Paolo Fraticelli

Vitamin D3 Affects Differentiation, Maturation, and Function of Human Monocyte-Derived Dendritic Cells

We studied the effects of 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3) on differentiation, maturation, and functions of dendritic cells (DC) differentiated from human monocytes in vitro in the presence of GM-CSF and IL-4 for 7 days. Recovery and morphology were not affected by 1α,25-(OH)2D3 up to 100 nM. DC differentiated in the presence of 10 nM 1α,25-(OH)2D3 (D3-DC) showed a marked decrease in the expression of CD1a, while CD14 remained elevated. Mannose receptor and CD32 were significantly increased, and this correlated with an enhancement of endocytic activity. Costimulatory molecules such as CD40 and CD86 were slightly decreased or nonsignificantly affected (CD80 and MHC II). However, after induction of DC maturation with LPS or incubation with CD40 ligand-transfected cells, D3-DC showed marginal increases in MHC I, MHC II, CD80, CD86, CD40, and CD83. The accessory cell function of D3-DC in classical MLR was also inhibited. Moreover, allogeneic T cells stimulated with D3-DC were poor responders in a second MLR to untreated DC from the same or an unrelated donor, thus indicating the onset of a nonspecific hyporesponsivity. In conclusion, our data suggest that 1α,25-(OH)2D3 may modulate the immune system, acting at the very first step of the immune response through the inhibition of DC differentiation and maturation into potent APC.

Fractalkine (CX3CL1) as an amplification circuit of polarized Th1 responses

Fractalkine (FKN, CX3CL1) is a membrane-bound CX3C chemokine induced by primary proinflammatory signals in vascular endothelial cells (ECs). Here we examined the role of FKN in polarized Th1 or Th2 responses. Proinflammatory signals, including LPS, IL-1, TNF, and CD40 ligand, induced FKN, as did IFN-gamma, which had synergistic activity with TNF. IL-4 and IL-13 did not stimulate the expression of FKN and markedly reduced induction by TNF and IFN-gamma. TNF alone or combined with IFN-gamma also induced release of soluble FKN, which was inhibited by IL-4 and IL-13. In light of this differential regulation of FKN by the master cytokines that control polarized responses, we analyzed the interaction of FKN with natural killer (NK) cells and polarized T-cell populations. NK cells expressed high levels of the FKN receptor CX3CR1 and responded to FKN. CX3CR1 was preferentially expressed in Th1 compared with Th2 cells. Th1 but not Th2 cells responded to FKN. By immunohistochemistry, FKN was expressed on ECs in psoriasis, a Th1-dominated skin disorder, but not in Th2-driven atopic dermatitis. Similarly, ECs in Mycobacterium tuberculosis granulomatous lymphadenitis, but not those in reactive lymph node hyperplasia or in Castelmans disease, showed immunoreactive FKN. These results indicate that regulated expression of FKN in ECs participates in an amplification circuit of polarized type I responses.

Preliminary classification criteria for the cryoglobulinaemic vasculitis

Background To develop preliminary classification criteria for the cryoglobulinaemic syndrome or cryoglobulinaemic vasculitis (CV). Methods Study part I developed a questionnaire for CV to be included in the formal, second part (study part II). Positivity of serum cryoglobulins was defined by experts as an essential condition for CV classification. In study part II, a core set of classification items (questionnaire, clinical and laboratory items, as agreed) was tested in three groups of patients and controls—that is, group A (new patients with the CV), group B (controls with serum cryoglobulins but lacking CV) and group C (controls without serum cryoglobulins but with features which can be observed in CV). Results In study part I (188 cases, 284 controls), a positive response to at least two of three selected questions showed a sensitivity of 81.9% and a specificity of 83.5% for CV. This questionnaire was employed and validated in study part II, which included 272 patients in group A and 228 controls in group B. The final classification criteria for CV, by pooling data from group A and group B, required the positivity of questionnaire plus clinical, questionnaire plus laboratory, or clinical plus laboratory items, or all the three, providing a sensitivity of 88.5% and a specificity of 93.6% for CV. By comparing data in group A versus group C (425 controls), the same classification criteria showed a sensitivity 88.5% and a specificity 97.0% for CV. Conclusion Classification criteria for CV were developed, and now need validation.

PTPN22 R620W polymorphism in the ANCA-associated vasculitides

OBJECTIVES PTPN22 is involved in T-cell activation and its R620W single-nucleotide polymorphism (SNP) has been shown to predispose to different autoimmune diseases. The aims of this study were to investigate the role of the PTPN22 R620W SNP in conferring susceptibility to the ANCA-associated vasculitides (AAVs), and to explore potential associations between the PTPN22 genotype and the disease manifestations. METHODS PTPN22 R620W SNP was genotyped in a cohort of 344 AAV patients [143 with granulomatosis with polyangiitis (Wegeners) (GPA), 102 with microscopic polyangiitis (MPA) and 99 with Churg-Strauss syndrome (CSS)] and in 945 healthy controls. RESULTS The frequency of the minor allele (620W) was significantly higher in GPA patients than in controls [P = 0.005, χ(2 )= 7.858, odds ratio (OR) = 1.91], while no statistically significant association was found with MPA or CSS. Among GPA patients, the 620W allele was particularly enriched in ANCA-positive patients as compared with controls (P = 0.00012, χ(2 )= 14.73, OR = 2.31); a particularly marked association was also found with ENT involvement (P = 0.0071, χ(2 )= 7.258, OR = 1.98), lung involvement (P = 0.0060, χ(2 )= 7.541, OR = 2.07) and skin manifestations of all kinds (P = 0.000047, χ(2 )= 16.567, OR = 3.73). CONCLUSION The PTPN22 620W allele confers susceptibility to the development of GPA (but not of MPA or CSS), and particularly of its ANCA-positive subset.

Validation of the classification criteria for cryoglobulinaemic vasculitis

OBJECTIVE The aim of this study was to validate the classification criteria for cryoglobulinaemic vasculitis (CV). METHODS Twenty-three centres were involved. New patients with CV (group A) and controls, i.e. subjects with serum cryoglobulins but lacking CV based on the gold standard of clinical judgment (group B) and subjects without cryoglobulins but with clinical features that can be observed in the course of CV (group C), were studied. Positivity of serum cryoglobulins was necessary for CV classification. Sensitivity and specificity of the criteria were calculated by comparing group A vs group B. The group A vs group C comparison was done to demonstrate the possible diagnostic utility of the criteria. RESULTS The study included 268 patients in group A, 182 controls in group B and 193 controls in group C (small vessel vasculitis, 51.8%). The questionnaire (at least 2/3 positive answers) showed 89.0% sensitivity and 93.4% specificity; the clinical item (at least 3/4 clinical involvement) showed 75.7% sensitivity and 89.0% specificity and the laboratory item (at least 2/3 laboratory data) showed 80.2% sensitivity and 62.4% specificity. The sensitivity and specificity of the classification criteria (at least 2/3 positive items) were 89.9% and 93.5%, respectively. The comparison of group A with group C demonstrated the clinical utility of the criteria in differentiating CV from CV mimickers. CONCLUSION Classification criteria for CV were validated in a second, large, international study confirming good sensitivity and specificity in a complex systemic disease.

Regional Implantation of Autologous Adipose Tissue-Derived Cells Induces a Prompt Healing of Long-Lasting Indolent Digital Ulcers in Patients with Systemic Sclerosis:

Digital ulcers (DUs) are a rather frequent and invalidating complication in systemic sclerosis (SSc), often showing a very slow or null tendency to heal, in spite of the commonly used systemic and local therapeutic procedures. Recently, stem cell therapy has emerged as a new approach to accelerate wound healing. In the present study, we have tentatively treated long-lasting and poorly responsive to traditional therapy SSc-related DUs by implantation of autologous adipose tissue-derived cell (ATDC) fractions. Fifteen patients with SSc having a long-lasting DU in only one fingertip who were unresponsive to intensive systemic and local treatment were enrolled in the study. The grafting procedure consisted of the injection, at the basis of the corresponding finger, of 0.5-1 ml of autologous ATDC fractions, separated by centrifugation of adipose tissue collected through liposuction from subcutaneous abdominal fat. Time to heal after the procedure was the primary end point of the study, while reduction of pain intensity and of analgesic consumption represented a secondary end point. Furthermore, the posttherapy variation of the number of capillaries, observed in the nailfold video capillaroscopy (NVC) exam and of the resistivity in the digit arteries, measured by high-resolution echocolor-Doppler, were also taken into account. A rather fast healing of the DUs was reached in all of the enrolled patients (mean time to healing 4.23 weeks; range 2-7 weeks). A significant reduction of pain intensity was observed after a few weeks (p < 0.001), while the number of capillaries was significantly increased at 3- and 6-month NVC assessment (p < 0.0001 in both cases). Finally, a significant after-treatment reduction of digit artery resistivity was also recorded (p < 0.0001). Even with the limitations related to the small number of patients included and to the open-label design of the study, the observed strongly favorable outcome suggests that local grafting with ATDCs could represent a promising option for the treatment of SSc-related DUs unresponsive to more consolidated therapies.

Retreatment regimen of rituximab monotherapy given at the relapse of severe HCV-related cryoglobulinemic vasculitis: Long-term follow up data of a randomized controlled multicentre study

OBJECTIVE To evaluate the efficacy and safety in the long term of a retreatment regimen with Rituximab (RTX) alone administered at clinical relapse in cryoglobulinemic vasculitis (CV). METHODS Thirty patients with severe HCV-related CV, previously enrolled in the multicentre Italian trial on RTX in the treatment of CV, were retrospectively evaluated after the end of the trial. All of them were managed with RTX alone at clinical relapse, if any. Disease activity at the last available follow up was defined as complete remission (absence of active disease), partial remission (response > 50% of at least one manifestation among glomerulonephritis, peripheral neuropathy or skin ulcers) or active disease. RESULTS The mean follow up after the first RTX cycle was 72.6 (20.4) months. After the end of the trial, 21/30 (70%) patients showed an active follow up [81.7 (10.9) months)], 3/30 (10%) lost follow up and 6/30 (20%) died. 12/21 (57.1%) patients were in complete disease remission, 5/21 (23.8%) showed a partial response and 4/21 (19%) had an active disease. 17/30 (56.7%) patients needed retreatment for relapse with a mean time to retreatment of 22.3 (12.1) months. Treatment survival of this regimen was 7.6 (0.3) years. Recurrent non-severe infections occurred in 3/30, with chronic hypogammaglobulinemia in 2/3 patients. CONCLUSIONS A long-term regimen of retreatment with RTX alone given at clinical relapse seems to be effective and safe in CV, with a low rate of infections and severe hypogammaglobulinemia.

Idiopathic hypereosinophilic syndrome presenting with severe vasculitis successfully treated with imatinib

Idiopathic hypereosinophilic syndrome (HES) is a rare disorder characterized by peripheral eosinophilia exceeding 1500/mm3, a chronic course, absence of secondary causes, and signs and symptoms of eosinophil-mediated tissue injury. One of the best-characterized forms of HES is the one associated with FIP1L1-PDGFRA gene rearrangement, which was recently demonstrated as responsive to treatment with the small molecule kinase inhibitor drug, imatinib mesylate. Here, we describe the case of a 51-year-old male, whose symptoms satisfied the clinical criteria for HES with cutaneous and cardiac involvement and who also presented with vasculitic brain lesions and retroperitoneal bleeding. Molecular testing, including fluorescence in situ hybridization, of bone marrow and peripheral blood showed no evidence of PDGFR rearrangements. The patient was initially treated with high-dose steroid therapy and then with hydroxyurea, but proved unresponsive to both. Upon subsequent initiation of imatinib mesilate, the patient showed a dramatic improvement in eosinophil count and progressed rapidly through clinical recovery. Long-term follow-up confirmed the efficacy of treatment with low-dose imatinib and with no need of supplemental steroid treatment, notwithstanding the absence of PDGFR rearrangement.

A painful diagnosis

Dr. Fraticelli: A 53-year-old man was admitted to our department for a 10-day history of widespread limb pain, worsening in the evening and sometimes so excruciating to make him feel faint. These symptoms were not triggered by particular events or behaviors, and not relieved by rest or particular positions. He did not report fever, weight loss, night sweats, joint pain or any other systemic symptom. The patient’s history was of notable complexity. He had been suffering from allergic asthma during his youth. Twenty years before the current evaluation, hypertrophic cardiomyopathy was reported on a routine echocardiography. In the recent years, he also developed a mild arterial hypertension. Two years before admission, the patient had an acute coronary syndrome secondary to a noteworthy three-vessel coronary disease. At that time he was treated only with medical therapy. Later on he had a syncope, consequent with third grade atrio-ventricular blockade, that required the implantation of a pacemaker with ICD. During the stay in the cardiac surgery ward, he developed progressive weakness of lower limbs, with reduction of the tendon reflexes and sensitivity. Basing on the results of electromyography and cerebrospinal fluid analysis, a diagnosis of Guillain–Barrè syndrome was made and a subsequent trial with intravenous immunoglobulin (Ig) infusion (0.4 g/kg/day for 5 days) was successful. One year before presentation, the patient was admitted to our ward for the acute onset of hemoptysis. The physical examination revealed the presence of mild respiratory crackles on auscultation of both lung bases and a mild sensitivity reduction in the lower limbs. The main laboratory findings were an increase in the serum creatinine levels (1.77 mg/dL) and mild hemoglobinuria and proteinuria (1 g/day) on urinalysis. A high-resolution computed tomography (HRCT) of the chest revealed the presence of a ground-glass pattern, suggestive of alveolitis, and the subsequent bronchoscopy with bronchoalveolar lavage showed the presence of hemosiderin-containing macrophages. Globally considered, the findings were suggestive of a systemic vasculitis with multisystem involvement. The presence of high titers of perinuclear anti-neutrophil cytoplasmic antibodies (p-ANCA) and the pauci-immune glomerulonephritis found on lightmicroscopy examination of the renal biopsy lead to the diagnosis of microscopic polyangiitis (MPA) (Fig. 1). The patient was treated with daily prednisone (starting from 50 mg/day) and one cycle of rituximab (375 mg/m g every week for 4 weeks [1]), followed by mycophenolate mofetil as steroid-sparing long-term treatment, achieving a consistent tapering of the prednisone dose. At the last follow-up, the disease appeared in remission. Ten months before our evaluation, the patient was admitted to the emergency department for a typical chest pain associated with cold sweating. After retrieval of abnormal ECG findings and elevation of cardiac enzymes, the patient underwent a coronary arteriography, which showed worsening of the known three-vessel stenosis. The & Paolo Fraticelli paolo.fraticelli@ospedaliriuniti.marche.it

SAT0353 Small airways involvement in scleroderma patients: results of a case-control study

Background Interstitial lung disease (ILD) and pulmonary hypertension are the leading cause of morbidity and mortality in systemic sclerosis (SSc). Although a ventilatory obstructive pattern, due to large airways impairment, has been rarely observed in SSc, a potential involvement of smaller airways (SA) has been suggested in previous reports. Recently, impulse oscillometry (IOS), a non-invasive forced oscillation technique, has been advocated as a valuable diagnostic tool for a sensitive assessment of SA. Objectives The main objectives of the present study was to investigate the prevalence of SA dysfunction by IOS in SSc patients compared to healthy controls, and to evaluate the correlation between SA dysfunction and selected radiological and clinical disease-related features. Methods Consecutive SSc patients were included in the present study according to eligibility criteria; controls were health volunteers. Both cases and controls underwent IOS measurements; cases also underwent pulmonary function tests and St. Georges respiratory questionnaire. Radiological features were assessed on the latest chest high resolution computed tomography (HRTC) scan available in the twelve months before study enrolment, evaluating for both SA signs of disease and ILD. A SA involvement at IOS was defined as R5-R20 ≥0.07 kPa/L/sec. Odds ratios and 95% confidence intervals for the IOS value was computed using multiple logistic regression models. Correlation between SA dysfunction and selected parameters were assessed using Pearsons correlation coefficient. Results 92 cases (M/F 14/78, mean age 57.06) and 84 controls (M/F 15/69, mean age 54.28) were included in the present study. The R5-R20≥0.07 kpa/L/sec was found in 20.65% of cases and in 3.57% of controls. The OR was 7.027 (95% CI 1.99–24.72, p<0.01). This value did not significantly change after the adjustment for confounding variables (OR a* 7.091). Correlations between R5–20 ≥0,07 kPa/L/s and selected parameters showed a significant inverse association with vital capacity (FVC) and forced expiratory volume in first second (FEV1) and a direct correlation with pulmonary artery systolic pressure estimated. With reference to cutaneous subtype a SA dysfunction was more prevalent in the limited form compared to the diffuse, respectively in 23% and 12%. Radiologic HRCT assessment of SA pathological features and ILD extent were provided for 77 patients: 19 (24.7%) presented at least one sign of SA disease. An underlying ILD was detected in 40 patients, characterized by NSIP pattern in 37. Conclusions A significant involvement of SA was found in a substantial proportion of SSc patients, compared to healthy controls. Moreover, this seemed to be associated with a more severe functional obstructive and restrictive impairment, and with higher PAPs values. Therefore, our findings suggests that SA may be a potential, less known, target of disease, and further studies are needed to assess prognostic and therapeutic implications of this pathologic feature. Disclosure of Interest None declared