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Contains fulltext : 294430.pdf (Publisher’s version ) (Open Access) BACKGROUND: Ta grade 3 (G3) non-muscle-invasive bladder cancer (NMIBC) is a relatively rare diagnosis with an ambiguous character owing to the presence of an aggressive G3 component together with the lower malignant potential of the Ta component. The European Association of Urology (EAU) NMIBC guidelines recently changed the risk stratification for Ta G3 from high risk to intermediate, high, or very high risk. However, prognostic studies on Ta G3 carcinomas are limited and inconclusive. OBJECTIVE: To evaluate the prognostic value of categorizing Ta G3 compared to Ta G2 and T1 G3 carcinomas. DESIGN, SETTING, AND PARTICIPANTS: Individual patient data for 5170 primary Ta-T1 bladder tumors from 17 hospitals were analyzed. Transurethral resection of the tumor was performed between 1990 and 2018. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Time to recurrence and time to progression were analyzed using cumulative incidence functions, log-rank tests, and multivariable Cox-regression models with interaction terms stratified by institution. RESULTS AND LIMITATIONS: Ta G3 represented 7.5% (387/5170) of Ta-T1 carcinomas of which 42% were classified as intermediate risk. Time to recurrence did not differ between Ta G3 and Ta G2 (p = 0.9) or T1 G3 (p = 0.4). Progression at 5 yr occurred for 3.6% (95% confidence interval [CI] 2.7-4.8%) of Ta G2, 13% (95% CI 9.3-17%) of Ta G3, and 20% (95% CI 17-23%) of T1 G3 carcinomas. Time to progression for Ta G3 was shorter than for Ta G2 (p

Item does not contain fulltext BACKGROUND: The pathological existence and clinical consequence of stage T1 grade 1 (T1G1) bladder cancer are the subject of debate. Even though the diagnosis of T1G1 is controversial, several reports have consistently found a prevalence of 2-6% G1 in their T1 series. However, it remains unclear if T1G1 carcinomas have added value as a separate category to predict prognosis within the non-muscle-invasive bladder cancer (NMIBC) spectrum. OBJECTIVE: To evaluate the prognostic value of T1G1 carcinomas compared to TaG1 and T1G2 carcinomas within the NMIBC spectrum. DESIGN, SETTING, AND PARTICIPANTS: Individual patient data for 5170 primary Ta and T1 bladder tumors from 17 hospitals in Europe and Canada were analyzed. Transurethral resection (TUR) was performed between 1990 and 2018. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Time to recurrence and progression were analyzed using cumulative incidence functions, log-rank tests, and multivariable Cox regression models stratified by institution. RESULTS AND LIMITATIONS: T1G1 represented 1.9% (99/5170) of all carcinomas and 5.3% (99/1859) of T1 carcinomas. According to primary TUR dates, the proportion of T1G1 varied between 0.9% and 3.5% per year, with similar percentages in the early and later calendar years. We found no difference in time to recurrence between T1G1 and TaG1 (p = 0.91) or between T1G1 and T1G2 (p = 0.30). Time to progression significantly differed between TaG1 and T1G1 (p < 0.001) but not between T1G1 and T1G2 (p = 0.30). Multivariable analyses for recurrence and progression showed similar results. CONCLUSIONS: The relative prevalence of T1G1 diagnosis was low and remained constant over the past three decades. Time to recurrence of T1G1 NMIBC was comparable to that for other stage/grade NMIBC combinations. Time to progression of T1G1 NMIBC was comparable to that for T1G2 but not for TaG1, suggesting that treatment and surveillance of T1G1 carcinomas should be more like the approaches for T1G2 NMIBC in accordance with the intermediate and/or high risk categories of the European Association of Urology NMIBC guidelines. PATIENT SUMMARY: Although rare, stage T1 grade 1 (T1G1) bladder cancer is still diagnosed in daily clinical practice. Using individual patient data from 17 centers in Europe and Canada, we found that time to progression of T1G1 cancer was comparable to that for T1G2 but not TaG1 cancer. Therefore, our results suggest that primary T1G1 bladder cancers should be managed with more aggressive treatment and more frequent follow-up than for low-risk bladder cancer.

The treatment options for metastatic soft tissue sarcomas (STSs) are limited. In most cases, immunotherapy with immune checkpoint inhibitors has not been successful so far. Macrophages dominate the immune landscape of STSs; thus, combinatorial strategies aiming at both tumor-infiltrating lymphocytes and macrophages may represent a particularly relevant treatment approach for metastatic or recurrent STSs.In this cohort study, 66 patients who underwent surgery for STSs were enrolled. Tumor cells and tumor-infiltrating immune cells were analyzed using flow cytometry and immunohistochemistry. In cell suspensions obtained from surgical resections, human T cells were activated by superparamagnetic polymer beads and cultured at a concentration of 0.3 × 10The most profound response to anti-CD47 therapy was observed in an undifferentiated pleiomorphic sarcoma which also displayed high expression of CD47 in the tumor microenvironment. Both anti-PD-1 and anti-CD47 therapies drastically increased the production of pro-inflammatory cytokines in the tumor microenvironment of STSs, but co-administration of both agents did not further increase cytokine secretion. Furthermore, all patient samples treated with a combination of both anti-PD-1 and anti-CD47 antibodies showed a dramatic reduction in cytokine secretion.Our findings suggest that anti-PD-1 and anti-CD47 therapies do not enhance each other, and the combined application of anti-PD-1 and anti-CD47 agents in vitro limits rather than potentiates their efficacy.

Background: Papillary urothelial neoplasm of low malignant potential (PUN-LMP) was introduced as a noninvasive, noncancerous lesion and a separate grade category in 1998. Subsequently, PUN-LMP was reconfirmed by World Health Organization (WHO) 2004 and WHO 2016 classifications for urothelial bladder tumors. Objectives: To analyze the proportion of PUN-LMP diagnosis over time and to determine its prognostic value compared to Ta-LG (low-grade) and Ta-HG (high-grade) carcinomas. To assess the intraobserver variability of an experienced uropathologist assigning (WHO) 2004/2016 grades at 2 time points. Materials and methods: Individual patient data of 3,311 primary Ta bladder tumors from 17 hospitals in Europe and Canada were available. Transurethral resection of the tumor was performed between 1990 and 2018. Time to recurrence and progression were analyzed with cumulative incidence functions, log-rank tests and multivariable Cox-regression stratified by institution. Intraobserver variability was assessed by examining the same 314 transurethral resection of the tumorslides twice, in 2004 and again in 2018. Results: PUN-LMP represented 3.8% (127/3,311) of Ta tumors. The same pathologist found 71/314 (22.6%) PUN-LMPs in 2004 and only 20/314 (6.4%) in 2018. Overall, the proportion of PUN-LMP diagnosis substantially decreased over time from 31.3% (1990-2000) to 3.2% (2000-2010) and to 1.1% (2010-2018). We found no difference in time to recurrence between the three WHO 2004/2016 Ta-grade categories (log-rank, P = 0.381), nor for LG vs. PUN-LMP (log-rank, P = 0.238). Time to progression was different for all grade categories (log-rank, P < 0.001), but not between LG and PUN-LMP (log-rank, P = 0.096). Multivariable analyses on recurrence and progression showed similar results for all 3 grade categories and for LG vs. PUN-LMP. Conclusions: The proportion of PUN-LMP has decreased to very low levels in the last decade. Contrary to its reconfirmation in the WHO 2016 classification, our results do not support the continued use of PUN-LMP as a separate grade category in Ta tumors because of the similar prognosis for PUN-LMP and Ta-LG carcinomas. (C) 2019 Elsevier Inc. All rights reserved.

Partially unfolded alpha-lactalbumin forms the oleic acid complex HAMLET, with potent tumoricidal activity. Here we define a peptide-based molecular approach for targeting and killing tumor cells, and evidence of its clinical potential (ClinicalTrials.gov NCT03560479). A 39-residue alpha-helical peptide from alpha-lactalbumin is shown to gain lethality for tumor cells by forming oleic acid complexes (alpha1-oleate). Nuclear magnetic resonance measurements and computational simulations reveal a lipid core surrounded by conformationally fluid, alpha-helical peptide motifs. In a single center, placebo controlled, double blinded Phase I/II interventional clinical trial of non-muscle invasive bladder cancer, all primary end points of safety and efficacy of alpha1-oleate treatment are reached, as evaluated in an interim analysis. Intra-vesical instillations of alpha1-oleate triggers massive shedding of tumor cells and the tumor size is reduced but no drug-related side effects are detected (primary endpoints). Shed cells contain alpha1-oleate, treated tumors show evidence of apoptosis and the expression of cancer-related genes is inhibited (secondary endpoints). The results are especially encouraging for bladder cancer, where therapeutic failures and high recurrence rates create a great, unmet medical need., Partially unfolded alpha-lactalbumin forms an oleic acid complex with antitumorigenic properties. Here, the authors define a structurally flexible, peptide-based oleate complex and report a phase I/II clinical trial where this complex is used to treat patients with bladder cancer.

Background: In the current European Association of Urology (EAU) non-muscle invasive bladder cancer (NMIBC) guideline, two classification systems for grade are advocated: WHO1973 and WHO2004/2016. Objective: To compare the prognostic value of these WHO systems. Design, setting, and participants: Individual patient data for 5145 primary Ta/T1 NMIBC patients from 17 centers were collected between 1990 and 2019. The median follow-up was 3.9 yr. Outcome measurements and statistical analysis: Univariate and multivariable analyses of WHO1973 and WHO2004/2016 stratified by center were performed for time to recurrence, progression (primary endpoint), cystectomy, and duration of survival, taking into account age, concomitant carcinoma in situ, gender, multiplicity, tumor size, initial treatment, and tumor stage. Harrell's concordance (C-index) was used for prognostic accuracy of classification systems. Results and limitations: The median age was 68 yr; 3292 (64%) patients had Ta tumors. Neither classification system was prognostic for recurrence. For a four-tier combination of both WHO systems, progression at 5-yr follow-up was 1.4% in lowgrade (LG)/G1, 3.8% in LG/G2, 7.7% in high grade (HG)/G2, and 18.8% in HG/G3 (log rank, p < 0.001). In multivariable analyses with WHO1973 and WHO2004/2016 as independent variables, WHO1973 was a significant prognosticator of progression (p < 0.001), whereas WHO2004/2016 was not anymore (p = 0.067). C-indices for WHO1973, WHO2004, and the WHO systems combined for progression were 0.71, 0.67, and 0.73, respectively. Prognostic analyses for cystectomy and survival showed results similar to those for progression. Conclusions: In this large prognostic factor study, both classification systems were prognostic for progression but not for recurrence. For progression, the prognostic value of WHO1973 was higher than that of WHO 2004/2016. The four-tier combination (LG/G1, LG/G2, HG/G2, and HG/G3) of both WHO systems proved to be superior, as it divides G2 patients into two subgroups (LG and HG) with different prognoses. Hence, the current EAU-NMIBC guideline recommendation to use both WHO classification systems remains correct. Patient summary: At present, two classification systems are used in parallel to grade non-muscle-invasive bladder tumors. Our data on a large number of patients showed that the older classification system (WHO1973) performed better in terms of assessing progression than the more recent (WHO2004/2016) one. Nevertheless, we conclude that the current guideline recommendation for the use of both classification systems remains correct, since this has the advantage of dividing the large group of WHO1973 G2 patients into two subgroups (low and high grade) with different prognoses. (c) 2020 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Background: The European Association of Urology (EAU) prognostic factor risk groups for non–muscle-invasive bladder cancer (NMIBC) are used to provide recommendations for patient treatment after transurethral resection of bladder tumor (TURBT). They do not, however, take into account the widely used World Health Organization (WHO) 2004/2016 grading classification and are based on patients treated in the 1980s. Objective: To update EAU prognostic factor risk groups using the WHO 1973 and 2004/2016 grading classifications and identify patients with the lowest and highest probabilities of progression. Design, setting, and participants: Individual patient data for primary NMIBC patients were collected from the institutions of the members of the EAU NMIBC guidelines panel. Intervention: Patients underwent TURBT followed by intravesical instillations at the physician's discretion. Outcome measurements and statistical analysis: Multivariable Cox proportional-hazards regression models were fitted to the primary endpoint, the time to progression to muscle-invasive disease or distant metastases. Patients were divided into four risk groups: low-, intermediate-, high-, and a new, very high-risk group. The probabilities of progression were estimated using Kaplan-Meier curves. Results and limitations: A total of 3401 patients treated with TURBT ± intravesical chemotherapy were included. From the multivariable analyses, tumor stage, WHO 1973/2004–2016 grade, concomitant carcinoma in situ, number of tumors, tumor size, and age were used to form four risk groups for which the probability of progression at 5 yr varied from 40%. Limitations include the retrospective collection of data and the lack of central pathology review. Conclusions: This study provides updated EAU prognostic factor risk groups that can be used to inform patient treatment and follow-up. Incorporating the WHO 2004/2016 and 1973 grading classifications, a new, very high-risk group has been identified for which urologists should be prompt to assess and adapt their therapeutic strategy when necessary. Patient summary: The newly updated European Association of Urology prognostic factor risk groups for non–muscle-invasive bladder cancer provide an improved basis for recommending a patient's treatment and follow-up schedule. The updated European Association of Urology prognostic factor risk groups for patients with non–muscle-invasive bladder cancer provide urologists with information that they should take into account when choosing a patient's treatment and scheduling follow-up.

Dent disease is an X-linked recessive renal tubular disorder characterized by proximal tubule dysfunction. Typical features include low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, rickets, and chronic renal failure. We present a case of a 6-year-old boy with nephrotic proteinuria without hypoalbuminemia or edema. His renal biopsy revealed focal segmental glomerulosclerosis (FSGS), some of the glomeruli were globally sclerotic. Hypercalciuria was present intermittently and urine protein electrophoresis showed low molecular weight protein fraction of 50%. The next generation sequencing identified pathogenic variant in OCRL gene causing Dent disease type 2. We report an uncommon histologic finding of FSGS in Dent disease type 2 and highlight the importance of protein content examination and genetic analysis for the proper diagnosis in these complicated cases.

Atypical haemolytic uraemic syndrome and steroid-resistant nephrotic syndrome are highly rare kidney diseases that can occur in childhood. In some cases, genetic variants may trigger these conditions, although in atypical haemolytic uraemic syndrome they mostly confer only a predisposition to the disease. Most variants causing atypical haemolytic uraemic syndrome were identified in genes encoding proteins regulating the complement pathway; on the other hand, there are approximately 58 genes encoding distinct proteins primarily causing steroid-resistant nephrotic syndrome. We present a child with steroid-resistant nephrotic syndrome and a confirmed homozygous c.966G > A, p.Trp322Ter pathogenic variant in DGKE. This variant was also found in compound with a novel DGKE heterozygous deletion c.171delG, p.Ser58Alafs*111 in a patient from our paediatric cohort with atypical haemolytic uraemic syndrome. Both cases presented with hypertension, nephrotic proteinuria and severe acute kidney injury followed by renal recovery; however, their renal histology was different. In this paper, we deal with the clinical course of children with disrupted DGKE, including the steroid-resistant nephrotic syndrome and atypical haemolytic uraemic syndrome overlap.

OBJECTIVE The methodology of cell blocks (CBs) has long been an integrated part of cytology. However, there are very few data on CBs derived from urine. Their main disadvantage is a lack of cellularity, which limits their broader clinical applicability. Factors affecting cellular adequacy in urine remain unclear. We assessed the impact of basic clinical and cytopathological factors on the adequacy of cellularity in urinary CBs. METHODS Freshly voided urine was collected from 401 consecutive individuals. Of these, 167 patients were diagnosed with urothelial carcinoma. The remaining 234 patients had various benign urological conditions. Papanicolaou classes were determined and CBs produced. Cellular adequacy was assigned to each CB (acellular, hypocellular, moderate cellularity, high cellularity), and moderately and highly cellular CBs were considered as adequate. Several factors were analysed to find any correlation with the adequacy of the cellularity. RESULTS In univariate analysis, seven factors significantly correlated with the adequacy of the CBs. In the multivariate model, positive sediment (OR = 3.7), female sex (OR = 2.7), positive urinary cytology (OR = 2.6) and positive leucocyturia (OR = 2.1) were independent predictors of adequate cellularity. Positive predictive value and negative predictive value of the model were 65.0% and 77.7%, respectively. CONCLUSIONS We determined four clinical and cytopathological factors which independently predict adequate cellularity in urinary CBs. Based on these results, several clinical situations have been proposed, in which the highest probability of adequate cellularity in urinary CBs can be achieved.

Surface functionalisations substantially influence the performance of drug delivery vehicles by improving their biocompatibility, selectivity and circulation in bloodstream. Herein, we present the study of in vitro and in vivo behaviour of a highly potent cytostatic alkaloid ellipticine (Elli) encapsulated in internal cavity of ferritin (FRT)-based nanocarrier (hereinafter referred to as FRTElli). In addition, FRTElli surface was functionalised with three different molecular coatings: two types of protective PAS peptides (10- or 20-residues lengths) with sequences comprising amino acids proline (P), alanine (A) and serine (S) (to form PAS-10-FRTElli or PAS-20-FRTElli, respectively), or polyethylene glycol (PEG-FRTElli). All three surface modifications of FRT disposed sufficient encapsulation efficiency of Elli with no premature cumulative release of cargo. Noteworthy, all tested surface modifications displayed beneficial effects on the in vitro biocompatibility. PAS-10-FRTElli exhibited markedly reduced uptake by macrophages compared to PAS-20-FRTElli, PEG-FRTElli or unmodified FRTElli. The exceptional properties of PAS-10-FRTElli were validated by an array of in vitro analyses including formation of protein corona, uptake efficiency or screenings of selectivity of cytotoxicity. In murine preclinical model bearing triple-negative breast cancer (MDA-MB-231) xenograft, compared to free Elli or FRTElli, PAS-10-FRTElli displayed enhanced accumulation of Elli within tumour tissue, while hampering the uptake of Elli into off-target tissues. Noteworthy, PAS-10-FRTElli led to decreased in vivo complement (C3) activation and protein corona formation. Taken together, presented in vivo results indicate that PAS-10-FRTElli represents a promising stealth platform for translation into clinical settings. info:eu-repo/semantics/publishedVersion

Objective Immunocytochemistry has attained a marginal role in urology so far. Combining the morphological and immunophenotypical changes of the urothelial cells retrieved from urine is a logical approach. The study aimed to analyse the diagnostic potential of immunocytological staining in the detection of high-grade and low-grade urothelial carcinoma. Methods Freshly voided urine was collected from 152 consecutive individuals, cytology classes were determined and cell blocks produced. A total of 77 patients were diagnosed with urothelial carcinoma and 75 patients had various benign urological conditions. Immunocytochemistry was performed using four antibodies: p53, MCM2, MCM5 and Ki-67. A diagnostic power to detect low grade and high-grade urothelial carcinoma was analysed for each antibody and their combinations with cytology. Results There were no significant differences between patients with low-grade tumours and control group. Antibodies p53 and Ki-67 slightly improved the sensitivity of urinary cytology while maintaining its specificity. The best negative predictive value was demonstrated in combinations of cytology and MCM5 (88.9%) and cytology, p53 and MCM5 (90.6%). In the diagnosis of high-grade tumours, all antibodies apart from MCM2 yielded better sensitivity and specificity than cytology alone (receiver operating characteristic curves: p53 = 0.853, MCM5 = 0.931, and Ki-67 = 0.895). Combined with cytology, the sensitivities went even higher for the cost of lower specificity. The best diagnostic performance was observed in the combination of MCM5 and Ki-67 (sensitivity = 96.2%; specificity = 80%). Conclusions Immunocytochemistry with p53, MCM5 and Ki-67 antibodies can improve the diagnostic power of urinary cytology in the detection and follow-up of urinary bladder urothelial carcinoma.

Proteinuria is a common manifestation of chronic kidney disease (CKD), and there is a high incidence of CDK and its complications following renal transplantation. However, little data are available on the association between proteinuria and graft/patient survival in the paediatric transplant population. The primary aim of this study was to investigate the associations between posttransplant proteinuria and graft/patient survival in children after renal transplantation. In this retrospective study, we screened all 91 children receiving renal allografts at a single institution between 1997 and 2007. The inclusion criteria were a functioning graft at 1 year posttransplant, data availability and no recurrence of focal-segmental glomerulosclerosis. The final cohort included 75 patients. Proteinuria was considered to be pathologic if the urinary protein/creatinine ratio was >30 mg/mmol. Donor and recipient characteristics, data on proteinuria, estimated glomerular filtration rate (eGFR) and rejection episodes were analysed. The most recent of the biopsies performed during the follow-up after 1 year posttransplant were analysed separately in the proteinuric group and the non-proteinuric group. Proteinuria at 1-year posttransplant was pathologic in 35 % of patients. The 5-year graft survival rate was significantly lower in the proteinuric group than in the non-proteinuric group (77 vs. 100 %; p

BACKGROUND The aim of this prospective single center study was to investigate the ability of urinary neutrophil gelatinase-associated lipocalin (NGAL) to distinguish acute rejection from other causes of acute kidney injury (AKI) in children after renal transplantation. METHODS Fifteen children fulfilled the inclusion criteria (acute kidney injury (AKI) with allograft biopsy, at least 21 days after renal transplantation, no sepsis) during 2013 - 2014 in our pediatric transplantation center. The mean age was 14.8  2.8, median time after renal transplantation was 0.4 years (range 0.1 - 3.8). Urinary NGAL was measured in spot urine by Chemiluminescent Microparticle Immunoassay technology. RESULTS Four patients had biopsy proven acute rejection (rejection group), eleven children had AKI of other cause (non-rejection group). The median urinary NGAL concentration in the rejection group was not significantly different from NGAL in the non-rejection group (7.3 ng/mL, range 3.0 - 42.3 vs. 8.6 ng/mL, range 3.4 - 54.7, p = 0.48). There was a significant negative correlation between eGFR and urinary NGAL concentrations (r = -0.77, p < 0.001). CONCLUSIONS Our small study suggests that in children after renal transplantation, urinary NGAL cannot be used as a specific marker for distinguishing acute rejection from other non-rejection causes of AKI. Urinary NGAL was mainly associated with graft function but not with the etiology of AKI.

Beta-lactam-associated acute tubulointerstitial nephritis (ATIN) is a rare condition in childhood. We report the case of an infant with penicillin-associated ATIN and concomitant acute pyelonephritis resulting in the development of severe acute kidney injury (AKI). The treatment consisted of penicillin suspension and appropriate AKI management, which required a short period of dialysis. Finally, full recovery and normalization of laboratory parameters occurred. We present here the first case of oral penicillin-associated ATIN in childhood.

Beta-lactam-associated acute tubulointerstitial nephritis (ATIN) is a rare condition in childhood. We report the case of an infant with penicillin-associated ATIN and concomitant acute pyelonephritis resulting in the development of severe acute kidney injury (AKI). The treatment consisted of penicillin suspension and appropriate AKI management, which required a short period of dialysis. Finally, full recovery and normalization of laboratory parameters occurred. We present here the first case of oral penicillin-associated ATIN in childhood.

Background Interleukin-35 (IL-35) is a newly described heterodimeric cytokine that belongs to the IL-12 family and consists of p35 (IL-12a) and EBI3 (IL-27b) subunits. IL-35 exerts immunomodulatory activities in several autoimmune inflammatory diseases. Objectives The aim of this study was to assess IL-35 expression in muscle tissue of patients with idiopathic inflammatory myopathies (IIM) and to compare serum levels of IL-35 in patients with IIM to healthy controls and asses potential association with activity of IIM. Methods The expression of IL-35 was studied in a series of 19 muscle biopsy samples of idiopathic inflammatory myopathies (9 dermatomyositis, 10 polymyositis) and 10 non-inflammatory control muscle biopsies from patients with myasthenia gravis.Serum levels of IL-35 were measured in 23 PM, 28 DM and 15 cancer associated myositis patients as well as in 40 healthy controls.Disease activity was evaluated by the Myositis Disease Activity Assessment Tool (MYOACT) and by serum muscle enzymes. Results Both IL-35 subunits were found in immune cells of the inflammatory infiltrates in IIM muscle biopsies, no immunoreactivity was observed in muscle tissue of control patients.IL-35 serum levels were increased in all IIM patients compared to healthy controls (p Conclusions IL-35 subunits are overexpressed in inflammatory infiltrates in muscle tissue of IIM patients and elevated circulating IL-35 levels correlate with several disease activity parameters. These data suggest potential role of IL-35 in the pathogenesis of inflammatory myopathies. Acknowledgement Supported by MHCR support for conceptual development of a research organization (023728) and BTCure (115142–2). Disclosure of Interest None declared

Background Interleukin-35 (IL-35) is a newly described hetero-dimeric cytokine of the IL-12 family. Studies of immunopathogenesis in idiopathic inflammatory myopathies (IIM) are of interest for future biological therapies. Objective To analyze expression of IL-35 in muscle biopsies and to study its effects on peripheral blood mononuclear cells (PBMC) in vitro to assess its pathogenetic and diagnostic value. Materials and methods Immunoexpression of IL-35 subunits (p35, EBI3) was studied immunohistochemically in 23 muscle biopsies of IIM (12 dermatomyositis, 11 polymyositis), 10 noninflammatory myopathies and 10 controls. PBMCs were stimulated with human recombinant IL-35 protein or TNF-α in vitro. Taq-Man RT-PCR was performed to analyze gene expression after 6hours and ELISA assays were used to confirm the protein secretion after 24 hours of stimulation. Results We showed localization of both IL-35 subunits in B-cells, T-cells and macrophages of the inflammatory infiltrates in IIM. No immunoreactivity was observed in controls. We found that gene expression of both IL-35 subunits were dose- and time-dependently induced by TNF-α in PBMCs. Conclusion Our data suggest pro-inflammatory properties of IL-35 and its potential role in the pathogenesis of IIM. The immunohistochemical expression of IL-35 might be also of diagnostic help in muscle biopsy.

Interleukin-35 (IL-35) is a recently described heterodimeric cytokine that belongs to the IL-12 family and consists of p35 (IL-12a) and EBI3 (IL-27b) subunits. The expression of IL-35 in humans is inducible in response to inflammatory stimuli. Increased IL-35 levels were documented in several autoimmune inflammatory diseases, suggesting a possible immunomodulatory role in their pathogenesis.The aim of this study was to explore a potential role of IL-35 in the pathogenesis of idiopathic inflammatory myopathies (IIM) by studying the expression of IL-35 subunits in muscle biopsy samples and by evaluating serum levels of IL-35 and their association with disease activity in IIM patients.The expression of IL-35 subunits was studied in serial sections of 9 muscle biopsy samples [4 polymyositis (PM), 5 dermatomyositis (DM)] and in 7 non-inflammatory control muscle biopsies. Serum levels of IL-35 were measured in 23 PM, 28 DM and 15 cancer associated myositis (CAM) patients as well as in 40 healthy controls. Disease activity was evaluated using the Myositis Disease Activity Assessment Tool (MDAAT) and by serum muscle enzymes.Expression of both IL-35 subunits was evident in the inflammatory infiltrates in IIM muscle biopsies, while no IL-35 expression was observed in control muscle samples. IL-35 serum levels were increased in all IIM patients compared to healthy controls [median 119.5 (range 32.1-1074.5) vs 36.2 (range 1.5-86.5) pg/ml, P 0.001]. There were no differences in IL-35 serum levels between myositis subgroups (DM, PM or CAM). Serum IL-35 levels correlated significantly with physician's assessment of global (r = 0.29, p = 0.021), muscle (r = 0.30, p = 0.017) and extramuscular (r = 0.30, p = 0.016) disease activity as well as creatine kinase (r = 0.26, p = 0.044) and lactate dehydrogenase (r = 0.40, p = 0.003) levels. There was a significant correlation with pulmonary activity in patients with interstitial lung disease (r = 0.39, p = 0.037). Serum IL-35 correlated negatively with duration of treatment (r = -34, p = 0.009).IL-35 is overexpressed in inflammatory infiltrates in muscle tissue and serum in IIM patients and there is correlation with several disease activity parameters. These data suggest potential role of locally produced IL-35 in the pathogenesis of inflammatory myopathies.