Relevance of distinct monocyte subsets to clinical course of ischemic stroke patients.

BACKGROUND AND PURPOSE: The most common strategy for treating patients with acute ischemic stroke is thrombolytic therapy, though only a few patients receive benefits because of the narrow time window. Inflammation occurring in the central nervous system (CNS) in association with ischemia is caused by immune cells including monocytes and involved in lesion expansion. If the specific roles of monocyte subsets in stroke can be revealed, they may become an effective target for new treatment strategies. METHODS: We performed immunological examinations of 36 consecutive ischemic stroke patients within 2 days of onset and compared the results with 24 age-matched patients with degenerative disorders. The stroke patients were repeatedly tested for the proportions of monocyte subsets in blood, and serum levels of pro- and anti-inflammatory cytokines immediately after admission, on days 3-7 and 12-16 after stroke onset, and on the day of discharge. In addition, immunological measurements were analyzed for relationships to stroke subtypes and complications, including progressive infarction (PI) and stroke-associated infection (SAI). RESULTS: Monocyte count was significantly increased from 0-16 days after stroke as compared to the controls (p<0.05). CD14(high)CD16(-) classical and CD14(high)CD16(+) intermediate monocytes were significantly increased from 0-7 and 3-16 days after stroke, respectively (p<0.05), whereas CD14 (dim)CD16(high) non-classical monocytes were decreased from 0-7 days (p<0.05). Cardioembolic infarction was associated with a persistent increase in intermediate monocytes. Furthermore, intermediate monocytes were significantly increased in patients with PI (p<0.05), while non-classical monocytes were decreased in those with SAI (p<0.05). IL-17A levels were positively correlated with monocyte count (r=0.485, p=0.012) as well as the percentage of non-classical monocytes (r=0.423, p=0.028), and negatively with that of classical monocytes (r=-0.51, p=0.007) during days 12-16. CONCLUSIONS: Our findings suggest that CD14(high)CD16(+) intermediate monocytes have a role in CNS tissue damage during acute and subacute phases in ischemic stroke especially in relation to cardioembolism

Time courses of monocyte subsets after stroke.

<p>(<b>A</b>) The overall number of circulating monocytes was increased. The percentages of (<b>B</b>) classical and (<b>C</b>) intermediate Mo were increased, whereas that of (<b>D</b>) non-classical Mo was decreased. Bars show the mean ± SEM. *p<0.05, t-test, compared to control. Control, n=24; day 0-2, n=36; day 3-7, n=35; day 12-16; n=33; day of hospital discharge, n=26.</p

Time courses of monocyte subsets regarding stroke complications.

<p>Classical and intermediate Mo were increased, while non-classical Mo were decreased. Patients were divided into (<b>A</b>) with or without PI, (<b>B</b>) excluding SAI, (<b>C</b>) and with or without SAI. *p<0.05, t test, compared to control. †p<0.05, t test, compared to other group in category. PI = progressing infarction, SAI = stroke-associated infection.</p

Stroke subtypes and intermediate monocytes after hyperacute phase of stroke.

<p>(<b>A</b>–<b>C</b>) A significantly elevated percentage of intermediate Mo in the CE group persisted until the day of discharge. *p<0.05, t-test, as compared to control. C: control; LAA: large artery atherosclerosis; CE: cardioembolism; SAO: small artery occlusion.</p

Analysis of monocyte subsets.

<p>(<b>A</b>) Monocytes were gated in a circle using forward scatter (FSC) and side scatter (SSC) plots. (<b>B</b>) Classical monocytes were identified by high expression of CD14 and no expression of CD16 (CD14^highCD16^-), intermediate monocytes by high expression of CD14 and various levels of positivity for the CD16 molecule (CD14^highCD16⁺), and non-classical monocytes by scant expression of CD14 and high expression of CD16 (CD14 ^dimCD16^high). The proportions of each subset were evaluated by comparing the number of cells (dots) in the individual compartment to the total number of monocytes enclosed in the circle designated as P1.</p

Dynamics of pro-inflammatory cytokines after stroke.

<p>(<b>A</b>) In the SAI group (closed diamond), serum IL-6 levels were elevated from 0–7 days after stroke, (<b>B</b>) while serum IL-17A levels were elevated in the PI group without SAI (closed square) from 3–7 days after stroke.</p

Stroke subtype and monocyte subsets from day 0-2 (hyperacute phase).

<p>(<b>A</b>) The overall monocyte counts in the LAA and CE groups were significantly increased as compared to the control. (<b>B</b>, <b>C</b>, <b>D</b>) The percentages for the monocyte subsets in the CE group were significantly changed as compared to the control. Bars show the mean ± SEM. *p<0.05, t-test, compared to control. †p<0.05, ANOVA, compared to CE. C: control; LAA: large artery atherosclerosis; CE: cardioembolism; SAO: small artery occlusion.</p

Safety and efficacy of eculizumab in anti-acetylcholine receptor antibody-positive refractory generalised myasthenia gravis (REGAIN): a phase 3, randomised, double-blind, placebo-controlled, multicentre study

Background Complement is likely to have a role in refractory generalised myasthenia gravis, but no approved therapies specifically target this system. Results from a phase 2 study suggested that eculizumab, a terminal complement inhibitor, produced clinically meaningful improvements in patients with anti-acetylcholine receptor antibody-positive refractory generalised myasthenia gravis. We further assessed the efficacy and safety of eculizumab in this patient population in a phase 3 trial. Methods We did a phase 3, randomised, double-blind, placebo-controlled, multicentre study (REGAIN) in 76 hospitals and specialised clinics in 17 countries across North America, Latin America, Europe, and Asia. Eligible patients were aged at least 18 years, with a Myasthenia Gravis-Activities of Daily Living (MG-ADL) score of 6 or more, Myasthenia Gravis Foundation of America (MGFA) class II\ue2\u80\u93IV disease, vaccination against Neisseria meningitides, and previous treatment with at least two immunosuppressive therapies or one immunosuppressive therapy and chronic intravenous immunoglobulin or plasma exchange for 12 months without symptom control. Patients with a history of thymoma or thymic neoplasms, thymectomy within 12 months before screening, or use of intravenous immunoglobulin or plasma exchange within 4 weeks before randomisation, or rituximab within 6 months before screening, were excluded. We randomly assigned participants (1:1) to either intravenous eculizumab or intravenous matched placebo for 26 weeks. Dosing for eculizumab was 900 mg on day 1 and at weeks 1, 2, and 3; 1200 mg at week 4; and 1200 mg given every second week thereafter as maintenance dosing. Randomisation was done centrally with an interactive voice or web-response system with patients stratified to one of four groups based on MGFA disease classification. Where possible, patients were maintained on existing myasthenia gravis therapies and rescue medication was allowed at the study physician's discretion. Patients, investigators, staff, and outcome assessors were masked to treatment assignment. The primary efficacy endpoint was the change from baseline to week 26 in MG-ADL total score measured by worst-rank ANCOVA. The efficacy population set was defined as all patients randomly assigned to treatment groups who received at least one dose of study drug, had a valid baseline MG-ADL assessment, and at least one post-baseline MG-ADL assessment. The safety analyses included all randomly assigned patients who received eculizumab or placebo. This trial is registered with ClinicalTrials.gov, number NCT01997229. Findings Between April 30, 2014, and Feb 19, 2016, we randomly assigned and treated 125 patients, 62 with eculizumab and 63 with placebo. The primary analysis showed no significant difference between eculizumab and placebo (least-squares mean rank 56\uc2\ub76 [SEM 4\uc2\ub75] vs 68\uc2\ub73 [4\uc2\ub75]; rank-based treatment difference \ue2\u88\u9211\uc2\ub77, 95% CI \ue2\u88\u9224\uc2\ub73 to 0\uc2\ub796; p=0\uc2\ub70698). No deaths or cases of meningococcal infection occurred during the study. The most common adverse events in both groups were headache and upper respiratory tract infection (ten [16%] for both events in the eculizumab group and 12 [19%] for both in the placebo group). Myasthenia gravis exacerbations were reported by six (10%) patients in the eculizumab group and 15 (24%) in the placebo group. Six (10%) patients in the eculizumab group and 12 (19%) in the placebo group required rescue therapy. Interpretation The change in the MG-ADL score was not statistically significant between eculizumab and placebo, as measured by the worst-rank analysis. Eculizumab was well tolerated. The use of a worst-rank analytical approach proved to be an important limitation of this study since the secondary and sensitivity analyses results were inconsistent with the primary endpoint result; further research into the role of complement is needed. Funding Alexion Pharmaceuticals