Antibody-mediated neutralization of galectin-3 as a strategy for the treatment of systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune, inflammatory and fibrotic disease with limited treatment options. Developing new therapies is therefore crucial to address patient needs. To this end, we focused on galectin-3 (Gal-3), a lectin known to be associated with several pathological processes seen in SSc. Using RNA sequencing of whole-blood samples in a cross-sectional cohort of 249 patients with SSc, Gal-3 and its interactants defined a strong transcriptomic fingerprint associated with disease severity, pulmonary and cardiac malfunctions, neutrophilia and lymphopenia. We developed new Gal-3 neutralizing monoclonal antibodies (mAb), which were then evaluated in a mouse model of hypochlorous acid (HOCl)-induced SSc. We show that two of these antibodies, D11 and E07, reduced pathological skin thickening, lung and skin collagen deposition, pulmonary macrophage content, and plasma interleukin-5 and -6 levels. Moreover, E07 changed the transcriptional profiles of HOCl-treated mice, resulting in a gene expression pattern that resembled that of control mice. Similarly, pathological pathways engaged in patients with SSc were counteracted by E07 in mice. Collectively, these findings demonstrate the translational potential of Gal-3 blockade as a therapeutic option for SSc.

S95021, a novel selective and pan-neutralizing anti interferon alpha (IFN-α) monoclonal antibody as a candidate treatment for selected autoimmune rheumatic diseases.

Increased interferon-α (IFN-α) production is a critical component in the pathophysiology of systemic lupus erythematosus (SLE) and other rheumatic autoimmune diseases. Herein, we report the characterization of S95021, a fully human IgG1 anti-IFN-α monoclonal antibody (mAb) as a novel therapeutic candidate for targeted patient populations. S95021 was expressed in CHOZN GS-/- cells, purified by chromatography and characterized by using electrophoresis, size exclusion chromatography and liquid chromatography-mass spectrometry. High purity S95021 was obtained as a monomeric entity comprising different charge variants mainly due to N-glycosylation. Surface plasmon resonance kinetics experiments showed strong association rates with all IFN-α subtypes and estimated KDs below picomolar values. Pan-IFN-α-binding properties were confirmed by immunoprecipitation assays and neutralization capacity with reporter HEK-Blue IFN-α/ß cells. S95021 was IFN-α-selective and exhibited superior potency and broader neutralization profile when compared with the benchmark anti-IFN-α mAbs rontalizumab and sifalimumab. STAT-1 phosphorylation and the type I IFN gene signature induced in human peripheral blood mononuclear cells by recombinant IFN-α subtypes or plasmas from selected autoimmune patients were efficiently reduced by S95021 in a dose-dependent manner. Together, our results show that S95021 is a new potent, selective and pan IFN-α-neutralizing mAb. It is currently further evaluated as a valid therapeutic candidate in selected autoimmune diseases in which the IFN-α pro-inflammatory pathway is dysregulated.

Methods for cartilage and subchondral bone histomorphometry.

This chapter presents the histological assessment of cartilage and bone of tibial plateaus, by procedures that have been applied and validated in two animal models of osteoarthritis: meniscectomized rats and guinea pigs. It starts from bone sampling, followed by all the steps of sample preparation from embedding to sectioning (without prior decalcification), staining, and mounting. Depending on the cartilage or bone components to be visualized, two dyes are described: safranin O and Goldner's trichrome. On these stained sections, various histomorphometric parameters are then quantified using the dedicated programs of an image analyzer. The following parameters are evaluated at the medial side of the tibia and are described at the levels of both cartilage (cartilage thickness, fibrillation index, proteoglycan content ratio based on safranin-O staining intensities and chondrocyte density) and bone (subchondral bone plate thickness).

Molecular characterization of the AMPA-receptor potentiator S70340 in rat primary cortical culture: whole-genome expression profiling.

To improve our understanding of the molecular events underlying the effects of positive allosteric modulators of the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (S)-AMPA-type glutamate receptors, gene expression profiles of primary cortical culture were measured by Agilent-Microarray technique under (S)-AMPA (1µM) stimulation for 0.5, 6, 24 and 48h in the presence or absence of S70340 (30µM), an allosteric potentiator of AMPA receptors. (S)-AMPA and S70340 treatment alone have little effect on gene expression whereas as early as 6h, their combination induced a large number of genes known to decrease apoptosis and mediate cell survival. Pathway analyses of (S)-AMPA+S70340 treatment-mediated gene expression from 6 to 48h further suggested the activation of cellular functions including neuron differentiation and neurite outgrowth. A proportion of genes implicated in these functions encode proteins involved in environmental cues and are expressed in growth cones, such as extracellular matrix component proteins and filopodia microfilament-associated proteins. Time course analysis of mRNA expression combined with in silico promoter analysis revealed an enrichment in the cAMP response element (CRE) among co-regulated genes. This study demonstrated that S70340-mediated AMPA potentialisation activated genes and functional processes involved in neuroprotective and cognitive effects and describes putative new functional biomarkers.

Early detection and monitoring of cartilage alteration in the experimental meniscectomised guinea pig model of osteoarthritis by 99mTc-NTP 15-5 scintigraphy.

PURPOSE: This study in the meniscectomised guinea pig aimed to demonstrate that the radiotracer (99m)Tc-NTP 15-5 would have pathophysiological validity for in vivo osteoarthritis imaging. METHODS: The specificity of (99m)Tc-NTP 15-5 for cartilage was determined in healthy animals (n = 13), by tissue radioactivity counting, joint autoradiography and scintigraphy. (99m)Tc-NTP 15-5 scintigraphy was performed at 20, 50, 80, 115, 130, 150 and 180 days after medial meniscectomy (n = 10 MNX) or sham operation (n = 5), and scintigraphic ratios (operated/contralateral) were calculated for femoral (F) and tibial (T) areas. F and T ratios were compared with those of (99m)Tc-MDP bone scintigraphy. At the study end-point, autoradiographic analysis of joint (99m)Tc-NTP 15-5 distribution and macroscopic scoring of cartilage integrity were performed. RESULTS: The high and specific accumulation of (99m)Tc-NTP 15-5 in normal cartilage (about 5.5 +/- 1.7 % of injected dose/g of tissue), which permitted joint imaging with high contrast, was affected by osteoarthritis. In the MNX group, (99m)Tc-NTP 15-5 accumulation in cartilage within the operated joint, relative to the contralateral joint, was observed to change in the same animals as pathology progressed. Although F and T ratios were significantly higher in MNX (F = 1.7 +/- 0.2; T = 1.6 +/- 0.1) than in shams (F = 1.0 +/- 0.1; T = 1.0 +/- 0.1) at day 50, they were significantly lower in MNX (F = 0.6 +/- 0.1; T = 0.7 +/- 0.1) than in shams (F = 1.0 +/- 0.1; T = 0.9 +/- 0.1) at day 180. No change in (99m)Tc-MDP uptake was observed over 6 months. Macroscopic analysis confirmed features of osteoarthritis only in MNX knees. CONCLUSION: These results in MNX guinea pigs provide additional support for the use of (99m)Tc-NTP 15-5 for in vivo imaging of osteoarthritis.

Effect of inhibition of matrix metalloproteinases on cartilage loss in vitro and in a guinea pig model of osteoarthritis.

OBJECTIVE: To study the effects of a matrix metalloproteinase (MMP) inhibitor (S-34219) on osteoarthritis (OA) cartilage cultures and in the meniscectomized guinea pig model of OA. METHODS: The inhibitory activity of S-34219 on MMPs and aggrecanase was studied by fluorimetry and immunoassay, respectively. The effects of S-34219 on proteoglycan and collagen degradation were studied in cultures of rabbit and human cartilage. Medial meniscectomy was performed on 29 Hartley male guinea pigs, and these animals were randomly allocated to 1 of 3 groups: a control meniscectomized group (MNXc) receiving the vehicle, or a meniscectomized group receiving either 10 mg/kg or 20 mg/kg S-34219, administered twice per day by oral gavage for 12 weeks from day 1 after surgery. An additional group comprised sham-operated animals. Tibial cartilage from the operated left knee was processed for histologic assessment of OA lesions. RESULTS: The 50% inhibitory concentration (IC(50)) of S-34219 on MMPs 1, 2, 3, 8, 9, and 13 was 55, 0.1, 0.5, 0.1, 0.03, and 0.2 nM, respectively; the IC(50) on aggrecanase 1 was 190 nM. In cultured rabbit cartilage, 100 nM S-34219 strongly inhibited MMP-dependent degradation of collagen and proteoglycans. A concentration 100 times higher was needed to inhibit aggrecanase-dependent degradation. In cultures of human OA cartilage, 100 nM S-34219 inhibited spontaneous type II collagen degradation by 66% and proteoglycan degradation by only 22%. For in vivo studies, treated groups were compared with the MNXc group and the results, expressed as the percentage variation versus MNXc, were as follows: in the 10 and 20 mg/kg groups, a significant decrease (P < 0.05) in global histologic score (-12% and -14%, respectively) was observed, and this was associated with a significant increase (P < 0.05) in cartilage thickness (+19% and +18%, respectively). Neither dose level changed the proteoglycan content. CONCLUSION: In both treated animal groups, S-34219 significantly prevented the loss of cartilage thickness, probably by inhibiting collagen breakdown that normally leads to the erosion of fibrillated superficial areas. The absence of a protective effect on glycosaminoglycan loss, both in vitro and in vivo, suggests that aggrecanases may have an important role in cartilage loss. This study reinforces the relevance of these models for testing chondroprotective drugs, and the potential role of dual inhibitors of collagenase and aggrecanase as disease-modifying drugs in the management of OA.