Amyotrophic lateral sclerosis transcriptomics reveals immunological effects of low-dose interleukin-2.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease causing upper and lower motor neuron loss and currently no effective disease-modifying treatment is available. A pathological feature of this disease is neuroinflammation, a mechanism which involves both CNS-resident and peripheral immune system cells. Regulatory T-cells are immune-suppressive agents known to be dramatically and progressively decreased in patients with amyotrophic lateral sclerosis. Low-dose interleukin-2 promotes regulatory T-cell expansion and was proposed as an immune-modulatory strategy for this disease. A randomized placebo-controlled pilot phase-II clinical trial called Immuno-Modulation in Amyotrophic Lateral Sclerosis was carried out to test safety and activity of low-dose interleukin-2 in 36 amyotrophic lateral sclerosis patients (NCT02059759). Participants were randomized to 1MIU, 2MIU-low-dose interleukin-2 or placebo and underwent one injection daily for 5 days every 28 days for three cycles. In this report, we describe the results of microarray gene expression profiling of trial participants' leukocyte population. We identified a dose-dependent increase in regulatory T-cell markers at the end of the treatment period. Longitudinal analysis revealed an alteration and inhibition of inflammatory pathways occurring promptly at the end of the first treatment cycle. These responses are less pronounced following the end of the third treatment cycle, although an activation of immune-regulatory pathways, involving regulatory T-cells and T helper 2 cells, was evident only after the last cycle. This indicates a cumulative effect of repeated low-dose interleukin-2 administration on regulatory T-cells. Our analysis suggested the existence of inter-individual variation amongst trial participants and we therefore classified patients into low, moderate and high-regulatory T-cell-responders. NanoString profiling revealed substantial baseline differences between participant immunological transcript expression profiles with the least responsive patients showing a more inflammatory-prone phenotype at the beginning of the trial. Finally, we identified two genes in which pre-treatment expression levels correlated with the magnitude of drug responsiveness. Therefore, we proposed a two-biomarker based regression model able to predict patient regulatory T-cell-response to low-dose interleukin-2. These findings and the application of this methodology could be particularly relevant for future precision medicine approaches to treat amyotrophic lateral sclerosis.

Repeated 5-day cycles of low dose aldesleukin in amyotrophic lateral sclerosis (IMODALS): A phase 2a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Low-dose interleukin-2 (ld-IL-2) enhances regulatory T-cell (Treg) function in auto-inflammatory conditions. Neuroinflammation being a pathogenic feature of amyotrophic lateral sclerosis (ALS), we evaluated the pharmacodynamics and safety of ld-IL-2 in ALS subjects. METHODS: We performed a single centre, parallel three-arm, randomised, double-blind, placebo-controlled study. Eligibility criteria included age < 75 years, disease duration < 5 years, riluzole treatment > 3 months, and a slow vital capacity ≥ 70% of normal. Patients were randomised (1:1:1) to aldesleukin 2 MIU, 1 MIU, or placebo once daily for 5 days every 4 weeks for 3 cycles. Primary outcome was change from baseline in Treg percentage of CD4+ T cells (%Tregs) following a first cycle. Secondary laboratory outcomes included: %Treg and Treg number following repeated cycles, and plasma CCL2 and neurofilament light chain protein (NFL) concentrations as surrogate markers of efficacy. Safety outcomes included motor-function (ALSFRS-R), slow vital capacity (SVC), and adverse event reports. This trial is registered with ClinicalTrials.gov, NCT02059759. FINDINGS: All randomised patients (12 per group), recruited from October 2015 to December 2015, were alive at the end of follow-up and included in the intent-to-treat (ITT) analysis. No drug-related serious adverse event was observed. Non-serious adverse events occurred more frequently with the 1 and 2 MIU IL-2 doses compared to placebo, including injection site reactions and flu-like symptoms. Primary outcome analysis showed a significant increase (p < 0·0001) in %Tregs in the 2 MIU and 1 MIU arms (mean [SD]: 2 MIU: +6·2% [2·2]; 1 MIU: +3·9% [1·2]) as compared to placebo (mean [SD]: -0·49% [1·3]). Effect sizes (ES) were large in treated groups: 2 MIU ES=3·7 (IC95%: 2·3-4·9) and 1 MIU ES=3·5 (IC95%: 2·1-4·6). Secondary outcomes showed a significant increase in %Tregs following repeated cycles (p < 0·0001) as compared to placebo, and a dose-dependent decrease in plasma CCL2 (p = 0·0049). There were no significant differences amongst the three groups on plasma NFL levels. INTERPRETATION: Ld-IL-2 is well tolerated and immunologically effective in subjects with ALS. These results warrant further investigation into their eventual therapeutic impact on slowing ALS disease progression. FUNDING: The French Health Ministry (PHRC-I-14-056), EU H2020 (grant #633413), and the Association pour la Recherche sur la SLA (ARSLA).

French legal approach to clinical research.

Since 1988, France has been committed to drafting laws regulating clinical research. These laws must both reflect general legal standards relating to personal data protection and patient information and comply with EU regulations, which are supra-national norms. The 2012 legislation known as "Jardé law" came into force in 2016 and distinguishes between 3 different types of research involving human subjects: category 1:interventional research implying an intervention on the patient which is not justified by their usual treatment. Category 2: interventional research, which does not focus on medicinal products and only entails minimal risks and constraints. Category 3: non-interventional research implying one or multiple acts or proceedings devoid of listed risks. These studies require preliminary favourable opinions from the French Ethical Research Committees (CPP), who are appointed by the State, and must ensure the protection of personal data. For the other types of studies (retrospective data, practice surveys), French legislation only requires that the protection of personal data is ensured. However, it is highly recommended to submit these studies to an Institutional Review Board (IRB) in order to confirm that human subjects are not involved and to obtain an ethical opinion in the event of a scientific journal submission. These laws are constantly evolving in order to comply with the various international recommendations and European regulations, which are binding in France.

Expression of P-glycoprotein (ABCB1) and Mrp1 (ABCC1) in adult rat brain: focus on astrocytes.

P-glycoprotein (P-gp, ABCB1) and the multidrug resistance-associated protein 1 (Mrp1, ABCC1) are two ATP-driven pumps that mediate the export of organic anions from cells and may confer cellular resistance to many cytotoxic hydrophobic drugs. Immunohistochemistry has shown that P-gp is expressed in rat brain capillary vessels forming the blood-brain barrier (BBB). Mrp1 mRNAs have been detected by RT-PCR in rat brain isolated capillaries. Although many studies have been published in this field, very little information is available on the expression, distribution and physiological functions of the two pumps in rat brain. To characterize the cerebral expression of both P-gp and Mrp1 transporters, we studied immunoreactions of rat brain sections with the two most commonly used antibodies: the monoclonal C219 (anti-P-gp) and the polyclonal 6KQ (anti-Mrp1). Immunological analyses revealed heterogeneity of the P-gp and Mrp1 expressions in rat brain. Indeed, choroidal and ependymal cells expressed Mrp1 rather than P-gp. However, tanycytes lining the third ventricle were strongly immunoreactive with both antibodies, suggesting a particular role for these cells in drug efflux mechanisms. Because of the detection of a 70-kDa component with 6KQ antibodies, immunoreactions obtained in rats were compared with these obtained in wild type and mrp1(-/-) mice. It showed that a positive reaction at the apical surface of the ependymal layer remained obvious, showing that 6KQ antibodies recognize an ependymal molecule, differing from the Mrp1. In addition, a continuous expression of C219-labeled epitopes, similar to endothelial labeling, was detected at the blood-brain barrier, whereas a discontinuous labeling, co-localized with glial fibrillary acidic protein (GFAP) immunostaining, was obtained with 6KQ antibodies. We showed that P-gp was preferentially expressed in the endothelial component and Mrp1 in the astroglial component of the blood-brain barrier. Moreover, Mrp1 was rather expressed than P-gp in parenchyma astrocytes and in glia limitans lining the meninges. These findings provide new insights into the cerebral distribution of two ABC transporters linked to multidrug resistance (MDR).

P-glycoprotein (ABCB1) but not multidrug resistance-associated protein 1 (ABCC1) is induced by doxorubicin in primary cultures of rat astrocytes.

At least two drug efflux pumps involved in multidrug resistance, P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (Mrp1), are expressed in rat astrocyte primary cultures. The aim of this study was to compare the expression of P-gp and Mrp1 in primary cultures exposed to 50 or 500 ng/mL doxorubicin (DOX). Among the two P-gp genes expressed in rodents, mdr1a and mdr1b, a time- and dose-dependent increase in mdr1b mRNA levels was revealed by northern blot analysis. This up-regulation was inhibited by actinomycin D and occurred as early as 2 h after exposure to 50 or 500 ng/mL DOX, whereas mdr1a and mrp1 transcripts were not modified by the DOX exposure. In addition, DOX also strongly enhanced, in a time- and dose-dependent manner, P-gp but not Mrp1 expression. Moreover, DOX raised the cellular efflux of vincristine, a substrate for both P-gp and Mrp1. This efflux was inhibited by the P-gp modulators PSC833 and GW918, but not by the Mrp1 modulator MK571. On the other hand, a 24-h exposure to 500 ng/mL DOX, but not 50 ng/mL DOX, induced apoptosis in primary cultures of rat astrocytes. Fumonisin B1, a ceramide synthase inhibitor, reduced DOX-induced apoptosis, suggesting that de novo synthesis of the ceramide regulatory pathway might be involved in DOX-induced apoptosis. Moreover, western blot analysis showed that fumonisin B1 was not able to decrease the overexpression of P-gp induced by DOX. Our results provide evidence that DOX up-regulates a functional P-gp in primary cultures of rat astrocytes and might cause astrocyte apoptosis via the ceramide pathway.

CSF-contacting epithelia in altered gravity: changes in apical structures and water channels expression.

Apical cytoskeletal structures and water channels are affected in both choroidal and ependymal cells lining the cerebral ventricles. Structural alterations and changes in expression of AQPI and AQP4, evaluated by immuno-cytochemistry and in situ hybridization confirm the impact of variations in gravity in CSF-lining epithelia.