An inhibitory monoclonal anti-protein antibody and an anti-peptide antibody share an epitope on rat cytochrome P-450 enzymes CYP1A1 and CYP1A2.

A monoclonal antibody, 12/2/3/2, which was raised against purified rat CYP1A1 recognises specifically rat and mouse CYP1A1 and CYP1A2, but not any cytochrome P-450 present in hepatic microsomal fractions from rabbit, guinea pig, hamster or human. By comparing the primary sequences of cytochromes P-450 to which 12/2/3/2 does and does not bind, 10 possible locations for its epitope were found. Of these, one was extremely hydrophilic and, hence, predicted to be the most antigenic in the native protein. An antibody was produced against the synthetic peptide corresponding to this region (Gly-Arg-Asp-Arg-Gln-Pro-Arg-Leu: residues 356-363 and 350-357 of rat CYP1A1 and CYP1A2, respectively). The antibody bound to rat, mouse and hamster CYP1A1 and to rat and mouse CYP1A2, but did not bind to any protein present in hepatic microsomal fractions from the rabbit, guinea pig or human. The binding of the anti-peptide antibody to CYP1A1 or CYP1A2 was partially antagonised by the monoclonal antibody. However, whereas the monoclonal antibody inhibited both CYP1A1- (aryl hydrocarbon hydroxylase) and CYP1A2-(high-affinity phenacetin O-deethylase) dependent monooxygenase activity, the anti-peptide antibody was without effect on these activities. Antigen denaturation by 8 M urea or 0.05% (w/v) SDS had no effect on binding of the anti-peptide antibody to cytochrome P-450, whilst binding of the monoclonal antibody was reduced by more than 1000-fold. The anti-peptide antibody partially antagonised the binding of 12/2/3/2 to urea-denatured but not native cytochrome P-450. These data suggest that whilst the complete binding site for the monoclonal antibody is discontinuous, sufficient of the epitope is linear, so that when the antigen is denatured the monoclonal antibody is still able to bind and this binding is antagonised by the anti-peptide antibody. However, inhibition of catalytic activity by the monoclonal antibody must require binding to discontinuous residues.

Short synthetic peptides exploited for reliable and specific targeting of antibodies to the C-termini of cytochrome P450 enzymes.

An antibody was raised against a synthetic peptide (Ser-Glu-Asn-Tyr-Lys-Asp-Asn) corresponding to residues 290-296 of the cytochrome P450 enzyme, CYP1A2, of both rat and mouse. A cysteine residue attached to the N-terminus of the peptide during synthesis allowed coupling in a specific orientation via the thiol group to the carrier protein, keyhole limpet haemocyanin. Antiserum raised in rabbits bound specifically to CYP1A2 in the rat and mouse. To determine those amino acid residues involved in binding of the antibody, related peptides of various lengths were synthesised and the binding of the antibody was determined by an enzyme-linked immunosorbent assay. These studies show that the minimum epitope is the C-terminal tripeptide sequence, Lys-Asp-Asn. Other than in rat and mouse CYP1A2, this tripeptide is found as an internal sequence in a large number of proteins including bovine fibronectin, chicken gizzard myosin heavy chain, and the P450 enzymes, rabbit CYP3A6 and human CYP3A4, but the antibody did not bind to any of these proteins. However, the antibody did bind to yeast glucose-6-phosphate dehydrogenase in which the tripeptide sequence is the C-terminus. Antibodies raised against a truncated peptide (Tyr-Lys-Asp-Asn), representing the C-terminal half of the peptide, also bound to glucose-6-phosphate dehydrogenase, but failed to bind to CYP1A2; thus although the C-terminal region of the peptide 290-296 is strongly immunogenic, it appears that it is not this population of antibodies that binds to CYP1A2. As antibodies were found to bind strongly to the C-terminus of glucose-6-phosphate dehydrogenase, the C-termini of proteins as targets for anti-peptide antibodies were investigated further by immunising rabbits with four 5-residue peptides which represent the C-termini of the P450 enzymes, CYP1A1, CYP1A2, CYP2E1 and CYP2A6. The peptides were coupled to keyhole limpet haemocyanin through their N-termini via cysteine residues added to the sequences. All four antisera bound specifically to their respective target proteins, as demonstrated by immunoblotting using hepatic microsomal fractions from rat, rabbit and human. It is suggested that this method of antibody production could be of general use for the reliable production of antisera against proteins where their sequence at the C-terminus is known, and such antibodies can be highly specific as they do not bind to internal sequences.

Identification of the epitope of an anti-peptide antibody which binds to CYP1A2 in many species including man.

An anti-peptide antibody was raised against the sequence Thr-Gly-Ala-Leu-Phe-Lys-His-Ser-Glu-Asn-Tyr-Lys which occurs at positions 283-294 in the rat cytochrome P450 enzyme CYP1A2. Compared with its binding to the peptide used for immunization, the antibody bound with only slightly reduced affinity to the truncated peptides Thr-Gly-Ala-Leu-Phe-Lys-His-Ser and Leu-Phe-Lys-His-Ser. However, binding to the peptide Ser-Glu-Asn-Tyr-Lys-Asp-Asn, which overlaps with the C-terminal region of the immunizing peptide, was very low. Thus, a major epitope for the anti-peptide antibody is Leu-Phe-Lys-His-Ser, which corresponds to a region of CYP1A2 that is conserved in many species. The antibody was tested by immunoblotting for its ability to bind to hepatic microsomal fractions from a number of species. Where possible animals were treated with compounds which induce CYP1A2 and the results compared with those with untreated animals. It was found that the antibody bound to rat, mouse, rabbit, hamster, guinea pig, pig, marmoset monkey and human CYP1A2. No evidence was found for binding to dog CYP1A2. The region corresponding to the major epitope at residues 286-290 of rat CYP1A2 was identical in mouse, hamster, rabbit and human CYP1A2. The sequence of marmoset and guinea pig CYP1A2 are not known but are predicted to be very similar to the sequence in the rat. The lack of binding of the antibody to dog CYP1A2 may be explained by two differences in this region compared with rat CYP1A2. Maximum inhibition of CYP1A2 activity by this antibody, as measured by high-affinity phenacetin O-deethylase activity, was 20%. This is in contrast to a previously described anti-peptide antibody directed to an adjacent region which caused 65% inhibition of this activity. Thus, the edge of an inhibitory region on the surface of cytochrome P450 has been identified. The ability of the antibody to bind to CYP1A2 from a number of animals should make this antibody of use for studying the levels of CYP1A2 apoprotein in many species.

Identification of the epitope of a monoclonal antibody which binds to several cytochromes P450 in the CYP1A subfamily.

The monoclonal antibody, 3/4/2, which was raised against purified rat cytochrome P450 isoenzyme 1A1 (CYP1A1) binds to cytochromes P4501A in many species. It was shown by immunoblotting that the antibody binds to CYP1A1 in microsomal fractions prepared from rat, mouse, rabbit, hamster and human. The antibody also binds to cytochrome P450 isoenzyme 1A2 in microsomal fractions prepared from rabbit and human, but not rat or mouse. Using purified isoenzymes in an enzyme-linked immunosorbent assay it was found that the affinity of binding to the two rabbit hydrocarbon-inducible isoenzymes is reduced compared with that for rat CYP1A1. Binding is not affected by denaturation of the antigens. The effects of chemical and enzymatic treatments on rat CYP1A1 showed that the epitope contains a trypsin-sensitive site that includes arginine, but lacks lysine. The epitope does not contain methionine, cysteine, aspartic acid or glutamic acid residues. In addition, digestion of the protein with cyanogen bromide produces a fragment of Mr 20,000 which contains the antibody binding site. By comparing the cross-reactivity of the antibody with the primary structures of CYP1A1 and 1A2 from the rat, mouse, rabbit and human, and by considering the results of the chemical and enzymatic treatments, it was possible to deduce the likely location and structure of the binding site of 3/4/2 on members of the CYP1A subfamily. It is concluded that the epitope for this antibody is Phe-Arg-His-Ser-Ser-Phe, which lies at positions 380-385 in rat CYP1A1. Further, it is predicted from a model of the tertiary structure of eukaryotic cytochrome P450 that a part of this binding site lies within a helix in the native protein.

Porcine enteric spirochete infections in the UK: surveillance data and preliminary investigation of atypical isolates.

Investigations into the possible causes of colitis and typhlocolitis were carried out on 98 pig units in the United Kingdom between 1997 and 1999. Brachyspira pilosicoli was identified most commonly, occurring as the suggested primary agent in 18% of the outbreaks but forming part of mixed infections in another 24% of outbreaks. The equivalent figures for other bacterial pathogens were: B. hyodysenteriae, 13% and 16%; Lawsonia intracellularis, 10% and 15%: Salmonella species, 6% and 12%; and Yersinia species, 4% and 10%. Unclassified Brachyspira species of unknown pathogenicity were identified in 12% of outbreaks. The 24 unclassified isolates divided into three groups on the basis of their phenotypic characteristics. In addition, there were 50 atypical Brachyspira species isolates that showed differences between their phenotypic characteristics and genetic identity based on sequence analysis of a section of the 23S rDNA gene. Four representative atypical isolates were found to be pathogenic as a result of an experimental oral challenge study in pigs.

Satisfaction with care and the utilization of dental services at a neighborhood health center.

The purpose of this research was to develop further information relevant to the problem of utilization of dental services at neighborhood health centers. The research objectives were: (1) A descriptive assessment of the utilization trends of the population served by a neighborhood health center; (2) Some determination of any possible correlation between failed appointments and patients' satisfaction with dental care; and, (3) Some exploration of possible reasons for non-utilization of scheduled dental appointments by a sample of patients from the neighborhood health center who report a history of previous dental treatment. From dental clinic daily appointment sheets, an assessment of utilization trends of a neighborhood health center population for a seven-month period (jon trends of a neighborhood health center population for a seven-month period (June 1, 1973 to December 31,1973) was obtained. In addition, analyses of patient treatment records and information obtained from personal interviews with 40 individuals from the health center population facilitated the completion of the second and third research objectives. The results indicated: (1) Compared to the findings of earlier research,-3 the reported ratio of broken to total appointments scheduled for this study population was substantially lower; (2) There is a rather strong relation between satisfaction with care and the utilization of dental services at the neighborhood health center studied; (3) Satisfaction with care is dependent upon a number of factors; and (4) Reasons given for nonutilization of scheduled dental services are practical ones, of which some are also reflected in the reasons given ofr dissatisfaction with dental care.

Investigations into field cases of porcine colitis with particular reference to infection with Serpulina pilosicoli.

Investigations into the possible causes of colitis and typhlocolitis were carried out on 85 pig units in the United Kingdom between 1992 and 1996. Serpulina pilosicoli was identified most commonly, occurring as the suggested primary agent on 21 (25 per cent) of the units but forming part of mixed infections on another 23 (27 per cent) of the units, the main co-infections being Yersinia pseudotuberculosis (eight units), proliferative enteropathy (six units), Salmonella species (four units) or Serpulina hyodysenteriae (two units). 'Atypical' Serpulina species, S hyodysenteriae, Salmonella typhimurium, Y pseudotuberculosis and Lawsonia intracellularis (proliferative enteropathy) were the suggested primary agents on seven, six, four, four and three units, respectively. Various combinations of mixed infections involving the latter organisms and other possibly incidental agents were recorded on another 10 units. Investigations on a further six units failed to detect any recognised pathogens. On units where S pilosicoli was the suggested primary agent, pigs ranging between 20 to 40 kg (eight to 16 weeks of age), but occasionally up to 50 kg, had diarrhoea and grew poorly over a period of two to three weeks. The prevalence was estimated to be between 5 and 15 per cent in affected batches, with a mortality of approximately 1 per cent. The clinical signs usually developed seven to 14 days after the moving and mixing of pigs. At postmortem examination, affected pigs had liquid contents in their colon, which contained accumulations of mucus in some chronic cases. Gross and histological lesions of colitis were prominent in the mid-spiral region of the colon. In mixed infections with Y pseudotuberculosis, Salmonella typhimurium or S hyodysenteriae, lesions were more extensive and affected the caecum as well as the colon. In the colon, lesions of proliferative enteropathy were usually confined to the proximal half of the ascending spiral but mixed infection with S pilosicoli caused more extensive colitis. Mixed infections were reported to prolong the time taken for pigs to recover naturally and to have a more detrimental effect on growth rates than S pilosicoli infection alone. Despite the successful treatment of batches of pigs with tiamulin or lincomycin, S pilosicoli infection persisted as a chronic problem on many units, with diarrhoea and colitis in successive batches of pigs unless prophylactic medication was used.

Hospital CEOs, CFOs, and nurse executives: opportunities for a new alliance.

This article examines the involvement of Utah acute care hospital nurse executives (NEs) in financial management roles. The authors surveyed NEs and their career supporters and hinderers. Findings suggest that NFs: 1. lack financial management skills, support, involvement, and satisfaction; 2. recognize financial management's importance and desire to improve performance; and 3. consider chief executive officers (CEOs) as their major supporters and chief financial officers (CFOs) their major hinderers in financial management. These "supporters" and "hinderers" of NEs showed consensus regarding the primacy of NEs' leadership and patient advocacy roles. These findings contrast with major professional association policy directives and expert opinions that advocate expanded financial management roles for NEs that will enable them to fully realize their executive potential. CEOs are positioned to establish norms that balance the traditional leadership-patient advocacy roles of NEs with newer financial management roles. CEOs can offer NEs and CFOs opportunities to improve NEs' financial management participation and performance. CEOs can provide empowerment and encourage CFOs to offer NEs "power tools" (for example, information, expertise, resources, and support). The three groups, however, must negotiate reasonable expectations for NEs in financial management and adequate preparation for these consequent responsibilities. Together, CEOs, CFOs, and NEs can successfully take hospitals into the future by leading them in ongoing learning and change.

Enrollee satisfaction with HMOs and its relationship with disenrollment.

Relying on 1997 data from a universe of 740 HMOs, this study uniquely documented, from the perspective of health plan administrators, rates of enrollee satisfaction and disenrollments. On the basis of various reporting totals per variable or indicator, the average level of satisfaction was 83.9%; the average number of disenrollments was 20,996 per plan. Among different datasets, an average of 18.9% members disenrolled per plan; an average of 10.2% were voluntary disenrollments; and an average of 18.3% were involuntary disenrollments. Plans with higher satisfaction enrollees had predominantly lower disenrollment rates, more enrollees likely to recommend plans to family or friends, fewer older enrollees, fewer male enrollees, and higher overall plan performance. To enhance the gaining and retaining of enrollees, plan administrators should closely monitor the various dimensions of satisfaction, such as services complement, quality of care, administrative efficiency, care management, enrollees' complaints, plan performance, appointment convenience, and waiting times.

Job satisfaction of selected categories of Utah hospital managers.

In a survey of managers in Utah hospitals, 85 percent responded overall satisfaction with their jobs. Surprisingly, women in the survey reported significantly greater satisfaction with their jobs than other respondents.

An opportunity for HMOs to use marketing to increase enrollee satisfaction.

PURPOSE: To identify the combination of marketing components (i.e., service, price, access, and promotion) of commercial health maintenance organizations (HMOs) that are related to overall enrollee satisfaction. The researchers focus on factors that commercial HMOs control directly--specifically, health care organization and financing. DESIGN: Descriptive (mail order). METHODOLOGY: This study uses national data provided by a major health benefits consulting firm, which collected data from a 1997 calendar year mail survey of HMO administrators. The administrators responded to an extensive survey, which tapped selected HMO marketing-mix components and the percentage of surveyed members who indicated satisfaction with their HMOs. To test hypotheses, researchers treated marketing-mix components as independent variables and enrollee satisfaction as the dependent variable. PRINCIPAL FINDINGS: This study found statistically significant relationships between overall satisfaction and HMO providers' quality; access, particularly to specialists and out-of-network providers; waiting times for physician services; customer service; and disease prevention/health promotion programs. The researchers did not find significant relationships between overall satisfaction and accreditation by the National Committee for Quality Assurance (NCQA), the presence of physician gatekeepers, numbers of providers, or financial indicators. The relationship between overall satisfaction and utilization was mixed. This study's findings are largely consistent with the literature, consumer- and professional-group position papers, and the President's Advisory Commission on Consumer Protection and Quality in the Health Care Industry. CONCLUSIONS: HMOs can use marketing as a way to address problems and pursue opportunities identified by enrollees. As these findings demonstrate, certain features of HMO design are more appealing to patients. By focusing on these preferences, HMOs can adopt a responsive market orientation that gives rise to more effective marketing mixes and hence improves enrollee satisfaction. With improved satisfaction, enrollees generate less need for government intervention through regulation or legislation.

Room at the top?

Nurse executives have joined hospital administrative teams, but are they accepted as fully integrated team executives? Learn how nurse executives and their influential colleagues view integration and its influences.

Methods for optimizing revenue in rural hospitals.

Rural hospitals have been failing over the last two decades, and one of the biggest reasons has been lack of attention paid to detail and accuracy in the coding and pricing of services rendered. Most research that has explored the problems of coding accuracy and its impact on reimbursement has focused on coding by medical record professionals, but many coding procedures are performed by "front line" lower-level employees working in a hospital's laboratory, radiology department, pharmacy, or other ancillary service departments. This article explains how rural hospitals can optimize their reimbursement and adhere to Medicare/Medicaid and other third-party payer regulations by training coders properly and by reviewing their pricing policies to make sure that prices charged accurately reflect the true cost of services.

Pharmacokinetics and pharmacodynamics of GS-9350: a novel pharmacokinetic enhancer without anti-HIV activity.

GS-9350 is a new chemical entity under development as a potent, mechanism-based inhibitor of human cytochrome P450 3A (CYP3A) isoforms. Its intended use is to increase the systemic exposure of coadministered agents that are metabolized by CYP3A enzymes. Unlike ritonavir, which is in current clinical use for this purpose, GS-9350 is devoid of anti-HIV activity. The pharmacokinetics of GS-9350 and its efficacy in increasing systemic exposure of the probe CYP3A substrate midazolam were examined in a study involving single- and multiple-dose escalations of GS-9350 from 50 to 400 mg. Single-dose escalation from 50 to 400 mg resulted in a 164-fold increase in GS-9350 exposure, whereas multiple-dose escalation in the dosage range of 50-300 mg resulted in a 47-fold increase in exposure. GS-9350 potently inhibited midazolam apparent clearance (95% reduction), similar in effect to ritonavir 100 mg. GS-9350 was generally well tolerated at all doses, and there was no evidence of dose-limiting toxicity. Establishing proof-of-concept, GS-9350 is currently under phase II development as a potential alternative to ritonavir for use with antiretroviral agents (including the HIV integrase inhibitor elvitegravir) that are often prescribed along with a "booster" drug.