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J H Pearce

Publications and source records attributed to J H Pearce.

At least 19 recordsLinked to original sources
PubMed2001

Induction of abnormal Chlamydia trachomatis by exposure to interferon-gamma or amino acid deprivation and comparative antigenic analysis.

Abnormal forms of Chlamydia trachomatis have been induced in vitro by a variety of methods including nutrient deprivation, addition of cytokines and addition of antibiotics. These forms have been shown to have altered morphology and infectivity and have been implicated in persistent infections in vivo although there is little direct evidence for their presence. Likely sites for abnormal forms in vivo are the genital tract and the synovial tissue of reactive arthritis patients, and T cells isolated from the synovial tissue have been shown to be specific for chlamydial antigens, in particular the Hsp60. Since T cell specificity is so important in reactive arthritis disease the antigenic composition of abnormal forms induced by Interferon-gamma and amino acid deprivation has been examined by western blotting in two strains of C. trachomatis belonging to different biovars. The degree of abnormality of the organisms was found to increase as the treatments became more severe. No simple patterns of antigenic changes were found and differences in the antigenic composition were seen in abnormal forms induced by the different treatments and also in the different strains.

Amino Acids↗
PubMed2000

Chlamydial development is adversely affected by minor changes in amino acid supply, blood plasma amino acid levels, and glucose deprivation.

This study has demonstrated the extreme sensitivity of Chlamydia trachomatis growing in McCoy cells to small changes in external amino acid supply. In the absence of cycloheximide, a decrease in the amino acid concentration of medium to 75% of control values was sufficient to induce the growth of enlarged chlamydial forms of reduced infectivity. Morphology became more distorted and the yield of infectious particles from inclusions declined as medium amino acid levels were further reduced. These events correlated with a general decline in intracellular amino acids, as measured by high-performance liquid chromatography, suggesting that chlamydiae require a minimum concentration of each amino acid for normal development. Cycloheximide enhanced the production of normal organisms and increased infectivity yield in media, suggesting that the drug increased the available pool of amino acids. This was supported by intracellular amino acid analyses. Aberrant forms with reduced infectivity were also induced during supply of infected cell cultures with medium containing blood plasma amino acid concentrations, supporting the proposal that nutrient levels in vivo could promote abnormal chlamydial development. Markedly abnormal forms were also observed during glucose deprivation, providing further evidence that aberrant development is a general stress-related response.

Amino Acids↗
PubMed2000

Amino acid transport into cultured McCoy cells infected with Chlamydia trachomatis.

Amino acid transport into McCoy cells infected with strains representative of the two major biovars of Chlamydia trachomatis has been studied to determine if uptake is increased during infection. Preliminary work suggested that the transport systems L, A/ASC (for neutral amino acid transport), N (for transport of Asn, Gln, and His) and y+ (for cationic amino acids) were present in McCoy cells. With lymphogranuloma venereum biovar strain 434, little difference in the influx of representative amino acids Trp, His, and Lys or the analogue 2-aminoisobutyric acid (AIB) was observed during infection. With trachoma biovar strain DK20, a small increase in the initial entry rate and equilibrium concentration of each amino acid was found. McCoy cells appear to have great capacity for concentrating amino acids, which might obviate the need for transport induction by chlamydiae under conditions favoring the growth of infectious organisms.

Amino Acids↗
PubMed1999

Peripheral blood T cell proliferative response to chlamydial organisms in gonococcal and non-gonococcal urethritis and presumed pelvic inflammatory disease.

OBJECTIVE: To study peripheral blood mononuclear cell (PBMC) proliferative response to Chlamydia trachomatis elementary bodies in (a) controls, (b) various stages of gonococcal (c) and non-gonococcal urethritis, and (d) women with a clinical diagnosis of pelvic inflammatory disease (PID). METHODS: We categorised 102 men presenting to a GUM clinic with urethritis by organisms (C trachomatis (CT) or Neisseria gonorrhoeae (NG) (both by culture), and whether it was their first (urethritis naive) or subsequent (urethritis experienced) attack. 23 women presenting to the clinic with a clinical diagnosis of PID were also investigated. We measured PBMC proliferative responses to C trachomatis (DK20--an oculogenital strain, serovar E), lysate of McCoy cells (used to propagate chlamydiae), and the recall antigen PPD. Controls were 37 men and women without present or past history of urethritis or chlamydial infection. Results were expressed as the ratio of the stimulation index (SI) obtained with DK20 compared with McCoy cells (DK index), and the ratio of the SI obtained with DK20 compared with PPD (PPD index). RESULTS: The median SI to DK20 in the urethritis was 12.7 which was significantly higher than the controls (7.6, p < 0.003). The median SI to the recall antigen PPD was similar in the urethritis patients (17.4) and the controls (22.4). All urethritis patient subgroups had a significantly higher DK index and PPD index than the controls. There was no difference in the PPD and DK index between urethritis naive and urethritis experienced patients and between the culture positive and culture negative urethritis subgroups. In PID patients only the PPD index was significantly higher than the controls. CONCLUSION: Men presenting with urethritis and women presenting with PID both have significantly greater peripheral blood mononuclear cell proliferative responses to the DK20 strain of C trachomatis than controls. A similar T cell proliferative response pattern in urethritis naive patients with either gonococcal or non-gonococcal urethritis could be because low sensitivity of CT culture failed to diagnose some cases of C trachomatis. However, it may also signify earlier exposure of the patients to chlamydial antigens (for example, C pneumoniae), cross reacting antigens such as heat shock proteins from other microbial species, or a "bystander" activation of chlamydia specific memory T cells trafficking through mucosal lymphoid tissue during urethritis. These results suggest evidence of T cell mediated response to C trachomatis cannot be used as a diagnostic tool.

Adult↗
PubMed1998

Isolation and characterisation of T lymphocytes from the urethra of patients with acute urethritis.

OBJECTIVES: To investigate local cellular immune responses in patients with acute urethritis. METHODS: We have established T cell lines from the urethral exudate and examined their phenotype by flow cytometry. As controls, T cell lines were cultured from first pass urine specimens of asymptomatic healthy individuals. RESULTS: Using interleukin 2 (IL-2) alone a T cell line was obtained on only one occasion. Following culture with IL-2, and subsequent expansion by a single stimulation with irradiated allogenic peripheral blood mononuclear cells (PBMC), phytohaemagglutinin (PHA), and IL-2, it was possible to establish T cell lines from 6/6 acute urethritis patients. T cell lines were also obtained from 4/12 controls subjects, but required repetitive rounds of stimulation with mitogen and allogeneic PBMC to produce sufficient cell numbers for analysis. Three of the patient T cell lines were dominated by T cells expressing the gamma delta receptor. CONCLUSION: The gamma delta T cell subset has been associated with immune responses at mucosal surfaces and has the ability to recognise certain bacterial antigens. The gamma delta T cell response may represent an important aspect of the immune response to organisms associated with acute urethritis.

Adult↗
PubMed1997

Identification and characterization of a DR4-restricted T cell epitope within chlamydia heat shock protein 60.

An epitope within the 60 kD Chlamydia trachomatis heat shock protein (hsp) 60, recognized by a HLA-DRB1*0401-restricted T cell clone from a reactive arthritis patient, has been characterized. Stimulatory peptides contained a nine amino acid sequence (residues 38-46) predicted by algorithm to confer strong binding to DRB1*0401, with valine in the P1 position. The overall length of the peptide was critical for efficient recognition; peptides with at least one residue N-terminal to the putative P1 position were markedly more stimulatory than a peptide whose N-terminal is the P1 valine. Optimal responses were seen with 14mer peptides having two to three amino acids N- and C-terminal to the core 9mer. The sequence of the defined epitope is identical in hsp60 from both C. trachomatis and C. pneumoniae. Since the latter is a common respiratory pathogen, patients infected with C. trachomatis may already be primed for responses to hsp60 by prior infection with C. pneumoniae. Such secondary responses are important in the pathogenesis of chlamydia-induced inflammatory diseases such as trachoma. Priming by infection with enteric organisms was considered because of the similarity of the epitope sequence in Escherichia coli hsp60. However, although an E. coli-related peptide was recognized, intact E. coli hsp60 was not, suggesting that the epitope is cryptic in E. coli hsp60. Human hsp60 has six amino acid differences from chlamydial hsp60 in the epitope sequence and was not recognized. Thus cross-reactive recognition of self hsp60 could not be implicated in the pathogenesis of chlamydia-induced reactive arthritis in this patient.

Adult↗
PubMed1996

Induction of alpha/beta interferon and dependent nitric oxide synthesis during Chlamydia trachomatis infection of McCoy cells in the absence of exogenous cytokine.

The propensity of two Chlamydia trachomatis strains (L2/434/Bu [biovar LGV] and E/DK20/ON [biovar trachoma]) to induce putative host defense responses upon infection of McCoy (mouse) cell cultures was examined. Both strains induced production of alpha/beta interferon and nitric oxide (NO) by McCoy cells. NO synthesis was mediated by the inducible isoform of NO synthase as indicated by the ability of cycloheximide or the arginine analog NG-monomethyl-L-arginine to abolish NO production; the extent of the response was dependent upon the dose of chlamydiae applied. Incubation of McCoy cells with chloramphenicol prior to infection reduced NO production by strain 434 but not by DK20, suggesting that initial chlamydial metabolism was essential to induction by the LGV strain. Antibody inhibition studies indicated that NO synthesis was dependent upon production of alpha/beta interferon and induction via lipopolysaccharide. Overall, our findings show that chlamydiae are capable of the induction of interferon and NO in murine fibroblasts in the absence of exogenous cytokines. However, the role of NO as an antichlamydial effector could not be clearly demonstrated since treatment with an arginine analog, while suppressing NO production, gave no consistent enhancement of infected cell numbers.

Animals↗
PubMed1996

Identification of 2 Chlamydia trachomatis antigens recognized by synovial fluid T cells from patients with Chlamydia induced reactive arthritis.

OBJECTIVE: To identify antigens of Chlamydia trachomatis recognized by synovial T cell clones from patients with reactive arthritis (ReA). METHODS: C. trachomatis specific T cell clones were isolated from synovial fluid of 2 patients with chlamydia induced ReA. The particular antigens/epitopes recognized by these clones were identified using T cell immunoblotting and testing recombinant chlamydial proteins and synthetic peptides. RESULTS: Two sets of clones were shown by immunoblotting to recognize antigens of roughly 60 and 18 kilodaltons (kDa) respectively. By testing recombinant chlamydial proteins these antigens were identified as the 57 kDa heat shock protein and the 18 kDa histone-like protein, Hc1. Mapping the epitope in Hc1 using synthetic peptides identified a peptide containing a sequence motif compatible with binding to HLA-DR1, the restricting antigen for the Hc1 specific clones. CONCLUSION: These are the first 2 chlamydial antigens to be identified as targets of the synovial T cell response in chlamydia induced ReA. Both have properties that are shared with target antigens identified in ReA induced by enteric infection and relevant to the pathogenesis of joint inflammation.

Adult↗
PubMed1993

Low-nutrient induction of abnormal chlamydial development: a novel component of chlamydial pathogenesis?

The intracellular development of chlamydiae in McCoy cells incubated in Eagle's minimal essential medium lacking all 13 amino acids was examined both by fluorescence and electron microscopy and by infectivity titration. Aberrant development occurred in almost all inclusions of strains of Chlamydia trachomatis and C. psittaci with the production of abnormal forms which differed in size, shape and internal structure from normal reticulate and elementary body forms. Detailed analysis of the response of C. trachomatis L2 strain 434 to graded reductions in amino acid level showed that infectivity was reduced and morphological abnormality increased as amino acid concentrations were lowered from 33 to 0% of amino acids present in minimal essential medium. Reversion of inclusions to normal and reappearance of infectious forms occurred on restoration of amino acids and further incubation. It is suggested that aberrant development may account for the presence in vivo of non-cultivable chlamydiae and that such development can arise via tryptophan deprivation mediated by local release of interferon gamma.

Amino Acids↗
PubMed1993

Chlamydia trachomatis Mip-like protein has peptidyl-prolyl cis/trans isomerase activity that is inhibited by FK506 and rapamycin and is implicated in initiation of chlamydial infection.

The Mip-like protein of Chlamydia trachomatis has sequence similarity with both the Mip protein of Legionella pneumophila, a virulence factor necessary for optimal intracellular infection, and FK506-binding proteins (FKBPs) of both prokaryotic and eukaryotic origin. FKBPs contain a site for peptidyl-prolyl cis/trans isomerase activity, which is blocked upon binding of the drugs, FK506 or rapamycin. In this paper we report that the recombinant chlamydial Mip-like protein exhibits a peptidyl-prolyl cis/trans isomerase activity which is inhibited by either rapamycin or FK506. To assess the role of the Mip-like protein in chlamydial infection, rapamycin or FK506 (25 microM), were used in either treatment of chlamydial organisms prior to inoculation, or were present at different intervals through the infection. Pretreatment of organisms alone reduced infectivity for McCoy cells by 30%, with inhibition rising to 80% on more prolonged exposure from 0 to 8h and 8 to 16h post-inoculation and declining thereafter. When drug was present during the developmental cycle at intervals from 0 to 24h post-inoculation abnormal chlamydiae were induced in residual inclusions. The results suggest that inhibition of the isomerase of the Mip-like protein interferes with one or more early events in the infective process that determine productive intracellular infection.

Amino Acid Sequence↗
PubMed1993

The Chlamydia trachomatis Mip-like protein is a lipoprotein.

The Mip-like protein of Chlamydia trachomatis is similar to the Mip protein of Legionella pneumophila and may be equally important for the initiation of intracellular infection. This article presents data which identify the chlamydial Mip-like protein as a lipoprotein. The amino acid sequence of the Mip-like protein contains a signal peptidase II recognition sequence, as is seen in procaryotic lipoproteins. Palmitic acid was incorporated into the recombinant chlamydial Mip-like protein. Globomycin, known to inhibit signal peptidase II, inhibited processing of the recombinant Mip-like protein. Labelling of chlamydial organisms with palmitic acid revealed incorporation into the native Mip-like protein.

Amino Acid Sequence↗
PubMed1993

Identification of T-cell stimulatory antigens of Chlamydia trachomatis using synovial fluid-derived T-cell clones.

Chlamydia trachomatis is a major cause of sexually transmitted disease, infertility and reactive arthritis in the Western world, and of trachoma in the developing world. There is evidence that the chronic inflammatory reaction seen in diseases associated with chlamydiae represents a delayed-type hypersensitivity response to chlamydial antigens. Little is known about which chlamydial antigens elicit T-cell responses yet such information could have important implications in terms of both immunopathological understanding of these diseases and immunoprophylaxis design. In this study, 61 chlamydia-specific T-cell clones have been produced from the synovial fluid of an individual with sexually acquired reactive arthritis (SARA). Ten clones have been characterized in detail and used to identify T-cell stimulatory antigens of chlamydiae by means of T-cell immunoblotting. Two distinct antigenic fractions have been identified, one recognized by three of the clones (molecular weight 18,000), the other recognized by six of the clones (molecular weight 30,000). The fractions are distinct from the major outer membrane protein, the 57,000 MW stress protein and the 60,000 MW cysteine-rich membrane protein of chlamydiae. The major histocompatibility complex (MHC) restriction of the response to these antigens differed: clones recognizing the 18,000 MW antigen required antigen-presenting cells expressing DR1 subtype DRB1*0101 or DRB1*0102 which only differ at amino acids 85 and 86 on the DR beta-chain; by contrast clones recognizing the 30,000 MW antigen were presented to only by antigen-presenting cells from DRB1*0101 individuals, reflecting extreme sensitivity of these clones to the polymorphism at positions 85 and 86 on the DR beta-chain.

Adult↗
PubMed1992

Chlamydia trachomatis Mip-like protein.

A 27 kDa Chlamydia trachomatis Mip-like protein with homology of a 175-amino-acid C-terminal fragment to the surface-exposed Legionella pneumophila mip-gene product has previously been described. In this paper the entire chlamydia Mip-like sequence of C. trachomatis serovar L2 (lymphogranuloma venereum (LGV) biovar) is presented. The sequence shows high similarity to the legionella Mip protein and its C-terminal region, like that of the legionella Mip, has high amino acid similarity to eukaryotic and prokaryotic FK506-binding proteins. The chlamydial mip-like gene was detected by polymerase chain reaction (PCR) in other C. trachomatis serovars and by sequencing of the mip-like genes of serovars B and E (trachoma biovar) was shown to be highly conserved within the two major biovars of C. trachomatis. Monoclonal and polyclonal antibodies raised against the recombinant Mip-like protein failed to demonstrate surface-exposed epitopes on infectious elementary bodies or reproductive reticulate body forms either by immunofluorescence or immuno-gold electron microscopy. However, a complement-dependent inhibition of up to 91% of infectivity for cell cultures was observed with antibodies to the N-terminal fragment of the Mip-like protein suggesting that antibody-accessible epitopes are present on infectious EBs.

Amino Acid Sequence↗
PubMed1991

Analysis of the human serological response to Chlamydia trachomatis 60-kDa proteins by two-dimensional electrophoresis and immunoblotting.

Earlier analyses of sera from humans and animals with chlamydial infections demonstrated a strong immune response to proteins of approximately 60 kDa. We have used two-dimensional (2D) electrophoresis followed by immunoblotting to more accurately define patient responses to the chlamydial 60-kDa cysteine-rich envelope protein (OMP2) and to the 57-kDa stress protein (SP) implicated in immunopathological damage. OMP2 and SP were separated on 2D gels by their distinct isoelectric points and identified by fluorography of [35S]-labelled proteins and cross-reaction with anti-mycobacterial antiserum, respectively. The majority of patients sera showed a strong reaction both to OMP2 and the SP (18/20 and 17/20 sera, respectively). Fewer sera (9/20 and 10/20 sera, respectively) reacted with two other polypeptides also present in the 60-kDa range by this analysis.

Antibodies, Bacterial↗
PubMed1991

Endocytic mechanisms utilized by chlamydiae and their influence on induction of productive infection.

The microfilament-disrupting drug cytochalasin D and, initially, inoculation at 20 degrees C were used to differentiate between phagocytosis (sensitive to both treatments) and pinocytosis (resistant to both treatments) to assess whether chlamydial uptake into McCoy cells occurred by one or both mechanisms and whether each could contribute to productive infection. Both treatments suppressed the infectivity of Chlamydia trachomatis L2/434/Bu and C. psittaci GPIC (the guinea pig inclusion conjunctivitis strain) following static inoculation by only 50%, indicating that there was simultaneous operation of both phagocytosis and pinocytosis during uptake that led to productive infection. Measurement of the entry of organisms by two separate assays established that both strains predominantly used a cytochalasin D-resistant (pinocytic) mechanism, implying that phagocytic uptake was coupled to a higher frequency of productive infection. Integration of the data on infectivity and entry allowed the potential for an organism to infect a host cell to be quantified. This synthesis revealed that for both strains the infectivity potential following phagocytic entry was ca. 10-fold greater than that following pinocytic entry. However, both entry mechanisms were exploited more efficiently by strain L2/434/Bu than by strain GPIC (unless the latter was inoculated with centrifugation), indicating that intrinsic strain properties are more important for infectivity potential than the endocytic mechanism utilized.

Cell Line↗
PubMed1990

Characterization of the cytochalasin D-resistant (pinocytic) mechanisms of endocytosis utilized by chlamydiae.

The cytochalasin D-resistant (pinocytic) portion of the entry of two chlamydia strains (Chlamydia trachomatis L2/434/Bu and Chlamydia psittaci GPIC [guinea pig inclusion conjunctivitis]) was examined. By ultrastructural criteria, few organisms of either strain were observed in association with coated host-cell plasma membrane during entry into McCoy cells; this argues against a coated-pit mechanism of entry. When association with a coated membrane was seen, coat material appeared to pinch off ahead of internalizing chlamydiae. However, entry of both strains was substantially reduced by cytosol acidification, a procedure shown to prevent coated-pit vesiculation (K. Sandvig, S. Olsnes, O. W. Petersen, and B. van Deurs, J. Cell Biol. 105:679-689, 1987). No conclusive evidence of displacement of the fluid-phase marker [3H]sucrose from constitutively forming endocytic vesicles was found. Indeed the entry of strain 434 (but not strain GPIC) was accompanied by the influx of a large volume of fluid, suggesting an inducible mechanism. Additionally, entry of strain 434 (but not strain GPIC) was partially inhibitable by amiloride, yet the drug had no effect on the entry of transferrin, a ligand known to enter solely via coated pits. Our findings endorse the view that chlamydial entry can occur via a pathway involving coated pits. However, the unusual morphology of entry and lack of fluid exclusion are consistent with a process whereby although chlamydiae are not fully enclosed by coat material, their entry is dependent on the vesiculation of coated pits. Furthermore, the data support the proposition that a significant proportion of the entry of strain 434 occurs via an inducible pathway independent of coated-pit uptake.

Ammonium Chloride↗