Showing papers by "Matija Tomšič published in 2018"

Clinical Pharmacogenetic Models of Treatment Response to Methotrexate Monotherapy in Slovenian and Serbian Rheumatoid Arthritis Patients: Differences in Patient's Management May Preclude Generalization of the Models

Abstract: Objectives: Methotrexate (MTX) is the first line treatment for rheumatoid arthritis (RA), but nevertheless 30% of patients experience MTX inefficacy. Our aim was to develop a clinical pharmacogenetic model to predict which RA patients will not respond to MTX monotherapy. We also assessed whether this model can be generalized to other populations by validating it on a group of Serbian RA patients. Methods: In 110 RA Slovenian patients, data on clinical factors and 34 polymorphisms in MTX pathway were analyzed by Least Absolute Shrinkage and Selection Operator (LASSO) penalized regression to select variables associated with the disease activity as measured by Disease Activity Score (DAS28) score after 6 months of MTX monotherapy. A clinical pharmacogenetic index was constructed from penalized regression coefficients with absolute value above 0.05. This index was cross-validated and also independently validated on 133 Serbian RA patients. Results: A clinical pharmacogenetic index for prediction of DAS28 after 6 months of MTX monotherapy in Slovenian RA patients consisted of DAS28 score at diagnosis, presence of erosions, MTX dose, Solute Carrier Family 19 Member 1 (SLC19A1) rs1051266, Solute Carrier Organic Anion Transporter Family Member 1B1 (SLCO1B1) rs2306283, Thymidylate Synthase (TYMS), and Adenosine Monophosphate Deaminase 1 (AMPD1) rs17602729. It correctly classified 69% of Slovenian patients as responders or nonresponders and explained 30% of variability in DAS28 after 6 months of MTX monotherapy. Testing for validity in another population showed that it classified correctly only 22.5% of Serbian RA patients. Conclusions: We developed a clinical pharmacogenetic model for DAS28 after 6 months of MTX monotherapy in Slovenian RA patients by combining clinical and genetic variables. The clinical pharmacogenetic index developed for Slovenian patients did not perform well on Serbian patients, presumably due to the differences in patients' characteristics and clinical management between the two groups.

X-Ray Diffraction and Computer Simulation Studies of the Structure of Liquid Aliphatic Aldehydes: From Propanal to Nonanal

Abstract: Synchrotron X-ray diffraction experiments and molecular dynamics simulations have been performed on simple aliphatic aldehydes, from propanal to nonanal. The performance of the OPLS all-atom interaction potential model for aldehydes has been assessed via direct comparison of simulated and experimental total scattering structure factors. In general, MD results reproduce the experimental data at least semi-quantitatively. However, a slight mismatch can be observed between the two datasets in terms of the position of the main diffraction maxima. Partial radial distribution functions have also been calculated from the simulation results. Clear differences could be detected between the various O-H partial radial distribution functions, depending on whether the H atom is attached to the carbon atom that is doubly bonded to the oxygen atom of the aldehyde group or not.

Naturally occurring antibodies against serum amyloid A reduce IL-6 release from peripheral blood mononuclear cells

Abstract: Serum amyloid A (SAA) is a sensitive inflammatory marker rapidly increased in response to infection, injury or trauma during the acute phase. Resolution of the acute phase and SAA reduction are well documented, however the exact mechanism remains elusive. Two inducible SAA proteins, SAA1 and SAA2, with their variants could contribute to systemic inflammation. While unconjugated human variant SAA1α is already commercially available, the variants of SAA2 are not. Antibodies against SAA have been identified in apparently healthy blood donors (HBDs) in smaller, preliminary studies. So, our objective was to detect anti-SAA and anti-SAA1α autoantibodies in the sera of 300 HBDs using ELISA, characterize their specificity and avidity. Additionally, we aimed to determine the presence of anti-SAA and anti-SAA1α autoantibodies in intravenous immunoglobulin (IVIg) preparations and examine their effects on released IL-6 from SAA/SAA1α-treated peripheral blood mononuclear cells (PBMCs). Autoantibodies against SAA and SAA1α had a median (IQR) absorbance OD (A450) of 0.655 (0.262–1.293) and 0.493 (0.284–0.713), respectively. Both anti-SAA and anti-SAA1α exhibited heterogeneous to high avidity and reached peak levels between 41–50 years, then diminished with age in the oldest group (51–67 years). Women consistently exhibited significantly higher levels than men. Good positive correlation was observed between anti-SAA and anti-SAA1α. Both anti-SAA and anti-SAA1α were detected in IVIg, their fractions subsequently isolated, and shown to decrease IL-6 protein levels released from SAA/SAA1α-treated PBMCs. In conclusion, naturally occurring antibodies against SAA and anti-SAA1α could play a physiological role in down-regulating their antigen and proinflammatory cytokines leading to the resolution of the acute phase and could be an important therapeutic option in patients with chronic inflammatory diseases.

Unexpected Composition Dependence of the First Sharp Diffraction Peak in an Alcohol-Aldehyde Liquid Mixture: n-Pentanol and Pentanal

Abstract: The total scattering structure factors of pure liquid n-pentanol, pentanal, and 5 of their mixtures, as determined by high energy synchrotron X-ray diffraction experiments, are presented. For the interpretation of measured data, molecular dynamics computer simulations are performed, utilizing 'all-atom' type force fields. The diffraction signals in general resemble each other over most of the monitored Q range above 1 $A^{-1}$, but the absolute values of the intensities of the small-angle scattering maximum ('pre-peak', 'first sharp diffraction peak'), around 0.6 $A^{-1}$, change in an unexpected fashion, non-linearly with the composition. MD simulations are not able to reproduce this low-Q behavior; on the other hand, they do reproduce the experimental diffraction data above 1 $A^{-1}$ rather accurately. Partial radial distribution functions are calculated based on the atomic coordinates in the simulated configurations. Inspection of the various O-O and O-H partial radial distribution functions clearly shows that both the alcoholic and the aldehydic oxygens form hydrogen bonds with the hydrogen atoms of the alcoholic OH-group.

X-ray diffraction and computer simulation studies of the structure of liquid aliphatic aldehydes: from propanal to nonanal

Abstract: Synchrotron X-ray diffraction experiments and molecular dynamics simulations have been performed on simple aliphatic aldehydes, from propanal to nonanal. The performance of the OPLS all-atom interaction potential model for aldehydes has been assessed via direct comparison of simulated and experimental total scattering structure factors. In general, MD results reproduce the experimental data at least semi-quantitatively. However, a slight mismatch can be observed between the two datasets in terms of the position of the main diffraction maxima. Partial radial distribution functions have also been calculated from the simulation results. Clear differences could be detected between the various O-H partial radial distribution functions, depending on whether the H atom is attached to the carbon atom that is doubly bonded to the oxygen atom of the aldehyde group or not.

Giant Cell Arteritis and Malignancy-More Than Just a Coincidence?

Abstract: BACKGROUND The association between malignancy and vasculitis is difficult to establish and is thought to be uncommon. Whereas there have been reports of increased incidence ofmalignancy in patients with giant cell arteritis (GCA), several studies found no increased risk. The objective of our study was to determine whether there was an increased incidence of malignancy in our cohort of prospectively followed patients with GCA.

Unexpected composition dependence of the first sharp diffraction peak in an alcohol-aldehyde liquid mixture: n-pentanol and pentanal

Abstract: The total scattering structure factors of pure liquid n-pentanol, pentanal, and 5 of their mixtures, as determined by high energy synchrotron X-ray diffraction experiments, are presented. For the interpretation of measured data, molecular dynamics computer simulations are performed, utilizing 'all-atom' type force fields. The diffraction signals in general resemble each other over most of the monitored Q range above 1 $A^{-1}$, but the absolute values of the intensities of the small-angle scattering maximum ('pre-peak', 'first sharp diffraction peak'), around 0.6 $A^{-1}$, change in an unexpected fashion, non-linearly with the composition. MD simulations are not able to reproduce this low-Q behavior; on the other hand, they do reproduce the experimental diffraction data above 1 $A^{-1}$ rather accurately. Partial radial distribution functions are calculated based on the atomic coordinates in the simulated configurations. Inspection of the various O-O and O-H partial radial distribution functions clearly shows that both the alcoholic and the aldehydic oxygens form hydrogen bonds with the hydrogen atoms of the alcoholic OH-group.

Analysis of Drug Effects on Primary Human Coronary Artery Endothelial Cells Activated by Serum Amyloid A

Abstract: Background. RA patients have a higher incidence of cardiovascular diseases compared to the general population. Serum amyloid A (SAA) is an acute-phase protein, upregulated in sera of RA patients. Aim. To determine the effects of medications on SAA-stimulated human coronary artery endothelial cells (HCAEC). Methods. HCAEC were preincubated for 2 h with medications from sterile ampules (dexamethasone, methotrexate, certolizumab pegol, and etanercept), dissolved in medium (captopril) or DMSO (etoricoxib, rosiglitazone, meloxicam, fluvastatin, and diclofenac). Human recombinant apo-SAA was used to stimulate HCAEC at a final 1000 nM concentration for 24 hours. IL-6, IL-8, sVCAM-1, and PAI-1 were measured by ELISA. The number of viable cells was determined colorimetrically. Results. SAA-stimulated levels of released IL-6, IL-8, and sVCAM-1 from HCAEC were significantly attenuated by methotrexate, fluvastatin, and etoricoxib. Both certolizumab pegol and etanercept significantly decreased PAI-1 by an average of 43%. Rosiglitazone significantly inhibited sVCAM-1 by 58%. Conclusion. We observed marked influence of fluvastatin on lowering cytokine production in SAA-activated HCAEC. Methotrexate showed strong beneficial effects for lowering released Il-6, IL-8, and sVCAM-1. Interesting duality was observed for NSAIDs, with meloxicam exhibiting opposite-trend effects from diclofenac and etoricoxib. This represents unique insight into specific responsiveness of inflammatory-driven HCAEC relevant to atherosclerosis.

P053 Serum amyloid a can modulate neutrophil surface expression of l-selectin and integrin alpha m

Abstract: Introduction Serum amyloid A (SAA) is one of the major acute phase proteins, elevated in the sera of newly diagnosed patients with giant cell arteritis (GCA).1 SAA was previously shown to activate neutrophils and recently, neutrophils have been recognised as active players in GCA pathogenesis, exhibiting changes in surface protein expression of l-selectin (CD62L) and integrin αM (CD11b) during therapy tapering.2 Objectives To determine the expression of CD62L and CD11b on neutrophils in peripheral blood of newly diagnosed, steroid naive GCA patients (day 0) vs. healthy blood donors (HBDs). We also aimed to examine the ability of SAA and SAA1α to activate neutrophils in whole blood of HBDs and GCA patients prior to receiving glucocorticoid therapy and 7 and 30 days after therapy. Methods Whole blood of 37 GCA patients and 17 HBDs was stained with anti-CD62L and anti-CD11b (eBioscience), lysed and analysed by flow cytometer (Miltenyi). Whole blood of 5 GCA and 6 HBDs was stimulated with 10 µM hrSAA and hrSAA1α for 20 min at 37°C, lysed and incubated on melting ice for 15 min. Samples were then stained with anti-CD62L and anti-CD11b. SAA in sera of GCA patients was measured by nephelometry. Results We observed higher neutrophil counts and surface expression of CD62L in peripheral blood of naive GCA patients compared to HBDs (p=0.006). Stimulation of whole blood with hrSAA or hrSAA1α significantly decreased CD62L and increased CD11b expression on neutrophils of HBDs and naive GCA patients. Whole blood stimulation with hrSAA/SAA1α caused significant attenuation of CD62L, while increasing CD11b expression on neutrophils of naive GCA patients (day 0) as compared to 7 and 30 days after therapy. Levels of SAA in GCA patients decreased after receiving therapy. Conclusions We show that hrSAA and hrSAA1α activate neutrophils implying tight adhesion and transendothelial migration. Interestingly however, in patients with GCA, increased SAA did not shed CD62L, as we observed elevation of both. The latter indicates potential initial attachment of neutrophils to activated endothelium, as a possible mechanism in GCA pathogenesis. References . 0‘Neill L, et al. Regulation of inflammation and angiogenesis in giant cell arteritis by acute-phase serum amyloid A. Arthritis Rheum2015;67:2447–56. . Nadkarni S, et al. Investigational analysis reveals a potential role for neutrophils in GCA disease progression. Circ Res2014;114:242–48. Acknowledgements The authors would like to thank the Slovenian Research Agency (ARRS) for providing funding for the National Research program P3-0314, as well as the Rotary Club Zgornji Brnik, Slovenia for contributing funding for the flow cytometer. We would also like to thank Prof. Mauro Peretti and Dr. Suchita Nadkarni from WHRI, Queen Mary, University of London for their support in setting up the protocols. Disclosure of interest None declared

P071 Autoantibodies against serum amyloid a reduce il-6 release from peripheral blood mononuclear cells

Abstract: Introduction Serum amyloid A (SAA) is a sensitive inflammatory marker rapidly increased during the acute phase, followed by a steady decline to physiological levels during resolution. While resolution and SAA reduction have been documented, the exact mechanism remains elusive. Although antibodies against SAA (anti-SAA) have been previously identified in healthy blood donors (HBDs) in smaller, preliminary studies, their potential function is still unclear.1,2 Objectives To detect anti-SAA and anti-SAA1α in the sera of 300 HBDs using ELISA, characterise their subclasses and avidity. Additionally, we aimed to evaluate their presence in intravenous immunoglobulin (IVIG) and potential effects on released IL-6 from SAA-treated peripheral blood mononuclear cells (PBMCs). Methods An in-house ELISA was adapted from Rosenau and Schur1 and developed2 for detection of anti-SAA and anti-SAA1α. Both antibody fractions were isolated from IVIG using MicroLink Protein Coupling Kit (Thermo Scientific). PBMCs were purified from 5 HBDs by density gradient centrifugation and stimulated with SAA or SAA1α (1.5 µg/ml) in the presence/absence of anti-SAA and anti-SAA1α for 5 hours, 37°C. IL-6 concentration was measured in supernatants by ELISA (Invitrogen). Results The median (IQR) absorbance in HBDs was 0.655 (0.262–1.293) for anti-SAA and 0.493 (0.284–0.713) for anti-SAA1α. Both anti-SAA and anti-SAA1α reached peak levels between 41–50 years and diminished with age, with women exhibiting significantly higher levels than men. Good positive correlation was observed between anti-SAA and anti-SAA1α. Both antibodies were prevalently of the IgG subclass, with heterogeneous to high avidity and were detected also in IVIG. Stimulation of PBMCs with SAA significantly induced IL-6 release (mean ±SD) (389.5±184.4 pg/ml) with levels decreasing significantly upon addition of 4.5 (131.4±44.4 pg/ml) or 9.0 µg/ml (118.1±57.4 pg/ml) anti-SAA. A similar trend was also found for SAA1α and anti-SAA1α. Conclusions Anti-SAA could play a physiological role in down-regulating proinflammatory activity of SAA and could represent an attractive, novel therapeutic option for patients with chronic inflammatory diseases. References . Rosenau BJ, Schur PH. Antibody to serum amyloid A. J Autoimmun2004;23:179–82. . Lakota K, Thallinger GG, Cucnik S, Bozic B, Mrak-Poljsak K, Ambrozic A, et al. Could antibodies against serum amyloid a function as physiological regulators in humans?Autoimmunity2011;44:149–58. Acknowledgements The authors would like to acknowledge funding from the Slovenian Research Agency (ARRS) for the National Research Program P3-0314. Disclosure of interest None declared

AB0299 The incidence rate and the management of early rheumatoid arthritis in a single centre interventional clinic

Abstract: Background In patients with rheumatoid arthritis (RA), early diagnosis and adherence to the treat to target recommendations (T2T) limit RA progression and improve patients’ quality of life.1 However, the implementation of T2T has always been a challenge, and real-life data are lacking. Slovenia has 40% less rheumatologists per capita than the European Union average, which makes the implementation of management guidelines even more challenging. Objectives To determine the incidence of RA and the proportion of patients with incident RA in whom first rheumatology assessment was done within the recommended time frame. Methods We analysed the prospectively collected data of adult patients diagnosed with RA during years 2014 to 2016 at the Rheumatology Department of the University Medical Centre Ljubljana, Slovenia. The department provides rheumatology services to a well-defined region with a population of 704.000 adult residents. Dates were recorded for inflammatory joint symptom onset, referral to rheumatologist, first rheumatologic assessment and initiation of DMARD therapy. The percentage of patients assessed by a rheumatologist and/or treated with a DMARD within 12 weeks of symptom onset and the median times for delay were then calculated. Results Between 1 January 2014 and 31 December 2016, 341 incident cases of RA (75% females, median age 61.9 (IQR 52–75.4) years) were identified, resulting in an annual incidence rate of 16/100.000 population (in females: 23.6/100.000; in males 8.3/100.000). Most patients (78.6%) were referred to our early interventional clinic. The median time from symptom onset to consultation was 12.9 (IQR 4.4–26.1) weeks, median time from referral to consultation was 1 (IQR 1–3) day. Median DMARD treatment delay was 16.6 (IQR 8.9–33.3) weeks. Within 12 weeks of symptom onset, 161 (47.2%) new RA patients were examined by a rheumatologist and 123 (36.1%) were started on DMARD therapy. Conclusions Our prospective data support the recent reports that uncovered a decrease in RA incidence.2 Moreover, despite the lack of rheumatologists and the heavily protracted nationwide waiting times for first rheumatologist assessment, our early interventional clinic enables us to recognise and manage substantial percentage of RA patients within the recommended time frame. References [1] Monti S, et al. Rheumatoid arthritis treatment: the earlier the better to prevent joint damage. RMD Open2015;1(Suppl 1):e000057. doi:10.1136/rmdopen-2015-000057 [2] Abhishek, et al. Rheumatoid arthritis is getting less frequent—results of a nationwide population-based cohort study. Rheumatology2017;56:36–744–7. Disclosure of Interest None declared