Genetic polymorphisms in IL-2, IL-10 and FOXP3 are associated with autoimmune neutropaenia in early childhood and autoantibody specificity in a Danish cohort.

Autoimmune neutropaenia (AIN) in early childhood is characterized by chronic neutropaenia and positivity for human neutrophil antibodies (HNA), resulting in the excessive destruction of neutrophils. The association between regulatory T cells (Tregs) and AIN has been described, and in this study, we investigated three Treg-associated genes, IL-2, IL-10 and FOXP3. The frequencies of three single nucleotide polymorphisms (SNPs) in IL-2 -330T>G (rs2069762), +114G>T (rs2069763) and IVS3-116 A>G (rs2069772), four SNPs in IL-10 -3575T>A (rs1800890), -1082G>A (rs1800896), -819 C>T (rs1800871) and -592 C>A (rs1800872) and three SNPs in FOXP3 -3499 A>G (rs3761547), -3279 C>A (rs3761548) and -924 A>G (rs2232365) were compared between 166 Danish AIN patients and 358 healthy controls. Disease association was observed for IL-2 IVS3-116 GG (p = 0.0081, OR = 0.35 [0.15-0.80]), IL-10 -3575 TT (p = 0.0078, OR = 1.71 [1.16-2.54]) and IL-10 -1082 AA (p = 0.014, OR = 1.76 [1.14-2.72]) in all patients and FOXP3 -924 (p = 0.0005, A OR = 0.41 [0.25-0.68] and G OR = 2.42 [1.46-4.01]) in male patients. None of the associations were linked to antibody specificity. Disease-associated haplotypes were observed in IL-2 and FOXP3. IL-2 -330T/+114 T/IVS3-116A was associated with anti-FcγRIIIb-positive patients (p = 0.012, OR = 2.07 [1.18-3.62]). FOXP3 -3499A/-3279C/-924A was associated with anti-HNA-1a-positive male patients (p = 0.016, OR = 0.41 [0.20-0.83]), and ACG was associated with female patients, both in the combined group (p = 0.006, OR = NA) and the anti-FcγRIIIb-positive group (p = 0.002, OR = NA). We conclude that our findings reveal a correlation between SNP in Treg-associated genes and AIN, indicating that AIN could be driven by dysfunction of immune homeostatic-evolving Tregs.

Genetic variations in low-to-medium-affinity Fcγ receptors and autoimmune neutropenia in early childhood in a Danish cohort.

Autoimmune neutropenia (AIN) in early childhood is caused by autoantibodies directed against antigens on the neutrophil membrane and is a frequent cause of neutropenia in children. Association of AIN with Fcγ receptor (FCGR) 3B variants is well described. In this study, we investigate genetic variations in the FCGR locus and copy number variation of FCGR3B. A total of 130 antibody-positive AIN patients, 64 with specific anti-HNA-1a antibodies and 66 with broad-reacting anti-FcγRIIIb antibodies, were genotyped with a multiplex ligation probe assay and compared with healthy controls. Positive findings were confirmed with real-time q-PCR. We determined copy numbers of the FCGR2 and FCGR3 genes and the following SNPs: FCGR2A Q62W (rs201218628), FCGR2A H166R (rs1801274), FCGR2B I232T (rs1050501), FCGR3A V176F (rs396991), haplotypes for FCGR2B/C promoters (rs3219018/rs780467580), FCGR2C STOP/ORF and HNA-1 genotypes in FCGR3B (rs447536, rs448740, rs52820103, rs428888 and rs2290834). Generally, associations were antibody specific, with all associations being representative of the anti-HNA-1a-positive group, while the only association found in the anti-FcγRIIIb group was with the HNA-1 genotype. An increased risk of AIN was observed for patients with one copy of FCGR3B; the HNA genotypes HNA-1a, HNA-1aa or HNA-1aac; the FCGR2A 166H and FCGR2B 232I variations; and no copies of FCGR2B 2B.4. A decreased risk was observed for HNA genotype HNA-1bb; FCGR2A 166R; FCGR2B 232T; and one copy of FCGR2B promoter 2B.4. We conclude that in our Danish cohort, there was a strong association between variation in the FCGR locus and AIN. The findings of different genetic associations between autoantibody groups could indicate the presence of two different disease entities and disease heterogeneity.

ABO, secretor, and Lewis carbohydrate histo-blood groups are associated with autoimmune neutropenia of early childhood in Danish patients.

BACKGROUND: Autoimmune neutropenia of early childhood (AIN) is caused by autoantibodies directed against antigens on the neutrophil membrane. The ABO, secretor, and Lewis histo-blood group systems control the expression of carbohydrate antigens and have previously been linked to autoimmune diseases. We aimed to investigate the association between genotypes and the risk of AIN in Danish patients. STUDY DESIGN AND METHODS: One hundred fifty-four antibody-positive AIN patients were included. Controls (n = 400) were healthy unrelated Danish blood donors. Molecular determination of ABO, secretor (FUT2), and Lewis (FUT3) genotypes were determined using real-time polymerase chain reaction (qPCR) or Sanger sequencing to infer the prevalence of Lewis antigens (Lea and Leb ) and secretor (SeSe or Sese) or nonsecretor (sese) phenotypes. RESULTS: Blood type O was more common in controls (46.8%) than in AIN patients (36.4%) (OR = 0.65; p = 0.028). Secretors of H Leb antigens were less frequent among AIN patients (25.2%) than controls (35.0%) (OR = 0.62; p = 0.037). DISCUSSION: ABO blood group antigens and the secretion of these antigens are associated with a diagnosis of AIN. The mechanism underlying the association between autoimmunity and interaction among ABO, secretor, and Lewis genotypes has not yet been elucidated, but several studies indicate a connection to the gut microbiota.

The development of an indel panel for microchimerism detection.

OBJECTIVES: The aim of the study was to create a simple assay for microchimerism detection independent of sex and without HLA genotyping. METHODS: The method is based on detection of insertion or deletions utilizing a multiplex PCR followed by fragment analysis by capillary electrophoresis, and probe-based qPCR assays. A total of 192 samples, taken either before pregnancy, during 1st trimester, or either during 2nd trimester or at miscarriage, obtained from a cohort of 97 female patients with either primary or secondary recurrent pregnancy loss, were screened for fetal microchimerism by the indel panel as well as an existing assay based on detection of the Y-chromosome marker; DYS14. RESULTS: The overall prevalence of DYS14 positive samples was 29% (55/192) whereas 32% (61/192) tested positive by the indel method. There was an overall agreement of 64% (122/192) between the results obtained by the two methods. A Fisher's Exact test showed no statistic significant difference in the prevalence of microchimerism detected by the two methods at any of the three times of sampling. The distribution of the number of positive wells detected by both methods were compared by a Mann-Whitney U test, which showed no statistically significant difference at any of the three times of sampling. CONCLUSION: The data indicates that microchimerism can be detected efficiently by the indel method. This makes it possible to detect both female and male cells without the need of HLA-genotyping. Furthermore, the indel method has potential to be implemented as a routine analysis. This will remove the sex bias in future explorations of the role microchimerism plays in health and disease.

Molecular pathways in patients with systemic lupus erythematosus revealed by gene-centred DNA sequencing.

OBJECTIVES: Systemic lupus erythematosus (SLE) is an autoimmune disease with extensive heterogeneity in disease presentation between patients, which is likely due to an underlying molecular diversity. Here, we aimed at elucidating the genetic aetiology of SLE from the immunity pathway level to the single variant level, and stratify patients with SLE into distinguishable molecular subgroups, which could inform treatment choices in SLE. METHODS: We undertook a pathway-centred approach, using sequencing of immunological pathway genes. Altogether 1832 candidate genes were analysed in 958 Swedish patients with SLE and 1026 healthy individuals. Aggregate and single variant association testing was performed, and we generated pathway polygenic risk scores (PRS). RESULTS: We identified two main independent pathways involved in SLE susceptibility: T lymphocyte differentiation and innate immunity, characterised by HLA and interferon, respectively. Pathway PRS defined pathways in individual patients, who on average were positive for seven pathways. We found that SLE organ damage was more pronounced in patients positive for the T or B cell receptor signalling pathways. Further, pathway PRS-based clustering allowed stratification of patients into four groups with different risk score profiles. Studying sets of genes with priors for involvement in SLE, we observed an aggregate common variant contribution to SLE at genes previously reported for monogenic SLE as well as at interferonopathy genes. CONCLUSIONS: Our results show that pathway risk scores have the potential to stratify patients with SLE beyond clinical manifestations into molecular subsets, which may have implications for clinical follow-up and therapy selection.

Association between human leukocyte antigens (HLAs) and human neutrophil antigens (HNAs) and autoimmune neutropenia of infancy in Danish patients.

BACKGROUND: Autoimmune neutropenia of infancy (AIN) is a frequent cause of neutropenia in children. The disease is caused by antibodies against epitopes on the immunoglobulin G (IgG) Fc receptor type 3b (FcγIIIb). We investigated the possible association of human neutrophil antigens (HNA), human leukocyte antigen (HLA)-DR, and HLA-DQ alleles with AIN and the association of these genotypes with the presence of autoantibodies. METHODS: Eighty AIN cases with a median age of 13.5 months were included. Controls were healthy unrelated Danish blood donors. Anti-HNA-1a autoantibodies were detected using a flow cytometric granulocyte immunofluorescence test (Flow-GIFT) with phenotyped donor cells for detection of antibody specificity. Molecular determination of HNA genotypes was determined using real-time polymerase chain reaction (q-PCR). High-resolution HLA-DRB1 and HLA-DQB1 were determined by next-generation sequencing. RESULTS: Antibodies against HNA-1a were detected in 51% (n = 41) of AIN patients, and anti-HNA-1b was detected in 3% (n = 2) of cases. In 46% of cases, the antibodies were anti-FcγIIIb-reactive. FCGR3B*01+,*02-,*03- was more common (odds ratio, 6.70; P < .0001), and FCGR3B*01-,*02+,*03- was less common (odds ratio, 0.30; P < .0001) among AIN cases. HNA-1a antibodies were significantly more frequent among AIN cases with the FCGR3B*01+,*02-,*03- genotype (odds ratio, 3.86; P < .007). The HLA-DRB1*14 - HLA-DQB1*05:03 haplotype was significantly more common (odds ratio, 7.44; P < .0001) in AIN patients. CONCLUSION: The HLA haplotype HLA-DRB1*14 - DQB1*05:03 is associated with Danish AIN cases. Among Danish AIN patients, anti-HNA-1a is the most common autoantibody, and the antibody is more common in cases with the FCGR3B*01+,*02-,*03- genotype.

Ficolin-3 Deficiency Is Associated with Disease and an Increased Risk of Systemic Lupus Erythematosus.

PURPOSE: Ficolin-3 deficiency is caused by a mutation (+1637delC) in the FCN3 gene. It is a rare condition and has been associated with both infection and autoimmune disease including systemic lupus erythematosus (SLE). Here we investigated if ficolin-3 deficiency is more frequent in patients than in controls and tried to identify a common phenotype among ficolin-3 deficient individuals. Since a significant part of patients identified with ficolin-3 deficiency was diagnosed with SLE, we explored whether the heterozygous state of the FCN3+1637delC variant represents a risk factor in the development of SLE. Further, we examined other possible causes of ficolin-3 deficiency when the FCN3+1637delC is not present. METHODS: A systematic literature search for studies measuring ficolin-3 was carried out. We examined 362 SLE patients and 596 controls for the presence of the variant FCN3+1637delC. We established assays for measurements of ficolin-3 and of auto-antibodies against ficolin-3. We sequenced the coding and non-coding regions of the FCN3 gene in an SLE patient with ficolin-3 deficiency not carrying the +1637delC. RESULTS: Ficolin-3 deficiency leads to an 8-time increased odds of having a disease (p < 0.05). Three out of nine patients with deficiency had SLE. The heterozygous state of the deficiency variant is not associated with increased risk of developing SLE (p = 0.18). CONCLUSION: By systematically reviewing the literature for the described cases of ficolin-3 deficiency, an autoimmune phenotype is emerging. Thirty-three percent of the ficolin-3 deficient patients had SLE. Heterozygosity for the FCN3 gene deletion causing the deficiency does not seem to be associated with the development of SLE.

Blood group genotyping of blood donors: validation of a highly accurate routine method.

BACKGROUND: In the past century, blood group determination using serology has been the standard method. Now, molecular methods are gaining traction, which provide additional and easily accessible information. Here we designed and validated a high-throughput extended genotyping setup. STUDY DESIGN AND METHODS: We developed 35 competitive allele-specific polymerase chain reaction assays for genotyping of blood donors. Samples from 1034 Danish blood donors were genotyped, and 45,314 red blood cell antigens and 6148 platelet antigens were predicted. Predicted phenotypes were compared with 16,119 serologic phenotypes. RESULTS: We found 62 discrepancies of which 43 were due to serology. After exclusion of the discrepancies caused by serology, the accuracy of genotyping was 99.9%. Of 17 discrepancies caused by the genotype, three were incorrect antigen-negative predictions and could potentially, as the solitary analysis, have caused an adverse transfusion reaction. CONCLUSION: We have established a robust and highly accurate blood group genotyping system with a very high capacity for screening blood donors. The system represents a significant improvement over the former serotyping-only procedure. Almost all new technology in medicine incurs increased costs, but the presented efficient genotyping system is a rare example of a significant qualitative and quantitative technologic progress that is also more cost-efficient than previous technologies.

Polymorphisms in Genes Coding for Cytokines, Mannose-Binding Lectin, Collagen Metabolism and Thrombophilia in Women with Cervical Insufficiency.

OBJECTIVE: To study the association between cervical insufficiency and single nucleotide polymorphisms in seven genes coding for pro- and anti-inflammatory cytokine-related factors, mannose-binding lectin 2 (MBL2), collagen1α1 (COL1A1), factor II and factor V Leiden genes. METHODS: In a case-control study, potential maternal biomarkers for cervical insufficiency were investigated in 30 women with a history of second-trimester miscarriage or preterm birth due to cervical insufficiency and in 70 control women. RESULTS: Homozygous carriers of the interleukin 6 (IL6) -174 genotype GG had an odds ratio (OR) of 3.1 [95% confidence interval (95% CI) 1.3-7.4, p = 0.01] and MBL2 genotypes coding for low or intermediate levels of plasma MBL had an OR of 3.3 (95% CI 1.2-9.0, p = 0.01) for cervical insufficiency compared with controls. Serum MBL levels were lower in women with cervical insufficiency than in controls (median 408 and 1,985 ng/ml, respectively, p < 0.01). CONCLUSIONS: Single nucleotide polymorphisms in the IL6 gene and the MBL2 gene and low MBL levels related to the latter polymorphism may increase the risk of preterm birth due to cervical insufficiency.

Mannose-binding lectin protein and its association to clinical outcomes in COPD: a longitudinal study.

BACKGROUND: Functional deficiency of mannose-binding lectin (MBL) may contribute to the pathogenesis of chronic obstructive pulmonary disease. We hypothesized that specific MBL2 gene polymorphisms and circulating MBL protein levels are associated with clinically relevant outcomes in the Predicting Outcome using systemic Markers In Severe Exacerbations of COPD PROMISE-COPD cohort. METHODS: We followed 277 patients with stable COPD GOLD stage II-IV COPD over a median period of 733 days (IQR 641-767) taking survival as the primary outcome parameter. Patients were dichotomized as frequent (≥ 2 AECOPD/year) or infrequent exacerbators. Serum MBL levels and single nucleotide polymorphisms of the MBL2 gene were assessed at baseline. RESULTS: The MBL2-HYPD haplotype was significantly more prevalent in frequent exacerbators (OR: 3.33; 95% CI, 1.24-7.14, p = 0.01). The median serum MBL concentration was similar in frequent (607 ng/ml, [IQR; 363.0-896.0 ng/ml]) and infrequent exacerbators (615 ng/ml, [IQR; 371.0-942.0 ng/ml]). Serum MBL was not associated with lung function characteristics or bacterial colonization in sputum. However, high serum MBL at stable state was associated with better survival compared to low MBL (p = 0.046, log rank test). CONCLUSIONS: In COPD, the HYPD haplotype of MBL2 gene is associated with frequent exacerbations and high serum MBL is linked to increased survival. The PROMISE-COPD study was registered at www.controlled-trials.com under the identifier ISRCTN99586989.

Structural genomic variation as risk factor for idiopathic recurrent miscarriage.

Recurrent miscarriage (RM) is a multifactorial disorder with acknowledged genetic heritability that affects ∼3% of couples aiming at childbirth. As copy number variants (CNVs) have been shown to contribute to reproductive disease susceptibility, we aimed to describe genome-wide profile of CNVs and identify common rearrangements modulating risk to RM. Genome-wide screening of Estonian RM patients and fertile controls identified excessive cumulative burden of CNVs (5.4 and 6.1 Mb per genome) in two RM cases possibly increasing their individual disease risk. Functional profiling of all rearranged genes within RM study group revealed significant enrichment of loci related to innate immunity and immunoregulatory pathways essential for immune tolerance at fetomaternal interface. As a major finding, we report a multicopy duplication (61.6 kb) at 5p13.3 conferring increased maternal risk to RM in Estonia and Denmark (meta-analysis, n = 309/205, odds ratio = 4.82, P = 0.012). Comparison to Estonian population-based cohort (total, n = 1000) confirmed the risk for Estonian female cases (P = 7.9 × 10(-4) ). Datasets of four cohorts from the Database of Genomic Variants (total, n = 5,846 subjects) exhibited similar low duplication prevalence worldwide (0.7%-1.2%) compared to RM cases of this study (6.6%-7.5%). The CNV disrupts PDZD2 and GOLPH3 genes predominantly expressed in placenta and it may represent a novel risk factor for pregnancy complications.

Lectin pathway of complement activation and relation with clinical complications in critically ill children.

BACKGROUND: Critically ill children are susceptible to nosocomial infections, which contribute to adverse outcomes. Deficiencies in the innate immunity lectin pathway of complement activation are implicated in a child's vulnerability to infections in conditions such as cancer, but the role during critical illness remains unclear. We hypothesized that low on-admission levels of the pathway proteins are, in part, genetically determined and associated with susceptibility to infectious complications and adverse outcomes. METHODS: We studied protein levels of mannose-binding lectin (MBL), H-ficolin and M-ficolin, three MBL-associated-serine proteases (MASPs) and MBL-associated protein (MAp44), and relation with functional genetic polymorphisms, in 130 healthy children and upon intensive care unit (ICU) admission in 700 critically ill children of a randomized study on glycemic control. RESULTS: Levels of MASP-1, MASP-2, MASP-3, and MAp-44 were lower and the levels of M-ficolin were higher in ICU patients on admission than those in matched healthy controls. Only a low on-admission MASP-3 level was independently associated with risk of new ICU infections and prolonged ICU stay, after correcting for other risk factors. On-admission MASP-3 varied with age, illness severity, and genetic variation. CONCLUSION: Low on-admission MASP-3 levels in critically ill children were independently associated with subsequent acquisition of infection and prolonged ICU stay. The biological explanation needs further investigation.

Comparison of real-time quantitative PCR and two digital PCR platforms to detect copy number variation in FCGR3B.

The importance of structural genetic variants, such as copy number variations (CNVs), in modulating human disease is being increasingly recognized. Several clinical conditions require investigation of human neutrophil antigen (HNA-1), which is encoded by the Fc gamma receptor IIIb gene (FCGR3B), including suspicion of neutropenia, infections, and proactive testing of blood component donors to reduce the potential risk in transfusion. In this study, we compared real-time quantitative polymerase chain reaction (qPCR) with two digital PCR (dPCR) platforms, namely droplet digital PCR and an array-based platform, to determine copy numbers (CNs) in FCGR3B. We initially tested 400 anonymous blood donors with qPCR using a commercially available TaqMan probe assay (Applied Biosystems) on a Quant Studio 12 Flex. CNs was determined for all 400 tested individuals with CNs ranging from zero to four. Zero copies were detected in 0.2% (1/400), one copy was detected in 3.8% (15/400), two copies were detected in 87.8% (351/400), three copies were detected in 8.0% (32/400), and four copies were detected in 0.2% (1/400) of tested individuals. From this cohort, we selected 32 donors with CNs from zero to four for analyses with Digital Real-Time PCR (dPCR) using Lab on an array (LOAA) on an On-Point analyzer from Optolane Technologies Inc. and the Droplet Digital PCR (ddPCR) platform from Bio-Rad Laboratories. We compared the obtained CNs of FCGR3B on the three platforms and found full concordance between the CNs obtained. We therefore conclude that all three platforms can be used for quantification of CNs for FCGR3B, and although dPCR has some advantages over qPCR, it was not necessary for reliably estimating CNs of the FCGR3B gene.

High-resolution HLA genotyping identifies risk alleles in both class I and II for primary autoimmune neutropenia in early childhood in a Danish cohort.

HLA studies in patients with autoimmune neutropenia (AIN) have shown very consistent results for the association with HLA class II alleles at low resolution. This study aimed to examine the association of both HLA class I and class II at high resolution to clarify the contribution of risk alleles to the disease. A total of 107 AIN patients were genotyped for six loci of HLA class I (HLA-A, -B and -C) and class II (HLA-DRB1, -DQB1, and -DPB1) genes by a high-resolution (3-field, 6-digit) analysis and compared with HLA typing of 1000 healthy controls. Compared with the controls, the allele frequencies were significantly higher in AIN patients for A*02:17:01G, C*01:02:01G, DRB1*10:01:01G, DRB1*14:01:01G, DRB1*16:01:01G, DQB1*05:02:01G, and DQB1*05:03:01G but lower significant for C*03:04:01G, DRB1*04:01:01G, DRB1*13:02:01G, DQB1*03:02:01G, and DQB1*06:04:01G. Frequently associated two-locus haplotypes were found to be DRB1*10:01:01G-DQB1*05:01:01G and DRB1*16:01:01G-DQB1*05:02:01G, while the S2 (Q- or D-KRAA) shared epitope (SE) was associated with lower risk. A unique association with HLA alleles was observed between patients with specific anti-HNA-1a antibodies and broad-reacting anti-FcγRIIIb. Anti-HNA-1a antibody-positive patients were associated with C*01:02:01G, DRB1*01:01:01G, DRB1*16:01:01G, DQB1*05:01:01G, DQB1*05:02:01G, DQB1*06:04:01G, and DPB1*10:01:01G; the two-locus haplotypes DRB1*01:01:01G-DQB1*05:01:01G and DRB1*16:01:01G-DQB1*05:02:01G; and the S3P (Q- or R-RRAA) SE. Anti-FcγRIIIb antibody-positive patients were associated with the alleles A*02:17:01G, DRB1*10:01:01G, and DQB1*05:02:01G; the haplotypes DRB1*10:01:01G-DQB1*05:01:01G and DRB1*11:01:02G-DQB1*05:02:01G; and the S3D (DRRAA) SE. The different associations regarding FcγRIIIb antibody specificities could indicate disease heterogeneity.

CD21 deficiency in 2 siblings and frequency of the associated mutation in the Danish population.

The clinical presentation of CD21 deficiency in 2 siblings caused by a novel mutation in the CD21 gene is reported, and the frequency of this mutation in the Danish population is explored. Successful treatment with IgG replacement in both patients with CD21 deficiency is also reported.

Single nucleotide polymorphisms and the risk of venous thrombosis: results from a Danish case-cohort study.

A number of single nucleotide polymorphisms (SNP) have been linked to higher risk of venous thromboembolism (VTE). We investigated the VTE risk associated with SNPs in the GP6 (rs1613662), SERPINC1 (rs2227589), F11 (rs2036914 and rs2289252), FGG (rs2066865), and F12 (rs1801020) genes. In F11, the CC genotype for rs2036914 and the CT and TT genotypes for rs2289252 were associated with a significantly higher VTE risk. A trend toward a thrombogenic effect was observed for the risk alleles of the GP6 and FGG SNPs. Risk estimates were unaffected by adjustments for blood type and F5 rs6025 (Factor V Leiden) mutation.

Susceptibility to leprosy is associated with M-ficolin polymorphisms.

PURPOSE: Mycobacterium leprae exploits complement activation and opsonophagocytosis to infect phagocytes. M-ficolin is encoded by the FCN1 gene and initiates the lectin pathway on monocyte surfaces. We investigated FCN1 promoter polymorphisms that could be responsible for the high interindividual variability of M-ficolin levels and for modulating leprosy susceptibility. METHODS: We genotyped rs2989727 (-1981 G > A), rs28909068 (-791 G > A), rs10120023 (-542 G > A), rs17039495 (-399 G > A), rs28909976 (-271IndelT), rs10117466 (-144C > A) and rs10858293 (+33 T > G) in 400 controls and 315 leprosy patients from Southern Brazil, and in 296 Danish healthy individuals with known M-ficolin levels. RESULTS: Ten haplotypes were identified with sequence-specific PCR and/or haplotype-specific sequencing. We found evidence for a protective codominant additive effect of FCN1*-542A-144C with leprosy in Euro-Brazilians (P=0.003, PBf =0.021, OR=0.243 [CI95% =0.083-0.71]), which was independent of age, ethnic group and gender effects (P=0.029). There was a trend for a positive association of the -399A variant in Afro-Brazilians (P=0.022, PBf =0.154, OR=4.151 [CI95% =1.115-15.454], as well as for a negative association of the FCN1*3A haplotype with lepromatous leprosy, compared with less severe forms of the disease (P=0.016, PBf =0.112, OR=0.324 [CI95% =0.123-0.858]). Danish individuals with this haplotype presented M-ficolin levels higher than the population average of circa 1,000 ng/ml, and -542A-144C, which is able to modify the recognition of transcription factors in silico, occurred in individuals with levels under the 25 percentil (P=0.031). CONCLUSIONS: Our data provide the first evidence that FCN1 polymorphisms are associated with leprosy. M-ficolin may represent a novel key to understand the immunopathogenesis of M. leprae infection.

A combination of the HLA-DRB1*03 phenotype and low plasma mannose-binding lectin predisposes to autoantibody formation in women with recurrent pregnancy loss.

Introduction: It is documented that a series of autoantibodies can be detected with increased frequency in women with recurrent pregnancy loss (RPL) and they may impact the pregnancy prognosis negatively. It is unknown whether the autoantibodies per se or the basic immune disturbances underlying autoantibody production, are the reason for this association. Our group has previously found that some genetically determined immunological biomarkers are associated with RPL and the same biomarkers are also in various degrees known to predispose to autoantibody production. The aim of this study was to clarify whether the RPL-associated immunogenetic biomarkers are associated with positivity for three major classes of autoantibodies associated with RPL. Methods: In 663 patients with RPL in whom we had results for HLA-DRB1 typing and plasma mannose-binding lectin (p-MBL) measurement, it was investigated whether there is a correlation between positivity for the autoantibodies: anticardiolipin antibodies, ß2 glycoprotein I antibodies, and lupus anticoagulant (jointly called antiphospholipid antibodies), thyroid-peroxidase antibodies, and antinuclear antibodies and each of the HLA-DRB1 alleles HLA-DRB1*03 or HLA-DRB1*07 either alone or in combination with low p-MBL defined as ≤500 µg/l. Results: Although slightly higher frequencies of positivity of two or more autoantibodies were seen in patients with either p-MBL ≤500 µg/l or being positive for HLA-DRB1*03, none were significantly associated. However, in patients with the combination of low p-MBL and HLA-DRB1*03, presence of at least one autoantibody was significantly more frequent than in patients with no such combination (OR= 2.4; 95% CI 1.2-5.0, p = 0.01). In an analysis of which autoantibodies were most strongly associated with the low p-MBL/HLA-DRB1*03 combination, antinuclear antibodies were significantly more frequent in these patients (OR 2.0; 95% CI 1.0-3.9, p=0.05) whereas the other autoantibodies were also positively but more weakly associated with this combination. Discussion: In conclusion, to clarify the pathogenetic background, underlying immunogenetic factors should be examined in autoantibody positive RPL patients (as well as other patients with autoimmune diseases) but the genetic background may be complex.

Campylobacter concisus from chronic inflammatory bowel diseases stimulates IL-8 production in HT-29 cells.

The emerging pathogen Campylobacter concisus has been isolated from patients with gastrointestinal diseases; however, it is also present in the gut of healthy individuals. The aim of this study was to compare IL-8 production in HT-29 cells after infection with C. concisus from different gastrointestinal disease phenotypes. Additionally, to investigate whether differentiation of isolates in genomospecies (GS1 and GS2) or presence of the zot gene, encoding the Zot toxin, affects IL-8 production. A total of 37 C. concisus isolates from patients with microscopic colitis (n = 20), ulcerative colitis (n = 5), Crohn's disease (n = 5), diarrhoea (n = 2) and from healthy controls (n = 5) were used. Intestinal HT-29 cells were infected and incubated for 24 h. Supernatants were subsequently removed and analysed for IL-8 by MILLIPLEX. All isolates were able to stimulate IL-8 production and IL-8 levels were higher than in non-infected HT-29 cells. No difference was observed between disease phenotypes or GS1 and GS2, whereas presence of the zot gene showed a tendency towards higher IL-8 production. Further investigations in other inflammatory and physiological models are needed to conclude whether C. concisus strains from different gastrointestinal disease phenotypes differ in pathogenic potential and play a part in gastrointestinal disease.

Characterization of circulating extracellular traps and immune responses to citrullinated LL37 in psoriasis.

Background: The DNA-binding peptide LL37 is a suspected autoantigen in psoriasis. It can be found in neutrophil extracellular traps (NETs) which have been suggested to play a role in the pathogenesis of the disease. Citrullination, the conversion of peptidyl-arginine into peptidyl-citrulline, can be implicated in the formation of NETs. We hypothesized that citrullination increases LL37 immunogenicity and that NETs are a source of LL37. Objectives: We aimed to characterize cytokine responses of B cells and T cells to native and citrullinated LL37 (citLL37) and determine the prevalence and composition of circulating NETs in patients with psoriasis and healthy blood donors (HDs). Methods: Mononuclear cells (MNCs) and serum were isolated from 20 HDs and 20 patients with psoriasis. The MNCs were stimulated with native LL37 and citLL37 and the proportion of cytokine-positive B cells and T cells was determined by flow cytometry. Circulating antibodies against native LL37 and citLL37 as well as circulating NETs were measured by ELISA, as was the content of LL37, citLL37, and IgG in the NETs. Results: CitLL37, but not native LL37, induced IFN-γ-production by T cells and B cells from psoriasis patients, as well as IL-10-production by the patients' CD4+ T cells. Serum from 40% of patients and 55% of HDs contained circulating NETs, of which 63% and 27%, respectively, contained LL37. Only two patients had NETs containing citLL37 and IgG antibodies were found in NETs from three patients and one HD. Post-hoc analysis of the cytokines produced by B cells and T cells after stimulation with citLL37 revealed two clusters of patients consisting of 10 high-responders and 9 low-responders. The high-responders were those that had circulating NETs in combination with an earlier age of onset of the disease. Conclusion: Citrullinated but not native LL37 elicits IFN-γ-responses by T cells and B cells from psoriasis patients, particularly those with circulating NETs and early disease onset, suggesting a role of citLL37 as an autoantigen in this subgroup of patients.