Objective:Patients are increasingly using and requesting LT4/LT3 combination treatment for persistent hypothyroid symptoms, but the efficacy and side effects of long-term therapy remain largely unexplored. This study aimed to describe the patient group experiencing a long-lasting impact of LT4/LT3 and evaluate their quality of life (QoL) and hypothyroid symptoms. Method:We performed a cross-sectional study of 66 hypothyroid patients who had previously initiated LT4/LT3 combination therapy. The patients were grouped by current treatment into patients still receiving LT4/LT3 treatment (T3 responders) and patients who had discontinued LT3 treatment due to lack of effect (T3 non-responders). ThyPRO was used to evaluate QoL, and a validated symptom score was used to assess hypothyroid symptoms. The paper describes a real-life study that depicts unsatisfied patients as they are met in an outpatient clinic. Results:The participants had a median age of 56 and had initiated LT4/LT3 combination therapy 5.4 years ago. Fifty-four patients still received LT4/LT3 therapy and 12 patients had discontinued LT3 treatment due to lack of effect. Patients in the T3 responder group experienced a QoL comparable to the background population. Surprisingly, symptom scores in the T3 responder group were at the same levels as seen in Danish females with overt hypothyroidism. Thyoid stimulating hormone (TSH) in the T3 responder group was less than 0.4 mU/L in 38% of patients, indicating overtreatment. Conclusion:LT4/LT3 treatment was well-tolerated with no side effects and high QoL, but patients still experienced many symptoms.

How does leukocyte subset distribution in peripheral blood during the window of implantation (WOI) differ between women with recurrent pregnancy loss (RPL) and fertile women? The distribution of leukocyte subsets differed between RPL and fertile controls. A multivariate model showed an AUC for RPL of 0.819 (p < 0.001) The immune system is suggested to be critical for implantation and pregnancy maintenance. Fluctuations in immune cell distributions occur throughout the menstrual cycle, and the levels during the WOI are suggested to be particularly important, as successful implantation may rely on a finely balanced immune response. Studies have reported that elevated pro-inflammatory responses, and altered proportions of natural killer (NK) cells, T regulatory (Treg)cells, and T-helper(Th)1/2/17 cells may contribute to RPL. However, the specific immune profiles of women with RPL compared to fertile women during the WOI remain poorly characterized, limiting the development of targeted therapeutic strategies. This cohort study included 37 women with RPL (≥2 pregnancy losses) following IVF/ICSI/FET (ART) and 38 fertile female controls. Blood samples were collected during the midluteal phase (WOI) corresponding to the day of embryo transfer and analyzed immediately for leukocyte subsets and ratios. Participants were 18–41 years old. The fertile female controls had regular cycles, no known systemic diseases, no history of pregnancy complications or infertility, and received no hormonal therapy. RPL patients underwent ART and placebo treatment in a placebo-controlled trial. Flow cytometry and Sysmex analyzer were used to measure leukocyte subsets including WBC, lymphocytes, neutrophils, monocytes, Treg-, Th1/2/17-, B-, and NK-cells. Progesterone measurement confirmed luteal phase in controls. Statistics included ROC curves and multivariate regression. The RPL group was significantly older (p = 0.004), had fewer live births (p = 0.041), and more pregnancy losses (p < 0.001) than controls. WBC and neutrophil counts were elevated (p = 0.009, p < 0.001) in RPL patients. The neutrophil-lymphocyte ratio (NLR) (2.71 vs. 1.68, p < 0.001) and monocyte-lymphocyte ratio (MLR) (0.30 vs. 0.23, p = 0.002) were significantly higher and the percentages of CD16-CD56bright NK cells (1.27 vs. 0.96, p = 0.032), Th17 cells (1.70 vs. 1.16, p = 0.006), and the Th17/Treg cell ratio (0.78 vs. 0.48, p = 0.002) were elevated in RPL patients compared to controls. NLR had the highest AUC (0.770) for identifying RPL patients in the univariate analysis, with an optimal cut-off of 2.12 (sensitivity of 83.8% and specificity of 62.5%). Using a multivariate model that included neutrophils, lymphocytes, CD3-CD16+CD56+ NK cells, CD3-CD16-CD56bright NK cells, the Th17/Treg ratio, and the NLR showed an AUC for RPL of 0.819 (sensitivity: 72.97%, specificity: 84.21%, positive predictive value: 81.82%, negative predictive value: 76.1%, p < 0.001). The small sample size and observational design limit generalizability and causal interpretation. Hormonal treatments related to ART in RPL patients may have influenced immune profiles, and it remains unclear whether the observed immune aberrations directly contribute to the increased risk of pregnancy loss in RPL patients. The results highlight potential systemic immune biomarkers for RPL, offering insights into its pathophysiology. If validated, these markers could aid in identifying patients with immune-mediated RPL, refine personalized therapies and improve treatment success rates in RPL patients. Yes

BACKGROUND AND OBJECTIVES:Human neutrophil antigen 2 (HNA-2), encoded by the CD177 gene, is considered one of the most important neutrophil antigens in human medicine, but molecular testing of CD177 is complicated and therefore not a standard procedure for investigating CD177 expression. CD177 expression can vary from 0% to 100%, and to date, the molecular basis for altered or non-expressed genes has not been determined. Reliance on phenotyping and crossmatching to investigate these neutropenic clinical cases is inconvenient for patients and demands substantial resources within the laboratory. The purpose of this study was therefore to test a new molecular testing approach based on long-read nanopore sequencing. MATERIALS AND METHODS:DNA from 44 Danish blood donors with different levels of CD177 expression, 22 of whom were found to be CD177 null, was selected as test samples. All the DNA was sequenced for the first eight exons and the beginning of exon 9 of CD177. RESULTS:All incidences of CD177 null cases could be associated with the known variant c.787A>T;p.K263X (rs20182172), and a correlation was observed between c.787A>T heterozygosity and a reduced expression of CD177, which is consistent with previously published findings. The c.1291G>A;p.G431R (rs78718189) variant was found to be linked to the atypical expression of CD177. The nanopore assay revealed a total of 14 variants in 7 exons in the 44 tested samples. CONCLUSION:On the basis of these observations, we conclude that long-read nanopore sequencing can be a reliable tool for the routine laboratory molecular testing of CD177.

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-Fc gamma RIIIb-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-Fc gamma RIIIb-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.

BACKGROUND:In 2010, Denmark was the first country to implement a targeted routine antenatal anti-D prophylaxis (tRAADP) program, offering fetal RHD genotyping to all nonimmunized D negative pregnant women. The program represented a shift from only postnatal prophylaxis to a combined antenatal and postnatal prophylaxis. This study aimed to evaluate the clinical effect of tRAADP in Denmark. STUDY DESIGN AND METHODS:This nationwide registry-based cohort study included all D negative women who gave birth between 2004-2020, identified through the National Medical Birth Register and the Departments of Clinical Immunology in Denmark. The clinical effect of tRAADP was assessed by comparing the incidence of new D immunization between 2004-2009 (non-tRAADP-cohort) and 2011-2018 (tRAADP-cohort). RESULTS:A total of 282 women were D immunized during pregnancy between 2004-2009 (non-tRAADP-cohort), and 167 between 2011-2018 (tRAADP-cohort). The incidence of new D immunization decreased from 0.46% (95% CI 0.41-0.52) in the non-tRAADP-cohort to 0.22% (95% CI 0.19-0.25) in the tRAADP-cohort. The risk reduction was statistically significant p < 0.001. Notably, in the tRAADP cohort 0.1% (95% CI 0.08-0.12) of new D immunizations occurred before the time of antenatal prophylaxis. DISCUSSION:tRAADP significantly reduced the incidence of new D immunization by more than half, thus demonstrating the expected effect. However, even with full adherence to the current program, some women with early fetomaternal hemorrhage (FMH) were still at risk. Future studies may evaluate the impact of administering an additional tRAADP dose earlier in the second trimester to prevent this.

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.

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.

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.

Pediatric PulmonologyVolume 59, Issue 4 p. 1103-1107 LETTER Surfactant protein D is associated with pulmonary manifestations of chronic graft-versus-host disease following hematopoietic stem cell transplantation Anne Mols Krarup MD, Anne Mols Krarup MD Department of Pediatrics and Adolescent Medicine, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Institute for Inflammation Research, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Contribution: ​Investigation, Data curation, Formal analysis, Visualization, Funding acquisition, Validation, Writing - review & editingSearch for more papers by this authorKatrine Kielsen MD, PhD, Katrine Kielsen MD, PhD orcid.org/0000-0002-7167-012X Department of Pediatrics and Adolescent Medicine, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Institute for Inflammation Research, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Contribution: Data curation, ​Investigation, Formal analysis, Supervision, Visualization, Writing - original draftSearch for more papers by this authorHilde Hylland Uhlving MD, PhD, Hilde Hylland Uhlving MD, PhD Department of Pediatrics and Adolescent Medicine, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Contribution: Conceptualization, Methodology, Data curation, ​Investigation, Supervision, Writing - review & editing, Funding acquisitionSearch for more papers by this authorRudi Steffensen PhD, Rudi Steffensen PhD Department of Clinical Immunology, Aalborg University Hospital, Aalborg, Denmark Contribution: Methodology, ​Investigation, Writing - review & editing, Resources, ValidationSearch for more papers by this authorMaria Ebbesen Sørum MD, PhD, Maria Ebbesen Sørum MD, PhD Department of Pediatrics and Adolescent Medicine, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Contribution: Funding acquisition, Writing - review & editing, Supervision, ​InvestigationSearch for more papers by this authorKim Gjerum Nielsen MD, DMSc, Kim Gjerum Nielsen MD, DMSc Department of Pediatrics and Adolescent Medicine, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Institute of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark Contribution: Writing - review & editing, Methodology, Supervision, ResourcesSearch for more papers by this authorFrederik F. Buchvald MD, PhD, Frederik F. Buchvald MD, PhD Department of Pediatrics and Adolescent Medicine, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Contribution: Methodology, Writing - review & editing, Supervision, ResourcesSearch for more papers by this authorGrith L. Sorensen MSc, PhD, DMSc, Grith L. Sorensen MSc, PhD, DMSc Department of Cancer and Inflammation, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark Contribution: Resources, ​Investigation, Validation, Writing - review & editing, MethodologySearch for more papers by this authorKlaus Gottlob Müller MD, PhD, DMSc, Corresponding Author Klaus Gottlob Müller MD, PhD, DMSc [email protected] Department of Pediatrics and Adolescent Medicine, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Institute for Inflammation Research, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Institute of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark CorrespondenceKlaus Gottlob Müller, MD, PhD, DMSc, Department of Pediatrics and Adolescent Medicine, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. Email: [email protected] Contribution: Conceptualization, Supervision, Project administration, Resources, Funding acquisition, Visualization, Writing - original draftSearch for more papers by this author Anne Mols Krarup MD, Anne Mols Krarup MD Department of Pediatrics and Adolescent Medicine, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Institute for Inflammation Research, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Contribution: ​Investigation, Data curation, Formal analysis, Visualization, Funding acquisition, Validation, Writing - review & editingSearch for more papers by this authorKatrine Kielsen MD, PhD, Katrine Kielsen MD, PhD orcid.org/0000-0002-7167-012X Department of Pediatrics and Adolescent Medicine, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Institute for Inflammation Research, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Contribution: Data curation, ​Investigation, Formal analysis, Supervision, Visualization, Writing - original draftSearch for more papers by this authorHilde Hylland Uhlving MD, PhD, Hilde Hylland Uhlving MD, PhD Department of Pediatrics and Adolescent Medicine, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Contribution: Conceptualization, Methodology, Data curation, ​Investigation, Supervision, Writing - review & editing, Funding acquisitionSearch for more papers by this authorRudi Steffensen PhD, Rudi Steffensen PhD Department of Clinical Immunology, Aalborg University Hospital, Aalborg, Denmark Contribution: Methodology, ​Investigation, Writing - review & editing, Resources, ValidationSearch for more papers by this authorMaria Ebbesen Sørum MD, PhD, Maria Ebbesen Sørum MD, PhD Department of Pediatrics and Adolescent Medicine, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Contribution: Funding acquisition, Writing - review & editing, Supervision, ​InvestigationSearch for more papers by this authorKim Gjerum Nielsen MD, DMSc, Kim Gjerum Nielsen MD, DMSc Department of Pediatrics and Adolescent Medicine, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Institute of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark Contribution: Writing - review & editing, Methodology, Supervision, ResourcesSearch for more papers by this authorFrederik F. Buchvald MD, PhD, Frederik F. Buchvald MD, PhD Department of Pediatrics and Adolescent Medicine, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Contribution: Methodology, Writing - review & editing, Supervision, ResourcesSearch for more papers by this authorGrith L. Sorensen MSc, PhD, DMSc, Grith L. Sorensen MSc, PhD, DMSc Department of Cancer and Inflammation, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark Contribution: Resources, ​Investigation, Validation, Writing - review & editing, MethodologySearch for more papers by this authorKlaus Gottlob Müller MD, PhD, DMSc, Corresponding Author Klaus Gottlob Müller MD, PhD, DMSc [email protected] Department of Pediatrics and Adolescent Medicine, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Institute for Inflammation Research, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Institute of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark CorrespondenceKlaus Gottlob Müller, MD, PhD, DMSc, Department of Pediatrics and Adolescent Medicine, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. Email: [email protected] Contribution: Conceptualization, Supervision, Project administration, Resources, Funding acquisition, Visualization, Writing - original draftSearch for more papers by this author First published: 11 January 2024 https://doi.org/10.1002/ppul.26836 Anne Mols Krarup and Katrine Kielsen shared first authorship. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat REFERENCES 1Uhlving HH, Bang CL, Christensen IJ, et al. Lung function after allogeneic hematopoietic stem cell transplantation in children: a longitudinal study in a population-based cohort. Biol Blood Marrow Transplant. 2013; 19: 1348-1354. 10.1016/j.bbmt.2013.06.005 PubMedWeb of Science®Google Scholar 2Duncan CN, Buonanno MR, Barry EV, Myers K, Peritz D, Lehmann L. Bronchiolitis obliterans following pediatric allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant. 2008; 41: 971-975. 10.1038/bmt.2008.19 CASPubMedWeb of Science®Google Scholar 3Sorensen GL. Surfactant protein D in respiratory and non-respiratory diseases. Front Med. 2018; 5: 18. 10.3389/fmed.2018.00018 Web of Science®Google Scholar 4Sørensen GL, Hjelmborg JB, Kyvik KO, et al. Genetic and environmental influences of surfactant protein D serum levels. Am J Physiol Lung Cell Mol Physiol. 2006; 290: L1010-L1017. 10.1152/ajplung.00487.2005 CASPubMedWeb of Science®Google Scholar 5Nakane T, Nakamae H, Kamoi H, et al. Prognostic value of serum surfactant protein D level prior to transplant for the development of bronchiolitis obliterans syndrome and idiopathic pneumonia syndrome following allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant. 2008; 42: 43-49. 10.1038/bmt.2008.73 CASPubMedWeb of Science®Google Scholar 6Lomas DA, Silverman EK, Edwards LD, et al. Serum surfactant protein D is steroid sensitive and associated with exacerbations of COPD. Eur Respir J. 2009; 34: 95-102. 10.1183/09031936.00156508 CASPubMedWeb of Science®Google Scholar 7Miller MR. Standardisation of spirometry. Eur Respir J. 2005; 26: 319-338. 10.1183/09031936.05.00034805 CASPubMedWeb of Science®Google Scholar 8Macintyre N. Standardisation of the single-breath determination of carbon monoxide uptake in the lung. Eur Respir J. 2005; 26: 720-735. 10.1183/09031936.05.00034905 CASPubMedWeb of Science®Google Scholar Volume59, Issue4April 2024Pages 1103-1107 ReferencesRelatedInformation

Recurrent pregnancy loss (RPL), defined as two or more pregnancy losses before the 24th week of gestation, affects 1%-3% of women worldwide. Approximately, 40% of RPL cases are secondary RPL (sRPL), where women have given birth before facing pregnancy losses. The underlying causes of RPL remain unclear, but immune-related factors may play a role. Previously, a randomised controlled trial using immunoglobulin (IVIG) in sRPL women with a history of four pregnancy losses performed in our RPL unit did not show significant effects of IVIG treatment overall. Yet, some evidence suggests potential benefits for a subset of sRPL patients. In the cohort used for the randomised controlled trial, we examined the role of maternal HLA class II-presented fetal HLA-derived epitopes in sRPL using the predicted indirectly recognisable HLA epitopes (PIRCHE-II) algorithm. In the placebo group, sRPL mothers with an anti-HLA antibody response had higher PIRCHE-II scores when having a live birth compared with sRPL women who experienced another pregnancy loss. This difference was not observed in the IVIG-treated group. Furthermore, as a proxy for T-cell memory, the number of overlapping peptides between the two paternal haplotypes in couples having live births without treatment displayed a larger number of overlapping peptides. This effect was primarily driven by class II-derived peptides. These results suggest that specific combinations of sRPL mothers and fathers, particularly those with an anti-HLA antibody response, may generate higher PIRCHE-II scores, which could contribute to successful live births. Understanding these immune interactions may provide insights for personalised diagnostic and therapeutic strategies in sRPL.

Molecular HLA typing techniques are currently undergoing a rapid evolution. While real‐time PCR is established as the standard method in tissue typing laboratories regarding allocation of solid organs, next generation sequencing (NGS) for high‐resolution HLA typing is becoming indispensable but is not yet suitable for deceased donors. By contrast, high‐resolution typing is essential for stem cell transplantation and is increasingly required for questions relating to various disease associations. In this multicentre clinical study, the TGS technique using nanopore sequencing is investigated applying NanoTYPE™ kit and NanoTYPER™ software (Omixon Biocomputing Ltd., Budapest, Hungary) regarding the concordance of the results with NGS and its practicability in diagnostic laboratories. The results of 381 samples show a concordance of 99.58% for 11 HLA loci, HLA‐A, ‐B, ‐C, ‐DRB1, ‐DRB3, ‐DRB4, ‐DRB5, ‐DQA1, ‐DQB1, ‐DPA1 and ‐DPB1. The quality control (QC) data shows a very high quality of the sequencing performed in each laboratory, 34,926 (97.15%) QC values were returned as ‘passed’, 862 (2.4%) as ‘inspect’ and 162 (0.45%) as ‘failed’. We show that an ‘inspect’ or ‘failed’ QC warning does not automatically lead to incorrect HLA typing. The advantages of nanopore sequencing are speed, flexibility, reusability of the flow cells and easy implementation in the laboratory. There are challenges, such as exon coverage and the handling of large amounts of data. Finally, nanopore sequencing presents potential for applications in basic research within the field of epigenetics and genomics and holds significance for clinical concerns.

Genetic variation in the FCGR3B gene is responsible for different variants of human neutrophil antigen 1 (HNA-1). Laboratory techniques currently utilized for routine HNA-1 genotyping, predominantly PCR-sequence-specific primer (PCR-SSP) and PCR-sequence-based typing (PCR-SBT), lack specificity for FCGR3B. This study compares the capabilities and limitations of existing technologies including an in-house TaqMan PCR, a commercial PCR-SSP test, PCR-SBT and multiplex ligation-dependent probe amplification (MLPA) with those of a long-read nanopore sequencing assay. Testing was performed with both related and unrelated Danish samples with different copy numbers and/or rare alleles. Long-read nanopore sequencing was validated by blind testing of ten English samples. The results showed that FCGR3B copy numbers correlate with a dose-dependent distribution of alleles that complicates genotyping by TaqMan PCR, PCR-SSP and PCR-SBT, due to co-amplification of the homologous FCGR3A gene. MLPA can correctly quantify the dose-dependent distribution but not detect novel variants. Long-read nanopore sequencing showed high specificity for FCGR3B and was able to detect dosage-dependent distribution, and rare and novel variants that were previously not described. Current HNA-1 genotyping methods cannot produce unambiguous allele-level results, whereas long-read nanopore sequencing has shown the potential to resolve observed ambiguities, identify new HNA-1 variants and allow definitive allele assignment.

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.

Autoimmune neutropenia (AIN) of early childhood is caused by autoantibodies against antigens on the neutrophil membrane. Human leukocyte antigens (HLA) have previously been associated with AIN. This study investigated HLA-DRB1 and HLA-DQB1 alleles in 160 antibody positive patients and compared with 1000 controls. Increased risk was observed for DRB1*10, DRB1*14, DRB1*16 and DQB1*05, and lower risk for DRB1*04, DRB1*13 and DQB1*03. Haplotypes with higher risk included: DRB1*10/DQB1*05, DRB1*14/DQB1*05 and DRB1*16/DQB1*05, while DRB1*04/DQB1*03, DRB1*07/DQB1*02, and DRB1*13/DQB1*06 were associated with lower risk. Associated HLA-DRB1 and –DQB1 differed between patients positive for anti-HNA-1a-specific antibodies and patients positive for broad reactive anti-FcγRIIIb antibodies. DRB1*01, DRB1*04 and DQB1*03 was only associated for anti-HNA-1a positive, and DRB1*10 was restricted to broad reactive anti-FcγRIIIb positive. Strong association between AIN and HLA-DRB1 and -DQB1 alleles and haplotypes suggested that they play a role in susceptibility or protection. Different associations regarding FcγRIIIb antibody specificities could indicate disease heterogeneity.

Chronic histocytic intervillositis (CHI) occurs in 5-6 of 10,000 pregnancies and is strongly associated with early and late miscarriages. The recurrence rate is very high. It is defined by infiltration of CD68+ histiocytes into the intervillous space of the placenta.A previous small study [1] found high levels of complement-activating anti-fetal HLA antibodies in women with CHI. Seven patients in our clinic had a concomitant diagnosis of recurrent pregnancy loss (RPL) and CHI diagnosed histologically in connection to second trimester pregnancy losses. The patients had blood samples taken for HLA antibody analysis, and their live firstborn children and dead fetuses had alleles determined in 11 HLA loci. Serum was screened for HLA antibodies and their complement activating properties. The patients had a median of 5 previous pregnancy losses. All patients were positive for HLA antibodies; in 6 cases specific for alleles carried by their firstborn children or a dead fetus. In 3 cases the specificity was against HLA-B alleles, in one case against an HLA-DRB1 allele, and in two cases they were directed against both HLA class I and II alleles. The levels of the antibodies were all very high with mean flourescense intensity (MFI) between 16,000 and 40,000 and all were complement-activating. MFI levels >7000 define strong HLA antibodies. Since histiocyte accumulation in the placenta and presence of very high levels of complement-activating anti-fetal HLA antibodies characterize women with CHI, it is likely that their pregnancy losses are caused by mechanisms similar to antibody-mediated allograft rejection.

BackgroundThe 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.ObjectivesWe 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).MethodsMononuclear 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.ResultsCitLL37, 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.ConclusionCitrullinated 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.

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, β2 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.