Francesca Calcaterra

Treatment with belimumab restores B cell subsets and their expression of B cell activating factor receptor in patients with primary Sjogren’s syndrome

OBJECTIVE The aim of this study was to investigate the biological effects of belimumab on B cells in the first phase II open-label trial with belimumab in patients with primary SS (pSS) (BELISS). METHODS Peripheral blood B cell subsets and their B cell activating factor-receptor (BAFF-R) expression were analysed by multicolour flow cytometry in 10 pSS patients either before or after 24 and 52 weeks of therapy with belimumab. Serum BAFF levels were analysed by ELISA. RESULTS At baseline, pSS patients showed a significant increase in circulating B cells compared with healthy donors matched for age and sex, with a predominant expansion of transitional and naive B cell subsets. pSS patients also showed higher serum BAFF levels and lower B cell BAFF-R expression. Therapy with belimumab in pSS patients induced a significant reduction in transitional and naive B cell subsets to levels similar to those observed in healthy donors. Furthermore, belimumab normalized BAFF-R expression in all B subsets comprised within the memory compartment. The restoration of B cell frequency and subset composition in response to belimumab was also associated with a decrease in serum levels of Ig, RF, ANAs, and with an increase in the C4 complement fraction. All of these belimumab-mediated effects were observed after 24 weeks of therapy and maintained until the end of the therapeutic protocol. CONCLUSION Taken together, our findings show that targeting BAFF with belimumab is successful in normalizing B cell frequency, phenotype and functions in pSS. TRIAL REGISTRATION clinicaltrials.gov; https://clinicaltrials.gov/; NCT01008982.

Comparison of Fibronectin and Collagen in Supporting the Isolation and Expansion of Endothelial Progenitor Cells from Human Adult Peripheral Blood.

Background Endothelial colony-forming cells (ECFCs), are circulating endothelial progenitor cells increasingly studied in various diseases because of their potential for clinical translation. Experimental procedures for their ex vivo culture still lack standardization. In particular two different extracellular matrix proteins, either fibronectin or collagen, are commonly used by different Authors for coating plastic plates, both allowing to obtain cells that have all the features of ECFCs. However, possible differences in the impact of each substrate on ECFCs have not been analysed, so far. Therefore, in this study we investigated whether fibronectin and collagen may differentially affect ECFC cultures. Methodology/Principal Findings ECFCs were isolated and cultured from peripheral blood mononuclear cells of healthy donors. The impact of fibronectin compared with collagen as the only variable of the experimental procedure was analysed separately in the phase of isolation of ECFC colonies and in the following phase of cell expansion. In the isolation phase, although similar frequencies of colonies were obtained on the two substrates, ECFC colonies appeared some days earlier when mononuclear cells were seeded on fibronectin rather than collagen. In the expansion phase, ECFCs cultured on collagen showed a longer lifespan and higher cell yields compared with ECFCs cultured on fibronectin, possibly related to the higher levels of IL-6 and IL-8 measured in their supernatants. ECFCs cultured on both substrates showed similar immunophenotype and ability for in vitro tube formation. Conclusions/Significance Overall, the results of this study indicate that, although both fibronectin and collagen efficiently sustain ECFC cultures, each of them brings some advantages within individual steps of the entire process. We suggest that colony isolation performed on fibronectin followed by cell expansion performed on collagen may represent a novel and the most efficient strategy to obtain ECFCs from adult peripheral blood samples.

MFSD2A Promotes Endothelial Generation of Inflammation-resolving Lipid Mediators and Reduces Colitis in Mice

BACKGROUND & AIMS Alterations in signaling pathways that regulate resolution of inflammation (resolving pathways) contribute to pathogenesis of ulcerative colitis (UC). The resolution process is regulated by lipid mediators, such as those derived from the ω-3 docosahexaenoic acid (DHA), whose esterified form is transported by the major facilitator superfamily domain containing 2A (MFSD2A) through the endothelium of brain, retina, and placenta. We investigated if and how MFSD2A regulates lipid metabolism of gut endothelial cells to promote resolution of intestinal inflammation. METHODS We performed lipidomic and functional analyses of MFSD2A in mucosal biopsies and primary human intestinal microvascular endothelial cells (HIMECs) isolated from surgical specimens from patients with active, resolving UC and healthy individuals without UC (controls). MFSD2A was knocked down in HIMECs with small hairpin RNAs or overexpressed from a lentiviral vector. Human circulating endothelial progenitor cells that overexpress MFSD2A were transferred to CD1 nude mice with dextran sodium sulfate-induced colitis, with or without oral administration of DHA. RESULTS Colonic biopsies from patients with UC had reduced levels of inflammation-resolving DHA-derived epoxy metabolites compared to healthy colon tissues or tissues with resolution of inflammation. Production of these metabolites by HIMECs required MFSD2A, which is required for DHA retention and metabolism in the gut vasculature. In mice with colitis, transplanted endothelial progenitor cells that overexpressed MFSD2A not only localized to the inflamed mucosa but also restored the ability of the endothelium to resolve intestinal inflammation, compared with mice with colitis that did not receive MFSD2A-overexpressing endothelial progenitors. CONCLUSIONS Levels of DHA-derived epoxides are lower in colon tissues from patients with UC than healthy and resolving mucosa. Production of these metabolites by gut endothelium requires MFSD2A; endothelial progenitor cells that overexpress MFSD2A reduce colitis in mice. This pathway might be induced to resolve intestinal inflammation in patients with colitis.

Bright expression of CD91 identifies highly activated human dendritic cells that can be expanded by defensins

CD91 is a scavenger receptor expressed by different immune cells and its ligands defensins have been demonstrated to contribute to immune responses against infections and tumours. We previously demonstrated that CD91 is expressed on human monocyte‐derived dendritic cells (moDCs) and that human defensins stimulate in vitro the activation of these cells. In this study, we observed that CD91 is expressed at different levels on two distinct moDC subsets: CD91dim and CD91bright moDCs. Although CD91bright moDCs represented a small proportion of total moDCs, this subset showed higher levels of activation and maturation markers compared with CD91dim moDCs. The frequency of CD91bright moDCs increased by ~ 50% after in vitro stimulation with recombinant human neutrophil peptide‐1 (rHNP‐1) and recombinant human β defensin‐1 (rHBD‐1), while lipopolysaccharide (LPS) stimulation decreased it by ~ 35%. Both defensins up‐regulated moDC expression of CD80, CD40, CD83 and HLA‐DR, although to a lower extent compared with LPS. Notably, upon culture with rHNP‐1 and rHBD‐1, CD91bright moDCs maintained their higher activation/maturation status, whereas this was lost upon culture with LPS. Our findings suggest that defensins promote the differentiation into activated CD91bright DCs and may encourage the exploitation of the CD91/defensins axis as a novel therapeutic strategy to potentiate antimicrobial and anti‐tumour immune response.

Lack of activation of peripheral blood dendritic cells in human pregnancies complicated by intrauterine growth restriction

INTRODUCTION The state of activation of dendritic cells (DCs) at the feto-maternal interface critically contributes to optimal decidual immune responses needed to support fetal-placental development. We recently demonstrated that during healthy pregnancy also peripheral blood DCs (PBDCs), which are easily accessible, are activated as well. In this study, to investigate a possible involvement of DCs in intrauterine growth restriction (IUGR), we evaluated whether PBDCs in pregnancy complicated by IUGR may be altered compared with PBDCs in healthy pregnancy. METHODS PBDCs from 12 pregnant women with primary IUGR, 21 healthy pregnant and 19 nonpregnant women were analyzed by flow cytometric analysis of whole-blood samples collected at a single time point. RESULTS The number of plasmacytoid PBDCs was significantly reduced in women with IUGR pregnancy. Myeloid and plasmacytoid PBDCs in IUGR lacked the state of activation (assessed as CD80, CD86, CD40 expression) and the shift to a proinflammatory pattern of cytokine production occurring during healthy pregnancy. DISCUSSION To our knowledge, this is the first study investigating the state of PBDC activation in IUGR pregnancy. Our results are in accordance with a previous study reporting a lower expression of activation and maturation markers by decidual DCs in IUGR placentas. CONCLUSIONS The reduced activation of PBDCs in IUGR pregnancy may possibly reflect a reduced activation of decidual DCs. If confirmed at the feto-maternal interface, the alterations of DCs described in IUGR pregnancy have the potential to negatively impact on vascular development during gestation. These observations may therefore broaden our understanding of IUGR pathogenesis.

Reduction of maternal circulating endothelial progenitor cells in human pregnancies with intrauterine growth restriction

INTRODUCTION Circulating endothelial progenitor cells (EPCs) may play a crucial role during pregnancy by sustaining adequate placentation and fetal growth. Unambiguous demonstration of EPC increase during pregnancy has been hampered so far by lack of standardized methods for EPC quantification. In this study we used the currently most accepted phenotype for EPC detection for investigating whether maternal circulating EPCs might increase during normal pregnancy and whether they may fail to increase in pregnancy complicated by idiopathic intrauterine growth restriction (IUGR), a leading cause of perinatal mortality and morbidity characterized by insufficient placental perfusion. METHODS Twenty-one non-pregnant women, 44 women during healthy pregnancy progression (9, 13 and 22 women in the first, second and third trimester, respectively) and 11 with pregnancy complicated by idiopathic IUGR were recruited in a cross-sectional study. EPCs in maternal blood were identified as CD45(dim)/CD34+ / KDR+ cells by flow cytometry. Plasmatic cytokines were measured by ELISA. RESULTS We observed a significant and progressive increase of EPCs in normal pregnancy, yet detectable in early pregnancy but even more pronounced in the third trimester. The increase of EPCs was impaired in IUGR-complicated pregnancies at comparable gestational age. The circulating levels of placental growth-factor and stromal-derived-factor-1 were significantly lower in IUGR than normal pregnancies, possibly contributing to EPC impairment. CONCLUSIONS EPC count in maternal circulation may have a great potential as a novel biomarker for pregnancy monitoring and may represent the target of novel therapeutic strategies designed to prevent adverse pregnancy outcomes often occurring in IUGR.

Enhanced phosphorylation of sphingosine and ceramide sustains the exuberant proliferation of endothelial progenitors in Kaposi sarcoma

Endothelial colony‐forming cells (ECFCs), a unique endothelial stem cell population, are highly increased in the blood of Kaposi sarcoma (KS) patients. KS‐derived ECFCs (KS‐ECFCs) are also endowed with increased proliferative and vasculogenic potential, thus suggesting that they may be precursors of KS spindle cells. However, the mechanisms underlying the increased proliferative activity of KS‐ECFCs remain poorly understood. Sphingosine‐1‐phosphate (S1P) and ceramide‐1‐phosphate (C1P) are metabolically interconnected sphingoid mediators crucial to cell proliferation. Here, we investigated the metabolism, release, and proliferative effects of S1P and C1P in KS‐ECFCs compared with control ECFCs (Ct‐ECFCs). Metabolic studies by cell labeling, chromatographic analyses, and digital autoradiography revealed that S1P and C1P biosynthesis and S1P secretion are all efficient processes in KS‐ECFCs, more efficient in KS‐ECFCs than Ct‐ECFCs. Quantitative PCR analyses demonstrated a significantly higher ceramide kinase and sphingosine kinase‐2 expression in KS‐ECFCs. Notably, also the expression of S1P1 and S1P3 receptors was augmented in KS‐ECFCs. Accordingly, treatment with exogenous C1P or S1P induced a significant, concentration‐dependent stimulation of KS‐ECFC proliferation, but was almost completely ineffective in Ct‐ECFCs. Hence, we identified C1P and S1P as autocrine/paracrine proliferative signals in KS‐ECFCs. A better understanding of the mechanisms that enhance S1P/C1P formation in KS‐ECFCs may yield effective therapeutic modalities.