Proteomic approach to profiling immune complex antigens in cerebrospinal fluid samples from patients with central nervous system autoimmune diseases

Background: Immune complexes (ICs) may clearly reflect immunological abnormalities caused by disease, especially for autoimmune diseases. Although ICs have been detected in cerebrospinal fluid (CSF) from patients with CNS autoimmune diseases, identities of antigens in such ICs have not been comprehensively determined. Methods: We used immune complexome analysis, in which nano-liquid chromatography-tandem mass spectrometry is employed to comprehensively identify antigens incorporated into ICs in biological fluids, to characterize ICs in CSF samples from patients with CNS autoimmune diseases, and to find disease-specific IC antigen to a certain CNS autoimmune disease. Also, we compared the IC antigens we identified with the reported CSF proteome or with the published plasma proteome to examine if the method is distinguished from the conventional CSF proteome analysis. Results: We identified 176 antigens in 78 CSF samples. We then assessed the overlaps among these antigens, the CSF proteome, and the plasma proteome; 140 of the 176 antigens were found to be exclusively detected by our method. Notably, IC-associated suprabasin in CSF was 100% specific to neuropsychiatric systemic lupus erythematosus (NPSLE). Conclusions: This report is the first to comprehensively identify the antigens incorporated into ICs in CSF. There was limited overlap between the antigens we identified and the CSF proteome or the plasma proteome; therefore, our method can be distinguished from the conventional CSF proteome analysis. Although the sensitivity of disease-specific IC-antigens detected in immune complexome analysis screening, the sensitivity may be improved by developing an ELISA method specifically for detecting the ICs. Immune complexome analysis of CSF may be a new and promising path to biomarker discovery for diagnosis and study for CNS autoimmune diseases

A Novel Highly Potent Autotaxin/ENPP2 Inhibitor Produces Prolonged Decreases in Plasma Lysophosphatidic Acid Formation <i>In Vivo</i> and Regulates Urethral Tension

<div><p>Autotaxin, also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), is a secreted enzyme that has lysophospholipase D activity, which converts lysophosphatidylcholine to bioactive lysophosphatidic acid. Lysophosphatidic acid activates at least six G-protein coupled recpetors, which promote cell proliferation, survival, migration and muscle contraction. These physiological effects become dysfunctional in the pathology of cancer, fibrosis, and pain. To date, several autotaxin/ENPP2 inhibitors have been reported; however, none were able to completely and continuously inhibit autotaxin/ENPP2 <i>in vivo</i>. In this study, we report the discovery of a highly potent autotaxin/ENPP2 inhibitor, ONO-8430506, which decreased plasma lysophosphatidic acid formation.</p><p>The IC₅₀ values of ONO-8540506 for lysophospholipase D activity were 6.4–19 nM for recombinant autotaxin/ENPP2 proteins and 4.7–11.6 nM for plasma from various animal species. Plasma lysophosphatidic acid formation during 1-h incubation was almost completely inhibited by the addition of >300 nM of the compound to human plasma. In addition, when administered orally to rats at a dose of 30 mg/kg, the compound demonstrated good pharmacokinetics in rats and persistently inhibited plasma lysophosphatidic acid formation even at 24 h after administration.</p><p>Smooth muscle contraction is a known to be promoted by lysophosphatidic acid. In this study, we showed that dosing rats with ONO-8430506 decreased intraurethral pressure accompanied by urethral relaxation. These findings demonstrate the potential of this autotaxin/ENPP2 inhibitor for the treatment of various diseases caused by lysophosphatidic acid, including urethral obstructive disease such as benign prostatic hyperplasia.</p></div

<i>In vitro</i> Inhibition of Plasma LysoPLD Activity.

<p>LysoPLD = lysophospholipase D.</p><p>IC₅₀ values of ONO-8430506 and other inhibitors for recombinant human ATX/ENPP2 when a synthetic fluorescent substrate (FS-3) or a biological substrate (16:0-LPC) was used are shown for (A). IC₅₀ values of ONO-8430506 for recombinant ATX/ENPP2 protein enzyme activity and LysoPLD activity in plasma samples from various species when incubated in the presence of a biological substrate 16:0-LPC are shown for (B). When 16:0-LPC was used as a substrate, the enzyme activity was determined based on the amount of choline generated. The IC₅₀ values means ± S.D. of three independent experiments.</p

Pharmacokinetics and Pharmacodynamics of the Inhibitor in Rats.

<p>Blood was collected at various time points after single oral administration of 3 or 30/kg ONO-8430506 to rats. The time course of changes in plasma concentration of ONO-8430506 (A), plasma <i>ex vivo</i> LysoPLD activity (B), and plasma concentrations of various LPAs (C) are shown. Plasma LysoPLD activity is shown relative to LysoPLD activity before administration of the compound. Quantify limit of each LPA from plasma was 5 ng/ml. Results are mean ± S.D. for three rats in each group.</p

<i>In vitro</i> Inhibition of LPA Formation by ONO-8430506 in Human and Rat Plasma.

<p>Human plasma (A) and rat plasma (B) were incubated at 37°C for 1 h. Plasma samples before and after incubation were immediately cooled on ice, LPA was extracted and five molecular species of LPA were quantified by LC-MS/MS. The diagrams on the left show the results from incubation of plasma alone, while the diagrams on the right show the inhibition of LPA formation when plasma was incubated with various concentrations of ONO-8430506. Results are means ± S.D. of four separate experiments in human plasma and two or three experiments in rat plasma.</p

ONO-8430506 regulates <i>in vivo</i> Rat Urethral Tension.

<p>Male SD rats were anesthetized with urethane and the vehicle, tamsulosin (α₁-blocker), or ONO-8430506 were administered intraduodenally. The histograms show the maximum percent decreases (%) in IUP (A and B) and maximum percent decreases in mean blood pressure (C and D) during 20-min after intraduodenal administration of each drug. Results are means ± S.E. for nine or ten rats per group. Comparisons between the treatment groups and vehicle control group were performed using the Dunnett's tests or Student's <i>t</i> tests. ## p<0.01; ### p<0.001 by Student's <i>t</i> test vs Vehicle. ** p<0.01, *** p<0.001 Dunnett test vs Vehicle. $<0.05 by Student's <i>t</i> test vs Tam 0.3. A correlation between the relative inhibition (vs. vehicle control) of plasma LysoPLD activity (%) by the compound and the decreases in IUP (%) at 20 min after intraduodenal administration are shown (E).</p