Potentiation of clopidogrel active metabolite formation by rifampicin leads to greater P2Y12 receptor blockade and inhibition of platelet aggregation after clopidogrel.

BACKGROUND: The thienopyridine P2Y(12) receptor antagonist clopidogrel reduces the risk of arterial thrombosis and individual pharmacodynamic responses to clopidogrel are believed to reflect the levels of active metabolite (AM) generated. Rifampicin increases the inhibitory effect of clopidogrel on platelet aggregation (PA). We studied the response to clopidogrel before and during administration of rifampicin in order to study the relationship between individual AM levels and P2Y(12) blockade. METHODS: Healthy volunteers received a 600-mg loading dose of clopidogrel followed by 75 mg daily for 7 days and, after a washout period and treatment with rifampicin [300 mg twice a day (b.i.d.)], received the same regimen of clopidogrel. Clopidogrel AM levels were determined over 4 h after the clopidogrel loading dose and unblocked P2Y(12) receptor number was assessed using a (33) P-2MeSADP binding assay. PA was measured by optical aggregometry with ADP and TRAP. RESULTS: Rifampicin enhanced clopidogrel AM production [area-under-the-curve (AUC): clopidogrel 89±22 ng h mL(-1) , clopidogrel+rifampicin 335±86 ng h mL(-1) , P<0.0001], and P2Y(12) blockade (unblocked receptors: clopidogrel 48±24, clopidogrel+rifampicin 4±2, P<0.0001) and reduced PA (5 µmol L(-1) ADP: clopidogrel 20±4, clopidogrel+rifampicin 5±2, P<0.01). Increasing numbers of unblocked receptors were required for an aggregation response with a decreasing concentration of ADP. PA induced by ADP 2 µmol L(-1) was particularly sensitive to low levels of receptor blockade. CONCLUSION: Potentiation of clopidogrel AM production by rifampicin leads to greater P2Y(12) blockade and consequently greater inhibition of PA. PA responses to low concentrations of ADP are more sensitive to P2Y(12) blockade.

Antibody prophylaxis and therapy against Nipah virus infection in hamsters.

Nipah virus (NiV), a member of the Paramyxoviridae family, causes a zoonotic infection in which the reservoir, the fruit bat, may pass the infection to pigs and eventually to humans. In humans, the infection leads to encephalitis with >40 to 70% mortality. We have previously shown that polyclonal antibody directed to either one of two glycoproteins, G (attachment protein) or F (fusion protein), can protect hamsters from a lethal infection. In the present study, we have developed monoclonal antibodies (MAbs) to both glycoproteins and assessed their ability to protect animals against lethal NiV infection. We show that as little as 1.2 mug of an anti-G MAb protected animals, whereas more than 1.8 mug of anti-F MAb was required to completely protect the hamsters. High levels of either anti-G or anti-F MAbs gave a sterilizing immunity, whereas lower levels could protect against a fatal infection but resulted in an increase in anti-NiV antibodies starting 18 days after the viral challenge. Using reverse transcriptase PCR, the presence of NiV in the different organs could not be observed in MAb-protected animals. When the MAbs were given after infection, partial protection (50%) was observed with the anti-G MAbs when the animals were inoculated up to 24 h after infection, but administration of the anti-F MAbs protected some animals (25 to 50%) inoculated later during the infection. Our studies suggest that immunotherapy could be used for people who are exposed to NiV infections.

Nipah virus: vaccination and passive protection studies in a hamster model.

Nipah virus, a member of the paramyxovirus family, was first isolated and identified in 1999 when the virus crossed the species barrier from fruit bats to pigs and then infected humans, inducing an encephalitis with up to 40% mortality. At present there is no prophylaxis for Nipah virus. We investigated the possibility of vaccination and passive transfer of antibodies as interventions against this disease. We show that both of the Nipah virus glycoproteins (G and F) when expressed as vaccinia virus recombinants induced an immune response in hamsters which protected against a lethal challenge by Nipah virus. Similarly, passive transfer of antibody induced by either of the glycoproteins protected the animals. In both the active and passive immunization studies, however, the challenge virus was capable of hyperimmunizing the vaccinated animals, suggesting that although the virus replicates under these conditions, the immune system can eventually control the infection.

Lingual thyroid--a threat to the airway.

The occurrence of a thyroid gland superficially placed on the pharyngeal portion of the tongue is rare, but poses problems to the patient and anaesthetist. This report describes a patient with a lingual thyroid and a history of problems associated with it that resulted in admission to the ICU and warnings about future intubation of the larynx. The patient underwent awake tracheal intubation using a standard fibreoptic assisted technique, and was advised that she purchase an appropriate Medic-Alert bracelet.

Characterization of a natural mutation in an antigenic site on the fusion protein of measles virus that is involved in neutralization.

Although measles virus is an antigenically monotypic virus, nucleotide sequence analysis of the hemagglutinin and nucleoprotein genes has permitted the differentiation of a number of genotypes. In contrast, the fusion (F) protein is highly conserved; only three amino acid changes have been reported over a 40-year period. We have isolated a measles virus strain which did not react with an anti-F monoclonal antibody (MAb) which we had previously shown to be directed against a dominant antigenic site. This virus strain, Lys-1, had seven amino acid changes compared with the Edmonston strain. We have shown that a single amino acid at position 73 is responsible for its nonreactivity with the anti-F MAb. With the same MAb, antibody-resistant mutants were prepared from the vaccine strain. A single amino acid change at position 73 (R-->W) was observed. The possibility of selecting measles virus variants in vaccinated populations is discussed.

Canine distemper virus DNA vaccination induces humoral and cellular immunity and protects against a lethal intracerebral challenge.

We have studied the immune responses to the two glycoproteins of the Morbillivirus canine distemper virus (CDV) after DNA vaccination of BALB/c mice. The plasmids coding for both CDV hemagglutinin (H) and fusion protein (F) induce high levels of antibodies which persist for more than 6 months. Intramuscular inoculation of the CDV DNA induces a predominantly immunoglobulin G2a (IgG2a) response (Th1 response), whereas gene gun immunization with CDV H evokes exclusively an IgG1 response (Th2 response). In contrast, the CDV F gene elicited a mixed, IgG1 and IgG2a response. Mice vaccinated (by gene gun) with either the CDV H or F DNA showed a class I-restricted cytotoxic lymphocyte response. Immunized mice challenged intracerebrally with a lethal dose of a neurovirulent strain of CDV were protected. However, approximately 30% of the mice vaccinated with the CDV F DNA became obese in the first 2 months following the challenge. This was not correlated with the serum antibody levels.

Antagonistic effects of FGF4 on BMP induction of apoptosis and chondrogenesis in the chick limb bud.

In an effort to define the roles of bone morphogenic proteins (BMPs) and fibroblast growth factors (FGFs) during chick limb development more closely, we have implanted beads impregnated with these growth factors into chick limb buds between stages 20 and 26. Embryos were sacrificed at the time the bone chondrocyte condensations first appear (stages 27-28). Implantation of beads containing BMPs at the earlier stages (20-22) caused apoptosis to occur, in the most severe cases leading to complete limb degeneration. Application of FGF4, either in the same, or in a different bead, prevented the BMP-induced apoptosis. We argue that the apoptosis observed on removal of the AER prior to stage 23 of development could be brought about by BMPs. The action of epithelial FGF in preventing BMP-mediated apoptosis in the mesenchyme would define a novel aspect of epithelial-mesenchymal interactions. Implanting the BMP4 beads into the core of the limb bud a day later (stages 25-26) caused intense chondrogenesis rather than apoptosis. FGF4 could again nullify this effect and by itself caused a reduction in bone size. This is the reverse of the functional relationship these growth factors have in mouse tooth specification (where it is BMP4 that inhibits the FGF8 function), and suggests that the balance between the effects of FGFs and BMPs could control the size of the chondrocyte precursor cell pool. In this way members of these two growth factor families could control the size of appendages when they are initially formed.

Inhibition of measles virus infection and fusion with peptides corresponding to the leucine zipper region of the fusion protein.

Measles virus (MV) infections are characterized by the induction of syncytia, i.e. the fusion of infected cells. Two MV proteins, the haemagglutinin (HA) and fusion (F) proteins, are involved in this process. Synthetic peptides representing two alpha-helical regions of the MV F protein were studied for their ability to inhibit MV fusion. A peptide corresponding to the leucine zipper region (amino acids 455-490) inhibited MV fusion, whereas a peptide to amino acids 148-177, corresponding to the amphipathic alpha-helix region, did not. Fusion inhibition was also obtained with vaccinia virus-expressed HA and F, a recent wild-type MV isolate and the closely related canine distemper virus, but not with mumps virus. The F455-490 peptide did not affect the synthesis of MV F or its transport to the cell membrane. Virus-cell attachment was unaffected, but haemolysis and virus entry into the cell were inhibited. In one-step growth curves the virus yield was unaffected.

Immunization with plasmid DNA encoding for the measles virus hemagglutinin and nucleoprotein leads to humoral and cell-mediated immunity.

We have evaluated the DNA vaccination strategy for measles virus (MV) hemagglutinin (HA) and nucleoprotein (NP) genes. Plasmids encoding either the MV, HA, or NP proteins inoculated intramuscularly into Balb/c mice induced both humoral and CTL class I restricted responses. Antibody responses were not increased by multiple inoculations. The major antibody isotype induced by both the HA and NP was IgG2a consistent with a Th1 response. In contrast, immunization with a plasmid which directed the synthesis of a partially secreted form of HA gave mainly IgG1 antibodies. When the amount of DNA was reduced for the HA plasmid (1 or 10 microg/animal), although the antibody was not induced, a CTL response was observed.

Analysis of the contribution of CTL epitopes in the immunobiology of morbillivirus infection.

In Balb/c (H-2d) mice, the nucleoprotein (NP) of measles virus (MV) induces a MHC class I restricted-CTL response to a single 9-amino-acid epitope (aa 281--289). This L(d)-restricted epitope is also present in the NP of the closely related canine distemper virus (CDV). To investigate whether this epitope is immunologically effective when it is present within the primary sequence of a nonviral protein, we have incorporated the 281--289 motif into the human CD36 protein. When cells are infected with vaccinia virus (VV) recombinants expressing this protein, CD36NP, the MV epitope is correctly processed and the cells are lysed by MVNP-specific CTLs. In vivo, VV-CD36NP induced CTLs which protected mice from a lethal dose of CDV, but did not block virus replication. The MVNP contains four other potential L(d)-restricted motifs. To investigate if these could be utilized in the absence of the dominant epitope, a mutant NP was produced in which one of the anchor residues in the aa 281--289 motif was mutated. Cells infected with a VV recombinant expressing this protein (VV-NP F289S) were only poorly lysed by MVNP-specific CTLs. Similarly, immunization of Balb/c mice with VV-NP F289S induced a lower level of CTL activity compared to the VV-NP, but the activity was now directed to three other epitopes. When mice were vaccinated with VV-NP F289S they were only partially protected from a lethal CDV challenge. The significance of these results for MV vaccine development is discussed.

Mode of entry of morbilliviruses.

The morbilliviruses have a restricted host range. This is probably dependent on the use of specific host cell receptors. In the present article, we have reviewed our approach to identify a host cell receptor for one of the morbilliviruses, measles virus and to elucidate the interaction between viral and cellular proteins during virus entry into the host cell.

Measles virus fusion: role of the cysteine-rich region of the fusion glycoprotein.

Measles virus (MV) fusion requires the participation of both the fusion (F) and hemagglutinin (H) glycoproteins. The canine distemper virus fusion protein (CDVF) cannot substitute for the measles virus fusion protein (MVF) in this process. Introduction of restriction enzyme sites into the cDNAs of CDVF and MVF by site-directed mutagenesis facilitated the production of chimeric F proteins which were tested for their capacity to give fusion when coexpressed with MVH. Fusion resulted when the amino-terminal half of the MVF cysteine-rich region was transferred to CDVF.

Establishment and characterisation of murine cells constitutively expressing the fusion, nucleoprotein and matrix proteins of measles virus.

To advance our understanding of the immunobiology of measles virus (MV) infections, we have investigated the possibility of establishing cell lines constitutively expressing the individual MV antigens. In contrast to previously published studies, we show that it is possible to establish cell lines expressing high levels of fusion (F), nucleoprotein (NP) and matrix (M) MV proteins. Once cloned, the cell lines were stable with high levels of expression for more than six months. The size and cell distribution of the NP and F proteins were similar to those observed in MV- or vaccinia-MV recombinant-infected cells. In contrast, the distribution of the M protein, although being similar to that of MV-infected cells, differed from that of Vaccinia-M recombinant virus-infected cells. Preliminary results suggest that these cell lines will be useful tools for studying the contribution of individual MV antigens to the cell-mediated immune response to this virus.

A leucine zipper structure present in the measles virus fusion protein is not required for its tetramerization but is essential for fusion.

The biological role of a leucine zipper motif present in the measles virus fusion (F) protein has been investigated. This motif is present in all paramyxovirus F proteins, all coronavirus spike proteins and many if not all retrovirus envelope proteins. By analogy to its role in certain transcription factors, it has been suggested that the motif may be responsible for the oligomerization of these viral membrane proteins. In this study, one, two or four heptadic leucines in the motif were substituted using site-directed mutagenesis. We found that fusion is prevented when all four heptadic leucines present in the motif are mutated whereas cellular transport and the oligomeric state of the F protein are unaffected.

Characterization of an R-binding site mediating the R-induced activation of the Epstein-Barr virus BMLF1 promoter.

In cells latently infected with Epstein-Barr virus, the switch from latency to productive infection is linked to the expression of two Epstein-Barr virus transcription factors called EB1 and R. R is an enhancer factor, and an R-responsive element (RRE) has been identified in the BMLF1 promoter. In this study, we have used bidirectional deletion mutagenesis to delineate the BMLF1 RRE (RRE-M) to a 44-bp sequence. We also show that R expressed from a recombinant vaccinia virus protects RRE-M against digestion by DNase I. Using mobility shift assays and dimethyl sulfate interferences, we have characterized the contact points between in vitro-translated R and the DNA. R binds in vitro to one site by simultaneously contacting two sequences within the site, which are separated by 8 bp: 5'-catGTCCCtctatcatGGCGCagac-3'. Site-directed mutagenesis of this sequence completely impaired the binding of R in vitro and rendered the BMLF1 promoter nonresponsive to R. The results suggest that the R-inducible BMLF1 enhancer is composed of a single R-binding site, called RRE-M.

Measles virus: both the haemagglutinin and fusion glycoproteins are required for fusion.

Vaccinia-measles recombinant viruses were used to examine the contribution of the individual measles virus glycoproteins in fusion. Although vaccinia virus recombinants expressing either the haemagglutinin or fusion proteins did not induce fusion in the cell lines examined, a double recombinant expressing both measles virus glycoproteins gave extensive syncytia in cells of human and simian origin. No fusion was observed in mouse, hamster or chicken cells. The fusion induced by the double recombinant could be specifically inhibited with either anti-fusion or anti-haemagglutinin monoclonal antibodies.

Characterization of an FMN-containing cyclohexanone monooxygenase from a cyclohexane-grown Xanthobacter sp.

A soluble cyclohexanone monooxygenase was purified 16.1-fold to homogeneity from a Xanthobacter sp. grown upon cyclohexane as sole source of carbon and energy. The native enzyme is a 50-kDa single polypeptide chain associated with FMN rather than FAD as flavin prosthetic group in a 1:1 stoichiometric relationship. The monooxygenase catalyses the transformation of cyclohexanone to the lactone 1-oxa-2-oxocycloheptane in an oxygen ring insertion reaction. Only related cycloalkanone substrates are accepted for oxygenation, no activity is shown towards straight-chain alkanones. Enzyme activity is strongly inhibited by sulphydryl-reactive agents, but is relatively insensitive to metal chelators, electron transport inhibitors and the metal ions Fe3+ and Cu2+. Cyclohexanone monooxygenase has Km values for cyclohexanone and NADPH of less than 0.5 microM and 12.5 microM respectively. Kinetic investigations under steady-state conditions demonstrate that the flavoprotein prosthetic group, FMN, is involved in the monooxygenase catalytic mechanism. The systematic name for the enzyme is cyclohexanone, NADPH:oxygen oxidoreductase (6-hydroxylating, 1,2-lactonizing) (EC 1.14.13.22).

Expression of measles virus nucleoprotein in Escherichia coli: use of deletion mutants to locate the antigenic sites.

Three distinct antigenic determinants on the nucleoprotein (NP) of measles virus were localized. These epitopes were defined by three monoclonal antibodies, one of which recognized all measles virus field strains examined, whereas the other two were variable. A measles virus NP cDNA subclone representing 502 of the 525 amino acids was cloned into a bacterial expression vector plasmid (pRIT) and expressed as a Protein A-NP fusion protein in Escherichia coli. The expressed protein reacted with all three monoclonal antibodies. A series of NP gene deletion was constructed in order to locate the antigenic sites. The antigenic site identified on all measles virus strains studied, which was designated site I, was located between amino acids 122 and 150. The two variable epitopes were located at the C terminus of the protein (site II at 457 to 476; site III at 519 to 525). The structural and biological implications of these observations are discussed.