Additional file 1: of Immune related adverse events associated with anti-CTLA-4 antibodies: systematic review and meta-analysis

Figures S5 to S7 Global immune-related adverse events (irAEs) with ipilimumab all dosage, 3 mg/kg, and 10 mg/kg for all grades and high grade. Figures S8 to S27 Organ-specific irAEs (endocrine, skin, gastrointestinal, and hepatic) for ipilimumab all dosage, 3 mg/kg, and 10 mg/kg and Tremelimumab, for all grades and high grade. Figures S28 to S31 Risk ratio of developing irAEs with ipilimumab at 10 mg/kg comparing with 3 mg/kg for organ-specific irAEs (gastrointestinal, skin, endocrine, and hepatic). Table S2 General characteristics of patients receiving anti-CTLA4 antibodies described in case reports. Table S3 Organ-specific irAEs. Table S4 Quality assessment. (DOCX 12329 kb

Analysis of hippocampal subfield volumes.

<p>(A) Segmentation of the hippocampus, divided into the dentate gyrus and the extended Ammon's horn (includes CA1, CA2, CA3, CA4 and subiculum), based on the mouse lemur brain atlas <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056593#pone.0056593-Lee1" target="_blank">[48]</a>. No correlation was found between age and total hippocampal volume (B), however, we observed a significant decrease with age of the normalized volume of the extended Ammon's horn (C), and a significant increase with age of the normalized volume of the dentate gyrus (D). (E–F) 3D views of hippocampal subfields in one young (E) and one old animal (F), showing the increased volume of the dentate gyrus and decreased volume of the Ammon's horn in the aged lemur as compared to the young one.</p

Schematic representation of the experimental design and pharmacological treatment protocols.

<p>A, general experimental plan showing phases of behavioural characterization: at baseline, during MPTP intoxication, upon L-DOPA chronic treatment for the development of dyskinesias, and after antidyskinetic treatment administration. Post-mortem analysis techniques are listed. Detail of the administration of L-DOPA and treatment with either IRC-082451 (IRC), amantadine (AMANT) or vehicle (VEH) in acute (B) and sub-chronic (C) treatment regimes.</p

Histological characterization of the nigrostriatal dopaminergic pathway in the all primate brains.

<p>Left column: representative coronal sections of healthy (CTRL, A), parkinsonian (MPTP, C), dyskinetic vehicle-treated (VEH, E) and dyskinetic IRC-treated (IRC, G) primate brains stained for tyrosine hydroxylase. Right column: areas of the substantia nigra where stereological counts were performed drawn on representative samples for each group of animals (B, CTRL; D, MPTP; F, VEH; H, IRC). Lower panel: histogram displaying TH-positive neuron number in the substantia nigra (I). Data are expressed as mean cell counts ± s.e.m. Unpaired Student <i>t</i> test (**, p<0.01).</p

RT-qPCR analysis of putaminal brain samples in healthy controls (CTRL), parkinsonian untreated (MPTP), dyskinetic vehicle-treated (VEH) and dyskinetic IRC-082451-treated non-human primates.

<p>Significant differences in mRNA levels were detected for FosB (A), cFOS (B) and ARC (C) when comparing dyskinetic IRC-treated and dyskinetic vehicle-treated animals. Data are expressed as mean ± s.e.m. One way ANOVA followed by a <i>post hoc</i> Fisher’s PLSD test (*, p<0.05; **, p<0.01).</p

Effect of a sub-chronic treatment with IRC-082451 on locomotor activity.

<p>The 5-day sub-chronic treatment with 5 mg/kg IRC was given concomitantly with L-DOPA and the total number of dyskinesias and distance travelled on day 5 are presented. A significant reduction in LID count was observed (A) together with an increase in spontaneous TDT (B) compared to the same animals under vehicle treatment. Data are presented as mean ± s.e.m. Unpaired Student <i>t</i> test (**, p<0.01).</p

¹⁸F-DOPA PET studies in IRC-082451-treated and vehicle-injected healthy animals.

<p>Representative results of one subject treated with both molecules on different days taking the occipital cortex as a reference region. Coronal PET image (upper left panel) and corresponding anatomical T2-weighted MRI images (upper right panel) showing the normal accumulation of the radiotracer in the caudate and putamen (A).Time activity curves (lower panel) demonstrate there is no significant difference between treatments in either the caudate-putamen complex (Put) or the occipital cortex (Ctx) curves. Data are expressed as mean ± s.e.m.</p

Effect of amantadine treatment on motor behaviour.

<p>Three different doses of amantadine were acutely injected together with L-DOPA and a significant antidyskinetic effect was observed at the 10 mg/kg dose (A). Ethovision analysis of the total distance travelled (TDT) shows there is no effect on locomotor activity of either dose of amantadine tested (B). Sub-chronic 5-day treatment with 10 mg/kg amantadine further confirms the antidyskinetic efficacy of the compound (C) and the lack of significant effect on locomotor activity compared to vehicle treatment (D). Data are presented as mean ± s.e.m. Unpaired Student <i>t</i> test (*, p<0.05).</p

Effect of a 4-day treatment with IRC-082451 (5 mg/kg/day).

<p>The sub-chronic treatment was given concomitantly with L-DOPA for 4 days and animals were filmed on the 5^th day (A). Total number of dyskinesias (B) and total distance travelled (C) over 6 h of film are presented and a significant reduction in LID counts is observed. Data are expressed as mean ± s.e.m. Unpaired Student <i>t</i> test (**, p<0.01).</p

Time-encoded video-based analysis of motor behaviour after acute treatment with escalating doses of IRC-082451.

<p>Quantification of L-DOPA-induced dyskinesias at 2.5, 5 and 10 mg/kg IRC-082451 compared to vehicle (0)(A). Total distance travelled (TDT) in meters as measured by Ethovision at different IRC doses compared to vehicle (B). Visualization of dyskinesia count output from The Observer software under vehicle (C) and after an acute 5 mg/kg IRC treatment (D). Horizontal lines represent a part of the body displaying dyskinesias whereas vertical coloured lines represent an individual dyskinetic event over time. Data are expressed as mean ± s.e.m. Unpaired Student <i>t</i> test (*, p<0.05; **, p<0.01).</p