S. Piantoni

Effects of Belimumab on Flare Rate and Expected Damage Progression in Patients With Active Systemic Lupus Erythematosus

To investigate effectiveness and safety of belimumab in patients with active systemic lupus erythematosus (SLE) in a clinical practice setting.

Belimumab decreases flare rate and hinders the expected damage progression in patients with active systemic lupus erythematosus.

To investigate effectiveness and safety of belimumab in patients with active systemic lupus erythematosus (SLE) in a clinical practice setting.

Vitamin D and antiphospholipid syndrome

Vitamin D (vitD) has been shown to have multiple immunomodulatory properties. Hypovitaminosis D has been described in many systemic autoimmune diseases. Antiphospholipid syndrome (APS), an autoimmune disease characterized by immune-mediated thrombosis and pregnancy loss, is a peculiar model for studying vitD, since these patients do not usually have a full-blown autoimmune disease, nor do they have particular restrictions regarding sun exposure. We assessed 25-OH vitD levels in 115 APS and 128 normal healthy donors (NHD) with the LIAISON® chemiluminescent immunoassay by DiaSorin (Italy). Median values were lower in APS patients than in NHD, with the greatest difference occurring during summertime (p < 0.01), suggesting that APS patients may be somehow prevented from vitD generation upon sun exposure. In our cohort, APS patients may have been instructed to use sunscreens in the presence of positive antinuclear antibodies (ANA). Comparing patients with positive and negative ANA, we found comparable vitD levels during the summer. By subdividing APS patients according to clinical features, thrombotic APS patients showed significantly lower levels than did pure obstetric APS patients (p < 0.01). In conclusion, our study confirms previous reports of hypovitaminosis D in APS patients, making them more similar to patients with other systemic autoimmune diseases than NHD. Hypovitaminosis D may be part of the mosaic of factors that determine autoimmunity, rather than a consequence of chronic disease and its treatment. The observation that patients with thrombotic APS, an aggressive phenotype, may be more deficient than those with exclusive obstetric manifestations fits well with the beneficial effects of vitD on thrombosis described both in vitro and in vivo. Therefore, there may be a rationale to assess the efficacy of vitD supplementation in APS patients.

Low levels of vitamin D are common in primary antiphospholipid syndrome with thrombotic disease.

The aim of this study was to assess vitamin D (vit.D) levels in patients with primary antiphospholipid syndrome (PAPS), the association between hypovitaminosis D and clinical manifestations, and the effect of vit.D supplementation on serum levels. Vit.D serum levels of 115 PAPS patients, classified according to the 2006 revised criteria at the Rheumatology Department, Brescia, and of 128 voluntary healthy donors (NHD) were tested in collaboration with DiaSorin (Saluggia, Italy) using the LIAISON chemiluminescent immunoassay. Clinical data were derived from clinical charts. Vit.D deficiency was more prevalent in PAPS than NHD (17% vs 5%). During the summer, vit.D levels were lower in PAPS than NHD (median 28 vs 40.1 ng/mL, P<0.01). PAPS were subdivided according to clinical characteristics (thrombotic vs obstetric). Both groups had lower vit.D levels compared to NHD. Thrombotic PAPS had significantly lower levels than obstetric PAPS (median 20.8 vs 33.3, P<0.01). Sixteen patients (14%) received oral 25-OH vit.D supplementation (average 400 UI/die), but 63% of them did not reach serum levels above 30 ng/mL. PAPS showed significantly lower levels of vit.D than NHD. Hypovitaminosis D was seen to cluster in patients with thrombosis which may suggest that the lack of vit.D could be one of the many factors involved in the thrombotic process. Low-dose supplementation did not seem to be effective in a small group of patients.

Relationship Between Different Subpopulations of Circulating CD4+ T-lymphocytes and Microvascular Structural Alterations in Humans

BACKGROUND Different components of the immune system, including innate and adaptive immunity (T-effector lymphocytes and T-regulatory lymphocytes—TREGs) may be involved in the development of hypertension. In addition, it was demonstrated in animal models that TREGs may prevent angiotensin II-induced hypertension and vascular injury/inflammation. However, no data are presently available in humans about possible relationships between T-lymphocyte subtypes and microvascular structural alterations. METHODS For this purpose, in the present study, we enrolled 24 normotensive subjects and 12 hypertensive patients undergoing an elective surgical intervention. No sign of local or systemic inflammation was present. All patients underwent a biopsy of subcutaneous fat during surgery. Subcutaneous small resistance arteries were dissected and mounted on a wire myograph and the media to lumen ratio (M/L) was calculated. In addition, retinal arteriolar structure was evaluated noninvasively by scanning laser Doppler flowmetry. Capillary density in the nailfold, dorsum of the finger, and forearm were evaluated by videomicroscopy. A peripheral blood sample was obtained before surgery for assessment of T-lymphocyte subpopulations by flow cytometry. RESULTS Significant negative correlations were observed between indices of microvascular structure (M/L of subcutaneous small arteries and wall to lumen ratio of retinal arterioles) and circulating TREG lymphocytes. A direct correlation was observed between M/L of subcutaneous small arteries and circulating Th17 lymphocytes. In addition, total capillary density was correlated with a TREG effector memory subpopulation. CONCLUSION Our data suggest that some lymphocyte subpopulations may be related to microvascular remodeling, confirming previous animal data, and opening therapeutic possibilities.

Relationship between different subpopulations of circulating CD4+ T lymphocytes and microvascular or systemic oxidative stress in humans

Abstract Background and objective: Different components of the immune system, including innate and adaptive immunity (T effector lymphocytes and T regulatory lymphocytes – TREGs) may be involved in the development of hypertension, vascular injury and inflammation. However, no data are presently available in humans about possible relationships between T-lymphocyte subtypes and microvascular oxidative stress. Our objective was to investigate possible relationships between T-lymphocyte subtypes and systemic and microvascular oxidative stress in a population of normotensive subjects and hypertensive patients. Patients and methods: In the present study we enrolled 24 normotensive subjects and 12 hypertensive patients undergoing an elective surgical intervention. No sign of local or systemic inflammation was present. All patients underwent a biopsy of subcutaneous fat during surgery. A peripheral blood sample was obtained before surgery for assessment of T lymphocyte subpopulations by flow cytometry and circulating indices of oxidative stress. Results: A significant direct correlation was observed between Th1 lymphocytes and reactive oxygen species (ROS) production (mainly in microvessels). Additionally, significant inverse correlations were observed between ROS and total TREGs, or TREGs subtypes. Significant correlations were detected between circulating indices of oxidative stress/inflammation and indices of microvascular morphology/Th1 and Th17 lymphocytes. In addition, a significant inverse correlation was detected between TREGs in subcutaneous small vessels and C reactive protein. Conclusions: Our data suggest that TREG lymphocytes may be protective against microvascular damage, probably because of their anti-oxidant properties, while Th1–Th17 lymphocytes seem to exert an opposite effect, confirming an involvement of adaptive immune system in microvascular damage.

CD8+ T Cell Profiles in Patients With Rheumatoid Arthritis—the Effects of Costimulation Blockade: Comment on the Article by Carvalheiro et al

To the Editor: We read with great interest the article by Carvalheiro and colleagues (1), in which they present data showing that CD81 T cells in patients with rheumatoid arthritis (RA) exhibit an effector phenotype, with expression of proinflammatory cytokines and cytolytic enzymes, including interferon-c (IFNc), interleukin-17 (IL-17), and granzyme B. These phenotypic and functional features of peripheral blood CD81 T cells were correlated with those observed in paired samples of synovial fluid and with the Disease Activity Score in 28 joints using the C-reactive protein level (DAS28-CRP), demonstrating the activated proinflammatory status of these lymphocytes in RA and suggesting a role for them in this disease. In their cross-sectional analysis, the authors did not identify any effect of concomitant antirheumatic drugs on the profiles of CD81 T cells. However, only 5 of 96 patients were treated with biologic drugs (all with tumor necrosis factor [TNF]–blocking agents), and this issue might be better addressed via a longitudinal followup study of CD81 T cell characteristics. We considered the T cell costimulatory blocker abatacept (CTLA-4Ig) the most appropriate candidate to modify the profile of T cells in RA (2), and report herein our observations on the effect of this treatment on CD81 T cells. The chemokine receptor CCR7 plays a crucial role with regard to homing in the T cell areas of secondary lymphoid organs. When CCR7 is down-modulated, as a result of repeated T cell stimulations, CCR7– T cells can be recruited in sites of inflammation where they can display their effector activities (3). Carvalheiro et al described a lower frequency of the CCR71CD81 T cells, with a phenotype characteristic of central memory T cells, in patients with RA as compared with healthy controls (1). This abnormality can be modified by treatment with abatacept: in 2010, we reported that in 20 patients with RA, the proportion of central memory (CCR71 CD45RA–) circulating CD81 T cells increased from a median of 11.5% of the total circulating CD81 T cells to 22.3% after 1 year of treatment (P 5 0.002) (4). The variation of CCR7 expression was correlated with that of DAS28-CRP (4). It was unclear, however, whether the results observed were related to a direct effect of abatacept or an indirect effect due to the reduction of disease activity. To evaluate this issue we studied the modification of the T cell phenotype after in vitro stimulation in 5 healthy controls and 10 RA patients before and after 12 months of abatacept therapy (9 of the RA patients were women, the median age was 58 years, 80% were rheumatoid factor positive, 80% were anti–citrullinated protein antibody positive, 7 had received previous treatment with TNF-blocking agents, and the median methotrexate dosage was 15 mg/week). Peripheral blood mononuclear cells (PBMCs) obtained after FicollHypaque gradient centrifugation were cryopreserved in liquid nitrogenous compounds. Paired samples obtained from the same patient before the start of abatacept therapy and after 12 months of abatacept therapy were thawed and cultured in the same experiment. PBMCs were left unstimulated or were stimulated for 5 days with CD3 (20 IU/ml) plus CD28 (1 lg/ml) (Beckman Coulter) at 378C temperature and 5% CO2 pressure, before membrane staining and flow cytometry analysis as previously described (4). As shown in Figure 1, after stimulation, a greater number of cells with the central memory phenotype (CCR71CD45RA–) were observed among CD81 and CD41 T cells in patients treated with abatacept for 12 months as compared with the same patients before the start of therapy and as compared with healthy controls. These results indicate that down-modulation of CCR7 expression after in vitro stimulation is reduced in T cells exposed in vivo to abatacept and suggest that this may be a direct effect of the drug. This observation helps to clarify the mechanism of action of abatacept on T cells, suggesting that in vivo it may act in the secondary lymphoid organ preventing down-modulation of CCR7 and cell efflux in the synovium. Analogous data were found in a study of experimental animals in which abatacept reduced the down-regulation of CD62L, another event needed to exit lymphoid organs (5). Accordingly, results from a study of an RA synovium/SCID mouse model suggested that abatacept acts by preventing T cell activation at a systemic level and does not act directly on the synovium (6). Moreover, our previous studies demonstrated that blocking the CD28 costimulatory pathway can prevent differentiation into the CD81 T cells effector phenotype: in our evaluation of 24 patients with RA we showed that the percentage of CD81 T cells producing IL-17 and IFNc significantly decreased

Decreased circulating T regulatory lymphocytes in obese patients undergoing bariatric surgery

Objective It has been previously demonstrated that T lymphocytes may be involved in the development of hypertension and microvascular remodeling, and that circulating T effector lymphocytes may be increased in hypertension. In particular, Th1 and Th 17 lymphocytes may contribute to the progression of hypertension and microvascular damage while T-regulatory (Treg) lymphocytes seem to be protective in this regard. However, no data is available about patients with severe obesity, in which pronounced microvascular alterations were observed. Design and methods We have investigated 32 severely obese patients undergoing bariatric surgery, as well as 24 normotensive lean subjects and 12 hypertensive lean subjects undergoing an elective surgical intervention. A peripheral blood sample was obtained before surgery for assessment of CD4+ T lymphocyte subpopulations. Lymphocyte phenotype was evaluated by flow cytometry in order to assess T-effector and Treg lymphocytes. Results A marked reduction of several Treg subpopulations was observed in obese patients compared with controls, together with an increased in CD4+ effector memory T-effector cells. Conclusion In severely obese patients, Treg lymphocytes are clearly reduced and CD4+ effector memory cells are increased. It may be hypothesized that they might contribute to the development of marked microvascular alterations previously observed in these patients.

PS5:95 Circulating angiogenic t-cells are reduced in patients with systemic lupus erythematosus with high disease activity and without known cardiovascular risk factors

Background Recent evidences underlined the central role of T-cells in the pathogenesis of Systemic Lupus Erythematosus (SLE) and in its cardiovascular complications.1 CD3 +CD31+CXCR4+angiogenic T-cells (Tang) have been identified as a T-cell subtype involved in the repair of damaged endothelium cooperating with endothelial progenitor cells.2 Tang were described as selectively expanded in the circulation of systemic sclerosis patients displaying peripheral vascular complications, as a reaction to an inefficient angiogenesis.3 Not much information is available on Tang in a SLE patients: in a recent study the percentage of circulating CD8 +Tang, but not CD4 +Tang, was higher in SLE than in healthy controls.4 However, in this study SLE patients with hypertension, dyslipidemia or smoking habit, factors which may influence Tang counts, were not excluded. The aim of this study was to characterise Tang in a cohort of patients with SLE without known cardiovascular risk factors. Methods Twenty female SLE patients with a recent disease onset (<5 years) and without traditional cardiovascular risk factors or previous events (age: median value=43 [25th-75th percentile=27–54] years) and 18 healthy controls (age: 40 [32–54] years) were enrolled. Phenotypic analysis of peripheral Tang lymphocytes was made by flow-cytometry. Disease activity was evaluated by SLEDAI-2K score. Results SLE patients were divided in two groups according with disease activity. Patients with SLEDAI-2K equal or higher than 6 were defined as patients with high disease activity (n:5). They had a lower percentage of circulating Tang in comparison with healthy controls (10 [8–15] vs 16 [14–23]% of CD3 +T cells, p=0.04). The result was confirmed in absolute number (83 [60–103] vs 242 [165–328] cell/microliter, p=0.04). SLE patients with low disease activity had levels of Tang which were intermediate between, and not significantly different from, healthy controls and patients with high disease activity. Conclusions Tang were reduced in our patients with active SLE, and no known cardiovascular risk factors, suggesting that this reduction was directly explained by disease activity. References . Mak A. J Immunol Res2014. . Hur J. Circulation2007. . Manetti M. PLoS One2017. . Miao J. Mediators Inflamm2016.

S7A:6 Baseline serum levels of baff or april are independent predictors of sledai response after 12 months of treatment with belimumab in patients with refractory systemic lupus erythematosus

Background Belimumab, a monoclonal antibody targeting BlyS (B lymphocyte stimulator), is used in refractory Systemic Lupus Erythematosus (SLE). Pivotal clinical trials showed that SLE patients with positive anti-dsDNA antibodies and reduced levels of C3 and/or C4 fractions were those more likely to be responders to treatment. Our study aims at exploring predictors of response to Belimumab in the post-marketing experience in consecutive SLE patients treated at a single centre. Methods Twenty-one patients received Belimumab intravenously at standard regimen (10 mg/kg at 0–15–30 days and then every 4 weeks). Anti-dsDNA were tested by Farr assay and C3/C4 levels by nephelometry. Biomarkers belonging to the TNF superfamily and related to B cell activity (BAFF, APRIL, sBCMA, sCD40L, sTACI, TWEAK) were tested by ELISA. All laboratory parameters were tested at baseline and every 6 months afterwards. SLE disease activity was assessed by SLEDAI-2K score. General linear modelling and correlation analysis were performed using SPSS. Results Enrolled patients were 2 males and 19 females with a median (25th-75th percentile) age of 38 (31–42) years. The disease duration at time of Belimumab start was 12 (8–19) years. The baseline SLEDAI score was 6 (4–9), the anti-dsDNA level was 26 (11–99) UI/ml, and their C3 and C4 level was 72 (56–86) and 9 (7–15) mg/dL, respectively. All the parameters of the TNF superfamily showed moderate/strong correlation (r values ranging from 0.543 and 0.989, p<0.01). With and without correction for different variables, BAFF and APRIL serum levels measured at the start of Belimumab treatment were the most robust predictors of relative SLEDAI reduction after 12 months of treatment (table 1). In contrast, C3, C4, anti-dsDNA, and SLEDAI were less likely to predict relative SLEDAI change at 12 month of Belimumab treatment (uncontrolled model: C3 p=0.410; C4 p=0.778; anti-dsDNA p=0.412) in this cohort of patients preselected for the treatment with Belimumab. Conclusions In this preselected ‘real-life’ cohort of refractory SLE patients fulfilling the requirements for Belimumab treatment baseline serum levels of BAFF or APRIL are independent predictors of response to treatment. Therefore, BAFF and APRIL could be useful for response estimation in patients qualifying for Belimumab treatment.Abstract S7A:6 Table 1 General linear modelling to calculate predictive value of baseline BAFF and APRIL levels for the relative change of SLEDAI at 12 month