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Biomedical subjects

R S Stephens

Publications and source records attributed to R S Stephens.

At least 19 recordsLinked to original sources
PubMed2001

Chlamydial genovar distribution after community wide antibiotic treatment.

Major outer membrane protein sequences, determined from Chlamydia-positive eye swab samples collected in 2 Egyptian villages, were used to analyze the epidemiology of trachoma in an endemic setting. Samples were collected during the 1999 Azithromycin in Control of Trachoma trial, in which residents of villages were mass treated with either oral azithromycin or topical tetracycline and were followed up for nearly 2 years. Three genovar families (A, Ba, and C) and 12 genovars were detected, with 2 genovars (A1 and Ba1) comprising almost 75% of the samples. The presence of >1 genovar within households was common, with > or =24% of households having >1 genovar. Evidence consistent with reinfection and persistence as mechanisms of communitywide continued presence of trachoma was provided by data for individuals infected with rare genovars.

Anti-Bacterial Agents↗
PubMed2001

Chlamydia outer membrane protein discovery using genomics.

Outer membrane proteins of microbial pathogens serve essential roles in engaging the host environment and can be important immunotherapeutic targets. Because of the difficulty of growing large quantities of chlamydiae suitable for biochemical fractionation, little was known about their outer membrane protein composition prior to the recent sequencing of the C. trachomatis and C. pneumoniae genomes. Using bioinformatic approaches to characterize chlamydial open reading frames, novel outer membrane proteins were predicted. Several of the predicted outer membrane proteins recently have been shown to be translated and localized to the surface of the chlamydial outer membrane.

Bacterial Outer Membrane Proteins↗
PubMed2001

Heparin-binding outer membrane protein of chlamydiae.

As an intracellular pathogen, the mechanism by which Chlamydia invade eukaryotic cells represents a cornerstone to understanding chlamydial biology. The ability of chlamydiae specifically to bind heparan sulphate or heparin and the association of this ability to bind and enter mammalian host cells was approached by searching experimentally for chlamydial outer membrane proteins that bind heparin. The 60 000 molecular weight cysteine-rich outer membrane complex protein, OmcB, bound heparin. The ability of OmcB to bind heparin was supported by mapping the region of the protein with heparin-binding capacity and demonstrating that an OmcB synthetic 20-mer peptide from this region specifically bound heparin. Surface localization of OmcB was shown using monospecific antisera specific to the 20-mer OmcB peptide that bound the surfaces of elementary bodies (EB) and by heparin-binding peptide cross-linking of EB surface proteins.

Amino Acid Sequence↗
PubMed2001

Substrate-specific diffusion of select dicarboxylates through Chlamydia trachomatis PorB.

Chlamydiae contain two porins, MOMP and PorB, that facilitate diffusion of solutes through the outer membrane. MOMP is a general porin that permits the diffusion of a wide variety of compounds including carbohydrates and amino acids. The relative inefficiency of PorB as a general porin and its low abundance in the outer membrane suggest that it may function as a substrate-specific porin. The tricarboxylic acid (TCA) cycle of chlamydiae is incomplete and to function would require the exogenous acquisition of 2-oxoglutarate or glutamate. A liposome-swelling assay for anions as well as an enzyme-linked liposome assay were used to demonstrate the efficient diffusion of dicarboxylates such as 2-oxoglutarate through PorB. These data demonstrate that PorB is a dicarboxylate-specific porin that may feed the chlamydial TCA cycle and provide chlamydiae with carbon and energy production intermediates.

Bacterial Outer Membrane Proteins↗
PubMed2001

Rapid, transient phosphatidylserine externalization induced in host cells by infection with Chlamydia spp.

Chlamydia organisms are obligate intracellular bacterial pathogens responsible for a range of human diseases. Persistent infection or reinfection with Chlamydia trachomatis leads to scarring of ocular or genital tissues, and Chlamydia pneumoniae infection is associated with the development of atherosclerosis. We demonstrate that C. trachomatis and C. pneumoniae infection in vitro elicits the externalization of the lipid phosphatidylserine on the surface of human epithelial, endothelial, granulocytic, and monocytic cells. Phosphatidylserine externalization is associated with cellular development, differentiation, and death. Infection-induced phosphatidylserine externalization was immediate, transient, calcium dependent, and infectious dose dependent and was unaffected by a broad-spectrum caspase inhibitor. Chlamydia-infected cells accelerated plasma clotting and increased the macrophage phagocytosis of infected cells that was phosphatidylserine dependent. The rapid externalization of phosphatidylserine by infected cells may be an important factor in the pathogenesis of chlamydial infections.

Apoptosis↗
PubMed2001

Expression of Chlamydia pneumoniae polymorphic membrane protein family genes.

The genome of the obligate intracellular bacterium Chlamydia pneumoniae CWL029 encodes a family of 21 proteins with predicted outer membrane localization. These polymorphic membrane proteins (Pmps) are heterogeneous in both amino acid sequence and predicted size but are unified by the conserved amino acid motifs GGAI and FXXN repeated in the N-terminal half of each protein. Reverse transcriptase PCR analysis showed that all pmp genes are transcribed. To determine whether all proteins are expressed, specific antisera were generated by immunization with mutually exclusive synthetic peptides representing each of the 21 predicted Pmps. Each antiserum reacted with, and was typically immunospecific for, the corresponding peptide immunogen by enzyme-linked immunosorbent assay. Western blot analyses of purified elementary bodies showed that 11 of the 21 Pmps were detectable. Attempts to demonstrate by Sarykosyl fractionation that the Pmps were localized to the outer membrane revealed that several of the Pmps were unstable and readily degraded. Analyses of additional C. pneumoniae strains showed that although some Pmps are conserved, others vary between strains, in both molecular weight and level of expression.

Amino Acid Sequence↗
PubMed2000

Design, expression and functional characterization of a synthetic gene encoding the Chlamydia trachomatis major outer membrane protein.

A synthetic gene coding for the Chlamydia trachomatis serovar L2 major outer membrane protein (MOMP) was designed, constructed and expressed in Escherichia coli. The native amino acid sequence was reverse translated and the resulting nucleotide combinations manipulated in order to evenly distribute 25 unique restriction sites along the length of the gene while retaining the native amino acid sequence. The synthetic gene was cloned into a T7 promoter-controlled plasmid (pET-3a) and the expressed product was analyzed to assess antigenicity, cellular localization and function. Monoclonal antibodies specific for native MOMP reacted to the expressed product by immunoblot. Outer membrane fractionation confirmed that the processed protein was located in the outer membrane. MOMP expressed in E. coli and present in the outer membrane was shown to function as a general diffusion porin. This system provides the means to produce readily modifiable MOMP either in purified form or as a membrane-associated protein, and so facilitate the investigation of its functional, structural and antigenic properties.

Amino Acid Sequence↗
PubMed2000

Characterization and functional analysis of PorB, a Chlamydia porin and neutralizing target.

A predicted protein (CT713) with weak sequence similarity to the major outer membrane protein (20.4% identity) in Chlamydia trachomatis was identified by Chlamydia genome analysis. We show that this protein is expressed, surface accessible, localized to the chlamydial outer membrane complex and functions as a porin. This protein, PorB, was highly conserved among different serovars, with nearly identical sequences between serovars D, B, C and L2. Sequence comparison between C. trachomatis and Chlamydia pneumoniae showed less conservation between species with 59.3% identity. Immunofluorescence staining with monospecific antisera to purified PorB revealed antigen localized within chlamydial inclusions and found throughout the developmental cycle. Antibodies to PorB neutralized infectivity of C. trachomatis in an in vitro neutralization assay confirming that PorB is surface exposed. As PorB was found to be in the outer membrane, as well as having weak structural characteristics similar to major outer membrane protein (MOMP) and other porins, a liposome-swelling assay was used to determine whether this protein had pore-forming capabilities. PorB had pore-forming activity and was shown to be different from MOMP porin activity.

Amino Acid Sequence↗
PubMed2000

Chlamydia-dependent biosynthesis of a heparan sulphate-like compound in eukaryotic cells.

One hypothesis for the mechanism of chlamydial interaction with its eukaryotic host cell invokes a trimolecular mechanism, whereby a Chlamydia-derived glycosaminoglycan bridges a chlamydial acceptor molecule and a host receptor enabling attachment and invasion. We show that a heparan sulphate-specific monoclonal antibody specifically binds a glycosa-minoglycan localized to the surface of the chlamydial organism and effectively neutralizes infectivity of both C. trachomatis and C. pneumoniae. In addition to the ability of this antibody to neutralize infectivity, direct visualization using immunofluorescence demonstrated staining of chlamydial organisms localized to the intracellular vacuole. The chlamydial-associated glycosaminoglycan was specifically labelled with [14C]-glucosamine, and the labelled compound was immunoprecipitated and resolved by gel electrophoresis. The chlamydial-associated glycosaminoglycan is a high-molecular-weight compound similar in size to heparin or heparan sulphate and was sensitive to cleavage by heparan sulphate lyase. These data demonstrate that a glucosamine-containing sulphated polysaccharide is produced within the intracellular vacuole containing chlamydiae and is a target for antibody-mediated neutralization of infectivity.

Animals↗
PubMed2000

Chlamydial genomics and vaccine antigen discovery.

Vaccine development for Chlamydia species has progressed on several research fronts, including knowledge of the components of the immune response required for immunity and resolution of infection as well as effective modes of delivery. The antigens required to elicit protective immune responses have not been identified. The full biologic potential for Chlamydia trachomatis and Chlamydia pneumoniae was exposed by the completion of the genome sequence for each organism. This knowledge will aid vaccine antigen discovery by facilitating the identification, testing, and evaluation of antigens by way of vaccine delivery approaches that elicit protective immunity.

Antigens, Bacterial↗
PubMed2000

Eukaryotic cell uptake of heparin-coated microspheres: a model of host cell invasion by Chlamydia trachomatis.

Using polystyrene microspheres coated with heparin or heparan sulfate, it was shown that coated microspheres specifically bound eukaryotic cells and were endocytosed by nonprofessional phagocytic cells. Coated microspheres displayed properties of binding to eukaryotic cells that were similar to those of chlamydiae, and the microspheres were competitively inhibited by chlamydial organisms. Endocytosis of heparin-coated beads resulted in the tyrosine phosphorylation of a similar set of host proteins as did endocytosis of chlamydiae; however, unlike viable chlamydial organisms, which prevent phagolysosomal fusion, endocytosed beads were trafficked to a lysosomal compartment. These findings suggest that heparin-coated beads and Chlamydia trachomatis enter eukaryotic cells by similar pathways.

Bacterial Adhesion↗
PubMed2000

Comparison of extended versus brief treatments for marijuana use.

Adult marijuana users (N = 291) seeking treatment were randomly assigned to an extended 14-session cognitive-behavioral group treatment (relapse prevention support group; RPSG), a brief 2-session individual treatment using motivational interviewing (individualized assessment and intervention; IAI), or a 4-month delayed treatment control (DTC) condition. Results indicated that marijuana use, dependence symptoms, and negative consequences were reduced significantly in relation to pretreatment levels at 1-, 4-, 7-, 13-, and 16-month follow-ups. Participants in the RPSG and IAI treatments showed significantly and substantially greater improvement than DTC participants at the 4-month follow-up. There were no significant differences between RPSG and IAI outcomes at any follow-up. The relative efficacy of brief versus extended interventions for chronic marijuana-using adults is discussed.

Adult↗
PubMed1999

Social anxiety and drinking in college students: a social cognitive theory analysis.

Evidence is reviewed that indicates that social anxiety is a significant motivation for drinking among college students. Although the link between social anxiety and alcohol consumption has been studied from a variety of perspectives, there has been little integration of data. Drawing from the alcohol and anxiety literature, the relationship between social anxiety and heavy drinking in college students is examined. Findings indicate a clear relationship between social anxiety and drinking that may be moderated by alcohol expectancies and self-efficacy beliefs specific to socially anxious situations. A social cognitive model is proposed to guide future research and intervention efforts. A better understanding of college students' reasons for drinking offers the possibility of improving prevention and treatment efforts designed to reduce excessive drinking.

Adult↗
PubMed1999

Cervical secretory immunoglobulin A in adolescent girls.

PURPOSE: To determine whether there are differences in levels of cervical secretory immunoglobulin A (sIgA) between adolescent girls in the secretory and proliferative phases of their menstrual cycle. METHODS: Sexually active adolescent girls (n = 117) at health maintenance organization (HMO) based adolescent medical clinic were recruited into the study. In addition to demographic and clinical data, cervical specimens were collected for sIgA measurement and gonorrhea culture, urine for chlamydia ligase chain reaction, and blood for progesterone levels. Subjects were classified as being in the proliferative phase or secretory phase of the menstrual cycle on the basis of their progesterone levels. RESULTS: The mean age of the subjects was 17.2 years old. There was no difference in the sIgA levels between those in the proliferative phase of their cycle (n = 45; mean sIgA level, 0.0055 mg/mL) and those in the secretory phase (n = 40; mean sIgA level, 0.0032 mg/mL) (p > .10). CONCLUSIONS: The secretory phase of the menstrual cycle does not appear to be associated with higher levels of sIgA in adolescent girls. These results suggest that adolescents with anovulatory cycles, i.e., those who lack a secretory phase, may not be at increased risk for genital tract infections such as chlamydia or gonorrhea.

Adolescent↗
PubMed1999

Comparative genomes of Chlamydia pneumoniae and C. trachomatis.

Chlamydia are obligate intracellular eubacteria that are phylogenetically separated from other bacterial divisions. C. trachomatis and C. pneumoniae are both pathogens of humans but differ in their tissue tropism and spectrum of diseases. C. pneumoniae is a newly recognized species of Chlamydia that is a natural pathogen of humans, and causes pneumonia and bronchitis. In the United States, approximately 10% of pneumonia cases and 5% of bronchitis cases are attributed to C. pneumoniae infection. Chronic disease may result following respiratory-acquired infection, such as reactive airway disease, adult-onset asthma and potentially lung cancer. In addition, C. pneumoniae infection has been associated with atherosclerosis. C. trachomatis infection causes trachoma, an ocular infection that leads to blindness, and sexually transmitted diseases such as pelvic inflammatory disease, chronic pelvic pain, ectopic pregnancy and epididymitis. Although relatively little is known about C. trachomatis biology, even less is known concerning C. pneumoniae. Comparison of the C. pneumoniae genome with the C. trachomatis genome will provide an understanding of the common biological processes required for infection and survival in mammalian cells. Genomic differences are implicated in the unique properties that differentiate the two species in disease spectrum. Analysis of the 1,230,230-nt C. pneumoniae genome revealed 214 protein-coding sequences not found in C. trachomatis, most without homologues to other known sequences. Prominent comparative findings include expansion of a novel family of 21 sequence-variant outer-membrane proteins, conservation of a type-III secretion virulence system, three serine/threonine protein kinases and a pair of parologous phospholipase-D-like proteins, additional purine and biotin biosynthetic capability, a homologue for aromatic amino acid (tryptophan) hydroxylase and the loss of tryptophan biosynthesis genes.

Amino Acid Sequence↗