Stakeholder analysis for the development of a community pharmacy service aimed at preventing cardiovascular disease.

BACKGROUND: Participatory approaches involving stakeholders across the health care system can help enhance the development, implementation and evaluation of health services. These approaches may be particularly useful in planning community pharmacy services and so overcome challenges in their implementation into practice. Conducting a stakeholder analysis is a key first step since it allows relevant stakeholders to be identified, as well as providing planners a better understanding of the complexity of the health care system. OBJECTIVES: The main aim of this study was to conduct a stakeholder analysis to identify those individuals and organizations that could be part of a leading planning group for the development of a community pharmacy service (CPS) to prevent cardiovascular disease (CVD) in Australia. METHODS: An experienced facilitator conducted a workshop with 8 key informants of the Australian health care system. Two structured activities were undertaken. The first explored current needs and gaps in cardiovascular care and the role of community pharmacists. The second was a stakeholder analysis, using both ex-ante and ad-hoc approaches. Identified stakeholders were then classified into three groups according to their relative influence on the development of the pharmacy service. The information gathered was analyzed using qualitative content analysis. RESULTS: The key informants identified 46 stakeholders, including (1) patient/consumers and their representative organizations, (2) health care providers and their professional organizations and (3) institutions and organizations that do not directly interact with patients but organize and manage the health care system, develop and implement health policies, pay for health care, influence funding for health service research or promote new health initiatives. From the 46 stakeholders, a core group of 12 stakeholders was defined. These were considered crucial to the service's development because they held positions that could drive or inhibit progress. Secondary results of the workshop included: a list of needs and gaps in cardiovascular care (n = 6), a list of roles for community pharmacists in cardiovascular prevention (n = 12) and a list of potential factors (n = 7) that can hinder the integration of community pharmacy services into practice. CONCLUSIONS: This stakeholder analysis provided a detailed picture of the wide range of stakeholders across the entire health care system that have a stake in the development of a community pharmacy service aimed at preventing CVD. Of these, a core group of key stakeholders, with complementary roles, can then be approached for further planning of the service. The results of this analysis highlight the relevance of establishing multilevel stakeholder groups for CPS planning.

Sex-specific time trends in first admission to hospital for peripheral artery disease in Scotland 1991-2007.

BACKGROUND: This study examined trends for all first hospital admissions for peripheral artery disease (PAD) in Scotland from 1991 to 2007 using the Scottish Morbidity Record. METHODS: First admissions to hospital for PAD were defined as an admission to hospital (inpatient and day-case) with a principal diagnosis of PAD, with no previous admission to hospital (principal or secondary diagnosis) for PAD in the previous 10 years. RESULTS: From 1991 to 2007, 41,593 individuals were admitted to hospital in Scotland for the first time for PAD. Some 23,016 (55.3 per cent) were men (mean(s.d.) age 65.7(11.7) years) and 18,577 were women (aged 70.4(12.8) years). For both sexes the population rate of first admissions to hospital for PAD declined over the study interval: from 66.7 per 100,000 in 1991-1993 to 39.7 per 100,000 in 2006-2007 among men, and from 43.5 to 29.1 per 100,000 respectively among women. After adjustment, the decline was estimated to be 42 per cent in men and 27 per cent in women (rate ratio for 2007 versus 1991: 0.58 (95 per cent confidence interval 0.55 to 0.62) in men and 0.73 (0.68 to 0.78) in women). The intervention rate fell from 80.8 to 74.4 per cent in men and from 77.9 to 64.9 per cent in women. The proportion of hospital admissions as an emergency or transfer increased, from 23.9 to 40.7 per cent among men and from 30.0 to 49.5 per cent among women. CONCLUSION: First hospital admission for PAD in Scotland declined steadily and substantially between 1991 and 2007, with an increase in the proportion that was unplanned.

High-titer recombinant adeno-associated virus production from replicating amplicons and herpes vectors deleted for glycoprotein H.

Production of high-titer rAAV is essential for in vivo clinical application. One limiting factor may be the failure of existing systems to replicate the packaging genome in such a way that expression of Rep and Cap proteins is coordinately amplified. DISC-HSV (disabled single-cycle virus) is a genetically modified herpes simplex virus (HSV) that by deletion of glycoprotein H (gH) is infectious only if propagated in a complementing cell line. In this study, we have used DISC-HSV as a helper for rAAV replication, and have simulated to some extent the amplication of the rep and cap genomes seen in wtAAV infection by incorporating both these and vector sequences in HSV amplicons. Facilitated production of AAV Rep and Cap proteins translates into a considerably improved recovery of rAAV, which transduces cells of the neuroretina in vivo with high efficiency. The potential for contamination with infectious herpes particles is eliminated by the use of noncomplementing (gH-) cell lines to propagate the virus, and by standard purification methods. The use of DISC-HSV and herpes-derived amplicons for production of rAAV may be a useful strategy for future in vivo studies and for clinical application.

A genetically inactivated herpes simplex virus type 2 (HSV-2) vaccine provides effective protection against primary and recurrent HSV-2 disease.

A glycoprotein H (gH)-deleted herpes simplex virus type 2 (HSV-2) was evaluated as a vaccine for the prevention of HSV-induced disease. This virus, which we term a DISC (disabled infectious single cycle) virus, can only complete one replication cycle in normal cells and should thus be safe yet still able to stimulate broad humoral and cell-mediated antiviral immune responses. A gH-deleted HSV-2 virus that has been tested as a vaccine in the guinea pig model of recurrent HSV-2 infection was constructed. Animals vaccinated with DISC HSV-2 showed complete protection against primary HSV-2-induced disease, even when challenged 6 months after vaccination. In addition, the animals were almost completely protected against recurrent disease. Even at low vaccination doses, there was a high degree of protection against primary disease. A reduction in recurrent disease symptoms was also observed following therapeutic vaccination of animals already infected with wild type HSV-2.

Construction and characterisation of a recombinant vaccinia virus expressing human papillomavirus proteins for immunotherapy of cervical cancer.

The presence and consistent expression of the genes encoding the human papillomavirus (HPV) E6 and E7 proteins in the great majority of cervical tumours presents the opportunity for an immunotherapeutic approach for control of the disease. This report describes the construction and characterisation of a recombinant vaccinia virus designed to express modified forms of the E6 and E7 proteins from HPV16 and HPV18, the viruses most commonly associated with cervical cancer. The recombinant virus (designated TA-HPV) was based on the Wyeth vaccine strain of vaccinia, and was shown to express the desired gene products. Studies in mice indicated that the recombinant virus was less neurovirulent than the parental virus and was capable of inducing an HPV-specific CTL response. This pre-clinical evaluation has provided a basis for the initiation of human trials in cervical cancer patients.

Induction of a protective immune response by mucosal vaccination with a DISC HSV-1 vaccine.

The vaccine potential of a genetically disabled Herpes Simplex Virus Type 1 virus (DISC HSV-1) was investigated in the guinea pig model of intravaginal (i.vag.) HSV-2 infection. Three mucosal vaccination routes, i.vag., intranasal (i.n.) and oral, were compared for their ability to protect guinea pigs from challenge with wild-type HSV-2. Each was effective, particularly the i.n. route, which almost completely abolished primary disease. This was accompanied by significantly lower challenge virus titres in vaginal swabs collected from the vaccinated animals. In all cases, vaccination with the inactivated virus preparation provided substantially less protection from disease than the live DISC HSV-1 by the equivalent route. Antibody levels in serum and vaginal washes were measured both after vaccination and challenge by ELISA and neutralization tests. The highest titres were observed following administration of the DISC HSV-1 vaccine by the i.n. route. Significant increases in IgA and IgG in vaginal wash fluids were also found in these vaccinated animals.

A recombinant vaccinia virus encoding human papillomavirus types 16 and 18, E6 and E7 proteins as immunotherapy for cervical cancer.

BACKGROUND: Human papillomavirus (HPV) infection, especially with type 16 or 18, is associated with cervical cancer. Two HPV proteins, E6 and E7, are consistently expressed in tumour cells. The objectives of the study were to examine the clinical and environmental safety and immunogenicity in the first clinical trial of a live recombinant vaccinia virus expressing the E6 and E7 proteins of HPV 16 and 18 (TA-HPV). METHODS: The study was an open label phase I/II trial in eight patients with late stage cervical cancer. The patients were vaccinated with a single dose of TA-HPV and kept in strict isolation to monitor local and systemic side-effects, environmental spread, and anti-E6/E7 immune responses. FINDINGS: Vaccination resulted in no significant clinical side-effects and there was no environmental contamination by live TA-HPV. Each patient mounted an antivaccinia antibody response and three of the eight patients developed an HPV-specific antibody response that could be ascribed to the vaccination. HPV-specific cytotoxic T lymphocytes, the effector mechanism most likely to be of therapeutic benefit, were detected in one of three evaluable patients. INTERPRETATION: Further studies to investigate the use ot TA-HPV for immunotherapy of cervical cancer are warranted.

Inhibition of the influenza virus RNA-dependent RNA polymerase by antisera directed against the carboxy-terminal region of the PB2 subunit.

The influenza virus RNA polymerase consists of a heterotrimeric complex of the PB1, PB2 and PA proteins, with the PB2 subunit responsible for recognizing 5' cap structures on the host cell RNAs used as primers for virus mRNA synthesis. To investigate further the role PB2 plays in mRNA synthesis, a set of polyclonal antisera raised against defined regions of the protein were tested for their ability to inhibit the virion transcriptase. All five sera were of sufficient titre to immunoprecipitate PB2 and four were capable of recognizing polymerase complexes containing PB1 and PA. However, only the serum raised against the carboxy terminus of PB2 (F5) substantially inhibited polymerase activity. This serum drastically reduced synthesis primed by globin mRNA, but only partially inhibited transcription primed by the dinucleotide ApG, or ApG and cap analogue. The preferential inhibition of globin-primed synthesis did not result from interference with cap recognition, as serum F5 did not reduce labelling of PB2 in a photoaffinity cap-binding assay. However, IgG and Fab fragments from F5 were found to inhibit virion endonuclease activity. This suggests that the C terminus of PB2 plays a crucial role in transcription initiation and implicates PB2 in endonuclease activity.

Challenges and progress in developing herpesvirus vaccines.

The biological complexities of the human herpesviruses and the wide range of diseases that they cause present many difficulties for vaccine development. Until recently, progress towards this aim has been slow; however, advances in immunology and molecular biology have yielded an exciting array of new approaches for vaccination that have shown promise in model systems. This explosion in technology, together with renewed appreciation of the public-health benefits of vaccination, has sparked a resurgence of interest in the development of new vaccines and several are in, or near, clinical trials in humans. These look set to have a major impact on the incidence of herpesvirus diseases in the future.

Protective vaccination against primary and recurrent disease caused by herpes simplex virus (HSV) type 2 using a genetically disabled HSV-1.

The vaccine potential of a mutant herpes simplex virus (HSV) type 1, with a deletion in the glycoprotein H (gH) gene, was evaluated. The virus requires a gH-expressing cell line for multi-cycle growth but can complete a single cycle of infection in noncomplementing cells. Such viruses, termed DISC (disabled infectious single cycle) viruses, should be safe, yet still able to stimulate humoral and cell-mediated responses against a broad range of virus antigens in vaccinated hosts. Prophylactic vaccination of guinea pigs with DISC HSV-1, by ear scarification or direct infection of the vaginal mucosa, afforded a high degree of protection against HSV-2-induced primary genital disease and reduced significantly the frequency of subsequent disease recurrence. There was also a trend toward reduced recurrence following therapeutic vaccination of animals already infected with HSV-2. DISC HSV vaccination, therefore, offers an effective route for control of HSV disease.

Ribosomal pausing during translation of an RNA pseudoknot.

The genomic RNA of the coronavirus infectious bronchitis virus contains an efficient ribosomal frameshift signal which comprises a heptanucleotide slippery sequence followed by an RNA pseudoknot structure. The presence of the pseudoknot is essential for high-efficiency frameshifting, and it has been suggested that its function may be to slow or stall the ribosome in the vicinity of the slippery sequence. To test this possibility, we have studied translational elongation in vitro on mRNAs engineered to contain a well-defined pseudoknot-forming sequence. Insertion of the pseudoknot at a specific location within the influenza virus PB1 mRNA resulted in the production of a new translational intermediate corresponding to the size expected for ribosomal arrest at the pseudoknot. The appearance of this protein was transient, indicating that it was a true paused intermediate rather than a dead-end product, and mutational analysis confirmed that its appearance was dependent on the presence of a pseudoknot structure within the mRNA. These observations raise the possibility that a pause is required for the frameshift process. The extent of pausing at the pseudoknot was compared with that observed at a sequence designed to form a simple stem-loop structure with the same base pairs as the pseudoknot. This structure proved to be a less effective barrier to the elongating ribosome than the pseudoknot and in addition was unable to direct efficient ribosomal frameshifting, as would be expected if pausing plays an important role in frameshifting. However, the stem-loop was still able to induce significant pausing, and so this effect alone may be insufficient to account for the contribution of the pseudoknot to frameshifting.

Effects of charged amino acid mutations on the bimolecular kinetics of reduction of yeast iso-1-ferricytochrome c by bovine ferrocytochrome b5.

The reduction of wild-type yeast iso-1-ferricytochrome c (ycytc) and several mutants by trypsin-solubilized bovine liver ferrocytochrome b5 (cytb5) has been studied under conditions in which the electron-transfer reaction is bimolecular. The effect of electrostatic charge modifications and steric changes on the kinetics has been determined by experimental and theoretical observations of the electron-transfer rates of ycytc mutants K79A, K'72A, K79A/K'72A, and R38A (K' is used to signify trimethyllysine (Tml)). A structurally robust Brownian dynamics (BD) method simulating diffusional docking and electron transfer was employed to predict the mutation effect on the rate constants. A realistic model of the electron-transfer event embodied in an intrinsic unimolecular rate constant is used which varies exponentially with donor-acceptor distance. The BD method quantitatively predicts rate constants over a considerable range of ionic strengths. Semiquantitative agreement is obtained in predicting the perturbing influence of the mutations on the rate constants. Both the experimentally observed rate constants and those predicted by BD descend in the following order: native ycytc > K79A > K'72A > K79A/K'72A. Variant R38A was studied at a different ionic strength than this series of mutations, and the theory agreed with experiment in predicting a smaller rate constant for the mutant. In all cases the predicted effect of mutation was in the correct direction, but not as large as that observed. The BD simulations predict that the two proteins dock through essentially a single domain, with a distance of closest approach of the two heme groups in rigid body docking typically around 12 A. Two predominant classes of complexes were calculated, the most frequent involving the quartet of cytb5/ycytc interactions, Glu48-Arg13, Glu56-Lys87, Asp60-Lys86, and heme-Tml72, having an average electrostatic energy of -13.0 kcal/mol. The second most important complexes were of the type previously postulated (Salemme, 1976; Mauk et al., 1986; Rodgers et al., 1988) with interactions Glu44-Lys27, Glu48-Arg13, Asp60-Tml72, and heme-Lys79 and having an energy of -6.4 kcal/mol. The ionic strength dependence of the bimolecular reaction rate was well reproduced using a discontinuous dielectric model, but poorly so for a uniform dielectric model.

Internal entry of ribosomes on a tricistronic mRNA encoded by infectious bronchitis virus.

mRNA3 specified by the coronavirus infectious bronchitis virus appears to be functionally tricistronic, having the capacity to encode three small proteins (3a, 3b, and 3c) from separate open reading frames (ORFs). The mechanism by which this can occur was investigated through in vitro translation studies using synthetic mRNAs containing the 3a, 3b, and 3c ORFs, and the results suggest that translation of the most distal of the three ORFs, that for 3c, is mediated by an unconventional, cap-independent mechanism involving internal initiation. This conclusion is based on several observations. A synthetic mRNA whose peculiar 5' end structure prevents translation of the 5'-proximal ORFs (3a and 3b) directs the synthesis of 3c normally. Translation of 3c, unlike that of 3a and 3b, was insensitive to the presence of the 5' cap analog 7-methyl-GTP, and it was unaffected by alteration of the sequence contexts for initiation on the 3a and 3b ORFs. Finally, an mRNA in which the 3a/b/c infectious bronchitis virus coding region was placed downstream of the influenza A virus nucleocapsid protein gene directed the efficient synthesis of 3c as well as nucleocapsid protein, whereas initiation at 3a and 3b could not be detected. Expression of the 3c ORF from this mRNA, however, was abolished when the 3a and 3b coding region was deleted, indicating that 3c initiation is dependent on upstream sequence elements which together may serve as a ribosomal internal entry site similar to those described for picornaviruses.

Mutational analysis of the "slippery-sequence" component of a coronavirus ribosomal frameshifting signal.

The ribosomal frameshift signal in the genomic RNA of the coronavirus IBV is composed of two elements, a heptanucleotide "slippery-sequence" and a downstream RNA pseudoknot. We have investigated the kinds of slippery sequence that can function at the IBV frameshift site by analysing the frameshifting properties of a series of slippery-sequence mutants. We firstly confirmed that the site of frameshifting in IBV was at the heptanucleotide stretch UUUAAAC, and then used our knowledge of the pseudoknot structure and a suitable reporter gene to prepare an expression construct that allowed both the magnitude and direction of ribosomal frameshifting to be determined for candidate slippery sequences. Our results show that in almost all of the sequences tested, frameshifting is strictly into the -1 reading frame. Monotonous runs of nucleotides, however, gave detectable levels of a -2/+1 frameshift product, and U stretches in particular gave significant levels (2% to 21%). Preliminary evidence suggests that the RNA pseudoknot may play a role in influencing frameshift direction. The spectrum of slip-sequences tested in this analysis included all those known or suspected to be utilized in vivo. Our results indicate that triplets of A, C, G and U are functional when decoded in the ribosomal P-site following slippage (XXXYYYN) although C triplets were the least effective. In the A-site (XXYYYYN), triplets of C and G were non-functional. The identity of the nucleotide at position 7 of the slippery sequence (XXXYYYN) was found to be a critical determinant of frameshift efficiency and we show that a hierarchy of frameshifting exists for A-site codons. These observations lead us to suggest that ribosomal frameshifting at a particular site is determined, at least in part, by the strength of the interaction of normal cellular tRNAs with the A-site codon and does not necessarily involve specialized "shifty" tRNAs.

Identification of two new polypeptides encoded by mRNA5 of the coronavirus infectious bronchitis virus.

The second smallest subgenomic messenger RNA, mRNA5, of the coronavirus infectious bronchitis virus includes in its "5' unique region" two separate open reading frames (5a and 5b), whose coding function has not so far been established, and thus it may represent a dicistronic messenger RNA. We report here that two polypeptides with the sizes expected for the 5a and 5b products can be synthesised by in vitro translation of a single artificial mRNA containing both the 5a and 5b ORFs. To establish whether these polypeptides represent genuine virus gene products, both the 5a and 5b coding sequences were expressed as bacterial fusion proteins, and these were used to raise monospecific antisera. Antisera raised against both the 5a and 5b-specific sequences recognized specifically proteins of the expected size in infectious bronchitis virus-infected chicken kidney and Vero cells, indicating that 5a and 5b do represent genuine virus genes, and suggesting that mRNA5 is indeed functionally dicistronic.

Association of the infectious bronchitis virus 3c protein with the virion envelope.

A highly purified radiolabeled preparation of the coronavirus infectious bronchitis virus (IBV) was analyzed, by immunoprecipitation with monospecific antisera, for the presence of a series of small virus proteins recently identified as the products of IBV mRNAs 3 and 5. One of these, 3c, a 12.4K protein encoded by the third open reading frame of the tricistronic mRNA3 was clearly detectable and was found to cofractionate with virion envelope proteins on detergent disruption of virus particles. These results, together with the hydrophobic nature of 3c and its previously demonstrated association with the membranes of infected cells, suggest strongly that 3c represents a new virion envelope protein, which may have counterparts in other coronaviruses.

A polycistronic mRNA specified by the coronavirus infectious bronchitis virus.

The third largest of the nested set of subgenomic mRNAs (mRNA3) from the coronavirus infectious bronchitis virus (IBV) contains three separate open reading frames (3a, 3b, and 3c) which are not present on the next smallest of the mRNAs, suggesting that this mRNA may be functionally polycistronic. However, although a protein product has been identified from the 3c open reading frame, to date the coding function of 3a and 3b has not been established. We present nucleotide sequence data suggesting that each of the three open reading frames is conserved in a variety of different IBV strains and further show, through the preparation of monospecific antisera against bacterial fusion proteins, that IBV-infected cells contain small amounts of the products of these ORFs. In vitro translation studies using synthetic mRNAs containing the 3a, 3b, and 3c open reading frames suggest strongly that all three proteins can be translated from a single molecular species, and expression studies carried out in intact cells support this conclusion. Thus mRNA3 of IBV appears to be functionally tricistronic.

Mutational analysis of the RNA pseudoknot component of a coronavirus ribosomal frameshifting signal.

The genomic RNA of the coronavirus IBV contains an efficient ribosomal frameshift signal at the junction of the overlapping 1a and 1b open reading frames. The signal is comprised of two elements, a heptanucleotide "slip-site" and a downstream tertiary RNA structure in the form of an RNA pseudoknot. We have investigated the structure of the pseudoknot and its contribution to the frameshift process by analysing the frameshifting properties of a series of pseudoknot mutants. Our results show that the pseudoknot structure closely resembles that which can be predicted from current building rules, although base-pair formation at the region where the two pseudoknot stems are thought to stack co-axially is not a pre-requisite for efficient frameshifting. The stems, however, must be in close proximity to generate a functional structure. In general, the removal of a single base-pair contact in either stem is sufficient to reduce or abolish frameshifting. No primary sequence determinants in the stems or loops appear to be involved in the frameshift process; as long as the overall structure is maintained, frameshifting is highly efficient. Thus, small insertions into the pseudoknot loops and a deletion in loop 2 that reduced its length to the predicted functional minimum did not influence frameshifting. However, a large insertion (467 nucleotides) into loop 2 abolished frameshifting. A simple stem-loop structure with a base-paired stem of the same length and nucleotide composition as the stacked stems of the pseudoknot could not functionally replace the pseudoknot, suggesting that some particular conformational feature of the pseudoknot determines its ability to promote frameshifting.

Analysis of the invariant Phe82 residue of yeast iso-1-cytochrome c by site-directed mutagenesis using a phagemid yeast shuttle vector.

A phagemid (pING4) carrying the yeast iso-1-cytochrome c gene was constructed which bears all the elements necessary for replication in yeast and bacteria and may be converted into a single-stranded form of DNA for site-directed mutagenesis and nucleotide sequencing. The recombinant vector was used to create a complete set of 19 amino acid changes at position 82, a phylogenetically conserved phenylalanine residue in mitochondrial cytochrome c. All the different forms of cytochrome c were functional in vivo, based upon their ability to support respiration when the mutant proteins were expressed in a yeast strain (otherwise devoid of cytochrome c) grown on non-fermentable carbon sources, with only the strain containing the Cys82 variant having a substantially decreased growth rate. These results are interpreted in terms of the available structural and functional information previously reported on a subset of cytochrome c proteins with mutations at position 82.