Optimized Benzalkonium Chloride Gel: A Potential Vaginal Microbicides

Objective To develop an optimized BZK gel with good pharmaceutical effect and less toxicity to vaginal mucosa. Methods Four methods as below were used to research the spermicidal activity of BZK gel： 1） in vitro spermicidal test; 2） in vivo spermicidal test in rabbits; 3） anti-fertility test in rabbits; 4） contraceptive test in rabbits. Mucosal irritation test was used in rats to evaluate the safety of optimized BZK gel. Microbiological assessments were used to research anti-STI pathogens （including treponema pallidum, neisseria gonorrhoeae, trichomona vaginalis, candida albicans, ureaplama urealyticum, herpes simplex virus type-2, chlamydiae trachomatis） effect of optimized BZK gel. Results In vitro spermicidal test, EC50 of BZK gel was 0.029mg/ml, a little higher than that of N-9 （0.019mg/ml）. The MIC of BZK gel was 0.25mg/ml, similar to that of N-9 （0.20mg/ml）. The vaginal mucosal irritation test in rats showed that 0.429% BZK gel showed only minimal vaginal irritation, and did not damage the vaginal epithelium or cause local inflammation in rats. Microbiological assessments showed that optimized BZK gel could inhibit or inactivate common ST1 pathogens even after 3-fold or 5-fold dilution. Conclusion Optimized BZK gel was an effective microbicides.

BASANT, a Polyherbal Safe Microbicide Eliminates HPV-16 in Women with Early Cervical Intraepithelial Lesions

Carcinoma cervix is a major cancer of women killing 510,000 women every year worldwide. Human papilloma virus (HPV) infection of cervical cells initiates the transformation of the cervical cells to malignant stage. HPV-16 is the most frequent type of HPV causing these changes. We report here the elimination of HPV-16 from the infected cells of all (11/11) women positive for HPV-16 by 30 intra-vaginal intakes of BASANT.

HIV/STIs and Pregnancy Prevention, Using a Cervical Barrier and Microbicide

Objectives: (A) HIV prevention, using a mechanical cervical barrier in combination with microbicide. (B) Prevention of pregnancy. (C) Shield the cervix to prevent sperm penetration and Gonorrhea, Chlamydia and HIV virus invasion. Methods: We investigated a new FDA approved cervical barrier FemCap (Figure 1). The FemCap is a contraceptive device that is designed with a unique delivery system for microbicides on its cervical and vaginal sides (Figure 4) to ensure better coverage, and retention of gel on the cervix and vagina. We also compared the acceptability and adherence with the FemCap, and retention of a stained vaginal lubricant when delivered with the FemCap versus the vaginal lubricant when delivered using a traditional vaginal applicator (Figure 2). We used the same vaginal applicator utilized in the CAPRISA 0041 study, to deliver Tenofovir microbicide. Thirty women compared the use of a vaginal applicator to deliver a high viscosity stained vaginal lubricant before and after intercourse, versus the FemCap to deliver the same lubricant once before intercourse. The acceptability and efficacy of this delivery system was evaluated. Results: Forty percent (12) women missed the application of the lubricant with the vaginal applicator before intercourse and 10% missed it after intercourse. Amongst FemCap users (3) women (10%) missed application of the vaginal lubricant before intercourse and all of them inserted it after intercourse. The stained gel was better retained over the cervix (Figure 5) by single application with the FemCap versus two applications with the traditional applicator (Figure 2). Conclusions: Women in this study preferred the FemCap due to elimination of leakage and the single application, method versus two applications with the traditional vaginal applicator. The use of the FemCap, can prevent pregnancy, HIV mother-to-child transmission, enhance compliance and retention of gel over the cervix and vagina that may potentially prevent STIs and increase the efficacy of Tenofovir.

The Motivations and Experiences of Young Women in a Microbicide Trial in the USA and Puerto Rico

Young women are an important target group in microbicide research, yet little is known about why they participate and stay in microbicide trials. Our study examined motivations for participating in a Phase I microbicide trial among 61 women ages 18 - 24 years in the continental USA and Puerto Rico. We also examined their perspectives on study participation. Participants underwent a semi-structured in-depth interview in which they were asked about factors that motivated enrollment and their experiences while participating. They also completed a Web-based Computer Assisted Self Interview in which they were asked to rate study burden (1 = low to 4 = high). Factors that motivated enrollment were altruism (29%), compensation (17%), a combination of altruism and compensation (37%) and free medical exams (17%). Factors that encouraged participants to stay in the study were study staff (95%), confirmation of good health (41%), and the opportunity to learn about their bodies (17%). Mean ratings of study burden ranged from 1.83 (having to travel to site) to 2.41 (colposcopy), indicating that participants were not highly bothered by visits or procedures. Although Phase I trials require invasive procedures, participants were not highly bothered by them and recognized them as necessary. Good relationships with staff and clear information about how procedures contribute to study goals may encourage participants to remain in trials. Young women may be motivated to enter microbicide trials by stressing the role they will play in discovering better HIV-prevention methods and highlighting the comprehensive preventive exams they will receive.

Establishing a Th17 based mouse model for preclinical assessment of the toxicity of candidate microbicides

Background To effectively block the invasion of human immunodeficiency virus （HIV）-1 on mucosal surface, vaginal anti-HIV-1 microbicides should avoid inflammatory responses and disruption of mucosa integrity because these will facilitate transepithelial viral penetration and replication. However, existing models fail to predict and evaluate vaginal mucosal toxicity induced by microbicides, and most importantly, they are unable to identify subtle or subclinical inflammatory reactions. This study was designed to develop a cost-effective in vivo model to evaluate microbicide safety in a preclinical study which can recapitulate the mucosal topical reaction.Methods A murine model was employed with nonoxynol-9 （N-9） as the topical stimulant within the vagina. Different concentrations of N-9 （1%, 3%, and 4%） were topically applied to the vagina for five consecutive days. A panel of inflammatory cytokines including interleukine-2 （IL-2）, IL-4, IL-6, IL-17A, interferon-Y （IFN-Y）, tumor necrosis factor-α （TNF-α）, and immuno-regulatory IL-10 were assayed in vaginal lavage. Cytokines were quantified by using cytometric bead array （CBA） and reverse transcript （RT） real-time PCR. Histopathological evaluation of vaginal tissues was conducted on hematoxylin-eosin stained slides and scored with a semi-quantitative system according to the severity of epithelial disruption, leucocyte infiltration, edema, and vascular injection. The association between the cytokines and histopathological scores was assessed by linear regression analysis.Results All three concentrations of N-9 induced inflammatory cytokine production. The 4% N-9 application resulted in a consistent production of cytokines in a time-dependent manner. The cytokines reached peak expression on day three with the exception of IL-4 which reached its peak on day one. Histopathological examination of 4% N-9 treated cervicovaginal tissues on day three showed intensive damage in four mice （sores： 10-13） and moderate damage in one mouse （score： 8）, which were significantly associated with both inflammatory cytokines IL-17A and IL-6 and anti-inflammatory cytokines IL-4 and IL-10. Interestingly, IL-17A showed significant positive association with inflammatory cytokine TNF-α （r=0.739; P ＜0.05）, anti-inflammatory cytokines IL-10 （r=0.804; P ＜0.01） and IL-4 （r=0.668; P ＜0.05）.Conclusions Our data demonstrate that a panel of cytokines （IL-17A, IL-6, IL-4 and IL-10） could be used as surrogate biomarkers to predict the histopathological damage. Th17 may play a central role in orchestrating inflammatory cytokine responses. This Th17 based mouse model is cost-effective and suitable to assess the toxicity of candidate microbicides in preclinical studies.

Phagocytosis: A Practical Approach for Evaluating Neutrophil Function in Health and Disease

Neutrophils, crucial players in the effector phase of the immune response, are recognized as important mediators of both innate and adaptive immune responses. Through the production of pro- and anti-inflammatory cytokines, they modulate the function of T and other lymphoid cells. Countless reports have highlighted the importance of these cells as efficient antimicrobial agents and annotated their involvement in the pathology of infectious and noninfectious diseases. The development of modern, sophisticated technologies has allowed the study of the functions of these cells in clinical settings. These advanced technologies include fluorescence-activated cell sorters, confocal microscopy, automated cell image analyzers, and live cell analysis instruments. Unfortunately, the cost of these modern instruments, maintenance, reagents, and the need for qualified technicians prohibit their use in low-income laboratories and universities in developing countries. With this in mind, we propose a series of basic tests that can be used in low-input clinical laboratories and universities to evaluate the function of neutrophils in health and disease. Our methodology allows us to assess in a practical and low-cost manner the functions of neutrophils in the phagocytic process, including opsonization, ingestion, ROI production (NBT reduction), myeloperoxidase content, phagosome-lysosome fusion, microbicidal activity, and NET production. Thus, under a disadvantageous ambiance, this may guide physicians in deciding whether a patient’s illness involves phagocytic defects without imposing a heavy financial burden.Graphical Abstract[-rId13-]

HIV microbicides： innovation and challenge

According to Joint United Nations Program on HIV/AIDS （UNAIDS） report in 2009, the estimated number of people living with HIV-1 is 33.4 million, and half of the infected adults （aged 15-49 years） are women, who acqluired HIV-1 mainly through heterosexual exposure. In China, HIV-1 transmission through sexual contact has also increased rapidly, and has reached over 70% in overall cases; heterosexual transmission accounted for 40% and men who had sex with man （MSM） for 32.0%.

Novel Antiviral Agents Targeting HIV Entry and Transmission

Studies of the mechanism of HIV entry and transmission have identified multiple new targets for drug development. A range of inhibitors have demonstrated potent antiretroviral activity by interfering with CD4-gp120 interaction,coreceptor binding or viral-cell fusion in preclinical and clinical studies. One of these agents,fusion inhibitor enfuvirtide,is already in clinical use. Here we review the progress in the development of specific entry inhibitors as novel therapeutics. The potential of entry inhibitors as topical microbicides to block HIV transmission is also discussed.

Degradation of naturally occurring and engineered antimicrobial peptides by proteases

We hypothesized that current antimicrobial peptides should be susceptible to proteolytic digestion. The antimicrobial peptides: Griffithinsin, RC-101, LL-37, LSA-5, PSC-RANTES and DJ007 were degraded by commercially available proteases. Two different species of anaerobic vaginal flora, Prevotella bivia and Porphyromonas asaccharolytica also degraded the materials. Griffithsin was resistant to digestion by 8 of the 9 proteases and the bacteria while LL-37 was the most sensitive to protease digestion. These data suggests most of the molecules may not survive for very long in the proteolytic rich environments in which they are intended to function.

<i>In vitro</i>activity of cationic peptides against <i>Neisseria gonorrhoeae</i>and vaginal <i>Lactobacillus</i>species: The effect of divalent cations

One of the new strategies for the prevention of HIV acquisition is the use of microbicides such as topical microbicides including antimicrobial and antiviral peptides. Ideally, new drug candidates should kill pathogens without determent to the normal bacterial flora considered important in health;such as hydrogen peroxide producing Lactobacillus species. The antimicrobial peptides LL-37 and LSA-5 were studied to determine their spectrum of activity against bacterial pathogens and normal flora organisms. The effects of divalent cations at biologically relevant concentrations were determined. We show the synthetic lytic peptide LSA-5 and the naturally occurring peptides LL-37 inactivate Neisseria gonorrhoeae but are less active against many normal flora members such as Lactobacillus species. Biologically relevant concentrations of calcium and magnesium prevented killing of sensitive strains. LSA-5 is more potent than LL-37, both are inhibited from killing sensitive strains by calcium and magnesium. Strains of Lactobacillus iners were killed by both microbicides even in the presence of the divalent cations. Antimicrobial peptides, such as LSA-5, have good potential for use in prevention of sexually transmitted disease, if formulated to sequester calcium and magnesium present in biological fluids.

Phenotypic differences between coryneform bacteria isolated from seminal fluid of healthy men and men with chronic prostatitis syndrome

We compared the potential phenotypic properties of coryneform bacteria associated with chronic prostatitis syndrome （CPS）, such as secretory inhibitor of lysozyme （SIL） and secretory inhibitor of platelet microbicidal protein （SIPMP）. A total of 110 clinical isolates of coryneform bacteria isolated from the seminal fluid of healthy men and men with CPS were tested. SIPMP production was tested by inhibiting platelet microbicidal protein （PMP） bioactivity against Bacillus subtilis, and was expressed as percentage of inhibition of PMP bactericidal activity. SIL production was tested by inhibiting lysozyme activity against Micrococcus lysodeikticus and was expressed in microgram per millilitre of inactivated lysozyme. A significantly higher proportion of CPS strains （58.7% vs. 19.2 %） was SIPMP-positive compared with non-CPS strains （P 〈 0.01）. Of the CPS strains tested, 77.8% were SIL-positive compared with 34% of the non- CPS isolates （P 〈 0.05）. These results suggest that the diagnosis of CPS should not rely solely on classical parameters, for example, the identification and counting of microorganisms, but the functional significance of these parameters must be estimated, possibly by the concentration of different bacterial substrains, detection of opportunistic microorganisms with pathogenic properties, such as pronounced resistance to the cationic antimicrobial peptides, and/or the ability to inhibit the antimicrobial host defence factors.

Distribution of secretory inhibitor of platelet microbicidal protein among urethral isolates with its correlation with prostatitis

Aim： To report the detection in vitro of secretory inhibitor of platelet microbicidal protein （SIPMP） phenotypes of urethral isolates along with a comparisonwith isolates from patients with or without chronic bacterial prostatitis （CBP）. Methods： Urethral isolates of Staphylococcus spp. （n = 64）, diphtheroids （n = 28）, micrococci （n = 15）, streptococci （n = 21）, Enterobacteriaceae （n = 9） and Enterococcusfaecalis （n = 19） from patients with or without CBP were tested. SIPMP production was tested by inhibition of platelet microbicidal protein （PMP） bioactivity against Bacillus subtilis and was expressed as percentage of inhibition of PMP bactericidal activity. Results： A significantly higher proportion of CBP-strains （57.78% vs. 16.67%） reduced PMP-induced killing of Bacillus subtilis than non- CBP strains did （P 〈 0.01）. SIPMP levels of staphylococci and Enterococcusfaecalis from the CBP group were significantly higher than those of the control group. Conclusion： These results suggest that SIPMP production is associated with the CBP source. Data from the present study might have significant implications for the understanding of the pathogenesis of CBP. （Asian J Androl 2008 Mar; 10： 189-192）.

UniPron is A Fully Effective Non-hormonal Reversible Contraceptive in Baboon Model (Papio Anubis)

Objective To determine the safety and efficacy of UniPron as a reversible contraceptive.Methods Vaginal swabs were obtained before and after UniPron administration, cultured onto appropriate culture media and bacteria identification was done based on type of media used, Gram stain reactions, colony morphology and biochemical tests. Vaginal biopsy tissues were processed using paraffin wax method, stained with hematoxylin and eosin and examined under light microscopy to determine the effect of the product on vaginal tissues. The effect of UniPron on sperm was examined by mixing the product with electroejaculated spermatozoa in vitro at different concentrations. For efficacy studies, male baboons of proven fertility were mated with UniPron treated or untreated females of proven fertility during the fertile stages. Results All the five females （100%） that were treated with UniPron did not conceive and they regained total fertility when the treatment was stopped while all the controls conceived. At a concentration of 40%, UniPron completely immobilized spermatozoa in an in-vitro system. UniPron mechanism of action was by lowering the vaginal pH and on application in baboon, the pH was lowered for at least 3 h after which it went back to normal.Conclusions As we plan for a study to test UniPron as a microbicide to prevent STIs including HIV, our current study has established that this novel product is effective in contraception and harmless to vaginal tissues and vaginal microbial flora in a baboon model （Papio anubis）.

Rabbit Defensin(NP-1) Gene Transformation of Wheat and In Vitro Microbicidal Activity Assay of Transgenic Plants

Monocot high expression vector pBARUNP1, harboring rabbit defensin(NP 1) gene and selective bar gene for resistance to the herbicide Basta, were constructed and then transferred into immature embryos of wheat (“Bobwhite” and “Zhong 60634”)via particle bombardment. Southern and RNA dot blots showed the stable integration and transcription of foreign NP 1 gene in the wheat genome. Furthermore, in vitro microbicidal activity assay indicated the proper translation of defensin. Crude protein extraction of transgenic plants exhibited to some extent cytotoxic to several pathogens including G. saubinetii, B. subtilis, E.coli, and A. tumefaciens.

Potentials of <i>Melastoma malabathricum</i>Linn. Flower and Fruit Extracts as Antimicrobial Infusions

Melastoma malabathricum Linn. is a shrub that belongs to the family Melastomataceae and a common herbal plant used in folk medicines to treat inflamed wounds. This study was carried out with the aim to evaluate the inhibitory activities of different concentrations of the M. malabathricum Linn. flower and fruit crude extracts against a variety of microorganisms. The inhibitory effects of both extracts were tested against the microorganisms using the disc diffusion method. The lowest concentrations of the extracts producing inhibition zones against the test microorganisms were used to determine their Minimum Inhibitory Concentrations (MICs) and Minimum Microbicidal Concentrations (MMCs). Both crude extracts showed strong inhibitory activities against Gram-positive bacteria. The range of MIC values for the crude flower and fruit extracts on all the bacteria tested were 12.5 to 100.0 mg/ml. Overall, Gram-positive bacteria were more susceptible to the crude extracts compared to Gram-negative species, potentiating a possible use of the extracts to inhibit or kill potential pathogens.

Distribution of secretory inhibitor of platelet microbicidal protein among anaerobic bacteria isolated from stool of children with diarrhea

AIM： To study the secretory inhibitor of platelet microbicidal protein （SIPMP） phenotypes of faecal anaerobic isolates from patients with diarrhea.METHODS： Faecal isolates of anaerobic bacteria （B. fragilis, n = 42; B. longum, n = 70; A. israelii, n = 21; E. lentum, n = 12） from children with diarrhea were tested. SIPMP production was tested by inhibition of platelet microbicidal protein （PMP） bioactivity against B. subtilis and was expressed as percentage of inhibition of PMP bactericidal activity.RESULTS： Among anaerobic isolates 80% of B. longum strains, 85.7% of A. israeli/strains, 50% of E. lentum strains and 92.86% of B. fragilis strains were SIPMP-positive. The isolated anaerobic organisms demonstrated SIPMP production at a mean level of 13.8% ± 0.7%, 14.7% ± 1.8%, 3.9% ± 0.9% （P 〈 0.05） and 26.8% ± 7.5% （P 〈 0.05） for bifidobacteria, A. israelii, E. lentum and B. fragilis, respectively.CONCLUSION： Data from the present study may have significant implications in understanding the pathogenesis of microecological disorders in the intestine, as well as for future improvement in the prevention and therapy of anaerobe-associated infections.

Synthesis of Poly(styrene-co-divinylbenzene)-supported Dichloro Cyanuric Acid

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Role of Lactic Acid Bacteria-Myeloperoxidase Synergy in Establishing and Maintaining the Normal Flora in Man

Lactic acid bacteria (LAB) are incapable of cytochrome synthesis and lack the heme electron transport mechanisms required for efficient oxygen-based metabolism. Consequently, LAB redox activity is flavoenzyme-based and metabolism is fermentative, producing lactic acid, and in many cases, hydrogen peroxide (H2O2). Despite this seeming metabolic limitation, LAB dominate in the normal flora of the mouth, vagina and lower gastrointestinal tract in man. Myeloperoxidase (MPO) is produced by the neutrophil leukocytes and monocytes that provide the innate phagocyte defense against infecting pathogens. MPO is unique in its ability to catalyze the H2O2-dependent oxidation of chloride (Cl-) to hypochlorite (OCl-). In turn, this OCl- directly reacts with a second H2O2 to produce singlet molecular oxygen (), a metastable electronic excitation state of oxygen with a microsecond lifetime that restricts its combustive reactivity within a submicron radius of its point of generation. Each day a healthy human adult produces about a hundred billion neutrophils containing about 4 femtograms MPO per neutrophil. Inflammatory states and G-CSF treatment increase both neutrophil production and the quantity of MPO per neutrophil. After a short circulating lifetime, neutrophils leave the blood and migrate into body spaces including the mouth, vagina, urinary tract, and gastrointestinal tract. Greater than a hundred thousand neutrophils are lavaged from the mouths of healthy humans;the quantity lavaged is proportional to the blood neutrophil count. MPO selectively and avidly binds to most Gram-positive and all Gram-negative bacteria tested, but LAB do not show significant MPO binding. Neutrophils migrating to normal flora sites release MPO into the LAB-conditioned milieu containing adequate acidity and H2O2 to support extra-phagocyte MPO microbicidal action. In combination, LAB plus MPO exert a potent synergistic microbicidal action against high MPO-binding microbes. This LAB-MPO synergy provides a mechanism for the establishment and maintenance of LAB in the normal flora of man.