Secretagogue effect of PDE4 inhibitor apremilast on human salivary gland organoids obtained from primary Sjögren's syndrome patients.

OBJECTIVES: The aim of the study was to culture vital salivary gland organoids obtained through labial or parotid biopsy of primary Sjögren's syndrome (pSS) patients in order to evaluate their morphological and functional features in basal condition and after stimulation with Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) activator forskolin and phosphodiesterase 4 (PDE4) inhibitor apremilast, their in vitro regenerative capacity and the immune-histological resemblance with original tissue. METHODS: Salivary gland tissues from five pSS patients were processed to obtain vital organoids; swelling assay and cell proliferation tests were performed after forskolin and apremilast application. Immunochemistry evaluation on original salivary gland tissue and corresponding organoids was performed, and secretomics analysis was conducted to assess their functional status. REULTS: After application of forskolin and apremilast, we observed organoid swelling after 30 minutes, compatible with a positive functional status and enhancement of saliva production. In 3 cases, apremilast induced organoid proliferation. All cases were positive for cytokeratin 14 (CK14) and most for cytokeratin 5 (CK5). All the cases were positive for amylase; its secretion, and thus functional status of organoids, was confirmed by its high concentration in the culture medium. A focal ductal differentiation was found in some cases, highlighted by epithelial membrane antigen (EMA) positivity. The more differentiated EMA positive areas were negative for the staminal marker CK14, showing a sort of "complementary staining". CONCLUSIONS: Our data highlighted that differentiated cells and vital functional organoids that recapitulate the development of original salivary glands can be obtained from pSS epithelium. For the first time, the direct stimulating effect of PDE4 inhibitor apremilast on pSS human salivary gland organoids is reported, opening new perspectives on targeting oral dryness with drugs that combine secretagogue and immunomodulatory effects.

Validation of thymic stromal lymphopoietin as a biomarker of primary Sjögren's syndrome and related lymphoproliferation: results in independent cohorts.

OBJECTIVES: Thymic stromal lymphopoietin (TSLP) has been implicated in primary Sjögren's syndrome (pSS) and related B-cell lymphoproliferation and lymphoma (NHL) by studies on salivary pathologic tissues and serum. The purpose of this work was to validate serum TSLP as biomarker of pSS and related lymphoproliferation by the study of two additional independent cohorts. METHODS: Serum TSLP was measured by ELISA in the original published Cohort-1 from Udine, Italy, including 91 patients. Two additional cohorts were then studied for validation: Cohort-2, including 4 sub-cohorts comprising 125 patients from the Universities of Roma, L'Aquila, Pisa and Perugia, belonging to the Italian SS Study Group (GRISS), and Cohort-3, including 59 patients from the University of Athens, Greece. Overall, 159 control subjects were enrolled. Active pSS-NHL, as well as pre-lymphomatous conditions, i.e. persistent salivary gland swelling and mixed cryoglobulinaemia, were investigated in detail. In addition, serum samples from pSS-NHL in complete remission were analysed (n=27). RESULTS: TSLP serum levels were confirmed to be significantly higher in pSS compared to controls in both Cohort-2 and Cohort-3, in particular in patients with lymphoproliferation. Serum TSLP was much higher in pSS pre-lymphomatous conditions. Finally, active NHL showed the highest TSLP serum levels, while in NHL in remission TSLP resulted undetectable or significantly lower than in benign pSS. CONCLUSIONS: By the study of independent cohorts, it was again demonstrated that serum TSLP levels are increased in pSS, above all in more advanced B-cell lymphoproliferation and NHL. Serum TSLP can therefore represent a novel biomarker for pSS-related lymphoproliferation.

Thymic stromal lymphopoietin expression from benign lymphoproliferation to malignant B-cell lymphoma in primary Sjögren's syndrome.

OBJECTIVES: To investigate the expression of thymic stromal lymphopoietin (TSLP) in primary Sjögren's syndrome (pSS), stratified according to the lymphoproliferative status, from a fully benign (fbSS) stage to myoepithelial sialadenitis (MESA) and to B-cell non-Hodgkin's lymphoma (NHL). METHODS: After initial serum studies in large numbers of pSS patients and in controls, TSLP was investigated also in pathologic salivary glands (SG) biopsies from 38 stratified pSS patients (13 fbSS; 13 MESA; 12 NHL) and from 13 controls with non-autoimmune sicca syndrome (nSS) by RT-PCR, immunohistochemistry and immunofluorescence. RESULTS: Significantly higher TSLP serum levels were shown in pSS than controls, increasing from fbSS to MESA and to NHL. In SG biopsies, TSLP-positive B lymphocytes increased with increasing lymphoproliferation, maximally in NHL, consistent with the detection of inducible TSLP long isoform (lfTSLP) mRNA only in MESA and NHL. By contrast, the constitutive TSLP short isoform (sfTSLP) mRNA showed no difference among subgroups. The TSLP expression by glandular epithelium declined with the progression from fbSS to MESA and to NHL. CONCLUSIONS: TSLP progressively increases from benign to malignant B-cell lymphoproliferation in pSS. The salivary epithelium expresses TSLP but, with the progression of lymphoproliferation, the B-cells may represent the major source of TSLP, in its long inducible isoform. A possible pathogenetic role of TSLP is herein hypothesised in pSS for the first time. Further analyses on TSLP, also as a biomarker of pSS and related lymphoproliferation, are worthwhile.

The TTTT B lymphocyte stimulator promoter haplotype is associated with good response to rituximab therapy in seropositive rheumatoid arthritis resistant to tumor necrosis factor blockers.

OBJECTIVE: To investigate the polymorphisms in the promoter region of the B lymphocyte stimulator (BLyS) gene as markers of response to rituximab (RTX) in rheumatoid arthritis (RA). METHODS: The study was first conducted in 152 Italian RA patients and then replicated in an additional 117 RA patients (73 Italian, 44 British). The European League Against Rheumatism response criteria were used to evaluate the response rate at months 4 and 6 after the first cycle of RTX, by means of the Disease Activity Score in 28 joints using the erythrocyte sedimentation rate; patients were classified according to the best response shown between months 4 and 6. BLyS promoter polymorphisms were analyzed by polymerase chain reaction followed by the analysis of the restriction fragments, BLyS promoter haplotypes were analyzed using the expectation-maximization algorithm, and BLyS serum levels were analyzed using enzyme-linked immunosorbent assay. Odds ratios (ORs) were calculated with 95% confidence intervals (95% CIs). RESULTS: The TTTT BLyS promoter haplotype appeared to be significantly associated with response to RTX only in the subset of seropositive patients (those positive for rheumatoid factor and/or anti-cyclic citrullinated peptide). The replication study confirmed that this association was limited to seropositive RA patients in whom treatment with anti-tumor necrosis factor (anti-TNF) agents had previously failed. In the whole series of seropositive patients in whom anti-TNF agents had previously failed, patients carrying the TTTT BLyS promoter haplotype were more prevalent in good responders (18 of 43 [41.9%]) than in moderate responders (20 of 83 [24.1%]) or in nonresponders (1 of 21 [4.8%]) (for good responders versus nonresponders, OR 14.4 [95% CI 1.77-117.39], P=0.0028). Furthermore, multivariate analysis selected the TTTT BLyS promoter haplotype as an independent marker of good response to RTX (for good responders versus nonresponders, OR 16.2 [95% CI 1.7-152.5], P=0.01; for good responders versus moderate responders and nonresponders combined, OR 3.1 [95% CI 1.2-7.8], P=0.02). The relationship between BLyS polymorphisms and BLyS serum levels remained unclear. CONCLUSION: BLyS promoter genotyping may be suitable for identifying seropositive RA patients who may have a good response to RTX after anti-TNF agents have failed.

Humoral and T-Cell Mediated Response after the Third Dose of mRNA Vaccines in Patients with Systemic Lupus Erythematosus on Belimumab.

OBJECTIVE: To evaluate humoral and T-cell cellular-mediated immune response after three doses of SARS-CoV-2 mRNA vaccines in patients with systemic lupus erythematosus (SLE) under Belimumab. PATIENTS AND METHODS: 12 patients on Belimumab and 13 age-matched healthy volunteers were recruited. Patients were in remission or in low disease activity, and they were taking no corticosteroids or only low doses. None of the patients and controls had detectable anti-SARS-CoV-2 antibodies due to previous exposure to the virus. All the patients received three doses of mRNA anti-SARS-CoV-2 vaccines and the humoral and cellular-mediated response were tested 4 weeks after the second dose (T0), 6 months after the second dose (T1) and 4 weeks after the third dose (T2). Comparison with the control group was performed at time T0 (i.e., 4 weeks after the second dose). Total anti-SARS-CoV-2 RBD antibodies were analyzed using a diagnostic assay, while cellular-mediated response was evaluated using the interferon-gamma release assay (IGRA). RESULTS: A humoral response was documented in all the patients at T0 (median 459; IQR 225.25-758.5), but the antibody titer significantly declined from T0 to T1 (median 44.7; IQR: 30.3-202; p = 0.0066). At T2, the antibody titer significantly increased from T1 (median 2500; IQR: 2500-2500), and it was not different from T0 (respectively p < 0.0001, p = 0.66). Cellular-mediated response significantly declined from T0 to T1 (p = 0.003) but not from T0 to T2 (p = 0.3). No differences were found between patients and controls at T0 as regards both humoral and cellular responses (p = 1.0 and p = 0.09 for humoral and cellular responses, respectively). CONCLUSION: The third dose of mRNA COVID-19 vaccine can restore both humoral and cellular immune response in SLE patients on Belimumab.

Chlamydophila psittaci subclinical infection in chronic polyarthritis.

OBJECTIVES: Recent evidence indicates that Chlamydophila psittaci (Cp) may establish chronic infections, which may promote autoimmunity and/or B cell lymphoproliferation. METHODS: The presence of a subclinical Cp infection was investigated in 293 patients with chronic inflammatory polyarthritis, including 175 patients with rheumatoid factor (RF)-positive and/or anti-CCP-positive rheumatoid arthritis (RA) and 118 with seronegative polyarthritis (46 RF-negative/anti-CCP-negative RA, 36 psoriatic arthritis and 36 undifferentiated spondyloarthritis). One hundred and eighty-five healthy controls were also investigated. The presence of Cp infection was assessed in peripheral blood mononuclear cells using several PCR protocols targeting different regions of the Cp genome (16S-23S spacer rRNA, OMP-A, and Gro-EL). The DNA of other Chlamydia species (C. Pneumoniae and C. Trachomatis) was also investigated. Amplicons were sequenced to confirm the specificity of PCR products. RESULTS: The presence of a subclinical chronic Cp infection was observed in a significantly higher percentage of patients with chronic polyarthritis (38/293; 13%) compared to healthy controls (1/185, 0.5%; OR=27.4, 95%CI:3.73-201.6, p<0.0001). Furthermore, the prevalence of Cp was higher in seronegative polyarthritis (23/118; 19.5%) than in seropositive RA patients (15/175; 7.4%; OR=2.58, 95%CI: 1.28-5.19, p=0.0078). The highest prevalence of Cp infection was found in RF/anti-CCP double-negative RA patients (13/46, 28.3%), followed by patients with psoriatic arthritis (6/36; 16.7%). No differences in age, sex, disease duration and undergoing therapies were noticed between Cp-positive and Cp-negative patients; nor between seropositive and seronegative patients. CONCLUSIONS: Cp may be an infectious trigger possibly involved in the pathogenesis of a fraction of inflammatory polyarthritis, particularly in seronegative patients.

Articular and Peripheral Nervous System Involvement Are Linked to the Long-Term Outcome in Primary Sjögren's Syndrome: The Relevance of Single Organ Manifestations Rather Than a Composite Score as Predictors.

Objective: The disease course in primary Sjögren's Syndrome (pSS) differs in different subsets of patients. The aim of this study was to clarify whether the pattern of organ involvement may improve the prediction of the very long-term disease outcome. Methods: We collected the data of 255 patients. The total European League Against Rheumatism (EULAR), EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI) score was compared with the pattern of organ involvement, as differentiated by the single ESSDAI domains: (i) at disease diagnosis, and (ii) in the follow-up, by verifying the appearance of new ESSDAI domains and/or the worsening of already active ESSDAI domains. Results: The mean follow-up duration was 9.1 ± 6.9 years. At disease diagnosis, only the articular activity at baseline could predict the long-term outcome of pSS detected at last follow-up visit, being protective in terms of stable or improved disease activity, as measured by ESSDAI [OR 2.9 (1.6-5.4), p = 0.01]. In the follow-up, the onset, and/or worsening of either the peripheral nervous system (PNS) domain (by multivariate and univariate analysis), or the biological domain (only by univariate analysis) correlated with a higher disease activity at the last visit [PNS domain: OR 5.9 (2.4-14.5), p < 0.0001; biological domain: OR 1.9 (1.0-3.8), p = 0.043]. A significantly higher number of patients with articular involvement were taking hydroxychloroquine at the last follow-up visits, if compared with patients without (41/130, 31.5 vs. 13/125, 10.4%, p < 0.0001). Conclusion: Single organ disease manifestations of SS, herein identified as the articular, PNS and biologic involvement, are relevant to predict the very long-term outcome in pSS.

The CC homozygosis of the -174G>C IL-6 polymorphism predicts a lower efficacy of rituximab therapy in rheumatoid arthritis.

Identification of genetic biomarkers of response to biologics in rheumatoid arthritis (RA) is a relevant issue. Being IL-6 a key cytokine for B cell survival, the interleukin-6 (IL-6) -174G>C and the IL-6 receptor (IL-6R) D358A gene polymorphisms were investigated in 158 RA patients treated with rituximab (RTX). One hundred and twenty-eight (81.0%) were RF positive and 126 (79.7%) were anti-CCP positive. Response to therapy was evaluated at the end of the sixth month after the first RTX infusion, by using both the EULAR and the ACR criteria. The possible relationship with IL-6 serum levels was also studied. By univariate analysis, lack of response by the EULAR criteria was more prevalent in RA patients with the IL-6 -174 CC genotypes (39.1%), than in the GC/GG patients (18.5%) (OR 2.83; 95%CI=1.10-7.27; p=0.031). A good response was noticed in only one patient (4.3%) with the IL-6 -174 CC genotype, while it was present in 24.4% of GG/GC cases (p=0.06). By stepwise multivariate analysis (including RA duration, baseline DAS28, baseline HAQ, RF status, anti-CCP status and IL-6 genotype as covariates), the IL-6 -174CC genotype was selected as an independent predictor of no response to RTX by both EULAR and ACR≥50 criteria, while the IL-6R polymorphism resulted as not associated. No definite association between gene polymorphisms and IL-6 serum levels was noticed. Present results suggest a possible role for IL-6 genotyping to better plan treatment with RTX in RA, and larger studies are worthwhile.

Transforming growth factor ß 869C/T and interleukin 6 -174G/C polymorphisms relate to the severity and progression of bone-erosive damage detected by ultrasound in rheumatoid arthritis.

INTRODUCTION: Single nucleotide polymorphisms (SNPs) of transforming growth factor ß (TGF-ß) and IL-6 genes (respectively, 869C/T and -174G/C) have been associated with radiographic severity of bone-erosive damage in patients with rheumatoid arthritis (RA). Musculoskeletal ultrasound (US) is more sensitive than radiography in detecting bone erosion. We analyzed the association between TGF-ß 869C/T and IL-6 -174G/C SNPs and bone-erosive damage, evaluated by US, in a cohort of patients with severely active RA. METHODS: Seventy-seven patients were enrolled before beginning anti-TNF treatment. Disease activity was measured using the disease activity score in 28 joints, and the clinical response was evaluated according to the European League Against Rheumatism response criteria. Rheumatoid factor (RF) and anticitrullinated protein/peptide antibodies (ACPAs) were detected. The 869C/T TGF-ß and -174G/C IL-6 SNPs were analyzed by PCR amplification. US was performed to assess the bone surfaces of metacarpophalengeal (MCP), proximal interphalangeal (PIP) and metatarsophalangeal (MTP) joints by obtaining multiplanar scans. According to the number of erosions per joint, a semiquantitative score ranging from 0 to 3 was calculated in each anatomical site to obtain a MCP total erosion score (TES), a PIP TES and a MTP TES, all ranging from 0 to 30, and a global patient TES calculated as the sum of these scores (range, 0 to 90). RESULTS: Patients carrying the TGF-ß 869TT genotype showed a statistically significant lower MTP TES than those with the CC or CT genotype (mean MTP TES ± standard deviation for 869TT 6.3 ± 5.7 vs. 869CC/CT 11.7 ± 7.8; P = 0.011). Interestingly, patients with the TT genotype showed dichotomous behavior that was dependent on autoantibody status. In the presence of ACPAs and/or RF, the TT genotype was associated with lower erosion scores at all anatomical sites compared with the CC and CT genotypes. Conversely, the same 869TT patients showed higher erosion scores in the absence of ACPAs or RF. CONCLUSIONS: In RA patients, TGF-ß 869C/T SNPs could influence the bone-erosive damage as evaluated by US. The serological autoantibody status (ACPAs and RF) can modulate this interaction.

Serum levels of anti-CCP antibodies, anti-MCV antibodies and RF IgA in the follow-up of patients with rheumatoid arthritis treated with rituximab.

Rheumatoid arthritis (RA) is characterized by the presence of circulating rheumatoid factor (RF) and anticitrullinated peptide antibodies (ACPA), which are positive in about 70-80% of patients. APCA have a higher specificity and therefore a higher diagnostic power than RF, but are less informative than RF in monitoring the course of the disease in patients under treatment. Recently, it has been reported that the anticitrullinated vimentin (a-MCV) antibody test can identify a particular subgroup of APCA that may be negative for anticyclic citrullinated peptide (a-CCP) antibodies. Concerning RF, the RF IgA isotype has been described as a more specific marker of erosive joint damage than total RF. The aim of our study was to monitor the levels of a-CCP, a-MCV, total RF and RF IgA in the follow-up of patients with RA treated with B-lymphocytedepletive rituximab (RTX), to detect any differences or peculiarities in patterns of these autoantibodies, especially in relation to their potential use as predictive markers of therapeutic response. We studied 30 patients with RA treated with RTX. All patients were previously unresponsive to at least 6 months of therapy with disease-modifying antirheumatic drugs (DMARDs; methotrexate, leflunomide, cyclosporine, chloroquine) and/or at least 6 months of therapy with anti-TNF biologics. The evaluation of response to RTX was made at month +6 using the EULAR criteria (DAS28). a-CCP, a-MCV, total RF and RF IgA were determined at baseline (before the first infusion of RTX) and after 1, 3 and 6 months. In serum samples obtained before treatment two cytokines essential for Blymphocyte proliferation, interleukin 6 (IL-6) and B-lymphocyte stimulator (BLyS) were also determined. In all patients a significant and consistent reduction in all the tested antibodies was found during follow-up, with no differences in respect of the degree of response to RTX. Of note, at baseline, generally a higher titre of all autoantibodies was seen in patients who then showed a better response to RTX. Finally, there were no differences in serum concentrations of IL-6 and BLyS in patients in relation to the presence or absence of the autoantibodies investigated, nor was there any significant correlation between the serum concentrations of the cytokines and the titres of the autoantibodies. Thus, neither a-MCV compared to a- CCP, nor RF IgA compared to routine total RF, provided any additional predictive information in the follow-up of patients with RA treated with RTX.

B-lymphocyte stimulator and a proliferation-inducing ligand serum levels in IgA-deficient patients with and without celiac disease.

IgA deficiency (IgAD) is the most common form of immunodeficiency and frequently associates with autoimmunity, especially with celiac disease (CD). The mechanisms underlying IgAD and the development of autoimmunity are still relatively unknown. Elevated B-lymphocyte stimulator (BLyS) and APRIL (a proliferation-inducing ligand) serum levels characterize several autoimmune diseases. We herein investigated BLyS and APRIL serum levels in IgAD patients with and without CD and compared these patients to CD patients with normal IgA and control patients (HBDs). Compared to HBDs, IgAD patients demonstrated a significant increase of BLyS (P < 0.0001) and APRIL (P = 0.003) levels, and no differences were seen between patients with or without CD. While BLyS appeared similarly overexpressed in IgAD and CD patients, APRIL was significantly increased only in IgAD patients. Because APRIL promotes IgA production, its overexpression may represent a physiological mechanism of compensation. BLyS upregulation may be involved in the increased risk of autoimmune disease development characterizing people carrying IgAD.