TPO, encoded by Thpo) and, to a lesser extent, erythropoietin (EPO) and granulocyte-macrophage colony-stimulating factor (GM-CSF) induced the release of SDF-1 from platelets, enhancing neovascularization through mobilization of CXCR4+VEGFR1+ hemangiocytes. Although revascularization of ischemic hindlimbs was partially diminished in mice deficient in both GM-CSF and G-CSF (Csf2−/−Csf3−/−), profound impairment in neovascularization was detected in sKitL-deficient Mmp9−/− as well as thrombocytopenic Thpo−/− and TPO receptor–deficient (Mpl−/−) mice. SDF-1–mediated mobilization and incorporation of hemangiocytes into ischemic limbs were impaired in Thpo−/−, Mpl−/− and Mmp9−/− mice. Transplantation of CXCR4+VEGFR1+ hemangiocytes into Mmp9−/− mice restored revascularization, whereas inhibition of CXCR4 abrogated cytokine- and VEGF-A–mediated mobilization of CXCR4+VEGFR1+ cells and suppressed angiogenesis. In conclusion, hematopoietic cytokines, through graded deployment of SDF-1 from platelets, support mobilization and recruitment of CXCR4+VEGFR1+ hemangiocytes, whereas VEGFR1 is essential for their angiogenic competency for augmenting revascularization. Delivery of SDF-1 may be effective in restoring angiogenesis in individuals with vasculopathies. PMID:16648859

CXCR4 Protein Epitope Mimetic Antagonist POL5551 Disrupts Metastasis and Enhances Chemotherapy Effect in Triple-Negative Breast Cancer.

Science.gov (United States)

Xiang, Jingyu; Hurchla, Michelle A; Fontana, Francesca; Su, Xinming; Amend, Sarah R; Esser, Alison K; Douglas, Garry J; Mudalagiriyappa, Chidananda; Luker, Kathryn E; Pluard, Timothy; Ademuyiwa, Foluso O; Romagnoli, Barbara; Tuffin, Gérald; Chevalier, Eric; Luker, Gary D; Bauer, Michael; Zimmermann, Johann; Aft, Rebecca L; Dembowsky, Klaus; Weilbaecher, Katherine N

2015-11-01

The SDF-1 receptor CXCR4 has been associated with early metastasis and poorer prognosis in breast cancers, especially the most aggressive triple-negative subtype. In line with previous reports, we found that tumoral CXCR4 expression in patients with locally advanced breast cancer was associated with increased metastases and rapid tumor progression. Moreover, high CXCR4 expression identified a group of bone marrow-disseminated tumor cells (DTC)-negative patients at high risk for metastasis and death. The protein epitope mimetic (PEM) POL5551, a novel CXCR4 antagonist, inhibited binding of SDF-1 to CXCR4, had no direct effects on tumor cell viability, but reduced migration of breast cancer cells in vitro. In two orthotopic models of triple-negative breast cancer, POL5551 had little inhibitory effect on primary tumor growth, but significantly reduced distant metastasis. When combined with eribulin, a chemotherapeutic microtubule inhibitor, POL5551 additively reduced metastasis and prolonged survival in mice after resection of the primary tumor compared with single-agent eribulin. Hypothesizing that POL5551 may mobilize tumor cells from their microenvironment and sensitize them to chemotherapy, we used a "chemotherapy framing" dosing strategy. When administered shortly before and after eribulin treatment, three doses of POL5551 with eribulin reduced bone and liver tumor burden more effectively than chemotherapy alone. These data suggest that sequenced administration of CXCR4 antagonists with cytotoxic chemotherapy synergize to reduce distant metastases. ©2015 American Association for Cancer Research.

TISSUE POLYPEPTIDE-SPECIFIC ANTIGEN - A DISCRIMINATIVE PARAMETER BETWEEN PROSTATE-CANCER AND BENIGN PROSTATIC HYPERTROPHY

NARCIS (Netherlands)

MARRINK, J; OOSTEROM, R; BONFRER, HMG; SCHRODER, FH; MENSINK, HJA

1993-01-01

The serum concentration of the cell proliferation marker TPS (tissue polypeptide-specific antigen) was compared with the tumour marker PSA (prostate specific antigen). PSA was found elevated in 50% of the benign prostatic hypertrophy (BPH) patients, in 88% of the patients with active prostate cancer

Over-expression of CXCR4, a stemness enhancer, in human blastocysts by low level laser irradiation

Directory of Open Access Journals (Sweden)

Mohammad Hossein Tahmasbi

2013-09-01

Full Text Available The key role of chemokine receptor CXCR4 in the maintenance of stemness property of stem cells has been shown recently. The low level laser irradiation (LLLI is being used currently in a wide variety of clinical cases as a therapeutic tool for wound healing, relieving pain and destroying tumor cells. The aim of this study was to evaluate the effect of LLLI mimicking low level laser therapy (LLLT on the expression level of CXCR4 gene a few hours after irradiation on human blastocysts. After the development of human embryos to the first grade blastocyst stage, they were irradiated with a low power Ga-Al-As laser at a continuous wavelength of 650 nm and a power output of 30 mW. The total RNA of the irradiated blastocysts and control groups were isolated in groups of 1x2 J/cm2, 2x2 J/cm2, 1x4 J/cm2 and 2x4 J/cm2 LLLI. Specific Real-Time PCR primers were designed to amplify all the two CXCR4 isoforms yet identified. RNA amplifications were done for all the groups. We showed for the first time that LLLI makes the human blastocysts to increase the expression level of CXCR4 a few hours after irradiation. Moreover, it was shown that two irradiation doses with one day interval can cause a significant increase in CXCR4 expression level in human blastocysts. This study revealed that LLLI could be a proliferation motivator for embryonic cell divisions through enhanced over-expression of CXCR4 level.

The prognostic value of CXC-chemokine receptor 2 (CXCR2) in gastric cancer patients

International Nuclear Information System (INIS)

Wang, Zhenglin; Liu, Hao; Shen, Zhenbin; Wang, Xuefei; Zhang, Heng; Qin, Jing; Xu, Jiejie; Sun, Yihong; Qin, Xinyu

2015-01-01

CXC chemokine receptor 2 (CXCR2) has been reported to play an important role in the proliferation and invasion of gastric cancer cells. The present study aims to investigate the impact of CXCR2 expression on the overall survival (OS) of gastric cancer patients after radical resection. Intratumoral CXCR2 expression was evaluated with immunohistochemistry on tissue microarrays containing tumor samples of 357 gastric cancer patients from a single center. CXCR2 expression levels were correlated to clinicopathological variables and OS. CXCR2 expression was mainly located in the cytoplasm of gastric carcinoma cells. High CXCR2 expression was associated with poor tumor differentiation (p = 0.021), increased tumor depth (p < 0.001), lymph node metastasis (p < 0.001), advanced TNM stage (p < 0.001) and short OS (p = 0.001). CXCR2 expression was an independent prognostic factor for OS (p = 0.001) in multivariate analysis, and could be combined with TNM stage to generate a predictive nomogram for clinical outcome in patients with gastric cancer. Intratumoral CXCR2 expression is a novel independent predictor for survival in gastric cancer patients. CXCR2 might be a promising therapeutic target of postoperative adjuvant treatment. The online version of this article (doi:10.1186/s12885-015-1793-9) contains supplementary material, which is available to authorized users

Silencing of CXCR4 inhibits tumor cell proliferation and neural invasion in human hilar cholangiocarcinoma.

Science.gov (United States)

Tan, Xin-Yu; Chang, Shi; Liu, Wei; Tang, Hui-Huan

2014-03-01

To evaluate the expression of CXC motif chemokine receptor 4 (CXCR4) in the tissues of patients with hilar cholangiocarcinoma (hilar-CCA) and to investigate the cell proliferation and frequency of neural invasion (NI) influenced by RNAi-mediated CXCR4 silencing. An immunohistochemical technique was used to detect the expression of CXCR4 in 41 clinical tissues, including hilar-CCA, cholangitis, and normal bile duct tissues. The effects of small interference RNA (siRNA)-mediated CXCR4 silencing were detected in the hilar-CCA cell line QBC939. Cell proliferation was determined by MTT. Expression of CXCR4 was monitored by quantitative real time polymerase chain reaction and Western blot analysis. The NI ability of hilar-CCA cells was evaluated using a perineural cell and hilar-CCA cell coculture migration assay. The expression of CXCR4 was significantly induced in clinical hilar-CCA tissue. There was a positive correlation between the expression of CXCR4 and lymph node metastasis/NI in hilar-CCA patients (philar-CCA. CXCR4 is involved in the invasion and proliferation of human hilar-CCA cell line QBC939, indicating that CXCR4 could be a promising therapeutic target for hilar-CCA.

Despite disorganized synapse structure, Th2 cells maintain directional delivery of CD40L to antigen-presenting B cells.

Science.gov (United States)

Gardell, Jennifer L; Parker, David C

2017-01-01

Upon recognition of peptide displayed on MHC molecules, Th1 and Th2 cells form distinct immunological synapse structures. Th1 cells have a bull's eye synapse structure with TCR/ MHC-peptide interactions occurring central to a ring of adhesion molecules, while Th2 cells have a multifocal synapse with small clusters of TCR/MHC interactions throughout the area of T cell/antigen-presenting cell interaction. In this study, we investigated whether this structural difference in the immunological synapse affects delivery of T cell help. The immunological synapse is thought to ensure antigen-specific delivery of cytolytic granules and killing of target cells by NK cells and cytolytic T cells. In helper T cells, it has been proposed that the immunological synapse may direct delivery of other effector molecules including cytokines. CD40 ligand (CD40L) is a membrane-bound cytokine essential for antigen-specific T cell help for B cells in the antibody response. We incubated Th1 and Th2 cells overnight with a mixture of antigen-presenting and bystander B cells, and the delivery of CD40L to B cells and subsequent B cell responses were compared. Despite distinct immunological synapse structures, Th1 and Th2 cell do not differ in their ability to deliver CD40L and T cell help in an antigen-specific fashion, or in their susceptibility to inhibition of help by a blocking anti-CD40L antibody.

Immunoassay of poliovirus antigens by single-radial-diffusion: development and characteristics of a sensitive autoradiographic zone size enhancement (ZE) technique

International Nuclear Information System (INIS)

Schild, G.C.; Wood, J.M.; Minor, P.D.; Dandawate, C.N.; Magrath, D.I.

1980-01-01

The reactions of polioviruses in single-radial-immunodiffusion (SRD) tests were investigated with a view to developing accurate and sensitive antigen assay systems. In direct SRD tests, the reactions were type-specific for polioviruses of types 1, 2 and 3 but the tests were of low sensitivity, being applicable only to the assay of virus concentrates. A novel autoradiographic zone size enhancement (ZE) test was developed which increased the sensitivity of the SRD assay 40-to 100-fold. The ZE test was dependent upon the ability of unlabelled poliovirus to co-migrate with the radioactive marker virus and so enhance the zone size detected autoradiographically. The areas of the autoradiographic zones were directly proportional to the concentration of unlabelled antigen. The ZE test was capable of detecting poliovirus D antigens in amounts corresponding to 10sup(3.3) to 10sup(4.3) TCID 50 of infectious virus. Studies with poliovirus type 3 strains indicated that the ZE test was narrowly strain-specific for the D-antigen of poliovirus type 3 strains when homologous type 3 D-antigen was used as radioactive marker, but broadly cross-reactive for the D-antigen of type 3 viruses when heterologous poliovirus type 3 D-antigen was used as marker. (author)

The diagnostic value of transrectal ultrasonography combined with prostate specific antigen density in prostate cancer

International Nuclear Information System (INIS)

Shen Weidong; Zha Yueqin; Wang Ajun; Hou Jianquan; Ouyang Jun

2008-01-01

Objective: To discuss the value of transrectal ultrasound (TRUS) and prostate specific antigen density (PSAD) and prostate specific antigen density of transition zone (PSATZ) for diagnosing prostate cancer. Methods: Chose cases of prostate cancer(PCa) and benign prostate hyperplasia(BPH), each was 19, all the eases were authenticated by pathology. Then compared the characteristic of prostate cancer with prostate specific antigen (PSA) and homologous PSAD, PSATZ. Results: Fourteen cases were discovered by ultrasound among the 19 PCa, the others were only diagnosed as BPH.Among the 14 cases, diffuse pathological changing was found in 1 patient, nodular changing in 13 patients (16 nodules were found). Among the 16 nodules, there were 13 hypoechoic nodules (75%) and 3 hyper echoic or compound echoic nodules (25%), and there were 13 nodules in outer zone and 3 nodules in transition zone.The PSA of PCa and BPH was 8.61-98.65 ng/ml [(48.79±25.34)ng/ml] and 0.58-28.36 ng/ml [(9.73±8.19)ng/ml]. There were no significant differences between the volume of prostate and prostate transition zone (P>0.05), but there were significant differences between the PSAD and PSATZ (P<0.01). That the PCa group was higher than that in the BPH group. Conclusion: It is higher sensitive but bess specific in diagonosis PCa by means of transrectal ultrasound. If it is combined with PSAD and PSATZ, the diagnostic rate of PCa is highly raised. (authors)

Chimeric antigen receptor (CAR-specific monoclonal antibody to detect CD19-specific T cells in clinical trials.

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Bipulendu Jena

Full Text Available Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63. We describe a novel anti-idiotype monoclonal antibody (mAb to detect CD19-specific CAR(+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1 was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19(+ tumor targets. This clone can be used to detect CD19-specific CAR(+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1 will be useful to investigators implementing CD19-specific CAR(+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.

Identification of antigen-specific human monoclonal antibodies using high-throughput sequencing of the antibody repertoire.

Science.gov (United States)

Liu, Ju; Li, Ruihua; Liu, Kun; Li, Liangliang; Zai, Xiaodong; Chi, Xiangyang; Fu, Ling; Xu, Junjie; Chen, Wei

2016-04-22

High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies. Copyright © 2016 Elsevier Inc. All rights reserved.

Alkylglycerols modulate the proliferation and differentiation of non-specific agonist and specific antigen-stimulated splenic lymphocytes.

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Linxi Qian

Full Text Available Alkylglycerols (AKGs are ether-linked glycerols derived from shark liver oil and found in small amounts in human milk. Previous studies showed that oral AKGs administration significantly increased the immune response in mice. The aim of the present study was to investigate the in vitro immunomodulatory effect of AKGs on stimulating splenic lymphocyte responses. C57BL/6 mice were immunized with hepatitis B surface antigen (HBsAg. Splenic B cells were purified and stimulated with anti-BCR and anti-CD38. Meanwhile, splenic CD4+ T cells were purified and stimulated with anti-CD3 and anti-CD28. For antigen specific stimulation, the purified CD4+ T cells were cocultured with HBsAg -pulsed dendritic cells. The stimulated lymphocytes were treated with different concentrations of AKGs. The cell proliferation was assessed by [3H]-thymidine incorporation assay. The maturation of B cells was assessed by examining the germline (GL transcription of IgG (γ1 mRNA expression, and the surface expressions of CD80/CD86 markers were examined by flow cytometry analysis. Th1/Th2 polarity was assessed by T-BET (Th1/GATA-3 (Th2 flow cytometry assay and by characteristic cytokines ELISA assay (TNF-α and IFN-γ for Th1; IL-4 and IL-10 for Th2. It was found that AKGs significantly increased the BCR/CD38 -stimulated B cell proliferation. The T cell proliferation in response to CD3/CD28 or specific antigen stimulation was also increased by AKGs. The transcriptional level of IgG (γ1 and the expressions of CD80/CD86 molecules were markedly increased by AKGs in BCR/CD38 -stimulated B cells. Meanwhile, the results showed that AKGs increased the expression of T-BET transcriptional factor and the production of Th1 cytokines (TNF-α and IFN-γ upon CD3/CD28 stimulation; whereas, levels of Th2 cytokines (IL-4 and IL-10 were decreased by AKGs. Our study demonstrated that AKGs can modulate immune responses by boosting the proliferation and maturation of murine lymphocytes in vitro.

Interaction of chemokine receptor CXCR4 in monomeric and dimeric state with its endogenous ligand CXCL12: coarse-grained simulations identify differences.

Science.gov (United States)

Cutolo, Pasquale; Basdevant, Nathalie; Bernadat, Guillaume; Bachelerie, Françoise; Ha-Duong, Tâp

2017-02-01

Despite the recent resolutions of the crystal structure of the chemokine receptor CXCR4 in complex with small antagonists or viral chemokine, a description at the molecular level of the interactions between the full-length CXCR4 and its endogenous ligand, the chemokine CXCL12, in relationship with the receptor recognition and activation, is not yet completely elucidated. Moreover, since CXCR4 is able to form dimers, the question of whether the CXCR4-CXCL12 complex has a 1:1 or 2:1 preferential stoichiometry is still an open question. We present here results of coarse-grained protein-protein docking and molecular dynamics simulations of CXCL12 in association with CXCR4 in monomeric and dimeric states. Our proposed models for the 1:1 and 2:1 CXCR4-CXCL12 quaternary structures are consistent with recognition and activation motifs of both partners provided by the available site-directed mutagenesis data. Notably, we observed that in the 2:1 complex, the chemokine N-terminus makes more steady contacts with the receptor residues critical for binding and activation than in the 1:1 structure, suggesting that the 2:1 stoichiometry would favor the receptor signaling activity with respect to the 1:1 association.

Cortisol increases CXCR4 expression but does not affect CD62L and CCR7 levels on specific T cell subsets in humans.

Science.gov (United States)

Besedovsky, Luciana; Linz, Barbara; Dimitrov, Stoyan; Groch, Sabine; Born, Jan; Lange, Tanja

2014-06-01

Glucocorticoids are well known to affect T cell migration, leading to a redistribution of the cells from blood to the bone marrow, accompanied by a concurrent suppression of lymph node homing. Despite numerous studies in this context, with most of them employing synthetic glucocorticoids in nonphysiological doses, the mechanisms of this redistribution are not well understood. Here, we investigated in healthy men the impact of cortisol at physiological concentrations on the expression of different migration molecules on eight T cell subpopulations in vivo and in vitro. Hydrocortisone (cortisol, 22 mg) infused during nocturnal rest when endogenous cortisol levels are low, compared with placebo, differentially reduced numbers of T cell subsets, with naive CD4(+) and CD8(+) subsets exhibiting the strongest reduction. Hydrocortisone in vivo and in vitro increased CXCR4 expression, which presumably mediates the recruitment of T cells to the bone marrow. Expression of the lymph node homing receptor CD62L on total CD3(+) and CD8(+) T cells appeared reduced following hydrocortisone infusion. However, this was due to a selective extravasation of CD62L(+) T cell subsets, as hydrocortisone affected neither CD62L expression on a subpopulation level nor CD62L expression in vitro. Corresponding results in the opposite direction were observed after blocking of endogenous cortisol synthesis by metyrapone. CCR7, another lymph node homing receptor, was also unaffected by hydrocortisone in vitro. Thus, cortisol seems to redirect T cells to the bone marrow by upregulating their CXCR4 expression, whereas its inhibiting effect on T cell homing to lymph nodes is apparently regulated independently of the expression of classical homing receptors. Copyright © 2014 the American Physiological Society.

Towards effective and safe immunotherapy after allogeneic stem cell transplantation: identification of hematopoietic-specific minor histocompatibility antigen UTA2-1

NARCIS (Netherlands)

Oostvogels, R.; Minnema, M. C.; van Elk, M.; Spaapen, R. M.; te Raa, G. D.; Giovannone, B.; Buijs, A.; van Baarle, D.; Kater, A. P.; Griffioen, M.; Spierings, E.; Lokhorst, H. M.; Mutis, T.

2013-01-01

Donor T cells directed at hematopoietic system-specific minor histoconnpatibility antigens (mHags) are considered important cellular tools to induce therapeutic graft-versus-tumor (GvT) effects with low risk of graft-versus-host disease after allogeneic stem cell transplantation. To enable the

Chemokine receptor CXCR4 downregulated by von Hippel-Lindau tumour suppressor pVHL

DEFF Research Database (Denmark)

Staller, Peter; Sulitkova, Jitka; Lisztwan, Joanna

2003-01-01

Organ-specific metastasis is governed, in part, by interactions between chemokine receptors on cancer cells and matching chemokines in target organs. For example, malignant breast cancer cells express the chemokine receptor CXCR4 and commonly metastasize to organs that are an abundant source of t...

Inhibition of human immunodeficiency virus replication by a dual CCR5/CXCR4 antagonist

DEFF Research Database (Denmark)

Princen, Katrien; Hatse, Sigrid; Vermeire, Kurt

2004-01-01

Here we report that the N-pyridinylmethyl cyclam analog AMD3451 has antiviral activity against a wide variety of R5, R5/X4, and X4 strains of human immunodeficiency virus type 1 (HIV-1) and HIV-2 (50% inhibitory concentration [IC(50)] ranging from 1.2 to 26.5 microM) in various T-cell lines, CCR5...... at the virus entry stage. AMD3451 dose-dependently inhibited the intracellular Ca(2+) signaling induced by the CXCR4 ligand CXCL12 in T-lymphocytic cells and in CXCR4-transfected cells, as well as the Ca(2+) flux induced by the CCR5 ligands CCL5, CCL3, and CCL4 in CCR5-transfected cells. The compound did...... not interfere with chemokine-induced Ca(2+) signaling through CCR1, CCR2, CCR3, CCR4, CCR6, CCR9, or CXCR3 and did not induce intracellular Ca(2+) signaling by itself at concentrations up to 400 microM. In freshly isolated monocytes, AMD3451 inhibited the Ca(2+) flux induced by CXCL12 and CCL4...

Prediction of site-specific interactions in antibody-antigen complexes: the proABC method and server.

KAUST Repository

Olimpieri, Pier Paolo

2013-06-26

MOTIVATION: Antibodies or immunoglobulins are proteins of paramount importance in the immune system. They are extremely relevant as diagnostic, biotechnological and therapeutic tools. Their modular structure makes it easy to re-engineer them for specific purposes. Short of undergoing a trial and error process, these experiments, as well as others, need to rely on an understanding of the specific determinants of the antibody binding mode. RESULTS: In this article, we present a method to identify, on the basis of the antibody sequence alone, which residues of an antibody directly interact with its cognate antigen. The method, based on the random forest automatic learning techniques, reaches a recall and specificity as high as 80% and is implemented as a free and easy-to-use server, named prediction of Antibody Contacts. We believe that it can be of great help in re-design experiments as well as a guide for molecular docking experiments. The results that we obtained also allowed us to dissect which features of the antibody sequence contribute most to the involvement of specific residues in binding to the antigen. AVAILABILITY: http://www.biocomputing.it/proABC. CONTACT: anna.tramontano@uniroma1.it or paolo.marcatili@gmail.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

RasGRP1, but not RasGRP3, is required for efficient thymic β-selection and ERK activation downstream of CXCR4.

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Dominic P Golec

Full Text Available T cell development is a highly dynamic process that is driven by interactions between developing thymocytes and the thymic microenvironment. Upon entering the thymus, the earliest thymic progenitors, called CD4(-CD8(- 'double negative' (DN thymocytes, pass through a checkpoint termed "β-selection" before maturing into CD4(+CD8(+ 'double positive' (DP thymocytes. β-selection is an important developmental checkpoint during thymopoiesis where developing DN thymocytes that successfully express the pre-T cell receptor (TCR undergo extensive proliferation and differentiation towards the DP stage. Signals transduced through the pre-TCR, chemokine receptor CXCR4 and Notch are thought to drive β-selection. Additionally, it has long been known that ERK is activated during β-selection; however the pathways regulating ERK activation remain unknown. Here, we performed a detailed analysis of the β-selection events in mice lacking RasGRP1, RasGRP3 and RasGRP1 and 3. We report that RasGRP1 KO and RasGRP1/3 DKO deficient thymi show a partial developmental block at the early DN3 stage of development. Furthermore, DN3 thymocytes from RasGRP1 and RasGRP1/3 double knock-out thymi show significantly reduced proliferation, despite expression of the TCRβ chain. As a result of impaired β-selection, the pool of TCRβ(+ DN4 is significantly diminished, resulting in inefficient DN to DP development. Also, we report that RasGRP1 is required for ERK activation downstream of CXCR4 signaling, which we hypothesize represents a potential mechanism of RasGRP1 regulation of β-selection. Our results demonstrate that RasGRP1 is an important regulator of proliferation and differentiation at the β-selection checkpoint and functions downstream of CXCR4 to activate the Ras/MAPK pathway.

SPECIFICITY OF ANTIBODY BOVINE ZONNA PELLUCIDAE 3 (ANTI-bZP3 TO RABBIT ZP3 BASED ON bZP3 AS CONTRACEPTIVE ANTIGENS

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Edwin Widodo

2010-06-01

Full Text Available Zonna pellucidae can be develop as antigen potential candidates based on reversible immunocontraceptive vaccines. Immunogenic sites of bovine zonna pellucidae 3 (bZP3 could stimulated the presence of anti-bZP3 which be located on rabbit ZP and inhibit sperm-egg interaction on fertilization process. Purpose of this research is to detect spesific binding anti-bZP3 to rabbit oocytes using dot blotting and ELISA method. Sub cutan induction of bZP3 with Freund's adjuvant, CFA (Complete Freund's Adjuvant for initial immunization and following by IFA (Incomplete Freund's Adjuvant at the 14th day and 39th day. Control female rabbit injected by Tris-Cl buffer diluted in Freund's adjuvant without bZP3 antigen. Rabbit serum injected to rat for producing Rat Anti Rabbit Anti-bZP3. This research concludes spesific binding of anti-bZP3 with increasing purple colour on dot blotting methods. Anti-bZP3 increasing on 24th day and 31th day and still until 48th day. Measurement with ELISA methods showed increased titer on OD405. Highest titer showed on 31th day post immunization. Anti-bZP3 synthetized by bZP3 induced on rabbit detectable by immunohistochemistry methods on late primary oocytes, early secondary oocytes, growing secondary oocytes, and oocytes on de Graaf folicular phase. Keywords: Dot blotting, ELISA, bZP3, anti-bZP3

Vaxfectin enhances antigen specific antibody titers and maintains Th1 type immune responses to plasmid DNA immunization.

Science.gov (United States)

Reyes, L; Hartikka, J; Bozoukova, V; Sukhu, L; Nishioka, W; Singh, G; Ferrari, M; Enas, J; Wheeler, C J; Manthorpe, M; Wloch, M K

2001-06-14

Antigen specific immune responses were characterized after intramuscular immunization of BALB/c mice with 5 antigen encoding plasmid DNAs (pDNAs) complexed with Vaxfectin, a cationic lipid formulation. Vaxfectin increased IgG titers for all of the antigens with no effect on the CTL responses to the 2 antigens for which CTL assays were performed. Both antigen specific IgG1 and IgG2a were increased, although IgG2a remained greater than IgG1. Furthermore, Vaxfectin had no effect on IFN-gamma or IL-4 production by splenocytes re-stimulated with antigen, suggesting that the Th1 type responses typical of intramuscular pDNA immunization were not altered. Studies with IL-6 -/- mice suggest that the antibody enhancement is IL-6 dependent and results in a correlative increase in antigen specific antibody secreting cells.

Human seminal proteinase and prostate-specific antigen are the ...

Indian Academy of Sciences (India)

https://www.ias.ac.in/article/fulltext/jbsc/033/02/0195-0207. Keywords. Kallikrein; prostate cancer biomarker; proteinase activity; seminal plasma; tumour proliferation and metastasis; therapeutic target. Abstract. Human seminal proteinase and prostate-specific antigen (PSA) were each isolated from human seminal fluid and ...

Role of CXCL12 and CXCR4 in normal cerebellar development and medulloblastoma.

Science.gov (United States)

Ozawa, Patricia Midori Murobushi; Ariza, Carolina Batista; Ishibashi, Cintya Mayumi; Fujita, Thiago Cezar; Banin-Hirata, Bruna Karina; Oda, Julie Massayo Maeda; Watanabe, Maria Angelica Ehara

2016-01-01

Chemokines and its receptors have significant impact on physiological and pathological processes and studies concerning their association with tumor biology are subject of great interest in scientific community. CXCL12/CXCR4 axis has been widely studied due to its significant role in tumor microenvironment, but it is also important to development and maintenance of tissues and organs, for example, in the brain and cerebellum. Studies have demonstrated that CXCL12 and CXCR4 are required for normal cerebellar development and that dysfunction in this pathway may be involved with medulloblastoma pathogenesis. In this context, a new molecular subgroup has been suggested based on the importance of the association between CXCR4 overexpression and sonic hedgehog subgroup. Treatment using CXCR4 antagonists showed significant results, evidencing the important role and possible therapeutic capacity of CXCR4 in MB. This review summarizes studies on MB cell biology, focusing on a chemokine-receptor axis, CXCL12/CXCR4, that may have implications for treatment strategies once it can improve life expectancy and reduce neurocognitive sequelae of patients with this neoplasia. © 2014 UICC.

Circulating Cxcr5-Expressing Cd8+T-Cells are Major Producers of Il-21 and Associate with Limited Hiv Replication.

Science.gov (United States)

Perdomo-Celis, Federico; Taborda, Natalia A; Rugeles, Maria T

2018-04-10

Despite advances made with the highly active anti-retroviral therapy (HAART) in the control of the human immunodeficiency virus 1 (HIV) infection, a cure has not been achieved due to the persistence of viral reservoirs. The major HIV reservoirs remain in the lymphoid follicles due to, among other factors, the partial absence of CD8T-cells in these structures. Recently, lymphoid follicle-confined and circulating CD8T-cells expressing the C-X-C chemokine receptor type 5 (CXCR5) were described, possessing antiviral mechanisms which could help to control HIV replication. and methods: By flow cytometry, we characterized the phenotype and function of circulating CXCR5-expressing CD8T-cells in HIV-infected patients with natural or HAART-induced control of HIV replication. Circulating CXCR5-expressing CD8T-cells exhibited low or null expression of the C-C chemokine receptor type 7 (CCR7) and had a transitional memory phenotype. Particular redistributions of CXCR5-expressing CD8T-cells were found in HIV-infected patients, and they were partially restored by HAART. The frequency of CXCR5CCR7CD8T-cells was higher in spontaneous HIV controllers and negatively correlated with plasma HIV RNA levels. Total and HIV-specific CXCR5CD8T-cells were major producers of interleukin-21, and this function was positively associated with their interferon-γ production. Circulating CXCR5-expressing CD8T-cells are associated with low level HIV replication, could be novel correlates of protection and potentially useful in the eradication of HIV reservoirs.

Manipulating the Lewis antigen specificity of the cholesterol-dependent cytolysin lectinolysin

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Sara eLawrence

2012-11-01

Full Text Available The cholesterol-dependent cytolysins (CDCs attack cells by punching large holes in their membranes. Lectinolysin from Streptococcus mitis is unique among CDCs due to the presence of an N-terminal lectin domain that enhances the pore-forming activity of the toxin. We recently determined the crystal structures of the lectin domain in complex with various glycans. These structures revealed the molecular basis for the Lewis antigen specificity of the toxin. Based on this information we have used in silico molecular modelling to design a mutant toxin, which we predicted would increase its specificity for Lewis y, an antigen found on the surface of cancer cells. Surprisingly, we found by surface plasmon resonance binding experiments that the resultant mutant lectin domain exhibited higher specificity for Lewis b antigens instead. We then undertook comparative crystallographic and molecular dynamics simulation studies of the wild-type and mutant lectin domains to understand the molecular basis for the disparity between the theoretical and experimental results. The crystallographic results revealed that the net number of interactions between Lewis y and wild-type versus mutant was unchanged whereas there was a loss of a hydrogen bond between mutant and Lewis b compared to wild-type. In contrast, the molecular dynamics studies revealed that the Lewis b antigen spent more time in the binding pocket of the mutant compared to wild-type and the reverse was true for Lewis y. The results of these simulation studies are consistent with the conclusions drawn from the surface plasmon resonance studies. This work is part of a program to engineer lectinolysin so that it will target and kill specific cells in human diseases.

Position-specific automated processing of V3 env ultra-deep pyrosequencing data for predicting HIV-1 tropism.

Science.gov (United States)

Jeanne, Nicolas; Saliou, Adrien; Carcenac, Romain; Lefebvre, Caroline; Dubois, Martine; Cazabat, Michelle; Nicot, Florence; Loiseau, Claire; Raymond, Stéphanie; Izopet, Jacques; Delobel, Pierre

2015-11-20

HIV-1 coreceptor usage must be accurately determined before starting CCR5 antagonist-based treatment as the presence of undetected minor CXCR4-using variants can cause subsequent virological failure. Ultra-deep pyrosequencing of HIV-1 V3 env allows to detect low levels of CXCR4-using variants that current genotypic approaches miss. However, the computation of the mass of sequence data and the need to identify true minor variants while excluding artifactual sequences generated during amplification and ultra-deep pyrosequencing is rate-limiting. Arbitrary fixed cut-offs below which minor variants are discarded are currently used but the errors generated during ultra-deep pyrosequencing are sequence-dependant rather than random. We have developed an automated processing of HIV-1 V3 env ultra-deep pyrosequencing data that uses biological filters to discard artifactual or non-functional V3 sequences followed by statistical filters to determine position-specific sensitivity thresholds, rather than arbitrary fixed cut-offs. It allows to retain authentic sequences with point mutations at V3 positions of interest and discard artifactual ones with accurate sensitivity thresholds.

Comparative analysis of monoclonal antibodies against prostate-specific membrane antigen (PSMA)

Czech Academy of Sciences Publication Activity Database

Tykvart, Jan; Navrátil, Václav; Sedlák, František; Corey, E.; Colombatti, M.; Fracasso, G.; Koukolík, F.; Bařinka, Cyril; Šácha, Pavel; Konvalinka, Jan

2014-01-01

Roč. 74, č. 16 (2014), s. 1674-1690 ISSN 0270-4137 R&D Projects: GA ČR GAP304/12/0847; GA MŠk LO1302; GA ČR GAP301/12/1513; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388963 ; RVO:86652036 Keywords : glutamate carboxypeptidase II * prostate -specific membrane antigen * folate hydrolase * NAALADase * Western blot * immunohistochemistry * ELISA * flow cytometry * surface plasmon resonance Subject RIV: CE - Biochemistry Impact factor: 3.565, year: 2014

Genetic engineering of chimeric antigen receptors using lamprey derived variable lymphocyte receptors

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Robert Moot

2016-01-01

Full Text Available Chimeric antigen receptors (CARs are used to redirect effector cell specificity to selected cell surface antigens. Using CARs, antitumor activity can be initiated in patients with no prior tumor specific immunity. Although CARs have shown promising clinical results, the technology remains limited by the availability of specific cognate cell target antigens. To increase the repertoire of targetable tumor cell antigens we utilized the immune system of the sea lamprey to generate directed variable lymphocyte receptors (VLRs. VLRs serve as membrane bound and soluble immune effectors analogous but not homologous to immunoglobulins. They have a fundamentally different structure than immunoglobulin (Ig-based antibodies while still demonstrating high degrees of specificity and affinity. To test the functionality of VLRs as the antigen recognition domain of CARs, two VLR-CARs were created. One contained a VLR specific for a murine B cell leukemia and the other contained a VLR specific for the human T cell surface antigen, CD5. The CAR design consisted of the VLR sequence, myc-epitope tag, CD28 transmembrane domain, and intracellular CD3ζ signaling domain. We demonstrate proof of concept, including gene transfer, biosynthesis, cell surface localization, and effector cell activation for multiple VLR-CAR designs. Therefore, VLRs provide an alternative means of CAR-based cancer recognition.

Effect of antigen on localization of immunologically specific B cells

International Nuclear Information System (INIS)

Ponzio, N.M.; Chapman, J.M.; Thorbecke, G.J.

1976-01-01

Studies were conducted to demonstrate homing of memory B cells to sites of antigen localization in lymph nodes, using functional criteria to detect local presence of memory cells at varying intervals after intravenous injection. Cell suspensions were prepared from spleens of donor mice injected with complete Freund's adjuvant. Recipient mice were injected with Escherichia coli endotoxin and immune or normal spleen cells and were gamma-irradiated. Results indicated that passively transferred unilateral B cell memory was established. The development over a period of several days of this difference between left and right lymph nodes suggests that recirculating memory B cells are being progressively selected by antigen in the lymph node, rather than that this difference is due to a specific exit of cells from the circulation towards the antigen

CXCR4(+) dendritic cells promote angiogenesis during embryo implantation in mice.

Science.gov (United States)

Barrientos, Gabriela; Tirado-González, Irene; Freitag, Nancy; Kobelt, Peter; Moschansky, Petra; Klapp, Burghard F; Thijssen, Victor L J L; Blois, Sandra M

2013-04-01

Early pregnancy is characterized by decidual adaption to the developing embryo involving angiogenesis and vascular growth. Failure of decidual vascular expansion is linked to diseases of pregnancy. Dendritic cells (DC) have been associated with vascular growth during early gestation, though it is unknown whether their capacity to modulate angiogenesis is ubiquitous to all DC subsets. Here, we show that DC normally found associated with the decidual vasculature co-express the C-X-C chemokine receptor type 4 (CXCR4). In addition, we demonstrate that impaired homing of CXCR4(+)DC during early gestation provoked a disorganized decidual vasculature with impaired spiral artery remodeling later in gestation. In contrast, adoptive transfer experiments provided evidence that CXCR4(+)DC are able to rescue early pregnancy by normalizing decidual vascular growth and delivery of pro-angiogenic factors, which results in adequate remodeling of the spiral arteries during placental development. Taken together, our results indicate an important role of CXCR4(+)DC in the regulation of decidual angiogenesis and highlight the importance of the CXCL12/CXCR4 pathway during this process, suggesting that this may represent a key pathway to evaluate during pregnancy pathologies associated with impaired vascular expansion.

Characterization of an antigenic site that contains a dominant, type-specific neutralization determinant on the envelope protein domain III (ED3) of dengue 2 virus

International Nuclear Information System (INIS)

Gromowski, Gregory D.; Barrett, Alan D.T.

2007-01-01

The surface of the mature dengue virus (DENV) particle consists of 90 envelope (E) protein dimers that mediate both receptor binding and fusion. The E protein ectodomain can be divided into three structural domains designated ED1, ED2, and ED3, of which ED3 contains the critical and dominant virus-specific neutralization sites. In this study the ED3 epitopes recognized by seven, murine, IgG1 DENV-2 type-specific, monoclonal antibodies (MAbs) were determined using site-directed mutagenesis of a recombinant DENV-2 ED3 (rED3) protein. A total of 41 single amino acid substitutions were introduced into the rED3 at 30 different surface accessible residues. The affinity of each MAb with the mutant rED3s was assessed by indirect ELISA and the results indicate that all seven MAbs recognize overlapping epitopes with residues K305 and P384 critical for binding. These residues are conserved among DENV-2 strains and cluster together on the upper lateral face of ED3. A linear relationship was observed between relative occupancy of ED3 on the virion by MAb and neutralization of the majority of virus infectivity (∼ 90%) for all seven MAbs. Depending on the MAb, it is predicted that between 10% and 50% relative occupancy of ED3 on the virion is necessary for virus neutralization and for all seven MAbs occupancy levels approaching saturation were required for 100% neutralization of virus infectivity. Overall, the conserved antigenic site recognized by all seven MAbs is likely to be a dominant DENV-2 type-specific, neutralization determinant

Protein-scaffold Directed Nanoscale Assembly of T Cell Ligands: Artificial Antigen Presentation with Defined Valency, Density and Ratio.

Science.gov (United States)

Smith, Mason R; Tolbert, Stephanie V; Wen, Fei

2018-05-07

Tuning antigen presentation to T cells is a critical step in investigating key aspects of T cell activation. However, existing technologies have limited ability to control the spatial and stoichiometric organization of T cell ligands on 3D surfaces. Here, we developed an artificial antigen presentation platform based on protein-scaffold directed assembly that allows fine control over the spatial and stoichiometric organization of T cell ligands on a 3D yeast-cell surface. Using this system, we observed that the T cell activation threshold on a 3D surface is independent of peptide-major histocompatibility complex (pMHC) valency, but instead determined by the overall pMHC surface density. When intercellular adhesion molecule 1 (ICAM-1) was co-assembled with pMHC, it enhanced antigen recognition sensitivity by 6-fold. Further, T cells responded with different magnitudes to varying ratios of pMHC and ICAM-1 and exhibited a maximum response at a ratio of 15% pMHC and 85% ICAM-1, introducing an additional parameter for tuning T cell activation. This protein-scaffold directed assembly technology is readily transferrable to acellular surfaces for translational research as well as large-scale T-cell manufacturing.

Antigen-specific and non-specific CD4+ T cell recruitment and proliferation during influenza infection

International Nuclear Information System (INIS)

Chapman, Timothy J.; Castrucci, Maria R.; Padrick, Ryan C.; Bradley, Linda M.; Topham, David J.

2005-01-01

To track epitope-specific CD4 + T cells at a single-cell level during influenza infection, the MHC class II-restricted OVA 323-339 epitope was engineered into the neuraminidase stalk of influenza/A/WSN, creating a surrogate viral antigen. The recombinant virus, influenza A/WSN/OVA II , replicated well, was cleared normally, and stimulated both wild-type and DO11.10 or OT-II TCR transgenic OVA-specific CD4 + T cells. OVA-specific CD4 T cells proliferated during infection only when the OVA epitope was present. However, previously primed (but not naive) transgenic CD4 + T cells were recruited to the infected lung both in the presence and absence of the OVA 323-339 epitope. These data show that, when primed, CD4 + T cells may traffic to the lung in the absence of antigen, but do not proliferate. These results also document a useful tool for the study of CD4 T cells in influenza infection

Epigenetic Silencing of CXCR4 Promotes Loss of Cell Adhesion in Cervical Cancer

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Suresh Singh Yadav

2014-01-01

Full Text Available In the network of chemokine signaling pathways, recent reports have described the SDF-1α/CXCR4 axis and its role in cancer progression and metastasis. Interestingly, we found downregulation of CXCR4 at both transcript and protein level in cervical cancer cell lines and primary tumors. We also found CXCR4 promoter hypermethylation in cervical cancer cell lines and primary biopsy samples. DNA hypomethylating drug 5-AZA-2′-deoxycytidine and histone deacetylase inhibitor Trichostatin A treatments in cell lines reactivate both CXCR4 transcription and protein expression. Cell adhesion assay demonstrated that autocrine SDF-1α promotes the loss of cell adhesion while paracrine SDF-1α predominantly protects the normal cervical cells from loss of cell adhesion. Cervical cancer cell line C-33A having increased expression of CXCR4 after TSA treatment showed increased cell adhesion by paracrine source of SDF-1α in comparison to untreated C-33A. These findings demonstrate the first evidence that epigenetic silencing of CXCR4 makes the cells inefficient to respond to the paracrine source of SDF-1α leading to loss of cell adhesion, one of the key events in metastases and progression of the disease. Our results provide novel insight of SDF-1α/CXCR4 signaling in tumor microenvironment which may be promising to further delineate molecular mechanism of cervical carcinogenesis.

Antigen-Specific Polyclonal Cytotoxic T Lymphocytes Induced by Fusions of Dendritic Cells and Tumor Cells

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Shigeo Koido

2010-01-01

Full Text Available The aim of cancer vaccines is induction of tumor-specific cytotoxic T lymphocytes (CTLs that can reduce the tumor mass. Dendritic cells (DCs are potent antigen-presenting cells and play a central role in the initiation and regulation of primary immune responses. Thus, DCs-based vaccination represents a potentially powerful strategy for induction of antigen-specific CTLs. Fusions of DCs and whole tumor cells represent an alternative approach to deliver, process, and subsequently present a broad spectrum of antigens, including those known and unidentified, in the context of costimulatory molecules. Once DCs/tumor fusions have been infused back into patient, they migrate to secondary lymphoid organs, where the generation of antigen-specific polyclonal CTL responses occurs. We will discuss perspectives for future development of DCs/tumor fusions for CTL induction.

The antigen presenting cells instruct plasma cell differentiation.

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Xu, Wei; Banchereau, Jacques

2014-01-06

The professional antigen presenting cells (APCs), including many subsets of dendritic cells and macrophages, not only mediate prompt but non-specific response against microbes, but also bridge the antigen-specific adaptive immune response through antigen presentation. In the latter, typically activated B cells acquire cognate signals from T helper cells in the germinal center of lymphoid follicles to differentiate into plasma cells (PCs), which generate protective antibodies. Recent advances have revealed that many APC subsets provide not only "signal 1" (the antigen), but also "signal 2" to directly instruct the differentiation process of PCs in a T-cell-independent manner. Herein, the different signals provided by these APC subsets to direct B cell proliferation, survival, class switching, and terminal differentiation are discussed. We furthermore propose that the next generation of vaccines for boosting antibody response could be designed by targeting APCs.

Antigen processing and remodeling of the endosomal pathway: requirements for antigen cross-presentation.

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Compeer, Ewoud Bernardus; Flinsenberg, Thijs Willem Hendrik; van der Grein, Susanna Geertje; Boes, Marianne

2012-01-01

Cross-presentation of endocytosed antigen as peptide/class I major histocompatibility complex complexes plays a central role in the elicitation of CD8(+) T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells capable of antigen cross-presentation, identification of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC), there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlights DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, maturation-induced endosomal sorting of membrane proteins, dynamic remodeling of endosomal structures and cell surface-directed endosomal trafficking. We will conclude with the description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.

Antigen processing and remodeling of the endosomal pathway: requirements for antigen cross-presentation.

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Ewoud Bernardus Compeer

2012-03-01

Full Text Available The cross-presentation of endocytosed antigen as peptide/class I MHC complexes plays a central role in the elicitation of CD8+ T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells (APC capable of antigen cross-presentation, description of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC, there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlight DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, recycling and maturation including the sorting of membrane proteins, dynamic remodeling of endosomal structures and cell-surface directed endosomal trafficking. We will conclude with description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.

Predictive value of prostate-specific antigen for prostate cancer

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Shepherd, Leah; Borges, Alvaro Humberto; Ravn, Lene

2014-01-01

INTRODUCTION: Although prostate cancer (PCa) incidence is lower in HIV+ men than in HIV- men, the usefulness of prostate-specific antigen (PSA) screening in this population is not well defined and may have higher false negative rates than in HIV- men. We aimed to describe the kinetics and predict......INTRODUCTION: Although prostate cancer (PCa) incidence is lower in HIV+ men than in HIV- men, the usefulness of prostate-specific antigen (PSA) screening in this population is not well defined and may have higher false negative rates than in HIV- men. We aimed to describe the kinetics...... and predictive value of PSA in HIV+ men. METHODS: Men with PCa (n=21) and up to two matched controls (n=40) with prospectively stored plasma samples before PCa (or matched date in controls) were selected. Cases and controls were matched on date of first and last sample, age, region of residence and CD4 count...... at first sample date. Total PSA (tPSA), free PSA (fPSA), testosterone and sex hormone binding globulin (SHBG) were measured. Conditional logistic regression models investigated associations between markers and PCa. Sensitivity and specificity of using tPSA >4 µg/L to predict PCa was calculated. Mixed...

Feasibility of minimally invasive radical prostatectomy in prostate cancer patients with high prostate-specific antigen. Feasibility and 1-year outcomes

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Do, M.; Ragavan, N.; Dietel, A.; Liatsikos, E.; Stolzenburg, J.U.; Anderson, C.; McNeill, A.

2012-01-01

Urologists are cautious to offer minimally invasive radical prostatectomy in prostate cancer patients with high prostate-specific antigen (and therefore anticipated to have locally advanced or metastatic disease) because of concerns regarding lack of complete cure after minimally invasive radical prostatectomy and of worsening of continence if adjuvant radiotherapy is used. A retrospective review of our institutional database was carried out to identify patients with prostate specific antigen (PSA) ≥20 ng/mL who underwent minimally invasive radical prostatectomy between January 2002 and October 2010. Intraoperative, pathological, functional and short-term oncological outcomes were assessed. Overall, 233 patients met study criteria and were included in the analysis. The median prostate-specific antigen and prostate size were 28.5 ng/mL and 47 mL, respectively. Intraoperative complications were the following: rectal injury (0.86%) and blood transfusion (1.7%). Early postoperative complications included prolonged (>6 days) catheterization (9.4%), hematoma (4.7%), deep venous thrombosis (0.86%) and lymphocele (5.1%). Late postoperative complications included cerebrovascular accident (0.4%) and anastomotic stricture (0.8%). Pathology revealed poorly differentiated cancer in 48.9%, pT3/pT4 disease in 55.8%, positive margins in 28.3% and lymph node disease in 20.2% of the cases. Adverse pathological findings were more frequent in patients with prostate-specific antigen >40 ng/mL and (or) in those with locally advanced disease (pT3/pT4). In 62.2% of the cases, adjuvant radiotherapy was used. At 1-year follow up, 80% of patients did not show evidence of biochemical recurrence and 98.8% of them had good recovery of continence. Minimally invasive radical prostatectomy might represent a reasonable option in prostate cancer patients with high prostate-specific antigen as a part of a multimodality treatment approach. (author)

Observations of pretreatment prostate-specific antigen doubling time in 107 patients referred for definitive radiotherapy

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Lee, W. Robert; Hanks, Gerald E.; Corn, Benjamin W.; Schultheiss, Timothy E.

1995-01-01

Purpose: To determine pretreatment prostate-specific antigen doubling times (PSADT) in patients referred for definitive radiotherapy. Methods and Materials: One hundred and seven patients with histologically proven nonmetastatic prostate cancer and an elevated prostate-specific antigen (PSA) who were referred for radiation therapy had three serum PSA values obtained prior to the start of definitive therapy. Prostate-specific antigen doubling times were calculated by linear regression. Results: Prostate-specific antigen values increased during the period of observation in 78 patients (73%). Forty-three patients (40%) had calculated PSADT of less than 2 years and of those patients with pretreatment serum PSA values of greater than 10 ng/mL more than 50% has calculated PSADT of less than 2 years. Conclusions: A significant minority of patients referred for radiotherapy have calculated PSADT of less than 2 years. The significance of this relatively fast growth rate is as yet undetermined, but suggests that patients referred for radiotherapy may have aggressive disease prior to treatment

Development of CXCR4 modulators by virtual HTS of a novel amide-sulfamide compound library.

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Bai, Renren; Shi, Qi; Liang, Zhongxing; Yoon, Younghyoun; Han, Yiran; Feng, Amber; Liu, Shuangping; Oum, Yoonhyeun; Yun, C Chris; Shim, Hyunsuk

2017-01-27

CXCR4 plays a crucial role in recruitment of inflammatory cells to inflammation sites at the beginning of the disease process. Modulating CXCR4 functions presents a new avenue for anti-inflammatory strategies. However, using CXCR4 antagonists for a long term usage presents potential serious side effect due to their stem cell mobilizing property. We have been developing partial CXCR4 antagonists without such property. A new computer-aided drug design program, the FRESH workflow, was used for anti-CXCR4 lead compound discovery and optimization, which coupled both compound library building and CXCR4 docking screens in one campaign. Based on the designed parent framework, 30 prioritized amide-sulfamide structures were obtained after systemic filtering and docking screening. Twelve compounds were prepared from the top-30 list. Most synthesized compounds exhibited good to excellent binding affinity to CXCR4. Compounds Ig and Im demonstrated notable in vivo suppressive activity against xylene-induced mouse ear inflammation (with 56% and 54% inhibition). Western blot analyses revealed that Ig significantly blocked CXCR4/CXCL12-mediated phosphorylation of Akt. Moreover, Ig attenuated the amount of TNF-α secreted by pathogenic E. coli-infected macrophages. More importantly, Ig had no observable cytotoxicity. Our results demonstrated that FRESH virtual high throughput screening program of targeted chemical class could successfully find potent lead compounds, and the amide-sulfamide pharmacophore was a novel and effective framework blocking CXCR4 function. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Differential expression and prognostic value of the chemokine receptor CXCR4 in bronchopulmonary neuroendocrine neoplasms

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Specht, Elisa; Wirtz, Ralph M.; Sayeg, Manal; Baum, Richard P.; Schulz, Stefan; Lupp, Amelie

2015-01-01

Introduction For many tumors, the overexpression of the chemokine receptor CXCR4 is associated with increased malignancy and poor patient outcomes. However, comprehensive data for neuroendocrine neoplasms of the lung are still lacking. Methods CXCR4 expression was evaluated in a panel of bronchopulmonary neuroendocrine neoplasms (BP-NEN) comprising typical carcinoids (n = 26), atypical carcinoids (n = 30), and small cell lung cancers (SCLC, n = 34). Samples were analyzed by immunohistochemistry using the novel monoclonal rabbit anti-human CXCR4 antibody UMB-2 and by qRT-PCR. The expression was correlated with clinical data and overall patient survival. Results CXCR4 was predominantly localized at the plasma membrane of the tumor cells. CXCR4 was expressed with a high intensity in almost all of the 30 SCLC samples. In contrast, it was detected infrequently and with low intensity in the typical carcinoid and atypical carcinoid samples. There was a significant correlation between the immunohistochemistry and qRT-PCR data. Additionally, there was a significant negative relationship between CXCR4 expression and overall survival. Conclusions With increasing malignancy, BP-NEN clearly differ in the extent of CXCR4 expression. As in other tumor entities, CXCR4 overexpression significantly correlates with negative patient outcome. Due to its particular high expression rate in SCLC, CXCR4 may serve as a promising new target for diagnostic and pharmacological intervention as well as for peptide receptor-based radionuclide therapy. PMID:25671300

Efficient T-cell surveillance of the CNS requires expression of the CXC chemokine receptor 3

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Christensen, Jeanette Erbo; Nansen, Anneline; Moos, Torben

2004-01-01

T-cells play an important role in controlling viral infections inside the CNS. To study the role of the chemokine receptor CXCR3 in the migration and positioning of virus-specific effector T-cells within the brain, CXCR3-deficient mice were infected intracerebrally with lymphocytic choriomeningitis......-cell-mediated immunopathology. Quantitative analysis of the cellular infiltrate in CSF of infected mice revealed modest, if any, decrease in the number of mononuclear cells recruited to the meninges in the absence of CXCR3. However, immunohistological analysis disclosed a striking impairment of CD8+ T-cells from CXCR3...

CXCR4 expression in papillary thyroid carcinoma: induction by nitric oxide and correlation with lymph node metastasis

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Yasuoka, Hironao; Kodama, Rieko; Hirokawa, Mitsuyoshi; Takamura, Yuuki; Miyauchi, Akira; Sanke, Tokio; Nakamura, Yasushi

2008-01-01

Metastasis to regional lymph nodes is a common step in the progression of cancer. Recent evidence suggests that tumor production of CXCR4 promotes lymph node metastasis. Nitric oxide (NO) may also increase metastatic ability in human cancers. Nitrite/nitrate levels and functional CXCR4 expression were assessed in K1 and B-CPAP papillary thyroid carcinoma (PTC) cells after induction and/or inhibition of NO synthesis. CXCR4 expression was also analyzed in primary human PTC. The relationship between nitrotyrosine levels, which are a biomarker for peroxynitrate formation from NO in vivo, CXCR4 expression, and lymph node status was also analyzed. Production of nitrite/nitrate and functional CXCR4 expression in both cell lines was increased by treatment with the NO donor DETA NONOate. The NOS inhibitor L-NAME eliminated this increase. Positive CXCR4 immunostaining was observed in 60.7% (34/56) of PTCs. CXCR4 expression was significantly correlated with nitrotyrosine levels and lymph node metastasis in human PTC. Our data indicate that NO stimulates CXCR4 expression in vitro. Formation of the NO biomarker nitrotyrosine was also correlated with CXCR4 expression and lymph node metastasis in human PTC. NO may induce lymph node metastasis via CXCR4 induction in papillary thyroid carcinoma

Prostatic specific antigen. From its early days until becoming a prostate cancer biomarker.

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Dellavedova, T

2016-01-01

Prostate-specific antigen (PSA) has been since the mid 80's the most commonly used biomarker for measuring current and future risk of prostate cancer, for its early detection and to measure response to treatments and detecting recurrence in all stages of the disease. PSA's early development came along with progress in the field of immunology, which allowed detection and study of antigens from different tissues and fluids when injecting them into rabbits to promote immune response. Rubin Flocks in 1960 was the first to investigate and discover prostate-specific antigens in benign and malignant tissue. Some years later, Hara, a Japanese forensic investigator, found 'gamma seminoprotein', that he used to detect human semen in rape cases. However, his work published in Japanese did not reach the Englishspeaking scientific community. In 1970 Ablin discovered both in prostatic fluid and tissue what he called "prostate-specific antigen", but he didn't characterize or describe it. Investigators Li and Beling, and Sensabaugh, approached the current PSA, but they were limited by available technology at that time. Dr T Ming Chu led a research team on prostate cancer in New York, USA and published their results in 1979. He finally received the patent for the discovery of "human purified prostate antigen" in 1984. Due to this work, the Food and Drug Administration (FDA), in USA, approved the use of PSA for monitoring recurrence after treatment. It was later known that PSA was not prostate-specific since it was produced in other tissues and fluids, but it was recognized that it was human species-specific. Works by Papsidero and Stamey showed new indications and utilities for PSA, but it was Catalona who first used it as a marker for prostate cancer in 1991. Thanks to these advances FDA authorized in 1994 the clinical use of PSA for early detection of prostate cancer.

Dissection of T-cell antigen specificity in human melanoma

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Andersen, Rikke Sick; Albæk Thrue, Charlotte; Junker, Niels

2012-01-01

Tumor-infiltrating lymphocytes (TIL) isolated from melanoma patients and expanded in vitro by interleukin (IL)-2 treatment can elicit therapeutic response after adoptive transfer, but the antigen specificities of the T cells transferred have not been determined. By compiling all known melanoma-as...... from different fragments of resected melanoma lesions. In summary, our findings provide an initial definition of T-cell populations contributing to tumor recognition in TILs although the specificity of many tumor-reactive TILs remains undefined....

CXCR7 is induced by hypoxia and mediates glioma cell migration towards SDF-1α

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Esencay, Mine; Sarfraz, Yasmeen; Zagzag, David

2013-01-01

Glioblastomas, the most common and malignant brain tumors of the central nervous system, exhibit high invasive capacity, which hinders effective therapy. Therefore, intense efforts aimed at improved therapeutics are ongoing to delineate the molecular mechanisms governing glioma cell migration and invasion. In order to perform the studies, we employed optimal cell culture methods and hypoxic conditions, lentivirus-mediated knockdown of protein expression, Western Blot analysis, migration assays and immunoprecipitation. We determined statistical significance by unpaired t-test. In this report, we show that U87MG, LN229 and LN308 glioma cells express CXCR7 and that exposure to hypoxia upregulates CXCR7 protein expression in these cell lines. CXCR7-expressing U87MG, LN229 and LN308 glioma cells migrated towards stromal-derived factor (SDF)-1α/CXCL12 in hypoxic conditions in the Boyden chamber assays. While shRNA-mediated knockdown of CXCR7 expression did not affect the migration of any of the three cell lines in normoxic conditions, we observed a reduction in the migration of LN229 and LN308, but not U87MG, glioma cells towards SDF-1α in hypoxic conditions. In addition, knockdown of CXCR7 expression in LN229 and LN308 glioma cells decreased levels of SDF-1α-induced phosphorylation of ERK1/2 and Akt. Inhibiting CXCR4 in LN229 and LN308 glioma cells that were knocked down for CXCR7 did not further reduce migration towards SDF-1α in hypoxic conditions and did not affect the levels of phosphorylated ERK1/2 and Akt. Analysis of immunoprecipitated CXCR4 from LN229 and LN308 glioma cells revealed co-precipitated CXCR7. Taken together, our findings indicate that both CXCR4 and CXCR7 mediate glioma cell migration towards SDF-1α in hypoxic conditions and support the development of therapeutic agents targeting these receptors

Immunity to tumour antigens.

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Li, Geng; Ali, Selman A; McArdle, Stephanie E B; Mian, Shahid; Ahmad, Murrium; Miles, Amanda; Rees, Robert C

2005-01-01

During the last decade, a large number of human tumour antigens have been identified. These antigens are classified as tumour-specific shared antigens, tissue-specific differentiation antigens, overexpressed antigens, tumour antigens resulting from mutations, viral antigens and fusion proteins. Antigens recognised by effectors of immune system are potential targets for antigen-specific cancer immunotherapy. However, most tumour antigens are self-proteins and are generally of low immunogenicity and the immune response elicited towards these tumour antigens is not always effective. Strategies to induce and enhance the tumour antigen-specific response are needed. This review will summarise the approaches to discovery of tumour antigens, the current status of tumour antigens, and their potential application to cancer treatment.

The prognostic significance of preoperative serum cancer antigen 15-3 levels in endometrial carcinomas

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Tas, Emre E.; Yavuz, Ayse F.

2017-01-01

Objectives: To determine the associations between serum cancer antigen 15-3 levels and prognostic factors in patients with endometrial carcinomas. Additionally, we investigated the clinical utility of serum cancer antigen 15-3 levels in the selection of low-risk patients with endometrioid type, tumor size <2 cm, myometrial invasion ≤50%, and histological grade 1-2. Methods: Ninety-six patients, who were surgically staged at Ankara Yildirim Beyazit University, Ankara, Turkey, between 2007 and 2016, were retrospectively analyzed. Demographic, clinical, and surgical characteristics were retrieved from the patients’ hospital records. A p<0.05 was considered significant. Results: Fifteen patients had advanced (≥Stage II) disease, 14 patients had Type 2 histology, 20 patients had Grade 3 tumors, 23 patients had lymphovascular space invasion, and 10 patients had positive lymph node involvement. Serum cancer antigen 15-3 levels were significantly higher in patients with advanced (≥Stage II) disease, Type 2 histology, Grade 3 tumors, lymp°hovascular space invasion, and positive lymph node involvement (p<0.05). Serum cancer antigen 15-3 levels were also significantly correlated with tumor size (p=0.006). Serum cancer antigen 15-3 levels were significantly lower (95% confidence interval: 0.57−0.79; p=0.03) in low-risk patients compared to other endometrial carcinoma patients. A cutoff of 25.0 IU/mL was used to identify high-risk patients with a specificity of 100%. Conclusion: Serum cancer antigen 15-3 levels significantly correlated with prognostic factors and were a useful diagnostic tool for endometrial carcinomas. PMID:29114696

The prognostic significance of preoperative serum cancer antigen 15-3 levels in endometrial carcinomas

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Emre E. Tas

2017-11-01

Full Text Available Objectives: To determine the associations between serum cancer antigen 15-3 levels and prognostic factors in patients with endometrial carcinomas. Additionally, we investigated the clinical utility of serum cancer antigen 15-3 levels in the selection of low-risk patients with endometrioid type, tumor size less than 2 cm, myometrial invasion ≤50%, and histological grade 1-2. Methods: Ninety-six patients, who were surgically staged at Ankara Yildirim Beyazit University, Ankara, Turkey, between 2007 and 2016, were retrospectively analyzed. Demographic, clinical, and surgical characteristics were retrieved from the patients’ hospital records. A p less than 0.05 was considered significant. Results: Fifteen patients had advanced (≥Stage II disease, 14 patients had Type 2 histology, 20 patients had Grade 3 tumors, 23 patients had lymphovascular space invasion, and 10 patients had positive lymph node involvement. Serum cancer antigen 15-3 levels were significantly higher in patients with advanced (≥Stage II disease, Type 2 histology, Grade 3 tumors, lymphovascular space invasion, and positive lymph node involvement (p less than 0.05. Serum cancer antigen 15-3 levels were also significantly correlated with tumor size (p=0.006. Serum cancer antigen 15-3 levels were significantly lower (95% confidence interval: 0.57−0.79; p=0.03 in low-risk patients compared to other endometrial carcinoma patients. A cutoff of 25.0 IU/mL was used to identify high-risk patients with a specificity of 100%. Conclusion: Serum cancer antigen 15-3 levels significantly correlated with prognostic factors and were a useful diagnostic tool for endometrial carcinomas.

Sequence similarity between the erythrocyte binding domain 1 of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals binding residues for the Duffy Antigen Receptor for Chemokines

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Garry Robert F

2011-01-01

Full Text Available Abstract Background The surface glycoprotein (SU, gp120 of the human immunodeficiency virus (HIV must bind to a chemokine receptor, CCR5 or CXCR4, to invade CD4+ cells. Plasmodium vivax uses the Duffy Binding Protein (DBP to bind the Duffy Antigen Receptor for Chemokines (DARC and invade reticulocytes. Results Variable loop 3 (V3 of HIV-1 SU and domain 1 of the Plasmodium vivax DBP share a sequence similarity. The site of amino acid sequence similarity was necessary, but not sufficient, for DARC binding and contained a consensus heparin binding site essential for DARC binding. Both HIV-1 and P. vivax can be blocked from binding to their chemokine receptors by the chemokine, RANTES and its analog AOP-RANTES. Site directed mutagenesis of the heparin binding motif in members of the DBP family, the P. knowlesi alpha, beta and gamma proteins abrogated their binding to erythrocytes. Positively charged residues within domain 1 are required for binding of P. vivax and P. knowlesi erythrocyte binding proteins. Conclusion A heparin binding site motif in members of the DBP family may form part of a conserved erythrocyte receptor binding pocket.

Isolation and characterization of NIH 3T3 cells expressing polyomavirus small T antigen

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Noda, T.; Satake, M.; Robins, T.; Ito, Y.

1986-01-01

The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of PSI2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 10 6 cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture

Role of GPER1, EGFR and CXCR1 in differentiating between malignant follicular thyroid carcinoma and benign follicular thyroid adenoma

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Zhao, Le; Zhu, Xiao-Yun; Jiang, Rong; Xu, Man; Wang, Ni; Chen, George G; Liu, Zhi-Min

2015-01-01

It is extremely difficult to discriminate between follicular thyroid carcinoma (FTC) and follicular thyroid adenoma (FTA) before surgery, because the morphologies of carcinoma cells and adenoma cells obtained by fine needle aspiration biopsy (FNAB) are similar. Molecular markers may be helpful on this issue. The purpose of this study was to assess the role of GPER1, EGFR and CXCR1 in differential diagnosis between FTC and FTA. GPER1, EGFR and CXCR1 mRNA expression levels were examined in 15 FTCs and 10 FTAs using real-time RT-PCR. FTC showed to have significantly increased mRNA levels of the three molecules compared to FTA (P FTA, respectively. Statistical analysis showed that GPER1, EGFR and CXCR1 protein expression were correlated with one another in FTC and concomitant high expression of the three molecules had stronger correlation with the occurrence of FTC than did each alone. The positive predictive values (PPV) for concomitant high expression of the three molecules for discriminating between FTC and FTA were 91.0% for GPER1/EGFR, 93.8% for GPER1/CXCR1, 92.3% for EGFR/CXCR1 and 98.2% for GPER1/EGFR/CXCR1, respectively. These results indicated that the evaluation of GPER1, EGFR and CXCR1 concomitant high expression may be helpful in differential diagnosis between FTC and FTA. PMID:26617848

CXCL10/CXCR3 signaling in glia cells differentially affects NMDA-induced cell death in CA and DG neurons of the mouse hippocampus

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van Weering, Hilmar R J; Boddeke, Hendrikus W G M; Vinet, Jonathan

2011-01-01

are far from understood. Here, we investigated the potential role for CXCL10/CXCR3 signaling in neuronal cell death and glia activation in response to N-methyl-D-aspartic acid (NMDA)-induced excitotoxicity in mouse organotypic hippocampal slice cultures (OHSCs). Our findings demonstrate that astrocytes...

Antigen detection systems

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Infectious agents or their constituent parts (antigens or nucleic acids) can be detected in fresh, frozen, or fixed tissues or other specimens, using a variety of direct or indirect assays. The assays can be modified to yield the greatest sensitivity and specificity but in most cases a particular m...

Antigen-specific tolerance of human alpha1-antitrypsin induced by helper-dependent adenovirus.

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Cerullo, V; McCormack, W; Seiler, M; Mane, V; Cela, R; Clarke, C; Rodgers, J R; Lee, B

2007-12-01

As efficient and less toxic virus-derived gene therapy vectors are developed, a pressing problem is to avoid immune response to the therapeutic gene product. Secreted therapeutic proteins potentially represent a special problem, as they are readily available to professional antigen-presenting cells throughout the body. Some studies suggest that immunity to serum proteins can be avoided in some mouse strains by using tissue-specific promoters. Here we show that expression of human alpha1-antitrypsin (AAT) was nonimmunogenic in the immune-responsive strain C3H/HeJ, when expressed from helper-dependent (HD) vectors using ubiquitous as well as tissue-specific promoters. Coadministration of less immunogenic HD vectors with an immunogenic first-generation vector failed to immunize, suggesting immune suppression rather than immune stealth. Indeed, mice primed with HD vectors were tolerant to immune challenge with hAAT emulsified in complete Freund's adjuvant. Such animals developed high-titer antibodies to coemulsified human serum albumin, showing that tolerance was antigen specific. AAT-specific T cell responses were depressed in tolerized animals, suggesting that tolerance affects both T and B cells. These results are consistent with models of high-dose tolerance of B cells and certain other suppressive mechanisms, and suggest that a high level of expression from HD vectors can be sufficient to induce specific immune tolerance to serum proteins.

Circulating CXCR5⁺CD4⁺ T Follicular-Like Helper Cell and Memory B Cell Responses to Human Papillomavirus Vaccines.

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Ken Matsui

Full Text Available Through the interaction of T follicular helper (Tfh cells and B cells, efficacious vaccines can generate high-affinity, pathogen-neutralizing antibodies, and memory B cells. Using CXCR5, CXCR3, CCR6, CCR7, PD1, and ICOS as markers, Tfh-like cells can be identified in the circulation and be classified into three functionally distinct subsets that are PD1+ICOS+, PD1+ ICOS-, or PD1-ICOS-. We used these markers to identify different subsets of CXCR5+CD4+ Tfh-like cells in response to highly immunogenic and efficacious vaccines for human papillomaviruses (HPV: Cervarix and Gardasil. In this small study, we used PBMC samples from 11 Gardasil recipients, and 8 Cervarix recipients from the Vaccine Research Center 902 Study to examine the induction of circulating Tfh-like cells and IgD-CD38HiCD27+ memory B cells by flow cytometry. PD1+ICOS+ CXCR3+CCR6-CXCR5+CD4+ (Tfh1-like cells were induced and peaked on Day (D 7 post-first vaccination, but not as much on D7 post-third vaccination. We also observed a trend toward increase in PD1+ICOS+ CXCR3-CCR6-CXCR5+CD4+ (Tfh2-like cells for both vaccines, and PD1+ICOS+ CXCR3-CCR6+CXCR5+CD4+ (Tfh17-like subset was induced by Cervarix post-first vaccination. There were also minimal changes in the other cellular subsets. In addition, Cervarix recipients had more memory B cells post-first vaccination than did Gardasil recipients at D14 and D30. We found frequencies of memory B cells at D30 correlated with anti-HPV16 and 18 antibody titers from D30, and the induction levels of memory B cells at D30 and PD1+ICOS+Tfh1-like cells at D7 post-first vaccination correlated for Cervarix. Our study showed that induction of circulating CXCR5+CD4+ Tfh-like subsets can be detected following immunization with HPV vaccines, and potentially be useful as a marker of immunogenicity of vaccines. However, further investigations should be extended to different cohorts with larger sample size to better understand the functions of these T

The CXC Chemokine Receptor 3 Inhibits Autoimmune Cholangitis via CD8+ T Cells but Promotes Colitis via CD4+ T Cells

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Qing-Zhi Liu

2018-05-01

Full Text Available CXC chemokine receptor 3 (CXCR3, a receptor for the C-X-C motif chemokines (CXCL CXCL9, CXCL10, and CXCL11, which not only plays a role in chemotaxis but also regulates differentiation and development of memory and effector T cell populations. Herein, we explored the function of CXCR3 in the modulation of different organ-specific autoimmune diseases in interleukin (IL-2 receptor deficiency (CD25−/− mice, a murine model for both cholangitis and colitis. We observed higher levels of CXCL9 and CXCL10 in the liver and colon and higher expression of CXCR3 on T cells of the CD25−/− mice compared with control animals. Deletion of CXCR3 resulted in enhanced liver inflammation but alleviated colitis. These changes in liver and colon pathology after CXCR3 deletion were associated with increased numbers of hepatic CD4+ and CD8+ T cells, in particular effector memory CD8+ T cells, as well as decreased T cells in mesenteric lymph nodes and colon lamina propria. In addition, increased interferon-γ response and decreased IL-17A response was observed in both liver and colon after CXCR3 deletion. CXCR3 modulated the functions of T cells involved in different autoimmune diseases, whereas the consequence of such modulation was organ-specific regarding to their effects on disease severity. Our findings emphasize the importance of extra caution in immunotherapy for organ-specific autoimmune diseases, as therapeutic interventions aiming at a target such as CXCR3 for certain disease could result in adverse effects in an unrelated organ.

Pretreatment antigen-specific immunity and regulation - association with subsequent immune response to anti-tumor DNA vaccination.

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Johnson, Laura E; Olson, Brian M; McNeel, Douglas G

2017-07-18

Immunotherapies have demonstrated clinical benefit for many types of cancers, however many patients do not respond, and treatment-related adverse effects can be severe. Hence many efforts are underway to identify treatment predictive biomarkers. We have reported the results of two phase I trials using a DNA vaccine encoding prostatic acid phosphatase (PAP) in patients with biochemically recurrent prostate cancer. In both trials, persistent PAP-specific Th1 immunity developed in some patients, and this was associated with favorable changes in serum PSA kinetics. In the current study, we sought to determine if measures of antigen-specific or antigen non-specific immunity were present prior to treatment, and associated with subsequent immune response, to identify possible predictive immune biomarkers. Patients who developed persistent PAP-specific, IFNγ-secreting immune responses were defined as immune "responders." The frequency of peripheral T cell and B cell lymphocytes, natural killer cells, monocytes, dendritic cells, myeloid derived suppressor cells, and regulatory T cells were assessed by flow cytometry and clinical laboratory values. PAP-specific immune responses were evaluated by cytokine secretion in vitro, and by antigen-specific suppression of delayed-type hypersensitivity to a recall antigen in an in vivo SCID mouse model. The frequency of peripheral blood cell types did not differ between the immune responder and non-responder groups. Non-responder patients tended to have higher PAP-specific IL-10 production pre-vaccination (p = 0.09). Responder patients had greater preexisting PAP-specific bystander regulatory responses that suppressed DTH to a recall antigen (p = 0.016). While our study population was small (n = 38), these results suggest that different measures of antigen-specific tolerance or regulation might help predict immunological outcome from DNA vaccination. These will be prospectively evaluated in an ongoing randomized, phase II trial.

The performance characteristics of prostate-specific antigen and prostate-specific antigen density in Chinese men.

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Teoh, Jeremy Yc; Yuen, Steffi Kk; Tsu, James Hl; Wong, Charles Kw; Ho, Brian Sh; Ng, Ada Tl; Ma, Wai-Kit; Ho, Kwan-Lun; Yiu, Ming-Kwong

2017-01-01

We investigated the performance characteristics of prostate-specific antigen (PSA) and PSA density (PSAD) in Chinese men. All Chinese men who underwent transrectal ultrasound-guided prostate biopsy (TRUS-PB) from year 2000 to 2013 were included. The receiver operating characteristic (ROC) curves for both PSA and PSAD were analyzed. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) at different cut-off levels were calculated. A total of 2606 Chinese men were included. For the ROC, the area under curve was 0.770 for PSA (P specificity of 14.1%, PPV of 29.5%, and NPV of 86.9%; PSAD of 0.12 ng ml-1 cc-1 had sensitivity of 94.5%, specificity of 26.6%, PPV of 32.8%, and NPV of 92.7%. On multivariate logistic regression analyses, PSA cut-off at 4.5 ng ml-1 (OR 1.61, 95% CI 1.05-2.45, P= 0.029) and PSAD cut-off at 0.12 ng ml-1 cc-1 (OR 6.22, 95% CI 4.20-9.22, Pprostate cancer detection on TRUS-PB. In conclusion, the performances of PSA and PSAD at different cut-off levels in Chinese men were very different from those in Caucasians. PSA of 4.5 ng ml-1 and PSAD of 0.12 ng ml-1 cc-1 had near 95% sensitivity and were significant predictors of prostate cancer detection in Chinese men.

Enzyme-labeled Antigen Method: Development and Application of the Novel Approach for Identifying Plasma Cells Locally Producing Disease-specific Antibodies in Inflammatory Lesions

International Nuclear Information System (INIS)

Mizutani, Yasuyoshi; Shiogama, Kazuya; Onouchi, Takanori; Sakurai, Kouhei; Inada, Ken-ichi; Tsutsumi, Yutaka

2016-01-01

In chronic inflammatory lesions of autoimmune and infectious diseases, plasma cells are frequently observed. Antigens recognized by antibodies produced by the plasma cells mostly remain unclear. A new technique identifying these corresponding antigens may give us a breakthrough for understanding the disease from a pathophysiological viewpoint, simply because the immunocytes are seen within the lesion. We have developed an enzyme-labeled antigen method for microscopic identification of the antigen recognized by specific antibodies locally produced in plasma cells in inflammatory lesions. Firstly, target biotinylated antigens were constructed by the wheat germ cell-free protein synthesis system or through chemical biotinylation. Next, proteins reactive to antibodies in tissue extracts were screened and antibody titers were evaluated by the AlphaScreen method. Finally, with the enzyme-labeled antigen method using the biotinylated antigens as probes, plasma cells producing specific antibodies were microscopically localized in fixed frozen sections. Our novel approach visualized tissue plasma cells that produced 1) autoantibodies in rheumatoid arthritis, 2) antibodies against major antigens of Porphyromonas gingivalis in periodontitis or radicular cyst, and 3) antibodies against a carbohydrate antigen, Strep A, of Streptococcus pyogenes in recurrent tonsillitis. Evaluation of local specific antibody responses expectedly contributes to clarifying previously unknown processes in inflammatory disorders

Effect of Heamolysis on Prostate-Specific Antigen

OpenAIRE

Sağlam, Hasan S.; Köse, Osman; Özdemir, Fatma; Adsan, Öztuğ

2012-01-01

Purpose. We have investigated the effect of haemolysis on free and total prostate-specific antigen (PSA) in daily clinical practice. Materials and Methods. Thirty-nine consecutive men were enrolled in this study. With an 18 gauge (G) needle 4 cc of blood samples were drawn from the right arm and 2 cc of it was expelled gently in a Vacutainer for regular PSA assay and the remaining was emptied into a second tube for complete haemolysis. Simultaneously 2 cc of more blood were taken with a 26 G ...

Interruption of CXCL13-CXCR5 axis increases upper genital tract pathology and activation of NKT cells following chlamydial genital infection.

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Janina Jiang

Full Text Available Regulation of immune responses is critical for controlling inflammation and disruption of this process can lead to tissue damage. We reported that CXCL13 was induced in fallopian tube tissue following C. trachomatis infection. Here, we examined the influence of the CXCL13-CXCR5 axis in chlamydial genital infection.Disruption of the CXCL13-CXCR5 axis by injecting anti-CXCL13 Ab to BALB/c mice or using Cxcr5-/- mice increased chronic inflammation in the upper genital tract (UGT; uterine horns and oviducts after Chlamydia muridarum genital infection (GT. Further studies in Cxcr5-/- mice showed an elevation in bacterial burden in the GT and increased numbers of neutrophils, activated DCs and activated NKT cells early after infection. After resolution, we noted increased fibrosis and the accumulation of a variety of T cells subsets (CD4-IFNγ, CD4-IL-17, CD4-IL-10 & CD8-TNFα in the oviducts. NKT cell depletion in vitro reduced IL-17α and various cytokines and chemokines, suggesting that activated NKT cells modulate neutrophils and DCs through cytokine/chemokine secretion. Further, chlamydial glycolipids directly activated two distinct types of NKT cell hybridomas in a cell-free CD1d presentation assay and genital infection of Cd1d-/- mice showed reduced oviduct inflammation compared to WT mice. CXCR5 involvement in pathology was also noted using single-nucleotide polymorphism analysis in C. trachomatis infected women attending a sub-fertility clinic. Women who developed tubal pathology after a C. trachomatis infection had a decrease in the frequency of CXCR5 SNP +10950 T>C (rs3922.These experiments indicate that disruption of the CXCL13-CXCR5 axis permits increased activation of NKT cells by type I and type II glycolipids of Chlamydia muridarum and results in UGT pathology potentially through increased numbers of neutrophils and T cell subsets associated with UGT pathology. In addition, CXCR5 appears to contribute to inter-individual differences in

The SDF-1–CXCR4 signaling pathway: a molecular hub modulating neo-angiogenesis

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Petit, Isabelle; Jin, David; Rafii, Shahin

2010-01-01

Pro-angiogenic bone marrow (BM) cells include subsets of hematopoietic cells that provide vascular support and endothelial progenitor cells (EPCs), which under certain permissive conditions could differentiate into functional vascular cells. Recent evidence demonstrates that the chemokine stromal-cell derived factor-1 (SDF-1, also known as CXCL12) has a major role in the recruitment and retention of CXCR4+ BM cells to the neo-angiogenic niches supporting revascularization of ischemic tissue and tumor growth. However, the precise mechanism by which activation of CXCR4 modulates neo-angiogenesis is not clear. SDF-1 not only promotes revascularization by engaging with CXCR4 expressed on the vascular cells but also supports mobilization of pro-angiogenic CXCR4+VEGFR1+ hematopoietic cells, thereby accelerating revascularization of ischemic organs. Here, we attempt to define the multiple functions of the SDF-1–CXCR4 signaling pathway in the regulation of neo-vascularization during acute ischemia and tumor growth. In particular, we introduce the concept that, by modulating plasma SDF-1 levels, the CXCR4 antagonist AMD3100 acutely promotes, while chronic AMD3100 treatment inhibits, mobilization of pro-angiogenic cells. We will also discuss strategies to modulate the mobilization of essential subsets of BM cells that participate in neo-angiogenesis, setting up the stage for enhancing revascularization or targeting tumor vessels by exploiting CXCR4 agonists and antagonists, respectively. PMID:17560169

Prostate specific antigen - brief update on its clinical use | Heyns ...

African Journals Online (AJOL)

Prostate specific antigen - brief update on its clinical use. ... (45 years in those with a family history of prostate cancer and – possibly – African men); ... PSA doubling time (the period it takes for the PSA to double) correlates with the prognosis ...

Establishment of immunoradiometric assay for free prostate-specific antigen

International Nuclear Information System (INIS)

Ma Lianxue

2009-01-01

An immunoradiometric assay (IRMA) of free prostate specific antigen (F-PSA) in serum was established. One monoclonal antibody against total PSA (T-PSA) was coated on the plastic tubes, the other against F-PSA was labeled with 125 I. The sensitivity of assay was 0.04 μg/L (n=20, +2s), the CVs were 2.9%-4.0% for the intra-assay and 3.5%-10.5% for the inter-assay and the average recovery was 102.7%. The correlative equation comparing with the FPSA-RIA (CIS BIO) is y=0.965 1 χ -0.001 1, and r=0.996 4. This F-PSA IRMA is a sensitive and precise method in detecting F-PSA and fit for the vitro assay. (authors)

The Hamster Model for Identification of Specific Antigens of Taenia solium Tapeworms

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Ochoa-Sánchez, Alicia; Jiménez, Lucía; Landa, Abraham

2011-01-01

Humans acquire taeniasis by ingesting pork meat infected with Taenia solium cysticerci, which are the only definitive hosts of the adult stage (tapeworm) and responsible for transmitting the human and porcine cysticercosis. Hence, detection of human tapeworm carriers is a key element in the development of viable strategies to control the disease. This paper presents the identification of specific antigens using sera from hamsters infected with T. solium tapeworms analyzed by western blot assay with crude extracts (CEs) and excretion-secretion antigens (E/S Ag) obtained from T. solium cysticerci and tapeworms and extracts from other helminthes as controls. The hamster sera infected with T. solium tapeworms recognized specific bands of 72, 48, 36, and 24 kDa, in percentages of 81, 81, 90, and 88%, respectively, using the T. solium tapeworms E/S Ag. The antigens recognized by these hamster sera could be candidates to improve diagnosis of human T. solium taeniasis. PMID:22253530

The Hamster Model for Identification of Specific Antigens of Taenia solium Tapeworms

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Alicia Ochoa-Sánchez

2011-01-01

Full Text Available Humans acquire taeniasis by ingesting pork meat infected with Taenia solium cysticerci, which are the only definitive hosts of the adult stage (tapeworm and responsible for transmitting the human and porcine cysticercosis. Hence, detection of human tapeworm carriers is a key element in the development of viable strategies to control the disease. This paper presents the identification of specific antigens using sera from hamsters infected with T. solium tapeworms analyzed by western blot assay with crude extracts (CEs and excretion-secretion antigens (E/S Ag obtained from T. solium cysticerci and tapeworms and extracts from other helminthes as controls. The hamster sera infected with T. solium tapeworms recognized specific bands of 72, 48, 36, and 24 kDa, in percentages of 81, 81, 90, and 88%, respectively, using the T. solium tapeworms E/S Ag. The antigens recognized by these hamster sera could be candidates to improve diagnosis of human T. solium taeniasis.

Immunization Elicits Antigen-Specific Antibody Sequestration in Dorsal Root Ganglia Sensory Neurons

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Gunasekaran, Manojkumar; Chatterjee, Prodyot K.; Shih, Andrew; Imperato, Gavin H.; Addorisio, Meghan; Kumar, Gopal; Lee, Annette; Graf, John F.; Meyer, Dan; Marino, Michael; Puleo, Christopher; Ashe, Jeffrey; Cox, Maureen A.; Mak, Tak W.; Bouton, Chad; Sherry, Barbara; Diamond, Betty; Andersson, Ulf; Coleman, Thomas R.; Metz, Christine N.; Tracey, Kevin J.; Chavan, Sangeeta S.

2018-01-01

The immune and nervous systems are two major organ systems responsible for host defense and memory. Both systems achieve memory and learning that can be retained, retrieved, and utilized for decades. Here, we report the surprising discovery that peripheral sensory neurons of the dorsal root ganglia (DRGs) of immunized mice contain antigen-specific antibodies. Using a combination of rigorous molecular genetic analyses, transgenic mice, and adoptive transfer experiments, we demonstrate that DRGs do not synthesize these antigen-specific antibodies, but rather sequester primarily IgG1 subtype antibodies. As revealed by RNA-seq and targeted quantitative PCR (qPCR), dorsal root ganglion (DRG) sensory neurons harvested from either naïve or immunized mice lack enzymes (i.e., RAG1, RAG2, AID, or UNG) required for generating antibody diversity and, therefore, cannot make antibodies. Additionally, transgenic mice that express a reporter fluorescent protein under the control of Igγ1 constant region fail to express Ighg1 transcripts in DRG sensory neurons. Furthermore, neural sequestration of antibodies occurs in mice rendered deficient in neuronal Rag2, but antibody sequestration is not observed in DRG sensory neurons isolated from mice that lack mature B cells [e.g., Rag1 knock out (KO) or μMT mice]. Finally, adoptive transfer of Rag1-deficient bone marrow (BM) into wild-type (WT) mice or WT BM into Rag1 KO mice revealed that antibody sequestration was observed in DRG sensory neurons of chimeric mice with WT BM but not with Rag1-deficient BM. Together, these results indicate that DRG sensory neurons sequester and retain antigen-specific antibodies released by antibody-secreting plasma cells. Coupling this work with previous studies implicating DRG sensory neurons in regulating antigen trafficking during immunization raises the interesting possibility that the nervous system collaborates with the immune system to regulate antigen-mediated responses. PMID:29755449

Immunization Elicits Antigen-Specific Antibody Sequestration in Dorsal Root Ganglia Sensory Neurons

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Manojkumar Gunasekaran

2018-04-01

Full Text Available The immune and nervous systems are two major organ systems responsible for host defense and memory. Both systems achieve memory and learning that can be retained, retrieved, and utilized for decades. Here, we report the surprising discovery that peripheral sensory neurons of the dorsal root ganglia (DRGs of immunized mice contain antigen-specific antibodies. Using a combination of rigorous molecular genetic analyses, transgenic mice, and adoptive transfer experiments, we demonstrate that DRGs do not synthesize these antigen-specific antibodies, but rather sequester primarily IgG1 subtype antibodies. As revealed by RNA-seq and targeted quantitative PCR (qPCR, dorsal root ganglion (DRG sensory neurons harvested from either naïve or immunized mice lack enzymes (i.e., RAG1, RAG2, AID, or UNG required for generating antibody diversity and, therefore, cannot make antibodies. Additionally, transgenic mice that express a reporter fluorescent protein under the control of Igγ1 constant region fail to express Ighg1 transcripts in DRG sensory neurons. Furthermore, neural sequestration of antibodies occurs in mice rendered deficient in neuronal Rag2, but antibody sequestration is not observed in DRG sensory neurons isolated from mice that lack mature B cells [e.g., Rag1 knock out (KO or μMT mice]. Finally, adoptive transfer of Rag1-deficient bone marrow (BM into wild-type (WT mice or WT BM into Rag1 KO mice revealed that antibody sequestration was observed in DRG sensory neurons of chimeric mice with WT BM but not with Rag1-deficient BM. Together, these results indicate that DRG sensory neurons sequester and retain antigen-specific antibodies released by antibody-secreting plasma cells. Coupling this work with previous studies implicating DRG sensory neurons in regulating antigen trafficking during immunization raises the interesting possibility that the nervous system collaborates with the immune system to regulate antigen-mediated responses.

Human Tregs Made Antigen Specific by Gene Modification: The Power to Treat Autoimmunity and Antidrug Antibodies with Precision

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Patrick R. Adair

2017-09-01

Full Text Available Human regulatory CD4+ T cells (Tregs are potent immunosuppressive lymphocytes responsible for immune tolerance and homeostasis. Since the seminal reports identifying Tregs, vast research has been channeled into understanding their genesis, signature molecular markers, mechanisms of suppression, and role in disease. This research has opened the doors for Tregs as a potential therapeutic for diseases and disorders such as multiple sclerosis, type I diabetes, transplantation, and immune responses to protein therapeutics, like factor VIII. Seminal clinical trials have used polyclonal Tregs, but the frequency of antigen-specific Tregs among polyclonal populations is low, and polyclonal Tregs may risk non-specific immunosuppression. Antigen-specific Treg therapy, which uses genetically modified Tregs expressing receptors specific for target antigens, greatly mitigates this risk. Building on the principles of T-cell receptor cloning, chimeric antigen receptors (CARs, and a novel CAR derivative, called B-cell antibody receptors, our lab has developed different types of antigen-specific Tregs. This review discusses the current research and optimization of gene-modified antigen-specific human Tregs in our lab in several disease models. The preparations and considerations for clinical use of such Tregs also are discussed.

MicroRNA-146a and AMD3100, two ways to control CXCR4 expression in acute myeloid leukemias

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Spinello, I; Quaranta, M T; Riccioni, R; Riti, V; Pasquini, L; Boe, A; Pelosi, E [Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome (Italy); Vitale, A; Foà, R [Department of Cellular Biotechnologies and Hematology, Division of Hematology, ‘Sapienza' University, Rome (Italy); Testa, U; Labbaye, C [Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome (Italy)

2011-06-01

CXCR4 is a negative prognostic marker in acute myeloid leukemias (AMLs). Therefore, it is necessary to develop novel ways to inhibit CXCR4 expression in leukemia. AMD3100 is an inhibitor of CXCR4 currently used to mobilize cancer cells. CXCR4 is a target of microRNA (miR)-146a that may represent a new tool to inhibit CXCR4 expression. We then investigated CXCR4 regulation by miR-146a in primary AMLs and found an inverse correlation between miR-146a and CXCR4 protein expression levels in all AML subtypes. As the lowest miR-146a expression levels were observed in M5 AML, we analyzed the control of CXCR4 expression by miR-146a in normal and leukemic monocytic cells and showed that the regulatory miR-146a/CXCR4 pathway operates during monocytopoiesis, but is deregulated in AMLs. AMD3100 treatment and miR-146a overexpression were used to inhibit CXCR4 in leukemic cells. AMD3100 treatment induces the decrease of CXCR4 protein expression, associated with miR-146a increase, and increases sensitivity of leukemic blast cells to cytotoxic drugs, this effect being further enhanced by miR-146a overexpression. Altogether our data indicate that miR-146a and AMD3100, acting through different mechanism, downmodulate CXCR4 protein levels, impair leukemic cell proliferation and then may be used in combination with anti-leukemia drugs, for development of new therapeutic strategies.

MicroRNA-146a and AMD3100, two ways to control CXCR4 expression in acute myeloid leukemias

International Nuclear Information System (INIS)

Spinello, I; Quaranta, M T; Riccioni, R; Riti, V; Pasquini, L; Boe, A; Pelosi, E; Vitale, A; Foà, R; Testa, U; Labbaye, C

2011-01-01

CXCR4 is a negative prognostic marker in acute myeloid leukemias (AMLs). Therefore, it is necessary to develop novel ways to inhibit CXCR4 expression in leukemia. AMD3100 is an inhibitor of CXCR4 currently used to mobilize cancer cells. CXCR4 is a target of microRNA (miR)-146a that may represent a new tool to inhibit CXCR4 expression. We then investigated CXCR4 regulation by miR-146a in primary AMLs and found an inverse correlation between miR-146a and CXCR4 protein expression levels in all AML subtypes. As the lowest miR-146a expression levels were observed in M5 AML, we analyzed the control of CXCR4 expression by miR-146a in normal and leukemic monocytic cells and showed that the regulatory miR-146a/CXCR4 pathway operates during monocytopoiesis, but is deregulated in AMLs. AMD3100 treatment and miR-146a overexpression were used to inhibit CXCR4 in leukemic cells. AMD3100 treatment induces the decrease of CXCR4 protein expression, associated with miR-146a increase, and increases sensitivity of leukemic blast cells to cytotoxic drugs, this effect being further enhanced by miR-146a overexpression. Altogether our data indicate that miR-146a and AMD3100, acting through different mechanism, downmodulate CXCR4 protein levels, impair leukemic cell proliferation and then may be used in combination with anti-leukemia drugs, for development of new therapeutic strategies