Use of cryopreserved cells for enabling greater flexibility in compound profiling.

Measurement of intracellular calcium release following agonist challenge within cells expressing the relevant membrane protein is a commonly used format to derive structure-activity relationship (SAR) data within a compound profiling assay. The Fluorometric Imaging Plate Reader (FLIPR) has become the gold standard for this purpose. FLIPR traditionally uses cells that are maintained in continuous culture for compound profiling of iterative chemistry campaigns. This supply dictates that assays can only be run on 4 of 5 weekdays, or alternative cell culture machinery is required such that plating can occur remotely at the weekend. The data reported here demonstrate that high-quality compound profiling data can be generated from the use of cryopreserved cells and that these cells can also be plated at various densities to generate equivalent data between 24 and 72 h post-plating. Hence, the authors report a method that allows data generation throughout the week and without the requirement of highly automated cell culture or continuous culture.

'Chemical condoms' for the prevention of HIV infection: evaluation of novel agents against SHIV(89.6PD) in vitro and in vivo.

BACKGROUND: Vaginal agents which are antiviral and/or inhibit the entry of HIV into the cell could prevent heterosexual transmission of HIV, and protect women who cannot negotiate condom use. METHODS: Four agents have been investigated for activity in vitro and in vivo against SHIV(89.6PD): two anionic polymers, dextrin-2-sulphate (D2S) and PRO 2000 (P2K), and two virucidal agents; a non-ionic detergent, nonoxynol-9 (N9) and a cyclic peptide ionophore, gramicidin-D (GD). All four agents were investigated in rhesus macaques, using an intra-vaginal challenge of two inoculations of 1 x 104 50% tissue culture infectious doses (TCID)50 of SHIV(89.6PD). RESULTS: D2S, P2K, GD and N9 all inhibited SHIV(89.6PD) in vitro. In vivo, three out of four control macaques were infected as judged by viral culture, seroconversion, DNA and RNA PCR; infection was confirmed in four out of eight macaques pre-treated with P2K, two out of four pre-treated with D2S, one out of four pre-treated with N9, two out of four pre-treated with GD and four out of four pre-treated with D2S + GD, a combination additive in vitro. INTERPRETATION: D2S and PRO-2000, novel inhibitors of HIV entry, showed evidence of protection in vivo, comparable to that seen with the virucide, N9. These data, together with the results of phase I and phase II studies in healthy women which have shown minimal toxicity, support plans for a phase III efficacy trial of chemically simple inhibitors of HIV entry with low toxicity, for the prevention of HIV infection in women.

Immunity in the female lower genital tract and the impact of HIV infection.

This study investigates the distribution of immunocompetent cells in the ectocervix, and cytokine and immunoglobulin (Ig) levels in cervicovaginal secretions to determine whether they are altered in asymptomatic human immunodeficiency virus (HIV) infection. Ectocervical biopsies from 10 HIV+ and 10 presumed HIV-ve women were studied by immunocytochemistry. Levels of Igs in cervicovaginal secretions were quantified by radial immunodiffusion (RID) and cytokine levels by ELISA. HIV+ women had significantly increased numbers of CD8+ lymphocytes resulting in reversal of the CD4:CD8 ratio. There was a significant increase in the proportion of activated CD8+ HLA-DR+ and CD4+ HLA-DR + lymphocytes, but not in CD8+ TIA-1+ cells. The epithelium of the cervix from HIV+ subjects showed a significant increase in both numbers of macrophages (CD68+) and proportions of activated macrophages (CD68+ HLA-DR+) compared to normal. The stroma contained increased proportions of inductive (D1+) and suppressive (D1+ D7+) macrophages but a decrease in effector phagocyte (D7+) proportions and Langerhans' cells. Significantly lower tumour necrosis factor (TNF)-alpha levels were observed in cervicovaginal secretions from HIV+ subjects. IgG levels were 4 times higher and IgM levels twice higher in cervicovaginal secretions from HIV+ women, compared to results from normal subjects. These results suggest a response within the CD8+ cells in HIV+ women, yet these cells may have a low cytolytic capacity. The raised proportions of HLA-DR+ and D1+ CD4+ macrophages could act as antigen-presenting cells (APC) for CD4+ CD45RO+ lymphocytes, and represent a local acquired response. However, the close juxtaposition of these cells offers the potential for them to act as a local reservoir of virus and promote its proliferation. The increase of IgG over sIgA in secretions of HIV+ subjects provides evidence suggesting a dysregulation of local humoral immunity.

A phase I study of a novel potential intravaginal microbicide, PRO 2000, in healthy sexually inactive women.

BACKGROUND: Although the male condom provides a reliable means of preventing HIV transmission, a broader choice of methods is required particularly in circumstances where the negotiation of condom use is difficult. Development of new products that may be effective as topical vaginal microbicides is the focus of a great deal of research activity currently. The novel agent PRO 2000, a naphthalene sulphonate derivative with in vitro activity against HIV and other sexually transmissible pathogens, is one such compound. We have studied the local and systemic safety and tolerance of a vaginal gel formulation of this agent at two concentrations (0.5% and 4%) over a 2 week period of daily exposure in two cohorts of healthy sexually abstinent women (one in London, UK, and the other in Antwerp, Belgium). METHODS: This was a randomised, placebo controlled, double blind, three arm clinical trial conducted on two sites. Macroscopic evidence of genital epithelial changes was sought using colposcopy and evidence of microscopic inflammation was acquired using high vaginal biopsy from predetermined sites (UK cohort only). Blood levels of PRO 2000 were measured and laboratory safety tests, including coagulation screens, were performed. The impact on vaginal ecology was also assessed. RESULTS: 73 women were enrolled across both sites (36 UK, 37 Belgium); 24, 24, 25 in the 4%, 0.5%, and placebo groups respectively. Of these, 70 completed 2 weeks' exposure to the study gel. Three (all in the 4% group) withdrew owing to adverse events which were possibly or probably gel related. Cervicovaginal abrasion was seen colposcopically in three subjects after 14 days of gel use (two in the 4% group and one in the placebo group). Genital ulceration was not seen during gel use in any of the subjects who completed the study. Histological evaluation of vaginal biopsy samples (36 women only) showed evidence of increased inflammatory signs in one participant of the 4.0% group. One volunteer in the placebo group had moderate inflammation at screening and at follow up. Severe inflammation was not seen among any of the subjects tested. Plasma levels of PRO 2000 and laboratory safety tests showed no evidence of systemic absorption. No impact was seen on normal vaginal ecology in the UK cohort where samples were taken 12 hours after the last gel application. CONCLUSION: In this phase I study PRO 2000 gel was found to be generally well tolerated with promising local and systemic safety profiles. The 0.5% gel was better tolerated than the 4% gel as fewer genital epithelial adverse events were seen in the former. Phase II studies are about to begin in sexually active women.

Effect on normal vaginal flora of three intravaginal microbicidal agents potentially active against human immunodeficiency virus type 1.

The effect on normal vaginal flora of three intravaginal microbicides potentially active against human immunodeficiency virus type 1 was examined. Volunteers received dextrin sulfate (D2S), nonoxynol-9 (N-9), or docusate sodium in separate placebo-controlled studies. High vaginal swabs were obtained for bacterial culture before and after microbicide application. D2S did not affect the vaginal flora. However, lactobacilli decreased by > or = 10(2) cfu/mL in 9 (56%) of 16 women given N-9 and in 5 (63%) of 8 women given docusate sodium. Women using N-9 were also significantly more likely to become colonized abnormally (usually with aerobic gram-negative rods) than were those using placebo, as were women using docusate sodium. Women with reduced lactobacilli were less likely to regain normal flora than were those whose lactobacilli were unaffected. However, coliform colonization occurred whether lactobacilli produced H2O2 or not. Continuous use of N-9 could induce susceptibility to urinary and gynecological infection. It is essential that potential microbicides are examined for activity against normal vaginal flora.

Safety study of nonoxynol-9 as a vaginal microbicide: evidence of adverse effects.

Nonoxynol-9 (N-9) is virucidal in vitro, and is therefore a candidate microbicide for preventing sexual transmission of HIV. However, the activity of N-9 is nonspecific, suggesting that virucidal levels may produce adverse effects including epithelial disruption, inflammation of the genital mucosa, or both. A randomized placebo controlled trial of daily use of 100 mg of N-9 took place for 1 week in 40 female volunteers. Outcome measures included symptoms, colposcopic and histologic changes in the genital tract, and impact on vaginal flora. Genital irritation was reported by 10 of the N-9 and 5 of the placebo group. Colposcopy showed erythema in 9 of the N-9 group and 2 of the placebo group. Histologic inflammation was found in 7 of the N-9 group and 2 of the placebo group. Inflammatory changes were characterized by patchy infiltration of the lamina propria predominantly with CD8+ lymphocytes and macrophages, in the absence of epithelial disruption. A transient reduction in numbers of lactobacilli was observed in 9 of the 15 women using N-9, and 6 of 18 women using placebo. N-9 used for 7 days in a standard spermicidal dose was associated with increased irritation, colposcopic and histologic evidence of inflammation and was more frequently associated with reduction in numbers of lactobacilli during gel use. The clinical significance of the recruitment of cells susceptible to HIV infection to the genital mucosa is unknown but raises concerns about the suitability of N-9 as a microbicide when given in this dose.

PIP: Since nonoxynol-9 (N-9) is virucidal in vitro, it is a candidate microbicide for preventing the sexual transmission of HIV. A randomized placebo-controlled trial of the daily use of 100 mg of N-9 was conducted for 1 week among 40 female volunteers aged 18-45 years. Genital irritation was reported by 10 of the N-9 users and 5 women in the placebo group. Colposcopy showed erythema in 9 of the N-9 group and 2 of the placebo group. Histologic inflammation was found in 7 of the N-9 group and 2 of the placebo group. Inflammatory changes in the women were characterized by patchy infiltration of the lamina propria mainly with CD8 lymphocytes and macrophages, in the absence of epithelial disruption. A transient reduction in the number of lactobacilli was observed in 9 of the 15 women using N-9, and 6 of the 18 women using placebo. N-9 used for 7 days in this standard spermicidal dose in the absence of sexual intercourse was therefore associated with increased irritation, colposcopic and histologic evidence of inflammation, and was more often associated with a reduction in the numbers of lactobacilli during gel use. The clinical significance of the recruitment of cells susceptible to HIV infection to the genital mucosa remains to be determined.

A placebo-controlled, double-blind prospective study in healthy female volunteers of dextrin sulphate gel: a novel potential intravaginal virucide.

A double-blind, placebo-controlled study was designed to evaluate the safety and tolerability of intravaginal dextrin sulphate (D2S) gel to assess its preliminary suitability as a potential vaginal virucide. Tolerability was assessed by questionnaire and patient interview. Colposcopy with vaginal biopsy was performed to assess the macroscopic and microscopic evidence of inflammation. The potential impact of the gel on normal vaginal flora was examined by quantitative lactobacilli culture with assessment of the ratio of peroxide to nonperoxide-producing organisms. Colposcopy revealed mild erythema in five of 24 subjects receiving active gel and in none of the 12 placebo recipients, but histology in all subjects revealed no evidence of inflammation. No impact on vaginal lactobacilli was found. We conclude that D2S gel is safe and well tolerated intravaginally at the dosing schedule used in this study.

Nonoxynol-9 in lubricated condoms. Results of a study in female prostitutes.

BACKGROUND AND OBJECTIVES: Debate continues on the efficacy and safety of intravaginal nonoxynol-9 for the prevention of horizontal transmission of human immunodeficiency and other sexually transmitted diseases. Little attention has been paid to the effects of nonoxynol-9 contained in the lubricant of many condoms. GOAL: To assess the tolerability of different levels of nonoxynol-9 in condom lubricants. STUDY DESIGN: Pilot, randomized, controlled trial in 70 female prostitutes. RESULTS: There was no association between dose of nonoxynol-9 and reported symptoms or signs of genital tract inflammation; an increased dose of nonoxynol-9 was associated with increased numbers of polymorphonuclear leukocytes on a vaginal wall smear. CONCLUSIONS: There is no recognized simple method of defining inflammation in the female genital tract. Future studies of the effects of low-dose nonoxynol-9 on the female genital tract require highly controlled exposures, plus colposcopy with or without vaginal biopsy to define inflammation.

PIP: It remains unclear whether the intravaginal application of nonoxynol-9 (N-9) safely prevents the transmission of HIV and other sexually transmitted diseases (STDs) during sexual intercourse. Some studies have suggested a protective effect, while others a potential adverse effect created through the inflammation and ulceration of the genital tract caused by N-9. N-9 is commonly found in the lubricating agents used on condoms. However, the dose in each condom is small relative to pessaries or spermicidal gels. 70 female prostitutes participated in a study to assess the tolerability of different levels of N-9 in condom lubricants. Women were excluded from participation if they had a STD, vaginal candidiasis or bacterial vaginosis, were pregnant, or were using intravaginal spermicides for contraception. Participants were asked to use the trial condoms for all vaginal intercourse for a 2-week period and to avoid using any other spermicidal preparation. 1636 identical condoms were used during the study, either lubricated with polyethyleneglycol, polyethyleneglycol with 2% N-9, or polyethyleneglycol with 4% N-9. No association was found between the dose of N-9 and reported symptoms or signs of genital tract inflammation, although an increased dose of N-9 was associated with increased numbers of polymorphonuclear leukocytes on a vaginal wall smear. Future studies of the effects of low-dose N-9 on the female genital tract require highly controlled exposures together with colposcopy with or without vaginal biopsy for confirmation.

Ethnic differences in women with HIV infection in Britain and Ireland. The study group for the mrc collaborative study of HIV infection in women.

OBJECTIVE: To examine ethnic differences in the socio-epidemiological and clinical characteristics of a cohort of women with HIV infection in Britain and Ireland. DESIGN AND METHODS: Analysis of baseline data (ethnic group, sexual history, likely route of HIV infection, reasons for HIV testing and first AIDS-defining disease) from 400 women with HIV infection recruited into a cohort study from 15 genitourinary medicine/HIV clinics in Britain and Ireland. RESULTS: Sixty-five per cent of women were white and 29% black African. Their median number of lifetime sexual partners was seven and three, respectively (P < 0.001). Ninety-three per cent of black African and 43% of white women were probably infected through sexual intercourse. Injecting drug use was the most likely route of infection in 55% of white women, but none of the black African women. Perceived risk (33%) or investigation of symptoms (26%) were the most common reasons for HIV testing. Seven per cent of white women and 16% of black African women (P < 0.001) had AIDS when HIV infection was diagnosed. The distribution of first AIDS-defining diagnoses differed (P = 0.001) by ethnic group. For white women, the most common disease was Pneumocystis carinii pneumonia; for black African women it was pulmonary tuberculosis. CONCLUSION: There are important differences between black African and white women in sexual history and route of transmission, disease stage at diagnosis and pattern of AIDS-defining diseases.

Evaluation of safety and efficacy of 3TC (lamivudine) in patients with asymptomatic or mildly symptomatic human immunodeficiency virus infection: a phase I/II study.

In a phase I/II study, 7 levels of 3TC therapy (from 0.5 to 20.0 mg/kg/day) were studied in 104 asymptomatic and mildly symptomatic human immunodeficiency virus-infected patients with CD4 cell counts < or = 400 x 10(6)/L. Mild and transient episodes of diarrhea, headache, fatigue, nausea, and abdominal pain were the most frequent events reported. No dose-limiting toxicities were observed. Small and transient increases in CD4 cell counts were detected during the first 4 weeks of treatment. These were followed by progressive declines during prolonged therapy. Sustained decreases in beta 2-microglobulin, neopterin, and p24 antigen levels were seen over the 52-week study. There was no consistent dose-response correlation for any surrogate marker. Penetration of 3TC into cerebrospinal fluid (CSF) was in the same range as reported for ddC and ddI; the mean CSF-to-serum ratio was 0.06. These findings indicate that 3TC exhibits an excellent safety profile and has antiretroviral activity at the dosages studied.

Safety and activity of saquinavir in HIV infection.

We evaluated saquinavir, an orally active, selective inhibitor of HIV proteinase, in a randomised, double-blind, dose-ranging study in 49 zidovudine-naive HIV-positive patients with few or no symptoms and CD4 cell counts of 500 or less. The study was designed to assess the antiviral activity and tolerability of saquinavir. Patients were randomised to receive 25, 75, 200, or 600 mg of saquinavir three times daily for 16 weeks. No serious adverse events occurred. CD4 cell counts showed a trend indicative of a dose response in favour of the 600 mg dosage, the maximum increase being seen around week 4. In none of the 8 patients with positive plasma viraemia at baseline did cultures become negative after treatment; peripheral blood mononuclear cell and plasma-viral load by culture and DNA and RNA PCR all showed a trend towards reduction at higher doses of saquinovir. Saquinavir was well tolerated in this group of previously untreated patients with few or no symptoms; this study shows that an HIV-proteinase inhibitor is active in HIV-infected patients.

Changing patterns of HIV-1 transmission in southern Brazil 1985-1991.

In order to describe the changing patterns of risk factors for HIV-1 transmission of patients using hospital services at an AIDS referral centre in Porto Alegre, southern Brazil, data on demographic characteristics, referral patterns and risk factors at time of first presentation were collected prospectively on 405 patients between October 1985 and September 1991. Overall HIV-related patient workload increased during the study period, as did the proportion of infected female patients seen (P < 0.05). Of all patients, 147 (36%) presented with symptomatic HIV disease and 77 (19%) presented with an AIDS defining condition; men were more likely to present with symptomatic disease than women. Approximately 156 (44%) of men were self-referred compared with 4 (8%) of the women (P < 0.0001). Of the 357 infected men, 82 (23%) were bisexuals; of the 26 heterosexually infected women, 7 (24%) had bisexual male partners. These data suggest the increasing importance of heterosexual HIV transmission in this hitherto 'low' prevalence area, with male bisexuals constituting an important route through which heterosexual females are being infected in this area. The data also suggest that heterosexual women in Southern Brazil do not perceive themselves to be at risk for HIV-1 infection.

PIP: Unprotected homosexual intercourse and IV drug use were originally described as the predominant routes of HIV transmission in South America. Recent data, however, indicate that heterosexual transmission is becoming increasingly important in the region. With the goal of describing the changing patterns of risk factors for HIV-1 transmission, the authors collected prospectively data on demographic characteristics, referral patterns, and risk factors of patients at the time of first presentation to hospital services at an AIDS referral center in Porto Alegre, southern Brazil. The 357 male and 48 female patients presented over the period October 1985-September 1991. The overall HIV-related patient workload increased during the study period, as did the proportion of infected female patients. 44% of the men and 8% of women were self-referred. 36% of patients presented with symptomatic HIV disease and 19% with an AIDS-defining condition, with the men more likely than women to present with symptomatic disease. 23% of the infected men were bisexual; 24% of the heterosexually infected women had bisexual male partners. These findings suggest the importance of heterosexual HIV transmission in this area, with male bisexuals serving as an important route through which heterosexual females are being infected in the area. The data also suggest that heterosexual women in southern Brazil do not perceive themselves as being at risk for HIV-1 infection.

Menstrual symptoms in women infected by the human immunodeficiency virus.

OBJECTIVE: To determine whether there is a higher prevalence of menstrual symptoms in women seropositive for the human immunodeficiency virus (HIV) compared to a matched control group and to examine the relation between menstrual symptomatology and immunosuppression. METHODS: In a cross-sectional study, 55 HIV-seropositive women and a matched control group underwent detailed gynecologic assessment. The prevalence of regular cycles, oligomenorrhea, amenorrhea, menorrhagia, dysmenorrhea, and dyspareunia was assessed in the two groups. Any association with clinical disease or CD4 lymphocyte count was sought. RESULTS: There were no significant differences in the prevalence of oligomenorrhea, amenorrhea, menorrhagia, dysmenorrhea, or dyspareunia between the groups. Furthermore, no differences were demonstrated between symptomatic and asymptomatic women infected by HIV, nor was any correlation found between CD4 lymphocyte count and menstrual loss or dysmenorrhea. CONCLUSION: Infection with HIV and related immunosuppression do not seem to have a clinically significant effect on menstruation.

HIV infection and the gynaecologist.

PIP: As women infected with human immunodeficiency virus (HIV) will soon account for 1 in 500 gynecological patients in Great Britain, gynecologists have an obligation to become informed about the transmission, clinical manifestations, and management of HIV. Women with HIV infection are at increased risk of lower genital tract neoplasia with extensive cervical, vaginal, and vulvar lesions. There is also a strong association between HIV infection and sexually transmitted diseases involving genital ulcerations. In fact, herpes, warts, and candidiasis may be the initial clinical presentation of HIV infection. Estradiol levels may fall in seropositive women, but, in general, menstruation and ovulation are maintained. Latex condoms remain the only contraceptive choice for HIV-infected women. Sufficient data have not been accumulated on the effectiveness of the female condom, but it has the potential of giving women in developing countries in particular greater control over HIV prevention. Condom use is essential even if both partners are HIV-positive since the acquisition of different HIV strains can accelerate disease progression. Given their higher risk of percutaneous in injury compared to other surgeons, gynecologists should use double gloving and blunt tipped needles and staples.^ieng

Women with HIV presenting at three London clinics between 1985-1992.

OBJECTIVE: To determine the changing patterns of HIV infection in women in three units in London. SUBJECTS: Three hundred and fifty seven HIV seropositive women who have attended outpatient clinics between 1984 and 1992. METHODS: A retrospective review of data obtained from a computerised database and supplemented by direct inspection of the notes. RESULTS: The number of newly identified women with HIV has risen steadily over the period of study with a significant shift towards a heterosexual mode of transmission. This is a reflection of increasing numbers of women from Sub-Saharan Africa rather than a rise in the incidence of HIV in women born in the UK. CONCLUSIONS: The increase in women infected by HIV remains predominantly restricted to women in "high risk" groups. Although encouraging, our data should be interpreted with caution since it suffers from the inherent bias of selective testing. Safer sex education and epidemiological surveillance should continue despite the apparent low risk to women born in the UK.