Extracellular matrix microarrays to study inductive signaling for endoderm specification.

During tissue development, stem and progenitor cells are faced with fate decisions coordinated by microenvironmental cues. Although insights have been gained from in vitro and in vivo studies, the role of the microenvironment remains poorly understood due to the inability to systematically explore combinations of stimuli at a large scale. To overcome such restrictions, we implemented an extracellular matrix (ECM) array platform that facilitates the study of 741 distinct combinations of 38 different ECM components in a systematic, unbiased and high-throughput manner. Using embryonic stem cells as a model system, we derived definitive endoderm progenitors and applied them to the array platform to study the influence of ECM, including the interactions of ECM with growth factor signaling, on the specification of definitive endoderm cells towards the liver and pancreas fates. We identified ECM combinations that influence endoderm fate decisions towards these lineages, and demonstrated the utility of this platform for studying ECM-mediated modifications to signal activation during liver specification. In particular, defined combinations of fibronectin and laminin isoforms, as well as combinations of distinct collagen subtypes, were shown to influence SMAD pathway activation and the degree of hepatic differentiation. Overall, our systematic high-throughput approach suggests that ECM components of the microenvironment have modulatory effects on endoderm differentiation, including effects on lineage fate choice and cell adhesion and survival during the differentiation process. This platform represents a robust tool for analyzing effects of ECM composition towards the continued improvement of stem cell differentiation protocols and further elucidation of tissue development processes. STATEMENT OF SIGNIFICANCE: Cellular microarrays can provide the capability to perform high-throughput investigations into the role of microenvironmental signals in a variety of cell functions. This study demonstrates the utility of a high-throughput cellular microarray approach for analyzing the effects of extracellular matrix (ECM) in liver and pancreas differentiation of endoderm progenitor cells. Despite an appreciation that ECM is likely involved in these processes, the influence of ECM, particularly combinations of matrix proteins, had not been systematically explored. In addition to the identification of relevant ECM compositions, this study illustrates the capability of the cellular microarray platform to be integrated with a diverse range of cell fate measurements, which could be broadly applied towards the investigation of cell fate regulation in other tissue development and disease contexts.

Using chlamydia positivity to estimate prevalence: evidence from the Chlamydia Screening Pilot in England.

Studies have suggested that positivity can be used to estimate the prevalence of Chlamydia trachomatis in large-scale chlamydia screening programmes. A recent pilot of opportunistic screening in England estimated that the prevalence among 16-24-year-old women in Portsmouth and Wirral was 9.8% and 11.2%, respectively. This study assessed the continued validity of positivity as an approximate for prevalence. We re-analysed data from the Chlamydia Screening Pilot to estimate positivity,calculated as total positive tests divided by total tests, and compared these estimates with the previously reported prevalence, measured as the number of women testing positive divided by the total number of women screened. Overall positivity was 9.4% in Portsmouth and 11.0% in the Wirral; these estimates were not statistically different from prevalence, regardless of health-care setting, age group or symptoms. We conclude that positivity can be used as a proxy for prevalence.

Screening for genital chlamydia infection: DNA amplification techniques should be the test of choice.

Our objective was to compare the sensitivities for the detection of Chlamydia trachomatis, of the ligase chain reaction (LCR) on first voided urine (FVU) specimens and enzyme immunoassay (EIA) on pooled endocervical/endourethral swabs from women and endourethral swabs from men. Men and women taking part in the UK chlamydia screening pilot were tested for chlamydia using LCR on a FVU. Patients attending genitourinary medicine clinics also had cervical and/or urethral swabs taken for chlamydia testing by EIA. In women, EIA on pooled swabs detected 575 of the 785 chlamydia positives and in men, EIA detected 209 of 351 positives. The sensitivity of EIA was 73% and 60% in women and men respectively. By using the EIA test, therefore, 27-40% of patients infected with chlamydia will be given a false negative result. We propose that it is unethical to use non-molecular testing in the future.

Opportunistic screening for genital chlamydial infection. I: acceptability of urine testing in primary and secondary healthcare settings.

OBJECTIVES: To determine the acceptability of opportunistic screening for Chlamydia trachomatis in young people in a range of healthcare settings. DESIGN: An opportunistic screening programme (1 September 1999 to 31 August 2000) using urine samples tested by ligase chain reaction (LCR). Data on uptake and testing were collected and in-depth interviews were used for programme evaluation. SETTING: General practice, family planning, genitourinary medicine clinics, adolescent sexual health clinics, termination of pregnancy clinics, and women's services in hospitals (antenatal, colposcopy, gynaecology and infertility clinics) in two health authorities (Wirral and Portsmouth and South East Hampshire). Main participants: Sexually active women aged between 16 and 24 years attending healthcare settings for any reason. MAIN OUTCOME MEASURES: Uptake data: proportion of women accepting a test by area, healthcare setting, and age; overall population coverage achieved in 1 year. Evaluation data: participants' attitudes and views towards opportunistic screening and urine testing. RESULTS: Acceptance of testing by women (16-24 years) was 76% in Portsmouth and 84% in Wirral. Acceptance was lower in younger women (Portsmouth only) and varied by healthcare setting within each site. 50% of the target female population were screened in Portsmouth and 39% in Wirral. Both the opportunistic offer of screening and the method of screening were universally acceptable. Major factors influencing a decision to accept screening were the non-invasive nature of testing and treatment, desire to protect future fertility, and the experimental nature of the screening programme. CONCLUSIONS: An opportunistic model of urine screening for chlamydial infection is a practical, universally acceptable method of screening.

Opportunistic screening for genital chlamydial infection. II: prevalence among healthcare attenders, outcome, and evaluation of positive cases.

OBJECTIVES: To determine the prevalence and treatment outcomes among young women screened opportunistically for genital Chlamydia trachomatis and to evaluate the impact of screening in those participating. DESIGN: An opportunistic screening programme (1 September 1999 to 31 August 2000) using urine samples, tested by ligase chain reaction (LCR). In-depth interviews were used for programme evaluation. SETTING: Screening was offered in two health authorities at general practice, family planning, genitourinary medicine (GUM), adolescent sexual health, termination of pregnancy clinics and women's services in hospitals (antenatal, colposcopy, gynaecology and infertility clinics). Main participants: Sexually active women (16-24 years) attending for any reason. MAIN OUTCOME MEASURES: Screening data: prevalence of infection by age and healthcare setting; proportion of positive patients attending for treatment. Evaluation data: participants' attitudes and views towards screening and follow up. RESULTS: In total, 16 930 women (16-24 years) were screened. Prevalence was higher in younger women (16-20) than those aged 21-24 years and was highly variable at different healthcare settings (range 3.4%-17.6%). Prevalence was approximately 9% in general practice. The role of the project health advisers in managing results and coordinating treatment of positive individuals was essential; the vast majority of all positives were known to be treated. Women felt that screening was beneficial. Improving awareness and education about sexually transmitted infections is required to alleviate negative reactions associated with testing positive for infection. CONCLUSIONS: Prevalence of infection outside GUM clinics is substantial and opportunistic screening using urine samples is an acceptable method of reaching individuals with infection who do not normally present at specialist clinics.

Opportunistic chlamydia screening; should positive patients be screened for co-infections?

This study examines the requirement for testing patients for other sexually transmitted infections (STIs) and bacterial vaginosis (BV) when diagnosed with genital chlamydia during opportunistic screening. Data were collected on all patients participating in the Department of Health chlamydia screening pilot study in Portsmouth. One thousand two hundred and forty-five women and 490 men with genital chlamydia were seen in Portsmouth genitourinary medicine (GUM) department. Of the women screened in GUM, 28% had coexisting STIs and 21% had BV. The corresponding figures for those initially screened in the community were 4% and 17%. An increased number of female sexual partners of male patients (76%) and male partners of female patients (55%) of the GUM group had co-infections; 58% of male partners from the community group had another STI. The increased morbidity associated with these infections warrants screening of all patients with chlamydia for other STIs and BV.

Cutting edge: differential requirements for Stat4 in expression of glycosyltransferases responsible for selectin ligand formation in Th1 cells.

A role for Stat4 in IL-12-induced up-regulation of selectin ligands on Th1 cells was explored. Th1 cells generated from Stat4(-/-) mice exhibited no IL-12-inducible P-selectin ligands, no up-regulation of core 2 beta1,6-glucosaminyltransferase I (C2GlcNAcT-I), and low levels of the Th1 transcription factor T-bet. In contrast, Stat4(-/-) Th1 cells exhibited only a partial defect in expression of IL-12-inducible E-selectin ligands and expressed equivalently high levels of alpha1,3-fucosyltransferase VII (FucT-VII) as wild-type Th1 cells. FucT-VII expression was induced by T cell activation, and was enhanced by IL-12 independently of Stat4, whereas C2GlcNAcT-I up-regulation was mediated exclusively by IL-12, acting through Stat4. These data show that FucT-VII and C2GlcNAcT-I are controlled through distinct pathways and imply the existence of at least one other IL-12-inducible glycosyltransferase required for E-selectin and possibly P-selectin ligand formation in Th1 cells.

Failed postnatal immunoprophylaxis for hepatitis B: characteristics of maternal hepatitis B virus as risk factors.

A retrospective case-control study was conducted to determine why some infants born full-term without obstetric intervention to hepatitis B e antigen (HBeAg)-seropositive mothers become infected by hepatitis B virus (HBV) despite having received passive-active immunoprophylaxis. Cases and controls comprised 12 hepatitis B surface antigen (HBsAg)-seropositive infants and 22 HBsAg-seronegative infants, respectively. Infants infected by putative vaccine-escape mutants were excluded. Risk factors, after adjustment for the level of maternal viremia, were the following allelic base changes in maternal HBV:C158, A328, G365, and A479 (P = .017, .005, .003, and .005, respectively). High-level maternal viremia (i.e., > or = 10(8) genome equivalents/mL) was a significant factor only after adjustment for G365 (P = .027). HBV DNA sequences recovered from one of the cases, the case's mother, and three infected contacts all had the high-risk mutations. Specific allelic mutations in maternal HBV and level of maternal viremia are potential predictors of vertical breakthrough infection.

An update on HIV-testing at a London sexually transmitted diseases clinic: long-term impact of the AIDS media campaigns.

Trends in the number of tests for HIV-1 antibody at a London sexually transmitted diseases clinic showed substantial changes between September 1985 and June 1988. From an average of 100 tests per month between September 1985 and August 1986, the average increased to 365 tests per month for September 1986 to August 1987. This levelled off at 243 tests per month between September 1987 and June 1988. The average number of positive tests per month between September 1985 and June 1988 remained constant, though the number of seropositive tests in females increased. Male and female populations displayed similar temporal patterns. The greatest increase was seen in the tests generated by heterosexual males and females with no other risk factors. This group generated 42% of the 8012 tests performed during the study period. The Clinic's catchment area predominantly included London and surrounding areas and temporal patterns were similar for residents from each of the Thames Regions. The temporal patterns observed coincided with periods of increased media attention on HIV infection/AIDS and involved campaigns by the popular press, television, Department of Health Education Authority. The increase in tests during the time of maximum media exposure, the subsequent plateau at a level substantially higher compared with the period preceding the media campaigns and the large increase in heterosexual males and females tested, all suggest that the campaigns have contributed to increasing awareness of HIV infection/AIDS as a major contemporary public health problem.

Risk factors for HIV-1 infection in a British population: lessons from a London sexually transmitted diseases clinic.

Between September 1985 and June 1988, 6923 people (4550 men and 2373 women) were tested for HIV-1 antibodies at a sexually transmitted disease clinic, London, UK. Of the 6923 individuals tested, 558 (8%) were seropositive, of whom 523 (94%) were men and 35 (6%) women. Of the seropositives, 84% were homosexual or bisexual men, 5% were intravenous drug users, 4% were heterosexual contacts of HIV seropositives, 4% had multiple risk factors, 2% were heterosexual contacts of central African partners and 1% were heterosexuals with no other risk factors. This prevalence pattern conforms to that observed in other industrialized nations. For the women, heterosexual intercourse with an HIV-infected partner, intravenous drug use and heterosexual contact with a partner from central Africa were the main risk factors for infection. The important risk factors among the men were heterosexual contact with an HIV-seropositive partner, being homosexual or bisexual, intravenous drug use and heterosexual contact with a resident from central Africa. heterosexual and homosexual transmission were implicated as the main routes for viral spread in this British population.

Intradermal hepatitis B vaccine in thalassaemia and sickle cell disease.

Thirty two patients with beta thalassaemia and sickle cell disease who were having regular blood transfusions were selected to test the efficacy and immunogenicity of low dose (2 micrograms or 0.1 ml) intradermal hepatitis B vaccine compared with the standard (20 micrograms or 1 ml) intramuscular dose. There was no significant difference in the rates of seroconversion, seroconversion had occurred in all patients by seven months. There were no significant differences in antibody titres between the intramuscular and intradermal groups at 1, 2, and 6 months. Although the titres were significantly higher in the intramuscular group at seven months and at 12-18 months, the antibody titre in the intradermal group did not fall below 10 IU/l. The results of this study suggest that low dose intradermal hepatitis B vaccination is an effective and economical way of stimulating an immune response in patients with beta thalassaemia and sickle cell disease.

Psychological aspects of recurrences of genital herpes.

Fifty-seven patients presenting with virologically confirmed first attacks of genital herpes were assessed for risk factors for time to the first recurrence. These factors included demographic details, personality traits, recent life events, herpes simplex biotype, frequency of orogenital intercourse and psychiatric illness as measured by the General Health Questionnaire (GHQ). This and our similar previous study (58 patients) were analysed separately and combined using a novel statistical technique (Cox's proportional hazards model). In the previous study there was a significant association only for GHQ score (p less than 0.02). For the present study age was the only statistical significant factor (the younger the patient the more likely a recurrence sooner (p less than 0.05), although there was a trend for high GHQ scorers to have recurrences sooner than low scorers. Data of the two studies combined showed that the only significant association with time to recurrence was the GHQ (p less than 0.02). These data support the notion that there is an association between psychiatric illness and recurrence of genital herpes.

The effect of indomethacin treatment on lymphocyte PHA reactivity in healthy controls and patients with lymphoma and sarcoidosis.

Lymphocytes from healthy volunteers who took indomethacin orally for 3 d showed an enhanced in vitro response to phytohaemagglutinin (PHA). When indomethacin was added in vitro, lymphocytes from healthy controls showed similar but more consistent increases of PHA responses than lymphocytes from patients with sarcoidosis or lymphoma. Moreover, the enhancing effect of indomethacin was by and large more pronounced in those patients who had relatively normal PHA lymphocyte responses in the absence of indomethacin, and the presence of indomethacin failed in most instances to increase subnormal responses to normal control levels. These findings are not consistent with the notion that increased prostaglandin synthesis is the principal cause of subnormal PHA reactivity of lymphocytes from patients with sarcoidosis or lymphoma.

Cutaneous adverse reactions to acyclovir: case reports.

Intravenous, oral, and topical formulations of acyclovir have been successful in treating genital herpes. We report on two patients who developed skin reactions while taking acyclovir, which resolved when treatment was stopped.

Treatment with acyclovir of genital herpes simplex virus infection complicated by eczema herpeticum.

Eczema herpeticum is a potentially serious disease that may be fatal. We report two cases of infection with genital herpes simplex virus (HSV) that were complicated by eczema herpeticum and were treated successfully with acyclovir.

Neonatal herpes simplex pneumonia.

A neonate with herpes simplex pneumonia is described. Herpes simplex infection should be considered in the differential diagnosis of pneumonia in newborn infants, even in the absence of clinically apparent herpes in the mother.