Type II restriction endonucleases from Helicobacter pylori include an enzyme with a novel recognition sequence.

Type II restriction endonuclease activities of Helicobacter pylori strain Roberts and of the type strain H. pylori NCTC 11637 were detected and analysed by conventional techniques. The endonucleases were partially purified, their optima for activity and their recognition and cleavage sites were determined. H. pylori (Roberts) contained at least two enzymes: HpyBI was an isoschizomer of RsaI (GT/AC) and HpyBII was of a novel specificity (GTN/NAC). H. pylori NCTC 11637 was found to contain an isoschizomer of EcoRV (HpyCI: GAT/ATC) and at least one other enzyme which was too unstable to characterise.

Detection of heavy metal ions at femtomolar levels using protein-based biosensors.

Sensors based on proteins (GST-SmtA and MerR) with distinct binding sites for heavy metal ions were developed and characterized. A capacitive signal transducer was used to measure the conformational change following binding. The proteins were overexpressed in Escherichia coli, purified, and immobilized in different ways to a self-assembled thiol layer on a gold electrode placed as the working electrode in a potentiostatic arrangement in a flow analysis system. The selectivity and the sensitivity of the two protein-based biosensors were measured and compared for copper, cadmium, mercury, and zinc ions. The GST-SmtA electrodes displayed a broader selectivity (sensing all four heavy metal ions) compared with the MerR-based ones, which showed an accentuated selectivity for mercury ions. Metal ions could be detected with both electrode types down to femtomolar concentration. The upper measuring limits, presumably due to near saturation of the proteins' binding sites, were around 10(-10) M. Control electrodes similarly constructed but based on bovine serum albumin or urease did not yield any signals. The electrodes could be regenerated with EDTA and used for more than 2 weeks with about 40% reduction in sensitivity.

Oral administration of a prodrug of the influenza virus neuraminidase inhibitor GS 4071 protects mice and ferrets against influenza infection.

We have recently described GS 4071, a carbocyclic transition-state analog inhibitor of the influenza virus neuraminidase, which has potent inhibitory activity comparable to that of 4-guanidino-Neu5Ac2en (GG167; zanamivir) when tested against influenza A virus replication and neuraminidase activity in vitro. We now report that GS 4071 is active against several strains of influenza A and B viruses in vitro and that oral GS 4104, an ethyl ester prodrug which is converted to GS 4071 in vivo, is active in the mouse and ferret models of influenza virus infection. Oral administration of 10 mg of GS 4104 per kg of body weight per day caused a 100-fold reduction in lung homogenate viral titers and enhanced survival in mice infected with influenza A or B viruses. In ferrets, a 25-mg/kg dose of GS 4104 given twice daily reduced peak viral titers in nasal washings and eliminated constitutional responses to influenza virus infection including fever, increased nasal signs (sneezing, nasal discharge, mouth breathing), and decreased activity. Consistent with our demonstration that the parent compound is highly specific for influenza virus neuraminidases, no significant drug-related toxicity was observed after the administration of oral dosages of GS 4104 of up to 800 mg/kg/day for 14 days in nonclinical toxicology studies with rats. These results indicate that GS 4104 is a novel, orally active antiviral agent with the potential to be used for the prophylaxis and treatment of influenza A and B virus infections.

Identification of GS 4104 as an orally bioavailable prodrug of the influenza virus neuraminidase inhibitor GS 4071.

GS 4071 is a potent carbocyclic transition-state analog inhibitor of influenza virus neuraminidase with activity against both influenza A and B viruses in vitro. GS 4116, the guanidino analog of GS 4071, is a 10-fold more potent inhibitor of influenza virus replication in tissue culture than GS 4071. In this study we determined the oral bioavailabilities of GS 4071, GS 4116, and their respective ethyl ester prodrugs in rats. Both parent compounds and the prodrug of the guanidino analog exhibited poor oral bioavailability (2 to 4%) and low peak concentrations in plasma (Cmaxs; Cmax <0.06 microg/ml). In contrast, GS 4104, the ethyl ester prodrug of GS 4071, exhibited good oral bioavailability (35%) as GS 4071 and high Cmaxs of GS 4071 (Cmax = 0.47 microg/ml) which are 150 times the concentration necessary to inhibit influenza virus neuraminidase activity by 90%. The bioavailability of GS 4104 as GS 4071 was also determined in mice (30%), ferrets (11%), and dogs (73%). The plasma of all four species exhibited high, sustained concentrations of GS 4071 such that at 12 h postdosing the concentrations of GS 4071 in plasma exceeded those necessary to inhibit influenza virus neuraminidase activity by 90%. These results demonstrate that GS 4104 is an orally bioavailable prodrug of GS 4071 in animals and that it has the potential to be an oral agent for the prevention and treatment of influenza A and B virus infections in humans.

Efficacy of 2'-deoxy-2'-fluororibosides against influenza A and B viruses in ferrets.

Single-dose treatments (5 to 40 mg/kg of body weight given intraperitoneally) of ferrets with 2'-deoxy-2'-fluoroguanosine or its prodrug, 2,6-diamino-purine-2'-fluororiboside, 1 h after infection with influenza A virus significantly inhibited replication of virus in the upper respiratory tract, resulting in amelioration of fever and nasal inflammation. Replication of virus in the lower respiratory tract was also reduced > 100-fold, but three doses were required to prevent replication in the lungs. In ferrets infected with influenza B virus, single-dose treatment (40 mg/kg given intraperitoneally) produced a similar but reduced response in comparison with that in ferrets infected with influenza A virus, indicating that dosing was not optimal for this virus.

Virulence of rimantadine-resistant human influenza A (H3N2) viruses in ferrets.

The influence of rimantadine-resistance mutations on the virulence of human H3N2 viruses in ferrets was examined. The similarities in virulence of the drug-resistant mutants with single amino acid substitutions at three different locations, 27, 30, and 31, within the M2 sequence and their corresponding sensitive wild-type isolates contrasted with differences in virulence between the three pairs of viruses. These data provide further evidence that rimantadine-resistant viruses that emerge during treatment of patients with the drug are unaltered both in their growth characteristics and virulence.

Nature of the endogenous pyrogen (EP) induced by influenza viruses: lack of correlation between EP levels and content of the known pyrogenic cytokines, interleukin 1, interleukin 6 and tumour necrosis factor.

Fever in influenza results from the release of endogenous pyrogen (EP) following virus-phagocyte interaction and its level correlates with the differing virulence of virus strains. However, the different levels of fever produced in ferrets by intracardial inoculation of EP obtained from the interaction of different virus strains with ferret of human phagocytes did not correlate with the levels of interleukin 1 (IL-1), IL-6 or tumour necrosis factor in the same samples as assayed by conventional in vitro methods. Hence, the EP produced by influenza virus appears to be different to these cytokines.

Influenza virus enhancement of membrane leakiness induced by staphylococcal alpha toxin, diphtheria toxin and streptolysin S.

Release of alpha-amino[14C]isobutyric acid from ferret Mpf cells was promoted by staphylococcal alpha toxin, diphtheria toxin and streptolysin S. This release was enhanced to a significant extent if the cells had been previously infected with influenza virus strain A/Puerto Rico/8/34 (PR8, H1N1), although infection with virus alone did not increase the release of radiolabel as compared with that from untreated cells; inactivated virus had a similar enhancing action. The mechanism of enhancement is unclear but it occurs between 0.5 and 2h post-inoculation and viral membrane/endosome membrane fusion is essential. Endotoxin had no effect on membrane permeability, either alone or with PR8. The relevance of these in vitro observations to the previously observed enhancement of toxin lethality by influenza virus in vivo is discussed.

Exacerbation of bacterial toxicity to infant ferrets by influenza virus: possible role in sudden infant death syndrome.

Of several toxins examined, only staphylococcal alpha and gamma toxin, endotoxin, and diphtheria toxins were lethal for 5-day-old ferrets. Their toxicities were enhanced in animals infected at 1 day old with influenza virus, from 3-fold with staphylococcal gamma toxin through 14-fold for staphylococcal alpha toxin, 84-fold for endotoxin, and 219-fold for diphtheria toxin. No increased viral replication occurred in any tissue; thus the effects of the toxins were exacerbated by the infection, not vice versa. Neonates died suddenly without clinical symptoms as in human babies dying from the sudden infant death syndrome (SIDS). Pathologic examination showed inflammation in the upper respiratory tract, lung edema and collapse, and early bronchopneumonia in the toxin- and influenza virus-treated animals but not in those treated with toxin or virus alone. Thus, bacterial toxins could play a role in SIDS, this being more likely with a concomitant influenza virus infection.

Mechanism of immunity to influenza: maternal and passive neonatal protection following immunization of adult ferrets with a live vaccinia-influenza virus haemagglutinin recombinant but not with recombinants containing other influenza virus proteins.

Neonatal ferrets are protected against infection with influenza virus by milk-derived anti-influenza virus IgG after suckling on an immune mother. Live vaccines protect better than killed vaccines despite their stimulation of lower maternal haemagglutination-inhibiting antibody levels. This suggests that antibody to virus proteins other than the haemagglutinin may also be involved. To investigate this, adult ferrets were immunized intradermally with live vaccinia-influenza virus recombinants each expressing one of the 10 influenza virus polypeptides. Adult ferrets immunized with a recombinant expressing the H3 haemagglutinin were completely protected, and also passively protected their offspring, against a live challenge with clone 7a of the reassortant influenza virus A/Puerto Rico/8/34-A/England/939/69 (H3N2), immunity being mediated by IgG antibody. However, ferrets immunized similarly with recombinants expressing the H1 haemagglutinin, neuraminidase (N1 or N2), polymerases (PB1, PB2 or PAC), matrix protein (M1 or M2), nucleoprotein (NP) or non-structural proteins (NS1 or NS2) were completely susceptible to the influenza virus.

Role of upper respiratory tract infection in the deaths occurring in neonatal ferrets infected with influenza virus.

Passive immunization of ferret neonates by colostrally-derived anti-influenza virus IgG did not entirely prevent infection when mothers were immunized with 1 or 2 doses of formalin inactivated vaccine with adjuvant (alhydrogel). Influenza virus replication was almost completely prevented in the lower respiratory tract but only slightly reduced in the upper respiratory tract leading to deaths in about 50% of the neonates. Such neonates showed at most only minor lesions in the lower respiratory tract but moderate to severe inflammatory changes in the upper respiratory tract of most animals. This supports previous results suggesting that deaths, reminiscent of the human sudden infant death syndrome (SIDS), may arise purely as a result of upper respiratory tract infection, possibly following obstruction of the airways.

Role of milk-derived IgG in passive maternal protection of neonatal ferrets against influenza.

Neonatal ferrets are protected against infection with influenza virus by colostral and milk-derived anti-influenza virus IgG after suckling on an immune mother. The levels of IgG elicited and then transmitted to neonates were similar when mothers were immunized by either live infection or killed vaccines. Maternal anti-influenza virus IgA and IgM appears not to cross the neonatal gut epithelium although both are present in maternal serum and milk.

Production of passive immunity in neonatal ferrets following maternal vaccination with killed influenza A virus vaccines.

Neonatal ferrets may be passively immunized following maternal vaccination with formalin-inactivated influenza A virus vaccine, but the level of protection from partial to complete depends upon the number of doses used to vaccinate the mother, the presence or absence of aluminum hydroxide adjuvant, whether or not the mothers were 'primed' by prior infection with a serologically heterologous type A virus, and the age of the neonate at challenge. Neonates were completely protected up to 2 weeks of age, but susceptibility returned to nasal epithelium at 5 weeks and to lung at 7 weeks. Mothers immunized up to 9 months previously also partially or completely protected their offspring, this correlating with the maternal serum haemagglutination-inhibition (HI) antibody titre at the time of neonatal challenge, not the duration of immunity.

Uptake of zinc and fluoride by several dentin components.

The uptake of zinc released from ZOE and Dispersalloy, and fluoride from ChemFil by different components of dentin was studied in vitro. These materials were placed over a 500-micron layer of the dentin fraction in a simulated cavity, and the zinc and fluoride levels in the fraction and underlying solution determined after 7 days. Parallel studies were also carried out in which these components, alone in simulated cavities, were placed over solutions containing different concentrations of zinc and fluoride and the uptake determined after 24 h. Zinc uptake was considerably greater by the inorganic than the organic fractions used in this study. Uptake from ZOE was disproportionately higher than from Dispersalloy presumably reflecting the fact that the zinc in ZOE is more loosely bound. In addition the high zinc levels in collagen beneath this material may be attributed to the binding of the eugenol component. In contrast fluoride uptake was generally highest by the organic fractions with relatively low uptake by the inorganic fractions which is in contrast to previous studies. Uptake of zinc and fluoride from solution by these fractions was consistent with these findings. Thus zinc is more strongly bound by the inorganic and fluoride by the organic fractions of dentin.

An in vitro study of the role of dentine in moderating the cytotoxicity of zinc oxide eugenol cement.

The protective role of different dentine fractions and of dentine slices in moderating the cytotoxicity of zinc oxide eugenol (ZOE) was investigated. The collagen fraction of dentine powder provided increased protection over intact powder. Slices of dentine offered greater protection probably by providing a physical barrier to the diffusion of eugenol which may also bind to the contents of the dentine tubules. With increasing thickness of the dentine slices this protection was increased. ZOE stimulated calcium release from dentine but in view of the low levels attained it is unlikely that this process has any significant effect on the protective role of dentine.

Zinc release from dental restorative materials in vitro.

Zinc release from a range of dental restorative materials was measured in the absence and presence of dentin over a 2-week period. The presence of dentin was designed to simulate the residual dentin in vivo. There were considerable differences in both the levels and pattern of zinc release for the different materials. In several cases the concentrations exceeded the level which has been reported cytotoxic to cells in culture. With dentin, little zinc passed into solution, and high concentrations were measured in the dentin. This may explain the discrepancy between in vivo toxicity tests using zinc-containing materials and previous in vitro tests which have not included dentin in the test system.