Biomedical subjects

P Fowler

Publications and source records attributed to P Fowler.

At least 37 records · Page 2Linked to original sources

PubMed1995

Induction in vitro of a primary human antiviral cytotoxic T cell response.

We report herein the successful priming of human anti-viral cytotoxic T cells (CTL) in vitro using two induction strategies based on the stimulation of peripheral blood mononuclear cells isolated from uninfected donors with synthetic viral peptides. The peptides used contain HLA-A2 binding motifs and have been identified as HLA-A2-restricted CTL epitopes in patients infected by the hepatitis B and C viruses. One approach uses repetitive long-term stimulation and the other uses bulk cultures containing large numbers of naive peripheral blood mononuclear cells. Both approaches successfully induce HLA-A2-restricted CTL specific for several viral epitopes. Some CTL recognize endogenously synthesized antigen on target cells infected with recombinant vaccinia virus expressing the corresponding viral proteins. This simple technique permits easy analysis of the primary human CTL repertoire, and may be exploitable for production of specific CTL effector cells for adoptive immunotherapy and dissection of the cellular and molecular requirements for priming of naive human CTL.

Amino Acid Sequence↗

PubMed1995

Developing outcome measures for ambulatory care--an application to asthma and diabetes.

Increasing emphasis is now being placed on the assessment of patient outcomes, both in evaluating medical interventions and in quality assurance initiatives. Clinicians, purchasers, managers and researchers need outcome measures that are valid, reliable and responsive. This paper describes the theory and practice underlying the development of outcome measures for two chronic conditions, asthma and diabetes, for application in ambulatory settings. Existing generic and condition-specific health status and health-related quality-of-life measures were administered to almost 1300 patients. The psychometric properties of these measures were examined to identify those that were of adequate validity and reliability in these population groups. Step-wise regression procedures were then used to identify a core set of scales that best predicted patients' general health perceptions, which could be used in measuring general health outcomes for each of these groups. These core sets consist of up to 40 items, spanning physical function, energy and vitality, emotional well-being and condition-specific aspects of health such as symptom control. Further analysis is being carried out to assess the responsiveness to change of these core item sets.

Activities of Daily Living↗

PubMed1995

Cytotoxic T lymphocyte response to hepatitis C virus-derived peptides containing the HLA A2.1 binding motif.

The HLA class I-restricted cytotoxic T lymphocyte (CTL) response is a major defense mechanism in viral infections. It has been suggested that the CTL response may contribute to viral clearance and liver cell injury during hepatitis C virus (HCV) infection. To test this hypothesis requires an understanding of the characteristics of HCV-specific cytotoxic effector cells and identification of the target antigens to which they respond. To begin this process we stimulated peripheral blood mononuclear cells (PBMC) from a group of HLA-A2 positive patients with chronic hepatitis C with a panel of 130 HCV-derived peptides containing the HLA-A2 binding motif. Effector cells were tested for their capacity to lyse HLA-A2-matched target cells that were either sensitized with peptide or infected with a vaccinia virus construct containing HCV sequences. Using this approach we have identified nine immunogenic peptides in HCV, three of which are derived from the putative core protein, three from the nonstructural (NS) 3 domain, two from NS4 and one from NS5. Selected responses were shown to be HLA-A2 restricted, mediated by CD8+ T cells and to recognize endogenously synthesized viral antigen. Unexpectedly, peptide-specific CTL responses could also be induced in sero-negative individuals, suggesting in vitro activation of naive CTL precursors. The precursor frequency of peptide-specific CTL was 10 to 100-fold higher in infected patients compared to uninfected controls, and the responses were greatly diminished by removal of CD45 RO+ (memory) T cells. Further quantitative studies are clearly required to establish whether a correlation exists between the HCV-specific CTL response and the clinical course of this disease. Definition of the molecular targets of the human CTL response to HCV creates this opportunity, and may also contribute to the development of a T cell-based HCV vaccine.

Amino Acid Sequence↗

PubMed1994

Extensive oxidative DNA damage in hepatocytes of transgenic mice with chronic active hepatitis destined to develop hepatocellular carcinoma.

A transgenic mouse strain that expresses the hepatitis B virus (HBV) large envelope protein in the liver was used to determine the extent of oxidative DNA damage that occurs during chronic HBV infection. This mouse strain develops a chronic necroinflammatory liver disease that mimics the inflammation, cellular hyperplasia, and increased risk for cancer that is evident in human chronic active hepatitis. When perfused in situ with nitroblue tetrazolium, an indicator for superoxide formation, the liver of transgenic mice displayed intense formazan deposition in Kupffer cells, indicating oxygen radical production, and S-phase hepatocytes were commonly seen adjacent to the stained Kupffer cells. Similar changes were not observed in nontransgenic control livers. To determine whether these events were associated with oxidative DNA damage, genomic DNA from the livers of transgenic mice and nontransgenic controls was isolated and examined for 8-oxo-2'-deoxyguanosine, an oxidatively modified adduct of deoxyguanosine. Results showed a significant, sustained accumulation in steady-state 8-oxo-2'-deoxyguanosine that started early in life exclusively in the transgenic mice and increased progressively with advancing disease. The most pronounced increase occurred in livers exhibiting microscopic nodular hyperplasia, adenomas, and hepatocellular carcinoma. Thus, HBV transgenic mice with chronic active hepatitis display greatly increased hepatic oxidative DNA damage. Moreover, the DNA damage occurs in the presence of heightened hepatocellular proliferation, increasing the probability of fixation of the attendant genetic and chromosomal abnormalities and the development of hepatocellular carcinoma.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1994

The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes.

The relationship between binding affinity for HLA class I molecules and immunogenicity of discrete peptide epitopes has been analyzed in two different experimental approaches. In the first approach, the immunogenicity of potential epitopes ranging in MHC binding affinity over a 10,000-fold range was analyzed in HLA-A*0201 transgenic mice. In the second approach, the antigenicity of approximately 100 different hepatitis B virus (HBV)-derived potential epitopes, all carrying A*0201 binding motifs, was assessed by using PBL of acute hepatitis patients. In both cases, it was found that an affinity threshold of approximately 500 nM (preferably 50 nM or less) apparently determines the capacity of a peptide epitope to elicit a CTL response. These data correlate well with class I binding affinity measurements of either naturally processed peptides or previously described T cell epitopes. Taken together, these data have important implications for the selection of epitopes for peptide-based vaccines, and also formally demonstrate the crucial role of determinant selection in the shaping of T cell responses. Because in most (but not all) cases, high affinity peptides seem to be immunogenic, our data also suggest that holes in the functional T cell repertoire, if they exist, may be relatively rare.

Amino Acid Sequence↗

PubMed1994

Development of a symptom based outcome measure for asthma.

Measuring symptom specific health outcome is complex, but the methodologies now exist to develop measures with the appropriate properties. As one element of a major programme to develop multidomain health outcome measures for chronic disease, a symptom based measure for asthma care has been developed for use in general practice and outpatient departments. This article outlines the development process, which used a framework recently described in the theoretical literature to show the constraints that scientific criteria place on the development of outcome measures and the means of overcoming such limiting factors. Although substantial effort is required to undertake a rigorous process of development, useful tools are the result. Two five item, symptom based outcome measures for adult asthma are described.

Ambulatory Care↗

PubMed1994

Is chest pain after sumatriptan oesophageal in origin?

3-5% of patients taking the 5HT1D agonist sumatriptan for migraine have chest discomfort, suggesting a cardiac origin. We have investigated an alternative explanation of an oesophageal cause in 24 volunteers after the subcutaneous administration of a supratherapeutic dose of sumatriptan (16 mg) or placebo in a randomised, double-blind crossover study. Sumatriptan did not alter the electrocardiogram but increased the amplitude (p < 0.001) and duration (p < 0.001) of oesophageal contractions without affecting velocity of propagation. Clinically abnormal motility was also increased (p = 0.001), and was more common in the 5 subjects with chest pain after sumatriptan. The effect of sumatriptan on oesophageal function provides an alternative explanation for the chest symptoms.

Adult↗

PubMed1994

Receptor interactions of the position 4 side chains of angiotensin II analogues: importance of aromatic ring quadrupole.

A triad of interacting groups (TyrOH-His-O2C) in angiotensin II (ANG II) has been postulated to create the tyrosinate anion pharmacophore (tyanophore) responsible for receptor activation/triggering (Biochim. Biophys. Acta 1991, 1065, 21). In the present study we investigated the effects on bioactivity of substituting the Tyr4 residue in [Sar1]ANG II with other anionic or electronegative amino acids, and with a number of aromatic amino acids lacking a hydroxyl group. [Sar1 Nva(delta-OH)4]ANG II, [Sar1 Nva(delta-OCH3)4]ANG II, [Sar1 Met4]ANG II, [Sar1 Gln4]ANG II, [Sar1 Glu4]ANG II and [Sar1 DL-Alg]ANG II had agonist activities in the rat isolated uterus assay of 4, 3, 19, 10, < 0.1 and < 0.1%, respectively, of that of ANG II. [Sar1 Nal4]ANG II, [Sar1 Pal4]ANG II, [Sar1 DL-Phg(4'-F)4]ANG II, [Sar1 Phe(4'-F)4]ANG II, [Sar1 Phe(F5)4]ANG II and [Sar1 His4]ANG II had agonist activities of 4.5, 7, < 0.1, 0.2, 1 and 0.6%, respectively. All peptides investigated were devoid of measurable antagonist activity except [Sar1 Phe(4'-F)4ANG II (pA2 = 7.7). These findings illustrate that anionic or electronegative aliphatic side chains replacing tyrosinate at position 4 can partially activate the angiotensin receptor. For ANG II analogues containing an aromatic amino acid other than Tyr at position 4, ligand binding and agonist activity are not dependent on the electronegativity or dipole moment of the aromatic ring, or on the ability of the 4' ring substituent to accept a proton.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Protein transfer of preformed MHC-peptide complexes sensitizes target cells to T cell cytolysis.

Recombinant GPI-anchored HLA-A2.1 (HLA-A2.1-GPI/beta 2m) was used as a protein transfer vehicle to deliver a hepatitis B virus antigenic peptide to the surfaces of cytotoxic T cell targets. Empty HLA-A2.1-GPI/beta 2m was first produced in D. melanogaster cotransfectants and immunoaffinity purified. Cell coating with HLA-A2.1-GPI/beta 2m was shown to occur rapidly, and to be protein concentration dependent. Protein-transferred HLA-A2.1-GPI/beta 2m effectively presented a hepatitis B virus peptide to peptide-specific HLA-A2.1-restricted T cell clones in cytotoxicity assays. Protein transfer of functional GPI-modified class I MHC-antigenic peptide complexes represents a novel strategy for delivering functional antigenic complexes to cell surfaces that bypasses limitations of gene transfer and permits control of antigenic peptide densities at cell surfaces.

Animals↗

PubMed1993

HLA A2 restricted cytotoxic T lymphocyte responses to multiple hepatitis B surface antigen epitopes during hepatitis B virus infection.

Inasmuch as the hepatitis B virus (HBV) is not directly cytopathic for the infected hepatocyte, it is generally presumed that viral clearance and liver cell injury during viral hepatitis are due to a CTL response to HBV encoded Ag presented by HLA class I molecules. We have previously examined the peripheral blood CTL response to two HBV nucleocapsid epitopes in patients with acute and chronic viral hepatitis, one of which is restricted by HLA-A2, whereas the other is dually restricted by HLA-A31 and Aw68. In this study, we defined the HLA-A2-restricted CTL response to the hepatitis B surface Ag (HBsAg) by using a panel of HBsAg-derived synthetic peptides containing the ideal HLA-A2.1 binding motif (-L------V). Several novel aspects of HBV immunobiology and pathogenesis are evident from this study. First, the peripheral blood CTL response to HBV-encoded Ag is remarkably polyclonal and multispecific in most patients with acute hepatitis. Indeed, HLA-A2-restricted CTL specific for as many as four envelope epitopes and one nucleocapsid epitope were found to be present simultaneously in individual patients with acute viral hepatitis. Second, HBV-specific CTL are not detectable in the peripheral blood in a minority of patients with acute hepatitis, nor have we detected a CTL response in any of the patients with chronic hepatitis that we have studied thus far. Although the cellular and molecular basis for CTL nonresponse remains to be determined, the data suggest that it may contribute to viral persistence. Third, the diversity and the specificity of the CTL response is determined in part by the coding sequence of the viral genome present in each infected patient. Indeed, the apparent nonresponse of some acutely infected patients to at least one HBsAg-specific CTL epitope actually reflects infection by a viral variant that contains a critical substitution in one of the anchor residues within the epitope. Finally, at a fundamental level, the data suggest that the presence of the HLA-A2.1-binding motif in a peptide may not be sufficient for binding; and the capacity of a peptide to bind the class I molecule does not guarantee that it will be immunogenic.

Amino Acid Sequence↗

PubMed1993

HLA-A31- and HLA-Aw68-restricted cytotoxic T cell responses to a single hepatitis B virus nucleocapsid epitope during acute viral hepatitis.

We have recently developed the technology to identify and characterize the human histocompatibility leukocyte antigen (HLA) class I-restricted, CD8+ cytotoxic T lymphocyte (CTL) response to hepatitis B virus (HBV)-encoded antigens in patients with acute viral hepatitis. CTL are expanded in vitro by stimulation with HBV-derived synthetic peptides and selected by restimulation with a panel of HLA-matched stable transfectants that express the corresponding HBV protein. We have recently reported the existence of an HLA-A2-restricted, CD8+ CTL response to an epitope located between residues 18 and 27 of the HBV nucleocapsid core antigen (HBcAg). We now report the discovery of a CTL epitope located between HBcAg residues 141 and 151 that completely overlaps a critical domain in the viral nucleocapsid protein that is essential for its nuclear localization and genome packaging functions as well as processing of the precore protein. The CTL response to this epitope is dually restricted by the HLA-A31 and HLA-Aw68 alleles, which, unexpectedly, appear to use a common binding motif based on the results of alanine substitution and competition analysis, and the binding properties of these two alleles predicted from their known primary sequence, and from the three-dimensional structure of HLA-Aw68. We have also demonstrated that the HBV-specific CTL response to this epitope is polyclonal during acute viral hepatitis, since these two restriction elements can present the HBcAg 141-151 epitope to independent CTL clones derived from a single patient; and that the CTL response is multispecific, since HLA-A2-restricted and HLA-Aw68-restricted CTL responses to HBcAg 18-27 and HBcAg 141-151, respectively, have been identified to coexist in another patient. The foregoing argue against the emergence of CTL escape mutants as a significant problem during HBV infection, especially at this locus, where mutations might be incompatible with viral replication. Finally, our data suggest an association between the HBV-specific CTL response and viral clearance, and they have implications for the design of immunotherapeutic strategies to terminate HBV infection in chronically infected patients.

Acute Disease↗

PubMed1993

Definition of a minimal optimal cytotoxic T-cell epitope within the hepatitis B virus nucleocapsid protein.

Residues 11 to 27 of the hepatitis B virus nucleocapsid antigen contain a cytotoxic T-cell epitope that is recognized by cytotoxic T cells from virtually all HLA-A2-positive patients with acute hepatitis B virus infection. Using panels of truncated and overlapping peptides, we now show that the optimal amino acid sequence recognized by cytotoxic T cells is a 10-mer (residues 18 to 27) containing the predicted peptide-binding motif for HLA-A2 and that this peptide can stimulate cytotoxic T cells able to recognize endogenously synthesized hepatitis B core antigen. Since patients with chronic hepatitis B virus infection fail to mount an efficient cytotoxic T-cell response to it, this epitope might serve as the starting point for the design of synthetic peptide-based immunotherapeutic strategies to terminate persistent viral infection.

Acute Disease↗

PubMed1992

Fine specificity of the human T cell response to hepatitis B virus core antigen.

The fine specificity of the human T cell response to the hepatitis B virus core antigen (HBcAg) was investigated in 23 patients with acute hepatitis B virus (HBV) infection using a panel of short synthetic peptides covering the entire core region. An immunodominant T cell epitope which was recognized by all except one patient, was identified within the core sequence 50-69. Two further important T cell recognition sites were represented by the amino acid sequences 1-20 and 117-131, which were stimulatory for the T cells of 69% and 73% of the patients, respectively. T cell recognition of the synthetic peptides was HLA class II restricted because the peptide-induced T cell proliferation was inhibited by anti-HLA class II but not by anti-HLA class I monoclonal antibodies. These findings may be relevant to the development of future preventive and therapeutic strategies against HBV infection.

Acute Disease↗

PubMed1992

Effect of sumatriptan, a new selective 5HT1-like agonist, on liquid gastric emptying in man.

Paired studies were carried out on 12 healthy male subjects to compare the effect of intravenous doses of the 5-HT1-like agonist sumatriptan (GR43175; 3 mg), 10 mg metoclopramide and saline control on the rate of gastric emptying of a radiolabelled liquid test-meal. Intravenous administration of metoclopramide accelerated gastric emptying by decreasing the lag period, while intravenous administration of sumatriptan delayed gastric emptying by increasing the lag period. The observation that sumatriptan causes a delay in gastric emptying in normal healthy volunteers, but relieves nausea and vomiting during migraine attacks, suggests that sumatriptan may be acting via a central mechanism to relieve symptoms of nausea and vomiting associated with migraine.

Adult↗

PubMed1992

Hepatitis B virus (HBV)-specific cytotoxic T-cell (CTL) response in humans: characterization of HLA class II-restricted CTLs that recognize endogenously synthesized HBV envelope antigens.

In this study, we show that CD4+, hepatitis B virus (HBV) envelope-specific T-cell clones produced by stimulation with a particulate antigen preparation are able to recognize and kill not only autologous antigen-presenting cells incubated with exogenous HBV envelope antigens but also autologous HLA class II-positive cells expressing endogenously synthesized HBV envelope antigens following infection with recombinant vaccinia viruses or transfection with recombinant Epstein-Barr virus expression vectors. Experiments with lysosomotropic agents and brefeldin A suggest that the endosomal compartment is likely involved in the processing of endogenously synthesized viral proteins for recognition by CD4+ T cells. Our study indicates that HBV envelope-specific, HLA class II-restricted CD4+ cytotoxic T lymphocytes can potentially participate in the immune clearance of HBV-infected cells and the pathogenesis of hepatocellular injury in hepatitis B.

Ammonium Chloride↗

PubMed1992

Hepatitis B virus (HBV)-specific cytotoxic T-cell response in humans: production of target cells by stable expression of HBV-encoded proteins in immortalized human B-cell lines.

To analyze the hepatitis B virus (HBV)-specific cytotoxic T-cell (CTL) response during acute and chronic viral hepatitis, target cells that express HBV-encoded antigens in the context of the appropriate HLA restriction element must be available for each subject studied. Since HBV is not infectious for human cells in vitro, such target cells must be produced by DNA-mediated gene transfer into cultured human primary cells or cell lines. For this purpose, we have developed a panel of Epstein-Barr virus-based episomal expression vectors containing each of the HBV open reading frames under the transcriptional control of the simian virus 40 early promoter. Transfection of Epstein-Barr virus-immortalized B-cell lines with this panel of recombinants consistently leads to stable expression of the HBV envelope, nucleocapsid, and polymerase proteins. The HBV X gene product is transiently expressed following transfection, but stable expression of this protein cannot be maintained on a long-term basis. To assess the suitability of this system for the identification of HBV-specific CTL in humans, a panel of EBO-HBV transfectants of defined HLA haplotype was used to monitor the HBV-specific CTL response in a patient with acute viral hepatitis type B. Transfectants that stably express the HBV nucleocapsid (core) antigen were found to serve as excellent targets for the detection of HLA class I-restricted CTL that recognize endogenously synthesized HBV core antigen in this patient; they were also successfully used to stimulate the specific expansion of these CTL in vitro.

B-Lymphocytes↗

PubMed1992

Outcome measures for primary health care: what are the research priorities?

A Delphi technique was used to ask general practitioners for their opinions as to which clinical problems and types of measure they thought most appropriate for the development of outcome measures for use in primary health care. The study comprised two rounds of postal questionnaires, targeted at general practitioners in academic departments throughout the United Kingdom and at trainers in the northern region, with the second questionnaire feeding back opinions from the first. Ninety eight participants suggested one or more areas in which outcome measures could be developed, giving a total of 451 suggestions. Consensus produced in the second round indicated that three clinical conditions were preferred for the development of outcome measures: asthma, diabetes and hypertension. Six categories of outcome measures were developed from the responses given in the first round--level of function, level of clinical control, incidence of complications, iatrogenic problems, patient understanding of a condition, and quality of life. Participants gave these measures different levels of importance according to the clinical problem in question. This Delphi study of doctors' opinions is a first step in the development process of appropriate, practicable measures of outcome for use in primary care and has achieved a degree of consensus among general practitioners.

Attitude of Health Personnel↗

PubMed1991

HLA class I-restricted human cytotoxic T cells recognize endogenously synthesized hepatitis B virus nucleocapsid antigen.

Knowledge of the immune effector mechanisms responsible for clearance of hepatitis B virus (HBV)-infected cells has been severely limited by the absence of reproducible systems to selectively expand and to characterize HBV-specific cytotoxic T lymphocytes (CTLs) in the peripheral blood of patients with viral hepatitis. By using a strategy involving sequential stimulation with HBV nucleocapsid synthetic peptides followed by autologous, or HLA class I-matched, HBV nucleocapsid transfectants, we now report the existence of CTLs able to lyse target cells that express endogenously synthesized HBV nucleocapsid antigen in the peripheral blood of patients with acute viral hepatitis B. The CTL response is HLA-A2 restricted, mediated by CD8-positive T cells, and specific for a single epitope, located between amino acid residues 11 and 27 of HBV core protein; these residues are shared with the secretable precore-derived hepatitis B e antigen. Equivalent lysis of target cells that express each of these proteins suggests that their intracellular trafficking pathways may intersect. The current report provides definitive evidence that HLA class I-restricted, CD8-positive CTLs that recognize endogenously synthesized HBV nucleocapsid antigen are induced during acute HBV infection in humans and establishes a strategy that should permit a detailed analysis of the role played by HBV-specific CTLs in the immunopathogenesis of viral hepatitis.

B-Lymphocytes↗