S. Vagnani

Risk factors for osteoporosis and fragility fractures in patients with systemic lupus erythematosus

Osteoporosis (OP) and fragility fractures (FFx) are a known comorbidity in patients with systemic lupus erythematosus (SLE). This work aimed at evaluating (1) the prevalence of OP and FFx in a cohort of SLE and (2) the risk factors associated with both OP and FFx. The following data were collected from clinical charts: age, sex, menopausal status (MP), body mass index, smoking habits, disease duration, daily dose and cumulative glucocorticoids (GCs), type of organ involvement, comorbidities and medications. Data on bone metabolism, calcium and vitamin D supplementation and treatment with bisphosphonates, teriparatide or denosumab were collected, together with bone mineral density (BMD) values (measured by dual-energy X-ray absorptiometry (DXA)) and history of FFx (occurred after the onset of SLE and unrelated to trauma). OP and reduced BMD were defined according to the WHO. 186 patients were included (women 175, men 11; mean age 46.4±13 years, mean disease duration 14.9±9 years). At their last visit, 97 patients (52.2%) had a reduced BMD and 52 (27.9%) had OP. 22 patients (11.8%), all women, had at least one FFx; six patients (27.3%) were pre-menopausal. On univariate analysis, age, cumulative dose of GC, MP, therapy with antiepileptics and chronic renal failure (CRF) were correlated with OP (p<0.03); age, total amount of GC, MP, CRF, anticoagulants (AC) and antiepileptic therapy were correlated with FFx (p<0.05). The multivariate logistic model confirmed a direct association of OP and age, MP and antiepileptic therapy (p≤0.01) and of FFx and age, chronic therapy with AC and antiepileptics (p<0.03). In conclusion, low BMD is frequently observed in SLE, and FFx are observed also in premenopausal patients. Together with traditional risk factors (age, MP and GC), CRF and chronic treatments with AC or antiepileptics seem to be associated with a higher risk profile for OP and FFx occurrence.

Sleep disorders and systemic lupus erythematosus.

Objective Sleep disturbances are often seen in rheumatic diseases, including systemic lupus erythematosus (SLE). However, the prevalence of sleep disorders in SLE as well as the contributing factors to their occurrence remain poorly understood. The aim of this paper is to review the clinical and psychobiological data on the relationship between sleep disturbances and SLE. Method We performed a systematic search of MEDLINE, EMBASE and PsychINFO, using MeSH headings and keywords for “sleep disorders” and “SLE.” Results Nine studies reporting the relationship between sleep disorders and SLE were found. Prevalence rates of sleep disorders ranged between 55% and 85%; differences in assessment techniques appeared to be a major source of this variability. In the majority of the studies an association between sleep disorders and disease activity, pain and fatigue has been reported. Psychosocial variables, depression, steroid use, and the role that sleep disruption has on pain, inflammation and cytokines, have been hypothesized as possible psychobiological factors. Conclusions Sleep disorders appear to occur in more than half of patients with SLE and appear to be associated with disease activity. Pain and fatigue are also related to sleep disorders. Among the hypotheses on the possible mechanisms underlining the association between sleep disorders and SLE, psychosocial/psychological factors, especially depression, were the most frequently reported.

The role of imaging in the evaluation of joint involvement in 102 consecutive patients with systemic lupus erythematosus

OBJECTIVE To assess the prevalence of joint involvement in consecutive patients with systemic lupus erythematosus (SLE) by means of clinical assessment, joint US and MRI and to evaluate the sensitivity and specificity of physician evaluation of joint involvement. METHODS At enrollment, patients underwent a complete physical examination including a 44-joint count, and hand deformities were scored. On the day of enrollment, each patient underwent a non-dominant hand-wrist ultrasound (US) examination and a non-dominant hand-wrist MRI study without contrast injection. RESULTS One hundred and two patients (F 95, M 7) were enrolled. By physician examination hand or wrist involvement was diagnosed in 23.5%. At least one pathological finding was revealed by US examination at wrist and/or hand joints in 55%. We found a low sensitivity (46.5%) with high specificity (93.2%) of the physician assessment for the evaluation of joint involvement. The MRI imaging showed at least one erosion in 47.3% patients at the hand and in 98.9% at the wrist; in healthy subjects erosions were found in 19.6% and 97.8% at the hand and wrist, respectively. CONCLUSIONS In conclusion, (i) physicians tend to underestimate the severity of joint involvement in SLE; (ii) US assessment shows a high prevalence of joint and tendon involvement; and (iii) the MRI evaluation shows a high prevalence of damage, suggesting that joint involvement in SLE could be more severe than expected.

Protective Effects of Quercetin on Rat Pial Microvascular Changes during Transient Bilateral Common Carotid Artery Occlusion and Reperfusion

The aim of this study was to assess the in vivo effects of quercetin on pial microvascular responses during transient bilateral common carotid artery occlusion (BCCAO) and reperfusion. Rat pial microcirculation was visualized by fluorescence microscopy through a closed cranial window. Pial arterioles were classified in five orders of branchings. Capillaries were assigned order 0, the smallest arterioles order 1, and the largest ones order 5. In ischemic rats, 30 min BCCAO and 60 min reperfusion caused arteriolar diameter decrease (by 14.5 ± 3.3% of baseline in order 2), microvascular leakage [0.47 ± 0.04, normalized gray levels (NGL)], leukocyte adhesion in venules (9 ± 2/100 μm venular length, v.l./30 s), and reduction of capillary perfusion (by 40 ± 7% of baseline). Moreover, at the end of BCCAO and reperfusion there was a significant increase in reactive oxygen species (ROS) formation when compared with baseline. Quercetin highest dose determined dilation in all arteriolar orders (by 40 ± 4% of baseline in order 2) and prevented microvascular permeability (0.15 ± 0.02 NGL), leukocyte adhesion (3 ± 1/100 μm v.l./30 s) as well as ROS formation, while capillary perfusion was protected. Inhibition of endothelial nitric oxide synthase (NOS) prior to quercetin reduced arteriolar dilation (order 2 diameter increase by 10.3 ± 2.5% of baseline) and caused permeability increase (0.29 ± 0.03 NGL); inhibition of neuronal NOS or inducible NOS did not affect quercetin-induced effects. Inhibition of guanylyl cyclase prior to quercetin reversed the quercetin’s effects on pial arteriolar diameter and leakage. In conclusion, quercetin was able to protect pial microcirculation from ischemia–reperfusion damage inducing arteriolar dilation likely by nitric oxide release. Moreover, quercetin scavenger activity blunted ROS formation preserving the blood–brain barrier integrity.

Rat Pial Microvascular Responses to Transient Bilateral Common Carotid Artery Occlusion and Reperfusion: Quercetin’s Mechanism of Action

The aim of the present study was to assess quercetin’s mechanism of action in rat pial microvessels during transient bilateral common carotid artery occlusion (BCCAO) and reperfusion. Rat pial microcirculation was visualized using fluorescence microscopy through a closed cranial window. Pial arterioles were classified in five orders of branchings. In ischemic rats, 30 min BCCAO and 60 min reperfusion caused arteriolar diameter decrease, microvascular leakage, leukocyte adhesion in venules, and reduction of capillary perfusion. Quercetin highest dose determined dilation in all arteriolar orders, by 40 ± 4% of baseline in order 2 vessels, and prevented microvascular permeability [0.15 ± 0.02 normalized gray levels (NGL)], leukocyte adhesion, and capillary failure. Protein kinase C (PKC) inhibition exerted by chelerythrine prior to quercetin attenuated quercetin-induced effects: order 2 arterioles dilated by 19.0 ± 2.4% baseline, while there was an increase in permeability (0.40 ± 0.05 NGL) and leukocyte adhesion with a marked decrease in capillary perfusion. Tyrosine kinase (TK) inhibition by tyrphostin 47 prior to quercetin lessened smaller pial arterioles responses, dilating by 20.7 ± 2.5% of baseline, while leakage increased (0.39 ± 0.04 NGL) sustained by slight leukocyte adhesion and ameliorated capillary perfusion. Inhibition of endothelium nitric oxide synthase (eNOS) by NG-nitro-L-arginine-methyl ester (L-NAME) prior to PKC or TK reduced the quercetin’s effects on pial arteriolar diameter and leakage. eNOS inhibition by L-NAME reduced quercetin effects on pial arteriolar diameter and leakage. Finally, combined inhibition of PKC and TK prior to quercetin abolished quercetin-induced effects, decreasing eNOS expression, while blocking ATP-sensitive potassium (KATP) channels by glibenclamide suppressed arteriolar dilation. In conclusion, the protective effects of quercetin could be due to different mechanisms resulting in NO release throughout PKC and TK intracellular signaling pathway activation.

Rat pial microvascular responses to melatonin during bilateral common carotid artery occlusion and reperfusion

Abstract: The present study assessed the in vivo rat pial microvascular responses induced by melatonin during brain hypoperfusion and reperfusion (RE) injury. Pial microcirculation of male Wistar rats was visualized by fluorescence microscopy through a closed cranial window. Hypoperfusion was induced by bilateral common carotid artery occlusion (BCCAO, 30 min); thereafter, pial microcirculation was observed for 60 min. Arteriolar diameter, permeability increase, leukocyte adhesion to venular walls, perfused capillary length (PCL), and capillary red blood cell velocity (VRBC) were investigated by computerized methods. Melatonin (0.5, 1, 2 mg/kg b.w.) was intravenously administered 10 min before BCCAO and at the beginning of RE. Pial arterioles were classified in five orders according to diameter, length, and branchings. In control group, BCCAO caused decrease in order 2 arteriole diameter (by 17.5 ± 3.0% of baseline) that was reduced by 11.8 ± 1.2% of baseline at the end of RE, accompanied by marked leakage and leukocyte adhesion. PCL and capillary VRBC decreased. At the end of BCCAO, melatonin highest dosage caused order 2 arteriole diameter reduction by 4.6 ± 2.0% of baseline. At RE, melatonin at the lower dosages caused different arteriolar responses. The highest dosage caused dilation in order 2 arteriole by 8.0 ± 1.5% of baseline, preventing leakage and leukocyte adhesion, while PCL and VRBC increased. Luzindole (4 mg/kg b.w.) prior to melatonin caused order 2 arteriole constriction by 12.0 ± 1.5% of baseline at RE, while leakage, leukocyte adhesion, PCL and VRBC were not affected. Prazosin (1 mg/kg b.w.) prior to melatonin did not significantly change melatonin’s effects. In conclusion, melatonin caused different responses during hypoperfusion and RE, modulating pial arteriolar tone likely by MT1 and MT2 melatonin receptors while preventing blood–brain barrier changes through its free radical scavenging action.

TNF-alpha inhibitors in Systemic Lupus Erythematosus. A case report and a systematic literature review

Abstract Joint involvement is a common manifestation of systemic lupus erythematosus (SLE) and is described as a non-erosive mild synovitis. However some SLE patients may present a more severe joint involvement requiring aggressive therapy. We describe the case of a SLE patient with a severe arthritis unresponsive to methotrexate, successfully treated with anti-TNF-alpha drug as induction therapy and we report the results of a systematic literature review on the use of TNF-alpha inhibitors in SLE.

Effects of bone marrow mesenchymal stem cells (BM-MSCs) on rat pial microvascular remodeling after transient middle cerebral artery occlusion

Previous studies have shown that the pial microcirculation remodeling improves neurological outcome after middle cerebral artery occlusion (MCAO), accompanied by higher expression of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS), modulating in vivo angiogenesis. This study was aimed to assess the effects of bone marrow mesenchymal stem cells (BM-MSCs) infused after MCAO on rat pial microcirculation. Animals were subjected to 2 h MCAO followed by BM-MSCs infusion into internal carotid artery. Pial microcirculation was observed at different reperfusion times by fluorescence microscopy. Geometric characteristics of arteriolar networks, permeability increase, leukocyte adhesion, perfused capillary density, VEGF, and endothelial nitric oxide synthase (e-NOS) expression were evaluated. Green fluorescent protein (GFP)-BM-MSCs were used to evaluate their distribution and cell phenotype development during reperfusion. BM-MSCs stimulated a geometric rearrangement of pial networks with formation of new anastomotic vessels sprouting from preexistent arterioles in the penumbra at 7–14–28 days of reperfusion. At the same time VEGF and eNOS expression increased. GFP-BM-MSCs appear to be involved in endothelial and smooth muscle cell programming in the infarcted area. In conclusion, transient MCAO induced pial vascular remodeling characterized by arteriolar anastomotic arcades (originated from preexistent arterioles in penumbra area) able to overlap the ischemic core supplying blood to the neuronal tissue. BM-MSCs appear to accelerate angiogenic processes facilitating new vessel formation; this mechanism was promoted by an increase in VEGF and eNOS expression.

Treatment with Allogenic Mesenchymal Stromal Cells in a Murine Model of Systemic Lupus Erythematosus

Objective Pre-clinical and uncontrolled studies in patients with systemic lupus erythematosus (SLE) showed that mesenchymal stromal cells (MSCs) have a potential therapeutic role in refractory cases. The optimal therapeutic strategy in these patients remain to be elucidated. Our aim was to test the hypothesis that repeated administrations of 1×106/kg body weight of allogenic MSCs, that is a significantly lower dosage with respect to the fixed 1×106 MSC used in animal models, can be effective in improving the clinical course of a murine SLE model. Methods Bone marrow derived MSCs were obtained from 12-week-old C57BL/6J mice. Seventy-five 8 weeks old female NZ mice were randomly assigned to receive via caudal vein the following alternative treatments: 1) single infusion of 106 MSCs/kg body weight at 18 weeks of age (NZs18) or at at 22 weeks of age (NZs22); 2) multiple monthly infusions of 106 MSCs/kg body weight starting at 18 weeks of age (NZM18) or at 22 weeks of age (NZM22); 3) saline infusions (NZc) Fifteen 8 weeks old C57BL/6J mice (Envigo, Huntingdon, UK) were used as untreated controls (C). Weekly, body weight was recorded and twenty-four hour urines were collected by metabolic cages for each animal; proteinuria was detected by dipstick analysis. At sacrifice, peripheral blood samples were collected from mice and anti-dsDNA antibodies were detected by enzyme immunoassorbent assay (ELISA) method using commercial kits. At sacrifice, kidneys were analyzed for histopathology and immunohistochemical analysis for B220, CD4, MPO, CD4+Foxp3, F40/80 infiltration was performed. Results Proteinuria occurrence was delayed NZS and NZM mice, no differences were observed in anti-dsDNA autoantibody titer among the groups at the different time-points; at 36 weeks, no significant differences were observed in term of nephritis scores. Inflammatory cells deposition (MPO and F4/80 positive cells) in NZM was significantly higher than in NZ and NZS. An overexpression of B lymphocytes (B220) was found in NZM while T regulatory cells (CD4+ Foxp3+ cells) were reduced in both NZS and NZM with respect to NZc. Conclusions Overall, our study failed to show a positive effect of a treatment with murine MSCs in this model and, for some aspects, even deleterious results seem to be observed.

AB0190 Treatment with Bone Marrow Mesenchymal Stromal Cells in a Murine Model of Systemic Lupus Erythematosus

Background In recent decades, cell-based therapies have emerged as a new therapeutic option for refractory and severe systemic lupus erythematosus (SLE) (1). Mesenchymal stromal cells (MSCs) could represent an excellent candidate but several aspects remain to be extensively investigated such as the most efficient treatment timing and dosage. Objectives The present research is aimed at comparing the effect of different protocols of allogenic MSCs infusions on clinical and serological parameters in a mouse model of SLE. Methods 35 female New Zealand Black/New Zealand White F1 (NZB/W) mice have been used as model of SLE and 10 C57BL/6J mice have been used as donors of allogenic bone marrow derived MSCs (BMMSCs). NZB/W mouse were divided into the following experimental groups:– 5 NZB/W treated with a single MSCs infusion at 20 weeks of age (NZB/W20)– 5 NZB/W treated with a single MSCs infusion at 24 weeks of age (NZB/W24)– 5 NZB/W treated with a single MSCs infusion at 32 weeks of age (NZB/W32)– 5 NZB/W treated with multiple MSCs infusions started at 20 weeks of age (NZB/WM20)– 5 NZB/W treated with multiple MSCs infusions started at 24 weeks of age (NZB/WM24)– 10 NZB/W mouse not treated as Controls (C) Proteinuria was recorded weekly and peripheral blood serum samples were collected at sacrifice to test for anti-dsDNA antibodies (Alpha Diagnostics International). At sacrifice, kidneys were harvested and sections were stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS). H&E and PAS stained sections were evaluated for glomerular, interstitial and vascular lesions according to a modified score described by Tao X et al (2). Results In untreated mice (C) proteinuria was detectable at 17 weeks of age (3.5±2.7 mg/day) and further increased until 35 weeks of age (12.5±3.5 mg/day). At 24 weeks, proteinuria was 1.8±2.3 mg/day in C group, 0.9±0.4 mg/day in NZB/W20, 0.7±0.5 mg/day in NZB/WM20. At 30 weeks proteinuria was 4.9±2 mg/day in untreated mice, 4.6±5 mg/day in NZB/W20, 3.2±0.2 mg/day in NZB/W24, 0.7±0.5 mg/day in NZB/WM20 and 0.8±0.4 mg/day in NZB/WM24. At 36 weeks proteinuria was 12.5±3.5 mg/day in C group, 8.5+6.1 mg/day in NZB/W20, 3.7+3.4 mg/day in NZB/W24, 9.4+5.1 mg/day in NZB/W32, 6.5±5 mg/day in NZB/WM20 (p=0.006 vs C group) and 4.5+2.5 mg/day in NZB/WM24 (P<0.0001 vs C group). The titers of the anti-dsDNA antibody did not change between groups. No significant differences in histological kidney scores were observed between single and multiple treatments. Conclusions Our results showed that multiple MSCs infusion are associated with a decrease of proteinuria; on the contrary both single and multiple treatments had no effect on autoantibodies production as well as on histological progression of the kidney disease. References Hugle T et al. stem cell transplantation for rheumatic autoimmune diseases. Arthritis Res Ther, 2008. Tao X et al. Therapeutic impact of the ethyl acetate extract of Tripterygium wilfordii Hook F on nephritis in NZB/W F1 mice. Arthritis ResTher, 2006. Disclosure of Interest None declared