Haemopoietic origin of myofibroblasts formed in the peritoneal cavity in response to a foreign body.

This study utilized both in vivo and in vitro techniques to investigate whether cells of bone marrow origin can differentiate into smooth muscle-like cells (myofibroblasts) with contractile filaments and proteins. Female C57BL/6 mice expressing the Ly5.2 antigen on the surface of their haemopoietic cells had four pieces of silastic tubing (3 x 0.5 mm outer diameter) or boiled blood clot (2-3 mm diameter) placed in their peritoneal cavity. After 3, 5, 7 and 14 days (n = 4/group) the implants were removed and those that had remained free-floating were processed for light microscopy, immunohistochemistry and electron microscopy. In the first 3-5 days, rounded cells adhered to the entire surface of the tubing then flattened. These cells stained with fluoresceinated antibodies to Ly5.2 showing that they were derived from haemopoietic cells. By 14 days the cells had become elongated and multilayered in a collagen matrix, forming a thick tissue capsule around the tubing or boiled clot. They contained contractile filaments and stained with antibodies to alpha-smooth muscle actin but no longer stained for Ly5.2. A separate set of female C57BL/6 Ly5.2 mice were X-irradiated to destroy bone marrow then immediately transfused with 10(6) nucleated bone marrow cells taken from the femur and tibia of a congenic strain of male mice expressing the Ly5.1 allele. Eight of the female mice with successful engraftment (80-99%) had silastic tubing implanted in the peritoneal cavity. After 14 days, in situ hybridization with Y chromosome probe confirmed the male donor, and thus bone marrow, origin of the elongated cells that formed the capsule. In vitro studies showed that cells of the murine macrophage cell lines RAW 264.7 and J774 express alpha-smooth muscle actin after exposure to the cytokine gamma-interferon in vitro. These data show that bone marrow-derived cells can differentiate into smooth muscle-like cells and raises the possibility that blood-derived cells may contribute to the development of fibro-proliferative vascular diseases such as atherosclerosis.

Cell surface proteoglycan expression by human periodontal cells.

Cell surface proteoglycans are known to be involved in many functions including interactions with components of the extracellular microenvironment and serve to influence cell shape, adhesion, proliferation, and differentiation. They also can act as co-receptors, to help bind and modify the action of various growth factors and cytokines. Despite their strategic location and relevance to cell function, few studies have considered the nature of the cell surface proteoglycans associated with cells of the periodontium. Due to the structural complexity and multiplicity of cell types in the periodontium, we have selected three different cell lines (gingival connective tissue fibroblast, periodontal ligament fibroblast, and osteoblast) which each represent the unique functions within the periodontium to study the expression of cell surface proteoglycans. We hypothesized that a number of cell surface proteoglycans will be expressed by human periodontal cells and these may be related to the source and function of the cell. Western blotting and RT-PCR methods were used to study the expression of five cell surface proteoglycans (syndecan-1, -2, -4, glypican and betaglycan) in three cell lines of human periodontal cells in vitro. Our results demonstrated the expression of protein cores for syndecan-1 (43 kDa), syndecan-2 (48 kDa), syndecan-4 (35 kDa), glypican (64 kDa), and betaglycan (100-110 kDa). RT-PCR results confirmed that all of these cells produced mRNA for the cell surface proteoglycans under study, of which syndecan-2 showed a significant difference in expression between the periodontal ligament fibroblasts, gingival fibroblasts and osteoblasts. We conclude that the presence of specific cell surface proteoglycans on periodontal cells implies a likely role for these molecules in cell-cell, cell-matrix interactions involved in periodontal disease and/or regeneration of the periodontium, of which they may have distinctive functions related to the source and function of these cells.

Single DNA sequence common to all chlamydial species employed for PCR detection of these organisms.

Chlamydial infection is responsible for a wide spectrum of diseases of the eye, genitourinary tract, and lung. This group of organisms is also implicated in the pathogenesis of coronary artery disease as well as arthritis. Since cross-species infection is widely reported (though probably underestimated), it is an advantage to have a rapid and reliable method to detect all forms of chlamydiae in patient samples. We have identified a 160/163-bp DNA fragment in Chlamydia which is highly conserved in all chlamydial species. A polymerase chain reaction method based on this sequence has been developed to detect, in clinical samples, chlamydiae which have been shown to be positive by fluorescent-staining immunoassay; this method can be utilized in combination with restriction endonuclease cleavage to identify individual chlamydial species. Thus we have developed a sensitive and rapid detection method and have used it on samples from patients with respiratory and genital infections.

Lipopolysaccharide biosynthesis genes in koala type I Chlamydia: cloning and characterization.

We showed in 1988 that there are two strains of Chlamydia psittaci which infect the koala (Phascolarctos cinereus). In order to further investigate the role of these chlamydial strains in pathogenesis, we have attempted to identify genes of koala type I strain chlamydia which are involved in the immunogenic response. Transformation of Escherichia coli with a plasmid containing a 6.3-kb fragment (pKOC-10) of C. psittaci DNA caused the appearance of a specific chlamydial lipopolysaccharide (LPS) epitope on the host strain. The smallest DNA fragment capable of inducing the expression of chlamydial LPS was an XbaI fragment, 2.4 kb in size (pKOC-5). DNA sequence analysis of the complete fragment revealed regions of high identity, at the amino acid level, to the gseA genes of C. pneumoniae, C. psittaci 6BC and C. trachomatis, and the kdtA gene of E. coli which code for transferases catalysing the addition of 3-deoxy-D-manno-octulosonic acid (Kdo) residues to lipid A. Two open reading frames (ORFs) of 1,314 and 501 nucleotides in size, within the 2.4-kb fragment, were evident, and mRNA species corresponding to these ORFs were detected by Northern analysis. Both ORF1 and ORF2 are required for the appearance of chlamydia-specific LPS on the surface of recombinant E. coli.

Genetic complementation of radiation response by 3' untranslated regions (UTR) of RNA.

The molecular basis of radiosensitivity was studied using a cDNA complementation approach to correct radiosensitivity in cells. Four cDNAs of sizes 1.6, 2.0, 2.2 and 2.5 kb were isolated that corrected several aspects of the phenotype of cells from patients with the human genetic disorder ataxia-telangiectasia, characterized by hypersensitivity to ionizing radiation. The criteria used to assess correction included cell viability, induced chromosome aberrations, G2 phase delay and induction of p53 after exposure to radiation. One cDNA (2.5 kb) was identified as the complete sequence of the RNA helicase p68, which was capable of correcting radiosensitivity based on two of the above four criteria, with p53 induction post irradiation being partially corrected. The 2.2 kb cDNA was shown to correspond to the complete sequence of arginyl tRNA synthetase and the other two cDNAs were identical to the 3' untranslated regions (UTR) of the transcription factor TFIIS (1.6 kb) and phospholipase A2 (2.0 kb) respectively. Additional transfections with the 3'UTR (198 nucleotides) of p68 RNA helicase and its inverse sequence revealed that the 3'UTR had the same complementation capacity as the full-length cDNA, whereas the inverse construct failed to complement radiosensitivity. These data provide additional support for a novel role for 3'UTRs in the regulation of gene expression.

Cloning and characterization of cDNA encoding a human arginyl-tRNA synthetase.

Arginyl-tRNA synthetase (ArgRS) plays a key role in protein synthesis as part of a multienzyme complex with a number of other aminoacyl-tRNA synthetase (aaRS) enzymes. We have isolated a full-length cDNA encoding ArgRS as part of a project on complementation of radiosensitivity in human cells with an Epstein-Barr Virus (EBV) vector-based human cDNA library. DNA sequence analysis identified an open reading frame of 1983 nucleotides with 87% homology to other mammalian ArgRS genes. The deduced amino acid (aa) sequence (661 aa) showed 87.7% identity to the Chinese hamster ovary (CHO) enzyme and 37.7% identity to the homologous Escherichia coli enzyme. Northern blot analysis revealed the presence of a single mRNA species of approx. 2.2 kb. The results described here demonstrate that ArgRS is highly conserved in mammalian cells and confirm the presence of a hydrophobic N-terminal region in the higher-molecular-weight complexed form of ArgRS.

Remarkable sequence relatedness in the DNA encoding the major outer membrane protein of Chlamydia psittaci (koala type I) and Chlamydia pneumoniae.

DNA encoding the major outer membrane protein (MOMP) of the koala type-I strain of Chlamydia psittaci (pathogen responsible for blindness and infertility in koalas) was cloned and sequenced. Comparison with momp gene sequences from other chlamydial species revealed a remarkable degree of homology (> 97%) with that of the human pathogen, Chlamydia pneumoniae. In comparison, the sequence only shared 75% DNA sequence homology with other C. psittaci members and 69% homology with C. trachomatis. The open reading frame consisted of 1167 bp encoding a 389-amino acid (aa) pre-MOMP including a leader sequence of 23 aa, similar to the C. pneumoniae gene. These genes were closely related even within the four variable domains (86-100% homology). Specific antibodies were capable of distinguishing between koala type I and C. pneumoniae. This very high degree of relatedness between C. pneumoniae, a human pathogen, and an individual strain of C. psittaci in the momp gene raises further questions on the host specificity, classification and evolutionary relationships of the different chlamydial species.

Comparison of type I and type II Chlamydia psittaci strains infecting koalas (Phascolarctos cinereus).

The native Australian marsupial Phascolarctos cinereus, otherwise known as the koala, is prone to infection by the obligate intracellular parasite Chlamydia psittaci, which causes ocular 'pink eye' and urogenital 'dirty tail' diseases. Several chlamydial DNA probes to both chromosomal and plasmid sequences were used to type by Southern blot analysis 51 samples taken from wild and captive koalas from habitats on the eastern seaboard of Australia as far apart as Queensland and Victoria. Two types of C. psittaci were observed and called types I and II. Type II was found more frequently than type I and occurred in both ocular and urogenital samples, while type I showed a strong but not absolute preference for ocular sites. Cross-hybridization analyses indicated that type I and type II had about 10% DNA sequence identity to each other. DNA analyses showed that type II was very closely related to some ovine and bovine chlamydiae but type I could not be related to any other C. psittaci strain available. Light and electron microscopic analyses of infected BGM monolayers revealed that the two strains were similar in morphological characteristics. The type I strain was considerably more infectious than the type II strain in BGM cells and in the yolk sacs of embryonated eggs. A PCR based assay detected both type I and type II koala chlamydiae in samples that had been negative by Southern blot and tissue culture and provided the first evidence that both types can occur simultaneously at the one site of infection.

Immuno-dot blot as a rapid diagnostic method for detection of chlamydial infection in koalas (Phasolarctos cinereus).

The sensitivity and specificity of an immunoscreening test for anti-chlamydial antibodies in koala (Phasolarctos cinereus) serum were determined after the adsorption of non-specific antibodies. The results of the test were compared with complement fixation tests, tissue culture, gene probe analysis and dot blot immunoscreening for host-borne chlamydial antigen. The immunoscreening test was the most sensitive test for the identification of chlamydial infection in koala serum samples, furthermore it was rapid, taking approximately 16 hours to complete, and inexpensive. However, for the assay of swab material from koalas, gene probe analysis remains the most sensitive method of detection of chlamydiae.

Some aspects of the immune response of koalas (Phascolarctos cinereus) and in vitro neutralization of Chlamydia psittaci (koala strains).

Western-blot analysis was used to study the reaction of koala antisera, two specific polyclonal antibodies and one monoclonal antibody, with chlamydial antigens in koalas infected with Chlamydia psittaci. The koala sera recognized four C. psittaci surface antigens, corresponding to the major outer membrane protein (39.5 kDa), 31 kDa protein, 18 kDa protein and lipopolysaccharide. The S25-23 LPS specific monoclonal antibody inhibited chlamydial infection (55-67%) with both koala strains (type I and type II). Both koala antiserum and rabbit polyclonal antibodies against either type of chlamydia significantly reduced the number of infected cells resulting from type II infections at a dilution of 1 in 20. Rabbit antiserum against type II was effective in neutralizing infection by type II elementary bodies, but was less effective against type I infection. In addition, no koala antiserum was effective in neutralizing type I infection.

Detection of Chlamydia psittaci in free-ranging koalas (Phascolarctos cinereus): DNA hybridization and immuno-slot blot analyses.

DNA-slot hybridization and immuno-slot blot analyses were compared for the detection of Chlamydia psittaci in crude swab material from free-ranging koalas. Immuno-slot blot analysis detected chlamydiae in 43 out of 68 koalas, with the sensitivity of the assay varying from 52 to 73% depending on the site of infection. Gene probe analysis was also used employing a genus-specific probe pCKO-10 isolated from a koala chlamydial gene library (ocular strain) and a plasmid probe pCKU cloned from a urogenital strain. The sensitivity of these two assays was comparable and they were considerably more efficient than the immuno-slot blot method for the detection of chlamydiae. Comparison of these data with a cell-culture method of detection, previously used with the same samples, demonstrated that gene probe analysis detected more positives than observed with cell culture. However, this appears to reflect more on the condition of the swab material rather than the sensitivity of the method.

Conserved DNA sequences in chlamydial plasmids.

Two 7.4-kb plasmids from Chlamydia psittaci have been cloned and characterized. These plasmids are quite distinct from the 6.2-kb C. psittaci and the C. trachomatis plasmids when compared by restriction endonuclease analysis. The plasmids show considerable cross-hybridization, with only a small region highly conserved and identified as a 4 X 22-bp tandemly repeated region. This sequence is identical in the two size categories of C. psittaci plasmids and differs from C. trachomatis plasmids by only 2 bp in the 22-bp motif. AT-rich clusters 5' to the repeat region which are present in C. trachomatis and Escherichia coli plasmids were absent from both classes of C. psittaci plasmids. Extensive regions are less highly conserved but show a sufficient degree of cross-hybridization to suggest that the plasmids are homologous.

Two distinct forms of Chlamydia psittaci associated with disease and infertility in Phascolarctos cinereus (koala).

While several diseases associated with Chlamydia psittaci infection have been reported in Phascolarctos cinereus (koala), it is still unclear whether one or more chlamydial strains are responsible. In this study, we provide evidence, obtained by restriction enzyme and gene probe analysis, that two quite distinct strains of C. psittaci infect koalas; one strain was isolated from the conjunctivae, and the other was isolated from the urogenital tract and the rectum. A gene probe, pFEN207, containing the coding sequence for an enzyme involved in the biosynthesis of the chlamydial genus-specific lipopolysaccharide antigen, and a separate probe, pCPML-4N, prepared from a DNA fragment of a koala-infecting strain of C. psittaci, were used to determine the patterns of hybridization in the koala-infecting strains; these patterns were found to be quite distinct from those observed with C. psittaci isolates from other animals. We also demonstrated by hybridization analysis with an avian strain plasmid that all three koala urogenital isolates contain a plasmid and that there is no evidence for the presence of a homologous plasmid in any of the ocular isolates.

Evaluation of an enzyme immunoassay test for the diagnosis of Chlamydia psittaci infection in free-ranging koalas (Phascolarctos cinereus) in southeastern Queensland, Australia.

The IDEIA Chlamydia Test, a commercially available antigen-capture enzyme-linked immunosorbent assay (ELISA) test, based on a monoclonal antibody for the detection of chlamydia in clinical specimens, was evaluated in a population of 65 free-ranging koalas in southeastern Queensland determined to be infected with Chlamydia psittaci. Compared to isolation of the organism in tissue culture, the sensitivity of the IDEIA test ranged from 3 to 11%, and the specificity from 90 to 97%. The results indicated that the IDEIA test is unsuitable for use as a diagnostic screening test for C. psittaci in free-ranging koalas.

Aspects of the epidemiology of Chlamydia psittaci infection in a population of koalas (Phascolarctos cinereus) in southeastern Queensland, Australia.

A population of free-ranging koalas in southeastern Queensland was examined to determine the prevalence of Chlamydia psittaci infections. Although C. psittaci was isolated from 46 of 65 (71%) koalas studied, only six (9%) of these had clinical signs of disease. Most adult females (82%) had back or pouch young present even though 67% of them were infected. There were no significant correlations between age, sex or site of sampling (urogenital versus conjunctival tissues) and the isolation of C. psittaci. No other important bacterial or fungal pathogens were isolated. The complement fixation test had a sensitivity of 7% and a specificity of 94% in detecting chlamydial infections, suggesting that it is unsuitable for use as a screening test. Chlamydia psittaci infection within this population appeared to represent a generally well-balanced host-parasite relationship and few animals had clinical signs of disease. Only four of 27 (15%) healthy koalas infected with C. psittaci followed for 24 wk after sampling developed eye disease or "dirty tail." Two koalas with keratoconjunctivitis recovered without treatment during the study period. Additional factors, including the stresses imposed by loss of habitat, may act to produce overt disease in koalas with latent C. psittaci infections.

Comparison of Chlamydia psittaci isolates by restriction endonuclease and DNA probe analyses.

DNAs from eight Chlamydia psittaci isolates (koala conjunctivitis, avian psittacosis, avian ornithosis, ovine abortion, ovine polyarthritis, sporadic bovine encephalomyelitis, and feline conjunctivitis) and one Chlamydia trachomatis isolate (lymphogranuloma venereum) were compared by restriction endonuclease and DNA probe analyses. Digestion with HindIII yielded a series of discrete fragments which allowed the differentiation of most isolates. A gene probe, pFEN207, which encodes the chlamydia-specific component of the lipopolysaccharide group antigen was used in Southern hybridizations. The probe was chlamydia specific and hybridized to a single BamHI fragment and multiple HindIII fragments in each isolate. The variation in size of the hybridizing fragments allowed easy differentiation of the isolates and may eventually lead to a meaningful subgrouping of the diverse group of disease agents presently included in the species C. psittaci.