Arbidol and Other Low-Molecular-Weight Drugs That Inhibit Lassa and Ebola Viruses.

Antiviral therapies that impede virus entry are attractive because they act on the first phase of the infectious cycle. Drugs that target pathways common to multiple viruses are particularly desirable when laboratory-based viral identification may be challenging, e.g., in an outbreak setting. We are interested in identifying drugs that block both Ebola virus (EBOV) and Lassa virus (LASV), two unrelated but highly pathogenic hemorrhagic fever viruses that have caused outbreaks in similar regions in Africa and share features of virus entry: use of cell surface attachment factors, macropinocytosis, endosomal receptors, and low pH to trigger fusion in late endosomes. Toward this goal, we directly compared the potency of eight drugs known to block EBOV entry with their potency as inhibitors of LASV entry. Five drugs (amodiaquine, apilimod, arbidol, niclosamide, and zoniporide) showed roughly equivalent degrees of inhibition of LASV and EBOV glycoprotein (GP)-bearing pseudoviruses; three (clomiphene, sertraline, and toremifene) were more potent against EBOV. We then focused on arbidol, which is licensed abroad as an anti-influenza drug and exhibits activity against a diverse array of clinically relevant viruses. We found that arbidol inhibits infection by authentic LASV, inhibits LASV GP-mediated cell-cell fusion and virus-cell fusion, and, reminiscent of its activity on influenza virus hemagglutinin, stabilizes LASV GP to low-pH exposure. Our findings suggest that arbidol inhibits LASV fusion, which may partly involve blocking conformational changes in LASV GP. We discuss our findings in terms of the potential to develop a drug cocktail that could inhibit both LASV and EBOV.IMPORTANCE Lassa and Ebola viruses continue to cause severe outbreaks in humans, yet there are only limited therapeutic options to treat the deadly hemorrhagic fever diseases they cause. Because of overlapping geographic occurrences and similarities in mode of entry into cells, we seek a practical drug or drug cocktail that could be used to treat infections by both viruses. Toward this goal, we directly compared eight drugs, approved or in clinical testing, for the ability to block entry mediated by the glycoproteins of both viruses. We identified five drugs with approximately equal potencies against both. Among these, we investigated the modes of action of arbidol, a drug licensed abroad to treat influenza infections. We found, as shown for influenza virus, that arbidol blocks fusion mediated by the Lassa virus glycoprotein. Our findings encourage the development of a combination of approved drugs to treat both Lassa and Ebola virus diseases.

Metabolite fingerprinting of barley whole seeds, endosperms, and embryos during industrial malting.

Samples of whole seeds, isolated endosperms including the aleurone layer and isolated embryos with attached scutellum from an industrial scale barley malting process (variety Braemar) were analysed for their water soluble metabolites by gas chromatography-mass spectrometry (GC-MS). 73 known metabolites and about 350 unknown signals were detected. Principal component analysis (PCA) showed a time dependent shift of sample profiles. Whole seeds and endosperm samples showed very similar patterns with nearly all compounds rising until the end of germination. In the embryos a maximum concentration of compounds was reached after 72-96 h of malting. Most concentrations decreased afterwards. The kilning step, namely the drying and roasting of germinated seeds, induced variable effects of increases, stability or decreases of metabolites and thereby separated kilned samples from germinated seeds in the PCA. A second barley cultivar (Quench) underwent the same malting and analysis procedures and gave nearly identical results. Fructose, malate, myo-inositol and raffinose exhibited the potential to serve as markers for specific developmental stages of seeds in both varieties. Biological markers represent targets for industrial process control. Their potential application would meet the maltsters' demand to flatten variances in germination properties and to produce equal composed malt by directed malting management.

Sensitivity of influenza viruses to zanamivir and oseltamivir: a study performed on viruses circulating in France prior to the introduction of neuraminidase inhibitors in clinical practice.

Influenza virus neuraminidase inhibitors (NAIs) were introduced in clinical practice in various parts of the world since 1999 but were only scarcely distributed in France. Prior to the generalization of zanamivir and oseltamivir utilization in our country, we decided to test a large panel of influenza strains to establish the baseline sensitivity of these viruses to anti-neuraminidase drugs, based upon a fluorometric neuraminidase enzymatic test. Our study was performed on clinical samples collected by practitioners of the GROG network (Groupe Régional d'Observation de la Grippe) in the south of France during the 2002-2003 influenza season. Out of 355 isolates tested in the fluorometric neuraminidase activity assay, 267 isolates could be included in inhibition assay against anti-neuraminidase drugs. Differences in IC50 range were found according to the subtype and the anti-neuraminidase drug. Influenza B and A/H1N1 viruses appeared to be more sensitive to zanamivir than to oseltamivir (mean B IC50 values: 4.19 nM versus 13 nM; mean H1N1 IC50 values: 0.92 nM versus 1.34 nM), while A/H1N2 and A/H3N2 viruses were more sensitive to oseltamivir than to zanamivir (mean H3N2 IC50 values: 0.67 nM versus 2.28 nM; mean H1N2 IC50 values: 0.9 nM versus 3.09 nM). Out of 128 N2 carrying isolates, 10 isolates had zanamivir or oseltamivir IC50 values in upper limits compared to their respective data range. Sequencing of the neuraminidase of these outliers N2 highlighted several mutations, but none of them were associated with resistance to neuraminidase inhibitors.

Neuraminidase assays.

Anti-neuraminidase (NA) antibodies (Ab) play a role in protection against influenza and in combination with anti-HA Ab they increase the protection in mice. To control the NA content of vaccines, which should improve vaccine standardisation and may benefit vaccine efficacy, a series of questions must be addressed: 1) The antigenic characterization of NA in vaccine strains and seed lots is based on the measurement of the enzymatic (E) activity using fetuin as substrate. The antigenic profile is established by inhibiting the E activity with post infectious ferret antisera. Overnight incubation ensures sensitivity, and fetuin substrate gives specificity by detection of variant specific antibodies. Several difficulties have to be overcome, such as the low level of E activity in MDCK grown viruses, and the lability of N1. 2) The NA protein content of the vaccines (in bulk or final product) can be measured by an ELISA capture test but the lability of the NA proteins at 4 degrees C must be checked. 3) The anti NA Ab response can be measured using a neuraminidase inhibition test. --The steric hindrance by HI antibodies does not exceed a titre of 20 in human sera. --Triton treatment of viruses reduces the steric hindrance in polyclonal sera and monoclonal antibodies but unmasks epitopes. 4) The correlations between neuraminidase inhibition, neutralization and protection, has been established in the mouse model, but remains to be shown in humans. 5) The use of a small fluorescent (MUN) or chemiluminescent (NA-STAR) substrate can be used for the rapid differentiation of N1 from N2 and NB, but not for the titration of protective NI antibodies.

Non-traumatic abdominal emergencies: imaging of acute intestinal obstruction.

The purpose of this paper is to give an overview of the main clinical questions in bowel obstruction, to discuss the value of various imaging modalities, including conventional radiography, ultrasound, and CT, to underline the impact of imaging in the management of patients with suspect bowel obstruction, and then to suggest a diagnostic triage in such patients.

Initial study of using a laminar fluid diffusion interface for sample preparation in high-performance liquid chromatography.

This report describes a new microfluidic device called the H Filter for sample preparation prior to HPLC. The H Filters make possible a diffusional transfer of an analyte from a sample stream into a stream of a "receiver" fluid. Existing mathematical models can be used for optimizing experimental conditions. The authors have selected the extraction of the antibiotic cephradine from blood to demonstrate the utility of the new device. The extracts of blood samples spiked with cephradine levels between 0.2 and 100 microg/ml were analyzed using a C8 reversed-phase column and UV detection at 260 nm. The HPLC results were in good agreement with theory. The recovery of 32.2+/-2.8% was uniform over the entire range of cephradine concentrations. The new method completely avoids the use of centrifuges, that is otherwise typical for most current methodologies for the preparation of blood samples prior to HPLC analysis.

[Acute appendicitis: to image or not to image?]. / Imagerie de l'appendicite: échographie, scanner ou rien du tout?

Acute appendicitis is frequently clinically suspected. However, about 50% of emergency room patients with such a diagnosis do not have acute appendicitis and between 20-25% of patients undergoing appendectomy based on clinical diagnosis have a normal appendix. On the other hand, if left untreated acute appendicitis may result in peritonitis. The purpose of this article is to review the indications for imaging patients with clinical suspicion of acute appendicitis, to describe the US and CT features of acute appendicitis, to review the advantages and limitations of US and CT, and to present the differential diagnosis to be considered in patients with right lower quadrant pain.

Expression of ACC oxidase genes differs among sex genotypes and sex phases in cucumber.

Ethylene has been implicated as a sex-determining hormone in cucumber: its exogenous application increases femaleness, and gynoecious genotypes were reported to produce more ethylene. In this study, three full-length ACC oxidase cDNAs were isolated from cucumber floral buds. RFLP analysis of a population that segregates for the F(femaleness) locus indicated that CS-ACO2 is linked to F at a distance of 8.7 cM. Expression of two of the genes, CS-ACO2 and CS-ACO3, was monitored in flowers, shoot tips and leaves of different sex genotypes. In situ mRNA hybridization indicated different patternsof tissue- and stage-specific expression of CS-ACO2 and CS-ACO3 in developing flowers. CS-ACO3 expression in mid-stage female flowers was localized to the nectaries, pistil and in the arrested staminoids, whereas CS-ACO2 transcript levels accumulated later and were found in placental tissue, ovary and staminoids. In male flowers, petals and nectaries expressed both genes, whereas ACO2 expression was strong in pollen of mature flowers. In young buds, strong expression was observed along developing vascular bundles. Four sex genotypes were compared for CS-ACO2 and CS-ACO3 expression in the shoot apex and young leaf. FF genotypes had higher transcript levels in leaves but lower levels in the shoot apex and in young buds, as compared to ff genotypes; the shoot-tip pattern is, therefore, inversely correlated with femaleness, and the possibility of a feedback inhibition mechanism underlying such correlation is discussed. The two CS-ACO genes studied displayed a differential response to ethrel treatment in different organs and sex genotypes, further demonstrating the complexity of the mechanisms controlling ethylene production during cucumber floral development.

Suitability of MDCK cells grown in a serum-free medium for influenza virus production.

MDCK cells have been adapted to grow in a serum-free environment using Ultra-MDCK medium (BioWHITTAKER). The growth of adapted U-MDCK cells was maintained for over a year without any reduction in growth rate or modification of cell karyotype; cells were scaled up to spinner culture using several microcarriers. The cells were shown to be a very good host for influenza A and B virus replication in both the presence and absence of trypsin in the infection medium. Trypsin-independent viruses replicated to high titres (10(7)-10(8) TCID50/ml) in U-MDCK cells, after selection through serial passages without trypsin. This virus progeny exhibited uncleaved and antigenically modified haemagglutinin compared with standard viruses grown with trypsin. Finally, large amounts of influenza A and B viruses were produced in U-MDCK cells grown on microcarriers under rod-stirred conditions using selected trypsin-independent variants.

Difficulties in standardizing the neuraminidase content of influenza vaccines.

To achieve better standardization of influenza vaccines, an ELISA immunocapture assay was developed for N2 neuraminidase quantification. This sensitive and highly specific assay was successfully applied to vaccine preparations produced in embryonated hens' eggs from 1992 to 1997 and to antigenically related viral suspensions produced in MDCK cells. A study of the neuraminidase activity of prototype A/H3N2 strains stored at 4 degrees C showed the gradual development of enzymatic instability from 1994 onwards, accompanied by antigenic modifications of the antigen. As the phenomenon was also more pronounced with the recombinant, the question arose of the standard of immunity provided when such viruses are used for vaccination. The antibodies inhibiting neuraminidase activity in vaccinated subjects were monitored in parallel using both complete virus and purified N2 NA. The study revealed the existence of an interference phenomenon which resulted in the titre of the N1 antibodies being overestimated. The interference was due to anti-HA antibodies impeding access to the substrate at the enzymatic site by steric hindrance.

Structural and chemical complementarity between antibodies and the crystal surfaces they recognize.

The sequences of the variable regions of three monoclonal antibodies with different specificities to cholesterol monohydrate and 1,4-dinitrobenzene crystals were determined. The structures of their binding sites were then modeled, based on homology to other antibodies of known structure. Two of these antibodies were previously shown to specifically recognize each one well-defined face of one of the crystals, out of a number of crystal faces of closely related structure. The binding site of the antibody which recognizes the stepped (301) face of the cholesterol crystal is predicted to assume the shape of a step with one hydrophobic and one hydrophilic side, complementary to the corresponding crystal surface. Within the step, the hydroxyl groups of five tyrosines are located such that they can interact with the hydroxyl and water molecules on the cholesterol crystal face, while hydrophobic contacts are made between the cholesterol backbone and hydrophobic amino acid sidechains. In contrast, the modeled binding site of the antibody which recognizes the flat (101) face of 1,4-dinitrobenzene crystals is remarkably flat. It is lined by aromatic and polar residues, that can make favorable contacts with the aromatic ring and nitro groups of the dinitrobenzene molecules, respectively.

A sensitive and specific ELISA immunocapture assay for rapid quantitation of influenza A/H3N2 neuraminidase protein.

Both HA and NA proteins elicit antibodies which have been shown to be capable of altering the course of infection. Nevertheless, while influenza virus vaccine standardization involves hemagglutinin (HA) and neuraminidase (NA) in terms of antigenic characterization, only HA protein quantitation is undertaken. An immunocapture ELISA (EIA) is described for N2 NA quantitation, based on the use of a highly specific monoclonal antibody (MAb) for capturing NA and an anti-NA antiserum for antigen detection. The amounts of NA in samples were deduced from the standard curve established by using purified NA. The NA-EIA is specific and detects as a little as 7 ng/ml. The capture and detector antibodies directed against A/Beijing/32/92 NA were shown to react with H3N2 prototype strains used in current influenza vaccines, provided that an antigenically matched reference NA is used as standard.

[Role of antineuraminidase antibodies in protection against influenza]. / Place des anticorps antineuraminidase dans la protection contre la grippe.

For improving the anti-influenza vaccination efficacy, the choice of strains carrying up dated neuraminidase antigen (NA) and the introduction of the optimal amount of NA antigen in the vaccine are critical. Monoclonal antibodies prepared against the neuraminidase N2 of A/Beijing/32/92 showed NA inhibition (NI) and neutralized (Nt) the cells infection by influenza virus either at an early stage (group 2 antibodies inhibit virus binding to cells) or at a late stage of infection (group 1 antibodies inhibit virus release). The specificity of the neutralization test is restricted to the homologous variant whereas the NI specificity is much broader. When both group 1 and group 2 antibodies are tested together, their neutralizing activity is significantly increased. The emergence in 1997 of an avian strain H5N1 in humans influenza infections at Hong Kong (Strain A/Hong-Kong/156/97) rose the threat of pandemic. The H5N1 strain carried H5 HA which is not recognized by the human immune system, but N1 might be related to other N1 antigens belonging to avian, swine and human strains. So we 1) characterized the N1 antigen from H5N1 in comparison with other known antigens, 2) we looked for anti N1 (H5N1) antibodies in humans according to the age and the vaccination status, 3) we checked the neutralizing activity of anti N1 antibodies. The N1 antigen (H5N1) appeared closely related to N1 from swine strains: Sw/31 correlated itself to the pandemic spanish virus (1918-19), and more recent swine isolates from 1982 and 1989. The anti N1 (H5N1) antibodies were present in sera collected from 75+ years old persons and these N1 antibodies were neutralizing H5N1 cells infection. Consequently, 75+ years old persons do not represent a priority group for vaccination in the case of H5N1 pandemic conditions.

Hybridoma growth in a new generation hollow fibre bioreactor: antibody productivity and consistency.

This paper analyses the performance of MAbMax(TM)/Tricentric(TM), a new generation hollow fibre bioreactor, for hybridoma growth and antibody productivity, the down stream processing of monoclonal antibody harvests throughout the run and the further control of antibody quality consistency. Handling and process parameters were optimised using a mouse hybridoma, IgG1(K) secretor, and then confirmed with several other hybridomas. Cells were kept at optimal viability during an unusually long period of time and a continuously high production of antibodies was detected over several months. Foetal bovine serum concentration was reduced to 1\% and the effects of weaning of cells from serum were monitored in terms of cell metabolism and antibody productivity. Antibody harvests collected at regular intervals throughout the run (2 to 12 weeks) were purified using affinity chromatography on a recombinant protein A/G matrix and then analysed in terms of antigen binding properties, isoelectric forms and oligosaccharide structures, in order 1) to control antibody quality consistency as a function of time and serum concentration and 2) to compare antibody characteristics as a function of culture conditions, in vitro bioreactor cultivation versus in vivo mouse ascite cultivation.

Monoclonal antibodies that specifically recognize crystals of dinitrobenzene.

Monoclonal antibodies have been elicited and selected after injection of crystals of 1,4-dinitrobenzene (1,4-DNB) and cholesterol monohydrate in mice. The reactivity of some of these antibodies to 1,4-DNB crystals, cholesterol monohydrate crystals, and other solid substrates has been characterized. Two of the antibodies selectively recognize 1,4-DNB crystal surfaces in an appropriately modified ELISA. They do not interact either with 1,4-DNB/BSA conjugates or with polystyrene and cholesterol monohydrate surfaces. They do interact with 1,2-DNB crystal surfaces, albeit with much lower reactivity. It is consequently suggested that these antibodies are not specific to the DNB molecule but rather to a repetitive motif of molecular moieties exposed at the crystal surfaces. Characterization of their binding regions may help to elucidate the interactions of antibodies with solid substrates and, in general, with antigens exposed on biological and artificial surfaces.

Monoclonal antibody recognition of cholesterol monohydrate crystal faces.

BACKGROUND: The immune system can elicit antibodies against a wide variety of antigens. We have proposed that crystal surfaces may also operate as antigens, binding specific antibodies. Here we exploit the crystal surfaces of cholesterol monohydrate to investigate antibody-surface recognition at the molecular level. RESULTS: Four monoclonal antibodies were selected. Two specifically interact with cholesterol monohydrate crystals, and one with 1,4-dinitrobenzene crystals. The fourth interacts nonselectively with various solid substrates. The relative reactivities of the four antibodies to the different surfaces of cholesterol monohydrate and to other surfaces were compared. The nonspecific antibody adsorbs mainly at imperfections. Of the two specific antibodies, one shows a clear preference for one set of faces, relative to others, the second adsorbs selectively at one face of cholesterol monohydrate crystals. CONCLUSIONS: Monoclonal antibodies can be selected that specifically bind to the crystal surfaces of cholesterol monohydrate. The binding sites of such antibodies appear to recognize a number of molecular moieties, exposed at the surface in a specific structural organization. Different antibodies recognize different structural organizations with varying degrees of selectivity. Antibody-crystal surface interactions may serve as convenient models for studies aimed at an understanding of the molecular bases of antibody recognition.

Simultaneous purification of influenza haemagglutinin and neuraminidase proteins by immunochromatography.

A new and rapid method for co-purification of haemagglutinin (HA) and neuraminidase (NA) proteins from influenza A/H3N2 viruses is described. Surface glycoproteins were first solubilized using a non-ionic detergent under high ionic strength conditions, then they were separated by chromatography on sepharose previously bound to monoclonal antibodies (MAbs) directed either against HA (IaH-chromatography) or against NA (IaN-chromatography). Depending on the protein specificity of the MAb immobilized on the column, HA or NA was bound to sepharose and the counterpart protein was free in the flow-through volume. IaH-chromatography and IaN-chromatography proved equally efficient in term of recoveries (> 75%) and purity (> or = 99%) of both HA and NA but differences appeared when considering functional and antigenic properties of pure proteins. Those properties were highly retained in IaH- and IaN-derived HA as well as in IaH-derived NA while IaN-NA was partially degraded. IaH-chromatography allowed the co-purification of HA and NA proteins in heterologous antigen-antibody system with a 50% rate of cross reactivity. IaH-HA and IaH-NA may be suitable for immunity studies, standardization of influenza vaccine and for diagnostic purposes.