Characterization and Biological Activities of In Vitro Digested Olive Pomace Polyphenols Evaluated on Ex Vivo Human Immune Blood Cells.

Olive pomace (OP) represents one of the main by-products of olive oil production, which still contains high quantities of health-promoting bioactive compounds. In the present study, three batches of sun-dried OP were characterized for their profile in phenolic compounds (by HPLC-DAD) and in vitro antioxidant properties (ABTS, FRAP and DPPH assays) before (methanolic extracts) and after (aqueous extracts) their simulated in vitro digestion and dialysis. Phenolic profiles, and, accordingly, the antioxidant activities, showed significant differences among the three OP batches, and most compounds showed good bioaccessibility after simulated digestion. Based on these preliminary screenings, the best OP aqueous extract (OP-W) was further characterized for its peptide composition and subdivided into seven fractions (OP-F). The most promising OP-F (characterized for its metabolome) and OP-W samples were then assessed for their potential anti-inflammatory properties in ex vivo human peripheral mononuclear cells (PBMCs) triggered or not with lipopolysaccharide (LPS). The levels of 16 pro-and anti-inflammatory cytokines were measured in PBMC culture media by multiplex ELISA assay, whereas the gene expressions of interleukin-6 (IL-6), IL-10 and TNF-α were measured by real time RT-qPCR. Interestingly, OP-W and PO-F samples had a similar effect in reducing the expressions of IL-6 and TNF-α, but only OP-W was able to reduce the release of these inflammatory mediators, suggesting that the anti-inflammatory activity of OP-W is different from that of OP-F.

Frequency and clinical correlates of antiphospholipid antibodies arising in patients with SARS-CoV-2 infection: findings from a multicentre study on 122 cases.

OBJECTIVES: COVID-19 features include disseminated intravascular coagulation and thrombotic microangiopathy indicating a hypercoagulable state. We aimed to investigate antiphospholipid antibodies (aPL) prevalence and clinical relationships in a large cohort of COVID-19 patients. METHODS: We analysed the prevalence and titres of serum aPL in 122 patients with COVID-19 and 157 with primary antiphospholipid syndrome (PAPS) and 91 with other autoimmune rheumatic diseases (oARD) for comparison. IgG/IgM anticardiolipin (aCL) and IgG/IgM anti-beta2glycoprotein I (ß2GPI) were assayed using homemade ELISA, IgA aCL and anti-ß2GPI by commercial ELISA kits and lupus anticoagulant (LAC) by multiple coagulation tests following updated international guidelines. RESULTS: Prevalence of IgG and IgM aCL and of IgG and IgM anti-ß2GPI across COVID-19 patients were 13.4%, 2.7%, 6.3% and 7.1%, being significantly lower than in PAPS (p<0.0001 for all). Frequency of IgG aCL and IgM anti-ß2GPI was comparable to oARD (13.4% vs. 13.2% and 7.1% vs. 11%, respectively), while IgG anti-ß2GPI and IgM aCL were lower (p<0.01). IgA aCL and IgA anti-ß2GPI were retrieved in 1.7% and 3.3% of COVID-19 patients, respectively. Positive LAC was observed in 22.2% COVID-19 vs. 54.1% of PAPS (p<0.0001) and 14.6% of oARD (p=0.21). Venous or arterial thromboses occurred in 18/46 (39.1%) COVID-19 patients and were not associated with positive aPL (p=0.09). CONCLUSIONS: Thrombosis is a frequent manifestation during COVID-19 infection. However, prevalence and titres of aPL antibodies or LAC were neither consistently increased nor associated with thrombosis when measured at a single timepoint, therefore not representing a suitable screening tool in the acute stage of disease.

Remission of hyperglycemia in spontaneously diabetic NOD mice upon transplant of microencapsulated human umbilical cord Wharton jelly-derived mesenchymal stem cells (hUCMS).

BACKGROUND: Our previous in vitro demonstration of the immunoregulatory effects of microencapsulated hUCMS on human peripheral blood mononuclear cells (PBMCs) extracted from patients with recent onset, type 1 diabetes mellitus (DM), prompted us to test our product for xenograft (TX) in non obese diabetic (NOD) mice with spontaneous DM. METHODS: We transplanted microencapsulated hUCMS into the peritoneal cavity of NOD mice with either severe or mild DM. Blood glucose (BG) levels were monitored following TX, in either basal or upon glucose stimulation. RESULTS: Only the NODs with mild DM showed full and sustained remission of hyperglycemia throughout 216 days post-TX, unlike recipients with severe DM, where no remission of hyperglycemia was attained, as reflected by erratic BG levels at all times. CONCLUSIONS: These data suggest that the stage of DM disease process in NOD mice, reflecting steady decline of residual b-cell mass, plays a pivotal role in determining the success of this cell therapy approach for treatment of DM.

Angiogenic T cells in primary Sjögren's syndrome: a double-edged sword?

OBJECTIVES: The mechanisms underlying increased cardiovascular risk in primary Sjögren's syndrome (pSS) remain unclear. Since the recently discovered angiogenic T cells (Tang) may participate in endothelial repair by cooperating with endothelial progenitor cells (EPC), we aimed to quantify and characterise Tang in the peripheral blood and minor salivary glands (MSG) of pSS patients. METHODS: Tang (CD3+CD31+CXCR4+) and EPC (CD34+CD133+VEGFR-2+) were quantified by flow cytometry in peripheral blood samples from 36 pSS patients and 20 healthy donors. Tang subsets were assessed on the basis of CD4, CD8 and CD28 expression. Labial MSG sections from 10 pSS patients and 12 non-pSS sicca syndrome controls were subjected to immunofluorescence staining to investigate the presence of Tang and the expression of the CXCR4-ligand stromal cell-derived factor-1 (SDF-1)/CXCL12. RESULTS: Circulating Tang cells were expanded and directly correlated to EPC in pSS. Both Tang and EPC directly correlated with disease activity as calculated with the EULAR Sjögren's syndrome disease activity index (ESSDAI). In pSS, the majority of Tang cells were CD4-CD8- double negative (DN) and lacked CD28 revealing a senescent phenotype. A subset of CD4+, CD8+ and DN Tang cells produced interleukin-17. Immunohistology revealed the exclusive presence of periductal and perivascular infiltrating Tang cells along with increased SDF-1/CXCL12 expression in pSS MSG compared to non-pSS sicca syndrome controls. CONCLUSIONS: In pSS, Tang cells are expanded in peripheral blood and infiltrate MSG. Tang may be novel actors in pSS-related endothelial dysfunction and glandular neo-angiogenesis and inflammation.

A Platelet's Guide to Synovitis.

BACKGROUND: Platelets have the ability to influence the immune system and the inflammatory process and may be strongly involved in the whole pathogenic process of chronic inflammatory joint diseases, such as rheumatoid arthritis. They may play a significant role even before the clinical onset of the disease, contributing to the loss of tolerance of the immune system and the induction of autoimmunity. Subsequently, they can interact with the most important cellular players involved in autoimmunity and inflammation, namely innate immunity cells and T cells and eventually contribute to the building of inflammation in the synovium, thus inducing the activation, migration, and proliferation of fibroblasts that eventually lead to joint damage. Due to their peculiar features, studying the behavior of platelets is a challenging task; however, platelets may prove to be valuable therapeutic targets in the future.

Anti-citrullinated alpha enolase antibodies, interstitial lung disease and bone erosion in rheumatoid arthritis.

Objectives: RA is an articular chronic inflammatory disease that in a subgroup of patients can also present with extra-articular manifestations (EAMs). Despite intense investigation on this topic, reliable biomarkers for EAMs are lacking. In recent years several ACPAs, including those targeting anti-citrullinated alpha enolase peptide-1 (anti-CEP-1), have been identified in patients with RA. Data about the ability of anti-CEP-1 to predict the development of erosive disease are confliciting and no evidence concerning their possible association with EAMs in RA is currently available. The aim of this study was to investigate the prevalence and significance of anti-CEP-1 with regard to the association with erosive disease and EAMs in a large cohort of patients with RA. Methods: Anti-CCP and anti-CEP-1 antibodies have been assessed on serum samples of RA patients, healthy donors and patients with SpA using commercially available ELISA kits. Results: Anti-CEP-1 antibodies are detectable in over 40% of RA patients and are associated with erosive RA and with RA-associated interstitial lung disease (ILD). Conclusion: Anti-CEP-1 antibodies may represent a useful biomarker for RA-associated ILD and erosive disease to be employed in clinical practice.

Prevalence and significance of anti-saccharomyces cerevisiae antibodies in primary Sjögren's syndrome.

OBJECTIVES: Saccharomyces cerevisiae is a common yeast used in the food industry. IgG and IgA antibodies against the phosphopeptidomannan of the S. cerevisiae cell wall (ASCA) are a well known marker of disease severity in Crohn's disease. Moreover, a number of studies assessed ASCA in several systemic and organ-specific autoimmune diseases postulating molecular mimicry as a possible link between ASCA and autoimmunity. However, since they have never been tested in primary Sjögren's syndrome (pSS), the purpose of this study was to investigate these antibodies in a large cohort of pSS patients, compared to healthy donors (HD), and their significance as potentially helpful biomarker in a clinical setting. METHODS: ASCA IgG+IgA were assessed with ASCA screen dot for Blue Diver instrument (Alphadia sa/nv, Belgium). The comparison between the aminoacid sequence of mannan of S. cerevisiae and well characterised auto-antigens peculiar to pSS (52kD and 60kD Ro/SSA, La/SSB) was performed with the Basic Local Alignment Search Tool (BLAST). RESULTS: The prevalence of ASCA in our pSS cohort was 4.8%. We also reported that the ASCA target protein has a high similarity with Ro60/SSA protein further supporting the molecular mimicry hypothesis. Finally, we observed that ASCA positivity is associated with pSS specific clinical and serological features. ASCA+ pSS patients displayed a triple combination of circulating anti-Ro52/SSA, anti-Ro60/SSA and anti-La/SSB antibodies, associated with low complement and cutaneous involvement. CONCLUSIONS: Our data suggest a possible pathogenic/prognostic significance of ASCA in pSS.

Novel Therapeutic Strategies in Primary Sjögren's Syndrome.

BACKGROUND: Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease mainly affecting exocrine glands. However, a subgroup of patients experiences extraglandular manifestations which worsens disease prognosis. To date evidence based guidelines for the management of pSS are lacking, hence the therapeutic approach is mainly based on expert opinion and data from other connective tissue diseases. In recent years, several studies have explored the efficacy and safety of biologic agents in pSS and after the failure of tumor necrosis factor inhibitors, the attention has been focused on compounds directly targeting B or T lymphocytes. The aim of this review article is to provide an overview of available data about B and T cell targeting in pSS and of future directions based on ongoing trials.

Mobilization of lymphatic endothelial precursor cells and lymphatic neovascularization in primary Sjögren's syndrome.

Although lymphatic neovascularization may be a key feature of chronic inflammation, it is almost unexplored in primary Sjögren's syndrome (pSS). A recent study revealed a pro-lymphangiogenic function of interleukin (IL)-17, a leading player in pSS pathogenesis. The aims of the study were to investigate lymphangiogenic mediators and lymphatic vasculature in pSS, as well as their possible association with IL-17. Circulating lymphatic endothelial precursor cells (LEPCs) and Th17 cells were enumerated in pSS patients and healthy donors. VEGF-C and IL-17 levels were assessed in paired serum samples. Lymphatic vasculature, VEGF-C/VEGF receptor (VEGFR)-3 and IL-17 were evaluated in pSS minor salivary glands (MSGs) and compared with normal and non-specific chronic sialadenitis (NSCS) MSGs. Circulating LEPCs were expanded in pSS and correlated with circulating Th17 cells, IL-17 and VEGF-C. In pSS MSGs, a newly formed lymphatic capillary network was found within periductal inflammatory infiltrates and the number of interlobular lymphatic vessels was significantly increased compared with normal and NSCS MSGs. Strong VEGF-C expression was detected in pSS ductal epithelial cells and periductal inflammatory cells. Numerous VEGFR-3(+) infiltrating mononuclear cells were exclusively observed in pSS MSGs. VEGFR-3 expression was strongly increased in lymphatic capillaries of pSS MSGs. IL-17(+) inflammatory cells were preferentially observed around lymphatic vessels in pSS MSGs. This study supports the notion that lymphvasculogenesis and lymphangiogenesis are active in pSS, thereby unmasking a novel aspect of disease pathogenesis. In addition, our results suggest another possible pathogenic role of IL-17 in pSS, further supporting its therapeutic targeting in this disease.

Restoration of t cell substes of patients with type 1 diabetes mellitus by microencapsulated human umbilical cord Wharton jelly-derived mesenchymal stem cells: An in vitro study.

Human umbilical cord Wharton jelly-derived mesenchymal stem cells (hUCMS) might apply to treating chronic autoimmune disorders, as already shown for Sjögren's syndrome, including type 1 diabetes mellitus (T1D). Since naked hUCMS grafts encountered restraints, we enveloped hUCMS, within immunoisolatory microcapsules (CpS-hUCMS), made of our endotoxin-free, clinical grade alginate. We then examined the vitro effects of interferon (IFN)-γ-pretreated CpS-hUCMS on Th17 and Treg of T1D patients (n=15) and healthy controls (n=10). Peripheral blood mononuclear cells (PBMCs) were co-cultured with PBMC/CpS-hUCMS: lymphocyte proliferation was assessed by carboxyfluorescein succinimidyl esther (CFSE) dilution assay, and phenotypic analysis of regulatory and effector Tc was also performed. Cytokine expression was performed by bead array and qPCR on IFN-γ-pretreated hUCMS before PBMCs co-culture. CpS-hUCMS restored a correct Treg/Th17 ratio, relevant to the T1D disease process. In summary, we have preliminarily developed a new biohybrid system, associated with immunoregulatory properties, that is ready for in vivo application.

Telocytes in minor salivary glands of primary Sjögren's syndrome: association with the extent of inflammation and ectopic lymphoid neogenesis.

It has been recently reported that telocytes, a stromal (interstitial) cell subset involved in the control of local tissue homeostasis, are hampered in the target organs of inflammatory/autoimmune disorders. Since no data concerning telocytes in minor salivary glands (MSGs) are currently available, aim of the study was to evaluate telocyte distribution in MSGs with normal architecture, non-specific chronic sialadenitis (NSCS) and primary Sjögren's syndrome (pSS)-focal lymphocytic sialadenitis. Twelve patients with pSS and 16 sicca non-pSS subjects were enrolled in the study. MSGs were evaluated by haematoxylin and eosin staining and immunofluorescence for CD3/CD20 and CD21 to assess focus score, Tarpley biopsy score, T/B cell segregation and germinal center (GC)-like structures. Telocytes were identified by immunoperoxidase-based immunohistochemistry for CD34 and CD34/platelet-derived growth factor receptor α double immunofluorescence. Telocytes were numerous in the stromal compartment of normal MSGs, where their long cytoplasmic processes surrounded vessels and encircled both the excretory ducts and the secretory units. In NSCS, despite the presence of a certain degree of inflammation, telocytes were normally represented. Conversely, telocytes were markedly reduced in MSGs from pSS patients compared to normal and NSCS MSGs. Such a decrease was associated with both worsening of glandular inflammation and progression of ectopic lymphoid neogenesis, periductal telocytes being reduced in the presence of smaller inflammatory foci and completely absent in the presence of GC-like structures. Our findings suggest that a loss of MSG telocytes might have important pathophysiological implications in pSS. The specific pro-inflammatory cytokine milieu of pSS MSGs might be one of the causes of telocyte loss.

Characterization of circulating endothelial microparticles and endothelial progenitor cells in primary Sjögren's syndrome: new markers of chronic endothelial damage?

OBJECTIVE: Chronic autoimmune diseases are associated with increased risk of cardiovascular death. Endothelial dysfunction represents the first stage of subclinical atherosclerosis and multiple factors contribute to endothelial injury. Among these, an altered balance between endothelial microparticle (EMP) release and endothelial progenitor cell (EPC) generation promotes endothelial dysfunction. The role of EMPs and EPCs in promoting endothelial damage in primary SS (pSS) has never been investigated. Our aim was to evaluate the role of EMPs and EPCs as markers of endothelial damage in pSS and their correlation with disease clinical and immunological features. METHODS: Circulating EMPs (CD31(+)/CD42(-)), true EPCs (CD34(+)/KDR(+)/CD133(+)) and mature EPCs (CD34(+)/KDR(+)/CD133(-)) were quantified by FACS analysis in 34 pSS patients and 18 age- and sex-matched controls. Correlation between EMP and EPC levels and parameters of disease activity and damage, clinical features and markers of immunological dysfunction was performed. RESULTS: Patients displayed higher EMP numbers with respect to healthy controls [HCs; mean 450 n/µl (S.D. 155) vs 231 (110), P < 0.0001]. EPC and mature EPC levels were higher in patients compared with HCs [mean 226 n/ml (S.D. 181) vs 69 (53), P < 0.001 and 166 (161) vs 36 (32), P < 0.0001, respectively). EMP levels directly correlated with disease duration from symptoms and diagnosis (ρ = 0.5, P < 0.01). Early EPCs inversely correlated with disease duration from symptoms (ρ = -0.5, P < 0.01) and diagnosis (ρ = -0.4, P < 0.05). CONCLUSION: This is the first demonstration of chronic endothelial fragmentation characterizing pSS. The reparative potentiality of the endothelial layer appears to be preserved in the earliest stages of disease. During the course of the disease, progressive exhaustion of the precursor endothelial pool may be hypothesized, leading to defective vascular layer restoration and endothelial dysfunction.

In vitro immunomodulatory effects of microencapsulated umbilical cord Wharton jelly-derived mesenchymal stem cells in primary Sjögren's syndrome.

OBJECTIVE: Human umbilical cord Wharton jelly-derived mesenchymal stem cells (hUCMS) are easy to retrieve in bulk. They may interact with immune cells by either cell contact or soluble factors. Little evidence is currently available on potential therapeutic application of hUCMS to systemic autoimmune disorders such as primary SS (pSS). We have recently developed an endotoxin-free alginate gel that can be used to microencapsulate different cell types for graft into non-immunosuppressed hosts. We aimed to assess the in vitro effects of IFN-γ-pretreated microencapsulated (CpS)-hUCMS on T cells of pSS. METHODS: Ten pSS patients and 10 healthy donors were selected. Peripheral blood mononuclear cells (PBMCs) were obtained from venous blood to establish co-cultures with CpS-hUCMS. Lymphocyte proliferation and phenotypic analysis was performed by flow cytometry and real-time PCR on IFN-γ-pretreated hUCMS was performed before PBMCs co-culture. RESULTS: We found that CpS-hUCMS suppress pSS T cell proliferation and restore the Treg/Th17 ratio, thereby possibly positively impacting the pSS disease process. CONCLUSION: We have developed a new biohybrid drug delivery system that now waits for clinical application in autoimmune diseases, including pSS.

Altered immunoregulation in rheumatoid arthritis: the role of regulatory T cells and proinflammatory Th17 cells and therapeutic implications.

In recent years several studies investigated the role of T lymphocyte subpopulations in the pathogenesis of rheumatoid arthritis (RA). Pathogenic Th17 cells mediate pannus growth, osteoclastogenesis, and synovial neoangiogenesis; hence they are key players in the development of the disease. On the other hand, regulatory T (Treg) cells are a T cell subset whose peculiar function is to suppress autoreactive lymphocytes. The imbalance between Th17 and Treg cells has been identified as a crucial event in the pathogenesis of RA. In addition, the effects of currently employed RA therapeutic strategies on these lymphocyte subpopulations have been extensively investigated. This review article aims to discuss current knowledge on Treg and Th17 cells in RA and possible implications of their therapeutic targeting in this disorder.

T Regulatory and T Helper 17 Cells in Primary Sjögren's Syndrome: Facts and Perspectives.

Historically, primary Sjögren's syndrome (pSS) was thought to be a T helper (h) 1 driven disease due to the predominance of CD4(+)T lymphocytes and their products in target organs and peripheral blood of patients. In the last decades, the identification of a number of T cell subsets, including Th17, T regulatory (Treg), and follicular helper T cells, challenged this long-standing paradigm and prompted to identify their role in pSS pathogenesis. In addition the impact of abnormal proinflammatory cytokine production, such as IL-6, IL-17, IL-22, and IL-23, has also attracted considerable attention. However, although several studies have been carried out in experimental models and patients with pSS, many aspects concerning the role of Treg cells and IL-17/Th17 cell system in pSS pathogenesis are not fully elucidated. In particular, the role played by different IL-17-producing T cell subsets as well as the effects of pharmacological therapies on Treg/Th17 cell balance represents an intriguing issue. The aim of this review article is to provide an overview of current knowledge on Treg cells and IL-17-producing T cells in pSS pathogenesis. We believe that these insights into pSS pathogenesis may provide the basis for successful therapeutic intervention in this disease.

CD4(-)CD8(-) T-cells in primary Sjögren's syndrome: association with the extent of glandular involvement.

OBJECTIVES: Growing evidence suggests that IL-17-producing T cells, lacking both CD4 and CD8 molecules and defined as double negative (DN) cells, play a pivotal role in the pathogenesis of a number of systemic autoimmune disorders. We recently demonstrated that this T-cell subset is expanded in the peripheral blood (PB) of patients with primary Sjögren's syndrome (pSS), produces IL-17 and accumulates in minor salivary glands (MSGs). We aimed to investigate glandular and PB DN T cells in early pSS in order to verify a possible correlation with MSGs histological patterns and clinical parameters. METHODS: Paired samples of PB mononuclear cells and MSGs from pSS patients were evaluated at the diagnosis by flow cytometry and immunofluorescence staining respectively. Histological analysis to identify histological scores, B/T cell segregation and the presence of germinal center (GC)-like structures was also performed. RESULTS: In early stages of pSS, circulating DN T cells appear to be not yet expanded and inversely correlated with circulating CD4(+)Th17 cells. The number of infiltrating DN T cells were associated with extent of glandular involvement, presence of GC-like structures and dryness symptoms and were inversely correlated with circulating DN T cells. CONCLUSIONS: Our findings suggest that DN T cells are actively involved in the pathogenic mechanisms leading to glandular dysfunction and damage in pSS and may play a role in ectopic lymphoneogenesis development occurring during the disease.

IL-17-producing CD4-CD8- T cells are expanded in the peripheral blood, infiltrate salivary glands and are resistant to corticosteroids in patients with primary Sjogren's syndrome.

OBJECTIVES: It has been recently observed that a T-cell subset, lacking of both CD4 and CD8 molecules and defined as double negative (DN), is expanded in the blood of patients with systemic lupus erythematosus, produces IL-17 and accumulates in the kidney during nephritis. Since IL-17 production is enhanced in salivary gland infiltrates of primary Sjögren's syndrome (SS) patients, we investigated whether DN T cells may be involved in the pathogenesis of salivary gland damage. METHODS: Phenotypic characterisation of peripheral blood mononuclear cells from SS patients and controls was performed by flow cytometry in freshly isolated and anti-CD3-stimulated cells. SS minor salivary glands were processed for immunofluorescence staining. RESULTS: CD3(+)CD4(-)CD8(-) DN T cells were major producers of IL-17 in SS and expressed ROR-γt. They were expanded in the peripheral blood, spontaneously produced IL-17 and infiltrated salivary glands. In addition, the expansion of αß-TCR(+) DN T cells was associated with disease activity. Notably, IL-17-producing DN T cells from SS patients, but not from healthy controls, were strongly resistant to the in vitro effect of dexamethasone. CONCLUSIONS: These findings appear to be of great interest since the identification of a peculiar T-cell subset with pro-inflammatory activity, but resistant to corticosteroids, in an autoimmune disorder such as SS may help to design new specific treatments for the disease.