Essential competencies in human resource management.
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Competency-Based Education↗
Biomedical subjects
J F Newman
Publications and source records attributed to J F Newman.
Managed care and ethical conflicts.
Managed care systems achieve efficiencies by rationing the resources used to deliver care. This rationing has led to widespread perceptions of patient abuse and neglect in the form of restricted access to care, denial of choice, and overall reduction in the quality of care delivered. Conflicts of interest and fundamental ethical principles are perceived as often compromised in the managed care environment. We argue that ethical issues are inherent in managed care and cannot be avoided. Recently, federal and state governments have passed legislation and regulations to deal with the issue.
Conflict of Interest↗
Delineation of a neutralizing subregion within the immunodominant epitope (GH loop) of foot-and-mouth disease virus VP1 which does not contain the RGD motif.
The major immunogenic site of foot-and-mouth disease virus (FMDV) is contained in a disordered loop comprising residues 134-158 of capsid protein VP1, located on the surface of the viral particle. Peptides corresponding to this sequence generally elicit protective levels of neutralizing antibodies in guinea pigs. In some instances, however, the level of neutralizing antibodies is low although the level of antibodies against the peptide, determined by ELISA, is as high as that in the sera with high neutralizing antibody titres. In an attempt to ascertain the reason for this difference, we have synthesized on a cellulose membrane 10 overlapping decapeptides, offset by one residue, covering the segment 141-159 of VP1 of two viruses belonging to serotypes A12 and O1, and tested them with guinea pig antisera raised against peptide 141-159, VP1 and FMDV particles (SPOTscan method). With type A, some peptides which were strongly positive with highly neutralizing antisera did not include the RGD triplet located at residues 145-147. In contrast, antisera with low neutralization titres reacted only with decapeptides which included the RGD motif. Moreover, peptide 147-156 coupled to keyhole limpet haemocyanin, but not peptide 141-149 coupled to the same carrier, elicited high levels of neutralizing antibodies in guinea pigs. In the case of serotype O, highly neutralizing antisera to virus reacted in ELISA with peptides 141-150 (containing the RGD motif) and 135-144 (located upstream from the RGD motif). The results suggest that the RGD triplet is not an indispensable constituent of peptides able to elicit a neutralizing antibody response against the virus.
Amino Acid Sequence↗
A universal virus inactivant for decontaminating blood and biopharmaceutical products.
Removal of virus infectivity from blood and biopharmaceutical products prepared from blood is an issue of considerable importance. Irrespective of the methods that are chosen it is vital that the biological activity of the product is not impaired. For blood and unfractionated plasma or serum, the problem is even more challenging. Selective inactivation of the genome is the key step in the preparation of killed virus vaccines. Imines have been used for more than 30 years for the preparation of inactivated foot-and-mouth disease virus vaccines without any evidence of survival of virus infectivity. Moreover, the immunogenicity of the virus is unimpaired. Viruses belonging to all the recognised families can be inactivated by imines. The biological properties of several proteins, including the cell growth-promoting factors in calf serum, are not impaired using conditions which ensure the inactivation of > 10(15) infectious units of poliovirus and foot-and-mouth disease virus (FMDV). Moreover, both viruses can be inactivated by imines at 4 degrees C, thus providing a method for removing infectivity from protein preparations which are unstable at higher temperatures. The mechanism by which FMDV is inactivated has been studied. We found that the RNA extracted from the virus after inactivation at 4 degrees C was not degraded and contained no hidden breaks but nevertheless was non-infectious. However, it could be amplified by PCR using primers corresponding to the gene coding for a portion of the viral RNA polymerase, but not from that coding for VP1, one of the structural proteins, showing that alteration of a base or bases had occurred in that region.
Animals↗
A universal virus inactivant for decontaminating blood and biopharmaceutical products.
Removal of virus infectivity from blood and biopharmaceutical products prepared from blood is an issue of considerable importance. For biopharmaceutical products, removal can usually be achieved by a series of fractionation steps or by inactivation with a suitable reagent. Irrespective of the methods that are chosen it is vital that the biological activity of the product is not impaired. For blood and unfractionated plasma or serum, the problem is even more challenging. Selective inactivation of the genome is the key step in the preparation of killed virus vaccines. Viruses belonging to all the recognised families can be inactivated by imines. In this paper it is shown that the biological properties of several proteins, including the cell growth-promoting factors in calf serum, are not impaired using conditions which ensure the inactivation of > 10(15) infectious units of poliovirus and foot-and-mouth disease virus (FMDV). Also shown is that both viruses can be inactivated by imines at 4 degrees C, thus providing a method for removing infectivity from protein preparations which are unstable at higher temperatures. The RNA extracted from FMDV inactivated at 4 degrees C was not degraded and contained no hidden breaks but nevertheless was non-infectious. However, it could be amplified by PCR using primers corresponding to the gene coding for a portion of the viral RNA polymerase, but not from that coding for VP1, one of the structural proteins, showing that alteration of a base or bases had occurred in that region. Surprisingly, it could be translated in the rabbit reticulocyte system although some of the products were different from those obtained with unmodified RNA.
Animals↗
Differentiating foot-and-mouth disease virus-infected from vaccinated animals with baculovirus-expressed specific proteins.
We had shown in preliminary studies with a small number of animals that antibodies against 2C could be detected in cattle and pigs which had been infected with FMDV but not in animals which had been vaccinated against the disease. To determine whether this test was generally applicable, sera from several hundred animals which had been vaccinated with different products in many countries have been tested in an ELISA using baculovirus expressed 2C. Our results show that only 1-2% of the sera gave a positive reaction by this method. In contrast, 100% of sera from convalescent animals gave a positive reaction. To be useful in differentiating between convalescent and vaccinated animals it is necessary to know how long these antibodies can be detected by our ELISA. We have determined the levels of antibodies against 2C and also other virus-specific proteins which are present in cattle and pigs following infection with FMDV. Our results show that levels of anti-3ABC antibodies could be detected by ELISA with baculovirus-expressed protein up to one year after infection. In contrast, the levels of anti-2C antibodies fell more rapidly than those against 3ABC indicating that the latter protein may be preferable for detecting convalescent animals. Nevertheless, we envisage that the final test format should include several virus-specific proteins to determine accurately the immune status of an animal.
Animals↗
A retro-inverso peptide corresponding to the GH loop of foot-and-mouth disease virus elicits high levels of long-lasting protective neutralizing antibodies.
Peptides corresponding to the immunodominant loop located at residues 135-158 on capsid protein VP1 of foot-and-mouth disease virus (FMDV) generally elicit high levels of anti-peptide and virus-neutralizing antibodies. In some instances, however, the level of neutralizing antibodies is low or even negligible, even though the level of anti-peptide antibodies is high. We have shown previously that the antigenic activity of peptide 141-159 of VP1 of a variant of serotype A can be mimicked by a retro-inverso (all-D retro or retroenantio) peptide analogue. This retro-inverso analogue induced greater and longer-lasting antibody titers than did the corresponding L-peptide. We now show that a single inoculation of the retro-inverso analogue elicits high levels of neutralizing antibodies that persist longer than those induced against the corresponding L-peptide and confer substantial protection in guinea pigs challenged with the cognate virus. In view of the high stability to proteases of retro-inverso peptide analogues and their enhanced immunogenicity, these results have practical relevance in designing potential peptide vaccines.
Amino Acid Sequence↗
Baculovirus expressed 2C of foot-and-mouth disease virus has the potential for differentiating convalescent from vaccinated animals.
Determining whether animals have been infected with foot-and-mouth disease virus or vaccinated is important because infected animals frequently become carriers of the virus, shed it intermittently and thus may be the source of new outbreaks of the disease. We had shown previously that the sera of convalescent animals contain antibodies to 2C, a highly conserved non-structural protein, whereas the sera of vaccinated animals do not. This is explained by observation that 2C is retained on the membranes of cells used for growing the virus for vaccine production. In contrast, the non-structural protein 3D, which is released into the medium, is not removed by centrifugation or filtration during vaccine production and therefore stimulates an immune response in both vaccinated and convalescent cattle. In this study we produced 2C and 3D in insect cells infected with recombinant baculoviruses. As demonstrated by serology and electron microscopy, 2C is also retained on the membranes of the insect cells. Both expressed proteins react with sera of convalescent animals, indicating that they are conformationally similar, but the 2C does not react with sera from vaccinated animals. The baculovirus expressed 2C appears to be a suitable antigen for the development of a reliable diagnostic test.
Animals↗
Foot-and-mouth disease virus and poliovirus particles contain proteins of the replication complex.
Nonstructural proteins 2C, 3CD, 3C, and 3D, and the cellular protein actin, are present in highly purified preparations of foot-and-mouth disease virus (FMDV) and poliovirus. They remain bound in variable amounts to the RNAs when the RNAs are extracted from the viruses with phenol or phenol-sodium dodecyl sulfate (SDS) and, for FMDV, when the RNA is released from the particles by a lowering of the pH below 7. RNA prepared by these methods is rapidly degraded at 37 degrees C, particularly in the presence of NH4+ ions, but hydrolysis can be prevented by antibody against Escherichia coli-expressed 3D, indicating that it is the RNA polymerase that has nuclease activity. In contrast, virion RNA from which the nonstructural proteins and actin have been removed by extraction with guanidine thiocyanate-phenol-chloroform or proteinase K-phenol is stable at 37 degrees C, although its specific infectivity is lower than that of the RNA extracted with phenol or phenol-SDS. The possible implications of the close association of replication complex proteins with the RNA in virus particles are discussed.
Animals↗
Absence of protein 2C from clarified foot-and-mouth disease virus vaccines provides the basis for distinguishing convalescent from vaccinated animals.
We have recently reported that cattle and pigs which have been vaccinated against foot-and-mouth disease can be distinguished from convalescent animals by the absence of antibodies to viral non-structural protein 2C (Lubroth and Brown, Res. Vet. Sci., 1995, 59, 70-78(1)). In this study, we show that the absence of 2C antibodies from the sera of vaccinated animals can be explained by the association of this viral protein with cellular debris which is separated from the virus harvest prior to inactivation of the supernatant for vaccine production. This serological marker can be of great value in countries where the disease occurs or in the veterinary regulatory arena when livestock are transported across borders, since it can be used to identify convalescent, persistently infected animals and vaccinates exposed to wild-type virus variants which have infected the vaccinated animals.
Animals↗
The cost of poor birth outcomes in employer-sponsored health plans.
OBJECTIVES: The authors examine the cost and incidence of poor birth outcomes in employer-sponsored health insurance plans. METHODS: An extensive study of national inpatient and outpatient claims data for prenatal, delivery, and postnatal care of nearly 59,000 mother-infant pairs was conducted. All maternal and infant costs incurred over a 2-year period were analyzed, and, furthermore, the longitudinal claims experience of a cohort of 20,000 mothers and infants was examined in detail. RESULTS: The study revealed that 25% of deliveries resulted in poor birth outcomes, which accounted for 40% of total costs over a 2-year period. Extrapolated nationwide, the net direct medical care cost of poor birth outcomes in employer plans has been estimated at approximately $5.6 billion for 1990, approximately 3% of aggregate after-tax corporate profits that year. CONCLUSIONS: Costs related to maternity and infant care are a major source of cost for employer-sponsored health insurance plans. Poor birth outcomes represent significantly higher cost for both the mother and infant at all stages of care-prenatal, at birth, and postnatal. To the extent that poor birth outcomes relate to maternal behavior and are preventable, their very high and protracted cost may justify substantial health promotion activity by employers and insurers.
Adult↗
Stabilising oral poliovaccine at high ambient temperatures.
The oral poliovaccine strains lose infectivity when they are stored above refrigerator temperatures. These losses increase rapidly at the high temperatures encountered in tropical countries, thus causing problems if the cold chain is inadequate. Attempts to minimise these losses have generally relied on the addition of salts such as MgCl2. In this paper we show that the fail in infectivity, which is due to the hydrolysis of the genomic RNA by the RNA polymerase within the virus particle, can be greatly inhibited by the suppression of this enzymatic activity. This can be achieved simply by reducing the pH below 7.
Capsid↗
Characterization of an acid-resistant mutant of foot-and-mouth disease virus.
A foot-and-mouth disease virus mutant which is stable at pH 6.4 has been isolated from a virus of serotype A. In contrast to the parent (P) virus, which gave a mixture of large and small plaques in BHK21 cells and in a bovine kidney cell line, the acid-resistant (AR) virus gave small plaques which did not increase markedly in size after 24 hr. The infectivity titer of the acid-resistant virus was about 100-fold lower in suckling mice than in BHK21 cells, whether the inoculation was made intraperitoneally or intracerebrally, whereas the parent virus gave similar titers in both systems. Furthermore, in mice the AR virus reached its end point two to three times more slowly. The diameter of the AR virus was almost 20% less than that of the P virus and it had a more distinct topography, but the two viruses cosedimented in sucrose gradients. However, the buoyant density in CsCl of the AR virus was slightly lower (1.42 compared with 1.43 g/cc) in coruns. The RNAs and capsid proteins of the two viruses gave similar profiles in sucrose gradients and by SDS-PAGE, respectively. However, isoelectric focusing of the capsid proteins revealed considerable differences between the two viruses. Whereas the P virus gave four protein bands, corresponding to VP1-VP4, the AR virus gave one band for VP4, two for VP3, two for VP2, and four for VP1. Sequence analysis of the genes coding for the capsid protein regions of the two viruses showed four changes (one silent), resulting in an Ala-3-->Ser substitution in VP1 and Glu-131-->Lys and Asp-133-->Ser substitutions in VP2.
Animals↗
Antigenic variants in a plaque-isolate of foot-and-mouth disease virus: implications for vaccine production.
The occurrence of many subtypes within a serotype of foot-and-mouth disease virus (FMDV) makes it difficult to control the disease by vaccination. Although inactivated vaccines are used successfully in many countries, the appearance in the field of antigenic variants against which the vaccines do not confer protection is a constant problem in vaccine manufacture. We had found previously a mixture of antigenic variants in a field isolate of serotype A12. In this report we demonstrate the presence of two variants in a plaque-isolate from this mixture. The second variant was detected only when the growth conditions were altered. Our observation points to the problems which may be encountered in the large scale growth of a virus for vaccine production.
Amino Acid Sequence↗
Stabilising oral poliovaccine at high ambient temperatures.
The oral poliovaccine strains lose infectivity when they are stored above refrigerator temperatures. These losses increase rapidly at the high temperatures encountered in tropical countries, thus causing problems if the cold chain is inadequate. Attempts to minimise these losses have generally relied on the addition of salts such as MgCl2. In this paper we show that the fall in infectivity, which is due to the hydrolysis of the genomic RNA by the RNA polymerase within the virus particle, can be greatly inhibited by the suppression of this enzymatic activity. This can be achieved simply by reducing the pH below 7.
DNA-Directed RNA Polymerases↗
Circular dichroism, molecular modeling, and serology indicate that the structural basis of antigenic variation in foot-and-mouth disease virus is alpha-helix formation.
Seven antigenic variants obtained from a single field isolate of foot-and-mouth disease virus, serotype A12, differ only at residues 148 and 153 in the immunodominant loop of viral protein VP1. Synthetic peptides corresponding to the region 141-160 are highly immunogenic. UV circular dichroism shows that (i) in aqueous solution the peptides are nearly identical, but in 100% trifluoroethanol they display helix-forming properties which correlate well with their serological crossreactivities for anti-peptide sera, and (ii) these properties are insensitive to substitutions at position 153, except for proline, but are highly sensitive to substitutions at position 148. This pattern can be explained by the effects of these substitutions on the amphiphilic character and positions of helices postulated in the region 146-156. Molecular models indicate that residues 147, 148, 150, 151, 153-155, and 157 are most likely to interact with residues of the antibody paratopes. The data are consistent with the existence of an inverse gamma-turn around Pro-153, and a beta-turn at the cell-attachment site at residues 145-147.
Amino Acid Sequence↗
Foot-and-mouth disease virus particles contain replicase protein 3D.
An antibody against the Escherichia coli-expressed RNA polymerase of foot-and-mouth disease virus (FMDV) reacts with the virus in ELISA and radioimmunoprecipitation experiments and with a protein of the disrupted virus particle in an immunoblot analysis. Treatment of the virus with trypsin, which cleaves capsid protein VP1 and a 56-kDa polypeptide present in trace amount in the particles, reduces the level of the reaction in ELISA and radioimmunoprecipitation and eliminates the immunoblot reaction. Electron microscopy showed that only approximately 20% of the virus particles reacted with the anti-polymerase antibody, whereas most reacted with an antibody against the immunodominant G-H loop of the virus. In the presence of ammonium ions, the expressed polymerase degrades the RNA of the virus into molecules sedimenting at approximately 12 S, indicating that it can act as a hydrolytic as well as a polymerizing enzyme. Moreover, the RNA in trypsin-treated virus particles is degraded when incubated at 37 degrees C, suggesting that the cleaved 56-kDa protein still possesses hydrolytic activity. In addition, the anti-polymerase antibody, which inhibits the polymerase activity of the E. coli-expressed protein, also partially inhibits the hydrolytic activity of the previously described endonuclease of the virus particle, suggesting that this enzyme is identical with the polymerase or forms part of it.
Antibodies, Viral↗
Function of minor polypeptides in foot-and-mouth disease virus and poliovirus.
Foot-and-mouth disease virus and poliovirus each contain several minor polypeptides, in addition to the four structural proteins. One of these, the viral RNA polymerase, can also act as a nuclease, hydrolysing the RNA and thus destroying viral infectivity. It is tightly bound to the RNA and may be the packaging signal for assembly of the particle.
Aphthovirus↗