Investigations into the significance of Nrf2 signalling and ubiquitination of proteins in Respiratory Syncytial Virus infection

Introduction and Objectives: Respiratory Syncytial Virus (RSV) is the commonest cause of bronchiolitis in infants. This negative strand RNA virus is known to infect and replicate in the airway epithelium. RSV infection induces elevated levels of reactive oxidative species and subsequent oxidative stress injury in the lungs. Nrf2, a transcription factor that regulates antioxidant protein expression, has an important role in preventing pulmonary oxidative damage. Sulforaphane is a potent, naturally occurring inducer of NRF2 found in vegetables such as broccoli. In this study we sought to determine whether Nrf2 induction by sulforaphane might reduce RSV replication in airway epithelial cells. We also selected six proteins including MAVS, DDX21, RPS10, prohibitin, annexin A1, HMGB1 from proteomics defining changes in their level of ubiquitination following RSV infection. Our aim was to determine which proteins change their level of polyubiquitination following the infection. This could help identify new biochemical pathways involved in the host defence or viral replication and new targets for potential therapeutic intervention. Method: BEAS2B cells were infected with RSV at MOI of 1 following pre-treatment with sulforaphane. Samples were harvested at time points 24 and 48 hours and analysed by Western Blotting for NrF2 and RSV. In addition, RT-qPCR was carried out for RSV quantification using an RSV N primer. A549 cells were infected with various concentrations of RSV (1:4-4:1). Samples were harvested at time point of 4 and 24 hours and analysed by Western Blotting using antibodies to ubiquitin and target proteins selected from ubiquitination proteomics data . Immunoprecipitation was used to confirm ubiquitination of these proteins and immunohistology to confirm their cellular localisation. Proteasome activity was inhibited using MG132 a specific, potent, reversible, and cell-permeable proteasome inhibitor. Results: Sulforaphane induced Nrf2 production in BEAS2Bs in a dose dependent manner. Results do not show that RSV replication is reduced in airway epithelial cells pretreated with Sulforaphane. However, preliminary data suggests that virus might have degrading effect on Nrf2. Western blots demonstrate changes in expression of target proteins and their ubiquitination following RSV infection and proteasome inhibition. Breakdown products of DDX21 and MAVS were detected in RSV infected samples. Conclusions: RSV infection changes expression of target proteins in A549 cells and might have influence on their ubiquitination, however, most probably it does not affect expression of Sulforaphane induced Nrf2. DDX21 and Nrf2 are likely to be degraded by the virus but results have to be confirmed in further experiments

Analysis of Clinical Symptoms and Laboratory Profiles in Children with Juvenile Idiopathic Arthritis in Malopolska Region (Poland) in the Years 2007-2010

Background: Juvenile idiopathic arthritis (JIA) is a heterogenic group of chronic inflammatory connective tissue diseases of unknown aetiology in children up to 16 years of age.Aim: The aim of this study was to analyse the incidence, clinical presentation and laboratory findings in children with JIA in Malopolska region.Materials and methods: A retrospective analysis included all children with JIA (N=251) hospitalized in the two reference rheumatology centres covering Malopolska region (Poland), between July 2007 and December 2010.Results: The annual incidence of JIA in Malopolska region was estimated at 9.5 per 100 000 children. Oligoarthritis (54.9%) was the most common category in all age groups with a tendency to decrease with age; from 71.4 % in children aged 1-6 years; 55.7% in aged 7-12 years to 39.3 % in aged 13-16 years. The frequency of polyarthritis and enthesitis-related arthritis was greater in adolescents (29.2 % and 22.5 %, respectively). HLA-B27 antigen and uveitis were most frequently found in children with enthesitis-related arthritis (58% and 18.5 %, respectively).Conclusions: The study suggests the improvement of diagnostic capacity of JIA during the last decade in Poland. In accordance with the existing data diverse clinical presentation of JIA categories and laboratory characteristics were proven

Original paper Prevalence of HLA-B27 antigen in patients with juvenile idiopathic arthritis

Introduction: Human leukocyte antigen B27 (HLA-B27) is considered as a risk factor for development of juvenile idiopathic arthritis (JIA). The aim of this study was to analyse the prevalence of HLA-B27 antigen in JIA categories and its influence on disease onset and response to conventional therapy. Material and methods : The retrospective analysis included 461 unselected children with JIA hospitalized in a single reference rheumatology centre between July 2007 and June 2012. The diagnosis was based on criteria by the International League of Association for Rheumatology. HLA-B27 was determined in 387 of all patients (84%) by hybridization of the amplified, labelled product to immobilize it on the microarray probe. Results: HLA-B27 antigen was found in 104 of 383 affected children (27.2%), 48 of 206 girls (23.3%), and 56 of 177 boys (31.6%) – most frequently in patients with enthesitis-related arthritis (71%), psoriatic arthritis (50%) and unclassified cases (86.7%). The age of JIA onset was slightly (by 1 year) but significantly different in patients with and without HLA-B27 antigen [11 (8.5–14) vs. 10 (5–13.5) years.; p < 0.001]. The use of disease-modifying antirheumatic drugs (DMARDs) and corticosteroids was more frequently clinically ineffective in HLA-B27 positive than negative patients (23.1% vs. 15.2%; p = 0.09). Patients with polyarthritis, systemic, and psoriatic arthritis more frequently received biological therapy. HLA-B27 positive patients with enthesitis-related arthritis received biological therapy more frequently than HLA-B27 negative ones (20.4% vs. 0, respectively; p = 0.09). Conclusions : HLA-B27 antigen is a strong risk factor for the development of enthesitis-related arthritis, and to a lesser extent for psoriatic arthritis and extended course of oligoarthritis. The presence of this antigen does not affect the disease onset but seems to predict resistance to therapy with disease-modifying drugs and corticosteroids

Patient with atypical chest pain and nephrotic range proteinuria finally diagnosed with non-secretory myeloma

Multiple myeloma is the second most common neoplastic disease of lymphoid tissue in adults. However, its atypical non secretory form, is quite rare. We present a case of 70 year old male with atypical chest pain and nephrotic range proteinuria fi nally diagnosed as non secretory myeloma who did not present any of characteristic fi ndings when admitted to hospital. Despite the unusual course the diagnosis was established quickly enough to provide a proper treatment. One should remember that occasionally patients do not match typical criteria necessary for diagnosing a disease

Beneficial Effect of Successful Simultaneous Pancreas-Kidney Transplantation on Plasma Profile of Metalloproteinases in Type 1 Diabetes Mellitus Patients

It is not fully elucidated whether the restoring of normal glucose metabolism after successful simultaneous pancreas-kidney transplantation (SPK) improves vascular wall morphology and function in type 1 diabetic (T1D) patients. Therefore, we compared arterial stiffness, assessed by pulse wave velocity (PWV), carotid intima-media thickness (IMT), and biomarkers of arterial wall calcification in T1D patients after SPK or kidney transplantation alone (KTA). In 39 SPK and 39 KTA adult patients of similar age, PWV, IMT, circulating matrix metalloproteinases (MMPs) and calcification biomarkers were assessed at median 83 months post transplantation. Additionally, carotid plaques were visualized and semi-qualitatively classified. Although PWV and IMT values were similar, the occurrence of atherosclerotic plaques (51.3 vs. 70.3%, p &lt; 0.01) and calcified lesions (35.9 vs. 64.9%, p &lt; 0.05) was lower in SPK patients. There were significantly lower concentrations of MMP-1, MMP-2, MMP-3, and osteocalcin in SPK subjects. Among the analyzed biomarkers, only logMMP-1, logMMP-2, and logMMP-3 concentrations were associated with log HbA1c. Multivariate stepwise backward regression analysis revealed that MMP-1 and MMP-3 variability were explained only by log HbA1c. Normal glucose metabolism achieved by SPK is followed by the favorable profile of circulating matrix metalloproteinases, which may reflect the vasoprotective effect of pancreas transplantation