Occurrence of Chlamydia trachomatis and Chlamydia pneumoniae in paediatric respiratory infections.

An emerging body of evidence suggests that half of asthma in both children and adults is associated with chronic lung infection. The aim of the present study was to determine the frequency of viable Chlamydia pneumoniae (Cp) and C. trachomatis (Ct) in the respiratory tracts of paediatric patients with chronic respiratory diseases. Bronchoalveolar lavage fluid (BALF) samples obtained from 182 children undergoing bronchoscopy for clinical reasons were assayed using PCR analysis, in vitro tissue culture and immunofluorescence staining for the presence of Cp and Ct. Chlamydia-specific DNA was detected by PCR in 124 (68%) out of 182 patients; 79 were positive for Cp, 77 positive for Ct and 32 for both organisms; 75 patients had cultivable Chlamydia. Ct DNA prevalence decreased, whereas Cp positivity generally increased with age. A total of 59 out of 128 asthma patients and 16 out of 54 nonasthmatics were Chlamydia culture positive. When the patients were divided into inflammatory versus noninflammatory airway disease, there were 69 (46%) out of 150 and six (18%) out of 32 BALF samples with cultivable Chlamydia, respectively. Viable Chlamydia pneumoniae and Chlamydia trachomatis occur frequently in children with chronic respiratory diseases and may be more prevalent in asthma patients. To the current authors' knowledge, this is the first report of viable Chlamydia trachomatis in the lungs of children.

Embryonic morphogenesis: role of fibrous lattice in the development of feathers and feather patterns.

In the morphogenesis of embryonic feather germs the formation of dermal cell groupings is associated with the development of a highly regular pattern of birefringence in the dermis. This birefringence is due to a lattice-like system of collagenous tracts along which dermal cells become progressively aligned and grouped in regularly spaced sites. The experimental results suggest that this fibrous lattice is of major significance in the morphogenesis of feather germs and in their characteristic pattern of distribution.

Antigen presentation using novel particulate organelles from halophilic archaea.

A presentation vehicle was developed based on particulate gas vesicles produced by halophilic archaea. Gas vesicle epitope displays were prepared using standard coupling methods or recombinant DNA technology. When presented in the context of gas vesicle preparations, either the hapten, TNP, or a model six amino acid recombinant insert in the outer gas vesicle protein, GvpC was rendered immunogenic. Assays to quantify humoral responses indicated that each preparation elicited strong antibody responses in the absence of exogenous adjuvant. Thus, each preparation elicited a humoral response when injected into mice and this response was long lived and exhibited immunologic memory. Recombinant gas vesicle preparations therefore constitute a new, self-adjuvanting carrier/display vehicle for presentation of an array of peptidyl epitopes.

Protein organization on the PHA inclusion cytoplasmic boundary.

Polyhydroxyalkanoate (PHA) cellular inclusions consist of polyesters, phospholipids, and proteins. Both the polymerase and the depolymerase enzymes are active components of the structure. Recently, proteins associated with these inclusions have been described in a number of bacterial species. In order to further clarify the structure and function of these proteins in relation to polymer inclusions, ultrastructural studies of isolated polymer inclusions were initiated. The surface boundary characteristics of polymer inclusions, produced by several genera of bacteria, two different Pseudomonas putida deletion mutants and by Escherichia coli recombinants, were examined. The recombinant E. coli carried either the PHB biosynthesis operon (phaCAB) from Ralstonia eutropha alone, or both this operon and a gene encoding an inclusion surface protein of R. eutropha (phaP). The results support two suggestions: (i) specific genes in the PHA gene cluster code for the proteins forming the surface boundary arrays which characterize the polymer inclusion; and (ii) transfer of such a gene would result in subcellular compartmentalization of accumulating polymer. Although the proteins appear to serve a similar function among different genera, nevertheless, the different surface proteins are encoded by a variety of non-homologous genetic sequences.

Investigation of the function of proteins associated to polyhydroxyalkanoate inclusions in Pseudomonas putida BMO1.

Polyhydroxyalkanoate (PHA) granule associated proteins from Pseudomonas oleovorans were purified and the N-terminal sequences of two major proteins migrating in sodium dodecyl sulfate polyacrylamide gels with a relative molecular mass of 18 and 43 kDa (GA1 and GA2, respectively) were analyzed. Radiolabeled degenerate probes deduced from these amino acid sequences were used to identify genomic DNA fragments from P. oleovorans and Pseudomonas putida encoding GA1 and GA2. DNA sequence analysis of the fragments obtained from P. putida revealed that the genes encoding these proteins were adjacent to phaC2 and ORF3, the PHA synthase II gene and an open reading frame of unknown function, respectively, found at the P. oleovorans and P. aeruginosa PHA synthase gene locus. The open reading frames encoding GA1, GA2 and ORF3 or smaller fragments beginning at GA1 were inactivated by chromosomal insertion of the Tn5 kanamycin resistance gene block (neo). When these mutants were grown on mineral salts agar media under nitrogen limitation, containing gluconate or decanoate as carbon sources, they appeared more translucent than the wild-type grown under similar conditions. Gas-chromatographic analysis of the cellular dry mass revealed that the mutant strains accumulated 30-50% less PHA than the P. putida wild type.

Intracellular depolymerase functionality and location in Pseudomonas oleovorans inclusions containing polyhydroxyoctanoate.

Microbial poly-3-hydroxyoctanoate inclusion bodies produced by Pseudomonas oleovorans when grown on n-octanoic acid, are complex macromolecular structures consisting of polyester, organized paracrystalline lattice arrays and lipids. While it is known that the polymer in the granules maintains its native, amorphous state while it is surrounded by the components of this complex, the precise functions of the various components during polymer production and utilization have yet to be established. By utilizing electron microscopy, SDS-PAGE, and gel filtration chromatography along with in vitro assays for depolymerase activity, the present study demonstrates that a protein species with molecular weight of approximately 32 kDa is the depolymerase protein of the polymer inclusion. When exogenous carbon was exhausted, cell viability required utilization of the stored polyester. Under these conditions, the concentration of the depolymerase increased while the concentrations of the polymerase decreased. Thus, the association of the depolymerase with the granules was shown to be under metabolic regulation relative to the polymerase. The results from the present studies show that careful manipulation of the substrate concentration can selectively, and differentially, alter the level of inclusion associated proteins as well as the quantity and quality of the polyester which is accumulated.

Intracellular depolymerase and polyhydroxyoctanoate granule integrity in Pseudomonas oleovorans.

When polyhydroxyoctanoate (PHO) was produced by Pseudomonas oleovorans during a regimen of intermittent feeding on octanoic acid, there was a significant change in both the polymer associated proteins and the composition of the enclosed polymer. The polymer granules were isolated with their protein coat intact and the enzymatic hydrolysis of the polymer within this cell free system was determined. The degradation rate for the PHO in these native granules reached a maximum of 1.17 mg/h at an optimum pH of 9 when incubated at 30 degrees C. A study of the effect of various inhibitors on depolymerase activity suggested that the enzyme most likely has disulfide linkages and serine residues at its active site. Ultrastructure studies suggested this loss of enzyme activity was correlated with significant organizational degeneration in the proteins associated with the PHO inclusion body. Once solubilized from the granule, the depolymerase itself remained enzymatically active, and addition of this released material to other granule preparations increased the rate of polymer granule degradation. Similarly, when colloidal suspensions of purified, amorphous PHO were placed in contact with that depolymerase, they also underwent rapid degradation. In contrast, when crystalline solvent-cast PHO films were placed in contact with this enzyme, no degradative activity was observed.

Some characteristics of a secreted chlamydial antigen recognized by IgG from C. trachomatis patient sera.

Chlamydia trachomatis serovars release a glycolipid antigen (GLXA) into the culture supernatant during the infective cycle. This antigen is recognized by IgG isolated from humans with a natural chlamydial infection; GLXA may be a major antigenic determinant of chlamydia. It can be immunopurified by molecular shift or affinity chromatography. Silver staining of SDS-PAGE gels demonstrates a pattern of bands that is essentially the same for preparations isolated by either method. GLXA can also be extracted from mature elementary bodies (EB). These preparations show the same pattern of silver staining bands, and the major bands are immunoreactive as shown by Western blot analysis. Isoelectric focusing studies demonstrate that purified GLXA has an acidic pI.

Characterization of an antigen secreted by Chlamydia-infected cell culture.

A soluble genus-specific chlamydial antigen has been isolated from the supernatants of cultures infected with Chlamydia trachomatis and from other sources. The antigen is a glycolipid that is secreted during the infective cycle. This exoglycolipid can be hydrolysed and fractionated into polysaccharide and lipid components. Both fractions retain antigenic activity. An immunodominant antigenic determinant of the lipid component contains fatty acids of C17 and C18:1. The polysaccharide immunodominant epitope gives rise to gulose when derivatives are formed. The secretion of the antigen into the media supernatant, the presence of gulose and the observed molecular weight are consistent with properties of alginate secreted by Gram-negative bacteria. Chemical analyses and SDS-PAGE indicate that the exoglycolipid is markedly different from LPS.

Association of caveolin with Chlamydia trachomatis inclusions at early and late stages of infection.

The mechanism by which the intracellular bacterial pathogen Chlamydia trachomatis enters eukaryotic cells is poorly understood. There are conflicting reports of entry occurring by clathrin-dependent and clathrin-independent processes. We report here that C. trachomatis serovar K enters HEp-2 and HeLa 229 epithelial cells and J-774A.1 mouse macrophage/monocyte cells via caveolin-containing sphingolipid and cholesterol-enriched raft microdomains in the host cell plasma membranes. First, filipin and nystatin, drugs that specifically disrupt raft function by cholesterol chelation, each impaired entry of C. trachomatis serovar K. In control experiments, filipin did not impair entry of the same organism by an antibody-mediated opsonic process, nor did it impair entry of BSA-coated microspheres. Second, the chlamydia-containing endocytic vesicles specifically reacted with antisera against the caveolae marker protein caveolin. These vesicles are known to become the inclusions in which parasite replication occurs. They avoid fusion with lysosomes and instead traffic to the Golgi region, where they intercept Golgi-derived vesicles that recycle sphingolipids and cholesterol to the plasma membrane. We also report that late-stage C. trachomatis inclusions continue to display high levels of caveolin, which they likely acquire from the exocytic Golgi vesicles. We suggest that the atypical raft-mediated entry process may have important consequences for the host-pathogen interaction well after entry has occurred. These consequences include enabling the chlamydial vesicle to avoid acidification and fusion with lysosomes, to traffic to the Golgi region, and to intercept sphingolipid-containing vesicles from the Golgi.

Examination of chlamydial glycolipid with monoclonal antibodies: cellular distribution and epitope binding.

A chlamydial glycolipid antigen (GLXA) is shed into the medium of C. trachomatis-infected cell cultures. This study screened monoclonal antibodies (mAb), prepared in different laboratories by immunization with embryonated egg propagated elementary bodies (EB), for their ability to bind with infected cells and to react with purified GLXA isolated from supernatants of infected McCoy cells. The fluorescent antibody (FA) staining pattern exhibited by a number of mAb indicated that they bound antigen present within the inclusion and at the inner membrane surface of infected cells; the observed pattern differs significantly from the distribution seen when anti-lipopolysaccharide (LPS) (mAb) were used. The staining pattern observed by immunofluorescence was confirmed and extended by ultrastructure studies of immunogold-labelled, infected human endometrial gland epithelial cells (HEGEC) and a human endometrial carcinoma-derived cell line (RL95-2). Additionally, the immunoelectron microscope studies revealed binding within the inclusion and on reticulate bodies, within the cell cytoplasm and at the surface of infected cells. The specificity of the reactive mAb, examined by molecular shift chromatography and isolated, affinity-purified GLXA, indicated that two mAb of the IgG isotype recognized an antigen which had been purified from tissue culture supernatants by affinity chromatography using an IgM mAb. The results suggest that GLXA is an important determinant whose role and function during in vitro and in vivo infections deserves further analyses.

Biochemical and functional antigenic mimicry by a polyclonal anti-idiotypic antibody for chlamydial exoglycolipid antigen.

A vaccine developed by using a genus-specific antigen (Ag) of Chlamydia trachomatis would elicit a wide range of protection against various chlamydial infections. We have produced an anti-idiotypic antibody (Ab2) in guinea pigs which, in rabbits, mimics the immunogenicity of a genus-specific exoglycolipid Ag (GLXA) of C trachomatis. Furthermore, the Ab2 fulfills the functional criteria of an 'internal image' of the nominal Ag: it inhibits the binding of the idiotypic (Id) monoclonal Ab (mAb1) to GLXA, and it induces in rabbits anti-anti-Id antibody (Ab3) which recognizes both the affinity-purified nominal Ag GLXA and whole organisms. Moreover, Ab3 induced by immunization of rabbits with guinea pig Ab2 neutralizes infectious heterologous chlamydiae and prevents in vitro and in vivo infection. Taken together, these results demonstrate functional and biochemical mimicry of the Ab2 for the chlamydial GLXA and suggest that anti-idiotypic Ab to GLXA is a potential candidate vaccine against chlamydia-related diseases.

The anti-idiotypic antibody to chlamydial glycolipid exoantigen (GLXA) protects mice against genital infection with a human biovar of Chlamydia trachomatis.

Despite more than three decades of anti-chlamydial vaccine research and improved vaccine strategies with new technologies, no vaccine candidate has protected against heterologous challenge, nor at more than one site of infection. The majority of experimental anti-chlamydial vaccines to date have targeted the chlamydial major outer membrane protein (MOMP). Many MOMP-directed vaccine candidates have been highly immunogenic, but have failed to protect against infectious challenge. We have extended our previous studies of a different anti-chlamydial vaccine, a monoclonal anti-idiotypic antibody (anti-Id; mAb2) which is a molecular mimic of the chlamydial glycolipid exoantigen (GLXA). The present studies demonstrate that the mAb2 vaccine is protective in a murine genital infection model utilizing a human urogenital strain. After either mucosal (oral or intranasal) or systemic (subcutaneous) immunization with the poly (lactide) encapsulated-mAb2 to GLXA, C3H/HeJ mice were significantly protected against topical vaginal challenge with Chlamydia trachomatis (K serovar; UW-31). Reduced vaginal shedding of organism and genital tract inflammation were associated with GLXA-specific and/or anti-EB neutralizing serum antibody. Our results demonstrate that the anti-Id (mAb2) vaccine is protective against an additional human biovar of C. trachomatis in C3H/HeJ mice, which are allogeneic to the source of mAb2 (BALB/c).