Overexpression of BAG3 (Bcl2-associated athanogene 3) in serum and skin of patients with systemic sclerosis.

OBJECTIVES: BAG3 (Bcl2-associated athanogene3) is able to induce the transformation of cancer-associated fibroblasts to alpha smooth muscle actin (a-SMA) positive (+) myofibroblasts. In systemic sclerosis (SSc), a-SMA+ myofibroblasts also play an important role in the progression of fibrosis in the skin and involved internal organs. The aim of the study was to investigate whether BAG3 is overexpressed in SSc and may be a biomarker of fibrogenesis. METHODS: BAG3 serum levels were measured in 106 patients with SSc, 47 with the limited (lc) and 59 the diffuse (dc) SSc, and in age- and sex-matched healthy controls (HC). BAG3 levels were then compared according to their clinical subset, nailfold video-capillaroscopic (NVC) patterns, interstitial lung disease (ILD, and correlated with modified Rodnan skin score (mRSS) and global disease activity. BAG3 expression was also investigated in skin biopsies of 8 dcSSc patients. RESULTS: BAG3 serum levels were significantly higher in dcSSc (143.3 pg/mL, 95%CI 78-208.5) than in HC (0.68 pg/mL, 95%CI 0.13-1.23), and were significantly higher in patients with late NVC pattern and ILD but did not correlate with disease activity and mRSS. Of note, BAG3 was strongly expressed in the skin biopsies of dcSSc patients. CONCLUSIONS: BAG3 is overexpressed in dcSSc patients and may contribute to skin and organ fibrosis by prompting the transition of fibroblasts into myofibroblasts and increasing their survival. Thus, BAG3 may play an important role in SSc fibrotic pathogenesis and be a potential biomarker of fibrosis. Further research on its role as a therapeutic target is warranted.

Cellular-Based Therapies in Systemic Sclerosis: From Hematopoietic Stem Cell Transplant to Innovative Approaches.

Systemic sclerosis (SSc) is a systemic disease characterized by autoimmune responses, vasculopathy and tissue fibrosis. The pathogenic mechanisms involve a wide range of cells and soluble factors. The complexity of interactions leads to heterogeneous clinical features in terms of the extent, severity, and rate of progression of skin fibrosis and internal organ involvement. Available disease-modifying drugs have only modest effects on halting disease progression and may be associated with significant side effects. Therefore, cellular therapies have been developed aiming at the restoration of immunologic self-tolerance in order to provide durable remissions or to foster tissue regeneration. Currently, SSc is recommended as the 'standard indication' for autologous hematopoietic stem cell transplantation by the European Society for Blood and Marrow Transplantation. This review provides an overview on cellular therapies in SSc, from pre-clinical models to clinical applications, opening towards more advanced cellular therapies, such as mesenchymal stem cells, regulatory T cells and potentially CAR-T-cell therapies.

Instruments for Outcome Evaluation of Specific Domains in Primary Sjögren's Syndrome.

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disorder characterized by very heterogeneous features. The spectrum of this disorder may vary from benign but disabling symptoms such as dryness, due to lachrymal and salivary involvement, pain and fatigue, to systemic, potentially severe, manifestations that may involve any organ. In recent decades, the arrival of biotechnological therapy has offered new opportunities for the treatment of this-until now-orphan disease. Currently, the possible use of these new drugs in therapeutic trials has made it necessary to have reliable outcome measures to evaluate their efficacy in this disease. A great effort has been made in multicenter, often multinational, studies to develop and validate instruments capable of assessing the different disease-related features. The adoption in therapeutic trials of the newly developed outcome measures aimed at assessing systemic features and patient reported symptoms has often yielded disappointing results. These negative data have been ascribed, on the one hand, to the trial design not being completely appropriate, and, on the other hand, to the fact that a single instrument may be not sufficient to cover the great clinical heterogeneity of the disease features. There is now growing belief that composite end points that include instruments that are able to assess the various aspects of the disease may be more properly and successfully used in future therapeutic trials.

Management of Sjögren's Syndrome: Present Issues and Future Perspectives.

In view of the new possibilities for the treatment of primary Sjögren's syndrome (pSS) given by the availability of new biotechnological agents targeting the various molecular and cellular actors of the pathological process of the disease, classification criteria aimed at selecting patients to be enrolled in therapeutic trials, and validated outcome measures to be used as response criteria to these new therapies, have been developed and validated in the last decades. Unfortunately, the therapeutic trials so far completed with these new treatments have yielded unsatisfactory or only partially positive results. The main issues that have been evoked to justify the poor results of the new therapeutic attempts are: (i) the extreme variability of the disease phenotypes of the patients enrolled in the trials, which are dependent on different underlying patterns of biological mechanisms, (ii) the fact that the disease has a long indolent course, and that most of the enrolled patients might already have irreversible clinical features. The advances in the research of new disease biomarkers that can better distinguish the different clinical phenotypes of patients and diagnose the disease in an earlier phase are also discussed.

The Role of Interferons in the Pathogenesis of Sjögren's Syndrome and Future Therapeutic Perspectives.

There is a great deal of evidence pointing to interferons (IFNs) as being key cytokines in the pathogenesis of different systemic autoimmune diseases, including primary Sjögren's syndrome (pSS). In this disease, a large number of studies have shown that an overexpression of type I IFN, the 'so-called' type I IFN signature, is present in peripheral blood mononuclear cells, and that this finding is associated with the development of systemic extra-glandular manifestations, and a substantial production of autoantibodies and inflammatory cytokines. In contrast, the absence or a milder expression of type I IFN signature and low level of inflammatory cytokines characterizes patients with a different clinical phenotype, where the disease is limited to glandular involvement and often marked by the presence of widespread pain and depression. The role of type II (IFNγ) in this subset of pSS patients, together with the potentially related activation of completely different immunological and metabolic pathways, are emerging issues. Expression of both types of IFNs has also been shown in target tissues, namely in minor salivary glands where a predominance of type II IFN signature appeared to have a certain association with the development of lymphoma. In view of the role played by IFN overexpression in the development and progression of pSS, inhibition or modulation of IFN signaling has been regarded as a potential target for the therapeutic approach. A number of therapeutic compounds with variable mechanisms of action have been tested or are under consideration for the treatment of patients with pSS.

Impact of COVID-19 outbreak in an Italian cohort of patients with systemic sclerosis.

BACKGROUND: Mortality rate in patients infected by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) can be related to the presence of comorbidities like diabetes, cardiovascular and pulmonary diseases. On the contrary, few data exist on the impact of CoronaVirus Disease 2019 (COVID-19) on patients with rheumatic disorders, namely in those having pulmonary involvement and treated with immunosuppressive agents. The present survey is aimed at knowing the impact of COVID-19 in a cohort of patients with systemic sclerosis (SSc). METHODS: Telephone interviews were carried out during the COVID-19 outbreak in patients with SSc followed in a Rheumatic Disease Unit in Italy. Patients were asked for confirmed SARS-CoV-2 infection, symptoms suggestive of COVID-19, and modification of their therapy. RESULTS: A total number of 526 patients with SSc were contacted and interviewed. Of them, 270 and 256 had limited cutaneous and diffuse cutaneous SSc, respectively. Interstitial lung disease (ILD) was present in 45% of patients and most of them (68.2%) were treated with immunosuppressive therapy. Only two patients were hospitalized for COVID-19-related pneumonia, and one of them died despite invasive ventilator support. An additional 11 patients reported flu-like symptoms compatible with a mild form of COVID-19. Nobody modified the therapy during the COVID-19 outbreak. CONCLUSION: Despite the large prevalence of ILD and immunosuppressive therapies, which can be considered risk factors for the occurrence and severity of incidental viral infections, the impact of COVID-19, in terms of mortality rate and morbidity, does not appear particularly severe in this large cohort of patients with SSc. Possible mechanisms influencing this figure are discussed.

High NEMO score values in nailfold videocapillaroscopy are associated with the subsequent development of ischaemic digital ulcers in patients with systemic sclerosis.

BACKGROUND: Nailfold videocapillaroscopy (NVC) is a feasible method that allows the observation of the microvascular changes that mark the course of systemic sclerosis (SSc). In previous studies, we demonstrated that the NEMO score, i.e. the cumulative number of microhaemorrhages and microthromboses, is a good indicator of the steady-state level and overtime changes of disease activity (DA) in SSc. OBJECTIVES: To verify whether high NEMO scores, which mirror a very active microvascular derangement in the fingers, may be associated with the subsequent development of ischaemic digital ulcers (IDUs). METHODS: The NEMO score was assessed at baseline (T0) in 98 patients with SSc, all classified according to the ACR-EULAR criteria. Of them, 90 were females, 48 had the limited and 50 had the diffuse cutaneous variant of SSc. Afterwards, the patients were closely followed up for 2 years, and the appearance of new IDUs recorded at any time of the follow-up. The T0-NEMO score values of patients who developed IDUs were compared to those of patients who did not. A receiver operating curve (ROC) was constructed, and the area under the curve (AUC) calculated by plotting the sensitivity and 1-specificity of the different NEMO score values in predicting the subsequent development of IDUs. RESULTS: During the follow-up, 38 out of 98 patients developed one or more IDUs. The NEMO score at T0 was significantly higher in those who developed IDUs with respect to those who did not [median 14.5 (95% CI 11.0-21.5) and 4.5 (95% CI 4.0-6.0), respectively, p < 0.0001]. The ROC curve derived from different T0-NEMO score values had an AUC of 0.79 (95% CI 0.69-0.86, p < 0.0001). A NEMO score of ≥ 12 had a sensitivity of 83.3% (95% CI 71.5-91.7) and a specificity of 63.2% (95% CI 46.0-78.2), with positive (P) and negative (N) predictive (PV) values of 58.9% (95% CI 44.7-72.2) and 85.6% (71.8-94.4), respectively. A NEMO score of ≥ 16 had a sensitivity of 95.0% (95% CI 86.1-99.0) and a NPV of 93.4% (77.5-99.2). CONCLUSIONS: Being a valid tool to measure DA levels in SSc, the NEMO score also appears to be closely related to the subsequent development of IDUs in this disease.

Autophagy occurs in lymphocytes infiltrating Sjögren's syndrome minor salivary glands and correlates with histological severity of salivary gland lesions.

BACKGROUNDS: The organization of minor salivary glands (MSG) infiltrates, in patients with Sjögren's syndrome (SS), associates with disease severity and progression. Aberrant regulation of lymphocyte autophagy is involved in autoimmunity, and in previous work, we provided the first evidence of upregulated autophagy in CD4+ T cells infiltrating SS MSG. The aim of this study was to further explore autophagy in SS infiltrating and circulating lymphocytes and to investigate its role in disease histopathological progression. METHODS: After collection of 20 SS MSG, the presence of lymphocyte aggregates (foci) and the formation of germinal center (GC)-like structures were observed by H&E and confirmed by immunohistochemistry. The expression of autophagy-related genes, Atg5 and MAP1LC3A, was detected by RT-PCR on microdissected salivary gland tissue and control tonsils. In MSG and tonsils, autophagic lymphocytes were identified by the detection of the autophagosome protein LC3B visualized as LC3 puncta staining by immunofluorescence. Peripheral blood autophagy was assessed by flow cytometry in SS and healthy controls (HC). RESULTS: Real-time PCR demonstrated higher expression in the autophagy genes Atg5 and MAP1LC3A in MSG GCs as compared to both small foci (p = 0.0075, p = 0.0002) and GCs from tonsils (p = 0.0001, p = 0.0037). In MSG, LC3 puncta staining was detectable on both CD3+ and CD20+ lymphocytes; in tonsils, LC3 puncta was almost undetectable on all lymphocytes. Compared to HC (n = 20), flow cytometry did not reveal any increase of autophagy in SS circulating lymphocytes (n = 30). CONCLUSIONS: In SS MSG, lymphocytes' autophagy is a feature of infiltrating T and B cells and is associated with histological severity. Interestingly, in MSG aberrant regulation of autophagy is detectable in GC-like structures possibly indicating its involvement in the development and persistence of the autoimmune process within the lesions.

Taking care of systemic sclerosis patients during COVID-19 pandemic: rethink the clinical activity.

COVID-19 outbreak has quickly spread worldwide, causing a high pressure on the health-care system. In Italy, from March 8, 2020, all the deferrable clinical activities have been suspended to increase the health care offer for COVID-19 patients. The hospital organization has been modified also in order to assure non-COVID-19 patients assistance. The Scleroderma Unit of ASST Pini-CTO Hospital, in Milan, in the region mostly hit by SARS-CoV-2 in Italy, follows more than 600 patients affected by systemic sclerosis (SSc). Patients with SSc need a close follow-up with a regular screening of organ involvement and frequent intravenous treatments. All SSc patients have been educated about ministerial directives to limit COVID-19 spread. The organization of our Scleroderma Unit has been quickly rethought to assure SSc patients assistance in safety for them and for health-care workers during urgent visits or infusion therapies. Using electronic way of communication with frequent virtual contact and guarantying home deliveries of some therapies, we allowed a continuity of care also outside the Hospital.

CXCL13 as biomarker for histological involvement in Sjögren's syndrome.

OBJECTIVES: SS is an autoimmune condition characterized by systemic B-cell activation, autoantibody production and ectopic germinal centres' formation within the salivary gland (SG). The extent of SG infiltrate has been proposed as a biomarker of disease severity. Plasma levels of CXCL13 correlate with germinal centres' activity in animal models and disease severity in SS, suggesting its potential use as a surrogate serum marker to monitor local B-cell activation. The aim of this study was to evaluate the potential role of CXCL13 as a biomarker of SG pathology in two independent SS cohorts. METHODS: 109 patients with SS were recruited at Sapienza University of Rome (Italy) (n = 60), or at Queen Elizabeth Hospital in Birmingham and Barts Health NHS Trust in London (n = 49). Both sera and matched minor SG biopsy were available. Sicca (n = 57) and healthy subjects' (n = 19) sera were used as control. RESULTS: CXCL13 serum level was higher in SS patients compared with controls. Correlations between its serum levels and a series of histomorphological parameters, including size of the aggregates and the presence germinal centres', were observed. CONCLUSION: Our data foster the use of CXCL13 to monitor the extent of local pathology in SS and its validation in longitudinal clinical studies.

Epidemiological profile and north-south gradient driving baseline systemic involvement of primary Sjögren's syndrome.

OBJECTIVE: To characterize the systemic phenotype of primary Sjögren's syndrome at diagnosis by analysing the EULAR-SS disease activity index (ESSDAI) scores. METHODS: The Sjögren Big Data Consortium is an international, multicentre registry based on worldwide data-sharing cooperative merging of pre-existing databases from leading centres in clinical research in Sjögren's syndrome from the five continents. RESULTS: The cohort included 10 007 patients (9352 female, mean 53 years) with recorded ESSDAI scores available. At diagnosis, the mean total ESSDAI score was 6.1; 81.8% of patients had systemic activity (ESSDAI score ≥1). Males had a higher mean ESSDAI (8.1 vs 6.0, P < 0.001) compared with females, as did patients diagnosed at <35 years (6.7 vs 5.6 in patients diagnosed at >65 years, P < 0.001). The highest global ESSDAI score was reported in Black/African Americans, followed by White, Asian and Hispanic patients (6.7, 6.5, 5.4 and 4.8, respectively; P < 0.001). The frequency of involvement of each systemic organ also differed between ethnic groups, with Black/African American patients showing the highest frequencies in the lymphadenopathy, articular, peripheral nervous system, CNS and biological domains, White patients in the glandular, cutaneous and muscular domains, Asian patients in the pulmonary, renal and haematological domains and Hispanic patients in the constitutional domain. Systemic activity measured by the ESSDAI, clinical ESSDAI (clinESSDAI) and disease activity states was higher in patients from southern countries (P < 0.001). CONCLUSION: The systemic phenotype of primary Sjögren's syndrome is strongly influenced by personal determinants such as age, gender, ethnicity and place of residence, which are key geoepidemiological players in driving the expression of systemic disease at diagnosis.

NEMO score in nailfold videocapillaroscopy is a good tool to assess both steady state levels and overtime changes of disease activity in patients with systemic sclerosis: a comparison with the proposed composite indices for this disease status entity.

BACKGROUND: In previous studies, we demonstrated that the NEMO score, i.e. the cumulative number of microhaemorrhages (MHEs) and microthromboses (MTs), observed in nailfold videocapillaroscopy was a good indicator of the steady state level of disease activity (DA) in patients with systemic sclerosis (SSc) when the European Scleroderma Study Group (EScSG) index was considered the gold standard. AIM OF THE STUDY: To verify whether the NEMO score could be (i) a valid tool to assess DA, even when the modified European Scleroderma Trials and Research (EUSTAR) index was considered to be the comparator, and (ii) a sensitive method to capture the DA overtime changes. PATIENTS AND METHODS: The NEMO score and the EScSG and EUSTAR indices were contemporarily assessed at baseline (T0) and after a follow-up of 4-56 months (T1) in 98 patients with SSc. The differences (Δ) between the T1 and T0 values of the NEMO score and the EScSG and EUSTAR indices were calculated and compared to each other. RESULTS: NEMO score values were very closely correlated with the corresponding values of the EScSG and EUSTAR indices both at T0 and T1 observations (p < 0.0001 in all cases with the exception of the correlation with EScSG values at T1 (p < 0.03)). The values of the two composite DA indices were also strictly related to each other in both T0 and T1 observations (p < 0.0001). Receiver operating characteristic (ROC) curve analysis showed the NEMO score had a good sensitivity and specificity in classifying patients with a predefined level of DA (scores ≥ 3.0 and ≥ 2.5 for the EScSG and EUSTAR indices, respectively, p < 0.0001 in both cases). Δ values of the NEMO score were significantly correlated with the corresponding values of both the EScSG and EUSTAR indices. Weighted Cohen's k level of agreement between Δ values of the NEMO score and those of the EScSG and EUSTAR indices was moderate (0.55 and 0.59, respectively). CONCLUSIONS: NEMO score proves to be a feasible, non-invasive, and valid tool to assess steady state levels and changes over time of DA in patients with SSc. Thus, it can represent an alternative or complementary method to measure this disease status entity in this disorder.

Gene Expression Profiles in Primary Sjögren's Syndrome With and Without Systemic Manifestations.

OBJECTIVE: To investigate the gene expression profile in patients with Sjögren's syndrome that is characterized by different clinical phenotypes. METHODS: RNA from peripheral blood mononuclear cells was purified in 8 patients with glandular features (GFs) and widespread pain (WP) and 11 with extraglandular manifestations (EGMs) and then was analyzed by hybridization on a human gene chip exploring more than 40,000 human genes. Differentially expressed genes (DEGs) in the two subgroups (ie, those with false discovery rate-corrected P values ≤ 0.01) with respect to 20 healthy controls have been submitted to functional classification using a Gene Ontology database and were mapped to define the networks of protein to protein interactions (PPIs). RESULTS: The enriched pathway analyses of DEGs and of the highly interconnected modules identified in the PPI networks showed that the pathological processes characterizing the two subgroups were substantially different. The predominant pathways in patients with EGMs are related to T- and B-cell activation, Toll-like receptor, interferon signaling, and apoptosis. Conversely, pathological processes related to pain transmission and modulation are preferentially operative in patients with GFs and WP. These data suggest that a neuroinflammatory pathway driven by cytokines and chemokines may play a central role in triggering WP features in this phenotype of patients. CONCLUSION: The present study supports the hypothesis that different biological pathways are operative in patients with primary Sjögren's syndrome with different clinical phenotypes. A better knowledge of these specific processes might help in tailoring more effective target therapies.

First Report of the Italian Registry on Immune-Mediated Congenital Heart Block (Lu.Ne Registry).

Objective: Neonatal Lupus (NL) is a rare syndrome caused by placental transfer of maternal anti-SSA/Ro and anti-La/SSB autoantibodies to the fetus. The rarity of this condition requires the establishment of multidisciplinary registries in order to improve our knowledge. Method: Inclusion criteria in this retrospective study were the maternal confirmed positivity for anti-SSA/Ro and/or anti-SSB/La antibodies, and the presence of II or III degree congenital heart block (CHB) in utero or neonatal period (up to 27 days after birth). Result: Eighty-nine cases of CHB were observed in 85 women with 88 pregnancies that occurred between 1969 and 2017. CHB was mostly detected in utero (84 cases, 94.2%), while five cases were observed in the neonatal period. A permanent pacemaker was implanted in 51 of 73 children born alive (69.8), whereas global mortality rate was 25.8% (23 cases): 16 in utero, five perinatal, and two during childhood. By univariate analysis, factors associated with fetal death were pleural effusion (p = 0.005, OR > 100; CI 95% 2.88->100 and hydrops (p = 0.003, OR = 14.09; CI 95% 2.01-122). Fluorinated steroids (FS) were administered in 71.4% pregnancies, and its use was not associated with better survival. Some centers treated all cases with fluorinated steroids and some centers did not treat any case. CHB was initially incomplete in 24 fetuses, and of them five cases of II degree block reverted to a lower degree block after treatments. Recurrence rate in subsequent pregnancies was 17.6% (3 out of 17). A prophylactic treatment was introduced in 10 of these 16 subsequent (58.8%) pregnancies, mostly with FS or high dose intravenous immunoglobulins. Conclusion: This is the first report from the Italian Registry of neonatal lupus/CHB. The live birth rate was nearly 80%, with nearly two thirds of the children requiring the implantation of a pacemaker. The management of fetuses diagnosed with CHB was heterogeneous across Italian Centers. The registry at present is mainly rheumatological, but involvement of pediatric cardiologists and gynecologists is planned.

Regional grafting of autologous adipose tissue is effective in inducing prompt healing of indolent digital ulcers in patients with systemic sclerosis: results of a monocentric randomized controlled study.

BACKGROUND: A randomized controlled trial (RCT) was performed to confirm preliminary uncontrolled data indicating that regional adipose tissue (AT) grafting (G) is effective in inducing ischemic digital ulcer (IDU) healing in patients with systemic sclerosis (SSc). PATIENTS AND METHODS: SSc patients with IDUs were randomized to be blindly treated with AT-G or a sham procedure (SP). AT-G consisted of injection, at the base of the finger with the IDU, of 0.5-1 ml AT after centrifugation of fat aspirate. The SP consisted of false liposuction and local injection of saline solution. The primary endpoint was to compare the cumulative prevalence of healed IDUs in the two groups within the following 8 weeks. RESULTS: AT-G and the SP were carried out in 25 and 13 patients, respectively. The two groups were comparable for age, gender, disease duration, and SSc subtypes. IDU healing was observed in 23/25 and 1/13 patients treated with AT-G and the SP, respectively (p < 0.0001). The 12 patients who received the unsuccessful SP underwent a rescue AT-G. In all of them, IDU healing was observed after 8 weeks of observation. It was noticeable that in the AT-G-treated patients a significant reduction of pain intensity (measured by visual analogue scale) was recorded after 4 and 8 weeks (p < 0.0001 in all cases). Similarly, a significant increase of capillary numbers in the affected finger was recorded by nailfold videocapillaroscopy after 4 and 8 weeks (p < 0.0001 in both cases). CONCLUSION: This RCT strongly confirms that AT-G is effective in inducing IDU healing in SSc patients. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03406988 . Registered retrospectively on 25 January 2018.

Autologous Hematopoietic Stem Cell Transplantation for Treatment of Systemic Sclerosis.

Systemic Sclerosis (SSc) is a complex autoimmune disease, characterized by high mortality and morbidity. The heterogeneity in terms of extent, severity, and rate of progression of skin and internal organ involvement gives rise to many difficulties in finding the optimal therapeutic interventions for SSc and, to date, no disease-modifying agents are available. In this scenario, it is not surprising that SSc was one of the first autoimmune diseases challenged with high-dose immunosuppressive treatment followed by autologous hematopoietic stem cell transplantation (AHSCT). In the last decades, AHSCT has emerged as a treatment option for refractory SSc through a reduction of the aberrant immune cells, followed by re-constitution of a new, self-tolerant immune system. After several case series and pilot studies, more recently three randomized controlled trials have shown a benefit in skin involvement, organ functions and quality of life measures in AHSCT compared to monthly cyclophosphamide. In addition, although AHSCT presents a certain risk of mortality, it has been shown that the overall survival is better, compared to the cyclophosphamide group. Current evidence suggests that SSc patients who are most likely to benefit from AHSCT are early, active, with rapidly progressing diffuse skin disease, and mild involvement of internal organs. As the studies have progressed, it has become evident the need for a more rigorous patient selection, the optimization of transplant and post-transplant procedures, and the intervention of multidisciplinary teams of specialists to increase the safety and efficacy of AHSCT in SSc.