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P Simmonds

Publications and source records attributed to P Simmonds.

210 records · Page 12Linked to original sources
PubMed1990

Analysis of sequence diversity in hypervariable regions of the external glycoprotein of human immunodeficiency virus type 1.

Nucleotide sequences in three hypervariable regions of the human immunodeficiency virus type 1 (HIV-1) env gene were obtained by sequencing provirus present in peripheral blood mononuclear cells of HIV-infected individuals. Single molecules of target sequences were isolated by limiting dilution and amplified in two stages by the polymerase chain reaction, using nested primers. The product was directly sequenced to avoid errors introduced by Taq polymerase during the amplification process. There was extensive variation between sequences from the same individual as well as between sequences from different individuals. Interpatient variability was markedly less in individuals infected from a common source. A high proportion of amino acid substitutions in the hypervariable regions altered the number and positions of potential N-linked glycosylation sites. Sequences in two hypervariable regions frequently contained short (3- to 15-bp) duplications or deletions, and by amplifying peripheral blood mononuclear cell DNA containing 10(2) or 10(3) proviral molecules and analyzing the product by high-resolution electrophoresis, the total number and abundance of distinct length variants within an individual could be estimated, providing a more comprehensive analysis of the variants present than would be obtained by sequencing alone. Sequences from many individuals showed frequent amino acid substitutions at certain key positions for neutralizing-antibody and cytotoxic T-cell recognition in the immunodominant loop. The rates of synonymous and nonsynonymous nucleotide substitution in the region of this and flanking regions indicate that strong positive selection for amino acid change is operating in the generation of antigenic diversity.

Amino Acid Sequence↗
PubMed1990

Concurrent evolution of human immunodeficiency virus type 1 in patients infected from the same source: rate of sequence change and low frequency of inactivating mutations.

Direct sequencing of segments of the envelope gene of human immunodeficiency virus type 1 proviruses in peripheral blood mononuclear cells has revealed that a cohort of hemophiliacs who were infected after exposure to a single common batch of factor VIII share closely related virus strains. Seventy-four sequences extending from hypervariable regions V4 through V5 from nine patients yielded a mean intrapatient nucleotide distance of 5.5%, while a mean of 4.2% was observed in 39 sequences of the V3 loop (six patients). Phylogenetic analysis revealed that sequences of six Edinburgh patients were particularly closely related and those from a patient infected in the United States were very distinct. The mean nucleotide distance among these six was 8.3%, while the mean distance from the U.S.-derived sequences was 25.5% in the V4-V5 region. The rate of sequence change across this patient group has been estimated to be 0.4% per year in the V4-V5 region and 0.5% per year in the V3 region, with at least a twofold range across patients. Only two inactivating nucleotide substitutions have been observed in a total of 42 kb of sequence obtained from the env and gag genes during this study.

Base Sequence↗
PubMed1990

Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers.

In human immunodeficiency virus (HIV)-infected individuals, the proportion of circulating mononuclear cells (PBMCs) which carry HIV provirus and the number of HIV proviral sequences per infected PBMC have been matters for conjecture. Using a double polymerase chain reaction which allows the detection of single molecules of provirus and a method of quantifying the provirus molecules, we have measured provirus frequencies in infected individuals down to a level of one molecule per 10(6) PBMCs. As a general rule, only a small proportion of PBMCs contain provirus (median value of samples from 12 patients, one per 8,000 cells), and most if not all of the infected cells carry a single provirus molecule. The frequency of provirus-carrying cells correlated positively both with the progression of the disease and with the success with which virus could be isolated from the same patients by cocultivation methods. Of seven asymptomatic (Centers for Disease Control stage II) patients, all but one contained one provirus molecule per 6,000 to 80,000 cells; of five Centers for Disease Control stage IV patients, all but one contained one provirus molecule per 700 to 3,300 cells. When considered in conjunction with estimates of the frequency of PBMCs that express viral RNA, our results suggest that either (i) the majority of provirus-containing cells are monocytes or (ii) most provirus-containing lymphocytes are transcriptionally inactive. We also present nucleotide sequence data derived directly from provirus present in vivo which we show is not marred by the in vitro selection of potential virus variants or by errors introduced by Taq polymerase. We argue from these data that, of the provirus present in infected individuals, the proportion which is defective is not high in the regions sequenced.

Base Sequence↗
PubMed1988

Femtogram detection of perfluorocarbon tracers using capillary gas chromatography-electron-capture negative ion chemical ionisation mass spectrometry.

An ultrasensitive and selective method has been developed for the detection of a range of perfluorocarbon tracers suitable for long-range atmospheric studies. Following direct injection onto a capillary column the perfluorocarbons are separated and detected using mass spectrometry with electron-capture negative ion chemical ionisation (ECNICI-MS) with methane as reagent gas and selected-ion monitoring. All nine perfluorocarbons tested underwent non-dissociative resonance electron capture to form the molecular anion which was the base peak in all cases. Using this technique, detection limits (signal-to-noise ratio of 2:1) of 3 and 2 fg were obtained for perfluoromethylcyclohexane and perfluoro-1,3-dimethylcyclohexane respectively. The corresponding detection limits using a conventional electron-capture detector were 8 and 9 fg respectively. In addition to increased sensitivity, the ECNICI-MS technique allows temperature programming for the simultaneous analysis of a range of perfluorocarbons and has superior selectivity being less prone to coeluting interferences. Furthermore the method is potentially one to two orders of magnitude more sensitive than the current gas chromatography-MS methods using 13C2H4 tracers.

Air Pollutants, Occupational↗
PubMed1988

HLA haplotype A1 B8 DR3 as a risk factor for HIV-related disease.

Of 32 patients exposed to a single batch of factor VIII contaminated with human immunodeficiency virus (HIV), 18 became antibody positive. Serial T cell subset analyses over the succeeding four years have shown a progressive decline in circulating T4 cells in those 18 but no change in the 14 who remain seronegative. 2 of the seroconverters have died and a further 7 have symptoms attributable to HIV infection. In the group as a whole, the HLA haplotype A1 B8 DR3 was weakly associated with an increased risk of seroconversion on exposure to the virus while, in those who seroconverted, it was strongly associated with a rapid decline in T4 cells and development of HIV-related symptoms within four years of infection.

Adult↗
PubMed1988

HIV antigen and antibody detection: variable responses to infection in the Edinburgh haemophiliac cohort.

Sequential serum samples from 18 haemophiliac patients exposed simultaneously to human immunodeficiency virus type 1 (HIV 1) in early 1984 were tested retrospectively for serological markers of infection. Assay for total antibodies to HIV established that the time to seroconversion might be as long as 110 days after exposure to contaminated factor VIII; serum samples were also tested by Western blotting, by enzyme linked immunosorbent assay (ELISA) for specific antibodies to envelope and core proteins, and for p24 antigen by two assay systems during the two years after infection. The studies showed that five of the 12 patients for whom serum samples obtained between exposure and seroconversion were available had transient p24 antigenaemia. Although amounts of total antibody to HIV and of antibodies to envelope proteins rose continuously during the two years of the study, amounts of antibody to the core protein were variable and tended to decline in patients who became symptomatic. Two patients had persistent p24 antigenaemia that began four months after seroconversion; these patients remained asymptomatic. One patient who developed the acquired immune deficiency syndrome (AIDS) had transient antigenaemia at the time of seroconversion but failed to show any antigen for the rest of the study; progression to AIDS was accompanied by an increase in antibodies to envelope proteins. Much of the variability in the course of infection with HIV must represent the differences in the susceptibility of the patients to infection.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Detection of antibody to viral proteins following primary infection with herpes simplex virus.

Sera from patients with primary genital infection with herpes simplex virus (HSV) were tested by immunoblotting (IB) and radioimmunoprecipitation (RIP) analysis to determine the protein targets of antibody elicited by infection. The two tests detected antibody to different antigens: IB primarily detected reactivity with p40, a phosphorylated capsid protein, and RIP detected antibody to glycoprotein B, a viral envelope component. Furthermore, RIP detected antibody at an earlier stage of infection then IB. A nondenaturing version of IB was developed and used to investigate the role that the solubilisation of antigen plays in the sensitivity of each test for antibodies with different specificities.

Animals↗
PubMedDate not supplied

Combination treatment of interferon alpha-2b and ribavirin in comparison to interferon monotherapy in treatment of chronic hepatitis C genotype 4 patients.

BACKGROUND AND AIM: Treatment of chronic hepatitis C patients, infected with genotype 4 with interferon-alpha yielded a limited response. Our aim was to compare the efficacy of interferon-alpha alone and in combination with ribavirin in chronic hepatitis C patients infected with genotype 4. PATIENTS: Fifty-two chronic hepatitis C patients (all males) infected with genotype 4, who had not received interferon, were randomized into 2 equal comparable groups. METHODS: Group I received interferon alpha-2b "Schering Plough" 3 MU, tiw combined with ribavirin (1000 mg/day). Group II received interferon alpha-2b alone in the same dose. Both groups were evaluated monthly, at the end of 24 weeks of treatment and 24 weeks later. Two patients were dropped from group I and one patient from group II. RESULTS: Biochemical response: at the end of treatment, a return to normal of ALT was obtained in 16/24 (66.7%) patients on combination therapy vs 8/25 (32%) patients on interferon alone (p = 0.0152). At the end of follow-up, a sustained response was achieved in 10/24 (41.7%) patients on combination therapy vs 4/25 (16%) patients on interferon (p = 0.0468). Virologic response: at the end of treatment, the rates of virological response were higher in the patients on combination therapy 9/24 (37.5%) than in those on interferon 4/25 (16%) (p = 0.0380). At the end of follow-up, loss of serum HCV RNA was reported in 5/24 (20.8%) patients on combination therapy vs 2/25 (8%) patients on interferon (p = 0.1916). Histologic response: mild histologic improvement was shown by a decrease in the inflammatory score, which was highest in patients in the combination group. CONCLUSIONS: In chronic hepatitis C patients infected with genotype 4, combination therapy with interferon-alpha and ribavirin was more effective than treatment with interferon monotherapy. At the end of the follow-up, about 50% of patients in both groups were still viraemic though their ALT remained normal.

Adult↗