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Biomedical subjects

J Braspenning

Publications and source records attributed to J Braspenning.

At least 19 recordsLinked to original sources

Substitution of doctors by nurses in primary care.

BACKGROUND: Demand for primary care services has increased in developed countries due to population ageing, rising patient expectations, and reforms that shift care from hospitals to the community. At the same time, the supply of physicians is constrained and there is increasing pressure to contain costs. Shifting care from physicians to nurses is one possible response to these challenges. The expectation is that nurse-doctor substitution will reduce cost and physician workload while maintaining quality of care. OBJECTIVES: Our aim was to evaluate the impact of doctor-nurse substitution in primary care on patient outcomes, process of care, and resource utilisation including cost. Patient outcomes included: morbidity; mortality; satisfaction; compliance; and preference. Process of care outcomes included: practitioner adherence to clinical guidelines; standards or quality of care; and practitioner health care activity (e.g. provision of advice). Resource utilisation was assessed by: frequency and length of consultations; return visits; prescriptions; tests and investigations; referral to other services; and direct or indirect costs. SEARCH STRATEGY: The following databases were searched for the period 1966 to 2002: Medline; Cinahl; Bids, Embase; Social Science Citation Index; British Nursing Index; HMIC; EPOC Register; and Cochrane Controlled Trial Register. Search terms specified the setting (primary care), professional (nurse), study design (randomised controlled trial, controlled before-and-after-study, interrupted time series), and subject (e.g. skill mix). SELECTION CRITERIA: Studies were included if nurses were compared to doctors providing a similar primary health care service (excluding accident and emergency services). Primary care doctors included: general practitioners, family physicians, paediatricians, general internists or geriatricians. Primary care nurses included: practice nurses, nurse practitioners, clinical nurse specialists, or advanced practice nurses. DATA COLLECTION AND ANALYSIS: Study selection and data extraction was conducted independently by two reviewers with differences resolved through discussion. Meta-analysis was applied to outcomes for which there was adequate reporting of intervention effects from at least three randomised controlled trials. Semi-quantitative methods were used to synthesize other outcomes. MAIN RESULTS: 4253 articles were screened of which 25 articles, relating to 16 studies, met our inclusion criteria. In seven studies the nurse assumed responsibility for first contact and ongoing care for all presenting patients. The outcomes investigated varied across studies so limiting the opportunity for data synthesis. In general, no appreciable differences were found between doctors and nurses in health outcomes for patients, process of care, resource utilisation or cost. In five studies the nurse assumed responsibility for first contact care for patients wanting urgent consultations during office hours or out-of-hours. Patient health outcomes were similar for nurses and doctors but patient satisfaction was higher with nurse-led care. Nurses tended to provide longer consultations, give more information to patients and recall patients more frequently than did doctors. The impact on physician workload and direct cost of care was variable. In four studies the nurse took responsibility for the ongoing management of patients with particular chronic conditions. The outcomes investigated varied across studies so limiting the opportunity for data synthesis. In general, no appreciable differences were found between doctors and nurses in health outcomes for patients, process of care, resource utilisation or cost. AUTHORS' CONCLUSIONS: The findings suggest that appropriately trained nurses can produce as high quality care as primary care doctors and achieve as good health outcomes for patients. However, this conclusion should be viewed with caution given that only one study was powered to assess equivalence of care, many studies had methodological limitations, and patient follow-up was generally 12 months or less. While doctor-nurse substitution has the potential to reduce doctors' workload and direct healthcare costs, achieving such reductions depends on the particular context of care. Doctors' workload may remain unchanged either because nurses are deployed to meet previously unmet patient need or because nurses generate demand for care where previously there was none. Savings in cost depend on the magnitude of the salary differential between doctors and nurses, and may be offset by the lower productivity of nurses compared to doctors.

Family Practice↗

Research methods used in developing and applying quality indicators in primary care.

Quality indicators have been developed throughout Europe primarily for use in hospitals, but also increasingly for primary care. Both development and application are important but there has been less research on the application of indicators. Three issues are important when developing or applying indicators: (1). which stakeholder perspective(s) are the indicators intended to reflect; (2). what aspects of health care are being measured; and (3). what evidence is available? The information required to develop quality indicators can be derived using systematic or non-systematic methods. Non-systematic methods such as case studies play an important role but they do not tap in to available evidence. Systematic methods can be based directly on scientific evidence by combining available evidence with expert opinion, or they can be based on clinical guidelines. While it may never be possible to produce an error free measure of quality, measures should adhere, as far as possible, to some fundamental a priori characteristics (acceptability, feasibility, reliability, sensitivity to change, and validity). Adherence to these characteristics will help maximise the effectiveness of quality indicators in quality improvement strategies. It is also necessary to consider what the results of applying indicators tell us about quality of care.

Consensus↗

A nuclear AAA-type ATPase (Rix7p) is required for biogenesis and nuclear export of 60S ribosomal subunits.

Ribosomal precursor particles are assembled in the nucleolus before export into the cytoplasm. Using a visual assay for nuclear accumulation of 60S subunits, we have isolated several conditional-lethal strains with defects in ribosomal export (rix mutants). Here we report the characterization of a mutation in an essential gene, RIX7, which encodes a novel member of the AAA ATPase superfamily. The rix7-1 temperature-sensitive allele carries a point mutation that causes defects in pre-rRNA processing, biogenesis of 60S ribosomal subunits, and their subsequent export into the cytoplasm. Rix7p, which associates with 60S ribosomal precursor particles, localizes throughout the nucleus in exponentially growing cells, but concentrates in the nucleolus in stationary phase cells. When cells resume growth upon shift to fresh medium, Rix7p-green fluorescent protein exhibits a transient perinuclear location. We propose that a nuclear AAA ATPase is required for restructuring nucleoplasmic 60S pre-ribosomal particles to make them competent for nuclear export.

Adenosine Triphosphatases↗

Low back pain in general practice: reported management and reasons for not adhering to the guidelines in The Netherlands.

BACKGROUND: Although guidelines for the management of low back pain have been published in the past decade, there is potential for further improvement in back pain care. AIM: To document the management of non-specific low back pain by general practitioners (GPs) in the Netherlands, to determine how this management of care is related to patient and physician factors, and to explore possible reasons for not adhering to the guidelines. METHOD: A prospective study was set up in which 57 GPs in 30 general practices completed a computerised questionnaire after each consultation for low back pain during a four-month period. RESULTS: Of 1640 back pain contacts, 1180 referred to non-specific low back pain. Diagnostic tests were ordered in 2% of first consultations and in 7% of follow-up consultations within one episode. The advice to stay active despite pain was given in 76% and 69% of these cases respectively. Patients were prescribed an analgesic in 53% and 41% of cases respectively (mainly NSAIDs [80%]). Patients were referred to a physiotherapist in 22% of first and in 50% of follow-up consultations. Older patients were physically examined less often, prescribed analgesics more often, and were told less often that staying active could benefit them. The advice to remain active was omitted more often when symptoms lasted longer. Only a small part of the variance in management was accounted for by patient characteristics or by differences between practices. CONCLUSION: The management of low back pain met the guidelines to a large extent. Management decisions were often related to characteristics in which the guidelines lack differentiation. Important reasons for non-adherence were perceived patients' preferences. Further implementation of guidelines will be difficult unless doctors' and patients' views are more explicitly known.

Adolescent↗

Kinetic selection of HPV 16 E6/E7-directed antisense nucleic acids: anti-proliferative effects on HPV 16-transformed cells.

The E6/E7-coding sequences of the human papillomavirus type 16 (HPV 16) were probed for kinetic accessibility in vitro by pools of catalytic antisense RNA. Only long-chain complementary RNA and very few antisense sequences with a 3' portion complementary to a 10 nt window within unspliced and spliced E6-coding target sequences showed fast annealing with k(ass) values of up to 10(4) M-1s-1 indicating that the majority of E6/E7 RNA sequences are unfavourable targets for antisense inhibitors and ribozymes. Fast-annealing antisense oligodeoxyribonucleotides directed against the window of 10 nt inhibited cell proliferation of HPV 16-transformed SiHa cells but not slow-annealing antisense species. Antisense RNA of several hundred nucleotides in length also showed significant anti-proliferative activity. Biological effects of antisense oligodeoxyribonucleotides were specific for the antisense sequence, could only be found in HPV-positive but not in HPV-negative cell lines, and were related to decreased levels of E7 protein and E6/E7-specific transcripts. This work suggests that HPV 16 E7/E6 sequences exhibit a low accessibility for antisense oligonucleotides. This can be overcome, however, by exploiting the relationship between fast annealing of antisense species and their increased efficacy in human cells.

Animals↗

E7-specific cytotoxic T cell tolerance in HPV-transgenic mice.

The "high-risk" human papillomavirus type 16 (HPV 16) is associated with the development of cervical cancer. Although the viral gene products E6 and E7 are constitutively expressed in HPV 16-associated lesions and therefore appear as candidate antigens for a specific immune response, the immune system fails to produce an efficient defence against tumor outgrowth in affected patients. Keratinocytes are the natural target cells of HPV infection. To investigate the E7-specific immune response in vivo, we used transgenic mice expressing the oncogenes E6 and E7 of HPV 16 under the control of the keratin 10 promoter in the suprabasal layers of the epidermis. This expression pattern closely reflects the viral early gene transcription that is observed in low grade cervical intraepithelial lesions (CIN). The transgene product E7 does not induce an immune response in these transgenic mice. However, upon vaccination anti-E7 antibodies were produced without causing signs of autoimmune disease. In contrast, E7-specific cytotoxic T lymphocytes (CTL) were not detected after immunization. From these results we conclude that in K10 HPV 16 E6/E7 transgenic mice the E7 transgene expression induces specific immunological tolerance on the CTL level.

Animals↗

Secretion of heterologous proteins from Schizosaccharomyces pombe using the homologous leader sequence of pho1+ acid phosphatase.

In this study we report the use of the S. pombe leader sequence of pho1+ acid phosphatase (Elliott et al., J. Biol. Chem. 216, 2916-2941, 1986) for the secretion of heterologous proteins into the medium. The green fluorescent protein (GFP) and the Human Papillomavirus (HPV) type 16 E7 protein are normally not secreted; fusion of the S. pombe pho1 leader peptide (SPL) to GFP and HPV 16 E7 resulted in an efficient secretion of these proteins although the latter contains a nuclear targeting sequence. These data suggest that SPL fused constructs could be applied for the production of other recombinant proteins using the S. pombe expression system. Furthermore, since GFP retains its intrinsic fluorescence during the secretion, this system may be useful to study the secretory pathway of fission yeast in vivo.

Acid Phosphatase↗

The CXXC Zn binding motifs of the human papillomavirus type 16 E7 oncoprotein are not required for its in vitro transforming activity in rodent cells.

The conserved region 3 (CR3) of the E7 protein of human papillomaviruses contains two CXXC motifs involved in zinc binding and in the homodimerization of the molecule. Studies have suggested that the intact CXXC motifs in the CR3 of HPV16 and HPV18 E7 are required for the in vitro transforming activity of these proteins. CR3 also contains a low affinity pRb binding site and is involved in the disruption of the E2F/Rb1 complex. E7 is structurally and functionally related to Adenovirus E1A protein, which also has two CXXC motifs in CR3. However, the Ad E1A transforming activity appears to be independent of the presence of such domains. In fact, this viral protein exists in vivo as two different forms of 289 and 243 amino acids. The shorter Ad E1A form (Ad E1A243), where both CXXC motifs are deleted by internal splicing, retains its in vitro transforming activity. We have investigated if the HPV16 E7 CR3 can be functionally replaced by the Ad E1A243 CR3, which lacks both CXXC motifs. A chimeric protein (E7/E1A243) containing the CR1 and CR2 of HPV16 E7 fused to the CR3 of Ad E1A 243 was constructed. The E7/E1A243 while not able to homodimerize in the S. cerevisiae two-hybrid system retains several of the properties of the parental proteins, HPV16 E7 and Ad E1A. It associates with the 'pocket' proteins, induces growth in soft agar of NIH3T3 cells and immortalizes rat embryo fibroblasts. These data suggest that the CXXC motifs in CR3 of E7 do not play a direct role in the transforming properties of this viral protein but probably are important for maintaining the correct protein configuration.

3T3 Cells↗

Antibodies against early proteins of human papillomaviruses as diagnostic markers for invasive cervical cancer.

Cervical cancer is the most prevalent tumor in developing countries and the second most frequent cancer among females worldwide. Specific human papillomaviruses (HPVs) and, most notably, HPV types 16 and 18 are recognized as being causally associated with this malignancy. Antibodies against early HPV proteins E6 and E7 have been found more often in patients with tumors than in controls. Existing peptide enzyme-linked immunosorbent assays (ELISAs) for the detection of anti-E6 and anti-E7 antibodies in human sera have low levels of sensitivity and specificity and thus are not suitable for use as diagnostic tools. Based on highly purified recombinant native proteins, we developed four sandwich ELISAs for the detection of antibodies against HPV type 16 and 18 E6 and E7 proteins. We demonstrate their sensitivities and high degrees of specificity for cervical cancer. Among a total of 501 serum specimens from unselected patients with invasive cervical cancer, 52.9% reacted positively in at least one of the four assays. In contrast, among 244 serum specimens from control subjects without cervical cancer, only 2 reactive serum specimens (0.8%) were found. For 19 of 19 antibody-positive patients, the HPV type indicated by seroreactivity was identical to the HPV DNA type found in the tumor, which also indicates a high degree of specificity for antibody detection with respect to HPV type. In a direct comparison of 72 serum specimens from patients with cervical cancer, 56% of the specimens reacted in at least one of the four protein ELISAs, whereas 40% reacted in at least one of seven peptide ELISAs covering the four antigens. These assays could be of value for the detection of invasive cervical cancer in settings in which cytology-based early tumor screening is not available, for the clinical management of patients diagnosed with cervical cancer, and for the immunological monitoring of E6 and E7 vaccination trials.

Adult↗

Chimeric papillomavirus-like particles.

We have constructed chimeric papillomavirus-like particles (CVLPs) by replacing the 34-carboxy-terminal amino acids of the HPV 16 L1 protein with various parts of the HPV 16 E7 protein. Chimeric proteins were expressed by recombinant baculoviruses and analyzed by electron microscopy for their ability to assemble into virus capsids. We were able to produce CVLPs in high efficiencies with inserts of up to 60 amino acids. CVLPs are able to induce a neutralizing antibody response, assayed by inhibition of hemagglutination of mouse erythrocytes. CVLPs are interacting with the putative receptor for papillomaviruses as they were shown to hemagglutinate mouse red blood cells and bind to and penetrate cells in vitro. As CVLPs follow a similar intracellular pathway as observed earlier for BPV VLPs, we speculate that CVLPs can be used to deliver peptides into mammalian cells in vitro and in vivo, possibly reaching the pathway for MHC class I presentation.

Amino Acid Sequence↗

The human papillomavirus type 16 E7 protein is associated with the nucleolus in mammalian and yeast cells.

In this study we show, by immunofluorescence and electron microscopy immuno-gold labelling, that the major transforming protein of Human Papillomavirus type 16 E7 is associated with the nucleolus of cells derived from the HPV16-positive cervical carcinoma line CaSki. The E7 nucleolar staining appeared to be cell cycle dependent, being considerably reduced in the G2 phase. The total level of the protein in the cell, however, remained constant during all phases. We also show that the cellular protein Rb1, which is targeted by E7, is localised in the nucleus and nucleolus in CaSki cells. Thus, it is possible that the presence of E7 in the nucleolus correlates with a hypothetical function(s) of Rb1 in this particular intranuclear compartment. The nucleolar localisation of HPV16 E7 protein was also observed in the fission yeast Schizosaccharomyces pombe, suggesting that a targeting mechanism of HPV16 E7 protein into the nucleolus is common to both mammalian and yeast systems. Nucleolar localisation of HPV16 E7 protein may be independent from Rb1 since no Rb1 related proteins have been identified in fission yeast.

Antibodies, Monoclonal↗

Priming of cytotoxic T lymphocytes by five heat-aggregated antigens in vivo: conditions, efficiency, and relation to antibody responses.

Mice were immunized i.p. with soluble or heat-denatured protein antigens [ovalbumin, beta-galactosidase, or recombinant E7 protein of human papilloma virus type 16 (HBV)]. Heat-denatured (100 degrees C) preparations of these proteins were able to induce cytotoxic T lymphocytes (CTL) that recognize cells expressing the respective genes, whereas native protein was either inefficient or required up to 30-fold higher doses. If the heat-treated proteins were separated into aggregated and soluble fractions by ultracentrifugation, only the aggregated fractions were able to induce specific CTL; this is probably because of the easier access to one of the major histocompatibility complex class I loading pathways for exogenous antigen. Addition of the adjuvant aluminium hydroxide (alum) to aggregated proteins abolished their ability to induce CTL; thus, a condition leading to a strong antibody response appeared to inhibit CTL induction. Interestingly, immunization with heat-denatured ovalbumin plus alum increased the IgM/IgG1 ratio compared to immunization with native ovalbumin and alum. Immunization of B6 mice transgenic for an HLA-A2/H-2K(b) hybrid gene with heat-denatured, recombinant HPV 16-E7 protein induced D(b)-restricted CTL specific for the peptide 49-57 of E7, indicating that this epitope is immunodominant over any A2-restricted E7 epitope in these mice. A whole influenza virus preparation heated to 100 degrees C or even autoclaved was still able to induce virus-specific CTL and BALB/c spleen cells heated to 100 degrees C could still cross-prime minor H-specific CTL in B6 mice, although with lower efficiency than fresh spleen cells. Thus, aggregated proteins can be considered as components for future vaccines.

Animals↗

A general purification protocol for E7 proteins from "high- and low-risk" human papillomavirus types expressed in the yeast Schizosaccharomyces pombe.

A purification protocol was developed to obtain human papillomavirus (HPV) type 16 E7 protein expressed in the yeast Schizosaccharomyces pombe. Only three chromatographic steps were necessary to purify the unfused HPV 16 E7 protein to homogeneity (95-99%) as shown by silver staining after polyacrylamide gel electrophoresis. Approximately 0.8 mg of highly purified E7 was obtained from 5 x 10(10) yeast cells. The purified HPV 16 E7 phosphoprotein (Ser 31/32) was refolded and assayed for functionality. Binding to the proteins Rb1 and p107 in vitro and induction of DNA synthesis after microinjection into serum-deprived NIH 3T3 cells suggest that the E7 protein retains some of its biological activities. Most importantly, the purification strategy is also applicable for different HPV 16 E7 mutants and for E7 proteins from other HPV types such as HPV 18 and 11.

Animals↗

p53-independent growth regulation of cervical cancer cells by the papillomavirus E6 oncogene.

Growth of cervical carcinoma cells depends on continuous expression of high risk type human papillomavirus oncogenes E6 and E7. E6 destabilizes p53, a tumor-suppressive transcription factor, which activates expression of the inhibitor of cell cycle progression p21 and other genes. E6-mediated p53 degradation can therefore result in cell cycle deregulation. It has, however, not yet been determined whether p53 inactivation is sufficient to provoke cell cycle progression in established cervical carcinoma cells. Moreover, it has not yet been clarified whether E6 confers additional p53-independent growth stimuli in cancer cells. To address these questions, we analysed p53 functions in SW 756 cervical cancer cells in which the expression of endogenous HPV 18 E6-E7 genes can be downregulated by dexamethasone. This results in significantly increased p53 levels and subsequent cell cycle arrest in the Gz phase. Surprisingly, p53 activities were suppressed rather than enhanced in these cervical cancer cells. However, if high risk papillomavirus type 16 E6 genes, including a mutant which does not degrade p53, were expressed in dexamethasone-treated SW 756 cells with suppressed endogenous HPV type 18 E6-E7 expression, the cells reentered the cell cycle even in the absence of a cooperating viral E7 gene. In contrast, the non oncogenic papillomavirus type 6 E6 gene did not release the cells from growth arrest under these conditions. These data indicate that suppression of p53 functions is not sufficient to provoke cell cycle progression in E6-E7-depleted cervical cancer cells and point to a p53-independent mitotic activity to oncogenic papillomavirus type E6 genes in cervical carcinoma cells.

Amino Acid Sequence↗