Cell migration in response to the amino-terminal fragment of urokinase requires epidermal growth factor receptor activation through an ADAM-mediated mechanism

BackgroundCell migration is an integral component of intimal hyperplasia development and proteases are pivotal in the process. Understanding the role of urokinase signaling within the cells of vasculature remains poorly defined. The study examines the role of amino-terminal fragment (ATF) of urokinase on a pivotal cross-talk receptor, epidermal growth factor receptor (EGFR). EGFR is transactivated by both G-protein-coupled receptors and receptor tyrosine kinases and is key to many of their responses. We hypothesize that A Disintegrin and Metalloproteinase Domains (ADAM) allows the transactivation of EGFR by ATF.ObjectiveTo determine the role of ADAM in EGFR transactivation by ATF in human vascular smooth muscle cells (VSMC) during cell migration.MethodsHuman coronary VSMC were cultured in vitro. Assays of EGFR phosphorylation were examined in response to ATF (10 nM) in the presence and absence of the matrix metalloprotease (MMP) inhibitor GM6001, the ADAM inhibitors TAPI-0 and TAPI-1, heparin binding epidermal growth factor (HB-EGF) inhibitor, CRM197, HB-EGF inhibitory antibodies, epidermal growth factor (EGF) inhibitory antibodies, and the EGFR inhibitor AG1478. The small interference ribonucleic acid (siRNA) against EGFR and ADAM-9, ADAM-10, ADAM-12, and adenoviral delivered Gbg inhibitor, βARKCT were also used.ResultsATF produced concentration-dependent VSMC migration (by wound assay and Boyden chamber), which was inhibited by increasing concentrations of AG1478. ATF was shown to induce time-dependent EGFR phosphorylation, which peaked at fourfold greater than control. Pre-incubation with the Gβγ inhibitor βARKCT inhibited EGFR activation by ATF. This migratory and EGFR response was inhibited by AG1478 in a concentration-dependent manner. Incubation with siRNA against EGFR blocked the ATF-mediated migratory and EGFR responses. EGFR phosphorylation by ATF was blocked by inhibition of MMP activity and the ligand HB-EGF. The presence of the ADAM inhibitors, TAPI-0 and TAPI-1 significantly decreased EGFR activation. EGFR phosphorylation by EGF was not interrupted by inhibition of MMP, ADAMs, or HB-EGF. Direct blockade of the EGFR prevented activation by both ATF and EGF. Incubation with siRNA to ADAM-9 and -10 significantly reduced HB-EGF release from VSMC and EGFR activation in response to ATF. The siRNA against ADAM-12 had no effect.ConclusionATF can induce transactivation of EGFR by an ADAM-mediated, HB-EGF-dependent process. Targeting a pivotal cross-talk receptor such as EGFR is an attractive molecular target to inhibit cell migration.Clinical RelevanceCell migration is an integral component of intimal hyperplasia development and proteases are pivotal in the process. Understanding the role of urokinase signaling within the cells of vasculature remains poorly defined. The study examines the role of ATF of urokinase on a pivotal cross-talk receptor, EGFR. EGFR is transactivated by both G-protein-coupled receptors and receptor tyrosine kinases and is key to many of their responses. ATF can induce transactivation of EGFR by an ADAM-mediated, HB-EGF-dependent process. Targeting a pivotal cross-talk receptor such as EGFR, which can be transactivated by both G-protein-coupled receptors and receptor tyrosine kinases is an attractive molecular target

Radiation arteritis: A contraindication to carotid stenting?

BackgroundCarotid artery stenting (CAS) for high-risk anatomic lesions is accepted practice. Neck irradiation and radiotherapy-induced arteritis are common indications. The clinical outcomes of CAS for radiation arteritis have been poorly defined.MethodsA prospective database of patients undergoing CAS at a tertiary referral academic medical center was maintained from 1999 to 2006. Patients undergoing primary carotid artery stenting for significant atherosclerotic (ASOD) and radiotherapy (XRT)-induced occlusive disease were analyzed. Life-table analyses were performed to assess time-dependent outcomes. Cox proportional hazard analysis or Fisher’s exact test was performed to identify factors associated with outcomes. Data are presented as the mean ± SEM unless otherwise indicated.ResultsDuring the study period, 150 patients underwent primary CAS, 75% with embolic protection. Fifty-eight percent were symptomatic. One hundred twenty-seven (85%) were treated for ASOD, and 23 (15%) had XRT. The 30-day all-cause mortality rate was 1% for ASOD and 0% for XRT (P = NS); overall survival at 3 years was equivalent. There was no significant difference in major adverse event rates as defined by the Stenting and Angioplasty with Protection in Patients at High Risk for Endarterectomy (SAPPHIRE) trial between the groups. The 3-year neurologic event-free rate was 85% for ASOD and 87% for XRT (P = NS). Late asymptomatic occlusions were seen only in XRT patients. The 3-year freedom from restenosis rate was significantly worse for the XRT group, at 20%, vs 74% for the ASOD group (P < .05). Likewise, the 3-year patency rate was also worse for the XRT group, at 91%, vs 100% for ASOD by Kaplan-Meier analysis (P < .05). No factor was predictive of occlusion or stenosis by Cox proportional hazards analysis.ConclusionCAS for radiation arteritis has poor long-term anatomic outcome and can present with late asymptomatic occlusions. These findings suggest that these patients require closer postoperative surveillance and raise the question of whether CAS is appropriate for carotid occlusive lesions caused by radiation arteritis

Biochemical properties of Paracoccus denitrificans FnrP:Reactions with molecular oxygen and nitric oxide

In Paracoccus denitrificans, three CRP/FNR family regulatory proteins, NarR, NnrR and FnrP, control the switch between aerobic and anaerobic (denitrification) respiration. FnrP is a [4Fe-4S] cluster containing homologue of the archetypal O2 sensor FNR from E. coli and accordingly regulates genes encoding aerobic and anaerobic respiratory enzymes in response to O2, and also NO, availability. Here we show that FnrP undergoes O2-driven [4Fe-4S] to [2Fe-2S] cluster conversion that involves up to 2 O2 per cluster, with significant oxidation of released cluster sulfide to sulfane observed at higher O2 concentrations. The rate of the cluster reaction was found to be ~6-fold lower than that of E. coli FNR, suggesting that FnrP can remain transcriptionally active under microaerobic conditions. This is consistent with a role for FnrP in activating expression of the high O2 affinity cytochrome c oxidase under microaerobic conditions. Cluster conversion resulted in dissociation of the transcriptionally active FnrP dimer into monomers. Therefore, along with E. coli FNR, FnrP belongs to the subset of FNR proteins in which cluster type is correlated with association state. Interestingly, two key charged residues, Arg140 and Asp154, that have been shown to play key roles in the monomer-dimer equilibrium in E. coli FNR are not conserved in FnrP, indicating that different protomer interactions are important for this equilibrium. Finally, the FnrP [4Fe-4S] cluster is shown to undergo reaction with multiple NO molecules, resulting in iron nitrosyl species and dissociation into monomers

Production of Potent Fully Human Polyclonal Antibodies against Ebola Zaire Virus in Transchromosomal Cattle

Polyclonal antibodies, derived from humans or hyperimmunized animals, have been used prophylactically or therapeutically as countermeasures for a variety of infectious diseases. SAB Biotherapeutics has successfully developed a transchromosomic (Tc) bovine platform technology that can produce fully human immunoglobulins rapidly, and in substantial quantities, against a variety of disease targets. In this study, two Tc bovines expressing high levels of fully human IgG were hyperimmunized with a recombinant glycoprotein (GP) vaccine consisting of the 2014 Ebola virus (EBOV) Makona isolate. Serum collected from these hyperimmunized Tc bovines contained high titers of human IgG against EBOV GP as determined by GP specific ELISA, surface plasmon resonance (SPR), and virus neutralization assays. Fully human polyclonal antibodies against EBOV were purified and evaluated in a mouse challenge model using mouse adapted Ebola virus (maEBOV). Intraperitoneal administration of the purified anti-EBOV IgG (100 mg/kg) to BALB/c mice one day after lethal challenge with maEBOV resulted in 90% protection; whereas 100% of the control animals succumbed. The results show that hyperimmunization of Tc bovines with EBOV GP can elicit protective and potent neutralizing fully human IgG antibodies rapidly and in commercially viable quantities

Niemann-Pick C1 Is Essential for Ebolavirus Replication and Pathogenesis In Vivo

ABSTRACT Recent work demonstrated that the Niemann-Pick C1 (NPC1) protein is an essential entry receptor for filoviruses. While previous studies focused on filovirus entry requirements of NPC1 in vitro, its roles in filovirus replication and pathogenesis in vivo remain unclear. Here, we evaluated the importance of NPC1, and its partner in cholesterol transport, NPC2, by using a mouse model of Ebolavirus (EBOV) disease. We found that, whereas wild-type mice had high viral loads and succumbed to EBOV infection, Npc1−/− mice were entirely free of viral replication and completely protected from EBOV disease. Interestingly, Npc1+/− mice transiently developed high levels of viremia, but were nevertheless substantially protected from EBOV challenge. We also found Npc2−/− mice to be fully susceptible to EBOV infection, while Npc1−/− mice treated to deplete stored lysosomal cholesterol remained completely resistant to EBOV infection. These results provide mechanistic evidence that NPC1 is directly required for EBOV infection in vivo, with little or no role for NPC1/NPC2-dependent cholesterol transport. Finally, we assessed the in vivo antiviral efficacies of three compounds known to inhibit NPC1 function or NPC1-glycoprotein binding in vitro. Two compounds reduced viral titers in vivo and provided a modest, albeit not statistically significant, degree of protection. Taken together, our results show that NPC1 is critical for replication and pathogenesis in animals and is a bona fide target for development of antifilovirus therapeutics. Additionally, our findings with Npc1+/− mice raise the possibility that individuals heterozygous for NPC1 may have a survival advantage in the face of EBOV infection

Impact of Chlamydia trachomatis in the reproductive setting: British Fertility Society Guidelines for practice

Chlamydia trachomatis infection of the genital tract is the most common sexually transmitted infection and has a world-wide distribution. The consequences of infection have an adverse effect on the reproductive health of women and are a common cause of infertility. Recent evidence also suggests an adverse effect on male reproduction. There is a need to standardise the approach in managing the impact of C. trachomatis infection on reproductive health. We have surveyed current UK practice towards screening and management of Chlamydia infections in the fertility setting. We found that at least 90% of clinicians surveyed offered screening. The literature on this topic was examined and revealed a paucity of solid evidence for estimating the risks of long-term reproductive sequelae following lower genital tract infection with C. trachomatis. The mechanism for the damage that occurs after Chlamydial infections is uncertain. However, instrumentation of the uterus in women with C. trachomatis infection is associated with a high risk of pelvic inflammatory disease, which can be prevented by appropriate antibiotic treatment and may prevent infected women from being at increased risk of the adverse sequelae, such as ectopic pregnancy and tubal factor infertility. Recommendations for practice have been proposed and the need for further studies is identified

Shaping a screening file for maximal lead discovery efficiency and effectiveness: elimination of molecular redundancy

High Throughput Screening (HTS) is a successful strategy for finding hits and leads that have the opportunity to be converted into drugs. In this paper we highlight novel computational methods used to select compounds to build a new screening file at Pfizer and the analytical methods we used to assess their quality. We also introduce the novel concept of molecular redundancy to help decide on the density of compounds required in any region of chemical space in order to be confident of running successful HTS campaigns

Alignment of the CMS silicon tracker during commissioning with cosmic rays

This is the Pre-print version of the Article. The official published version of the Paper can be accessed from the link below - Copyright @ 2010 IOPThe CMS silicon tracker, consisting of 1440 silicon pixel and 15 148 silicon strip detector modules, has been aligned using more than three million cosmic ray charged particles, with additional information from optical surveys. The positions of the modules were determined with respect to cosmic ray trajectories to an average precision of 3–4 microns RMS in the barrel and 3–14 microns RMS in the endcap in the most sensitive coordinate. The results have been validated by several studies, including laser beam cross-checks, track fit self-consistency, track residuals in overlapping module regions, and track parameter resolution, and are compared with predictions obtained from simulation. Correlated systematic effects have been investigated. The track parameter resolutions obtained with this alignment are close to the design performance.This work is supported by FMSR (Austria); FNRS and FWO (Belgium); CNPq, CAPES, FAPERJ, and FAPESP (Brazil); MES (Bulgaria); CERN; CAS, MoST, and NSFC (China); COLCIENCIAS (Colombia); MSES (Croatia); RPF (Cyprus); Academy of Sciences and NICPB (Estonia); Academy of Finland, ME, and HIP (Finland); CEA and CNRS/IN2P3 (France); BMBF, DFG, and HGF (Germany); GSRT (Greece); OTKA and NKTH (Hungary); DAE and DST (India); IPM (Iran); SFI (Ireland); INFN (Italy); NRF (Korea); LAS (Lithuania); CINVESTAV, CONACYT, SEP, and UASLP-FAI (Mexico); PAEC (Pakistan); SCSR (Poland); FCT (Portugal); JINR (Armenia, Belarus, Georgia, Ukraine, Uzbekistan); MST and MAE (Russia); MSTDS (Serbia); MICINN and CPAN (Spain); Swiss Funding Agencies (Switzerland); NSC (Taipei); TUBITAK and TAEK (Turkey); STFC (United Kingdom); DOE and NSF (USA)