Protocols for collecting mouse PDL cells and bone marrow cells, differentiation, and data analysis.

Periodontal ligament cells (PDLCs) and macrophages in bone marrow cells have been widely used to investigate novel therapeutic agents to treat periodontitis. Here, we present a protocol for collecting primary mouse PDLCs and bone marrow cells. We detail steps for culturing and differentiation for both cell types and review data analysis for in vitro experiments using primary PDLCs and bone marrow cells. This protocol can be used to explore the impact of novel therapeutic agents using in vitro experiments. For complete details on the use and execution of this protocol, please refer to Sirisereephap et al.1.

A novel macrolide-Del-1 axis to regenerate bone in old age.

Aging is associated with increased susceptibility to chronic inflammatory bone loss disorders, such as periodontitis, in large part due to the impaired regenerative potential of aging tissues. DEL-1 exerts osteogenic activity and promotes bone regeneration. However, DEL-1 expression declines with age. Here we show that systemically administered macrolide antibiotics and a non-antibiotic erythromycin derivative, EM-523, restore DEL-1 expression in 18-month-old ("aged") mice while promoting regeneration of bone lost due to naturally occurring age-related periodontitis. These compounds failed to induce bone regeneration in age-matched DEL-1-deficient mice. Consequently, these drugs promoted DEL-1-dependent functions, including alkaline phosphatase activity and osteogenic gene expression in the periodontal tissue while inhibiting osteoclastogenesis, leading to net bone growth. Macrolide-treated aged mice exhibited increased skeletal bone mass, suggesting that this treatment may be pertinent to systemic bone loss disorders. In conclusion, we identified a macrolide-DEL-1 axis that can regenerate bone lost due to aging-related disease.

Hypoxia-inducible factor affects hepatitis B virus transcripts and genome levels as well as the expression and subcellular location of the hepatitis B virus core protein.

Globally, a chronic-hepatitis B virus (HBV) infection is the leading cause of hepatocellular carcinoma (HCC). The transcription factor hypoxia-inducible factor 1 (HIF1) is often elevated in HCC, including HBV-associated HCC. Previous studies have suggested that the expression of the HIF1 subunit, HIF1α, is elevated in HBV-infected hepatocytes; however, whether HIF1 activity affects the HBV lifecycle has not been fully explored. We used a liver-derived cell line and ex vivo cultured primary hepatocytes as models to determine how HIF1 affects the HBV lifecycle. We observed that HIF1 elevates HBV RNA transcript levels, core protein levels, core protein localization to the cytoplasm, and HBV genome replication. Attenuating the transcription activity of HIF1 blocked HIF1-mediated effects on the HBV lifecycle. Our studies show that HIF1 regulates various stages of the HBV lifecycle in hepatocytes and could be a therapeutic target for blocking HBV replication and the development of HBV-associated diseases.

Phospholipid content, expression and support of thrombin generation of neonatal platelets.

AIM: Newborns have, despite low clotting factors and poor in vitro platelet function, a well functioning haemostasis. We investigated whether phospholipids (PL) in neonatal platelets differ from those in adult platelets in their exposure on the platelet surface, and their effect on thrombin generation. METHODS: The effect of newborn and adult platelets on thrombin generation (TG) was measured by means of calibrated automated thrombography (CAT), and in a purified system. In addition, clotting times were measured. Phosphatidylserine (PS)-exposure was measured by flow cytometry. The amount of PL was determined by means of mass-spectrometry (Materials and Methods section in Supporting Information online). RESULTS: In comparison with adults the clotting times in platelet-rich plasma of newborns were less shortened by adding calciumionophore. No differences in the support of TG between neonatal and adult platelets were found by means of CAT. In the purified system, TG was increased by ionophor-stimulated platelets but no difference was evident between newborn and adult platelets. Flow cytometric analysis showed no difference between adult and newborn platelets. Results of mass-spectrometry showed a very similar pattern of phospholipid-content of adult and newborn platelets. CONCLUSION: Our results do not provide any evidence that a different phospholipid-expression of neonatal platelets may alter TG in neonates.

Pediatric patients with congenital heart disease: thrombin generation measured by calibrated automated thrombography.

The aim of this study was to investigate the possible suitability of the calibrated automated thrombography to determine the coagulation status of pediatric patients with congenital heart disease. Thrombin generation was measured in 60 patients with congenital heart disease using the calibrated automated thrombography and compared to data using standard coagulation parameters such as prothrombin, antithrombin, tissue factor pathway inhibitor, prothrombin fragment 1.2 (F 1.2), and activated partial thromboplastin time. A significant positive correlation was observed between prothrombin and the endogenous thrombin potential (P < 0.01; r = 0.295) as well as between prothrombin and peak height (P < 0.01; r = 0.581). A significant negative correlation was seen between tissue factor pathway inhibitor and endogenous thrombin potential (P < 0.01; r = -0.480) and between tissue factor pathway inhibitor and peak height (P < 0.01; r = -0.234). No statistically significant correlation was found between antithrombin and parameters of continuous thrombin generation. Significant correlation was seen neither between activated partial thromboplastin time and F1.2 nor between activated partial thromboplastin time and prothrombin. The data presented here indicate that calibrated automated thrombography measurements determine thrombin generation more accurately and therefore reflect better the coagulation status of pediatric patients with congenital heart disease then standard global coagulation assays such as activated partial thromboplastin time.

Thrombin generation before and after multicomponent blood collection.

BACKGROUND: Apheresis technology has made tremendous progress up to the development of automated blood component collection, which offers increased efficiency in donor blood use, but the concern about the contact of donor blood with artificial surfaces remains. Activation of the hemostatic system is a major issue in this context and is controversial. The aim of this study was to estimate the effect of apheresis on continuous thrombin generation (TG), representing a new tool to examine the overall function of the plasmatic clotting system. STUDY DESIGN AND METHODS: Twenty-six voluntary blood donors, fulfilling the law requirement for apheresis donation, participated in the study. Two units of platelets (6 x 10(11)) and 1 unit of red cells (250 mL; hematocrit level, 80%) were collected using two types of cell separators (Amicus, Fenwal, Inc.; and Trima Accel, Gambro BCT). Each donor underwent collection on both apheresis systems with at least 8 weeks in between. Samples of blood were collected before, immediately after, and 48 hours after apheresis. TG was measured using a slow fluorogenic substrate by means of calibrated automated thrombography (CAT). RESULTS: CAT data changed only slightly, and no significant changes were seen before, immediately after, and 48 hours after apheresis (p > 0.05). The variables did not differ significantly between the two different apheresis systems (p > 0.05). CONCLUSION: Using a CAT-based technique, no change in variables of continuous TG were observed, suggesting that multicomponent blood collection did not lead to severe alterations in the hemostatic system of the donors.

Calibrated automated thrombin generation in normal uncomplicated pregnancy.

Pregnancy is associated with substantial changes in the haemostatic system and a six-fold higher incidence of venous thromboembolism. Conventional global tests, such as prothrombin time and activated partial thromboplastin time, do not definitely detect this hypercoagulable condition. We investigated whether the changes in haemostatic system during pregnancy are reflected in the calibrated automated thrombography (CAT). Thrombin generation was measured in platelet-poor plasma (PPP) of 150 healthy pregnant women without any pregnancy associated diseases by means of CAT. In addition, prothrombin (FII), antithrombin (AT), protein S, protein C, tissue factor pathway inhibitor (TFPI), plasminogen activator inhibitor-1 (PAI-1), thrombin-antithrombin complex (TAT), and prothrombin fragments 1+2 (F1+2) were measured. Endogenous thrombin potential (ETP) and peak of thrombin generation increased significantly with gestational weeks, while lag time and time to peak remained unchanged. A significant increase of PAI-1, TFPI, F1+2 and TAT as well as a significant decrease of free protein S, protein S antigen, and protein S activity was observed. Levels of AT and protein C remained stable during pregnancy. Division of population in trimester of pregnancy and analysis of differences between the trimesters showed rather similar results. Our study shows that endogenous thrombin potential does increase with duration of normal uncomplicated pregnancy. Whether parameters of continuous thrombin generation will correlate with thrombembolic disease remains to be shown.

Thrombin generation determined by calibrated automated thrombography (CAT) in pediatric patients with congenital heart disease.

INTRODUCTION: Thrombin generation was studied in pediatric patients with congenital heart disease (CHD) undergoing cardiac surgery using the calibrated automated thrombography (CAT) in terms of the lag time until the onset of thrombin formation, time to thrombin peak maximum (TTP), endogenous thrombin potential (ETP), and thrombin peak height. The possible suitability to determine the coagulation status of these patients was investigated. MATERIALS AND METHODS: CAT data of 40 patients with CHD (age range from newborn to 18 years) were compared to data using standard coagulation parameters such as prothrombin (FII), antithrombin (AT), tissue factor pathway inhibitor (TFPI), prothrombin fragment 1.2 (F 1.2), thrombin-antithrombin (TAT), activated partial thromboplastin time (aPTT), and prothrombin time (PT). RESULTS: A significant positive correlation was seen between ETP and FII (p<0.01; r=0.369), as well as between peak height and F II (p<0.01; r=0.483). A significant negative correlation was seen between ETP and TFPI values (p<0.05; r=-0.225) while no significant correlation was seen between peak height and TFPI. A significant negative correlation was seen between F 1.2 generation and ETP (p<0.05; r=-0.254) and between F 1.2 generation and peak height (p<0.05; r=-0.236). No correlation was seen between AT and ETP or peak. CONCLUSIONS: Our data indicate that CAT is a good global test reflecting procoagulatory and inhibitory factors of the hemostatic system in pediatric patients with CHD.