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Objective Vedolizumab has been shown to reduce the antibody response to an oral cholera, but not intramuscular hepatitis B vaccine in healthy subjects (Wyant, Gut 2015). The generation of an immune response to an oral antigen (Ag) begins at inductive sites such as Peyer’s patches (PP) followed by cell migration to effector sites in the gut. The integrin α4β7 is known to play a major role in cell trafficking to gut-associated lymphoid tissues. This study assessed whether MT-102, a potent and selective small molecule α4β7 inhibitor has similar effects on immunizations to those reported in the clinic for vedolizumab. Methods An in vivo immunization model system was established to determine the effects of MT-102, dosed via osmotic minipump, on the development of a gut-specific immune response. Mice were orally immunized with cholera toxin (CTX), and anti-CTX antibodies were quantified in fecal and plasma samples. The impact of MT-102 on lymphocytes within the PP of immunized mice was determined by flow cytometry. Results MT-102 delayed the production of anti-CTX IgA and IgG antibodies as measured in fecal samples without perturbing total gut IgA levels. Furthermore, MT-102 also inhibited the levels of Ag-specific antibodies in the plasma following repeated CTX oral immunizations. Blocking α4β7 induced a decrease in the frequency of B cells in the PP of treated mice compared to the control group. Conclusion α4β7-specific inhibitor, MT-102, effectively diminished the mucosal antibody response to an orally delivered Ag in mice. These results are consistent with what has been reported clinically in vedolizumab-treated subjects and thus establishes that potent small molecule inhibitors of α4β7 can replicate the pharmacology of an antibody.

The 3′ ends of metazoan microRNAs (miRNAs) are initially defined by the RNase III enzymes during maturation, but subsequently experience extensive modifications by several enzymatic activities. For example, terminal nucleotidyltransferases (TENTs) elongate miRNAs by adding one or a few nucleotides to their 3′ ends, which occasionally leads to differential regulation of miRNA stability or function. However, the catalytic entities that shorten miRNAs and the molecular consequences of such shortening are less well understood, especially in vertebrates. Here, we report that poly(A)-specific ribonuclease (PARN) sculpts the 3′ ends of miRNAs in human cells. By generating PARN knockout cells and characterizing their miRNAome, we demonstrate that PARN digests the 3′ extensions of miRNAs that are derived from the genome or attached by TENTs, thereby effectively reducing the length of miRNAs. Surprisingly, PARN-mediated shortening has little impact on miRNA stability, suggesting that this process likely operates to finalize miRNA maturation, rather than to initiate miRNA decay. PARN-mediated shortening is pervasive across most miRNAs and appears to be a conserved mechanism contributing to the 3′ end formation of vertebrate miRNAs. Our findings add miRNAs to the expanding list of noncoding RNAs whose 3′ end formation depends on PARN.

INTRODUCTION: The leukocyte adhesion cascade is important for the maintenance of homeostasis and the ability of immune cells to access sites of infection and inflammation. Despite much work identifying the molecular components of the cascade, and numerous simulations to predict the relationship between molecule density, identity, and adhesion, these relationships have not been measured experimentally. METHODS: Using surfaces functionalized with recombinant ICAM-1 and/or E-selectin along with immobilized SDF-1α, we used a flow chamber to measure rates of tethering, rolling and arrest of primary naïve human CD4(+) T lymphocytes on different surface densities of ligand. RESULTS: Cells required a minimum level of ligand density to progress beyond tethering. E-selectin and ICAM-1 were found to have a synergistic relationship in promoting cell arrest. Surfaces with both ligands had the highest levels of arrest, while surfaces containing only E-selectin hindered the cell’s ability to progress beyond rolling. In contrast, surfaces of ICAM-1 allowed only tethering or arrest. Cells maintained constant rolling velocity and time to stop over large variations in surface density and composition. In addition, surface densities of only O(10(1)) sites/μm(2) allowed for rolling while surface densities of O(10(2)) sites/μm(2) promoted arrest, approximately equal to previously determined simulated values. CONCLUSIONS: We have systematically and experimentally mapped out the state diagram of T-cell adhesion under flow, directly demonstrating the quantitative requirements for each dynamic state of adhesion, and showing how multiple adhesion molecules can act in synergy to secure arrest.

Specific gene silencing through RNA interference (RNAi) holds great promise as the next-generation therapeutic development platform. Previously, we have shown that branched, tripodal interfering RNA (tiRNA) structures could simultaneously trigger RNAi-mediated gene silencing of three target genes with 38 nt-long guide strands associated with Argonaute 2. Herein, we show that the branched RNA structure can trigger effective gene silencing in Dicer knockout cell line, demonstrating that the Dicer-mediated processing is not required for tiRNA activity. The finding of this study confirms the flexibility of the structure of RNAi triggers as well as the length of the guide strand in RNAi-mediated gene silencing.

BackgroundIntegrin αvβ8 activates TGFβ in immune cells. αvβ8 inhibitors have been shown to potentiate immune checkpoint blockade (ICB) in preclinical models [1]. Radioimmunotherapy (RIT) induces immunogenic cell death and antigen presentation, however it concurrently activates immunosuppressive pathways. Interestingly, αvβ8 immunosuppressive activity was implicated in radiotherapy resistance [2]. We have explored whether antagonizing αvβ8 overcomes the suppressive effect of TGFβ and restores anti-tumor immunity in advanced ICB and RIT resistant tumors.MethodsEfficacy was evaluated after combination treatment with low dose radiation, αvβ8 (clone C6D4) and PD-1 (clone J43) mAb in an advanced CT26 colon cancer syngeneic mouse model. Mice were treated at tumor volume of >120 mm3 and euthanized at 2,000 mm3. Flow cytometry and transcriptomic analysis were used to assess the mechanism of action. Tumor volumes are presented as mean±SEM. Statistics were performed by one-way ANOVA, or log-rank test. Bone marrow derived dendritic cell (BMdDC) cultures were isolated from C57BL/6 mice.ResultsCell death, including radiation-induced apoptosis, induced immunoregulatory and maturation program in a population of ex vivo cultured BMdDC, recently described as mregDC/DC3 [3,4]. mregDC/DC3 signature was associated with increased αvβ8 expression, suggesting a role of this integrin in inducing an immunosuppressive phenotype.A CT26 model was established to mimic the progression of late-stage tumors and was unresponsive to radiation, ICB and RIT. In CT26 implanted mice, αvβ8 is expressed on tumor stoma, and is not detectable on cancer cells. Addition of αvβ8 mAb to RIT markedly increased tumor regression (P=0.0067) and survival (PAbstract 595 Figure 1Complete response (CR) with improved survival when αvβ8 inhibition is added to RIT in CT26 syngeneic model of colorectal cancer in an advanced, ICB and RIT unresponsive stage. (A) Effect of combination therapy with low dose radiation (small animal radiation research platform (SARRP) at 5 Gray (Gy) on the day of staging (day 10)), PD-1 mAb (10 mg/kg twice weekly for 2 weeks) and αvβ8 mAb (7 mg/kg three times weekly for 3 weeks) measured by tumor burden. 5Gy+PD-1 and 5Gy+αvβ8 has a minimal effect on tumor growth inhibition showing slight improvement relative to radiation alone (5Gy+IgG). Addition of αvβ8 antagonism (5Gy+αvβ8+PD-1) improves anti-tumor responses leading to CR in 8 of 10 mice. (B) Kaplan-Meier Curve presenting time to progression. 5Gy+IgG improved survival over monotherapy with either αvβ8 or PD1 mAb. 5Gy+αvβ8+PD-1 resulted in a profound improvement of the survival over all other treatment conditionsConclusionsInhibition of αvβ8 in combination with RIT eradicated an advanced tumor, unresponsive to the respective monotherapies or conventional RIT. The anti-tumor effect was driven by enhancement of adaptive immunity, improvement of DC function and reduced tumor tolerance. These data provide evidence that αvβ8 inhibition enhances RIT and may be effective against ICB refractory tumors.ReferencesReszka-Blanco NJ,Yadav V, Krumpoch M, Cappellucci L, Cui D, Dowling JE, et al., Inhibition of integrin αvβ8 enhances immune checkpoint induced anti-tumor immunity by acting across immunologic synapse in syngeneic models of breast cancer. AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1559.Jin S, Lee WC, Aust D, Pilarsky C, Cordes N, β8 integrin mediates pancreatic cancer cell radiochemoresistance. Mol Cancer Res. 2019; 17(10): 2126–2138.Maier B, Leader AM, Chen ST, Tung N, Chang C, LeBerichel J, et al., A conserved dendritic-cell regulatory program limits antitumour immunity. Nature. 2020; 580 (7802): 257–262.Garris CS, Arlauckas SP, Kohler RH, Trefny MP, Garren S, Piot C, Engblom C, et al., Successful anti-PD-1 cancer immunotherapy requires T cell-dendritic cell crosstalk involving the cytokines IFN-γ and IL-12. Immunity. 2018; 49(6): 1148–1161.Dodagatta-Marri E, Ma H-Y, Liang B, Li J, Meyer DS, Chen S-Y, et al., Integrin αvβ8 on T cells suppresses anti-tumor immunity in multiple models and is a promising target for tumor immunotherapy. Cell Report. 2021; 36(1): 109309Ethics ApprovalAll animal work was approved by the site Institutional Animal Care and Use Committee and was performed in conformance with the Guide for the Care and Use of Laboratory Animals within an AAALAC-accredited program. Humane euthanasia criteria were predetermined on the basis of body weight and defined clinical observations.

DROSHA is the catalytic subunit of the Microprocessor complex, which initiates microRNA (miRNA) maturation in the nucleus by recognizing and cleaving hairpin precursors embedded in primary transcripts. However, accumulating evidence suggests that not all hairpin substrates of DROSHA are associated with the generation of functional small RNAs. By targeting those hairpins, DROSHA regulates diverse aspects of RNA metabolism across the transcriptome, serves as a line of defense against the expression of potentially deleterious elements, and permits cell fate determination and differentiation. DROSHA is also versatile in the way that it executes these noncanonical functions, occasionally depending on its RNA-binding activity rather than its catalytic activity. Herein, we discuss the functional and mechanistic diversity of DROSHA beyond the miRNA biogenesis pathway in light of recent findings.

Leukocyte adhesion is important for the proper functioning of the immune system. While leukocyte homing is mediated by adhesion receptors, the activation of these receptors is modulated by intracellular signaling molecules. In Leukocyte Adhesion Deficiency Type 3, the loss of the kindlin-3 prevents the activation of Leukocyte Function-associated Antigen-1 (LFA-1), which leads to a defect in adhesion, causing recurrent infections and bleeding disorders. Here, we use Integrated Signaling Adhesive Dynamics, a computer model of leukocyte rolling and adhesion combined with a simulated intracellular signaling cascade, to predict the response of T cells to depletion of kindlin-3. Our model predicts that cell adhesion is hypersensitive to the amount of kindlin-3 in the cell, while the rolling velocity is independent of kindlin-3 concentration. In addition, our simulation predicted that the time to stop, an important metric of adhesion, would increase with decreasing kindlin-3 expression. These predictions were confirmed experimentally in experiments using Jurkat cells with reduced expression of kindlin-3. These results suggest that Adhesive Dynamics is a versatile tool for quantifying adhesion in the immune response and predicting the effects of engineering cellular components.

Introduction We explored whether integrin αvβ8 inhibition potentiates immune checkpoint blockade (ICB) in syngeneic orthotopic models of breast cancer. Integrin αvβ8 mediates cell type specific and tissue localized activation of TGFβ1/3 to regulate the immune system. For example, αvβ8 expressed on dendritic cells (DC) in the intestine has been shown to be a key mediator of tolerance, maintaining gut immunologic homeostasis. Methods Efficacy was evaluated in combination with anti-PD-1 in EMT6 and PyMT breast cancer syngeneic mouse models. A potent αvβ8 inhibitor was orally administered at 60mg/kg BID for 21 days. Anti-αvβ8 or non-isoform specific anti-TGFβ mAbs were dosed TIWx3 at 7 and 10mg/kg, respectively. Tumor volumes are presented as mean±SEM. Statistics were performed by t test, one-way ANOVA, or log-rank test. Flow cytometry and transcriptome analysis on bulk and single-cell levels were used to assess the mechanism of action in EMT6. Results A similar αvβ8 expression pattern on DC, macrophages and regulatory T cells (Treg) was observed in mouse models and human tumors. Combination of oral αvβ8 inhibitor with anti-PD-1 was efficacious in the primary ICB resistant EMT6 model and resulted in superior tumor regression during treatment (p=0.0003) and improved survival with 5/12 complete responders relative to 0/12 in anti-PD-1 alone. Across studies the αvβ8 inhibitor phenocopied the results obtained with αvβ8 and TGFβ mAbs (n=3 independent studies). Complete responders re-challenged 89 days after treatment with EMT6 or 4T1 tumors showed no EMT6 tumor growth, suggesting the combination induced long-term immunologic memory. Analysis of tumors by flow cytometry showed combination resulted in increased CD8 T cell infiltrates (p=0.0006), T cell activation (CD8+CD69+, p=0.0194) and IFN-γ expression (CD8+IFN-γ+, p=0.0021). Single cell transcriptomic analysis of lymph nodes showed that αvβ8 inhibition potentiated DC co-stimulation (Cd40, Cd83/6) and migration (Ccr7, Cxcl16, Ccl22). Moreover, combination treatment led to tumor infiltrated Treg dysfunction including downregulation of Ctla4, Il10 and Tigit, and upregulation of Ifn-γ. The observation of these anti-tolerance and pro-inflammatory signatures in DC and Treg has not been described previously. Anti-tumor efficacy was driven by immune-mediated mechanisms as confirmed by a CD8 depletion study. Efficacy was confirmed in PyMT breast cancer model. Conclusions An αvβ8 inhibitor in combination with anti-PD-1 showed efficacy in syngeneic mouse models, supported by increased T cell infiltrates and evidence of reduced tumor tolerance. These results show that an orally administered αvβ8 targeted inhibitor is a potent modulator of anti-tumor immune response acting across the immunologic synapse, and is a promising therapeutic approach to ICB refractory tumors. Citation Format: Natalia J. Reszka-Blanco, Vinod Yadav, Megan Krumpoch, Laura Cappellucci, Dan Cui, James E. Dowling, Elizabeth Gwara, Bryce Harrison, Dooyoung Lee, Fu-Yang Lin, Lia Luus, Meghan Monroy, Terence I. Moy, Eugene Nebelitsky, Qi Qiao, Andrew Sullivan, Dawn Troast, Blaise Lippa, Bruce Rogers, Adrian S. Ray. Inhibition of integrin αvβ8 enhances immune checkpoint induced anti-tumor immunity by acting across immunologic synapse in syngeneic models of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1559.

Objective: MORF-057 is an orally bioavailable, selective, and potent small molecule inhibitor of α4β7 integrin being developed for inflammatory bowel diseases (IBD) that is currently in phase 1 clinical testing. We have previously presented work that characterized its nonclinical pharmacologic profile. The current study integrates data to generate a pharmacokinetic (PK) and pharmacodynamic (PD) model of MORF-057. Methods: To determine in vivo potency, MORF-057 was tested in murine gut homing assays and the PD response was determined relative to the non-protein bound drug in mouse plasma. A cell adhesion assay (CAA) for α4β7 was refined to enable detection of picomolar-level sensitivity. Murine receptor occupancy (RO) assays for α4β7 and α4β1 were established under physiologic conditions, and MORF-057 was evaluated for its potency and selectivity in fresh mouse whole blood. These datasets were used to build and validate predictive models of PD response. Results: MORF-057 strongly inhibited the homing of α4β7hi cells to murine gut lymphoid tissues with an IC90 of 7.9 nM. MORF-057 showed high potency in CAA with an IC90 of 8.8 nM. Similarly, RO assays confirmed MORF-057 to be a highly potent inhibitor of α4β7 in mouse whole blood with an IC90 of 20.5 nM and over 1500-fold selectivity vs. α4β1. The predictive models built upon these datasets revealed a strong PK-PD relationship of α4β7 inhibitors in vivo. Conclusions: We observed consistently high potency of MORF-057 across multiple assay platforms. Integrated modeling based on these assays, particularly the RO assay, successfully predicted the PD response to MORF-057. These data begin to establish the relationship between PK, target engagement, and PD with MORF-057.

Essentials Baseline coagulation activity can be detected in non-bleeding state by in vivo biomarker levels. A detailed mathematical model of coagulation was developed to describe the non-bleeding state. Optimized model described in vivo biomarkers with recombinant activated factor VII treatment. Sensitivity analysis predicted prothrombin fragment 1 + 2 and D-dimer are regulated differently. SummaryBackground Prothrombin fragment 1 + 2 (F1 + 2), thrombin–antithrombin III complex (TAT) and D-dimer can be detected in plasma from non-bleeding hemostatically normal subjects or hemophilic patients. They are often used as safety or pharmacodynamic biomarkers for hemostatis-modulating therapies in the clinic, and provide insights into in vivo coagulation activity. Objectives To develop a quantitative systems pharmacology (QSP) model of the blood coagulation network to describe in vivo biomarkers, including F1 + 2, TAT, and D-dimer, under non-bleeding conditions. Methods The QSP model included intrinsic and extrinsic coagulation pathways, platelet activation state-dependent kinetics, and a two-compartment pharmacokinetics model for recombinant activated factor VII (rFVIIa). Literature data on F1 + 2 and D-dimer at baseline and changes with rFVIIa treatment were used for parameter optimization. Multiparametric sensitivity analysis (MPSA) was used to understand key proteins that regulate F1 + 2, TAT and D-dimer levels. Results The model was able to describe tissue factor (TF)-dependent baseline levels of F1 + 2, TAT and D-dimer in a non-bleeding state, and their increases in hemostatically normal subjects and hemophilic patients treated with different doses of rFVIIa. The amount of TF required is predicted to be very low in a non-bleeding state. The model also predicts that these biomarker levels will be similar in hemostatically normal subjects and hemophilic patients. MPSA revealed that F1 + 2 and TAT levels are highly correlated, and that D-dimer is more sensitive to the perturbation of coagulation protein concentrations. Conclusions A QSP model for non-bleeding baseline coagulation activity was established with data from clinically relevant in vivo biomarkers at baseline and changes in response to rFVIIa treatment. This model will provide future mechanistic insights into this system.

INTRODUCTION: The arrest of rolling T lymphocytes at specific locations is crucial to proper immune response function. We previously developed a model of chemokine-driven integrin activation, termed integrative signaling adhesive dynamics (ISAD). In addition, we have shown that loss of diacylglycerol kinase (DGK) leads to a gain of function regarding adhesion under shear flow. We undertook this study to understand the sensitivity of adhesion to perturbations in other signaling molecules. METHODS: We adapted multi-parametric sensitivity analysis (MPSA) for use in our ISAD model to identify important parameters, including initial protein concentrations and kinetic rate constants, for T lymphocyte arrest. We also compared MPSA results to those obtained from a single parametric sensitivity analysis. RESULTS: In addition to the previously shown importance of DGK in lymphocyte arrest, PIP(2) cleavage and Rap1 activation are crucial in determining T cell arrest dynamics, which agree with previous experimental findings. The l-selectin density on the T lymphocyte surface also plays a large role in determining the distance rolled before arrest. Both the MPSA and single-parametric method returned similar results regarding the most sensitive kinetic rate constants. CONCLUSION: We show here that the regulation of the amount of second messengers are, in general, more critical for determining T lymphocyte arrest over the initial signaling proteins, highlighting the importance of amplification of signaling in cell adhesion responses. Overall, this work provides a mechanistic insight of the contribution of key pathways and components, thus may help to identify potential therapeutic targets for drug development against immune disorders. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12195-019-00575-2) contains supplementary material, which is available to authorized users.

Epigenetic gene regulation is essential for developmental processes. Eggless (Egg), the Drosophila orthologue of the mammalian histone methyltransferase, SETDB1, is known to be involved in the survival and differentiation of germline stem cells and piRNA cluster transcription during Drosophila oogenesis; however the detailed mechanisms remain to be determined. Here, using high-throughput RNA sequencing, we investigated target genes regulated by Egg in an unbiased manner. We show that Egg plays diverse roles in particular piRNA pathway gene expression, some long non-coding RNA expression, apoptosis-related gene regulation, and Decapentaplegic (Dpp) signaling during Drosophila oogenesis. Furthermore, using genetic and cell biological approaches, we demonstrate that ectopic upregulation of dpp caused by loss of Egg in the germarium can trigger apoptotic cell death through activation of two pro-apoptotic genes, reaper and head involution defective. We propose a model in which Egg regulates germ cell differentiation and apoptosis through canonical and noncanonical Dpp pathways in Drosophila oogenesis.

A number of therapeutics have been developed or are under development aiming to modulate the coagulation network to treat various diseases. We used a systems model to better understand the effect of modulating various components on blood coagulation. A computational model of the coagulation network was built to match in-house in vitro thrombin generation and activated Partial Thromboplastin Time (aPTT) data with various concentrations of recombinant factor VIIa (FVIIa) or factor Xa added to normal human plasma or factor VIII-deficient plasma. Sensitivity analysis applied to the model revealed that lag time, peak thrombin concentration, area under the curve (AUC) of the thrombin generation profile, and aPTT show different sensitivity to changes in coagulation factors' concentrations and type of plasma used (normal or factor VIII-deficient). We also used the model to explore how variability in concentrations of the proteins in coagulation network can impact the response to FVIIa treatment.

The structural flexibility of RNA interference (RNAi)-triggering nucleic acids suggests that the design of unconventional RNAi trigger structures with novel features is possible. Here, we report a cross-shaped RNA duplex structure, termed quadruple interfering RNA (qiRNA), with multiple target gene silencing activity. qiRNA triggers the simultaneous down-regulation of four cellular target genes via an RNAi mechanism. In addition, qiRNA shows enhanced intracellular delivery and target gene silencing over conventional siRNA when complexed with jetPEI, a linear polyethyleneimine (PEI). We also show that the long antisense strand of qiRNA is incorporated intact into an RNA-induced silencing complex (RISC). This novel RNA scaffold further expands the repertoire of RNAi-triggering molecular structures and could be used in the development of therapeutics for various diseases including viral infections and cancer.

The nuclear RNase III enzyme DROSHA interacts with its cofactor DGCR8 to form the Microprocessor complex, which initiates microRNA (miRNA) maturation by cleaving hairpin structures embedded in primary transcripts. Apart from its central role in the biogenesis of miRNAs, DROSHA is also known to recognize and cleave miRNA-like hairpins in a subset of transcripts without apparent small RNA production. Here, we report that the human DROSHA transcript is one such noncanonical target of DROSHA. Mammalian DROSHA genes have evolved a conserved hairpin structure spanning a specific exon–intron junction, which serves as a substrate for the Microprocessor in human cells but not in murine cells. We show that it is this hairpin element that decides whether the overlapping exon is alternatively or constitutively spliced. We further demonstrate that DROSHA promotes skipping of the overlapping exon in human cells independently of its cleavage function. Our findings add to the expanding list of noncanonical DROSHA functions.

Background Three quinuclidine derivatives (FRM-1, FRM-2 and FRM-3) were subject to significant mass loss to cellular retention in Caco-2 permeation experiments. The apparent permeability coefficient (Papp) calculated with either 'sink' (Papp,sink) or 'non-sink' (Papp,nonsink) method was significantly biased. As a result, a simplified 3-compartmental distribution model was applied in this study to derive the 'intrinsic' Papp (Papp,int) and to understand the impact of cellular retention on estimating Papp and active efflux ratio (ER) values. Methods Time-courses of the amount of test compounds in the donor, receiver and cells were determined in the presence and absence of bafilomycin A1 (BFA, 100 nM) and / or cyclosporine A (CsA, 10 .M). A mathematical model was constructed to describe the mass transfer of test compounds among three compartments. The temporal profiles of directional Papp,sink, Papp,nonsink and the corresponding of ER values were compared with the counterpart parameters derived from data-fitting to the mathematical model. Simulations were performed for a better understanding of experimental observations. Results The mass recovery of test compounds deteriorated with incubation time and was direction dependent. Based on the directional Papp,sink values, the resulting ER is close to unity for FRM-1, and approximately 2 and 3.5 for FRM-2 and FRM-3. Treatment with BFA considerably enhanced mass recovery for FRM-1 and FRM-3 (by 5- and 2-fold) but elicited no impact on FRM-2, while ER values largely unchanged. Expectedly, Papp,nonsink was higher than Papp,sink, but the resulting ER was lower in most cases. In contrast, the model-derived Papp,int was much greater than the values of Papp,sink and Papp,nonsink. The model also quantitatively unveiled the respective contributions of lysosomal sequestration and nonspecific binding to the cellular retention of the compounds. Conclusion Our work reveals the different mechanisms involved in cellular retention of these quinuclidine derivatives, and more importantly, demonstrates the value of kinetic analyses with mathematical modeling in minimizing the bias in Papp estimation when assumptions for conventional calculations are violated.

MicroRNAs (miRNAs) are key posttranscriptional regulators of gene expression involved in diverse biological pathways in bilateral animals and plants. The key to understanding the biological function of a miRNA is to identify its regulatory targets. Although a few miRNA targets have been identified genetically, the rapidly expanding list of miRNAs has necessitated genome-wide tools for identifying target mRNAs, and a number of computational and experimental approaches have consequently emerged. Some of these approaches have also provided insights into the mechanistic aspects of miRNA-mediated regulation, another intensely debated area in the miRNA field. Here, we review several emerging features of miRNA-target interactions in animals and genome-wide approaches for probing those interactions.

Rapid arrest of T cells at target sites upon engagement of chemokine receptors is crucial to the proper functioning of the immune system. Although T-cell arrest always occurs under hydrodynamic forces in vivo, most studies investigating the molecular mechanisms of arrest have been performed under static conditions. While the requirement of the adapter protein SLP-76 (Src homology 2-domain containing leukocyte-specific phosphoprotein of 76 kDa) in TCR-induced integrin activation has been demonstrated, its role in chemokine-triggered T-cell adhesion is unknown. Using a flow chamber system, we show that SLP-76 plays an important role in regulating the transition from tethering and rolling to firm adhesion of T cells under physiological shear flow in response to CXCL12α (stromal cell-derived factor-1α); SLP-76-deficient primary T cells exhibited defective adhesion with a significant decrease in the number of firmly arrested cells. We further demonstrate the N-terminal phosphotyrosines of SLP-76 play a critical role in T-cell adhesion under flow. These findings reveal a novel role for SLP-76 in CXCR4-mediated T lymphocyte trafficking.

Platelet spreading is critical for hemostatic plug formation and thrombosis. However, the detailed dynamics of platelet spreading as a function of receptor-ligand adhesive interactions has not been thoroughly investigated. Using reflection interference contrast microscopy, we found that both adhesive interactions and PAR4 activation affect the dynamics of platelet membrane contact formation during spreading. The initial growth of close contact area during spreading was controlled by the combination of different immobilized ligands or PAR4 activation on fibrinogen, whereas the growth of the total area of spreading was independent of adhesion type and PAR4 signaling. We found that filopodia extend to their maximal length and then contract over time; and that filopodial protrusion and expansion were affected by PAR4 signaling. Upon PAR4 activation, the integrin α(IIb)β(3) mediated close contact to fibrinogen substrata and led to the formation of ringlike patterns in the platelet contact zone. A systematic study of platelet spreading of GPVI-, α(2)-, or β(3)-deficient platelets on collagen or fibrinogen suggests the integrin α(2) is indispensable for spreading on collagen. The platelet collagen receptors GPVI and α(2) regulate integrin α(IIb)β(3)-mediated platelet spreading on fibrinogen. This work elucidates quantitatively how receptor-ligand adhesion and biochemical signals synergistically control platelet spreading.

Dendritic cells (DCs) respond to chemotactic signals to migrate from sites of infection to secondary lymphoid organs where they initiate the adaptive immune response. The key chemokines directing their migration are CCL19, CCL21, and CXCL12, but how signals from these chemokines are integrated by migrating cells is poorly understood. Using a microfluidic device, we presented single and competing chemokine gradients to murine bone-marrow derived DCs in a controlled, time-invariant microenvironment. Experiments performed with counter-gradients revealed that CCL19 is 10–100-fold more potent than CCL21 or CXCL12. Interestingly, when the chemoattractive potencies of opposing gradients are matched, cells home to a central region in which the signals from multiple chemokines are balanced; in this region, cells are motile but display no net displacement. Actin and myosin inhibitors affected the speed of crawling but not directed motion, whereas pertussis toxin inhibited directed motion but not speed. These results provide fundamental insight into the processes that DCs use to migrate toward and position themselves within secondary lymphoid organs.

Summary. Background: In resting platelets, endothelial cell specific adhesion molecule (ESAM) is located in alpha granules, increasing its cell surface expression following platelet activation. However, the function of ESAM on platelets is unknown. Objective: To determine whether ESAM has a role in thrombus formation. Methods and results: We found that following platelet activation ESAM localizes to the junctions between adjacent platelets, suggesting a role for this protein in contact-dependent events that regulate thrombus formation. To test this hypothesis we examined the effect of ESAM deletion on platelet function. In vivo, ESAM−/− mice achieved more stable hemostasis than wild-type mice following tail transection, and developed larger thrombi following laser injury of cremaster muscle arterioles. In vitro, ESAM−/− platelets aggregated at lower concentrations of G protein-dependent agonists than wild-type platelets, and were more resistant to disaggregation. In contrast, agonist-induced calcium mobilization, αIIbβ3 activation, alpha-granule secretion and platelet spreading, were normal in ESAM-deficient platelets. To understand the molecular mechanism by which ESAM regulates platelet activity, we utilized a PDZ domain array to identify the scaffold protein NHERF-1 as an ESAM binding protein, and further demonstrated that it associates with ESAM in both resting and activated platelets. Conclusions: These findings support a model in which ESAM localizes to platelet contacts following platelet activation in order to limit thrombus growth and stability so that the optimal hemostatic response occurs following vascular injury.

The adapter protein SH2 domain-containing leukocyte protein of 76 kDa (SLP-76) is an essential mediator of signaling from the T-cell antigen receptor (TCR). We report here that SLP-76 also mediates signaling downstream of integrins in T cells and that SLP-76-deficient T cells fail to support adhesion to integrin ligands. In response to both TCR and integrin stimulation, SLP-76 relocalizes to surface microclusters that colocalize with phosphorylated signaling proteins. Disruption of SLP-76 recruitment to the protein named LAT (linker for activation of T cells) inhibits SLP-76 clustering downstream of the TCR but not downstream of integrins. Conversely, an SLP-76 mutant unable to bind ADAP (adhesion and degranulation-promoting adapter protein) forms clusters following TCR but not integrin engagement and fails to support T-cell adhesion to integrin ligands. These findings demonstrate that SLP-76 relocalizes to integrin-initiated signaling complexes by a mechanism different from that employed during TCR signaling and that SLP-76 relocalization corresponds to SLP-76-dependent integrin function in T cells.

l -selectin on leukocytes is critical in leukocyte tethering and adhesion to inflamed endothelium and lymphocyte homing to lymphoid organs. The spatial distribution of l -selectin on leukocytes controls cellular adhesive function in hydrodynamic shear. How l -selectin changes its position on the cell membrane remains an open question, but a possible candidate is shear stress encountered on the cell surface. Here we demonstrate shear-induced l -selectin polarization on the membrane during the process of centrifugation of resting neutrophils via immunofluorescent microscopy. It was found that randomly distributed l -selectin on neutrophils moves to a polar cap at one end of the cell after centrifugation (300 × g for 2 min) without inflammatory stimuli. This l -selectin redistribution under shear was predicted by Monte Carlo simulations that show how convection dominates over diffusion, leading to l -selectin cap formation during centrifugation at 280 × g or during leukocyte adhesion to the endothelial wall at 1 dyn/cm 2 . Those results point to a role for shear stress in the modulation of l -selectin distribution, and suggest a possible alternate mechanism and reinterpretation of previous in vitro studies of l -selectin mediated adhesion of neutrophils isolated via centrifugation.

We describe a successful alliance between a university and several high schools. The alliance is centered on a laboratory experiment constructed by students and faculty. The experiment involves sophisticated concepts and equipment not readily available in high schools. Much of the experiment is directly related to the science and mathematics learned in high school, with opportunities to extend their understanding by applying it to a research experience. The experiment is in plasma physics, but a similar alliance can be implemented in any area of science. Although the number of high school students affected by any one alliance is small, the impact is potentially large in the scientific life of a participating student or teacher.

The stabilization of optimum pH for cells can cause a higher erythropoietin (EPO) production rate and a good growth rate with the prolonged culture span in recombinant Chinese hamster ovary (r-CHO) cells. Our strategy for stabilizing the optimum pH in this study is to reduce the lactate production by adding sodium lactate to a culture medium. When 40 mM sodium lactate was added, a specific growth rate was decreased by approximately 22% as compared with the control culture. However the culture longevity was extended to 187 h, and more than a 2.7-fold increase in a final accumulated EPO concentration was obtained at 40 mM of sodium lactate. On the condition that caused the high production of EPO, a specific glucose consumption rate and lactate production rate decreased by 23.3 and 52%, respectively. Activity of lactate dehydrogenase (LDH) in r-CHO cells increased and catalyzed the oxidation of lactate to pyruvate, together with the reverse reaction, at the addition of 40 mM sodium lactate. The addition of 40 mM sodium lactate caused the positive effects on a cell growth and an EPO production in the absence of carbon dioxide gas as well as in the presence of carbon dioxide gas by reducing the accumulation of lactate.

T lymphocyte arrest within microvasculature is an essential process in immune surveillance and the adaptive immune response. Integrins and chemokines coordinately regulate when and where T cells stop under flow via chemokine-triggered inside-out activation of integrins. Diacylglycerol kinases (DGKs) regulate the levels of diacylglycerol (DAG) which in turn determine the activation of guanine nucleotide exchange factors (GEFs) and Ras proximity 1 (Rap1) molecules crucial to the activation of integrin lymphocyte function-associated antigen 1 (LFA-1). However, how the level of DGK regulates chemokine-stimulated LFA-1-mediated T cell arrest under flow is unknown. Using a combination of experiment and computational modeling, we demonstrate that DGKζ is a crucial regulator of CXCL12-triggered T cell arrest on surfaces presenting inter-cellular adhesion molecule 1 (ICAM-1). Using flow chamber assays, we found that the deficiency of DGKζ in T cells significantly increased firm arrest to ICAM-1-coated substrates and shortened the time to stop without altering the rolling velocity. These results suggest that DGKζ levels affect LFA-1-mediated T cell firm arrest, but not P-selectin-mediated rolling during CXCL12 stimulation. We accurately simulated the role of DGKζ in firm arrest of T cells computationally using an Integrated-Signaling Adhesive Dynamics (ISAD). In the absence of DGK catalytic reaction, the model cells rolled for a significantly shorter time before arrest, compared to when DGK molecules were present. Predictions of our model for T cell arrest quantitatively match experimental results. Overall these results demonstrate that DGKζ is a negative regulator of CXCL12-triggered inside-out activation of LFA-1 and firm adhesion of T cells under shear flow.

The pattern of T lymphocyte homing is hypothesized to be controlled by combnations of chemokine receptors and complementary chemokines. Here, we use numerical simulation to explore the relationship among chemokine potency and concentration, signal transduction, and adhesion. We have developed a form of Adhesive Dynamics – a mechanically accurate stochastic simulation of adhesion – that incorporates stochastic signal transduction using the next subvolume method. We show that using measurable parameter estimates derived from a variety of sources, including signaling measurements that allow us to train parameter values, we can readily simulate approximate time scales for T lymphocyte arrest. We find that adhesion correlates with total chemokine receptor occupancy, not the frequency of occupation, when multiple chemokine receptors feed through a single G-protein. A general strategy for selective T-lymphocyte recruitment appears to require low affinity chemokine receptors. For a single chemokine receptor, increases in multiple cross-reactive chemokines can lead to an overwhelming increase in adhesion. Overall, the methods presented here provide a predictive framework for understanding chemokine control of T-lymphocyte recruitment.

Mammalian platelets are small, anuclear circulating cells that form tightly adherent, shear-resistant thrombi to prevent blood loss after vessel injury. Platelet thrombi that form in coronary and carotid arteries also underlie common vascular diseases such as myocardial infarction and stroke and are the target of drugs used to treat these diseases. Birds have high-pressure cardiovascular systems like mammals but generate nucleated thrombocytes rather than platelets. Here, we show that avian thrombocytes respond to many of the same activating stimuli as mammalian platelets but are unable to form shear-resistant aggregates ex vivo. Avian thrombocytes are larger than mammalian platelets, spread less efficiently on collagen, and express much lower levels of the α2bβ3 integrin required for aggregate formation, features predicted to make thrombocyte aggregates less resistant than platelets are to the high fluid shear forces of the arterial vasculature. In vivo carotid vessel injury stimulates the formation of occlusive platelet thrombi in mice but not in the size- and flow-matched carotid artery of the Australian budgerigar. These studies indicate that unique physical and molecular features of mammalian platelets enable them to form shear-resistant arterial thrombi, an essential element in the pathogenesis of human cardiovascular diseases.

We recently reported that Semaphorin 4D (Sema4D) and its receptors are expressed on the platelet surface and showed that Sema4D(−/−) mice have a selective defect in collagen-induced platelet aggregation and an impaired vascular injury response. Here we investigated the mechanisms involved, tested the role of platelet-platelet contacts in Sema4D-mediated events, and examined the relationship between Sema4D-dependent signaling and integrin αIIbβ3 outside-in signaling. The results show that spleen tyrosine kinase (Syk) activation, an early step in collagen signaling via the glycoprotein VI (GPVI)/FcRγ complex, is greatly reduced in Sema4D(−/−) platelets and can be restored by adding soluble Sema4D. Earlier events, including FcRγ phosphorylation, occur normally; later events are impaired. In contrast, when engagement of αIIbβ3 was blocked, Sema4D(−/−) and control platelets were indistinguishable in assays of Syk activation, adhesion, spreading on collagen, and activation of αIIbβ3. Finally, we found that, unlike the Sema4D knockout, αIIbβ3 blockade inhibited FcRγ phosphorylation and that stimulating aggregation with Mn2+ failed to normalize Syk activation in the absence of Sema4D. Collectively, these results show that αIIbβ3 and Sema4D jointly promote collagen responses by amplifying Syk activation, partly by forming integrin-mediated contacts that enable the binding of Sema4D to its receptors and partly through integrin outside-in signaling. These 2 processes are interdependent, but distinguishable.

A strategy for treating inflammatory disease is to create micro-particles with the adhesive properties of leukocytes. The underlying rheology of deformable adhesive microspheres would be an important factor in the adhesive performance of such particles. In this work the effect of particle deformability on the selectin-mediated rolling of polymer vesicles (polymersomes) is evaluated. The rheology of the polymersome membrane was modulated by cross-linking unsaturated side-chains within the hydrophobic core of the membrane. Increased membrane rigidity resulted in decreased rates of particle recruitment rather than decreased average rolling velocities. Reflective interference contrast microscopy of rolling vesicles confirmed that neither flaccid nor rigid vesicles sustained close contacts with the substrate during rolling adhesion. A variable-shear rate parallel-plate flow chamber was employed to evaluate individual vesicles rolling on substrates under different flow conditions. Analysis of the trajectories of single flaccid vesicles revealed several distinct populations of rolling vesicles; however, some of these populations disappear when the vesicle membranes are made rigid. This work shows that membrane mechanics affects the capture, but not the rolling dynamics, of adherent leuko-polymersomes.

Here we accurately recreate the mechanical shedding of L-selectin and its effect on the rolling behavior of neutrophils in vitro using the adhesive dynamics simulation by incorporating the shear-dependent shedding of L-selectin. We have previously shown that constitutively expressed L-selectin is cleaved from the neutrophil surface during rolling on a sialyl Lewis x-coated planar surface at physiological shear rates without the addition of exogenous stimuli. Utilizing a Bell-like model to describe a shedding rate which presumably increases exponentially with force, we were able to reconstruct the characteristics of L-selectin-mediated neutrophil rolling observed in the experiments. First, the rolling velocity was found to increase during rolling due to the mechanical shedding of L-selectin. When most of the L-selectin concentrated on the tips of deformable microvilli was cleaved by force exerted on the L-selectin bonds, the cell detached from the reactive plane to join the free stream as observed in the experiments. In summary, we show through detailed computational modeling that the force-dependent shedding of L-selectin can explain the rolling behavior of neutrophils mediated by L-selectin in vitro.

Precise spatial and temporal regulation of cell adhesion and de-adhesion is critical for dynamic lymphocyte migration. Although a great deal of information has been learned about integrin lymphocyte function–associated antigen (LFA)-1 adhesion, the mechanism that regulates efficient LFA-1 de-adhesion from intercellular adhesion molecule (ICAM)-1 during T lymphocyte migration is unknown. Here, we show that nonmuscle myosin heavy chain IIA (MyH9) is recruited to LFA-1 at the uropod of migrating T lymphocytes, and inhibition of the association of MyH9 with LFA-1 results in extreme uropod elongation, defective tail detachment, and decreased lymphocyte migration on ICAM-1, without affecting LFA-1 activation by chemokine CXCL-12. This defect was reversed by a small molecule antagonist that inhibits both LFA-1 affinity and avidity regulation, but not by an antagonist that inhibits only affinity regulation. Total internal reflection fluorescence microscopy of the contact zone between migrating T lymphocytes and ICAM-1 substrate revealed that inactive LFA-1 is selectively localized to the posterior of polarized T lymphocytes, whereas active LFA-1 is localized to their anterior. Thus, during T lymphocyte migration, uropodal adhesion depends on LFA-1 avidity, where MyH9 serves as a key mechanical link between LFA-1 and the cytoskeleton that is critical for LFA-1 de-adhesion.

Cell rolling is an important physiological and pathological process that is used to recruit specific cells in the bloodstream to a target tissue. This process may be exploited for biomedical applications to capture and separate specific cell types. One of the most commonly studied proteins that regulate cell rolling is P-selectin. By coating surfaces with this protein, biofunctional surfaces that induce cell rolling can be prepared. Although most immobilization methods have relied on physisorption, chemical immobilization has obvious advantages, including longer functional stability and better control over ligand density and orientation. Here we describe chemical methods to immobilize P-selectin covalently on glass substrates. The chemistry was categorized on the basis of the functional groups on modified glass substrates: amine, aldehyde, and epoxy. The prepared surfaces were first tested in a flow chamber by flowing microspheres functionalized with a cell surface carbohydrate (sialyl Lewis(x)) that binds to P-selectin. Adhesion bonds between P-selectin and sialyl Lewis(x) dissociate readily under shear forces, leading to cell rolling. P-selectin immobilized on the epoxy glass surfaces exhibited enhanced long-term stability of the function and better homogeneity as compared to that for surfaces prepared by other methods and physisorbed controls. The microsphere rolling results were confirmed in vitro with isolated human neutrophils. This work is essential for the future development of devices for isolating specific cell types based on cell rolling, which may be useful for hematologic cancers and certain metastatic cancer cells that are responsive to immobilized selectins.

We present a novel method to create cavities in PDMS that is simple and exhibits wide process latitude allowing control over the radius of curvature to form shallow concave pits or deep spherical cavities.

In previous work, we have described the adhesive capture of circulating stem cells to surfaces coated with adhesive selectin protein, both in vitro and in vivo. Here we describe PDMS surfaces microfabricated to contain an array of square 80 × 80 × 80 micron cavities. These cavities are intended to provide a local bioreactor environment to culture stem cells over extended periods of time, while sheltered from the higher shear stresses of the surrounding blood flow external of the cavities. In this paper we present in vitro flow experiments with polymeric, blood cell-sized microspheres, showing the creation of stable vortices within the microscale cavities. Computational fluid dynamics (CFD) was performed to predict the velocity field within the cavity, and for comparison with experimentally determined microsphere velocities. Future work will establish the ability to place local chemoattract molecules within the cavity interior, and the ability to accumulate viable stem cells within these cavities.

It is known that the coagulation factor levels change with age. In this work, we have applied a quantitative systems pharmacology (QSP) model of the coagulation network to predict whether age related changes in coagulation factor levels will impact dose response for PF-05320907 on various pharmacodynamic endpoints. PF-05230907 (PF-907) is a variant of FXa, in which the conformational transition from zymogen to active protease is impaired. Binding to activated factor V facilitates its transition to the active conformation, rescues procoagulant activity and is hypothesized to localize PF-907 hemostatic effect to sites of hemorrhage. It is currently in development for the indication of intracerebral hemorrhage (ICH). Pharmacokinetic (PK) and pharmacodynamic (PD) data are available from the healthy subjects phase 1 study of single dose escalation of intravenous bolus infusion of PF-907. The next study for PF-907 will be conducted in ICH patients, who will have a much higher median age than the median age in the healthy subjects in the Phase 1 study. Our group has implemented a QSP model for the coagulation network to enable integrated understanding of all the data and underlying pharmacology1. The model has been optimized to describe in vitro biomarker changes including; thrombin generation assay (TGA), activated partial thromboplastin time (aPTT) and prothrombin time (PT) as well as in vivo biomarker changes including prothrombin fragment 1+2 (PF1+2), thrombin-anti-thrombin III complex (TAT) and D-dimer. In this simulation study, we used the model to first describe biomarker changes with treatment of FXa variant in hemostatic normal subjects and then used model simulations to predict the behavior of important biomarkers in an older ICH population. A single compartment PK model for PF-907 was first established to describe the PK data obtained from the phase 1 study. The PK model was then combined with the QSP model to predict biomarker changes following PF-907 treatment. Comparison with observed clinical data showed that the model adequately predicted dose-dependent change in TGA parameters, aPTT, PF1+2, TAT and D-dimer. In addition, the model also predicted that there would be no change in PT, which was consistent with observed first in human results with the PF-907 treatment. After model validation using FIH data, the model was then used to predict biomarker changes for older subjects using literature reported changes in baseline levels of coagulation factors for subjects over a period of 40 years. The simulation predicted minimal shifts in the PD responses suggesting that the dose-response to PF-907 may not change significantly between young and older populations. The model, however, did not consider other characteristics beyond coagulation factor level changes in older populations, which may impact the safety profile of PF-907 treatment. In summary, this study indicates that it is possible to predict the response of a hemostatic agent with a QSP model. Following validation, the model can also extrapolate from a standard subject to new patient populations and indicates that no dose adjustment due to age is required. Reference 1. Nayak, S., Lee, D., Patel-Hett, S., Pittman, D., Martin, S., Heatherington, A., Vicini, P. and Hua, F. (2015), Using a Systems Pharmacology Model of the Blood Coagulation Network to Predict the Effects of Various Therapies on Biomarkers. CPT: Pharmacometrics & Systems Pharmacology. doi:10.1002/psp4.50 Disclosures Nayak: Pfizer Inc: Employment. Lee:Pfizer Inc.: Employment. Arkin:Pfizer Inc: Employment. Martin:Pfizer Inc: Employment. Heatherington:Pfizer Inc.: Employment. Denney:Pfizer Inc.: Employment. Vicini:Pfizer Inc.: Employment. Hua:Pfizer Inc: Employment.

The interaction of L-selectin expressed on leukocytes with endothelial cells leads to capture and rolling and is critical for the recruitment of leukocytes into sites of inflammation. It is known that leukocyte activation by chemoattractants, the change of osmotic pressure in cell media, or cross-linking of L-selectin all result in rapid shedding of L-selectin. Here we present a novel mechanism for surface cleavage of L-selectin on neutrophils during rolling on a sialyl Lewis x-coated surface that involves mechanical force. Flow cytometry and rolling of neutrophils labeled with Qdot(R)-L-selectin antibodies in an in vitro flow chamber showed that the mechanical shedding of L-selectin occurs during rolling and depends on the amount of shear applied. In addition, the mechanical L-selectin shedding causes an increase in cell rolling velocity with rolling duration, suggesting a gradual loss of L-selectin and is mediated by p38 mitogen-activated protein kinase activation. Thus, these data show that mechanical force induces the cleavage of L-selectin from the neutrophil surface during rolling and therefore decreases the adhesion of cells to a ligand-presenting surface in flow.

Leukocyte recruitment to sites of inflammation is initiated by adhesive tethering and rolling on the activated vascular wall under shear flow. Although the binding kinetics between adhesion molecules is primarily responsible for leukocyte recruitment, we have found that hydrodynamic interactions between cells near adhesive surfaces affect leukocyte behavior in the initial recruiting step onto the vascular endothelial wall. Previous experimental evidence has shown that cell–cell physical adhesion is one multicellular mechanism that accelerates cell recruitment to an adhesive surface. Our laboratory has shown, however, that hydrodynamic collisions between cells induce the capture of free-stream cells to a ligand-presenting surface under shear flow, verified by monitoring leukocytes in postcapillary venules, as well as by using carbohydrate-coated microspheres or neutrophils in flow chambers. We also demonstrate that neighboring cells roll more slowly as two rolling cells become closer due to cell–cell and cell–surface hydrodynamic interactions, not only in vitro but also in vivo. To explain and probe these phenomena, we developed the multipartlcle adhesive dynamics (MAD) theoretical simulation that fuses an adhesive dynamics model with a boundary integral calculation of suspension flow. Our simulation results show good agreement with experiments measuring binary cell capture and pair rolling velocity. In this chapter, therefore, we compile evidence that a distinct hydrodynamic mechanism of cell-surface interaction in blood flow plays a significant role in the inflammation cascade.

Coagulation is a process crucial to stop bleeding from a damaged vessel. The network is comprised of a complex interplay of various pro-coagulant and anti-coagulant factors. Several systems models for the coagulation pathway have been published, as a way to investigate the pathway complexity. Most published models describe the thrombin generation profile (TGA) or prothrombin time (PT) following extrinsic pathway activation by tissue factor, or activated partial prothrombin time (aPTT) through activation of the intrinsic pathway by contact activation. In the clinical setting, thrombin-anti-thrombin complex (TAT) and prothrombin fragment 1+2 (PF1+2) are often used as biomarkers for in vivo coagulation activity in the non-bleeding state. Even in the absence of any treatment, there are detectable levels of PF1+2 and TAT in healthy volunteers indicating low level coagulation activity exists in the normal baseline, non-bleeding state. In this study, we developed a mathematical model for coagulation to describe the baseline activity of PF1+2 and TAT. We then used the model to understand the impact of the coagulation pathway activity during non-bleeding state on thrombin generation activated by tissue factor. First, a coagulation model from Hockin et al. (J Biol Chem. 2002;277(21):18322-33) was modified to describe internally generated data for thrombin generation and aPTT modulation following addition of various concentrations of recombinant factor VIIa or plasma derived factor Xa to normal or hemophilic human plasma. Next, protein synthesis and degradation were incorporated into the model and platelet-dependent reactions were tuned down to describe the baseline coagulation activity in non-bleeding healthy subjects. Using a simulated annealing algorithm, the new parameters were optimized to fit published data for PF1+2 and TAT in healthy volunteers and changes of PF1+2 following treatment with eptacog alfa (recombinant factor VIIa). In the model, the baseline coagulation activities can be described by a very low level of tissue factor, which is much lower than the detection limit of a regular ELISA method. Using this baseline model, we tested the effect of this baseline coagulation activity on tissue factor-activated thrombin generation, and found that very small baseline levels of activated enzymes significantly shorten the lag time of thrombin generation, but did not affect the peak thrombin. We also performed a global sensitivity analysis to identify key proteins in the coagulation network whose modulation will have the biggest impact on PF1+2 and TAT levels. Disclosures Lee: Pfizer Inc.: Employment. Nayak:Pfizer Inc.: Employment. Pittman:Pfizer Inc.: Employment. Arkin:Pfizer Inc.: Employment. Martin:Pfizer Inc.: Employment. Heatherington:Pfizer Inc.: Employment. Vicini:Pfizer Inc.: Employment. Hua:Pfizer Inc: Employment.

It was examined whether hepatocyte cell lines can be used for ammonia removal in mammalian cell cultures. It was found that there exists a critical ammonium concentration level for each hepatocyte cell to remove ammonia. Among the cells tested in this work, primary hepatocytes showed the strongest ammonia removal capability if ammonium concentration is higher than the critical level. However, primary hepatocytes lost the liver function gradually and finally died after 2-3 weeks. Because of this limitation, primary hepatocytes were not appropriate to be used for ammonia removal in long-term cultures. Hep G2 cells, which are immortal, also showed a strong ammonia removal activity. The ammonia removal activity of Hep G2 cells depended on the concentration of ammonium in the medium, as in the case of primary hepatocytes. However, urea could not be detected in the course of ammonia removal by Hep G2 cells. Instead of urea, Hep G2 cells secreted glutamine into the culture medium. The capacity for ammonia removal was higher in the absence than in the presence of glutamine. Thus we checked the activity of glutamine synthetase in the Hep G2 cells. The level of glutamine synthetase activity increased with the addition of ammonium chloride. This result accounts for the ammonium concentration dependency of Hep G2 cells in ammonia removal and glutamine synthesis. Furthermore Hep G2 cells could grow well in the absence of glutamine, which was necessarily required in mammalian cell cultures. These results prove that glutamine formation serves as the primary mechanism of detoxifying ammonia in hepatocyte cell lines as expected. In addition, it was demonstrated that ammonium level could be reduced 38% and that erythropoietin production increased 2-fold in the mixed culture of Hep G2 and recombinant CHO cells.