Fluorescent-labeled Antibodies Research Articles

Screening for Potential HLA-DRB1-Restricted T-Cell Epitopes in Factor VIII Using Peptide Microarrays

Therapeutic factor VIII (FVIII) infusions provoke CD4 T-cell proliferation and cytokine secretion leading to neutralizing anti-FVIII antibodies (inhibitors) in a substantial fraction of hemophilia A patients, leading to bleeding that is difficult to control. T-cell stimulation occurs when FVIII peptides bind to HLA-DRB1 proteins and are presented on the surface of antigen presenting cells, followed by T-cell recognition of these peptide-HLA-DRB1 complexes. Our laboratory is interested in identifying immunodominant, HLA-restricted T-cell epitopes in FVIII, in order to identify and then modify these immunological “hotspots” that cause inhibitors to develop. We have previously mapped several immunodominant HLA-restricted T-cell epitopes in FVIII recognized by the immune systems of inhibitor subjects, using HLA-DRB1 tetramers loaded with 20-mer FVIII peptides. However, because FVIII is such a large protein (>2,000 amino acid residues), methods more efficient than systematic mapping using overlapping peptides spanning its sequence would be useful. This study focuses on the binding of FVIII peptides to recombinant extracellular domains of 10 HLA-DRB1 proteins, which together represent HLA-DRB1 alleles found in >50% of the U.S population. PepStarTM (JPT Peptide Technologies) microarrays containing triplicate sets of FVIII peptides immobilized on glass slides via a flexible linker were incubated with HLA-DRB1 proteins at several dilutions, followed by incubation with a fluorescent-labeled antibody that recognizes the peptide-bound conformations of HLA-DRB1 proteins, and the slides were washed and read on a microarray scanner. The intensities of fluorescent signals from the triplicate sets of spots were quantified and also displayed as heat maps. Fluorescent signals ranged from background levels (indicating no binding) up to the detector saturation range. Between 20-40% of the FVIII peptides produced fluorescent signals indicating peptide binding to each of the HLA-DRB1 proteins tested, including positive control peptides containing FVIII and non-FVIII epitopes with known HLA-DRB1 restriction. The percentage of apparently high-affinity binding interactions was much lower. T-cell epitopes are generally 10-14 amino acid residues long. Therefore, strong signals from pairs of 20-mer peptides with a 12-residue overlapping sequence localized the FVIII sequence fitting into the HLA-DRB1 binding groove to the overlapping region, while disparate signals from other pairs of overlapping sequences localized the HLA-DRB1 binding sequence to the non-overlapping regions. For subsets of the FVIII peptides, the signal strengths were compared to (a) affinities (IC50 values) derived from quantitative, competition peptide-HLA-DRB1 binding assays, and (b) results of computer prediction algorithms MHCPanII and Propred. Peptides that bound strongly to one or more HLA-DRB1 protein were prioritized for further testing. This microarray-based screening procedure is an efficient method to generate manageable lists of potential T-cell epitopes in large antigenic proteins such as FVIII, where systematic epitope mapping using HLA-DRB1 tetramers loaded with peptides spanning the entire sequence is not feasible, due to both the high cost of such experiments and the blood volumes that can reasonably be requested from patient volunteers. DisclosuresNo relevant conflicts of interest to declare.

A new one-step antigen heterologous homogeneous fluorescence immunoassay for progesterone detection in serum

A new homogeneous immunoassay for the detection of progesterone was developed to measure its concentration in human serum. We utilized the weak cross-reactivity of a monoclonal anti-progesterone antibody to an analog molecule (in this case β-estradiol) to create a mixture, in which the fluorescence-labeled antibody (AbF) and quencher-labeled BSA-estradiol (eBSAq) were at optimized equilibrium. At this stage, most antibodies were bound to eBSAq and the fluorescence of AbF was quenched. After adding samples containing free progesterone to the system, these would replace the eBSAq at the antigen-binding site. The fluorescence would be released. In contrast to conventional competitive immunoassays, the fluorescence signal increases with increasing progesterone concentration, greatly simplifying detection and calibration. The performance of the assay was very simple; there was only one mixing step; and other hormones like testosterone, estradiol or dehydroepiandrosterone (DHEA) do not interfere the assay. A wide linear range from 0.1µg/L to 100µg/L was achieved in buffer, with a LOD of 0.1µg/L. In human serum the LOD was 5µg/L, and the linear range was 5–500µg/L. For this assay it is important to find the right combination of antibody and cross-reactive antigen. If such a combination could be defined, it is conceivable to apply this assay to a wide range of analytes.

Circulating Plasma Cells in Newly Diagnosed Symptomatic Multiple Myeloma As a Prognostic Marker for Patients with Standard-Risk Cytogenetics

Multiple Myeloma (MM) is a clonal B-cell disorder characterized by accumulation of malignant plasma cells (PCs) in the bone marrow (BM). Circulating PCs can be detected in the peripheral blood of a significant proportion of patients with MM and their presence is a well-known prognostic factor. Indeed, the appearance of circulating PCs in the blood could indicate relative indipendence from adhesion to the microenvironment, thus implying more aggressive disease. In this study, we examined the relationship between the number of PCs and citogenetic risk in patients with newly diagnosed MM.We analyzed peripheral blood from patients with Monoclonal Gammopathy of Undetermined Significance (MGUS; n=15), Smoldering Myeloma (SM; n=28), Solitary Plasmacytomas (SP; n=3) and active Multiple Myeloma (MM; n=105). These patients were followed by the U.O.C. Ematologia at the "Mazzoni" Hospital from January 2006 to December 2013, with a median follow-up of 25 months. We analyzed clinical, laboratory and cytogenetic data of patients with active MM. However, cytogenetic analysis was not evaluable for 15 patients. The number of circulating PCs was detected by flow cytometry using a simple two-colours approach. Cells were stained with fluorescence-labeled CD38 and CD45 antibodies and 50,000 events were acquired and analyzed for each patient. PCs were identified by gating on CD38bright+/CD45- cells.Using a receiver operating characteristics (ROC) analysis, we assessed that ³41circulating PCs is the optimal cut-off for defining poor prognosis.The 8-years probability of Overall Survival (OS) and Progression-Free Survival (PFS) in patients with <41 and ³41circulating PCs, was 32% vs 8% (p=0.017) and 29% vs 0% (p=0.0008), respectively. Patients with high-risk cytogenetics (n=24) had poor prognosis, independently of circulating PCs (PC<41 vs PC³41: OS=0% vs OS=16%, p=n.s.; PFS=0% vs 17%, p=n.s.). Patients with standard-risk cytogenetics (n=66) showed a better prognosis associated to a lower number of circulating PCs (PC<41 vs PC³41: OS=36% vs 10%, p=0.026; PFS=37% vs 0%, p=0.0001). These data were confirmed by multivariate analysis (Cox model) for the subgroup with standard-risk cytogenetics, in which the presence of ³41 circulating PCs, older age, DS stage >I and lack of maintenance therapy, adversely affected OS and PFS.All patients with SP showed no circulating PCs. In all cases of MGUS or SM, circulating PCs, when detected, were <20.In summary, our results suggest that the quantification of circulating PCs by flow cytometry could provide useful prognostic information in newly diagnosed MM patients with standard-risk cytogenetics. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Neutrophils Promote Hematopoiesis Via a Novel Mechanism Involving Leucine Rich α-2 Glycoprotein (LRG)

Leucine-rich α2 glycoprotein (LRG), the founding member of the leucine-rich repeat superfamily of proteins, was initially identified in serum more than 30 years ago, but its biologic function has remained elusive. A role for LRG has been implicated in inflammation and angiogenesis. Our laboratory previously identified cDNA and genomic clones for human and murine LRG, and showed that ectopically expressed LRG localizes to the granule compartment in transfected myeloid cell lines and promotes their granulocytic differentiation. We also demonstrated that expression of LRG is transcriptionally regulated during neutrophil granulocyte differentiation in a manner similar to that reported for genes encoding the different subsets of neutrophil granule proteins. The presence of LRG in primary neutrophils and a role for LRG in hematopoiesis, however, have not been previously described. Based on our prior studies in transfected myeloid cell lines, we considered the tantalizing possibility that LRG is a novel neutrophil granule protein that is secreted extracellularly upon neutrophil activation to modulate hematopoiesis. To investigate this, we examined LRG in primary human neutrophils isolated from healthy volunteers. Immunoblot analysis of whole cell lysates from neutrophils (97% purity) identified a higher molecular weight LRG species in neutrophils (62 kDa) compared to serum (50 kDa); our data demonstrate the difference in apparent molecular weight is due to differential glycosylation. Immunofluorescence microscopy using antibodies to human LRG and antibodies to the neutrophil granule proteins myeloperoxidase (MPO), lactoferrin (LF), and matrix metalloproteinase 9 (MMP-9, also known as gelatinase), along with fluorescently-labeled secondary antibodies, demonstrated the presence of LRG in the cytoplasm of neutrophils in a compartment corresponding to LF. ELISA and immunoblot analyses of subcellular fractions from isolated neutrophils prepared by nitrogen cavitation demonstrated the presence of LRG in LF-containing fractions as well as some MMP-9-containing fractions, consistent with localization of LRG to the secondary/tertiary granule compartment. Neutrophil exocytosis assays using ionomycin, phorbol-12-myristate 13-acetate, and f-Met-Leu-Phe as stimulants also indicated that LRG is co-released with LF and MMP9, but not with MPO. Notably, LRG secreted from activated neutrophils could bind cytochrome c as reported for LRG purified from serum. Recent reports that LRG can also bind to the TGFβR1 receptor on endothelial cells prompted us to investigate the effects of LRG on TGFβ signaling in hematopoietic cells. LRG significantly antagonized the inhibitory effect of TGFβ on HL-60 cell proliferation (n=3; p<0.05) and also on colony growth of human hematopoietic progenitor cells. When LRG was added to hematopoietic progenitor cells cultured in TGFβ-containing Methocult (SF H4436, serum free), a 50% increase in CFU-GMs was observed. Collectively, these data suggest a novel mechanism whereby neutrophils modulate hematopoiesis in the microenvironment via extracellular release of LRG, and invoke an additional role for neutrophils in innate immunity that has not previously been reported DisclosuresNo relevant conflicts of interest to declare.

Surface profiles of live colorectal cancer cells and tumor infiltrating lymphocytes from surgical samples correspond to prognostic categories

Extensive surface profiles of colorectal cancer (CRC) cells and tumor infiltrating lymphocytes (TIL) have been obtained from 45 surgical resection samples. Live cells were captured on an antibody microarray and stained with fluorescently-labeled antibodies. Minimal panels of 11 CRC antigens (CD13, CD24, CD26, CD49d, CD138, CD166, CA-125, CA19-9, EGFR, Galectin-4 and HLA-DR) and 11 T-cell antigens (CD10, CD11b, CD11c, CD25, CD31, CD95, CD151, CD181, Galectin-4, CA19-9, TSP-1) provide signatures for relapse and survival. Hierarchical clustering of profiles from CRC cells and TIL identified groups of patients for survival, systemic relapse and death. The groups from CRC and TIL profiles for systemic relapse showed 79.2% concordance, enabling prediction of relapse after surgery. The results demonstrate communication between CRC cells and TIL.

FITC-conjugated cyclic RGD peptides as fluorescent probes for staining integrin αvβ3/αvβ5 in tumor tissues.

This study sought to evaluate FITC-conjugated cyclic RGD peptides (FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2) as fluorescent probes for in vitro assays of integrin αvβ3/αvβ5 expression in tumor tissues. FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were prepared, and their integrin αvβ3/αvβ5 binding affinity was determined using the displacement assay against 125I-echistatin bound to U87MG glioma cells. IC50 values of FITC-Galacto-RGD2, FITC-3P-RGD2, and FITC-RGD2 were calculated to be 28 ± 8, 32 ± 7, and 89 ± 17 nM, respectively. The integrin αvβ3/αvβ5 binding affinity followed a general trend: FITC-Galacto-RGD2 ∼ FITC-3P-RGD2 > FITC-RGD2. The xenografted tumor-bearing models were established by subcutaneous injection of 5 × 106 tumor cells into shoulder flank (U87MG, A549, HT29, and PC-3) or mammary fat pad (MDA-MB-435) of each athymic nude mouse. Three to six weeks after inoculation, the tumor size was 0.1–0.3 g. Tumors were harvested for integrin αvβ3/αvβ5 staining, as well as hematoxylin and eosin (H&E) staining. Six human carcinoma tissues (colon cancer, pancreatic cancer, lung adenocarcinoma, squamous cell lung cancer, gastric cancer, and esophageal cancer) were obtained from recently diagnosed cancer patients. Human carcinoma slides were deparaffinized in xylene, rehydrated with ethanol, and then used for integrin αvβ3/αvβ5 staining, as well as H&E staining. It was found that the tumor staining procedures with FITC-conjugated cyclic RGD peptides were much simpler than those with the fluorescence-labeled integrin αvβ3 antibodies. Since FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were able to co-localize with the fluorescence-labeled integrin β3 antibody, their tumor localization and tumor cell binding are integrin αvβ3-specific. Quantification of the fluorescent intensity in five xenografted tumors (U87MG, MDA-MB-435, A549, HT29, and PC-3) and six human carcinoma tissues revealed an excellent linear relationship between the relative integrin αvβ3/αvβ5 expression levels determined with FITC-Galacto-RGD2 and those obtained with the fluorescence-labeled anti-human integrin β3 antibody. There was also an excellent linear relationship between the tumor uptake (%ID/g) of 99mTc-3P-RGD2 (an integrin αvβ3/αvβ5-targeted radiotracer) and the relative integrin αvβ3/αvβ5 expression levels from the quantification of fluorescent intensity in the tumor tissues stained with FITC-Galacto-RGD2. These results suggest that FITC-conjugated cyclic RGD peptides might be useful to correlate the in vitro findings with the in vivo imaging data from an integrin αvβ3/αvβ5-targeted radiotracer. The results from this study clearly showed that the FITC-conjugated cyclic RGD peptides (particularly FITC-3P-RGD2 and FITC-Galacto-RGD2) are useful fluorescent probes for assaying relative integrin αvβ3/αvβ5 expression levels in tumor tissues.

Evaluation of E-cadherin, β-catenin and vimentin protein expression using quantitative immunohistochemistry in nasopharyngeal carcinoma patients.

Aberrant expression of proteins involved in epithelial-to-mesenchymal transition have been described in various cancers. In this retrospective study, we sought to evaluate E-cadherin, β-catenin and vimentin protein expression in non-metastatic nasopharyngeal (NPC) patients treated with curative intent, examine their relationship with each other, and with clinical outcome measures. Pre-treatment formalin-fixed paraffin-embedded biopsies of 140 patients treated between January 2000 and December 2007 were assembled into a tissue microarray (TMA). Automated quantitative immunohistochemistry (AQUA®) was performed on sequential TMA sections stained with fluorescent-labeled antibodies against E-cadherin, β-catenin and vimentin. Cox proportional hazards regression was used to estimate the effect of cytoplasmic vimentin, cytoplasmic E-cadherin, β-catenin nuclear/cytoplasmic ratio expression on overall survival and disease-free survival. The average age of the patients was 51.7 years (SD=12.1; range 18-85), 66% were male, 71% had a KPS ≥ 90% at the start of treatment and 65% had stage III/IV disease. After adjusting for performance status, WHO and stage, high E-cadherin levels over the 75th percentile were found to produce a significantly increased risk for both a worse overall survival (HR = 2.53, 95% CI 1.21, 5.27) and disease free survival (DFS; HR = 2.14, 95%CI 1.28, 3.59). Vimentin levels over the first quartile produced an increased risk for a worse DFS (HR = 2.21, 95% CI 1.11, 4.38). No association was seen between β-catenin and survival. In this cohort of NPC patients, higher levels of E-cadherin and higher levels of vimentin were associated with worse outcomes. Further work is needed to understand the role of these epithelial mesenchymal transition proteins in NPC.

Abstract 4938: In vivo visualization using two-photon laser scanning microscopy of anti-c-MET antibody in peritoneal metastatic gastric cancer

Abstract Background: c-MET (hepatocyte growth factor receptor) gene is overexpressed in approximately 20% of gastric cancer. Onartuzumab, a MET-selective humanized one-armed monoclonal antibody, has been studied in metastatic Met-positive non-small cell lung cancer and expected to be introduced to MET-positive gastric cancer. Two-photon laser scanning microscopy (TPLSM) has revolutionized in vivo real-time imaging because of the benefits of higher resolution, increased tissue penetration, and reduced photodamage. We established a method of intravital imaging for intra-abdominal organs using TPLSM with an organ stabilizing system (Japanese Patent No.5268282). Aim: To visualize the binding of fluorescence labeled-antibody to its receptor of cancer cells in peritoneal metastatic xenografts of living mice using a TPLSM. Method: Human gastric cancer cell lines (NUGC4) were inoculated into the peritoneal cavity of green fluorescent protein (GFP) expressing nude mice to make macroscopic peritoneal metastases. In vitro study, the binding of Zenon Alexa Fluor 594 labeled anti-c-MET antibody to viable NUGC4 cells was imaged using fluorescence microscopy. In vivo study, the binding of fluorescence labeled-antibody to viable NUGC4 cells of peritoneal metastatic xenografts was imaged using a TPLSM. Result: Zenon Alexa Fluor 594 labeled anti-c-MET antibody (red) was bound to the surface of viable NUGC4 cells, and clearly visualized under a fluorescence microscopy. Peritoneal metastases with label-free NUGC4 cells (black) and murine stromal cells (green) were imaged under TPLSM in vivo real-time. Fluorescence labeled-antibody could be visualized within tumor vessels of peritoneal metastases after its intravenous administration. Thereafter, label-free NUGC4 cells were identified by the binding of fluorescence labeled- anti-c-MET antibody to c-MET of NUGC4 cells. Conclusion: We could visualize fluorescence labeled-antibody in peritoneal metastases of gastric cancer in living mice using a TPLSM. The binding of fluorescence labeled- anti-c-MET antibody to c-MET of viable NUGC4 cells was clearly imaged at high magnification and high resolution in vivo real-time. It is valuable to develop a method of in vivo optical pathology for therapeutic monoclonal antibodies using MPM on metastatic tumor xenografts. Citation Format: Shozo Ide, Koji Tanaka, Tadanobu Shimura, Masato Okigami, Satoru Kondo, Takahito Kitajima, Hiroki Imaoka, Yoshinaga Okugawa, Susumu Saigusa, Yuji Toiyama, Hiromi Yasuda, Masaki Ohi, Yasuhiro Inoue, Toshimitsu Araki, Keiichi Uchida, Yasuhiko Mohri, Masato Kusunoki. In vivo visualization using two-photon laser scanning microscopy of anti-c-MET antibody in peritoneal metastatic gastric cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4938. doi:10.1158/1538-7445.AM2014-4938

Abstract 2071A: Anti-VEGF treatment in orthotopic breast cancer xenografts: Dynamic contrast-enhanced micro-CT correlates with 3D multispectral fluorescence histology

Abstract Aim: Here, we evaluate the ability of dynamic contrast-enhanced micro-computed tomography (DCE micro-CT) in combination with 3D fluorescence ultramicroscopy (UM) to assess the early physiological and morphological treatment response of orthotopic breast tumors to anti-angiogenic drug therapy. Methods: Non-invasive DCE micro-CT was performed on breast cancer xenografts (KPL-4) that were treated twice with an antibody against the vascular endothelial growth factor (VEGF) or a control antibody. Tumor physiology was assessed in different tumor regions of interests measuring the relative blood volume (rBV) and the permeability-surface area product (PS). Parametric maps were calculated on a voxel-by-voxel basis to evaluate intra-tumor vascular heterogeneity. Mean physiological measurements were compared with morphological measures of the tumor vascular architecture (microvessel density (MVD) and relative vessel volume (rVV)) as obtained by 3-dimensional fluorescence UM. Additionally, vessel leakage was assessed by UM quantifying the penetration strength of a co-injected fluorescence-labeled therapeutic antibody into the tumor tissue. Results: Examination of the parametric maps revealed significantly different spatial patterns of intra-tumoral blood supply (rBV) between anti-VEGF-treated tumors and control. While a characteristic rim vascularization (high rBV values in the tumor periphery) was observed in the control tumors, treated tumors showed a widely homogeneous blood supply. Compared with high-resolution UM, these physiological rBV maps showed excellent agreement with the morphological appearance of the 3D tumor vascular architecture. Assessment of the mean physiology showed a significant decrease of rBV (p&amp;lt;0.01) and PS (p&amp;lt;0.05) in the tumor periphery after anti-angiogenic treatment. The reduction of rBV correlated well with a significant reduction of rVV (p&amp;lt;0.01) in the corresponding tumor region. The decrease in PS was found consistent with a significantly reduced antibody uptake (p&amp;lt;0.01) in the tumor tissue after treatment. No treatment effect was observed by tumor volume. Conclusions: DCE micro-CT used along with UM provides comprehensive and complementary information of the physiological and morphological anti-angiogenic treatment response in breast cancer xenografts. This technology may help to improve current standard methods in the assessment of anti-angiogenic drug efficacy in preclinical drug development. Citation Format: Thomas Pöschinger, Anja Renner, Fabian Eisa, Michael Dobosz, Robert Brauweiler, Willi A. Kalender, Werner Scheuer. Anti-VEGF treatment in orthotopic breast cancer xenografts: Dynamic contrast-enhanced micro-CT correlates with 3D multispectral fluorescence histology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2071A. doi:10.1158/1538-7445.AM2014-2071A

Generic epitaxial graphene biosensors for ultrasensitive detection of cancer risk biomarker

A generic electrochemical method of ‘bioreceptor’ antibody attachment to phenyl amine functionalized graphitic surfaces is demonstrated. Micro-channels of chemically modified multi-layer epitaxial graphene (MLEG) have been used to provide a repeatable and reliable response to nano-molar (nM) concentrations of the cancer risk (oxidative stress) biomarker 8-hydroxydeoxyguanosine (8-OHdG). X-ray photoelectron spectroscopy, Raman spectroscopy are used to characterize the functionalized MLEG. Confocal fluorescence microscopy using fluorescent-labelled antibodies indicates that the anti-8-OHdG antibody selectively binds to the phenyl amine-functionalized MLEG’s channel. Current–voltage measurements on functionalized channels showed repeatable current responses from antibody–biomarker binding events. This technique is scalable, reliable, and capable of providing a rapid, quantitative, label-free assessment of biomarkers at nano-molar (<20 nM) concentrations in analyte solutions. The sensitivity of the sensor device was investigated using varying concentrations of 8-OHdG, with changes in the sensor’s channel resistance observed upon exposure to 8-OHdG. Detection of 8-OHdG concentrations as low as 0.1 ng ml−1 (0.35 nM) has been demonstrated. This is five times more sensitive than reported enzyme linked immunosorbent assay tests (0.5 ng ml−1).

Circulating Tumor Cells in Peripheral and Pulmonary Venous Blood Predict Poor Long-Term Survival in Surgically Resected Non-Small Cell Lung Cancer Patients

ABSTRACT Aim: We tested the hypothesis that the circulating tumor cells (CTCs) in preoperative peripheral blood (PPB) and intraoperative pulmonary venous blood (IPVB) could predict poor long-term survival in surgically resected NSCLC patients. Methods: CTCs were separated from the blood using magnetic beads coated with antibody against epithelial-cell adhesion molecule (EpCAM) through magnetic activated cell sorting (MACS). The CTCs were quantified with fluorescence-labeled antibodies against pan-cytokeratin through flow cytometry. CTCs were prospectively quantified in PPB and IPVB in 23 consecutive stage I-IIIA patients with surgically resected NSCLC. Association between CTCs and prognosis of these patients was evaluated after 5-year follow-up. Results: In the NSCLC patients, outcomes were assessed according to levels of CTCs at surgery, and compared with CTCs detected in benign pulmonary diseases, and healthy volunteers, where the mean and 95% CI of CTCs counts were all 5 CTCs/15mL in PPB and >50 CTCs/15mL in IPVB. Univariate Cox proportional-hazards regression analysis showed that CTCs count in PPB or IPVB was an independent risk factor for tumor-free and overall survivals. The high risk group of patients had a shorter median tumor-free survival (22 months vs. >60.0 months, P 60 months, P Conclusions: CTCs count in PPB and IPVB was an independent risk factor for tumor-free and overall survival in surgically resected NSCLC patients. Disclosure: All authors have declared no conflicts of interest.

Flow Cytometric Determination of the Expression of gp 51 Protein of Bovine Leukaemia Virus in Experimentally Infected Sheep

Abstract The study was performed on lambs experimentally infected with bovine leukaemia virus (BLV). The presence of BLV antibodies in sera of infected animals was detected by agar gel immunodiffusion test and ELISA. Proviral DNA was detected by PCR and nested PCR. Dual-colour flow cytometry analysis was performed with the use of specific monoclonal antibodies against lymphocyte CD markers and gp51 viral envelope protein, followed by incubation with fluorescent-labelled secondary antibodies conjugated with FITC or PE. Gp51 viral envelope protein was detected in tumours caused by BLV infection. The BLV infection resulted in depletion of CD4+ lymphocytes, increase in CD8+ lymphocytes, and decrease in CD4+ to CD8+ ratio in infected sheep. Proliferation of IgM+ CD19+ cells was also detected. These cells had an immature character without tendency to differentiate, and their vitality was prolonged. Flow cytometry enabled detection of gp51 expression in sheep blood lymphocytes at the early stages of the infection, before detection of serum antibodies using ELISA.

PYGM expression analysis in white blood cells: A complementary tool for diagnosing McArdle disease?

McArdle disease is caused by an inherited deficiency of the enzyme myophosphorylase, resulting in exercise intolerance from childhood and acute crises of early fatigue and contractures. In severe cases, these manifestations can be accompanied by rhabdomyolysis, myoglobinuria, and fatal renal failure. Diagnosis of McArdle disease is based on clinical diagnostic tests, together with an absence of myophosphorylase activity in skeletal muscle biopsies and genetic analysis of the myophosphorylase-encoding gene, PYGM. The recently reported association between myophosphorylase and Rac1 GTPase in a T lymphocyte cell line prompted us to study myophosphorylase expression in white blood cells (WBCs) from 20 healthy donors and 30 McArdle patients by flow cytometry using a fluorescent-labeled PYGM antibody. We found that T lymphocytes expressed myophosphorylase in healthy donors, but expression was significantly lower in McArdle patients (p<0.001). PYGM mRNA levels were also lower in white blood cells from McArdle patients. Nevertheless, in 13% of patients (who were either heterozygotes or homozygotes for the most common PYGM pathogenic mutation among Caucasians (p.R50X)), the percentage of myophosphorylase-positive white blood cells was not different compared with the control group. Our findings suggest that analysis of myophosphorylase expression in white blood cells might be a useful, less-invasive, complementary test for diagnosing McArdle disease.

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Real-time imaging of endothelial cell-cell junctions during neutrophil transmigration under physiological flow.

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Simultaneous flow cytometric analysis of IFN-γ and CD4 mRNA and protein expression kinetics in human peripheral blood mononuclear cells during activation.

The application of fluorescently-labeled antibodies for flow cytometric identification and characterization of specific cell types within heterogeneous populations by their protein expression profile is well established. While detection of proteins is informative, concomitant transcript analysis in the same cells would provide a more complete and comprehensive view of intracellular signaling events. We recently reported on the efficient detection of RNA in suspension cells for flow cytometric analysis. The improved RNA flow cytometry procedure described here allows for the specific labeling of multiple RNA species, and is compatible with antibody-based targeting of extracellular and intracellular antigens for multiplexing purposes. To show proof of concept, human peripheral blood mononuclear cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin for a maximum of 5 h, during which their CD4 and interferon-gamma (IFN-γ) transcript and protein levels were monitored. Substantial and increasing numbers of IFN-γ mRNA+ cells were detected within 30 min after initiation of induction, while IFN-γ protein+ cells could only be discerned at 1 h and beyond. Surprisingly, resting lymphocytes contained less CD4 mRNA but more of the protein per cell compared with monocytes, revealing a difference in the relationship of transcript and protein levels in these two cell types. We additionally applied monensin, which is commonly used to block cytokine secretion, and found that IFN-γ mRNA can still be analyzed consistently using the improved RNA flow cytometry staining method. Notably, a subset of IFN-γ mRNA(-)/protein+ cells that were not observed in the absence of monensin became apparent at the 5-h mark. This subset probably represents cells that have accumulated IFN-γ protein, but no longer transcribe mRNA. Collectively, the results described here exemplify how the improved RNA flow cytometry labeling procedure can be applied to simultaneously assess mRNA and protein dynamics to gain insight into the regulation of gene transcription and translation in individual cells.

In vivomolecular imaging of HER2 expression in a rat model of Barrett's esophagus adenocarcinoma

Human epidermal growth factor receptor 2 (HER2) is involved in the malignant progression of several human cancers, including esophageal adenocarcinoma (EAC). The purpose of this study was to evaluate HER2 overexpression and to explore the feasibility of confocal laser endomicroscopy for in vivo molecular imaging of HER2 status in an animal model of Barrett's-related EAC. Rats underwent esophagojejunostomy with gastric preservation. At 30 weeks post-surgery, the esophagus of 46 rats was studied; endoscopic and histological findings were correlated with HER2 immunofluorescence on excised biopsies and gross specimens. At this age, 23/46 rats developed Barrett's esophagus (BE), and 6/46 had cancer (four EAC and two squamous cell carcinomas). A significant overexpression of HER2 was observed in esophageal adenocarcinoma compared with normal squamous esophagus (9.4-fold) and BE (6.0-fold). AKT and its phosphorylated form were also overexpressed in cancer areas. Molecular imaging was performed at 80 weeks post-surgery in four rats after tail injection of fluorescent-labeled anti-HER2 antibody. At this age, 3/4 rats developed advance adenocarcinoma and showed in vivo overexpression of HER2 by molecular confocal laser endomicroscopy with heterogeneous distribution within cancer; no HER2 signal was observed in normal or Barrett's tissues. Therefore, HER2 overexpression is a typical feature of the surgical induced model of EAC that can be easily quantified in vivo using an innovative mini-invasive approach including confocal endomicroscopy; this approach may avoid limits of histological evaluation of HER2 status on 'blinded' biopsies.

Measurement of autophagy by flow cytometry.

In recent years, flow cytometry has been used to detect the presence of autophagy mainly by the fluorescent antibody labeling of the autophagy marker, the microtubule associated protein LC3-II. Here we describe the indirect antibody labeling of LC3-II in cells displaying drug-induced autophagy by the use of rapamycin and chloroquine, as well as cells undergoing serum starvation. Although the mechanism of action of LysoTracker dyes is not fully understood, lysosomal mass increases during the autophagic process to enable the cell to produce autolysosomes. Given that LC3-II and LysoTracker are measuring different biological events in the autophagic process, they surprisingly both up-regulated during autophagic process. This approach shows that although LysoTracker dyes do not specifically label lysosomes or autophagosomes within the cell, they allow the simultaneous measurement of an autophagy related process and other live cell functions, which is not possible with the standard LC3-II antibody technique.

Hapten-Grafted Graphene as a Transducer for Homogeneous Competitive Immunoassay of Small Molecules

A hapten-grafted graphene-based biosensor by integrating both the graphene nanosheets and immunoassay sensing technologies was developed for ultrasensitive homogeneous competitive immunoassay of small molecules. The structure of hapten-grafted graphene avoids the activity loss of biomolecules immobilized onto the graphene surface and is beneficial to preserve the binding affinity between small molecule and its specific antibody. The sandwich structure formed between hapten-grafted graphene nanosheets and fluorescence-labeled antibody increases the quenching efficiency of the organic dye, thereby resulting in high signal-to-background ratios and improved sensitivity for Bisphenol A (BPA) detection. On the basis of fluorescence resonance energy transfer (FRET) and homogeneous competitive immunoassay mechanism, high BPA concentrations in the sample reduce the amount of fluorescence-labeled anti-BPA antibody bound to graphene-BPA nanosheets, thus resulting in remarkable fluorescence signals. The linear quantification of BPA over concentration ranges from 0.5 to 50 nM with a detection limit determined as 0.12 nM. These findings show that the proposed method provides a powerful tool for the rapid and sensitive detection of small molecules in biological and environmental samples.

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (“antibody feeding”) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.