Clinical trials with glycoprotein IIB/IIIA antagonists - No benefit without bleeding?

As the glycoprotein GPIIb/IIIa receptor is the final common pathway in platelet aggregation, antagonists of this receptor cause a profound inhibition of aggregation induced by any agonist. The short-term efficacy and safety of GPIIb/IIIa antagonists in patients undergoing coronary angioplasty was demonstrated with murine 7E3 Fab, but this antibody was immunogenic. Abciximab is a chimeric human-mouse monoclonal antibody that is less immunogenic. The first major trial with a GPIIb/IIIa antagonist was the EPIC trial with abciximab, which showed that abciximab reduced the ischemic complications of coronary balloon angioplasty and atherectomy in high-risk patients, but increased the risk of bleeding. Subsequent studies showed that using less concurrent heparin reduced bleeding. Abciximab also reduced the rate of revascularization. Further studies have shown that the benefits of abciximab extended to all patients undergoing angioplasty (EPILOG), including patients with unstable angina (CAPTURE) and acute myocardial infarction (RAPPORT). Clinical trials with eptifibatide and tirofiban have failed to demonstrate benefit, at the doses used, in angioplasty. Abciximab and eptifibatide, but not oral xemilofiban, improve the safety of the coronary stenting procedure. Shortterm intravenous treatment with lamifiban, eptifibatide or tirofiban is beneficial in acute coronary syndromes (unstable angina, non-Q wave myocardial infarction). Orally active GPIIb/IIIa antagonists are being developed for use in acute coronary syndromes and myocardial infarction. However, no benefit has been shown with lefradafiban in acute coronary syndromes and sibrafiban and orbofiban are harmful. Eptifibatide, lamifiban and abciximab improve coronary patency in myocardial infarction, and long-term trials of GPIIb/IIIa antagonists are being conducted in acute myocardial infarction. Abciximab can cause thrombocytopenia, and all the GPIIb/IIIa antagonists increase the incidence of bleeding, but there is no excess of intracranial hemorrhage. (C) 2001 Prous Science. All rights reserved.

Production of the baculovirus-expressed dengue virus glycoprotein NS1 can be improved dramatically with optimised regimes for fed-batch cultures and the addition of the insect moulting hormone, 20-Hydroxyecdysone

A perennial problem in recombinant protein expression is low yield of the product of interest. A strategy which has been shown to increase the production of baculovirus-expressed proteins is to utilise fed-batch cultures. One disadvantage of this approach is the time-consuming task of optimising the feeding strategy. Previously, a statistical optimisation routine was applied to develop a feeding strategy that increased the yield of beta-Galactosidase (beta-Gal) by 2.4-fold (Biotechnol. Bioeng, 59 (1998) 178). This involves the single addition of nutrient concentrates (amino acids, lipids. glucose and yeastolate ultrafiltrate) into Sf9 cell cultures grown in SF900II medium. In this study, it is demonstrated that this optimised fed-batch strategy developed for a high-yielding intracellular product beta-Gal could be applied successfully to a relatively low-yielding glycosylated and secreted product such as the dengue virus glycoprotein NS1. Optimised batch infections yielded 4 mug/ml of NS1 at a peak cell density of 4.2 x 10(6) cells/ml. In contrast. optimised fed-batch infections exhibited a 3-fold improvement in yield, with 12 mug ml of NS1 produced at a peak cell density of 11.3 x 10(6) cells/ml. No further improvements in yield were recorded when the feed volumes were doubled and the peak cell density was increased to 23 x 10(6) cells/ml, unless the cultures were stimulated by the addition of 4 mug/ml of 20-Hydroxyecdysone (an insect moulting hormone). In this case, the NS1 yield was increased to 20 mug/ml. which was nearly 5-fold higher than optimised batch cultures. (C) 2002 Elsevier Science B.V. All rights reserved.

Synthesis and characterization of rabies virus glycoprotein-tagged amphiphilic cyclodextrins for siRNA delivery in human glioblastoma cells: in vitro analysis

In man brain cancer is an aggressive, malignant form of tumour, it is highly infiltrative in nature, is associated with cellular heterogeneity and affects cerebral hemispheres of the brain. Current drug therapies are inadequate and an unmet clinical need exists to develop new improved therapeutics. The ability to silence genes associated with disease progression by using short interfering RNA (siRNA) presents the potential to develop safe and effective therapies. In this work, in order to protect the siRNA from degradation, promote cell specific uptake and enhance gene silencing efficiency, a PEGylated cyclodextrin (CD)-based nanoparticle, tagged with a CNS-targeting peptide derived from the rabies virus glycoprotein (RVG) was formulated and characterized. The modified cyclodextrin derivatives were synthesized and co-formulated to form nanoparticles containing siRNA which were analysed for size, surface charge, stability, cellular uptake and gene-knockdown in brain cancer cells. The results identified an optimised co-formulation prototype at a molar ratio of 1:1.5:0.5 (cationic cyclodextrin:PEGylated cyclodextrin:RVG-tagged PEGylated cyclodextrin) with a size of 281±39.72nm, a surface charge of 26.73±3mV, with efficient cellular uptake and a 27% gene-knockdown ability. This CD-based formulation represents a potential nanocomplex for systemic delivery of siRNA targeting brain cancer.

Evaluation of glycoprotein B genotypes and load of CMV infecting blood leukocytes on prognosis of AIDS patients

BACKGROUND: Cytomegalovirus (CMV) remains an important pathogen to immunocompromised patients even in the era of HAART. The present study aimed at evaluating the influence of CMV viral load and its gB genotypes on AIDS patients' outcome. METHODS: Blood samples of 101 AIDS patients were collected and tested for HIV load, CD4 - cell count and opportunistic pathogens, including CMV. Semi-nested PCRs were run to detect CMV genome and in the positive samples, gB genotyping and CMV load were established using enzymatic restriction and real time PCR, respectively. All patients were clinically followed for four years. RESULTS: In thirty patients (31%) CMV was detected and all fatal cases (n = 5) occurred in this group of patients (p = 0.007), but only two patients had CMV disease (1.9%). However, viral load was not statistically associated with any analyzed parameter. The most frequently observed CMV genotype was gB2 (45.16%) followed by gB3 (35.48%). gB2 genotype was more frequently found in patients with CD4-cell counts under 200 cells/mm³ (p = 0.0017), and almost all fatal cases (80%) had gB2 genotype. CONCLUSIONS: Our study suggests that CMV and its polymorphisms in biologically relevant genes, such as the gB encoding ORF, may still influence the prognosis and outcome of AIDS patients. The gB2 genotype was associated to patient's bad outcome.

Comparative study of lymphocytes from individuals that were vaccinated and unvaccinated against the pandemic 2009-2011 H1N1 influenza virus in Southern Brazil

ABSTRACTINTRODUCTION:While no single factor is sufficient to guarantee the success of influenza vaccine programs, knowledge of the levels of immunity in local populations is critical. Here, we analyzed influenza immunity in a population from Southern Brazil, a region with weather conditions that are distinct from those in the rest of country, where influenza infections are endemic, and where greater than 50% of the population is vaccinated annually.METHODS:Peripheral blood mononuclear cells were isolated from 40 individuals. Of these, 20 had received the H1N1 vaccine, while the remaining 20 were unvaccinated against the disease. Cells were stimulated in vitro with the trivalent post-pandemic influenza vaccine or with conserved major histocompatibility complex I (MHC I) peptides derived from hemagglutinin and neuraminidase. Cell viability was then analyzed by [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide)]-based colorimetric assay (MTT), and culture supernatants were assayed for helper T type 1 (Th1) and Th2-specific cytokine levels.RESULTS:Peripheral blood lymphocytes from vaccinated, but not unvaccinated, individuals exhibited significant proliferation in vitro in the presence of a cognate influenza antigen. After culturing with vaccine antigens, cells from vaccinated individuals produced similar levels of interleukin (IL)-10 and interferon (IFN)-γ, while those from unvaccinated individuals produced higher levels of IFN-γ than of IL-10.CONCLUSIONS:Our data indicate that peripheral blood lymphocytes from vaccinated individuals are stimulated upon encountering a cognate antigen, but did not support the hypothesis that cross-reactive responses related to previous infections can ameliorate the immune response. Moreover, monitoring IL-10 production in vaccinated individuals could comprise a valuable tool for predicting disease evolution.

Concomitant Use of Glycoprotein IIb/IIIa Inhibitor and Streptokinase after Unsuccessful Rescue Angioplasty

A 38-year-old man with acute myocardial infarction in the lower wall affecting the right ventricle underwent thrombolytic treatment with streptokinase. Approximately 2 hours after the thrombolytic treatment started, he presented with signs of coronary reocclusion. He underwent emergency cineangiocoronariography that revealed that his right coronary artery was completely occluded by a clot. He unsuccessfully underwent angioplasty and stent implantation. After the concomitant use of glycoprotein IIb/IIIa inhibitor, coronary TIMI III flow was achieved without additional dilations, and he was discharged from the hospital 5 days later with no further complications.

Impact of cultivation conditions on N-glycosylation of influenza A virus hemagglutinin, on quasispecies composition, and on immunogenicity of virus preparations

Magdeburg, Univ., Fak. für Verfahrens- und Systemtechnik, Diss., 2014

Demyelination in brain cell aggregate cultures, induced by a monoclonal antibody against the myelin/oligodendrocyte glycoprotein (MOG).

Previous work has shown that aggregate cultures prepared from fetal rat telencephalon and grown in a chemically defined medium offer a useful model to study developmental processes such as myelin synthesis. Since compact myelin is formed in these cultures, we investigated the possibility to use this culture system to study demyelinating mechanisms. In particular, we examined the effect of a monoclonal antibody (8-18C5) directed against the myelin/oligodendrocyte glycoprotein (MOG). We found that addition of anti-MOG antibodies and complement to aggregate cultures led to a highly significant decrease in myelin basic protein (MBP) content and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) specific activity. These results indicate that, in our culture system, anti-MOG antibodies have a strong demyelinating effect.

Simultaneous LC-MS/MS quantification of P-glycoprotein and cytochrome P450 probe substrates and their metabolites in DBS and plasma.

BACKGROUND: An LC-MS/MS method has been developed for the simultaneous quantification of P-glycoprotein (P-gp) and cytochrome P450 (CYP) probe substrates and their Phase I metabolites in DBS and plasma. P-gp (fexofenadine) and CYP-specific substrates (caffeine for CYP1A2, bupropion for CYP2B6, flurbiprofen for CYP2C9, omeprazole for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4) and their metabolites were extracted from DBS (10 µl) using methanol. Analytes were separated on a reversed-phase LC column followed by SRM detection within a 6 min run time. RESULTS: The method was fully validated over the expected clinical concentration range for all substances tested, in both DBS and plasma. The method has been successfully applied to a PK study where healthy male volunteers received a low dose cocktail of the here described P-gp and CYP probes. Good correlation was observed between capillary DBS and venous plasma drug concentrations. CONCLUSION: Due to its low-invasiveness, simple sample collection and minimal sample preparation, DBS represents a suitable method to simultaneously monitor in vivo activities of P-gp and CYP.

Impact of genetic polymorphisms in cytomegalovirus glycoprotein B on outcomes in solid-organ transplant recipients with cytomegalovirus disease.

BACKGROUND:It is unknown whether specific viral polymorphisms affect in vivo therapeutic response in patients with cytomegalovirus (CMV) disease. Polymorphisms in the CMV glycoprotein B (gB) gene allow discrimination of 4 distinct genotypes (gB1-gB4). We assessed the influence of gB genotypes on the clinical and virologic outcome of CMV disease. METHODS:Solid-organ transplant recipients enrolled in a multicenter trial of CMV disease treatment (VICTOR study) were included in this study. CMV gB genotyping was performed using quantitative real-time polymerase chain reaction at day 0 (start of antiviral therapy). RESULTS:Among 239 patients with CMV disease, the prevalence of gB strain types was 26% for gB1, 10% for gB2, 10% for gB3, and 5% for gB4, whereas mixed infections were present in 49%. Donor-seropositive/recipient-seropositive patients were more likely to have mixed gB infection than donor-seropositive/recipient-seronegative patients (40% vs. 12%; P = .001). Median baseline viral loads were higher and time to viral eradication was longer ( P = .006 and P = .026 , respectively) for mixed infection versus infection with a single genotype. In a multivariate model, mixed gB infection was a significant predictor of failure to eradicate virus by day 21 (mixed vs single genotype; odds ratio, 2.66; 95% confidence interval, 1.31-5.38; P = .007 ) after controlling for baseline viral load, CMV serostatus at baseline, ganciclovir resistance, and antiviral treatment. No effect of gB genotype was seen on virologic or clinical CMV recurrence. CONCLUSIONS:No specific gB genotype appears to confer a specific CMV virulence advantage. However, mixed gB genotype infections are associated with higher viral loads and delayed viral clearance.

Viral envelope glycoprotein processing by proprotein convertases.

The proprotein convertases (PCs) are a family of nine mammalian enzymes that play key roles in the maintenance of cell homeostasis by activating or inactivating proteins via limited proteolysis under temporal and spatial control. A wide range of pathogens, including major human pathogenic viruses can hijack cellular PCs for their own purposes. In particular, productive infection with many enveloped viruses critically depends on the processing of their fusion-active viral envelope glycoproteins by cellular PCs. Based on their crucial role in virus-host interaction, PCs can be important determinants for viral pathogenesis and represent promising targets of therapeutic antiviral intervention. In the present review we will cover basic aspects and recent developments of PC-mediated maturation of viral envelope glycoproteins of selected medically important viruses. The molecular mechanisms underlying the recognition of PCs by viral glycoproteins will be described, including recent findings demonstrating differential PC-recognition of viral and cellular substrates. We will further discuss a possible scenario how viruses during co-evolution with their hosts adapted their glycoproteins to modulate the activity of cellular PCs for their own benefit and discuss the consequences for virus-host interaction and pathogenesis. Particular attention will be given to past and current efforts to evaluate cellular PCs as targets for antiviral therapeutic intervention, with emphasis on emerging highly pathogenic viruses for which no efficacious drugs or vaccines are currently available.