Silvana Zeni

X Chromosome Monosomy: A Common Mechanism for Autoimmune Diseases

The majority of human autoimmune diseases are characterized by female predominance. Although sex hormone influences have been suggested to explain this phenomenon, the mechanism remains unclear. In contrast to the role of hormones, it has been suggested, based on pilot data in primary biliary cirrhosis, that there is an elevation of monosomy X in autoimmune disease. Using peripheral white blood cells from women with systemic sclerosis (SSc), autoimmune thyroid disease (AITD), or healthy age-matched control women, we studied the presence of monosomy X rates using fluorescence in situ hybridization. We also performed dual-color fluorescence in situ hybridization analysis with a chromosome Y α-satellite probe to determine the presence of the Y chromosome in the monosomic cells. In subsets of patients and controls, we determined X monosomy rates in white blood cell subpopulations. The rates of monosomy X increased with age in all three populations. However, the rate of monosomy X was significantly higher in patients with SSc and AITD when compared with healthy women (6.2 ± 0.3% and 4.3 ± 0.3%, respectively, vs 2.9 ± 0.2% in healthy women, p < 0.0001 in both comparisons). Importantly, X monosomy rate was more frequent in peripheral T and B lymphocytes than in the other blood cell populations, and there was no evidence for the presence of male fetal microchimerism. These data highlight the thesis that chromosome instability is common to women with SSc and AITD and that haploinsufficiency for X-linked genes may be a critical factor for the female predominance of autoimmune diseases.

Prognostic model based on nailfold capillaroscopy for identifying Raynaud's phenomenon patients at high risk for the development of a scleroderma spectrum disorder: PRINCE (Prognostic Index for Nailfold Capillaroscopic Examination)

OBJECTIVE To construct a prognostic index based on nailfold capillaroscopic examinations that is capable of predicting the 5-year transition from isolated Raynauds phenomenon (RP) to RP secondary to scleroderma spectrum disorders (SSDs). METHODS The study involved 104 consecutive adult patients with a clinical history of isolated RP, and the index was externally validated in another cohort of 100 patients with the same characteristics. Both groups were followed up for 1-8 years. Six variables were examined because of their potential prognostic relevance (branching, enlarged and giant loops, capillary disorganization, microhemorrhages, and the number of capillaries). RESULTS The only factors that played a significant prognostic role were the presence of giant loops (hazard ratio [HR] 2.64, P = 0.008) and microhemorrhages (HR 2.33, P = 0.01), and the number of capillaries (analyzed as a continuous variable). The adjusted prognostic role of these factors was evaluated by means of multivariate regression analysis, and the results were used to construct an algorithm-based prognostic index. The model was internally and externally validated. CONCLUSION Our prognostic capillaroscopic index identifies RP patients in whom the risk of developing SSDs is high. This model is a weighted combination of different capillaroscopy parameters that allows physicians to stratify RP patients easily, using a relatively simple diagram to deduce the prognosis. Our results suggest that this index could be used in clinical practice, and its further inclusion in prospective studies will undoubtedly help in exploring its potential in predicting treatment response.

Improving outcome prediction of systemic sclerosis from isolated Raynaud’s phenomenon: role of autoantibodies and nail-fold capillaroscopy

OBJECTIVE A simple weighted prognostic algorithm, based on capillaroscopy and autoantibodies, is developed to classify patients at different risk of transition from isolated RP to SSc within 5 years from the screening visit. METHODS Two hundred and eighty-eight of 768 patients with isolated RP who underwent capillaroscopy were recruited. The prognostic contributions of capillaroscopic findings (giant loops, haemorrhages and the number of capillaries) and SSc-associated autoantibodies (ACAs, anti-topo I and ANAs) were assessed in a semi-parametric regression models suitable for competing risks. A prognostic index was built by a bagging technique. A structured tree approach was used to extract simple classificatory rules that can be directly interpreted. RESULTS Thirty-four transitions from isolated RP to SSc and 42 to other CTDs were observed. All of the chosen variables had a substantial prognostic impact. A complex non-linear prognostic pattern was observed for capillaries, with the risk of developing SSc increasing as the number of loops decreased. The presence of ANAs had a strong impact on prognosis [hazard ratio (HR) = 9.70], which was increased by the presence of ACA (HR = 3.94; P < 0.001). A weighted prognostic classification for the development of SSc was constructed using capillary number, giant loops and ANAs. The prognostic discrimination was satisfactory (Harrells C-index = 0.86). CONCLUSION Our prognostic model is based on easy-to-obtain features (i.e. the number of capillaries, giant loops and ANAs) and could be used to facilitate clinical decision making in the screening phase, and may also have important implications for stratifying patients into risk groups for future clinical assessment.

Interleukin-17A+ cell counts are increased in systemic sclerosis skin and their number is inversely correlated with the extent of skin involvement

OBJECTIVE Levels of interleukin-17A (IL-17A) have been found to be increased in synovial fluid from individuals with systemic sclerosis (SSc). This study was undertaken to investigate whether IL-17A-producing cells are present in affected SSc skin, and whether IL-17A exerts a role in the transdifferentiation of myofibroblasts. METHODS Skin biopsy samples were obtained from the involved skin of 8 SSc patients and from 8 healthy control donors undergoing plastic surgery. Immunohistochemistry and multicolor immunofluorescence techniques were used to identify and quantify the cell subsets in vivo, including IL-17A+, IL-4+, CD3+, tryptase-positive, α-smooth muscle actin (α-SMA)-positive, myeloperoxidase-positive, and CD1a+ cells. Dermal fibroblast cell lines were generated from all skin biopsy samples, and quantitative polymerase chain reaction, Western blotting, and solid-phase assays were used to quantify α-SMA, type I collagen, and matrix metalloproteinase 1 (MMP-1) production by the cultured fibroblasts. RESULTS IL-17A+ cells were significantly more numerous in SSc skin than in healthy control skin (P = 0.0019) and were observed to be present in both the superficial and deep dermis. Involvement of both T cells and tryptase-positive mast cells in the production of IL-17A was observed. Fibroblasts positive for α-SMA were found adjacent to IL-17A+ cells, but not IL-4+ cells. However, IL-17A did not induce α-SMA expression in cultured fibroblasts. In the presence of IL-17A, the α-SMA expression induced in response to transforming growth factor β was decreased, while MMP-1 production was directly enhanced. Furthermore, the frequency of IL-17A+ cells was higher in the skin of SSc patients with greater severity of skin fibrosis (lower global skin thickness score). CONCLUSION IL-17A+ cells belonging to the innate and adaptive immune system are numerous in SSc skin. IL-17A participates in inflammation while exerting an inhibitory activity on myofibroblast transdifferentiation. These findings are consistent with the notion that IL-17A has a direct negative-regulatory role in the development of dermal fibrosis in humans.

Feasibility of Different Capillaroscopic Measures for Identifying Nailfold Microvascular Alterations

OBJECTIVE To ascertain the most reliable and relevant capillaroscopic measurements of nailfold videocapillaroscopy (NVC) by analyzing their inter- and intraobserver agreement and predictive value. METHODS We studied 217 subjects (110 with Raynauds phenomenon under ongoing evaluation, and 107 with connective tissue diseases) by evaluating the number of capillaries, intercapillary distances, avascular areas, capillary disorganization, capillary loop length, capillary width, percentage of minor abnormalities (tortuous, crossed, or enlarged capillaries), and major abnormalities (giant, bushy, meandering, or branching capillaries), microhemorrhage, skin transparency, and subpapillary plexus visibility. Every finger of both hands was examined. All of the measurements were made by 2 observers under blinded conditions. RESULTS A total of 877 nailfold images were analyzed. The number of capillaries/mm, interpeak distance, and avascular areas were poorly discriminant, with no statistical differences between their areas under the receiver operating characteristic curve; their reproducibility and repeatability were good, except for the intercapillary distance. Minor abnormalities were observed in 75% of the cases and major abnormalities in 34%; the inter- and intraobserver agreement concerning the major abnormalities was almost perfect. There was very good inter- and intraobserver agreement regarding the analysis of capillary disorganization and hemorrhages. CONCLUSIONS This study shows that NVC can be useful in quantitatively and reproducibly recording various parameters. We suggest that combining the parameters showing the greatest reliability and prognostic value may be the best means of analyzing NVC images.

X Monosomy in Female Systemic Lupus Erythematosus

Abstract:  Systemic lupus erythematosus (SLE) is a systemic autoimmune disease, predominantly occurring in women of childbearing age. SLE, like several other autoimmune diseases, is characterized by a striking female predominance and, although sex hormone influences have been suggested as an explanation for this phenomenon, definitive data are still unavailable. Our group recently reported an increased X monosomy in lymphocytes of women, affected with primary biliary cirrhosis (PBC), systemic sclerosis (SSc), and autoimmune thyroiditis (AITD) in comparison to healthy women, thus suggesting the involvement of this chromosome in female predominance and in the deregulation of the immune system that characterizes autoimmunity. We have now evaluated X monosomy rates in SLE using fluorescence in situ hybridization (FISH) on peripheral mononuclear white blood cells (PBMCs) from female patients compared to healthy age‐matched controls. In addition, because of a previous finding of microchimerism as a pathogenetic cause of a number of autoimmune diseases, we investigated the presence of cells carrying the Y chromosome. We did not identify an increased X monosomy in women with SLE compared to controls (P= 0.3960, SLE vs. HCs, Students t‐test), thus suggesting that a different mechanism of immune deregulation might be predominant in the female population of patients with SLE.

Evaluation of nailfold videocapillaroscopic abnormalities in patients with systemic lupus erythematosus.

Objective:Nailfold capillaroscopy (NFC) has been shown to have a remarkable value in the differential diagnosis of connective tissue diseases. In fact, NFC patterns reflect the microvascular changes that may play a significant role in pathogenesis. The aims of this study were to determine in patients with systemic lupus erythematosus (SLE) the prevalence of NFC patterns, to evaluate any association with clinical features and laboratory parameters. Materials and Methods:One hundred twenty-three patients with SLE were included in this retrospective study. Video NFC parameters were analyzed in each patient. In all cases, the following parameters were evaluated: capillary arrangement, density, size, and shape. Results:In patients with SLE, major capillary abnormalities were frequently observed (44 of 123 = 35.8%); however, no specific pattern was noted. There was a significant correlation between the SLEDAI index and the severity of capillary abnormalities (P < 0.0001). Pathologic capillary abnormalities were also significantly increased in SLE with positive anti-U1-RNP antibodies (P < 0.05). Conclusion:NFC may be a useful method to evaluate the microvascular changes in patients with SLE, and the presence of major capillary abnormalities seems to herald a more severe clinical course of the illness.

Brief report: successful pregnancies but a higher risk of preterm births in patients with systemic sclerosis: an Italian multicenter study

OBJECTIVE To assess fetal and maternal outcomes in women with systemic sclerosis (SSc). METHODS Prospectively collected data on 99 women with SSc from 25 Italian centers were analyzed retrospectively. Women with SSc were observed during 109 pregnancies (from 2000 to 2011), and outcomes were compared to those in the general obstetric population (total of 3,939 deliveries). The maternal age at conception was a mean ± SD 31.8 ± 5.3 years, and the median disease duration at conception was 60 months (range 2-193 months). RESULTS SSc patients, compared to the general obstetric population, had a significantly increased frequency of preterm deliveries (25% versus 12%) and severe preterm deliveries (<34 weeks of gestation) (10% versus 5%), intrauterine growth restriction (6% versus 1%), and babies with very-low birth weight (5% versus 1%). Results of multivariable analysis showed that corticosteroid use was associated with preterm deliveries (odds ratio [OR] 3.63, 95% confidence interval [95% CI] 1.12-11.78), whereas the use of folic acid (OR 0.30, 95% CI 0.10-0.91) and presence of anti-Scl-70 antibodies (OR 0.26, 95% CI 0.08-0.85) were protective. The disease remained stable in most SSc patients, but there were 4 cases of progression of disease within 1 year from delivery, all in anti-Scl-70 antibody-positive women, 3 of whom had a disease duration of <3 years. CONCLUSION Women with SSc can have successful pregnancies, but they have a higher-than-normal risk of preterm delivery, intrauterine growth restriction, and babies with very-low birth weight. Progression of the disease during or after pregnancy is rare, but possible. High-risk multidisciplinary management should be standard for these patients, and pregnancy should be avoided in women with severe organ damage and postponed in women with SSc of recent onset, particularly if the patient is positive for anti-Scl-70 antibodies.

VASCULAR IMPAIRMENT IN PATIENTS WITH PRIMARY BILIARY CIRRHOSIS

Experimental and clinical observation suggest that patients with primary biliary cirrhosis (PBC) have endothelial dysfunction. Postischemic digital blood flow, nailfold capillaroscopy, von Willebrand factor (vWf) and tissue-type plasminogen activator (t-PA) plasma levels were examined in 59 PBC patients. Forty-six subjects (15 with liver diseases other than PBC, 11 hypercholesterolemics, 20 healthy subjects) served as controls. PBC versus healthy controls (209.8 +/- 1.4% and 16.54 +/- 1.44 ng/ml vs. 120.2 +/- 1.4% and 9.91 +/- 1.49 ng/ml; p<0.001) and related to bilirubin (r = 0.38, p<0.02; r = 0.47, p<0.0005, respectively). vWf was also increased in other liver diseases (249.9 +/- 1.7%; p<0.001) and related to bilirubin (r = 0.59, p<0.05). Postischemic finger blood flow negatively correlated with vWf(p<0.05 or less). Our data indicate that PBC patients have microvascular disease. Whether vessels other than those of the fingers were involved remained unclear. vWf and t-PA might reflect a dysfunction of teh hepatic vascular endothelium.

N-TproBNP as biomarker in systemic sclerosis

Systemic sclerosis (SSc) is a connective tissue disorder characterized by tissue fibrosis affecting the skin and internal organs, fibroproliferative vasculopathy, and autoimmune activation. SSc still heralds a poor prognosis with significant morbidity and mortality. Early detection of organ involvement is critical as currently available treatments are most effective when started early. Many candidate biomarkers have been investigated in the past two decades. However, despite the enormous efforts, no accurate tool to predict the pattern of organ involvement and to assess disease activity has been yet identified. The N-terminal fragment of probrain natriuretic peptide (N-TproBNP) is a neurohormone released by ventricular myocytes in response to pressure overload. N-TproBNP is highly relevant for diagnosis, prognosis, and prediction of pulmonary arterial hypertension in SSc. Moreover, several studies support its potential benefit for cardiac assessment of scleroderma patients. Conversely, the role of N-TproBNP as surrogate marker of pulmonary fibrosis and skin involvement is much less clear. We provide an extensive review of the studies that have previously investigated the role of N-TproBNP as candidate biomarker in scleroderma manifestations, presenting also the findings of a recent study we conducted in a cohort of 87 SSc patients.