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.

Early treatment with hydroxychloroquine prevents the development of endothelial dysfunction in a murine model of systemic lupus erythematosus.

INTRODUCTION: Accelerated atherosclerosis is one of the major causes of morbidity in patients with systemic lupus erythematosus (SLE). Endothelial dysfunction (ED) is considered an early marker of atherosclerosis. It is a reversible alteration, thus representing an attractive target for prevention strategies against cardiovascular disease. Studies have shown that ED occurs in patients with SLE even in the absence of severe, active disease. Hydroxychloroquine (HCQ) is widely used in SLE to control disease activity, but its use is also associated with an improvement in long-term prognosis. Beyond the beneficial effect in well-established disease, our hypothesis is that treatment with HCQ might have a beneficial impact on ED prevention in SLE. The aim of this study was to assess the impact of early treatment with HCQ on ED in a murine model of SLE. METHODS: Twelve-week-old NZB/W F1 (NZ) and C57BL/6 J mice (controls) were allocated to receive HCQ or vehicle for 6, 12, or 18 weeks. Proteinuria and anti-double-stranded DNA autoantibodies were determined. ED was assessed in mesenteric arteries (pressurized myography). Nitric oxide (NO) availability and reactive oxygen species (ROS) production were evaluated. Vascular ROS production was measured with dihydroethidium (DHE) fluorescent dye. RESULTS: Starting from 18 weeks of age, NZ mice showed a progressive reduction in NO availability, which was normalized by ascorbic acid and apocynin in the up to 24-week-old group, and partly ameliorated in older animals. HCQ administration normalized the NO availability in the up to 24-week-old group, with a partial amelioration in the 30-week-old group. DHE analysis revealed a progressive increment of vascular ROS generation among NZ groups, which was prevented by apocynin. Similarly, in the NZ HCQ-treated group, vascular ROS production was abrogated. CONCLUSIONS: The ED that characterizes this mouse model of SLE is caused by the nicotinamide adenine dinucleotide phosphate oxidase-driven ROS excess. Very early treatment with HCQ is able to exert vascular protection via an antioxidant effect.

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.

Leukopenia, lymphopenia, and neutropenia in systemic lupus erythematosus: Prevalence and clinical impact--A systematic literature review.

OBJECTIVE: To systematically review the available evidence to evaluate (1) the prevalence and degree of leukopenia, lymphopenia, and neutropenia in patients with systemic lupus erythematosus (SLE), (2) whether these conditions carry a major infection risk for patients, and (3) whether a treatment with colony stimulating factors (CSF) can be an effective and safe option in SLE patients with leukopenia. MATERIAL AND METHODS: MedLine and Embase were searched by including MeSH terms, text words, and subheadings "systemic lupus erythematosus," "leukopenia" (first search), and "colony stimulating factor" (second search). Inclusion and exclusion criteria were a priori defined and two reviewers screened the retrieved articles for selection criteria; data from the included studies were recorded in ad hoc standard forms; the results were synthesized and transported to evidence tables. RESULTS: A total of 17 articles were included in the systematic literature review: nine articles were retrieved for the first research question and 11 for the second while no articles satisfied the inclusion criteria for the third research question. The prevalence of leukopenia is reported in 22-41.8% of cases and lymphopenia is reported cumulatively from 15% to 82% of the patients while neutropenia is described in 20-40% of the patients. There is no evidence of a significant association between overall reduction of white blood cells and infection occurrence while some studies found a strong association between low lymphocytes/neutrophils count and the risk of major infections. Only case reports and case series have been found to investigate the safety of CSF in SLE patients. CONCLUSIONS: The results of this systematic literature review are inconclusive for many aspects related to the original research questions and highlight the need for further studies. Indeed, the strength of the evidence is not sufficiently robust to draw specific recommendations on how to balance between the need to treat the patient with SLE with immunosuppressive drugs and the risk of severe infections.

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.

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

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.

The diagnosis and classification of undifferentiated connective tissue diseases.

The term undifferentiated connective tissue disease (UCTD) refers to unclassifiable systemic autoimmune diseases which share clinical and serological manifestations with definite connective tissue diseases (CTDs) but not fulfilling any of the existing classification criteria. In this review we will go through the more recent evidence on UCTD and we will discuss in what extent the availability of new criteria for the CTDs could interfere with the "UCTD concept". The development of criteria able to identify early phases of defined CTD, may help in the differentiation of stable UCTD form their early stages and may offer a valuable guide to the treating physician to set up appropriate follow up schedules as well as therapeutic protocols. This simplified subset of CTD could offer a model to study clinic pathological correlations as well as the role of possible environmental factors in the development of autoimmunity.

The diagnosis and classification of mixed connective tissue disease.

The term "mixed connective tissue disease" (MCTD) concerns a systemic autoimmune disease typified by overlapping features between two or more systemic autoimmune diseases and the presence of antibodies against the U1 small nuclear ribonucleoprotein autoantigen (U1snRNP). Since the first description of this condition in 1972, the understanding of clinical manifestations and long-term outcome of MCTD have significantly advanced. Polyarthritis, Raynaud's phenomenon, puffy fingers, lung involvement and esophageal dysmotility are the most frequently reported symptoms among the different cohorts during the course of the disease. Moreover, in recent years a growing interest has been focused on severe organ involvement such as pulmonary arterial hypertension and interstitial lung disease which can accrue during the long-term follow-up and can still significantly influence disease prognosis. Over the last years, significant advances have been made also in disease pathogenesis understanding and a central pathogenetic role of anti-U1RNP autoantibodies has clearly emerged. Although controversies on disease definition and classification still persist, MCTD identifies a group of patients in whom increased surveillance for specific manifestations and prognostic stratification became mandatory to improve patient's outcomes.

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

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 (V(RBC) ) 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 V(RBC) 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 V(RBC) 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 V(RBC) 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.