Follow-up SARS-CoV-2 serological study of a health care worker cohort following COVID-19 booster vaccination.

BACKGROUND: Studies have shown that Omicron breakthrough infections can occur at higher SARS-CoV-2 antibody levels compared to previous variants. Estimating the magnitude of immunological protection induced from COVID-19 vaccination and previous infection remains important due to varying local pandemic dynamics and types of vaccination programmes, particularly among at-risk populations such as health care workers (HCWs). We analysed a follow-up SARS-CoV-2 serological survey of HCWs at a tertiary COVID-19 referral hospital in Germany following the onset of the Omicron variant. METHODS: The serological survey was conducted in January 2022, one year after previous surveys in 2020 and the availability of COVID-19 boosters including BNT162b2, ChAdOx1-S, and mRNA-1273. HCWs voluntarily provided blood for serology and completed a comprehensive questionnaire. SARS-CoV-2 serological analyses were performed using an Immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA). Antibody levels were reported according to HCW demographic and occupational characteristics, COVID-19 vaccination and SARS-CoV-2 infection history, and multivariate linear regression was used to evaluate these associations. RESULTS: In January 2022 (following the fourth COVID-19 wave in Germany including the onset of the Omicron variant), 1482/1517 (97.7%) HCWs tested SARS-CoV-2 seropositive, compared to 4.6% in December 2020 (second COVID-19 wave). Approximately 80% had received three COVID-19 vaccine doses and 15% reported a previous laboratory-confirmed SARS-CoV-2 infection. SARS-CoV-2 IgG geometric mean titres ranged from 335 (95% Confidence Intervals [CI]: 258-434) among those vaccinated twice and without previous infection to 2204 (95% CI: 1919-2531) among those vaccinated three times and with previous infection. Heterologous COVID-19 vaccination combinations including a mRNA-1273 booster were significantly associated with the highest IgG antibody levels compared to other schemes. There was an 8-to 10-fold increase in IgG antibody levels among 31 HCWs who reported a SARS-CoV-2 infection in May 2020 to January 2022 after COVID-19 booster vaccination. CONCLUSIONS: Our findings demonstrate the importance of ongoing COVID-19 booster vaccination strategies in the context of variants such as Omicron and despite hybrid immunity from previous SARS-CoV-2 infections, particularly for at-risk populations such as HCWs. Where feasible, effective types of booster vaccination, such as mRNA vaccines, and the appropriate timing of administration should be carefully considered.

Vascular Endothelial Growth Factor as Potential Biomarker for COVID-19 Severity.

INTRODUCTION: COVID-19 is characterized by immunological responses to viral replication and coherent with endothelitis, microvascular disturbance of lung vasculature and coagulopathy. Vascular Endothelial Growth Factor (VEGF) is a proangiogenic mediator regulating endothelial changes. It is induced by proinflammatory signaling and hypoxia. We sought to determine whether VEGF levels differ between SARS-CoV-2-positive patients of different disease severity and whether VEGF might be useful in risk stratification. METHODS: After retrospective screening of all SARS-CoV-2-positive patients treated in Unfallkrankenhaus Berlin in 2020, we included those with documented VEGF measurement. We extracted laboratory values and clinical parameters. An exploratory data analysis was performed to detect possible relations between VEGF level and clinical disease features. RESULTS: We included 167 SARS-CoV-2-positive patients of which 139 suffered from COVID-19. Seventy-one of the COVID-19 patients had to be treated in the intensive care unit (ICU), those patients exhibited higher VEGF levels than those being admitted to normal wards (535 vs 279 pg/L, P < .001). APACHE-2 (Acute Physiology And Chronic Health Evaluation Score) correlated with mortality and patients with high values showed higher VEGF concentrations on admission (456 vs 875 pg/L, p = 0.006). Receiver operating characteristic analytic revealed that the occurrence of organ dysfunctions like acute respiratory distress syndrome (ARDS), shock, or acute kidney injury could be predicted by VEGF. It was significantly higher in patients who later died compared to survivors (637 vs 389 pg/mL, P = 0.041) and predicted mortality with same accuracy as established markers. In our cohort, association of VEGF above 277 pg/L on admission with risk of ARDS could be confirmed in logistic regression adjusting for possible confounding factors (odds ratio 3.1, 95% confidence interval: 1.34-7.7). DISCUSSION: Even though there are several limitations to this retrospective study it revealed that in COVID-19 patients VEGF can contribute to the prediction of necessity of ICU, mortality and the prediction of ARDS, kidney injury or shock. Its use in risk stratification and potential pathogenetic involvement should be further investigated.

Longitudinal SARS-CoV-2 seroepidemiological investigation among healthcare workers at a tertiary care hospital in Germany.

BACKGROUND: SARS-CoV-2 cases in Germany increased in early March 2020. By April 2020, cases among health care workers (HCW) were detected across departments at a tertiary care hospital in Berlin, prompting a longitudinal investigation to assess HCW SARS-CoV-2 serostatus with an improved testing strategy and associated risk factors. METHODS: In May/June and December 2020, HCWs voluntarily provided blood for serology and nasopharyngeal/oropharyngeal (NP/OP) samples for real-time polymerase chain reaction (PCR) and completed a questionnaire. A four-tiered SARS-CoV-2 serological testing strategy including two different enzyme-linked immunosorbent assays (ELISA) and biological neutralization test (NT) was used. ELISA-NT correlation was assessed using Pearson's correlation coefficient. Sociodemographic and occupational factors associated with seropositivity were assessed with multivariate logistic regression. RESULTS: In May/June, 18/1477 (1.2%) HCWs were SARS-CoV-2 seropositive, followed by 56/1223 (4.6%) in December. Among those tested in both, all seropositive in May/June remained seropositive by ELISA and positive by NT after 6 months. ELISA ratios correlated well with NT titres in May/June (R = 0.79) but less so in December (R = 0.41). Those seropositive reporting a past SARS-CoV-2 positive PCR result increased from 44.4% in May/June to 85.7% in December. HCWs with higher occupational risk (based on profession and working site), nurses, males, and those self-reporting COVID-19-like symptoms had significantly higher odds of seropositivity. CONCLUSIONS: This investigation provides insight into the burden of HCW infection in this local outbreak context and the antibody dynamics over time with an improved robust testing strategy. It also highlights the continued need for effective infection control measures particularly among HCWs with higher occupational risk.

Relation of α2-Antiplasmin Genotype and Genetic Determinants of Fibrinogen Synthesis and Fibrin Clot Formation with Vascular Endothelial Growth Factor Level in Axial Spondyloarthritis.

OBJECTIVE: Coagulation and fibrinolysis are interrelated with the expression of vascular endothelial growth factor (VEGF), which frequently is increased in axial spondyloarthritis (axSpA). We tested whether (i) α2-antiplasmin (A2AP) Arg6Trp, (ii) fibrinogen, factor XIII A-subunit or B-subunit genotypes are associated with VEGF levels and assessed whether the known association between elevated VEGF and radiographic spinal progression in axSpA depends on genetic background. METHODS: One hundred and eighty-six axSpA patients from the German Spondyloarthritis Inception Cohort were genotyped, characterized for VEGF levels, and statistically analyzed. The association between VEGF and radiographic spinal progression was assessed in dependence on genetic background in stratified analyses. RESULTS: A2AP 6Trp carriage was associated with VEGF elevation (OR: 2.37, 95% CI: 1.06-5.29) and VEGF levels (6Trp, 455 ± 334 pg/mL; 6Arg/Arg, 373 ± 293 pg/mL; p < 0.008). Association between elevated VEGF and radiographic spinal progression in axSpA (OR: 3.11, 95% CI: 1.02-8.82) depended remarkably on the fibrinogen (FGA) genotype. When considering axSpA patients with elevated VEGF, in FGA rs6050A>G wild types, 42.1% of patients (8 of 19) progressed, while in G-allele carriers, no radiographic progression happened (0 of 13) (p < 0.04). CONCLUSIONS: The A2AP Arg6Trp genotype seems to influence VEGF levels in axSpA. The predictive value of VEGF elevations in respect of radiographic spinal progression in axSpA depends on FGA genotypes.

Sialic acid methylation refines capillary electrophoresis laser-induced fluorescence analyses of immunoglobulin G N-glycans of ovarian cancer patients.

Alterations in IgG N-glycosylation coincide with the development of a number of diseases including cancer and could potentially be used as diagnostic markers. CE-LIF of 8-aminopyrene-1,3,6-trisulfonic acid labeled N-glycans is a well-established rapid method to characterize IgG N-glycans that needs only low amounts of starting material. However,sialylated N-glycans have short migration times due to their negative charge. As a result,some of them are not well resolved and co-migrate with neutral glycans. In this work, we neutralized the negative charge of sialic acids by methylation and optimized the protocol using the commercially available disialylated biantennary oligosaccharide (A2G2S2). IgGN-glycans isolated from healthy human serum were then analyzed using this method. We could demonstrate that co-migration of A2, FA2G2S1, and FA2B[3]G1S1 was prevented,which allowed an accurate quantification of these N-glycans. Finally, we investigated the IgG N-glycan profiles of patients suffering from ovarian cancer using the conventional and methylation methods.With both methods, we observed an increase of agalactosylated structures that was accompanied by a decrease in digalactosylated structures. Finally, using the methylation protocol, we could further demonstrate an increase of A2, which was technically impossible with the conventional method.

Identification of 34 N-glycan isomers in human serum by capillary electrophoresis coupled with laser-induced fluorescence allows improving glycan biomarker discovery.

Alterations in glycosylation have been observed in many human diseases and specific changes in glycosylation have been proposed as relevant diagnostic information. Capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) is a robust method to quantify desialylated N-glycans that are labeled with 8-aminopyrene-1,3,6-trisulfonic acid prior to analysis. To date, only a maximum of 12 glycan structures, the most abundant ones, have been identified by CE-LIF to characterize glycome modulations of total serum in the course of the diseases. In most forms of cancer, findings using CE-LIF were limited to the increase of triantennary structures carrying a Lewis(x) epitope. In this work, we identified 32 linkage and positional glycan isomers in healthy human serum using exoglycosidase digestions as well as standard glycoproteins, for which we report the assignment of novel structures. It was possible to identify and quantify 34 glycan isomers in the serum of primary epithelial ovarian cancer patients (EOC). Reduced levels of diantennary structures and of high-mannose 5 were statistically significant in the EOC samples, and also, elevated branching as well as increased antennary fucosylation were observed. For the first time, we could demonstrate that not only antennary fucosylation was of relevance in tetraantennary structures but also core-fucosylated tetraantennary N-glycans were statistically increased in EOC patients. The results of the current study provide an improved dataset to be used in glycan biomarker discovery.

Polyclonal Anti-D Antibodies Significantly Reduce the Rate of Miscarriages in Rh(D) positive Women with Recurrent Pregnancy loss.

Background: Macrophages play a key role in all environmental conditions surrounding pregnancy. Coating of autologous red blood cells (RBCs) with polyclonal antibodies to Rh(D) antigen may result in an immunomodulation and improved outcome in Rh(D) positive women with recurrent pregnancy loss (RPL). Methods: A total of 60 Rh(D) positive women (age 23 to 45 years) with a history of RPL and ineffective treatment with low molecular weight heparin (LMWH) and/or aspirin were included in this retrospective study. In addition to this treatment, Anti-D (300 µg) was given subcutaneously to each woman either prior to pregnancy and/or two times within 12 weeks of gestation. Results: Treatment with Anti-D in non-responders to heparin/aspirin resulted in successful pregnancies in 67% of all cases. The remaining women had only aborts (23%) or did not become pregnant (10%). None of the treated women has developed anemia due to this treatment or any other significant adverse reaction. The rate of successful pregnancies does not appear to be influenced by the administration of: Anti-D prior to pregnancy, age, thrombophilia or previous alive births. Conclusion: The improved outcome following the administration of Anti-D in women with RPL might be explained by immune modulations induced by different immune reactions including polarization of decidual macrophages. The results obtained in this study clearly indicate that Anti-D is safe and highly effective in treatment of Rh(D) positive women with RPL. However, further studies are required to support our results and to find out the optimal dose and timing of Anti-D administration.

Intestinal Microbiota Reduction Followed by Fasting Discloses Microbial Triggering of Inflammation in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) synovitis is dominated by monocytes/macrophages with inflammatory patterns resembling microbial stimulation. In search of triggers, we reduced the intestinal microbiome in 20 RA patients (open label study DRKS00014097) by bowel cleansing and 7-day fasting (≤250 kcal/day) and performed immune monitoring and microbiome sequencing. Patients with metabolic syndrome (n = 10) served as a non-inflammatory control group. Scores of disease activity (DAS28/SDAI) declined within a few days and were improved in 19 of 20 RA patients after breaking the fast (median ∆DAS28 = -1.23; ∆SDAI = -43%) or even achieved remission (DAS28 < 2.6/n = 6; SDAI < 3.3/n = 3). Cytometric profiling with 46 different surface markers revealed the most pronounced phenomenon in RA to be an initially increased monocyte turnover, which improved within a few days after microbiota reduction and fasting. Serum levels of IL-6 and zonulin, an indicator of mucosal barrier disruption, decreased significantly. Endogenous cortisol levels increased during fasting but were insufficient to explain the marked improvement. Sequencing of the intestinal microbiota indicated that fasting reduced potentially arthritogenic bacteria and changed the microbial composition to species with broader metabolic capabilities. More eukaryotic, predominantly fungal colonizers were observed in RA, suggesting possible involvement. This study demonstrates a direct link between the intestinal microbiota and RA-specific inflammation that could be etiologically relevant and would support targeted nutritional interventions against gut dysbiosis as a causal therapeutic approach.

Fibrinogen and factor XIII A-subunit genotypes interactively influence C-reactive protein levels during inflammation.

OBJECTIVE: Fibrinogen is a target of autoimmune reactions in rheumatoid arthritis (RA). Fibrin(ogen) derivatives are involved in inflammatory processes and the generation of a stable fibrin network is necessary for sufficient inflammation control. As the density and stability of fibrin networks depend on complex interactions between factor XIIIA (F13A) and fibrinogen genotypes, the authors studied whether these genotypes were related to C-reactive protein (CRP) levels during acute-phase reactions. METHODS: Association between α-fibrinogen (FGA), ß-fibrinogen (FGB) and F13A genotypes with CRP levels was tested in two cohorts with longitudinal CRP measurements. Discovery and replication cohorts consisted of 288 RA (913 observations) and 636 non-RA patients (2541 observations), respectively. RESULTS: Genotype FGB -455G>A (rs1800790) was associated with CRP elevations (≥ 10 mg/l) in both cohorts (RA, OR per allele 0.69, p=0.0007/P(adj)<0.015; non-RA, OR 0.70, p=0.0004/p(adj)<0.02; combined, OR 0.69, p<10(-5)/p(adj)=0.001). Genotype F13A 34VV (rs5985) was conditional for the association of FGB -455G>A with CRP as indicated by a clear restriction on F13A 34VV individuals and a highly significant heterogeneity between F13A 34VV and F13A 34L genotypes (p<10(-5), p(adj)=0.001). In both cohorts, mean CRP levels significantly declined with ascending numbers of FGB -455A alleles. Genotype FGA T312A (rs6050) exhibited opposite effects on CRP compared with FGB -455G>A. Again, this relation was dependent on F13A V34L genotype. CONCLUSION: Novel genetic determinants of CRP completely unrelated to previously known CRP regulators were identified. Presumably, these haemostatic gene variants modulate inflammation by influencing fibrin crosslinking. These findings could give new perspectives on the genetic background of inflammation control.

Coronavirus Disease 2019-Related Alterations of Total and Anti-Spike IgG Glycosylation in Relation to Age and Anti-Spike IgG Titer.

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been affecting the world since January 2020 and has caused millions of deaths. To gain a better insight into molecular changes underlying the COVID-19 disease, we investigated here the N-glycosylation of three immunoglobulin G (IgG) fractions isolated from plasma of 35 severe COVID-19 patients, namely total IgG1, total IgG2, and anti-Spike IgG, by means of MALDI-TOF-MS. All analyses were performed at the glycopeptide level to assure subclass- and site-specific information. For each COVID-19 patient, the analyses included three blood withdrawals at different time-points of hospitalization, which allowed profiling longitudinal alterations in IgG glycosylation. The COVID-19 patients presented altered IgG N-glycosylation profiles in all investigated IgG fractions. The most pronounced COVID-19-related changes were observed in the glycosylation profiles of antigen-specific anti-Spike IgG1. Anti-Spike IgG1 fucosylation and galactosylation showed the strongest variation during the disease course, with the difference in anti-Spike IgG1 fucosylation being significantly correlated with patients' age. Decreases in anti-Spike IgG1 galactosylation and sialylation in the course of the disease were found to be significantly correlated with the difference in anti-Spike IgG plasma concentration. The present findings suggest that patients' age and anti-S IgG abundance might influence IgG N-glycosylation alterations occurring in COVID-19.

Middle Meningeal Artery Embolization Minimizes Burdensome Recurrence Rates After Newly Diagnosed Chronic Subdural Hematoma Evacuation (MEMBRANE): study protocol for a randomized controlled trial.

BACKGROUND: Chronic subdural hematoma (cSDH) is the most common complication of mild traumatic brain injury demanding neurosurgery in high-income countries. If undetected and untreated, cSDH may increase intracranial pressure and cause neurological deficiencies. The first-line intervention of choice is burr hole trepanation and hematoma evacuation. However, any third patient may experience rebleeding, demanding craniotomy with excess morbidity. Adjunct endovascular embolization of the frontal and parietal branches of the middle meningeal artery (MMA) is a promising approach to avoid relapse and revision but was hitherto not studied in a randomized trial. METHODS: MEMBRANE is an investigator-initiated, single-center, randomized controlled trial. Male, female, and diverse patients older than 18 years scheduled for surgical evacuation of a first cSDH will be assigned in a 1:1 fashion by block randomization to the intervention (surgery plus endovascular MMA embolization) or the control group (surgery alone). The primary trial endpoint is cSDH recurrence within 3 months of follow-up after surgery. Secondary endpoints comprise neurological deficits assessed by the modified Rankin Scale (mRS) and recurrence- or intervention-associated complications during 3 months of follow-up. Assuming a risk difference of 20% of rebleeding and surgical revision, a power of 80%, and a drop-out rate of 10%, 154 patients will be enrolled onto this trial, employing an adaptive O'Brien-Fleming approach with a planned interim analysis halfway. DISCUSSION: The MEMBRANE trial will provide first clinical experimental evidence on the effectiveness of endovascular embolization of the MMA as an adjunct to surgery to reduce the risk of recurrence after the evacuation of cSDH. TRIAL REGISTRATION: German Clinical Trials Registry (Deutsches Register Klinischer Studien [DRKS]) DRKS00020465. Registered on 18 Nov 2021. CLINICALTRIALS: gov NCT05327933 . Registered on 13 Apr 2022.

Heparin or aspirin or both in the treatment of recurrent abortions in women with antiphospholipid antibody (syndrome).

PURPOSE OF REVIEW: Presence of antiphospholipid antibodies (aPL) is associated with unsuccessful pregnancy outcome. Based on an early concept of aPL-induced thrombophilia and placental thrombosis, antithrombotic interventions have been used to reduce incidence of miscarriage in antiphospholipid antibody syndrome (APS). The aim of this review is to summarize current knowledge on pathogenesis of miscarriage in APS and the impact of different antithrombotic therapy strategies. RECENT FINDINGS: Pathogenetic concepts on miscarriage in APS comprise aPL-mediated cell activation, disturbance of coagulation along with increased complement activation. There is increasing evidence that heparin exerts its effect by inhibiting complement activation rather than by its anticoagulation capacity. In this regard, the outcome of pregnancies in APS has considerably improved by the invention of therapies using combinations of aspirin, unfractionated heparin (UFH) and/or low molecular weight heparin (LMWH). However, there is no clear evidence as to which treatment regimen should be preferred. Some studies indicate superiority of aspirin plus heparin over aspirin-only. Whether heparin-only treatment would confer equal effects and whether UFH and LMWH were of comparable efficacy currently is unknown. SUMMARY: Treatment with aspirin and heparin decreases the risk of miscarriages in APS. Well designed trials as well as better patients-at-risk profiling are warranted that identify which treatment strategy should be favored and whether detailed characterization of aPL specificities could help in individualizing therapy.

Association of Genetic Polymorphisms of Fibrinogen, Factor XIII A-Subunit and α2-Antiplasmin with Fibrinogen Levels in Pregnant Women.

Fibrinogen synthesis is stimulated by proinflammatory triggers and depends on α-, ß- and γ-fibrinogen (FGA, FGB, FGG) genotypes. Constellations of fibrinogen, factor XIII A-subunit (F13A) and α2-antiplasmin (A2AP) genotypes predisposing for dense fibrin gels with high antifibrinolytic capacity (e.g., FGB rs1800790 A-allele carriage in F13A 34Val/Val or A2AP 6Arg/Arg wildtypes) are related with reduced inflammation. As both relationships are likely to influence each other, we tested whether the association of fibrinogen genotypes with fibrinogen levels is influenced by F13A and A2AP genotypes in a population under proinflammatory stress. In total, 639 women were followed during pregnancy (2218 observations). The relationship between fibrinogen genotypes and levels was statistically assessed in univariate and multivariate analyses without and with stratification for F13A Val34Leu and A2AP Arg6Trp. Strong associations with fibrinogen levels could be found for FGB rs1800790G > A, FGA rs2070016T > C and FGG rs1049636T > C. For FGB rs1800790G > A and FGA rs2070016T > C, this relationship significantly depended on F13A Val34Leu and A2AP Arg6Trp genotypes. Specifically, in F13A 34Val/Val wildtypes, carriage of FGB rs1800790A was related to significantly lower fibrinogen levels compared with FGB rs1800790GG wildtypes (p < 0.01). For A2AP 6Arg/Arg wildtypes, a comparable relationship could be found (p < 0.04). As these genotype constellations related to lower fibrinogen levels have previously been shown to be associated with reduced inflammatory activity, these findings suggest that the influence of fibrinogen, F13A and A2AP genotypes on inflammation could affect the control of fibrinogen levels and vice versa.

Predictive value of C-reactive protein for radiographic spinal progression in axial spondyloarthritis in dependence on genetic determinants of fibrin clot formation and fibrinolysis.

OBJECTIVE: Genetic determinants of fibrin clot formation and fibrinolysis have an impact on local and systemic inflammatory response. The aim of the present study was to assess whether coagulation-related genotypes affect the predictive value of C-reactive protein (CRP) in regards of radiographic spinal progression in axial spondyloarthritis (axSpA). METHODS: Two hundred and eight patients with axSpA from the German Spondyloarthritis Inception Cohort were characterised for genotypes of α-fibrinogen, ß-fibrinogen (FGB) and γ-fibrinogen, factor XIII A-subunit (F13A) and α2-antiplasmin (A2AP). The relation between CRP levels and radiographic spinal progression defined as worsening of the modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS) by ≥2 points over 2 years was assessed in dependence on the respective genetic background in logistic regression analyses. RESULTS: Overall, CRP was associated with mSASSS progression ≥2 points: time-averaged CRP ≥10 mg/L, OR: 3.32, 95% CI 1.35 to 8.13. After stratification for coagulation-related genotypes, CRP was strongly associated with mSASSS progression in individuals predisposed to form loose, fibrinolysis-susceptible fibrin clots (FGB rs1800790GG, OR: 6.86, 95% CI 2.08 to 22.6; A2AP 6Trp, OR: 5.86, 95% CI 1.63 to 21.0; F13A 34Leu, OR: 8.72, 95% CI 1.69 to 45.1), while in genotypes predisposing to stable fibrin clots, the association was absent or weak (FGB rs1800790A, OR: 0.83, 95% CI 0.14 to 4.84; A2AP 6Arg/Arg, OR: 1.47, 95% CI 0.35 to 6.19; F13A 34Val/Val, OR: 1.72, 95% CI 0.52 to 5.71). CONCLUSIONS: Elevated CRP levels seem to be clearly associated with radiographic spinal progression only if patients are predisposed for loose fibrin clots with high susceptibility to fibrinolysis.

Fibrinogen, factor XIII and α2-antiplasmin genotypes are associated with inflammatory activity and anti-citrullinated protein antibodies.

BACKGROUND: Fibrin(ogen) derivatives, crosslinked fibrin and fibrinolysis play important roles in inflammation and are involved in pathogenesis of rheumatoid arthritis (RA). About 2/3 of RA patients exhibit anti-citrullinated protein antibodies (ACPA) that target deiminated fibrinogen. Genetic variants of ß-fibrinogen (FGB) (rs1800790G>A) and factor XIII A-subunit (F13A) Val34Leu (rs5985) are known to influence interactively inflammatory processes. It is hypothesized that predisposition for dense fibrin clots is related to better inflammation control. METHODS: To test this hypothetical model a cohort of 924 patients (288 RA and 636 non-RA patients) (3545 observations) was genotyped for FGB (rs1800790G>A, rs1800788C>T), α-fibrinogen (FGA) (rs6050A>G, rs2070006G>A, rs2070016T>C), γ-fibrinogen (FGG) (rs1049636T>C), F13A Val34Leu (rs5985) and α2-antiplasmin (A2AP) Arg6Trp (rs2070863). Genotype constellations potentially predisposing for dense fibrin clots were defined and their relation to inflammatory activity as measured by C-reactive protein (CRP) and disease activity score of 28 joints (DAS28) was assessed in univariate and multivariate analyses. The relation of these genotype constellations with presence of ACPA was tested. RESULTS: Genotype constellations involving FGB rs1800790G>A and FGA rs2070016T>C were inversely associated with CRP levels (≥10 mg/L) (OR: 0.49, P < 10-8/7adj = 0.0001; OR: 0.52, P < 0.0005/Padj = 0.01). In RA, both genotype constellations were observed with higher frequencies of low disease activity (DAS28 ≤ 3.2) (OR: 2.66, P = .009; OR 2.78, P = .01) and lower frequencies of high disease activity (DAS28>5.1) (OR: 0.52, P < .03, OR: 0.42, P = .01). Associations with CRP depended on A2AP 6Arg/Arg genotype known to be necessary for optimal anti-fibrinolytic capacity (P = .001). Finally, Genotype constellations involving FGB rs1800790G>A and FGA rs2070016T>C were found to be associated with ACPA-positivity in RA (OR: 2.18, P < .03; OR: 1.95, P = .09). CONCLUSIONS: These results support the hypothesis that genotypes, which increase fibrin clot density and anti-fibrinolytic capacity, reduce inflammatory activity and are related to humoral autoimmunity in RA.

Is there a 'window of opportunity' for intervention to reduce risk of coronary artery disease in SLE?

Patients with systemic lupus erythematosus (SLE) have a substantially enhanced risk for cardiovascular complications, especially coronary artery disease (CAD). Evidence from a recent study by Urowitz et al. indicates that the incidence not only of classic CAD risk factors but also of nontraditional CAD risk factors increases within the first 3 years after onset of SLE. The data indicate that patients with SLE require careful management of hypertension and hypercholesterolemia, smoking cessation, and increased physical activity in order to reduce the risk of CAD. Intensive immune intervention is likely to be related to reduction of risk from nontraditional CAD risk factors, but it is not known if this treatment approach also affects classic CAD risk factors. Although established in patients with rheumatoid arthritis in recent years, the effect of immune intervention on classic risk factors for CAD needs to be evaluated in patients with SLE, through either clinical studies or international registries.

Hypogalactosylation of immunoglobulin G in rheumatoid arthritis: relationship to HLA-DRB1 shared epitope, anticitrullinated protein antibodies, rheumatoid factor, and correlation with inflammatory activity.

BACKGROUND: Galactosylation of immunoglobulin G (IgG) is reduced in rheumatoid arthritis (RA) and assumed to correlate with inflammation and altered humoral immunity. IgG hypogalactosylation also increases with age. To investigate dependencies in more detail, we compared IgG hypogalactosylation between patients with RA, patients with axial spondyloarthritis (axSpA), and healthy control subjects (HC), and we studied it in RA on the background of HLA-DRB1 shared epitope (SE), anticitrullinated protein antibodies (ACPA), and/or rheumatoid factor (RF) status. METHODS: Patients with RA (n = 178), patients with axSpA (n = 126), and HC (n = 119) were characterized clinically, and serum IgG galactosylation was determined by capillary electrophoresis. Markers of disease activity, genetic susceptibility, and serologic response included C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), DAS28, SE, HLA-B27, ACPA, and RF. Expression of glycosylation enzymes, including beta 1-4 galactosyltransferase (B4GALT3) activity, were estimated from transcriptome data for B-cell development (GSE19599) and differentiation to plasma cells (GSE12366). RESULTS: IgG hypogalactosylation was restricted to RA and associated with increasing CRP levels (p < 0.0001). In axSpA, IgG hypogalactosylation was comparable to HC and only marginally increased upon elevated CRP. Restriction to RA was maintained after correction for CRP and age. Treatment with sulfasalazine resulted in significantly reduced IgG hypogalactosylation (p = 0.003) even after adjusting for age, sex, and CRP (p = 0.009). SE-negative/ACPA-negative RA exhibited significantly less IgG hypogalactosylation than all other strata (vs SE-negative/ACPA-positive, p = 0.009; vs SE-positive/ACPA-negative, p = 0.04; vs SE-positive/ACPA-positive, p < 0.02); however, this indicated a trend only after Bonferroni correction for multiple testing. In SE-positive/ACPA-negative RA IgG hypogalactosylation was comparable to ACPA-positive subsets. The relationship between IgG hypogalactosylation and disease activity was significantly different between strata defined by SE (CRP, p = 0.0003, pBonferroni = 0.0036) and RF (CRP, p < 0.0001, pBonferroni < 0.0012), whereas ACPA strata revealed only a nonsignificant trend (p = 0.15). Gene expression data indicated that the key enzyme for galactosylation of immunoglobulins, B4GALT3, is expressed at lower levels in B cells than in plasma cells. CONCLUSIONS: Increased IgG hypogalactosylation in RA but not in axSpA points to humoral immune response as a precondition. Reduced B4GALT3 expression in B cells compared with plasma cells supports relatedness to early B-cell triggering. The differential influence of RA treatment on IgG hypogalactosylation renders it a potential diagnostic target for further studies.

Sequencing-based typing of HLA.

Human leukocyte antigen (HLA) typing is largely performed by use of PCR-based techniques in patients that require stem cell transplantation. In this chapter, HLA typing by sequencing-based typing techniques is described.

A Prothrombotic Score Based on Genetic Polymorphisms of the Hemostatic System Differs in Patients with Ischemic Stroke, Myocardial Infarction, or Peripheral Arterial Occlusive Disease.

BACKGROUND: While twin studies indicate a genetic component in arterial thrombosis such as ischemic stroke, myocardial infarction (MI), or peripheral arterial occlusive disease (PAOD), the clinical relevance of hemostatic polymorphisms in arterial thrombosis is a matter of debate. METHODS: We analyzed the prevalence of 13 hemostatic polymorphisms [PAI-1, PLAT, F5 (including factor V Leiden and HR2 haplotype), F2, F7, F13A, FGB, TFPI, THBD, MTHFR, ACE, and ITGA2] in patients referred to a tertiary referral center. A "prothrombotic score" was calculated by dividing the number of risk-increasing polymorphisms for thrombosis minus the number of risk-lowering polymorphisms (F7 and F13A) by the number of polymorphisms tested. RESULTS: Datasets of 144 patients with prior ischemic stroke (mean age 44 ± 13 years; 65% female) were compared to 62 patients with MI or PAOD (mean age 54 ± 14 years; 47% female). The prothrombotic score was lower in MI and PAOD patients compared to stroke patients [odds ratios 2.7 (95% confidence intervals 1.1-6.2)]. Frequencies of individual polymorphisms did not differ between both groups. CONCLUSION: Patients with MI or PAOD had a lower burden of prothrombotic mutations compared to patients with prior stroke, indicating that a prothrombotic state might play a different role in distinct forms of arterial thrombosis.