Activated NK cells cause placental dysfunction and miscarriages in fetal alloimmune thrombocytopenia.

Miscarriage and intrauterine growth restriction (IUGR) are devastating complications in fetal/neonatal alloimmune thrombocytopenia (FNAIT). We previously reported the mechanisms for bleeding diatheses, but it is unknown whether placental, decidual immune cells or other abnormalities at the maternal-fetal interface contribute to FNAIT. Here we show that maternal immune responses to fetal platelet antigens cause miscarriage and IUGR that are associated with vascular and immune pathologies in murine FNAIT models. Uterine natural killer (uNK) cell recruitment and survival beyond mid-gestation lead to elevated NKp46 and CD107 expression, perforin release and trophoblast apoptosis. Depletion of NK cells restores normal spiral artery remodeling and placental function, prevents miscarriage, and rescues hemorrhage in neonates. Blockade of NK activation receptors (NKp46, FcɣRIIIa) also rescues pregnancy loss. These findings shed light on uNK antibody-dependent cell-mediated cytotoxicity of invasive trophoblasts as a pathological mechanism in FNAIT, and suggest that anti-NK cell therapies may prevent immune-mediated pregnancy loss and ameliorate FNAIT.Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a gestational disease caused by maternal immune responses against fetal platelets. Using a FNAIT mouse model and human trophoblast cell lines, here the authors show that uterine natural killer cell-mediated trophoblast apoptosis contributes to FNAIT pathogenesis.

Urinary DNA Methylation Biomarkers for Noninvasive Prediction of Aggressive Disease in Patients with Prostate Cancer on Active Surveillance.

PURPOSE: Patients with prostate cancer on active surveillance are monitored by repeat prostate specific antigen measurements, digital rectal examinations and prostate biopsies. A subset of patients on active surveillance will later reclassify with disease progression, prompting definitive treatment. To minimize the risk of under treating such patients on active surveillance minimally invasive tests are urgently needed incorporating biomarkers to identify patients who will reclassify. MATERIALS AND METHODS: We assessed post-digital rectal examination urine samples of patients on active surveillance for select DNA methylation biomarkers that were previously investigated in radical prostatectomy specimens and shown to correlate with an increasing risk of prostate cancer. Post-digital rectal examination urine samples were prospectively collected from 153 men on active surveillance who were diagnosed with Gleason score 6 disease. Urinary sediment DNA was analyzed for 8 DNA methylation biomarkers by multiplex MethyLight assay. Correlative analyses were performed on gene methylation and clinicopathological variables to test the ability to predict patient risk reclassification. RESULTS: Using backward logistic regression a 4-gene methylation classifier panel (APC, CRIP3, GSTP1 and HOXD8) was identified. The classifier panel was able to predict patient reclassification (OR 2.559, 95% CI 1.257-5.212). We observed this panel to be an independent and superior predictor compared to current clinical predictors such as prostate specific antigen at diagnosis or the percent of tumor positive cores in the initial biopsy. CONCLUSION: We report that a urine based classifier panel of 4 methylation biomarkers predicts disease progression in patients on active surveillance. Once validated in independent active surveillance cohorts, these promising biomarkers may help establish a less invasive method to monitor patients on active surveillance programs.

Fetal and neonatal alloimmune thrombocytopenia.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is an alloimmune disorder resulting from platelet opsonization by maternal antibodies that destroy fetal platelets. The major risk of FNAIT is severe bleeding, particularly intracranial hemorrhage. Miscarriage has also been reported but the incidence requires further study. Analogous to adult autoimmune thrombocytopenia (ITP), the major target antigen in FNAIT is the platelet membrane glycoprotein (GP)IIbIIIa. FNAIT caused by antibodies against platelet GPIbα or other antigens has also been reported, but the reported incidence of the anti-GPIbα-mediated FNAIT is far lower than in ITP. To date, the maternal immune response to fetal platelet antigens is still not well understood and it is unclear why bleeding is more severe in FNAIT than in ITP. In this review, we introduce the pathogenesis of FNAIT, particularly those new discoveries from animal models, and discuss possible improvements for the diagnosis, therapy, and prevention of this devastating disease.

Maternal anti-platelet ß3 integrins impair angiogenesis and cause intracranial hemorrhage.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening disease in which intracranial hemorrhage (ICH) is the major risk. Although thrombocytopenia, which is caused by maternal antibodies against ß3 integrin and occasionally by maternal antibodies against other platelet antigens, such as glycoprotein GPIbα, has long been assumed to be the cause of bleeding, the mechanism of ICH has not been adequately explored. Utilizing murine models of FNAIT and a high-frequency ultrasound imaging system, we found that ICH only occurred in fetuses and neonates with anti-ß3 integrin-mediated, but not anti-GPIbα-mediated, FNAIT, despite similar thrombocytopenia in both groups. Only anti-ß3 integrin-mediated FNAIT reduced brain and retina vessel density, impaired angiogenic signaling, and increased endothelial cell apoptosis, all of which were abrogated by maternal administration of intravenous immunoglobulin (IVIG). ICH and impairment of retinal angiogenesis were further reproduced in neonates by injection of anti-ß3 integrin, but not anti-GPIbα antisera. Utilizing cultured human endothelial cells, we found that cell proliferation, network formation, and AKT phosphorylation were inhibited only by murine anti-ß3 integrin antisera and human anti-HPA-1a IgG purified from mothers with FNAIT children. Our data suggest that fetal hemostasis is distinct and that impairment of angiogenesis rather than thrombocytopenia likely causes FNAIT-associated ICH. Additionally, our results indicate that maternal IVIG therapy can effectively prevent this devastating disorder.

Platelets and platelet alloantigens: Lessons from human patients and animal models of fetal and neonatal alloimmune thrombocytopenia.

Platelets play critical roles in hemostasis and thrombosis. Emerging evidence indicates that they are versatile cells and also involved in many other physiological processes and disease states. Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life threatening bleeding disorder caused by fetal platelet destruction by maternal alloantibodies developed during pregnancy. Gene polymorphisms cause platelet surface protein incompatibilities between mother and fetus, and ultimately lead to maternal alloimmunization. FNAIT is the most common cause of intracranial hemorrhage in full-term infants and can also lead to intrauterine growth retardation and miscarriage. Proper diagnosis, prevention and treatment of FNAIT is challenging due to insufficient knowledge of the disease and a lack of routine screening as well as its frequent occurrence in first pregnancies. Given the ethical difficulties in performing basic research on human fetuses and neonates, animal models are essential to improve our understanding of the pathogenesis and treatment of FNAIT. The aim of this review is to provide an overview on platelets, hemostasis and thrombocytopenia with a focus on the advancements made in FNAIT by utilizing animal models.

Novel multiplex MethyLight protocol for detection of DNA methylation in patient tissues and bodily fluids.

Aberrant DNA methylation is a hallmark of cancer and is an important potential biomarker. Particularly, combined analysis of a panel of hypermethylated genes shows the most promising clinical performance. Herein, we developed, optimized and standardized a multiplex MethyLight assay to simultaneously detect hypermethylation of APC, HOXD3 and TGFB2 in DNA extracted from prostate cancer (PCa) cell lines, archival tissue specimens, and urine samples. We established that the assay is capable of discriminating between fully methylated and unmethylated alleles with 100% specificity and demonstrated the assay as highly accurate and reproducible as the singleplex approach. For proof of principle, we analyzed the methylation status of these genes in tissue and urine samples of PCa patients as well as PCa-free controls. These data show that the multiplex MethyLight assay offers a significant advantage when working with limited quantities of DNA and has potential applications in research and clinical settings.