P2Y11 Agonism Prevents Hypoxia/Reoxygenation- and Angiotensin II-Induced Vascular Dysfunction and Intimal Hyperplasia Development.

Vascular dysfunction in cardiovascular diseases includes vasomotor response impairments, endothelial cells (ECs) activation, and smooth muscle cells (SMCs) proliferation and migration to the intima. This results in intimal hyperplasia and vessel failure. We previously reported that activation of the P2Y11 receptor (P2Y11R) in human dendritic cells, cardiofibroblasts and cardiomyocytes was protective against hypoxia/reoxygenation (HR) lesions. In this study, we investigated the role of P2Y11R signaling in vascular dysfunction. P2Y11R activity was modulated using its pharmacological agonist NF546 and antagonist NF340. Rat aortic rings were exposed to angiotensin II (AngII) and evaluated for their vasomotor response. The P2Y11R agonist NF546 reduced AngII-induced vascular dysfunction by promoting EC-dependent vasorelaxation, through an increased nitric oxide (NO) bioavailability and reduced AngII-induced H2O2 release; these effects were prevented by the use of the P2Y11R antagonist NF340. Human vascular SMCs and ECs were subjected to AngII or H/R simulation in vitro. P2Y11R agonist modulated vasoactive factors in human ECs, that is, endothelial nitric oxide synthase (eNOS) and endothelin-1, reduced SMC proliferation and prevented the switch towards a synthetic phenotype. H/R and AngII increased ECs secretome-induced SMC proliferation, an effect prevented by P2Y11R activation. Thus, our data suggest that P2Y11R activation may protect blood vessels from HR-/AngII-induced injury and reduce vascular dysfunctions. These results open the way for new vasculoprotective interventions.

ADS-J1 inhibits semen-derived amyloid fibril formation and blocks fibril-mediated enhancement of HIV-1 infection.

Semen-derived enhancer of viral infection (SEVI) is composed of amyloid fibrils that can greatly enhance HIV-1 infectivity. By its cationic property, SEVI promotes viral sexual transmission by facilitating the attachment and internalization of HIV-1 to target cells. Therefore, semen-derived amyloid fibrils are potential targets for microbicide design. ADS-J1 is an anionic HIV-1 entry inhibitor. In this study, we explored an additional function of ADS-J1: inhibition of SEVI fibril formation and blockage of SEVI-mediated enhancement of viral infection. We found that ADS-J1 bound to an amyloidogenic peptide fragment (PAP248-286, comprising amino acids 248 to 286 of the enzyme prostatic acid phosphatase), thereby inhibiting peptide assembly into amyloid fibrils. In addition, ADS-J1 binds to mature amyloid fibrils and antagonizes fibril-mediated enhancement of viral infection. Unlike cellulose sulfate, a polyanion that failed in clinical trial to prevent HIV-1 sexual transmission, ADS-J1 shows no ability to facilitate fibril formation. More importantly, the combination of ADS-J1 with several antiretroviral drugs exhibited synergistic effects against HIV-1 infection in semen, with little cytotoxicity to vaginal epithelial cells. Our results suggest that ADS-J1 or a derivative may be incorporated into a combination microbicide for prevention of the sexual transmission of HIV-1.

Emergent HIV technology: urban Tanzanian women's narratives of medical research, microbicides and sexuality.

In response to the growing HIV epidemic in Africa in the 1990s, microbicide technologies emerged from discourses of empowerment and imaginings of the sexual lives and agency of African women. This draws on an anthropological enquiry which explored narratives from Tanzanian women who participated in a microbicide clinical trial. In the context of the HIV epidemic in Tanzania, women's lives were full of uncertainty and insecurity and their sexual lives were situated in a wider discourse of urban women's sexuality linked to morality and power. Their narratives revealed that women participated in the trial to seek knowledge as well as to 'try' the gel. In relation to their concerns about sexual health, the gel was experienced as cleansing as well as enhancing sexual desire and pleasure. The idea of empowerment imbued in the gel and transported to the women through the clinical trial was meaningful to the women, and this and ideas of sexual health and pleasure suggest future and hopeful possibilities for such HIV prevention technologies. However, if made widely available the potential for enhanced inequalities and further intensified surveillance of women's sexual lives must be considered.

A novel poly-naphthol compound ST104P suppresses angiogenesis by attenuating matrix metalloproteinase-2 expression in endothelial cells.

Angiogenesis, the process of neovascularization, plays an important role in physiological and pathological conditions. ST104P is a soluble polysulfated-cyclo-tetrachromotropylene compound with anti-viral and anti-thrombotic activities. However, the functions of ST104P in angiogenesis have never been explored. In this study, we investigated the effects of ST104P in angiogenesis in vitro and in vivo. Application of ST104P potently suppressed the microvessels sprouting in aortic rings ex vivo. Furthermore, ST104P treatment significantly disrupted the vessels' development in transgenic zebrafish in vivo. Above all, repeated administration of ST104P resulted in delayed tumor growth and prolonged the life span of mice bearing Lewis lung carcinoma. Mechanistic studies revealed that ST104P potently inhibited the migration, tube formation and wound closure of human umbilical endothelial cells (HUVECs). Moreover, ST104P treatment inhibited the secretion and expression of matrix metalloproteinase-2 (MMP-2) in a dose-dependent manner. Together, these results suggest that ST104P is a potent angiogenesis inhibitor and may hold potential for treatment of diseases due to excessive angiogenesis including cancer.

Upregulated protein arginine methyltransferase 1 by IL-4 increases eotaxin-1 expression in airway epithelial cells and participates in antigen-induced pulmonary inflammation in rats.

Protein arginine methyltransferases (PRMTs), catalyzing methylation of both histones and other cellular proteins, have emerged as key regulators of various cellular processes. This study aimed to identify key PRMTs involved in Ag-induced pulmonary inflammation (AIPI), a rat model for asthma, and to explore the role of PRMT1 in the IL-4-induced eosinophil infiltration process. E3 rats were i.p. sensitized with OVA/alum and intranasally challenged with OVA to induce AIPI. The expressions of PRMT1-6, eotaxin-1, and CCR3 in lungs were screened by real-time quantitative PCR. Arginine methyltransferase inhibitor 1 (AMI-1, a pan-PRMT inhibitor) and small interfering RNA-PRMT1 were used to interrupt the function of PRMT1 in A549 cells. In addition, AMI-1 was administrated intranasally to AIPI rats to observe the effects on inflammatory parameters. The results showed that PRMT1 expression was mainly expressed in bronchus and alveolus epithelium and significantly upregulated in lungs from AIPI rats. The inhibition of PRMTs by AMI-1 and the knockdown of PRMT1 expression were able to downregulate the expressions of eotaxin-1 and CCR3 with the IL-4 stimulation in the epithelial cells. Furthermore, AMI-1 administration to AIPI rats can also ameliorate pulmonary inflammation, reduce IL-4 production and humoral immune response, and abrogate eosinophil infiltration into the lungs. In summary, PRMT1 expression is upregulated in AIPI rat lungs and can be stimulated by IL-4. Intervention of PRMT1 activity can abrogate IL-4-dependent eotaxin-1 production to influence the pulmonary inflammation with eosinophil infiltration. The findings may provide experimental evidence that PRMT1 plays an important role in asthma pathogenesis.

Bridging the gap between preclinical and clinical microbicide trials: blind evaluation of candidate gels in murine models of efficacy and safety.

BACKGROUND: Despite significant protection in preclinical studies, cellulose sulfate (CS) failed to protect women against HIV-1/2 and was associated with a trend toward increased HIV-1 acquisition in one of the clinical trials. These results highlight the need for preclinical tests more predictive of clinical outcomes. The objective of this study was to test coded vaginal gels, including CS, in murine models of safety and efficacy to determine the models' utility for evaluating future products. METHODS: Four coded formulations, including 6% CS, 2% PRO 2000 and two placebo gels, were administered intravaginally to medroxyprogesterone-treated mice and their ability to prevent genital herpes (efficacy) or to alter the susceptibility to low dose HSV challenge (safety) was determined. Nonoyxnol-9 served as a positive toxicity control. RESULTS: CS and PRO 2000 significantly protected mice from genital herpes following infection with a laboratory or clinical isolate of HSV-2 introduced in buffer (p<0.001). However, protection was reduced when virus was introduced in seminal plasma. Moreover, mice were significantly more susceptible to infection with low doses of HSV-2 when challenged 12 h after the 7th daily dose of CS or nonoxynol-9 (p<0.05). The increased susceptibility was associated with alterations in epithelial architecture. CONCLUSIONS: CS prevented genital herpes when present at the time of viral challenge, but increased the rate of infection when gel was applied daily for 7 days with a vaginal wash prior to viral inoculation. The findings presumably reflect altered epithelial architecture, which may have contributed to the trend towards increased HIV observed clinically.

Critically engaging: integrating the social and the biomedical in international microbicides research.

Randomized controlled trials and critical social theory are known not to be happy bedfellows. Such trials are embedded in a positivist view of the world, seeking definitive answers to testable questions; critical social theory questions the methods by which we deem the world knowable and may consider experiments in the biomedical sciences as social artifacts. Yet both of these epistemologically and methodologically divergent fields offer potentially important advances in HIV research. In this paper, we describe collaboration between social and biomedical researchers on a large, publicly funded programme to develop vaginal microbicides for HIV prevention. In terms of critical engagement, having integrated and qualitative social science components in the protocol meant potentially nesting alternative epistemologies at the heart of the randomized controlled trial. The social science research highlighted the fallibility and fragility of trial data by demonstrating inconsistencies in key behavioural measurements. It also foregrounded the disjuncture between biomedical conceptions of microbicides and the meanings and uses of the study gel in the context of users' everyday lives. These findings were communicated to the clinical and epidemiological members of the team on an ongoing basis via a feedback loop, through which new issues of concern could also be debated and, in theory, data collection adjusted to the changing needs of the programme. Although critical findings were taken on board by the trialists, a hierarchy of evidence nonetheless remained that limited the utility of some social science findings. This was in spite of mutual respect between clinical epidemiologists and social scientists, equal representation in management and coordination bodies, and equity in funding for the different disciplines. We discuss the positive role that social science integrated into an HIV prevention trial can play, but nonetheless highlight tensions that remain where a hierarchy of epistemologies exists alongside competing paradigms and priorities.

"One teabag is better than four": Participants response to the discontinuation of 2% PRO2000/5 microbicide gel in KwaZulu-Natal, South Africa.

INTRODUCTION: The Microbicides Development Programme evaluated the safety and effectiveness of 0.5% and 2% PRO2000/5 microbicide gels in reducing the risk of vaginally acquired HIV. In February 2008 the Independent Data Monitoring Committee recommended that evaluation of 2% PRO2000/5 gel be discontinued due to futility. The Africa Centre site systematically collected participant responses to this discontinuation. METHODS: Clinic and field staff completed field reports using ethnographic participant observation techniques. In-depth-interviews and focus group discussions were conducted with participants discontinued from 2% gel. A total of 72 field reports, 12 in-depth-interviews and 3 focus groups with 250 women were completed for this analysis. Retention of discontinued participants was also analysed. Qualitative data was analysed using NVivo 2 and quantitative data using STATA 10.0. RESULTS: Participants responded initially with fear that discontinuation was due to harm, followed by acceptance after effective messaging, and finally with disappointment. Participants reported that their initial fear was exacerbated by being contacted and advised to visit the clinic for information about the closure. Operational changes were subsequently made to the contact procedures. By incorporating feedback from participants, messages were continuously revised to ensure that information was comprehensible and misconceptions were addressed quickly thereby enabling participants to accept the discontinuation. Participants were disappointed that 2% PRO2000/5 was being excluded as a HIV prevention option, but also that they would no longer have access to gel that improved their sexual relationships with their partners and assisted condom negotiations. In total 238 women were discontinued from gel and 185 (78%) went on to complete their scheduled follow-up period. DISCUSSION: The use of qualitative social science techniques allowed the site team to amend operational procedures and messaging throughout the discontinuation period. This proved instrumental in ensuring that the discontinuation was successfully completed in a manner that was both understandable and acceptable to participants. TRIAL REGISTRATION: Current Controlled Trials. ISRCTN64716212.

A novel strategy for inducing enhanced mucosal HIV-1 antibody responses in an anti-inflammatory environment.

Prophylactic vaccination against HIV-1 sexual transmission will probably require antibody elicitation at genital mucosal surfaces. However, HIV-1 envelope glycoprotein (Env)-based antigens are weakly immunogenic, particularly when applied mucosally. The polyanion PRO 2000 is safe for human vaginal application, and thus may represent a potential formulating agent for vaginal delivery of experimental vaccine immunogens. Based upon its biochemical properties, we hypothesized that PRO 2000 might enhance mucosal immunogenicity of HIV-1 envelope glycoprotein (Env)-based antigens, promoting local and systemic immune responses. Vaginal immunization with Env-PRO 2000 resulted in significantly increased titres of Env-specific mucosal IgA and IgG in mice and rabbits, respectively, compared to Env alone, revealing modest but significant mucosal adjuvant activity for PRO 2000. In vitro, PRO 2000 associated with Env, protecting the glycoprotein from proteolytic degradation in human vaginal lavage. Unexpectedly, PRO 2000 antagonized TLR4 activation, suppressing local production of inflammatory cytokines. Since inflammation-mediated recruitment of viral target cells is a major risk factor in HIV-1 transmission, the immune modulatory and anti-inflammatory activities of PRO 2000 combined with its intravaginal safety profile suggests promise as an HIV-1 mucosal vaccine formulating agent.

Gelling medical knowledge: innovative pharmaceuticals, experience, and perceptions of efficacy.

As new pharmaceutical products to combat the acquisition of HIV are produced, their clinical efficacy is determined through large-scale clinical trials. Trial participants, however, also independently evaluate the effectiveness of these technologies. During a phase III microbicide clinical trial in Johannesburg, South Africa, female participants acknowledged that although the gel had not yet been clinically proven to be efficacious, they believed that it was capable of healing infections, cleansing the vagina, increasing fertility, and preventing HIV. These responses were informed by experiences of gel use coupled with ideas regarding the flow of bodily fluids and the removal of dirt for bodily cleanliness and the maintenance of health. Examining participant responses to the gel provides insight into the relationship between knowledge and experience when utilizing previously unfamiliar biotechnologies.

Microbicides Development Programme: design of a phase III trial to measure the efficacy of the vaginal microbicide PRO 2000/5 for HIV prevention.

BACKGROUND: With 2.5 million new HIV infections per year, effective preventive methods against HIV are urgently needed, especially in sub-Saharan Africa. MDP301 is an ongoing trial of the vaginal microbicide PRO 2000/5 being conducted by the Microbicides Development Programme. The main objective of the trial is to determine the efficacy and safety of 0.5% and 2% concentrations of PRO 2000/5 gel compared to placebo in preventing vaginally acquired HIV infection. METHODS/DESIGN: MDP301 is a multicentre randomised placebo-controlled Phase III trial. The design was informed by pre-trial feasibility and pilot studies. The choice of trial population, assessments and endpoints are discussed along with statistical and ethical considerations. Adaptations to the design were made during the conduct of the trial; these included closing a study arm and changing the timing of the primary endpoint. DISCUSSION: The development of effective microbicide products remains one of the strongest hopes for new biomedical prevention tools. MDP301 is the largest Phase III microbicide trial to date, with 9404 enrolments, and is scheduled for completion in September 2009. Results are expected towards the end of 2009.

PRO-2000, an antimicrobial gel for the potential prevention of HIV infection.

Indevus Pharmaceuticals Inc, under license from Paligent Inc, is developing PRO-2000, an antimicrobial gel for the prevention of HIV infection. The company is also investigating its potential to prevent the transmission of other sexually transmitted diseases. In February 2005, Indevus launched a pivotal phase III trial for the prevention of HIV infection in women. At that time, further phase III trials in 12,000 African women were scheduled to begin in 2005. A second phase III trial began for the prevention of sexually transmitted infections, including HIV, herpes, Chlamydia and gonorrhea, in Africa in October 2005.

Intravitreal injection of the heparin analog 5-amino-2-naphthalenesulfonate reduces retinal neovascularization in mice.

The effect of the heparin analog 5-amino-2-naphthalenesulfonate (5-amino-2-NMS) on retinal neovascularization was investigated in the mouse model for oxygen-induced retinopathy (OIR). From postnatal day 7 (P7) until P12, mice were kept in a 75% oxygen environment. On P12, they received an intravitreal injection of 10mM 5-amino-2-NMS in one eye and PBS as control substance in the fellow eye. The animals were intracardially perfused with fluorescein-dextran solution on P17. Retinal whole mounts were prepared and ischemic retinopathy was evaluated in 30 animals using a standardized retinopathy score. A single intravitreal injection of 5-amino-2-NMS reduces significantly angioproliferative changes (blood vessel tufts, extra-retinal neovascularization, and blood vessel tortuosity) compared to the contralateral control eye (p=0.025). The median retinopathy score (maximal 13) for the 5-amino-2-NMS treated eyes was 6 versus 8 for the control eyes. 5-Amino-2-NMS binds to the heparin-binding site of FGF1 and FGF2 and thus may be a promising substance for the local treatment of retinal neovascularization.

The non-nucleoside antiviral, BAY 38-4766, protects against cytomegalovirus (CMV) disease and mortality in immunocompromised guinea pigs.

New antiviral drugs are needed for the treatment of cytomegalovirus (CMV) infections, particularly in immunocompromised patients. These studies evaluated the in vitro and in vivo activity of the non-nucleosidic CMV inhibitor, BAY 38-4766, against guinea pig cytomegalovirus (GPCMV). Plaque reduction assays indicated that BAY 38-4766 was active against GPCMV, with an IC(50) of 0.5muM. Yield reduction assays demonstrated an ED(90) and ED(99) of 0.4 and 0.6muM, respectively, of BAY 38-4766 against GPCMV. Guinea pigs tolerated oral administration of 50mg/kg/day of BAY 38-4766 without evidence of biochemical or hematologic toxicity. Plasma concentrations of BAY 38-4766 were high following oral dosing, with a mean peak level at 1-h post-dose of 26.7mg/ml (n=6; range, 17.8-35.4). Treatment with BAY 38-4766 reduced both viremia and DNAemia, as determined by a real-time PCR assay, following GPCMV infection of cyclophosphamide-immunosuppressed strain 2 guinea pigs (p<0.05, Mann-Whitney test). BAY 38-4766 also reduced mortality following lethal GPCMV challenge in immunosuppressed Hartley guinea pigs, from 83% (20/24) in placebo-treated guinea pigs, to 17% (4/24) in BAY 38-4766-treated animals (p<0.0001, Fisher's exact test). Mortality differences were accompanied by reduction in DNAemia in Hartley guinea pigs. Based upon its favorable safety, pharmacokinetic, and therapeutic profiles, BAY 38-4766 warrants further investigation in the GPCMV model.

Inhibition of Neisseria gonorrhoeae genital tract infection by leading-candidate topical microbicides in a mouse model.

The development of effective vaginal microbicides is paramount in the fight against the spread of sexually transmitted infections. Preclinical testing of candidate microbicides for the prevention of gonorrhea has been seriously hindered by the lack of an animal model. We assessed the efficacy of 7 promising formulated agents--CarraGuard, Ushercell, [poly]sodium 4-styrene sulfonate (T-PSS), PRO 2000, ACIDFORM, cellulose acetate phthalate (CAP), and BufferGel--by use of a mouse model of Neisseria gonorrhoeae genital tract infection. Mice received test agent, relevant placebo, or no treatment, followed by intravaginal N. gonorrhoeae challenge. N. gonorrhoeae colonization was tested by vaginal culture. CarraGuard, Ushercell, and T-PSS demonstrated significant protection, compared with control agents and no treatment. PRO 2000, ACIDFORM, and CAP showed significant protection, compared with no treatment but not compared with respective control agents. Mice that received BufferGel were provided significant protection, compared with untreated control mice; no placebo was tested. The findings of the present study suggest that topical agents may effectively reduce N. gonorrhoeae infection and that further evaluation is warranted.