Generation of human antibodies targeting human platelet antigen (HPA)-1a.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a condition during pregnancy, which can lead to thrombocytopenia and a bleeding tendency with intracranial hemorrhage (ICH) being the most concerning complication in the fetus or neonate. An incompatibility between human platelet antigen (HPA)-1a accounts for the majority of FNAIT cases. Binding of HPA-1a-specific alloantibodies to their target on fetal platelets and endothelial cells can induce apoptosis of megakaryocytes, disrupt platelet function, and impair angiogenesis. Currently, there is no screening program to identify pregnancies at risk for severe disease. A better understanding of HPA-1a-specific antibody heterogeneity in FNAIT could aid in identifying pathogenic antibody properties linked to severe disease. STUDY DESIGN AND METHODS: This study aimed to isolate HPA-1a-specific B-cells from an HPA-1a-alloimmunized pregnant woman. Using fluorescently labeled HPA-1a-positive platelets, single B-cells were sorted and cultured for 10 days to stimulate antibody production. Subsequently, supernatants were tested for the presence of antibodies by enzyme-linked immunosorbent assay and their reactivity towards HPA-1a-positive platelets. Amplification and sequencing of variable regions allowed the generation of monoclonal antibodies using a HEK-Freestyle-based expression system. RESULTS: Three platelet-specific B-cells were obtained and cloned of which two were specific for HPA-1a, named D- and M-204, while the third was specific for HLA class I, which was named L-204. DISCUSSION: This study outlined an effective method for the isolation of HPA-1a-specific B-cells and the generation of monoclonal antibodies. Further characterization of these antibodies holds promise for better understanding the pathogenic nature of alloantibodies in FNAIT.

Chemoselective Heterogeneous Hydrogenation of Sulfur Containing Quinolines under Mild Conditions.

Sulfur, alongside oxygen and nitrogen, holds a prominent position as one of the key heteroatoms in nature and medicinal chemistry. Its significance stems from its ability to adopt different oxidation states, rendering it valuable as both a polarity handle and a hydrogen bond donor/acceptor. Nevertheless, the poisonous nature of its free electron pairs makes sulfur containing substrates inaccessible for many catalytic protocols. Strong and (at low temperatures) irreversible chemisorption to the catalyst's surface is in particular detrimental for heterogeneous catalysts, possessing only few catalytically active sites. Herein, we present a novel heterogeneous Ru-S catalyst that tolerates multiple sulfur functionalities, including thioethers, thiophenes, sulfoxides, sulfones, sulfonamides, and sulfoximines, in the hydrogenation of quinolines. The utility of the products was further demonstrated by subsequent diversifications of the sulfur functionalities.

Children Newly Diagnosed with Fetal and Neonatal Alloimmune Thrombocytopenia: Neurodevelopmental Outcome at School Age.

OBJECTIVE: To evaluate the neurodevelopmental outcome at school age in children newly diagnosed with fetal and neonatal alloimmune thrombocytopenia (FNAIT). STUDY DESIGN: This observational cohort study included children diagnosed with FNAIT between 2002 and 2014. Children were invited for cognitive and neurological testing. Behavioral questionnaires and school performance results were obtained. A composite outcome of neurodevelopmental impairment (NDI) was used, defined, and subdivided into mild-to-moderate and severe NDI. Primary outcome was severe NDI, defined as IQ <70, cerebral palsy with Gross Motor Functioning Classification System level ≥ III, or severe visual/hearing impairment. Mild-to-moderate NDI was defined as IQ 70-85, minor neurological dysfunction or cerebral palsy with Gross Motor Functioning Classification System level ≤ II, or mild visual/hearing impairment. RESULTS: In total, 44 children were included at a median age of 12 years (range: 6-17 years). Neuroimaging at diagnosis was available in 82% (36/44) of children. High-grade intracranial hemorrhage (ICH) was detected in 14% (5/36). Severe NDI was detected in 7% (3/44); two children had high-grade ICH, and one had low-grade ICH and perinatal asphyxia. Mild-to-moderate NDI was detected in 25% (11/44); one child had high-grade ICH, and eight children were without ICH, yet for two children, neuroimaging was not performed. Adverse outcome (perinatal death or NDI) was 39% (19/49). Four children (9%) attended special needs education, three of whom had severe NDI and one had mild-to-moderate NDI. Total behavioral problems within the clinical range were reported in 12%, which is comparable with 10% in the general Dutch population. CONCLUSION: Children who are newly diagnosed with FNAIT are at increased risk for long-term neurodevelopmental problems, even those without ICH. TRIAL REGISTRATION: The study was registered at ClinicalTrials.gov (Identifier: NCT04529382).

Natural history of human platelet antigen 1a-alloimmunised pregnancies: a prospective observational cohort study.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a rare disease that untreated can lead to intracranial haemorrhage or death. The natural history of FNAIT is still unclear; therefore, the benefits of screening cannot be estimated and no routine screening is yet in place. We aimed to assess the incidence of clinically detectable FNAIT among pregnancies in human platelet antigen-1a (HPA-1a)-immunised women. METHODS: We did a prospective observational cohort study of pregnant women negative for rhesus D (RhD) and rhesus c (Rhc) antigens, without age limits, who underwent routine antenatal screening for red cell antibodies at 27 weeks' gestation and were typed for HPA-1a between March 1, 2017, and May 1, 2020. HPA-1a-negative women were tested for HPA alloantibodies. Health-care professionals were masked to all test results. The main outcome was the proportion of neonates with severe, clinically detectable FNAIT, defined as having an intracranial bleed, organ bleed, or bleeding-related death observed during pregnancy or within the first week of life. Cases of clinically detectable FNAIT not categorised as severe were categorised as mild. This study is registered with ClinicalTrials.gov (NCT04067375). FINDINGS: Of 153 106 women typed for HPA-1a, 3722 (2·4%) were negative for HPA-1a. 913 HPA-1a-negative women gave informed consent, underwent HPA-1a antibody screening, and were included in the study. Anti-HPA-1a antibodies were detected in 85 HPA-1a-negative participants, among whom three with HPA-1a-negative fetuses and one with a previous child with FNAIT were excluded. As controls, 820 HPA-1a-negative, non-immunised pregnancies and 2704 randomly selected pregnancies of women negative for RhD and Rhc who were typed HPA-1a positive were included. Of 81 fetuses included, one (1·2%) was diagnosed with severe HPA-1a-mediated intracranial haemorrhage and three (3·7%) had mild FNAIT. Gravidity and parity did not seem to be risk factors for HPA-1a immunisation. 73 (90·1%) of 81 HPA-1a-immunised women were positive for HLA-DRB3*01:01. INTERPRETATION: Our data suggest that, without intervention, the incidence of major clinically detectable bleeding in FNAIT is estimated as 11 (95% CI 0-32) per 10 000 HPA-1a-negative pregnancies. These findings imply that severe bleeding is a rare event that potentially could be prevented by a screening programme. FUNDING: Landsteiner Foundation for Blood Transfusion Research and Sanquin.

Postnatal treatment for children with fetal and neonatal alloimmune thrombocytopenia: a multicentre, retrospective, cohort study.

BACKGROUND: Children affected by fetal and neonatal alloimmune thrombocytopenia (FNAIT) are at risk of severe intracranial haemorrhage. Management in the postnatal period is based on sparse evidence. We aimed to describe the contemporary management and outcomes of patients with FNAIT in high-income countries. METHODS: In this multicentre, retrospective, cohort study, we set up a web-based registry for the collection of deidentified data on the management and course of neonates with FNAIT. Eight centres from seven countries (Australia, Norway, Slovenia, Spain, Sweden, the Netherlands, and the USA) participated. Eligibility criteria comprised neonates with FNAIT being liveborn between Jan 1, 2010, and Jan 1, 2020; anti-human platelet antigen (HPA) alloantibodies in maternal serum; confirmed maternal and fetal HPA incompatibility; and bleeding detected at antenatal ultrasound, neonatal thrombocytopenia (<150 × 109 platelets per L), or both in the current or previous pregnancy. Clinical data were retrieved from local medical records of the first neonatal admission and entered in the registry. The key outcome was the type of postnatal treatment given to neonates with FNAIT. Other outcomes were daily median platelet counts in the first week of life, median platelet count increment after first unmatched versus first matched transfusions, and the proportion of neonates with mild or severe bleeding. FINDINGS: 408 liveborn neonates with FNAIT were entered into the FNAIT registry, of whom 389 from Australia (n=74), Norway (n=56), Slovenia (n=19), Spain (n=55), Sweden (n=31), the Netherlands (n=138), and the USA (n=16) were included in our analyses. The median follow-up was 5 days (IQR 2-9). More neonates were male (241 [64%] of 379) than female (138 [36%]). Severe thrombocytopenia (platelet count <50 × 109 platelets per L) was reported in 283 (74%) of 380 neonates, and extreme thrombocytopenia (<10 × 109 platelets per L) was reported in 92 (24%) neonates. Postnatal platelet count nadir was higher in the no-treatment group than in all other groups. 163 (42%) of 389 neonates with FNAIT received no postnatal treatment. 207 (53%) neonates received platelet transfusions, which were either HPA-unmatched (88 [43%] of 207), HPA-matched (84 [41%]), or a combination of both (35 [17%]). The proportion of neonates who received HPA-matched platelet transfusions varied between countries, ranging from 0% (Slovenia) to 63% (35 of 56 neonates; Norway). Postnatal intravenous immunoglobulin treatment was given to 110 (28%) of 389 neonates (alone [n=19] or in combination with platelet transfusions [n=91]), with the proportion receiving it ranging from 12% (17 of 138 neonates; the Netherlands) to 63% (ten of 16 neonates; the USA) across countries. The median platelet increment was 59 × 109 platelets per L (IQR 35-94) after HPA-unmatched platelet transfusions and 98 × 109 platelets per L (67-134) after HPA-matched platelet transfusions (p<0·0001). Severe bleeding was diagnosed in 23 (6%) of 389 liveborn neonates, with one having a severe pulmonary haemorrhage and 22 having severe intracranial haemorrhages. Mild bleeding was diagnosed in 186 (48%) neonates. INTERPRETATION: Postnatal management of FNAIT varies greatly between international centres, highlighting the absence of consensus on optimal treatments. Our data suggest that HPA-matched transfusions lead to a larger median platelet count increment than HPA-unmatched transfusions, but whether HPA matching is also associated with a reduced risk of bleeding remains unknown. FUNDING: Sanquin.

Long-term neurodevelopmental outcome in children after antenatal intravenous immune globulin treatment in fetal and neonatal alloimmune thrombocytopenia.

BACKGROUND: Children with fetal and neonatal alloimmune thrombocytopenia face increased risk of intracranial hemorrhage potentially leading to developmental impairment. To prevent intracranial hemorrhage, pregnant women with alloantibodies against fetal platelets are often treated with intravenous immunoglobulin. Intravenous immunoglobulin seems effective in vastly reducing the risk of fetal or neonatal bleeding complications. However, information on long-term neurodevelopment of these children is lacking. OBJECTIVE: This study aimed to evaluate long-term neurodevelopmental outcome in children with fetal and neonatal alloimmune thrombocytopenia who were treated with intravenous immunoglobulin antenatally. STUDY DESIGN: An observational cohort study was performed, including children of mothers treated with intravenous immunoglobulin during pregnancy because a previous child was diagnosed with fetal and neonatal alloimmune thrombocytopenia. Children were invited for a follow-up assessment including standardized cognitive and neurologic tests. The parents were asked to complete a behavioral questionnaire and school performance reports. The primary outcome was severe neurodevelopmental impairment, defined as severe cognitive impairment (intelligence quotient <70), cerebral palsy with Gross Motor Function Classification System Level ≥3, bilateral blindness, and/or bilateral deafness (requiring amplification). The secondary outcome was mild to moderate neurodevelopmental impairment, defined as either mild to moderate cognitive impairment (intelligence quotient <85), cerebral palsy with Gross Motor Function Classification System Level ≤2, minor neurologic dysfunction, vision loss, and/or hearing loss. RESULTS: Between 2003 and 2017, 51 children were live-born after antenatal intravenous immunoglobulin treatment. One family moved abroad and was therefore not eligible for inclusion. In total, 82% (41/50) of the eligible cases were included for neurodevelopmental assessment at a median age of 9 years and 8 months. Severe neurodevelopmental impairment was not detected. The incidence of mild to moderate neurodevelopmental impairment was 14% (6/41; 95% confidence interval, 6%-29%). The children's mean cognitive score, behavioral scores, and academic achievement were not different from those observed in the Dutch norm groups. Neuroimaging was performed in 90% (37/41) of cases. Severe intracranial hemorrhage was diagnosed in 2 cases (5%), one antenatally before the start of intravenous immunoglobulin and the other case 1 day after birth. Both cases had a normal neurodevelopmental outcome. CONCLUSION: The risk of neurodevelopmental impairment in children whose mothers were treated for fetal and neonatal alloimmune thrombocytopenia with antenatal intravenous immunoglobulin is comparable to that reported in the general population.

Clinical characteristics of human platelet antigen (HPA)-1a and HPA-5b alloimmunised pregnancies and the association between platelet HPA-5b antibodies and symptomatic fetal neonatal alloimmune thrombocytopenia.

Fetal neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal alloantibodies directed against the human platelet antigens (mostly HPA-1a or HPA-5b) of the (unborn) child and can lead to severe bleeding. Anti-HPA-1a-mediated FNAIT shows a severe clinical outcome more often than anti-HPA-5b-mediated FNAIT. Given the relatively high prevalence of anti-HPA-5b in pregnant women, the detection of anti-HPA-5b in FNAIT-suspected cases may in some cases be an incidental finding. Therefore we investigated the frequency of anti-HPA-5b-associated severe bleeding in FNAIT. We performed a retrospective nationwide cohort study in cases with clinical suspicion of FNAIT. HPA antibody screening was performed using monoclonal antibody-specific immobilisation of platelet antigens. Parents and neonates were typed for the cognate antigen. Clinical data were collected by a structured questionnaire. In 1 864 suspected FNAIT cases, 161 cases (8·6%) had anti-HPA-1a and 60 (3·2%) had anti-HPA-5b. The proportion of cases with severe bleeding did not differ between the cases with anti-HPA-1a (14/129; 11%) and anti-HPA-5b (4/40; 10%). In multigravida pregnant women with a FNAIT-suspected child, 100% (81/81) of anti-HPA-1a cases and 79% (38/48) of anti-HPA-5b cases were HPA-incompatible, whereas 86% and 52% respectively were expected, based on the HPA allele distribution. We conclude that anti-HPA-5b can be associated with severe neonatal bleeding symptoms. A prospective study is needed for true assessment of the natural history of anti-HPA-5b mediated FNAIT.

Placental Complement Activation in Fetal and Neonatal Alloimmune Thrombocytopenia: An Observational Study.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a disease that causes thrombocytopenia and a risk of bleeding in the (unborn) child that result from maternal alloantibodies directed against fetal, paternally inherited, human platelet antigens (HPA). It is hypothesized that these alloantibodies can also bind to the placenta, causing placental damage. This study aims to explore signs of antibody-mediated placental damage in FNAIT. We performed a retrospective study that included pregnant women, their newborns, and placentas. It comprised 23 FNAIT cases, of which nine were newly diagnosed (14 samples) and 14 were antenatally treated with intravenous immune globulins (IVIg) (21 samples), and 20 controls, of which 10 had anti-HLA-class I antibodies. Clinical information was collected from medical records. Placental samples were stained for complement activation markers (C1q, C4d, SC5b-9, and mannose-binding lectin) using immunohistochemistry. Histopathology was examined according to the Amsterdam criteria. A higher degree of C4d deposition was present in the newly diagnosed FNAIT cases (10/14 samples), as compared to the IVIg-treated FNAIT cases (2/21 samples, p = 0.002) and anti-HLA-negative controls (3/20 samples, p = 0.006). A histopathological examination showed delayed maturation in four (44%) placentas in the newly diagnosed FNAIT cases, five (36%) in the IVIg-treated FNAIT cases, and one in the controls (NS). C4d deposition at the syncytiotrophoblast was present in combination with low-grade villitis of unknown etiology in three newly diagnosed FNAIT cases that were born SGA. We conclude that a higher degree of classical pathway-induced complement activation is present in placentas from pregnancies with untreated FNAIT. This may affect placental function and fetal growth.

HIP (HPA-screening in pregnancy) study: protocol of a nationwide, prospective and observational study to assess incidence and natural history of fetal/neonatal alloimmune thrombocytopenia and identifying pregnancies at risk.

INTRODUCTION: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) may lead to severe fetal or neonatal bleeding and/or perinatal death. Maternal alloantibodies, targeted against fetal human platelet antigens (HPAs), can result thrombocytopenia and bleeding complications. In pregnancies with known immunisation, fetal bleeding can be prevented by weekly maternal intravenous immunoglobulin infusions. Without population-based screening, immunisation is only detected after birth of an affected infant. Affected cases that might have been prevented, when timely identified through population-based screening. Implementation is hampered by the lack of knowledge on incidence, natural history and identification of pregnancies at high risk of bleeding. We designed a study aimed to obtain this missing knowledge. METHODS AND ANALYSIS: The HIP (HPA-screening in pregnancy) study is a nationwide, prospective and observational cohort study aimed to assess incidence and natural history of FNAIT as well as identifying pregnancies at high risk for developing bleeding complications. For logistic reasons, we invite rhesus D-negative or rhesus c-negative pregnant women, who take part in the Dutch population-based prenatal screening programme for erythrocyte immunisation, to participate in our study. Serological HPA-1a typing is performed and a luminex-based multiplex assay will be performed for the detection of anti-HPA-1a antibodies. Results will not be communicated to patients or caregivers. Clinical data of HPA-1a negative women and an HPA-1a positive control group will be collected after birth. Samples of HPA-1a immunised pregnancies with and without signs of bleeding will be compared with identify parameters for identification of pregnancies at high risk for bleeding complications. ETHICS AND DISSEMINATION: Ethical approval for this study has been obtained from the Medical Ethical Committee Leiden-The Hague-Delft (P16.002). Study enrolment began in March 2017. All pregnant women have to give informed consent for testing according to the protocol. Results of the study will be disseminated through congresses and publication in relevant peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT04067375.

Bidirectional tumor/stroma crosstalk promotes metastasis in mesenchymal colorectal cancer.

Patients with the mesenchymal subtype colorectal cancer (CRC) have a poor prognosis, in particular patients with stroma-rich tumors and aberrant SMAD4 expression. We hypothesized that interactions between SMAD4-deficient CRC cells and cancer-associated fibroblasts provide a biological explanation. In transwell invasion assays, fibroblasts increased the invasive capacity of SMAD4-deficient HT29 CRC cells, but not isogenic SMAD4-proficient HT29 cells. A TGF-ß/BMP-specific array showed BMP2 upregulation by fibroblasts upon stimulation with conditioned medium from SMAD4-deficient CRC cells, while also stimulating their invasion. In a mouse model for experimental liver metastasis, the co-injection of fibroblasts increased metastasis formation of SMAD4-deficient CRC cells (p = 0.02) but not that of SMAD4-proficient CRC cells. Significantly less metastases were seen in mice co-injected with BMP2 knocked-down fibroblasts. Fibroblast BMP2 expression seemed to be regulated by TRAIL, a factor overexpressed in SMAD4-deficient CRC cells. In a cohort of 146 stage III CRC patients, we showed that patients with a combination of high stromal BMP2 expression and the loss of tumor SMAD4 expression had a significantly poorer overall survival (HR 2.88, p = 0.04). Our results suggest the existence of a reciprocal loop in which TRAIL from SMAD4-deficient CRC cells induces BMP2 in fibroblasts, which enhances CRC invasiveness and metastasis.