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1.  Identification of Personal Lubricants That Can Cause Rectal Epithelial Cell Damage and Enhance HIV Type 1 Replication in Vitro 
AIDS Research and Human Retroviruses  2011;27(9):1019-1024.
Abstract
Over-the-counter personal lubricants are used frequently during vaginal and anal intercourse, but they have not been extensively tested for biological effects that might influence HIV transmission. We evaluated the in vitro toxicity anti-HIV-1 activity and osmolality of popular lubricants. A total of 41 lubricants were examined and compared to Gynol II and Carraguard as positive and negative controls for toxicity, respectively. Cytotoxicity was assessed using the XTT assay. The MAGI assay with R5 and X4 HIV-1 laboratory strains was used to evaluate antiviral activity. The effect of the lubricants on differentiated Caco-2 cell monolayers (transepithelial electrical resistance, TEER) was also measured. None of the lubricants tested showed significant activity against HIV-1. Surprisingly, four of them, Astroglide Liquid, Astroglide Warming Liquid, Astroglide Glycerin & Paraben-Free Liquid, and Astroglide Silken Secret, significantly enhanced HIV-1 replication (p<0.0001). A common ingredient in three of these preparations is polyquaternium-15. In vitro testing of a chemically related compound (MADQUAT) confirmed that this similarly augmented HIV-1 replication. Most of the lubricants were found to be hyperosmolar and the TEER value dropped approximately 60% 2 h after exposure to all lubricants tested. Cells treated with Carraguard, saline, and cell controls maintained about 100% initial TEER value after 2–6 h. We have identified four lubricants that significantly increase HIV-1 replication in vitro. In addition, the epithelial damage caused by these and many other lubricants may have implications for enhancing HIV transmission in vivo. These data emphasize the importance of performing more rigorous safety testing on these products.
doi:10.1089/aid.2010.0252
PMCID: PMC3161103  PMID: 21309617
2.  In Vitro and Ex Vivo Testing of Tenofovir Shows It Is Effective As an HIV-1 Microbicide 
PLoS ONE  2010;5(2):e9310.
Background
Tenofovir gel has entered into clinical trials for use as a topical microbicide to prevent HIV-1 infection but has no published data regarding pre-clinical testing using in vitro and ex vivo models. To validate our findings with on-going clinical trial results, we evaluated topical tenofovir gel for safety and efficacy. We also modeled systemic application of tenofovir for efficacy.
Methods and Findings
Formulation assessment of tenofovir gel included osmolality, viscosity, in vitro release, and permeability testing. Safety was evaluated by measuring the effect on the viability of vaginal flora, PBMCs, epithelial cells, and ectocervical and colorectal explant tissues. For efficacy testing, PBMCs were cultured with tenofovir or vehicle control gels and HIV-1 representing subtypes A, B, and C. Additionally, polarized ectocervical and colorectal explant cultures were treated apically with either gel. Tenofovir was added basolaterally to simulate systemic application. All tissues were challenged with HIV-1 applied apically. Infection was assessed by measuring p24 by ELISA on collected supernatants and immunohistochemistry for ectocervical explants. Formulation testing showed the tenofovir and vehicle control gels were >10 times isosmolar. Permeability through ectocervical tissue was variable but in all cases the receptor compartment drug concentration reached levels that inhibit HIV-1 infection in vitro. The gels were non-toxic toward vaginal flora, PBMCs, or epithelial cells. A transient reduction in epithelial monolayer integrity and epithelial fracture for ectocervical and colorectal explants was noted and likely due to the hyperosmolar nature of the formulation. Tenofovir gel prevented HIV-1 infection of PBMCs regardless of HIV-1 subtype. Topical and systemic tenofovir were effective at preventing HIV-1 infection of explant cultures.
Conclusions
These studies provide a mechanism for pre-clinical prediction of safety and efficacy of formulated microbicides. Tenofovir was effective against HIV-1 infection in our algorithm. These data support the use of tenofovir for pre-exposure prophylaxis.
doi:10.1371/journal.pone.0009310
PMCID: PMC2824823  PMID: 20174579
3.  Reformulated tenofovir gel for use as a dual compartment microbicide 
Objectives
Coital use of 1% tenofovir gel was shown to be modestly effective at preventing HIV transmission when applied vaginally in the CAPRISA 004 trial. Because the gel is hyperosmolar, which would reduce the integrity of the epithelium and induce fluid movement into the lumen, rectal use may not be acceptable. This study evaluated the pre-clinical safety and efficacy of a reformulated (reduced osmolality) tenofovir gel product.
Methods
Reduced glycerine (RG)-tenofovir gel was compared with the original tenofovir gel for physiochemical characteristics, product safety and anti-HIV-1 activity.
Results
The formulations were similar in all characteristics except for osmolality and spreadability/firmness. The RG-tenofovir gel had a 73% lower osmolality, a 29.6% increase in spreadability and a 27% decrease in firmness as compared with the original tenofovir gel. When applied to epithelial cell monolayers, tenofovir gel showed a transient reduction in the transepithelial resistance while the RG-tenofovir gel did not. Both gels retained ectocervical and colorectal explant viability. However, tenofovir gel treatment resulted in epithelial stripping that was absent after RG-tenofovir gel treatment of the polarized explants. Anti-HIV-1 activity was confirmed by lack of HIV-1 infection in polarized explants treated with either gel as compared with the control explants.
Conclusions
Reducing the osmolality of the tenofovir gel resulted in improved epithelial integrity, which suggests better safety upon rectal use. The improved gel safety did not compromise drug release or anti-HIV-1 activity. These data support the use of this gel as a dual compartment microbicide.
doi:10.1093/jac/dks173
PMCID: PMC3417689  PMID: 22581908
HIV prevention; rectal microbicide; formulation; preclinical testing; safety
4.  Comparative evaluation of tableting compression behaviors by methods of internal and external lubricant addition: Inhibition of enzymatic activity of trypsin preparation by using external lubricant addition during the tableting compression process 
AAPS PharmSci  2001;3(3):32-42.
This study evaluated tableting compression by using internal and external lubricant addition. The effect of lubricant addition on the enzymatic activity of trypsin, which was used as a model drug during the tableting compression process, was also investigated. The powder mixture (2% crystalline trypsin, 58% crystalline lactose, and 40% microcrystalline cellulose) was kneaded with 5% hydroxypropyl cellulose aqueous solution and then granulated using an extruding granulator equipped with a 0.5-mm mesh screen at 20 rpm. After drying, the sample granules were passed through a 10-mesh screen (1680 μm). A 200-mg sample was compressed by using 8-mm punches and dies at 49, 98, 196, or 388 MPa (Mega Pascal) at a speed of 25 mm/min. The external lubricant compression was performed using granules without lubricant in the punches and dies. The granules were already dry coated by the lubricant. In contrast, the internal lubricant compression was performed using sample granules (without dry coating) containing 0.5% lubricant. At 98 MPa, for example, the compression level using the external lubricant addition method was about 13% higher than that for internal addition. The significantly higher compressing energy was also observed at other MPas. By comparison, the friction energy for the external addition method calculated based on upper and lower compression forces was only slightly larger. The hardness of tablets prepared using the internal addition method was 34% to 48% lower than that for the external addition method. The total pore volume of the tablet prepared using the external addition method was significantly higher. The maximum ejection pressure using the no-addition method (ie, the tablet was prepared using neither dry-coated granules nor added lubricant) was significantly higher than that of other addition methods. The order was as follows: no addition, external addition, and then internal addition. The ejection energy (EE) for internal addition was the lowest; for no addition, EE was the highest. In the dissolution test, the tablets obtained using external addition immediately disintegrated and showed faster drug release than those prepared using internal addition. This result occurred because the water penetration rate of the tablet using the external addition was much higher. The trypsin activity in tablets prepared using the external addition method was significantly higher than that produced using the internal addition method at the same pressure. All these results suggest that the external addition method might produce a fast-dissolution tablet. Because the drug will be compressed using low pressure only, an unstable bulk drug may be tableted without losing potency.
doi:10.1208/ps030320
PMCID: PMC2751015  PMID: 11741271
Tableting; Trypsin; Preparation; Compression; Dissolution
5.  Effect of Vaginal Lubricants on Natural Fertility 
Obstetrics and gynecology  2012;120(1):44-51.
Objective
Over-the-counter vaginal lubricants have been shown to negatively affect in vitro sperm motility. The objective of this study was to estimate the effect of vaginal lubricant use during procreative intercourse on natural fertility.
Methods
Women aged 30–44 years with no history of infertility who had been trying to conceive for less than 3 months completed a baseline questionnaire on vaginal lubricant use. Subsequently, women kept a diary to record menstrual bleeding, intercourse, and vaginal lubricant use and conducted standardized pregnancy testing for up to 6 months. Diary data were used to determine the fertile window and delineate lubricant use during the fertile window. A proportional hazards model was used to estimate fecundability ratios with any lubricant use in the fertile window considered as a time-varying exposure.
Results
Of the 296 participants, 75 (25%) stated in their baseline questionnaire that they use vaginal lubricants while attempting to conceive. Based on daily diary data, 57% of women never used a lubricant, 29% occasionally used a lubricant, and 14% used a lubricant frequently. Women who used lubricants during the fertile window had similar fecundability to those women who did not use lubricants (fecundability ratio 1.05, 95% CI: 0.59, 1.85) after adjusting for age, partner race, and intercourse frequency in the fertile window.
Conclusion
Lubricants are commonly used by couples during procreative intercourse. Lubricant use during procreative intercourse does not appear to reduce the probability of conceiving.
doi:10.1097/AOG.0b013e31825b87ae
PMCID: PMC3427535  PMID: 22914390
6.  Covert Use, Vaginal Lubrication, and Sexual Pleasure: A Qualitative Study of Urban U.S. Women in a Vaginal Microbicide Clinical Trial 
Archives of sexual behavior  2009;39(3):748-760.
Using data from a U.S. clinical safety trial of tenofovir gel, a candidate microbicide, we explored the intersection of sexual pleasure and vaginal lubrication to understand whether and under what circumstances women would use a microbicide gel covertly with primary partners. This study question emerged from acceptability research in diverse settings showing that even though future microbicides are extolled as a disease prevention method that women could use without disclosing to their partners, many women assert they would inform their primary partner. Participants (N = 84), stratified by HIV-status and sexual activity (active vs. abstinent), applied the gel intra-vaginally for 14 days. At completion, quantitative acceptability data were obtained via questionnaire (N = 79) and qualitative data via small group discussions (N = 15 groups, 40 women). Quantitatively, 71% preferred a microbicide that could not be noticed by a sex partner and 86% experienced greater vaginal lubrication during application of the gel. Based on our analysis of the qualitative data, we suggest that women would find it more acceptable to use a microbicide covertly in primary relationships if they believed that the method was truly unnoticeable. Our findings also showed that women’s assessment of the possibility of discreet, if not covert, use was strongly related to their perception of how a microbicide’s added vaginal lubrication would influence their own and their partner’s pleasure, as well as their partner’s experience of his sexual performance. A microbicide that increases pleasure for both partners could potentially be used without engendering opposition from primary partners.
doi:10.1007/s10508-009-9509-3
PMCID: PMC2855760  PMID: 19636696
Sexual pleasure; HIV/STI prevention; vaginal Microbicide; pregnancy prevention
7.  Use of Saliva as a Lubricant in Anal Sexual Practices Among Homosexual Men 
Objectives
Compared with other sexually active adults, men who have sex with men (MSM) are more frequently infected with several pathogens including cytomegalovirus, hepatitis B virus, and Kaposi sarcoma-associated herpesvirus. Because one common element between these organisms is their presence in saliva, we evaluated saliva exposure among MSM in a heretofore relatively unrecognized route—via use of saliva as a lubricant in anal sex.
Methods
MSM in a San Francisco population–based cohort were interviewed regarding use of saliva by the insertive partner as a lubricant in various anal sexual practices.
Results
Among 283 MSM, 87% used saliva as a lubricant in insertive or receptive penile–anal intercourse or fingering/fisting at some point during their lifetime; 31%–47% did so, depending upon the act, in the prior 6 months. Saliva use as a lubricant was more common among younger men and among HIV-infected men when with HIV-infected partners. Even among MSM following safe sex guidelines by avoiding unprotected penile–anal intercourse, 26% had anal exposure to saliva via use as a lubricant.
Conclusions
Among MSM, use of saliva as a lubricant is a common, but not ubiquitous, practice in anal sex. The findings provide the rationale for formal investigation of whether saliva use in this way contributes to transmission of saliva-borne pathogens in MSM.
doi:10.1097/QAI.0b013e31819388a9
PMCID: PMC3975591  PMID: 19131893
homosexual; saliva; lubricant; anal intercourse
8.  Condoms and condiments: compatibility and safety of personal lubricants and their use in Africa 
Previous research on the use of personal lubricants for sexual intercourse is limited and has primarily focused on condom compatibility and breakage, with only recent limited assessment of lubricant safety and possible epidemiologic implications. This article discusses the global evidence of lubricant compatibility with latex condoms and biological safety of lubricants, as well as documentation of lubricant use and current guidelines for HIV prevention programming in Africa. Data on lubricant compatibility with condoms are less available than commonly realized, and many lubricant products may not have been thoroughly tested for safety due to flexible regulatory environments. Recent laboratory and study findings from microbicides research also suggest that some water-based lubricants may have safety issues. Some African populations are using several types of lubricants, especially oil-based petroleum jellies, and receive little evidence-based guidance. More research is needed from the medical community to guide prevention programming.
doi:10.7448/IAS.16.1.18531
PMCID: PMC3708353  PMID: 23841994
lubricants; condoms; HIV prevention; Africa
9.  Effects of feminine hygiene products on the vaginal mucosal biome 
Microbial Ecology in Health and Disease  2013;24:10.3402/mehd.v24i0.19703.
Background
Over-the-counter (OTC) feminine hygiene products come with little warning about possible side effects. This study evaluates in-vitro their effects on Lactobacillus crispatus, which is dominant in the normal vaginal microbiota and helps maintain a healthy mucosal barrier essential for normal reproductive function and prevention of sexually transmitted infections and gynecologic cancer.
Methods
A feminine moisturizer (Vagisil), personal lubricant, and douche were purchased OTC. A topical spermicide (nonoxynol-9) known to alter the vaginal immune barrier was used as a control. L. crispatus was incubated with each product for 2 and 24h and then seeded on agar for colony forming units (CFU). Human vaginal epithelial cells were exposed to products in the presence or absence of L. crispatus for 24h, followed by epithelium-associated CFU enumeration. Interleukin-8 was immunoassayed and ANOVA was used for statistical evaluation.
Results
Nonoxynol-9 and Vagisil suppressed Lactobacillus growth at 2h and killed all bacteria at 24h. The lubricant decreased bacterial growth insignificantly at 2h but killed all at 24h. The douche did not have a significant effect. At full strength, all products suppressed epithelial viability and all, except the douche, suppressed epithelial-associated CFU. When applied at non-toxic dose in the absence of bacteria, the douche and moisturizer induced an increase of IL-8, suggesting a potential to initiate inflammatory reaction. In the presence of L. crispatus, the proinflammatory effects of the douche and moisturizer were countered, and IL-8 production was inhibited in the presence of the other products.
Conclusion
Some OTC vaginal products may be harmful to L. crispatus and alter the vaginal immune environment. Illustrated through these results, L. crispatus is essential in the preservation of the function of vaginal epithelial cells in the presence of some feminine hygiene products. More research should be invested toward these products before they are placed on the market.
doi:10.3402/mehd.v24i0.19703
PMCID: PMC3758931  PMID: 24009546
Lactobacillus; L. crispatus; cytokines; Interleukin-8; Nonoxynol-9; vaginal microbiota; vaginal epithelial colonization; mucosal immunity
10.  Interactive Cytokine Regulation of Synoviocyte Lubricant Secretion 
Tissue Engineering. Part A  2009;16(4):1329-1337.
Cytokine regulation of synovial fluid (SF) lubricants, hyaluronan (HA), and proteoglycan 4 (PRG4) is important in health, injury, and disease of synovial joints, and may also provide powerful regulation of lubricant secretion in bioreactors for articulating tissues. This study assessed lubricant secretion rates by human synoviocytes and the molecular weight (MW) of secreted lubricants in response to interleukin (IL)-1β, IL-17, IL-32, transforming growth factor-beta 1 (TGF-β1), and tumor necrosis factor-alpha (TNF-α), applied individually and in all combinations. Lubricant secretion rates were assessed using ELISA and binding assays, and lubricant MW was assessed using gel electrophoresis and Western blotting. HA secretion rates were increased ∼40-fold by IL-1β, and increased synergistically to ∼80-fold by the combination of IL-1β + TGF-β1 or TNF-α + IL-17. PRG4 secretion rates were increased ∼80-fold by TGF-β1, and this effect was counterbalanced by IL-1β and TNF-α. HA MW was predominantly <1 MDa for controls and individual cytokine stimulation, but was concentrated at >3 MDa after stimulation by IL-1β + TGF-β1 + TNF-α to resemble the distribution in human SF. PRG4 MW was unaffected by cytokines and similar to that in human SF. These results contribute to an understanding of the relationship between SF cytokine and lubricant content in health, injury, and disease, and provide approaches for using cytokines to modulate lubricant secretion rates and MW to help achieve desired lubricant composition of fluid in bioreactors.
doi:10.1089/ten.tea.2009.0210
PMCID: PMC2862605  PMID: 19908966
11.  A study on risk factors associated with inconsistent condom and lubricant use among men who have sex with men in central Karnataka, India 
The Australasian Medical Journal  2011;4(10):469-473.
Background
Among the sexual minority groups, the Men who have Sex with Men (MSM) community is a large and scattered network. Sexual activity among MSM is frequent and often unplanned. STI and HIV are major medical problems faced by this vulnerable group. Stigma and discrimination towards this group result in poor access to preventive services that encourage condom and lubricant usage.
Method
A cross-sectional, community-based study of 309 MSM was carried out in the Davangere district between December 2008 and February 2010. Participants were identified in three stages: cruising venue identification and mapping; determining eligibility and willingness to participate; and recruitment to the study. Consecutive sampling was used to recruit the participants with the help of a snowball technique, obtaining informed and written consent.
Results
Of the participants 79.61% and 88.03% reported inconsistent use of condom and lubricant during the three months prior to the interview, respectively. In multivariate analysis, middle socioeconomic class, sex in a public place and increased frequency of sex were significantly associated with inconsistent condom use. Whereas, practising both types of anal sex (receptive and insertive), not using a condom during the last sexual encounter and increased frequency of sex were significantly associated with inconsistent lubricant use.
Conclusion
Many social and behavioural factors are involved in the inconsistent use of condom and lubricant among MSM. Preventive programmes must identify these factors in order to target consistent condom and lubricant use among the MSM community.
doi:10.4066/AMJ.2011.563
PMCID: PMC3562872  PMID: 23386862
Inconsistent condom use; Men who have Sex with Men; Human Immunodeficiency Virus; Sexual risk behaviour
12.  Frequency, Patterns and Preferences of Lubricant Use During Anal Intercourse Within Male Sexual Partnerships in Lima, Peru: Implications for a Rectal Microbicide HIV Prevention Intervention 
AIDS care  2012;25(5):579-585.
Understanding current practices of lubricant use during anal intercourse can help to assess the contexts for the introduction of topical rectal microbicides as an HIV prevention tool for men who have sex with men (MSM). We used quantitative and qualitative methods to assess: current patterns of lubricant use; preferred characteristics of commercial lubricant formulations; and social and behavioral contexts of lubricant use within male sexual partnerships in Lima, Peru. Between 2007 and 2008, we conducted a quantitative behavioral survey with 547 MSM followed by qualitative individual and group interviews with 36 MSM from Lima, Peru. Approximately half of all participants in the quantitative survey (50.3%) reported using commercial lubricant during intercourse occasionally or consistently during the preceding two months, with lack of availability at the time of intercourse the most commonly reported reason for non-use. No clear preferences regarding the color, smell, taste, or viscosity of commercial lubricants were identified, and all participants who reported using a commercial lubricant used the same product (“Love-Lub”). In the qualitative analysis, participants characterized lubricant use as a sexual practice consistently controlled by the receptive partner, who typically obtained and applied lubricant independently, with or without the consent of the insertive partner. Quantitative findings supported this differential pattern of lubricant use, with men who reported sexual identities or roles consistent with receptive anal intercourse, including unprotected receptive intercourse, more likely to report lubricant use than MSM who claimed an exclusively insertive sexual role. Given the social, behavioral, and biological factors contributing to increased vulnerability for HIV and STI acquisition by the receptive partner in anal intercourse, delivery of a topical rectal microbicide as a lubricant formulation could provide an important HIV prevention resource for at-risk MSM in Lima, Peru.
doi:10.1080/09540121.2012.726335
PMCID: PMC3556347  PMID: 23082796
13.  Increasing safer sexual behavior among Lao kathoy through an integrated social marketing approach 
BMC Public Health  2011;11:872.
Background
Although HIV prevalence has remained low in Laos thus far, there is reason to be concerned that Lao male-to-female (MtF) transgender persons (kathoy) and their partners may facilitate the spread of HIV. Little is known about how to most effectively reach kathoy with HIV prevention programming. This paper evaluates an intervention with Lao kathoy with the objective of increasing safe sex with regular and casual partners.
Methods
Quantitative surveys were administered in November 2004 (n = 288) and June 2006 (n = 415) using time location sampling at venues where kathoy were known to congregate. Respondents were aged 15-35 and from three urban centers in Laos. UNIANOVA tests were used to compare baseline and follow-up survey data and to evaluate the impact of PSI's kathoy-specific interventions on items that changed significantly over time.
Results
Exposure to the intervention was associated with higher levels of condom use at last anal sex with casual partners and greater use of water-based lubricant. Exposure was also linked to improved perceptions of product availability for condoms and water-based lubricant. Knowledge about the importance of consistent condom use improved over time as well as the need to use condoms with regular partners. Some HIV knowledge decreased over time and the intention to use condoms with casual partners when water-based lubricant is available also declined.
Conclusions
Study results demonstrate the feasibility of reaching kathoy with an integrated social marketing approach; combining product promotion, peer education, and other types of interpersonal communication. The approach was successful at increasing condom use with casual partners and water-based lubricant use, but the importance of using condoms along with water-based lubricant must be emphasized and modified strategies are required for improving condom use with boyfriends. Future messages should emphasize consistent condom use with all types of partners as well as improve knowledge and correct misconceptions about HIV and AIDS, STIs, condom use, and lubricant use. It is also important that authorities create an enabling environment to support such interventions and help foster behavior change.
doi:10.1186/1471-2458-11-872
PMCID: PMC3257214  PMID: 22087632
14.  Diaphragm and lubricant gel for prevention of HIV acquisition in southern African women: a randomised controlled trial 
Lancet  2007;370(9583):251-261.
Summary
Background
Female-controlled methods of HIV prevention are urgently needed. We assessed the effect of provision of latex diaphragm, lubricant gel, and condoms (intervention), compared with condoms alone (control) on HIV seroincidence in women in South Africa and Zimbabwe.
Methods
We did an open-label, randomised controlled trial in HIV-negative, sexually active women recruited from clinics and community-based organisations, who were followed up quarterly for 12–24 months (median 21 months). All participants received an HIV prevention package consisting of pre-test and post-test counselling about HIV and sexually transmitted infections, testing, treatment of curable sexually transmitted infections, and intensive risk-reduction counselling. The primary outcome was incident HIV infection. This study is registered with ClinicalTrials.gov, number NCT00121459.
Findings
Overall HIV incidence was 4.0% per 100 woman-years: 4.1% in the intervention group (n=2472) and 3.9% in the control group (n=2476), corresponding to a relative hazard of 1.05 (95% CI 0.84–1.32, intention-to-treat analysis). The proportion of women using condoms was significantly lower in the intervention than in the control group (54% vs 85% of visits, p<0.0001). The proportions of participants who reported adverse events (60% [1523] vs 61% [1529]) and serious adverse events (5% [130] vs 4% [101]) were similar between the two groups.
Interpretation
We observed no added protective benefit against HIV infection when the diaphragm and lubricant gel were provided in addition to condoms and a comprehensive HIV prevention package. Our observation that lower condom use in women provided with diaphragms did not result in increased infection merits further research. Although the intervention seemed safe, our findings do not support addition of the diaphragm to current HIV prevention strategies.
doi:10.1016/S0140-6736(07)60950-7
PMCID: PMC2442038  PMID: 17631387
15.  Progenitor cell therapy for traumatic brain injury: effect of serum osmolarity on cell viability and cytokine production 
Regenerative medicine  2010;5(1):65.
Introduction
The potential translation of mesenchymal stem cell (MSC) therapy into a multimodal protocol for traumatic brain injury requires evaluation of viability and cytokine production in a hyperosmolar environment. Optimization of MSC therapy requires delivery to the target area without significant loss of cellular function or viability. No model evaluating the potential efficacy of MSC therapy at varying osmolarities currently exists.
Methods
Rat MSCs were characterized with flow cytometric immunophenotyping. MSCs (passage 3) were placed in culture with multipotent adult progenitor cell media at varying osmolarities (250, 270, 290, 310, 330, 350 and 370 mOsm) potentially found with hypertonic saline infusion. After culture for 24 h, cellular viability was measured using flow cytometry (n = 6). Next, brain tissue supernatant was harvested from both normal rat brains and injured brains 6 h after cortical injury. Subsequently, MSCs were placed in culture with multipotent adult progenitor cell media ± 20% normal brain or injured brain supernatant (at the aforementioned osmolarities) and allowed to remain in culture for 24 h (n = 11). At this point, media supernatant cytokine levels were measured using a multiplex cytokine assay system.
Results
MSCs showed no clinically significant difference in viability at 24 h. MSCs cultured with 20% injured brain supernatant showed an decrease in proinflammatory cytokine production (IL-1α and IL-1β) with increasing osmolarity. No difference in anti-inflammatory cytokine production (IL-4 and IL-10) was observed.
Conclusion
Progenitor cell therapy for traumatic brain injury may require survival and activity in a hyperosmolar environment. Culture of MSCs in such conditions shows no clinically significant effect on cell viability. In addition, MSC efficacy could potentially be enhanced via a decrease in proinflammatory cytokine production. Overall, a multimodal traumatic brain injury treatment protocol based upon MSC infusion and hypertonic saline therapy would not negatively affect progenitor cell efficacy and could be considered for multicenter clinical trials.
doi:10.2217/rme.09.73
PMCID: PMC2823122  PMID: 20017695
cytokines; inflammation; mesenchymal stem cells; osmolarity
16.  Safety and Efficacy of tenofovir / IQP-0528 combination gels – a dual compartment microbicide for HIV-1 prevention 
Antiviral research  2012;96(2):221-225.
Tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor and IQP-0528 is a nonnucleoside reverse transcriptase inhibitor that also blocks virus entry. TFV and IQP-0528 alone have shown antiviral activity as microbicide gels. Because combination therapy will likely be more potent than mono-therapy, these drugs have been chosen to make a combination microbicide gel containing 2.5% TFV/1% IQP-0528. Safety and efficacy testing was done to evaluate five prototype combination gels. The gels retained TZM-bl cell and ectocervical and colorectal tissue viability. Further, the epithelium of the ectocervical and colorectal tissue remained intact after a 24 hour exposure. The ED50 calculated from the formulations for IQP-0528 was ~32 nM and for TFV was ~59 nM and their inhibitory activity was not affected by semen. The ED50 of TFV in the combination gels was ~100-fold lower than when calculated for the drug substance alone reflecting the activity of the more potent IQP-0528. When ectocervical and colorectal tissue were treated with the combination gels, HIV-1 p24 release was reduced by ≥1 log10 and ≥2 log10, respectively. Immunohistochemistry for the ectocervical tissues treated with combination gels showed no HIV-1 infected cells at study end. With the increased realization of receptive anal intercourse among heterosexual couples often in conjunction with vaginal intercourse, having a safe and effective microbicide for both mucosal sites is critical. The safety and efficacy profiles of the gels were similar for ectocervical and colorectal tissues suggesting these gels have the potential for dual compartment use.
doi:10.1016/j.antiviral.2012.08.004
PMCID: PMC3501576  PMID: 22940075
HIV prevention; combination microbicide; rectal microbicide; pyrimidinedione; topical gel
17.  Preventing Friction Induced Chondrocyte Apoptosis: A Comparison of Human Synovial Fluid and Hylan G-F 20 
The Journal of rheumatology  2012;39(7):1473-1480.
Objectives
Symptomatic osteoarthritis (OA) is a common painful disease with limited treatment options. A rising number of OA patients have been treated with intraarticular injections of hyaluronic acid, including the high molecular weight hylan G-F 20, which is injected following arthrocentesis. This study investigated the effectiveness of hylan G-F 20 to lower coefficient of friction (COF) and prevent chondrocyte apoptosis in vitro.
Methods
A disc-on-disc bovine cartilage bearing was used to measure the static and kinetic COF when lubricated with hylan G-F 20, human synovial fluid (HSF) and phosphate buffered saline (PBS). Following friction testing, we stained paraffin embedded sections of these cartilage bearings for activated caspase-3, a marker of apoptosis.
Results
Bearings lubricated with hylan G-F 20 had kinetic COF values that were similar to bearings lubricated with PBS, but significantly higher than those lubricated with HSF. There were no significant differences in static COF values in bearings lubricated with hylan G-F 20 as compared to PBS or HSF. However, bearings lubricated with HSF had a significantly lower static COF values compared to bearings lubricated with PBS. The mean percentage of caspase-3 positive chondrocytes in the superficial and upper intermediate zones of bearings lubricated with hylan G-F 20 were significantly higher when compared to bearings lubricated with HSF or unloaded controls, but significantly lower than those lubricated with PBS.
Conclusion
These findings indicate that joint lubrication may prevent chondrocyte apoptosis by lowering the COF. Furthermore, removal of synovial fluid prior to hylan G-F 20 injection may be detrimental to cartilage health.
doi:10.3899/jrheum.111427
PMCID: PMC3605976  PMID: 22660808
articular cartilage; chondrocyte; apoptosis; synovial fluid; hylan
18.  Acceptability and Use of Sexual Barrier Products and Lubricants among HIV-Seropositive Zambian Men 
AIDS Patient Care and STDs  2008;22(12):1015-1020.
Abstract
This study assessed the acceptability and preference for sexual barrier and lubricant products among men in Zambia following trial and long-term use. It also examined the role of men's preferences as facilitators or impediments to product use for HIV transmission reduction within the Zambian context. HIV-seropositive and - serodiscordant couples were recruited from HIV voluntary counseling and testing centers in Lusaka between 2003 and 2006; 66% of those approached agreed to participate. HIV seropositive male participants participated in a product exposure group intervention (n = 155). Participants were provided with male and female condoms and vaginal lubricants (Astroglide® [BioFilm, Inc., Vista, CA] & KY® gels [Johnson & Johnson, Langhorne, PA], Lubrin® suppositories [Kendwood Therapuetics, Fairfield, NJ]) over three sessions; assessments were conducted at baseline, monthly over 6 months and at 12 months. At baseline, the majority of men reported no previous exposure to lubricant products or female condoms and high (79%) levels of consistent male condom use in the last 7 days. Female condom use increased during the intervention, and male condom use increased at 6 months and was maintained over 12 months. The basis for decisions regarding lubricant use following product exposure was most influenced by a preference for communicating with partners; participant preference for lubricant products was distributed between all three products. Results illustrate the importance of development of a variety of products for prevention of HIV transmission and of inclusion of male partners in interventions to increase sexual barrier product use to facilitate barrier acceptability and use in Zambia.
doi:10.1089/apc.2007.0212
PMCID: PMC2929379  PMID: 19072108
19.  The Diaphragm and Lubricant Gel for Prevention of Cervical Sexually Transmitted Infections: Results of a Randomized Controlled Trial 
PLoS ONE  2008;3(10):e3488.
Background
We evaluated the effectiveness of the Ortho All-Flex Diaphragm, lubricant gel (Replens®) and condoms compared to condoms alone on the incidence of chlamydial and gonococcal infections in an open-label randomized controlled trial among women at risk of HIV/STI infections.
Methods
We randomized 5045 sexually-active women at three sites in Southern Africa. Participants who tested positive for curable STIs were treated prior to enrollment as per local guidelines. Women were followed quarterly and tested for Chlamydia trachomatis (CT) or Neisseria gonorrhoeae (GC) infection by nucleic-acid amplification testing (Roche Amplicor®) using first-catch urine specimens. STIs detected at follow-up visits were treated. We compared the incidence of first infection after randomization between study arms in both intent-to-treat (ITT) and per-protocol populations.
Findings
Baseline demographic, behavioral and clinical characteristics were balanced across study arms. Nearly 80% of participants were under 35 years of age. Median follow-up time was 21 months and the retention rate was over 93%. There were 471 first chlamydia infections, 247 in the intervention arm and 224 in the control arm with an overall incidence of 6.2/100 woman-years (wy) (relative hazard (RH) 1.11, 95% Confidence Interval (CI): 0.93–1.33; p = 0.25) and 192 first gonococcal infections, 95 in the intervention arm and 97 in the control arm with an overall incidence of 2.4/100wy (RH 0.98, 95%CI: 0.74–1.30; p = 0.90). Per protocol results indicated that when diaphragm adherence was defined as “always use” since the last visit, there was a significant reduction in the incidence of GC infection among women randomized to the intervention arm (RH 0.61, 95%CI: 0.41–0.91, P = 0.02).
Interpretation
There was no difference by study arm in the rate of acquisition of CT or GC. However, our per-protocol results suggest that consistent use of the diaphragm may reduce acquisition of GC.
Trial Registration
ClinicalTrials.gov NCT00121459 [NCT00121459]
doi:10.1371/journal.pone.0003488
PMCID: PMC2567030  PMID: 18941533
20.  Same-sex sexual behavior of men in Kenya: Implications for HIV prevention, programs, and policy 
Facts, Views & Vision in ObGyn  2012;4(4):285-294.
Unprotected anal sex has long been recognized as a risk factor for HIV transmission among men who have sex with men (MSM). In Africa, however, general denial of MSM existence and associated stigma discouraged research. To address this gap in the literature, partners conducted the first behavioral surveys of MSM in Kenya. The first study was to assess HIV risk among MSM in Nairobi, and the second study a pre-post intervention study of male sex workers in Mombasa. The 2004 behavioral survey of 500 men in Mombasa revealed that MSM were having multiple sexual partners and failed to access appropriate prevention counseling and care at Kenya clinics. A 2006 capture-recapture enumeration in Mombasa estimated that over 700 male sex workers were active, after which a pre-intervention baseline survey of 425 male sex workers was conducted. Awareness of unprotected anal sex as an HIV risk behavior and consistent condom use with clients was low, and use of oil-based lubricants high. Based on this information, peer educators were trained in HIV prevention, basic counseling skills, and distribution of condoms and lubricants. To assess impact of the interventions, a follow-up survey of 442 male sex workers was implemented in 2008. Exposure to peer educators was significantly associated with increased consistent condom use, improved HIV knowledge, and increased use of water-based lubricants. These results have provided needed information to the Government of Kenya and have informed HIV prevention interventions.
PMCID: PMC3987482  PMID: 24753921
HIV prevention; men who have sex with men; male sex workers; Africa; condoms; surveys
21.  Mapracorat, a novel selective glucocorticoid receptor agonist, inhibits hyperosmolar-induced cytokine release and MAPK pathways in human corneal epithelial cells 
Molecular Vision  2010;16:1791-1800.
Purpose
Increasing evidence suggests that tear hyperosmolarity is a central mechanism causing ocular surface inflammation and damage in dry eye disease. Mapracorat (BOL-303242-X) is a novel glucocorticoid receptor agonist currently under clinical evaluation for use in the treatment of dry eye disease. This study assessed the anti-inflammatory effects of mapracorat in an in vitro osmotic stress model which mimics some of the pathophysiological changes seen in dry eye.
Methods
Human corneal epithelial cells were cultured in normal osmolar media (317 mOsM) or 440 mOsM hyperosmolar media for 24 h. Luminex technology was used to determine the effect of mapracorat on hyperosmolar-induced cytokine release. Effects of mapracorat on mitogen-activated protein kinase (MAPK) phosphorylation were determined by cell based ELISA. Effects of mapracorat on nuclear factor kappa B (NFκB) and activator protein-1 (AP-1) transcriptional activity were assessed by reporter gene assay. Dexamethasone was used as a control.
Results
Hyperosmolar conditions induced release of the pro-inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) from cultured human corneal epithelial cells, and altered the phosphorylation state of p38 and c-Jun N-terminal kinase (JNK) and transcriptional activity of NFκB and AP-1. Incubation of cells with mapracorat inhibited hyperosmolar-induced cytokine release with comparable activity and potency as dexamethasone. This inhibition was reversed by the glucocorticoid receptor antagonist mifepristone (RU-486). Increased phosphorylation of p38 and JNK caused by hyperosmolarity was inhibited by mapracorat. Mapracorat also significantly decreased the hyperosmolar-induced increase in NFκB and AP-1 transcriptional activity.
Conclusions
Mapracorat acts as a potent anti-inflammatory agent in corneal epithelial cells challenged with osmotic stress, with comparable activity to the traditional steroid dexamethasone. These in vitro data suggest that mapracorat may be efficacious in the treatment of dry eye disease.
PMCID: PMC2932489  PMID: 20824100
22.  Characterization of Commercially Available Vaginal Lubricants: A Safety Perspective 
Pharmaceutics  2014;6(3):530-542.
Vaginal lubricants are widely used by women to help solve intercourse difficulties or as enhancers, but recent reports raise questions about their safety. Twelve commercially available gel products were tested for pH value, pH buffering capacity, osmolality and cytotoxicity relevant to vaginal delivery. Obtained data were analyzed in light of the recent Advisory Note by the World Health Organization (WHO) for personal lubricants to be concomitantly used with condoms. Results showed that most products do not comply with pH and osmolality recommended standards, thus posing a potential hazard. Four products presented values of osmolality around three-times higher than the maximum acceptable limit of 1200 mOsm/kg. In vitro cell testing further identified substantial cytotoxicity even at 1:100 dilutions for three products, contrasting with no significant effect of up to at least a 1:5 dilution of a Universal Placebo gel. However, no direct correlation between these last results and pH or osmolality was found, thus suggesting that the individual toxicity of specific formulation components plays an important role in the outcome of a particular product. Although further assessment is required, these results highlight potential safety issues related to the formulation of commercially available vaginal lubricants.
doi:10.3390/pharmaceutics6030530
PMCID: PMC4190534  PMID: 25247884
vaginal drug delivery; microbicides; buffer capacity; osmolality; cytotoxicity
23.  Attitudes of men in an Australian male tolerance study towards microbicide use 
Sexual health  2008;5(3):273-278.
Background
Vaginal microbicides are in development to provide new options for prevention of STIs. Although promoted as a female initiated product, men may influence the decision to use a microbicide and the way that it is used, so it is important to explore their views.
Methods
Men (n=36) enrolled in a 7-day, phase 1 clinical safety trial of SPL7013 Gel were interviewed pre and post-use of the gel. The trial did not include use of the gel during sex. Interviews were digitally-recorded and transcribed verbatim, and analysed using a framework approach.
Results
The men (mean age 37 years) were interested in the idea of vaginal microbicides, had little knowledge about them, and varied beliefs about how they work. They tended to assess microbicide use in relation to condoms and lubricants. Many would want a microbicide to be as effective as condoms. Participants did not anticipate difficulties discussing use with their partners. Many thought that a microbicide would be less intrusive than condoms; some anticipated that the lubricating properties might enhance sexual pleasure. Some anticipated using a microbicide with a condom or with a lubricant, and a few raised questions about the timing of use and use during different types of sexual activity.
Conclusions
No major barriers to microbicide use were found in this sample of Australian men, who anticipated being willing to use them if they are shown to be safe and effective. Our findings should help to inform the design of further studies as well as future information materials and anticipatory guidance.
PMCID: PMC3777800  PMID: 18771643
microbicides; acceptability; sexuality; males
24.  The slippery slope: Lubricant Use and Rectal Sexually Transmitted Infections: a newly identified risk 
Sexually transmitted diseases  2012;39(1):59-64.
Background
Use of lubricant products is extremely common during receptive anal intercourse (RAI) yet has not been assessed as a risk for acquisition of sexually transmitted infections (STIs).
Methods
From 2006–2008 a rectal health and behavior study was conducted in Baltimore and Los Angeles as part of the UCLA Microbicide Development Program (NIAID IPCP# #0606414). Participants completed questionnaires and rectal swabs were tested for Neisseria gonorrhoeae and Chlamydia trachomatis with the Aptima Combo 2 assay and blood was tested for syphilis (for RPR and TPHA with titer) and HIV. Of those reporting lubricant use and RAI, STI results were available for 380 participants. Univariate and multivariate regressions assessed associations of lubricant use in the past month during RAI with prevalent STIs.
Results
Consistent lubricant use during RAI in the past month was reported by 36% (137/380) of participants. Consistent past month lubricant users had a higher prevalence of STI than inconsistent users (9.5% vs. 2.9%; p=0.006). In a multivariable logistic regression model testing positive for STI was associated with consistent use of lubricant during RAI in the past month (adjusted Odds Ratio (AOR) 2.98 (95%CI 1.09, 8.15) after controlling for age, gender, study location, HIV status, and numbers of RAI partners in the past month.
Conclusions
Findings suggest some lubricant products may increase vulnerability to STIs. Because of wide use of lubricants and their potential as carrier vehicles for microbicides, further research is essential to clarify if lubricant use poses a public health risk.
doi:10.1097/OLQ.0b013e318235502b
PMCID: PMC3244680  PMID: 22183849
rectal sexually transmitted infections; lubricants; rectal health
25.  Limbal Stem Cell Transplantation 
Executive Summary
Objective
The objective of this analysis is to systematically review limbal stem cell transplantation (LSCT) for the treatment of patients with limbal stem cell deficiency (LSCD). This evidence-based analysis reviews LSCT as a primary treatment for nonpterygium LSCD conditions, and LSCT as an adjuvant therapy to excision for the treatment of pterygium.
Background
Clinical Need: Condition and Target Population
The outer surface of the eye is covered by 2 distinct cell layers: the corneal epithelial layer that overlies the cornea, and the conjunctival epithelial layer that overlies the sclera. These cell types are separated by a transitional zone known as the limbus. The corneal epithelial cells are renewed every 3 to 10 days by a population of stem cells located in the limbus.
Nonpterygium Limbal Stem Cell Deficiency
When the limbal stem cells are depleted or destroyed, LSCD develops. In LSCD, the conjunctival epithelium migrates onto the cornea (a process called conjunctivalization), resulting in a thickened, irregular, unstable corneal surface that is prone to defects, ulceration, corneal scarring, vascularization, and opacity. Patients experience symptoms including severe irritation, discomfort, photophobia, tearing, blepharospasm, chronic inflammation and redness, and severely decreased vision.
Depending on the degree of limbal stem cell loss, LSCD may be total (diffuse) or partial (local). In total LSCD, the limbal stem cell population is completed destroyed and conjunctival epithelium covers the entire cornea. In partial LSCD, some areas of the limbus are unharmed, and the corresponding areas on the cornea maintain phenotypically normal corneal epithelium.
Confirmation of the presence of conjunctivalization is necessary for LSCD diagnosis as the other characteristics and symptoms are nonspecific and indicate a variety of diseases. The definitive test for LSCD is impression cytology, which detects the presence of conjunctival epithelium and its goblet cells on the cornea. However, in the opinion of a corneal expert, diagnosis is often based on clinical assessment, and in the expert’s opinion, it is unclear whether impression cytology is more accurate and reliable than clinical assessment, especially for patients with severe LSCD.
The incidence of LSCD is not well understood. A variety of underlying disorders are associated with LSCD including chemical or thermal injuries, ultraviolet and ionizing radiation, Stevens-Johnson syndrome, multiple surgeries or cryotherapies, contact lens wear, extensive microbial infection, advanced ocular cicatricial pemphigoid, and aniridia. In addition, some LSCD cases are idiopathic. These conditions are uncommon (e.g., the prevalence of aniridia ranges from 1 in 40,000 to 1 in 100,000 people).
Pterygium
Pterygium is a wing-shaped fibrovascular tissue growth from the conjunctiva onto the cornea. Pterygium is the result of partial LSCD caused by localized ultraviolet damage to limbal stem cells. As the pterygium invades the cornea, it may cause irregular astigmatism, loss of visual acuity, chronic irritation, recurrent inflammation, double vision, and impaired ocular motility.
Pterygium occurs worldwide. Incidence and prevalence rates are highest in the “pterygium belt,” which ranges from 30 degrees north to 30 degrees south of the equator, and lower prevalence rates are found at latitudes greater than 40 degrees. The prevalence of pterygium for Caucasians residing in urban, temperate climates is estimated at 1.2%.
Existing Treatments Other Than Technology Being Reviewed
Nonpterygium Limbal Stem Cell Deficiency
In total LSCD, a patient’s limbal stem cells are completely depleted, so any successful treatment must include new stem cells. Autologous oral mucosal epithelium transplantation has been proposed as an alternative to LSCT. However, this procedure is investigational, and there is very limited level 4c evidence1 to support this technique (fewer than 20 eyes examined in 4 case series and 1 case report).
For patients with partial LSCD, treatment may not be necessary if their visual axis is not affected. However, if the visual axis is conjunctivalized, several disease management options exist including repeated mechanical debridement of the abnormal epithelium; intensive, nonpreserved lubrication; bandage contact lenses; autologous serum eye drops; other investigational medical treatments; and transplantation of an amniotic membrane inlay. However, these are all disease management treatments; LSCT is the only curative option.
Pterygium
The primary treatment for pterygium is surgical excision. However, recurrence is a common problem after excision using the bare sclera technique: reported recurrence rates range from 24% to 89%. Thus, a variety of adjuvant therapies have been used to reduce the risk of pterygium recurrence including LSCT, amniotic membrane transplantation (AMT), conjunctival autologous (CAU) transplantation, and mitomycin C (MMC, an antimetabolite drug).
New Technology Being Reviewed
To successfully treat LSCD, the limbal stem cell population must be repopulated. To achieve this, 4 LSCT procedures have been developed: conjunctival-limbal autologous (CLAU) transplantation; living-related conjunctival-limbal allogeneic (lr-CLAL) transplantation; keratolimbal allogeneic (KLAL) transplantation; and ex vivo expansion of limbal stem cells transplantation. Since the ex vivo expansion of limbal stem cells transplantation procedure is considered experimental, it has been excluded from the systematic review. These procedures vary by the source of donor cells and the amount of limbal tissue used. For CLAU transplants, limbal stem cells are obtained from the patient’s healthy eye. For lr-CLAL and KLAL transplants, stem cells are obtained from living-related and cadaveric donor eyes, respectively.
In CLAU and lr-CLAL transplants, 2 to 4 limbal grafts are removed from the superior and inferior limbus of the donor eye. In KLAL transplants, the entire limbus from the donor eye is used.
The recipient eye is prepared by removing the abnormal conjunctival and scar tissue. An incision is made into the conjunctival tissue into which the graft is placed, and the graft is then secured to the neighbouring limbal and scleral tissue with sutures. Some LSCT protocols include concurrent transplantation of an amniotic membrane onto the cornea.
Regulatory Status
Health Canada does not require premarket licensure for stem cells. However, they are subject to Health Canada’s clinical trial regulations until the procedure is considered accepted transplantation practice, at which time it will be covered by the Safety of Human Cells, Tissues and Organs for Transplantation Regulations (CTO Regulations).
Review Strategy
The Medical Advisory Secretariat systematically reviewed the literature to assess the effectiveness and safety of LSCT for the treatment of patients with nonpterygium LSCD and pterygium. A comprehensive search method was used to retrieve English-language journal articles from selected databases.
The GRADE approach was used to systematically and explicitly evaluate the quality of evidence and strength of recommendations.
Summary of Findings
Nonpterygium Limbal Stem Cell Deficiency
The search identified 873 citations published between January 1, 2000, and March 31, 2008. Nine studies met the inclusion criteria, and 1 additional citation was identified through a bibliography review. The review included 10 case series (3 prospective and 7 retrospective).
Patients who received autologous transplants (i.e., CLAU) achieved significantly better long-term corneal surface results compared with patients who received allogeneic transplants (lr-CLAL, P< .001; KLAL, P< .001). There was no significant difference in corneal surface outcomes between the allogeneic transplant options, lr-CLAL and KLAL (P = .328). However, human leukocyte antigen matching and systemic immunosuppression may improve the outcome of lr-CLAL compared with KLAL. Regardless of graft type, patients with Stevens-Johnson syndrome had poorer long-term corneal surface outcomes.
Concurrent AMT was associated with poorer long-term corneal surface improvements. When the effect of the AMT was removed, the difference between autologous and allogeneic transplants was much smaller.
Patients who received CLAU transplants had a significantly higher rate of visual acuity improvements compared with those who received lr-CLAL transplants (P = .002). However, to achieve adequate improvements in vision, patients with deep corneal scarring will require a corneal transplant several months after the LSCT.
No donor eye complications were observed.
Epithelial rejection and microbial keratitis were the most common long-term complications associated with LSCT (complications occurred in 6%–15% of transplantations). These complications can result in graft failure, so patients should be monitored regularly following LSCT.
Pterygium
The search yielded 152 citations published between January 1, 2000 and May 16, 2008. Six randomized controlled trials (RCTs) that evaluated LSCT as an adjuvant therapy for the treatment of pterygium met the inclusion criteria and were included in the review.
Limbal stem cell transplantation was compared with CAU, AMT, and MMC. The results showed that CLAU significantly reduced the risk of pterygium recurrence compared with CAU (relative risk [RR], 0.09; 95% confidence interval [CI], 0.01–0.69; P = .02). CLAU reduced the risk of pterygium recurrence for primary pterygium compared with MMC, but this comparison did not reach statistical significance (RR, 0.48; 95% CI, 0.21–1.10; P = .08). Both AMT and CLAU had similar low rates of recurrence (2 recurrences in 43 patients and 4 in 46, respectively), and the RR was not significant (RR, 1.88; 95% CI, 0.37–9.5; P = .45). Since sample sizes in the included studies were small, failure to detect a significant difference between LSCT and AMT or MMC could be the result of type II error. Limbal stem cell transplantation as an adjuvant to excision is a relatively safe procedure as long-term complications were rare (< 2%).
GRADE Quality of Evidence
Nonpterygium Limbal Stem Cell Deficiency
The evidence for the analyses related to nonpterygium LSCD was based on 3 prospective and 7 retrospective case series. Thus, the GRADE quality of evidence is very low, and any estimate of effect is very uncertain.
Pterygium
The analyses examining LSCT as an adjuvant treatment option for pterygium were based on 6 RCTs. The quality of evidence for the overall body of evidence for each treatment option comparison was assessed using the GRADE approach. In each of the comparisons, the quality of evidence was downgraded due to serious or very serious limitations in study quality (individual study quality was assessed using the Jadad scale, and an assessment of allocation concealment and the degree of loss to follow-up), which resulted in low- to moderate-quality GRADE evidence ratings (low-quality evidence for the CLAU and AMT and CLAU and MMC comparisons, and moderate-quality evidence for the CLAU and CAU comparison).
Ontario Health System Impact Analysis
Nonpterygium Limbal Stem Cell Deficiency
Since 1999, Ontario’s out-of-country (OOC) program has approved and reimbursed 8 patients for LSCTs and 1 patient for LSCT consultations. Similarly, most Canadian provinces have covered OOC or out-of-province LSCTs. Several corneal experts in Ontario have the expertise to perform LSCTs.
As there are no standard guidelines for LSCT, patients who receive transplants OOC may not receive care aligned with the best evidence. To date, many of the patients from Ontario who received OOC LSCTs received concurrent AMTs, and the evidence from this analysis questions the use of this procedure. In addition, 1 patient received a cultured LSCT, a procedure that is considered investigational. Many patients with LSCD have bilateral disease and therefore require allogeneic transplants. These patients will require systemic and topical immunosuppression for several years after the transplant, perhaps indefinitely. Thus, systemic side effects associated with immunosuppression are a potential concern, and patients must be monitored regularly.
Amniotic membrane transplantation is a common addition to many ocular surface reconstruction procedures, including LSCT. Amniotic membranes are recovered from human placentas from planned, uneventful caesarean sections. Before use, serological screening of the donor’s blood should be conducted. However, there is still a theoretical risk of disease transmission associated with this procedure.
Financial Impact
For the patients who were reimbursed for OOC LSCTs, the average cost of LSCT per eye was $18,735.20 Cdn (range, $8,219.54–$33,933.32). However, the actual cost per patient is much higher as these costs do not include consultations and follow-up visits, multiple LSCTs, and any additional procedures (e.g., corneal transplants) received during the course of treatment OOC. When these additional costs were considered, the average cost per patient was $57,583 Cdn (range, $8,219.54–$130,628.20).
The estimated average total cost per patient for performing LSCT in Ontario is $2,291.48 Cdn (range, $951.48–$4,538.48) including hospital and physician fees. This cost is based on the assumption that LSCT is technically similar to a corneal transplant, an assumption which needs to be verified. The cost does not include corneal transplantations, which some proportion of patients receiving a LSCT will require within several months of the limbal transplant.
Pterygium
Pterygium recurrence rates after surgical excision are high, ranging from 24% to 89%. However, according to clinical experts, the rate of recurrence is low in Ontario. While there is evidence that the prevalence of pterygium is higher in the “pterygium belt,” there was no evidence to suggest different recurrence rates or disease severity by location or climate.
Conclusions
Nonpterygium Limbal Stem Cell Deficiency
Successful LSCTs result in corneal re-epithelialization and improved vision in patients with LSCD. However, patients who received concurrent AMT had poorer long-term corneal surface improvements. Conjunctival-limbal autologous transplantation is the treatment option of choice, but if it is not possible, living-related or cadaveric allogeneic transplants can be used. The benefits of LSCT outweigh the risks and burdens, as shown in Executive Summary Table 1. According to GRADE, these recommendations are strong with low- to very low-quality evidence.
Benefits, Risks, and Burdens – Nonpterygium Limbal Stem Cell Deficiency
Short- and long-term improvement in corneal surface (stable, normal corneal epithelium and decreased vascularization and opacity)
Improvement in vision (visual acuity and functional vision)
Long-term complications are experienced by 8% to 16% of patients
Risks associated with long-term immunosuppression for recipients of allogeneic grafts
Potential risk of induced LSCD in donor eyes
High cost of treatment (average cost per patient via OOC program is $57,583; estimated cost of procedure in Ontario is $2,291.48)
Costs are expressed in Canadian dollars.
GRADE of recommendation: Strong recommendation, low-quality or very low-quality evidence
benefits clearly outweigh risks and burdens
case series studies
strong, but may change if higher-quality evidence becomes available
Pterygium
Conjunctival-limbal autologous transplantations significantly reduced the risk of pterygium recurrence compared with CAU. No other comparison yielded statistically significant results, but CLAU reduced the risk of recurrence compared with MMC. However, the benefit of LSCT in Ontario is uncertain as the severity and recurrence of pterygium in Ontario is unknown. The complication rates suggest that CLAU is a safe treatment option to prevent the recurrence of pterygium. According to GRADE, given the balance of the benefits, risks, and burdens, the recommendations are very weak with moderate quality evidence, as shown in Executive Summary Table 2.
Benefits, Risks, and Burdens – Pterygium
Reduced recurrence; however, if recurrence is low in Ontario, this benefit might be minimal
Long-term complications rare
Increased cost
GRADE of recommendation: Very weak recommendations, moderate quality evidence.
uncertainty in the estimates of benefits, risks, and burden; benefits, risks, and burden may be closely balanced
RCTs
very weak, other alternatives may be equally reasonable
PMCID: PMC3377549  PMID: 23074512

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