Nipocalimab in Early-Onset Severe Hemolytic Disease of the Fetus and Newborn.

BACKGROUND: In early-onset severe hemolytic disease of the fetus and newborn (HDFN), transplacental transfer of maternal antierythrocyte IgG alloantibodies causes fetal anemia that leads to the use of high-risk intrauterine transfusions in order to avoid fetal hydrops and fetal death. Nipocalimab, an anti-neonatal Fc receptor blocker, inhibits transplacental IgG transfer and lowers maternal IgG levels. METHODS: In an international, open-label, single-group, phase 2 study, we assessed treatment with intravenous nipocalimab (30 or 45 mg per kilogram of body weight per week) administered from 14 to 35 weeks' gestation in participants with pregnancies at high risk for recurrent early-onset severe HDFN. The primary end point was live birth at 32 weeks' gestation or later without intrauterine transfusions as assessed against a historical benchmark (0%; clinically meaningful difference, 10%). RESULTS: Live birth at 32 weeks' gestation or later without intrauterine transfusions occurred in 7 of 13 pregnancies (54%; 95% confidence interval, 25 to 81) in the study. No cases of fetal hydrops occurred, and 6 participants (46%) did not receive any antenatal or neonatal transfusions. Six fetuses received an intrauterine transfusion: five fetuses at 24 weeks' gestation or later and one fetus before fetal loss at 22 weeks and 5 days' gestation. Live birth occurred in 12 pregnancies. The median gestational age at delivery was 36 weeks and 4 days. Of the 12 live-born infants, 1 received one exchange transfusion and one simple transfusion and 5 received only simple transfusions. Treatment-related decreases in the alloantibody titer and IgG level were observed in maternal samples and cord blood. No unusual maternal or pediatric infections were observed. Serious adverse events were consistent with HDFN, pregnancy, or prematurity. CONCLUSIONS: Nipocalimab treatment delayed or prevented fetal anemia or intrauterine transfusions, as compared with the historical benchmark, in pregnancies at high risk for early-onset severe HDFN. (Funded by Janssen Research and Development; UNITY ClinicalTrials.gov number, NCT03842189.).

The CD177 c.1291A Allele Leads to a Loss of Membrane Expression and Mimics a CD177-Null Phenotype.

CD177 is a glycosyl phosphatidyl inositol (GPI)-linked, neutrophil-specific glycoprotein that in 3-5% of normal individuals is absent from all neutrophils. The molecular mechanism behind the absence of CD177 has not been unravelled completely. Here, we analyse the impact of the recently described CD177 c.1291G>A variant on CD177 expression. Recombinant CD177 c.1291G>A was expressed in HEK293F cells and its expression on the cell surface, inside the cell, and in the culture supernatant was investigated. The CD177 c.1291G>A protein was characterised serologically and its interaction with proteinase 3 (PR3) was demonstrated by confocal laser scanning microscopy. Our experiments show that CD177 c.1291G>A does not interfere with CD177 protein biosynthesis but affects the membrane expression of CD177, leading to very low copy numbers of the protein on the cellular surface. The mutation does not interfere with the ability of the protein to bind PR3 or human polyclonal antibodies against wild-type CD177. Carriers of the c.1291G>A allele are supposed to be phenotyped as CD177-negative, but the protein is present in soluble form. The presence of CD177 c.1291A leads to the production of an unstable CD177 protein and an apparent "CD177-null" phenotype.

Fetal and neonatal alloimmune thrombocytopenia: No evidence of systemic inflammation as a modulator of disease severity. Could placental inflammation be key?

In fetal/neonatal alloimmune thrombocytopenia (FNAIT), maternal alloantibodies against paternal human platelet antigens (HPA) cross the placenta and lead to platelet destruction. The extent of thrombocytopenia varies among neonates, and inflammation may constitute an important trigger. A set of stable inflammatory markers was measured in serum samples from neonates with low platelet counts, of which n = 50 were diagnosed with FNAIT due to anti-HPA-1a antibodies and n = 50 were thrombocytopenic without detectable maternal HPA antibodies. Concentrations of C-reactive protein, soluble CD14, procalcitonin, and sFlt-1 did not differ between the two cohorts. There was no correlation between C-reactive protein or soluble CD14 and the platelet count, but a negative correlation between procalcitonin concentrations and the neonatal platelet count in both cohorts. sFlt-1 concentration and the platelet count were correlated in FNAIT cases exclusively. None of the inflammatory markers was statistically different between cases with and without intracranial haemorrhage. We were unable to identify systemic inflammation as a relevant factor for thrombocytopenia in FNAIT. The antiangiogenic enzyme sFlt-1, released by the placenta, did correlate with the platelet count in FNAIT cases. Our findings may give rise to the hypothesis that placental inflammation rather than systemic inflammation modulates disease severity in FNAIT.

Anti-human platelet antigen-5b antibodies and fetal and neonatal alloimmune thrombocytopenia; incidental association or cause and effect?

Most cases of fetal and neonatal thrombocytopenia (FNAIT) are caused by maternal anti-human platelet antigen-1a antibodies (anti-HPA-1a). Anti-HPA-5b antibodies are the second most common antibodies in suspected FNAIT cases. Given the high prevalence of anti-HPA-5b antibodies in pregnant women delivering healthy newborns, the association with FNAIT may be coincidental. This review of the literature related to FNAIT using the MEDLINE database was conducted according to PRISMA guidelines. A retrospective analysis of a single-centre cohort of 817 suspected FNAIT cases was conducted. The pooled prevalence of anti-HPA-5b antibodies in unselected pregnant women of European descent was 1.96% (n = 3113), compared with 3.4% (n = 5003) in women with suspected FNAIT. We found weak evidence that a small proportion of pregnant women presenting with anti-HPA-5b antibodies will give birth to a newborn with mild thrombocytopenia. The neonatal platelet counts were not different between suspected FNAIT cases (n = 817) with and without maternal anti-HPA-5b antibodies. The prevalence of maternal anti-HPA-5b antibodies was not different between neonates with intracranial haemorrhage and healthy controls. The current experimental and epidemiological evidence does not support the hypothesis that anti-HPA-5b antibodies cause severe thrombocytopenia or bleeding complications in the fetus or newborn.

Recommendation for validation and quality assurance of non-invasive prenatal testing for foetal blood groups and implications for IVD risk classification according to EU regulations.

BACKGROUND AND OBJECTIVES: Non-invasive assays for predicting foetal blood group status in pregnancy serve as valuable clinical tools in the management of pregnancies at risk of detrimental consequences due to blood group antigen incompatibility. To secure clinical applicability, assays for non-invasive prenatal testing of foetal blood groups need to follow strict rules for validation and quality assurance. Here, we present a multi-national position paper with specific recommendations for validation and quality assurance for such assays and discuss their risk classification according to EU regulations. MATERIALS AND METHODS: We reviewed the literature covering validation for in-vitro diagnostic (IVD) assays in general and for non-invasive foetal RHD genotyping in particular. Recommendations were based on the result of discussions between co-authors. RESULTS: In relation to Annex VIII of the In-Vitro-Diagnostic Medical Device Regulation 2017/746 of the European Parliament and the Council, assays for non-invasive prenatal testing of foetal blood groups are risk class D devices. In our opinion, screening for targeted anti-D prophylaxis for non-immunized RhD negative women should be placed under risk class C. To ensure high quality of non-invasive foetal blood group assays within and beyond the European Union, we present specific recommendations for validation and quality assurance in terms of analytical detection limit, range and linearity, precision, robustness, pre-analytics and use of controls in routine testing. With respect to immunized women, different requirements for validation and IVD risk classification are discussed. CONCLUSION: These recommendations should be followed to ensure appropriate assay performance and applicability for clinical use of both commercial and in-house assays.

Sex-specific differences in plasma levels of FXII, HK, and FXIIa-C1-esterase inhibitor complexes in community-acquired pneumonia.

Sex-dependent differences in immunity and coagulation play an active role in the outcome of community-acquired pneumonia (CAP). Contact phase proteins act at the crossroads between inflammation and coagulation thus representing a point of convergence in host defense against infection. Here, we measured the levels of factor XII (FXII), FXIIa-C1 esterase inhibitor (C1INH) complexes, and high-molecular-weight kininogen (HK) in plasma of patients with CAP and correlated them to clinical disease severity. Levels of FXIIa-C1INH/albumin ratio were elevated, irrespective of sex, in plasma of patients with CAP (n = 139) as compared with age-matched donors (n = 58). No simultaneous decrease in FXII levels, indicating its consumption, was observed. Stratification by sex revealed augmented FXII levels in plasma of women with CAP as compared with sex-matched donors yet no apparent differences in men. This sex-specific effect was, however, attributable to lower FXII levels in female donors relative to men donors. Plasma estradiol levels mirrored those for FXII. Levels of HK/albumin ratio were decreased in CAP plasma as compared with donors, however, after stratification by sex, this difference was only observed in women and was related to higher HK/albumin values in female donors as opposed to male donors. Finally, strong negative correlation between plasma levels of HK/albumin ratio and CAP severity, as assessed by CRB65 score, in males and females was observed. Our study identifies sex-dependent differences in plasma levels of the contact phase proteins in elderly subjects that may contribute to specific clinical outcomes in CAP between men and women.

Characterization of CD177-reactive iso- and auto-antibodies.

BACKGROUND: CD177 is a surface protein on neutrophils and a main mediator for the surface expression of proteinase 3 (PR3). Its functions are largely unknown. At least three types of antibodies have been described to target CD177: isoantibodies, which are formed in CD177-null individuals as a result of an immune reaction following transfusion or pregnancy; autoantibodies present in sera from patients with autoimmune neutropenia; and antineutrophil cytoplasmic antibodies in sera from patients with glomerulonephritis with polyangiitis. In this study, we aimed to compare the binding characteristics of auto- and iso-antibodies to optimize their detectability in the neutrophil serology laboratory. STUDY DESIGN AND METHODS: The reactivity of iso- and auto-antibodies against CD177 was studied using granulocytes, "native" CD177/PR3 complex, and recombinant CD177 or PR3. RESULTS: All iso- and auto-antibodies were reactive with CD177/PR3 when immobilized with monoclonal antibody (moab) 7D8. Seventy-five percent of autoantibodies, but none of the isoantibodies, did not react with CD177/PR3 immobilized with moab MEM166. The majority of autoantibodies did not react with recombinant CD177, whereas most isoantibodies tested positive. DISCUSSION: Our results suggest that iso- and auto-antibodies against CD177 target different epitopes. Isoantibodies mainly target CD177 alone, while the majority of autoantibodies target a native epitope present on the neutrophil surface, but absent from recombinant CD177 which lacks PR3. Moab MEM166 binds to the native epitope and hinders the binding of CD177 autoantibodies. The results may help to design diagnostic strategies, especially for the identification of autoantibodies.

ADAR1 Is Required for Dendritic Cell Subset Homeostasis and Alveolar Macrophage Function.

RNA editing by adenosine deaminases acting on dsRNA (ADAR) has become of increasing medical relevance, particularly because aberrant ADAR1 activity has been associated with autoimmunity and malignancies. However, the role of ADAR1 in dendritic cells (DC), representing critical professional APCs, is unknown. We have established conditional murine CD11c Cre-mediated ADAR1 gene ablation, which did not induce general apoptosis in CD11c+ cells but instead manifests in cell type-specific effects in DC subpopulations. Bone marrow-derived DC subset analysis revealed an incapacity to differentiate CD103 DC+ in both bulk bone marrow and purified pre-DC lineage progenitor assays. ADAR1 deficiency further resulted in a preferential systemic loss of CD8+/CD103+ DCs, revealing critical dependency on ADAR1, whereas other DC subpopulations were moderately affected or unaffected. Additionally, alveolar macrophages were depleted and dysfunctional, resembling pulmonary alveolar proteinosis. These results reveal an unrecognized role of ADAR1 in DC subset homeostasis and unveils the cell type-specific effects of RNA editing.

Incidental diagnosis of leukocyte adhesion deficiency type II following ABO typing.

Individuals with the Bombay phenotype (Oh) in the ABO blood group system do not express the H, A, and B antigens but have no clinical symptoms. Bombay phenotype with clinical symptoms has been described in leukocyte adhesion deficiency type II (LAD II), a fucosylation disorder caused by mutations in SLC35C1. Only few LAD II patients have been described so far. Here we describe an additional patient, a 22-year old male, born to unrelated parents, presenting with inflammatory skin disease, periodontitis, growth, and mental retardation, admitted to the department of dentistry for treatment under general anesthesia. Pre-operative routine investigations revealed the presence of the Bombay phenotype (Oh). Genomic sequencing identified two novel triplet deletions of the SLC35C1 gene. Functional investigations confirmed the diagnosis of LAD II. Therapy with oral fucose led to the disappearance of the chronic skin infections and improvements in behavior and attention span.

Maternal antibodies against paternal class I human leukocyte antigens are not associated with foetal and neonatal alloimmune thrombocytopenia.

The causative role of maternal, anti-human leukocyte antigen (anti-HLA) class I antibodies in foetal and neonatal alloimmune thrombocytopenia (FNAIT) remains controversial. Furthermore, in FNAIT cases caused by anti-human platelet antigen-1a (anti-HPA-1a) antibodies, the possible additive effect of maternal anti-HLA class I antibodies on outcomes is unclear. Among 817 mother-father-neonate trios of suspected FNAIT, we assessed the possible association of maternal anti-HLA class I antibodies with neonatal platelet count, and the incidence of FNAIT caused by anti-HPA-1a antibodies. In 144 FNAIT cases caused by anti-HPA-1a antibodies, we investigated the possible association of maternal anti-HLA class I antibodies with neonatal platelet count, birth weight, and the incidence of intracranial haemorrhage (n = 16). Maternal anti-HLA class I antibodies were not associated with neonatal platelet count in suspected cases of FNAIT. There was no significant interaction between the presence of anti-HLA class I antibodies and anti-HPA-1a antibodies. In FNAIT cases caused by anti-HPA-1a antibodies, there was no association between the presence of anti-HLA class I antibodies and neonatal platelet count, birth weight, or occurrence of intracranial haemorrhage. This study's findings do not support the concept that maternal anti-HLA class I antibodies represent a risk factor of FNAIT or disease severity.

Primary structure of human neutrophil antigens 1a and 1b.

BACKGROUND: Neutrophil specific Fcγ receptor IIIb (CD16b) is a low-affinity IgG receptor. Its polymorphic variants are associated with human neutrophil antigens (HNA). HNA-1a and HNA-1b differ in four amino acids. Immunization can lead to the production of alloantibodies. The exact contribution of four amino acid exchanges for the formation of HNA-1a, -1b epitopes is currently unknown. STUDY DESIGN AND METHODS: Permutation of each polymorphic amino acid from wild-type CD16b cDNA constructs was performed and expressed on HEK293 cells. All 16 receptor variants were produced and tested against 19 well-characterized HNA antisera in an antigen capture assay. RESULTS: Analyzing the reaction pattern revealed that anti-HNA-1a antibodies can bind whenever asparagine (N) is present in position 65, regardless of the three other positions (CD16b *N**). Anti-HNA-1b antibodies can bind when serine (S) is present in position 36 (CD16b S***), when N is present in position 82 (CD16b **N*), or both (CD16b S*N*). CD16b variants with N65 and S36 and/or N82 (such as CD16b SNN*) bind both, anti-HNA-1a and anti-HNA-1b alloantibodies. If these specific amino acids are missing (as in CD16b RSD*), no antibodies will bind. CONCLUSION: Whereas the primary structure of HNA-1a and HNA-1b usually differs in four amino acids, epitope composition is not "antithetical". N65 alone determines the presence of HNA-1a, and S36 and/or N82 determine the presence of HNA-1b. Amino acid 106 does not participate in epitope formation. Our findings are of specific relevance when a HNA-1 phenotype is predicted from a genotype.

Naturally occurring point mutation Cys460Trp located in the I-EGF1 domain of integrin ß3 alters the binding of some anti-HPA-1a antibodies.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by the destruction of platelets in the fetus or newborn by maternal platelet alloantibodies, mostly against human platelet antigen (HPA)-1a. Recent studies indicate that two anti-HPA subtypes exist: Type I reacts with epitopes residing on the plexin-semaphorin-integrin (PSI) and type II with plexin-semaphorin-integrin/integrin epidermal growth factor 1 (I-EGF1) domains of the ß3 integrin. Here, we evaluated whether a Cys460Trp mutation in the I-EGF1 domain found in a patient with Glanzmann thrombasthenia can alter the binding of anti-HPA-1a. METHODS: Stable HEK293 cell lines expressing wild-type and mutant αIIbß3 and αvß3 were generated to prove the reactivity of different antibodies against HPA-1a. RESULTS: Flow cytometry analysis of wild-type (Cys460) and mutant (Trp460) expressed on HEK293 cells showed equal surface expression of αIIbß3 and αvß3. When tested with mutant αIIbß3 cells, reduced binding was observed in Type II but not in Type I anti-HPA-1a. These results could be confirmed with platelets carrying Cys460Trp mutation. Interestingly, reduced binding of Type I antibodies was detected with mutant αvß3 cells. Both antibody types were found in maternal sera from FNAIT cases by an antigen-capture assay with use of HEK293 transfected cells. CONCLUSIONS: These observations confirm the existence of Type I and Type II anti-HPA-1a. Furthermore, this study underlines different immunogenicity of HPA-1a antigen(s) residing on either αIIbß3 or αvß3. Further analysis of FNAIT cases from mothers having a fetus with and without intracranial bleedings with use of such an approach may highlight the functional relevance of different anti-HPA-1a subtypes.

CD71 surface analysis of T cells: a simple alternative for extracorporeal photopheresis quality control.

BACKGROUND: Extracorporeal photopheresis (ECP) is a leukapheresis-based cellular therapy that is used with increasing frequency worldwide to treat various T-cell-mediated diseases. Currently, the inhibition of T-cell proliferation after photopheresis is analysed frequently using time-consuming assays including radioactive thymidine assays or carboxyfluorescein succinimidyl ester (CFSE) staining. We investigated whether simple surface T-cell staining using surrogate markers of T-cell proliferation can replace time-consuming measurement of T-cell proliferation in ECP quality control. STUDY DESIGN AND METHODS: T-cell activation markers were investigated by flow cytometry after ECP. Candidates were validated by direct comparison with the classical CFSE T-cell proliferation inhibition test and apoptosis staining. Finally, surface T-cell staining was performed in patient samples in comparison with classical methods. RESULTS: CD71 expression exhibited the fastest and most robust upregulation, which was detectable as early as 6-8 h after T-cell stimulation and almost completely abrogated by ECP. In a direct comparison with the CFSE T-cell proliferation assay, suppression of CD71 expression after ECP was almost identical and detectable as early as 16 h after stimulation in peripheral blood mononuclear cells of healthy donors. Furthermore, in direct comparison with classical apoptosis staining, the inhibition delta of CD71 after ECP was significantly higher. Moreover, in patients under T-cell suppressive therapy, T-cell-dependent CFSE and CD71 assays exhibited decreased sensitivity to detect ECP treatment and were inferior in comparison to apoptosis staining. CONCLUSION: Surface CD71 analysis represents a very simple quality control alternative to detect ECP-mediated T-cell proliferation inhibition in normal PBMC samples devoid of T-cell suppressive drugs.

Targeted antenatal anti-D prophylaxis for RhD-negative pregnant women: a systematic review.

BACKGROUND: All non-sensitized Rhesus D (RhD)-negative pregnant women in Germany receive antenatal anti-D prophylaxis without knowledge of fetal RhD status. Non-invasive prenatal testing (NIPT) of cell-free fetal DNA in maternal plasma could avoid unnecessary anti-D administration. In this paper, we systematically reviewed the evidence on the benefit of NIPT for fetal RhD status in RhD-negative pregnant women. METHODS: We systematically searched several bibliographic databases, trial registries, and other sources (up to October 2019) for controlled intervention studies investigating NIPT for fetal RhD versus conventional anti-D prophylaxis. The focus was on the impact on fetal and maternal morbidity. We primarily considered direct evidence (from randomized controlled trials) or if unavailable, linked evidence (from diagnostic accuracy studies and from controlled intervention studies investigating the administration or withholding of anti-D prophylaxis). The results of diagnostic accuracy studies were pooled in bivariate meta-analyses. RESULTS: Neither direct evidence nor sufficient data for linked evidence were identified. Meta-analysis of data from about 60,000 participants showed high sensitivity (99.9%; 95% CI [99.5%; 100%] and specificity (99.2%; 95% CI [98.5%; 99.5%]). CONCLUSIONS: NIPT for fetal RhD status is equivalent to conventional serologic testing using the newborn's blood. Studies investigating patient-relevant outcomes are still lacking.

Non-invasive risk-assessment and bleeding prophylaxis with IVIG in pregnant women with a history of fetal and neonatal alloimmune thrombocytopenia: management to minimize adverse events.

INTRODUCTION: In pregnant women with a history of fetal and neonatal alloimmune thrombocytopenia (FNAIT), prenatal intervention in subsequent pregnancies may be required to prevent fetal bleeding. Several invasive and non-invasive protocols have been published: amniocentesis for fetal genotyping, fetal blood sampling for the determination of fetal platelet count, intrauterine platelet transfusions, and weekly maternal i.v. immunoglobulin (IVIG) infusion with or without additional corticosteroid therapy. This is the first retrospective study that report the experience with a non-invasive protocol focused on side effects of maternal IVIG treatment and neonatal outcome. METHODS: Pregnant women with proven FNAIT in history and an antigen positive fetus were treated with IVIG (1 g/kg/bw) every week. To identify potential IVIG-related hemolytic reactions isoagglutinin titer of each IVIG lot and maternal blood count were controlled. IVIG-related side effects were prospectively documented and evaluated. Furthermore, ultrasound examination of the fetus was performed before starting IVIG administration and continued regularly during treatment. Outcome of the index and subsequent pregnancy was compared. Corresponding data of the newborns were analyzed simultaneously. RESULTS: IVIG was started at 20 weeks of gestation (median). Compared to the index pregnancy, platelet counts of the newborns were higher in all cases. No intracranial hemorrhage occurred (Index pregnancies: 1 case). Platelet counts were 187 × 109/l (median, range 22-239, 95% CI) and one newborn had mild bleeding. No severe hemolytic reaction was observed and side effects were moderate. CONCLUSION: Among pregnant women with FNAIT history, the use of non-invasive fetal risk determination and maternal IVIG resulted in favorite outcome of all newborns. Invasive diagnostic or therapeutic procedures in women with a history of FNAIT should be abandoned.

Potential of Next-Generation Sequencing in Noninvasive Fetal Molecular Blood Group Genotyping.

Hemolytic disease of the fetus and newborn and fetal and neonatal alloimmune thrombocytopenia are caused by maternal antibodies against fetal alloantigens on red blood cells or platelets that are inherited from the father. After transplacental transport to the fetal circulation, antibodies of the IgG class may cause severe fetal anemia or bleeding complications. The indication for noninvasive fetal blood group genotyping is given if a clinically relevant antibody is detected in a pregnant woman and if the father is heterozygous (or unknown) for the implicated blood group allele. This mini-review will focus on the advantages and current limitations of next-generation sequencing (NGS) for noninvasive diagnosis of fetal blood groups which is, in contrast to fetal aneuploidy screening, proposed only by some research groups. Targeted massively parallel sequencing of short DNA fragments from maternal cell-free plasma samples enables counting of fetal alleles for many single nucleotide polymorphisms in parallel. This information can be utilized for estimation of the fetal fraction of cell-free DNA (cfDNA) as well as detection of the paternal blood group allele in question. Adherence to a cut-off of ≥4% fetal fraction for reporting conclusive results is recommended to avoid false-negative results due to low fetal fraction. For screening purposes of fetal RHD in RhD-negative pregnant women, real-time PCR methods are very well established. However, for diagnostic purposes, the targeted amplicon-based NGS approach has the inherent capability to estimate the fetal fraction of cfDNA. In the future, improving the accuracy of NGS by consensus sequencing of single cfDNA molecules may enable reliable fetal blood group genotyping already in the first trimester of pregnancy.

Glycoprotein V is a relevant immune target in patients with immune thrombocytopenia.

Platelet autoantibody-induced platelet clearance represents a major pathomechanism in immune thrombocytopenia (ITP). There is growing evidence for clinical differences between anti-glycoprotein IIb/IIIa and anti-glycoprotein Ib/IX mediated ITP. Glycoprotein V is a well characterized target antigen in Varicella-associated and drug-induced thrombocytopenia. We conducted a systematic study assessing the prevalence and functional capacity of autoantibodies against glycoprotein V. A total of 1140 patients were included. In one-third of patients, platelet-bound autoantibodies against glycoproteins Ib/IX, IIb/IIIa, or V were detected in a monoclonal antibody immobilization of platelet antigen assay; platelet-bound autoantiglycoprotein V was present in the majority of samples (222 out of 343, 64.7%). Investigation of patient sera revealed the presence of free autoantibodies against glycoprotein V in 13.5% of these patients by an indirect monoclonal antibody immobilization of platelet antigen assay, but in 39.6% by surface plasmon resonance technology. These antibodies showed significantly lower avidity (association/dissociation ratio 0.32±0.13 vs 0.73±0.14; P<0.001). High- and low-avidity antibodies induced comparable amounts of platelet uptake in a phagocytosis assay using CD14+ positively-selected human macrophages [mean phagocytic index, 6.81 (range, 4.75-9.86) vs 6.01 (range, 5.00-6.98); P=0.954]. In a NOD/SCID mouse model, IgG prepared from both types of anti-glycoprotein V autoantibodies eliminated human platelets with no detectable difference between the groups from the murine circulation [mean platelet survival at 300 minutes, 40% (range, 27-55) vs 35% (16-46); P=0.025]. Our data establish glycoprotein V as a relevant immune target in immune thrombocytopenia. We would suggest that further studies including glycoprotein V will be required before ITP treatment can be tailored according to platelet autoantibody specificity.

The nonconservative CD177 single-nucleotide polymorphism c.1291G>A is a genetic determinant for human neutrophil antigen-2 atypical/low expression and deficiency.

BACKGROUND: Human neutrophil antigen-2 (HNA-2) is exclusively expressed on neutrophils. HNA-2-deficient individuals (HNA-2 null) are susceptible to produce isoantibodies. The nonsense CD177 coding single-nucleotide polymorphism (SNP) c.787A>T has been demonstrated as the primary genetic mechanism for HNA-2 deficiency. We hypothesized that the other genetic variants also contribute to HNA-2 expression variation and deficiency. STUDY DESIGN AND METHODS: The deficiency, density, and percentage of HNA-2 antigen on neutrophils from 292 healthy blood donors were determined in flow cytometry. CD177 genotypes were determined by genomic DNA sequence analyses. The full-length CD177 cDNAs were amplified and sequenced. Additionally, the whole CD177 genomic sequence in eight HNA-2-null immunized women and four HNA-2-positive donors were analyzed with next-generation sequencing. The associations of CD177 SNP genotypes with HNA-2 expression variation were statistically analyzed. RESULTS: A functional CD177 SNP c.1291G>A was identified in the current study. Atypical (trimodal) HNA-2 expression phenotype was consistently observed in donors carrying the heterozygous c.1291G/A genotype. Phenotype-genotype analyses of SNP c.787A>T and SNP c.1291G>A revealed that all homozygous 787T-1291G (TG/TG) genotype donors were HNA-2 null in healthy blood donors. On the other hand, five of eight HNA-2-immunized females were homozygous for the 787T-1291G (TG/TG) genotype while the other three HNA-2-immunized females had the 787T-1291G/787A-1291A (TG/AA) genotype and the lowest HNA-2 expression was observed in healthy subjects with the 787T-1291G/787A-1291A (TG/AA) and 787A-1291A/787A-1291A (AA/AA) genotype. CONCLUSION: The CD177 SNP c.1291G>A is a genetic determinant for the atypical and low HNA-2 expression, which also contributes to HNA-2 deficiency phenotype.

Molecular and Functional Characterization of Fcγ Receptor IIIb-Ligand Interaction: Implications for Neutrophil-Mediated Immune Mechanisms in Malaria.

The Fcγ receptor IIIb (FcγRIIIb) is a low-affinity receptor of IgG and is essential in neutrophil-mediated effector functions. Different allelic forms of FcγRIIIb carrying human neutrophil antigen (HNA-1a, -1b, -1c, and -1d) have been identified. Here, we have generated stable transfected HEK293 cell lines expressing HNA-1aa, -1bb, and -1bc. Of these, cells expressing HNA-1bc interacted significantly stronger (binding affinities, 2.277 versus 0.743) with human IgG than cells expressing the HNA-1aa or -1bb alloforms. The higher affinity of IgG toward the HNA-1c alloform was confirmed using neutrophils derived from German blood donors. Neutrophils from HNA-1abc-phenotyped individuals bound IgG significantly stronger (1.825 versus 0.903) than did neutrophils from HNA-1ab-typed individuals. These findings were confirmed by surface plasmon resonance (SPR) analysis demonstrating that recombinant HNA-1bc had a higher affinity (dissociation constant [Kd ], 7.24 × 10-6 M) than recombinant HNA-1bb (Kd , 1.15 × 10-5 M) against normal IgG. Finally, we demonstrated that Plasmodium falciparum merozoites opsonized with human IgG affinity purified against P. falciparum glutamate-rich protein (GLURP) enhanced stronger reactive oxygen species (ROS) emission in neutrophils obtained from HNA-1abc donors than in neutrophils from HNA-1ab donors. Collectively, these results indicate that the amino acid substitution Ala78Asp resulting in the HNA-1c allotype leads to higher affinity toward human IgG, enhancement of neutrophil activation, and possibly effective clearance of malaria by intracellular ROS.

Mini photopheresis for refractory chronic graft-versus-host disease in children and adolescents.

BACKGROUND: Extracorporeal photopheresis (ECP) has been established to treat graft-versus-host disease. Our mini ECP technique (mini-ECP) allows for treatment of patients with GVHD and contraindications for classical ECP or low body weight. The safety and efficacy of applying ECP for the long-term treatment of chronic GVHD (cGVHD) have not been described. STUDY DESIGN AND METHODS: A retrospective analysis of mini-ECP treatments for children and adolescents with cGVHD was performed. Mini-ECP with 100 to 200 mL of whole blood was used to treat 14 patients. The median age at the start of treatment was 7 years (range, 1-17 years), and median body weight was 20 kg (range, 8-53 kg). A total of 703 mini-ECP treatments was performed. The median number of treatments per patient was 35 (range, 8-129), and median treatment duration was 11 months (range, 1.4-28.5 months). RESULTS: Mini-ECP was well tolerated. Four adverse events occurred in three patients, and two of them were related to the ECP procedure. Complete or partial responses were observed in 10 patients. Steroids were discontinued in seven patients and tapered in three others. Responses were seen in the skin, mouth, gastrointestinal tract, and eyes. CONCLUSION: Mini-ECP represents a less invasive ECP alternative for low-body-weight patients with cGVHD and contraindications for apheresis.