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Biomedical subjects

P Timms

Publications and source records attributed to P Timms.

At least 19 recordsLinked to original sources
PubMed2003

A mathematical model for the investigation of the Th1 immune response to Chlamydia trachomatis.

Chlamydia are bacterial pathogens of humans and animals causing the important human diseases trachoma, sexually transmitted chlamydial disease and pneumonia. Of the human chlamydial diseases, sexually transmitted disease caused by Chlamydia trachomatis is a major public health concern. Chlamydia trachomatis replicates intracellularly and is characterised by a complex developmental cycle. Chlamydia is susceptible to humoral and cell-mediated immunity. Here we investigate the Th1 cell-mediated immune response against Chlamydia-infected cells as the response changes over the chlamydial developmental cycle. We suggest a form for the immune response over one developmental cycle by modelling the change in the number of intracellular chlamydial particles and assume peptides are presented in proportion to the number of replicating forms of chlamydial particles. We predict, perhaps non-intuitively, that persistent Chlamydia should be induced and forced not to return to the lytic cycle. We also suggest that extending the length of the time of the lytic cycle will effectively decrease the required efficacy of the Th1 response to eliminate the pathogen. We produce plots of active disease progression, control and clearance for varying levels of Th1 effectiveness.

Chlamydia Infections↗
PubMed2002

Screening semen from koalas (Phascolarctos cinereus) for Chlamydia species by PCR.

Artificial insemination is a valuable method for facilitating genetic exchange between captive colonies of koalas (Phascolarctos cinereus) and for the maintenance of genetically important remnant populations. However, to reduce potential disease transmission, their semen needs to be screened for venereal diseases caused by organisms such as Chlamydia species. Semen samples from 11 koalas, eight of them with clinical signs of cystitis, were examined for the presence of Chlamydia by an optimised PCR assay. Chlamydia was detected in semen from seven of the 11 animals.

Animals↗
PubMed2002

Reduced levels of gamma-interferon secretion in response to chlamydial 60 kDa heat shock protein amongst women with pelvic inflammatory disease and a history of repeated Chlamydia trachomatis infections.

Peripheral lymphocytes in uninfected fertile controls, women with various histories of Chlamydia trachomatis infection, pelvic inflammatory disease (PID) and infertility not due to C. trachomatis infection (endometriosis) were cultured overnight with PHA mitogen and the 60 kDa chlamydial heat shock protein. Plasma samples were then assayed for levels of gamma-interferon and IL-10 using a commercial ELISA system. Women with PID and those with a history of multiple C. trachomatis infections showed reduced gamma-interferon production in response to cHSP60, not seen in women infected only once and those with infertility due to other causes (endometriosis). Secretion of IL-10 in response to cHSP60 did not vary significantly across the various patient groups, though all patients showed elevated levels of total IL-10 compared with uninfected controls.

Bacterial Outer Membrane Proteins↗
PubMed2002

Novel PCR-EIA method for the detection of Chlamydia pneumoniae in respiratory specimens.

We report the development of a microtitre plate-based PCR-EIA assay (ELAHA; Enzyme Linked Amplification and Hybridization Assay) for the sensitive and specific detection of Chlamydia pneumoniae in sputum samples from patients with chronic obstructive airways disease (COAD). Following PCR amplification of a segment of the chlamydial heat shock 60 protein gene, the 587 bp sized amplicon is captured onto the streptavidin coated surface of a microtitre plate using a C. pneumoniae specific biotinylated probe and the level of captured product is subsequently determined via a colorimetric reaction using an automated plate reader. The ELAHA is a simple, rapid and inexpensive method for detection of low levels of infectious agents and is readily adaptable to current clinical laboratory equipment. The assay was evaluated with a cohort of hospital respiratory patients: (i) COAD patients with acute exacerbation, (ii) COAD patients without exacerbation (stable) and (iii) a non-respiratory control group. The ELAHA produced 6/12 (50%) C. pneumoniae positives in the COAD with exacerbation group, 3/13 (23%) positives in the COAD without exacerbation group and only 1/6 (17%) positives in the control non-respiratory group. This sensitive and robust PCR-EIA method can provide clinically relevant diagnostic evidence of current C. pneumoniae infection contributing to serious respiratory tract diseases such as COAD.

Case-Control Studies↗
PubMed2001

Differential expression of ompA, ompB, pyk, nlpD and Cpn0585 genes between normal and interferon-gamma treated cultures of Chlamydia pneumoniae.

A common feature of many chlamydial infections is that they are often asymptomatic and may persist for long periods of time if left untreated. In addition to the well recognized lytic stage of the chlamydial developmental cycle, evidence is now emerging to support a persistent phase in the cycle in which the reticulate bodies are morphologically abnormal, viable but non-infectious and presumably also have altered gene expression patterns. We used an RT-PCR approach to study the differential levels of gene transcription for 14 genes (16SrRNA, ompA, ompB, omcB, 76 kDa, gseA, pmp1, gltX, hsp60, yaeT, pyk, nlpD, Cpn0585, Cpn1046) between normal and IFN-gamma treated Chlamydia pneumoniae cell cultures. Even though the level of morphologically abnormal reticulate bodies in our IFN-gamma treated cultures was low (approximately 10% morphologically discernible, although presumably a larger percentage were in the persistent state but not yet morphologically altered) we identified five genes (ompA, ompB, pyk, nlpD, Cpn0585) that were clearly upregulated when compared to normal cultures. This gene transcript profile may be characteristic of a general stress state in Chlamydia, induced by IFN-gamma treatment in this case, but perhaps more widely induced in other in vitro and in vivo situations.

Chlamydophila pneumoniae↗
PubMed2001

Peptide immunization of guinea pigs against Chlamydia psittaci (GPIC agent) infection induces good vaginal secretion antibody response, in vitro neutralization and partial protection against live challenge.

Immunization of female guinea pigs with a chimeric peptide consisting of variable domain IV (VDIV) and a region known as GP8 from the major outer membrane protein of Chlamydophila caviae, formerly Chlamydia psittaci guinea pig inclusion conjunctivitis strain, was performed to assess whether humoral immune responses could be elicited in the reproductive tracts of immunized animals. The C. caviae strain is able to cause a sexually transmitted infection in the guinea pig that closely parallels C. trachomatis infections in humans. The best anti-VDIV antibody response in vaginal secretions was achieved by intraperitoneal priming with subsequent intravaginal boosting (P < 0.001). Dot-blot analyses of vaginal secretions confirmed that these anti-VDIV antibodies, produced against a linear peptide, were able to recognize and bind to whole conformational C. caviae elementary bodies. Following live intravaginal challenge with C. caviae, a significant reduction in the intensity (P = 0.01) and an apparent reduction in the duration of the infection was evident between the guinea pigs immunized with VDIV-GP8 and non-immunized controls.

Animals↗
PubMed2001

Solid-phase amplification and detection: a single-tube diagnostic assay for infectious agents.

BACKGROUND: We report the development of an enzyme-linked immunosorbent assay-like single-tube assay for the detection of infectious agents in a microtiter tray format. METHODS AND RESULTS: The method, sequential nucleic acid amplification and capture (SNAAC), combines amplification with hybridization of the product to a surface/matrix-bound oligonucleotide probe. After amplification of the target sequence using species-specific primers, one of which contains a detection tag such as fluorescein or biotin, a denaturation and hybridization cycle is performed. This allows capture by an oligonucleotide that is covalently bound to the surface of a microtiter tray well or other support. After washing to remove unincorporated solution-phase oligonucleotide bearing the detection tag, the level of captured product is determined through a colorimetric reaction using an automated plate reader. We show the value and utility of the SNAAC detection method using cloned sequences of the important human respiratory pathogen Chlamydia pneumoniae. CONCLUSIONS: SNAAC is a simple, rapid, and inexpensive method for the detection of low levels of infectious agents that is readily adaptable to current clinical laboratory equipment, thus avoiding the need to develop or purchase new instrumentation.

Biotin↗
PubMed2001

Koala biovar of Chlamydia pneumoniae infects human and koala monocytes and induces increased uptake of lipids in vitro.

We examined the ability of the koala biovar of Chlamydia pneumoniae to infect both Hep-2 cells and human monocytes and the effect of infection on the formation of foam cells. The koala biovar produced large inclusions in both human and koala monocytes and in Hep-2 cells. Koala C. pneumoniae induced foam cell formation with and without added low-density lipoprotein, in contrast to TW183, which produced increased foam cell formation only in the presence of low-density lipoprotein.

Animals↗
PubMed2001

Ultrastructural study of Chlamydia pneumoniae in a continuous-infection model.

We have established an in vitro model of long-term continuous Chlamydia pneumoniae infection in HEp-2 cells. Using transmission electron microscopy, we demonstrated the presence of spontaneous abnormal chlamydial inclusions similar in appearance to the persistent chlamydial forms induced in vitro by treatment with cytokines or antibiotics or by nutrient deprivation.

Chlamydophila Infections↗
PubMed2001

Development of ligase-assisted spacer addition for the measurement of microsatellites.

Conventional methods for detecting differences in microsatellite repeat lengths rely on electrophoretic fractionation on long denaturing polyacrylamide gels, a time-consuming and labor-intensive method. Therefore, there is a need for the development of new and rapid approaches to routinely detect such length polymorphisms. The advent of techniques allowing the coupling of DNA molecules to solid surfaces has provided new prospects in the area of mutation detection. We describe here the development and optimization of the ligase-assisted spacer addition (LASA) method, a novel and rapid procedure based on an ELISA format to measure microsatellite repeat lengths. The LASA assay was successfully applied to a set of 11 bird samples to assess its capabilities as a genotyping method.

Animals↗
PubMed2000

Chlamydia trachomatis infection: incidence, health costs and prospects for vaccine development.

Chlamydia trachomatis infection is now the most common sexually transmitted disease worldwide. World Health Organisation figures estimated that 89 million new cases of genital Chlamydia infections occurred in 1995, highlighting the worldwide prevalence of infections and the economic burden on healthcare delivery. A number of methods have been developed for detection of chlamydial infection, which vary in sensitivity and specificity. No single method has yet gained general acceptance and in many countries Chlamydia infections are not reported, suggesting that the above figures may be an underestimate of the problem. As yet there is no consensus as to what constitutes a protective immune response against genital Chlamydia infection. Studies in animal models have shown that cell-mediated immunity, both Th1-driven macrophage activation and cytotoxic T cell responses, as well as antibody can mediate protection at different stages of the chlamydial life cycle. A successful vaccine would probably need to elicit both a systemic cell-mediated immune response to limit/resolve established infections and a mucosal IgA response to reduce bacterial shedding and the resulting spread of infection to partners of infected individuals. The immune response to Chlamydia, either through natural infection or following immunisation, also has the potential to enhance inflammation and to act as a driving force for constant mutation in the variable regions of the major outer membrane protein. As a result a constant prevalence of infection is maintained even in an immune population through the emergence of new allelic variants. Immune responses against antigens such as the 60 kDa heat shock protein can exacerbate inflammation through molecular mimicry and must not be elicited as a result of vaccination. Thus there are many challenges for the development of a successful vaccine which must elicit immunity against multiple serovars while at the same time minimising damaging pro-inflammatory immune responses.

Animals↗
PubMed2000

Genetic diversity and gene flow among southeastern Queensland koalas (Phascolarctos cinereus).

Habitat fragmentation and destruction associated with the rapid urban and rural development of southeast Queensland presents an immediate threat to the survival of koala populations within this region. A sensitive method combining heteroduplex analysis (HDA) with temperature gradient gel electrophoresis (TGGE) was optimized to detect within-species variation in a mitochondrial DNA (mtDNA) control-region fragment, approximately 670 bp in length, from the koala. Eight different haplotypes were characterized in koalas, of which four were novel. Analysis of mtDNA diversity in 96 koalas from five populations in southeast Queensland revealed that the number of haplotypes in a single population ranged from one to five, with an average within-population haplotype diversity of 0.379 +/- 0.016, and nucleotide diversity of 0.22 +/- 0.001%. Nucleotide divergence between populations averaged 0.09 +/- 0.001% and ranged from 0.00 to 0.14%. Significant genetic heterogeneity was observed among most populations, suggesting that koala populations may be spatially structured along matrilines, although this may not be universal. The limited distribution of the central phylogenetic haplotype suggested the possibility of historical population bottlenecks north of the Gold Coast, while the presence of two highly divergent haplotypes at the Moreton site may indicate the occurrence of one or more undocumented translocation events into this area.

Animals↗
PubMed2000

Detection of Chlamydia pneumoniae DNA and antigen in the circulating mononuclear cell fractions of humans and koalas.

Chlamydia pneumoniae is a common respiratory pathogen of humans which, in addition to causing disease at the respiratory site, has recently been linked to disease at other body sites. If C. pneumoniae does contribute to disease at nonrespiratory sites, then it must have a mechanism by which it reaches these sites. We analyzed the peripheral blood mononuclear cell (PBMC) fractions from 60 healthy human blood donors for the presence of C. pneumoniae DNA (by ompA PCR) and chlamydial antigens (by genus- and species-specific monoclonal antibody staining). Ten of the sixty (16. 7%) blood donors were C. pneumoniae positive by PCR, and all 10 of these PCR-positive individuals' samples demonstrated specific staining with anti-C. pneumoniae monoclonal antibodies. The only other host naturally infected with C. pneumoniae is the koala, in which the bacterium also causes respiratory infections. We demonstrated the presence of C. pneumoniae DNA and antigens in the PBMC fractions of 30% of 20 koalas tested. Our finding of C. pneumoniae-infected PBMCs in koalas as well as humans suggests that the ability to infect PBMCs and to disseminate from the respiratory site is not restricted to the human biovar of C. pneumoniae but is a general characteristic of this chlamydial species.

Animals↗
PubMed2000

Identification and mapping of sigma-54 promoters in Chlamydia trachomatis.

The first sigma(54) promoters in Chlamydia trachomatis L2 were mapped upstream of hypothetical proteins CT652.1 and CT683. Comparative genomics indicated that these sigma(54) promoters and potential upstream activation binding sites are conserved in orthologous C. trachomatis D, C. trachomatis mouse pneumonitis strain, and Chlamydia pneumoniae (CWL029 and AR39) genes.

Base Sequence↗
PubMed1999

Development of a quantitative gene expression assay for Chlamydia trachomatis identified temporal expression of sigma factors.

Chlamydia trachomatis is an important human pathogen which possesses a unique bi-phasic developmental cycle. We used lightcycler methodology to quantitatively measure gene transcript levels in C. trachomatis strain L2. By measuring 16S rRNA transcript levels, we determined C. trachomatis L2 to have a generation time of approximately 3 h and an inclusion burst size of 200-300 particles. The three chlamydial sigma factor genes rpoD (sigma66), rpsD (sigma28) and rpoN (sigma54) exhibited different patterns of temporal expression. rpoD was central to early chlamydial development, whereas rpsD and rpoN were temporally expressed, coinciding with elementary body (EB) to reticulate body (RB) conversion and RB to EB conversion, respectively.

Cell Line↗
PubMed1999

Epizootiology of Chlamydia infections in two free-range koala populations.

The prevalence of Chlamydia pecorum and Chlamydia pneumoniae infections in two free-range koala populations was assessed using genus-specific PCR combined with species-specific DNA probe hybridisation. Population A had a very high overall level of chlamydial infection (85%) with significantly more of these infections being due to C. pecorum (73%) compared to C. pneumoniae (24%). The second population had a much lower prevalence of infection (10%) with equal levels of both species. An important finding of this study was that. while five of 24 C. pecorum-infected koalas had clinical signs of the disease (both ocular and urogenital sites), none out of seven C. pneumoniae-infected koalas had signs of clinical disease. This suggests that C. pecorum may be the more pathogenic of the two chlamydial species infecting this host. The level of infection (assessed by intensity of the specific hybridisation signal) also differed between chlamydial species, with C. pecorum infections ranging from low to high grade whereas C. pneumoniae infections were always low grade. When the age of infected koalas was examined, 58% of young, sexually immature koalas were found to have C. pecorum infections, increasing to 100% of koalas in the older age groups. This suggests that, in this population at least, young koalas are readily infected with C. pecorum from their mothers. While the infection levels with C. pneumoniae were too low to be statistically significant, again, sexually immature koalas were found to be infected. The recent separation of chlamydial infections in koalas into two species is beginning to indicate different epizootiologies for koala C. pecorum compared to koala C. pneumoniae.

Age Factors↗