Effect of Andexanet-TFPI Interaction on in Vitro Thrombin Formation and Coagulation Markers in the TF-Pathway

Abstract Background: Tissue factor pathway inhibitor (TFPI) plays a major role in regulation of tissue factor (TF)-initiated coagulation in a FXa-dependent manner. Andexanet alfa (AnXa) is a modified, recombinant, catalytically inactive form of human FXa being developed to reverse the anticoagulant activity of FXa inhibitors in patients during episodes of major bleeding. As a modified FXa, AnXa retained high binding affinity to fXa inhibitors, including TFPI. Our previous in vitro studies demonstrated that AnXa reverses rivaroxaban-induced inhibition of thrombin generation (TG) initiated by either the extrinsic or intrinsic pathways. However, the thromboelastography (TEG) results showed that AnXa-TFPI interaction enhanced thrombin formation at low TF conditions, which suggested potential increase in the fibrin degradation product in the presence of recombinant tissue plasminogen activator (rtPA) (Lu, G., et al, ASH2016). In the current study, we further characterized the differential effect of AnXa on thrombin formation and clot lysis via the two different pathways, and correlated the TG and TEG parameters with the coagulation markers in the TF-initiated reaction in plasma, including prothrombin fragment (F1+2), thrombin-antithrombin complex (TAT), and fibrin degradation product (D-dimer). Methods: TF-initiated TG in human plasma was measured using a calibrated automated thrombogram (TF-CAT) and the PPP-reagent (5 pM TF, Diagnostica Stago). Non-TF-initiated TG was measured using CAT and an aPTT reagent, Actin FS (Siemens Healthcare) as the activator (Actin FS-CAT). Clot formation was measured in plasma using a TEG 5000 analyzer (Haemonetics). TF (1 pM) and Actin FS (1:240 final dilution) were used as the activators with or without rtPA (150 ng/mL). Time-dependent thrombin formation and clot lysis were monitored by quenching samples at various times (0, 2.5, 5, 10, 15, 30, 60 min) following initiation of the reaction by TF (1 pM) in plasma with or without rtPA (150 ng/mL) and AnXa (4.0 µM). Corn trypsin inhibitor (CTI, 50 µg/mL) was added to block contact activation. F1+2, TAT and D-dimer were quantified according to manufacturers' recommendations. Results: The effect of AnXa on TF-CAT and Actin FS-CAT were compared side-by-side with AnXa at low (0 - 20 nM) and high (0 - 4.0 µM) concentration ranges. AnXa (5 nM) increased TF-CAT parameters sensitive to TFPI activity (i.e., peak thrombin), with less effect on endogenous thrombin potential (ETP). No further increase in these parameters was observed at AnXa therapeutic concentrations (e.g., 2.0 and 4.0 µM), consistent with the expected effect of AnXa on the activity of TFPI, which is present at low plasma concentration (~2.4 nM). In contrast, AnXa did not significantly affect the Actin FS-CAT parameters. The effect of AnXa-TFPI interaction on coagulation and fibrinolytic pathways in human plasma were compared using TEG, with or without rtPA. AnXa (4.0 µM) reduced the TF-TEG-R parameter (lag time, equivalent to clotting time) and resulted in an increased area under the fibrinolytic profile with rtPA as previously shown. In contrast, AnXa and the buffer controls had similar profiles in the Actin FS-TEG assay under similar conditions, with or without rtPA (150 ng/mL) in the current studies, suggesting lack of an AnXa-TFPI effect. We further investigated the effect of AnXa-TFPI interaction on coagulation markers following TF (1pM)-initiated thrombin formation in plasma. AnXa (4 µM) increased F1+2 and TAT levels at early time points following initiation (2.5, 5, 10, 15 min), but had no difference at or after 30 min, compared to buffer control. A similar pattern was observed for D-dimer level when exogenous rtPA (150 ng/mL) was added. Compared to buffer control, AnXa caused elevation of D-dimer level at early time points but had no difference at or after 30 min following initiation of the reaction. Conclusions: AnXa-TFPI interaction can modulate the TF/TFPI activity in the initiation phase of the extrinsic coagulation pathway and increase parameters sensitive to TFPI activity in the TF-CAT and TF-TEG assays, but has minimal effect on the later phase of the reactions and the overall thrombin formation potential. Consistent with clinical observations, AnXa-TFPI interaction increased the F1+2 and TAT levels, as well as D-dimer level in the presence of rtPA in the early phase of the reaction following initiation of the reaction by TF. Disclosures Lu: Portola Pharmaceuticals, Inc.: Employment. Lin: Portola Pharmaceuticals, Inc.: Employment. Curnutte: 3-V Biosciences: Equity Ownership; Sea Lane Biotechnologies: Consultancy; Portola Pharmaceuticals, Inc.: Employment, Equity Ownership, Patents & Royalties. Conley: Portola Pharmaceuticals, Inc.: Employment, Equity Ownership, Patents & Royalties.

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Therapeutic Rationale and Clinical Development of TNT009, an Upstream Classical Pathway Inhibitor, for Cold Agglutinin Disease

Blood ◽

10.1182/blood.v126.23.3560.3560 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3560-3560 ◽

Cited By ~ 1

Author(s):

Ulrich Jaeger ◽

Eileen L Rose ◽

Andrew Singh ◽

Sebastian H A Feickert ◽

Simon Panzer ◽

...

Keyword(s):

Complement Activation ◽

Research Funding ◽

Cold Agglutinin ◽

Serum Samples ◽

Employment Equity ◽

Equity Ownership ◽

Human Rbcs ◽

D Dimer

Abstract Cold agglutinin disease (CAD) is an autoimmune hemolytic anemia (AIHA) characterized by the presence of autoantibodies (cold agglutinins) that bind red blood cells (RBCs) and activate the classical complement pathway (CP). We have previously shown in vitro that in contrast to C5 inhibition, inhibition of the CP specific protease C1s prevents complement opsonin deposition on cold agglutinin-sensitized RBCs and protects them from phagocytosis, underscoring the necessity to block upstream CP activity (Shi et al., Blood, 2014). Based on the strong scientific rationale and nonclinical data, a Phase 1 clinical trial for TNT009, a monoclonal antibody (mAb) inhibitor of C1s, has commenced at the Medical University of Vienna, Austria. Phase 1a consists of healthy volunteer cohorts in single- and multiple-ascending dose protocols for which interim study results will be presented. In the integrated protocol design of Phase 1b, TNT009 will be dosed in patients with diseases in which pathological CP activity has been implicated, including CAD, warm AIHA and additional non-hematologic indications. In anticipation of the clinical trial, we initiated a screening campaign in Vienna to find prospective CAD patients with serological markers of anemia and hemolysis. To date, plasma and serum samples have been collected from 15 CAD patients. Serum samples from 10 patients induce robust complement activation (C3b/iC3b deposition and/or hemolysis) on AET-treated human RBCs incubated in the patient's own complement-containing serum. In contrast to isotype control (IC), 100 mcg/mL of TNT003 (mouse parental mAb of TNT009), showed near complete inhibition of patient serum mediated C3b/iC3b deposition (90 ± 4%, n = 10; p< 1 x 10-5) and hemolysis (93 ± 5 %, n = 9; p< 1 x 10-5) (Fig. 1). To further support the rationale of C1s inhibition in CAD, we asked whether serological signs of anemia and hemolysis were associated with evidence of increased in vivo CP activity in patient samples. We first examined how well experimental laboratory results agreed with standard clinical readouts. We found good concordance between patient sample induced C3 deposition on RBCs (FACS) and clinical C3 DAT scores (p< .05). Furthermore, IgM staining on RBCs incubated in patient samples (FACS) correlated well with cold agglutinin titers determined in the clinic (p < .001). Next, we observed that the extent of in vitro hemolysis correlated with C3d DAT scores (p< .05), LDH levels (p< .05), and bilirubin levels (p= .05). The agreement between the results from our in vitro patient sample-induced hemolysis assay with serologicalmarkers of complement activity, hemolysis and anemia used in the clinic suggest that our in vitro paradigm serves as a good model for in vivo complement activity in CAD patients. We then measured plasma C4 levels and CP activity in CAD serum samples (Wieslab Classical Pathway ELISA). We found that plasma C4 positively correlated with hemoglobin levels (p = .05). Additionally, we found an inverse correlation between serum CP activity and reticulocyte count (p < .05) and bilirubin levels (p = .05). These data demonstrate that in vivo consumption of the CP and its components (low CP activity, low C4) is associated with markers of anemia and hemolysis (low hemoglobin, high reticulocyte counts, high bilirubin). Finally, an emerging literature calls attention to an increased thromboembolic risk in AIHA, similar to that seen in patients with other hemolytic anemias such as paroxysmal nocturnal hemoglobinuria. We therefore measured D-dimer levels and found it significantly elevated in CAD patient plasma compared to healthy controls (p < .0001). Preliminary analyses show an inverse correlation of C4 and D-dimer in patient plasma (p < .05) suggesting that in vivo CP activity may contribute to the elevated thromboembolic risk in patients (Fig. 2). On-going analyses for other markers of thrombosis, in addition to other experimental approaches to assess the hypercoagulable state in these patients will seek to corroborate this finding. The successful identification of CAD patients with altered complement and hematological profiles provides a unique opportunity to assess proof-of-concept early in the clinical development of TNT009. Figure 1. TNT009 Parental mAb (TNT003) Inhibits CAD Serum Mediated Complement Activation on AET-Treated Human RBCs Figure 1. TNT009 Parental mAb (TNT003) Inhibits CAD Serum Mediated Complement Activation on AET-Treated Human RBCs Figure 2. Elevated D-dimer Correlates with Lower C4 Levels in CAD Patient Plasma Figure 2. Elevated D-dimer Correlates with Lower C4 Levels in CAD Patient Plasma Disclosures Jaeger: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; True North Therapeutics, Inc.: Research Funding; Hoffmann La Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Rose:True North Therapeutics, Inc.: Employment, Equity Ownership. Singh:True North Therapeutics, Inc.: Employment, Equity Ownership. Jilma:True North Therapeutics, Inc.: Consultancy, Research Funding. Gilbert:True North Therapeutics, Inc.: Employment, Equity Ownership. Panicker:True North Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties.

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Phenotype Does Not Always Equal Function: HDAC Inhibitors and UM171, but Not SR1, Lead to Rapid Upregulation of CD90 on Non-Engrafting CD34+CD90-Negative Human Cells

Blood ◽

10.1182/blood.v130.suppl_1.659.659 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 659-659

Author(s):

Kevin A. Goncalves ◽

Megan D. Hoban ◽

Jennifer L. Proctor ◽

Hillary L. Adams ◽

Sharon L. Hyzy ◽

...

Keyword(s):

In Vitro Culture ◽

Small Molecule ◽

Hdac Inhibitors ◽

Employment Equity ◽

Nsg Mice ◽

Cd34 Cells ◽

Equity Ownership ◽

Human Hscs

Abstract Background. The ability to expand human hematopoietic stem cells (HSCs) has the potential to improve outcomes in HSC transplantation and increase the dose of gene-modified HSCs. While many approaches have been reported to expand HSCs, a direct comparison of the various methods to expand transplantable HSCs has not been published and clinical outcome data for the various methods is incomplete. In the present study, we compared several small molecule approaches reported to expand human HSCs including HDAC inhibitors, the aryl hydrocarbon antagonist, SR1, and UM171, a small molecule with unknown mechanism, for the ability to expand phenotypic HSC during in vitro culture and to expand cells that engraft NSG mice. Although all strategies increased the number of phenotypic HSC (CD34+CD90+CD45RA-) in vitro, SR1 was the most effective method to increase the number of NOD-SCID engrafting cells. Importantly, we found that HDAC inhibitors and UM171 upregulated phenotypic stem cell markers on downstream progenitors, suggesting that these compounds do not expand true HSCs. Methods. Small-molecules, SR1, HDAC inhibitors (BG45, CAY10398, CAY10433, CAY10603, Entinostat, HC Toxin, LMK235, PCI-34051, Pyroxamide, Romidepsin, SAHA, Scriptaid, TMP269, Trichostatin A, or Valproic Acid) and UM171 were titrated and then evaluated at their optimal concentrations in the presence of cytokines (TPO, SCF, FLT3L, and IL6) for the ability to expand human mobilized peripheral blood (mPB)-derived CD34+ cells ex vivo . Immunophenotype and cell numbers were assessed by flow cytometry following a 7-day expansion assay in 10-point dose-response (10 µM to 0.5 nM). HSC function was evaluated by enumeration of colony forming units in methylcellulose and a subset of the compounds were evaluated by transplanting expanded cells into sub-lethally irradiated NSG mice to assess engraftment potential in vivo . All cells expanded with compounds were compared to uncultured or vehicle-cultured cells. Results. Following 7 days of expansion, SR1 (5-fold), UM171 (4-fold), or HDAC inhibitors (>3-35-fold) resulted in an increase in CD34+CD90+CD45RA- number relative to cells cultured with cytokines alone; however, only SR1 (18-fold) and UM171 (8-fold) demonstrated enhanced engraftment in NSG mice. Interestingly, while HDAC inhibitors and UM171 gave the most robust increase in the number and frequency of CD34+CD90+CD45RA- cells during in vitro culture, these methods were inferior to SR1 at increasing NSG engrafting cells. The increase in CD34+CD90+CD45RA- cells observed during in vitro culture suggested that these compounds may be generating a false phenotype by upregulating CD90 and down-regulating CD45RA on progenitors that were originally CD34+CD90-CD45RA+. We tested this hypothesis by sorting CD34+CD90-CD45RA+ cells and culturing these with the various compounds. These experiments confirmed that both HDAC inhibitors (33-100 fold) and UM171 (28-fold) led to upregulation of CD90 on CD34+CD90-CD45RA+ cells after 4 days in culture. Since approximately 90% of the starting CD34+ cells were CD90-, these data suggest that most of the CD34+CD90+CD45RA- cells in cultures with HDAC inhibitors and UM171 arise from upregulation of CD90 rather than expansion of true CD34+CD90+CD45RA- cells and may explain the disconnect between in vitro HSC phenotype and NSG engraftment in vivo . This was further confirmed by evaluation of colony forming unit frequency of CD34+CD90-CD45RA+ cells after culture with compounds. Conclusions. We have showed that AHR antagonism is optimal for expanding functional human HSCs using the NSG engraftment model. We also demonstrated that UM171 and HDAC inhibitors upregulate phenotypic HSC markers on downstream progenitors. This could explain the discrepancy between impressive in vitro phenotypic expansion and insufficient functional activity in the NSG mouse model. Therefore, these data suggest caution when interpreting in vitro expansion phenotypes without confirmatory functional transplantation data, especially as these approaches move into clinical trials in patients. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership. Hoban: Magenta Therapeutics: Employment, Equity Ownership. Proctor: Magenta Therapeutics: Employment, Equity Ownership. Adams: Magenta Therapeutics: Employment, Equity Ownership. Hyzy: Magenta Therapeutics: Employment, Equity Ownership. Boitano: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

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Highly Differentiated Anti-CD47 Antibody, AO-176, Potently Inhibits Hematologic Malignancies Alone and in Combination

Blood ◽

10.1182/blood-2019-126298 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1844-1844

Author(s):

John Richards ◽

Myriam N Bouchlaka ◽

Robyn J Puro ◽

Ben J Capoccia ◽

Ronald R Hiebsch ◽

...

Keyword(s):

Multiple Myeloma ◽

Combination Therapy ◽

Tumor Growth ◽

Growth Inhibition ◽

Tumor Cells ◽

Tumor Growth Inhibition ◽

Employment Equity ◽

Equity Ownership

AO-176 is a highly differentiated, humanized anti-CD47 IgG2 antibody that is unique among agents in this class of checkpoint inhibitors. AO-176 works by blocking the "don't eat me" signal, the standard mechanism of anti-CD47 antibodies, but also by directly killing tumor cells. Importantly, AO-176 binds preferentially to tumor cells, compared to normal cells, and binds even more potently to tumors in their acidic microenvironment (low pH). Hematological neoplasms are the fourth most frequently diagnosed cancers in both men and women and account for approximately 10% of all cancers. Here we describe AO-176, a highly differentiated anti-CD47 antibody that potently targets hematologic cancers in vitro and in vivo. As a single agent, AO-176 not only promotes phagocytosis (15-45%, EC50 = 0.33-4.1 µg/ml) of hematologic tumor cell lines (acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, and T cell leukemia) but also directly targets and kills tumor cells (18-46% Annexin V positivity, EC50 = 0.63-10 µg/ml) in a non-ADCC manner. In combination with agents targeting CD20 (rituximab) or CD38 (daratumumab), AO-176 mediates enhanced phagocytosis of lymphoma and multiple myeloma cell lines, respectively. In vivo, AO-176 mediates potent monotherapy tumor growth inhibition of hematologic tumors including Raji B cell lymphoma and RPMI-8226 multiple myeloma xenograft models in a dose-dependent manner. Concomitant with tumor growth inhibition, immune cell infiltrates were observed with elevated numbers of macrophage and dendritic cells, along with increased pro-inflammatory cytokine levels in AO-176 treated animals. When combined with bortezomib, AO-176 was able to elicit complete tumor regression (100% CR in 10/10 animals treated with either 10 or 25 mg/kg AO-176 + 1 mg/kg bortezomib) with no detectable tumor out to 100 days at study termination. Overall survival was also greatly improved following combination therapy compared to animals treated with bortezomib or AO-176 alone. These data show that AO-176 exhibits promising monotherapy and combination therapy activity, both in vitro and in vivo, against hematologic cancers. These findings also add to the previously reported anti-tumor efficacy exhibited by AO-176 in solid tumor xenografts representing ovarian, gastric and breast cancer. With AO-176's highly differentiated MOA and binding characteristics, it may have the potential to improve upon the safety and efficacy profiles relative to other agents in this class. AO-176 is currently being evaluated in a Phase 1 clinical trial (NCT03834948) for the treatment of patients with select solid tumors. Disclosures Richards: Arch Oncology Inc.: Employment, Equity Ownership, Other: Salary. Bouchlaka:Arch Oncology Inc.: Consultancy, Equity Ownership. Puro:Arch Oncology Inc.: Employment, Equity Ownership. Capoccia:Arch Oncology Inc.: Employment, Equity Ownership. Hiebsch:Arch Oncology Inc.: Employment, Equity Ownership. Donio:Arch Oncology Inc.: Employment, Equity Ownership. Wilson:Arch Oncology Inc.: Employment, Equity Ownership. Chakraborty:Arch Oncology Inc.: Employment, Equity Ownership. Sung:Arch Oncology Inc.: Employment, Equity Ownership. Pereira:Arch Oncology Inc.: Employment, Equity Ownership.

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Clinical Pharmacodynamic Markers and Combinations with SY1425 (tamibarotene) in a Genomically-Defined Subset of Non-APL AML

Blood ◽

10.1182/blood.v128.22.2898.2898 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2898-2898

Author(s):

Michael R McKeown ◽

Christopher Fiore ◽

Emily Lee ◽

Matthew L Eaton ◽

Christian C. Fritz

Keyword(s):

Transcription Factor ◽

Cell Lines ◽

Binding Sites ◽

Clinical Studies ◽

High Cell ◽

Preclinical Models ◽

Employment Equity ◽

Equity Ownership ◽

Maximal Induction

Abstract SY-1425, a potent and selective agonist of the retinoic acid receptor RARα, is being investigated in a Ph2 trial in a novel genomically-defined subset of non-APL AML and MDS patients (clinicaltrials.gov NCT02807558). RARa is a nuclear hormone receptor and transcription factor that regulates genes involved in cell differentiation and proliferation. We identified a super-enhancer (SE) at the RARA locus, the gene encoding RARa, in a subset of primary non-APL AML blasts. Preclinical models demonstrated a correlation between the presence of a RARA SE and sensitivity to SY-1425, providing the rationale for clinical investigation. Further research has investigated pharmacodynamics (PD) markers and combinations of drugs to support clinical development of SY-1425. In this study we identified DHRS3mRNA induction as a measure of RARα target engagement with SY-1425. We also demonstrated synergy in preclinical models with SY-1425 and hypomethylating agents. Since RARα is a transcription factor that regulates target genes when bound by a retinoid, we characterized the dynamic expression changes of a panel of RARA enhancer- high and - low non-APL AML cell lines (hereafter referred to as RARA-high and -low) in response to SY-1425 treatment. DHRS3 showed the largest expression increase following treatment in 3 RARA-high cell lines, with a range of 29 to 115 fold. In contrast, there was a much lower DHRS3 induction in 3 RARA-low cell lines (range of 1.6 to 6.1 fold). Induction was found to be both time- and dose-dependent with maximal induction at approximately 6 hours and half maximal induction near the EC50 for the anti-proliferative effect in RARA-high cell lines. DHRS3 encodes dehydrogenase/reductase (SDR family) member 3, a metabolic enzyme involved in maintaining cellular retinol homeostasis and had previously been shown to be induced by retinoids. Thus, DHRS3induction in tumor cells represents a potentially useful PD marker for clinical studies of SY-1425. To better understand the mechanism of induction of DHRS3 by SY-1425 we examined the chromosomal localization of RARα as well as the epigenomic state of the DHRS3 locus by ChIP-seq for RARα and H3K27 acetylation, the latter being an indicator of active enhancers and promoters. In the untreated state, OCI-AML3 (a typical RARA-high AML cell line) was found to have multiple RARα binding sites both within and distal to the DHRS3 gene but minimal H3K27 acetylation. Following treatment with SY-1425, the level of H3K27 acetylation at DHRS3 increased, resulting in the formation of a SE. Moreover, the SE encompassed the RARα binding sites, consistent with the model in which SY-1425 converts RARα into an activator of DHRS3expression. Similar results were seen for the CD38 locus in which SY-1425 treatment increased expression, H3K27 acetylation, and RARα binding. CD38 is a cell surface antigen and marker of myeloid maturation readily analyzed by FACS analysis, suggesting it could be an additional PD marker to be used in clinical studies. Indeed, it was found that SY-1425 induced CD38 cell surface expression at similar levels in RARA-high AML cell lines and the NB-4 APL cell line, but not in RARA-low cell lines. We also investigated combinations of SY-1425 with approved or investigational AML and MDS agents in in vitro and in vivo models to inform future clinical studies and to further explore potential PD markers unique to the combined action of the drugs. Several standard of care agents and drugs in current development were found to have synergistic interactions with SY-1425 in RARA-high but not RARA-low cell lines. In particular, azacitidine and decitabine each showed strong in vitro synergy with SY-1425. Evaluation of SY-1425 plus azacitidine in a RARA-high PDX model of non-APL AML demonstrated a better response compared to either agent alone. Additional genome-wide ChIP-seq and expression studies of RARA-high cells treated with various combinations are being investigated to identify optimal PD markers for these combinations. These studies support the use of DHRS3 mRNA induction in tumor cells as a PD marker in the recently initiated Ph2 study of SY-1425 in genomically-defined non-APL AML and MDS patients (clinicaltrials.gov NCT02807558) and further exploration as a PD marker for future combination studies. Disclosures McKeown: Syros Pharmaceuticals: Employment, Equity Ownership. Fiore:Syros Pharmaceuticals: Employment, Equity Ownership. Lee:Syros Pharmaceuticals: Employment, Equity Ownership. Eaton:Syros Pharmaceuticals: Employment, Equity Ownership. Fritz:Syros Pharmaceuticals: Employment, Equity Ownership.

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F-actin serves as a template for cytokeratin organization in cell free extracts

Journal of Cell Science ◽

10.1242/jcs.115.7.1373 ◽

2002 ◽

Vol 115 (7) ◽

pp. 1373-1382 ◽

Cited By ~ 2

Author(s):

Kari L. Weber ◽

William M. Bement

Keyword(s):

Time Course ◽

Early Time ◽

Dynamic Imaging ◽

Actin Network ◽

Intact Cells ◽

Time Points ◽

Egg Extracts ◽

Actin Cables

The microtubule, F-actin, and intermediate filament systems are often studied as isolated systems, yet the three display mutual interdependence in living cells. To overcome limitations inherent in analysis of polymer-polymer interactions in intact cells, associations between these systems were assessed in Xenopus egg extracts. In both fixed and unfixed extract preparations, cytokeratin associated with F-actin cables that spontaneously assembled in the extracts. Time-course experiments revealed that at early time points cytokeratin cables were invariably associated with F-actin cables,while at later time points they could be found without associated F-actin. In extract samples where F-actin assembly was prevented, cytokeratin formed unorganized aggregates rather than cables. Dynamic imaging revealed transport of cytokeratin by moving F-actin as well as examples of cytokeratin release from F-actin. Experimental alteration of F-actin network organization by addition of α-actinin resulted in a corresponding change in the organization of the cytokeratin network. Finally, pharmacological disruption of the F-actin network in intact, activated eggs disrupted the normal pattern of cytokeratin assembly. These results provide direct evidence for an association between F-actin and cytokeratin in vitro and in vivo, and indicate that this interaction is necessary for proper cytokeratin assembly after transition into the first mitotic interphase of Xenopus.

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An in vitro transcription analysis of early responses of the human immunodeficiency virus type 1 long terminal repeat to different transcriptional activators

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.1883-1893.1991 ◽

1991 ◽

Vol 11 (4) ◽

pp. 1883-1893

Author(s):

Y C Li ◽

J Ross ◽

J A Scheppler ◽

B R Franza

Keyword(s):

Human Immunodeficiency Virus ◽

Protein Synthesis ◽

Early Time ◽

In Vitro Transcription ◽

Jurkat Cells ◽

Time Points ◽

Immunodeficiency Virus ◽

Hiv 1

In this report we introduce a simple, fast, and reliable method to prepare whole cell or nuclear extracts from small numbers of cells. These extracts were used to study transcriptional activation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in vitro. Our results revealed that the time courses of activation of extracts derived from cells stimulated with the mitogenic lectin phytohemagglutinin (PHA) or with the tumor promoter phorbol 12-myristate 13-acetate (PMA) are different. PMA induces a rapid onset of increased in vitro transcription from the HIV-1 LTR, while PHA causes a slow and sustained response. The biochemical relevance of protein synthesis inhibition by cycloheximide treatment of cells was investigated. In these studies, PMA induction of a change in in vitro transcriptional activity is not dependent on protein synthesis. Cycloheximide alone is insufficient to induce activation. Oligonucleotide-mediated site-directed mutagenesis demonstrated that mutation of the TATA box in the LTR ablated initiation of both basal-level transcription and activation by extracts from cells stimulated with PMA. Surprisingly, mutation of both kappa B sites in the LTR reduced but did not eliminate the in vitro response to extracts prepared at early time points after PHA or PMA stimulation of Jurkat cells. The reduction was greater in extracts derived from cells treated with PMA. Deletion analysis of the HIV-1 LTR revealed at least one region (-464 to -252) capable of suppressing in vitro transcription in extracts from Jurkat cells stimulated by PMA. This result is consistent with early studies of the HIV-1 LTR in transient transfection assays. We therefore have been able to observe distinct regulatory events at early time points after cells are exposed to agents known to induce transcription of both the HIV-1 LTR reporter gene constructs and the HIV-1 provirus itself.

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Developmental Pathways Pervade Stem Cell Responses to Evolving Extracellular Matrices of 3D Bioprinted Microenvironments

Stem Cells International ◽

10.1155/2018/4809673 ◽

2018 ◽

Vol 2018 ◽

pp. 1-15

Author(s):

Quyen A. Tran ◽

Visar Ajeti ◽

Brian T. Freeman ◽

Paul J. Campagnola ◽

Brenda M. Ogle

Keyword(s):

Stem Cell ◽

Cell Differentiation ◽

Human Mesenchymal Stem Cells ◽

Early Time ◽

Stem Cell Differentiation ◽

Model Systems ◽

Ecm Remodeling ◽

Time Points ◽

3D Microenvironment

Developmental studies and 3D in vitro model systems show that the production and engagement of extracellular matrix (ECM) often precede stem cell differentiation. Yet, unclear is how the ECM triggers signaling events in sequence to accommodate multistep process characteristic of differentiation. Here, we employ transcriptome profiling and advanced imaging to delineate the specificity of ECM engagement to particular differentiation pathways and to determine whether specificity in this context is a function of long-term ECM remodeling. To this end, human mesenchymal stem cells (hMSCs) were cultured in 3D bioprinted prisms created from ECM proteins and associated controls. We found that exogenous ECM provided in 3D microenvironments at early time points impacts on the composition of microenvironments at later time points and that each evolving 3D microenvironment is uniquely poised to promote stem cell differentiation. Moreover, 2D cultures undergo minimal ECM remodeling and are ill-equipped to stimulate pathways associated with development.

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The role of microRNA-3085 in chondrocyte function

Scientific Reports ◽

10.1038/s41598-020-78606-6 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Linh Le ◽

Lingzi Niu ◽

Matthew J. Barter ◽

David A. Young ◽

Tamas Dalmay ◽

...

Keyword(s):

Feedback Inhibition ◽

Interleukin 1 ◽

Early Time ◽

Null Cell ◽

Time Points ◽

Vitro Analysis ◽

In Vitro Analysis ◽

Cultured Chondrocytes

AbstractMicroRNAs have been shown to play a role in cartilage development, homeostasis and breakdown during osteoarthritis. We previously identified miR-3085 in humans as a chondrocyte-selective microRNA, however it could not be detected by Northern blot. The aim of the current study was to prove that miR-3085 is a microRNA and to investigate the function of miR-3085 in signaling pathways relevant to cartilage homeostasis and osteoarthritis. Here, we confirm that miR-3085 is a microRNA and not another class of small RNA using (1) a pre-miR hairpin maturation assay, (2) expression levels in a Dicer null cell line, and (3) Ago2 pulldown. MicroRNA-3085-3p is expressed more highly in micromass than monolayer cultured chondrocytes. Transfection of miR-3085-3p into chondrocytes decreases expression of COL2A1 and ACAN, both of which are validated as direct targets of miR-3085-3p. Interleukin-1 induces the expression of miR-3085-3p, at least in part via NFκB. In a feed-forward mechanism, miR-3085-3p then potentiates NFκB signaling. However, at early time points after transfection, its action appears to be inhibitory. MyD88 has been shown to be a direct target of miR-3085-3p and may be responsible for the early inhibition of NFκB signaling. However, at later time points, MyD88 knockdown remains inhibitory and so other functions of miR-3085-3p are clearly dominant. TGFβ1 also induces the expression of miR-3085-3p, but in this instance, it exerts a feedback inhibition on signaling with SMAD3 and SMAD4 shown to be direct targets. This in vitro analysis shows that miR-3085-3p functions in chondrocytes to induce IL-1-signaling, reduce TGFβ1 signaling, and inhibit expression of matrix genes. These data suggest that miR-3085-3p has a role in chondrocyte function and could contribute to the process of osteoarthritis.

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Fibrin fragment D-dimer and fibrinogen B beta peptides in plasma as markers of clot lysis during thrombolytic therapy in acute myocardial infarction

Blood ◽

10.1182/blood.v76.7.1341.1341 ◽

1990 ◽

Vol 76 (7) ◽

pp. 1341-1348 ◽

Cited By ~ 29

Author(s):

CM Lawler ◽

EG Bovill ◽

DC Stump ◽

DJ Collen ◽

KG Mann ◽

...

Keyword(s):

Myocardial Infarction ◽

Degradation Product ◽

Degradation Products ◽

Tissue Type ◽

Clot Lysis ◽

Fibrin Degradation Product ◽

Fibrin Degradation Products ◽

Group 2 ◽

D Dimer ◽

Group 1

Abstract The validity of markers in plasma of in vitro thrombolysis was investigated in 12 patients with extensive fibrinogen breakdown (greater than 80%, group 1) and in 12 patients with minimal breakdown (less than 20%, group 2). The patients were treated with 100 mg of recombinant tissue-type plasminogen activator (rt-PA) in the “Thrombolysis in Myocardial Infarction II” (TIMI II) trial. Cross- linked fibrin degradation product levels were measured with two variant enzyme-linked immunosorbent assays (ELISAs), both using a fibrin fragment D-dimer specific capture antibody. In one instance, a tag antibody was used that cross-reacts with fibrinogen (pan-specific tag ELISA); in the other, the tag antibody was specific for fibrin fragment D (fibrin-specific tag ELISA). Apparent concentrations of cross-linked fibrin degradation products at baseline were within normal limits with both assays in most patients. At 8 hours after rt-PA infusion, the measured cross-linked fibrin degradation products were increased about twofold to fourfold in group 2 with both assays. However, in group 1, levels were significantly higher with the pan-specific tag ELISA (5.8 +/- 4.2 micrograms/mL) compared with the fibrin-specific tag ELISA (1.5 +/- 1.3 micrograms/mL). This observation was most likely a result of detection of fibrinogen degradation products in the pan-specific ELISA. Apparent levels of fibrinopeptide B beta 1–42, a marker of fragment X formation, increased during thrombolysis from 4.2 +/- 2.8 pmol/mL to 2,000 +/- 230 pmol/mL in group 1 and from 4.1 +/- 2.1 pmol/mL to 300 +/- 43 pmol/mL in group 2, and were correlated significantly with the extent of fibrinogen breakdown (r = -0.8). Fibrinopeptide beta 15–42 levels increased from 4.3 +/- 3 pmol/mL to 70 +/- 19 pmol/mL in group 1, but did not increase in group 2. The apparent increase in group 1 could be explained by cross-reactivity of fibrinopeptide B beta 1–42 in the fibrinopeptide beta 15–42 assay. We conclude that cross-linked fibrin degradation product levels as measured with a pan-specific tag ELISA and fibrinopeptide beta 15–42 levels as measured with certain monoclonal antibody-based ELISA are influenced by the extent of fibrinogen degradation. Fibrinopeptide B beta 1–42 is a marker specific for fibrinogen breakdown. Cross-linked fibrin degradation product levels, measured with a fibrin-specific tag ELISA, appear to be markers specific for thrombolysis. Consequently, assays similar to the fibrin- specific tag ELISA may provide more accurate information when correlated with clinical endpoints.

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Single Cell Profiling of B Cell Receptor Signaling and Apoptosis Networks in Chronic Lymphocytic Leukemia: Association with in Vitro Sensitivity to Fludarabine Monophosphate (F-ara-A).

Blood ◽

10.1182/blood.v114.22.1263.1263 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1263-1263

Author(s):

Erik Evensen ◽

Adam Palazzo ◽

Ying-Wen Huang ◽

Alessandra Cesano ◽

Laura Z. Rassenti ◽

...

Keyword(s):

Hydrogen Peroxide ◽

B Cell ◽

Single Cell ◽

Phosphatase Activity ◽

Cell Receptor ◽

B Cell Receptor ◽

Employment Equity ◽

Bcr Signaling ◽

Equity Ownership

Abstract Abstract 1263 Poster Board I-285 Background In conjunction with antigen-driven responses, ligand-independent signaling (termed tonic signaling) through both the pre-B cell receptor and B-cell receptor has an important role in B cell development, maturation and survival. In addition to the recognized role of CD79 alpha and CD79 beta BCR signaling, tyrosine phosphatases can impact tonic BCR signaling (Wienands et al. PNAS, 93 p.7865 (1996), Monroe Nat. Rev. Immunol. 6 p.283 (2006)). We previously subjected chronic lymphocytic leukemia (CLL) cells with modulators of BCR signaling and monitored their responses using flow cytometry-based Single Cell Network Profiling (SCNP). Of the many signaling modulators studied, hydrogen peroxide treatment (a general inhibitor of tyrosine phosphatase activity) augmented BCR signaling in a subset of CLL patient samples evaluated. In the remaining samples there was an apparent lack of response to hydrogen peroxide. These data suggested that differential phosphatase activity proximal to BCR signaling was driving the biology of these two patient groups. Objectives Studies were designed to evaluate whether there were any associations between tonic and/or ligand-dependent BCR signaling and in vitro sensitivity to fludarabine, as well as whether such response profiles showed a relationship to the hydrogen peroxide-dependent signaling we observed previously. Methods 23 CLL samples and 7 healthy PBMCs were treated with anti-m alone, hydrogen peroxide alone or the combination for 10 minutes. Separate aliquots of the same sample were exposed to F-ara-A for 48 hours. SCNP was carried out on gated B cells with quantitation of single cell measures of intracellular phosphorylated kinases and adaptor proteins downstream of the BCR. Additionally, the relative activation status of several protein markers of the apoptotic cascade (cytoplasmic cytochrome C, cleaved caspase 3, and cleaved PARP) was measured. Results As previously observed, CLL samples could be segregated into one of two groups exhibiting either responsive or refractory signaling after exposure to hydrogen peroxide alone. Moreover, responsive signaling in CLL cells was correlated in that all the measured components of the canonical B cell receptor network (p-Lyn, p-Syk, p-BLNK, p-PLC-gamma-2, p-Erk and p-Akt) showed the same phosphorylation response: either augmented in unison, or not activated at all. In vitro F-ara-A treatment (48 hours in the presence of 1mM F-ara-A) of parallel samples from these same CLL patients identified distinct populations of apoptosis responsive and refractory cells. Surprisingly, the capacity of patient samples to show augmented BCR signaling in response to hydrogen peroxide was associated prominently with the ability of cells in these patients to exhibit apoptotic proficiency to F-ara-A in vitro. This implies a link between mechanisms governing apoptosis in these CLL cells, survival pathways, and cell states that govern the role of phosphatase activity and BCR signaling potential. Conclusions This study reveals a link between tonic BCR signaling and regulation of apoptosis pathways. This suggests that the subgroup of CLL patients with active phosphatase activity (which suppresses BCR responses) have cell populations that are responsive to F-ara-A, a standard drug in CLL therapy. Conversely, the presence of CLL cells in a patient sample that remain unresponsive to hydrogen peroxide repression of phosphatase activity appear to identify patient samples which cannot undergo apoptosis in response to in vitro F-Ara-A exposure. The clinical implications of this work will be the focus of future translational studies. Disclosures Evensen: Nodality Inc.: Employment, Equity Ownership. Palazzo:Nodality Inc.: Employment, Equity Ownership. Huang:Nodality Inc.: Employment, Equity Ownership. Cesano:Nodality Inc.: Employment, Equity Ownership. Fantl:Nodality, Inc.: Employment, Equity Ownership.

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