Multiple Species of Endothelial Microparticles (EMP) Released from Different Membrane Protein Clustering Domains Exhibit Distinctive Phenotypes and Activities.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1802-1802 ◽  
Author(s):  
Wenche Jy ◽  
Joaquin J. Jimenez ◽  
Lucia M. Mauro ◽  
Carlos Bidot ◽  
Lawrence L. Horstman ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Membrane Protein ◽  
Fluorescence Microscopy ◽  
Cell Interaction ◽  
Color Flow ◽  
Multiple Species ◽  
Normal Controls

Abstract INTRODUCTION: We have previously shown that EMP comprise multiple species of vesicles released from endothelial cells (EC) upon stimulation. However, the mechanism underlying EMP release is not clear, nor is their functional role. We postulated that EMP release is initiated by formation of discrete clusters of membrane proteins, each of which may release distinctive EMP characterized by the predominant protein in the cluster or raft. Therefore, each such subspecies may have distinctive activities in cell interaction or other function. In this study, we employed flow cytometry to investigate this postulated mechanism, and compared in vitro with in vivo findings. METHODS: EMP were prepared by incubating renal endothelial cells (EC) with 10 ng/mL of TNF for 24hr. Two-color flow cytometry was used to analyze the phenotypic composition of the resulting EMP, the markers used including CD31, CD62E, CD51, CD54, annexinV (AnV), tissue factor (TF), and lectin Ulex europaeus I (Ulex). Fluorescence microscopy was used to study membrane protein movement and clustering. RESULTS:(1) Phenotypic composition of EMP was evaluated in culture supernatants by flow cytometry, first by the number detected with each marker. Expressed in millions/mL, they were: by Ulex, 280; AnV, 52; CD54, 48; CD62E, 46; CD31, 34; TF, 36; and CD51, 8.(2) Two-color technique was used to establish the degree to which more than 1 marker (antigen) was present on the same EMP. It was found that only a small fraction (<5%) of CD54+ or CD62E+ EMP were also positive for CD31, and vice versa.(3) Cell interactions: Incubating the EMP mixture with neutrophils resulted in selective binding of CD54+ and CD62E+ EMP to the neutrophils and loss of 95% and 70% of free CD54+ and CD62E+ EMP, respectively, from the cell-free supernatants. EMP positive for the other markers showed little binding to leukocytes. These data confirm subspecies of EMP with little overlap of markers and differing affinity for leukocytes. (4) Fluorescence microscopy: Upon EC stimulation, a time-dependent movement of surface markers CD31 and CD54 resulted in their clustering to different locations prior to shedding of vesicles. Majority of vesicles were seen to shed from these clusters. This process may explain how EC can release multiple subspecies of EMP. (5a) In vivo: Levels of CD54+ EMP were always low or nearly undetectable in plasma from patients or normal controls. However, high levels of CD54+ EMP/leukocyte conjugates were found in several thrombotic and inflammatory disorders. This is consistent with in vitro findings. (5b) In vivo total MP: Study of plasma from 26 normal controls showed that MP measured by Ulex were about 3 to 4-fold higher than if measured by AnV. The majority of Ulex+ MP were negative for AnV. SUMMARY:Our data support the hypothesis that upon activation or apoptosis, EC developed multiple membrane protein clusters as a prelude to EMP release.EMP species released from these membrane clusters exhibit distinctive phenotypes and activities such as leukocyte binding.AnV has been widely used a marker for total MP, but this will miss MP not expressing AnV. We show that the lectin marker Ulex gives the highest counts of MP, in vitro and in vivo, suggesting that Ulex may be a better proxy than AnV for defining total MP.

Circulating CD62E Occurs in Soluble Form as Well as Bound to Endothelial Microparticles (EMP): Functional Differences in Platelet Aggregation in Thrombotic Thrombocytopenic Purpura (TTP).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2624-2624
Author(s):  
Joaquin J. Jimenez ◽  
Wenche Jy ◽  
Lucia M. Mauro ◽  
Michael N. Markou ◽  
George W. Burke ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Platelet Aggregation ◽  
Acute Phase ◽  
Critical Role ◽  
In Vivo Studies ◽  
Soluble Form ◽  
Dependent Manner

Abstract Injured endothelial cells (EC) are believed to play a critical role in the pathophysiology of TTP. Soluble markers of endothelial disturbance measured by enzyme-linked immunoassay (ELISA) have been found elevated in TTP. We have recently demonstrated an increase in the release of CD31/42b- EMP, and CD62E+ EMP. Moreover, we have observed that CD62E+ EMP also express vWF. The aim of this study was to quantitate soluble (s) vs. EMP-bound CD62E (bCD62E) in vitro and in vivo, in relation to the functional activity of vWF+ EMP. METHODS: Brain and renal microvascular endothelial cells (MVEC) were cultured and treated with 10ng/mL TNF-α to induce activation, or deprived of serum and growth factors (GFD) to induce apoptosis. Culture supernatants were collected and evaluated in a time-dependent manner. For in vivo studies, platelet-poor plasma was obtained from 4 TTP patients during the acute phase and upon remission. Filtration through 0.1μm, which retains most EMP, was employed to discriminate between (s) and bCD62E. sCD62E was measured by ELISA post-filtration and bCD62E by ELISA pre-filtration. Additionally, CD62E+ and CD62E+/vWF+ EMP were measured by flow cytometry. To assess pro-aggregatory function, EMP were added to washed platelets in the presence of 1 mg/mL ristocetin and aggregates were measured by flow cytometry. RESULTS: In vitro: Activation did not induce release of sCD62E at 3 hours, although bCD62E was present (1.5±0.5X106 EMP/mL). At 6 hours, some sCD62E was detected in the filtrate (0.09±0.02 ng/mL), but most was present in the unfiltered medium (3.5±0.85 ng/mL), signifying that the majority was bCD62E, confirmed by a doubling of CD62E+ EMP (3.0±0.6X106/mL). Subsequently, sCD62E levels were 1.0±0.2 ng/mL at 12 hr, 3.5±0.7 ng/mL at 18 hr, and 5±0.9 ng/mL at 24 hr. In contrast, EMP counts at 12, 18 and 24 hours were 4.6±1, 7±1.3 and 9±1.8 X106/mL (p=0.01, p=0.01, p=0.02, respectively). For all time periods, 40-60% of CD62E were positive for vWF. In control or GFD cultures, there was not a significant increase in sCD62E or CD62E+ EMP at any time period. MVEC from renal gave similar results. In acute TTP plasma samples, CD62E measured by ELISA was significantly increased (65±22 ng/mL) vs. remission (30±6 ng/mL). bCD62E accounted for 50% in acute and 15% in remission. CD62E+/vWF+ EMP were significantly elevated in plasma from acute TTP patients vs. remission (15±4.5 vs. 3±0.5, p=0.01). Sample filtration resulted in a decrease of &gt;95% EMP in both acute and remission TTP plasma. MVEC-derived CD62E+/vWF+ EMP resulted in a dose-dependent increase in platelet aggregation. Additionally, plasma from 4 TTP patients with elevated CD62E+/vWF+ EMP obtained during the acute phase enhanced the formation of platelet aggregates by 48±12% (p=0.02) above remission plasma with low EMP counts. CONCLUSIONS: The results demonstrate that CD62E heretofore regarded as a soluble marker of endothelial dysfunction, in reality exists in both a soluble and EMP-bound form. Indeed, this distinction is highly relevant because CD62E+ EMP also express vWF and are pro-aggregatory to platelets. These EMP have been shown to be elevated during the acute phase of TTP and decrease upon remission. Thus, CD62E+/vWF+ EMP may be active participants in the formation of platelet-rich thrombi in TTP.

scholarly journals Insulin uptake across the luminal membrane of the rat proximal tubule in vivo and in vitro

2009 ◽  
Vol 296 (5) ◽  
pp. F1227-F1237 ◽  
Author(s):  
Pavel Kolman ◽  
Angelo Pica ◽  
Nicolas Carvou ◽  
Alan Boyde ◽  
Shamshad Cockcroft ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Microscopy ◽  
Proximal Tubule ◽  
Apical Membrane ◽  
Fluorescence Signal ◽  
Pt Cell ◽  
Insulin Uptake ◽  
Rat Proximal Tubule

We visualized insulin uptake in vivo across the apical membrane of the rat proximal tubule (PT) by confocal microscopy; we compared it with in vitro findings in a rat PT cell line (WKPT) using fluorescence microscopy and flow cytometry. Surface tubules were observed in vivo with a 633-nm single laser-illuminated real-time video-rate confocal scanning microscope in upright configuration for optical sectioning below the renal capsule. Fields were selected containing proximal and distal tubules; Cy5-labeled insulin was injected twice (the second time after ∼140 min) into the right jugular vein, and the fluorescence signal (at 650–670 nm) was recorded. Fluorescence was detected almost immediately at the brush-border membrane (BBM) of PT cells only, moving inside cells within 30–40 min. As a measure of insulin uptake, the ratio of the fluorescence signal after the second injection to the first doubled (ratio: 2.11 ± 0.26, mean ± SE, n = 10), indicating a “priming,” or stimulating, effect of insulin on its uptake mechanism at the BBM. This effect did not occur after pretreatment with intravenous lysine (ratio: 1.03 ± 0.07, n = 6; P < 0.01). Cy2- or Cy3-labeled insulin uptake in a PT cell line in vitro was monitored by 488-nm excitation fluorescence microscopy using an inverted microscope. Insulin localized toward the apical membrane of these cells. Semiquantitative analysis of insulin uptake by flow cytometry also demonstrated a priming effect (upregulation) on insulin internalization in the presence of increasing amounts of insulin, as was observed in vivo; moreover, this effect was not seen with, or affected by, the similarly endocytosed ligand β2-glycoprotein.

Abstract 1461: BLADE Is A Novel Membrane Protein That Regulates Endothelial Apoptosis and Consequently Angiogenesis Both In Vitro and In Vivo

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Koji Ikeda ◽  
Ritsuko Nakano ◽  
Thomas Quertermous ◽  
Hiroaki Matsubara
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Membrane Protein ◽  
Umbilical Vein ◽  
Hepatoma Cell ◽  
Endothelial Cell Migration ◽  
Late Time ◽  
Endothelial Apoptosis

Endothelial apoptosis is a pivotal process for angiogenesis during embryogenesis as well as postnatal. Here, we identified a novel gene, termed BLADE, that regulates endothelial apoptosis and consequently angiogenesis. BLADE was highly expressed in a variety of human cultured endothelial cells such as endothelial cells from umbilical vein (HUVEC), aorta, coronary artery and microvessels while no expression was observed in non-endothelial cells including HeLa cell, hepatoma cell and vascular smooth muscle cells. Furthermore, BLADE was expressed predominantly in blood vessels during embryogenesis, suggesting its role in angiogenesis. Amino acid sequence analysis revealed that BLADE was a novel membrane protein with no conserved domain reported before. BLADE expression was altered by treatment with VEGF, TNF-alpha and TGF-beta in HUVEC. Knockdown of BLADE in HUVEC using short interference RNA resulted in significant reduction of endothelial apoptosis. In contrast, BLADE knockdown affected neither endothelial cell migration nor proliferation. Among factors associated with apoptosis, Bcl-2 expression was drastically increased in HUVEC by BLADE-knockdown. Further analysis revealed that this increase of Bcl-2 is due to reduced ubiquitination and less subsequent degradation of Bcl-2. Inhibition of Bcl-2 completely abolished the anti-apoptotic effect of BLADE-knockdown. In vitro tube-formation of HUVEC on Matrigel was reduced concomitantly with reduced apoptosis by BLADE-knockdown at early time (12h). Nevertheless, significantly more tubes were preserved at late time (96h) by BLADE-knockdown. Moreover, in vivo angiogenesis assessed by Matrigel-plug was dramatically enhanced by BLADE-knockdown. Taken together, BLADE is a novel factor regulating endothelial apoptosis as well as angiogenesis both in vitro and in vivo. Detailed analysis of BLADE will provide new insights in the regulation of endothelial apoptosis, and in the molecular link between endothelial apoptosis and angiogenesis. Because BLADE is highly preferentially expressed in endothelial cells, inhibition of BLADE might be an ideal approach to enhance endothelial cell survival and angiogenesis without affecting the survival of other type of cells.

Fucoidan down Regulates the Expression of CXCR4 In Vitro on Human Hematopoietic CD34+ Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3384-3384
Author(s):  
Mohammad R. Irhimeh ◽  
Robert E. Nordon ◽  
Kap-Hyoun E. Ko ◽  
Helen Fitton ◽  
Raymond M. Lowenthal
Keyword(s):  
Flow Cytometry ◽  
Clinical Study ◽  
Factorial Design ◽  
Color Flow ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Negative Effect ◽  
Vitro Effect

Abstract Sulfated glycans such as fucoidan have been shown to induce rapid hematopoietic progenitor stem cell (HPCs) mobilization in mice and primates. Our previous clinical study has demonstrated up-regulation of CXCR4 on peripheral CD34+ cells in volunteers who had received oral doses of 75% pure fucoidan. The aim of this study was to investigate the in vitro effects of fucoidan on a cytokine mediated CD34+ cell expansion system and to characterize the fucoidan effects on CD34+ proliferation and differentiation using a factorial design experiment and flow cytometry. Isolex selected CD34+ cells harvested from multiple myeloma patients were used in the study. There is institutional approval for the in vitro growth of CD34+ cells from mobilized peripheral blood. Fractional (25−1 resolution V, 16 runs, 2 blocks, E=ABCD) and full (24, 16 runs, 2 blocks) factorial design experiments were used to investigate the effects of depyrogenated fucoidan (DP-GFS) and cytokines on the growth of CD34+ cells. The cells were grown in 24 well dishes (1 ml/well) at an inoculum density of 20,000 cells/ml in serum free media and cultured for 10 days in a cytokine cocktail including G-CSF, FL, SCF, TPO and SDF1 each at two levels 100 ng/ml or 1000 ng/ml. DP-GFS was used in the cultures at four different levels including 0, 10, 100 and 500 μg/ml. Cells were then harvested and analyzed using three color flow cytometry and a panel of antibodies that includes CD15, CD14, CD41a, CD38, CD34, and CXCR4. The in vitro effect of DP-GFS on CXCR4 expression was opposite to that found in vivo using 75% fucoidan. At 100 ng/ml and at saturating levels of FL, SCF, TPO and G-CSF (1000 ng/ml), DP-GFS at 10 μg/ml was observed to reduce the number of CD34+ cells produced per input CD34+ cell (1.09±0.07 versus 0.84±0.05, n=16, p=0.002) and have a negative effect on the percentage of CD34+ cells that express CXCR4 (87.2±1.8 versus 77.4±2.3, n=16, p=0.001). There was no significant effect of DP-GFS on the total cellular output nor other cell subsets examined (CD41a+CD14−, CD15+, CD14+). Our in vitro results are more consistent with a recent clinical study that demonstrates down-regulation of CXCR4 receptors on CD34+ cells in G-CSF or GM-CSF mobilized blood (Dlubek et al, 2006). We hypothesize that fucoidan acts in vitro and in vivo via different mechanisms.

scholarly journals A cell based assay for evaluating binding and uptake of an antibody using hepatic nonparenchymal cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuki Noguchi ◽  
Kazuhisa Ozeki ◽  
Hiroaki Takesue ◽  
Hidetaka Akita
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Liver Sinusoidal Endothelial Cells ◽  
Nonparenchymal Cells ◽  
A Cell ◽  
Hepatic Nonparenchymal Cells ◽  
Titration Assay ◽  
Parenchymal Cells

AbstractEvaluation of the binding and uptake of an antibody in liver non-parenchymal cells (NPC), including liver sinusoidal endothelial cells, is important for revealing its pharmacokinetic (PK) behavior, since NPC has important roles in eliminating an antibody from the blood via the Fc fragment of IgG receptor IIB (FcγRIIB). However, there is currently no in vitro quantitative assay using NPC. This study reports on the development of a cell-based assay for evaluating the binding and uptake of such an antibody using liver NPC of mice and monkeys. In mice, the FcγRIIB-expressing cells were identified in the CD146-positive and CD45-negative fraction by flow cytometry. A titration assay was performed to determine the PK parameters, and the obtained parameter was comparable to that determined by the fitting of the in vivo PK. This approach was also extended to NPC from monkeys. The concentration-dependent binding and uptake was measured to determine the PK parameters using monkey NPC, the FcγRIIB-expressing fraction of which was identified by CD31 and CD45. The findings presented herein demonstrate that the in vitro liver NPC assay using flow cytometry is a useful tool to determine the binding and uptake of biologics and to predict the PK.

Inhibition of P-Selectin and PSGL-1 Using Humanized Monoclonal Antibodies Increases the Sensitivity of Multiple Myeloma Cells to Proteasome Inhibitors

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4758-4758
Author(s):  
Barbara Muz ◽  
Feda Azab ◽  
Pilar De La Puente ◽  
Scott A Rollins ◽  
Richard Alvarez ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Endothelial Cells ◽  
Monoclonal Antibodies ◽  
Stromal Cells ◽  
Half Life ◽  
Adherent Cells ◽  
Humanized Monoclonal Antibodies

Abstract Introduction: Multiple myeloma (MM) is a plasma cell malignancy localized in the bone marrow (BM). Despite the introduction of novel therapies more than 70% of MM patients relapse. We have previously shown that inhibition of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) play a key role on proliferation of MM and that its inhibition with small-molecule inhibitors sensitized MM cells to therapy. However, these small molecule inhibitors had low specificity to P-selectin and showed poor pharmacokinetics. In this study, we tested inhibition of P-selectin and PSGL-1 using functionally blocking monoclonal antibodies to sensitize MM cells to therapy. Methods: The humanized monoclonal antibodies anti-P-selectin (SelG1) and anti-PSGL-1 (SelK2) were obtained from Selexys Pharmaceuticals. Endothelial cells, stromal cells derived from MM patients, and MM cell lines (MM1s, H929, OPM1 and RPMI8226) were used for in vitro expression, proliferation, apoptosis and immuno-blotting assays. The expression of P-selectin and PSGL-1 were tested by the interaction of SelG1 (5-20µg/mL) and SelK2 (5-20µg/mL) antibodies with endothelial cells, stromal cells and MM cells for 1hr, followed by addition of a secondary-FITC antibody and flow cytometry analysis. For adhesion assay, cells were treated with increasing concentrations of SelG1 and SelK2 for 1hr; pre-labeled MM cells were then applied to unlabeled endothelial or stromal cells for 1hr, non-adherent cells were washed, and adherent cells were analyzed by a fluorescent reader. For proliferation, MM1s-GFP+ cells cultured alone, with stroma, or with endothelial cells; and were treated with, or without, bortezomib and carfilzomib, in the presence, or absence, of SelG1 and SelK2 antibodies, and proliferation was determined by flow cytometry. Protein expression associated with survival, apoptosis and cell cycle signaling was analyzed by western blotting. For in vivo, MM1s-Luc-GFP cells were injected intravenously into SCID mice and tumor progression followed for 4 weeks. Anti-mouse-P-selectin antibody was used to inhibit P-selectin in the mouse endothelial cells and stroma. Likewise, SelK2 and anti-mouse PSGL-1 were used to inhibit PSGL-1 on human MM cells in the mouse microenvironment, respectively. Mice were divided into 6 groups (1) vehicle treated control, (2) anti-mouse-P-selectin (5mg/kg) alone, (3) SelK2 (5mg/kg) and anti-mouse-PSGL-1 (5mg/kg) alone, (4) bortezomib (0.5mg/kg) alone, (5) a combination of anti-mouse-P-selectin and bortezomib, and (6) a combination of SelK2 (5mg/kg), anti-mouse-PSGL-1 (5mg/kg) and bortezomib. Anti-mouse-P-selectin, anti-mouse–PSGL-1, SelK2 and bortezomib were administered intraperitoneally twice a week. Results: The half-life of SelG1 in a Phase I clinical study was previously shown to be 363 hours while the half-life of SelK2 in a primate study was approximately 100 hours. P-selectin expression was detected on endothelial and stromal cells using the SelG1 antibody, while no expression was found on MM cells. PSGL-1 was highly expressed on MM cells as well as on endothelial cells and stromal cells as detected by SelK2 monoclonal antibody. Inhibition of P-selectin and PSGL-1 with SelG1 or SelK2, respectively, decreased MM cell adhesion to endothelial and stromal cells, and decreased the proliferation of MM cells induced by stromal and endothelial cells. Similarly, inhibition of the interaction between P-selectin and PSGL-1 sensitized MM cells to bortezomib and carfilzomib in vitro. In vivo results demonstrated that inhibition of the P-selectin or PSGL-1 as single agents delay tumor growth compared to non-treated mice and that it enhances the effect of bortezomib. Conclusions: These results demonstrate that inhibition of P-selectin and PSGL-1 by SelG1 and SelK2 antibodies, respectively, disrupts the interaction between MM cells and BM microenvironment and decreases adhesion and proliferation of MM cells. Moreover, inhibition of P-selectin and PSGL-1 increased the sensitivity of MM cells to bortezomib and carfilzomib in vitro, and to bortezomib in vivo. These data provides a basis for future clinical trials for sensitization of refractory MM patients to therapy by inhibition of P-selectin and PSGL-1 using SelG1 and SelK2 monoclonal antibodies. Disclosures Rollins: Selexys: Employment. Alvarez:Selexys: Employment. Kawar:Selexys: Employment. Azab:Selexys: Research Funding.

Novel Method to Detect Minimal Residual Disease in Multiple Myeloma Predicts Recurrence Better Than CD138-Based Models

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2985-2985
Author(s):  
Barbara Muz ◽  
Feda Azab ◽  
Pilar De La Puente ◽  
Justin King ◽  
Micah John Luderer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Research Funding ◽  
Mononuclear Cells ◽  
Residual Disease ◽  
Tumor Burden ◽  
New Method ◽  
Color Flow ◽  
Cd38 Expression

Abstract Introduction: Diagnosis, responses to treatment, minimal residual disease (MRD) and circulating tumor cells (CTC) in multiple myeloma (MM) are all detected by the gold-standard marker CD138 flow cytometry-based method. However, the presence of clonogenic CD138-negative MM cells and hypoxia-driven CD138 downregulation were shown previously. In this study, we found that CD138 is significantly downregulated in MRD and CTC populations in MM, thus we developed a novel two-color flow cytometry-based set of biomarkers (independent of CD138) to detect MRD and CTC in MM. Methods: We created a MRD mouse model by treating MM-bearing mice with a high dose of bortezomib. We then tested the effect of hypoxia and drug treatment on the expression of different markers, including CD138. Therefore, to detect MM cells we utilized CD38-APC antibody, followed by the exclusion of non-MM CD38-expressing cells such as T cells, monocytes, NK cells, B cells and dendritic cells using (CD3, CD14, CD16, CD19 and CD123)-V450 antibodies, respectively. To verify the ability of the new method to selectively detect MM cells, mononuclear cells from peripheral blood (PB) from healthy patients and MM cell lines (hypoxic and normoxic) were stained by the antibody cocktail and analyzed by flow cytometry. Next, we compared the sensitivity of the traditional CD138 method to the new method in detecting (a) normoxic or hypoxic MM cells spiked into bone marrow (BM) cells in vitro, (b) normoxic or hypoxic MM cells spiked into PB in vitro, (c) MM1s-GFP+ cells in mice with different tumor burden in the BM in vivo, (d) circulating MM1s-GFP+ in the PB in vivo, (e) MRD cells in the BM samples from 16 patients with complete remission (CR) or very good partial response (VGPR), and (F) CTC in 12 progressive MM patients. We further aimed to predict time to progression (TTP) in 16 patients with complete remission based on the detection of MRD in these patients using the new method and compared with flow cytometry-based CD138 or histology. Results: In vivo, we found that bortezomib-treated MRD cells were hypoxic, compared to a progressive vehicle-treated cells. CD138 expression in these cells was significantly decreased, but CD38 expression was unchanged. In vitro expression of CD138 was decreased due to hypoxia and bortezomib treatment, whereas CD38 expression was unchanged. Furthermore, we developed a new method using an antibody cocktail, where the MM cell population is defined as CD38+/CD3-/CD14-/CD16-/CD19-/CD123- (APC+ and V450-). In vitro, the new method detected 100% of hypoxic and normoxic MM cells, and less than 0.5% of mononuclear cells from the PB or BM of healthy donors. In contrast, CD138+ cells failed to detect 50% of hypoxic MM cells and 10-25% of normoxic cells. In vivo, the amount of cells detected by the new method directly correlated with the number of MM1s-GFP+ cells detected in the BM with a range between 0-60%, with a correlation coefficient (slope) of 0.99 and R2 of 0.999. The CD138 detected only a fraction of the MM1s-GFP+ population (<10%) even in mice with a high BM tumor burden. In addition, the new method detected close to a 100% of the circulating MM1-GFP+ cells in vivo, while the CD138 marker detected less than 1% of the circulating MM1s-GFP+ cells. In patients, the new method detected 0.5-8% MRD cells in the BM of 16 patients with CR or VGPR (which were defined as CD138-negative), and 0.1-1.8% CTC in the PB of 12 progressive MM patients. In contrast, CD138 marker detected less than 0.5% of MRD cells in the BM of the CR and VGPR patients, and less than 0.1% of CTCs in the PB of the progressive patients. Furthermore, we found that, while CD138 and histology failed to predict recurrence in CR patients, the new method successfully detected CD138-negative MM population whose prevalence in the BM inversely correlated with TTP in MM patients defined as CR based on CD138 and histology. Conclusions: We confirmed that CD138 expression is variable on MM cells, and that it is downregulated in MRD and CTC populations, and that it was not effective in detecting these particular populations in MM. Furthermore, we developed a novel two-color flow cytometry-based biomarker-set to detect MM cells independent of CD138. The new methods detected close to a 100% of all MM cells in vitro and in vivo, including MRD and CTC. Moreover, the new method detected a CD138-negative MRD and CTC in MM patients, and the prevalence of this population inversely correlated with TTP in MM patients. Disclosures Vij: Celgene, Onyx, Takeda, Novartis, BMS, Sanofi, Janssen, Merck: Consultancy; Takeda, Onyx: Research Funding. Azab:Verastem: Research Funding; Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner.

Abstract 154: Atherosclerosis-specific CD4 T Cells Use the Chemokine CCL5 and Its Receptor CCR5 to Home to Mature Atherosclerotic Lesions in Mice

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Jie Li ◽  
Klaus Ley
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Transcriptomic Analysis ◽  
Cell Phenotype ◽  
Color Flow ◽  
Cell Homing ◽  
T Cell Homing

Introduction: T cell adaptive immunity is involved in the pathogenesis of atherosclerosis, but the phenotype of T cell subset and chemokine receptor that regulates effector T cell trafficking to atherosclerotic aortas is unknown. By multi-color flow cytometry, we discovered that >40% of T cells in the atherosclerotic aortas express CCR5. We hypothesize that CCR5 plays an important role in T cell recruitment to atherosclerotic aortas and CCR5+ T cells play an important roll in the pathogenesis of atherosclerosis. Methods: T cell phenotype in the aorta of Apoe -/- mice on western diet 3-5 months was analyzed by multi-color flow cytometry. T cell homing assay were done in vitro by incubating 400,000 T eff from CD45.1Apoe -/- mice with explanted Apoe -/- aortas 12 to 18 hrs with media alone, CCL5 neutralizing antibody (0.5 ug/ml) or PTX (300ng/ml), and in vivo by adoptively transferring splenocytes from Apoe -/- Ccr5 -/- and dsRedApoe -/- mice at 1:1 ratio into CD45.1Apoe -/- recipients. 24 hours later, recipient aortas were digested and analyzed by flow cytometry. To visualize T cell proximity to APCs in the aorta by 2 photon imaging, T eff from Apoe -/- mice were labeled with SNARF before incubation with explanted aortas from CD11c-YFP+Apoe -/- mice in the above 3 conditions. In vitro T cell suppression assay was done by incubating 10 4 CD4+CD25- T cells with α-CD3 mAb and 5x10 4 irradiated splenic APCs and decreasing amount of Treg or CCR5Teff. Transcriptomic analysis is done following SMARTseq II prortocol from Illumina. Results: 43±6% of αβ T cells in the atherosclerotic aortas express CCR5, significantly higher than that in aortas of wild-type C57BL/6 mice (10±7%) or Apoe -/- mice on normal diet (7±6%). CCR5 and its ligand CCL5 play the major role in regulating T cell homing to atherosclerotic aortas in vitro and in vivo . Interestingly, at late stage of disease, the CCR5+ T cells in the atherosclerotic plaques are all FoxP3 + IFN-γ + T-bet + CD25 - CD44 hi CD62L lo (CCR5Teff) and do not suppress T cell proliferation. CCR5Teffs are only found in the aorta and para-aortic lymph nodes of Apoe -/- mice but not in the spleen or other organs. Transcriptomic analysis shows that CCR5Teff is more similar to Teff rather than Tregs.

Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

1997 ◽  
Vol 77 (05) ◽  
pp. 0975-0980 ◽  
Author(s):  
Angel Gálvez ◽  
Goretti Gómez-Ortiz ◽  
Maribel Díaz-Ricart ◽  
Ginés Escolar ◽  
Rogelio González-Sarmiento ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Platelet Adhesion ◽  
Umbilical Vein ◽  
Procoagulant Activity ◽  
Experimental Conditions ◽  
Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

Epinephrine Induces von Willebrand Factor Release from Cultured Endothelial Cells: Involvement of Cyclic AMP-dependent Signalling in Exocytosis

1997 ◽  
Vol 77 (06) ◽  
pp. 1182-1188 ◽  
Author(s):  
Ulrich M Vischer ◽  
Claes B Wollheinn
Keyword(s):  
Endothelial Cells ◽  
Adenylate Cyclase ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Free Calcium ◽  
Camp Content ◽  
Von Willebrand ◽  
Willebrand Factor

Summaryvon Willebrand factor (vWf) is released from endothelial cell storage granules after stimulation with thrombin, histamine and several other agents that induce an increase in cytosolic free calcium ([Ca2+]i). In vivo, epinephrine and the vasopressin analog DDAVP increase vWf plasma levels, although they are thought not to induce vWf release from endothelial cells in vitro. Since these agents act via a cAMP-dependent pathway in responsive cells, we examined the role of cAMP in vWf secretion from cultured human umbilical vein endothelial cells. vWf release increased by 50% in response to forskolin, which activates adenylate cyclase. The response to forskolin was much stronger when cAMP degradation was blocked with IBMX, an inhibitor of phosphodiesterases (+200%), whereas IBMX alone had no effect. vWf release could also be induced by the cAMP analogs dibutyryl-cAMP (+40%) and 8-bromo-cAMP (+25%); although their effect was weak, they clearly potentiated the response to thrombin. Epinephrine (together with IBMX) caused a small, dose-dependent increase in vWf release, maximal at 10-6 M (+50%), and also potentiated the response to thrombin. This effect is mediated by adenylate cyclase-coupled β-adrenergic receptors, since it is inhibited by propranolol and mimicked by isoproterenol. In contrast to thrombin, neither forskolin nor epinephrine caused an increase in [Ca2+]j as measured by fura-2 fluorescence. In addition, the effects of forskolin and thrombin were additive, suggesting that they act through distinct signaling pathways. We found a close correlation between cellular cAMP content and vWf release after stimulation with epinephrine and forskolin. These results demonstrate that cAMP-dependent signaling events are involved in the control of exocytosis from endothelial cells (an effect not mediated by an increase in [Ca2+]i) and provide an explanation for epinephrine-induced vWf release.

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