Atif Saghir

Testosterone undecanoate improves sexual function in men with type 2 diabetes and severe hypogonadism: results from a 30‐week randomized placebo‐controlled study

To evaluate the sexual function response to 30 weeks’ treatment with long‐acting testosterone undecanoate (TU) or placebo in 199 men with type 2 diabetes and either severe or mild hypogonadism (HG).

Detection and characterisation of bone destruction in murine rheumatoid arthritis using statistical shape models

HighlightsArticulated statistical shape model represents variability of normal bone shapes.Bone shape pathologies are identified as deviations from the model statistics.3D colour‐coded renderings of a given sample show the magnitudes of deformations.Individual erosions and malformations are segmented and their severity quantified.Objective evidence shown for different deformations in different arthritis types. &NA; Rheumatoid arthritis (RA) is an autoimmune disease in which chronic inflammation of the synovial joints can lead to destruction of cartilage and bone. Pre‐clinical studies attempt to uncover the underlying causes by emulating the disease in genetically different mouse strains and characterising the nature and severity of bone shape changes as indicators of pathology. This paper presents a fully automated method for obtaining quantitative measurements of bone destruction from volumetric micro‐CT images of a mouse hind paw. A statistical model of normal bone morphology derived from a training set of healthy examples serves as a template against which a given pathological sample is compared. Abnormalities in bone shapes are identified as deviations from the model statistics, characterised in terms of type (erosion / formation) and quantified in terms of severity (percentage affected bone area). The colour‐coded magnitudes of the deviations superimposed on a three‐dimensional rendering of the paw show at a glance the severity of malformations for the individual bones and joints. With quantitative data it is possible to derive population statistics characterising differences in bone malformations for different mouse strains and in different anatomical regions. The method was applied to data acquired from three different mouse strains. The derived quantitative indicators of bone destruction have shown agreement both with the subjective visual scores and with the previous biological findings. This suggests that pathological bone shape changes can be usefully and objectively identified as deviations from the model statistics. Graphical abstract Figure. No caption available.

Testosterone replacement therapy: improved sexual desire and erectile function in men with type 2 diabetes following a 30-week randomized placebo-controlled study

Although testosterone replacement treatment (TRT) can improve sexual function in many hypogonadal (HG) men with type 2 diabetes (T2DM), some show either no improvement or only in a limited number of domains. Indeed, it is often difficult for the clinician to offer an indication of the likely efficacy of TRT as little data exist on the proportion of TRT‐treated men who will demonstrate improvement in domains such as sexual desire (SxD) and erectile function (EF). We describe in men with T2DM: firstly, the likelihood of improved sexual desire (SxD) and erectile function (EF) following TRT at various time points, and secondly, if probability of SxD change predicted likelihood of subsequent EF change. During a 30‐week randomized controlled study of testosterone undecanoate (TU), 199 T2DM men with HG (189 men completing) identified from primary care registers (placebo (P): 107, TU: 92) were stratified using baseline total testosterone (TT)/free testosterone (FT) into Mild (TT 8.1–12 nmol/L or FT 0.18–0.25 nmol/L) and Severe HG groups (TT ≤8 nmol/L and FT ≤0.18 nmol/L) and placebo (P)‐ and TU‐treated groups. Associations between TU, SxD and EF were investigated using chi‐square and logistic regression analysis. The proportion of men with improved SxD after 6 weeks and EF improvement after 30 weeks was significantly higher following TU treatment compared to P, this particularly evident in Severe HG men. Changes in SxD and EF were significantly associated in all groups. Logistic regression showed that SxD change at 6 weeks predicted of EF change after 30 weeks. Our study confirms TRT leads to changes in SxD and EF at different time points and suggests SxD and EF changes are related. SxD change after 6 weeks predicting EF change at 30 weeks is possibly a useful clinical finding.

TNFα depleting therapy improves fertility and animal welfare in TNFα-driven transgenic models of polyarthritis when administered in their routine breeding:

Transgenic tumour necrosis factor alpha (TNFα)-driven models of polyarthritis such as the TNFΔARE mouse have proven to be invaluable in delineating aspects of inflammatory disease pathophysiology in humans. Unfortunately, the onset of joint destruction and inflammation in these models represents a significant detriment to breeding management. We examined whether TNFα depleting therapy ‘infliximab’ might represent a significant refinement in routine breeding. Clinical scores of joint inflammation were assessed in TNFΔARE males receiving either infliximab (10 mg/kg) or saline by twice-weekly intraperitoneal injection. Joint histology and bone morphology were assessed by histological analysis and micro-computed tomography (CT), respectively. Analysis of breeding was examined retrospectively in TNFΔARE males prior to, and following, regular introduction of infliximab. Clinical scores of inflammation were significantly reduced in TNFΔARE males receiving infliximab (control 6.6 arbitrary units [AU] ± 0.88 versus infliximab 4.4 AU ± 1.4; P < 0.05), while measures of pannus invasion and bone erosion by histology and micro-CT were markedly reduced. In the breeding groups, TNFΔARE males receiving infliximab injections sired more litters over their breeding lifespan (control 1.69 ± 0.22 versus infliximab 3.00 ± 0.19; P < 0.005). Furthermore, prior to infliximab, TNFΔARE males had a 26% risk of failing to sire any litters. This was reduced to 7% after the introduction of infliximab. This study is the first to report that regular administration of infliximab is effective at suppressing disease activity and improving animal welfare in TNFΔARE animals. In addition, we have shown that infliximab is highly efficacious in improving breeding behaviour and increasing the number of litters sired by TNFΔARE males.

02.07 Prophylactic treatment with pepitem inhibits onset of collagen induced arthritis and pepitem therapy reduces disease severity

Background The inappropriate recruitment and retention of T-cells into the joint is a cardinal feature of RA, yet the molecular mechanisms underpinning this remain unclear. We recently showed that patients with Rheumatoid arthritis (RA) have a defect in a newly identified immuno-protective check-point (adiponectin-PEPITEM axis) that normally limits T-cell trafficking during inflammation. Here we examined the therapeutic potential of PEPITEM in a murine model of arthritis. Methods Collagen induced arthritis (CIA) was trigger in DBA-1 mice by immunisation with bovine type II collagen. Synthetic PEPITEM was administered by daily injections starting at day 21 (prior to disease onset) or at the first signs of inflammation. Disease onset and severity were evaluated daily. Bone morphology and leukocyte infiltration were assessed by microCT, immunohistochemistry, flow cytometry and qPCR. Results Administration of synthetic PEPITEM prior to disease onset inhibited the development of CIA. We observed a significant reduction in disease incidence, clinical score, leukocyte infiltration and bone erosion when compared to control peptide treated mice. Excitingly, PEPITEM therapy administered at the first signs of inflammation in CIA also reduced clinical score, leukocyte infiltration and bone erosion when compared to control mice. Whilst we observed fewer leukocytes in the synovium, PEPITEM therapy did not alter leukocyte trafficking through the draining lymph node. Interestingly, in both approaches, we detected higher levels of the FOXP3 transcript, and TReg cells, in PEPITEM treated mice than in control animals. Conclusions This leads to the exciting possibility that targeting PEPTIEM represents a valid therapeutic approach for treating T-cell mediated diseases, such as RA. Funding This work was funded by a Pfizer I-CRP research grant.

04.23 Identification of a novel subset of pathogenic stromal cells with key effector functions in tissue inflammation and damage

Background Stromal cells are key effector cells in tissue inflammation and damage. Despite their importance these cells are yet to be targeted therapeutically. We have previously identified a distinct subset of stromal cells defined by their expression of a cassette of cell surface proteins including Podoplanin/gp38 (PDPN), Fibroblast activation protein (FAP) and Cadherin-11. The aim of this study was to determine the pathogenic role of these cells in tissue inflammation. Methods Salivary gland inflammation was induced by intra-ductal administration of a replication-deficient adenovirus resulting in tertiary lymphoid structure (TLS) formation as previously described.1 Polyarthritis was induced using the KRN serum transfer model as previously described.2 To delete FAP-expressing cells we used a model of conditional depletion of FAP-expressing cells, in which FAP-DTR mice were treated with Diphtheria Toxin Disease severity was assessed clinically, histologically and by MicroCT and a combination of immunofluorescence, quantitative RT-PCR and flow cytometry was performed on digested salivary glands and synovial membrane tissue. Results Tissue resident stromal cells dynamically express FAP, PDPN and cadherin-11 during inflammation. This cassette of cell surface markers defined a population of stromal cells that expand during inflammation by in situ proliferation. In the salivary gland, these pathogeneic cells are found surrounding ectopic lymphoid aggregates where they produce lymphoid chemokines and survival factors. In the synovium, they are localised to the synovial lining layer and pannus tissue where they invade articular cartilage and bone. Selective deletion of FAP expressing cells resulted in a defect in lymphoid chemokine production, decreased number of infiltrating lymphocytes and severely inhibited TLO formation in the salivary gland. In the joint, therapeutic deletion significantly attenuated synovial inflammation, inflammatory cell infiltration, reduced the severity of arthritis and protected against joint damage. Conclusions We have identified a pathogenic subset of stromal cells defined by their expression of common cassette of cell surface markers. Whilst the phenotype of these cells is common to inflammation our data suggests their function is both tissue and disease specific. References 1. Bombardieri, Barone, et al. JI. 2012. 2. Huang, et al. Arthrits Rheumatol2014.

06.16 Platelet-derived clec-2 and its ligand podoplanin (gp38) inhibit synovial inflammation

Background During synovial inflammation, platelets and their associated microparticles escape from the synovial microvasculature and provide pro-inflammatory factors leading to the activation of synovial fibroblasts (SF) that actively contribute to joint damage.1 In patients with rheumatoid arthritis (RA), SF up-express the surface protein Podoplanin (PDPN), a ligand for CLEC-2.2,3 Although the function of PDPN is still poorly understood, recent data suggest that the PDPN/CLEC-2 axis can modulate cellular responses. Within the RA synovium, platelets are considered the sole source of CLEC-2.3 Despite these observations, clear experimental approaches that explore the role of PDPN/CLEC-2 interactions in RA pathogenesis are lacking. Materials and methods PDPN expression by freshly isolated mouse synoviocytes was measured by flow cytometry (FACS) during joint inflammation and after resolution. PDPN expressing cells were characterised by FACS and quantitative PCR. Arthritis severity in the absence of CLEC-2 was assessed in the global tamoxifen-inducible Clec1b deleting mice (ie, Rosa26-Ert2Cre x Clec1bFlox/Flox) as well as in the platelet-specific Clec1b deleting mice (ie, Pf4-Cre x Clec1bFlox/Flox). Wild-type mice with auto-immune arthritis were injected with anti-PDPN antibody. The disease was monitored by body weight, clinical scores, ankle and paw thicknesses. The synovium cellular contents from arthritic animals were analysed by FACS and cartilage damage quantified by histology while bone erosion and remodelling were monitored by MicroCT. Results Joint inflammation triggered PDPN up-expression on a pro-inflammatory SF subset with concurrent accumulation of PDPN+ anti-inflammatory macrophages. These populations disappeared with the resolution of inflammation. In the absence of CLEC-2, arthritis was more severe and led to more pronounced bone erosion and remodelling. Mice treated with an agonist anti-PDPN antibody were partially protected from induced auto-immune arthritis as demonstrated by their clinical features and reduced leucocyte infiltrations into the joints. Conclusions We provide the first in vivo evidence that the PDPN/CLEC-2 interaction restrains arthritis as ablation of platelet-derived CLEC-2 leads to worse arthritis, bone erosion and remodelling. Accordingly, mimicking PDPN/CLEC-2 interactions with an agonist anti-PDPN restrains auto-immune arthritis in mice. These observations suggest that platelets, known for promoting joint inflammation, also contribute to the suppression of arthritis in a CLEC-2 dependent manner. The mechanisms underlying this anti-inflammatory process are currently under investigation. References 1. Boilard E. et al. Nature Review Rheumatol. 2012;8:534–42. 2. Ekwall A.K. et al. Arthritis Res Ther. 2011;13:R40. 3. Del Rey M.J. et al. PLoS One.2014;9:e0099607.

A1.17 Podoplanin and its ligand CLEC-2 restrain synovial inflammation

Background and objectives During synovial inflammation, platelets and their microparticles escape from the vasculature to fuel the synovial membrane with pro-inflammatory factors leading to the activation of synovial fibroblasts (SF) that actively contribute to joint damage.1 Patients with rheumatoid arthritis (RA) show an up-regulation of surface protein Podoplanin (PDPN) on SF.2,3 Although the function of PDPN is still poorly understood, recent data suggest that PDPN ligation to its ligand CLEC-2 can modulate cellular responses. Within the RA synovium, platelets are considered the sole source of CLEC-2.3 Despite these observations, clear experimental approaches that explore the role of PDPN/CLEC-2 interactions in RA are lacking. Materials and methods PDPN expression by freshly isolated mouse synoviocytes was measured by flow cytometry during joint inflammation and after resolution. Tamoxifen-inducible Clec1b deletion mice (TIC mice) were used to assess the disease severity in absence of CLEC-2. CLEC-2 deletion was confirmed on circulating platelets by flow cytometry. Arthritis was induced by anti-collagen antibodies and LPS injections. The disease severity was monitored by body weight, clinical scores, ankle and paw thicknesses. Bone erosion and bone remodelling were studied by MicroCT scans and 3D reconstructions. Results Joint inflammation triggers PDPN up-regulation on SF and an accumulation of PDPN+ leucocytes in the synovium. These high levels of PDPN expression disappear when inflammation resolved. In absence of CLEC-2, arthritis is more severe, bone erosion and bone remodelling are more pronounced. Conclusions In this work, we provide the first in vivo evidence that PDPN/CLEC-2 interactions act to restrain arthritis by showing that ablation of CLEC-2 expression leads to worse arthritis, bone erosion and bone remodelling. These observations suggest that platelets, known for promoting joint inflammation, also contribute to the suppression of arthritis in a CLEC-2 dependent manner. The mechanisms underlying this anti-inflammatory process are currently under investigation. References Boilard E, et al. Nat Rev Rheumatol. 2012;8:534–542 Ekwall AK, et al. Arthritis Res Ther. 2011;13:R40 Del Rey MJ, et al. PLoS One. 2014;9:e0099607

THU0035 A Key Role for Platelet-Derived Clec-2 in the Regulation of Synovial Inflammation

Background It is becoming clear that platelets play a more significant role in the immune system than previously recognized1-3. Furthermore they are known to play a role in the induction of inflammation but the mechanisms by which they do so remain unclear. Both CLEC-2, which is highly expressed by platelets, and its ligand PDPN (gp38) are found in the joints of patients with rheumatoid arthritis (RA)4. However little is known about the role of CLEC-2-PDPN axis in synovial inflammation. Objectives To determine whether platelet-derived CLEC-2 induces synovial inflammation by triggering PDPN-expressing fibroblasts to adopt a pro-inflammatory phenotype. Methods Using flow cytometry we analyzed the PDPN expression in biopsies from RA patients as well as in mouse joints using the TNFΔare/+ and the anti-collagen antibodies induced arthritis (CAIA) models. We also induced arthritis in tamoxifen-induced CLEC-2 deficient mice to monitor the disease onset and resolution. After resolution, bones erosion and remodelling was assessed by micro-CT. We generated mouse PDPNPos. and PDPNNeg. synovial fibroblasts (SF) that we co-cultured with wild-type or CLEC-2 deficient platelets. After incubation pro- and anti-inflammatory makers were measured at the mRNA (qPCR) and the protein (Luminex) levels. Results In human and mouse synovium, high levels of PDPN expression are found on SF as well as on a subset of haematopoietic cells. PDPN expression on mouse SF and myeloid cells is dynamic and depends on the level of inflammation. In vivo studies in which CLEC-2 is selectively deleted in platelets show that arthritis is more severe in absence of platelet-derived CLEC-2 suggesting that platelet-derived CLEC-2 plays a protective role in physiological conditions. However, in vitro co-culture data indicate that platelet-derived CLEC-2 induce SF to adopt a pro-inflammatory phenotype. This discrepancy between our in vitro and in vivo data might be explained by the fact that platelet-derived CLEC-2 induces myeloid cells to adopt an anti-inflammatory phenotype. Conclusions Our observations suggest that CLEC-2-PDPN interactions play a dual role in arthritis depending on whether they occur between platelets and fibroblasts or platelets and macrophages. References Benezech C, et al. CLEC-2 is required for development and maintenance of lymph nodes. Blood. 2014;123:3200-3207. Acton SE, et al. Dendritic cells control fibroblastic reticular network tension and lymph node. Nature. 2014;514:498-502 LID - 410.1038/nature13814 [doi]. Astarita JL, et al. The CLEC-2-podoplanin axis controls the contractility of fibroblastic reticular cells and lymph node microarchitecture. Nat Immunol. 2014:doi: 10.1038/ni.3035. Del Rey MJ, et al. Clinicopathological correlations of podoplanin (gp38) expression in rheumatoid synovium and its potential contribution to fibroblast platelet crosstalk. PLoS One. 2014;9:e99607. Disclosure of Interest None declared