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We aimed to apply and compare the QRISK3 and the adjusted Global AntiPhospholipid Syndrome (APS) Score (aGAPSS) in a cohort of systemic lupus erythematosus (SLE) patients, with and without a concomitant diagnosis of APS, in order to assess their augmented risk of developing cardiovascular diseases (CVDs).Patients (25-85 yo) with a diagnosis of SLE and/or of Secondary APS (SAPS) were included. QRISK3 was calculated using the official online calculator; aGAPSS using the validated point-values based on aPL-profile and independent risk factors.The cohort included 142 SLE patients: 34 SAPS (23.9%) and 108 SLE patients without APS (76.1%).When considering all the cohort, patients with cerebrovascular/coronary events showed higher values of aGAPSS (10.1 ± 6.2 vs. 5.8 ± 6.1; p = 0.007), but not of the QRISK3. Furthermore, a significant association was observed between the occurrence of these events and high-risk aGAPSS: p = 0.03 for aGAPSS≥8, p = 0.01 for aGAPSS ≥9, p = 0.008 for aGAPSS ≥10. aGAPSS strongly correlated with the occurrence of any thrombotic event, both at the uni- and multivariate analysis (p = 0.012 and p = 0.009). Male gender also resulted to positively correlate with the occurrence of any thrombotic event at both uni- and multivariate analysis (p = 0.017 and p = 0.03). Focusing on aPL-profile, regardless the diagnosis, we found a statistical significance only for aGAPSS (aPL+ =9.6 ± 6.3 vs. aPL- = 4.1 ± 5.1; p 0.001).Despite QRISK3 being more accurate than traditional risk score in predicting CVD risk in SLE patients, aGAPSS appears to be the most valuable tool for this purpose.

Background Patients on B-cell depleting agents may have a suboptimal response to vaccination, placing them at a higher risk of contracting SARS-CoV-2 or suffering from a more severe prognosis. Indeed, available data on pre-exposure prophylaxis with tixagevimab/cilgavimab (Evusheld) in subjects with glomerular diseases (GD) who received rituximab is limited. Methods We conducted a prospective study analyzing the safety and efficacy of tixagevimab/cilgavimab for pre-exposure prophylaxis in patients with GD who received rituximab in the previous 12 months. Rate of symptomatic infections and hospitalizations were compared to patients with GD treated with rituximab who refused to receive tixagevimab/cilgavimab. Results Tixagevimab/cilgavimab was administered to 22 patients (12 females, mean age 58,4 ± 19,6 years) with GD diagnoses including membranous nephropathy, lupus nephritis, ANCA-associated vasculitis and focal segmental glomerulosclerosis. No patient treated with tixagevimab/cilgavimab experienced symptomatic infection with SARS-CoV-2 during the follow-up (mean observation time follow-up was 112 ± 23 days), while 11 out 28 controls (39,3%) reported a symptomatic infection (p = 0001), requiring hospitalization in 2 cases. Reported adverse events were mild, namely self-limiting headache (4), discomfort at the injection site (3), flu-like symptoms/myalgia (3), and fever (1). No serious adverse event, (e.g. cardiac events, anaphylaxis) was reported. Conclusion Pre-exposure prophylaxis with tixagevimab/cilgavimab (Evusheld) seems safe and lowering of about 40% the risk of symptomatic SARS-CoV-2 infection in vaccinated subjects with GD who received anti-CD20 therapy. Possible applications in the subset of patients who need immunosuppressive therapy, especially with Rituximab, in a pandemic setting might be envisaged.

We aimed to investigate the epidemiology, the clinical and laboratory characteristics of the pediatric involvement of antiphospholipid syndrome (APS), by performing a review of the current evidence and reviewing local experience in the Northwest Italy. To achieve this, we performed a detailed literature search to identify articles describing clinical and laboratory characteristics of pediatric APS. In concomitance, we conducted a registry-based study collecting data from the Piedmont and Aosta Valley Rare Disease Registry including pediatric patients diagnosed with APS in the last 11 years. The literature review led to inclusion of six articles with a total of 386 pediatric patients (65% females, 50% with systemic lupus erythematosus (SLE) as concomitant diagnosis). Rates of venous and arterial thrombosis were 57 and 35%, respectively. “Extra-criteria manifestations” included mostly hematologic and neurologic involvement. Almost one-quarter of patients (19%) reported recurrent events and 13% manifested as catastrophic APS. A total of 17 pediatric patients (mean age 15.1 ± 2.8, 76% female) developed APS in the Northwest of Italy. In 29% of cases, SLE was a concomitant diagnosis. Deep vein thrombosis was the most frequent manifestation (28%) followed by catastrophic APS (6%). The estimated prevalence of pediatric APS in Piedmont and Aosta Valley Region is 2.5/100,000 people, whereas the estimated annual incidence is 0.2/100,000 inhabitants. In conclusion, clinical manifestations of pediatric APS seem to be more severe and with a high prevalence of noncriteria manifestations. International efforts are needed to better characterize this condition and to develop new specific diagnostic criteria to avoid missed/delayed diagnosis in children with APS.

(1) Background: Immunological laboratory testing is known to be complex, and it is usually performed in tertiary referral centers. Many criticalities affect diagnostic immunological testing, such as limited availability, the need for specifically trained laboratory staff, and potential difficulties in collecting blood samples, especially in the most vulnerable patients, i.e., the elderly and children. For this reason, the identification of a new feasible and reliable methodology for autoantibody detection is urgently needed. (2) Methods: We designed a systematic review to investigate the available literature on the utilization of saliva samples for immunological testing. (3) Results: A total of 170 articles were identified. Eighteen studies met the inclusion criteria, accounting for 1059 patients and 671 controls. The saliva collection method was mostly represented by passive drooling (11/18, 61%), and the most frequently described methodology for antibody detection was ELISA (12/18, 67%). The analysis included 392 patients with rheumatoid arthritis, 161 with systemic lupus erythematosus, 131 with type 1 diabetes mellitus, 116 with primary biliary cholangitis, 100 with pemphigus vulgaris, 50 with bullous pemphigoids, 49 with Sjogren syndrome, 39 with celiac disease, 10 with primary antiphospholipid syndromes, 8 with undifferentiated connective tissue disease, 2 with systemic sclerosis, and 1 with autoimmune thyroiditis. The majority of the reviewed studies involved adequate controls, and saliva testing allowed for a clear distinction of patients (10/12 studies, 83%). More than half of the papers showed a correlation between saliva and serum results (10/18, 55%) for autoantibody detection, with varying rates of correlation, sensitivity, and specificity. Interestingly, many papers showed a correlation between saliva antibody results and clinical manifestations. (4) Conclusions: Saliva testing might represent an appealing alternative to serum-based testing for autoantibody detection, considering the correspondence with serum testing results and the correlation with clinical manifestations. Nonetheless, standardization of sample collection processing, maintenance, and detection methodology has yet to be fully addressed.

The interest of extra-criteria antiphospholipid antibodies is growing, especially in patients negative for conventional antibodies. In this study we aimed to assess the clinical utility of anti-phosphatidyl-serine/prothrombin antibodies (aPS/PT) testing in patients negative for Beta2-Glycoprotein 1(β2GPI)-dependent tests, for identifying antiphospholipid syndrome (APS) patients that developed cerebrovascular events (CVE). When screening APS patients attending our center, out of 119 aPS/PT IgG/IgM-positive patients, thus patients negative for aβ2GPI and aCL, 42 patients (35%) tested negative for β2GPI-dependent tests and were tested with thrombin generation assay (TGA). Ten patients (24%), with isolated aPS/PT IgG/IgM, had a history of CVE. Lupus anticoagulant (LA)-positive test was more frequently observed in patients with CVE (8/22 vs. 2/20; p = 0.045). Out of the 10 patients who experienced CVE, 3 patients were aPS/PT IgG positive (all LA positive), and 8 patients were aPS/PT IgM positive (6/8 LA positive). One patient was positive for both aPS/PT IgG and IgM. LA-positive patients had only high titers of aPS/PT IgG/IgM, all of them being ≥ 80 U/ml, while the 2 LA-negative patients were aPS/PT IgM positive with medium titers [40–60 U/ml]. LA-positive patients had significantly altered TGA profile when compared to those who were LA negative, considering all TGA parameters. LA-positive patients had significantly higher tLag (8.4 ± 3.3 min vs. 6.6 ± 1.8 min; p = 0.046), higher tPeak (14 ± 4.3 min vs. 11 ± 2.7 min; p = 0.015) and lower Peak (207 ± 152 nM vs. 356.3 ± 104.7 nM; p p = 0.033). The use of aPS/PT might be of help in identifying patients with CVE and APS, as also confirmed by TGA testing.

Purpose Genotype–phenotype correlation in congenital 21 hydroxylase deficiency is strong but by no means absolute. Indeed, clinical and hormonal features may vary among patients carrying similar CYP21A2 mutations, suggesting that modifier genes may contribute to the phenotype. Aim of the present study was to evaluate whether polymorphisms in the p450 oxidoreductase (POR) gene may affect clinical features in patients with 21 hydroxylase deficiency Methods Sequencing of the POR gene was performed in 96 patients with 21 hydroxylase deficiency (49 classic, 47 non-classic) and 43 control subjects. Results Prevalence of POR polymorphisms in patients with 21 hydroxylase was comparable to controls and known databases. The rs2228104 polymorphism was more frequently associated with non-classic vs classic 21 hydroxylase deficiency (allelic risk 7.09; 95% C.I. 1.4–29.5, p Conclusions Polymorphisms in the POR gene are associated with clinical features of 21 hydroxylase deficiency both as regards predisposition to classic vs non-classic forms and severity of classic adrenal hyperplasia.

AimTo apply thrombin generation assay (TGA) in a large cohort of antiphospholipid antibodies (aPL)-positive patients.Material and methods108 patients were tested with TGA and lupus anticoagulant (LA) testing and divided according to their aPL profile. Briefly, 21 patients were positive for anti-phosphatidylserine (aPS)/prothrombin (PT) IgG/IgM (group1), 29 for anti-ß2-glycoprotein-I (aβ2GPI) and anti-cardiolipin (aCL) IgG/IgM (group2), 31 for aPS/PT, aβ2GPI and aCL IgG/IgM (group3), 27 for aPS/PT and/or aβ2GPI+aCL IgM at low-titres (group4). 31 healthy donors (HDs) and 24 controls treated with vitamin K antagonists (VKA) were included.ResultsThe most deranged TGA and LA profiles were observed in tetra-positive patients (group3) that differed significantly to the other groups, thus those with isolated, double or triple aPL-positivity. Moreover, when comparing the TGA profile of all antiphospholipid syndrome (APS) patients, aPL-carriers, HDs and VKA controls, we observed that the aPL+ patients (especially APS) showed a distinctive profile that allowed to distinguish them from the other groups with significantly higher tLag and tPeak, and lower Peak and area under the curve.When focusing on APS clinical manifestations, patients with a high-risk profile (group3) showed significant differences from those presenting low-titres aPL (group 4) regarding the number of venous events (p=0.04), recurrence of any thrombotic event (p=0.01), of arterial events (5 vs 0, p=0.02), the occurrence of TIA (p=0.04), DVT (p=0.02) and, when analysing extracriteria manifestations, of peripheral artery disease (p=0.04).ConclusionsTGA seems a valuable approach to stratify aPL+ patients according to their risk profile. The differences among different populations of autoantibodies specificities could be considered a translational validation of the increased thrombotic risk of patients with triple or tetra aPL-positivity.

3beta-hydroxysteroid dehydrogenase type II deficiency (HSD3B2 deficiency), a rare form of congenital adrenal hyperplasia (CAH), is characterized by varying degrees of salt loss and incomplete masculinization in males and mild virilization or normal external genitalia in females. We report the case of a patient (46XY) showing salt loss and incomplete masculinization, markedly elevated levels of 17OHP (17 hydroxyprogesterone), ACTH (Adreno Cortico Tropic Hormone), testosterone and delta4androstenedione (delta4A), low levels of cortisol and absence of bone skeletal alterations that frequently characterize POR (Cytochrome P450 oxidoreductase) deficiency. Mutation analysis by Sanger sequencing of the HSD3B2 gene showed that the patient presented with a compound heterozygote for two novel variants c.370A>G p.Ser124Gly and c.308-6 G>A. The two HSD3B2 gene variants were also present in the patient’s older brother showing only incomplete masculinization. The in silico analysis revealed a probable damaging effect of c.370A>G p.Ser124Gly: residue p.Ser124 is highly conserved among species and seems to be located in the catalytic site of the enzyme, playing a pivotal role in NAD(H) binding to its substrate. Intronic c.308-6G>A variant is predicted to be likely pathogenic; the substitution seems to cause a change in the splice acceptor site located 6bp downstream of the variant. The two siblings seem to be affected by 3β-HSD2 deficiency; nevertheless, the two novel variants are likely to cause variable expressivity of the disease.

ObjectivesWhen treating Behçet’s disease (BD), anti-tumor necrosis factor (TNF)-α agents have become a second-line therapy when conventional immunosuppressive drugs have failed. However, in the case of failure of treatment with anti-TNFα drugs, further options are limited. Based on previous reports of the efficacy of vedolizumab (VDZ) in inflammatory bowel diseases, we decided to administer VDZ to treat a patient with intestinal BD.MethodsWe present the case of a 49-year-old female patient with BD. Her clinical manifestations included erythema nodosum, oro-genital ulcers, positive Pathergy test, positive HLA-B51, and biopsy-proven intestinal BD. The patient was unsuccessfully treated with conventional immunosuppressive and several biological agents.ResultsTreatment with VDZ was started intravenously at a dose of 300 mg at 0, 2, and 6 weeks and then every 4 weeks.After the second dose of VDZ, the patient reported a marked improvement of intestinal BD and a concomitant amelioration of arthralgia, erythema nodosum lesions and aphthosis. Clinical remission was achieved at 6 months after starting VDZ.ConclusionVDZ might represent a valid option to treat patients with BD who are non-responsive to standard treatments or anti-TNFα agents, particularly, those cases with intestinal involvement.

Background: The gold standard for diagnosis of lupus nephritis (LN) is still represented by renal biopsy, and serological prognostic biomarkers are still lacking. Dickkopf homolog-3 (DKK3) has been suggested as a marker of tissue fibrosis in different conditions; however, its role in autoimmune diseases needs to be elucidated. Here, we investigated the prognostic role of DKK3 in systemic lupus erythematosus (SLE) patients with and without LN, assessing its changes in relation to kidney function, flares, and interstitial fibrosis. Methods: Overall, 132 SLE patients (57 with LN) were included and prospectively followed up for at least 36 months. DKK3 was measured in serum at baseline. Biopsies were evaluated for glomerular involvement, interstitial fibrosis, and tubular atrophy. Results: Patients with biopsy-proven LN had significantly higher levels of DKK3 than those without (median [min–max]: 215 ng/mL [81–341] vs. 21.1 ng/mL [1–69], p < 0.01). DKK3 levels were associated with prevalent chronic kidney diseases (OR: 4.31 [C.I. 2.01–6.61] per DKK3 doubling, p < 0.01), higher chronicity index at biopsy (1.75 [1.51–2.77] per DKK3 doubling, p < 0.01), and flares rate (OR: 1.45 [C.I. 1.1–5.71] per DKK3 doubling, p < 0.044). Conclusions: While kidney biopsy still represents the gold standard for diagnostic and prognostic assessment in LN, DKK3 could represent an additional prognostic tool to monitor SLE patients and guide therapeutic choices.

To support the management of rheumatoid arthritis (RA) patients treated with tofacitinib, we designed the TuTOR (tailoring tofacitinib oral therapy in rheumatoid arthritis) mobile app. The impact of the app on medical adherence was evaluated using a crossover design alternating a paper-diary and the TuTOR App. Twenty patients with RA (mean age at inclusion, 59 ± 13 years) were included in the study. A statistically significant decrease in DAS28 was observed since the first month of therapy (mean DAS28 at baseline, 3.9 ± 1 vs. 1° month 3.1 ± 1, p = 0.0016). Similarly, the numerical rating scale (NRS) of perceived activity of disease and subjective fatigue progressively decreased. No differences were reported in DAS28 or NRS between the TuTOR app and the paper-diary groups. A significant decrease was observed in HAQ during the follow-up (baseline 1.38 ± 1.11 vs. six months 0.83 ± 0.9; p = 0.01). When filling out the self-reporting questionnaires, most of the patients (82%) preferred the TuTOR App helping them to remember to take the pills. Furthermore, 82% of patients used the app regularly (vs. 53% for the paper diary). Three patients suspended tofacitinib due to gastrointestinal intolerance. Both digital and paper devices can help maximize adherence to therapy; however, the TuTOR app was preferred by the patients for its simplicity and immediacy.

Patients with antiphospholipid syndrome (APS) present with clinical features of recurrent thrombosis and pregnancy morbidity and persistently test positive for the presence of antiphospholipid antibodies (aPL). At least one clinical (vascular thrombosis or pregnancy morbidity) and one lab-based (positive test result for lupus anticoagulant, anticardiolipin antibodies and/or anti-β2-glycoprotein 1 antibodies) criterion have to be met for a patient to be classified as having APS. Nevertheless, the clinical variety of APS encompasses additional signs and symptoms, potentially affecting any organ, that cannot be explained exclusively by a prothrombotic state. Those manifestations, also known as extra-criteria manifestations, include haematologic (thrombocytopenia and haemolytic anaemia), neurologic (chorea, myelitis and migraine) manifestations as well as the presence of livedo reticularis, nephropathy and valvular heart disease. The growing body of evidence describing the clinical aspect of the syndrome has been paralleled over the years by emerging research interest focusing on the development of novel biomarkers that might improve the diagnostic accuracy for APS when compared to the current aPL tests. This review will focus on the clinical utility of extra-criteria aPL specificities. Besides, the promising role of a new technology using particle based multi-analyte testing that supports aPL panel algorithm testing will be discussed. Diagnostic approaches to difficult cases, including real-world case studies investigating the diagnostic added value of extra criteria aPL, particularly anti-phosphatidylserine/prothrombin, will also be examined.

Antiphospholipid syndrome (APS) is the most common acquired thrombophilia. The clinical manifestations of APS are mainly vascular thrombosis (venous and/or arterial) and/or recurrent pregnancy morbidity with the concomitant persistent presence of antiphospholipid antibodies (aPL). Therefore, the goals of the treatment of patients with APS are reducing the pregnancy morbidity and/or the prevention of thrombotic events during the follow-up. Optimal treatment of APS has long been discussed, due to the heterogeneity of the clinical manifestations and the consequent plurality in the medical specialties involved in managing this condition. This review summarizes the available evidence on primary thromboprophylaxis in aPL-positive individuals with no prior thrombotic events, secondary prophylaxis in patients with positive history for thrombotic events, the management of refractory or difficult cases and the current strategies for the management of APS during pregnancy.

BACKGROUND Antiphospholipid antibodies (aPL) are known to increase the risk of obstetrical complications. However, aPL significance and prevalence in women with late-onset pregnancy complications (LO-PC) need further clarification. OBJECTIVES To investigate the prevalence of aPL in a cohort of women who experienced LO-PC and to compare it with a cohort of uneventful pregnancies. METHODS One hundred pregnant women who experienced LO-PC, had a low risk for chromosomal abnormalities, and absence of fetal abnormalities were recruited from August 2018 to August 2019. One hundred women with uneventful pregnancy were included as controls. aPL testing was performed on serum samples derived from prenatal screening test and included both criteria and "extra criteria" aPL. RESULTS Patients with LO-PC had significantly higher aPL prevalence when compared with controls (31/100 [31%] vs 10/100 [10%]; P

Background For a number of persons with rare diseases (RDs) a definite diagnosis remains undiscovered with relevant physical, psychological and social consequences. Undiagnosed RDs (URDs) require other than specialised clinical centres, outstanding molecular investigations, common protocols and dedicated actions at national and international levels; thus, many “Undiagnosed RDs programs” have been gradually developed on the grounds of a well-structured multidisciplinary approach. Methods The Italian Undiagnosed Rare Diseases Network (IURDN) was established in 2016 to improve the level of diagnosis of persons with URD living in Italy. Six Italian Centres of Expertise represented the network. The National Centre for Rare Diseases at the Istituto Superiore di Sanità coordinates the whole project. The software PhenoTips was used to collect the information of the clinical cases. Results One hundred and ten cases were analysed between March 2016 and June 2019. The age of onset of the diseases ranged from prenatal age to 51 years. Conditions were predominantly sporadic; almost all patients had multiple organs involvements. A total of 13/71 family cases were characterized by WES; in some families more than one individual was affected, so leading to 20/71 individuals investigated. Disease causing variants were identified in two cases and were associated to previously undescribed phenotypes. In 5 cases, new candidate genes were identified, although confirmatory tests are pending. In three families, investigations were not completed due to the scarce compliance of members and molecular investigations were temporary suspended. Finally, three cases (one familial) remain still unsolved. Twelve undiagnosed clinical cases were then selected to be shared at International level through PhenomeCentral in accordance to the UDNI statement. Conclusions Our results showed a molecular diagnostic yield of 53,8%; this value is comparable to the diagnostic rates reported in other international studies. Cases collected were also pooled with those collected by UDNI International Network. This represents a unique example of global initiative aimed at sharing and validating knowledge and experience in this field. IURDN is a multidisciplinary and useful initiative linking National and International efforts aimed at making timely and appropriate diagnoses in RD patients who still do not have a confirmed diagnosis even after a long time.

Background:Antiphospholipid Syndrome (APS) is an autoimmune disease whose precise aetiology is still unknown, but the high heterogeneity of its manifestations and clinical course is presumably due to the occurrence of different mechanisms and alterations at different levels and pathways [1]. The first genetic studies in APS focused primarily on the human leukocytes antigen system region, but more recent data highlighted a role of other genes in APS susceptibility, primarily those involved in the immune response and in the haemostatic process.Objectives:We aimed to deepen the investigation of APS genetic background starting from a case of familial APS, analysing two siblings with thrombotic APS (Table 1), both triple positive for antiphospholipid antibodies (aPL).Table 1.Main clinical and laboratory characteristics of the patients included in the study.PatientAgeaPL ProfileRelevant Clinical History1 (F)51Triple positive (LA, aCL IgG, aβ2GPI IgG)Two episodes of ischemic stroke, one episode of CAPS (renal thrombotic microangiopathy, visual impairment, ischemic stroke)2 (M)47Triple positive (LA, aCL IgG, aβ2GPI IgG)Three episodes of deep vein thrombosis, regardless ongoing well conducted therapy vitamin k antagonist and additional retinal vein thrombosisLA: lupus anticoagulant; aCL: anti-cardiolipin antibodies; aβ2GPI: anti- β2 glycoprotein I antibodies; CAPS: catastrophic APS.Methods:Genomic DNA was extracted from peripheral blood and the samples underwent Whole Exome Sequencing (WES). Sequencing was done on a 100X coverage, and reads have been aligned to the human reference genome (GRCh37/hg19 assembly) using the Burrows–Wheeler Alignment tool (BWA). The mean sequencing depth on target regions was 170X for patient 1, 205X for patient 2, moreover, 99.50% of the targeted bases had at least 10X coverage for all the three donors. The resulting single nucleotide polymorphisms (SNPs) have been analysed through a step-by-step process based on their frequency population (using Genome Aggregation Database), their predicted effects on the protein (using VarSome) and a literature research about the genes carrying them. Moreover, genes previously associated with a pro-thrombotic tendency and with APS have been analysed in the two patients.Results:Starting from more than 120000 SNPs for each patients, the analysis led to reduce the list of SNPs of interest to 27 missense mutations. The complete literature research regarding the genes carrying these mutations allowed to further reduce the number of selected genes, focusing on those that exert a role potentially involved in APS pathogenesis and development. In particular, these genes (PLA2G6, HSPG2, BCL3, ZFAT, ATP2B2, CRTC3 and ADCY3) take part in the immune response and the vascular homeostasis. The list of the DNA missense variants of interest found in our cases of familial APS is resumed in Figure 2.Figure 2.List of DNA missense variants of interest found in patient 1 and 2. Genes potentially involved in APS pathogenesis and development are highlighted in bold.No mutations on genes known to be associated with a pro-thrombotic state (F5, F2, MTHFR, F13A1, PROC, PROS1, FGB and SERPINE1), or on genes previously associated with APS (B2GPI, PF4V1, SELP, TLR2, TLR4, GP Ia, GP1BA, F2R, F2RL1, TFPI, F3, VEGFA, FLT1, and TNF) have been found in the WES analysis.Conclusion:To some extent, this can be seen as a proof of concept of the complexity of APS. Efforts to interpret the genetic risk factors involved in the heterogeneous clinical features of the syndrome, for instance, the integration of WES and network-based approaches might help to identify and stratify patients at risk of developing APS.References:[1]Iuliano A, Galeazzi M, Sebastiani GD. Antiphospholipid syndrome’s genetic and epigenetic aspects. Autoimmun Rev. 2019;18(9).Disclosure of Interests:None declared

Background:Risk stratification in patients with antiphospholipid antibodies (aPL) remains a clinical challenge [1].Objectives:We aimed to evaluate the role of Thrombin Generation Assay (TGA) in distinguishing various populations of aPL positive patients (with and without lupus anticoagulant - LA) and its association with β2GPI-dependent and anti-phosphatidyl-serine/prothrombin(aPS/PT) antibodies.Methods:One-hundred-and-eight patients were tested with TGA and divided as follows: 21 patients with aPS/PT IgG/IgM (Group 1), 29 with aβ2GPI IgG/IgM (Group 2), 31 with aPS/PT and aβ2GPI IgG/IgM (Group 3), 27 with aPS/PT and/or aβ2GPI IgM low-titers (Group 4). Table 1 resumes the clinical characteristics of the APS patients (excluding aPL asymptomatic). Thirty-one healthy donors (HDs) and 24 controls treated with VKA were also included.Results:The most deranged TGA and LA profile was observed in patients with both aPS/PT and aβ2GPI when compared to those with an isolated positivity for aPS/PT or aβ2GPI and patients with aPS/PT and/or aβ2GPI IgM at low titres (Figure 1). Similarly, patients with aPS/PT and/or aβ2GPI at medium/high titres presented with the higher rate of clinical manifestations.When comparing the TGA curves of APS patients, asymptomatic aPL positive (aPL+) subjects, HDs and controls treated with VKA, we observed that aPL+ patients (particularly those with a confirmed diagnosis of APS) showed a characteristic profile.Differences among groups were confirmed also when comparing APS clinical manifestations. When comparing Group 1 and Group 4 we found significant differences with respect to the number of thrombotic events (21vs.15,pWhen analysing the cumulative frequency of extra-criteria APS manifestations, Group 1,2 and 3 were comparable, while comparing Group 3 and those positive for aβ2GPI and/or aPS/PT IgM at low titres (Group 4) we found a statistically significant difference (71%vs13%,pConclusion:TGA seems a valuable approach to stratify aPL+ patients according to their risk profile. The differences among groups and different populations of autoantibodies specificities obtained from this test can be considered a translational validation of the increased thrombotic risk of patients with triple or tetra aPL positivity.References:[1]Miyakis S et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J. Thromb. Haemost. 2006;4(2):295–306.Table 1.“Classical” and “Extra-criteria” APS clinical manifestations of each group (considering solely patients with a confirmed diagnosis of APS).Group 1: isolated aPS/PT+ IgG/IgM (16)Group 2: isolated aβ2GPI+ IgG/IgM(20)Group 3: aPS/PT+ and aβ2GPI+ IgG/IgM (24)Group 4: aPS/PT+ and/or aβ2GPI+ IgM low titres (15)APS clinical manifestationsThrombosis (Y/N), n (%)14 (87,5%)16 (80%)21 (87,5%)13 (86,6%)N of thrombotic events21233615Arterial thrombosis (Y/N), n (%)10 (62,5%)9 (45%)14 (58,3%)11 (73,3%)Arterial events, n13102112Venous thrombosis (Y/N), n (%)6 (37,5%)10 (50%)11 (45,8%)3 (20%)Venous events, n913153Pregnancy morbidity, n (%)3 (18,8%)4 (20%)3 (12,5%)2 (13,3%)Recurrent thrombosis (Y/N), n (%)3 (18,8%)3 (15%)7 (29,1%)0Livedo reticularis, n (%)1 (6,2%)1 (5%)4 (16,6%)0Thrombocytopenia, n (%)4 (25%)4 (20%)6 (25%)2 (13,3%)Valvular, n (%)1 (6,2%)02 (8,3%)0Peripheral artery disease, n (%)03 (15%)4 (16,6%)0Diffuse alveolar hemorrhage, n (%)001 (4,1%)0Figure 1.Graphical representation of the differences between TGA and LA profiles between groups.Disclosure of Interests:None declared

Background: A beneficial effect of rituximab (RTX) on focal segmental glomerulosclerosis (FSGS) in pediatric patients or in transplant recipients has been reported in isolated cases. However, the use of RTX in adult patients with idiopathic FSGS needs further investigation. Methods: Eight patients who had biopsy-proven FSGS (63.9 ± 14.0, range 40-81 years, 4 women, 4 men) with major risk factors precluding corticosteroids or conventional immunosuppression were treated with a high dose of RTX (8 weekly doses of 375 mg/m2) and prospectively followed up for at least 2 years (29.1 ± 8.8 months, range 24-42 months). Results: RTX failed to improve proteinuria in 7 out of 8 patients, who had persistent nephrotic proteinuria. In one case, a rapidly deteriorating renal function was also observed. Only one patient showed an improvement in renal function and a remarkable reduction in proteinuria. There were no differences in clinical or laboratory characteristics or in the CD20 B lymphocyte count after RTX between the responder and the 7 nonresponder patients. Conclusions: Only a minority (1 of 8) in our series of adult patients with FSGS showed positive effects of high doses of RTX. Future studies are warranted to investigate more promising therapeutic options in the management of FSGS.

Introduction: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by highly heterogeneous clinical manifestations and a multi systemic involvement. SLE is prone to relapses and remissions, which makes the management challenging. Moreover, conventional therapies may have considerable side-effects, which highlights the demand for new therapeutic strategies. The progress towards a better understanding of the underlying pathogenesis has led to the development of biological therapies targeted directly against crucial molecular mediators of SLE.Areas covered: In this review we analyzed available biological therapies, phase II and III trials and upcoming new therapies that are currently under development for SLE treatment.Expert opinion: The complex pathogenesis of SLE is far from being fully understood. Evidence towards a better understanding of the pathogenic pathways, the cellular and molecular mediators involved in SLE are emerging. Henceforth, new biological therapeutic ...

Patients with congenital adrenal hyperplasia (CAH) due to 21 hydroxylase deficiency present a strong genotype-phenotype correlation but variability in clinical features among patients carrying similar CYP21A2 mutations suggests that polygenic traits contribute to the phenotype. Aim of the present study is to evaluate whether polymorphisms in P450 oxoreductase (POR), a microsomal cofactor to 21 hydroxylase, contribute to the phenotype of patients with CYP21A2 mutations. Methods: Leukocyte DNA from 97 patients with CAH (49 classic, 48 non classic) due to CYP21A2 mutations and 43 control subjects were sequenced for POR, gene variants identified and their association with clinical features 21-hydroxylase deficiency assessed. Results: Several intronic and exonic variants in POR were identified, all in Hardy-Weinberg equilibrium in controls. Interestingly, patients with classic CAH carrying the rs2286822 polymorphism (CT/TT vs CC in intron 11) presented more severe Prader stage (81 vs 67%, p

Background Systemic lupus erythematosus (SLE) is a complex and potentially life-threatening autoimmune disease characterized by a wide range of manifestations. Renal involvement represents a major cause of morbidity and mortality and lupus nephritis (LN) remains a major therapeutic challenge.1 Therefore, selecting appropriate outcome measures for clinical trials represent a crucial step in order to identify appropriate drug regimens for LN treatment. The aim of this study was to systematically review the available literature to compare renal primary outcome measures used in randomized controlled trials (RCTs) for both the induction and the maintenance therapy in biopsy proven LN setting. Methods A detailed literature search was applied a priori to Ovid-MEDLINE (2000–2018),clinicaltrail.gov and to abstracts from the EULAR and ACR/ARHP Annual Meetings (table 1). Results A total of 3922 patients with LN were identified from 28 RCTs. Mean follow-up period was 17.3±15.9 months. Patients were distributed, based on the presence of active biopsy-proven LN, as follow: 717 (23.6%) patients presented class III LN, 1681 (55.3%) class IV, 448 (14.7%) class V, and 195 (6.4%) class IV+V. Twenty-three RCTs (92%) were designed to investigate induction therapy for active LN, while 5 (8%) for maintenance. Twenty-five out of 28 (89.3%) trials considered ‘complete remission’ (CR) as the primary outcome, and 3 (10.7%) studies used ‘time to renal flare/time to treatment failure’ as primary endpoint. When analyzing the definition of CR based on the inclusion of urine protein excretion, renal function (including serum creatinine, serum albumine, glomerular filtration rate, and/or estimated glomerular filtration rate), urinary sediment, and the SELENA-SLEDAI, we found that only 1 (4%) trial considered all these variables, 13 (52%) comprehended 3 items, 9 (36%) 2 items, and 2 (8%) included only one variable. In detail, protein excretion was included as a variable computed to define CR in 23 (92%) studies (with range of UPCR raging from 3.2 to 0.3), renal function in 22 (88%), urinary sediment in 13 (52%), and SELENA-SLEDAI in 2 (8%) of the analyzed trials. Conclusions We report an overall heterogeneity of the primary endpoints used in RTC in LN patients. With the armamentarium of treatments for LN potentially expanding, a better homogeneity in the choice of clinical outcomes is warranted to improve the comparability of the results, potentially leading to a more personalized approach to LN management. Funding Source(s): None Reference Houssiau FA, Lauwerys BR. Current management of lupus nephritis. BestPract Res Clin Rheumatol 2013;27:31928.

As in many autoimmune diseases, the pathogenesis of the antiphospholipid syndrome (APS) is the result of a complex interplay between predisposing genes and triggering environmental factors, leading to a loss of self-tolerance and immune-mediated tissue damage. While the first genetic studies in APS focused primarily on the human leukocytes antigen system (HLA) region, more recent data highlighted the role of other genes in APS susceptibility, including those involved in the immune response and in the hemostatic process. In order to join this intriguing debate, we analyzed the single-nucleotide polymorphisms (SNPs) derived from the whole exome sequencing (WES) of two siblings affected by APS and compared our findings with the available literature. We identified genes encoding proteins involved in the hemostatic process, the immune response, and the phospholipid metabolism (PLA2G6, HSPG2, BCL3, ZFAT, ATP2B2, CRTC3, and ADCY3) of potential interest when debating the pathogenesis of the syndrome. The study of the selected SNPs in a larger cohort of APS patients and the integration of WES results with the network-based approaches will help decipher the genetic risk factors involved in the diverse clinical features of APS.

B cells (BC) play a critical role in systemic lupus erythematosus (SLE). BC depletion therapy still remains an attractive option, despite the disappointing results of randomized controlled trials (RTCs).Twelve patients with SLE [3 males, mean age 43.8 yrs (25-55)] with severe multiorgan involvement all including kidney (3 patients with Class IV, 4 with Class III/V and 5 with Class V, according to the International Society of Nephrology/Renal Pathology Society glomerulonephritis classification), skin lesions [10], severe polyarthralgias with arthritis [10], polyserositis [2], and lymphadenopathy [5] have been prospectively treated with an intensified B cell depletion therapy (IBCDT) protocol due to their resistance or intolerance to previous therapy (six cases) or as a front line immunosuppressive treatment in 6 women with unsatisfactory therapeutic compliance or as a specific request of a short-time immunosuppression for gestational perspectives.Rituximab (RTX) 375 mg/sm on days 1, 8, 15, 22, and 2 more doses after 1 and 2 months, associated with 2 IV administrations of 10mg/kg of cyclophosphamide and 3 methylprednisolone pulses (15mg/kg) followed by oral prednisone (0.8 mg/kg/day, rapidly tapered to 5mg/day by the end of the 3rd month after RTX). No further immunosuppressive maintenance therapy has been given.Patients had been followed-up for a mean of 44.5 (24-93)months. Significant decreases (p0.05) were found in the levels of ESR (baseline mean value: 55.0mm; 3 months: 36; end of follow-up: 13), anti-dsDNA antibodies (baseline: 185 U; 3 months: 107; end of follow-up: 15), and proteinuria (baseline: 4.9 g/24h; 3 months: 0.97; end of follow-up: 0.22). C4 values (baseline 11 mg/dl) significantly increased (p0.05) after 3 months (22 mg/dl) and at the end of the follow-up (20mg/dl). Of the 12 patients, 9 (75%) have remained well after one cycle of IBCDT, with no flare (mean 51.6 months [25-93]). Three patients relapsed after 36, 41, and 72 months, respectively. Following re-treatment, they again showed complete remission over 18-48 months of observation.A promising role of RTX in an intensified protocol of induction therapy can be envisaged in patients for whom avoiding immunosuppressive maintenance therapy and sparing steroids are particularly appealing. Moreover, our data confirm in one of the longest follow-up available, the opportunity to reconsider the regimens of BL depletion in the treatment of the most severe or refractory forms of SLE despite the disappointing results of RCTs.

Rheumatoid Arthritis (RA) is a complex systemic autoimmune disease in which various cell types are involved. Among them, neutrophils have been recognized as important players in the onset and the progression of RA. The pathogenic role of neutrophils in RA lies in the alteration of several processes, including increased cell survival and migratory capacity, abnormal inflammatory activity, elevated oxidative stress and an exacerbated release of neutrophil extracellular traps. Through these mechanisms, neutrophils can activate other immune cells, thus perpetuating inflammation and leading to the destruction of the cartilage and bone of the affected joint. Given the considerable contribution of neutrophils to the pathophysiology of RA, several studies have attempted to clarify the effects of various therapeutic agents on this subtype of leukocyte. To date, recent studies have envisaged the role of new molecules on the pathogenic profile of neutrophils in RA, which could represent novel targets in future therapies. In this review, we aim to review the pathogenic role of neutrophils in RA, the effect of conventional treatments and biologic therapies, and the new, potential targets of neutrophil-derived molecules for the treatment of RA.

Minimal change disease (MCD) accounts for 15% of adult nephrotic syndrome (NS) cases. Adult-MCD patients may have more severe clinical features than pediatric patients. In children, Rituximab (RTX) has been used since 2006 to treat frequently relapsing NS. In adults, data about the efficacy of RTX for MCD are limited. We report our experience on the use of RTX in adult biopsy-proven MCD. Our series includes 6 adult patients (2 males and 4 females), age 45-73 years, treated with RTX (4 weekly doses of 375 mg/m2). Proteinuria decreased from 11,2 (23-4.8) g/24 hours to 0.6 (0-2) g/24 hours after 6 months, and to 0.4 (0-1, 4) g/24 h in the 4 pts with the longer follow-up. Creatinine decreased from 1.95 (0.5-5) mg/dl to 0.88 (0.6-1.3) mg/l. Five patients achieved a complete renal remission, while in 1 pt proteinuria decreased by 75%. RTX successfully depleted CD19 lymphocytes in 100% of pts for at least 6 months. No clinically relevant adverse events have been observed. This case series shows a remarkable efficacy of RTX in treatment of MCD. RTX can be an attractive alternative both in recurrent forms and in induction-therapy of MCD. RTX may be preferentially used in patients at a high risk of development of the adverse effects of corticosteroids and should be considered as an alternative option in patients with recurrent NS. Additional data are needed to inform clinical practice on how best to use RTX in this patient population, so that definitive randomized trials can be planned.

The immune system is delegated to defend the body from attacks from outside or inside. Many diseases can affect immune system reducing its ability to defend self or inducing an abnormal response against external or internal antigens. Rare diseases affecting immune system present some issue in common with other rare diseases and some peculiarities due to the huge variability in the disease's expression. However, a correct estimation of the epidemiology of rare disorders is necessary for evaluating the prognosis and the responses to new therapies, for planning proper public health services, and finally to establish fair and sustainable prices for innovative medicines. Due to the enormous number of different rare immunological diseases, in this chapter we are going to analyse some of them that can be considered paradigmatic of the various expressions of disease.

Background Salivary and serum levels of interleukin-6 (IL-6) and interleukin-8 (IL-8) have previously been studied in oral cancer with conflicting results. Methods We designed a controlled study to assess the correlation between pretreatment salivary and serum levels of IL-6 and IL-8, and all-cause survival and cancer recurrence in oral cancer patients. Results Fifty-two oral cancer patients and 52 healthy control cases were selected. In univariate analysis, salivary IL-6 and IL-8 seemed to be more expressed in cases (pConclusions To date, this is the largest prospective controlled study that has analyzed the pretreatment salivary and serum levels of IL-6 and IL-8 in oral cancer patients, suggesting salivary IL-6 as a possible prognostic biomarker. But further validation in a larger sample is still necessary.

Bloodstream infection (BSI) and associated sepsis represent a major source of mortality in industrialized countries. Prompt treatment with targeted antibiotics affects both the financial impact and the clinical outcome of BSI: every hour gained in initiating the correct antimicrobial therapy significantly increases the probability of patient survival. However, the current standard-of-care, which depends on blood culture-based diagnosis, are often unable to provide such a fast response. Fast and sensitive molecular techniques for the detection of sepsis-related pathogens from primary blood samples are strongly needed. The aim of this study was to assess the usefulness of the IRIDICA BAC BSI Assay, a PCR/ESI-MS-based technology for the early diagnosis of bloodstream infections from primary blood samples in critical patients. This evaluation has been performed by comparison with the traditional culture-based methods. The study was performed on a total of 300 prospective whole blood specimens obtained from patients suspected of sepsis, admitted to enrolling ER units from The Greater Romagna Area. The overall concordance between the two techniques was of 86%, with a calculated sensitivity of 76% and an assay specificity of 90%. The clinical significance of discrepant results was evaluated reviewing the patients' clinical records and the results of additional relevant microbiological tests. The data here obtained support the ability of the IRIDICA BAC BSI Assay to identify a broad range of bacteria directly from primary whole blood samples, within eight hours. This might allow a timely administration of a suitable treatment.

Objectives We aim to evaluate the safety of performing percutaneous native kidney biopsy (PKB) as an outpatient procedure (implying an observation period of 6 hours) compared with the traditional inpatient policy. Design, setting, participants and measurements Group I, in whom PKB was performed in the outpatient department (2012–2016) and followed by 6 hours’ observation period and then by regular outpatient visits and group II, in whom PKB was performed and followed by at least 1 day hospital admission. Group II included retrospectively retrieved patients who underwent PKB in our Institution between January 2000 and November 2012 as an inpatient procedure. All biopsies were performed by a single nephrologist following a structured protocol. Results 462 biopsies were reviewed, 210 (45.5%) of patients were women and the mean age was 54.7±17.9 years. One hundred and twenty-nine (27.9%) of these biopsies were performed in outpatients. A total of 36 (7.8%) of patients developed a complication, and of those, 9 (1.9%) suffered for a major complication (arteriovenous fistula (six cases, 1.2%), ischaemic stroke (2; 0.4%), thromboembolic pulmonary embolism (1; 0.2%)) and 27 (5.8%) for minor(macroscopic haematuria (12 cases, 2.6%), haematomas on sonography not requiring intervention (15 cases, 3.2%)). When comparing the complication rate between groups I and II, no statical difference was observed. When analysing together both groups, after multivariate analysis, serum creatinine >3 mg/dL (OR 2.03, 95% CI 1.18 to 6.81) and known severe hypertension (OR 2.01, 95% CI 1.2 to 4.7) were found to be independent risk factors for minor and major complications, respectively. Conversely, we found no association of risk with the number of biopsy passes, gender, age, diagnosis, presence of haematuria before the kidney biopsy nor the degree of proteinuria. Conclusions Outpatient biopsy could be a valuable, safe and perhaps cost-effective method of obtaining diagnostic renal tissue in the majority of patients.

Background Renal involvement is common in patients with systemic autoimmune conditions, mainly systemic lupus erythematosus (SLE) and vasculitis, including cryoglobulinemia. Despite the advances in percutaneous kidney biopsy (PKB) techniques and overall improved safety of the procedure, clinically significant bleeding complications do occur. Objectives to investigate the safety of performing percutaneous native kidney biopsy (PKB) as an outpatient procedure (implying an observation period of 6 hrs) compared to the traditional inpatient policy in patients with systemic autoimmune conditions. Methods Group I, in whom PKB was performed in the outpatient department (2012–2016) and followed by 6 hours9 observation period and then by regular outpatient visits and group II, in whom PKB was performed and followed by at least 1-day hospital admission. Group II included retrospectively retrieved patients who underwent PKB in our Institution between January 2000 and November 2012 as in patient procedure. All biopsies were performed by a single nephrologist following a structured protocol. Results A total of 81 biopsies (group I and group II) were included in this study, 44 (54%) of patients were female and the mean age was 49.9±17.6 years. Twenty-six per cent of biopsies were performed for the diagnostic workup of nephrotic range proteinuria, 21% for rapidly progressive renal insufficiency, and the remaining 53% for non-nephrotic proteinuria and/or hematuria. No patient suffered for a major complication and only 3 (3.7%) patients (one with cryoglobulinemic vasculitis and 2 with ANCA associated vasculits) developed a minor complication, including gross hematuria in one case and sub-capsular perinephric hematoma on sonography not requiring intervention in 2 patients Conclusions The lack of major complications and the very limited rate of minor bleeding support that outpatient biopsy could be a valuable, safe, and perhaps cost-effective method of obtaining diagnostic renal tissue in the majority of patients with systemic autoimmune diseases. Disclosure of Interest None declared

17α-Hydroxylase deficiency is an uncommon type of congenital adrenal hyperplasia (CAH) caused by mutations in the CYP17A1 gene encoding both 17α-hydroxylase and 17,20-lyase, essential for sex steroids production. Main clinical features include lack of pubertal development, hypertension, and hypokalemia. We report the first case of a 46,XX female homozygote for the p.Glu331del mutation in the CYP17A1 gene showing an atypical clinical presentation. She was evaluated the first time for primary amenorrhea and delayed puberty in the presence of low levels of androgens, 17β-estradiol, serum cortisol, and high levels of progesterone and gonadotropins. After puberty, the patient did not show hypocortisolism and/or hypertension. She started estrogen therapy for pubertal induction, followed by ethinylestradiol/gestodene with clinical and biochemical stability during the follow-up period. At the age of 40 years, she developed hypokalemia and clinical signs of hypocortisolism. Oral corticosteroid treatment was started showing a prompt clinical improvement. Modeling analysis predicted the main outcome of the E331 deletion to impair cytochrome b5 binding, according to a major effect on the enzyme’s lyase activity. These data broaden the molecular and clinical spectrum of CAH caused by 17α-hydroxylase deficiency and adds to current genotype–phenotype correlations.

// Dario Roccatello 1,2,* , Savino Sciascia 1,2,* , Daniela Rossi 1 , Laura Solfietti 1 , Roberta Fenoglio 2 , Elisa Menegatti 1 and Simone Baldovino 1 1 Department of Clinical and Biological Sciences, Center of Research of Immunopathology and Rare Diseases, Coordinating Center of the Network for Rare Diseases of Piedmont and Aosta Valley, S. Giovanni Bosco Hospital and University of Turin, Turin, Italy 2 Nephrology and Dialysis Unit, S. Giovanni Bosco Hospital and University of Turin, Turin, Italy * These authors have equally contributed to this manuscript Correspondence to: Dario Roccatello, email: // Keywords : mixed cryoglobulinemia, HCV-associated cryoglobulinemic vasculitis, HCV associated membranoproliferative glomerulonephritis, polyneuropathies, necrotizing skin ulcers Received : October 07, 2016 Accepted : March 09, 2017 Published : April 09, 2017 Abstract Mixed cryoglobulinemia syndrome (MC) is a systemic vasculitis involving kidneys, joints, skin, and peripheral nerves. While many autoimmune, lymphoproliferative, and neoplastic disorders have been associated with this disorder, hepatitis C virus (HCV) is known to be the etiologic agent in the majority of patients. Therefore, clinical research has focused on anti-viral drugs and, more recently, on the new, highly potent Direct-acting Antiviral Agents (DAAs). These drugs assure sustained virologic response (SVR) rates >90%. Nevertheless, data on their efficacy in patients with HCV-associated cryoglobulinemic vasculitis are disappointing, possibly due to the inability of the drugs to suppress the immune-mediated process once it has been triggered. Despite the potential risk of exacerbation of the infection, immunosuppression has traditionally been regarded as the first-line intervention in cryoglobulinemic vasculitis, especially if renal involvement is severe. Biologic agents have raised hopes for more manageable therapeutic approaches, and Rituximab (RTX), an anti CD20 monoclonal antibody, is the most widely used biologic drug. It has proved to be safer than conventional immunosuppressants, thus substantially changing the natural history of HCV-associated cryoglobulinemic vasculitis by providing long-term remission, especially with intensive regimens. The present review focuses on the new therapeutic opportunities offered by the combination of biological drugs, mainly Rituximab, with DAAs.

The immune system is delegated to defend the body from attacks from outside or inside. Many diseases can affect immune system reducing its ability to defend self or inducing an abnormal response against external or internal antigens. Rare diseases affecting immune system present some issue in common with other rare diseases and some peculiarities due to the huge variability in the disease’s expression. However, a correct estimation of the epidemiology of rare disorders is necessary for evaluating the prognosis and the responses to new therapies, for planning proper public health services, and finally to establish fair and sustainable prices for innovative medicines. Due to the enormous number of different rare immunological diseases, in this chapter we are going to analyse some of them that can be considered paradigmatic of the various expressions of disease.

// Dario Roccatello 1,2,* , Savino Sciascia 1,2,* , Daniela Rossi 1 , Mirella Alpa 1 , Carla Naretto 1 , Massimo Radin 1 , Roberta Fenoglio 2 , Simone Baldovino 1,2 and Elisa Menegatti 1 1 Department of Clinical and Biological Sciences, Center of Research of Immunopathology and Rare Diseases, Coordinating Center of the Network for Rare Diseases of Piedmont and Aosta Valley, Department of Clinical and Biological Sciences, University of Turin, Turin, Italy 2 Nephrology and Dialysis Unit, S. Giovanni Bosco Hospital and University of Turin, Turin, Italy * These authors have equally contributed to this manuscript Correspondence to: Dario Roccatello, email: // Keywords : rituximab, systemic vasculitis, ANCA-associated vasculitis, granulomatosis with polyangiitis, microscopic polyangiitis, Immunology and Microbiology Section, Immune response, Immunity Received : March 10, 2017 Accepted : April 19, 2017 Published : May 23, 2017 Abstract Background: ANCA associated vasculitides (AAV) often present with a chronic relapsing course. Relapse leads to increased immunosuppressive exposure and consequent toxicity. While two randomized controlled trials have shown rituximab (RTX) to be the most effective induction treatment in patients with relapsing disease, the optimal treatment duration and RTX dose remain debated. Whether to administer a maintenance dose to every patient, at a fixed time interval or on the basis of B cell count and ANCA titre or only when disease manifestations do occur is still debated as well. Methods: 11 patients (5 with granulomatosis with polyangiitis, 4 with microscopic polyangiitis-MPA-, and 2 with eosinophilic granulomatosis with polyangiitis -EGPA-) intolerant or refractory to conventional therapies including cyclophosphamide were enrolled. All patients received the so called “improved 4+2” RTX scheme as a rescue therapy. Following RTX administration, immunosuppressive drugs were rapidly tapered and no immunosuppressive maintenance therapy had been given. Results: After a mean follow-up of 85 months since the “4+2” RTX protocol: four out of 11 patients (37%, 1 EGPA and 3 MPA, all MPO-positive) remained in remission after one cycle of “4+2” RTX infusion protocol with no relapse for a median 66 months [60-108]). Seven relapsing patients were re-treated once with RTX (again as monotherapy with the same protocol) after a median of 54 months (24-96). Following re-treatment, they again showed complete remission over a median of 32 months (12-96) of observation. Conclusion: In one of the longest-term observation (85 months) studies, sustained clinical remission without immunosuppressive maintenance therapy (and a negligible dose of prednisone since the 5 th month) was obtained by the “4 + 2” RTX infusion protocol in patients with ANCA-associated vasculitis intolerant or refractory to conventional therapy.

Idiopathic membranous nephropathy (MN) is a common immune-mediated glomerular disease and the main cause of nephrotic syndrome (NS) in Caucasian adults. Rituximab (RTX) has been reported to safely reduce proteinuria in patients with primary MN and severe NS. However, the effects of RTX treatment on T-cells including regulatory T-cells (Treg) in MN have not been fully determined.Seventeen patients [mean age 67 (29-86) years, 6 women, 11 men] with biopsy-proven MN, and persistent proteinuria3.5 g/24h were prospectively enrolled and received RTX, 375 mg/m(2) (iv) on days 1, 8, 15 and 22. Changes in circulating B and T cell homeostasis were examined in the peripheral blood by flow-cytometry studies; serum levels of IL-35 were measured using a high-sensitivity ELISA kits (baseline, at month 3, 6, 9 and 12).Patients had been followed-up for a mean of 36.3 months (24-48). Proteinuria decreased from 5.6 (3.5-8) g/24h to 2.4 (0.06-13) g/24h at 6 months (p0.05) and to 1.3 (0.06-8) at 12 months (p0.01), respectively after therapy with RTX. Four patients received a 2nd course of RTX (one at 6 months because of persistent NS, and three at 12, 18, or 30 months for relapse). The three relapsing patients became proteinuria-free (0.5 g/24h) in the following 6 months. Serum creatinine remained stable during the follow-up: median 1mg/dl (0.7-1.6) at 12 months and 1.1 (0.7-1.7) at 24 months as compared to 1 (0.5-2.4) at baseline. At 6 months after RTX, complete remission (CR) was observed in 7 patients, partial remission (PR) in 4, while 6 were non responders (NR) non responder (NR). At the end of the follow-up, 14 patients were in CR, 1 in PR, while 2 were NR. In the T-cell compartment, upon detection of B cell depletion, there was an increase in Treg up to 10-fold when comparing baseline and at month 12 (mean ± SD 1.2 ± 0.6%, and 5.8 ± 0.7% p=0.02, respectively). When stratifying patients in responders (CR+PR) and NRs at month 12, we observed a significant increase in Treg cells from month 6 which persisted till 12 months only in the responder group (5.5 ± 0.6% and 1.1 ± 0.6%, p=0.04, respectively in responders and NRs). A statistically significant decrease in the levels of active T-lymphocytes (HLA-DR+CD8+ cells) was observed, with a maximum reached at 12 months after treatment with RTX [6 ± 1.1% baseline, 4.7 ± 1.7% at 6 months (p=0.043) and 1.5 ± 1.4% at 12 months (p=0.05)]. A marked increase in IL-35 levels [defined as delta40% (serum values at 6 months minus baseline values)] was seen in 68% of the patients who achieved clinical response (CR or PR) at 12 month, but in none of the patients who failed to respond (p=0.034).Our findings and data from literature support the idea that RTX can be envisaged as a first-line therapy for patients at risk of progression because of persistent NS due to idiopathic MN. Insights into the putative T cell-related mechanisms of action have been discussed.