[Risk factors for delayed gastric emptying after laparoscopic pancreaticoduodenectomy: a single-center experience of 1 000 cases].

Objective: To explore the causes and summarize the treatment experience for clinically relevant delayed gastric emptying(DGE) after laparoscopic pancreaticoduodenectomy(LPD). Methods: The clinical data of 1 000 patients who underwent LPD in the Department of Liver Transplantation and Hepatobiliary Surgery,Shandong Provincial Hospital Affiliated to Shandong First Medical University between March 2017 and September 2022 was retrospectively collected. There were 640 males and 360 females,with an age of (60.1±11.4)years(range: 13 to 93 years),and 590 patients were older than 60 years. Depending on the severity of DGE,patients were divided into a clinically relevant DGE group and a 0/A grade DGE group. The comparison between the two groups was performed by the χ2 test,Fisher's exact probability method,t test or the rank sum test,and the effects of various treatment strategies for clinically relevant DGE were evaluated. Results: LPD was conducted successfully in all 1 000 patients,with a surgical time of (344.8±103.6)minutes(range:160 to 450 minutes) and intraoperative blood loss (M(IQR)) of 100 (150) ml(range:50 to 1 000 ml). A total of 74 patients(7.4%) developed clinically relevant DGE. Compared to those in the 0/A grade DGE group,patients in the clinically relevant DGE group had a higher preoperative body mass index of ((24.9±3.5)kg/m2 vs. (23.9±3.3)kg/m2,t=-2.419,P=0.016),more postoperative bile leakage(51.4%(38/74) vs. 10.8%(100/926)),pancreatic fistula(59.5%(44/74) vs. 22.9%(212/926)),abdominal infection(74.3%(55/74) vs.14.6%(135/926)),and abdominal bleeding(43.2%(32/74) vs. 11.3%(105/926))(all P<0.05). Among these patients,10 cases(13.5%) received enteral nutrition treatment,22 cases(29.7%) received parenteral nutrition treatment,and 42 cases(56.8%) received a combination of enteral and parenteral nutrition treatment. The time for patients to return to a normal diet was 21(14)days (range: 8 to 85 days). Compared to those who received only enteral(23.5(27.0)days) or parenteral nutrition treatment(15.5(11.0)days),patients who received a combination of enteral and parenteral nutrition treatment(25.5(31.0)days) had a longer time to return to a normal diet (Z=20.019,P<0.01). Among the 60 patients who developed secondary DGE,48 cases(80.0%) received ultrasound-guided puncture and drainage treatment,while 12 cases(20.0%) only received anti-infection treatment. The patients in the non-puncture drainage group had a longer time to return to a normal diet than those in the puncture drainage group (26.5(12.5)days vs. 20.0(11.0)days, Z=-2.369,P=0.018). Conclusions: Patients with clinically relevant DGE after LPD had a higher proportion of postoperative complications such as pancreatic fistula,biliary fistula and abdominal infection. A combination of enteral and parenteral nutrition treatment is needed for patients with a long-term course of DGE."Smooth" drainage and ani-infectious therapy could contribute to the recovery of DGE.

Tp47-Induced Monocyte-Derived Microvesicles Promote the Adherence of THP-1 Cells to Human Umbilical Vein Endothelial Cells via an ERK1/2-NF-κB Signaling Cascade.

The Treponema pallidum membrane protein Tp47 induces immunocyte adherence to vascular cells and contributes to vascular inflammation. However, it is unclear whether microvesicles are functional inflammatory mediators between vascular cells and immunocytes. Microvesicles that were isolated from Tp47-treated THP-1 cells using differential centrifugation were subjected to adherence assays to determine the adhesion-promoting effect on human umbilical vein endothelial cells (HUVECs). Intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) levels in Tp47-induced microvesicle (Tp47-microvesicle)-treated HUVECs were measured, and the related intracellular signaling pathways of Tp47-microvesicle-induced monocyte adhesion were investigated. Tp47-microvesicles promoted THP-1 cell adhesion to HUVECs (P < 0.01) and upregulated ICAM-1 and VCAM-1 expression in HUVECs (P < 0.001). The adhesion of THP-1 cells to HUVECs was inhibited by anti-ICAM-1 and anti-VCAM-1 neutralizing antibodies. Tp47-microvesicle treatment of HUVECs activated the extracellular signal-regulated kinase 1/2 (ERK1/2) and NF-κB signaling pathways, whereas ERK1/2 and NF-κB inhibition suppressed the expression of ICAM-1 and VCAM-1 and significantly decreased the adhesion of THP-1 cells to HUVECs. IMPORTANCE Tp47-microvesicles promote the adhesion of THP-1 cells to HUVECs through the upregulation of ICAM-1 and VCAM-1 expression, which is mediated by the activation of the ERK1/2 and NF-κB pathways. These findings provide insight into the pathophysiology of syphilitic vascular inflammation.

Recombinant Treponema pallidum protein Tp47 promoted the phagocytosis of macrophages by activating NLRP3 inflammasome induced by PKM2-dependent glycolysis.

BACKGROUND: Glycolysis is a critical pathway in cellular glucose metabolism that provides energy and participates in immune responses. However, whether glycolysis is involved in NOD-like receptor family protein 3 (NLRP3) inflammasome activation and phagocytosis of macrophages in response to Treponema pallidum infection remains unclear. OBJECTIVES: To investigate the role of glycolysis in activating the NLRP3 inflammasome for regulating phagocytosis in macrophages in response to T. pallidum protein Tp47 and its associated mechanisms. METHODS: Interactions between activation of the NLRP3 inflammasome and phagocytosis and the role of glycolysis in Tp47-treated macrophages were investigated through experiments on peritoneal macrophages and human monocytic cell line-derived macrophages. RESULTS: Activation of phagocytosis and NLRP3 inflammasome were observed in Tp47-treated macrophages. Treatment with NLRP3 inhibitor MCC950 or si-NLRP3 attenuated Tp47-induced phagocytosis. Glycolysis and glycolytic capacity were enhanced by Tp47 stimulation in macrophages, and a change in the levels of glycolytic metabolites (phosphoenolpyruvate, citrate and lactate) was induced by Tp47 in macrophages. Inhibition of glycolysis with 2-deoxy-D-glucose, a glycolysis inhibitor, decreased the activation of NLRP3. Expression of M2 isoform of pyruvate kinase (PKM2), an enzyme catalysing a rate-limiting reaction in the glycolytic pathway, was upregulated in Tp47-stimulated macrophages. Inhibition of PKM2 with shikonin or si-PKM2 decreased glycolysis and NLRP3 activation. CONCLUSION: Tp47 promotes phagocytosis in macrophages by activating the NLRP3 inflammasome, which is induced by the enhancement of PKM2-dependent glycolysis.

Effects of dietary pyrroloquinoline quinone disodium supplementation on inflammatory responses, oxidative stress, and intestinal morphology in broiler chickens challenged with lipopolysaccharide.

This study was conducted to investigate the effects of pyrroloquinoline quinone disodium (PQQ·Na2) on inflammatory responses, oxidative stress, and intestinal morphology of broiler chickens challenged with lipopolysaccharide (LPS). A 2 × 2 factorial arrangement in a complete randomized design experiment was used to study the effect of dietary PQQ·Na2 (0 or 1 mg/kg) on broiler chickens with or without a challenge with LPS. A total of two hundred eighty-eight 1-day-old Arbor Acre broiler chickens were randomly assigned to 4 treatments with 6 replicate cages of 12 birds per cage. All experimental broilers were injected intraperitoneally with 0.5 mg/kg body weight of either Escherichia coli LPS or sterile saline at 16, 18, and 20 d of age. Results showed that injecting LPS significantly increased the concentrations of interleukin-1beta (IL-1ß) in serum of birds on day 20 and day 21. Meanwhile, LPS injection increased (P < 0.05) the relative mRNA expression of interleukin-6 (IL-6) in the duodenal mucosa of broilers on day 21. However, dietary supplementation with PQQ·Na2 decreased (P < 0.05) the concentration of IL-6 in serum of birds on day 20 and the levels of IL-1ß, IL-6, and interleukin-10 (IL-10) in serum of broiler chickens on day 21. Besides, supplementation of PQQ·Na2 within diet decreased (P < 0.05) the mRNA expressions of IL-1ß and IL-10 in the duodenal mucosa of birds on day 20. Relative to saline injection, the activity of glutathione peroxidase (GSH-Px) in serum and the activities of total superoxide dismutase (T-SOD) and catalase (CAT) in liver were found to be lower (P < 0.05) in broilers after LPS challenge on day 21. However, birds fed with PQQ·Na2 showed higher (P < 0.05) GSH-Px activity in serum and higher (P < 0.05) T-SOD activities in liver on day 21 and day 42. Pyrroloquinoline quinone disodium also significantly attenuated the LPS-induced decreases in villus height to crypt depth ratio in the duodenum of broilers. In conclusion, dietary PQQ·Na2 supplementation significantly exerted protective effects on inflammation damage and oxidant stress of broilers under LPS challenge by regulating the expression of inflammatory cytokines (IL-1ß, IL-6, and IL-10) and activities of antioxidant enzymes (GSH-Px, T-SOD, and CAT). Moreover, dietary PQQ·Na2 supplementation significantly ameliorated the LPS-impaired intestinal morphology in broilers. Therefore, it has been considered that PQQ·Na2 can be used as a potential feed additive in broiler production.

Effects of main cereal type and feed form on production performance, egg quality and egg sanitary indices of laying hens.

1. The experiment was conducted to evaluate the effects of cereal types (maize or wheat) and feed forms (pelleted or mash feed) on production performance, egg quality and egg sanitary indices in laying hens.2. Three hundred and sixty hens (Jinghong No. 1) at 18 weeks of age were randomly assigned to four treatments with six replicates of 15 hens per replicate according to a 2 × 2 factorial design with two cereal types (maize or wheat) and two feed forms (pelleted or mash feed).3. Compared with the wheat-based diet, the maize-based diet improved (P < 0.05) average egg weight of laying hens. Yolk colour of hens fed with the maize-based diet was higher (P < 0.05) in comparison to those fed the wheat-based diet, while Haugh units were lower (P < 0.05) for the maize-based treatment. Egg mass and average daily feed intake of hens fed the pelleted diet were higher (P < 0.05) than of those fed the mash diet. However, the mash diet improved (P < 0.05) yolk colour compared with the pelleted diet. The percentage of dirty eggs for the wheat-based diet was higher (P < 0.05) than for the maize-based diet. The percentage of dirty eggs was higher (P < 0.05) in birds fed the pelleted diet compared with those fed the mash diet. There were interactions (P < 0.05) between cereal type and feed form, with regard to average egg weight and shell thickness.4. In conclusion, dietary cereal type affected average egg weight, yolk colour and Haugh units in eggs, while feed form influenced egg mass, average daily feed intake and yolk colour.

A Multiple-Cell Microenvironment in a 3-Dimensional System Enhances Direct Cellular Reprogramming Into Hepatic Organoids.

OBJECTIVES: The difficulty in proliferation and availability and the rapid loss functions of primary human hepatocytes highlight the need to develop an alternative, preferably renewable source of human induced hepatocytes in regenerative medicine. Liver organoids generated on a multiple-cell microenvironment in a 3-dimensional (3D) system can provide a highly efficient solution to this issue. METHODS: Human hepatocytes were induced from fibroblasts by the lentiviral expression of FOXA3, HNF1A, and HNF4A. Together with these induced hepatocytes, human umbilical vein endothelial cells and mesenchymal stem cells in a 3D system were used to produce liver organoids. Liver-related gene and protein expression of liver organoids and induced hepatocytes were tested using a 2-dimensional (2D) system. RESULTS: Liver organoids notably increased the expression of hepatic transcription factors, marker genes, transporter genes, and liver metabolism enzyme genes, while it decreased the specific gene expression of fibroblasts. Liver organoids expressed comparable liver-specific proteins, such as ALB, AAT, and HNF4A in the 3D system. CONCLUSION: Direct reprogramming in multiple-cell microenvironments in 3D systems is more controllable and efficient than cell reprogramming in 2D systems. Liver organoids have the potential for use in disease modeling, pharmaceutical applications, and cellular transplantation.

Development of Individualized Induced Pluripotent Stem Cells From Fibroblasts of Keloid Lesions in Patients.

OBJECTIVE: Presently, interesting research related to induced pluripotent stem cells (iPSCs) is emerging. However, the development of new therapies and techniques for treatment of refractory diseases is still required in dermatology. We are exploring novel methods to provide stem cell therapy and elucidate research mechanisms underlying troublesome diseases by reprogramming iPSCs from the fibroblasts of keloid lesions from patients in vitro. METHOD: Here, we identified the expression of fibroblastic genes in the fibroblast derived from diseased individuals. Corresponding iPSCs were then produced by transfecting patient fibroblasts with non-modified RNA cocktails, expressing OCT4, SOX2, KLF4, cMYC, NANOG, and LIN28 reprogramming factors. The pluripotency of these patient-derived iPSCs was identified by immunocytochemistry, real-time quantitative polymerase chain reaction, and teratoma formation in vivo in non-obese diabetic/severe combined immunodeficiency mice. RESULTS: All iPSCs derived from patients significantly expressed the pluripotent transcription factors and could be expanded in vitro. Furthermore, induction of terminal differentiation in long-term culture and the capability of forming embryonic bodies to differentiate into all 3 germ layers in vivo were confirmed in immune-deficient mice. CONCLUSION: Fibroblasts from a keloid patient were successfully reprogrammed to iPSCs in vitro. This reprogramming may provide a basis for the production of individualized modified artificial skin to prevent rejections after xenogeneic skin transplantation and trauma through autologous skin transplantation. These cells can also offer a new platform for research on mechanisms underlying skin diseases and personal medical applications.

Prognostic significance of CD44 variant 2 upregulation in colorectal cancer.

BACKGROUND: CD133 and CD44 are putative cancer stem cell (CSC) markers in colorectal cancer (CRC). However, their clinical significance is currently unclear. Here, we evaluated primary CRC cell isolates to determine the significance of several CSC markers, including CD133 and CD44, as predictors of tumourigenesis and prognosis. METHODS: CD133- and CD44-positive cells from fresh clinical samples of 77 CRCs were selected by flow cytometric sorting and evaluated for tumourigenicity following subcutaneous transplantation into NOD/SCID mice. Cancer stem cell marker expression was examined in both xenografts and a complementary DNA library compiled from 167 CRC patient samples. RESULTS: CD44(+), CD133(+) and CD133(+)CD44(+) sub-populations were significantly more tumourigenic than the total cell population. The clinical samples expressed several transcript variants of CD44. Variant 2 was specifically overexpressed in both primary tumours and xenografts in comparison with the normal mucosa. A prognostic assay using qRT-PCR showed that the CD44v2(high) group (n=84, 5-year survival rate (5-OS): 0.74) had a significantly worse prognosis (P=0.041) than the CD44v2(low) group (n=83, 5-OS: 0.88). CONCLUSIONS: CD44 is an important CSC marker in CRC patients. Furthermore, CRC patients with high expression of CD44v2 have a poorer prognosis than patients with other CD44 variants.

The development of humanized liver with Rag1 knockout rats.

BACKGROUND: The animal model with humanized liver is useful for testing drug metabolism and toxicity in preclinical studies. A mouse model has been reported in which the liver was repopulated more than 90% with human hepatocytes; however, in the rat, the target is far from being reached. In this study, we attempt to develop a humanized liver model with an immunodeficient rat. METHODS: Rag1 knockout rats were treated with neonatal thymectomy. At 3 and 4 weeks of age, they were injected with hepatotoxin retrorsine; 2 weeks after, the animals were subjected to 70% partial hepatectomy and transplanted with immature human hepatocytes via portal vein. The recipients were also treated with anti-asialo GM1 antibody weekly from the day before transplantation and were injected with FK506 every 3 days after transplantation. RESULTS: In Rag1 knockout rats, B lymphocytes were deleted almost completely in peripheral blood. However, T and natural killer (NK) lymphocytes were kept present. When they were treated additionally with neonatal thymectomy for T-lymphocyte deletion and suppressed neutralized NK lymphocytes with anti-asialo GM1, B, T, and NK cells in lymphocytes were reduced to very low levels of 0.75%, 1.58%, and 0.26%, respectively. After transplanting human donor hepatocytes into retrorsine-treated recipient livers, at week 3 the human cell-derived hepatic colonies were expanded in the recipient liver and the liver repopulation rate with human hepatocytes reached approximately 17%. The human hepatocyte-specific genes, albumin, CYP3A4, CYP2C18, and CYP2C9, also could be detected in the recipient rat. CONCLUSION: It is possible to generate a chimera animal with humanized liver in a novel severely immunodeficient rat model.

Evidence of a sophisticatedly heterogeneous population of human umbilical vein endothelial cells.

Induction and promotion of angiogenesis play a role in a diverse range of physiologic and pathophysiologic processes that are especially relevant to the field of regenerative medicine. For assessing vasculogenesis and neo-angiogenesis, identifying angiogenic factors, angiocrine factors, and vascular niche, facilitating tissue-repair and tumor growth, efficiently generating induced pluripotent stem cells, and coculturing with organ-specific stem cells, isolation and characterization of the subpopulation of human umbilical vein endothelial cells (HUVECs) and their endothelial progenitor cells (EPCs) are needed. In this study, primary HUVECs were collected from fresh umbilical cords and fractionated and characterized with the use of flow cytometry. Clonal colony assay showed that endothelial colony-forming units in culture frequently existed in fresh HUVECs. Antigenic profiling demonstrated that undifferentiated EPCs in HUVECs had normal endothelial marker CD31 with a subpopulation of cells positive for hematopoietic stem cell marker CD34 and c-Kit. With continuing passages, EPC markers CD34 and vascular endothelial growth factor receptor 2 expression decreased dramatically. Moreover, a distinct subpopulation with different proliferative capability and angiogenesis from the early-passage HUVECs was shown. In conclusion, it is possible to isolate accurately and to enrich EPCs or hematoangioblast-like cells from a heterogeneous population of HUVECs, and to explore the differential process with flow cytometry for further investigations.

Self-organization of human hepatic organoid by recapitulating organogenesis in vitro.

BACKGROUND: Careful orchestration among endodermal epithelial, endothelial, and mesenchymal cells initiate liver organogenesis prior to vascular function. Nonparenchymal endothelial or mesenchymal cells not only form passive conduits, but also establish an organogenic stimulus. Herein, we have evaluated the potential roles of primitive endothelial and mesenchymal cells toward hepatic organization in vitro. METHODS: To track the cellular movements and localization, we retrovirally transduced enhanced green fluorescence protein and kusabira orange into human fetal liver cells (GFP-hFLCs) and human umbilical vein endothelial cells (KO-HUVECs), respectively. GFP-hFLCs were cocultivated with KO-HUVECs and human mesenchymal stem cells (hMSCs) under conventional two-dimensional (2D) conditions. RESULTS: Even under 2D culture, fetal liver, endothelial, and mesenchymal cells self-organized into a macroscopically visible three-dimensional (3D) organoid. Time-lapse confocal imaging showed dynamic cellular organizations of GFP-hFLCs and KO-HUVECs. Endothelial cells organized into patterned clusters wrapping fetal liver cells, forming vessel-like lumens inside. Mesenchymal cells supported the generated organoid from outside. CONCLUSION: Generation of whole organ architecture remains a great challenge so far. Our preliminary results showed that recapitulation of primitive cellular interactions during organogenesis elicit the intrinsic self-organizing capacity to form hepatic organoids. Future studies to define precise conditions mimicking organogenesis may ultimately lead to the generation of a functional liver for transplantation and for other applications such as drug development.

Generation of functional human vascular network.

BACKGROUND: One of the major obstacles in regenerating thick, complex tissues such as the liver is their need for vascularization, which is essential to maintain cell viability during tissue growth and to induce structural organization. Herein, we have described a method to engineer a functional human vascular network. METHODS: Enhanced green fluorescence protein-labeled human umbilical vein endothelial cells (GFP-HUVECs) were cocultivated with kusabira orange-labeled human mesenchymal stem cells (KO-hMSCs) inside a collagen/fibronectin matrix. Premature vascular network formation was visualized by fluorescence microscopy imaging. Furthermore, constructs prevascularized in vitro were implanted into a transparency window in immunodeficient mice. RESULTS: Following several days of cultivation, GFP-HUVECs formed vessel-like structures that were stabilized by pericytes differentiated from KO-hMSCs. After implantation in vivo, the patency of human vascular structures was proved by rhodamine dextran infusion. These functional vascular structures remained for over 2 months. DISCUSSION: Vascularization is the key challenge to organ generation. We successfully generated human vascular networks inside a matrix. Integration of parenchymal cells using our engineering technique should facilitate future efforts to reconstitute vascularized human organ systems in vitro.

Latent membrane protein-1 of Epstein - Barr virus increases sensitivity to arsenic trioxide-induced apoptosis in nasopharyngeal carcinoma cell.

BACKGROUND AND AIM: Epstein - Barr virus (EBV)-encoded LMP1 is suggested to have an important role in the pathogenesis and development of nasopharyngeal carcinoma (NPC). Our previous study showed that As2O3 exhibited growth inhibition of NPC in animal model. Here, we further explore whether LMP1 is involved in As2O3 anticancer effects in NPC cell line. METHODS: Both the stable expressing LMP1 cell line HNE1-LMP1 and its parental cell line HNE1 without LMP1 expression were used as in vitro models to assess arsenic trioxide effect. Both cell lines were treated with As2O3 for 72 h. The median inhibition concentration (IC50) was assessed by the MTT assay. Apoptosis was observed by phase-contrast microscopy and TUNEL staining. The alteration of telomere lengths was detected by Southern blotting. RESULTS: IC50 for As2O3 in HNE1-LMP1 cells and HNE1 cells was 2.22 and 5.09 micromol/L, respectively. After exposure to 2 and 4 micromol/L As2O3 for 72 h, the apoptotic index in HNE1-LMP1 was 26.27 -/+ 1.3 and 49.13 -/+ 1.4%, respectively. On the contrary, in HNE1 cells the apoptotic index was 12.6 -/+ 0.9 and 33.20 -/+ 1.3%, respectively. As compared with parental cell line HNE1, HNE1-LMP1 cells were more sensitive to growth inhibition and apoptosis (p < 0.001). The elongation of telomere length was also found in HNE1-LMP1 cells. Meanwhile, longer telomeres in HNE1-LMP1 cells failed to maintain telomere stabilization, instead, it prone to be shortened when exposure to As2O3, as comparing with HNE1 cells. CONCLUSION: LMP1 plays important role in enhancing NPC cell response to As2O3. The elongation of telomere length induced by LMP1 may contribute to the mechanisms of As2O3 sensitivity.

Clonal identification and characterization of self-renewing pluripotent stem cells in the developing liver.

Using flow cytometry and single cell-based assays, we prospectively identified hepatic stem cells with multilineage differentiation potential and self-renewing capability. These cells could be clonally propagated in culture where they continuously produced hepatocytes and cholangiocytes as descendants while maintaining primitive stem cells. When cells that expanded in vitro were transplanted into recipient animals, they morphologically and functionally differentiated into hepatocytes and cholangiocytes with reconstitution of hepatocyte and bile duct structures. Furthermore, these cells differentiated into pancreatic ductal and acinar cells or intestinal epithelial cells when transplanted into pancreas or duodenal wall. These data indicate that self-renewing pluripotent stem cells persist in the developing mouse liver and that such cells can be induced to become cells of other organs of endodermal origin under appropriate microenvironment. Manipulation of hepatic stem cells may provide new insight into therapies for diseases of the digestive system.

A role for thrombin receptor signaling in endothelial cells during embryonic development.

The coagulation protease thrombin triggers fibrin formation, platelet activation, and other cellular responses at sites of tissue injury. We report a role for PAR1, a protease-activated G protein-coupled receptor for thrombin, in embryonic development. Approximately half of Par1-/- mouse embryos died at midgestation with bleeding from multiple sites. PAR1 is expressed in endothelial cells, and a PAR1 transgene driven by an endothelial-specific promoter prevented death of Par1-/- embryos. Our results suggest that the coagulation cascade and PAR1 modulate endothelial cell function in developing blood vessels and that thrombin's actions on endothelial cells-rather than on platelets, mesenchymal cells, or fibrinogen-contribute to vascular development and hemostasis in the mouse embryo.

Role of thrombin signalling in platelets in haemostasis and thrombosis.

Platelets are critical in haemostasis and in arterial thrombosis, which causes heart attacks and other events triggered by abnormal clotting. The coagulation protease thrombin is a potent activator of platelets ex vivo. However, because thrombin also mediates fibrin deposition and because multiple agonists can trigger platelet activation, the relative importance of platelet activation by thrombin in haemostasis and thrombosis is unknown. Thrombin triggers cellular responses at least in part through protease-activated receptors (PARs). Mouse platelets express PAR3 and PAR4 (ref. 9). Here we show that platelets from PAR4-deficient mice failed to change shape, mobilize calcium, secrete ATP or aggregate in response to thrombin. This result demonstrates that PAR signalling is necessary for mouse platelet activation by thrombin and supports the model that mouse PAR3 (mPAR3) does not by itself mediate transmembrane signalling but instead acts as a cofactor for thrombin cleavage and activation of mPAR4 (ref. 10). Importantly, PAR4-deficient mice had markedly prolonged bleeding times and were protected in a model of arteriolar thrombosis. Thus platelet activation by thrombin is necessary for normal haemostasis and may be an important target in the treatment of thrombosis.

Clonal expansion of hepatic stem/progenitor cells following flow cytometric cell sorting.

Although hepatic stem cells are believed to exist and play a critical role in developing and regenerating liver, little is known about their cell surface specificity or differentiation capabilities. To make prospective studies of hepatic stem cells possible, we established an in vitro culture system for identification and characterization of hepatic stem/progenitor cells. By combining this culture system with fluorescence activated cell sorting (FACS), a population of cells that were capable of forming large colonies and providing their descendants for relative longer period was isolated from fetal mouse livers. These data suggest that hepatic stem/progenitor cells with high proliferative potential are existent in the developing mouse liver, and that they are enriched by using flow cytometry.