Background The kallikrein-kinin system (KKS) is a complex biochemical pathway that plays a crucial role in regulating several physiological processes, including inflammation, coagulation, and blood pressure. Dysregulation of the KKS has been associated with several pathological conditions such as hereditary angioedema (HAE), hypertension, and stroke. Developing an accurate quantitative model of the KKS may provide a better understanding of its role in health and disease and facilitate the rapid and targeted development of effective therapies for KKS-related disorders. Objectives Here, we present a novel, detailed mechanistic model of the plasma KKS, elucidating the processes of Factor XII (FXII) activation, the kallikrein feedback loop, cleavage of high molecular weight kininogen leading to bradykinin (BK) production, and the impact of inhibitors. Methods The model incorporates both surface and solution-phase reactions of all proteins in the KKS, describing how binding site concentration affects the rate of surface reactions. The model was calibrated and validated using a variety of published and in-house experimental datasets, which encompass a range of dextran sulphate (DXS) concentrations to initiate contact activation and various KKS inhibitors to block bradykinin production. Results Our mathematical model showed that a trace amount of activated FXII is required for subsequent FXII activation. The model also reveals a bell-shaped curve relationship between the activation of the KKS and the number of DXS surface binding sites. Simulations of BK generation in healthy and HAE plasma demonstrated the impact of C1 esterase inhibitor (C1inh) deficiency via increased peak BK levels and accelerated formation in HAE plasma. The efficacy of KKS inhibitors, such as CSL312, ecallantide, and C1inh, was also evaluated, with CSL312 showing the most potent inhibition of BK generation. Conclusions The present model represents a valuable framework for studying the intricate interactions within the plasma KKS and provides a better understanding of the mechanism of action of various KKS-targeted therapies.

ABSTRACT Human serum albumin (HSA) has a long circulatory half-life owing, in part, to interaction with the neonatal Fc receptor (FcRn or FCGRT) in acidic endosomes and recycling of internalised albumin. Vascular endothelial and innate immune cells are considered the most relevant cells for FcRn-mediated albumin homeostasis in vivo. However, little is known about endocytic trafficking of FcRn–albumin complexes in primary human endothelial cells. To investigate FcRn–albumin trafficking in physiologically relevant endothelial cells, we generated primary human vascular endothelial cell lines from blood endothelial precursors, known as blood outgrowth endothelial cells (BOECs). We mapped the endosomal system in BOECs and showed that BOECs efficiently internalise fluorescently labelled HSA predominantly by fluid-phase macropinocytosis. Pulse-chase studies revealed that intracellular HSA molecules co-localised with FcRn in acidic endosomal structures and that the wildtype HSA, but not the non-FcRn-binding HSAH464Q mutant, was excluded from late endosomes and/or lysosomes. Live imaging revealed that HSA is partitioned into FcRn-positive tubules derived from maturing macropinosomes, which are then transported towards the plasma membrane. These findings identify the FcRn–albumin trafficking pathway in primary vascular endothelial cells, relevant to albumin homeostasis.

Human Complement Receptor 1 (HuCR1) is a potent membrane-bound regulator of complement both in vitro and in vivo, acting via interaction with its ligands C3b and C4b. Soluble versions of HuCR1 have been described such as TP10, the recombinant full-length extracellular domain, and more recently CSL040, a truncated version lacking the C-terminal long homologous repeat domain D (LHR-D). However, the role of N-linked glycosylation in determining its pharmacokinetic (PK) and pharmacodynamic (PD) properties is only partly understood. We demonstrated a relationship between the asialo-N-glycan levels of CSL040 and its PK/PD properties in rats and non-human primates (NHPs), using recombinant CSL040 preparations with varying asialo-N-glycan levels. The clearance mechanism likely involves the asialoglycoprotein receptor (ASGR), as clearance of CSL040 with a high proportion of asialo-N-glycans was attenuated in vivo by co-administration of rats with asialofetuin, which saturates the ASGR. Biodistribution studies also showed CSL040 localization to the liver following systemic administration. Our studies uncovered differential PD effects by CSL040 on complement pathways, with extended inhibition in both rats and NHPs of the alternative pathway compared with the classical and lectin pathways that were not correlated with its PK profile. Further studies showed that this effect was dose dependent and observed with both CSL040 and the full-length extracellular domain of HuCR1. Taken together, our data suggests that sialylation optimization is an important consideration for developing HuCR1-based therapeutic candidates such as CSL040 with improved PK properties and shows that CSL040 has superior PK/PD responses compared with full-length soluble HuCR1.

The neonatal Fc receptor (FcRn) is responsible for the recycling of endocytosed albumin. Here we reveal the spatiotemporal dynamics of intracellular albumin–FcRn interactions required for recycling in living primary macrophages by fluorescence lifetime imaging microscopy and raster image correlation spectroscopy.

Background and Aims: Replacement FIX therapy (rIX) is an effective treatment for hemophilia B even with undetectable levels in the blood 1. However, the mechanistic reason for hemostasis with low plasma levels is not well understood. There is growing evidence that FIX interactions with one or multiple binding partners (BP), may play a significant role in the exposure and hemostatic efficacy of rIX 2,3. The aim of this study is to explore this hypothesis by comparing the plasma PK, tissue biodistribution, and in vivo endpoints of different rIX variants using a mouse QSP model.

Background We have recently reported on a recombinant von Willebrand factor (VWF) D ' D3 albumin fusion protein (rD ' D3-FP) developed to extend the half-life of coagulation factor VIII (FVIII) for the treatment of hemophilia A. Based on predictive modelling presented in this study, we hypothesized that modifying rD ' D3-FP to improve FVIII interaction would reduce exchange with endogenous VWF and provide additional FVIII half-life benefit. Objectives The aim of this study was to identify novel rD ' D3-FP variants with enhanced therapeutic efficacy in extending FVIII half-life. Methods Through both directed mutagenesis and random mutagenesis using a novel mammalian display platform, we identified novel rD ' D3-FP variants with increased affinity for FVIII (rVIII-SingleChain) under both neutral and acidic conditions and assessed their ability to extend FVIII half-life in vitro and in vivo. Results In rat preclinical studies, rD ' D3-FP variants with increased affinity for FVIII displayed enhanced potency, with reduced dose levels required to achieve equivalent rVIII-SingleChain half-life extension. In cell-based imaging studies in vitro, we also demonstrated reduced dissociation of rVIII-SingleChain from the rD ' D3-FP variants within acidic endosomes and more efficient co-recycling of the rD ' D3-FP/rVIII-SingleChain complex via the FcRn recycling system. Conclusions In summary, at potential clinical doses, the rD ' D3-FP variants provide marked benefits with respect to dose levels and half-life extension of co-administered FVIII, supporting their development for use in the treatment of hemophilia A.

The neonatal Fc receptor (FcRn) rescues albumin and IgG from degradation following endocytosis and thereby extends the half-life of these plasma proteins. However, the pathways for the uptake of these soluble FcRn ligands, and the recycling itinerary of the FcRn-ligand complexes, have not been identified in primary cells. Here, we have defined the recycling of human albumin and IgG in primary mouse macrophages selectively expressing the human FcRn. Albumin is internalised by macropinocytosis; in the absence of FcRn, internalised albumin is rapidly degraded, while in the presence of FcRn albumin colocalises to SNX5-positive membrane domains and is partitioned into tubules emanating from early macropinosomes for delivery in transport carriers to the plasma membrane. Soluble monomeric IgG was also internalised by macropinocytosis and rapidly recycled by the same pathway. In contrast, the fate of IgG bound to surface Fcγ receptors differed from monomeric IgG endocytosed by macropinocytosis. Overall, our findings identify a rapid recycling pathway for FcRn ligands from early macropinosomes to the cell surface of primary cells.

A novel mechanism for extending the circulatory half-life of coagulation factor VIII (FVIII) has been established and evaluated preclinically. The FVIII binding domain of von Willebrand factor (D'D3) fused to human albumin (rD'D3-FP) dose dependently improved pharmacokinetics parameters of coadministered FVIII in all animal species tested, from mouse to cynomolgus monkey, after IV injection. At higher doses, the half-life of recombinant FVIII (rVIII-SingleChain) was calculated to be increased 2.6-fold to fivefold compared with rVIII-SingleChain administered alone in rats, rabbits, and cynomolgus monkeys, and it was increased 3.1-fold to 9.1-fold in mice. Sustained pharmacodynamics effects were observed (ie, activated partial thromboplastin time and thrombin generation measured ex vivo). No increased risk of thrombosis was observed with coadministration of rVIII-SingleChain and rD'D3-FP compared with rVIII-SingleChain alone. At concentrations beyond the anticipated therapeutic range, rD'D3-FP reduced the hemostatic efficacy of coadministered rVIII-SingleChain. This finding might be due to scavenging of activated FVIII by the excessive amount of rD'D3-FP which, in turn, might result in a reduced probability of the formation of the tenase complex. This observation underlines the importance of a fine-tuned balance between FVIII and its binding partner, von Willebrand factor, for hemostasis in general.

We investigate the capacity of published numerical models of thrombin generation to reproduce experimentally observed threshold behavior under conditions in which diffusion and/or flow are important. Computational fluid dynamics simulations incorporating species diffusion, fluid flow, and biochemical reactions are compared with published data for thrombin generation in vitro in 1) quiescent plasma exposed to patches of tissue factor and 2) plasma perfused through a capillary coated with tissue factor. Clot time is correctly predicted in individual cases, and some models qualitatively replicate thrombin generation thresholds across a series of tissue factor patch sizes or wall shear rates. Numerical results suggest that there is not a genuine patch size threshold in quiescent plasma-clotting always occurs given enough time-whereas the shear rate threshold observed under flow is a genuine physical limit imposed by flow-mediated washout of active coagulation factors. Despite the encouraging qualitative results obtained with some models, no single model robustly reproduces all experiments, demonstrating that greater understanding of the underlying reaction network, and particularly of surface reactions, is required. In this direction, additional simulations provide evidence that 1) a surface-localized enzyme, speculatively identified as meizothrombin, is significantly active toward the fluorescent thrombin substrate used in the experiments or, less likely, 2) thrombin is irreversibly inhibited at a faster-than-expected rate, possibly explained by a stimulatory effect of plasma heparin on antithrombin. These results highlight the power of simulation to provide novel mechanistic insights that augment experimental studies and build our understanding of complex biophysicochemical processes. Further validation work is critical to unleashing the full potential of coagulation models as tools for drug development and personalized medicine.

Shear induced lift and drag forces on a small, neutrally buoyant, rigid spherical particle, moving close to a wall (l/a = 1.2) are examined. These wall induced forces are computed numerically in a linear flow for low particle and shear Reynolds numbers (Rep ≤ 1 & Reγ ≤ 1). Mesh resolution and domain size dependencies for low Reynolds numbers are examined. Decomposed slip and shear hydrodynamic forces are analysed separately for a fixed and moving particle. Numerical results are compared with theoretically predicted forces for Reγ, Rep « 1. The deviations of lift forces from theoretical values demonstrate a strong shear rate dependence near the wall for finite Reγ values. Results presented will aid in developing a comprehensive wall-induced lift model valid for a range of finite Reynolds numbers to analyze the cross stream migration of particles in wall bounded flows.

Humic substances (HSs) are considered as natural colloidal particles or polyelectrolytes and represent a big portion of recalcitrant organic carbon in the terrestrial environment. HSs have more than 50% of elemental carbon with hydrophobic aliphatic and aromatic moieties as well as hydrophilic polar and charged groups. In soil, sediments and water bodies, HSs interact with inorganic and/or organic pollutants and control their fate depending on the charging and aggregation of HSs. Considering the importance of HSs in the natural environmental condition, we focused to investigate the charging and aggregation behavior of humic substances (HSs) in the presence of hydrophobic monovalent cations, namely, tetraphenylphosphonium TPP. Three types of HSs standard Suwannee river fulvic acid (SRFA), Suwannee river humic acid (SRHA), and Leonardite humic acid (LHA) with different aromaticity were used in this investigation. The hydrophobic counter ion TPP induced the charge reversal of the HSs and also formed the large visible aggregates of all the HSs at lower pH. The iso-electric point (IEP) or charge reversal pH of LHA was higher than that of SRFA and SRHA in every concentrations of TPPCl (tetraphenylphosphonium chloride), demonstrating the strong hydrophobic interaction between HSs, especially LHA, and TPP. The large remarkable aggregation was observed in the case of LHA with TPP, indicating the existence of stronger hydrophobic attraction among LHAs and TPP. Microscopic observation and the size determination by dynamic light scattering were carried out to confirm the aggregation. The HS aggregates showed fractal structure with values of fractal dimension Df between 2-2.2 in unstirred conditions, revealing that the HS aggregates with TPP were formed via cluster-cluster aggregation with restructuring. The fractal dimension Df increased to 2.8-2.9 in stirring conditions, and the aggregates deemed to be formed by breakage and the regrowth between smaller aggregates and larger aggregates.

Hereditary angioedema (HAE) is a rare genetic disorder with 4 known disease types that are all clinically characterized by local swellings of the skin (ie, edema of the extremities, genitals, and face), abdominal pain attacks, and, occasionally, life-threatening attacks of laryngeal edema.1Bork K. Pasteurized C1 inhibitor concentrate in hereditary angioedema: pharmacology, safety, efficacy and future directions.Expert Rev Clin Immunol. 2008; 4: 13-20Crossref PubMed Scopus (37) Google Scholar In the event of an HAE attack, therapeutic options include the intravenous administration of C1-inhibitor (C1-INH) concentrate,1Bork K. Pasteurized C1 inhibitor concentrate in hereditary angioedema: pharmacology, safety, efficacy and future directions.Expert Rev Clin Immunol. 2008; 4: 13-20Crossref PubMed Scopus (37) Google Scholar, 2Gompels M.M. Lock R.J. Abinun M. Bethune C.A. Davies G. Grattan C. et al.C1 inhibitor deficiency: consensus document.Clin Exp Immunol. 2005; 139: 379-394Crossref PubMed Scopus (379) Google Scholar kallikrein inhibitors (eg, Ecallantide), and a bradykinin (BK) receptor antagonist (eg, Icatibant). Despite a number of acute treatment options for HAE attacks, the prophylactic treatment of HAE remains an area of unmet medical need. Current prophylactic treatments include intravenously administered C1-INH (CINRYZE), and the recently approved subcutaneous C1-INH product HAEGARDA. Factor XII (FXII) is the principal initiator of the plasma contact system and autoactivates to FXIIa upon contact with a negatively charged surface. Activation of FXII leads to the production of the proinflammatory mediator BK through the kallikrein-kinin system (KKS). The binding of BK to BK type 2 receptors activates various intracellular signaling pathways that dilate vessels, induce chemotaxis of neutrophils, and increase vascular permeability and fluid efflux.3Björkqvist J. Jämsä A. Renné T. Plasma kallikrein: the bradykinin-producing enzyme.Thromb Haemost. 2013; 110: 399-407Crossref PubMed Scopus (106) Google Scholar BK production is increased during HAE attacks and is thought to be the primary mediator of swelling in HAE.4Nussberger J. Cugno M. Amstutz C. Cicardi M. Pellacani A. Agostoni A. Plasma bradykinin in angio-oedema.Lancet. 1998; 351: 1693-1697Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar, 5Nussberger J. Cugno M. Cicardi M. Agostoni A. Local bradykinin generation in hereditary angioedema.J Allergy Clin Immunol. 1999; 104: 1321-1322Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar Like BK, plasma levels of FXIIa have also been shown to increase in patients during HAE attacks.6Csuka D. Veszeli N. Imreh É. Zotter Z. Skopál J. Prohászka Z. et al.Comprehensive study into the activation of the plasma enzyme systems during attacks of hereditary angioedema due to C1-inhibitor deficiency.Orphanet J Rare Dis. 2015; 10: 132Crossref PubMed Scopus (33) Google Scholar, 7Cugno M. Cicardi M. Coppola R. Agostoni A. Activation of factor XII and cleavage of high molecular weight kininogen during acute attacks in hereditary and acquired C1-inhibitor deficiencies.Immunopharmacology. 1996; 33: 361-364Crossref PubMed Scopus (62) Google Scholar However, although FXII is key to the initiation of the plasma contact system, it has not been explored as a target for the treatment of HAE. We previously reported the generation of a fully human recombinant antibody (3F7) that specifically and potently inhibited FXIIa and prevented contact-mediated thrombosis without impairing hemostasis in mouse and rabbit models.8Larsson M. Rayzman V. Nolte M.W. Nickel K.F. Björkqvist J. Jämsä A. et al.A factor XIIa inhibitory antibody provides thromboprotection in extracorporeal circulation without increasing bleeding risk.Sci Transl Med. 2014; 6: 222ra17Crossref PubMed Scopus (232) Google Scholar In this study, we investigate the potential for 3F7, and its affinity matured variant CSL312, to modulate KKS-mediated BK production and subsequent edema formation in a number of in vitro assays and in vivo models. We first examined the effect of 3F7 on mast cell heparin-initiated activation of the KKS using a murine passive cutaneous anaphylaxis model.9Oschatz C. Maas C. Lecher B. Jansen T. Björkqvist J. Tradler T. et al.Mast cells increase vascular permeability by heparin-initiated bradykinin formation in vivo.Immunity. 2011; 34: 258-268Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar 3F7 administered intraperitoneally 4 hours before antigen challenge dose-dependently inhibited ear skin edema, with approximately 90% inhibition observed at 25 mg/kg of 3F7 (Fig 1, A and B). Benchmarking studies were performed to compare the efficacy of 3F7 with the HAE drugs Icatibant (a BK receptor 2 antagonist) and Ecallantide (a plasma kallikrein inhibitor). When administered 10 minutes before antigen challenge, Icatibant and Ecallantide treatment inhibited edema by approximately 40% and 65%, respectively, compared with approximately 90% inhibition with 3F7 (Fig 1, C). Although 3F7 maintained marked inhibition of the passive cutaneous anaphylaxis response when administered 4 hours before antigen injection, the other inhibitors administered at the same time frame displayed reduced efficacy (Fig 1, D), likely because of faster serum clearance. These results highlight the potential of anti-FXIIa antibodies to provide prolonged protection from edema, with significance for prophylactic management of HAE. The effect of 3F7 was also investigated in a C1-INH−/− murine model of angiotensin-converting enzyme inhibitor–induced angioedema. Captopril treatment increases vascular permeability, which can be quantitated by extraction of coadministered Evans blue dye from the colons of treated mice, and was greater in C1-INH−/− than in their wild-type littermates (Fig 1, E). 3F7 administered before captopril treatment ablated vascular permeability increase at doses of 2.5 mg/kg or greater (Fig 1, F). To enhance the potency of 3F7, affinity maturation was undertaken and a lead antibody clone (CSL312) was selected for further development. Compared with the parental 3F7 antibody,8Larsson M. Rayzman V. Nolte M.W. Nickel K.F. Björkqvist J. Jämsä A. et al.A factor XIIa inhibitory antibody provides thromboprotection in extracorporeal circulation without increasing bleeding risk.Sci Transl Med. 2014; 6: 222ra17Crossref PubMed Scopus (232) Google Scholar CSL312 displays a 44-fold increase in affinity to activated βFXIIa (KD = 140 pM) and improved potency in an activated partial thromboplastin time (aPTT) assay with human plasma (see Fig E1 in this article's Online Repository at www.jacionline.org). Although displaying picomolar affinity to activated FXII, CSL312 binding to zymogen FXII is a weaker interaction with rapid dissociation (see Fig E2 in this article's Online Repository at www.jacionline.org). CSL312 specificity for human FXII was tested against a panel of relevant human serine proteases (FVIIa, FIXa, FXa, FXIa, kallikrein, tissue plasminogen activator, activated protein C, and urokinase plasminogen activator) using in vitro chromogenic activity assays. At enzyme to antibody ratios of up to 1:500, no IC50 concentration was reached for any protease tested (data not shown). Prothrombin time was unaltered at any CSL312 concentration tested (data not shown). CSL312 potently inhibits FXIIa chromogenic activity following dextran sulfate (DXS) activation of diluted plasma from normal, HAE types I/II, HAE with normal C1-INH (nC1-INH-HAE), and acquired angioedema with C1-INH deficiency (C1-INH-AAE) donors (see Fig E3 in this article's Online Repository at www.jacionline.org), with maximal FXIIa inhibition observed at 10 μg/mL for all plasma samples tested. A small but statistically significant increase in FXII levels was observed in patients with C1-INH-HAE type I and nC1-INH-HAE when compared with normal donors. An LC-MS/MS assay was developed to measure the concentration of BK in undiluted plasma. Using DXS-activated pooled healthy human plasma, CSL312 completely inhibited BK generation at concentrations of more than 133 nM (Fig 2, A). When compared against clinically approved HAE therapeutics (C1-INH and Ecallantide), CSL312 displayed the most potent inhibition (Fig 2, A). This assay was also used to investigate the efficacy of CSL312 with 48 HAE plasma samples and 4 cynomolgus plasma samples (Fig 2, B). Here, it can be seen that all HAE and cynomolgus samples were completely protected against DXS-mediated activation, with most HAE samples requiring less than 200 nM (30 μg/mL) of CSL312 for complete inhibition and no observed difference in the efficacy of CSL312 between HAE types. Cynomolgus plasma samples require a slightly higher CSL312 concentration (267 nM) for complete protection. The pharmacokinetic and pharmacodynamic profile of CSL312 following intravenous infusion to cynomolgus monkeys was investigated. The pharmacokinetics of CSL312 in cynomologus monkeys were typical for an mAb (Fig 2, C). The beta phase constituted approximately 40% of total clearance, and the terminal half-life was approximately 9.1 days, with neither parameter showing any significant nonlinearity over the dose range (3, 9, 27 mg/kg), as determined by ANOVA. CSL312 administration was well tolerated and a dose-dependent and prolonged increase in aPTT was observed in animals treated with CSL312 (Fig 2, D). There was no increase in prothrombin time at any doses tested (data not shown). In this study, we show that specific antibody-mediated inhibition of FXII protease activity effectively reduced edema formation in animal models and prevented BK formation following contact activation of normal, HAE (C1-INH-HAE type I/II and HAE with normal C1-INH), and acquired angioedema plasma samples. CSL312 displayed a long plasma half-life in cynomolgous monkeys of 9.1 days and prolonged inhibition of FXIIa activity following a single dose over the course of a 36-day study. Coupled with the ability to formulate at high concentrations (>100 mg/mL), it is well suited to subcutaneous prophylactic application in HAE with potential dosing regimens of once every 2 weeks, or less frequently. We are currently undertaking a phase I clinical study with CSL312 in normal human volunteers and aim to investigate the efficacy of CSL312 in patients with HAE in the near future. 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The neonatal Fc receptor (FcRn) has a pivotal role in albumin and IgG homeostasis. Internalized IgG captured by FcRn under acidic endosomal conditions is recycled to the cell surface where exocytosis and a shift to neutral pH promote extracellular IgG release. Although a similar mechanism is proposed for FcRn-mediated albumin intracellular trafficking and recycling, this pathway is less well defined but is relevant to the development of therapeutics exploiting FcRn to extend the half-life of short-lived plasma proteins. Recently, a long-acting recombinant coagulation factor IX-albumin fusion protein (rIX-FP) has been approved for the management of hemophilia B. Fusion to albumin potentially enables internalized proteins to engage FcRn and escape lysosomal degradation. In this study, we present for the first time a detailed investigation of the FcRn-mediated recycling of albumin and the albumin fusion protein rIX-FP. We demonstrate that following internalization via FcRn at low pH, rIX-FP, like albumin, is detectable within the early endosome and rapidly (within 10-15 min) traffics into the Rab11+ recycling endosomes, from where it is exported from the cell. Similarly, rIX-FP and albumin taken up by fluid-phase endocytosis at physiological pH traffics into the Rab11+ recycling compartment in FcRn-positive cells but into the lysosomal compartment in FcRn-negative cells. As expected, recombinant factor IX (without albumin fusion) and an FcRn interaction-defective albumin variant localized to the lysosomal compartments of both FcRn-expressing and nonexpressing cells. These results indicate that FcRn-mediated recycling via the albumin moiety is a mechanism for the half-life extension of rIX-FP observed in clinical studies.

Tanka et al., Microbial Immunol 29(10) 1985, pp. 959-972 (Abstract). Tanka et al., Microbial Immunol, 30(4), 1986, pp. 373-388 (Abstract). Fuji et al., J. Immunol 137(5) 1986, pp. 1552-1556. Sameleon et al., PNAS, 80, 1983, pp. 6972-6976. Dower, Mel Immunol, 22, 1985, pp. 937–947. Dower, J. Immunol 132, 1984, p. 751. Lymphokine Res. 1(3), 1982-Summary of the 3rd Inter national Lymphokine Workshop. (11 Patent Number:

Many membrane cargoes undergo endocytosis and intracellular recycling to the plasma membrane via the early endosomes and the recycling endosomes. However whether specific sorting signals are required for transport from early endosomes to recycling endosomes is not known and the current view is that transport to the recycling endosomes is by a passive default process. Here we show that the cytoplasmic tail of the neonatal Fc receptor (FcRn) contains discrete signals for endocytosis and for sorting to the recycling endosomes. The FcRn cytoplasmic tail has previously been shown to contain the unusual WISL motif for AP2/clathrin-mediated endocytosis. By analysing FcRn mutants and CD8/FcRn chimeric molecules, we have identified an extended WISL sequence (GLPAPWISL) which promotes sorting from the early endosomes to the recycling endosomes. The insertion of GLPAPWISL into the cytoplasmic tail of CD8 resulted in efficient endocytosis and trafficking to the recycling endosomes, with only low levels detected in the late endosomes. Replacement of the highly conserved GLAPAP sequence within the GLPAPWISL motif with alanine residues resulted in endocytosis of the CD8/FcRn chimera to the early endosomes which was then trafficked predominantly to the late endosomes rather than the recycling endosomes. These studies demonstrate that signals within the cytoplasmic domains of membrane cargo can mediate active transport from early to recycling endosomes.

Spatio-temporal regulation of intracellular signalling networks is key to normal cellular physiology; dysregulation of which leads to disease. The family of three mammalian tribbles proteins has emerged as an important controller of signalling via regulating the activity of mitogen activated protein kinases (MAPK), the PI3-kinase induced signalling network and E3 ubiquitin ligases. However, the importance of potential redundancy in the action of tribbles and how the differences in affinities for the various binding partners may influence signalling control is currently unclear. We report that tribbles proteins can bind to an overlapping set of MAPK-kinases (MAPKK) in live cells and dictate the localisation of the complexes. Binding studies in transfected cells reveal common regulatory mechanisms and suggest that tribbles and MAPKs may interact with MAPKKs in a competitive manner. Computational modelling of the impact of tribbles on MAPK activation suggests a high sensitivity of this system to changes in tribbles levels, highlighting that these proteins are ideally placed to control the dynamics and balance of activation of concurrent signalling pathways.

Hematopoiesis is a multistage process involving the differentiation of stem and progenitor cells into distinct mature cell lineages. Here we present Haemopedia, an atlas of murine gene-expression data containing 54 hematopoietic cell types, covering all the mature lineages in hematopoiesis. We include rare cell populations such as eosinophils, mast cells, basophils, and megakaryocytes, and a broad collection of progenitor and stem cells. We show that lineage branching and maturation during hematopoiesis can be reconstructed using the expression patterns of small sets of genes. We also have identified genes with enriched expression in each of the mature blood cell lineages, many of which show conserved lineage-enriched expression in human hematopoiesis. We have created an online web portal called Haemosphere to make analyses of Haemopedia and other blood cell transcriptional datasets easier. This resource provides simple tools to interrogate gene-expression-based relationships between hematopoietic cell types and genes of interest.

Summary There is increasing evidence that activation of inflammatory responses in a variety of tissues is mediated co‐operatively by the actions of more than one cell type. In particular, the monocyte has been implicated as a potentially important cell in the initiation of inflammatory responses to Toll‐like receptor (TLR)‐activating signals. To determine the potential for monocyte‐regulated activation of tissue cells to underpin inflammatory responses in the vasculature, we established cocultures of primary human endothelial cells and monocytes and dissected the inflammatory responses of these systems following activation with TLR agonists. We observed that effective activation of inflammatory responses required bidirectional signalling between the monocyte and the tissue cell. Activation of cocultures was dependent on interleukin‐1 (IL‐1). Although monocyte‐mediated IL‐1β production was crucial to the activation of cocultures, TLR specificity to these responses was also provided by the endothelial cells, which served to regulate the signalling of the monocytes. TLR4‐induced IL‐1β production by monocytes was increased by TLR4‐dependent endothelial activation in coculture, and was associated with increased monocyte CD14 expression. Activation of this inflammatory network also supported the potential for downstream monocyte‐dependent T helper type 17 activation. These data define co‐operative networks regulating inflammatory responses to TLR agonists, identify points amenable to targeting for the amelioration of vascular inflammation, and offer the potential to modify atherosclerotic plaque instability after a severe infection.