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.

Expression of the hepatitis C virus structural proteins in mammalian cells induces morphology similar to that in natural infection

Like many positive-strand RNA viruses, replication of the hepatitis C virus (HCV) is associated with cytoplasmic membrane rearrangements. However, it is unclear which HCV Proteins induce these ultrastructural features. This work examined the morphological changes induced by expression of the HCV structural proteins, core, E1 and E2, expressed from a Semliki Forest Virus (SFV) recombinant RNA replicon. Electron microscopy of cells expressing these proteins showed cytoplasmic vacuoles containing membranous and electron-dense material that were distinct from the type I cytoplasmic vacuoles induced during SFV replicon replication. Immunogold labelling showed that the core and E2 proteins localized to the external and internal membranes of these vacuoles. At times were also associated with some of the internal amorphous material. Dual immunogold labelling with antibodies raised against the core protein and against an endoplasmic reticulum (ER)-resident protein (protein disulphide isomerase) showed that the HCV-induced vacuoles were associated with ER-labelled membranes. This report has identified an association between the HCV core and E2 proteins with induced cytoplasmic vacuoles which are morphologically similar to those observed in HCV-infected liver tissue, suggesting that the HCV structural proteins may be responsible for the induction of these vacuoles during HCV replication in vivo.

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.

Prediction of Golgi Type II membrane proteins based on their transmembrane domains

Motivation: A major issue in cell biology today is how distinct intracellular regions of the cell, like the Golgi Apparatus, maintain their unique composition of proteins and lipids. The cell differentially separates Golgi resident proteins from proteins that move through the organelle to other subcellular destinations. We set out to determine if we could distinguish these two types of transmembrane proteins using computational approaches. Results: A new method has been developed to predict Golgi membrane proteins based on their transmembrane domains. To establish the prediction procedure, we took the hydrophobicity values and frequencies of different residues within the transmembrane domains into consideration. A simple linear discriminant function was developed with a small number of parameters derived from a dataset of Type II transmembrane proteins of known localization. This can discriminate between proteins destined for Golgi apparatus or other locations (post-Golgi) with a success rate of 89.3% or 85.2%, respectively on our redundancy-reduced data sets.

Computational differentiation of N-terminal signal peptides and transmembrane helices

Signal peptides and transmembrane helices both contain a stretch of hydrophobic amino acids. This common feature makes it difficult for signal peptide and transmembrane helix predictors to correctly assign identity to stretches of hydrophobic residues near the N-terminal methionine of a protein sequence. The inability to reliably distinguish between N-terminal transmembrane helix and signal peptide is an error with serious consequences for the prediction of protein secretory status or transmembrane topology. In this study, we report a new method for differentiating protein N-terminal signal peptides and transmembrane helices. Based on the sequence features extracted from hydrophobic regions (amino acid frequency, hydrophobicity, and the start position), we set up discriminant functions and examined them on non-redundant datasets with jackknife tests. This method can incorporate other signal peptide prediction methods and achieve higher prediction accuracy. For Gram-negative bacterial proteins, 95.7% of N-terminal signal peptides and transmembrane helices can be correctly predicted (coefficient 0.90). Given a sensitivity of 90%, transmembrane helices can be identified from signal peptides with a precision of 99% (coefficient 0.92). For eukaryotic proteins, 94.2% of N-terminal signal peptides and transmembrane helices can be correctly predicted with coefficient 0.83. Given a sensitivity of 90%, transmembrane helices can be identified from signal peptides with a precision of 87% (coefficient 0.85). The method can be used to complement current transmembrane protein prediction and signal peptide prediction methods to improve their prediction accuracies. (C) 2003 Elsevier Inc. 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.

Nanomedicines, a strategy to evade multidrug resistance

Multidrug resistance is a major problems associated with cancer chemotherapy. Efflux transports is one of the numerous mechanisms involved in multidrug resistance. P-glycoprotein is a transmembrane protein, responsible for drug efflux, which decreases drugs intracellular bioavailability, consequently decreasing their efficacy against cancer. Cancer growth and dissemination depends on the expression of transcriptional factors such as, Twist. Among other features, this protein is related with cells chemoresistance possible by regulation of multidrug resistance pathways including the P-glycoprotein expression. The herein study proposes to demonstrate if paclitaxel entrapped nanoparticles is an effective system in evading multidrug resistance mechanisms and if functionalization of a specific antibody against cancer stem cells receptors (anti-CD44v6) has the capability to target selectively these cells increasing nanoparticles efficacy. Therefore solid lipid nanoparticles were prepared and a breast cancer cell line (MDA-MB-436) was exposed to them in order to assess unloaded nanoparticles cytotoxic effects, increased pharmacologic efficacy of loaded nanoparticles relative to the free drug and their ability to evade multidrug resistance. The proposed solid lipid nanoparticles system proved to be capable of efficiently evading multidrug resistance mechanisms; however no improvement was added when these nanoparticles were functionalized with the antibody in the in vitro studies. However, the nanoparticles system is effective against multidrug resistance mechanisms.

Fluorescence-based approaches towards the assessment of structural and functional alterations in lysosomes and vacuoles induced by lactoferrin and acetic acid

Dissertação de mestrado em Biofísica e Bionanossistemas

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.

transmembrane adaptor protein SIT regulates T-cell development and homeostasis

SIT, T-cell, homeostasis, development, signalling, adaptor proteins

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.