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.

High-resolution mapping of serovar-specific and common antigenic determinants of the major outer membrane protein of Chlamydia trachomatis.

The principal surface protein antigen of Chlamydia trachomatis is the major outer membrane protein (MOMP). The MOMP is antigenically complex. Among the 15 serovars of C. trachomatis, mAbs define serovar-, subspecies-, and species-specific determinants on MOMP. The molecular basis of the antigenic diversity of these proteins is reflected in amino acid variable sequence domains. We have mapped the dominant topographic antigenic determinants of MOMP that are defined by mAbs. Using recombinant DNA approaches we have identified the linear distribution of two antigenic domains. One domain contains a serovar-specific determinant and the other contains subspecies- and species-specific determinants. These antigenic domains correspond to two amino acid sequence variable domains. Synthetic peptides were immunogenic and these resolved the serovar-specific determinant within a 14-amino acid peptide. The subspecies- and species-specific determinants were overlapping within a 16-amino acid peptide.

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.

HIV associated pulmonary emphysema: a review of the literature and inquiry into its mechanism.

Chronic lung diseases are increasingly recognised complications of the human immunodeficiency virus (HIV) infection and acquired immune deficiency syndrome (AIDS). Of these, pulmonary emphysema, characterised by permanent destruction of the lung parenchyma distal to the terminal bronchioles accompanied by various degrees of inflammation, is emerging as a distinct source of morbidity for patients infected with HIV. Similarly, HIV is now frequently cited as a susceptibility factor for the development of emphysema, independent of cigarette smoking status. The presence of common coexistent confounding factors that may predispose patients to chronic lung injury such as drugs, opportunistic infections and malnutrition, limits the scope of studies of direct mechanisms involved in HIV associated emphysematous lung disease. We review the clinical studies supporting a direct association between HIV infection and emphysema. Recent developments in the basic understanding of HIV infection and emphysema are also reviewed, since they may aid in understanding the pathobiology of HIV associated emphysema. The authors emphasise how HIV infection may affect cytotoxic lymphocyte activation, lung capillary endothelial cell injury and apoptosis, sphingolipid imbalance and oxidative stress in the lung. A better understanding of the pathogenesis of HIV associated pulmonary emphysema may provide clues and therapeutic targets that have broader application in this disease, including cigarette smoke induced emphysema.

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.

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.

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.

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.

Direct fluorescent monoclonal antibody stain for rapid detection of infant Chlamydia trachomatis infections.

A method of direct fluorescent antibody staining for rapid diagnosis of Chlamydia trachomatis infections in infants is described. This method utilized a fluorescein-conjugated species-specific monoclonal antibody to C trachomatis for detecting chlamydial elementary bodies in smears of the conjunctiva, nasopharynx, oropharynx, anus, and vagina. The sensitivity of direct fluorescent antibody staining was compared with isolation of the organisms in McCoy cells. Thirty-nine infants with purulent conjunctivitis were studied. Diagnosis of C trachomatis conjunctivitis was correctly made by smear in all 16 infants when inflamed eyes were sampled. Positive smears were obtained from 12/14 culture-positive and 4/16 culture-negative nasopharyngeal specimens from infants with chlamydial conjunctivitis. All nasopharyngeal cultures and smears from infants with nonchlamydial conjunctivitis were negative. These results indicate that the direct smear test is a sensitive and specific test for diagnosing C trachomatis infection of the eye and nasopharynx in infants, and this test can be completed within one hour of specimen collection.

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.

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.

Treating adult marijuana dependence: a test of the relapse prevention model.

Men (n = 161) and women (n = 51) seeking treatment for marijuana use were randomly assigned to either a relapse prevention (RP; G.A. Marlatt & J.R. Gordon, 1985) or a social support (SSP) group discussion intervention. Data collected for 12 months posttreatment revealed substantial reductions in frequency of marijuana use and associated problems. There were no significant differences between the cognitive-behavioral RP intervention and the SSP group discussion conditions on measures of days of marijuana use, related problems, or abstinence rates. Men in the RP condition were more likely than men in the SSP condition to report reduced use without problems at 3-month follow-up. Posttreatment increases in problems associated with alcohol did not appear to relate to reduced marijuana use. Results are discussed in terms of the need for further research with marijuana-dependent adults and the efficacy of RP.

Adult marijuana users seeking treatment.

In an effort to study the efficacy of attracting and intervening with adult marijuana users, 290 men and 92 women were screened for participation in a treatment-outcome study focused on marijuana cessation. The well-educated, self-referred sample reported using marijuana on 79 of the past 90 days before testing. Indices of the severity of marijuana abuse and general psychopathology were in the clinical range for a majority of Ss. Ss who did not report evidence of alcohol or other drug abuse (n = 144) reported less severe consequences of marijuana use and experienced less general psychological distress than Ss who also reported lifetime (n = 165) or current abuse (n = 73) of other substances in addition to marijuana. The findings indicate the need for clinical research targeting adults who are dependent on marijuana.

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.

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.

Responses to depressed interpersonal behavior: mixed reactions in a helping role.

We placed 144 female subjects in a helping role and randomly assigned them to interact with a confederate in a 3 X 3 X 2 X 2 (Psychopathology X Blaming X Advice Seeking X Sex of Confederate) factorial design. In order to study behaviors that mediate interpersonal responses to depression, male and female confederates enacted depressed, anxious, or normal roles and blamed themselves, others, or no one for their problems. The confederates requested advice in half of the conditions. Results indicated that depressed confederates were rejected more on questionnaire measures; however, depressed confederates received more conversational advice and support from subjects than did the equally disturbed anxious confederates. The self-blaming and advice-seeking manipulations did not interact with depression to produce more negative reactions in subjects. There was no evidence of a negative mood induction in subjects, nor did the sex of the confederate have important interpersonal consequences. Results are discussed in terms of theoretical and methodological issues in studies of interpersonal factors in depression.

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.

Verbal report methods in clinical research on alcoholism: response bias and its minimization.

Verbal report procedures, such as interviews, tests and questionnaires, have become the dominant method to obtain clinical data on alcohol abuse and its modification through treatment. The extent to which this method provides reliable and valid information for research purposes, and how its accuracy and usefulness can be enhanced, is examined. A review of methodological studies in the alcohol literature shows that although the information obtained from alcoholics and heavy drinkers tends to be reliable and valid, there can be considerable variability in accuracy, depending on the sensitivity of the information sought, the specificity of the validation criteria, the personal characteristics of the respondents and the demand characteristics of the task. It is suggested that the question of whether verbal report procedures are valid or invalid is less important than the issue of how they can be improved to the point that confidence can be placed in their findings. To facilitate this process, methodological techniques likely to enhance validity are reviewed.

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.

Testing the abstinence violation effect construct with marijuana cessation.

It has been proposed that internal, stable, and global attributions for the cause of a lapse following a period of abstinence and concomitant feelings of guilt and loss of control increased the probability of a return to regular substance use. The Abstinence Violation Effect (AVE) hypotheses were tested in a sample of 75 adult marijuana users who reported a lapse into marijuana use following completion of either a relapse prevention (RP) or social support group treatment aimed at abstinence. Results showed that more internal, stable, and global attributions for the cause of the lapse and perceived loss of control were related significantly to concurrently reported relapse. Further, internal and global attributions predicted marijuana use during the subsequent 6 months. Results are discussed in terms of support for the AVE construct, treatment implications, and the failure of the RP treatment to modify reactions to a lapse.