Background: Delayed graft function (DGF) following kidney transplantation is associated with inferior short- and long-term transplant outcomes and is not preventable by current therapies. The complement pathway has been proposed as a target for DGF prevention, as it majorly contributes to ischemia-reperfusion injury (IRI) underlying DGF. We addressed the impact of blocking the classical (CP) and lectin (LP) complement pathways at the level of complement factor C2, thereby leaving the alternative (AP) complement pathway intact, in a rat model of kidney transplantation. Methods: Recipient rats received a kidney exposed to a long cold ischemia time and were treated intravenously with anti-C2 monoclonal antibody (mAb) or placebo control in the peri-transplantation period. Serum creatinine levels and blood urea were analyzed at Day 3 post-transplant. Transplanted kidneys from surviving animals were histologically examined at Day 14. As the involvement of the three distinct complement pathways in IRI is species dependent, we further assessed the relative importance of the CP/LP in IRI-induced complement activation on different human endothelial cells in vitro using a hypoxic chamber. Results: Blocking C2 activity significantly improved serum creatinine and blood urea nitrogen levels 3 days post-transplant compared with those kidneys treated with placebo. Furthermore, kidney health at 2 weeks post-transplant improved, as indicated by a reduced incidence and/or severity of IRI-related histopathological findings, including tubular regeneration, kidney enlargement, and discoloration, which were associated with reduced kidney weight. In several human endothelial cell types, C3 fixation was consistently dependent on C2 presence and could be prevented by anti-C2 mAb treatment in a dose-dependent manner. Conclusions: We confirmed in preclinical models that C2 is a suitable target to prevent complement-induced IRI and subsequent DGF after kidney transplant.

BACKGROUND:Multifocal motor neuropathy (MMN) is a rare, chronic immune-mediated polyneuropathy characterized by asymmetric distal limb weakness. An important feature of MMN is the presence of IgM antibodies against gangliosides, in particular GM1 and less often GM2. Antibodies against GM1 bind to motor neurons (MNs) and cause damage through complement activation. The involvement of Schwann cells (SCs), expressing GM1 and GM2, in the pathogenesis of MMN is unknown.METHODS:Combining the data of our 2007 and 2015 combined cross-sectional and follow-up studies in Dutch patients with MMN, we evaluated the presence of IgM antibodies against GM1 and GM2 in serum from 124 patients with MMN and investigated their binding to SCs and complement-activating properties. We also assessed the relation of IgM binding and complement deposition with clinical characteristics.RESULTS:Thirteen out of 124 patients (10%) had a positive ELISA titer for IgM anti-GM2. Age at onset of symptoms was significantly lower in MMN patients with anti-GM2 IgM. IgM binding to SCs correlated with IgM anti-GM2 titers. We found no correlation between IgM anti-GM2 titers and MN binding or with IgM anti-GM1 titers. IgM binding to SCs decreased upon pre-incubation of serum with soluble GM2, but not with soluble GM1. IgM anti-GM2 binding to SCs correlated with complement activation, as reflected by increased C3 fixation on SCs and C5a formation in the supernatant.CONCLUSION:Circulating IgM anti-GM2 antibodies define a subgroup of patients with MMN that has an earlier onset of disease. These antibodies probably target SCs specifically and activate complement, similarly as IgM anti-GM1 on MNs. Our data indicate that complement activation by IgM antibodies bound to SCs and MNs underlies MMN pathology.

BACKGROUND AND PURPOSE:Complement factor C2 is a potential therapeutic target in immune-mediated neuropathies. However, literature suggests that classical complement pathway activation may proceed to C3 in the absence of C2, a so-called "C2 bypass." Here, we evaluated a C2 bypass mechanism during complement activation by pathogenic human IgM from patients with immune-mediated neuropathies. METHODS:IgM autoantibodies from 51 patients with multifocal motor neuropathy (MMN) or anti-myelin-associated glycoprotein (MAG) neuropathy (AMN) were used to activate complement in ex vivo disease models. C2 bypass was evaluated using C2-depleted (C2D) serum and a therapeutic anti-C2 antibody. RESULTS:In two different disease models of MMN, IgM anti-GM1 and IgM anti-GM2 autoantibodies from MMN patients were bound to induced pluripotent stem cell-derived motor neurons and Schwann cells, respectively, and fixed C3 upon incubation with fresh serum. C3 fixation was inhibited by anti-C2 and did not occur with C2D serum. Similarly, in an AMN model, IgM anti-MAG antibodies were incubated with fresh serum fixed C3, which in all cases was abrogated in the absence of C2 or in the presence of anti-C2. CONCLUSIONS:In ex vivo disease models of MMN and AMN, complement activation by IgM autoantibodies from 51 patients was in all cases dependent on C2 and was inhibited by an antihuman C2 antibody. No evidence of a C2 bypass mechanism was found.

Peripheral neuropathy is a frequent complication of type 2 diabetes mellitus (T2DM). We investigated whether human islet amyloid polypeptide (hIAPP), which forms pathogenic aggregates that damage pancreatic islet β cells in T2DM, is involved in T2DM-associated peripheral neuropathy. In vitro, hIAPP incubation with sensory neurons reduced neurite outgrowth and increased levels of mitochondrial reactive oxygen species. hIAPP-transgenic mice, which have elevated plasma hIAPP levels without hyperglycemia, developed peripheral neuropathy as evidenced by pain-associated behavior and reduced intraepidermal nerve fiber (IENF) density. Similarly, hIAPP Ob/Ob mice, which have hyperglycemia in combination with elevated plasma hIAPP levels, had signs of neuropathy, although more aggravated. In wild-type mice, intraplantar and intravenous hIAPP injections induced long-lasting allodynia and decreased IENF density. Non-aggregating murine IAPP, mutated hIAPP (pramlintide), or hIAPP with pharmacologically inhibited aggregation did not induce these effects. T2DM patients had reduced IENF density and more hIAPP oligomers in the skin compared with non-T2DM controls. Thus, we provide evidence that hIAPP aggregation is neurotoxic and mediates peripheral neuropathy in mice. The increased abundance of hIAPP aggregates in the skin of T2DM patients supports the notion that hIAPP is a potential contributor to T2DM neuropathy in humans.

Background Idiopathic non-histaminergic acquired angioedema (InH-AAE) does not respond to anti-histamines and is possibly mediated by bradykinin. Objective To investigate the efficacy and safety of recombinant human C1-esterase inhibitor (rhC1-INH), for prophylaxis of InH-AAE, and to evaluate contact system parameters as biomarkers for attacks. Methods A prospective, open-label study of patients with InH-AAE with > 2 AE attacks/month. rhC1-INH (50 IU/kg was administered intravenously; maximum 4200 IU) twice weekly for 2 months, preceded and followed by a one-month period of observation. The primary endpoint was a >50% reduction in attack frequency. C1INH-function and plasma levels of high molecular weight kininogen, factor XII, plasma prekallikrein, C4, cleaved kininogen, d-dimer were evaluated as potential biomarkers. Post-trial follow-up was evaluated in all patients. Results Six patients (mean age 41 years; four female) were enrolled. One patient showed a reduction in attack frequency of 84% (3 versus 19) during rhC1-INH treatment, and showed clinical response to plasma-derived C1-INH but not to omalizumab post-trial. Restarting rhC1-INH treatment resulted in a similarly rapid response. The other 5 patients showed no improvement. No major adverse events were reported. None of the measured biomarkers were related to treatment response. Post-trial, omalizumab was administered to four patients, of which two reported response. Conclusion rhC1-INH treatment was effective in 1 of 6 InH-AAE patients, suggesting a bradykinin-dependent mechanism of attacks in this patient. Response to omalizumab or tranexamic acid during follow-up in a number of the patients points to a heterogeneous pathogenesis of InH-AAE disease requiring a personalised treatment approach.

Background and ObjectivesTo determine the role of complement in the disease pathology of multifocal motor neuropathy (MMN), we investigated complement activation, and inhibition, on binding of MMN patient-derived immunoglobulin M (IgM) antibodies in an induced pluripotent stem cell (iPSC)-derived motor neuron (MN) model for MMN.MethodsiPSC-derived MNs were characterized for the expression of complement receptors and membrane-bound regulators, for the binding of circulating IgM anti-GM1 from patients with MMN, and for subsequent fixation of C4 and C3 on incubation with fresh serum. The potency of ARGX-117, a novel inhibitory monoclonal antibody targeting C2, to inhibit fixation of complement was assessed.ResultsiPSC-derived MNs moderately express the complement regulatory proteins CD46 and CD55 and strongly expressed CD59. Furthermore, MNs express C3aR, C5aR, and complement receptor 1. IgM anti-GM1 antibodies in serum from patients with MMN bind to MNs and induce C3 and C4 fixation on incubation with fresh serum. ARGX-117 inhibits complement activation downstream of C4 induced by patient-derived anti-GM1 antibodies bound to MNs.DiscussionBinding of IgM antibodies from patients with MMN to iPSC-derived MNs induces complement activation. By expressing complement regulatory proteins, particularly CD59, MNs are protected against complement-mediated lysis. Yet, because of expressing C3aR, the function of these cells may be affected by complement activation upstream of membrane attack complex formation. ARGX-117 inhibits complement activation upstream of C3 in this disease model for MMN and therefore represents an intervention strategy to prevent harmful effects of complement in MMN.

New therapeutic approaches to resolve persistent pain are highly needed. We tested the hypothesis that manipulation of cytokine receptors on sensory neurons by clustering regulatory cytokine receptor pairs with a fusion protein of interleukin (IL)-4 and IL-10 (IL4-10 FP) would redirect signaling pathways to optimally boost pain-resolution pathways. We demonstrate that a population of mouse sensory neurons express both receptors for the regulatory cytokines IL-4 and IL-10. This population increases during persistent inflammatory pain. Triggering these receptors with IL4-10 FP has unheralded biological effects, because it resolves inflammatory pain in both male and female mice. Knockdown of both IL4 and IL10 receptors in sensory neurons in vivo ablated the IL4-10 FP-mediated inhibition of inflammatory pain. Knockdown of either one of the receptors prevented the analgesic gain-of-function of IL4-10 FP. In vitro, IL4-10 FP inhibited inflammatory mediator-induced neuronal sensitization more effectively than the combination of cytokines, confirming its superior activity. The IL4-10 FP, contrary to the combination of IL-4 and IL-10, promoted clustering of IL-4 and IL-10 receptors in sensory neurons, leading to unique signaling, that is exemplified by activation of shifts in the cellular kinome and transcriptome. Interrogation of the potentially involved signal pathways led us to identify JAK1 as a key downstream signaling element that mediates the superior analgesic effects of IL4-10 FP. Thus, IL4-10 FP constitutes an immune-biologic that clusters regulatory cytokine receptors in sensory neurons to transduce unique signaling pathways required for full resolution of persistent inflammatory pain.

Due to the increasing number of therapeutic monoclonal antibodies (mAbs) used in the clinic, there is an increasing need for robust analytical methods to quantify total mAb concentrations in human plasma for clinical studies and therapeutic drug monitoring. We developed an easy, rapid, and robust sample preparation method for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The method was validated for infliximab (IFX), rituximab (RTX), cetuximab (CTX), dupilumab (DPL), dinutuximab (DNX), vedolizumab (VDZ), and emicizumab (EMZ). Saturated ammonium sulfate (AS) was used to precipitate immunoglobulins in human plasma. After centrifugation, supernatant containing albumin was decanted, and the precipitated immunoglobulin fraction was re-dissolved in buffer containing 6M guanidine. This fraction was then completely denatured, reduced, alkylated, and trypsin digested. Finally, signature peptides from the seven mAbs were simultaneously quantified on LC-MS/MS together with their internal standards stable isotopically labeled peptide counterparts. The linear dynamic ranges (1 – 512 mg/L) of IFX, CTX, RTX, and EMZ showed excellent (R2 > 0.999) linearity and those of DPL, DNX, and VDZ showed good (R2 > 0.995) linearity. The method was validated in accordance with the EMA guidelines. EDTA plasma, sodium citrate plasma, heparin plasma, and serum yielded similar results. Prepared samples were stable at room temperature (20°C) and at 5°C for 3 days, and showed no decline in concentration for all tested mAbs. This described method, which has the advantage of an easy, rapid, and robust pre-analytical sample preparation, can be used as a template to quantify other mAbs in human plasma or serum.

Background: Activation of the classical and lectin pathway of complement may contribute to tissue damage and organ dysfunction of antibody-mediated diseases and ischemia-reperfusion conditions. Complement factors are being considered as targets for therapeutic intervention. Objective: We sought to characterize ARGX-117, a humanized inhibitory monoclonal antibody against complement C2. Methods: The mode-of-action and binding characteristics of ARGX-117 were investigated in detail. Furthermore, its efficacy was analyzed in in vitro complement cytotoxicity assays. Finally, a pharmacokinetic/pharmacodynamic study was conducted in cynomolgus monkeys. Results: Through binding to the Sushi-2 domain of C2, ARGX-117 prevents the formation of the C3 proconvertase and inhibits classical and lectin pathway activation upstream of C3 activation. As ARGX-117 does not inhibit the alternative pathway, it is expected not to affect the antimicrobial activity of this complement pathway. ARGX-117 prevents complement-mediated cytotoxicity in in vitro models for autoimmune hemolytic anemia and antibody-mediated rejection of organ transplants. ARGX-117 exhibits pH- and calcium-dependent target binding and is Fc-engineered to increase affinity at acidic pH to the neonatal Fc receptor, and to reduce effector functions. In cynomolgus monkeys, ARGX-117 dose-dependently reduces free C2 levels and classical pathway activity. A 2-dose regimen of 80 and 20 mg/kg separated by a week, resulted in profound reduction of classical pathway activity lasting for at least 7 weeks. Conclusions: ARGX-117 is a promising new complement inhibitor that is uniquely positioned to target both the classical and lectin pathways while leaving the alternative pathway intact.

PEGylation of lipid-based nanoparticles and other nanocarriers is widely used to increase their stability and plasma half-life. However, either pre-existing or de novo formed anti-PEG antibodies can induce hypersensitivity reactions and accelerated blood clearance through binding to the nanoparticle surfaces, leading to activation of the complement system. In this study, we investigated the consequences and mechanisms of complement activation by anti-PEG antibodies interacting with different types of PEGylated lipid-based nanoparticles. By using both liposomes loaded with different (model) drugs and LNPs loaded with mRNA, we demonstrate that complement activation triggered by anti-PEG antibodies can compromise the bilayer/surface integrity, leading to premature drug release or exposure of their mRNA contents to serum proteins. Anti-PEG antibodies also can induce deposition of complement fragments onto the surface of PEGylated lipid-based nanoparticles and induce the release of fluid phase complement activation products. The role of the different complement pathways activated by lipid-based nanoparticles was studied using deficient sera and/or inhibitory antibodies. We identified a major role for the classical complement pathway in the early activation events leading to the activation of C3. Our data also confirm the essential role of amplification of C3 activation by alternative pathway components in the lysis of liposomes. Finally, the levels of pre-existing anti-PEG IgM antibodies in plasma of healthy donors correlated with the degree of complement activation (fixation and lysis) induced upon exposure to PEGylated liposomes and mRNA-LNPs. Taken together, anti-PEG antibodies trigger complement activation by PEGylated lipid-based nanoparticles, which can potentially compromise their integrity, leading to premature drug release or cargo exposure to serum proteins.

OBJECTIVES:Granzymes are serine proteases involved in eliminating tumour cells and virally infected cells. In addition, extracellular granzyme levels are elevated in inflammatory conditions, including several types of infection and autoimmune diseases, such as rheumatoid arthritis (RA). While GrA and GrB have been associated with RA, a role for the other three granzymes (GrH, GrK, and GrM) in this disease remains unclear. Here, we aimed to investigate the presence and role of GrM and GrK in serum and synovial fluid of patients with RA, psoriatic arthritis, and osteoarthritis.METHODS:Granzyme levels were determined in serum, synovial fluid, peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) of RA patients and relevant control groups. In addition, the link between GrM and inflammatory cytokines in synovial fluid was investigated.RESULTS:Serum GrM and GrK levels were not affected in RA. GrM, but not GrK, levels were elevated in synovial fluid of RA patients. GrM was mainly expressed by cytotoxic lymphocytes in SFMCs with a similar expression pattern as compared with PBMCs. Intra-articular GrM expression correlated with IL-25, IL-29, XCL1, and TNFα levels. Intriguingly, purified GrM triggered the release of IL-29 (IFN-λ1) from human fibroblasts in vitro.CONCLUSIONS:These data indicate that GrM levels are increased in RA synovial fluid and that GrM can stimulate proinflammatory IL-29 release from fibroblasts, suggesting a role of GrM in the pathogenesis of RA.

Hereditary angioedema (HAE) is a potentially life-threatening disorder caused by dangerous attacks of bradykinin-mediated tissue swelling. Plasma kallikrein (PKa) is a critical player in this disease. It cleaves high-molecular-weight kininogen (HK) into a cleaved form of HK (cHK), which liberates bradykinin. In addition, it contributes to plasminogen activation and accelerates factor XII activation. The oral PKa inhibitor ATN-249 is under clinical development as a treatment for HAE, which is an appealing alternative to injectable therapies. This compound was recently investigated in a phase I ascending dose study in healthy participants (ACTRN12618000430235).1Kalfus I, Offman E, McDonald A. Pharmacokinetics and safety of ATN-249, a novel oral plasma kallikrein inhibitor for hereditary angioedema. Poster presented at: Western Society of Allergy, Asthma and Immunology 2019 Annual Scientific Session. January 20-24, 2019; Wailea, Hawaii.Google Scholar,2Kalfus I. McDonald A. Qian S. Potency, selectivity, and exposure evaluation of ATN-249, a new oral kallikrein inhibitor for hereditary angioedema [abstract].J Allergy Clin Immunol. 2017; 139: AB378Abstract Full Text Full Text PDF Google Scholar Here, we have investigated the biologic activity of ATN-249 in plasma of these subjects. As bradykinin production is difficult to directly determine, we investigated the effects of ATN-249 on triggered PKa activity and cHK generation instead. The study was a phase I randomized, double-blind, placebo-controlled single–ascending dose study. After providing written informed consent, 48 healthy males received a single oral dose of ATN-249 (50, 100, 150, 200, 400, or 800 mg). Each dose cohort consisted of 8 subjects: 6 were given active compound and 2 were given placebo. Plasma samples were collected before the dose and at 2, 4, 9, 12, and 24 hours after intake. Threshold stimulation of PKa activity in plasma has value in the diagnostics of bradykinin-dependent HAE.3Lara-Marquez M.L. Christiansen S.C. Riedl M.A. Herschbach J. Zuraw B.L. Threshold-stimulated kallikrein activity distinguishes bradykinin- from histamine-mediated angioedema.Clin Exp Allergy. 2018; 48: 1429-1438Crossref PubMed Scopus (23) Google Scholar Similar approaches have been used to demonstrate the biologic activity of other HAE therapies.4Cornpropst M. Collis P. Collier J. Babu Y.S. Wilson R. Zhang J. et al.Safety, pharmacokinetics, and pharmacodynamics of avoralstat, an oral plasma kallikrein inhibitor: phase 1 study.Allergy. 2016; 71: 1676-1683Crossref PubMed Scopus (9) Google Scholar, 5Aygören-Pürsün E. Bygum A. Grivcheva-Panovska V. Magerl M. Graff J. Steiner U.C. et al.Oral plasma kallikrein inhibitor for prophylaxis in hereditary angioedema.N Engl J Med. 2018; 379: 352-362Crossref PubMed Scopus (64) Google Scholar, 6Zhang J. Kellogg D. Wilson R. Harman L. Bantia S. Babu Y.S. A simple, sensitive and selective fluorogenic assay to monitor kallikrein activity in activated plasma [abstract].J Allergy Clin Immunol. 2012; 131: AB210Google Scholar However, the level to which PKa needs to be inhibited to effectively protect HK remains uncertain. We recently developed an ELISA-based assay to detect cHK in plasma of patients with HAE.7Hofman Z.L.M. de Maat S. Suffritti C. Zanichelli A. van Doorn C. Sebastian S.A.E. et al.Cleaved kininogen as a biomarker for bradykinin release in hereditary angioedema.J Allergy Clin Immunol. 2017; 140: 1700-1703Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar On the basis of this assay, we developed a method to investigate the drug activity of ATN-249 (for a full description, see the Methods section in this article’s Online Repository at www.jacionline.org). Stimulation of plasma with 2.5 μM ellagic acid for 3 minutes leads to consistent cHK production (see Fig E1, A and B in this article’s Online Repository at www.jacionline.org). For comparison, we monitored PKa activity in plasma with a fluorogenic substrate (see Fig E1, C). All 48 study subjects provided predose samples. Triggered PKa activity and cHK generation in this sample collection varied widely (Fig 1, A and C [the amount of converted PKa substrate after 15 minutes is shown]). We next examined these parameters on an individual basis for each donor in samples that were consecutively collected over 24 hours within the group of placebo-treated subjects (n = 12). This analysis revealed that each healthy placebo-treated subject generated a comparable amount of PKa activity (mean SD, 1.0 × 106 relative fluorescence units) and cHK (mean SD, 6.15% cHK) (Fig 1, B and D). Surprisingly, there was a large interindividual variation between subjects. As can be expected, there was a moderately positive correlation between triggered PKa activity and cHK generation (Fig 1, E [consecutive data of all placebo-treated subjects]). In the study subjects who received ATN-249, triggered PKa activity and cHK generation were transiently depressed. We examined the data after normalization to correct for interindividual variation (ie, 100% signifies the amount of cHK that a subject formed before drug intake). ATN-249 intake transiently attenuated triggered PKa activity in all groups 2 hours after intake, with a mean decrease in PKa activity ranging from 92.5% (95% CI, 72.2%-112.7%) for the 800 mg dose to 47.1 % (95% CI, 20.4%-73.8%) for the 50 mg dose. We observed a decrease in triggered PKa activity that lasted until 12 hours after intake in the groups with doses between 150 and 400 mg and until 24 hours for 800 mg (Fig 2, A). Decreased cHK generation in the group treated with 800 mg of ATN-249 was observed from 2 hours after intake (a mean decrease in cHK generation of 41.1% (95% CI, 22.3%-59.9%) and remained statistically significant until 9 hours after intake (Fig 2, B). In groups that received lower doses (150-400 mg), transiently decreased cHK generation was seen until 4 hours after intake. A subanalysis of data at 2 hours after ATN-249 intake showed a dose-dependent inhibition of PKa activity that was accompanied by decreased cHK generation (Fig 2, C [compound data of all time points during treatment in Fig E2 and individual tracings of study subjects in Fig E3, both of which are available in this article’s Online Repository at www.jacionline.org]). In this study, we demonstrated the biologic effect of the oral PKa inhibitor ATN-249 in the plasma of healthy male subjects on ex vivo triggered PKa activity and cHK generation. To our knowledge, we are first to report considerable interindividual variability in these assays, arguing against a generalized cutoff value for PKa activity or plasma cHK levels for HAE management. It will be of high interest to further investigate variables that might influence the observed interindividual variation, including sex, age, and lifestyle-related factors. A limitation of this study is that we used ellagic acid as a trigger for contact activation. This nonphysiologic agent is routinely used in coagulation diagnostics but is most probably not representative for the (currently unidentified) trigger of bradykinin production in patients with HAE. It is likely that the endogenous triggers for PKa activity in HAE are milder, and as a result, the therapeutic efficacy of ATN-249 may be larger than our data suggest. PKa activity assays can be rapidly performed, which is favorable for clinical diagnostics.8Kaplan A.P. Maas C. The search for biomarkers in hereditary angioedema.Front Med (Lausanne). 2017; 4: 206Crossref PubMed Scopus (29) Google Scholar However, we propose that there is value in determining the level of HK protection in plasma by directly investigating this molecule. We found that ATN-249 inhibits triggered PKa activity more efficiently than triggered cHK generation. There are several explanations for this mismatch: first, although active-site inhibitors may efficiently compete with low-affinity synthetic fluorogenic tripeptide substrates for the active site of PKa, the real aim of these agents is to protect its natural target, HK, against cleavage. This mechanism depends on a natural multisite binding interaction between PKa and HK, which is much more challenging to disrupt. Second, most plasma coagulation factors, including plasma prekallikrein, are present at higher levels in plasma than are required for normal function.9Bird J.E. Smith P.L. Wang X. Schumacher W.A. Barbera F. Revelli J.P. Seiffert D. Effects of plasma kallikrein deficiency on haemostasis and thrombosis in mice: Murine Ortholog of the Fletcher Trait.Thromb Haemost. 2012; 107: 1140-1150Crossref Scopus (61) Google Scholar As a result, a high degree of PKa inhibition is required to protect HK from cleavage and prevent bradykinin production. Our data support this: whereas a single dose of 800 mg of ATN-249 inhibits triggered PKa activity by 92% (95% CI, 72%-112%), cHK generation was inhibited by 41% (95% CI, 22%-60%) (Fig 2, C). It is of high interest to identify the extent of PKa inhibition and HK protection that are required for therapeutic management of HAE, and it cannot be excluded that this needs to be determined on an individual basis. In conclusion, we propose that strategies to monitor triggered PKa and cHK generation hold promise for personalized medicine in patients with HAE. We thank Ms Wariya Sanrattana for preparing the artwork. BSA, ellagic acid (EA) (E2250), dextran sulfate ([average] molecular weight, 500,000 g/mol), skimmed milk powder, NaCl, Na2HPO4, NaH2PO4, and Tween-20 were obtained from Sigma-Aldrich (St. Louis, Mo). The 96-well flat-bottom MaxiSorp microtitre plates, FluoroNunc black polystyrene microtitre plates, 0.8-mL polyprolypene deepwell storage plates (AB-0765), and storage plate cap strips (AB0981) were obtained from Thermo Scientific (Waltham, Mass). The 1.2-mL polypropylene tubes were obtained from BRAND GMBH + CO KG (Wertheim, Germany). β-Factor XIIa, was obtained from Enzyme Research Laboratories (South Bend, Ind). 3,3′,5,5′-Tetramethyl-benzidine was obtained from Tebu Bio (Heerhugowaard, The Netherlands). Streptavidin poly–horseradish peroxidase was obtained from Sanquin Blood Supply, Reagents Division (Amsterdam, The Netherlands). Phe-Pro-Arg-chloromethylketone (PPACK) was purchased from Haematologic Technologies (Essex Junction, Vt). Anti-cleaved kininogen specific monoclonal variable domain of a heavy-chain-only antibody (VhH)-D1 and biotinylated monoclonal anti-kininogen VhH-H4 were produced in our laboratory as previously published.E1Hofman Z.L.M. de Maat S. Suffritti C. Zanichelli A. van Doorn C. Sebastian S.A.E. et al.Cleaved kininogen as a biomarker for bradykinin release in hereditary angioedema.J Allergy Clin Immunol. 2017; 140: 1700-1703Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar The substrate Z-Phe-Arg-AMC·HCl (I-1160) was obtained from Bachem (Bubendorf, Switzerland). Sodium citrate blood collection tubes were obtained from Becton Dickinson Vacutainers (Vianen, The Netherlands). The SpectraMax M4 Microplate reader used for absorbance measurements and the SpectraMax iD3 used for for fluorescence measurements were obtained from Molecular Devices (San Jose, Calif). A randomized, double-blind, placebo-controlled single–ascending dose study was performed with ATN-249 (ACTRN12618000430235). After giving written informed consent, 48 healthy male participants (age 18-55 years) were divided into 6 ascending dose cohorts. Each cohort consisted of 8 participants. Six participants received a single oral dose of ATN-249 and the 2 remaining participants received control treatment consisting of hard gelatin capsules identical to those containing ATN-249. ATN-249 was administered in capsules following overnight fasting of at least 10 hours, and ascending doses were administered per cohort as follows: 50 mg, 100 mg, 150 mg, 200 mg, 400 mg, or 800 mg of ATN-249. Serial blood draws were collected in sodium citrate blood collection tubes (3.2% [wt/vol]) before the dose and at 2, 4, 9, 12, and 24 hours after intake. Samples were centrifuged at 2000 g for 10 minutes at 4°C, and plasma was collected and stored at –80°C. Before the analyses, plasma samples were thawed at 37°C and briefly vortexed, and 50-μL aliquots were stored in deepwell storage plates at –80°C until analysis. Normal pooled plasma (NPP) was collected under the approval of the University Medical Center Utrecht ethical committee. Blood was collected following standard procedures in sodium citrate (3.2% [wt/vol]) blood collection tubes from approximately 50 healthy volunteers who signed written informed consent. Shortly after collection, the tubes were centrifuged twice at 2000 g for 10 minutes, and the plasma was stored at –80°C. Samples underwent 1 extra freeze-thaw cycle when being pooled. We set out to determine a trigger condition for plasma cHK generation that is sensitive to PKa inhibition. We performed titration experiments with EA (0-20 μM) to activate NPP. NPP in 1.2-mL polypropylene tubes was prewarmed at 37°C for at least 5 minutes before activation. Predilutions of ellagic acid were prepared in HEPES-buffered saline (HBS) (10 mmol/L of HEPES, 150 mmol/L of NaCl, 1 mmol/L of MgSO4, and 5 mmol/L of KCl [pH 7.4]) and were prewarmed at 37°C for at least 5 minutes before activation. NPP (100 μL) was activated by adding prediluted EA (100 μL), followed by vigorous resuspending. After 1, 5, or 10 minutes, activation was stopped with PPACK (final concentration, 150 μM) by diluting samples 4-fold in PBS-1 (127.9 mM NaCl, 6.2 mM Na2HPO4, and 3.7 mM NaH2PO4 [pH 7.0]) supplemented with 0.1% (vol/vol) Tween-20 and 1% (wt/vol) skimmed milk (modified PBS with Tween-20 [MPBST]), as well as 200 μM PPACK. The samples were diluted further in MPBST containing 50 μM PPACK to a 141-fold dilution. We coated 96-well-flat-bottom microtiter plates with 5 μg/mL of VhH D1 (50 μL per well) in PBS-2 (137 mM NaCl, 2.7 mM KCl, 9.2 mM Na2HPO4, and 1.76 mM KH2PO4] [pH 7.4]) overnight at 4°C. The plates were blocked for 2 hours with 150 μL of MPBST per well while shaking at 600 rpm at room temperature (RT). For the assay standards, NPP was activated by incubating 9 volumes of NPP with 1 volume of β-factor XIIa in PBS-2 (final concentration, 1 μg/mL) at 37°C for 10 minutes. Next, activated plasma was further diluted to a 141-fold final dilution in mPBST, with 50 μM PPACK to stop enzymatic activity. Nonactivated plasma samples were prepared in the same way (but without adding β-factor XIIa to PBS-2). These diluted nonactivated and activated plasma samples were mixed together at various ratios to generate a standard curve with a cHK range from 0% to 100%. Plasma samples from study subjects (50-μL aliquots) were thawed at 37°C, vortexed, and prewarmed at 37°C for 10 minutes before activation. Plasma samples were activated by adding 50 μL of prewarmed EA (final concentration, 2.5 μM) to the plasma followed by vigorous resuspending. After 3 minutes, contact activation was stopped by 4-fold sample dilution into MPBST containing 200 μM PPACK. The samples were further diluted in MPBST containing 50 μM PPACK to a 141-fold final dilution. Dextran sulfate (final concentration, 0.5 μg/mL in PBS-2) was added to the samples and the samples were vigorously mixed on a plate vortex, and incubated for 10 minutes. Hereafter, samples were vigorously mixed on a plate vortex and incubated on the plates with immobilized capture VhH D1 in duplicate (50 μL per well) for 1 hour while shaking at 600 rpm at RT. Next, the plates were washed 3 times with 150 μL of PBS-1 with 0.1% (vol/vol) Tween-20 (PBST). Detection VhH H4-biotin (2 μg/mL in MPBST, 25 μM PPACK) was added (50 μL per well) and incubated for 1 hour while shaking at 600 rpm at RT. Plates were washed 3 times with 150 μL of PBST per well. Next, 50 μL of streptavidin poly–horeseradish peroxidase (0.5 μg/mL in MPBST and 25 μM PPACK) per well was added and incubated for 30 minutes while shaking at 600 rpm at RT. Plates were washed 3 times with 150 μL of PBST. Next, 100 μL of 3,3′,5,5′-tetramethyl-benzidine per well was added and substrate conversion was measured at 650 nm for 20 minutes, with absorbance measured every minute. A calibration curve was generated in GraphPad Prism 7.0 software (GraphPad Software, La Jolla, Calif) by using a sigmoidal 4PL fit model on which the test samples were interpolated. We blocked 96-well flat-bottom black plates with 150 μL of HBS containing 0.1% BSA (wt/vol) at 37°C for 20 minutes on a plate heater. Plasma (NPP or study samples) was thawed at 37°C for 5 to 10 minutes and vortexed, after which 15 μL was added to the plates. Next, I-1160 (20 μL per well prediluted in HBS to a final concentration of 50 μM) was added, followed by EA (15 μL per well prediluted in HBS to a final concentration of 2.5 μM). Substrate conversion was immediately monitored at 360/460 (excitation/emission) every minute for 30 minutes by an iD3 Spectramax plate reader at 37°C, with samples shaken before every read. Relative fluorescent units after 15 minutes were used for data analysis. Data were analyzed by using GraphPad Prism 7 software. Column Statistics was used for descriptive statistics and normality testing, and a sigmoidal 4PL fit model was used to interpolate samples. The Dunnett multiple comparisons test was used for analysis of effect on cHK generation and PKa activity. The Spearman rank test was used to calculate correlation between cHK and PKa. The Kruskal-Wallis test was used for differences among consecutive samples of study subjects receiving placebo. In addition to analysis of the cHK values, cHK values normalized to predose cHK levels for individual study subjects were analyzed, where relative cHK level = cHK at time point x/predose cHK × 100. Likewise, the relative fluorescence units (RFUs) of PKa substrate conversion were normalized to the predose RFU for individual study subjects as follows: Relative PKa activity = RFU at time point x/predose RFU × 100.

BACKGROUND:Chronic spontaneous urticaria (CSU) is characterized by recurrent itchy weals and/or angioedema and is believed to be driven by mast cell activation. It was shown that excessive mast cell activation during anaphylaxis initiates contact activation, resulting in bradykinin release. Evidence for bradykinin release was never demonstrated in CSU.OBJECTIVE:To study biomarkers of bradykinin release in CSU.METHODS:Plasma samples of CSU patients were collected during routine visits at the outpatient clinic. Cleaved high molecular weight kininogen (cHK) was used as a biomarker for bradykinin release. cHK, factor XIIa-C1-inhibitor (FXIIa-C1-INH), kallikrein-C1-INH, plasmin-antiplasmin (PAP) complexes and soluble urokinase-type plasminogen activator receptor (suPAR) levels were determined by ELISA. Clinical data and data on tryptase levels were collected from medical records. cHK levels were compared to previously determined levels in hereditary angioedema (HAE).RESULTS:One hundred seventeen samples from 88 CSU patients and 28 samples from healthy controls were analysed. Median cHK level in CSU was 9.1% (range: 1.4%-21.5%), significantly increased compared to healthy controls (median 6.0% range: 0%-19.9%; P = .0005) and comparable to HAE (n = 46, median 10.3%, range 0%-44.3%, P > .9999). cHK levels normalized in patients during disease remission (median 6.5% range 1.5%-20.8%) but were not dependent on the presence of angioedema, acute angioedema attacks or response to antihistamines. Surprisingly, cHK levels were inversely correlated to serum tryptase (r = -0.65 P = .0137). C1-INH complexes and suPAR levels were not elevated in patients compared to healthy controls. PAP-complex levels in patients were elevated compared to healthy controls but there was no correlation between PAP-complex and cHK levels.CONCLUSIONS:cHK levels are elevated in symptomatic CSU patients compared to healthy controls, indicating increased bradykinin production. Increased cHK levels are not limited to patients with angioedema.CLINICAL RELEVANCE:If elevated bradykinin generation has clinical implications in the pathology of CSU is open to debate.

The addition of rabbit anti-human thymocyte globulin (ATG) to the conditioning regimen prior to allogeneic hematopoietic cell transplantation has significantly reduced the risk of graft- versus -host disease (GvHD) and graft failure. However, ATG has a small therapeutic window. Overexposure of ATG post-HCT hampers T cell immune reconstitution and has been associated with increased relapse rates and viral reactivations, whereas underexposure has been associated with an increased incidence of GvHD, both of which lead to increased mortality. Therapeutic drug monitoring of T cell binding ATG plasma levels provides a means to optimize dosing for patients at high risk for graft failure to ensure timely T cell immune reconstitution and subsequently increase survival chances. This manuscript describes the first liquid chromatography tandem-mass spectrometry (LC-MS/MS) method to quantify the pharmacologically active fraction of polyclonal ATG in plasma. This was achieved through immunoaffinity purification of active ATG from plasma with Jurkat T cells. After the binding and washing, samples were eluted, denatured, and trypsin-digested. Signature peptides originating from the IgG constant chain were measured with LC-MS/MS. Critical method parameters were optimized, and the method was successfully validated following European Medicines Agency (EMA) guidelines. The method covered the therapeutic range of ATG and was validated at a lower limit of quantification (LLOQ) of 1 AU/mL with an overall CV and bias of 11.8% and − 2.5%, respectively. In conclusion, we developed a LC-MS/MS-based method to quantify active polyclonal rabbit ATG in human plasma. We suggest that this novel assay can be used to monitor and optimize dosing of ATG in clinical practice.

Fibrosis is a condition shared by numerous inflammatory diseases. Our incomplete understanding of the molecular mechanisms underlying fibrosis has severely hampered effective drug development. CXCL4 is associated with the onset and extent of fibrosis development in multiple inflammatory and fibrotic diseases. Here, we used monocyte-derived cells as a model system to study the effects of CXCL4 exposure on dendritic cell development by integrating 65 longitudinal and paired whole genome transcriptional and methylation profiles. Using data-driven gene regulatory network analyses, we demonstrate that CXCL4 dramatically alters the trajectory of monocyte differentiation, inducing a novel pro-inflammatory and pro-fibrotic phenotype mediated via key transcriptional regulators including CIITA. Importantly, these pro-inflammatory cells directly trigger a fibrotic cascade by producing extracellular matrix molecules and inducing myofibroblast differentiation. Inhibition of CIITA mimicked CXCL4 in inducing a pro-inflammatory and pro-fibrotic phenotype, validating the relevance of the gene regulatory network. Our study unveils that CXCL4 acts as a key secreted factor driving innate immune training and forming the long-sought link between inflammation and fibrosis.

ObjectiveAn ideal disease modifying osteoarthritis drug (DMOAD) has chondroprotective, anti-inflammatory, and analgesic effects. This study describes the production and characterization of a canine IL4-10 fusion protein (IL4-10 FP) and evaluates its in vivo DMOAD activity in a canine model of osteoarthritis (OA).DesignThe canine Groove model was used as an in vivo model of degenerative knee OA. Six weeks after OA induction dogs were intra-articularly injected weekly, for ten weeks, with either IL4-10 FP or phosphate buffered saline (PBS). In addition to the use of human IL4-10 FP, canine IL4-10 FP was developed and characterized in vitro, and tested in vivo. Force plate analysis (FPA) was performed to analyze joint loading as a proxy measure for pain. After ten weeks dogs were euthanized and cartilage and synovial tissue samples were analyzed by histochemistry (OARSI scores) and biochemistry (cartilage proteoglycan turnover).ResultsRepetitive intra-articular injections with human IL4-10 FP led to antibody formation, that blocked its functional activity. Therefore, a canine IL4-10 FP was developed, which completely inhibited LPS-induced TNFα production by canine blood cells, and increased proteoglycan synthesis of canine cartilage in vitro (p = 0.043). In vivo, canine IL4-10 FP restored the, by OA impaired, joint loading (p = 0.002) and increased cartilage proteoglycan content (p = 0.029).ConclusionsThis first study on the potential DMOAD activity upon prolonged repeated treatment with IL4-10 FP demonstrates that a species-specific variant has anti-inflammatory and chondroprotective effects in vitro and chondroprotective and analgesic effects in vivo. These data warrant further research on the DMOAD potential of the IL4-10 FP.

Antihistamines are the most prescribed therapy in recurrent idiopathic angioedema, yet little is known about their efficacy. Herein, we report on clinical improvement with antihistamine therapy in 120 patients evaluating angioedema attack frequency. A high incidence (36%) of antihistamine refractory cases was observed. Forty percent of patients on antihistamine prophylaxis suffered from 1 or more angioedema attacks per month. Our findings stress the need for additional treatment options for recurrent idiopathic angioedema.

Fibrosis is a condition shared by numerous inflammatory diseases. Our incomplete understanding of the molecular mechanisms underlying fibrosis has severely hampered effective drug development. CXCL4 is associated with the onset and extent of fibrosis development in systemic sclerosis, a prototypic inflammatory and fibrotic disease. Here, we integrated 65 paired longitudinal transcriptional and methylation profiles from monocyte-derived cells with/without CXCL4 exposure. Using data-driven gene regulatory network analyses, we demonstrate that CXCL4 dramatically alters the trajectory of monocyte differentiation, inducing a novel pro-inflammatory and pro-fibrotic phenotype mediated via key transcriptional regulators including CIITA. CXCL4 exposed monocyte-derived cells directly trigger a fibrotic cascade by producing ECM molecules and inducing myofibroblast differentiation. Inhibition of CIITA mimicked CXCL4 in inducing a pro-inflammatory and pro-fibrotic phenotype, validating the relevance of the gene regulatory network. Our study unveils CXCL4 as a key secreted factor driving innate immune training and forming the long-sought link between inflammation and fibrosis.

Coagulation factor XII (FXII) drives production of the inflammatory peptide bradykinin. Pathological mutations in the F12 gene, which encodes FXII, provoke acute tissue swelling in hereditary angioedema (HAE). Interestingly, a recently identified F12 mutation, causing a W268R substitution, is not associated with HAE. Instead, FXII-W268R carriers experience cold-inducible urticarial rash, arthralgia, fever, and fatigue. Here, we aimed to investigate the molecular characteristics of the FXII-W268R variant. We expressed wild type FXII (FXII-WT), FXII-W268R, and FXII-T309R (which causes HAE), as well as other FXII variants in HEK293 freestyle cells. Using chromogenic substrate assays, immunoblotting, and ELISA, we analyzed expression media, cell lysates, and purified proteins for FXII activation. Recombinant FXII-W268R forms increased amounts of intracellular cleavage products that are also present in expression medium and display enzymatic activity. The active site-incapacitated variant FXII-W268R/S544A reveals that intracellular fragmentation is largely dependent on autoactivation. Purified FXII-W268R is highly sensitive to activation by plasma kallikrein and plasmin, compared with FXII-WT or FXII-T309R. Furthermore, binding studies indicated that the FXII-W268R variant leads to the exposure of a plasminogen-binding site that is cryptic in FXII-WT. In plasma, recombinant FXII-W268R spontaneously triggers high-molecular-weight kininogen cleavage. Our findings suggest that the W268R substitution influences FXII protein conformation and exposure of the activation loop, which is concealed in FXII-WT. This results in intracellular autoactivation and constitutive low-grade secretion of activated FXII. These findings help to explain the chronically increased contact activation in carriers of the FXII-W268R variant.