Purification and characterization of the major iron-regulated protein expressed by pathogenic Neisseriae.

This report describes a method to purify the major iron-regulated protein (MIRP) expressed by N. gonorrhoeae and N. meningitidis. This purification procedure involves maximal expression of the MIRP by growing the organisms on iron-limited media; cellular disruption by sonication followed by centrifugal fractionation; selective solubilization of the MIRP with the cationic detergent hexadecyltrimethylammonium bromide; cation-exchange chromatography in the presence of this detergent; and gel filtration chromatography. The MIRP purified by this technique migrates as a single band when analyzed by SDS-PAGE. The purified MIRP displayed an unusually basic isoelectric point, this value being greater than 9.35. Further biochemical analysis revealed the highly conserved nature of this protein isolated from the two pathogenic species of the genus Neisseria. For example, the amino acid composition of the meningococcal and gonococcal MIRPs were nearly identical and amino terminal sequence analysis showed that both shared the identical primary sequence through residue 48. Surprisingly, the first five NH2-terminal residues of the MIRPs exhibited homology with the first five residues of the gonococcal porin, protein I. Purified preparations of the MIRP exhibited a characteristic pink color reminiscent of the basic iron-binding protein lactoferrin. This observation coupled with the property of iron-regulation prompted us to analyze purified MIRP for iron-content. Approximately 0.5 mol iron per 1 mol of MIRP was detected. This study is the first to show that iron is associated with the MIRP, a property that may implicate this protein as playing a direct role in neisserial iron assimilation. While the precise function of the MIRP is not known, the availability of this protein in pure and biologically relevant quantities will allow further studies to elucidate its pathobiologic function.

Relative efficiency of chlamydia screening in non-clinical settings in two California counties.

We examined the relative efficiency of non-clinical sites to screen for chlamydia in youth and young adults. Chlamydia screening targeting youth (under 30 years of age) was performed at non-clinical sites in high-morbidity neighbourhoods of two California counties. Venues were subdivided into community outreach, schools, parenting centres and drug treatment/correctional facilities. Relative efficiency was estimated with multivariate Poisson regression using incidence of chlamydia per person-hours labour adjusting for strategy and county. Among the 1514 youths screened, the overall prevalence of chlamydia was 5.5%. By venue, the highest prevalence was in drug treatment/correction facilities at 11.1% followed by parenting centres at 6.3%, community outreach at 4.9% and schools at 4.6%. Drug treatment/correctional facilities were the most labour time efficient at 9.9 person-hours per chlamydia case. Schools and parenting centres had the lowest cost per screen at 0.9 person-hours per screen. Adjusted relative labour time efficiency (chlamydia cases per paid person-hour) was significantly higher in schools, 2.0 (95% confidence interval [CI] 1.0-4.2), parenting centres, 3.2 (95% CI 1.6-6.6) and drug treatment/correctional facilities, 2.9 (95% CI 1.0-7.8), compared with community outreach. In conclusion, parenting centres and drug treatment centres and correctional facilities are the most efficient venues for chlamydia screening.

Using strain typing to characterise a fluoroquinolone-resistant Neisseria gonorrhoeae transmission network in southern California.

OBJECTIVE: We investigated the initial outbreak of fluoroquinolone-resistant Neisseria gonorrhoeae (QRNG) in southern California with analysis of transmission using strain typing. METHODS: Surveillance for QRNG was conducted between 2000 and 2002 in southern California, including epidemiology and strain typing by a combination of antibiogram, auxotype, serovar, Lip type and amino acid alteration patterns in the quinolone-resistance determining region of GyrA and ParC. Combining epidemiological data with strain typing, we describe the emergence of QRNG outbreak strains using risk factor analysis and transmission networks. RESULTS: Two outbreak strains accounted for 82% of isolates. Both strains required proline, were Lip type 17c, had amino acid alterations 91> Phe in GyrA and 87> Arg in ParC, but they differed by their serovar, IB-3C8 versus IB-2H7, 2G2. Outbreak strains were positively associated with men who have sex with men (MSM), adjusted odds ratio (AOR) 23.9 (95% confidence interval (CI) 2.2 to 261) and negatively associated with travel history: AOR 0.05, (95% CI 0.0 to 0.6). Network analysis demonstrated that 17 cases were connected by sexual contacts and/or public venues including bars, bathhouses/sex clubs, and internet sites. CONCLUSIONS: QRNG may have become established among Californian MSM through an identified transmission network of southern Californian bars, bathhouses and internet sites.

The effects of estrogens and antiestrogens on hormone-responsive human breast cancer in long-term tissue culture.

We have established or characterized six lines of human breast cancer maintained in long-term tissue culture for at least 1 year and have examined these lines for estrogen responsiveness. One of these cell lines, MCF-7, shows marked stimulation of macromolecular synthesis and cell division with physiological concentrations of estradiol. Antiestrogens are strongly inhibitory, and at concentrations greater than 3 X 10(-7) M they kill cells. Antiestrogen effects are prevented by simultaneous treatment with estradiol or reversed by addition of estradiol to cells incubated in antiestrogen. Responsive cell lines contain high-affinity specific estradiol receptors. Antiestrogens compete with estradiol for these receptors but have a lower apparent affinity for the receptor than estrogens. Stimulation of cells by estrogens is biphasic, with inhibition and cell death at concentrations of 17beta-estradiol or diethylstilbestrol exceeding 10(-7) M. Killing by high concentrations of estrogen is probably a nonspecific effect in that we observe this response with 17alpha-estradiol at equivalent concentrations and in the otherwise unresponsive cells that contain no estrogen receptor sites.

The effects of glucocorticoids and progesterone on hormone-responsive human breast cancer in long-term tissue culture.

Glucocorticoids, at physiological concentration, inhibit cell division and thymidine incorporation in three lines of human breast cancer maintained in long-term tissue culture. At steroid concentrations sufficient to inhibit thymidine incorporation 50%, little or no effect is seen on protein synthesis 48 hr after hormone addition. All three of these lines are shown to have glucocorticoid receptors demonstrable by competitive protein binding assays. Receptors are extensively characterized in one line by sucrose density gradient analysis and binding specificity studies. Good correlation between receptor-binding specificity and biological activity is found except for progesterone, which binds to glucocorticoid receptor but is noninhibitory. Cross-competition and quantification studies demonstrate a separate receptor for progesterone. This receptor has limited binding specificities restricted largely to progestational agents, whereas the glucocorticoid receptor bound both glucocorticoids and progesterone. Two other human breast cancer lines neither contain glucocorticoid receptor nor are inhibited by glucocorticoids. It is concluded that in some cases glucocorticoids can directly limit growth in human breast cancer in vitro without requiring alterations in other trophic hormones.

The effects of androgens and antiandrogens on hormone-responsive human breast cancer in long-term tissue culture.

We have examined five human breast cancer cell lines in continuous tissue culture for androgen responsiveness. One of these cell lines shows a 2- to 4-fold stimulation of thymidine incorporation into DNA, apparent as early as 10 hr following androgen addition to cells incubated in serum-free medium. This stimulation is accompanied by an acceleration in cell replication. Antiandrogens [cyproterone acetate (6-chloro-17alpha-acetate-1,2alpha-methylene-4,6-pregnadiene-3,20-dione) and R2956 (17beta-hydroxy-2,2,17alpha-trimethoxyestra-4,9,11-triene-1-one)] inhibit both protein and DNA synthesis below control levels and block androgen-mediated stimulation. Prolonged incubation (greater than 72 hr) in antiandrogen is lethal. The MCF- cell line contains high-affinity receptors for androgenic steroids demonstrable by sucrose density gradients and competitive protein binding analysis. By cross-competition studies, androgen receptors are distinguishable from estrogen receptors also found in this cell line. Concentrations of steroid that saturate androgen receptor sites in vitro are about 1000 times lower than concentrations that maximally stimulate the cells. Changes in quantity and affinity of androgen binding to intact cells at 37 degrees as compared with usual binding techniques using cytosol preparation at 0 degrees do not explain this difference between dissociation of binding and effect. However, this difference can be explained by conversion of [3H]-5alpha-dihydrotestosterone to 5alpha-androstanediol and more polar metabolites at 37 degrees. An examination of incubation media, cytoplasmic extracts and crude nuclear pellets reveals probable conversion of [3H]testosterone to [3H]-5alpha-dihydrotestosterone. Our data provide compelling evidence that some human breast cancer, at least in vitro, may be androgen dependent.

Effects of estrone, estradiol, and estriol on hormone-responsive human breast cancer in long-term tissue culture.

The effects of estrone, estradiol, and estriol on MCF-7 human breast cancer are compared. In this estrogen-responsive cell line, all three estrogens are capable of inducing equivalent stimulation of amino acid and nucleoside incorporation. Estriol is capable of partially overcoming antiestrogen inhibition with Tamoxifen (lCl 46474), even when antiestrogen is present in 1000-fold excess. Antiestrogen effects are completely overcome by 100-fold less estriol. Studies of metabolism of estrogens by MCF-7 cells revealed no conversion of estriol to either estrone or estradiol. All three steroids bind to a high-affinity estrogen receptor found in these cells. The apparent dissociation constant is lower for estradiol than for estrone and estriol, but all three bind to an equal number of sites when saturating concentrations are used. Tritiated estrogens used in binding studies were shown to be radiochemically pure. We conclude that estriol can bind to estrogen receptor and stimulate human breast cancer in tissue culture. Our data do not support an antiestrogenic role for estriol in human breast cancer.

Sexually transmitted disease (STD) and HIV risk in heterosexual adults attending a public STD clinic: evaluation of a randomized controlled behavioral risk-reduction intervention trial.

OBJECTIVE: To evaluate the efficacy of a cognitive/behavioral skills-building intervention to prevent sexually transmitted diseases (STD) in high-risk heterosexual adults. DESIGN: A randomized controlled trial with assessments at baseline, and at 3 and 5 months. SETTING: San Francisco STD Clinic. PATIENTS: A total of 399 patients were randomly assigned to a four-session, individual, multi-component, cognitive/behavioral intervention (n = 199), or a brief standardized counseling session offered to all patients (n = 200). INTERVENTION: Based on the AIDS Risk-Reduction Model, the aims of the intervention were to increase prevention knowledge, reduce high-risk psychosocial factors, and build decision-making and communication skills to modify sexual behaviors. MAIN OUTCOME MEASURES: The primary outcome of interest was STD. The secondary outcome was number of risky sexual activities. RESULTS: There were no differences between the intervention (13%) and control (11%) groups in their acquisition of STD. Among men, condom use increased more at 3 months in the intervention group than the control group (56.8 versus 42.3%; P < 0.05). In addition, the mean number of sexual partners without condom use was lower in the intervention group than in the control group at 5 months (0.6 versus 0.9; P < 0.01). CONCLUSIONS: The results suggest that a cognitive/behavioral, skills-building intervention consisting of individual, multiple sessions and follow-up assessments can be implemented and evaluated with high-risk heterosexually active adults attending public STD clinics. Our intervention did not have a significant impact on STD, although it had some impact on behavior in men, but not in women.

Red man syndrome: inadvertent administration of an excessive dose of rifampin to children in a day-care center.

A cluster of toxic reactions among children inadvertently given excessive doses of rifampin for chemoprophylaxis of invasive Haemophilus influenzae disease in a day-care center was investigated. In all 19 children, who received five times the therapeutic dose of rifampin, dramatic adverse reactions developed. A striking, "glowing" red discoloration of the skin and facial or periorbital edema were found to be the hallmarks of rifampin toxicity. These clinical signs of acute toxicity contrast sharply with the adverse side effects of rifampin reported with therapeutic doses.

Detection of meningitis epidemics in Africa: a population-based analysis.

Portions of sub-Saharan Africa are subject to major epidemics of meningococcal meningitis that require early detection and rapid control. We evaluated the usefulness of weekly meningitis rates derived from active surveillance data in Burkina Faso for detecting a meningitis epidemic. By analysing the rates of disease in 40 x 40km2 areas within a study region of Burkina Faso, we found that a threshold of 15 cases/100,000/week averaged over 2 weeks was 72-93% sensitive and 92-100% specific in detecting epidemics exceeding 100 cases/100,000/year. During epidemic periods, the positive predictive value of this threshold approached 100% for detecting local epidemics. Additionally, meningitis incidence was proportional to village size, with villages greater than 8000 having the highest disease rates during a major group A meningococcal epidemic in 1983-1984. Despite the rudimentary nature of surveillance data available in many developing countries, these data can be used to detect the early emergence of meningitis epidemics. Additional studies are needed to determine the relevance of this approach for detecting epidemics.

Features of sexually transmitted disease services important to African American adolescents.

OBJECTIVE: To determine the relative importance of various features of health clinics when African American adolescents consider seeking care for sexually transmitted diseases (STDs). DESIGN: Confidential interviewer-administered telephone survey. SETTING: A predominantly low-income, African American neighborhood in San Francisco, Calif. PARTICIPANTS: Random sample of African American adolescents aged 12 to 17 years; 302 (76.6%) of 394 identified eligible adolescents participated. MAIN OUTCOME MEASURES: Items and scales measuring adolescents' sense of the importance of the attributes of the provider (alpha = .58), availability of services (alpha = .61), and perceived confidentiality of health services from family (alpha = . 72) when deciding where to seek care for possible STDs. RESULTS: More than 90% (90.4%) of subjects rated items relating to provider attributes as being highly important when they consider where they would seek care for an STD; between 62.5% and 82.7% rated availability items as being highly important; and between 38.6% and 60.8% rated items pertaining to confidentiality as being highly important. Greater importance was placed on provider attributes and confidentiality by female than male adolescents. The importance placed on provider attributes and confidentiality increased as adolescents aged. CONCLUSIONS: Low-income, African American adolescents place great importance on provider attributes, less importance on availability, and even less importance on confidentiality when deciding where to seek health care for a possible STD. Health care providers and organizations need to be aware of these adolescent preferences to better promote screening and treatment of STDs in this population.

Plasma cell endometritis in women with symptomatic bacterial vaginosis.

OBJECTIVE: To evaluate the endometrial microbiology and histopathology in women with symptomatic bacterial vaginosis but no signs or symptoms of upper genital tract disease or other vaginal or cervical infections. METHODS: Endometrial biopsies were performed on 41 women complaining of vaginal discharge or pelvic pain at a sexually transmitted disease clinic. These women had neither culture nor serologic evidence of Neisseria gonorrhoeae or Chlamydia trachomatis infection. Twenty-two women with bacterial vaginosis diagnosed by Gram stain examination of vaginal fluid, but with neither signs nor symptoms of upper genital tract infection, were compared with 19 women who had no evidence of bacterial vaginosis on vaginal fluid Gram stain. Endometrial biopsies were evaluated for histopathologic evidence of plasma cell endometritis and were cultured for N gonorrhoeae, C trachomatis, aerobic and anaerobic bacteria, Mycoplasma species, and Ureaplasma urealyticum. RESULTS: Ten of 22 women with bacterial vaginosis had plasma cell endometritis, compared with one of 19 controls (odds ratio [OR] 15, 95% confidence interval [CI] 2-686; P < .01). Bacterial vaginosis-associated organisms were cultured from the endometria of nine of 11 women with and eight of 30 women without plasma cell endometritis (OR 12.4, 95% CI 2-132; P = .002). CONCLUSION: Plasma cell endometritis was frequently present in women with bacterial vaginosis and without other vaginal or cervical infections. This suggests the possibility of an association between bacterial vaginosis and nonchlamydial, nongonococcal, upper genital tract infection.

Chlamydia trachomatis infections.

Chlamydia trachomatis infections are the most common bacterial cause of sexually transmitted disease in the United States. Although precise incidence of infection is not known, it has been calculated that more than 4 million chlamydial infections occur each year. This article discusses the epidemiology of sexually transmitted chlamydial infections, the spectrum of clinical manifestations and their sequelae, the laboratory diagnosis of genital infections, and antibiotic treatment, emphasizing the significance of these issues for control efforts in the United States.

Evaluation of Syva's enzyme immunoassay for the detection of Chlamydia trachomatis in urogenital specimens.

A newly developed microwell enzyme immunosorbent assay (EIA) system by Syva Company (Palo Alto, CA) can detect Chlamydia trachomatis in < 3 hr. It uses a polyclonal antibody to chlamydial lipopolysaccharide and end points are determined with a spectrophotometer. Three clinical trial sites (University of California Medical Center, San Francisco, CA; University of Washington, Seattle, WA; and Louisiana State University Medical Center, New Orleans, LA), compared this EIA with tissue culture (TC) for identifying Chlamydia in urogenital specimens. Overall prevalence by TC was 10.4% (136 of 1306). When tests were compared with TC (using vials or microtiter plates and a fluorescent antibody stain), we found an EIA sensitivity of 93.4% (127 of 136) and a specificity of 98.1% (1148 of 1170). This EIA has a performance profile that is, at the very least, comparable with other nonculture methods for diagnosing genital tract infections with C. trachomatis.

The impact of health care market changes on local decision making and STD care: experience in three counties.

INTRODUCTION: In 1993 health care reform including universal coverage appeared imminent. Some county health departments elected to discontinue provision of direct services including sexually transmitted disease (STD) care. County A moved in this direction with final clinic closure in 1996. Coincidentally, two other counties elected to continue their STD treatment services. These events have created a "natural experiment" in which to evaluate the contrasting strategies among three counties. METHODS: This report describes the changes in local delivery over a three-year period (1993-1995). Measurements were carried out in three counties in two states. STD program capacity, service delivery, and morbidity rates for STDs (chlamydia and gonorrhea) were monitored in each study county. Quantitative data were complemented by a qualitative patient survey at each site. RESULTS: Capacity changed dramatically in County A as compared with the other two over the three-year period. Major declines in STD clinic visits (-43%) and laboratory testing (-46%) occurred. A major drop in reported STD incidence (-23% for chlamydia and -49% for gonorrhea) also occurred, including a drop in public provider reporting. CONCLUSIONS: In County A, a decision to end delivery of personal health services led to a divestiture in STD service delivery and a decline in all measured parameters. Etiology of concurrent declined in reported STDs is unclear. Possibly decreased public services led to a spurious decline in reporting. Alternatively, an overall strategy of shifting care to private providers has succeeded in reducing disease. Public health surveillance may be less accurate in such settings.

Failure to disclose HIV risk among gay and bisexual men attending sexually transmitted disease clinics.

We analyzed data from a multisite study of 1,063 gay or bisexual men attending sexually transmitted disease clinics to evaluate factors predicting failure to disclose human immunodeficiency virus (HIV) risk behaviors to clinic staff and the extent of such failure. We compared data from a brief screening assessment on unprotected anal and oral sex with data on the same behaviors from a subsequent detailed interview. We also compared behavioral data from screening and the interview with data on diagnoses of rectal gonorrhea abstracted from medical charts. Of 523 men reporting unprotected anal sex at interview, 29% failed to report this behavior at screening. Men failing to disclose unprotected anal sex were also less likely to disclose engaging in unprotected oral sex. Among men reporting no unprotected anal sex, either at screening or interview, 1.6% were diagnosed with rectal gonorrhea. Logistic regression analyses comparing men who did and did not disclose at screening having engaged in unprotected anal sex showed that men who failed to disclose reported greater involvement in gay organizations, greater perceived peer support for condoms, fewer episodes of unprotected anal sex in the last four months, and lower rates of substance abuse treatment. Our data suggest that men who failed to disclose may have lower risk levels, and may be more integrated into the gay community. Brief interviews, as opposed to detailed ones, also may underestimate incidence of unsafe sex. Where feasible, HIV risk assessment and counseling and laboratory screening should be routinely provided to all clinic attendees, regardless of self-reports.

Structure of outcome beliefs in condom use. The Project RESPECT Study Group.

To study the structure of beliefs about condom use outcomes, the authors derived and tested 4 psychosocial hypothetical models: (a) a 2-factor model of the personal and social outcomes of condom use; (b) a 2-factor model of the pros and cons of the behavior; (c) a 3-factor model (i.e., physical, self-evaluative, and social) of outcome expectancies; and (d) a thematic 4-factor model of the protection, self-concept, pleasure, and interaction implications of the behavior. All 4 models were studied with a confirmatory factor analysis approach in a multisite study of 4,638 participants, and the thematic solution was consistently the most plausible. Self-concept and pleasure were most strongly associated with attitudes toward using condoms, intentions to use condoms, and actual condom use, whereas protection and interaction generally had little influence.

Acute bacterial meningitis in children and adults. A perspective.

Bacterial meningitis is an acute and serious illness associated with significant morbidity and mortality. Although methods to make the diagnosis are relatively simple and effective antimicrobial agents are available, bacterial meningitis accounts for more than 2000 reported deaths per year in the United States. The majority of cases and deaths occur in otherwise healthy individuals.

Crack cocaine use and high-risk behaviors among sexually active black adolescents.

The recent spread of crack cocaine use among inner-city teenagers has been accompanied by dramatic increases in juvenile delinquency and sexually transmitted diseases (STDs) among teenagers. This study examined the prevalence of five factors which promote STDs, including human immunodeficiency virus (HIV), among a sample of sexually active black adolescent crack users and non-users from the San Francisco Bay Area. Significant differences were observed between these groups with respect to history of engaging in sexual intercourse under the influence of drugs or alcohol, exchanging sexual favors for drugs or money, condom use in the most recent sexual encounter, and having five or more sexual partners in the last year. Approximately 63% of all respondents reported engaging in at least one of these risk behaviors. In multiple logistic regression analysis, reporting one or more of these STD/HIV risk behaviors was significantly associated with crack use and having one or more relatives who used drugs. Intervention efforts need to address both individual and environmental risk factors in order to reduce teens' risk for STDs, including HIV.

PIP: Researchers combined data from two surveys conducted in the summer of 1988 to examine the association between sexually transmitted diseases (STD) risk behaviors and crack cocaine use among sexually active, black teenagers. All of the subjects lived in an inner city neighborhood in the San Francisco Bay Area in California. The Centers for Disease Control had earlier found a large increase in gonorrhea among black teenagers in San Francisco. Crack users were more likely the nonusers to have sexual intercourse under the influence of drugs or alcohol (43% vs. 11% for boys and 46% vs. 14% for girls; p .001), to take part in sexual acts placing them at risk for STD in exchange for money or drugs (29% vs. 4% for boys and 25% vs. 0 for girls; p .001), and to have more sexual partners in the last year (11.1 vs. 5.3 partners for boys; p .01 and 4.5 vs. 2.5 partners for girls; insignificant). Female nonusers were more likely to have used a condom during last sexual intercourse than were female crack users (39% vs. 18%; p .01). For both groups of boys, condom use during the last sexual act was at the same low rate (about 25%). Approximately 63% of all adolescents admitted to having at least one sexual risk behavior. Crack cocaine use and having at least one relative who used drugs were the greatest predictors of STD risk behavior (odds ratios, 2.2 and 1.97; p .001 and .01, respectively). An urban anthropology study in the same neighborhoods showed that the teenagers lived in an area conducive to high-risk behaviors including drug use common among friends and relatives, more than one sexual partner/year, high prevalence of STDs, and low condom use. The findings from these studies indicated that public health workers planning to implement interventions to prevent or reduce the risk of teenage drug use and/or HIV infection must consider both individual and environmental influences.

Using intervention theory to model factors influencing behavior change. Project RESPECT.

Project RESPECT was a multisite randomized trial comparing three clinic-based interventions' ability to increase condom use and prevent infection with HIV and sexually transmitted diseases. Because Project RESPECT had guiding concepts that determined the content of the sessions, the authors investigated how the intervention operated using these theoretical variables. Growth curve analysis and structural equation modeling estimated the correlation between intentions toward condom use and self-reports of condom use and isolated the treatment effects on mediating variables--attitudes, self-efficacy, and social norms--that predict intentions. The correlations between intentions and behavior exceeded .70 for both genders, justifying the emphasis on intentions. Project RESPECT was effective through changing attitudes and self-efficacy for females in both counseling interventions. For males, only enhanced counseling had significant effects on these two mediator variables.