CD45 Antibody Research Articles

Recombinant anti-human and anti-porcine CD14 antibodies

Recombinant anti-human and anti-porcine CD14 antibodies

Monocyte activation is a feature of common variable immunodeficiency irrespective of plasma lipopolysaccharide levels

Common variable immunodeficiency disorders (CVID), the most frequent cause of symptomatic primary immunodeficiency, are defined by impaired antibody production. Notwithstanding, T cell activation and granulomatous manifestations represent the main causes of CVID morbidity even in patients receiving immunoglobulin (Ig) G replacement therapy. Additionally, gut pathology is a frequent feature of CVID. In this study, we investigated monocyte imbalances and their possible relationship with increased microbial translocation in CVID patients. Monocyte subsets were defined according to CD14 and CD16 expression levels and evaluated in terms of human leucocyte antigen D-related (HLA-DR), CD86 and programmed death-1 molecule ligand 1 (PD-L1) expression by flow cytometry, in parallel with the quantification of plasma lipopolysaccharide (LPS) and serum levels of soluble CD14 (sCD14), LPS-binding protein (LBP) and anti-LPS antibodies. CVID patients (n=31) featured significantly increased levels of serum sCD14 and an expansion of CD14(bright) CD16(+) monocytes in direct correlation with T cell and B cell activation, the latter illustrated by the frequency of the CD21(low) CD38(low) subset. Such alterations were not observed in patients lacking B cells due to congenital agammaglobulinaemia (n=4). Moreover, we found no significant increase in circulating LPS or LBP levels in CVID patients, together with a relative preservation of serum anti-LPS antibodies, in agreement with their presence in commercial IgG preparations. In conclusion, CVID was associated with monocyte imbalances that correlated directly with T cell activation markers and with B cell imbalances, without an association with plasma LPS levels. The heightened monocyte activated state observed in CVID may represent an important target for complementary therapeutic strategies.

NAD+-dependent Sirtuin 1 and 6 Proteins Coordinate a Switch from Glucose to Fatty Acid Oxidation during the Acute Inflammatory Response

The early initiation phase of acute inflammation is anabolic and primarily requires glycolysis with reduced mitochondrial glucose oxidation for energy, whereas the later adaptation phase is catabolic and primarily requires fatty acid oxidation for energy. We reported previously that switching from the early to the late acute inflammatory response following TLR4 stimulation depends on NAD(+) activation of deacetylase sirtuin 1 (SirT1). Here, we tested whether NAD(+) sensing by sirtuins couples metabolic polarity with the acute inflammatory response. We found in TLR4-stimulated THP-1 promonocytes that SirT1 and SirT 6 support a switch from increased glycolysis to increased fatty acid oxidation as early inflammation converts to late inflammation. Glycolysis enhancement required hypoxia-inducing factor-1α to up-regulate glucose transporter Glut1, phospho-fructose kinase, and pyruvate dehydrogenase kinase 1, which interrupted pyruvate dehydrogenase and reduced mitochondrial glucose oxidation. The shift to late acute inflammation and elevated fatty acid oxidation required peroxisome proliferator-activated receptor γ coactivators PGC-1α and β to increase external membrane CD36 and fatty acid mitochondrial transporter carnitine palmitoyl transferase 1. Metabolic coupling between early and late responses also required NAD(+) production from nicotinamide phosphoryltransferase (Nampt) and activation of SirT6 to reduce glycolysis and SirT1 to increase fatty oxidation. We confirmed similar shifts in metabolic polarity during the late immunosuppressed stage of human sepsis blood leukocytes and murine sepsis splenocytes. We conclude that NAD(+)-dependent bioenergy shifts link metabolism with the early and late stages of acute inflammation.

Interleukin-10 inhibits lipopolysaccharide-induced upregulation of tissue factor in canine peripheral blood monocytes

The potentially fatal hemostatic disorder of disseminated intravascular coagulation (DIC) is initiated in bacterial sepsis by lipopolysaccharide (LPS)-induced tissue factor (TF) expression on monocytes. Interleukin-10 (IL-10) is a potent inhibitory cytokine that downregulates monocyte inflammatory and procoagulant responses. We hypothesized that canine recombinant IL-10 (rIL-10) would inhibit LPS-induced TF upregulation on canine monocytes in a dose-dependent manner. Canine peripheral blood mononuclear cells (PBMC), obtained by double-density gradient centrifugation, and monocytes, purified from PBMC by immunomagnetic bead separation with an anti-canine CD14 antibody (Ab), were stimulated in suspension with LPS (0.1–1000ng/mL) for various times. Recombinant IL-10 (10–5000pg/mL) was added with LPS or up to 2h later. Tissue factor procoagulant activity was measured by cleavage of a chromogenic substrate by activated Factor X generated by the TF-factor VII complex. We found that rIL-10, when given concurrently or 1h after LPS, strongly inhibited LPS-induced TF procoagulant activity in canine PBMC and monocytes. This inhibition was dose-dependent and blocked by an anti-canine IL-10 Ab. Our results indicate that rIL-10 effectively inhibits LPS-induced TF upregulation in canine monocytes and could potentially be useful in limiting the development of DIC in dogs with endotoxemia.

Abstract 384: Interleukin-6 Mediates the Altered Platelet Function Associated with Experimental Colonic Inflammation

Patients with inflammatory bowel disease (IBD) are susceptible to microvascular thrombosis and thromboembolism. The increased incidence of thrombosis is accompanied by enhanced coagulation and abnormalities in platelet function. Clinical studies have also revealed alterations in platelet activation, enhanced platelet-leukocyte interaction, and elevated plasma levels of prothrombotic cytokines, such as IL-6. This study was directed towards determining whether: 1) experimental colitis, induced by 6 days of dextran sodium sulfate (DSS) ingestion, is associated with platelet activation and the formation of platelet-leukocyte aggregates (PLAs), 2) IL-6 deficiency alters these responses to DSS colitis, and 3) the platelet abnormalities observed in DSS mice can be recapitulated by chronic infusion of murine recombinant IL-6. Flow cytometry was used to characterize platelet function in heparin-anticoagulated whole blood. Platelets were identified by characteristic light scattering and membrane expression of CD41. Platelet activation was monitored using the expression of an activation epitope of GPIIb/IIIa integrin (with JON/A antibody). The combination of CD41, CD45.2, Gr-1, F4/80 and isotype control antibodies were used to detect and quantify aggregates of leukocytes, neutrophils and monocytes with platelets in control, wild type (WT) colitic, IL-6 -/- colitic, and WT mice implanted with IL-6 loaded Alzet osmotic minipumps (for 6 days). Our results indicate that DSS colitis is associated with increased numbers of activated platelets and the formation of aggregates of leukocytes (PLA), neutrophils (PNA) and monocytes (PMA) with platelets. These platelet responses to experimental colitis were largely undetected in IL-6 -/- mice. Chronic infusion (at a rate that yielded plasma IL-6 levels similar to those detected in DSS colitic mice) of IL-6 recapitulated the increased platelet activation and formation of PLA, PNA, and PMA observed in DSS-colitic mice. Collectively, these findings show that the altered platelet function detected in human IBD can be reproduced in an animal model of colonic inflammation and that interleukin-6 plays a critical role in the genesis of these platelet abnormalities in the setting of experimental IBD.

Abstract P034: Bone Marrow--Derived Cells Are Not an Essential Component in Cardiosphere Formation

Background: Cardiospheres (CS) are composed of heterogeneous population of cells but it is unknown whether bone marrow derived cells are an essential cell component in CS formation. Methods: Chimera mice were generated by transplantation of bone morrow cells from GFP transgenic mice to irradiated C57BL mice. Mice were randomized into 3 groups 5 months after transplantation: 1) myocardial infarction; 2) sham operated; 3) un-operated (n=5/group). Hearts were harvested 2-weeks post-surgery. Cardiac explants were cultured and putative cardiosphere forming cells (CFCs) (small cells migrating out from the explants) were collected 14 days later and reseeded on new culture dishes for CS formation. The number of CS from each heart was counted at 3 days. CS cell composition was analyzed by FACS. To further analyze the role of bone marrow derived CD45+ cells in forming CS, CD45+ cells was isolated from CFCs by CD45 antibody coated immunomagnetic beads. The number of CS formed from 1×10 5 putative CFCs, CFCs without CD45+ cells and CD45+ cells from CFCs (n=6-9/cell type) respectively were also counted at 3 days in culture. Results: Compared to sham (122± 23/heart) and un-operated hearts (18± 5/heart), infarcted hearts formed more CS (357± 64/heart, P<0.01). In all groups, irrespective of any surgery, 18.4± 4.5% of cells in CS co-expressed GFP and CD45, indicating they originated in bone marrow. Low percentage of bone marrow stem/progenitor cells (3.9% Sca-1+GFP+CD45+ and 1% c-Kit+GFP+CD45+ cells) were detected in CS, but a high percentage of cells within CS were cardiac stem/progenitor cells (26.3± 9.4% cells were Sca-1+GFP-CD45-, 0.10± 0.04% c-Kit+GFP-CD45-). Depleting CD45+ cells from putative CFCs actually increased the formation of CS (67±10 CS/1×10 5 cells) compared to un-depleted CFCs (51± 6 CS/1×10 5 cells, P<0.0001). Purified CD45+ cells from CFCs did not form CS in culture. Conclusion: Myocardial infarction increases the formation of CS in culture. Bone marrow derived CD45+ cells make up a small percentage of CS, but are not necessary for CS formation.

C-Reactive Protein Induces Release of Both Endothelial Microparticles and Circulating Endothelial Cells In Vitro and In Vivo: Further Evidence of Endothelial Dysfunction

Inflammation is pivotal in atherosclerosis. A key early event in atherosclerosis is endothelial dysfunction. C-reactive protein (CRP), the prototypic marker of inflammation in humans, is a risk marker for cardiovascular disease, and there is mounting evidence to support its role in atherothrombosis. CRP has been shown to promote endothelial dysfunction both in vitro and in vivo. Emerging biomarkers of endothelial dysfunction include circulating endothelial cells (CECs) and endothelial microparticles (EMPs). However, there is a paucity of data examining the effect of CRP on CEC and EMP production in vitro and in vivo. In this report, we treated human aortic endothelial cells (HAECs) with increasing concentrations of CRP (0-50 μg/mL) or boiled CRP. We counted CECs and EMPs by flow cytometry. Although CRP treatment resulted in a significant increase in release of both CECs and EMPs, boiled CRP failed to have an effect. Pretreatment of HAECs with sepiapterin or diethylenetriamine NONOate, both of which preserve nitric oxide (NO), resulted in attenuation of CRP's effects on CECs and EMPs. CD32 and CD64 blocking antibodies but not CD16 antibody or lectin-like oxidized LDL receptor 1 small interfering RNA (LOX-1 siRNA) prevented CRP-induced production of CECs and EMPs. Furthermore, delivery of human CRP to Wistar rats compared with human serum albumin resulted in significantly increased CECs and EMPs, corroborating the in vitro findings. We provide novel data that CRP, via NO deficiency, promotes endothelial dysfunction by inducing release of CECs and EMPs, which are biomarkers of endothelial dysfunction.

The Effect of ICAM-1 Antibody Therapy in the SCID-Hu Mouse Model Using Primary Myeloma Cells

Abstract 2914 Background:Myeloma (MM) - stromal cell-cell interactions play a crucial role in MM cell growth, survival, and drug resistance. Intercellular adhesion molecule-1 (ICAM-1) is up-regulated in different cancers, including MM, and represents one of the major adhesion molecules mediating tumor-stromal cell-cell contacts. Previous investigations have shown that ICAM antibodies induce antitumor activity in SCID mice xenografted with MM cell lines, likely involving Fc :Fc gamma receptor dependent anti-tumor mechanisms. We have examined the anti-MM activity in the SCID-hu mouse model of a specific humanized ICAM1 antibody (BI-505, Bio-Invent, Sweden) using primary MM cells. Materials and Methods:The expression of ICAM1 was examined in plasma cells from 22 donors, 351 newly diagnosed MMs, and 9 MM cell lines using Affymetrix U133 Plus2 chips. Human fetal femurs and tibias, obtained at 17 to 22 gestational weeks, were cut into fragments and implanted subcutaneously in 16 SCID mice (SCID-hu) at age 6 to 8 weeks. Four weeks after bone implantation, approximately 5 ×106 CD138+ plasma cells from MM patients were injected directly into the human bone of the SCID-hu mice in final volume of 30 to 40μl of phosphate-buffered saline(PBS). Human immunoglobin (hIg) levels by ELISA were used as an indicator of myeloma cell growth. When hIg level reached 10μg/mL or higher in 2 consecutive samples after 4 weeks of injection of the tumor cells, the mice were divided into four groups (n=4), Control group (not injected MM cells and no drug treatment); the other 3 groups were injected with MM cells: the isotype control group (isotype IgG 2mg/kg, i.p., twice weekly), BI-505 group (2mg/kg, i.p., twice weekly) and bortezomib group (1mg/kg, i.p., twice weekly). Bone remolding was detected by X-radiography and by measuring bone mineral density (BMD). Tumor cells were detected by immunohistochemistry using the CD138 antibody. Results:High expression of ICAM1 was observed in normal plasma cells, primary MM cells and MM cell lines. With a follow- up of 10 weeks, the tumor burden in the mice treated with BI-505 or bortezomib was significantly lower compared with the isotype control group (p: BI505 < 0.01; p: bortezomib < 0.01). Also, the number of MM tumor cells stained with the CD138 antibody was significantly less in samples treated with either BI-505 or bortezomib than in the isotype control group (p < 0.01). In addition, 6 weeks after injection of tumor cells, X-rays showed severe bone resorption in the isotype-control group, while there was no obvious bone resorption in the fetal bones after treatment with BI-505 or bortezomib. The BMD (0.0715±0.006 g/cm2) of isotype control was significantly lower than that in other 3 groups including control: 0.1278±g/cm2, BI-505 group: 0.102±0.0064 g/cm2, and bortezomib group: 0.106±0.0059g/cm2. Importantly, the number of TRAP-positive cells was significantly higher in the isotype control group than in the other 3 groups (p < 0.01). Conclusion:ICAM1 expression presents in all subtypes of myeloma. The ICAM1 antibody BI-505 significantly inhibits primary MM cell growth and bone destruction in the SCID-hu mice to the same degree as bortezomib, indicating its clinical applicability in therapy of MM. Disclosures:No relevant conflicts of interest to declare.

Mycobacterium Tuberculosis infection and Tissue Factor Expression in Macrophages

Abstract 1198Tissue factor (TF), the primary initiator of the coagulation cascade, is normally absent from cells that regularly come in contact with blood such as endothelial cells and monocytes yet could be induced in these cells in a variety of pathological conditions. The aberrant expression of TF by cells of the monocyte/macrophage lineage is thought to be a major contributor to thrombotic disorders and inflammation. Macrophages play a central role in the innate immune response essential for host defense against pathogenic infections. Tuberculosis (TB), a fatal disease caused by Mycobacterium tuberculosis (Mtb), affects nearly one third of the world’s population. A number of studies have reported the presence of thrombotic complications, particularly disseminated intravascular coagulation (DIC) in TB patients. However, it is unclear how Mtb infection causes DIC or other thrombotic disorders as mycobacteria are not known to produce endotoxins or exotoxins that otherwise initiate the clotting cascade. In the present study, we have investigated whether Mtb infection induces TF expression in macrophages and various host and pathogenic factors responsible for TF expression. We have tested the effect of live virulent Mtb H37Rv, gamma-irradiated Mtb H37Rv (γ-Mtb) and various components derived from Mtb H37Rv on TF expression in macrophages. Exposure of human monocyte-derived macrophages (MDM) to live virulent Mtb H37Rv (at 1 to 5 bacteria/macrophage) or γ-Mtb H37Rv (10 μg/ml) markedly increased TF expression in MDM (5 to 20-fold increase compared to untreated MDM). TF expression in macrophages in response to Mtb is predominantly higher in CD14hi (CD14+) macrophages. Although CD14loCD16hi (CD16+) macrophages also expressed TF in response to Mtb, the level of TF induction in these cells was much lower. γ-Mtb induced TF expression in macrophages much more robustly than E.coli-derived LPS. The γ-Mtb-mediated induction of TF expression in macrophages peaked around 9 h and was sustained throughout 48 h, which markedly varies from the kinetics of LPS-induced TF expression, which peaks between 3–6 h and then reduced down to basal level by 18–24 h. To identify the potential macrophage receptor(s) responsible for the induction of TF by Mtb, MDM were pre-treated with antibodies against CD14 and Toll-like receptors (TLR) before they were exposed to γ-Mtb. Although CD14 antibodies markedly inhibited LPS-induced TF activity, they only suppressed Mtb-induced TF activity minimally. Pretreatment of MDMs with TLR2 or TLR4 antibodies had no significant effect on γ-Mtb-induced TF expression. However, combining the CD14 antibodies together with TLR2 and TLR4 antibodies showed nearly a 50% reduction in γ-Mtb-induced TF activity. These data indicate that a cooperative action of multiple receptors and signaling pathways may be responsible for the robust and sustained induction of TF expression. In order to identify the specific component(s) of γ-Mtb that are responsible for TF induction, we have treated MDMs with various subcellular fractions or purified components derived from Mtb H37Rv. The whole cell lysate of Mtb, cell wall, cell membrane and culture filterate proteins induced TF activity to varying degrees. Of all the purified components tested, the mycobacterial cell wall core component mycolyl arabinogalactan peptidoglycan (mAGP), phosphatidylinositol mannoside-6 (PIM6) and lipomannan (LM) elicited induction of TF protein and activity in the order of mAGP > PIM6 > LM. It is interesting to note that the treatment of MDMs with individual components, mycolic acid, arabinogalactan and peptidoglycan of mAGP complex did not induce TF expression, indicating that the structure of mAGP may be required for recognition by pattern recognition receptors on macrophages. In summary, our data show that Mtb induces TF expression in macrophages, probably through a novel mechanism. Although Mtb-induced TF expression in monocytes/macrohages may lead to systemic thrombotic disorders through the release of TF containing microparticles, it may be the localized and sustained expression of TF on macrophages that internalize the infectious bacteria at the site of infection in lung is responsible for granuloma formation in tuberculosis patients that prevents the spread of the bacteria. Disclosures:No relevant conflicts of interest to declare.

Induction of Megakaryocytes From Fibroblasts by p45NF-E2/Maf

Abstract 908The determinant factors leading from stem cells to megakaryocytes (MK) and the subsequent release of platelets have yet to be identified. Here we report that a combination of transcription factors p45NF-E2, MafG, and MafK converts mouse fibroblast cell line 3T3 and adult human dermal fibroblasts into MKs. We first performed screening for MK-inducing factors using the preadipocyte cell line 3T3-L1 known to be able to differentiate into MKs in a previously defined MK lineage induction media containing thrombopoietin (Zauli G et al, Blood 1997). Gene expression levels of candidate transcription factors were compared between 3T3 cells that do not differentiate into MKs and 3T3-L1 cells using quantitative real-time PCR. The expressions of p45NF-E2 and CEBPα were undetectable in 3T3 cells, whereas expression was seen in 3T3-L1 cells. Both cell lines had similar expression levels of GATA2, RUNX1, FOG1, and PPARγ and undetectable levels of GATA1. 3T3 cells transfected with p45NF-E2, its binding protein Maf, or CEBPα, and 3T3 cells transfected with any combination of these genes, were cultured in MK lineage induction media for 8 days. MK differentiation was assessed using CD41 expression in MK-sized cells by flow cytometry analysis. Frequency of CD41 expression in the 3T3 cells varied based on transgenic factors expressed: 19 ± 12% (p45NF-E2); 25 ± 13% (p45NF-E2/MafG/MafK); 18 ± 3% (p45NF-E2/MafG/MafK/CEBPα); 13 ± 2% (p45NF-E2/CEBPα); and 4 ± 1% (CEBPα). These results suggest that p45NF-E2 with or without Maf are defined factors for reprogramming of 3T3 fibroblast to MKs. We then tested whether other adult non-hematopoietic tissues could be forced into megakaryopoiesis by ectopic expression of p45NF-E2 and Maf. Adult human dermal fibroblasts (Cell Applications) were transfected with p45NF-E2/Maf genes and cultured in MK lineage induction media for 12 days. Large-sized cells were isolated using a 2-step density BSA gradient. By flow cytometry analysis, >99% of these cells expressed CD41. DNA ploidy of the induced MKs (iMKs) ranged from 2N to 16N with a mean of 4N. We then infused 5 × 105 large-sized iMKs into NOG mouse after induction of mild thrombocytopenia in them at day 7 after irradiation with 2.0 Gy. Tail vein samples were obtained from the tested female recipient NOG mice before and after 5 min, 30 min, 90 min, and 3h post iMK infusion. Flow cytometry analysis was performed on each sample stained with PE-conjugated anti-mouse CD41 and FITC-conjugated anti-human CD41 antibody. Human CD41-positive platelet-sized cells in the sample from before and after 5 min, 30 min, 90 min, and 3h represented 0.9%, 0.5%, 2.1%, 3.3%, and 4.0%, respectively, of the entire platelet pool. To examine whether the iMK-derived platelets were incorporated into ex vivo thrombus formation, FITC-anti human CD41 antibody-labeled blood sample from iMK-infused thrombocytopenic NOG mouse was perfused on type I collagen-coated chip under flow condition (Total Thrombus-formation Analyzing System). After perfusion for 10 min, we observed that cells expressing human CD41 were incorporated into thrombi on collagen surface. These findings demonstrate that p45NF-E2 is a key determinant factor for megakaryopoiesis and thrombopoiesis, and is sufficient to lead to platelet formation from a number of non-hematopoietic adult tissues. Generation of iMKs from fibroblasts could have important implications for studying the mechanisms of MK differentiation and platelet production. Disclosures:No relevant conflicts of interest to declare.

Comparison of Immunophenotypic and Genotypic Profiles of Neoplastic Plasma Cells by Use of Flow Cytometric Immunophenotyping and FISH Analysis Following Plasma Cell Enrichment,

Abstract 3925According to The European Myeloma Network(1), FISH analysis of plasma cell myeloma (multiple myeloma, or MM) should be performed on samples enriched for plasma cells or an assay that allows for identification and study of plasma cells apart from other cells should be utilized. We enriched 400 bone marrow samples sent to our laboratory from May-September 2010 using CD138 antibody microbeads for plasma cells and then performed FISH utilizing probes for CEP3, CEP7, CEP9, CEP11, IgH breakapart, IgH/CCND1 (11;14), IgH/MAF (14;16), IgH/FGFR3(4;14), Rb1/13q, and TP53/CEP17. The genotypic profiles were then compared to the immunophenotypic profiles of monoclonal plasma cells derived from flow cytometric immunophenotyping for the following antibodies: CD38, CD19, CD56, CD117, CD20, HLA DR, CD45 and CD10.We identified genetic abnormalities in 90.6% (184/203) of the samples with monoclonal plasma cell percentages of 0.1% or more as detected by concurrent flow cytometric analysis. The most common identified genetic abnormality was aneuploidy {55.5% (222/400)}, either as a sole finding or in combination with translocations or gene loss. The aneuploid samples were further found to be hyperdipoid {23.75% (95/400)}, hypodiploid {15.75% (63/400)}, or polypoid {16.00% (64/400)}. IgH rearrangement was the seond most common abnormality and was seen in 30.2% of the 400 cases. The IgH rearrangements and frequencies were as follows: 11.25% (45/400) with t(11;14), 5.5% (22/400) with t(4;14), 3.0% (12/400) with 14;16, and 10.5% (42/100) with IgH translocation with partners unknown. Deletion of chromosome 13q/monosomy 13 was observed in 26% (104/400) of cases. This was an isolated finding in 3/400 (0.75%), observed with aneuploidy in 71/400 (17.75%), and seen in combination with IgH rearrangement in 64/400 (16.0%). There was a strong association between del (13q14) and IgH rearrangement, especially with t(4;14) and t(14;16). Loss of TP53 was observed in 3.8% (15/400) of cases. This was an isolated finding in 0/400 (0%), observed with aneuploidy in 14/400 (3.5%), and seen in combination with IgH rearrangement in 8/400 (2%).We explored the relationship between identified genetic abnormalities and respective immunophenotypic findings for the cases. Our results showed that t(4;14) was significantly associated with expression of CD56 (17/17 positive, 100%) and the absence of CD117 (76.5% negative, 13/17) and CD20 (82.4% negative, 14/17). On the other hand, t(14;16) was only rarely seen in combination with CD56 expression (88.9% negative, 8/9) or CD20 expression (100% negative, 9/9), but was highly associated with CD117 expression (66.7% positive, 6/9). t(11;14) was associated with CD20 expression (21.9% positive, 7/32), CD56 expression (34.4% positive, 11/32), and CD117 expression (37.5% positive, 12/32). Additionally, our results found that both CD56 and CD117 expression correlate with aneuploidy. Disclosures:No relevant conflicts of interest to declare.

Detection of apoptotic circulating tumor cells in advanced pancreatic cancer following 5-fluorouracil chemotherapy

The objective of the present study was to evaluate CA19-9 and CK8/18 expression patterns in pancreatic cancer cell lines induced by 5-fluorouracil (5-Fu), and in circulating tumor cells (CTCs) in peripheral blood of patients previously untreated with advanced pancreatic cancer. Furthermore, the goal was to test the relationship of dynamic of CTCs with the effects of the first cycle of chemotherapy. To accomplish this study, CD45 antibody coated beads were used to discard white blood cells in peripheral blood. This was done in combination with CA19-9-Alexa488 and CK8/18-Alexa594 immunofluorescence staining to identify CTCs in circulation from 41 advanced pancreatic cancer patients, before and after chemotherapy. The PL45 pancreatic cancer cell line was incubated with 20 µmmol/L 5-Fu for 12 and 24 hours to induce apoptosis, and the expression patterns of CA19-9 and CK8/18 were measured, and the extent of apoptosis was evaluated. Subsequently, apoptotic cells and CTCs were measured. Of 41 patients with stage III and Ⅳ pancreatic cancer, 80.5% were detected with more than two CTCs in 7.5mL peripheral blood before any therapy and the median number of CTCs was 16.8±16.0 (0-59). After 7 days by the first cycle of 5-Fu chemotherapy, only 29.3% (12/41) of these patients were detected more than two CTCs in 7.5mL peripheral blood and the median number of CTCs was 3.8±7.8 (0-40) (P=0.000). And no CTCs were detected in 20 healthy donors from 7.5mL peripheral blood (P=0.000). Apoptotic CTCs were detected after advanced pancreatic patients were administered the first cycle of chemotherapy. Punctate, granular, and bubble-like morphologies with CA19-9 and CK8/18 staining were found and may reflect apoptosis in pancreatic cancer cells and CTCs. Apoptotic CTCs may indicate the efficacy of chemotherapy in pancreatic cancer patients. However, further studies are required to follow up these findings.

The therapeutic human CD38 antibody daratumumab improves the anti-myeloma effect of newly emerging multi-drug therapies

The therapeutic human CD38 antibody daratumumab improves the anti-myeloma effect of newly emerging multi-drug therapies

Control of Megakaryocyte Expansion and Bone Marrow Fibrosis by Lysyl Oxidase

Lysyl oxidase (LOX), a matrix cross-linking protein, is known to be selectively expressed and to enhance a fibrotic phenotype. A recent study of ours showed that LOX oxidizes the PDGF receptor-β (PDGFR-β), leading to amplified downstream signaling. Here, we examined the expression and functions of LOX in megakaryocytes (MKs), the platelet precursors. Cells committed to the MK lineage undergo mitotic proliferation to yield diploid cells, followed by endomitosis and acquisition of polyploidy. Intriguingly, LOX expression is detected in diploid-tetraploid MKs, but scarce in polyploid MKs. PDGFR-BB is an inducer of mitotic proliferation in MKs. LOX inhibition with β-aminopropionitrile reduces PDGFR-BB binding to cells and downstream signaling, as well as its proliferative effect on the MK lineage. Inhibition of LOX activity has no influence on MK polyploidy. We next rationalized that, in a system with an abundance of low ploidy MKs, LOX could be highly expressed and with functional significance. Thus, we resorted to GATA-1(low) mice, where there is an increase in low ploidy MKs, augmented levels of PDGF-BB, and an extensive matrix of fibers. MKs from these mice display high expression of LOX, compared with control mice. Importantly, treatment of GATA-1(low) mice with β-aminopropionitrile significantly improves the bone marrow fibrotic phenotype, and MK number in the spleen. Thus, our in vitro and in vivo data support a novel role for LOX in regulating MK expansion by PDGF-BB and suggest LOX as a new potential therapeutic target for myelofibrosis.

Proinflammatory cytokines and CD14 expression in mammary tissue of cows following intramammary inoculation of Panax ginseng at drying off

The lack of efficacy of conventional strategies for the maintenance of healthy udders in domestic cattle has prompted studies on the use of immunomodulators or biological response modifiers (BRM) for this purpose. These compounds are agents that modify the host's response to pathogens leading to beneficial effects on disease outcome. The objective of this study was to evaluate the effects of a single intramammary infusion of Panax ginseng (GS) extract on the amount of pro-inflammatory cytokines and the number of monocytes/macrophages present in bovine mammary tissues at drying off . Eight mammary quarters from six nonpregnant cows in late lactation were infused with 10 mL of GS (3 mg/mL), six quarters were treated with 10 mL of placebo (vehicle alone) and six quarters were maintained as uninoculated controls. The analyses of tumor necrosis factor-alpha (TNF-α) by immunohistochemistry revealed that the production of this proinflammatory cytokine significantly increased ( P < 0.05) in the inoculated mammary glands of cows following BRM inoculation, whereas the interleukin-1 alpha (IL-1α) and IL-6 staining area was not affected by BRM treatment. The number of monocytes/macrophages detected with CD14 antibody was significantly higher ( P < 0.05) in BRM-treated quarters than in placebo and uninoculated control quarters. These results indicated an immunomodulator potential of the BRM used. The beneficial effect of the extract could be used as alternative therapy in the control of mastitis at drying off, either alone or in conjunction with dry cow antibiotic therapy.

Human mesenchymal stromal cells express CD14 cross‐reactive epitopes

Mesenchymal stromal cells (MSCs) do not express a unique definite epitope or marker gene. As such, minimal criteria were recently established for defining multipotent MSC. These criteria include expression of CD73, CD90, CD105, and a lack of hematopoietic marker expression. However, we detected binding of a CD14 antibody on bone marrow- and placenta-derived MSC and investigated the staining of CD14 antibodies on these MSC in more detail. The MSC were isolated from human bone marrow and placenta tissue, expanded, characterized by quantitative RT-PCR, flow cytometry, and immunocytochemistry and differentiated to generate osteoblasts, chondrocytes, and adipocytes. The CD14-cross-reactive MSCs were enriched by cell sorting. Human peripheral blood mononuclear cells, fibroblasts, and hematopoietic cell lines served as controls. Utilizing four different clones of CD14 monoclonal antibodies, we found that three CD14 reagents stained the MSC. Two CD14 antibodies (HCD14 and M5E2) clearly marked the CD90(+) MSC population with distinct intensities, clone 134 620 generated a shift in flow cytometry histograms, but clone MΦP9 did not stain MSC. Transcripts encoding CD14 or the CD14 protein were not detected in MSC. We confirm that bone marrow- and placenta-derived MSC do not express CD14 and that the CD14 antibody MΦP9 discriminates between monocytes and MSC more efficiently than the other antibodies employed here. This investigation does not contradict previous work but provides a more accurate characterization of MSC.

Active Site-labeled Prothrombin Inhibits Prothrombinase in Vitro and Thrombosis in Vivo

Mouse and human prothrombin (ProT) active site specifically labeled with D-Phe-Pro-Arg-CH(2)Cl (FPR-ProT) inhibited tissue factor-initiated thrombin generation in platelet-rich and platelet-poor mouse and human plasmas. FPR-prethrombin 1 (Pre 1), fragment 1 (F1), fragment 1.2 (F1.2), and FPR-thrombin produced no significant inhibition, demonstrating the requirement for all three ProT domains. Kinetics of inhibition of ProT activation by the inactive ProT(S195A) mutant were compatible with competitive inhibition as an alternate nonproductive substrate, although FPR-ProT deviated from this mechanism, implicating a more complex process. FPR-ProT exhibited ∼10-fold more potent anticoagulant activity compared with ProT(S195A) as a result of conformational changes in the ProT catalytic domain that induce a more proteinase-like conformation upon FPR labeling. Unlike ProT and ProT(S195A), the pathway of FPR-ProT cleavage by prothrombinase was redirected from meizothrombin toward formation of the FPR-prethrombin 2 (Pre 2)·F1.2 inhibitory intermediate. Localization of ProT labeled with Alexa Fluor® 660 tethered through FPR-CH(2)Cl ([AF660]FPR-ProT) during laser-induced thrombus formation in vivo in murine arterioles was examined in real time wide-field and confocal fluorescence microscopy. [AF660]FPR-ProT bound rapidly to the vessel wall at the site of injury, preceding platelet accumulation, and subsequently to the thrombus proximal, but not distal, to the vessel wall. [AF660]FPR-ProT inhibited thrombus growth, whereas [AF660]FPR-Pre 1, lacking the F1 membrane-binding domain did not bind or inhibit. Labeled F1.2 localized similarly to [AF660]FPR-ProT, indicating binding to phosphatidylserine-rich membranes, but did not inhibit thrombosis. The studies provide new insight into the mechanism of ProT activation in vivo and in vitro, and the properties of a unique exosite-directed prothrombinase inhibitor.

Redistribution and Hemostatic Action of Recombinant Activated Factor VII Associated with Platelets

Clinical evidence accumulated from hemophilic patients during prophylaxis with recombinant activated factor VII (rFVIIa) suggests that the duration of the hemostatic action of rFVIIa exceeds its predicted plasma half-life. Mechanisms involved in this outcome have not been elucidated. We have investigated in vitro the redistribution of rFVIIa in platelets from healthy donors, patients with FVII deficiency, and one patient with Bernard-Soulier syndrome. Platelet-rich plasma was exposed to rFVIIa (3 to 60 μg/mL). Flow cytometry, immunocytochemistry, and coagulation tests were applied to detect and quantify rFVIIa. The hemostatic effect of rFVIIa associated to platelets was evaluated using perfusion models. Our studies revealed a dose-dependent association of rFVIIa to the platelet cytoplasm with redistribution into the open canalicular system, and α granules. Mechanisms implicated in the internalization are multiple, involve GPIb and GPIV, and require phospholipids and cytoskeletal assembly. After platelet activation with thrombin, platelets exposed rFVIIa on their membrane. Perfusion studies revealed that the presence of 30% of platelets containing FVIIa improved platelet aggregate formation and enhanced fibrin generation (P < 0.01 versus control). Our results indicate that, at therapeutic concentrations, rFVIIa can be internalized into platelets, where it is protected from physiological clearance mechanisms and can still promote hemostatic activity. Redistribution of rFVIIa into platelets may explain the prolonged prophylactic effectiveness of rFVIIa in hemophilia.

Monocytes induce proximal tubular epithelial-mesenchymal transition through NF-kappa B dependent upregulation of ICAM-1

Inflammatory cell infiltration plays a key role in the pathogenesis of tubulointerstitial damage in chronic renal diseases. In addition to secreting the profibrotic cytokines, monocytes themselves have been demonstrated to be directly associated with renal fibrogenesis. However, how infiltrating monocytes interact with resident cells and the underlying mechanisms remain elusive. In this study we investigated the effects of monocytes on phenotypic changes of human proximal tubular HK-2 cells. The typical epithelial cell morphology of HK-2 cells disappeared after co-culture with monocytes, accompanied by decreased E-cadherin expression, and increased α-SMA and fibronectin expression, suggesting that HK-2 cells undergo epithelial-mesenchymal transition (EMT). Further analysis revealed that the effects were dependent on direct contact of the two types of cells as conditioned medium had no effects. Interestingly, administration of CD18 antibody directly inhibited this process. Furthermore, by microarray and RT-PCR we found that NF-kB signaling may play a role in this process and blockade of this signaling pathway in HK-2 cells could inhibit ICAM-1 expression and EMT phenotypes. Taken together, these findings suggest that monocytes infiltration could directly induce EMT of HK-2 cells via upregulation ICAM-1 through NF-kB signaling pathway.

Abstract 5225: Quantification of circulating tumor cell aggregates in breast cancer patient blood using an enrichment-free detection method

Abstract Circulating tumor cells (CTCs) have been implicated as a potential population of cells that may seed metastasis in cancer patients. In order to characterize and understand this population of cells, an enrichment-free immunofluorescence detection method was developed by our lab. This method take whole patient blood and classifies cells based on cytokeratin (CTCs) and CD45 (WBCs) antibody staining. Using this CTC detection method, a population of cells residing in tumor cell aggregates was observed in blood draws from Stage IV breast cancer patients. It was previously observed in ex vivo mouse experiments that homotypic aggregate formation of tumor cells may increase metastatic efficiency (Glinsky, et al., Cancer Research, 2003). In this work, we seek to determine if a significant proportion of CTCs reside in homotypic aggregates in patient blood by enumerating CTC aggregates from the blood draws of 23 Stage IV breast cancer patients and 16 healthy donor patients. Preliminary analysis measuring associations of CTCs with other nucleated cells found in patient blood draws revealed that ∼43% of cytokeratin-positive (CK+) cells found in breast cancer patient samples localize with either other CK+ or white blood cells (WBCs) versus 14% in healthy donor patient samples. Furthermore, ∼19% of WBCs were found to be associated with either CK+ or other WBCs in breast cancer patient samples versus ∼7% in healthy donor patient samples. Pathological review confirming CK+ cells as CTCs revealed that of the CTCs detected in patient blood after pathological review, ∼2% of CTCs were found as homotypic aggregates versus ∼0.07% in healthy donor samples (p=0.0005). Four breast cancer patients were found to have no CTC aggregates. Two breast cancer patients had significantly higher than average CTC aggregate percentages (∼6% and ∼7%). Patients will continue to be monitored to measure any differences in disease outcome of patients with high incidence of CTC aggregates versus patients with low incidence of CTC aggregates and healthy donors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5225. doi:10.1158/1538-7445.AM2011-5225