A cell biology approach to microbial pathogenesis.
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Antigen Presentation↗
Biomedical subjects
R S Stephens
Publications and source records attributed to R S Stephens.
Clinical application of a homogeneous colorimetric assay for tear lysozyme.
PURPOSE: Tear lysozyme and tear lactoferrin are enzymes synthesized by the lacrimal gland. Their concentration in human tears reflects tear gland function. Tear gland dysfunction can lead to ocular surface disease. We developed a colorimetric lysozyme assay. The objective of this study was to determine the diagnostic power and the clinical application of this assay that allows rapid and precise quantification of tear lysozyme. METHODS: Tear specimens of 120 eyes (30 Sjögren's patients and 30 controls) were collected using standardized filter paper discs. Tear lysozyme concentration was determined using p-nitrophenyl penta-N-acetyl-beta-chitopentaoside as substrate in the colorimetric assay. The results were compared to clinical findings and to two commonly used tests, the Micrococcus agar diffusion assay for tear lysozyme and the tear lactoferrin immunodiffusion assay. RESULTS: The colorimetric assay showed a good dose-response relationship. The use of the assay as a method of diagnosing aqueous tear deficiency, using the clinical findings and the medical history as gold standard, demonstrated 85% sensitivity and 92% specificity. The results of the colorimetric assay when compared with the Micrococcus agar diffusion assay showed a linear relationship of r=0.77; when compared with the lactoferrin immunoassay r=0.73. CONCLUSIONS: The colorimetric assay is simple to perform and does not require sophisticated laboratory equipment and personnel. Results can be precisely quantified within one hour after tear collection. The diagnostic power of the test is comparable to previously reported assays for lysozyme and lactoferrin and will be useful in the diagnosis of ocular surface disease.
Adolescent↗
Computational analysis of the polymorphic membrane protein superfamily of Chlamydia trachomatis and Chlamydia pneumoniae.
Whole sequence genome analysis is invaluable in providing complete profiles of related proteins and gene families. The genome sequences of the obligate intracellular bacteria Chlamydia trachomatis and Chlamydia pneumoniae both encode proteins with similarity to several 90-kDa Chlamydia psittaci proteins. These proteins are members of a large superfamily, C. trachomatis with 9 members and C. pneumoniae with 21 members. All polymorphic membrane protein (Pmp) are heterogeneous, both in amino acid sequence and in predicted size. Most proteins have apparent signal peptide leader sequences and hence are predicted to be localized to the outer membrane. The unifying features of all proteins are the conserved amino acid motifs GGAI and FXXN repeated in the N-terminal half of each protein. In both genomes, the pmp genes are clustered at various locations on the chromosome. Phylogenetic analysis suggests six related families, each with at least one C. trachomatis and one C. pneumoniae orthologue. One of these families has seen prolific expansion in C. pneumoniae, resulting in 13 protein paralogues. The maintenance of orthologues from each species suggests specific functions for the proteins in chlamydial biology.
Amino Acid Motifs↗
Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis.
Analysis of the 1,042,519-base pair Chlamydia trachomatis genome revealed unexpected features related to the complex biology of chlamydiae. Although chlamydiae lack many biosynthetic capabilities, they retain functions for performing key steps and interconversions of metabolites obtained from their mammalian host cells. Numerous potential virulence-associated proteins also were characterized. Several eukaryotic chromatin-associated domain proteins were identified, suggesting a eukaryotic-like mechanism for chlamydial nucleoid condensation and decondensation. The phylogenetic mosaic of chlamydial genes, including a large number of genes with phylogenetic origins from eukaryotes, implies a complex evolution for adaptation to obligate intracellular parasitism.
Aerobiosis↗
Further evidence for the involvement of human chromosome 6p24 in the aetiology of orofacial clefting.
Chromosomal translocations affecting the 6p24 region have been associated with orofacial clefting. Here we present a female patient with cleft palate, severe growth retardation, developmental delay, frontal bossing, hypertelorism, antimongoloid slant, bilateral ptosis, flat nasal bridge, hypoplastic nasal alae, protruding upper lip, microretrognathia, bilateral, low set, and posteriorly rotated ears, bilateral microtia, narrow ear canals, short neck, and a karyotype of 46,XX,t(6;9)(p24;p23). The translocation chromosomes were analysed in detail by FISH and the 6p24 breakpoint was mapped within 50-500 kb of other breakpoints associated with orofacial clefting, in agreement with the assignment of such a locus in 6p24. The chromosome 9 translocation breakpoint was identified to be between D9S156 and D9S157 in 9p23-p22, a region implicated in the 9p deletion syndrome.
Chromosome Mapping↗
College on problems of drug dependence meeting, Puerto Rico (June 1996) marijuana use and dependence.
Discoveries concerning an endogenous cannabinoid system and observations of dramatic increases in marijuana use among youth in the United States have fueled a recent increase in basic and clinical research to better understand and treat marijuana dependence. At the annual meeting of the College on Problems of Drug Dependence (Puerto Rico, 1996) a symposium 'Marijuana Use: Basic Mechanisms, Epidemiology, and Clinical Issues' reviewed a number of important areas of ongoing research that address marijuana dependence. Overviews and original research were presented regarding the development of dependence (preclinical and clinical research), motivational effects (laboratory models), the epidemiology of dependence and its development, clinical management of marijuana use among patients seeking treatment for other drugs of abuse, and treatment for adult marijuana dependence. This paper summarizes the symposium presentations and provides discussion of recent scientific developments concerning marijuana use and dependence.
Adult↗
Secretion of proinflammatory cytokines by epithelial cells in response to Chlamydia infection suggests a central role for epithelial cells in chlamydial pathogenesis.
Chlamydia species infect epithelial cells at mucosal surfaces, and are major causes of sexually transmitted diseases. Infection is characterized by inflammation which is exacerbated upon reinfection, ultimately leading to tissue damage and scarring. Although central for the development of disease manifestations, little is known about the mechanisms that initiate and sustain the inflammatory response to Chlamydia. Infection of cervical and colonic epithelial cells with Chlamydia trachomatis and Chlamydia psittaci is shown in the present studies to upregulate mRNA expression and secretion of the proinflammatory cytokines IL-8, GRO alpha, GM-CSF, and IL-6. In contrast to the rapid, but transient, cytokine induction following infection with other invasive bacteria, the epithelial cytokine response to Chlamydia was delayed until 20-24 h after infection, persisted throughout the chlamydial growth cycle (2-4 d), and required bacterial protein synthesis. Moreover, epithelial cell lines and primary endocervical epithelial cells released IL-1alpha after Chlamydia infection, and increased secretion of the proinflammatory cytokines could be inhibited by anti-IL-1alpha. This suggests that IL-1alpha, released following lysis of infected epithelial cells, may amplify the inflammatory response by stimulating additional cytokine production by noninfected neighboring cells. These findings suggest a novel pathophysiologic concept wherein the acute host response to Chlamydia at mucosal surfaces is primarily initiated and sustained by epithelial cells, the first and major targets of chlamydial infection.
Actins↗
Chlamydia trachomatis glycosaminoglycan-dependent and independent attachment to eukaryotic cells.
Chlamydia trachomatis consists of two biovars, lymphogranuloma venereum (LGV) and trachoma, that differ in their infectivity in vivo and in vitro. Although addition of exogenous heparin or heparan sulfate in vitro effectively inhibits infectivity of both biovars and inhibits LGV biovar attachment to host cells, trachoma biovar attachment was only modestly inhibited (approximately 30%) by exogenous heparin. To dissect the relationship of heparin inhibition of attachment and infectivity, a heparan sulfate lyase (heparitinase) was used to treat organisms and evaluated for changes in attachment and infectivity. In contrast to heparitinase-treated LGV biovar organisms that lose their ability to attach and infect, treatment of trachoma biovar organisms with a concentration of heparitinase sufficient to reduce trachoma biovar infectivity by > 90%, only inhibited attachment to host cells by approximately 40%. Significantly, attachment could be fully restored for heparitinase-treated organisms of both biovars with exogenous heparan sulfate; however, the coating of the trachoma biovar organisms with heparan sulfate rendered the trachoma biovar similar to the phenotype of the LGV biovar by > 90% sensitivity to heparin inhibition of attachment. These data suggest that the LGV biovar used predominantly a heparin-inhibitable mechanism for attaching to host cells, whereas the trachoma biovar used a heparin-independent means in addition to a heparin-dependent mechanism to adhere to host cells. Once attached, the trachoma biovar, nevertheless, relied on the heparin-dependent pathway to enter host cells.
Animals↗
Antigen conformation dependence of Chlamydia trachomatis infectivity neutralization.
Chlamydia trachomatis infections cause the most common notifiable diseases in the United States, reflecting the successful adaptation of these organisms to persist in their obligate human host population. Antigenic variation of the quantitatively predominant major outer membrane protein (MOMP) is considered to play an important role in this adaptation as a means of immune evasion. The relative capacity of murine polyvalent sera produced following infection, recovery, and challenge to neutralize infectivity was highly serovar-specific and dependent upon thermolabile antigens. The structural complexity of these immunodominant antigens was mimicked by chlamydial MOMP expressed in Escherichia coli, as antibodies that neutralize infectivity by recognition of conformation-dependent antigens were specifically removed from sera following absorption using MOMP expressed in E. coli.
Animals↗
Cross-reactive cytotoxic T-lymphocyte-mediated lysis of Chlamydia trachomatis- and Chlamydia psittaci-infected cells.
Cells infected with Chlamydia trachomatis are lysed by CD8+ T cells in vitro. The ability of C. trachomatis-elicited spleen cells to lyse target cells infected with other chlamydial strains was determined by measuring lysis by immune spleen cells of targets infected with three strains of C. trachomatis and two strains of C. psittaci. C. trachomatis (lymphogranuloma venereum [LGV])-elicited immune murine spleen cells lysed target cells infected with other C. trachomatis serovars, although with lower sensitivity than they lysed LGV-infected target cells. Additionally, target cells infected with C. psittaci were lysed by C. trachomatis-elicited immune spleen cells. Notably, C. psittaci-infected cells were lysed with greater efficiency than were cells infected with the C. trachomatis strain used to elicit the immune spleen cells. The lysis of C. psittaci-infected cells was characterized further and could be only partially accounted for by CD8+ T-cell-mediated lysis, the remaining lysis being due to an antigen-nonspecific component. These results indicate that mechanisms of immunologically mediated lysis differ between C. trachomatis- and C. psittaci-infected cells. This has important implications for the interpretation of results obtained with C. psittaci models of infection and immune resolution, particularly as they may be extrapolated to C. trachomatis.
Animals↗
Structural requirements of heparin binding to Chlamydia trachomatis.
Heparin is a functional and structural analog of the Chlamydia trachomatis heparan sulfate-like attachment ligand that mediates infectivity by bridging chlamydiae to eukaryotic cells. The binding of heparin to the Chlamydia organism's surface was characterized by a direct binding assay. Although for two C. trachomatis biovars the binding by heparin was saturable, trachoma biovar organisms bound twice the amount of heparin than lymphogranuloma venereum biovar organisms. To prove the structural nature of the heparan sulfate-like ligand interactions, a range of heparin-derived oligosaccharides and sulfation-modified species of heparin were compared for their ability to compete with [3H]heparin for binding to chlamydial organisms and for inhibition of chlamydial attachment and infection of eukaryotic host cells. The assays revealed that a decasaccharide was the minimal chain length required to effectively bind C. trachomatis organisms, compete with the host cell receptor and rescue infectivity. In addition, a moderately sulfated adhesin analog, N-desulfated, N-acetylated heparin, was able to compete with chlamydial organisms for host cell receptors, whereas this derivative could not compete with [3H]heparin for binding to chlamydial organisms. These results indicate that the specificity of the eukaryotic cell receptor and the chlamydial surface acceptor differ in their fine-structure requirements of ligand binding, and that the size and sulfation density of the heparan sulfate-like ligand each contribute to its ability to bind and bridge chlamydiae to eukaryotic cells.
Animals↗
Recall of original serologic response after challenge with homologous and heterologous Chlamydia trachomatis serovars.
Chlamydia trachomatis serovar-specific major outer membrane protein (MOMP) antigens are important targets of immune neutralization in vitro, and natural immunity to infection is associated with serovar specificity. Reinfection, often by different serovars, plays an essential role in chlamydial disease pathogenesis. By use of a murine model, the anamnestic serologic response was characterized following priming and challenge inoculations using 6 different serovars. The serologic response was evaluated using synthetic peptides representing MOMP variant segments (VS) 1,2,3, and 4 antigenic same serovar resulted in serologic responses to homologous VS1 peptides. After challenge with a different serovar, anti-VS1 serologic responses were often elicited with specificity to both the priming serovar and the challenge serovar. The recall of serologic response to the original serovar was typically dependent upon the antigenic relationship of the 2 serovars.
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↗
Problems in the application of the Addiction Severity Index (ASI) in rural substance abuse services.
The aim of this research was to test the utility of the Addiction Severity Index (ASI) in a rural community substance abuse outpatient treatment center and to predict future alcohol and drug problems based on ASI information. Substance abuse counselors used the ASI to assess the problems of 89 adult clients at intake, and research staff assessed drug use outcomes 3 months later. There were significant improvements between intake and follow-up on the scores on the alcohol, drug, legal, and family sections of the ASI. Scores on the alcohol abuse, drug abuse, and medical sections of the ASI predicted some of the variance (16%) in drug and alcohol use outcomes, but psychiatric, medical, legal, and employment problems were not significant predictors. There was some difficulty in obtaining adequate reliability in counselors' severity ratings for alcohol problems. The relationship of counselors' severity ratings to the more objective composite scores varied substantially by subsection of the ASI. The improvements in alcohol and drug use indices appeared to be more related to subjective appraisals of problems related to use rather than due to changes in drug-using behavior. Implications of these findings and recommendations for further research using the ASI are discussed.
Adult↗
Self-efficacy and marijuana cessation: a construct validity analysis.
Hypotheses regarding the relationships between self-efficacy for avoiding marijuana use and theoretically related measures were examined in a sample of 161 men and 51 women who sought treatment aimed at marijuana cessation. Theoretically proposed sources of efficacy judgments showed stronger univariate and multivariate relationships with efficacy for avoiding marijuana use after treatment than before treatment. The cognitive-behavioral relapse prevention treatment resulted in marginally greater self-efficacy, compared with a nonbehavioral treatment, but the link between coping skill training and efficacy was ambiguous. Efficacy contributed incrementally to the prediction of posttreatment marijuana use beyond efficacy source variables, but it did not completely mediate the effects of those sources of efficacy judgments. Predictive validity was stronger for frequency of posttreatment marijuana use than for abstinence status. The need for better assessment of the efficacy construct and potential revisions in efficacy theory as applied to substance use are discussed.
Adult↗
CD8+ T lymphocyte-mediated lysis of Chlamydia-infected L cells using an endogenous antigen pathway.
Intracellular bacterial pathogens have evolved to either grow in the nutrient-rich cytoplasm or remain sequestered within a vacuole. One potentially important selective advantage for growth within a vacuole may be evasion of cell-mediated detection and cytolysis. To address this question we used the endosomally confined bacterium Chlamydia trachomatis, which naturally infects epithelial cells, to examine CTL-mediated lysis of nonphagocytic cells. CTL-mediated lysis of infected target cells was detected, although the increased expression of ICAM-1 by transfection was required. The elimination of CD8+ T cells or addition of brefeldin A or cycloheximide eliminated specific cytolysis, whereas conversely, treatment with chloroquine or ammonium chloride had only minor effects. These results implicate endogenous Ag processing for Chlamydia-specific cytolysis. This work demonstrates CTL-mediated lysis of cells infected with an intracellular bacterium that inhibits lysosomal fusion and is confined to an endosomal vacuole.
Animals↗
Measurement of human tear lysozyme using a novel synthetic substrate.
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Colorimetry↗
Molecular mimicry and Chlamydia trachomatis infection of eukaryotic cells.
A new experimental model for microbe-host-cell interaction is proposed in which a molecular mimic of heparan sulfate is used by Chlamydia to attach to the mammalian cell surface. A heparan-sulfate-like ligand, bound to the surface of Chlamydia, mediates infectivity by bridging the microorganism and mammalian cell receptors.
Animals↗