Citrullinated and carbamylated proteins in extracellular microvesicles from plasma of patients with rheumatoid arthritis.

OBJECTIVES: To investigate the expression of citrullinated and carbamylated proteins in extracellular microvesicles (EMVs) from RA patients. METHODS: We enrolled 24 RA naïve for biological therapy and 20 healthy donors (HD), matched for age and sex. For each patient, laboratory and clinical data were recorded and clinical indexes were measured (Clinical Disease Activity Index, Simplified Disease Activity Index, DAS28). EMVs in RA patients and HD were purified from plasma and measured by nanoparticle tracking analysis (NanoSight). Further, EMVs were incubated with anti-citrullinated/carbamylated proteins antibodies and processed by flow cytometry and western blot to evaluate the expression of citrullinated/carbamylated antigens. RESULTS: NanoSight revealed a significant increase of EMVs in RA compared with HD. Moreover, cytofluorimetric analysis showed a significative higher expression of citrullinated antigens on EMVs' surface in RA than donors, while no substantial difference was found in the expression of carbamylated antigens. These data were confirmed by western blot which identified vimentin, glycolytic enzyme alpha-enolase 1 and collagen type II as the main citrullinated and carbamylated proteins carried by EMVs. Finally, a relevant correlation between the expression of citrullinated antigens and disease activity was found. CONCLUSIONS: The results of this study suggest an involvement of EMVs in the pathogenesis of RA by inducing autoimmunity.

KLRG1 is reduced on NK cells in SLE patients, inversely correlates with disease activity and is modulated by hydroxychloroquine in vitro.

OBJECTIVES: Killer cell lectin-like receptor G 1 (KLRG1), a transmembrane receptor with inhibitory capacity expressed in human immune cells, emerged as a novel susceptibility gene for systemic lupus erythematosus (SLE). The aim of this study was to investigate the expression of KLRG1 in SLE patients compared to healthy controls (HC) on both NK and T cells and to evaluate its possible involvement in SLE pathogenesis. METHODS: Eighteen SLE patients and twelve healthy controls were enrolled. Peripheral blood mononuclear cells (PBMCs) from these patients were phenotypically characterized by immunofluorescence and flow cytometry. The effect of the hydroxychloroquine (HCQ) in vitro on KLRG1 expression and its signaling mediated functions in NK cells were analyzed. RESULTS: KLRG1 expression was significantly reduced on the analyzed immune cell populations in SLE patients compared to HC, especially on total NK cells. Moreover, expression of KLRG1 on total NK cells inversely correlated with the SLEDAI-2K. A direct association between KLRG1 expression on NK cells and patients' treatment with HCQ was observed. In vitro treatment with HCQ increased KLRG1 expression on NK cells. In HC, KLRG1+ NK cells showed reduced degranulation and IFNγ production, while in SLE patient, this inhibition occurred only for the IFNγ production. CONCLUSION: With this study we revealed a reduced expression and an impaired function of KLRG1 on NK cells in SLE patients. These results suggest a possible role of KLRG1 in the pathogenesis of SLE and as a novel biomarker of this disease.

Clinical and histological features of patients with primary Sjögren's syndrome and autoimmune thyroiditis: a national multicentre cross-sectional study.

OBJECTIVES: Primary Sjögren's syndrome (pSS) is frequently associated with autoimmune thyroiditis (AT). The aim of this study was to evaluate the prevalence of AT in a national cohort of pSS and to describe the clinical and histological phenotype of patients with pSS and associated AT. METHODS: In this multicentre cross-sectional study, data from 2546 pSS were collected and the presence of AT was reported. In a subgroup, the histology of minor salivary glands was evaluated. Differences between pSS with and without AT were evaluated. RESULTS: A concomitant pSS and AT was detected in 19.6% of cases. Patients with pSS and AT displayed a lower prevalence of lymphoma, male sex and disease-modifying anti-rheumatic drugs (DMARDs) use and a higher prevalence of fibromyalgia, coeliac disease and hypergammaglobulinaemia. Multivariable analysis confirmed a higher prevalence of fibromyalgia and coeliac disease and lower use of DMARDs. In a subgroup of patients (n=232), a significantly higher focus score and number of foci was detected in pSS without AT (n=169) as compared to pSS with AT (n=54). CONCLUSIONS: This is the largest study evaluating the coexistence of pSS and AT. We confirm a high association between pSS and AT and describe the presence of a different phenotype characterized by a higher rate of celiac disease and fibromyalgia. Although not significant, the lower prevalence of both lymphoma and intake of DMARDs, along with a significantly lower focus score and number of foci, possibly suggest a more favourable outcome in concomitant pSS and AT which further deserve future investigations.

mRNA expression analysis confirms CD44 splicing impairment in systemic lupus erythematosus patients.

BACKGROUND: Systemic Lupus Erythematosus (SLE) is a complex chronic autoimmune disease characterized by several immunological alterations. T cells have a peculiar role in SLE pathogenesis, moving from the bloodstream to the peripheral tissues, causing organ damage. This process is possible for their increased adherence and migration capacity mediated by adhesion molecules, such as CD44. Ten different variant isoforms of this molecule have been described, and two of them, CD44v3 and CD44v6 have been found to be increased on SLE T cells compared to healthy controls, being proposed as biomarkers of disease and disease activity. The process of alternative splicing of CD44 transcripts is not fully understood. We investigated the mRNA expression of CD44v3 and CD44v6 and also analyzed possible CD44 splicing regulators (ESRP1 molecule and rs9666607 CD44 polymorphism) in a cohort of SLE patients compared to healthy controls. METHODS: This study involved 18 SLE patients and 18 healthy controls. Total RNA and DNA were extracted by peripheral blood mononuclear cells. The expression study was conducted by quantitative RT-polymerase chain reaction, using SYBR Green protocol. Genotyping of rs9666607 SNP was performed by direct sequencing. RESULTS: CD44v6 mRNA expression was higher in SLE patients compared to healthy controls (p = 0.028). CD44v3/v6 mRNA ratio in healthy controls was strongly unbalanced towards isoform v3 compared to SLE patients (p = 0.002) and decreased progressively from healthy controls to the SLE patients in remission and those with active disease (p = 0.015). The expression levels of CD44v3 and CD44v6 mRNA correlated with the disease duration (p = 0.038, Pearson r = 0.493 and p = 0.038, Pearson r = 0.495, respectively). Splicing regulator ESRP1 expression positively correlated with CD44v6 expression in healthy controls (p = 0.02, Pearson r = 0.532) but not in SLE patients. The variant A allele of rs9666607 of CD44 was associated with higher level of global CD44 mRNA (p = 0.04) but not with the variant isoforms. CONCLUSIONS: In SLE patients, the increase in CD44v6 protein correlates with a higher transcript level of this isoform, confirming an impairment of CD44 splicing in the disease, whose regulatory mechanisms require further investigation.

Up-regulation of autophagy by etanercept treatment results in TNF-induced apoptosis reduction in EA.hy926 endothelial cell line.

OBJECTIVES: Rheumatoid arthritis (RA) is an autoimmune systemic inflammatory disease associated with a high prevalence of atherosclerosis. Endothelial dysfunction has emerged as a potentially valuable prognostic tool in predicting the development of atherosclerosis. Tumour necrosis factor (TNF) is the main cytokine involved in RA pathogenesis, exerting a pro-atherogenic role. TNF-inhibitors are effective treatments in RA, also improving endothelial function. Regarding this, no experimental data are known about the involvement of etanercept. We investigated the contribution of TNF to endothelial dysfunction and the effect of in vitro treatment with etanercept, with a special focus on autophagy and apoptosis pathways. METHODS: Autophagy and apoptosis were evaluated by Western blot and flow cytometry in EA.hy926 endothelial cells treated with TNF alone or in combination with etanercept for 24h. RESULTS: Blocking autophagy, TNF was able to induce endothelial cell apoptosis. Co-treatment with etanercept reverted this effect, up-regulating the autophagy pathway. CONCLUSIONS: Our results confirm the protective role of etanercept, by restoring autophagy on TNF-induced endothelial damage.

Increased eryptosis in patients with primary antiphospholipid syndrome (APS): a new actor in the pathogenesis of APS.

OBJECTIVES: Antiphospholipid syndrome (APS) is an autoimmune disease characterised by a hypercoagulable state and the presence of antiphospholipid antibodies (aPL). During the mechanism of red blood cells (RBCs) death, called eryptosis, RBCs can adhere to vascular wall participating in the development of a pro-thrombotic state. It is known that enhanced eryptosis contributes to several pathological conditions but the role of this process in APS has not been investigated yet. We analysed spontaneous eryptosis in a cohort of APS patients and aPL carriers (asymptomatic subjects with positive aPL tests). The effect on eryptosis of antibodies (Abs) purified from serum of APS patients and aPL carriers was also investigated. METHODS: In this study, 30 patients with primary APS (PAPS) and 17 aPL carriers were recruited. Twenty healthy donors (HD) and 13 patients affected by autoimmune haemolytic anaemia (AHIA) were also recruited. RBCs were incubated with PAPS and aPL carriers Abs, purified by ammonium sulfate precipitation. Levels of eryptosis were analysed by flow cytometry. RESULTS: In vitro Abs from APS patients induced eryptosis in RBCs isolated from HD after 4 h of culture. On the contrary, Abs from aPL carriers had no effect on the percentage of phosphatidylserine-exposing RBCs. Ex vivo, APS patients showed higher levels of spontaneous eryptosis compared to HD and aPL carriers. CONCLUSIONS: In this study, we demonstrated a potential new aspect of APS pathogenesis based on the ability of Abs isolated from APS patients, not identified in aPL carriers, to stimulate eryptosis suggesting a possible contribution of this process in the clinical manifestations of APS.

Homocysteinylated alpha 1 antitrypsin as an antigenic target of autoantibodies in seronegative rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is a chronic autoimmune disease and rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA) are the most frequently detected autoantibodies (autoAbs). To date, more than 20% of RA cases are still defined as seronegative forms (seronegative RA, SN-RA). The aim of this study was to identify new antigenic targets of autoAbs in RA patients, which can also be recognized in SN-RA. Using a proteomic approach, we tested sera from SN-RA patients by analyzing synovial fluid (SF) proteins from these patients. Sera from SN-RA patients revealed a strong reactive spot, corresponding to alpha 1 antitrypsin (A1AT). Reverse-phase nanoliquid chromatography and tandem mass spectrometry (Matrix Assisted Laser Desorption/Ionization-Time Of Flight, MALDI-TOF/TOF) confirmed the presence of A1AT in SF and showed that homocysteinylation was one of the post-translational modifications of A1AT. Homocysteinylated (Hcy)-A1AT immunoprecipitated from SN-RA patients' SFs and in vitro modified Hcy-A1AT were used as antigens by Enzyme-Linked ImmunoSorbent Assay (ELISA) to test the presence of specific autoAbs in sera from 111 SN-RA patients, 132 seropositive (SP)-RA patients, and from 95 patients with psoriatic arthritis, 40 patients with osteoarthritis, and 41 healthy subjects as control populations. We observed that a large portion of SN-RA patients (75.7%), and also most of SP-RA patients' sera (87.1%) displayed anti-Hcy-A1AT autoAbs (anti-HATA). Native A1AT was targeted at a lower rate by SP-RA patients autoAbs, while virtually no SN-RA patients' sera showed the presence of anti-native A1AT autoAbs. In conclusion, anti-HATA can be considered potential biomarkers for RA, also in the SN forms. The discovery of novel autoAbs targeting specific autoantigens can represent higher clinic significance for all RA patients' population.

Caffeine intake influences disease activity and clinical phenotype in systemic lupus erythematosus patients.

OBJECTIVE: Caffeine, one of the most widely consumed products in the world, seems to interact with multiple components of the immune system by acting as a non-specific phosphodiesterase inhibitor. In vitro dose-dependent treatment with caffeine down-regulates mRNA levels of key inflammation-related genes in peripheral blood mononuclear cells. So far, no robust data are available about the possible contribution of caffeine in systemic lupus erythematosus (SLE). The aim of this study was to evaluate the impact of caffeine consumption on SLE-related disease phenotype and activity, in terms of clinimetric assessment and cytokine serum levels. METHODS: We performed a cross-sectional study, enrolling consecutive patients and reporting their clinical and laboratory data. Disease activity was assessed by SLE Disease Activity Index 2000 (SLEDAI-2K). Caffeine intake was evaluated by a 7-day food frequency questionnaire, including all the main sources of caffeine. As previously reported, patients were divided into four groups according to the daily caffeine intake: <29.1 mg/day (group 1), 29.2-153.7 mg/day (group 2), 153.8-376.5 mg/day (group 3) and >376.6 mg/day (group 4). At the end of questionnaire filling, blood samples were collected from each patient to assess cytokine levels. These were assessed by using a panel by Bio-Plex assays to measure the levels of IL-6, IL-10, IL-17, IL-27, IFNγ, IFNα and BLyS. RESULTS: We enrolled 89 consecutive SLE patients. We observed a negative correlation between caffeine consumption and disease activity, measured with SLEDAI-2K. A significantly higher prevalence of lupus nephritis, neuropsychiatric involvement, haematological manifestations, hypocomplementaemia and anti-dsDNA positivity was observed in patients with a low intake of caffeine. Furthermore, patients with a low intake of caffeine were more frequently treated with glucocorticoids. Regarding cytokine analysis, a negative correlation between daily caffeine consumption and serum level of IFNγ was found (p = 0.03, r = -0.2); furthermore, patients with a high intake of caffeine showed lower serum levels of IFNα (p = 0.02), IL-17 (p = 0.01) and IL-6 (p = 0.003). CONCLUSIONS: In this report we demonstrated the impact of caffeine on SLE disease activity status, as confirmed by the inverse correlation between its intake and both SLEDAI-2K values and cytokine levels. Moreover, patients with a low caffeine consumption seem to have a more severe disease phenotype.

Assessment of semaphorin 3A and its role in bone remodelling in a group of ankylosing spondylitis patients.

OBJECTIVES: Several molecules are involved in the pathogenesis of new bone formation in ankylosing spondylitis (AS). The aim of the present study was to evaluate serum levels of semaphoring 3A in AS and to investigate any correlations with radiographic damage, disease activity, function and treatment. METHODS: AS patients who fulfilled the modified New York criteria were enrolled for this study. Healthy subjects were also enrolled as control group. BASDAI, ASDAS-CRP, BASMI, BASFI, patients and physician VAS, C-reactive protein and erythrocyte sedimentation rate were evaluated at baseline visit. Radiographs of the spine and pelvis performed within six months from the enrolment in the study were collected in all patients. Spinal damage was assessed using the mSASSS. Serum concentrations of semaphorin3A were assessed at baseline and after four months of therapy in patients who started an anti-TNF. RESULTS: Twenty healthy subjects and forty AS patients were enrolled in the study. Of these patients, 15 started anti-TNF therapy the day of baseline visit. Semaphorin3A serum concentrations [median (25th-75th)] were similar in AS patients [0.26 (0.20-0.31) ng/ml] and controls [0.28 (0.26-0.3) ng/ml; p=ns). No significant correlation was found between semaphorin 3A serum levels and radiographic damage index. Semaphorin 3A serum levels positively correlated with ESR values (rho=0.37, p=0.049) and with disease activity assessed by the physician VAS (rho=0.47, p<0.01). No differences were found in the semaphorin3A serum levels after 4 months, compared to baseline values. CONCLUSIONS: The results of the present study could contribute to the intriguing topic of bone remodelling in AS.

Autoantibodies specific to D4GDI modulate Rho GTPase mediated cytoskeleton remodeling and induce autophagy in T lymphocytes.

T lymphocytes from patients with Systemic Lupus Erythematosus (SLE) display multiple abnormalities, including increased cell activation, abnormal cell death by apoptosis and impairment of autophagy pathway. In the present study we report the presence of specific antibodies to D4GDI, a small GTPase family inhibitor, in a significant percentage (46%) of SLE patient sera. We also found a significant association between the presence of these autoantibodies and hematologic manifestations occurring in these patients. Investigating the possible implication of anti-D4GDI autoantibodies in SLE pathogenesis or progression, we found that these antibodies were capable of binding D4GDI expressed at the lymphocyte surface and triggering a series of subcellular events, including Rho GTPase activation. These antibodies were also able to induce autophagy in T cells from both healthy donors and SLE patients, but only those negative to these antibodies. We can conclude that anti-D4GDI autoantibodies could be capable of triggering important responses in T cells such as cytoskeleton remodeling and autophagy pathway and that, in SLE patients, the chronic exposure to these specific autoantibodies could lead to the selection of autophagy-resistant T cell clones contributing to the pathogenesis of the disease.

Autophagy as a pathogenic mechanism and drug target in lymphoproliferative disorders.

Autophagy represents a key mechanism of cytoprotection that can be activated by a variety of extracellular and intracellular stresses and allows the cell to sequester cytoplasmic components and damaged organelles, delivering them to lysosomes for degradation and recycling. However, the autophagy process has also been associated with the death of the cell. It has been demonstrated to be constitutive in some instances and inducible in others, and the idea that it could represent a pathogenetic determinant as well as a possible prognostic tool and a therapeutic target in a plethora of human diseases has recently been considered. Among these, cancer represents a major one. In this review, we recapitulate the critical implications of autophagy in the pathogenesis, progression, and treatment of lymphoproliferative disorders. Leukemias and lymphomas, in fact, represent paradigmatic human diseases in which advances have recently been made in this respect.

The Glycan Ectodomain of SARS-CoV-2 Spike Protein Modulates Cytokine Production and Expression of CD206 Mannose Receptor in PBMC Cultures of Pre-COVID-19 Healthy Subjects.

The ability of recombinant, SARS-CoV-2 Spike (S) protein to modulate the production of two COVID-19 relevant, pro-inflammatory cytokines (IL-6 and IFN-γ) in PBMC cultures of healthy, pre-COVID-19 subjects was investigated. We observed that cytokine production was largely and diversely modulated by the S protein depending on antigen or mitogen stimulation, as well as on the protein source, insect (S-in) or human (S-hu) cells. While both proteins co-stimulated cytokine production by polyclonally CD3-activated T cells, PBMC activation by the mitogenic lectin Concanavalin A (Con A) was up-modulated by S-hu protein and down-modulated by S-in protein. These modulatory effects were likely mediated by the S glycans, as demonstrated by direct Con A-S binding experiments and use of yeast mannan as Con A binder. While being ineffective in modulating memory antigenic T cell responses, the S proteins and mannan were able to induce IL-6 production in unstimulated PBMC cultures and upregulate the expression of the mannose receptor (CD206), a marker of anti-inflammatory M2 macrophage. Our data point to a relevant role of N-glycans, particularly N-mannosidic chains, decorating the S protein in the immunomodulatory effects here reported. These novel biological activities of the S glycan ectodomain may add to the comprehension of COVID-19 pathology and immunity to SARS-CoV-2.

Role of autophagy in immunity and autoimmunity, with a special focus on systemic lupus erythematosus.

Autophagy is a lysosome-mediated catabolic process that allows cells to degrade unwanted cytoplasmic constituents and to recycle nutrients. Autophagy is also involved in innate and adaptive immune responses, playing a key role in interactions against microbes, in antigen processing for major histocompatibility complex (MHC) presentation, and in lymphocyte development, survival, and proliferation. Over recent years, perturbations in autophagy have been implicated in a number of diseases, including autoimmunity. Systemic lupus erythematosus (SLE) is a multifactorial disease characterized by autoimmune responses against self-antigens generated by dying cells. Genome-wide association studies have linked several single-nucleotide polymorphisms (SNPs) in the autophagy-related gene Atg5 to SLE susceptibility. Loss of Atg5-dependent effects, including clearance of dying cells and cell antigen presentation, might contribute to the autoimmunity and inflammation associated with SLE. Moreover, activation of the mammalian target of rapamycin (mTOR), a key player in the autophagy regulation, has recently been demonstrated in SLE, confirming an altered autophagy pathway in this disease. In the present review, we summarize the autophagy mechanisms, their molecular regulation, and their relevance in immunity and autoimmunity. The potential of targeting autophagy pathway in SLE, by developing innovative therapeutic approaches, has finally been discussed.

T lymphocytes from patients with systemic lupus erythematosus are resistant to induction of autophagy.

Autophagy, the cytoprotection mechanism that takes place under metabolic impairment, has been implicated in the pathogenesis of autoimmunity. Here, we investigated the spontaneous and induced autophagic behavior of T lymphocytes from patients with systemic lupus erythematosus (SLE) compared with that of T lymphocytes from healthy donors by measuring the autophagy marker microtubule-associated protein 1 light chain 3 (LC3)-II. No significant differences in spontaneous autophagy were found between T lymphocytes from patients with SLE and from healthy donors, apart from CD4(+) naive T cells from patients with SLE in which constitutively higher levels of autophagy (P<0.001) were detected. At variance, whereas treatment of T lymphocytes from healthy donors with serum IgG from patients with SLE resulted in a 2-fold increase in LC3-II levels (P<0.001), T lymphocytes from SLE patients were resistant to autophagic induction and also displayed an up-regulation of genes negatively regulating autophagy, e.g., α-synuclein. These findings could open new perspectives in the search for pathogenetic determinants of SLE progression and in the development of therapeutic strategies aimed to recover T-cell compartment homeostasis by restoring autophagic susceptibility.

Autoantibodies to estrogen receptor α interfere with T lymphocyte homeostasis and are associated with disease activity in systemic lupus erythematosus.

OBJECTIVE: Estrogens influence many physiologic processes and are also implicated in the development or progression of numerous diseases, including autoimmune disorders. Aberrations of lymphocyte homeostasis that lead to the production of multiple pathogenic autoantibodies, including autoantibodies specific to estrogen receptor (ER), have been detected in the peripheral blood of patients with systemic lupus erythematosus (SLE). This study was undertaken to assess the presence of both anti-ERα and anti-ERß antibodies in sera from patients with SLE, to analyze the effect of these antibodies on peripheral blood T lymphocyte homeostasis, and to evaluate their role as determinants of disease pathogenesis and progression. METHODS: Anti-ER antibody serum immunoreactivity was analyzed by enzyme-linked immunosorbent assay in samples from 86 patients with SLE and 95 healthy donors. Phenotypic and functional analyses were performed by flow cytometry and Western blotting. RESULTS: Anti-ERα antibodies were present in 45% of the patients with SLE, whereas anti-ERß antibodies were undetectable. In healthy donors, anti-ERα antibodies induced cell activation and consequent apoptotic cell death in resting lymphocytes as well as proliferation of anti-CD3-stimulated T lymphocytes. A significant association between anti-ERα antibody values and clinical parameters, i.e., the SLE Disease Activity Index and arthritis, was found. CONCLUSION: Our data suggest that anti-ERα autoantibodies interfere with T lymphocyte homeostasis and are significantly associated with lupus disease activity.

Renal Stem Cells, Renal Resistive Index, and Neutrophil Gelatinase Associated Lipocalin Changes After Revascularization in Patients With Renovascular Hypertension and Ischemic Nephropathy.

BACKGROUND: Percutaneous transluminal renal angioplasty (PTRA) with or without stenting is the gold standard therapy in patients with atherosclerotic renal artery stenosis (aRAS). However, therapeutic success depends on the correct timing of revascularization and the reversibility of the renal damage. MATERIALS AND METHODS: We report a case series of patients treated with PTRA for renovascular hypertension and ischemic nephropathy. We measured bilateral renal resistive index (RRI), circulating renal stem cells (RSC), and Neutrophil Gelatinase Associated Lipocalin (NGAL) at baseline and after PTRA at different time points to understand their changes in post-revascularization. RESULTS: At baseline, the studied patients (n = 5) had different RSC levels. After PTRAs, all patients showed an improvement in blood pressure, while renal function varied differently within the studied subjects. RRI > 0.75 at baseline and the absence of NGAL decrease after PTRAs were associated with post-PTRA renal function worsening, despite an increase of RSC in all patients. CONCLUSION: Although limited to a few patients, our observation allowed the exploration of the behaviour of the studied parameters in different degrees of renal ischemia. This revealed different disease models suggesting the importance of further investigations in larger and homogeneous cohorts to confirm that a greater basal RSC percentage, low RRI values before PTRA, and a post-revascularization NGAL reduction could be related to better renal outcomes in aRAS patients.

Differential effects of the glycolysis inhibitor 2-deoxy-D-glucose on the activity of pro-apoptotic agents in metastatic melanoma cells, and induction of a cytoprotective autophagic response.

2-Deoxy-D-glucose (2DG) is a synthetic glucose analogue that inhibits glycolysis and blocks cancer cell growth. In this report, we evaluated the role of 2DG in the induction of cell death in human metastatic melanoma cells. We have also examined the effects of 2DG in combined treatments with four different pro-apoptotic agents: (i) Temozolomide (TMZ), a chemotherapic drug commonly used to treat metastatic melanoma, (ii) Pyrimethamine (Pyr), a pro-apoptotic antifolate drug recently reappraised in cancer therapy, (iii) Cisplatin (CisPt), a drug capable of directly binding to DNA ultimately triggering apoptosis of cancer cells and (iv) the kinase inhibitor Staurosporine (STS), a prototypical inducer of mitochondria-mediated apoptosis. We found that 2DG per se: (i) induced a cell cycle arrest in G(0) /G(1) , (ii) promoted autophagy, (iii) was ineffective in inducing apoptosis in association with the chemotherapic drug TMZ, whereas (iv) it was synergistic with CisPt and STS pro-apoptotic drugs through a mechanism involving changes of mitochondrial homeostasis. Conversely, (v) 2DG hindered the pro-apoptotic effects of Pyr via a mechanism involving either the block of cell cycle in G(0) /G(1) or the modification of the free radical production of the cell, i.e., decreasing the production of reactive oxygen species (ROS) and increasing the production of reactive nitrogen species (RNS). Moreover, a clear-cut autophagic response involving endoplasmic reticulum remodelling was detectable. Since autophagic cytoprotection has been suggested to contribute to the induction of chemoresistance, these results could provide useful clues as concerns the use of 2DG as anticancer agent in combinatory protocols.

Phase II study of sorafenib in patients with relapsed or refractory lymphoma.

The safety and activity of the multikinase inhibitor sorafenib were investigated in patients with relapsed or refractory lymphoproliferative disorders who received sorafenib (400 mg) twice daily until disease progression or appearance of significant clinical toxicity. The primary endpoint was overall response rate (ORR). Biomarkers of sorafenib activity were analysed at baseline and during treatment. Thirty patients (median age, 61 years; range, 18-74) received a median of 4 months of therapy. Grade 3-4 toxicities included hand/foot skin reactions (20%), infections (12%), neutropenia (20%) and thrombocytopenia (14%). Two patients achieved complete remission (CR), and two achieved partial remission (PR) for an ORR of 13%. Stable disease (SD) and progressive disease (PD) was observed in 15 (50%) and 11 patients (37%), respectively. The median overall survival (OS) for all patients was 16 months. For patients who achieved CR, PR and SD, the median time to progression and OS was 5 and 24 months, respectively. Compared with patients with PD, responsive patients had significantly higher baseline levels of extracellular signal-regulated kinase phosphorylation and autophagy and presented a significant reduction of these parameters after 1 month of therapy. Sorafenib was well tolerated and had a clinical activity that warrants development of combination regimens.

Autophagy in Rheumatic Diseases: Role in the Pathogenesis and Therapeutic Approaches.

Autophagy is a lysosomal pathway for the degradation of damaged proteins and intracellular components that promotes cell survival under specific conditions. Apoptosis is, in contrast, a critical programmed cell death mechanism, and the relationship between these two processes influences cell fate. Recent evidence suggests that autophagy and apoptosis are involved in the self-tolerance promotion and in the regulatory mechanisms contributing to disease susceptibility and immune regulation in rheumatic diseases. The aim of this review is to discuss how the balance between autophagy and apoptosis may be dysregulated in multiple rheumatic diseases and to dissect the role of autophagy in the pathogenesis of rheumatoid arthritis, systemic lupus erythematosus, and Sjögren's syndrome. Furthermore, to discuss the potential capacity of currently used disease-modifying antirheumatic drugs (DMARDs) to target and modulate autophagic processes.

Belimumab Decreases Autophagy and Citrullination in Peripheral Blood Mononuclear Cells from Patients with Systemic Lupus Erythematosus.

Belimumab (BLM) is a B lymphocyte stimulator (BLyS) inhibitor approved for the treatment of systemic lupus erythematosus (SLE). Autophagy is a cell survival mechanism involved in the pathogenesis of SLE. Citrullination is a post-translational modification catalyzed by peptidylarginine deiminase (PAD) enzymes. Autophagy and citrullination may generate neoepitopes, evoking an autoimmune response. No previous studies have investigated the connection of these processes, and how BLM could affect them, in SLE. Ex vivo autophagy and protein citrullination were analyzed by western blot in lysates from 26 SLE patients' PBMCs at baseline and after 2, 4, and 12 weeks of BLM administration, and from 16 healthy donors' PBMCs. Autophagic PBMCs were identified by the immunofluorescent detection of the autophagy-associated proteins LC3B (LC3 puncta) and LAMP-1. Autophagosome accumulation was evaluated in CD14- (PBLs) and CD14+ (monocytes) SLE cells. The presence of the BLyS receptors BAFF-R, BCMA, and TACI on SLE CD4+, CD8+ T cells and monocytes, as well as serum IL-18 levels, was also assessed. Following BLM administration, we observed a decrease in autophagy and citrullination, with a lowering of LC3-II, citrullinated vimentin, and PAD4 expression levels in PBMCs from SLE patients. LC3-II levels showed a correlation with the SLE Disease Activity Index 2000 (SLEDAI-2K) after 12 weeks of therapy. The LC3B/LAMP-1 analysis confirmed the reduction in autophagy. A lesser autophagosome accumulation occurred in PBLs and monocytes which, in turn, seemed to be the main cellular populations contributing to autophagy. A reduction in patients' serum IL-18 concentrations occurred. CD4+ and CD8+ cells weakly expressed BAFF receptors; monocytes expressed only BAFF-R. BLM could impact on autophagy and citrullination, offering an opportunity for a deeper understanding of these mechanisms in SLE, and a possible tool for the clinical management of SLE.