Porphyromonas gingivalis amount in the tongue biofilm is associated with erosive arthritis in systemic lupus erythematosus.

BACKGROUND: Several data have demonstrated the occurrence of erosive arthritis in Systemic Lupus Erythematosus (SLE) patients. However, a few studies have focused on the pathogenic mechanisms involved in this feature. The implication of oral pathogens has been proved in Rheumatoid Arthritis: in particular, Porphyromonas gingivalis (Pg), by inducing citrullination, could trigger autoimmune response. Here, we evaluated amount of Pg on the tongue in a cohort of SLE patients with arthritis, focusing on the association with the erosive phenotype. METHODS: SLE patients with arthritis were enrolled. DAS28 was applied to assess activity. Erosive damage was evaluated by ultrasound at level of MCP (metacarpophalangeal) and PIP (proximal interphalangeals) joints. All subjects underwent a tongue cytologic swab in order to quantify the amount of Pg (real-time PCR). The bacterium expression was obtained from the ratio between the patient's DNA amount and that obtained from healthy subjects. RESULTS: 33 patients were enrolled (M/F 3/30; median age 47 years, IQR 17; median disease duration 216 months, IQR 180): 12 of them (36.4%) showed erosive damage, significantly associated with ACPA positivity (p = 0.03) and higher values of DAS28 (p = 0.01). A mean ratio of 19.7 ± 31.1 was found for Pg amount. Therefore, we used Pg mean values as threshold, identifying two groups of patients, namely, highPg and lowPg. Erosive damage was significantly more frequent in highPg patients in comparison with lowPg (60.0% vs 26.0%, p = 0.001). Furthermore, highPg patients showed higher prevalence of skin manifestations, serositis, and neurological involvement (p = 0.005, p = 0.03, p = 0.0001, respectively). CONCLUSION: The possible contribution of oral microbiota in SLE erosive arthritis was here evaluated for the first time, finding a significant association between erosive damage and higher expression of Pg at tongue level.

Lack of cross-reactivity between rheumatoid factor IgM and anti-S1 receptor binding domain of SARS-CoV-2 IgM: a case-control study.

OBJECTIVES: Since the onset of the COVID-19 outbreak, concern has been raised about reliability of SARS-CoV-2 serological tests in people with serum positivity for rheumatoid factor (RF), due to its ability to interfere during tests carried out with immunoassay techniques, leading to false positive results. The aim of this study was to analyse, on sera from RF seropositive rheumatoid arthritis (RA) patients, the interference between RF IgM and anti-S1 RBD IgM. METHODS: The study was conducted on consecutive patients affected by RF seropositive RA and, as control group, COVID-19 patients with SARS-CoV-2 pneumonia hospitalised at Sapienza University of Rome from April 2020 and April 2021. Serum samples from COVID-19 patients during their hospitalisation were collected, while RA subjects' samples were harvested prior to the onset of the COVID-19 pandemic. All samples were tested for RF IgM using nephelometry and ELIA, and for anti-S1 RBD IgM by ELISA. RESULTS: Forty RF seropositive RA and 42 COVID-19 patients were enrolled. In all RA patients, both nephelometric assay and ELIA showed RF IgM positivity, while only one patient of the control group tested positive for RF IgM by nephelometric assay and ELIA. IgM directed to S1 RBD were not detected in sera of RA patients, while all COVID-19 patients presented anti-S1 RBD IgM (median anti-S1 RBD IgM COVID-19 vs. RA: 368.5 IU/mL, IQR 654 IU/mL vs. 18.45 IU/mL, IQR 20 IU/mL; p<0.0001). CONCLUSIONS: This study confirmed the lack of cross-reactivity between RF and anti-S1 RBD IgM, offering to clinicians a valuable tool for a better management of RA patients undergoing SARSCoV-2 serological tests.

Erosive arthritis in systemic lupus erythematosus: application of cluster analysis.

OBJECTIVES: In the present study, we applied cluster analysis (CA) in SLE patients with joint involvement to identify which disease subset most commonly develops erosive damage. METHODS: We collected clinical and laboratory data of SLE patients with a clinical history of joint involvement (arthritis/arthralgia). Ultrasonographic assessment was performed at level of MCPs and PIPs joints, to identify erosive arthritis, defined as the presence of erosions in at least one joint. Moreover, we detected RF, ACPA anti-CarP, and Dkk1 serum levels. We applied an unsupervised hierarchical CA to identify the aggregation of patients into different subgroups sharing common characteristics in terms of clinical and laboratory phenotypes. RESULTS: CA included 112 SLE patients (M/F 6/106; median age 45 years, IQR 17; median disease duration 96 months, IQR 165). Arthritis was observed in 82 patients (73.2%) and inflammatory arthralgia in 30 (26.8%). US-detected erosive arthritis was observed in 29 patients (25.9%). CA on clinical and laboratory features allowed the identification of four main clusters: in particular erosive arthritis was located in a cluster including renal and neuropsychiatric involvement, serositis, positivity for ACPA, anti-Carp, anti-Sm, anti-RNP, detectable levels of Dkk1. CONCLUSIONS: The application of CA made it possible to better characterise SLE phenotype including erosive arthritis. In particular, feature-driven CA leads to the identification of a more aggressive disease, due to a common pathogenic mechanism.

The Prostacyclin Analogue Iloprost Modulates CXCL10 in Systemic Sclerosis.

The prostacyclin analogue iloprost is used to treat vascular alterations and digital ulcers, the early derangements manifesting in systemic sclerosis (SSc), an autoimmune disease leading to skin and organ fibrosis. Bioindicator(s) of SSc onset and progress are still lacking and the therapeutic approach remains a challenge. The T helper 1 (Th1) chemokine interferon (IFN)γ-induced protein 10 (IP-10/CXCL10) associates with disease progression and worse prognosis. Endothelial cells and fibroblasts, under Th1-dominance, release CXCL10, further enhancing SSc's detrimental status. We analyzed the effect of iloprost on CXCL10 in endothelial cells, dermal fibroblasts, and in the serum of SSc patients. Human endothelial cells and dermal fibroblasts activated with IFNγ/Tumor Necrosis Factor (TNF)α, with/without iloprost, were investigated for CXCL10 secretion/expression and for intracellular signaling cascade underlying chemokine release (Signal Transducer and Activator of Transcription 1, STAT1; Nuclear Factor kappa-light-chain-enhancer of activated B cells, NF-kB; c-Jun NH2-terminal kinase, JNK: Phosphatidyl-Inositol 3-kinase (PI3K)/protein kinase B, AKT; Extracellular signal-Regulated Kinase 1/2, ERK1/2). CXCL10 was quantified in sera from 25 patients taking iloprost, satisfying the American College of Rheumatology (ACR)/European Alliance of Associations for Rheumatology (EULAR) 2013 classification criteria for SSc, and in sera from 20 SSc sex/age-matched subjects without therapy, previously collected. In human endothelial cells and fibroblasts, iloprost targeted CXCL10, almost preventing IFNγ/TNFα-dependent cascade activation in endothelial cells. In SSc subjects taking iloprost, serum CXCL10 was lower. These in vitro and in vivo data suggest a potential role of iloprost to limit CXCL10 at local vascular/dermal and systemic levels in SSc and warrant further translational research aimed to ameliorate SSc understanding/management.

Thrombospondin 1 and 2 along with PEDF inhibit angiogenesis and promote lymphangiogenesis in intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: The microenvironment of intrahepatic cholangiocarcinoma (iCCA) is hypovascularized, with an extensive lymphatic network. This leads to rapid cancer spread into regional lymph nodes and the liver parenchyma, precluding curative treatments. Herein, we investigated which factors released in the iCCA stroma drive the inhibition of angiogenesis and promote lymphangiogenesis. METHODS: Quantitative proteomics was performed on extracellular fluid (ECF) proteins extracted both from cancerous and non-cancerous tissues (NCT) of patients with iCCA. Computational biology was applied on a proteomic dataset to identify proteins involved in the regulation of vessel formation. Endothelial cells incubated with ECF from either iCCA or NCT specimens were used to assess the role of candidate proteins in 3D vascular assembly, cell migration, proliferation and viability. Angiogenesis and lymphangiogenesis were further investigated in vivo by a heterotopic transplantation of bone marrow stromal cells, along with endothelial cells in SCID/beige mice. RESULTS: Functional analysis of upregulated proteins in iCCA unveils a soluble angio-inhibitory milieu made up of thrombospondin (THBS)1, THBS2 and pigment epithelium-derived factor (PEDF). iCCA ECF was able to inhibit in vitro vessel morphogenesis and viability. Antibodies blocking THBS1, THBS2 and PEDF restored tube formation and endothelial cell viability to levels observed in NCT ECF. Moreover, in transplanted mice, the inhibition of blood vessel formation, the de novo generation of the lymphatic network and the dissemination of iCCA cells in lymph nodes were shown to depend on THBS1, THBS2 and PEDF expression. CONCLUSIONS: THBS1, THBS2 and PEDF reduce blood vessel formation and promote tumor-associated lymphangiogenesis in iCCA. Our results identify new potential targets for interventions to counteract the dissemination process in iCCA. LAY SUMMARY: Intrahepatic cholangiocarcinoma is a highly aggressive cancer arising from epithelial cells lining the biliary tree, characterized by dissemination into the liver parenchyma via lymphatic vessels. Herein, we show that the proteins THBS1, THBS2 and PEDF, once released in the tumor microenvironment, inhibit vascular growth, while promoting cancer-associated lymphangiogenesis. Therefore, targeting THBS1, THBS2 and PEDF may be a promising strategy to reduce cancer-associated lymphangiogenesis and counteract the invasiveness of intrahepatic cholangiocarcinoma.

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.

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.

Association between antibodies to carbamylated proteins and subclinical atherosclerosis in rheumatoid arthritis patients.

BACKGROUND: Rheumatoid arthritis (RA) patients carry a high risk of cardiovascular morbidity and mortality. The excess of cardiovascular disease cannot be entirely explained by traditional risk factors and the immune system contributes to the development of atherosclerosis. Moreover, post-translational modifications such as citrullination and carbamylation have been linked to inflammation and atherosclerosis. Anti-carbamylated proteins antibodies (anti-CarP) are a new subset of autoantibodies identified in RA patients. This study aimed to investigate a possible association between anti-CarP and subclinical atherosclerosis in RA patients. METHODS: We enrolled RA patients and normal healthy controls (NHS) without known cardiovascular risk factors or heart disease. Cardiovascular risk was assessed using the Modified Systemic Coronary Risk Evaluation (mSCORE). Anti-CarP were investigated by a solid phase "home-made" ELISA. Anti-citrullinated protein antibodies (ACPA) and Rheumatoid Factor (RF) were investigated by ELISA assays. Subclinical atherosclerosis was evaluated by brachial artery Flow-Mediated Dilatation (FMD) and Carotid Intima-Media Thickness (c-IMT) while arterial stiffness by Ankle-Brachial Index (ABI) and Cardio-Ankle Vascular Index (CAVI). RESULTS: We enrolled 50 RA patients (34 F and 16 M, mean age 58.4 ± 13.1 years, mean disease duration 127 ± 96.7 months) and 30 age and sex matched NHS. According to the mSCORE, 58% of patients had a low risk, 32% a moderate and 8% a high risk for cardiovascular disease. FMD was significantly lower in RA patients than in NHS (5.6 ± 3.2 vs 10.7 ± 8.1%; p < 0.004) and CAVIs significantly higher in a RA patients compared to NHS (left CAVI 8.9 ± 1.7 vs 8.1 ± 1.5; p < 0.04 for and right CAVI 8.8 ± 1.6 vs 8.0 ± 1.4; p < 0.04 for the). ABI and c-IMT did not differ between the two populations. The multivariate regression analysis showed a significant association of anti-CarP antibodies with FMD, left and right CAVI and both c-IMT (r = 1.6 and p = 0.05; r = 1.7 and p = 0.04; r = 2.9 and p = 0.05; r = 1.5 and p = 0.03; r = 1.1 and p = 0.03 respectively). CONCLUSIONS: This study confirms that RA patients, without evidence of cardiovascular disease or traditional risk factors, have an impaired endothelial function. Moreover, we found an association with anti-CarP antibodies suggesting a possible contribution of these autoantibodies to endothelial dysfunction, the earliest stage of atherosclerosis. Besides ultrasound assessment, anti-CarP should be assessed in RA patients and considered an additional cardiovascular risk factor.

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.

Autoantibodies specific to a peptide of ß2-glycoprotein I cross-react with TLR4, inducing a proinflammatory phenotype in endothelial cells and monocytes.

ß(2)-glycoprotein I (ß(2)GPI) is the major antigenic target for antiphospholipid Abs. Anti-ß(2)GPI Abs are a heterogeneous population of Igs targeting all domains of the molecule. Abs specific to ß(2)GPI domain I are strongly associated with thrombosis and obstetric complications. In the present study, we sought to understand the possible pathogenic mechanism for this subset of anti-ß(2)GPI Abs, investigating their potential cross-reactivity with other self-proteins involved in inflammatory or coagulant events. We compared the amino acid sequence of the ß(2)GPI domain I with human proteins in a protein databank and identified a peptide sharing 88% identity with an epitope of human TLR4. A high percentage of patients with antiphospholipid syndrome (41%) and systemic lupus erythematosus (50%) presented serum IgG specific to this peptide. Anti-ß(2)GPI peptide Abs binding the TLR4 were able to induce NF-κB activation in HEK293 cells that were stably transfected with the TLR4 gene. Anti-ß(2)GPI peptide Abs induced activation of TLR4 and triggered interleukin-1 receptor-associated kinase phosphorylation and NF-κB translocation, promoting VCAM expression on endothelial cells and TNF-α release by monocytes. In conclusion, our observations suggest a novel pathogenic mechanism in the TLR4 stimulation by anti-ß(2)GPI peptide Abs that links adaptive immune responses with innate immunity in antiphospholipid syndrome and systemic lupus erythematosus.

Autoantibody Recognition of Natural and Homocysteinylated Alpha-1 Antitrypsin: Indirect Enzyme-Linked Immunosorbent Assay (ELISA) Quantification in Sera.

In this chapter, indirect enzyme-linked immunosorbent assays (ELISAs) to quantitatively measure autoantibodies directed to human natural and homocysteinylated alpha-1 antitrypsin (anti-AATA and anti-HAATA, respectively) in serum are described. The illustrated ELISA protocols are slightly different, since the two protein forms have different biochemical features and, consequently, different affinity for the matrix (polystyrene microplate wells), so that specific experimental conditions have to be performed for the quantification of the serum antibody recognition.These procedures can be carried out to evaluate the anti-AATA and the anti-HAATA levels, testing serum samples, for research use.

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.

Does Patisiran Reduce Ocular Transthyretin Synthesis? A Pilot Study of Two Cases.

BACKGROUND: Variant transthyretin-mediated amyloidosis (ATTR-v) is a well-characterized disease affecting the neurologic and cardiovascular systems. Patisiran has been approved for neurologic involvement as it reduces hepatic synthesis of transthyretin (TTR). Eye involvement is a lateonset feature increasing the risk of glaucoma and cataracts in patients. AIMS: The aim of this case series was to assess whether patisiran can effectively reduce TTR synthesis in such a barrier-protected organ as the eye. METHODS: Two patisiran-treated ATTR-v patients underwent serum and aqueous humor sampling to measure TTR levels detected by SDS-PAGE and immunoblotting. Serum samples were compared to healthy control (HC), whereas aqueous humor samples were compared to non-amyloidotic subjects affected by cataracts and glaucoma. RESULTS: Serum TTR levels representative of hepatic synthesis were sharply lower in treated patients if compared to the HC (-87.5% and -93.75%, respectively). Aqueous humor TTR levels showed mild-tono reduction in treated patients compared to non-amyloidotic subjects with cataracts (-34.9% and +8.1%, respectively) and glaucoma (-41.1% and -2.1%). CONCLUSION: Patisiran does not seem to be as effective in inhibiting ocular TTR synthesis as it is in inhibiting hepatic synthesis. Re-engineering the envelope could allow the drug to target RPE cells thus avoiding any ocular involvement.

Vimentin/cardiolipin complex as a new antigenic target of the antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by arterial and venous thrombosis, recurrent abortions, and antiphospholipid antibodies (aPL). However, it is possible to find patients with clinical signs of APS who persistently test negative for aPL (seronegative APS, or SN-APS). The aim of this study was to identify new antigenic target(s) of autoantibodies in APS patients, which may also be recognized in SN-APS. We tested sera from patients with SN-APS with a proteomic approach by analyzing endothelial cell-surface membrane proteins. Sera from SN-APS patients revealed 2 reactive spots corresponding to vimentin, a protein that is shown to bind cardiolipin in vitro. Antivimentin/cardiolipin antibodies were tested in 29 SN-APS patients, 40 APS patients, 30 patients with systemic lupus erythematosus, 30 with rheumatoid arthritis, 30 with venous or arterial thrombosis, and 32 healthy control patients. We observed that not only a large proportion of SN-APS patients but also almost all the APS patients displayed the presence of antivimentin/cardiolipin antibodies. To verify the possible pathogenic role of these autoantibodies, we demonstrated that affinity-purified antivimentin/cardiolipin antibodies induced interleukin receptor-associated kinase phosphorylation and nuclear factor-κB activation in endothelial cells. Our results prompt to identify vimentin as a "new" cofactor for aPL, which may represent a useful tool mainly in SN-APS patients.