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Background: von Willebrand factor (VWF) is a glycoprotein that plays an important role in primary hemostasis. VWF is synthesized and stored in endothelial cells (ECs) and megakaryocytes/platelets. Plasma VWF is primarily derived from ECs and is generally believed to be essential for hemostasis. VWF synthesized in megakaryocytes is stored in platelet α-granules, from which it is released following platelet activation. The relative contribution of VWF stored in ECs or megakaryocytes/platelets or present in plasma to hemostasis is not clear., Objectives: We investigated whether EC-derived VWF plays the major role in hemostasis while the contribution of platelet-derived VWF is negligible, or if platelet-derived VWF also significantly contributes to hemostasis., Methods and Results: Mice expressing VWF only in ECs (EC-VWF) or platelets (Plt-VWF) were created by reciprocal bone marrow transplantation between C57BL/6J (WT) and VWF knockout mice (VWF-/-). Plasma VWF levels in EC-VWF were similar to WT. Plt-VWF mice had a trace amount of VWF in their plasma while VWF levels in platelet lysate were comparable to WT. Tail bleeding time was normal in EC-VWF. Interestingly, Plt-VWF showed partially corrected bleeding time and significantly decreased blood loss volume compared with VWF-/-. Adhesion of platelets perfused over immobilized collagen under shear stress was significantly higher in both EC-VWF and Plt-VWF compared with VWF-/-., Conclusion: VWF synthesized in ECs is sufficient to support hemostasis in VWF-/- mice, and VWF produced in megakaryocytes/platelets can also contribute to hemostasis in the absence of EC-derived VWF., (© 2012 International Society on Thrombosis and Haemostasis.)

Intravenous infusion studies in humans suggest that both von Willebrand factor (VWF) and factor VIII (FVIII) remain intravascular in contrast to other coagulation proteins. We explored whether infusion of VWF and FVIII by either intraperitoneal (i.p.) or subcutaneous (s.c.) injection would result in efficient absorption of these large proteins into the vascular circulation. FVIII(null) or VWF(null) mice were infused with plasma-derived or recombinant VWF and/or FVIII by i.p., s.c., or intravenous (i.v.) injection. Both VWF and FVIII were absorbed into the blood circulation after i.p. injection with a peak between 2 and 4 h at levels similar to those observed in mice infused intravenously. In contrast, neither VWF nor FVIII was detected in the plasma following s.c. injection. Although i.v. injection achieved peak plasma levels quickly, both human VWF and FVIII rapidly decreased during the first 2 h following i.v. injection. Following both i.v. and i.p. infusion of VWF, the multimeric structure of circulating VWF was similar to that observed in the infusate. These results demonstrate that both VWF and FVIII can be efficiently absorbed into the blood circulation following i.p., but not s.c. injection, indicating that i.p. administration could be an alternative route for VWF or FVIII infusion., (© 2012 Blackwell Publishing Ltd.)

Purpose: To explore the technical feasibility of double contrast percutaneous transhepatic cholangiographic CT (DC-PCT-CT) in patients with bile duct obstruction., Methods: Seven patients with bile duct obstructive diseases were studied, including 5 males and 3 females, ranging in age from 24 yrs to 74 yrs (average: 47.7 yrs). There were 5 cases of hilar cholangiocarcinoma, 1 case of sclerosing cholangitis, and 1 case of malignant transformation of adenoma at the distal end of the common bile duct. PTC was carried out initially, involving injection of 30 ml 4.5-6.0 mgl iohexol. After the bile duct system was filled, CT scan was performed, and further followed by enhanced CT with intravenous injection of 300 mgl/ml contrast agent. Arterial phase, venous phase, and parenchymal phase acquisitions were obtained. Raw CT images were viewed and multiplanar reconstruction (MPR), maximum intensity projection (MIP), and volume rendering (VR) image post-processing were performed., Results: DC-PCT-CT was performed successfully and bile duct drainage was carried out. Mild lesion enhancement was demonstrated in three cases in arterial phase, while all seven cases demonstrated enhancement of various degrees in venous phase.The lesions lead to track-like, asymmetrical or irregular bile duct obstructive narrowing, and in one case intra-luminal filling defect. Reliable diagnosis was suggested in all cases. MPR, MIP and VR images were useful in demonstrating precise lesion location and for surgical planning., Conclusion: In patients with bile duct obstruction, DC-PTC-CT is a feasible technique offering both important diagnostic value and drainage application.

Donor pancreatic lymph node cells (PLNC) protect islet transplants in Non-obese diabetic (NOD) mice. We hypothesized that induced FoxP3(+) regulatory T cells (Tregs) were required for long-term islet engraftment. NOD or NOD.NON mice were treated with ALS (antilymphocyte serum) and transplanted with NOR islets +/-PLNC (5 × 10(7) ). In vivo proliferation and expansion of FoxP3(+) Tregs was monitored in spleen and PLN from ALS- and ALS/PLNC-treated recipient mice. Anti-CD25 depletion was used to determine the necessity of Tregs for tolerance. FoxP3(+) numbers significantly increased in ALS/PLNC-treated recipients compared to ALS-treated mice. In ALS/PLNC-treated mice, recipient-derived Tregs localized to the transplanted islets, and this was associated with intact, insulin-producing β cells. Proliferation and expansion of FoxP3(+) Tregs was markedly increased in PLNC-treated mice with accepted islet grafts, but not in diabetic mice not receiving PLNC. Deletion of Tregs with anti-CD25 antibodies prevented islet graft tolerance and resulted in rejection. Adoptive transfer of Tregs to secondary NOD.scid recipients inhibited autoimmunity by cotransferred NOD effector T cells. Treg expansion induced by ALS/PLNC-treatment promoted long term islet graft survival. Strategies leading to Treg proliferation and localization to the transplant site represent a therapeutic approach to controlling recurrent autoimmunity., (© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Obliterative airway disease (OAD) due to chronic alloantigen rejection remains a major challenge for long-term graft survival in lung transplantation. It is known that superagonistic CD28-specific monoclonal antibody JJ316 (supCD28 MAb) has the ability to induce regulatory T cells (Tregs) efficiently. Here we used a rat orthotopic tracheal transplantation model to investigate the effects of supCD28 MAb on expanding Tregs in vivo and its application in suppression of acute and chronic airway allograft rejection. SupCD28 MAb administration revealed a significant increase in the CD4+CD25+ T cells, CD4+FoxP3+ T cells, and CD4+CD25+ FoxP3+ T cells population among CD4+ T cells in spleen, peripheral blood, as well as cervical lymph nodes. The allografts from animals treated with supCD28 MAb showed significantly less airway obliteration and rejection of the respiratory epithelium compared with allografts of the mouse immunoglobulin G-treated group on the 5th day and the 60th day after transplantation. Overall, our data demonstrated that an intraperitoneally administrated low dose of supCD28 MAb was sufficient to induce Treg cell expansion in vivo and was effective in protecting the airway graft from early rejection and chronic OAD development. These findings provide the basis for new therapies to prevent OAD and perhaps rejection of allografts in other human transplantations., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objective: The effect of local administration of hirudin on random pattern skin flap survival was investigated in a porcine model., Methods: Three random pattern skin flaps (4 × 14 cm) were created on each flank of five Chinese minipigs. The experimental group (10 flaps) received 20 antithrombin units of hirudin, injected subdermally into the distal half immediately after surgery and on days 1 and 2; a control group (10 flaps) was injected with saline and a sham group (10 flaps) was not injected. All flaps were followed for 10 days postoperatively., Results: Macroscopically, the congested/necrotic length in the experimental group was significantly decreased compared with the other two groups by day 3. Histopathological evaluation revealed venous congestion and inflammation in the control and sham groups from day 1, but minimal changes in the experimental group. By day 10, the mean ± SD surviving area was significantly greater in the experimental group (67.6 ± 2.1%) than in the control (45.2 ± 1.4%) or sham (48.3 ± 1.1%) groups., Conclusions: Local administration of hirudin can significantly increase the surviving area in overdimensioned random pattern skin flaps, in a porcine model.

Relapsing polychondritis (RP) is a rare autoimmune disease that affects cartilage throughout the body, causing episodic and progressive inflammation. Although rare, RP has diverse acute and subacute nervous system complications, which may sometimes precede systemic manifestations. Here, we report four patients with RP who presented with meningoencephalitis or meningitis without infectious aetiology. In addition, we review the literature for this disease with regard to clinical manifestations and treatment options., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Expression profiles of microRNAs (miRNAs) can shape the repertoire of proteins expressed in development, differentiation and diseases. This study aimed to identify miRNA profile of articular cartilage at different developmental stages in rats., Methods: Three small RNA libraries were constructed from the femoral head cartilage of Sprague-Dawley (SD) rats at postnatal day 0, day 21 and day 42 and sequenced by a deep sequencing approach. Then a bioinformatics approach was employed to distinguish genuine miRNAs from small RNAs represented in the mass sequencing data. The expression of indicated miRNAs was determined by stem-loop RT-qPCR to valuate the consistency with Solexa sequencing., Results: Two hundred and fifty-eight of 310 known miRNA and miRNA* genes were organized into 91 compact clusters. Two hundred and forty-six miRNAs were detected in all three small RNA libraries of rat articular cartilage. Forty-six, fifty-two and fifty-six miRNA* genes were identified from three small RNA libraries, respectively, and 86 novel miRNA candidate genes were found simultaneously. In addition, 23 known miRNAs were up-regulated (fold change ≥ 4); six were down-regulated (fold change ≤ -4) during articular cartilage development. The predicted targets of differentially expressed miRNAs were locally secreted factors and transcription factors that regulate proliferation and differentiation of chondrocytes. The same expression tendency of indicated miRNAs during articular cartilage development stages was observed by using Solexa sequencing and stem-loop RT-qPCR., Conclusion: Our study provided a unique opportunity to decipher how the elaboration of the miRNA repertoire contributes to the development process of articular cartilage., (Copyright © 2011 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

This study examined the response of serum biomarkers of cardiac and skeletal muscle damage at rest and after a routine workout of 21 km run in 12 male adolescent (16.2±0.6 years) long-distance runners. Biomarkers of cardiac [troponins (cTnT, cTnI), creatine kinase MB mass (CK-Mbmass)] and skeletal muscle [creatine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and hydroxybutyrate dehydrogenase (HBD)] damage were assayed at rest, 2, 4 and 24 h post-exercise. At rest, cTnT and cTnI were not detectable; however, CK, CK-MBmass, AST, ALT and HBD were above corresponding clinical cut-off values. Post-exercise significant elevations above rest were observed for all biomarkers, except ALT, 2 and 4 h following the run, and remained elevated in cTnI, CK, CK-MBmass, LDH and AST 24 h post-workout. A significant increase in data points above clinical cut-off values from rest to post-exercise was reported for cTnT, cTnI and CK at 2 and 4 h, and in cTnI and CK 24 h post-exercise. In conclusion, a 21 km run in adolescent runners increased post-exercise biomarkers of cardiac and skeletal muscle damage., (© 2010 John Wiley & Sons A/S.)

Osteosarcoma (OS) is the most common primary malignancy of bone. There is a critical need to identify the events that lead to the poorly understood mechanism of OS development and metastasis. The goal of this investigation is to identify and characterize a novel marker of OS progression. We have established and characterized a highly metastatic OS subline that is derived from the less metastatic human MG63 line through serial passages in nude mice via intratibial injections. Microarray analysis of the parental MG63, the highly metastatic MG63.2 subline, as well as the corresponding primary tumors and pulmonary metastases revealed insulin-like growth factor binding protein 5 (IGFBP5) to be one of the significantly downregulated genes in the metastatic subline. Confirmatory quantitative RT-PCR on 20 genes of interest demonstrated IGFBP5 to be the most differentially expressed and was therefore chosen to be one of the genes for further investigation. Adenoviral mediated overexpression and knockdown of IGFBP5 in the MG63 and MG63.2 cell lines, as well as other OS lines (143B and MNNG/HOS) that are independent of our MG63 lines, were employed to examine the role of IGFBP5. We found that overexpression of IGFBP5 inhibited in vitro cell proliferation, migration and invasion of OS cells. Additionally, IGFBP5 overexpression promoted apoptosis and cell cycle arrest in the G1 phase. In an orthotopic xenograft animal model, overexpression of IGFBP5 inhibited OS tumor growth and pulmonary metastases. Conversely, siRNA-mediated knockdown of IGFBP5 promoted OS tumor growth and pulmonary metastases in vivo. Immunohistochemical staining of patient-matched primary and metastatic OS samples demonstrated decreased IGFBP5 expression in the metastases. These results suggest 1) a role for IGFBP5 as a novel marker that has an important role in the pathogenesis of OS, and 2) that the loss of IGFBP5 function may contribute to more metastatic phenotypes in OS.

Blockade of the interactions between CD28/CTLA-4 and their ligands, CD80 (B7, B7.1)/CD86 (B70, B7.2), is an attractive means to induce antigen-specific peripheral tolerance in autoimmune disease and organ transplantation. In this study, we generated and characterized a monoclonal antibody (Clone 4E5) against human CD80. 4E5 could recognize both human and mouse CD80 and suppress mixed lymphocyte reaction in vitro. To investigate their potency for clinical use, we further administrated 4E5 to a mouse lupus-like disease model (C57BL/J6) induced by Pristane. 4E5 could inhibit the immune response and attenuate the severity of lupus-like disease. The data showed 4E5 function and suggested that blockade of CD80/CD28 co-stimulatory signal pathway with 4E5 is a promising strategy to decelerate the progression of lupus-like disease and other autoimmune diseases.

Background/aim: The clinical characteristics of POEMS (polyneuropathy, organomegaly, endocrinopathy, M-protein and skin changes) syndrome in China are largely unknown. This work thus studied the clinical manifestations of POEMS syndrome in China., Methods: We retrospectively reviewed the medical records of 82 patients with POEMS syndrome in our hospital and made a comparison with those reported outside China., Results: There were 82 patients. Forty (49%) were 45 years old or younger. Sensorimotor deficits were the common initial symptoms. The clinical manifestations are as follows: (i) peripheral neuropathy and abnormal electromyogram were seen in all patients (100%); (ii) organomegaly was present in 72 patients (88%); 61 of them (74%) had splenomegaly; (iii) endocrinopathy was present in 74 cases (90%); hypothyroidism was seen in 51 of 70 patients (73%); (iv) 60 patients (73%) had monoclonal plasmaproliferative disorder; only 22 of 40 (55%) had M-protein; (v) skin changes were seen in 71 patients (87%); (vi) 68 patients (83%) had oedema and effusions; of these, hydropericardium was seen in 23 patients (28%); (vii) 35 of 55 patients (64%) had abnormal electrocardiogram and only 21 of 46 (46%) had bone lesions in X-ray., Conclusions: POEMS syndrome in China has its own distinctive features, parts of which are commoner in the young people, the higher frequency of splenomegaly, hypothyroidism, hydropericardium and abnormal electrocardiogram, as well as the lower M-protein and bone lesions in X-ray., (© 2011 The Authors. Internal Medicine Journal © 2011 Royal Australasian College of Physicians.)

Background: The ACCENT group previously established disease-free survival (DFS) with 2 or 3 years median follow-up to predict 5 year overall survival (5 year OS) in stage II and III colon cancer. ACCENT further proposed (1) a stronger association between DFS and OS in stage III than II, and (2) 6 or 7 years necessary to demonstrate DFS/OS surrogacy in recent trials. The relationship between end-points in trials with oral fluoropyrimidines, oxaliplatin and irinotecan is unknown., Methods: Associations between the treatment effect hazard ratios (HRs) on 2 and 3 years DFS, and 5 and 6 years OS were examined in 6 phase III trials not included in prior analyses from 1997 to 2002. Individual data for 12,676 patients were analysed; two trials each tested oxaliplatin, irinotecan and oral treatment versus 5-FU/LV., Findings: Overall association between 2/3 year DFS and 5/6 year OS HRs was modest to poor (simple R² measures: 0.58-0.76, model-based R²: 0.17-0.49). In stage III patients, the association increased (model-based R² ≥ 0.79). Observed treatment effects on 2 year DFS accurately 5/6 year OS effects overall and in stage III patients., Interpretation: In recent trials of cytotoxic chemotherapy, 2 or 3 years DFS HRs are highly predictive of 5 and 6 years OS HRs in stage III but not stage II patients. In all patients the DFS/OS association is stronger for 6 year OS, thus at least 6 year follow-up is recommended to assess OS benefit. These data support DFS as the primary end-point for stage III colon cancer trials testing cytotoxic agents., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Proapoptotic BH3 interacting domain death agonist (Bid), a BH3-only Bcl-2 family member, is situated at the interface between the DNA damage response and apoptosis, with roles in death receptor-induced apoptosis as well as cell cycle checkpoints following DNA damage.(1, 2, 3) In this study, we demonstrate that Bid functions at the level of the sensor complex in the Atm and Rad3-related (Atr)-directed DNA damage response. Bid is found with replication protein A (RPA) in nuclear foci and associates with the Atr/Atr-interacting protein (Atrip)/RPA complex following replicative stress. Furthermore, Bid-deficient cells show an impaired response to replicative stress manifest by reduced accumulation of Atr and Atrip on chromatin and at DNA damage foci, reduced recovery of DNA synthesis following replicative stress, and decreased checkpoint kinase 1 activation and RPA phosphorylation. These results establish a direct role for the BH3-only Bcl-2 family member, Bid, acting at the level of the damage sensor complex to amplify the Atr-directed cellular response to replicative DNA damage.

Background: The cancer stem cell hypothesis suggests that neoplastic clones are maintained exclusively by a small subpopulation of cells, which have indefinite proliferation and differentiation potentials and give rise to phenotypically diverse cancer cells. Cancer stem cells have been isolated by their ability to efflux Hoechst 33342 dye and are referred to as the 'side population' (SP)., Methods and Results: The Hoechst efflux assay was used to isolate and characterize the SP from murine D121 lung carcinoma cells. Here, we demonstrated that D121-SP cells contain cancer stem cell characteristics, that is, upregulation of the transcription factors SOX2 and Oct 4 in D121-SP cells. In addition, the migration of D121-SP was decreased, and apoptosis of D121-SP was upregulated following knocking down of SOX2 in D121 cells. Importantly, downregulation of SOX2 in D121 cells markedly suppressed their metastatic potential in syngeneic mice., Conclusions: These results suggest that the SP is an enriched source of lung tumour cells with stem cell properties and that SOX2 has an important role in maintaining stem cell properties and functions that may be a potential target for effective lung cancer therapy.

Objectives: Post-exercise cardiac troponin T (cTnT) release has been widely reported in adult athletes but limited data is available for adolescents. The aim of this study was to determine the incidence and magnitude of cTnT appearance in a large group of adolescent runners, and to determine any association between cTnT release and personal characteristics of adolescents., Methods: We recruited 63 adolescent runners (mean±SD: age 16.4±1.5 years; 10 females) who all completed a simulated half-marathon race (an all-out 21-km run) during routine training. Personal data collected included age, training history, 21-km run performance as well as pre-post exercise serum cTnT levels. Serum cTnT was assayed using a 3rd generation assay., Results: At pre-exercise, cTnT concentrations were below the 0.01 µg/L cTnT detection limit of assay in 58/63 runners. The post-exercise cTnT level (range: <0.01-1.36 µg/L) was significantly (p<0.001) greater than that of the pre-exercise (range: <0.01-0.02 µg/L). After the exercise, 57 (90%) and 44 (70%) subjects had cTnT concentrations above the detection: 0.01, and clinical thresholds: 0.05 µg/L, respectively. Post-exercise cTnT was inversely correlated with training years (r=-0.25, p<0.05) and age (r=-0.31, p<0.05). Compared with runners who had trained for ≥ 3 years, runners with less training experience demonstrated increased post-race cTnT levels (p<0.01)., Conclusion: cTnT increases are virtually universal among adolescent runners following a 21-km run during routine training and can reach levels typically diagnostic for acute myocardial infarction potentially initiating diagnostic dilemmas. Adolescents with less training experience had higher post-exercise cTnT.

Microtubule dynamics is essential for many vital cellular processes such as in intracellular transport, metabolism, and cell division. Evidences demonstrate that α-synuclein may associate with microtubular cytoskeleton and its major component, tubulin. In the present study, the molecular interaction between α-synuclein and tubulin was confirmed by GST pull-down assay and co-immunoprecipitation. The interacting regions within α-synuclein with tubulin were mapped at the residues 60-100 of α-synuclein that is critical for the binding activity with tubulin. Microtubule assembly assays and sedimentation tests demonstrated that α-synuclein influenced the polymerization of tubulin in vitro, revealing an interacting region-dependent feature. Confocal microscopy detected that exposures of α-synuclein proteins inhibited microtubule formation in the cultured cells, with a length-dependent phenomenon. Our data highlight a potential role of α-synuclein in regulating the microtubule dynamics in neurons. The association of α-synuclein with tubulin may further provide insight into the biological and pathophysiological function of synuclein.

We have developed a multi-disciplinary approach combining molecular biology, delivery technology, combinatorial chemistry and reversible masking to create improved systemic, targeted delivery of plasmid DNA while avoiding nonspecific uptake in vivo. We initially used a well-characterized model targeting the asialolglycoprotein receptor in the liver. Using our bilamellar invaginated vesicle (BIV) liposomal delivery system with reversible masking, we increased expression in the liver by 76-fold, nearly equaling expression in first-pass organs using non-targeted complexes, with no expression in other organs. The same technology was then applied to efficiently target delivery to a human tumor microenvironment model. We achieved efficient, targeted delivery by attachment of specific targeting ligands to the surface of our BIV complexes in conjunction with reversible masking to bypass nonspecific tissues and organs. We identified ligands that target a human tumor microenvironment created in vitro by co-culturing primary human endothelial cells with human lung or pancreatic cancer cells. The model was confirmed by increased expression of tumor endothelial phenotypes including CD31 and vascular endothelial growth factor-A, and prolonged survival of endothelial capillary-like structures. The co-cultures were used for high-throughput screening of a specialized small molecule library to identify ligands specific for human tumor-associated endothelial cells in vitro. We identified small molecules that enhanced the transfection efficiency of tumor-associated endothelial cells, but not normal human endothelial cells or cancer cells. Intravenous (i.v.) injection of our targeted, reversibly masked complexes into mice, bearing human pancreatic tumor and endothelial cells, specifically increased transfection to this tumor microenvironment approximately 200-fold. Efficacy studies using our optimized targeted delivery of a plasmid encoding thrombospondin-1 eliminated tumors completely after five i.v. injections administered once every week.

Doppel (Dpl) is a recently identified prion (PrP)-like protein due to the structural and biochemical similarities, however, its natural function and pathogenic role in neurodegenerative diseases remains unclear. To investigate the possible pathogenic pathway of Dpl and its structural analog for cell apoptosis, mammalian expressing recombinant plasmids containing human PRND gene encoding the full-length Dpl and a truncated human PRNP gene deleting the sequences encoding the peptide from aa 32 to 121 (PrPDelta32-121) were generated. MTT assays showed the cell viabilities of the human neuroblastoma cell line SH-SY5Y receiving Dpl and PrPDelta32-121 expressing plasmids were remarkably lower. Obvious apoptosis phenomena were observed to be associated with the cells transient expressing Dpl and PrPDelta32-121, including reduced mitochondrial transmembrane potential (psim), decreased pro-caspase-3 quantity, more numbers of annexin V- and annexin V/PI-double-stained cells and depressed Bcl-2 level. Moreover, we also found that the Dpl- and PrPDelta32-121-induced cytotoxicities and relevant apoptotic events in SH-SY5Y cells could be fully antagonized by co-expression of the human full-length PrP. These data highly indicate that cytotoxicity induced by the expression of Dpl and truncated PrP in neural derived cells are closely related with the apoptosis process, probably triggering the mitochondrial pathway. It also implies that the cell-benefit activity of the full-length PrP may result from its anti-apoptosis capacity.

Ketamine, a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist, is associated with accelerated neuronal apoptosis in the developing rodent brain. In this study, postnatal day (PND) 7 rats were treated with 20 mg/kg ketamine or saline in six successive doses (s.c.) at 2-h intervals. Brain frontal cortical areas were collected 6 h after the last dose and RNA isolated and hybridized to Illumina Rat Ref-12 Expression BeadChips containing 22,226 probes. Many of the differentially expressed genes were associated with cell death or differentiation and receptor activity. Ingenuity Pathway Analysis software identified perturbations in NMDA-type glutamate, GABA and dopamine receptor signaling. Quantitative polymerase chain reaction (Q-PCR) confirmed that NMDA receptor subunits were significantly up-regulated. Up-regulation of NMDA receptor mRNA signaling was further confirmed by in situ hybridization. These observations support our working hypothesis that prolonged ketamine exposure produces up-regulation of NMDA receptors and subsequent over-stimulation of the glutamatergic system by endogenous glutamate, triggering enhanced apoptosis in developing neurons., (Published by Elsevier Ltd.)

We present the apparent mass measurements at the bottom of granular packings for different bead and silo sizes. The redirection parameter K in Janssen theory is found to increase with the ratios of the diameter of the silo to the bead. We attribute this feature to the friction between the beads and the confining wall of silo; it is the role of friction that leads to variations in the shielding of vertical stresses as well as pressure screening.

Background: The interaction of glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) initiates platelet adhesion, and simultaneously triggers intracellular signaling cascades leading to platelet aggregation and thrombus formation. Some of the signaling events are similar to those occurring during apoptosis, however, it is still unclear whether platelet apoptosis is induced by the GPIbalpha-VWF interaction., Objectives: To investigate whether the GPIbalpha-VWF interaction induces platelet apoptosis and the role of 14-3-3zeta in apoptotic signaling., Methods: Apoptotic events were assessed in platelets or Chinese hamster ovary (CHO) cells expressing wild-type (1b9) or mutant GPIb-IX interacting with VWF by flow cytometry or western blotting., Results: Ristocetin-induced GPIbalpha-VWF interaction elicited apoptotic events in platelets, including phosphatidylserine exposure, elevations of Bax and Bak, gelsolin cleavage, and depolarization of mitochondrial inner transmembrane potential. Apoptotic events were also elicited in platelets exposed to pathologic shear stresses in the presence of VWF; however, the shear-induced apoptosis was eliminated by the anti-GPIbalpha antibody AK2. Furthermore, apoptotic events occurred in 1b9 cells stimulated with VWF and ristocetin, but were significantly diminished in two CHO cell lines expressing mutant GPIb-IX with GPIbalpha truncated at residue 551 or a serine-to-alanine mutation at the 14-3-3zeta-binding site in GPIbalpha., Conclusions: This study demonstrates that the GPIbalpha-VWF interaction induces apoptotic events in platelets, and that the association of 14-3-3zeta with the cytoplasmic domain of GPIbalpha is essential for apoptotic signaling. This finding may suggest a novel mechanism for platelet clearance or some thrombocytopenic diseases.

The androgen receptor (AR) plays a crucial role in the physiological and pathological functions of androgen. As a transcription factor, the AR modulates androgen activity by regulating the transcription of target genes that are involved in numerous physiological functions and pathological disorders, such as acne vulgaris, androgenetic alopecia, benign prostate hyperplasia (BPH), and prostate cancers. Although many natural and synthetic curcumin analogues have been reported to possess anticancer activity through a common cytotoxic property against proliferating tumor cells, none has been reported to inhibit cancer cell growth through a more specific mechanism or target in the cancer cells. Recently, new curcumin analogues were studied extensively regarding their synthesis, structure-activity (i.e., anticancer activity) relationships, and mechanism of action. These compounds, such as ASC-J9 and its analogues (3 and 4), have now been shown to inhibit prostate cancer proliferation through a novel mechanism of enhancing AR degradation.

Thrombotic diseases or fatalities have been reported to occasionally occur under conditions of hypergravity although the mechanism is still unclear. To investigate the effect of hypergravity on platelets that are the primary players in thrombus formation, platelet rich plasma (PRP) or washed platelets were exposed to hypergravity at 8 G for 15 minutes. No platelet aggregation was induced by 8 G alone, whereas ristocetin or collagen-induced platelet aggregation was significantly increased. The number of platelets adherent to immobilized fibrinogen and the area of platelets spreading on von Willbrand factor (VWF) matrix were increased simultaneously. Flow cytometry assay indicated that integrin alphaIIbbeta3 was partially activated in 8 G-exposed platelets, but there was no significant difference in P-selectin surface expression between platelets treated with 8 G and 1 G control. The results indicate that hypergravity leads to human platelet hyperactivity, but fails to incur essential platelet activation events, suggesting a novel mechanism for thrombotic diseases occurring under hypergravitional conditions.

The acrosome reaction has long been thought to be induced by the zona pellucida. Here we report the identification and function of a novel human sperm glycosylphosphatidylinositol (GPI)-anchored membrane protein, NYD-SP8. The release of the protein during sperm-egg interaction and its binding to the cumulus, the first layer of egg investment, elicits cross-talk between the gametes and produces calcium dependant release of progesterone, which lead to the acrosome reaction. An in vivo mouse model of NYD-SP8 immunization is also established showing a reduced fertility rate. Thus, contrary to accepted dogma, our study demonstrates for the first time that, prior to reaching the zona pellucida, sperm may release a surface protein that acts on the cumulus cells leading to the acrosome reaction, which may be important for determining the outcome of fertilization.

Aims: To clarify the taxonomic status of strain ZG0656 and analyse the effects of its acarviostatin products on blood glucose levels in mammalian systems., Methods and Results: Our program to screen for new alpha-amylase inhibitors led to the isolation of strain ZG0656. The polyphasic taxonomic study revealed that strain ZG0656 represents a novel variation of Streptomyces coelicoflavus, for which we propose the name S. coelicoflavus var. nankaiensis. Four chemically distinct alpha-amylase inhibitors, acarviostatins I03, II03, III03 and IV03, were isolated from strain ZG0656. Acarviostatins III03 and IV03 are both novel oligomers. All four acarviostatins are mixed noncompetitive porcine pancreas alpha-amylase inhibitors. Acarviostatin III03 is the most potent alpha-amylase inhibitor known to date. Moreover, in the in vitro and in vivo experiments, acarviostatins III03 showed significant inhibition of starch hydrolysis and glucose transfer to blood., Conclusions: Strain ZG0656 is a novel variation of S. coelicoflavus, whose products are novel effective alpha-amylase inhibitors. Among the products, acarviostatins III03 could significantly depress blood glucose levels in mammalian systems and be developed towards a possible therapeutic agent for diabetes., Significance and Impact of the Study: Acarviostatin III03 is the most potent alpha-amylase inhibitor known to date. The oligomer will benefit the research on the relationship between alpha-amylase and various inhibitors and will offer more choices in diabetes treatments.

Objective: Abnormal maturation and ossification of the endplate chondrocytes play a central role in the pathogenesis of degenerative disorders of the cervical spine. It is widely held that insulin like growth factor-1 (IGF-1) stimulates chondrocyte proliferation and inhibits chondrocyte terminal differentiation both in vitro and in vivo. However, the mechanism underlying such regulation is not fully understood. The present study aimed to determine the role of IGF-1 on the mRNA expression of collagen type II, alpha 1 (Col2a1) and matrix metallopeptidase 13 (MMP-13) in rat endplate chondrocytes. The possible pathways that transduce IGF-1 effects such as phosphatidylinositol-3 (PI-3)-kinase (PI3K) and mitogen activated protein kinase (MAPK) were also investigated in these cells., Methods: Cultured endplate chondrocytes harvested from rat cervical spines were treated with IGF-1 (100ng/ml), and the changes in Col2a1 and MMP-13 mRNA were monitored with real-time polymerase chain reaction (PCR). MMP-13 activity was also assayed. Activation of signaling proteins was evaluated by western blot analysis. Cells were also treated with pharmacological agents that block PI3K and MAPK signaling pathways., Results: IGF-1 increased Col2a1 mRNA expression in rat endplate chondrocytes in a time- and dose-dependent manner. IGF-1 treatment resulted in a fourfold increase of Col2a1 mRNA with the effect maximizing at 24h. In contrast, IGF-1 treatment for 24h caused a roughly 50% reduction in MMP-13 mRNA. Similar effects were seen on the protein levels of type II collagen (col2) and MMP-13. Consistent with these results, IGF-1 also repressed MMP-13 activity. IGF-1 activated both the PI3K and the extracellular signal-regulated kinase (ERK) pathways as evidenced by phosphorylation of either Akt or ERK1/2 (respectively). The PI3K inhibitor Wartmannin significantly inhibited the IGF-1 effect on Col2a1 mRNA expression but did not affect IGF-1-induced repression of MMP-13 expression. In contrast, the ERK/MAPK inhibitor PD98059 significantly inhibited the effect of IGF-1 on MMP-13 mRNA repression and enhanced IGF-1-induced Col2a1 mRNA expression., Conclusions: In rat endplate chondrocytes the PI3K pathway mainly transduces IGF-1 effect on col2 expression while the ERK pathway mediates IGF-1 effect on MMP-13 expression.

Objective: To assess the clinical features of nervous system (NS) involvement in patients with ANCA-associated vasculitides (AAV), including microscopic polyangiitis (MPA), Wegener's granulomatosis (WG), and Churg-Strauss syndrome (CSS)., Methods: One hundred and seventy-nine patients admitted to Peking Union Medical College Hospital from 1995 to 2008, including 93 cases of MPA, 61 cases of WG, and 25 cases of CSS, were enrolled in this study. Medical charts including demographic data, clinical features, laboratory findings, treatments and outcomes were systematically reviewed., Results: NS involvements were observed in 36.6% of MPA, 50.8% of WG, and 76.0% of CSS patients. Peripheral neuropathy predominated in each type of AAV. In CSS and MPA, the majority was mononeuritis multiplex and distal symmetrical polyneuropathy, whereas, differently, 64.5% of WG patients with NS involvement had cranial neuropathy. Central nervous system (CNS) involvement accounted for 21.1%, 29.4%, and 32.3% of neuropathy respectively in CSS, MPA and WG patients, including arachnoid hemorrhage, cerebrovascular neuro-pathy, meningitis, and diffuse brain damage. 157 (87.7%) AAV patients responded to treatment with high dose of prednisone plus immunosuppressants. Thirteen (14.0%) MPA and four (6.6%) WG patients died. The leading causes of death were diffuse alveolar hemorrhage (DAH) (6, 35.3%) and infection (6, 35.3%). No patient died directly of neuropathy., Conclusion: NS involvement was common in AAVs and the characteristic of NS involvement was different among MPA, WG and CSS patients. DAH and infection instead of NS damage remained the leading causes of death in AAVs.

Mitochondrial defects including reduction of a key mitochondrial tricarboxylic acid cycle enzyme alpha-ketoglutarate-dehydrogenase complex (KGDHC) are characteristic of many neurodegenerative diseases. KGDHC consists of alpha-ketoglutarate dehydrogenase, dihydrolipoyl succinyltransferase (E2k), and dihydrolipoamide dehydrogenase (Dld) subunits. We investigated whether Dld or E2k deficiency influences adult brain neurogenesis using immunohistochemistry for the immature neuron markers, doublecortin (Dcx) and polysialic acid-neural cell adhesion molecule, as well as a marker for proliferation, proliferating cell nuclear antigen (PCNA). Both Dld- and E2k-deficient mice showed reduced Dcx-positive neuroblasts in the subgranular zone (SGZ) of the hippocampal dentate gyrus compared with wild-type mice. In the E2k knockout mice, increased immunoreactivity for the lipid peroxidation marker, malondialdehyde occurred in the SGZ. These alterations did not occur in the subventricular zone (SVZ). PCNA staining revealed decreased proliferation in the SGZ of E2k-deficient mice. In a transgenic mouse model of Alzheimer's disease, Dcx-positive cells in the SGZ were also reduced compared with wild type, but Dld deficiency did not exacerbate the reduction. In the malonate lesion model of Huntington's disease, Dld deficiency did not alter the lesion-induced increase and migration of Dcx-positive cells from the SVZ into the ipsilateral striatum. Thus, the KGDHC subunit deficiencies associated with elevated lipid peroxidation selectively reduced the number of neuroblasts and proliferating cells in the hippocampal neurogenic zone. However, these mitochondrial defects neither exacerbated certain pathological conditions, such as amyloid precursor protein (APP) mutation-induced reduction of SGZ neuroblasts, nor inhibited malonate-induced migration of SVZ neuroblasts. Our findings support the view that mitochondrial dysfunction can influence the number of neural progenitor cells in the hippocampus of adult mice.

Cardiac troponin release is generally found in adult athletes after continuous-type endurance exercises or sport competitions. The purpose of this study was to investigate whether the physical stress experienced by adolescents while playing basketball, an intense, intermittent-type sport, could induce transient elevations of the serum cardiac troponin T (cTnT) and I (cTnI). Serum cTnT and cTnI levels in 10 male adolescent players (age 15.0 +/- 0.7 yr) were assessed immediately before and at 2, 4 and 24 h after a game randomly selected from a preseason basketball-training program. At 4 h following the game, serum cTnT levels in four of the ten subjects were above the cutoff of 0.01 ng . ml (-1) for myocardial injury. Two of these four subjects had values higher than the acute myocardial infarction cutoff of 0.05 ng . ml (-1). In three of the four subjects, the serum cTnI was above the cutoff of 0.06 ng . ml (-1) for myocardial injury. Nevertheless, serum cardiac troponins at 24 h had returned to pre-exercise levels. These findings suggest that the physical stress encountered during intense, intermittent-type sports could cause release of cardiac troponins in some adolescents at low risk for cardiac disease.

Setting: Urban inner city human immunodeficiency virus (HIV) clinic., Objective: To evaluate tuberculin skin testing (TST) and QuantiFERON-TB Gold (QFT-G) testing in an HIV-infected population relative to the presence of risk factors for latent tuberculosis infection (LTBI)., Design: Cross-sectional analysis of the response of a whole blood gamma interferon release assay to early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) antigens and TST relative to known risk factors for LTBI., Results: Of 207 subjects enrolled, four were excluded due to missing data and three specimens yielded discordant results. Ten specimens were indeterminate due to inadequate response to mitogen. All indeterminate results occurred in subjects with CD(4) counts <200 cells/mm(3). Eleven QFT-G results and 13 TST results were positive. The concordance between TST and QFT-G was poor (kappa 0.38). QFT-G results were more likely than TST to be associated with risk factors for LTBI., Conclusions: QFT-G, but not TST, showed a statistically significant association between the number of risk factors for LTBI and a positive result (OR 1.6). QFT-G testing may be more useful than TST in individuals with HIV infection.

Secreted protein acidic and rich in cysteine (SPARC) is an extracellular glycoprotein expressed in several solid cancers, including malignant gliomas, upon adoption of metastatic or invasive behaviors. SPARC expression in glioma cells promotes invasion and survival under stress, the latter process dependent on SPARC activation of AKT. Here we demonstrate that downregulation of SPARC expression with short interfering RNA (siRNA) in glioma cells decreased tumor cell survival and invasion. SPARC siRNA reduced the activating phosphorylation of AKT and two cytoplasmic kinases, focal adhesion kinase (FAK) and integrin-linked kinase (ILK). We determined the contributions of FAK and ILK to SPARC effects using SPARC protein and cell lines engineered to overexpress SPARC. SPARC activated FAK and ILK in glioma cells previously characterized as responsive to SPARC. Downregulation of either FAK or ILK expression inhibited SPARC-mediated AKT phosphorylation, and targeting both FAK and ILK attenuated AKT activation more potently than targeting either FAK or ILK alone. Decreased SPARC-mediated AKT activation correlated with a reduction in SPARC-dependent invasion and survival upon the downregulation of FAK and/or ILK expression. These data further demonstrate the role of SPARC in glioma tumor progression through the activation of intracellular kinases that may provide novel therapeutic targets for advanced cancers.

Thiamine-dependent enzymes are diminished in multiple neurodegenerative diseases. Thiamine deficiency (TD) reduces the activity of thiamine dependent-enzymes [e.g., the alpha-ketoglutarate dehydrogenase complex (KGDHC)], induces regional selective neurodegeneration and serves as a model of a mild impairment of oxidative metabolism. The current experiments tested whether changes in KGDHC protein subunits (E1k, E2k and E3) or activity or message levels underlie the selective loss of neurons in particular brain regions. Thus, TD-induced changes in these variables in the brain region most vulnerable to TD [the sub-medial thalamic nucleus (SmTN)] were compared to those in a region that is relatively resistant to TD (cortex) at stages of TD when the neuron loss in SmTN is not present, minimal or severe. Impaired motor performance on rotarod was apparent by 8 days of TD (-32%) and was severe by 10 days of TD (-97%). At TD10, the overall KGDHC activity measured by an in situ histochemical staining method declined 52% in SmTN but only 20% in cortex. Reductions in the E2k and E3 mRNA in SmTN occurred as early as TD6 (-28 and -18%, respectively) and were more severe by TD10 (-61 and -66%, respectively). On the other hand, the level of E1k mRNA did not decline in SmTN until TD10 (-48%). In contrast, TD did not alter mRNA levels of the subunits in cortex at late stages. Western blots and immunocytochemistry revealed different aspects of the changes in protein levels. In SmTN, the immunoreactivity of E1k and E3 by Western blotting increased 34 and 40%, respectively, only at TD8. In cortex, the immunoreactivity of the three subunits was not altered. Immunocytochemical staining of brain sections from TD10 mice indicated a reduction in the immunoreactivity of all subunits in SmTN, but not in cortex. These findings demonstrate that the response of the KGDHC activity, mRNA and immunoreactivity of E1k, E2k and E3 to TD is region and time dependent. Loss of KGDHC activity in cortex is likely related to post-translational modification rather than a loss of protein, whereas in SmTN transcriptional and post-translational modifications may account for diminished KGDHC activity. Moreover, the earlier detection in TD induced-changes of the transcripts of KGDHC indicates that transcriptional modification of the two subunits (E2k and E3) of KGDHC may be one of the early events in the cascade leading to selective neuronal death.

Segregation in vertically vibrated binary granular mixtures with the same size is studied experimentally. The partially segregated state occurring in this system is observed carefully. We find that the characteristic of the partially segregated state is that the lighter particles tend to rise and form a pure layer on the top of the system while the heavier particles and some of the lighter ones stay at the bottom and form a mixed layer. The ratio of the thickness of the pure top layer to that of the whole system can be taken as an order parameter, which describes the degree of the segregation quantitatively and is useful in the investigation of the system. By use of it, we find that the segregation state is only dependent on the density ratio of the two kinds of particles. The dependent of the segregation on the vibration frequency is also studied by use of this order parameter, and finally, two typical phase diagrams are given.

Understanding the elusive catalytic role of titanium-based additives on the reversible hydrogenation of complex hydrides is an essential step toward developing hydrogen storage materials for the transport sector. Improved bulk diffusion of hydrogen is one of the proposed effects of doping sodium alanate with TiCl3, and here we study hydrogen dynamics in doped and undoped Na3AlH6 using a combination of density functional theory calculations and quasielastic neutron scattering. The hydrogen dynamics is found to be vacancy mediated and dominated by localized jump events, whereas long-range bulk diffusion requires significant activation. The fraction of mobile hydrogen is found to be small for both undoped and doped Na3AlH6, even at 350 K, and improved hydrogen diffusion as a result of bulk-substituted titanium is found to be unlikely. We also propose that previously detected low-temperature point defect motion in sodium alanate could result from vacancy-mediated sodium diffusion.

Objective: The present study aimed to investigate the modulation of membrane-bound intercellular adhesion molecule-1 (mICAM-1) and soluble ICAM-1 (sICAM-1) expression by tumor necrosis factor-alpha (TNFalpha) in human osteoarthritic (OA) osteoblasts., Methods: Cultured human primary osteoblasts were stimulated with increasing concentrations of human recombinant TNFalpha. Expression of mICAM-1 and sICAM-1 was evaluated by immunocytochemistry, enzyme-linked immunosorbent assay and semi-quantitative reverse transcriptase-polymerase chain reaction. In addition, we investigated the molecular mechanisms underlying ICAM-1 induction by TNFalpha, focusing on the activation of the mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) pathways., Results: Our data showed that TNFalpha dose-dependently increased mICAM-1 and sICAM-1 expression at the protein and mRNA levels in OA osteoblasts. The inhibitor of de novo mRNA synthesis, actinomycin D, suppressed TNFalpha-induced mICAM-1 and sICAM-1 expression. Upon examination of the signaling components, we found that TNFalpha was a potent activator of p38, p44/42, p54/46 MAPK, and IkappaBalpha (IkappaBalpha). The chemical inhibitors of p38, p44/42 MAPK, and NF-kappaB blocked TNFalpha-induced mICAM-1 expression but not that of sICAM-1. Transfection experiments revealed that p38 MAPK or IkappaB kinase alpha (IKKalpha) overexpression enhanced TNFalpha-induced mICAM-1 production. Furthermore, osteoblasts treatment with a chemical inhibitor of metalloproteinase-9 (MMP-9) activity, a proteolytic enzyme involved in ICAM-1 cleavage, evoked a significant 25% decrease of TNFalpha-induced sICAM-1 release., Conclusion: Taken together, these findings illustrate the central role played by TNFalpha in the regulation of ICAM-1. We suggest that TNFalpha differentially regulates sICAM-1 and mICAM-1 expression and that sICAM-1 release involves, in part, the proteolytic cleavage of mICAM-1 by MMP-9. The capacity of the MMP-9 inhibitor to prevent sICAM-1 production may be useful for the development of novel therapeutic approaches relevant to OA.

Background: Previous studies from our laboratory have demonstrated that lineage-targeted synthesis of factor VIII (FVIII) under the direction of the platelet-specific integrin alphaIIb gene promoter (2bF8) can correct the murine haemophilia A phenotype even in the presence of high titer inhibitory antibodies in a transgenic mouse model., Objective: In this study, we assessed the efficacy of using a genetic therapy approach to correct haemophilia A in FVIII-deficient (FVIII(null)) mice by transplantation of bone marrow (BM) transduced with a lentivirus (LV)-based gene transfer cassette encoding 2bF8., Results: Functional FVIII activity (FVIII:C) was detected in platelet lysates from treated mice and the levels were similar to 2bF8 heterozygous transgenic mice. Mice transplanted with 2bF8 LV-transduced BM survived tail clipping and we did not detected inhibitory or non-inhibitory FVIII antibodies over the period of this study (11 months). Furthermore, BM transferred from the primary transplant recipients into FVIII(null) secondary recipients demonstrated sustained platelet-FVIII expression leading to correction of the haemophilia A phenotype showing that gene transfer occurred within long-term repopulating haematopoietic stem cells., Conclusions: These results demonstrate that ectopic expression of FVIII in platelets by lentivirus-mediated bone marrow transduction/transplantation may be a promising strategy for gene therapy of haemophilia A in humans.

Programmed death-1 ligand-1 (PD-L1), a member of the B7 family of costimulatory molecules, plays an important role in the regulations of the cellular and humoral immune responses. In this study, two mouse anti-human PD-L1 monoclonal antibodies named 10E10 and 2H11 were successfully generated and further characterized. Monoclonal antibody 10E10 bound to distinct PD-L1 epitope comparing an available anti-PD-L1 monoclonal antibody on a series of malignant cell lines, activated T lymphocytes, B lymphocytes and dendritic cells. Then, by using immunohistochemistry staining with monoclonal antibody 2H11, the expression of PD-L1 was found in human gastric carcinoma specimens but not in normal or gastric adenoma tissues. Additional data show that PD-L1 can be regarded as a decisive factor in evaluating gastric carcinoma prognosis and anti-human PD-L1 monoclonal antibody 10E10 could inhibit T-cell apoptosis induced by tumor-associated PD-L1. Taken together, these results showed that the two functional mouse anti-human PD-L1 monoclonal antibodies we generated might be of great value for further exploration of the costimulatory molecule regulating network and immunointervention for tumor immunotherapy.

In this study, the authors document the characteristics of South Asian (SA) cerebrovascular patients. A retrospective medical record review comparing SA (n = 99) and European-American (n = 106) patients was performed. SA patients were younger and had a greater prevalence of diabetes, but lower prevalences of hyperlipidemia and tobacco use. SA patients experienced a 75% lower risk of cardiogenic infarctions, but a threefold increased risk of intracranial atherothrombosis. Risk factor modifications and secondary prevention strategies may differ for SA patients.

Aim: This study was designed to examine the appearance and clearance characteristics of serum cardiac troponin T (cTnT) and I (cTnI) in junior runners after an exhaustive 21-km run. The dependence of the alternations of the cardiac troponins on the runners' training status reflected in training years, running ability and physiological profile at ventilatory threshold (Th(vent)) was also examined., Methods: Ten trained male adolescents (16.2+/-0.6 years) performed 21-km run and graded treadmill exercise with maximum effort on two different days. cTnT and cTnI immediately before and 2, 4 and 24 hours after exercise were measured in the 21-km trial. The parameters at Thvent including the running speed (Th(vent)-Speed) and VO2 (Th(vent)-VO2) were identified in the graded exercise trial., Results: Serum cTnT and cTnI in 6 of the 10 subjects measured 4 hours after the 21-km run were above the myocardial injury cutoffs of 0.03 and 0.09 ng x mL(-1), respectively, but returned towards pre-exercise levels within 24 hours that were not in the same manner observed in the clinical situation of myocardial infraction. The 6 subjects' training status was generally lower than that of the rest 4. Further, subjects' 4-h serum cTnT and cTnI were negatively related to their training years, Th(vent)-Speed and Th(vent)-VO2, and positively related to their personal best in half- and full-marathon races (r2 = or > 36%, n = 10)., Conclusions: Such findings implied that clinicians based the diagnosis of long-distance run-induced myocardial infraction in novice junior runners upon the transient postexercise elevations of cardiac troponins alone should be with caution.

von Willebrand factor (VWF) performs a critical function in platelet binding at the site of vascular injury and also serves as the carrier protein for coagulation factor FVIII (FVIII), protecting it from proteolytic degradation in plasma. Both proteins undergo rapid, regulated release in response to DDAVP administration in patients with mild hemophilia A or von Wille-brand disease. Here, we attempt to summarize our current understanding of the establishment of the regulated storage pool of VWF and FVIII. The data presented indicate that regulated secretion of both proteins occurs only if there is endogenous synthesis of FVIII together with VWF.

OX40, a membrane-bound molecule of the tumor-necrosis-factor-receptor superfamily, is a critical costimulatory receptor during the immune response. Here, we newly generated two specific mouse antihuman OX40 monoclonal antibodies (mAbs) (2G2 and 1F7), whose specificities are quite different from the available OX40 mAb (ACT35) by competition assay. It was also found that both mAbs could enhance the proliferation, activation and differentiation of T lymphocytes primed by anti-CD3 mAb. These results evidenced that both were functional antihuman OX40 mAbs. Furthermore, stained by 2G2 and 1F7, FCM and immunohistochemistry detected the constitutive expression of OX40 on tumor cell lines from epithelium, breast cancer and glioma tissues. Meanwhile, the non-tumor tissues (thyroid gland, stomach gland) were also found OX40 expression. These results suggested that OX40 is not only expressed in activated T cells, but also in some tumors as well as normal gland tissues. Such expression pattern indicated that OX40 may be a valuable surface antigen in unveiling its expression and function outside the immune system. Briefly, these novel antibodies may contribute to the evaluation of the mechanism of tumor metastasis and eventually shed light on further study of tumor immunotherapy and autoimmune diseases.

Reticulons (RTNs) are a group of integral membrane proteins that have a uniquely conserved C-terminal domain named RHD. In mammalian genomes, transcripts are produced from four genes, rtn1 to rtn4, under the regulation of tissue or cell-type-specific expression. The presence of alternative promoters for gene expression and multiple cryptic splicing sites have resulted in large numbers of genes/proteins that are classified among the reticulon family. Although this family exists in almost all eukaryotes, only the rtn4 gene product, Nogo (RTN4), has gained relatively more in-depth attention. Despite predominant localization in the endoplasmic reticulum, Nogo on the cell surface appears to play a critical role as an inhibitory molecule for axonal growth and regeneration in humans and rodents. Recently, studies have expanded the biological functions of RTNs to other facets including modulating the enzymatic activity of beta-secretase in Alzheimer's disease. In this review, we summarize the accumulated findings concerning the structural and functional aspects of RTNs and speculate on their linkage to the pathogenesis of neurodegenerative diseases.

2IgB7-H3 has recently been identified as a new member of the B7 family. Its expression at the protein level remains largely unknown due to the lack of the specific monoclonal antibody (mAb). To characterize the expression of 2IgB7-H3, we newly generated two mouse antihuman 2IgB7-H3 mAbs (4H7 and 21D4). We found the constitutive expression of 2IgB7-H3 on a series of tumor cell lines. Furthermore, the expression was examined on monocyte-derived dendritic cells (Mo-DCs) and DCs from CD34(+) hematopoietic progenitor cells (HPC) by means of mAb staining. The results showed that 2IgB7-H3 was expressed on Mo-DCs at a high and stable level during differentiation in vitro. With the maturation of DCs from CD34(+) HPCs, the expression of the molecule was upregulated. However, the 2IgB7-H3 was not expressed on fresh isolated T and B lymphocytes, monocytes, or CD34(+) HPCs. These results suggested that 2IgB7-H3 may be a valuable surface antigen for the detection of DCs.

Objective: To assess visual search and recognition of roadside targets and safety errors during a landmark and traffic sign identification task in drivers with Alzheimer's disease., Methods: 33 drivers with probable Alzheimer's disease of mild severity and 137 neurologically normal older adults underwent a battery of visual and cognitive tests and were asked to report detection of specific landmarks and traffic signs along a segment of an experimental drive., Results: The drivers with mild Alzheimer's disease identified significantly fewer landmarks and traffic signs and made more at-fault safety errors during the task than control subjects. Roadside target identification performance and safety errors were predicted by scores on standardised tests of visual and cognitive function., Conclusions: Drivers with Alzheimer's disease are impaired in a task of visual search and recognition of roadside targets; the demands of these targets on visual perception, attention, executive functions, and memory probably increase the cognitive load, worsening driving safety.

Objective: To determine trabecular and subchondral bone metabolic changes in experimental canine osteoarthritis (OA)., Methods: OA was induced in 19 dogs by transection of the anterior cruciate ligament (ACL) of the right knee through a stab wound. Dogs were sacrificed at 8 (n=7) and 12 weeks (n=12) after surgery. Non-operated normal dogs (n=6) were used as controls. After sacrifice, samples were obtained from the weight-bearing area of medial tibial plateaus. Explants and cell cultures were prepared from subchondral and trabecular bone. Osteocalcin (Oc), cellular alkaline phosphatase (ALPase), urokinase plasminogen-activator (uPA), prostaglandin E2 (PGE2), metalloproteinase (MMP) and nitric oxide (NO) were measured using standard procedures., Results: ALPase production was significantly increased only at week 12 in subchondral and trabecular bone, while an increase in Oc was noted at week 8. uPA and MMP activity were increased significantly at week 12 in subchondral bone, while PGE2 levels were significantly higher in subchondral and trabecular bone at week 12 compared to normal. A decrease in NO production appeared late at week 12 in trabecular bone, whereas NO levels from subchondral bone were significantly increased compared to normal at week 8., Discussion: Intense bone remodeling takes place in both subchondral and trabecular bone in the knee following ACL transection. This process seems to occur around week 12, although Oc and NO appeared to be involved earlier at 8 weeks. These results suggest that not only subchondral but also trabecular bone metabolism is altered in this OA model.

The extracorporeal shock wave lithotripsy (ESWL) has come into wide use due to its non-invasive advantage. However, as for the shock wave propagation in human body in relation to practical lithotripsy, there are a few studies on numerical simulation based on models conforming to practical situations. In this paper we report a numerical method of finite difference time domain (FDTD) for analyzing ultrasonic pulse nonlinear propagation in human body tissues and demonstrated it through an experiment of Reichenberger's ESWL in water.

Cyanobacterial blooms that generate microcystins (MCs) are being increasingly recognized as a potent health hazard in aquatic ecosystems. However, immunomodulation induced by cyanotoxins has not been well documented. This paper reports the in vivo data on the immune disorder caused by crude microcystin (MC) extract of cyanobacteria blooms collected from Taihu Lake, China, with respect to cytokine mRNA levels. Using reverse-transcriptional polymerase chain reaction (RT-PCR), the expression of multiple cytokines, including proinflammatory (IL-1beta, TNF-alpha, and IL-6) and Th1/Th2-related cytokines (IL-2, IL-4 and IL-10), was evaluated following the cyanobacteria blooms extract containing MCs (CBE) exposure at four doses of 23, 38, 77, 115 mg lyophilized algae cells/kg body weight. The results showed that the mRNA levels of TNF-alpha, IL-1beta, IL-2 and IL-4 decreased significantly following injection of all doses as compared to the control (LPS or ConA only), while the IL-6 level was unaffected. Contrast to this decrease, the level of IL-10 mRNA was, however, transiently up regulated following injection of the lowest dose of CBE. The distinct patterns of expression of these cytokines suggested a modulation of cytokine network, the essential component of the host immune system. We further developed a mathematical model to simulate the interaction of T helper cell subsets and related cytokines, which proved to be a good approach to study the kinetics of the interaction of cells and cytokines in microcystin immunosuppression.

The platinum compound oxaliplatin has been shown to be an effective chemotherapeutic agent for the treatment of colorectal cancer. In this study, we investigate the molecular mechanisms of action of oxaliplatin to identify means of predicting response to this agent. Exposure of colon cancer cells to oxaliplatin resulted in G2/M arrest and apoptosis. Immunofluorescent staining demonstrated that the apoptotic cascade initiated by oxaliplatin is characterised by translocation of Bax to the mitochondria and cytochrome c release into the cytosol. Oxaliplatin treatment resulted in caspase 3 activation and oxaliplatin-induced apoptosis was abrogated by inhibition of caspase activity with z-VAD-fmk, but was independent of Fas/FasL association. Targeted inactivation of Bax or p53 in HCT116 cells resulted in significantly increased resistance to oxaliplatin. However, the mutational status of p53 was unable to predict response to oxaliplatin in a panel of 30 different colorectal cancer cell lines. In contrast, the expression profile of these 30 cell lines, assessed using a 9216-sequence cDNA microarray, successfully predicted the apoptotic response to oxaliplatin. A leave-one-out cross-validation approach was used to demonstrate a significant correlation between experimentally observed and expression profile predicted apoptosis in response to clinically achievable doses of oxaliplatin (R=0.53; P=0.002). In addition, these microarray experiments identified several genes involved in control of apoptosis and DNA damage repair that were significantly correlated with response to oxaliplatin.