Serum neurofilament light for detecting disease activity in individual patients in multiple sclerosis: A 48-week prospective single-center study.

BACKGROUND: Serum neurofilament light (sNfL) reflects neuroaxonal damage and is now used as an outcome in treatment trials of relapsing-remitting multiple sclerosis (RRMS). However, the diagnostic properties of sNfL for monitoring disease activity in individual patients warrant further investigations. METHOD: Patients with suspected relapse and/or contrast-enhancing lesions (CELs) were consecutively included and performed magnetic resonance imaging (MRI) of the brain at baseline and weeks 28 and 48. Serum was obtained at baseline and 2, 4, 8, 16, 24, and 48 weeks. Neurofilament light concentration was measured using Single molecule array technology. RESULTS: We included 44 patients, 40 with RRMS and 4 with clinically isolated syndrome. The median sNfL level peaked at 2 weeks post-baseline (14.6 ng/L, interquartile range (IQR); 9.3-31.6) and reached nadir at 48 weeks (9.1 ng/L, IQR; 5.5-15.0), equivalent to the median sNfL of controls (9.1 ng/L, IQR; 7.4-12). A baseline Z-score of more than 1.1 (area under the curve; 0.78, p < 0.0001) had a sensitivity of 81% and specificity of 70% to detect disease activity. CONCLUSION: One out of five patients with relapse and/or CELs did not change significantly in post-baseline sNfL levels. The utility of repeated sNfL measurements to monitor disease activity is complementary rather than a substitute for clinical and MRI measures.

A phase I, dose-escalation study of TB-403, a monoclonal antibody directed against PlGF, in patients with advanced solid tumours.

BACKGROUND: TB-403 (RO 5323441), a humanised monoclonal antibody, is a novel antiangiogenesis agent directed against placental growth factor. The safety, pharmacokinetics (PK), and antitumour activity of TB-403 were assessed in a phase I, dose-escalation study in patients with advanced solid tumours. METHODS: Patients in sequential dose groups received either weekly doses of 1.25, 5.0, or 10 mg kg(-1) or doses of 20 or 30 mg kg(-1) every third week. RESULTS: Twenty-three patients were enrolled and received TB-403. The most common adverse events (AEs) were fatigue, constipation, pyrexia, dyspnoea, and nausea. One serious AE, a lung embolus in a patient with non-small cell lung cancer treated with 10 mg kg(-1) weekly, was deemed possibly related to TB-403. No dose-limiting toxicities were observed, and a maximum-tolerated dose was not reached. The PK parameters were dose linear and the terminal half-life values ranged from 9 to 14 days. Six patients exhibited stable disease for at least 8 weeks. Two patients, (oesophageal squamous cell carcinoma and pancreatic adenocarcinoma) both treated with 5 mg kg(-1) weekly, remained stable for 12 months. CONCLUSION: TB-403 treatment in this patient population is well tolerated, with a safety profile distinct from that of vascular endothelial growth factor-axis inhibitors.

Characterization of the EGF-like module pair 3-4 from vitamin K-dependent protein S using NMR spectroscopy reveals dynamics on three separate time scales and extensive effects from calcium binding.

Protein S, a cofactor of anticoagulant activated protein C, exhibits three high-affinity Ca(2+)-binding sites in a region comprising four EGF modules. The EGF 3-4 module pair constitutes the smallest fragment that retains one high-affinity Ca(2+)-binding site and is therefore useful for investigation of the structural basis of the unusually high-affinity Ca(2+) binding compared to other EGF-containing proteins characterized so far. Extensive chemical shift effects caused by Ca(2+) binding to the EGF 3-4 module pair are observed, particularly from Ca(2+) binding to the high-affinity site in EGF 4. Ca(2+) binding to the high-affinity site in EGF 4 and the low-affinity site in EGF 3 is associated with slow and fast exchange on the NMR time-scale, respectively. We show the presence of two isoforms, characterized by a cis or trans Lys 167-Pro 168 peptide bond, that do not convert on time scales that were accessible to the experiments (k(ex) < 0.2 s(-1)). Both conformers have similar Ca(2+) affinities and backbone dynamics. Further, broadening of (1)H resonances involving residues in the major beta-sheet of EGF 3 and (15)N exchange terms, primarily in the N-terminal part of the protein, indicate the presence of slow exchange on a microsecond to millisecond time scale. (15)N spin relaxation data suggest that the module pair has a well-defined relative orientation between EGF modules 3 and 4 and has a significantly anisotropic rotational diffusion tensor in solution.

1H, 15N and (13)C assignments and secondary structure of the EGF-like module pair 3-4 from vitamin K-dependent protein S.

Vitamin K-dependent protein S, which is a cofactor for activated protein C and thus important for down-regulation of the coagulation cascade, contains several Ca(2+)-binding sites with unusually high affinity. The 89 amino acid fragment constituting the third and fourth epidermal growth factor-like (EGF) modules of protein S is the smallest fragment that retains high-affinity Ca(2+) binding and is therefore useful for investigating the structural basis of this property. Heteronuclear multidimensional nuclear magnetic resonance experiments were used to obtain extensive assignments of the (1)H, 15N and (13)C resonances of the module pair with one Ca(2+) bound in EGF 4. In addition, nearly complete assignments of the (1)H resonances of the isolated Ca(2+)-free EGF 3 module were obtained. The assignment process was complicated by broadening of several resonances, spectral heterogeneity caused by cis-trans isomerisation of the peptide bond preceding Pro-168, and dimerisation. Analysis of weighted average secondary chemical shifts, (3)J(HNHalpha) coupling constants, and NOE connectivities suggest that both EGF modules in this fragment adhere to the classical secondary structure of EGF modules, consisting of one major and one minor anti-parallel beta-sheet.

Calcium-binding EGF-like modules in coagulation proteinases: function of the calcium ion in module interactions.

Epidermal growth factor (EGF)-like modules are involved in protein-protein interactions and are found in numerous extracellular proteins and membrane proteins. Among these proteins are enzymes involved in blood coagulation, fibrinolysis and the complement system as well as matrix proteins and cell surface receptors such as the EGF precursor, the low density lipoprotein receptor and the developmentally important receptor, Notch. The coagulation enzymes, factors VII, IX and X and protein C, all have two EGF-like modules, whereas the cofactor of activated protein C, protein S, has four EGF-like modules in tandem. Certain of the cell surface receptors have numerous EGF modules in tandem. A subset of EGF modules bind one Ca(2+). The Ca(2+)-binding sequence motif is coupled to a sequence motif that brings about beta-hydroxylation of a particular Asp/Asn residue. Ca(2+)-binding to an EGF module is important to orient neighboring modules relative to each other in a manner that is required for biological activity. The Ca(2+) affinity of an EGF module is often influenced by its N-terminal neighbor, be it another EGF module or a module of another type. This can result in an increase in Ca(2+) affinity of several orders of magnitude. Point mutations in EGF modules that involve amino acids which are Ca(2+) ligands result in the biosynthesis of biologically inactive proteins. Such mutations have been identified, for instance, in factor IX, causing hemophilia B, in fibrillin, causing Marfan syndrome, and in the low density lipoprotein receptor, causing hypercholesterolemia. In this review the emphasis will be on the coagulation factors.

EGF-like module pair 3-4 in vitamin K-dependent protein S: modulation of calcium affinity of module 4 by module 3, and interaction with factor X.

Calcium-binding epidermal growth factor (EGF)-like modules are found in numerous extracellular and membrane proteins involved in such diverse processes as blood coagulation, lipoprotein metabolism, determination of cell fate, and cell adhesion. Vitamin K-dependent protein S, a cofactor of the anticoagulant enzyme activated protein C, has four EGF-like modules in tandem with the three C-terminal modules each harbouring a Ca(2+)-binding consensus sequence. Recombinant fragments containing EGF modules 1-4 and 2-4 have two Ca(2+)-binding sites with dissociation constants ranging from 10(-8) to 10(-5) M. Module-module interactions that greatly influence the Ca(2+) affinity of individual modules have been identified. As a step towards an analysis of the structural basis of the high Ca(2+) affinity, we expressed the Ca(2+)-binding EGF pair 3-4 from human protein S. Correct folding was shown by (1)H NMR spectroscopy. Calcium-binding properties of the C-terminal module were determined by titration with chromophoric chelators; binding to the low-affinity N-terminal site was monitored by (1)H-(15)N NMR spectroscopy. At physiological pH and ionic strength, the dissociation constants for Ca(2+) binding were 1.0x10(-6) M and 4. 8x10(-3) M for modules 4 and 3, respectively, i.e. the calcium affinity of the C-terminal site was about 5000-fold higher than that of the N-terminal site. Moreover, the Ca(2+) affinity of EGF 4, in the pair 3-4, was about 9000-fold higher than that of synthetic EGF 4. The EGF modules in protein S are known to mediate the interaction with factor Xa. We have now found modules 3-4 to be involved in this interaction. However, the individual modules 3 and 4 manifested no measurable activity.

Characterization of recombinant epidermal growth factor (EGF)-like modules from vitamin-K-dependent protein S expressed in Spodoptera cells--the cofactor activity depends on the N-terminal EGF module in human protein S.

Epidermal growth factor (EGF)-like modules in protein S, a physiological anticoagulant protein that functions as a cofactor to activated protein C, have been expressed in Spodoptera cells using baculovirus. EGF modules 1-3, 1-4, 2-3 and 2-4 were produced on a preparative scale. The isolated modules were more than 95% homogeneous, as judged by sequence determination. 45Ca2+-ligand blotting experiments indicated that recombinant proteins that contained the fourth EGF module, i.e. EGF 1-4 and 2-4, bound Ca2+ with high affinity. The 45Ca2+-ligand blotting results, together with results of competitive binding experiments using monoclonal antibodies as structural probes, indicated that the recombinant proteins had been folded to a native conformation. EGF modules 1-3 and 1-4 inhibited the interaction between activated protein C and protein S, whereas modules 2-3 and 2-4 had no effect on this interaction. It is thus apparent that EGF module 1 is crucial for the interaction between protein S and activated protein C. Moreover, EGF modules 1-4 were approximately 10-fold more effective in inhibiting the interaction than modules 1-3, suggesting a very weak interaction between module 4 and activated protein C or that this module is important to keep module 1 in a conformation that is optimal for interaction with activated protein C.

The high affinity calcium-binding sites in the epidermal growth factor module region of vitamin K-dependent protein S.

Vitamin K-dependent protein S, a cofactor of the anticoagulant enzyme-activated protein C, has four epidermal growth factor (EGF)-like modules, all of which have one partially hydroxylated Asp (EGF 1; beta-hydroxyaspartic acid) or Asn (EGF 2, 3, and 4; beta-hydroxyasparagine) residue. The three C-terminal modules have a typical Ca2+ binding sequence motif that is usually present in EGF modules with hydroxylated Asp/Asn residues. Using the chromophoric Ca2+ chelators Quin 2 and 5,5'-Br2BAPTA, we have now determined the Ca2+ affinity of recombinant fragments containing EGF modules 1-3, 1-4, 2-3, and 2-4. EGF modules 1-4 and 2-4 each contains two very high affinity Ca2+-binding sites, i.e. with dissociation constants ranging from 10(-10) to 10(-8) M in the absence of salt and from 10(-8) to 10(-6) M in the presence of 0.15 M NaCl. In contrast, in EGF 1-3 and EGF 2-3, the Ca2+ affinity is 2-4 orders of magnitude lower. EGF 4 thus appears to have the highest Ca2+ affinity, and furthermore it seems to influence the Ca2+ affinity of its immediate N-terminal neighbor EGF 3 by a factor of approximately 230. In addition, EGF 4 seems to influence the Ca2+ affinity of EGF 2 by a factor of approximately 25. The Ca2+ affinity of the binding sites in EGF modules 3 and 4 in fragments EGF 1-4 and EGF 2-4 is 10(3)-10(5)-fold higher than in the corresponding isolated modules, implying important contributions to the Ca2+ affinity of each module from interactions with neighboring modules. This difference is much higher than the approximately 10-fold difference previously found in similar comparisons of EGF modules from fibrillin. However, the modules studied in protein S and fibrillin appear to have the similar Ca2+ ligands. The structural basis for the difference in Ca2+ affinity is not yet understood.

Calcium-binding properties of the third and fourth epidermal-growth-factor-like modules in vitamin-K-dependent protein S.

Protein S is a plasma glycoprotein requiring vitamin K for normal biosynthesis and functioning as a cofactor of activated protein C, a regulator of blood coagulation. Protein S contains four modules that are similar to the epidermal growth factor (EGF) precursor. Qualitative Ca2+-binding experiments have indicated that the EGF-module region of bovine protein S harbors high-affinity Ca2+-binding sites. We have chemically synthesized the third and fourth EGF modules from human protein S, which both have the sequence motif associated with Ca2+-binding and Asp/Asn beta-hydroxylation. Both modules were folded to a native conformation, as judged by immunochemical experiments and NMR spectroscopy. Ca2+ binding to the modules was monitored with 1H-NMR spectroscopy. At physiological pH and 0.15 M NaCl, each module was found to have a single Ca2+-binding site with low affinity, i.e. Kd values of 6.1 mM for the third and 8.6 mM for the fourth EGF module. At low salt conditions the Ca2+ affinities are 5.2 mM and 0.6 mM, respectively. This Ca2+ affinity is similar to that of the isolated N-terminal EGF module from coagulation factors IX and X. The very high affinity Ca2+ binding to the EGF-module region of protein S thus appears to be due to the influence of neighboring modules.