Anne Troldborg

Polymorphisms within Toll-like receptors are associated with systemic lupus erythematosus in a cohort of Danish females

OBJECTIVES Toll-like receptors (TLRs) are pattern-associated receptors in innate immunity that may be involved in the recognition of self-antigens and the production of pathogenic autoantibodies. This study was undertaken to examine whether polymorphisms of TLR genes are associated with SLE and to determine the expression of various TLRs in peripheral blood mononuclear cells (PBMCs) of patients with SLE. METHODS The TLR polymorphisms in a cohort of 143 Danish lupus patients and 432 healthy Danish blood donors were analysed. Groups were age matched. Genotyping for the TLR single-nucleotide polymorphisms (SNPs) was performed using Sequenom Multiplex technology. In addition, the mRNA expression of TLRs in PBMCs from 56 SLE patients and 56 healthy controls was studies by quantitative real-time PCR. RESULTS We found a genetic association with SLE and three SNPs located within the TLR3, TLR8 and TLR9 genes (rs3775291, P = 0.006; rs37648, P = 0.013; rs352143, P < 0.02). Furthermore, the relative TLR7, TLR8, IFN-α and LY6E mRNA expression levels were significantly higher in SLE patients than in healthy controls (P < 0.0001, P < 0.0001, P = 0.0004 and P < 0.0001, respectively). CONCLUSION These results obtained from a female lupus population of Danish ancestry suggest that variations in TLR3, TLR8 and TLR9 genes are implicated in the pathogenesis of the disease. If these polymorphisms are associated with innate immune dysfunction they may add to the growing field of theoretically well founded new therapeutic targets.

Lectin Complement Pathway Proteins in Healthy Individuals

Since the discovery of the lectin pathway of complement activation, numerous clinical cohorts have been examined for one or more proteins, with the intention of uncovering the functions of the proteins or with the aim of discovering new biomarkers or diagnostic tools. To unveil the abnormal, it is pivotal to know the normal. Our aim was to describe the concentrations of the 11 known proteins of the lectin pathway in serum and plasma and to uncover possible gender differences, age and diurnal variations, which must be taken into account for investigation in different cohorts. We examined the concentrations of all lectin pathway proteins mannan‐binding lectin (MBL), H‐ficolin, L‐ficolin, M‐ficolin, collectin‐K1, collectin‐L1, MBL‐associated serine protease 2 (MASP‐2), MASP‐3, MBL‐associated protein of 44 kDa (MAp44) and MAp19 in 300 Danish blood donors in serum and ethylenediamine tetraacetic acid (EDTA) plasma in established assays, and we further developed a new assay to measure MASP‐1 in the same samples. We found significant differences in concentrations between serum and plasma for all proteins except for MBL and MASP‐3. H‐ficolin, M‐ficolin and MAp19 displayed convincing diurnal variation. H‐ficolin, in particular, halved from morning to the middle of the night. There were gender differences for most proteins, whereas age did not seem to influence concentration. The present study underlines the necessity of considering which material to use, correct matching and a trial design that takes the nature of the protein into account in order for the outcome of cohort studies to have significant relevance.

A non-synonymous single-nucleotide polymorphism in the gene encoding Toll-like Receptor 3 (TLR3) is associated with sero-negative Rheumatoid Arthritis (RA) in a Danish population

BackgroundIt has been suggested that polymorphisms in Toll-like Receptors (TLRs) are associated with Rheumatoid Arthritis (RA), but the implicated alleles have differed between studies. The aim of this investigation was to explore whether polymorphisms of TLR genes are associated with RA in a predominantly Caucasian population from Denmark using a case–control approach.FindingsDNA samples (3 university hospital outpatient clinics) were obtained from patients with RA (n = 704) and healthy controls (n = 639) in a Danish population. TLR single nucleotide polymorphisms (SNPs) were selected based on the previously reported associations with chronic autoimmune diseases. Genotyping for the TLR SNPs was performed using Sequenom Multiplex technology.We identified one SNP in TLR3, [(rs3775291, P = 0.02, OR (95% CI) 1.31 (1.1087-1.5493)] significantly associated with the whole RA cohort. Subgroup analysis according to IgM rheumatoid factor (RF) and anti-cyclic citrinullated peptide (CCP) status suggested a significant association of sero-negative RA with the rs3775291 A allele and disease activity in this subset.ConclusionThese observations on a RA population of Danish ancestry suggest that variations in the TLR3 locus may be implicated in the pathogenesis of sero-negative RA. Since this TLR3 SNP has previously been associated with systemic lupus erythematous (SLE), the present findings support the notion that TLR3 genetic variants may represent a common risk factor in different chronic inflammatory conditions, including RA and SLE.

Levels in plasma of the serine proteases and associated proteins of the lectin pathway are altered in patients with systemic lupus erythematosus.

Objective. To examine whether proteins of the lectin pathway of the complement system are involved in systemic lupus erythematosus (SLE) pathogenesis. Methods. Using time-resolved immunofluorometric assays, plasma levels of mannan-binding lectin (MBL)-associated serine proteases 1 (MASP-1), MASP-2, MASP-3, MBL-associated protein of 19 kDa (MAp19), and MAp44 were determined in 58 patients with SLE and 65 healthy controls (HC). Results. Plasma concentrations in patients with SLE were higher than HC regarding MASP-1, MASP-3, and MAp44 (p < 0.0001, 0.0003, and 0.0013). Complement factor 3 correlated negatively and anti-dsDNA positively with levels of MAp19 (p = 0.0035, p = 0.0133). Conclusion. In SLE, plasma levels of MASP and MAp are altered and associated with SLE characteristics, supporting a role in SLE pathogenesis.

Human endogenous retroviral genetic element with immune suppressive activity in both human autoimmune diseases and experimental arthritis

Human endogenous retroviruses (HERVs) are remnants of past retroviral infections in the human genome and have been implicated in different aspects of human biology. The aim of this study was to identify HERVs that are associated with the pathogenesis of rheumatic diseases such as systemic lupus erythematosus (SLE).

AB0082 Anti-Inflammatory Activity of A Human Endogenous Retroviral Genetic Element in Experimental Arthritis

Background Human Endogenous Retroviruses (HERVs) are ancient retroviral integrations fixed in the genome of humans. As for all retroviruses, HERVs contain three protein coding genes: gag, pol and env. The retroviral envelope proteins can influence human biology through their inherent immune suppressive activity (1) localized to a secondary protein structure (the ISU domain) in the envelope protein. Our previous research has shown that the env-59-protein of HERV-H is negatively correlated to the inflammatory marker IL-6 in SLE patients, and has an inhibitory effect on stimulated PBMCs from both RA and SLE patients. We aimed to investigate, if this effect could be translated from in vitro to in vivo experiments. Objectives To examine the anti-inflammatory potential of a peptide based on the ISU domain of HERV-H ENV-59 in the SKG mouse model (2). Methods At 7–11 weeks of age, female SKG mice were randomized to treatment groups. Arthritis was induced with mannan. After disease induction, the animals were treated daily with subcutaneous injections of a synthetically developed peptide (Env59-ISU), a scrambled peptide or saline. Two 4-week studies were performed. In study one, we wished to demonstrate an anti-inflammatory effect of Env59 ISU. Three groups of 5 mice were tested (group #1: 4 μg peptide/day, group #2:40 μg peptide/day, group #3: saline). Study two aimed to confirm the results of study one. Forty mice were randomized to 5 treatment groups (group 1: saline, group 2: 1μg/peptide/day, group 3: 4μg/peptide/day, group 4: 20 μg/peptide/day and group 5: 4 μg/scrambled-peptide/day). SKG arthritis was assessed and scoring was performed independently by two persons and blinded to treatment group. Hind paws from two mice were fixated in 70% ethanol, and subsequently embedded undecalcified in methylmethacrylate. Tissue sections were stained with Masson-Golden trichrome. Results In study one, we found significantly lower arthritis score in Env59-treated mice compared to those treated with saline. Group #3 receiving 4μg peptide had the lowest average arthritis score throughout the trial (Fig.1), which was confirmed by study two showing, that the 4μg peptide- treated group (group 3) had significantly lower arthritis score compared to saline and the scrambled peptide. The pattern observed in the arthritis score was confirmed using serum amyloid A3 (SAA3) as a biomarker for inflammation and using histology in mice treated with saline compared to peptide-treated mice.Figure 1 Conclusions We demonstrate that a synthetic peptide corresponding to the sequence of Env59-ISU has anti-inflammatory effect in a chronic inflammatory mouse model of RA and propose the peptide as a potential new treatment for inflammatory diseases. References Tolstrup M et al. Anti-inflammatory effect of a retrovirus-derived immunosuppressive peptide in mouse models. BMC Immunol. 2013;14:51 Sakaguchi N et al. Altered thymic T-cell selection due to a mutation of the ZAP-70 gene causes autoimmune arthritis in mice. Nature. 2003 Nov 27;426(6965):454–60. Disclosure of Interest None declared

Evidence for the Use of Tranexamic Acid in Subarachnoid and Subdural Hemorrhage: A Systematic Review

Abstract Despite neurosurgical treatment, a subarachnoid hemorrhage (SAH) can cause a debilitating stroke. The case fatality rate ranges from 27 to 44%, and many survivors are left permanently disabled; therefore, the need for improved treatment is obvious. Furthermore, subdural hemorrhages (SDHs) have a cumulative recurrence rate of 14% in the first year and are potentially fatal. The aim of this systematic review is to assess and discuss the evidence for the role of tranexamic acid (TXA) in treatment of SAH and SDH. Systematic literature searches were performed in MEDLINE, Embase, Scopus, and Web of Science (1946‐2016). The inclusion criteria were TXA‐treated group and control group; SAH or SDH verified by imaging, intraoperatively or at autopsy; human subjects; English; and an objective outcome. The search terms matched 443 records. Eight studies including nontraumatic SAH patients and one study including traumatic intracranial bleeding met the inclusion criteria. Comparing TXA‐treated nontraumatic patients with controls, we found an overall odds ratio (OR) for rebleeding of 0.41 (95% confidence interval [CI]: 0.26‐0.64), six studies reported the mortality rate with an overall OR of 0.88 (95% CI: 0.68‐1.12), four studies reported no statistically significant difference on the Glasgow Outcome Scale, and one study showed a statistically significant increased risk of cerebral ischemia, whereas seven studies found no statistically significant difference. No studies including SDH patients met the inclusion criteria. TXA reduced the overall risk of rebleeding following nontraumatic SAH. A nonsignificant reduction in mortality was demonstrated in nontraumatic SAH without substantial indication of increased risk of ischemic lesions.

The lectin pathway and coagulation in lung cancer patients undergoing lobectomy – A randomised controlled trial

BACKGROUND Lectin pathway proteases activate coagulation and may theoretically play a role in the increased thrombosis risk in cancer, which is especially high during surgery. AIMS To investigate lectin pathway proteins during lung cancer surgery, the influence of low molecular weight heparin (LMWH) on lectin pathway proteins, and correlations between lectin pathway proteins and coagulation. METHODS Fifty lung cancer patients undergoing video-assisted thoracoscopic surgery lobectomy were randomised to LMWH, n = 26, or no anticoagulant (control), n = 24. Pre-, intra- and postoperative lectin pathway protein concentrations (mannose-binding lectin (MBL), H- and M-ficolin, collectin liver-1, MBL-associated serine protease (MASP)-1, -2 and -3, MBL-associated proteins MAp44 and MAp19) were assessed using a time-resolved immunofluorometric assay, and fibrinogen, fibrin d-dimer, and thrombin generation were analysed. RESULTS For all proteins except MASP-1, concentrations decreased during surgery in both groups; most markedly M-ficolin which decreased 49% (median: 2836 [quartiles:2297-3505] to 1424 [1187-2199] ng/ml) (LMWH group) and 43% (2974 [2539-3510] to 1685 [1391-2076] ng/ml) (control group), while MBL decreased 12% (1936 [823-2801] to 1702 [676-2830] ng/ml) (LMWH) and 23% (1526 [250-2412] to 1175 [229-1947]) (controls). No differences in postoperative change were observed between groups except for MAp19 (p = 0.03) which decreased 9% (401 [337-467] to 364 [288-416] ng/ml) (LMWH) vs 28% (370 [272-468] to 268 [212-379] ng/ml) (controls). No correlation was found between lectin pathway proteins and coagulation (r = -0.23-0.28, p > 0.06) except for M-ficolin and fibrinogen (r = 0.29-0.36, p = 0.01-0.04). CONCLUSION Lectin pathway proteins were influenced by surgery but not by LMWH. No consistent correlation was found between lectin pathway proteins and coagulation.

The Lectin Pathway of Complement Activation in Patients with Systemic Lupus Erythematosus

Objective. The pathogenesis of systemic lupus erythematosus (SLE) involves complement activation. Activation of complement through the classical pathway (CP) is well established. However, complement activation through pattern recognition not only happens through the CP, but also through the lectin pathway (LP). We investigated the hypothesis that the LP is activated in SLE and involved in the pathogenesis of the disease. Methods. Using immunoassays developed in-house, we measured concentrations of LP proteins in a cohort of 372 patients with SLE and 170 controls. We estimated complement activation measuring total C3, and investigated whether LP protein concentrations were associated with complement activation and disease activity. Protein changes and disease activity over time were assessed in a cohort of 52 patients with SLE followed with repeated samples over a 5-year period. Results. Concentrations of LP proteins in SLE were altered compared with controls. The differences observed in LP proteins associated with complement activation were reflected by a decrease in total C3. The pattern recognition molecules (M-ficolin, CL-L1, and CL-K1), the serine protease (MASP-3), and the associated protein (MAp19) displayed a negative correlation with disease activity. Changes in MASP-2 concentrations over time correlated significantly with increased disease activity. Association between active proteinuria and serum concentration was observed for MASP-3 and MAp19. Conclusion. In patients with SLE, we measured specific changes in LP proteins that are associated with complement activation and disease activity, indicating that the LP is activated in patients with SLE. These novel findings substantiate the involvement of the LP in SLE.

The complement lectin pathway after cardiac arrest

The lectin pathway (LP) of the complement system may initiate inflammatory reactions when body tissue is altered. We aimed to investigate the levels of the LP proteins in out‐of‐hospital cardiac arrest patients, and to compare these with healthy individuals. Furthermore, we aimed to clarify whether the duration of targeted temperature management influenced LP protein levels, and we further examined whether LP proteins were associated with 30‐day mortality. We included 82 patients resuscitated from out‐of‐hospital cardiac arrest. The patients were randomly assigned to 24 or 48 hours of targeted temperature management at 33 ± 1°C. Blood samples were obtained 22, 46 and 70 hours after target temperature was reached. Levels of the LP proteins (mannan‐binding lectin [MBL], M‐ficolin, H‐ficolin, collectin liver 1 [CL‐L1], MBL‐associated serine protease 1 [MASP‐1], MASP‐2, MASP‐3 and MBL‐associated protein of 44 kDa [MAp44]) were measured using time‐resolved immunofluorometric assays. Data from 82 gender matched healthy individuals were used for comparison. Levels of CL‐L1, MASP‐1, MASP‐2 and MAp44 were significantly higher, whereas M‐ficolin levels were significantly lower in cardiac arrest patients compared with healthy individuals. MASP‐2, MASP‐3 and M‐ficolin levels changed significantly when comparing 24 and 48 hours of targeted temperature management. The LP protein levels were not different between 30‐day survivors and non‐survivors after cardiac arrest. The differences in LP protein levels between patients and healthy individuals may indicate that cardiac arrest patients have an activated LP. Overall, the LP protein levels were not influenced by the duration of targeted temperature management, and the levels were not associated with 30‐day mortality.