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1.  A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation 
Zhao, Huaying | Ghirlando, Rodolfo | Alfonso, Carlos | Arisaka, Fumio | Attali, Ilan | Bain, David L. | Bakhtina, Marina M. | Becker, Donald F. | Bedwell, Gregory J. | Bekdemir, Ahmet | Besong, Tabot M. D. | Birck, Catherine | Brautigam, Chad A. | Brennerman, William | Byron, Olwyn | Bzowska, Agnieszka | Chaires, Jonathan B. | Chaton, Catherine T. | Cölfen, Helmut | Connaghan, Keith D. | Crowley, Kimberly A. | Curth, Ute | Daviter, Tina | Dean, William L. | Díez, Ana I. | Ebel, Christine | Eckert, Debra M. | Eisele, Leslie E. | Eisenstein, Edward | England, Patrick | Escalante, Carlos | Fagan, Jeffrey A. | Fairman, Robert | Finn, Ron M. | Fischle, Wolfgang | de la Torre, José García | Gor, Jayesh | Gustafsson, Henning | Hall, Damien | Harding, Stephen E. | Cifre, José G. Hernández | Herr, Andrew B. | Howell, Elizabeth E. | Isaac, Richard S. | Jao, Shu-Chuan | Jose, Davis | Kim, Soon-Jong | Kokona, Bashkim | Kornblatt, Jack A. | Kosek, Dalibor | Krayukhina, Elena | Krzizike, Daniel | Kusznir, Eric A. | Kwon, Hyewon | Larson, Adam | Laue, Thomas M. | Le Roy, Aline | Leech, Andrew P. | Lilie, Hauke | Luger, Karolin | Luque-Ortega, Juan R. | Ma, Jia | May, Carrie A. | Maynard, Ernest L. | Modrak-Wojcik, Anna | Mok, Yee-Foong | Mücke, Norbert | Nagel-Steger, Luitgard | Narlikar, Geeta J. | Noda, Masanori | Nourse, Amanda | Obsil, Tomas | Park, Chad K. | Park, Jin-Ku | Pawelek, Peter D. | Perdue, Erby E. | Perkins, Stephen J. | Perugini, Matthew A. | Peterson, Craig L. | Peverelli, Martin G. | Piszczek, Grzegorz | Prag, Gali | Prevelige, Peter E. | Raynal, Bertrand D. E. | Rezabkova, Lenka | Richter, Klaus | Ringel, Alison E. | Rosenberg, Rose | Rowe, Arthur J. | Rufer, Arne C. | Scott, David J. | Seravalli, Javier G. | Solovyova, Alexandra S. | Song, Renjie | Staunton, David | Stoddard, Caitlin | Stott, Katherine | Strauss, Holger M. | Streicher, Werner W. | Sumida, John P. | Swygert, Sarah G. | Szczepanowski, Roman H. | Tessmer, Ingrid | Toth, Ronald T. | Tripathy, Ashutosh | Uchiyama, Susumu | Uebel, Stephan F. W. | Unzai, Satoru | Gruber, Anna Vitlin | von Hippel, Peter H. | Wandrey, Christine | Wang, Szu-Huan | Weitzel, Steven E. | Wielgus-Kutrowska, Beata | Wolberger, Cynthia | Wolff, Martin | Wright, Edward | Wu, Yu-Sung | Wubben, Jacinta M. | Schuck, Peter
PLoS ONE  2015;10(5):e0126420.
Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.
PMCID: PMC4440767  PMID: 25997164
2.  Bat and pig IFN-induced transmembrane protein 3 restrict cell entry by influenza virus and lyssaviruses 
The Journal of General Virology  2015;96(Pt 5):991-1005.
IFN-induced transmembrane protein 3 (IFITM3) is a restriction factor that blocks cytosolic entry of numerous viruses that utilize acidic endosomal entry pathways. In humans and mice, IFITM3 limits influenza-induced morbidity and mortality. Although many IFITM3-sensitive viruses are zoonotic, whether IFITMs function as antiviral restriction factors in mammalian species other than humans and mice is unknown. Here, IFITM3 orthologues in the microbat (Myotis myotis) and pig (Sus scrofa domesticus) were identified using rapid amplification of cDNA ends. Amino acid residues known to be important for IFITM3 function were conserved in the pig and microbat orthologues. Ectopically expressed pig and microbat IFITM3 co-localized with transferrin (early endosomes) and CD63 (late endosomes/multivesicular bodies). Pig and microbat IFITM3 restricted cell entry mediated by multiple influenza haemagglutinin subtypes and lyssavirus glycoproteins. Expression of pig or microbat IFITM3 in A549 cells reduced influenza virus yields and nucleoprotein expression. Conversely, small interfering RNA knockdown of IFITM3 in pig NPTr cells and primary microbat cells enhanced virus replication, demonstrating that these genes are functional in their species of origin at endogenous levels. In summary, we showed that IFITMs function as potent broad-spectrum antiviral effectors in two mammals – pigs and bats – identified as major reservoirs for emerging viruses.
PMCID: PMC4631062  PMID: 25614588
3.  Phosgene Exposure: A Case of Accidental Industrial Exposure 
Journal of Medical Toxicology  2013;10(1):51-56.
Phosgene is a rare exposure with strong clinical implications. We report a phosgene exposure that resulted in the patient's death.
Case Report
A 58 year-old man arrived to the emergency department 1 hour after exposure to phosgene with complaints of a sore throat. Initial vital signs were blood pressure 175/118 mmHg, heart rate 98/min, respirations 12/min, and oxygen saturation of 93% on room air. Physical exam revealed few scattered rhonchi, without signs of distress. Initial arterial blood gases (ABG's) revealed pH 7.42, pCO2 43 mmHg, pO2 68 mmHg, HCO3 27 meq/L, and oxygen saturation of 93% on room air. Initial chest x-ray 2 hours after the exposure demonstrated clear lung fields. Approximately 2.5 hours after the exposure, he began complaining of dyspnea, restlessness and his oxygen saturation dropped below 90%. He received nebulized albuterol, 1 gram intravenous methylprednisolone, and 100 % oxygen via face mask. Minimal improvement was noted and he was intubated. The post intubation chest x-ray, 3.5 hours after the exposure, revealed diffuse alveolar infiltrates. Acetylcysteine, terbutaline, and IV steroids were administered without improvement. The patient died 30 hours after exposure.
There are many misunderstandings concerning phosgene due to its rare presentation. Traditional treatment modalities are often unproven in human trials and were unsuccessful in this case.
This case highlights the significant toxicity that results from phosgene exposure and the challenges of the limited treatment modalities. There is concern for the use of this agent in chemical terrorism.
PMCID: PMC3951639  PMID: 23842907
Phosgene; ARDS; Chemical warfare; Pulmonary edema
4.  Antiviral therapies against Ebola and other emerging viral diseases using existing medicines that block virus entry 
F1000Research  2015;4:30.
Emerging viral diseases pose a threat to the global population as intervention strategies are mainly limited to basic containment due to the lack of efficacious and approved vaccines and antiviral drugs. The former was the only available intervention when the current unprecedented Ebolavirus (EBOV) outbreak in West Africa began. Prior to this, the development of EBOV vaccines and anti-viral therapies required time and resources that were not available. Therefore, focus has turned to re-purposing of existing, licenced medicines that may limit the morbidity and mortality rates of EBOV and could be used immediately. Here we test three such medicines and measure their ability to inhibit pseudotype viruses (PVs) of two EBOV species, Marburg virus (MARV) and avian influenza H5 (FLU-H5). We confirm the ability of chloroquine (CQ) to inhibit viral entry in a pH specific manner. The commonly used proton pump inhibitors, Omeprazole and Esomeprazole were also able to inhibit entry of all PVs tested but at higher drug concentrations than may be achieved in vivo. We propose CQ as a priority candidate to consider for treatment of EBOV.
PMCID: PMC4431382  PMID: 26069727
ebola; emerging viral disease; avian influenze; H5N1; Marburg; chloroquine; omemprazole; esomeprazole
5.  Antiviral therapies against Ebola and other emerging viral diseases using existing medicines that block virus entry 
F1000Research  2015;4:30.
Emerging viral diseases pose a threat to the global population as intervention strategies are mainly limited to basic containment due to the lack of efficacious and approved vaccines and antiviral drugs. The former was the only available intervention when the current unprecedented Ebolavirus (EBOV) outbreak in West Africa began. Prior to this, the development of EBOV vaccines and anti-viral therapies required time and resources that were not available. Therefore, focus has turned to re-purposing of existing, licenced medicines that may limit the morbidity and mortality rates of EBOV and could be used immediately. Here we test three such medicines and measure their ability to inhibit pseudotype viruses (PVs) of two EBOV species, Marburg virus (MARV) and avian influenza H5 (FLU-H5). We confirm the ability of chloroquine (CQ) to inhibit viral entry in a pH specific manner. The commonly used proton pump inhibitors, Omeprazole and Esomeprazole were also able to inhibit entry of all PVs tested but at higher drug concentrations than may be achieved in vivo. We propose CQ as a priority candidate to consider for treatment of EBOV.
PMCID: PMC4431382  PMID: 26069727
ebola; emerging viral disease; avian influenze; H5N1; Marburg; chloroquine; omemprazole; esomeprazole
6.  Multiplex Evaluation of Influenza Neutralizing Antibodies with Potential Applicability to In-Field Serological Studies 
Journal of Immunology Research  2014;2014:457932.
The increased number of outbreaks of H5 and H7 LPAI and HPAI viruses in poultry has major public and animal health implications. The continuous rapid evolution of these subtypes and the emergence of new variants influence the ability to undertake effective surveillance. Retroviral pseudotypes bearing influenza haemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins represent a flexible platform for sensitive, readily standardized influenza serological assays. We describe a multiplex assay for the study of neutralizing antibodies that are directed against both influenza H5 and H7 HA. This assay permits the measurement of neutralizing antibody responses against two antigenically distinct HAs in the same serum/plasma sample thus increasing the amount and quality of serological data that can be acquired from valuable sera. Sera obtained from chickens vaccinated with a monovalent H5N2 vaccine, chickens vaccinated with a bivalent H7N1/H5N9 vaccine, or turkeys naturally infected with an H7N3 virus were evaluated in this assay and the results correlated strongly with data obtained by HI assay. We show that pseudotypes are highly stable under basic cold-chain storage conditions and following multiple rounds of freeze-thaw. We propose that this robust assay may have practical utility for in-field serosurveillance and vaccine studies in resource-limited regions worldwide.
PMCID: PMC4101955  PMID: 25101305
7.  Single Domain Antibody Multimers Confer Protection against Rabies Infection 
PLoS ONE  2013;8(8):e71383.
Post-exposure prophylactic (PEP) neutralizing antibodies against Rabies are the most effective way to prevent infection-related fatality. The outer envelope glycoprotein of the Rabies virus (RABV) is the most significant surface antigen for generating virus-neutralizing antibodies. The small size and uncompromised functional specificity of single domain antibodies (sdAbs) can be exploited in the fields of experimental therapeutic applications for infectious diseases through formatting flexibilities to increase their avidity towards target antigens. In this study, we used phage display technique to select and identify sdAbs that were specific for the RABV glycoprotein from a naïve llama-derived antibody library. To increase their neutralizing potencies, the sdAbs were fused with a coiled-coil peptide derived from the human cartilage oligomeric matrix protein (COMP48) to form homogenous pentavalent multimers, known as combodies. Compared to monovalent sdAbs, the combodies, namely 26424 and 26434, exhibited high avidity and were able to neutralize 85-fold higher input of RABV (CVS-11 strain) pseudotypes in vitro, as a result of multimerization, while retaining their specificities for target antigen. 26424 and 26434 were capable of neutralizing CVS-11 pseudotypes in vitro by 90–95% as compared to human rabies immunoglobulin (HRIG), currently used for PEP in Rabies. The multimeric sdAbs were also demonstrated to be partially protective for mice that were infected with lethal doses of rabies virus in vivo. The results demonstrate that the combodies could be valuable tools in understanding viral mechanisms, diagnosis and possible anti-viral candidate for RABV infection.
PMCID: PMC3748109  PMID: 23977032
8.  Production, characterization, and antigen specificity of recombinant 62-71-3, a candidate monoclonal antibody for rabies prophylaxis in humans 
The FASEB Journal  2013;27(5):2055-2065.
Rabies kills many people throughout the developing world every year. The murine monoclonal antibody (mAb) 62-71-3 was recently identified for its potential application in rabies postexposure prophylaxis (PEP). The purpose here was to establish a plant-based production system for a chimeric mouse-human version of mAb 62-71-3, to characterize the recombinant antibody and investigate at a molecular level its interaction with rabies virus glycoprotein. Chimeric 62-71-3 was successfully expressed in Nicotiana benthamiana. Glycosylation was analyzed by mass spectroscopy; functionality was confirmed by antigen ELISA, as well as rabies and pseudotype virus neutralization. Epitope characterization was performed using pseudotype virus expressing mutagenized rabies glycoproteins. Purified mAb demonstrated potent viral neutralization at 500 IU/mg. A critical role for antigenic site I of the glycoprotein, as well as for two specific amino acid residues (K226 and G229) within site I, was identified with regard to mAb 62-71-3 neutralization. Pseudotype viruses expressing glycoprotein from lyssaviruses known not to be neutralized by this antibody were the controls. The results provide the molecular rationale for developing 62-71-3 mAb for rabies PEP; they also establish the basis for developing an inexpensive plant-based antibody product to benefit low-income families in developing countries.—Both, L., van Dolleweerd, C., Wright, E., Banyard, A. C., Bulmer-Thomas, B., Selden, D., Altmann, F., Fooks, A. R., Ma, J. K.-C. Production, characterization, and antigen specificity of recombinant 62-71-3, a candidate monoclonal antibody for rabies prophylaxis in humans.
PMCID: PMC3633812  PMID: 23371065
plant biotechnology; molecular pharming; PEP; tobacco
9.  Helix 11 Dynamics is Critical for Constitutive Androstane Receptor Activity 
The constitutive androstane receptor (CAR) transactivation can occur in the absence of exogenous ligand and this activity is enhanced by agonists TCPOBOP and meclizine. We use biophysical and cell-based assays to show that increased activity of CAR(TCPOBOP) relative to CAR(meclizine) corresponds to a higher affinity of CAR(TCPOBOP) for the steroid receptor coactivator-1. Additionally, steady-state fluorescence spectra suggest conformational differences between CAR(TCPOBOP):RXR and CAR(meclizine):RXR. Hydrogen/deuterium exchange (HDX) data indicate that the CAR activation function 2 (AF-2) is more stable in CAR(TCPOBOP):RXR and CAR(meclizine):RXR than in CAR:RXR. HDX kinetics also show significant differences between CAR(TCPOBOP):RXR and CAR(meclizine):RXR. Unlike CAR(meclizine):RXR, CAR(TCPOBOP):RXR shows a higher overall stabilization that extends into RXR. We identify residues 339–345 in CAR as an allosteric regulatory site with a greater magnitude reduction in exchange kinetics in CAR(TCPOBOP):RXR than CAR(meclizine):RXR. Accordingly, assays with mutations on CAR at leucine-340 and leucine-343 confirm this region as an important determinant of CAR activity.
PMCID: PMC3032979  PMID: 21220114
allosterism; coactivator; H/D exchange; ITC; nuclear receptor; spectroscopy
10.  Henipavirus Neutralising Antibodies in an Isolated Island Population of African Fruit Bats 
PLoS ONE  2012;7(1):e30346.
Isolated islands provide valuable opportunities to study the persistence of viruses in wildlife populations, including population size thresholds such as the critical community size. The straw-coloured fruit bat, Eidolon helvum, has been identified as a reservoir for henipaviruses (serological evidence) and Lagos bat virus (LBV; virus isolation and serological evidence) in continental Africa. Here, we sampled from a remote population of E. helvum annobonensis fruit bats on Annobón island in the Gulf of Guinea to investigate whether antibodies to these viruses also exist in this isolated subspecies. Henipavirus serological analyses (Luminex multiplexed binding and inhibition assays, virus neutralisation tests and western blots) and lyssavirus serological analyses (LBV: modified Fluorescent Antibody Virus Neutralisation test, LBV and Mokola virus: lentivirus pseudovirus neutralisation assay) were undertaken on 73 and 70 samples respectively. Given the isolation of fruit bats on Annobón and their lack of connectivity with other populations, it was expected that the population size on the island would be too small to allow persistence of viruses that are thought to cause acute and immunising infections. However, the presence of antibodies against henipaviruses was detected using the Luminex binding assay and confirmed using alternative assays. Neutralising antibodies to LBV were detected in one bat using both assays. We demonstrate clear evidence for exposure of multiple individuals to henipaviruses in this remote population of E. helvum annobonensis fruit bats on Annobón island. The situation is less clear for LBV. Seroprevalences to henipaviruses and LBV in Annobón are notably different to those in E. helvum in continental locations studied using the same sampling techniques and assays. Whilst cross-sectional serological studies in wildlife populations cannot provide details on viral dynamics within populations, valuable information on the presence or absence of viruses may be obtained and utilised for informing future studies.
PMCID: PMC3257271  PMID: 22253928
11.  Regulation of CCL2 Expression by an Upstream TALE Homeodomain Protein-Binding Site That Synergizes with the Site Created by the A-2578G SNP 
PLoS ONE  2011;6(7):e22052.
CC Chemokine Ligand 2 (CCL2) is a potent chemoattractant produced by macrophages and activated astrocytes during periods of inflammation within the central nervous system. Increased CCL2 expression is correlated with disease progression and severity, as observed in pulmonary tuberculosis, HCV-related liver disease, and HIV-associated dementia. The CCL2 distal promoter contains an A/G polymorphism at position -2578 and the homozygous -2578 G/G genotype is associated with increased CCL2 production and inflammation. However, the mechanisms that contribute to the phenotypic differences in CCL2 expression are poorly understood. We previously demonstrated that the -2578 G polymorphism creates a TALE homeodomain protein binding site (TALE binding site) for PREP1/PBX2 transcription factors. In this study, we identified the presence of an additional TALE binding site 22 bp upstream of the site created by the -2578 G polymorphism and demonstrated the synergistic effects of the two sites on the activation of the CCL2 promoter. Using chromatin immunoprecipitation (ChIP) assays, we demonstrated increased binding of the TALE proteins PREP1 and PBX2 to the -2578 G allele, and binding of IRF1 to both the A and G alleles. The presence of TALE binding sites that form inverted repeats within the -2578 G allele results in increased transcriptional activation of the CCL2 distal promoter while the presence of only the upstream TALE binding site within the -2578 A allele exerts repression of promoter activity.
PMCID: PMC3132772  PMID: 21760952
12.  Coreceptor and Cytokine Concentrations May Not Explain Differences in Disease Progression Observed in HIV-1 Clade A and D Infected Ugandans 
PLoS ONE  2011;6(5):e19902.
The use of cellular coreceptors and modulation of cytokine concentrations by HIV to establish a productive infection is well documented. However, it is unknown whether the expression of these proteins affects the course of HIV clade A and D disease, reported to have different progression rates.
Methodology/Principal Findings
We investigated whether the number of CD4+ T-cells expressing CCR5 or CXCR4, the density of these coreceptors and concentrations of specific immune proteins linked to HIV pathogenesis vary between individuals infected with HIV clade A or D. We undertook additional analyses stratifying participants by early (CD4>500 cells/µl) or late (CD4<200 cells/µl) disease stage. Whole blood samples were taken from 50 HIV-1 infected individuals drawn from cohorts in rural south-west Uganda. Late stage participants had less than half the number of CD4+/CCR5+ T-cells (p = 0.0113) and 5.6 times fewer CD4+/CXCR4+ cells (p<0.0001) than early stage participants. There was also a statistically significant difference in the density of CXCR4 on CD4+ cells between clade A and D infected early stage participants (142 [A] vs 84 [D]; p = 0.0146). Across all participants we observed significantly higher concentration of Th1 cytokines compared to Th2 (66.4 vs 23.8 pg/ml; p<0.0001). Plasma concentrations of IFNγ and IL-2 were 1.8 and 2.4 fold lower respectively in Late-D infected participants compared to Late-A participants. MIP-1β levels also decreased from 118.0 pg/ml to 47.1 pg/ml (p = 0.0396) as HIV disease progressed.
We observed specific alterations in the abundance of CD4+/CCR5+ and CD4+/CXCR4+ T-cells, and concentrations of immune proteins across different HIV clades and as infection progresses. Our results suggest that these changes are unlikely to explain the observed differences in disease progression between subtype A and D infections. However, our observations further the understanding of the natural progression of non-clade B HIV infection and how the virus adapts to exploit the host environment.
PMCID: PMC3104992  PMID: 21655330
13.  Management and Treatment of Temporomandibular Disorders: A Clinical Perspective 
A temporomandibular disorder (TMD) is a very common problem affecting up to 33% of individuals within their lifetime. TMD is often viewed as a repetitive motion disorder of the masticatory structures and has many similarities to musculoskeletal disorders of other parts of the body. Treatment often involves similar principles as other regions as well. However, patients with TMD and concurrent cervical pain exhibit a complex symptomatic behavior that is more challenging than isolated TMD symptoms. Although routinely managed by medical and dental practitioners, TMD may be more effectively cared for when physical therapists are involved in the treatment process. Hence, a listing of situations when practitioners should consider referring TMD patients to a physical therapist can be provided to the practitioners in each physical therapist's region. This paper should assist physical therapists with evaluating, treating, insurance billing, and obtaining referrals for TMD patients.
PMCID: PMC2813497  PMID: 20140156
Dentistry; Physical Therapy; Temporomandibular Disorders; Temporomandibular Joint
14.  A robust lentiviral pseudotype neutralisation assay for in-field serosurveillance of rabies and lyssaviruses in Africa 
Vaccine  2009;27(51):7178-7186.
The inflexibility of existing serological techniques for detection of rabies in surveillance constrains the benefit to be gained from many current control strategies. We analysed 304 serum samples from Tanzanian dogs for the detection of rabies antibodies in a pseudotype assay using lentiviral vectors bearing the CVS-11 envelope glycoprotein. Compared with the widely used gold standard fluorescent antibody virus neutralisation assay, a specificity of 100% and sensitivity of 94.4% with a strong correlation of antibody titres (r = 0.915) were observed with the pseudotype assay. To increase the assay's surveillance specificity in Africa we incorporated the envelope glycoprotein of local viruses, Lagos bat virus, Duvenhage virus or Mokola virus and also cloned the lacZ gene to provide a reporter element. Neutralisation assays using pseudotypes bearing these glycoproteins reveal that they provide a greater sensitivity compared to similar live virus assays and will therefore allow a more accurate determination of the distribution of these highly pathogenic infections and the threat they pose to human health. Importantly, the CVS-11 pseudotypes were highly stable during freeze–thaw cycles and storage at room temperature. These results suggest the proposed pseudotype assay is a suitable option for undertaking lyssavirus serosurveillance in areas most affected by these infections.
PMCID: PMC2789314  PMID: 19925950
Rabies virus; Lyssavirus; Africa; Pseudotype
15.  Emerging Technologies for the Detection of Rabies Virus: Challenges and Hopes in the 21st Century 
The diagnosis of rabies is routinely based on clinical and epidemiological information, especially when exposures are reported in rabies-endemic countries. Diagnostic tests using conventional assays that appear to be negative, even when undertaken late in the disease and despite the clinical diagnosis, have a tendency, at times, to be unreliable. These tests are rarely optimal and entirely dependent on the nature and quality of the sample supplied. In the course of the past three decades, the application of molecular biology has aided in the development of tests that result in a more rapid detection of rabies virus. These tests enable viral strain identification from clinical specimens. Currently, there are a number of molecular tests that can be used to complement conventional tests in rabies diagnosis. Indeed the challenges in the 21st century for the development of rabies diagnostics are not of a technical nature; these tests are available now. The challenges in the 21st century for diagnostic test developers are two-fold: firstly, to achieve internationally accepted validation of a test that will then lead to its acceptance by organisations globally. Secondly, the areas of the world where such tests are needed are mainly in developing regions where financial and logistical barriers prevent their implementation. Although developing countries with a poor healthcare infrastructure recognise that molecular-based diagnostic assays will be unaffordable for routine use, the cost/benefit ratio should still be measured. Adoption of rapid and affordable rabies diagnostic tests for use in developing countries highlights the importance of sharing and transferring technology through laboratory twinning between the developed and the developing countries. Importantly for developing countries, the benefit of molecular methods as tools is the capability for a differential diagnosis of human diseases that present with similar clinical symptoms. Antemortem testing for human rabies is now possible using molecular techniques. These barriers are not insurmountable and it is our expectation that if such tests are accepted and implemented where they are most needed, they will provide substantial improvements for rabies diagnosis and surveillance. The advent of molecular biology and new technological initiatives that combine advances in biology with other disciplines will support the development of techniques capable of high throughput testing with a low turnaround time for rabies diagnosis.
PMCID: PMC2745658  PMID: 19787037
16.  HIV blocking antibodies following immunisation with chimaeric peptides coding a short N-terminal sequence of the CCR5 receptor 
Vaccine  2008;26(45):5752-5759.
The chemokine receptor CCR5 is required for cellular entry by many strains of HIV, and provides a potential target for molecules, including antibodies, designed to block HIV transmission. This study investigates a novel approach to stimulate antibodies to CCR5. Rabbits were immunised with chimaeric peptides which encode a short fragment of the N-terminal sequence of CCR5, as well as an unrelated T cell epitope from Tetanus toxoid. Immunisation with these chimaeric peptides generates a strong antibody response which is highly focused on the N-terminal CCR5 sequence. The antibody to the chimaeric peptide containing an N-terminal methionine also recognises the full length CCR5 receptor on the cell surface, albeit at higher concentrations. Further comparison of binding to intact CCR5 with binding to CCR5 peptide suggest that the receptor specific antibody generated represents a very small fragment of the total anti-peptide antibody. These findings are consistent with the hypothesis that the N-terminal peptide in the context of the intact receptor has a different structure to that of the synthetic peptide. Finally, the antibody was able to block HIV infection of macrophages in vitro. Thus results of this study suggest that N-terminal fragments of CCR5 may provide potential immunogens with which to generate blocking antibodies to this receptor, while avoiding the dangers of including T cell auto-epitopes.
PMCID: PMC2670972  PMID: 18765264
CCR5; Antibody; Chimaeric peptide; Blocking antibodies
17.  Prep1/Pbx2 Complexes Regulate CCL2 Expression Through the −2578 Guanine Polymorphism 
Genes and immunity  2008;9(5):419-430.
CC-chemokine ligand 2 (CCL2) is the major chemoattractant protein that recruits monocytes to sites of inflammation and increased expression of CCL2 is associated with numerous inflammatory diseases including human immunodeficiency virus-associated dementia (HIV-D). The −2578 guanine polymorphism in the CCL2 promoter has been associated with increased expression of CCL2 as well as pathogenesis of HIV-D; however, the molecular mechanism of regulation is unknown. We propose a molecular model for −2578G regulated CCL2 expression in astrocytes, which are major producers of CCL2 in the brain. The −2578G polymorphism creates a consensus binding site for the transcriptional regulator Prep1, which along with binding partner Pbx2, preferentially binds the −2578G allele. CCL2 promoters harboring the G allele under unstimulated conditions exhibit a lower basal activity compared to the ancestral A allele. Upon IL-1β stimulation, Prep1/Pbx2 complexes maintain the ability to bind −2578G alleles, yet transcription levels from promoters that harbor the A allele or G allele are equally activated, suggesting that the −2578 region does not influence CCL2 transcription under pro-inflammatory conditions. Therefore, promoters that harbor the −2578G allele undergo a higher fold induction and by extension, individuals homozygous for −2578G would be expected to exhibit hyper-responsive CCL2 phenotypes during periods of inflammation.
PMCID: PMC2570563  PMID: 18480829
TALE; CCL2; SNP; Promoter; Prep1; Pbx2
18.  Duffy Antigen Receptor for Chemokines Mediates trans-Infection of HIV-1 from Red Blood Cells to Target Cells and Affects HIV-AIDS Susceptibility 
Cell host & microbe  2008;4(1):52-62.
Duffy antigen receptor for chemokines (DARC) expressed on red blood cells (RBCs) influences plasma levels of HIV-1-suppressive and proinflammatory chemokines such as CCL5/RANTES. DARC is also the RBC receptor for Plasmodium vivax. Africans with DARC −46C/C genotype, which confers a DARC-negative phenotype, are resistant to vivax malaria. Here, we show that HIV-1 attaches to RBCs via DARC, effecting trans-infection of target cells. In African Americans, DARC −46C/C is associated with 40% increase in the odds of acquiring HIV-1. If extrapolated to Africans, ∼11% of the HIV-1 burden in Africa may be linked to this genotype. After infection occurs, however, DARC-negative RBC status is associated with slower disease progression. Furthermore, the disease-accelerating effect of a previously described CCL5 polymorphism is evident only in DARC-expressing and not in DARC-negative HIV-infected individuals. Thus, DARC influences HIV/AIDS susceptibility by mediating trans-infection of HIV-1 and by affecting both chemokine-HIV interactions and chemokine-driven inflammation.
PMCID: PMC2562426  PMID: 18621010
19.  Investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison 
The Journal of General Virology  2008;89(Pt 9):2204-2213.
Cross-neutralization between rabies virus (RABV) and two European bat lyssaviruses (EBLV-1 and -2) was analysed using lentiviral pseudotypes as antigen vectors. Glycoprotein (G-protein) cDNA from RABV challenge virus standard-11 (CVS-11) and EBLV-1 and -2 were cloned and co-expressed with human immunodeficiency virus (HIV) or murine leukemia virus (MLV) gag–pol and packageable green fluorescent protein (GFP) or luciferase reporter genes in human cells. The harvested lentiviral (HIV) vector infected over 40 % of baby hamster kidney (BHK) target cells, providing high-titre pseudotype stocks. Tests on blinded antibody-positive (n=15) and -negative (n=45) sera, predetermined by the fluorescent antibody virus neutralization (FAVN) test approved by the World Health Organization (WHO) and Office International des Epizooties (OIE), revealed that the CVS-11 pseudotype assay had 100 % concordance with FAVN and strongly correlated with neutralization titres (r2=0.89). Cross-neutralization tests using sera from RABV-vaccinated humans and animals on pseudotypes with CVS-11, EBLV-1 and EBLV-2 envelopes showed that the relative neutralization titres correlated broadly with the degree of G-protein diversity. Pseudotypes have three major advantages over live-virus neutralization tests: (i) they can be handled in low-biohazard-level laboratories; (ii) the use of reporter genes such as GFP or β-galactosidase will allow the assay to be undertaken at low cost in laboratories worldwide; (iii) each assay requires <10 μl serum. This robust microassay will improve our understanding of the protective humoral immunity that current rabies vaccines confer against emerging lyssaviruses, and will be applicable to surveillance studies, thus helping to control the spread of rabies.
PMCID: PMC2886951  PMID: 18753230
20.  Thermodynamic Characterization of the Interaction between CAR/RXR and SRC-1 Peptide by Isothermal Titration Calorimetry† 
Biochemistry  2007;46(3):862-870.
The constitutive androstane receptor (CAR) enhances transcription of specific target genes that regulate several metabolic pathways. CAR functions as an obligate heterodimer with the retinoid-X-receptor (RXR). Also part of the active receptor complex is the steroid receptor coactivator-1 (SRC-1) which interacts with the receptor complex via specific receptor interaction domains (RIDs). A peptide derived from the SRC-1 RID2 is used to study the thermodynamic properties of the interaction with the CAR/RXR ligand binding domain (LBD) complex. In the absence of ligands for both CAR and RXR, coactivator peptide binding to the CAR/RXR heterodimer is characterized by favorable enthalpy change and unfavorable entropy change. The addition of the CAR agonist, TCPOBOP, increases affinity for coactivator by decreasing unfavorable entropy and increasing the favorable intrinsic enthalpy of the interaction. The RXR ligand, 9-cis RA, generates a second SRC-1 site on RXR and increases affinity by improving the entropic component of binding. There is an additional increase in affinity for one of the two sites in the presence of both ligands. The change in heat capacity (ΔCp) is also investigated. A twofold difference in ΔCp is observed between liganded and unliganded CAR/RXR. The observed thermodynamic parameters for SRC-1 peptide binding to liganded and apo CAR/RXR as well as the difference in the ΔCp data provide evidence that the apo CAR/RXR heterodimer is conformationally mobile. The more favorable enthalpic contribution for TCPOBOP-bound CAR/RXR indicates that pre-formation of the binding site improves the complementarity of the coactivator-receptor interaction.
PMCID: PMC2518310  PMID: 17223708

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