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This chapter describes the principles for selection of antigenic peptides for the development of anti-peptide antibodies suitable for microarray-based multiplex affinity assays and optional mass spectrometry detection. The methods described here are mostly applicable to small- and medium-scale multiplex affinity assay and microarrays. Although the same principles of peptide selection may also be applied to larger-scale arrays (with 100+ features), informatics software and printing methods may well differ. Due to the sheer number of proteins/peptides to be processed and analyzed, dedicated software with high processing capacity and enterprise-level array robotics may be required for larger-scale efforts. This report aims to provide practical advice to those seeking to develop or use arrays with up to ~100 different peptide or protein features.

Background Fluoroquinolones have been associated with increased risk of tendinopathy and Achilles tendon rupture (ATR), especially in patients over 60 years of age. Methods A retrospective study was carried out including patients over 60 years of age with ATR attended in our centre over the period 2000–2017. Results We identified 44 patients with RTA, of whom 18% (8/44) had been previously treated with fluoroquinolones, with a mean age at diagnosis of ATR of 77.37 years and concomitant corticotherapy in 4 of them. In 7 patients, the rupture was spontaneous and all required surgical management. A significantly higher frequency of smoking, concomitant corticotherapy and spontaneous ruptures were found in the group treated with fluoroquinolones. Conclusions ATR is an adverse event that can occur in patients over 60 years of age treated with fluoroquinolones, so an adequate risk-benefit assessment should be carried out in this population, especially in the presence of associated risk factors.

espanolIntroduccion Las fluoroquinolonas han sido asociadas con aumento del riesgo de tendinopatia y rotura del tendon de Aquiles (RTA), especialmente en pacientes mayores de 60 anos. Metodos Se llevo a cabo un estudio retrospectivo en el que se incluyo a los pacientes mayores de 60 anos con RTA atendidos en nuestro centro durante el periodo 2000-2017. Resultados Se identifico a 44 pacientes con RTA, de los cuales 18% (8/44) habian sido tratados previamente con fluoroquinolonas, con una media de edad al diagnostico de RTA de 77,37 anos y corticoterapia concomitante en 4 de ellos. En 7 pacientes la rotura fue espontanea y todas requirieron tratamiento quirurgico. Se encontro una frecuencia significativamente mayor de tabaquismo, corticoterapia concomitante y roturas espontaneas en el grupo tratado con fluoroquinolonas. Conclusiones La RTA es un evento adverso que puede ocurrir en pacientes mayores de 60 anos tratados con fluoroquinolonas, por lo que deberia realizarse una adecuada evaluacion relacion riesgo-beneficio en esta poblacion, especialmente en presencia de factores de riesgo asociados. EnglishBackground Fluoroquinolones have been associated with increased risk of tendinopathy and Achilles tendon rupture (ATR), especially in patients over 60 years of age. Methods A retrospective study was carried out including patients over 60 years of age with ATR attended in our centre over the period 2000-2017. Results We identified 44 patients with RTA, of whom 18% (8/44) had been previously treated with fluoroquinolones, with a mean age at diagnosis of ATR of 77.37 years and concomitant corticotherapy in 4 of them. In 7 patients, the rupture was spontaneous and all required surgical management. A significantly higher frequency of smoking, concomitant corticotherapy and spontaneous ruptures were found in the group treated with fluoroquinolones. Conclusions ATR is an adverse event that can occur in patients over 60 years of age treated with fluoroquinolones, so an adequate risk-benefit assessment should be carried out in this population, especially in the presence of associated risk factors.

The solubility of saturated fatty acids is essential for the design and operation of separation and purification units, since crystallization is the main method used in these cases. In this work, differential scanning calorimetry (DSC) was used to determine the solubility of lauric, palmitic, and stearic acid in alcoholic solvents (ethanol, n-propanol and 2-propanol). Comparison with literature data confirmed the efficacy and precision of the DSC technique. For the three saturated fatty acids evaluated, the solubility was highest in 2-propanol and lowest in ethanol. Activity coefficient models (Margules, Wilson, and nonrandom two-liquids models) were correlated with the experimental data, showing good agreement.

We compared the diagnostic performance of the standard method (Kato-Katz) with two recently developed methods (Mini-FLOTAC and Flukefinder) for the detection and quantification of Fasciola hepatica eggs in human stool. Uninfected human stool samples were artificially spiked with F. hepatica eggs to reach final concentrations of 14, 28, 41, or 96 eggs per gram of stool (epg). Only Flukefinder showed 100% sensitivity in all but the samples with the lowest concentration of eggs (14 epg), in which it had a sensitivity of 60%. Each of the methods underestimated the true fecal egg counts (FECs), Flukefinder resulting in the most biased egg counts (egg counts 0.18 times lower than the expected FECs). Only the Flukefinder resulted in more precise results (coefficient of variance < 30%) from FECs of 96 epg onward. The outcome of this study indicates that the Flukefinder is a useful alternative diagnostic method for human fascioliasis in stool.

Liquid–liquid equilibrium (LLE) data were obtained for polyethylene glycol (PEG)-salt aqueous two-phase system (ATPS) at 298.15, 318.15 and 338.15 K. Two salts, copper sulfate and manganese sulfate, and PEG with different molecular weights, 2 000, 4000 (manganese sulfate only), and 6 000, were used. The effects of the molecular weight of PEG and temperature on phase separation and the salting-out effect were studied. For all aqueous two-phase systems, the increase in the polymer molecular weight provides a larger biphasic region. The temperature promoted an increase in the biphasic region, and the slope of tie-lines tended to decrease with increasing temperature. The Setschenow-type equation was used to evaluate the salting-out effect of MnSO4 and CuSO4, as well as the temperature and molecular weight of the PEG influence on the phase formation. The rank order of salting-out coefficients is Mn2+ > Cu+2 since the cation Mn2+ is more kosmotropic than Cu2+. NRTL and UNIQUAC models have been used to fit the 18 experimental LLE data. The interaction parameters of the models were estimated. The results show that the quality of fitting is better with the UNIQUAC model.

Background: Patients with Rheumatoid Arthritis (RA) have been traditionally treated with other TNF inhibitors (TNFis) after the withdrawal of a first TNFi (cycling). However, due to the increase in the biological therapy options, changing to a biologic agent with different mechanism of action (switching) is an alternative choice. More studies are needed to clarify the role of both strategies in order to guide treatment decisions. Objectives: To analyze the survival of the second biological drug, TNFi or biologic with a different mechanism of action, in patients with RA non-responders to the first TNFi. To evaluate factors associated with the survival of the second-line therapy. Methods: A retrospective, longitudinal, observational study was performed, which included patients diagnosed of RA and treated with biological therapies between 2008 and 2017, who discontinue a first-line TNFi and started a second-line biological therapy. The demographic and clinical data were obtained from their medical records. Kaplan Meier and Log-rank survival analysis were performed, as well as Cox regression to identify related factors. Results: 69 patients were identified, 14 men (20.3%) with a mean age at the beginning of the treatment of 52.68 ± 19.79 years. Demographic and clinical data are shown in the table. The main cause of withdrawal of the first TNFi was secondary failure (47.8%), followed by side effects (34.8%) and primary failure (13%). Cycling was performed in 34 patients (49.3%) and switching in 35 (50.7%). During the follow-up, the main causes of withdrawal of the second-line treatment were primary and secondary failure (10.1% in both cases). The survival analysis of the 42 patients who presented a primary or secondary failure to the first TNFi was stratified according to the cause of withdrawal of the second biological therapy. When cycling was chosen, the average treatment survival was 8.8 months (CI95%:4.8-12.8) whereas for the switching option it was 23 months (CI95%:5.4-40.5), being the differences statistically significant (p=0.049). Age was shown as a protective factor (HR=0.86, CI95%:0.76-98) and the positivity of Rheumatoid Factor (RF) as a risk factor (HR=4.76, CI95%:1.39-16.30) for the withdrawal of the second biological therapy. No differences were found in the withdrawal rate due to adverse effects among patients who performed switching or cycling (11.42% vs 5.88%, p=0.41). Conclusion: Switching strategy showed a greater drug survival time and was considered as a better option after the failure of a first TNFi. RF positivity was associated with an increased risk for the withdrawal of the second-line biological therapy. Reference: [1] Todoerti M, Favalli E, Iannone F, Olivieri I, Benucci M, Cauli A, Mathieu A, Santo L, Minisola G, Lapadula G, Bucci R, Gremese E, Caporali R. Switch or swap strategy in rheumatoid arthritis patients failing TNF inhibitors? Results of a modified Italian Expert Consensus. Rheumatology. 2018; 57: vii42–vii53 Disclosure of Interests: None declared

Background Fluoroquinolones (FQ) are a class of broad-spectrum antibiotics whose use has spread as they are considered safe and well tolerated drugs. Among its musculoskeletal side effects, Achilles tendon rupture (ATR) is a well-known complication that can be disabling, arising recent interest from the pharmacovigilance system after evaluating the reported side effects. Objectives To describe the epidemiological and clinical features of patients diagnosed with FQ-associated ATR in a Spanish tertiary hospital. Methods A retrospective observational study was performed, which included all patients older than 60 years who were diagnosed of ATR in our center during the period 2000-2017, identifying patients who had been previously treated with FQ. The demographic, clinical and outcome data were obtained from their medical records. Results During the study period, 44 patients with ATR were identified, 8 (14.6%) of them previously treated with FQ. In this group of patients, the mean age at diagnosis of ATR was 77.37 ± 9.54 years, being male 6 (75%). Four of them (50%) received concomitant treatment with corticosteroids (CS) and one patient had undergone kidney transplantation due to nephroangiosclerosis. Seven patients (87.5%) were treated with Levofloxacin and one case received Ciprofloxacin, all of them orally. The indication for FQ treatment in half of the cases was acute bronchitis and in the other half exacerbations of underlying respiratory pathology (chronic obstructive pulmonary disease and diffuse interstitial lung disease). The mean duration of treatment with FQ was 6.16 ± 2.4 days, while the mean time from the start of treatment to the diagnosis of ATR was 19.25 ± 14.83 days. In seven patients (87.5%) the rupture was spontaneous, while one patient presented traumatic rupture (low impact traumatism). 87.5% of the ruptures were total ruptures and all cases required surgical treatment, without recurrence reported. The comparison of the characteristics of patients with ATR who had or not received treatment with FQ is shown in the table, identifying significant differences in favor of a higher percentage of patients who were smokers, received concomitant treatment with CS and had spontaneous rupture in the group of patients who had received FQ. Conclusion The Achilles tendon is the most frequent location of tendinopathy associated with FQ, being affected in 95% of cases. The risk factors associated to an increased risk to develop FQ-associated ATR includes age over 60 years, male gender, chronic treatment with CS and organ transplantation, all these being present in our cases. Despite being a relatively frequent adverse event, it is underdiagnosed and the risk of ATR is not usually assessed when indicating FQ treatment. It is important to perform a risk/benefit assessment, specially in patients with associated risk factors, because most ruptures are complete and require surgical treatment, and may be a potential cause of disability. References [1] Bidell M, Lodise T. Fluoroquinolone-Associated Tendinopathy: Does Levofloxacin Pose the Greatest Risk?. Pharmacotherapy. 2016;36(6):679-93. [2] Godoy-Santos A, Bruschini H, Cury J, Srougi M, De Cesar N, Fonseca L, Maffulli N. Fluoroquinolones and the Risk of Achilles Tendon Disorders: Update on a Neglected Complication. Urology. 2018;113:20-25. Disclosure of Interests None declared

Background The relationship between subacromial syndrome (SS) and the presence of calcifications in the thickness of the superspinatus tendon is complex. It is undeniable that the presence of a solid body hinders the movement of the rotator cuff through the subacromial space and to some extent contributes to the trapping that conditions pain and functional limitation, but, on the other hand, calcifications in the thickness of the supraspinatus tendon are incidental findings that have no clinical significance or whose presence precedes the painful syndrome in months or years. We understand that in a SS related to calcification, it contributes as an irreducible agent that conditions an inflammatory response of the tendon when it attempts to cross the subacromial space, however, there are no studies available that explain this relationship from a sonoanatomic perspective. Objectives To determine the extent to which the disposition and characteristics of a calcification of the supraspinatus tendon, or of the tendon itself, are modified when an SS is triggered. Methods A quasi-experimental, “before-after” study was conducted with individuals who presented a SS and in whom incidental calcification in the TSE had been previously diagnosed in an ultrasound study. Demographic data and static images from the ultrasound studies of each patient were compared. The explanatory variable was time (before-after) and the response variables in image were: (1) Increased tendon thickness measured at 30mm from the deep edge of the acromion (GROSORB), (2) Increased tendon thickness measured at the height of the middle of the longitudinal axis of the calcification (GROSORC), (3) Longitudinal axis of the calcification (LONGC), (4) Thickness or height of the calcification, when this was measurable (ALTC), and (5) Distance between the calcification and the deep end of the acromion (DISTC). In addition, two indices were determined: (a) Increased tendon thickness in the calcification zone (GROSORC/GROSORB) and (b) proportion of tendon thickness occupied by calcification (ALTC/GROSORC). The measurements were carried out using the software of the equipment in which the original and subsequent study was made (GE Logiq e; Toshiba Nemio XG). Results The “before” ultrasounds were performed between 2012 and 2016 and the “after” ultrasounds between 2015 and 2018. The time fashion between the two ultrasounds for each patient was 2 years. Thirty-three subjects were included, with an average age of 47.5 SD 7.6 years. Proportion of women 66.6%. Proportion of dominant shoulders (51.1%). As shown in table 1, the most significant changes were the thickness of the tendon measured at the height of the middle of the longitudinal axis of calcification and the increase of this measure with respect to the thickness measured at 30mm from the deepest region of acromion (GROSORC/GROSORB). Although the calcification height also showed a statistically significant increase, it could only be measeured in 20 of the 33 subjects. In the correlation tests, the GROSORC/GROSORB index increase correlated strongly with the DISTC (Coef. Pearson 0.686, P Conclusion The most important factor that explains the development of an SS associated with calcification is the increase in tendon thickness at the height of the calcification itself and, at the same time, this measure increases when the calcification is more lateralized and the more elongated its longitudinal diameter. These modifications could have implications when deciding the therapeutic management of these processes. Disclosure of Interests None declared

Background: Systemic Juvenile Idiopathic Arthritis (SJIA) and adult onset Still’s disease are rare diseases of unknown etiology that share several clinical and laboratory features. The pathogenesis of these two diseases is complex and multifactorial, with an important role of innate immunity: activation of neutrophils and macrophages and increased levels of cytokines dependent on the activation of the inflammosome. A proportion of these patients will develop a Macrophage activation syndrome (MAS), a serious and life-threatening complication. Objectives: To identify immunological markers that allow a differential diagnosis between patients with inactive SJIA, active SJIA or MAS. To evaluate if there are differences between biomarkers in SJIA and adult onset Still’s disease Methods: Observational, prospective and multicenter study. Inclusion criteria: patients with SJIA or adult onset Still’s disease, followed in Paediatric or Adult Rheumatology Department from 5 Madrid hospitals. Group 1: Patients with active SJIA or adult onset Still’s disease +/- diagnosis of secondary MAS at the first visit, have been followed-up performing a second clinical and analytical visit at 3 months. Group 2: Patients with inactive SJIA or adult onset Still’s. Clinical and analytical data were collected, PBMCs were obtained before and after treatment, and several leukocyte subtypes were evaluated by flow cytometry (FACS). The intracytoplasmic expression of TNF-α, IL-1b and IL-6 in monocytes was determined by flow cytometry before and after stimulation with lipopolysaccharides (LPS) in the presence of monensin (Golgi inhibitor). In addition, IL-18 levels were determined by ELISA in all visits. Results: A total of 25 patients were included (18 SJIA and 7 adult onset Still’s diseases), 2 of them being excluded from the analysis because exclusions criteria. 30,4% patients presented MAS at the first visit (5 SJIA and 2 adult onset Still’s disease) and other 2 at some time during the disease. The median of IL-18 was 296.1 (CI 108.3-346.0) in patients who have never had MAS and 899.85 (CI: 397.32-1095.6) in the other group. Patients with active MAS had a CD4/CD8 ratio close to the lower limit (1.1 +/- 0.1 SD) reflecting a decrease in the CD4 + population and an increase in CD8 +. On the other hand, the rest of the groups presented a CD4/CD8 ratio of 2.6 +/- 0.5 SD in patients without disease activity and 2.0 +/- 0.7 SD in patients with active SJIA or adult onset Still’s disease but without MAS. No significant differences were found between patients with SJIA or adult onset Still’s disease. Conclusion: The results of this study show that the analysis of three determinations:%CD4,%CD8 and the CD4/CD8 ratio can help in the early detection of patients with MAS secondary to SJIA or adult onset Still’s disease. The determination of IL-18 has been in our study a marker of MAS, similar to several published studies. No significant differences were found in the immunological tests between patients with SJIA and adult onset Still’s disease with active or inactive disease. Reference: [1] Weiss ES et all Interleukin-18 diagnostically distinguishes and pathogenically promotes human and murine macrophage activation syndrome. Blood. 2018 Mar 29;131(13):1442-1455 Disclosure of Interests: None declared

Background: The pathological class of lupus nephritis (LN) may change to a different class during the course of the disease. Renal biopsy is repeated is repeated in many patients during a flare but there is there is no agreement about systematically recommending them because proliferative lesions on their original biopsy rarely switch to a pure nonproliferative nephritis during a flare. However, renal rebiopsy may be useful in some cases to make appropriate adjustments or changes of treatment. Objectives: To analyze the impact of renal rebiopsy on the therapeutic approach in patients with previous histological diagnosis of LN who experience a worsening in the clinical parameters of renal involvement. Methods: Retrospective study of patients with histological diagnosis of NL subjected to at least one renal biopsy. We studied the demographic, clinical, histopathological variables of the first and subsequent renal biopsies, received treatment and the therapeutic modifications in relation to the result of the rebiopsies. Results: We analyzed 35 patients diagnosed with LN between 1978 and 2017. 9 of them had been rebiopsied at least on one occasion and made a total of 11 rebiopsies (7 patients with a rebiopsy and 2 patients with 2 rebiopsies). All patients were female and Caucasian, except for a Hispanic woman, with a mean age at the time of the rebiopsy of 31 ± 12 years (14-55). The mean serum creatinine at the time of the first re-biopsy was 0.8 ± 0.17 mg/dl (0.5-1.06) and in the second, 1.18 ± 0.05 mg/dl (1.15-1.23). The fundamental indication for the rebiopsy was the increase in proteinuria, up to non-nephrotic range in 64% of the patients and within the nephrotic range in 36%. In comparison with the previous biopsy, 3 of the rebiopsies (27%) showed evolution from a non-proliferative to a proliferative form (from II to III, from II to IV and from V to V + IV). 4 of the rebiopsies (36%) started from a proliferative class and changed class but within these forms (3 from IV to III and and 1 from III to IV). The remaining 4 rebiopsies (27%) showed no change in the histological type. Regarding the baseline biopsy, we observed a decrease in the index of activity of the rebiopsies (5.4 ± 2.2 vs 3.4 ± 2.5, p = 0.017) and an increase in the chronicity index (0.8 ± 0.7 vs 2.9 ± 3.2, p = 0.027). In all cases, therapeutic modifications were carried out. In 9 cases (82%) the immunosuppression was increased and in two of them (18%) it was decreased. Conclusion: The repetition of renal biopsy in cases of LN with clinical data of renal deterioration is relevant. The change of histological class and the evolution of activity and chronicity indexes support the decision to increase immunosuppression and are fundamental to diminish it. References [1] Narvaez J, et al. The value of repeat biopsy in lupus nephritis flares. Medicine (Baltimore). 2017;96:e7099. Disclosure of Interests: None declared

Background:Tapering and withdrawal of biological DMARDs (bDMARDs) is often attempted in Juvenile Idiopathic Arthritis (JIA) patients but data about response to re-treatment after an unsuccessful tapering are scarce.Objectives:Our aim is to assess response to re-treatment after a disease flare during tapering or withdrawal of bDMARDs and to describe re-treatment strategies.Methods:A retrospective, descriptive study was conducted in a cohort of JIA patients followed up in a referral hospital and who had received bDMARDs between 2000 and 2019. All JIA patients with at least one flare during tapering or withdrawal were included. Response to re-treatment was defined as achieving Wallace criteria for remission for 6 months.Results:A total of 142 flares during tapering or withdrawal were identified and included. In 3/142 (2.1%) patients bDMARDS were not re-started after a flare, 1 due to patient refusal, 2 due to unplanned pregnancy. Main characteristics according to tapering strategies are reported in Table 1.Table 1.Main characteristics according to tapering strategies. Apso, Psoriatic Arthritis; ERA, Enthesitis related Arthritis; OligE, Oligoarticular Extended; OligP, oligoarticular persistent; Poly RF+, polyarticular rheumatoid factor positive; PolyRF-, polyarticular rheumatoid factor negative; sJIA, Systemic JIA; Und, Undifferenciated.TaperingWithdrawalCases, n (%)80 (57.5)59 (42.4)Female, n (%)57 (71.3)36 (61)Age at diagnosis,years, median (IQR)5 (2-11)4 (2-11)Age at start of tapering or withdrawal, years, median (IQR)17 (11-25)16 (8-29)Categories, n (%)OligP19 (23.8)12 (20.3)OligE15 (18.8)13 (22)sJIA10 (12.5)11 (18.6)ERA12 (15)8 (13.6)Apso7 (8.8)6 (10.2)Poly RF+9 (11.3)4 (6.8)Poly RF-7 (8.8)3 (5.1)Und1 (1.3)2 (3.4)Uveitis, n (%)24 (30)16 (27.1)Laboratory values, n (%)- RF15 (18.8)5 (8.5)- ANA45 (56.3)33 (55.9)cDMARDs at start of tapering, n (%)40 (50)20 (33.9)bDMARD target, n (%)- TNF inhibitors63 (78.8)45 (76.3)- IL6-blockers14 (17.5)5 (8.5)- IL1-blockers2 (2.5)7 (11.9)- Rituximab1 (1.3)2 (3.4)Line of bDMARD, n (%)- First50 (62.5)45 (76.3)- Second18 (22.5)10 (16.9)- Third5 (6.3)2 (3.4)- Forth6 (7.5)2 (3.4)- Fifth1 (1.3)0 (0)Time in bDMARD treatment, months median (IQR)28.4 (13.3-52.5)28.2 (13.9-44.6)Remission prior tapering or withdrawal,months median (IQR)8 (6-12)8 (6-12)Fifty-nine out of 80 (73.8%) tapering cases were on remission at 6 months (6m) and 42/80 (52.5%) at 12 months (12m). Regarding dosage schedule adjustments, interval widening was the most frequent in 70/80 (87.5%) followed by combined strategy in 7/80 (8.8%) and lower dosage in 3/80 (3.8%). Median remaining dosage administrated was 54.9% IQR(50-75). Median time to flare was 14.9 months IQR (5.6-24.7). Median time to re-start of bDMARDs was 0 months min-max(0-13).After withdrawal of bDMARDs 23/59 (39%)cases at 6m and 14/59 (23.7%) cases at 12m were on remission. Median time to flare was 4.7 months IQR (2.8-11.4). Median time to re-start of bDMARDs was 1 month min-max (1-6).Most frequent re-treatment strategies were re-start of standard dosage [tapering 42/80 (52.5%), withdrawal 34/59 (57.6%)], re-start of previous effective dosage [tapering 28/80 (35%), withdrawal 11/59 (18.6%)], change of bDMARD [tapering 6/80 (7.5%), withdrawal 6/59 (10.2), re-start of bDMARD and cDMARD [tapering 4/80 (5%), withdrawal 6/59 (10.2)] and re-start of cDMARD [tapering 0/80 (0), withdrawal 1/59 (1.7%)].Overall response to re-treatment was 75/80 (93.8%) and 53/58 (91.4%) among tapering and withdrawal, respectively. In 12/139 (8.6%) patients bDMARD was changed, achieving remission in 11/12 (91.7%) cases. No factors were associated with response to re-treatment and there were no differences between re-treatment strategies and re-achieving remission (p=0.596).Conclusion:Response to re-treatment was high (92.7%), no risk factors were identified. Most frequent retreatment strategy was re-start of standard bDMARD dosage in 76/139 (54.7%) followed up by restart of previous effective bDMARD dosage in 39/139 (28.1%).Disclosure of Interests:None declared

Background:Biological therapy (BT) has changed the treatment and perspectives of JIA patients but little is known about when is the best moment to start BT and the impact of this prompt iniciation.Objectives:To analyze the response to BT of Juvenile Idiophatic Arthritis (JIA) patients according to the time when the BT was started.Methods:A retrospective, descriptive study was conducted on JIA patients followed up in a referal hospital that started BT up to 24 months after diagnosis from 2000 to 2018. Disease activity was measured, at 2 years after diagnosis, according to Wallace criteria for remission (absence of: active arthritis, active uveitis, fever, rash or any other manifestation attributable to JIA, normal CRP and ESR, PGA indicating no active disease) for at least 6 months.Results:55 JIA patients that started BT up to 24 months from diagnosis were analyzed. 69,1% were girls with a median age at diagnosis of 8 years old IQR(3-13), median age at the start of BT of 9 years old IQR(3-13). Regarding JIA categories: 25,5% were Oligoarticular Persistent (OligP), 18,2% Systemic JIA (sJIA), 16,4% Entesitis related Arthritis (ERA), 12,7% Psoriatic Arthritis (APso) and Polyarticular RF- (PolyRF-), 5,5% Oligoarticular Extended (OligE) and Polyarticular RF+ (PolyRF+), 3,6% Undifferentiated (Und). 20% of patients had uveitis during followup. Conventional DMARD (cDMARD) was indicated in 83,6% of patients (95,7% Methotrexate) at diagnosis [median 0 months IQR(0-2,3)]. At the end of followup (2 years) only 30,9% of patients continued with cDMARDs. The main causes of discontinuation were: adverse events (46,7%), remission (36,7%). TNF inhibitors were precribed in 81,8% of patients and 18,2% of patients recieved two BT during the first 2 years from diagnosis. 54,5% of BT were indicated during the first 6 months from diagnosis, 27,3% from 7 to 12 months, 12,7% from 13 to 18 months, 5,5% from 19 to 24 months.After 2 years from diagnosis, 78,2% of patients were on remission and 21,8% active. Among patients with active disease: 75% had arthritis, 16,7% had uveitis and 8,3% had both. There were no differences regarding disease activity among patients with uveitis and neither taking cDMARDs. Regarding JIA categories: 66,7% of OligE, 57,1% of PolyRF- and 57,1% of APso patients were active at 2 years from diagnosis when compared to the other categories (p=0.004).Patients on remission at 24 months from diagnosis started sooner the BT than active patients [CI 95% (0,46-8,29) p=0,029]. The time when the BT was started was correlated to the activity at 2 years (K= 0,294 p=0,029). When the BT was prescribed after 7,5months from diagnosis it was correlated, in a COR curve, with a higher probability of active disease at 2 years (S= 0,67 E= 0,63). There was a correlation, among patients on remission at 2 years, between prompt start of BT and less time to reach remission (K= -0,345 p=0,024). Patients with active disease at 2 years, regardless of moment of BT iniciation, required more BT during follow-up (p=0,002).Conclusion:Prompt iniciation of BT was correlated with a better outcome. JIA patients that started BT early after diagnosis had a higher probability of remission after 2 years. Starting BT after 7,5 months was correlated with a higher probability of active disease at 2 years. Active disease at 24 months was correlated with persistent active disease during follow-up.Disclosure of Interests:None declared

Background:Juvenile Idiopathic Arthritis (JIA) is a heterogeneous group of pediatric diseases. Different response to biological treatment (BT) has been reported according to disease subtype.Objectives:To analyze the prescription and withdrawal of BT in JIA patients with focus on JIA category.Methods:A retrospective observational study was conducted on JIA patients followed in a referal hospital and who had received at least one BT between 1999 and 2019.Results:130 JIA patients were analyzed: 29 (22,4%) were Oligoarticular Persistent (OligP), 22 (16,9%) Enthesitis related Arthritis (ERA), 20 (15,4%) Systemic (sJIA), 19 (14,6%) Polyarticular RF- (PolyRF-), 14 (10,8%) Polyarticular RF+(PolyRF+), 13 (10%) Oligoarticular-Extended (OligE), 11 (8,4%) Psoriatic Arthritis (APso) and 2 (1,5%) Undifferentiated (Und).The main characteristics are summarized in table 1.The first line BT most frequently indicated was Etanercept up to 40% in all the categories except for ERA, where the most frequent BT was Adalimumab and sJIA, where the most frequent BT was Anakinra. The time between diagnosis and start of BT was different among the categories (p=0,007). In the Und category, the time until BT was the shortest (median: 1 month), since both patients had coxitis, followed by APso [median: 9 months IQR(1-57)] and sJIA [median: 17,5 months IQR(0,3-146,8)].The survival of the first BT was different among the categories (p=0,006): 94,7% of the ERA continue receiving the first BT, followed by 76,2% of OligP and 50% of PolyRF+ and APso. Only 42% of sJIA continue on the first BT prescribed [up to 53,3% were TNF inhibitors (TNFi)]. The categories with less retention of the first BT were: OligE (25%); PolyRF- (27,3%) and Und (0%). The most frequent cause of discontinuation, among these categories, was secondary failure.In the survival analysis between categories, there were differences on OligP (p=0,004), OligE (p=0,042) and PolyRF- (p=0,017). Tocilizumab and Adalimumab were the BT with highest survival with regards to Infliximab, Etanercept, Rituximab (OligE, PolyRF-), Abatacept (OligE, PolyRF-) and Certolizumab (OligP). The survival rate of IL1 inhibitiors and IL6 inhibitiors was higher regarding to TNFi in sJIA patients (p=0,013).Conclusion:Taking into account JIA category is mandatory to choose BT and to understand the response and discontinuation of BT. OligE and PolyRF - showed a high rate of change of the first BT related to secondary failure of Etanercept and Infliximab when compared to Adalimumab and Tocilizumab, as described in the survival analysis. The category with the highest retention of the first BT was ERA. UND patients started sooner BT due to the presence of coxitis. In sJIA, IL1 inhibitors and IL6 inhibitors were superior to TNFi in the survival analysis, as reported in existing literature.Table:Disclosure of Interests:None declared

Background:Patients with Rheumatoid Arthritis (RA) ≥65 years old constitute an important and not very well studied group. Even though the course of the disease may be similar to that of younger patients, treatment is usually less aggressive given the limited information on efficacy, especially of biological treatments, in this age group.Objectives:To describe the characteristics of patients with RA ≥65 years old who started a second biological agent. To compare the survival of this second-line treatment between patients ≥65 and Methods:Retrospective, observational and longitudinal study. Patients diagnosed of RA, who started a second biological agent between 2000 and 2019, who discontinued a first-line TNF inhibitor, were included. Demographic, clinical and analytical data were obtained. The sample was divided in 2 groups: Results:157 patients were identified, 42 (26.8%) were ≥65 years old. In this group, 73.8% were women, with a mean age at the beginning of second biological treatment of 71.43±4.76 years. Demographic and clinical data of ≥65 years old patients are shown in the table. The most frequent second biological agent was Rituximab (23.8%), followed by Adalimumab (21.4%) and Tocilizumab (19%). 76.2% of patients had a disease-modifying drug associated, being Methotrexate the most frequent (45.2%). Discontinuation of second biological agent occurred in 30 patients (71.42%) ≥65 years old, which is similar to the percentage found in patients Conclusion:The survival of second biological agent after the failure of a first TNF inhibitor in patients ≥65 years is similar to the survival in younger patients, although there was a higher percentage of adverse effects in the first group. Rituximab and Abatacept showed a higher survival in patients ≥65 years. Smoking, erosions and diabetes mellitus were associated with an increased risk for the withdrawal of the second-line biological therapy.References:[1]Richter M, Matteson E, DavisIII J, Achenbach S, Crowson C. Comparison of Biologic Discontinuation in Patients With Elderly-Onset Versus Younger-Onset Rheumatoid Arthritis. ACR Open Rheumatology. 2019; 1(10): 627–631.Variablesn= 42 (mean ± SD or %)Age at diagnosis (years)56.48 ± 9.94Age at the beginning of the treatment (years)71.43 ± 4.76Women31 (73.8%)Smokers11 (26.2%)RF (+)35 (83.3%)ACPA (+)30 (71.4%)Erosions29 (69%)Arterial hypertension21 (50%)Diabetes mellitus4 (9.5%)Dyslipidemia11 (26.2%)Disease-modifying drugMethotrexate19 (45.2%)Leflunomide10 (23.8%)Sulfasalazine1 (2.4%)Hydroxychloroquine0 (0%)InfectionsRespiratory infection3 (7.1%)Skin/Soft tissues infections2 (4.8%)Herpes zoster1 (2.4%)Disclosure of Interests:None declared

Background:Several studies have proposed that the immunosenescence of elderly patients with Rheumatoid Arthritis (RA) in treatment with biological therapies could eliminate the need for concomitant immunosuppression with disease-modifying drugs (DMARDs), due to a probable lower production of anti-drug antibodies; however, the evidence is limited.Objectives:To compare the characteristics of patients with RA who started a second biological agent, according to age groups. To analyse second biological agent survival and its relationship with DMARDs.Methods:Retrospective, observational and longitudinal study. Patients with RA who started a second biologic between 2000 and 2019, who discontinued a first-line TNF inhibitor, were included. Demographic, clinical and analytical data were obtained. The sample was divided in 2 groups: Results:156 patients were included. 83.3% were women, with a mean age at the beginning of second biological treatment of 54.64±13.54 years. 22 patients (14.1%) were ≥70 years. Comparative analysis is detailed in table 1: patients ≥70 years presented a longer time from diagnosis to the start of biological treatment, and a higher prevalence of hypertension and diabetes mellitus. The main cause of withdrawal in this group was adverse events (46.67%) while in younger patients was treatment failure (25.27% primary failure, 29.66% secondary failure). The most frequent biological agent in ≥70 years was Rituximab (27.26%) while in Table 1.Variable≥70 years = 22 (mean ± SD or %)pAge at diagnosis (years)40.5 ± 12.358.8 ± 8.9Age at the beginning of the treatment (years)51.28 ± 11.4475.14 ± 3.5Time since diagnosis (years)10.65 ± 8.2016.27 ± 9.090.003Women113 (84.33%)17 (77.27%)0.373Smokers29 (21.64%)2 (9.09%)0.320Rheumatoid factor positive109 (81.34%)17 (77.27%)0.770Anti-CCP positive114 (90.48%)14 (82.35%)0.390Erosions92 (70.23%)16 (76.19%)0.576Arterial hypertension28 (21.37%)14 (66.67%)Diabetes mellitus3 (2.24%)4 (18.18%)Retirement91 (67.91%)15 (68.18%)0.980Infections10 (7.46%)3 (13.64%)0.397Second biological agent withdrawal causePrimary failure23 (25.27%)3 (20%)0.242Secondary failure27 (29.66%)3 (20%)Adverse events25 (27.47%)7 (46.67%)Remission2 (2.20%)0 (0%)Exitus3 (3.30%)0 (0%)Neoplasia3 (3.30%)0 (0%)Table 2.Disease-modifying drug≥70 years oldpMethotrexate72 (53.73%)9 (40.91%)0.667Leflunomide22 (16.42%)5 (22.73%)Sulfasalazine2 (1.49%)1 (4.55%)Hydroxychloroquine6 (4.48%)0 (0%)At least two of the above7 (5.22%)1 (4.55%)Conclusion:DMARD concomitant treatment has been related to a higher second biological treatment survival. This beneficial effect was not observed in RA patients ≥70 years of age whose second biological agent withdrawal cause was failure. In this age group, withdrawal related to adverse events was more frequent.References:[1]Kalden JR, Schulze-Koops H. Immunogenicity and loss of response to TNF inhibitors: implications for rheumatoid arthritis treatment. Nature reviews Rheumatology. 2017;13(12):707-718.Disclosure of Interests:None declared

Background:Biological treatment (BT) has changed perspectives of JIA patients. Increasing data from real life experience have been reported.Objectives:To compare drug survival, safety and efficacy of BT in patients with Juvenile Idiopathic Arthritis (JIA).Methods:A retrospective observational study was conducted on JIA patients followed in a referal hospital and who had received at least one BT between 1999 and 2019.Results:218 BT in 130 JIA patients were analyzed. 67.7% were women with a median age at diagnosis of 8 years old IQR (3-13) and a median age at the beginning of the BT of 15 years old IQR(7.8-21). 21.5% of the patients had uveitis during follow-up. BT were indicated due to: arthritis(73.9%), uveitis(10.1%), arthritis and uveitis(2.7%), systemic activity(8.3%) and macrophage activation syndrome (1,8%).There were 130 BT started in 1st line, 55 in 2nd line, 20 in 3rd line, 10 in 4th line and 3 in 5th line.The 1st line BT most frequently indicated was Etarnecept(ETN) up to 40%, followed by 30% Adalimumab(ADA) and 16,2% Infliximab(INF). The median duration of the 1st line was 51 months IQR (14-109,3). However, 53.8% of the 1st line BT were swiched: 28.3% due to adverse events, 25.7% due to 1° failure and 25.7% due to 2° failure. The BT that were discontinued were: INF (76.2%) and Anakinra (ANAK) (75%) due to adverse events and ETN (59.6%) due to 1° and 2° failure. 55 patients started a 2nd BT: 43.6% received ADA and 20% Tocilizumab (TCZ) with a median duration of 43 months IQR (12-90). 22 of 55 BT required a change: 75% of ETN and 59% of INF prescribed in 2nd line were discotinued. The causes were: 40% 1° failure, 28% 2° failure and 12% remission. In 1st line 87,6% of patients received TNF inhibitors, 74% mantained the target in 2nd line. In 3rd line TCZ was the most frequent BT. 71.5% of patients continue on BT. BT was withdrawn in 20 of 130 patients due to remission (40%), adverse events (30%), and pregnancy (10%).In the analysis by decades, 80 BT (36.7%) were started from 1999 to 2008 and 138 BT (63.3%) from 2009 to 2019. In the 1st decade ETN and INF were the most frequently prescribed and in the 2nd decade, ADA and TCZ (p In the survival analysis, TCZ and ADA were the BT with the highest survival (p=0.001). Of the 31 patients that started TCZ, 61.3% continue on TCZ, with a median duration of 46 months IQR(25-99) and 36/68(52,9%) still on ADA with a median duration of 61,5 months IQR(30.5-98).Conclusion:42.3% of patients required more than one BT. Since the onset of the BT there has been a change in prescription, probably related to the emerge of new targets and the evidence provided by clinical trials and guidelines. TCZ and ADA were the BT with the highest survival rate. On the other hand, INF and ANAK were the ones with the lowest survival rate. The most common causes of BT change in 1st line were adverse events in relation to INF and ANAK. In 2nd line there was a high rate of change in those patients who maintained TNFi, related to 1° failure.Disclosure of Interests:None declared

Background and Aims: \ud Cultured cell suspensions have been the preferred model to study the apoplast as well as to monitor metabolic and cell cycle-related changes. Previous work showed that methyl jasmonate (MeJA) inhibits leaf growth in a CORONATINE INSENSITIVE 1 (COI1)-dependent manner, with COI1 being the jasmonate (JA) receptor. Here, the effect of COI1 overexpression on the growth of stably transformed arabidopsis cell cultures is described.\ud \ud Methods: \ud Time-course experiments were carried out to analyse gene expression, and protein and metabolite levels.\ud \ud Key Results: \ud Both MeJA treatment and the overexpression of COI1 modify growth, by altering cell proliferation and expansion. DNA content as well as transcript patterns of cell cycle and cell wall remodelling markers were altered. COI1 overexpression also increases the protein levels of OLIGOGALACTURONIDE OXIDASE 1, BETA-GLUCOSIDASE/ENDOGLUCANASES and POLYGALACTURONASE INHIBITING PROTEIN2, reinforcing the role of COI1 in mediating defence responses and highlighting a link between cell wall loosening and growth regulation. Moreover, changes in the levels of the primary metabolites alanine, serine and succinic acid of MeJA-treated Arabidopsis cell cultures were observed. In addition, COI1 overexpression positively affects the availability of metabolites such as β-alanine, threonic acid, putrescine, glucose and myo-inositol, thereby providing a connection between JA-inhibited growth and stress responses.\ud \ud Conclusions: \ud This study contributes to the understanding of the regulation of growth and the production of metabolic resources by JAs and COI1. This will have important implications in dissecting the complex relationships between hormonal and cell wall signalling in plants. The work also provides tools to uncover novel mechanisms co-ordinating cell division and post-mitotic cell expansion in the absence of organ developmental control.

Background Immune checkpoint inhibitors (ICI), a type of immunotherapy which block negative co-stimulation of T-cells, have surfaced as an important alternative of treatment for advanced malignancies. Unfortunately, these agents have been associated with immune-related adverse events (irAE). Objectives To analyse the frequency, type and outcome of irAE in Spanish patients treated with ICI. Methods We carried out a retrospective, observational and longitudinal study, including adult patients diagnosed with cancer and treated with ICI followed in our centre (Spanish university hospital) from October 2012 to December 2017. Demographics, clinical, therapeutic and outcome date were collected from their medical records. Results We included 125 patients, 66.4% males, all them Caucasians, with a mean age at diagnosis 63 years (range:42–81). The most common types of cancers were lung carcinoma (61.6%) and melanoma (12.8%). The ICI most frequently used was nivolumab (29.6%), followed by pembrolizumab (8.8%) and atezolizumab (8.8%), as monotherapy. It was also common, combinations, or the use of consecutive way with different drugs. Occurrence of irAE was reported in 51 patients (table 1) and the systems more frequently involved were gastrointestinal tract, endocrine glands and skin. Most of these cases were treated steroids at high doses but a case of colitis required infliximab. Withdrawal of immunotherapy was required in almost half of the adverse events (45%). During the follow-up period there were 5 deaths associated with irAE. Conclusions The frequency and type of irAE associated ICI in our series were similar to those reported in previous studies, except for rheumatological events, being arthritis and sicca syndrome the most frequently reported in other series. These adverse events are not rare and may be a potential cause of morbity in these patients, so it is important to recognise them early and treat properly, with the rheumatologist being one of the specialists involved. Disclosure of Interest None declared

Paraneoplastic polyarthritis is an inflammatory arthritis, is usually seronegative, and has a temporal and pathophysiological relationship with an underlying malignancy. Although head and neck tumors may be a cause of paraneoplastic polyarthritis, its association with tongue carcinoma has not been previously reported. We present the case of a 69-year-old man who was a former smoker and presented with polyarthritis since 2 months in the wrists, proximal interphalangeal joints, knees, and elbows, with increased levels of acute-phase reactants; negativity for rheumatoid factor, anticitrullinated cyclic peptide antibody, and antinuclear antibody; and negative results for crystals and microorganisms in the synovial fluid. Cervical computed tomography and posterior rhinoscopy were performed, which detected an asymptomatic lesion on the base of the tongue, whose biopsy was compatible with nonkeratinizing squamous cell carcinoma. Polyarthritis did not respond to glucocorticoids at medium doses (oral prednisone 20 mg/day) but progressively resolved after the initiation of antineoplastic therapy.

Orientador : Prof. Dr. Marcos R. Mafra Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Engenharia de Alimentos. Defesa : Curitiba, 09/08/2016 Inclui referências : f. 59-64 Resumo: A qualidade dos alimentos está relacionada com a presença de compostos lipídicos por conferirem textura, sabor, nutrição e densidade calórica. Os principais constituintes dos lipídeos são os ácidos graxos. Devido à importância nutricional nos alimentos, tem sido contínua a busca por um maior conhecimento das propriedades físico-químicas dos ácidos graxos e também sobre a sua influência na qualidade dos alimentos. Os ácidos graxos mais abundantes na natureza são o ácido láurico seguido do ácido palmítico. Os ácidos graxos são amplamente empregados em sistemas térmicos, assim como matéria-prima na fabricação de sabonetes, cosméticos, produtos farmacêuticos e alimentares. Os processos de separação e/ou purificação dos ácidos graxos se concentram na cristalização e na extração líquido-líquido. A solubilidade é, portanto, uma propriedade fundamental para o projeto e operação destas unidades. As técnicas convencionais de determinação da solubilidade são em geral demoradas, suscetíveis a erros, além de despenderem um grande volume de amostra. Desta forma, este trabalho tem como objetivo o desenvolvimento de uma metodologia para determinação da solubilidade de ácidos graxos saturados em solventes orgânicos, baseada na calorimetria exploratória diferencial, DSC, tendo sido avaliado também o efeito antissolvente da água nestes sistemas. Para avaliação da solubilidade do ácido palmítico e do ácido láurico foram selecionados os solventes, etanol, 2-propanol e n-propanol. O efeito antissolvente foi avaliado no etanol e no 2-propanol. Na avaliação dos sistemas empregando solventes puros houve destaque para o solvente 2-propanol, no qual ambos os ácidos graxos estudados apresentaram maior solubilidade. O efeito antissolvente da água foi significativo quando a concentração de água na mistura solvente + água foi próxima a 30 % (mol/mol). Os modelos de van't Hoff e Apelblat apresentaram boa correlação com os dados experimentais para todos os sistemas avaliados. Este trabalho demonstrou a adequação da técnica de DSC, utilizada na determinação da solubilidade de ácidos graxos, quando comparada às técnicas convencionais, além de ter contribuído na determinação de dados de solubilidade ainda inéditos na literatura. Palavras-chave: Ácido Palmítico. Ácido Láurico. Modelo de van't Hoff. Modelo de Apelblat. DSC. Abstract: The quality of the foods is related to the presence of lipid compounds to confer texture, flavor, nutrition and caloric density. The main constituents of lipids are fatty acids. Due to nutritional importance in the foods, the search for more knowledge of physicochemical properties of fatty acids and also about their influence on the quality of food is increasing. The most abundant in nature fatty acids are lauric acid followed by palmitic acid. The fatty acids are widely used in thermal systems, as well as raw material in manufacturing soaps, cosmetics, pharmaceuticals and foods. The separation processes and/or purification of fatty acids are concentrated in the crystallization and liquid-liquid extraction. The solubility is therefore an essential property for the design and operation of these units. The conventional techniques for determining solubility are generally time consuming, error prone, and expend a large volume of sample. Thus, this work aims to develop a methodology for determining the solubility of saturated fatty acids in solvents organics, based on differential scanning calorimetry, DSC, also been reported the anti-solvent effect of water in these systems. To evaluate the solubility of palmitic acid and lauric acid were selected the solvents ethanol, 2-propanol and n-propanol. The effect anti-solvent was assessed in ethanol and 2-propanol. In the evaluation of systems using pure solvents, excelled the solvent 2-propanol, in which both studied fatty acids had higher solubility. The anti-solvent effect of water was significant when the concentration of water in the mixture solvent + water was close to 30 % (mol/mol). van't Hoff and Apelblat models presented well with the experimental data for all systems evaluated. This study demonstrated the appropriateness of the technical DSC used for the determination of solubility of fatty acids, when compared to conventional techniques, besides contributing to determine solubility unpublished data in the literature. Keywords: Palmitic Acid. Lauric acid. van’t Hoff model. Apelblat model. DSC.

This chapter describes the principles of selection of antigenic peptides for the development of anti-peptide antibodies for use in microarray-based multiplex affinity assays and also with mass-spectrometry detection. The methods described here are mostly applicable to small to medium scale arrays. Although the same principles of peptide selection would be suitable for larger scale arrays (with 100+ features) the actual informatics software and printing methods may well be different. Because of the sheer number of proteins/peptides to be processed and analyzed dedicated software capable of processing all the proteins and an enterprise level array robotics may be necessary for larger scale efforts. This report aims to provide practical advice to those who develop or use arrays with up to ~100 different peptide or protein features.

This chapter describes the principles of selection of antigenic peptides for the development of anti-peptide antibodies for use in microarray-based multiplex affinity assays and also with mass-spectrometry detection. The methods described here are mostly applicable to small to medium scale arrays. Although the same principles of peptide selection would be suitable for larger scale arrays (with 100+ features) the actual informatics software and printing methods may well be different. Because of the sheer number of proteins/peptides to be processed and analyzed dedicated software capable of processing all the proteins and an enterprise level array robotics may be necessary for larger scale efforts. This report aims to provide practical advice to those who develop or use arrays with up to ~100 different peptide or protein features.

In this paper, we report the preparation of an inorganic–organic nanocomposite, obtained by the in situ oxidative polymerization of pyrrole inside the layers of α-Sn(HPO 4 ) 2 ·H 2 O (α-SnP). The polymerization was carried out by Cu 2+ ions, previously incorporated in the α-SnP structure, by an ion-exchange process. The Cu 2+ exchanged materials and the nanocomposites obtained after pyrrole polymerization were characterized by XRD, FTIR, UV-Vis, Raman and EPR spectroscopy. The characterization results suggest the formation of polymer chains between the α-SnP layers.