CDK8/19 inhibition triggers a switch from mitosis to endomitosis in cord blood megakaryocytes.

Neonatal and adult megakaryocytes differ in proliferative capacity and ploidy levels, and neonatal and adult platelets differ in function, gene expression, and protein content. The mechanisms underlying these differences are incompletely understood. CDK8 and CDK19 are transcriptional kinases part of the CDK-mediator complex, which regulates gene transcription in a cell-specific manner. We discovered that cortistatin A, a potent highly selective inhibitor of CDK8/CDK19, significantly reduced cell expansion and increased ploidy in cord blood-derived megakaryocytes. These phenotypic changes were associated with gene expression changes that partially overlapped developmentally regulated genes. These findings might have relevance for the management of developmental megakaryocyte disorders.

The Correlation between Epicardial Adipose Tissue Thickness Measured by Echocardiography and P-Wave Dispersion and Atrial Fibrillation.

Background: Recent studies have indicated a close relationship between the thickness of epicardial adipose tissue (EAT) and the occurrence as well as persistence of atrial fibrillation (AF). However, the pathogenesis of this association is still in the exploratory stage. The aim of this study is to explore the correlation EAT, as measured by echocardiography, and P-wave dispersion (Pd) in the context of atrial fibrillation. Additionally, the study seeks to analyze the utility of EAT at different anatomical sites in identifying individuals who are predisposed to atrial fibrillation. Methods: A total of 136 subjects were enrolled and categorized into groups based on the guidelines: paroxysmal atrial fibrillation group (PAF group), persistent atrial fibrillation group (AF group), and non-atrial fibrillation group. Comprehensive clinical data, including general information and medications that could impact the occurrence of atrial fibrillation, were gathered for all patients. Echocardiography was employed to measure the maximum EAT thickness near the apex of the heart on the anterior right ventricular wall and near the base of the right ventricle for each participant. Pd values were computed for each patient based on standard 12-lead synchronous electrocardiogram (ECG). The study involved comparing the disparity in EAT thickness between the two specified sites across the three groups. Additionally, correlation analyses were performed to assess the relationship between EAT thickness at the two sites and Pd. Regression analysis was applied to explore potential risk factors for atrial fibrillation. The diagnostic value of EAT at each site in predicting atrial fibrillation was evaluated using Receiver Operating Characteristic curve (ROC) analysis. Results: EAT thickness of the anterior wall near the apex of the heart and near the base of the right ventricle were significantly positively correlated with Pd (p < 0.05), EAT thickness near the base and left atrial diameter were independent risk factors for atrial fibrillation (OR = 13.673, 95% CI 2.819~66.316, p = 0.001; OR = 2.294, 95% CI 1.020~5.156, p = 0.045). ROC analysis showed that the area under the curve of EAT thickness near the heart base was 0.723, and the best threshold for predicting the occurrence of AF was 1.05 cm. Conclusions: The echocardiography-measured epicardial adipose tissue thickness, particularly in proximity to the heart base, exhibits a significant correlation with Pd. Notably, EAT thickness near the heart base demonstrates superior predictive capability for atrial fibrillation compared to thickness near the apex.

Serum sex hormones correlate with pathological features of papillary thyroid cancer.

PURPOSE: Sex hormones are thought to be responsible for the unique gender differences in papillary thyroid cancer(PTC). Most previous studies on these have focused on the expression of estrogen receptors, or have been limited to animal studies. The aim of our study was to explore the relationship between serum sex hormones and the pathological features of PTC in the clinical setting, as further evidence of the role of sex hormones in PTC. METHODS: Retrospective data analysis of patients who underwent thyroid surgery at the Department of Thyroid Surgery, Nanjing Drum Tower Hospital from January 2022 to September 2022 Correlation between serum sex hormone and pathological features was analyzed in male patients and in menopausal female patients. Serum sex hormones include luteinizing hormone(LH), follicle stimulating hormone(FSH), estradiol(E2), total testosterone(TT), progesterone(P), and prolactin(PRL). Tumor pathological characteristics include the number and size of tumor, presence of extrathyroidal extension(ETE), presence of lymph node metastasis(LNM). RESULTS: Preoperative serum E2 in male patients was positively correlated with tumor size in PTC, LH was negatively correlated with LNM, while TT and P were negatively correlated with ETE. Similar findings were not observed in menopausal female patients. CONCLUSION: We observed that serum sex hormones correlate with the pathological features of PTC in male patients, for the first time in a clinical study. High serum estrogens may be a risk factor for PTC, while androgens are the opposite. This somewhat corroborates previous research and provides new variables for future PTC prediction models.

Quantitative label-free mass spectrometry reveals content and signaling differences between neonatal and adult platelets.

BACKGROUND: Recent clinical studies have shown that transfusions of adult platelets increase morbidity and mortality in preterm infants. Neonatal platelets are hyporesponsive to agonist stimulation, and emerging evidence suggests developmental differences in platelet immune functions. OBJECTIVES: This study was designed to compare the proteome and phosphoproteome of resting adult and neonatal platelets. METHODS: We isolated resting umbilical cord blood-derived platelets from healthy full-term neonates (n = 8) and resting blood platelets from healthy adults (n = 6) and compared protein and phosphoprotein contents using data-independent acquisition mass spectrometry. RESULTS: We identified 4770 platelet proteins with high confidence across all samples. Adult and neonatal platelets were clustered separately by principal component analysis. Adult platelets were significantly enriched in immunomodulatory proteins, including ß2 microglobulin and CXCL12, whereas neonatal platelets were enriched in ribosomal components and proteins involved in metabolic activities. Adult platelets were enriched in phosphorylated GTPase regulatory enzymes and proteins participating in trafficking, which may help prime them for activation and degranulation. Neonatal platelets were enriched in phosphorylated proteins involved in insulin growth factor signaling. CONCLUSION: Using label-free data-independent acquisition mass spectrometry, our findings expanded the known neonatal platelet proteome and identified important differences in protein content and phosphorylation between neonatal and adult platelets. These developmental differences suggested enhanced immune functions for adult platelets and presence of molecular machinery related to platelet activation. These findings are important to understanding mechanisms underlying key platelet functions as well as the harmful effects of adult platelet transfusions given to preterm infants.

Phosphoproteomics reveals content and signaling differences between neonatal and adult platelets.

Background and Objective: Recent clinical studies have shown that transfusions of adult platelets increase morbidity and mortality in preterm infants. Neonatal platelets are hyporesponsive to agonist stimulation, and emerging evidence suggests developmental differences in platelet immune functions. This study was designed to compare the proteome and phosphoproteome of resting adult and neonatal platelets. Methods: We isolated resting umbilical cord blood-derived platelets from healthy full term neonates (n=9) and resting blood platelets from healthy adults (n=7), and compared protein and phosphoprotein contents using data independent acquisition mass spectrometry. Results: We identified 4745 platelet proteins with high confidence across all samples. Adult and neonatal platelets clustered separately by principal component analysis. Adult platelets were significantly enriched for immunomodulatory proteins, including ß2 microglobulin and CXCL12, whereas neonatal platelets were enriched for ribosomal components and proteins involved in metabolic activities. Adult platelets were enriched for phosphorylated GTPase regulatory enzymes and proteins participating in trafficking, which may help prime them for activation and degranulation. Neonatal platelets were enriched for phosphorylated proteins involved in insulin growth factor signaling. Conclusions: Using state-of-the-art mass spectrometry, our findings expanded the known neonatal platelet proteome and identified important differences in protein content and phosphorylation compared with adult platelets. These developmental differences suggested enhanced immune functions for adult platelets and presence of a molecular machinery related to platelet activation. These findings are important to understanding mechanisms underlying key platelet functions as well as the harmful effects of adult platelet transfusions given to preterm infants.

Unique Features of Nanomaterials and their Combination Support Applications in Orthodontics.

Nanotechnology is a rapidly evolving field with numerous biological applications and is becoming increasingly significant due to its immense potential to enhance the properties of orthodontic and biomaterials. It is employed in various emerging areas of orthodontics, focusing on improving the performance of diverse orthodontic appliances and accessories, as well as nanoelectromechanical systems (NEMS) and nanorobots. Nevertheless, the biocompatibility and cytotoxicity of nanomaterials in orthodontic applications require further investigation. This paper reviews the latest applications of nanomaterials in orthodontics, elucidates their unique features and synergistic applications in orthodontics, and outlines prospective developments in the field.

Erratum: MicroRNA-101-3p suppresses cell proliferation, invasion and enhances chemotherapeutic sensitivity in salivary gland adenoid cystic carcinoma by targeting Pim-1.

[This corrects the article on p. 3015 in vol. 5, PMID: 26693056.].

Fetal vs adult megakaryopoiesis.

Fetal and neonatal megakaryocyte progenitors are hyperproliferative compared with adult progenitors and generate a large number of small, low-ploidy megakaryocytes. Historically, these developmental differences have been interpreted as "immaturity." However, more recent studies have demonstrated that the small, low-ploidy fetal and neonatal megakaryocytes have all the characteristics of adult polyploid megakaryocytes, including the presence of granules, a well-developed demarcation membrane system, and proplatelet formation. Thus, rather than immaturity, the features of fetal and neonatal megakaryopoiesis reflect a developmentally unique uncoupling of proliferation, polyploidization, and cytoplasmic maturation, which allows fetuses and neonates to populate their rapidly expanding bone marrow and blood volume. At the molecular level, the features of fetal and neonatal megakaryopoiesis are the result of a complex interplay of developmentally regulated pathways and environmental signals from the different hematopoietic niches. Over the past few years, studies have challenged traditional paradigms about the origin of the megakaryocyte lineage in both fetal and adult life, and the application of single-cell RNA sequencing has led to a better characterization of embryonic, fetal, and adult megakaryocytes. In particular, a growing body of data suggests that at all stages of development, the various functions of megakaryocytes are not fulfilled by the megakaryocyte population as a whole, but rather by distinct megakaryocyte subpopulations with dedicated roles. Finally, recent studies have provided novel insights into the mechanisms underlying developmental disorders of megakaryopoiesis, which either uniquely affect fetuses and neonates or have different clinical presentations in neonatal compared with adult life.

Association between morphological features of necrotizing pancreatitis on endoscopic ultrasound and outcomes of the endoscopic transmural step-up approach.

OBJECTIVE: To investigate the association between necrotic collections on endoscopic ultrasound (EUS) and outcomes of the endoscopic transmural step-up approach in necrotizing pancreatitis (NP). METHODS: Adult NP patients who had undergone endoscopic transmural step-up approach, endoscopic transmural drainage or endoscopic transmural necrosectomy, were retrospectively enrolled, and divided into groups 1, 2 and 3 based on the amount of solid necrotic debris (quantified as a percentage of the total collection size of <30%, 30%-50%, and >50%). RESULTS: A total of 134 patients were included, of whom 52, 59 and 23 patients were categorized into groups 1, 2 and 3. Patients with more solid necrotic debris required more necrosectomy sessions (group 3 vs group 2 vs group 1: 2.0 vs 1.0 vs 1.0, P < 0.001), were more likely to experience stent occlusion (group 3 vs group 2 vs group 1: 34.8% vs 16.9% vs 9.6%, P = 0.011), and had a longer hospitalization (group 3 vs group 2 vs group 1: 40.0 d vs 28.0 d vs 25.5 d, P = 0.015). High procalcitonin level (adjusted odds ratio [aOR] 6.14, 95% confidence interval [CI] 1.40-26.94, P = 0.016) and any organ failure (aOR 11.51, 95% CI 2.42-54.78, P = 0.002) were independently associated with clinical failure of endoscopic transmural step-up approach. CONCLUSIONS: More solid necrotic debris on EUS is related to more necrosectomy sessions, higher incidence of stent occlusion and longer hospitalization. A nomogram combining procalcitonin and any organ failure performs well in predicting clinical failure of endoscopic transmural step-up approach.

Iron status influences the response of cord blood megakaryocyte progenitors to eltrombopag in vitro.

Eltrombopag (ELT) is a thrombopoietic agent approved for immune thrombocytopenia and also a potent iron chelator. Here we found that ELT exhibited dose-dependent opposing effects on in vitro megakaryopoiesis: low concentrations (≤6 µM, ELT6) stimulated megakaryopoiesis, but high concentrations (30 µM, ELT30) suppressed megakaryocyte (MK) differentiation and proliferation. The suppressive effects of ELT30 were reproduced by other iron chelators, supporting iron chelation as a likely mechanism. During MK differentiation, committed MK progenitors (CD34+/CD41+ and CD34-/CD41+ cells) were significantly more sensitive than undifferentiated progenitors (CD34+/CD41- cells) to the suppressive effects of ELT30, which resulted from both decreased proliferation and increased apoptosis. The antiproliferative effects of ELT30 were reversed by increased iron in the culture, as were the proapoptotic effects when exposure to ELT30 was short. Because committed MK progenitors exhibited the highest proliferative rate and the highest sensitivity to iron chelation, we tested whether their iron status influenced their response to ELT during rapid cell expansion. In these studies, iron deficiency reduced the proliferation of CD41+ cells in response to all ELT concentrations. Severe iron deficiency also reduced the number of MKs generated in response to high thrombopoietin concentrations by ∼50%, compared with iron-replete cultures. Our findings support the hypothesis that although iron deficiency can stimulate certain cells and steps in megakaryopoiesis, it can also limit the proliferation of committed MK progenitors, with severity of iron deficiency and degree of thrombopoietic stimulation influencing the ultimate output. Further studies are needed to clarify how megakaryopoiesis, iron deficiency, and ELT stimulation are clinically interrelated.

Development of gates to measure the immature platelet fraction in C57BL/6J mice using the Sysmex XN-V series multispecies hematology analyzer.

The immature platelet fraction (IPF) is a measure of newly released platelets, which has been used as a marker of platelet production in multiple human studies but is not widely available in multispecies analyzers. We developed gates to measure the IPF in diluted and undiluted murine blood samples on the Sysmex XN-1000V multispecies hematology analyzer. IPF gates were created using undiluted and diluted (1/10) blood samples obtained from adult and newborn (postnatal day 10, P10) C57BL/6J wild-type (WT) mice, and from 3 murine models of thrombocytopenia: c-MPL-/- mice, which lack the thrombopoietin receptor (hyporegenerative); antibody-mediated thrombocytopenia; and acute inflammation-induced thrombocytopenia. P10 mice were chosen because, at their size, we could consistently obtain (by terminal phlebotomy) the blood volume needed to run an undiluted sample. The undiluted blood IPF gate successfully differentiated between mechanisms of thrombocytopenia in both adult and P10 mice. For diluted samples, 2 IPF gates were generated: a thrombocytopenic (T) gate, which performed well in samples with platelet counts (PCs) <800 × 109/L in adult mice and <500 × 109/L in newborn mice, and a non-thrombocytopenic (NT) gate, which performed well in samples with PCs above these thresholds. PCs and IPFs measured in diluted blood using these gates agreed well with those measured in undiluted blood and had good reproducibility. These diluted gates allow for the accurate measurement of PCs and IPFs in small (10 µL) blood volumes, which can be obtained easily from adult and newborn mice as small as P1 to assess platelet production serially.

[Spatial niches of dominant zooplankton species in Yueqing Bay, Zhejiang Province]. / æµ™æ±Ÿä¹æ¸ æ¹¾æµ®æ¸¸åŠ¨ç‰©ç©ºé—´ç”Ÿæ€ä½.

The index of dominance (S), average crowding (X*), niche breadth (Bi), and niche overlap (Qik ) of dominant zooplankton species were calculated using data collected from four zooplankton surveys from May 2016 to February 2017 in Yueqing Bay, Zhejiang Province. The results showed that there were 17 dominant zooplankton species (with S＞0.02). The niche breadth values of those dominant species differed greatly and were positively correlated with S. The niche overlaps of zooplanktons were extremely low. The total amount of species pairs with niche overlap higher than 0.6 (Qik＞0.6) were 25 in Yueqing Bay, which represented 18.4% of the total pairs. Results from the redundancy analysis showed that the distribution of dominant zooplankton species was mainly affected by temperature and salinity, which caused ecological differentiation of zooplankton species.

Pooling, room temperature, and extended storage time increase the release of adult-specific biologic response modifiers in platelet concentrates: a hidden transfusion risk for neonates?

BACKGROUND: Adult donor platelets (PLTs) are frequently transfused to prevent or stop bleeding in neonates with thrombocytopenia. There is evidence for PLT transfusion-related morbidity and mortality, leading to the hypothesis on immunomodulatory effects of transfusing adult PLTs into neonates. Candidate factors are biologic response modifiers (BRMs) that are expressed at higher rates in adult than in neonatal PLTs. This study investigated whether storage conditions or preparation methods impact on the release of those differentially expressed BRMs. STUDY DESIGN AND METHODS: Pooled PLT concentrates (PCs) and apheresis PCs (APCs) were stored under agitation for up to 7 days at room temperature (RT) or at 2 to 8°C. The BRMs CCL5/RANTES, TGFß1, TSP1, and DKK1 were measured in PCs' supernatant, lysate, and corresponding plasma. PLT function was assessed by light transmission aggregometry. RESULTS: Concerning the preparation method, higher concentrations of DKK1 were found in pooled PCs compared to APCs. In supernatants, the concentrations of CCL5, TGFß1, TSP1, and DKK1 significantly increased, both over standard (≤4 days) and over extended storage times (7 days). Each of the four BRMs showed an up to twofold increase in concentration after storage at RT compared to cold storage (CS). There was no difference in the aggregation capacity. CONCLUSION: This analysis shows that the release of adult-specific BRMs during storage is lowest in short- and CS APCs. Our study points to strategies for reducing the exposure of sick neonates to BRMs that can be specifically associated to PLT transfusion-related morbidity.

Changes in megakaryopoiesis over ontogeny and their implications in health and disease.

A growing body of research has made it increasingly clear that there are substantial biological differences between fetal/neonatal and adult megakaryopoiesis. Over the last decade, studies revealed a developmentally unique uncoupling of proliferation, polyploidization, and cytoplasmic maturation in neonatal MKs that results in the production of large numbers of small, low ploidy, but mature MKs during this period of development, and identified substantial molecular differences between fetal/neonatal and adult MKs. This review will summarize our current knowledge on the developmental differences between fetal/neonatal and adult MKs, and recent advances in our understanding of the underlying molecular mechanisms, including newly described developmentally regulated pathways and miRNAs. We will also discuss the implications of these findings on the ways MKs interact with the environment, the response of neonates to thrombocytopenia, the pathogenesis of Down syndrome-transient myeloproliferative disorder (TMD), and the developmental stage specific-manifestations of congenital amegakaryocytic thrombocytopenia.

Outcomes of endoscopic sphincterotomy vs open choledochotomy for common bile duct stones.

BACKGROUND: Endoscopic sphincterotomy (EST) for the management of common bile duct stones (CBDS) is used increasingly widely because it is a minimally invasive procedure. However, some clinical practitioners argued that EST may be complicated by post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (PEP) and accompanied by a higher recurrence of CBDS than open choledochotomy (OCT). Whether any differences in outcomes exist between these two approaches for treating CBDS has not been thoroughly elucidated to date. AIM: To compare the outcomes of EST vs OCT for the management of CBDS and to clarify the risk factors associated with stone recurrence. METHODS: Patients who underwent EST or OCT for CBDS between January 2010 and December 2012 were enrolled in this retrospective study. Follow-up data were obtained through telephone or by searching the medical records. Statistical analysis was carried out for 302 patients who had a follow-up period of at least 5 years or had a recurrence. Propensity score matching (1:1) was performed to adjust for clinical differences. A logistic regression model was used to identify potential risk factors for recurrence, and a receiver operating characteristic (ROC) curve was generated for qualifying independent risk factors. RESULTS: In total, 302 patients undergoing successful EST (n = 168) or OCT (n = 134) were enrolled in the study and were followed for a median of 6.3 years. After propensity score matching, 176 patients remained, and all covariates were balanced. EST was associated with significantly shorter time to relieving biliary obstruction, anesthetic duration, procedure time, and hospital stay than OCT (P < 0.001). The number of complete stone clearance sessions increased significantly in the EST group (P = 0.009). The overall incidence of complications and mortality did not differ significantly between the two groups. Recurrent CBDS occurred in 18.8% (33/176) of the patients overall, but no difference was found between the EST (20.5%, 18/88) and OCT (17.0%, 15/88) groups. Factors associated with CBDS recurrence included common bile duct (CBD) diameter > 15 mm (OR = 2.72; 95%CI: 1.26-5.87; P = 0.011), multiple CBDS (OR = 5.09; 95%CI: 2.58-10.07; P < 0.001), and distal CBD angle ≤ 145° (OR = 2.92; 95%CI: 1.54-5.55; P = 0.001). The prediction model incorporating these factors demonstrated an area under the receiver operating characteristic curve of 0.81 (95%CI: 0.76-0.87). CONCLUSION: EST is superior to OCT with regard to time to biliary obstruction relief, anesthetic duration, procedure time, and hospital stay and is not associated with an increased recurrence rate or mortality compared with OCT in the management of CBDS.

Thrombocytopenia is associated with severe retinopathy of prematurity.

Retinopathy of prematurity (ROP) is characterized by abnormal retinal neovascularization in response to vessel loss. Platelets regulate angiogenesis and may influence ROP progression. In preterm infants, we assessed ROP and correlated with longitudinal postnatal platelet counts (n = 202). Any episode of thrombocytopenia (<100 × 109/l) at ≥30 weeks postmenstrual age (at onset of ROP) was independently associated with severe ROP, requiring treatment. Infants with severe ROP also had a lower weekly median platelet count compared with infants with less severe ROP. In a mouse oxygen-induced retinopathy model of ROP, platelet counts were lower at P17 (peak neovascularization) versus controls. Platelet transfusions at P15 and P16 suppressed neovascularization, and platelet depletion increased neovascularization. Platelet transfusion decreased retinal of vascular endothelial growth factor A (VEGFA) mRNA and protein expression; platelet depletion increased retinal VEGFA mRNA and protein expression. Resting platelets with intact granules reduced neovascularization, while thrombin-activated degranulated platelets did not. These data suggest that platelet releasate has a local antiangiogenic effect on endothelial cells to exert a downstream suppression of VEGFA in neural retina. Low platelet counts during the neovascularization phase in ROP is significantly associated with the development of severe ROP in preterm infants. In a murine model of retinopathy, platelet transfusion during the period of neovascularization suppressed retinopathy.

Effects of Short-term High Dose Atorvastatin on Left Ventricular Remodeling in Patients with First Time Attack of Anterior Acute Myocardial Infarction.

Objects The aim of this trial was to evaluate the effect of short-term high-dose atorvastatin therapy on levels of high-sensitivity C-reactive protein (hs-CRP), malonaldehyde (MDA), endothelin-1(ET-1), matrix metalloproteinases (MMPs), and left ventricular (LV) remodeling in patients with first time attack of acute anterior myocardial infarction (AAMI) .Methods A hundred and three patients with first time attack of AAMI who underwent successful primary percutaneous coronary intervention were randomized to receive atorvastatin 40 mg once daily for 1 week followed by 20 mg once daily (intensive treatment group, IT group, n=49), or atorvastatin 20 mg once daily (standard treatment group, ST group, n=54). Plasma levels of hs-CRP, MDA, ET-1, MMP-2 and MMP-9 were measured on admission, at 1 week, 2 weeks and 6 months follow up and compared between the IT group and ST group. Echocardiography was performed on admission, at 2 week, and 1 year follow up. The left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV) and left ventricular ejection fraction (LVEF) were measured at each echocardiographic examination and compared between the IT group and ST group.Results Plasma levels of hs-CRP (F=7.718, P=0.009), ET-1 (F=7.882, P=0.006), MMP-9 (F=4.834, P=0.028) and pro-BNP (F=4.603, P=0.032) were significantly lower at 1 week after initial onset of AAMI in the IT group compared with the ST group. The changes of LVEDV, LVESV, and LVEF at the 1 year follow-up from the admission did not differ between the IT group and the ST group (t=0.722, P=0.444; t=1.228, P=0.221; t=1.354, P=0.187, repectively).Conclusions Short-term high-dose atorvastatin treatment for AAMI was associated with lower hs-CRP, ET-1 and MMP-9 levels compared to the standard dose treatment. However, this beneficial effect is not likely to related to the left ventricular remodeling.

Developmental differences between newborn and adult mice in response to romiplostim.

Thrombocytopenia is frequent among sick neonates. While most cases are transient, some neonates experience prolonged and severe thrombocytopenia. These infants often pose diagnostic and therapeutic challenges, and may receive large numbers of platelet transfusions. Romiplostim (ROM) is a thrombopoietin (TPO)-receptor-agonist approved for treatment of adults with chronic immune thrombocytopenia (ITP). The immature platelet fraction (IPF) is a novel measure of newly produced platelets, which could aid with the diagnostic evaluation of thrombocytopenic neonates. This study had the following two objectives: (1) compare the response of newborn and adult mice to escalating doses of ROM in vivo and (2) assess the correlation between IPF and megakaryocyte (MK) mass in newborn and adult treated and untreated mice. In the first set of studies, newborn (day 1) and adult mice received a single subcutaneous (SC) dose of ROM ranging from 0 to 300 ng/g, and platelet counts were followed every other day for 14 days. Both sets of mice responded with dose-dependent platelet and IPF increases, peaking on days 5-7 post-treatment, but neonates had a blunted response (2.1-fold compared to 4.2-fold maximal increase in platelet counts, respectively). On day 5 post-treatment with 300 ng/g ROM, MKs in the bone marrow (BM) and spleen of adult mice were significantly increased in numbers and size (p < 0.0001 for both) compared to controls. MKs in the spleen and BM (but not liver) of treated neonates also increased in number, but not in size. The immature platelet count (IPC, calculated as IPF x platelet count) was highly correlated with the MK number and size in neonatal and adult BM and spleen, but not neonatal liver. The lack of response of neonatal liver MKs was not due to a cell-intrinsic reduced responsiveness to TPO, since neonatal liver progenitors were more sensitive to murine TPO (mTPO) in vitro than adult BM progenitor. In vivo treatment of newborn mice with high mTPO doses or with higher doses of ROM (900 ng/g) resulted in peak platelet counts approaching 3-fold of controls. Taken together, our data indicate that newborn mice are less responsive to ROM than adult mice in vivo, due to a combination of likely pharmacokinetic differences and developmental differences in the response of MKs to thrombopoietic stimulation, evidenced by neonatal MKs increasing in numbers but not in size. PK/PD studies in human infants treated with ROM are warranted.

Developmental Stage-Specific Manifestations of Absent TPO/c-MPL Signalling in Newborn Mice.

Congenital amegakaryocytic thrombocytopaenia (CAMT) is a disorder caused by c-MPL mutations that impair thrombopoietin (TPO) signalling, resulting in a near absence of megakaryocytes (MKs). While this phenotype is consistent in adults, neonates with CAMT can present with severe thrombocytopaenia despite normal MK numbers. To investigate this, we characterized MKs and platelets in newborn c-MPL ­/­ mice. Liver MKs in c-MPL ­/­ neonates were reduced in number and size compared with wild-type (WT) age-matched MKs, and exhibited ultrastructural abnormalities not found in adult c-MPL ­/­ MKs. Platelet counts were lower in c-MPL ­/­ compared with WT mice at birth and did not increase over the first 2 weeks of life. In vivo biotinylation revealed a significant reduction in the platelet half-life of c-MPL ­/­ newborn mice (P2) compared with age-matched WT pups, which was not associated with ultrastructural abnormalities. Genetic deletion of the pro-apoptotic Bak did not rescue the severely reduced platelet half-life of c-MPL ­/­ newborn mice, suggesting that it was due to factors other than platelets entering apoptosis early. Indeed, adult GFP+ (green fluorescent protein transgenic) platelets transfused into thrombocytopenic c-MPL ­/­ P2 pups also had a shortened lifespan, indicating the importance of cell-extrinsic factors. In addition, neonatal platelets from WT and c-MPL ­/­ mice exhibited reduced P-selectin surface expression following stimulation compared with adult platelets of either genotype, and platelets from c-MPL ­/­ neonates exhibited reduced glycoprotein IIb/IIIa (GPIIb/IIIa) activation in response to thrombin compared with age-matched WT platelets. Taken together, our findings indicate that c-MPL deficiency is associated with abnormal maturation of neonatal MKs and developmental stage-specific defects in platelet function.