Health, health promotion, and homelessness.
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Delivery of Health Care↗
Biomedical subjects
P Timms
Publications and source records attributed to P Timms.
Characterization of the koala biovar of Chlamydia pneumoniae at four gene loci--ompAVD4, ompB, 16S rRNA, groESL spacer region.
Koalas are infected with two species of Chlamydia, C. pecorum and C. pneumoniae. While it is known that significant genetic diversity occurs in the C. pecorum strains infecting koalas, very little is known about the C. pneumoniae strains that infect this host. In the current study, 10 isolates of koala C. pneumoniae were analysed at four gene loci and found to be different to both the human and horse C. pneumoniae strains at all loci (biovar differences ranging from 0.3% at groESL up to 9.0% at ompAVD4). All koala biovar isolates studied were found to be 100% identical at ompAVD4 (all 10 isolates) and at ompB (all three isolates) gene. This lack of allelic polymorphisms at ompAVD4 has now been observed for koala C. pneumoniae, human C. pneumoniae, guinea pig inclusion conjuctivitis C. psittaci and feline conjuctivitis C. psittaci and may be correlated to a lack of antibody response to the chlamydial major outer membrane protein (MOMP) in these same strain/host combinations. This study also provides the first documented case of natural C. pneumoniae infection causing a severe and extended respiratory episode in a captive koala population. This captive episode is in contrast to most free-range observations in which koala C. pneumoniae is rarely documented as causing respiratory, ocular or urogenital tract disease.
Animals↗
Plasma levels of matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinases -1 and -2 (TIMP-1 and TIMP-2) as noninvasive markers of liver disease in chronic hepatitis C: comparison using ROC analysis.
As chronic liver disease progresses, an imbalance occurs between synthesis and breakdown of extracellular matrix (ECM). Matrix metalloproteinases (MMPs) are involved in degrading ECM while tissue inhibitors of metalloproteinases (TIMPs) prevent their fibrolytic action. We determined if plasma levels of MMP-2, TIMP-1 and TIMP-2 are related to liver histology in patients with chronic hepatitis C. Plasma MMP-2, TIMP-1 and TIMP-2 levels were measured in 43 patients with chronic hepatitis C. Plasma levels of MMP-2, TIMP-1 and TIMP-2 and serum ALT were correlated with liver biopsy score and specificity and sensitivity of the assays in detecting advanced liver disease were calculated from ROC analysis. Plasma TIMP-1 was significantly correlated with histological activity index (r = 0.45), portal inflammation (r = 0.48), periportal necrosis (r = 0.34) and focal necrosis (r = 0.38). Plasma TIMP-2 was significantly correlated with fibrosis (r = 0.43) and confluent necrosis (r = 0.41). Using ROC analysis both TIMP-1 and TIMP-2 had significant diagnostic ability in detecting advanced liver disease (Area under the curve 0.73 for both, p 0.015 and 0.036 respectively). A normal plasma TIMP-1 excluded advanced liver disease. Neither plasma MMP-2 or serum ALT were related to fibrosis or to histological activity index. With increased severity of liver disease in chronic hepatitis C there is increased plasma levels of TIMPs -1 and -2. Plasma TIMP-1 and TIMP-2 are sensitive and to a lesser extent specific in detecting advanced liver disease in chronic hepatitis C and could be used in preference to serum ALT.
Adult↗
Efficiency of transcription from promoter sequence variants in Lactobacillus is both strain and context dependent.
The introduction of consensus -35 (TTGACA) and -10 (TATAAT) hexamers and a TG motif into the Lactobacillus acidophilus ATCC 4356 wild-type slpA promoter resulted in significant improvements (4.3-, 4.1-, and 10.7-fold, respectively) in transcriptional activity in Lactobacillus fermentum BR11. In contrast, the same changes resulted in decreased transcription in Lactobacillus rhamnosus GG. The TG motif was shown to be important in the context of weak -35 and -10 hexamers (L. fermentum BR11) or a consensus -10 hexamer (L. rhamnosus GG). Thus, both strain- and context-dependent effects are critical factors influencing transcription in Lactobacillus.
Bacterial Proteins↗
Chlamydia pneumoniae in a free-ranging giant barred frog (Mixophyes iteratus) from Australia.
The koala biovar of Chlamydia pneumoniae was identified in lung tissue from a sick, free-ranging giant barred frog (Mixophyes iteratus) by using electron microscopy, C. pneumoniae-specific fluorescent-antibody staining, cell culture, and sequencing of the ompA, ompB and 16S rRNA genes. This is the first report of a chlamydial strain infecting both a homeotherm and a poikilotherm and only the fourth host (in addition to humans, koalas, and horses) to be naturally infected with this species of Chlamydia. The frog had severe, chronic, mononuclear pneumonia and nonregenerative anemia and pancytopenia.
Animals↗
Randomly amplified polymorphic DNA variation in populations of eastern Australian koalas, Phascolarctos cinereus.
Randomly amplified polymorphic DNA (RAPD) variation in populations of the koala, Phascolarctos cinereus, was investigated, revealing significant differences in the level of diversity between southern and northern regions of eastern Australia. Of the 20 polymorphic RAPD markers identified in koalas, 4-7 were polymorphic in southern populations, while 12-17 were polymorphic in northern populations. Analysis of molecular variance revealed a significant difference in the estimated variance between koalas from northern and those from southern regions (P < 0.001), where populations from the north were greater than twice as variable as their southern cousins. The total genetic diversity observed was attributed to regional differences (30.91%), population differences within a region (11.77%), and differences among individuals within a population (57.32%). For the within-region analyses, a large proportion of the genetic diversity was attributable to individual differences within a population, 80.34% for the north and 91.23% for the south. These results demonstrate that RAPD markers are useful for determining population structure among koalas.
Animals↗
Genetic variation in captive koalas (Phascolarctos cinereus): parentage determination and individual identification.
Highly repeatable randomly amplified polymorphic DNA (RAPD) markers were developed for parentage studies in the koala (Phascolarctos cinereus). Of the 25 RAPD primers screened, 5 (20.0%) produced 32 repeatable polymorphic RAPD bands (average/primer = 6.4 +/- 4.2). A high level of polymorphism was observed for each group of koalas (Featherdale, 71.9%; Lone Pine, 84.4%). All 25 koalas could be uniquely identified using either RAPD or microsatellite markers. Of the 32 RAPD markers generated in koalas, 25 were informative for parentage analyses. These RAPD markers successfully determined both parents to three offspring and a male parent to a fourth offspring. Paternity analysis (where the female parent is known) succeeded in assigning the correct male parent to seven offspring. Our RAPD-PCR method generates informative genetic markers that are useful for parentage determination and individual identification of captive koalas. This would provide genetic analysis to zoos and wildlife parks as a low-cost alternative to the more expensive microsatellite markers.
Animals↗
Failure to detect Chlamydia pneumoniae in atherosclerotic plaques of Australian patients.
Chlamydia pneumoniae is a recently reported, but common, respiratory tract pathogen. The organism has been detected by electron microscopy, immunocytochemistry, polymerase chain reaction (PCR) and recently culture within atherosclerotic plaques, suggesting a possible association between C. pneumoniae infection and atherosclerosis. Interestingly this association has not been found by all researchers. We examined 17 carotid endarterectomy specimens, 16 carotid arteries and 16 coronary arteries from autopsy specimens. They were examined by PCR for the presence of C. pneumoniae. In none of the 49 atherosclerotic samples examined was C. pneumoniae detected. The sensitivity of our PCR assay was rigorously tested and found to detect consistently fewer than ten elementary bodies. The association between C. pneumoniae and atherosclerosis is intriguing but has not yet been demonstrated in Australian patients.
Arteriosclerosis↗
Chlamydia pneumoniae DNA is not detectable within sarcoidosis tissue.
Sarcoidosis is a granulomatous disease of unknown etiology. Recent studies have suggested the possibility of a bacterial origin with Chlamydia pneumoniae being one of the many bacteria considered. The aim of this study was to use the polymerase chain reaction (PCR) in an attempt to identify C. pneumoniae within fresh/frozen sarcoidosis tissue. Tissue from 20 sarcoidosis patients and 17 controls was evaluated. DNA was extracted from all tissue specimens and PCR amplified with primers specific for C. pneumoniae. All study tissues were negative for the presence of DNA sequences from C. pneumoniae. These findings could not be attributed to PCR inhibition or to lack of sensitivity of the PCR assay. The negative finding suggests either that there is no involvement between C. pneumoniae and sarcoidosis or that, having incited granulomata formation, it is no longer present in detectable amounts.
Adult↗
The phylogeny of Ureaplasma urealyticum based on the mba gene fragment.
Sequencing of mba gene fragments of reference strains of Ureaplasma urealyticum serovars 1, 3, 6, 14 and 8, in addition to 33 clinical U. urealyticum isolates is reported. A phylogenetic tree deduced from alignment of these sequences clearly demonstrates two major clusters (confidence limit 100%), which equate to the parvo and T960 biovars, and five types, which have been designated mba 1, 3, 6, 8 and X. These relationships are supported by bootstrap analysis. Polymorphisms within the mba fragment of types mba 1, 3 and 6 were used to define nine subtypes (mba 1a, 1b, 3a, 3b, 3c, 3d, 3e, 6a and 6b), thus facilitating high resolution typing of U. urealyticum. Inclusion of reference strains for serovars 1, 3, 6 and 8 in the mba typing scheme showed that the results of this analysis are broadly consistent with currently accepted serotyping. In addition, a ure gene fragment from nine of the clinical isolates was amplified and sequenced. Comparisons of the sequences clearly distinguished the two biovars of U. urealyticum; however, this fragment was invariant within the parvo biovar. This study has shown that the mba sequence can reveal the fine details of the relationships between U. urealyticum isolates and also supports the significant evolutionary gap between the two biovars.
Bacterial Proteins↗
Comparison of PCR, nested PCR, and random amplified polymorphic DNA PCR for detection and typing of Ureaplasma urealyticum in specimens from pregnant women.
A PCR assay, using three primer pairs, was developed for the detection of Ureaplasma urealyticum, parvo biovar, mba types 1, 3, and 6, in cultured clinical specimens. The primer pairs were designed by using the polymorphic base positions within a 310- to 311-bp fragment of the 5' end and upstream control region of the mba gene. The specificity of the assay was confirmed with reference serovars 1, 3, 6, and 14 and by the amplified-fragment sizes (81 bp for mba 1, 262 bp for mba 3, and 193 bp for mba 6). A more sensitive nested PCR was also developed. This involved a first-step PCR, using the primers UMS-125 and UMA226, followed by the nested mba-type PCR described above. This nested PCR enabled the detection and typing of small numbers of U. urealyticum cells, including mixtures, directly in original clinical specimens. By using random amplified polymorphic DNA (RAPD) PCR with seven arbitrary primers, we were also able to differentiate the two biovars of U. urealyticum and to identify 13 RAPD-PCR subtypes. By applying these subtyping techniques to clinical samples collected from pregnant women, we established that (i) U. urealyticum is often a persistent colonizer of the lower genital tract from early midtrimester until the third trimester of pregnancy, (ii) mba type 6 was isolated significantly more often (P = 0.048) from women who delivered preterm than from women who delivered at term, (iii) no particular ureaplasma subtype(s) was associated with placental infections and/or adverse pregnancy outcomes, and (iv) the ureaplasma subtypes most frequently isolated from women were the same subtypes most often isolated from infected placentas.
Cervix Uteri↗
ABC of mental health. Mental health on the margins.
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Attitude of Health Personnel↗
Protein A as a fusion partner for the expression of heterologous proteins in Lactobacillus.
An expression system based on the Staphylococcus aureus protein A gene (spa) was developed to allow the production and export of proteins in Lactobacillus. Plasmid shuttle vectors were constructed that carried the eZZ gene, a synthetic gene based on the Protein A gene (spa) but lacking the carboxy-terminal membrane-anchoring region. A gene fusion was created between the eZZ gene and the VD4 region of a chlamydial major outer-membrane protein gene. Expression studies demonstrated the recognition of the spa regulatory signals by several Lactobacillus, with the recombinant protein being expressed (from 0.1 microgram of EZZVD4 fusion protein per ml in L. plantarum up to 10 micrograms of EZZ protein per ml in L. fermentum) and exported (levels up to 20% in L. fermentum) in several Lactobacillus strains.
Escherichia coli↗
Interference with vancomycin fluorescence polarisation immunoassay in a nonuraemic patient.
Commercially available immunoassays for vancomycin have been reported to overestimate serum vancomycin concentrations by varying degrees in patients with renal impairment, particularly those on peritoneal dialysis, by interference with crystalline degradation product-1 (CDP-1). To date this interference has not been reported in any other patient population. This report describes vancomycin fluorescence polarisation immunoassay (FPIA) interference in a patient with only mildly impaired renal function. At this time it is not possible to confirm that the interference was caused by CDP-1.
Anti-Bacterial Agents↗
The role of Ureaplasma urealyticum in adverse pregnancy outcome.
We investigated Ureaplasma urealyticum genital tract colonization rates in an Australian population to determine whether colonization was associated with adverse pregnancy outcome. Women attending an antenatal clinic were evaluated for lower genital tract colonization at their first antenatal visit (162 women) and at 28 weeks' gestation (120 women). Placentas from 92 women were cultured. U. urealyticum was the predominant isolate from the lower (57.4%) and upper (17.4%) genital tract in this population of pregnant women. U. urealyticum was a persistent colonizer during mid-trimester of pregnancy (in 88% of women colonized) whereas M. hominis, G. vaginalis, and Group B streptococcus were present as transient flora of the lower genital tract. Lower genital tract colonization during pregnancy was not directly associated with adverse pregnancy outcome. However preterm delivery in afebrile, asymptomatic women, could possibly be associated with chorioamnionitis (4 of 16 preterm births). Screening of women with a history of preterm birth may prevent upper genital tract infections and preterm delivery.
Adult↗
Identification and characterization of a basic cell surface-located protein from Lactobacillus fermentum BR11.
Extraction of Lactobacillus fermentum BR11 cells with 5 M LiCl yielded a preparation containing a single predominant polypeptide with an apparent molecular mass of 32 kDa. A clone encoding an immunoreactive 32-kDa polypeptide was isolated from a pUC18 library of L. fermentum BR11 DNA by screening with an antiserum raised against whole cells of L. fermentum BR11. Sequence determination of the insert in the clone revealed a complete 795-bp open reading frame (ORF) that defines a 28,625-Da polypeptide (BspA). N-terminal sequencing of the LiCl-extracted polypeptide from L. fermentum BR11 confirmed that it is the same as the cloned BspA. BspA was found to have a sequence similar to those of family III of the bacterial solute-binding proteins. The sequences of two ORFs upstream of bspA are consistent with bspA being located in an operon encoding an ATP-binding cassette-type uptake system. Unusually, BspA contains no lipoprotein cleavage and attachment motif (LXXC), despite its origin in a gram-positive bacterium. Biotin labelling and trypsin digestion of whole cells indicated that this polypeptide is exposed on the cell surface. The isoelectric point as predicted from the putative mature sequence is 10.59. It was consequently hypothesized that the positively charged BspA is anchored by electrostatic interaction with acidic groups on the cell surface. It was shown that BspA could be selectively removed from the surface by extraction with an acidic buffer, thus supporting this hypothesis.
Amino Acid Sequence↗
Cytotoxic-T-lymphocyte-mediated cytolysis of L cells persistently infected with Chlamydia spp.
Persistent chlamydial infections have been proposed as a means whereby chlamydiae evade immune resolution of infection. Such a mechanism would require evasion not only of the humoral immune responses but also of cell-mediated immune responses. We hypothesized that if such a mechanism is important, persistently infected cells should not be recognized by cytotoxic T cells. Persistent infections were simulated in vitro by treatment of Chlamydia trachomatis- or Chlamydia psittaci-infected cells with gamma interferon (IFN-gamma), penicillin, or tryptophan depletion. Cultures were examined for induction of a chlamydial stress response (measured by transcription of groesl RNA) and for the effects on viability, infectivity, morphology, and immune recognition. Although both IFN-gamma and penicillin induced aberrant chlamydial morphology and growth, we did not find evidence that these treatments elicited a classical stress response. In addition, T-cell-mediated lysis of Chlamydia-infected target cells treated with IFN-gamma or penicillin or grown in tryptophan-deficient media was examined. The immune cell-mediated lysis of these treated infected cells demonstrated that despite the effects of these compounds on chlamydial growth and development, the infected cells continued to be efficiently recognized and killed by cytotoxic T cells. Thus, it seems unlikely that these in vitro models of persistence represent functional mechanisms to evade immune clearance.
Base Sequence↗
Lactobacilli: vehicles for antigen delivery to the female urogenital tract.
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Animals↗