Memory T cells possess an innate-like function in local protection from mucosal infection.

Mucosal infections pose a significant global health burden. Antigen-specific tissue-resident T cells are critical to maintaining barrier immunity. Previous studies in the context of systemic infection suggest that memory CD8+ T cells may also provide innate-like protection against antigenically unrelated pathogens independent of T cell receptor engagement. Whether bystander T cell activation is also an important defense mechanism in the mucosa is poorly understood. Here, we investigated whether innate-like memory CD8+ T cells could protect against a model mucosal virus infection, herpes simplex virus 2 (HSV-2). We found that immunization with an irrelevant antigen delayed disease progression from lethal HSV-2 challenge, suggesting that memory CD8+ T cells may mediate protection despite the lack of antigen specificity. Upon HSV-2 infection, we observed an early infiltration, rather than substantial local proliferation, of antigen-nonspecific CD8+ T cells, which became bystander-activated only within the infected mucosal tissue. Critically, we show that bystander-activated CD8+ T cells are sufficient to reduce early viral burden after HSV-2 infection. Finally, local cytokine cues within the tissue microenvironment after infection were sufficient for bystander activation of mucosal tissue memory CD8+ T cells from mice and humans. Altogether, our findings suggest that local bystander activation of CD8+ memory T cells contributes a fast and effective innate-like response to infection in mucosal tissue.

Physician attitudes and knowledge on prophylactic salpingectomy in perimenopausal patients.

PURPOSE: Eighty percent of the approximately 500,000 hysterectomies performed annually in the US are for benign indications. There is lack of consensus regarding concurrent removal of fallopian tubes and/or ovaries. Ovarian cancer risk reduction is the principal benefit but the adverse consequences of ovarian removal can include vasomotor disturbance, vaginal dryness, cardiovascular disease, osteoporosis, and cognitive decline. Emerging evidence on the role of fallopian tubes in ovarian carcinogenesis and the consequences of oophorectomy have led the American College of Obstetricians-Gynecologists (ACOG) to recommend bilateral salpingectomy with ovarian conservation during benign hysterectomy for women at population risk for ovarian cancer. METHODS: Five hundred members of the ACOG Collaborative Ambulatory Research Network (CARN) were randomly selected to participate in this survey study. RESULTS: 165 completed the survey (35.3% response rate). Most respondents reported that a family history of breast, ovarian or colon cancer and patient age influence their decision to offer salpingectomy more than 75% of the time. Factors that a majority of respondents reported discussing during counseling included possible ovarian cancer risk reduction, surgical menopause, severity of symptoms, and the effects on bone and cardiovascular health. The respondents mean score for the knowledge-based questions was only 1.7 (±0.92) out of 4 points. CONCLUSION: Several factors may affect decision making for prophylactic salpingectomy at the time of hysterectomy however paramount among these is cancer risk reduction. Most physicians found it difficult to discuss and implement a change in care for patients with preconceived notions of ovarian preservation or removal.

The human memory T cell compartment changes across tissues of the female reproductive tract.

Memory CD4 T cells in tissues fulfill numerous functions that are critical for local immune homeostasis and protection against pathogens. Previous studies have highlighted the phenotypic and functional heterogeneity of circulating and tissue-resident memory CD4 T cells across different human tissues such as skin, lung, liver, and colon. Comparatively little is known in regard to memory CD4 T cells across tissues of the female reproductive tract (FRT). We examined CD4 T cells in donor-matched vaginal, ecto- and endocervical tissues, which differ in mucosal structure and exposure to external environmental stimuli. We hypothesized that this could be reflected by tissue-specific differences in the memory CD4 T cell compartment. We found differences in CD4 subset distribution across these tissues. Specifically, CD69+CD103+ CD4 T cells were significantly more abundant in vaginal than cervical tissues. In contrast, the transcriptional profiles of CD4 subsets were fairly conserved across FRT tissues. CD69+CD103+ CD4 T cells showed a TH17 bias independent of tissue niche. Our data suggest that FRT tissues affect T cell subset distribution but have limited effects on the transcriptome of each subset. We discuss the implications for barrier immunity in the FRT.

HSV-2 Infection Enhances Zika Virus Infection of Primary Genital Epithelial Cells Independently of the Known Zika Virus Receptor AXL.

Zika virus (ZIKV) is transmitted to people by bite of an infected mosquito and by sexual contact. ZIKV infects primary genital epithelial cells, the same cells targeted by herpes simplex virus 2 (HSV-2). HSV-2 seroprevalence is high in areas where ZIKV is endemic, but it is unknown whether HSV-2 increases the risk for ZIKV infection. Here, we found that pre-infecting female genital tract epithelial cells with HSV-2 leads to enhanced binding of ZIKV virions. This effect did not require active replication by HSV-2, implying that the effect results from the immune response to HSV-2 exposure or to viral genes expressed early in the HSV-2 lifecycle. Treating cells with toll-like receptor-3 ligand poly-I:C also lead to enhanced binding by ZIKV, which was inhibited by the JAK-STAT pathway inhibitor ruxolitinib. Blocking or knocking down the well-studied ZIKV receptor AXL did not prevent binding of ZIKV to epithelial cells, nor prevent enhanced binding in the presence of HSV-2 infection. Blocking the α5 integrin receptor did not prevent ZIKV binding to cells either. Overall, our results indicate that ZIKV binding to genital epithelial cells is not mediated entirely by a canonical receptor, but likely occurs through redundant pathways that may involve lectin receptors and glycosaminoglycans. Our studies may pave the way to new interventions that interrupt the synergism between herpes and Zika viruses.

Washington State Surgeon Perspectives on Quality Measures in the Surgical Care of Stress Urinary Incontinence in Women.

INTRODUCTION: No consensus exists on outcomes that define high quality care in female stress urinary incontinence management. A working group of surgeons from diverse health care settings in Washington State who treat stress urinary incontinence was convened through a state level quality collaborative. Preliminary questions were developed and focus groups conducted to obtain surgeon input and perspectives on stress urinary incontinence surgery quality measures to guide future research. METHODS: Washington State surgeons who perform sling surgery were recruited via email. Focus groups were convened via teleconference using preliminary questions as discussion points. Participants were surgeons from a variety of locations and practice types. Focus groups were led by a trained moderator, recorded and transcribed verbatim, and qualitatively analyzed using inductive content analysis. RESULTS: Eight urologists and 6 gynecologists from 5 academic, 4 private practice and 5 hospital based settings participated in 3 focus groups. The 4 emergent concepts derived, were a perceived need to individualize rather than standardize the management approach to recurrent and persistent stress urinary incontinence, a need to establish and validate shared decision making tools for synthetic mesh procedures, a need to define risk factors and treatment strategies for unique populations, including those with mixed urinary incontinence, younger patients and those with concomitant prolapse, and an enthusiasm to identify practical and clinically meaningful quality measures. CONCLUSIONS: Focus groups with clinical experts on quality of stress urinary incontinence care identified several priority topics for future study. Further work is needed to refine research prioritization on this important area of women's health.

Perineal Laceration and Episiotomy Repair Using a Beef Tongue Model.

Introduction: Declining rates of operative vaginal deliveries and routine episiotomy in obstetric practice, along with rising cesarean section rates, have decreased OB/GYN resident experience with episiotomy repair and obstetric anal sphincter injuries (OASIS). Simulation models are valuable educational tools in procedural training. Several models have been reported, each with its own limitations and benefits. Methods: We developed a 1-hour workshop to teach novice OB/GYN residents perineal laceration repair skills on a modified beef tongue model. The model required 5-10 minutes to assemble following written and video instruction, and learners had 30-50 minutes to practice using learner instructions. Learners were evaluated using a procedure checklist and global objective structured assessment of technical skills. To evaluate the session, we surveyed current faculty and residents, as well as residency graduates. Results: Between 2008 and 2017, an estimated 82 OB/GYN residents participated in this activity, and 95 participants and facilitators received the survey. Forty-one (59%) respondents agreed that this model was similar to repairing OASIS in clinical practice. Our trainees reported that the optimal time for simulated OASIS repair was the R2 and R3 years; however, 90% of respondents felt residents should be offered this simulation yearly. Discussion: Based on our survey of trainees, graduates, and faculty, we created a realistic simulated OASIS repair training, despite the limitation that the model lacked a rectum. Learners reported an interest in repeating the simulation frequently during residency to augment their clinical experience and increase perceived competence in third- and fourth-degree laceration repair by their graduation.

Mechanisms of Endogenous HIV-1 Reactivation by Endocervical Epithelial Cells.

Pharmacological HIV-1 reactivation to reverse latent infection has been extensively studied. However, HIV-1 reactivation also occurs naturally, as evidenced by occasional low-level viremia ("viral blips") during antiretroviral treatment (ART). Clarifying where blips originate from and how they happen could provide clues to stimulate latency reversal more effectively and safely or to prevent viral rebound following ART cessation. We studied HIV-1 reactivation in the female genital tract, a dynamic anatomical target for HIV-1 infection throughout all disease stages. We found that primary endocervical epithelial cells from several women reactivated HIV-1 from latently infected T cells. The endocervical cells' HIV-1 reactivation capacity further increased upon Toll-like receptor 3 stimulation with poly(I·C) double-stranded RNA or infection with herpes simplex virus 2 (HSV-2). Notably, acyclovir did not eliminate HSV-2-induced HIV-1 reactivation. While endocervical epithelial cells secreted large amounts of several cytokines and chemokines, especially tumor necrosis factor alpha (TNF-α), CCL3, CCL4, and CCL20, their HIV-1 reactivation capacity was almost completely blocked by TNF-α neutralization alone. Thus, immunosurveillance activities by columnar epithelial cells in the endocervix can cause endogenous HIV-1 reactivation, which may contribute to viral blips during ART or rebound following ART interruption.IMPORTANCE A reason that there is no universal cure for HIV-1 is that the virus can hide in the genome of infected cells in the form of latent proviral DNA. This hidden provirus is protected from antiviral drugs until it eventually reactivates to produce new virions. It is not well understood where in the body or how this reactivation occurs. We studied HIV-1 reactivation in the female genital tract, which is often the portal of HIV-1 entry and which remains a site of infection throughout the disease. We found that the columnar epithelial cells lining the endocervix, the lower part of the uterus, are particularly effective in reactivating HIV-1 from infected T cells. This activity was enhanced by certain microbial stimuli, including herpes simplex virus 2, and blocked by antibodies against the inflammatory cytokine TNF-α. Avoiding HIV-1 reactivation could be important for maintaining a functional HIV-1 cure when antiviral therapy is stopped.

A pro-inflammatory CD8+ T-cell subset patrols the cervicovaginal tract.

The immune system of the cervicovaginal tract (CVT) must balance immunosurveillance and active immunity against pathogens with maintenance of tolerance to resident microbiota and to fetal and partner antigens for reproductive purposes. Thus, we predicted that CVT immunity is characterized by distinctive features compared to blood and other tissue compartments. Indeed, we found that CVT CD8+ T-cells had unique transcriptional profiles, particularly in their cytokine signature, compared to that reported for CD8+ T-cells in other tissue sites. Among these CVT CD8+ T-cells, we identified a CD69- CD103- subset that was characterized by reduced migration in response to tissue-exit signals and higher pro-inflammatory potential as compared to their blood counterpart. These inflammatory mucosal CD8+ T-cells (Tim) were increased in frequency in the CVT of individuals with chronic infection, pointing to a potential role in perpetuating inflammation. Our findings highlight the specialized nature of immunity within the CVT and identify Tim cells as potential therapeutic targets to tame tissue inflammation upon chronic infection.

Stress Urinary Incontinence Surgery in Washington State Before and After Introduction of the Mesh Midurethral Sling.

OBJECTIVES: Mesh midurethral slings (MUSs) are safe, effective treatments for female stress urinary incontinence (SUI), but many companies have ceased production because of controversies surrounding transvaginal mesh. To determine if introduction of MUS has increased the complication rate associated with SUI surgery, we compared women undergoing SUI surgery in the MUS era to those who had surgery prior its introduction. METHODS: This was a retrospective cohort study of a statewide hospital discharge database. Stress urinary incontinence surgeries from 1987 to 1996 and 2007 to 2013 were identified using International Classification of Diseases, Ninth Revision codes. RESULTS: A total of 30,723 SUI surgeries were performed during the study periods. After 2006, slings accounted for 91.8% of SUI surgeries. Patients were older (54.5 vs 53.0 years, P < 0.001) and sicker (22.6% vs 9.7% had ≥1 comorbid condition, P < 0.0001). Blood transfusion was more common in the MUS era (1.2% vs 0.4%, P < 0.001) however, other complications were either similar between groups or less common in the MUS era including 30-day readmission (2.5% vs 2.4%, P = 0.543), reoperation for urinary retention (0.1% vs 0.2%, P < 0.0375), and wound infection (0.1% vs 0.5%, P < 0.001), despite more concomitant prolapse surgeries (69.0 vs 26.9%, P < 0.001) and hysterectomies (53.0 vs 35.4%, P < 0.001) in the MUS era. Hospital stays were shorter after 2006 (1.0 vs 3.0 days, P < 0.001), and fewer women required reoperation for SUI within 2 years (0.5% vs 1.8%, P < 0.001). CONCLUSIONS: Following introduction of MUS, women who underwent SUI surgery were slightly older with more medical comorbidities yet did not appear to experience increased surgical complications. Fewer women underwent reoperation for recurrent SUI, and hospital stays were shorter, suggesting an improvement in care. This study supports the continued availability and use of MUSs.

Provision of Reproductive Healthcare to Women with Disabilities: A Survey of Obstetrician-Gynecologists' Training, Practices, and Perceived Barriers.

Purpose: The purpose of this study was to document current awareness, attitudes, and training regarding the care of women with disabilities by obstetrician-gynecologists (ob-gyns) and explore barriers that may explain observed discrepancies in care. Methods: One thousand ob-gyns, including 500 members of the Collaborative Ambulatory Research Network (CARN), were surveyed on practice accessibility, training, awareness, barriers, beliefs, comfort, challenges, practices, contraceptive counseling, and preconception/pregnancy counseling. Results: CARN, 49.0%, and non-CARN, 19.4%, members completed the survey for an overall response rate of 33.9%. Most respondents indicated feeling "somewhat" (57.5%) or "very" (21.9%) aware of the special healthcare needs of women with disabilities. Only 17.2%, however, received any information or training on the provision of healthcare to women with disabilities. Eighty-one percent agreed somewhat or strongly that women with disabilities are less likely to receive comprehensive reproductive healthcare. Respondents who provided contraceptive counseling (94.3%) initiated it with women of reproductive age who did not have a disability more frequently than those who had a disability. Finally, only 19.3% felt "definitely" adequately equipped to manage the pregnancies of women with disabilities. Conclusion: Women with disabilities require reproductive healthcare no less than women without disabilities; however, the evidence consistently identifies disparities. This study suggests that while ob-gyn providers are aware of these issues, they lack adequate training and resources to provide equal care.

Cryopreservation of human mucosal tissues.

BACKGROUND: Cryopreservation of leukocytes isolated from the cervicovaginal and colorectal mucosa is useful for the study of cellular immunity (see Hughes SM et al. PLOS ONE 2016). However, some questions about mucosal biology and sexually transmitted infections are better addressed with intact mucosal tissue, for which there is no standard cryopreservation protocol. METHODS AND FINDINGS: To find an optimal preservation protocol for mucosal tissues, we tested slow cooling (1°C/min) with 10% dimethylsulfoxide (designated "cryopreservation") and fast cooling (plunge in liquid nitrogen) with 20% dimethylsulfoxide and 20% ethylene glycol ("vitrification"). We compared fresh and preserved human cervicovaginal and colorectal tissues in a range of assays, including metabolic activity, human immunodeficiency virus infection, cell phenotype, tissue structure by hematoxylin-and-eosin staining, cell number and viability, production of cytokines, and microbicide drug concentrations. Metabolic activity, HIV infectability, and tissue structure were similar in cryopreserved and vitrified vaginal tissues. However, vitrification led to poor cell recovery from the colorectal mucosa, with 90% fewer cells recovered after isolation from vitrified colorectal tissues than from cryopreserved. HIV infection rates were similar for fresh and cryopreserved ectocervical tissues, whereas cryopreserved colorectal tissues were less easily infected than fresh tissues (hazard ratio 0.7 [95% confidence interval 0.4, 1.2]). Finally, we compared isolation of cells before and after cryopreservation. Cell recoveries were higher when cells were isolated after freezing and thawing (71% [59-84%]) than before (50% [38-62%]). Cellular function was similar to fresh tissue in both cases. Microbicide drug concentrations were lower in cryopreserved explants compared to fresh ones. CONCLUSIONS: Cryopreservation of intact cervicovaginal and colorectal tissues with dimethylsulfoxide works well in a range of assays, while the utility of vitrification is more limited. Cell yields are higher from cryopreserved intact tissue pieces than from thawed cryopreserved single cell suspensions isolated before freezing, but T cell functions are similar.

Benefits and Barriers to Teaching Medical Students in an Ob-Gyn Clinic.

INTRODUCTION: As the US health-care system has evolved over the past decade, access to obstetric care in rural communities has declined, and there has been a challenge in retaining obstetrics and gynecology (OB-GYN) providers to train the next generation of physicians. The current pilot study sought to identify the factors that influence faculty who train medical students within the field of OB-GYN with the hope of influencing recruitment and retention of providers for the future. METHODS: Clinical OB-GYN faculty within the University of Washington School of Medicine regional medical education program were surveyed about practice patterns and beliefs regarding medical student training as part of a pilot study on provider recruitment and retention. RESULTS: Fifty-seven eligible respondents completed the survey. Most (88.9%) reported their hospitals encourage student participation in patient care. Students in their practices participate in many aspects of patient care, including conducting exams (96.2%) and participating in the operating room (94.3%). The majority found the rewarding aspects of teaching medical students to be intellectual stimulation (90.9%), continuing the tradition of medical teaching (87.5%), and the intrinsic satisfaction of teaching (83.6%). Challenging aspects of teaching included reduced reimbursement (40%) and the student/workload (63.6%). DISCUSSION: Medical student education continues to rely on a generation's medical professionals to impart their knowledge to the next. We hope that with a better understanding of the benefits of participation and minimization of the challenges, we can perpetuate this tradition despite the uncertainty in our health-care system.

New Partner Recruitment to Rural Versus Urban Ob-Gyn Practices: A Survey of Practicing Ob-Gyns.

PURPOSE: The purpose of this pilot study was to investigate the recruitment efforts of practicing obstetrics and gynecology (ob-gyns) from rural and urban practices. METHOD: The authors surveyed practicing ob-gyns from 5 states in the Pacific Northwest in 2016 about their background, practice setting, practice profile, partner recruitment, and retention. RESULTS: Seventy-three patients completed the study (53.2% response rate). Thirty-seven percent of respondents work in an urban practice and 43% have a rural practice, with the remainder in a suburban setting. A majority of the respondents attempted to recruit a new partner in the past 5 years. Respondents were most interested in experience and diversity in new recruits. Urban respondents, however, were more interested in hiring those with specialized skills (χ2 = 7.842, P = .02) than rural providers who were more interested in partners familiar with their community (χ2= 7.153, P = .03). Reasons most often cited to leave their practice were reimbursement, limited social/marital options, and workload, other than rural providers who more often also cited lack of access to specialty care (χ2= 13.256, P = .001). Rural providers were more likely to cite marital and family status as an advantage to recruitment, whereas urban and suburban providers were more often neutral. CONCLUSIONS: Reduced access to care has led to significant health disparities for women living in rural communities. Understanding which providers are most likely to be successful in these settings might help preserve access as our health-care systems evolves.

Obstetricians and gynecologists' opinions about the Affordable Care Act and their expectations about how it will impact their practice.

As the primary healthcare providers for women, obstetrician-gynecologists' (OB/GYNs) experiences with and opinions about the Affordable Care Act (ACA) are important to understand. An online survey was sent to 1000 randomly selected OB/GYNs who were members of the American College of Obstetricians and Gynecologists (ACOG) in 2014. Of those, 523 opened the email and 163 responded (31% participation rate). Data were collected August 2014-October 2014 and analyzed in 2015-2016. Support for the ACA was widely distributed, with the largest subset of the sample (about 21%) in the "very supportive" category. Opinions of the ACA were more supportive than they were in a previous study conducted in 2011. When given a list of possible positive and negative impacts of the ACA on their practice, roughly 1 in 5 reported that the ACA increased work-related stress (28%), decreased total profits (22%), and lowered career satisfaction (22%), whereas 8.6% reported that the ACA increased quality of care. Around half of the providers thought that their newly insured patients would have the same level of education (42%) and numeric ability (55%) as their current patients. Almost all respondents (87%) indicated that it is at least slightly important for patients to understand their numeric likelihood of risk (such as numeric risk information from medications, treatments, and other procedures you might prescribe) -31% think it is extremely important and 44% think it is moderately important.

Attitudes and Beliefs Regarding the Utility of Robotically Assisted Gynecologic Surgery Among Practicing Gynecologists.

While use of robotic-assisted surgery has increased rapidly, little is known about the attitudes and beliefs of practicing gynecologists regarding the utility of the technology. We surveyed a large sample of gynecologists to examine their attitudes and beliefs about the benefits, utility, and factors driving use of robotic-assisted gynecologic surgery. A 51-item survey was mailed to 600 fellows or junior fellows of the American College of Obstetricians and Gynecologists. The survey included questions on use of robotic surgery, decision-making, and beliefs regarding the technology. Responses were stratified based on whether the respondent used robotic surgery or not. A total of 310 responses were received including 27.8% who used robotic surgery in their practices. Hysterectomy was the most commonly performed procedure. Opinions about the use and effectiveness of robotic procedures varied based on whether an individual was a robot user. Eighty-two percentage of robot users and 21% of nonrobot users believed robotic surgery provided benefits over laparoscopic (p < .0001). Among both groups, the ability to increase access to minimally invasive surgery and marketing were believed to be the greatest drivers of use of robotic surgery. Attitudes and beliefs about the effectiveness of robotic gynecologic surgery are highly variable among clinicians.

Cryopreservation of Human Mucosal Leukocytes.

BACKGROUND: Understanding how leukocytes in the cervicovaginal and colorectal mucosae respond to pathogens, and how medical interventions affect these responses, is important for developing better tools to prevent HIV and other sexually transmitted infections. An effective cryopreservation protocol for these cells following their isolation will make studying them more feasible. METHODS AND FINDINGS: To find an optimal cryopreservation protocol for mucosal mononuclear leukocytes, we compared cryopreservation media and procedures using human vaginal leukocytes and confirmed our results with endocervical and colorectal leukocytes. Specifically, we measured the recovery of viable vaginal T cells and macrophages after cryopreservation with different cryopreservation media and handling procedures. We found several cryopreservation media that led to recoveries above 75%. Limiting the number and volume of washes increased the fraction of cells recovered by 10-15%, possibly due to the small cell numbers in mucosal samples. We confirmed that our cryopreservation protocol also works well for both endocervical and colorectal leukocytes. Cryopreserved leukocytes had slightly increased cytokine responses to antigenic stimulation relative to the same cells tested fresh. Additionally, we tested whether it is better to cryopreserve endocervical cells on the cytobrush or in suspension. CONCLUSIONS: Leukocytes from cervicovaginal and colorectal tissues can be cryopreserved with good recovery of functional, viable cells using several different cryopreservation media. The number and volume of washes has an experimentally meaningful effect on the percentage of cells recovered. We provide a detailed, step-by-step protocol with best practices for cryopreservation of mucosal leukocytes.

Determination of the Membrane Permeability to Water of Human Vaginal Mucosal Immune Cells at Subzero Temperatures Using Differential Scanning Calorimetry.

To study mucosal immunity and conduct HIV vaccine trials, it is important to be able to cryopreserve mucosal specimens and recover them in functional viable form. Obtaining a good recovery depends, in part, on cooling the cells at the appropriate rate, which is determined by the rate of water transport across the cell membrane during the cooling process. In this study, the cell membrane permeabilities to water at subzero temperatures of human vaginal mucosal T cells and macrophages were measured using the differential scanning calorimetry method proposed by Devireddy et al. in 1998. Thermal histograms were measured before and after cell lysis using a Slow-Fast-Fast-Slow cooling program. The difference between the thermal histograms of the live intact cells and the dead lysed cells was used to calculate the temperature-dependent cell membrane permeability at subzero temperatures, which was assumed to follow the Arrhenius relationship, [Formula: see text], where Lpg is the permeability to water at the reference temperature (273.15 K). The results showed that Lpg = 0.0209 ± 0.0108 µm/atm/min and Ea = 41.5 ± 11.4 kcal/mol for T cells and Lpg = 0.0198 ± 0.0102 µm/atm/min and Ea = 38.2 ± 10.4 kcal/mol for macrophages, respectively, in the range 0°C to -40°C (mean ± standard deviation). Theoretical simulations predicted that the optimal cooling rate for both T cells and macrophages was about -3°C/min, which was proven by preliminary immune cell cryopreservation experiments.

A study of the osmotic characteristics, water permeability, and cryoprotectant permeability of human vaginal immune cells.

Cryopreservation of specimens taken from the genital tract of women is important for studying mucosal immunity during HIV prevention trials. However, it is unclear whether the current, empirically developed cryopreservation procedures for peripheral blood cells are also ideal for genital specimens. The optimal cryopreservation protocol depends on the cryobiological features of the cells. Thus, we obtained tissue specimens from vaginal repair surgeries, isolated and flow cytometry-purified immune cells, and determined fundamental cryobiological characteristics of vaginal CD3(+) T cells and CD14(+) macrophages using a microfluidic device. The osmotically inactive volumes of the two cell types (Vb) were determined relative to the initial cell volume (V0) by exposing the cells to hypotonic and hypertonic saline solutions, evaluating the equilibrium volume, and applying the Boyle van't Hoff relationship. The cell membrane permeability to water (Lp) and to four different cryoprotective agent (CPA) solutions (Ps) at room temperature were also measured. Results indicated Vb values of 0.516 V0 and 0.457 V0 for mucosal T cells and macrophages, respectively. Lp values at room temperature were 0.196 and 0.295 µm/min/atm for T cells and macrophages, respectively. Both cell types had high Ps values for the three CPAs, dimethyl sulfoxide (DMSO), propylene glycol (PG) and ethylene glycol (EG) (minimum of 0.418 × 10(-3) cm/min), but transport of the fourth CPA, glycerol, occurred 50-150 times more slowly. Thus, DMSO, PG, and EG are better options than glycerol in avoiding severe cell volume excursion and osmotic injury during CPA addition and removal for cryopreservation of human vaginal immune cells.

Optimizing viable leukocyte sampling from the female genital tract for clinical trials: an international multi-site study.

BACKGROUND: Functional analysis of mononuclear leukocytes in the female genital mucosa is essential for understanding the immunologic effects of HIV vaccines and microbicides at the site of HIV exposure. However, the best female genital tract sampling technique is unclear. METHODS AND FINDINGS: We enrolled women from four sites in Africa and the US to compare three genital leukocyte sampling methods: cervicovaginal lavages (CVL), endocervical cytobrushes, and ectocervical biopsies. Absolute yields of mononuclear leukocyte subpopulations were determined by flow cytometric bead-based cell counting. Of the non-invasive sampling types, two combined sequential cytobrushes yielded significantly more viable mononuclear leukocytes than a CVL (p<0.0001). In a subsequent comparison, two cytobrushes yielded as many leukocytes (∼ 10,000) as one biopsy, with macrophages/monocytes being more prominent in cytobrushes and T lymphocytes in biopsies. Sample yields were consistent between sites. In a subgroup analysis, we observed significant reproducibility between replicate same-day biopsies (r = 0.89, p = 0.0123). Visible red blood cells in cytobrushes increased leukocyte yields more than three-fold (p = 0.0078), but did not change their subpopulation profile, indicating that these leukocytes were still largely derived from the mucosa and not peripheral blood. We also confirmed that many CD4(+) T cells in the female genital tract express the α4ß7 integrin, an HIV envelope-binding mucosal homing receptor. CONCLUSIONS: CVL sampling recovered the lowest number of viable mononuclear leukocytes. Two cervical cytobrushes yielded comparable total numbers of viable leukocytes to one biopsy, but cytobrushes and biopsies were biased toward macrophages and T lymphocytes, respectively. Our study also established the feasibility of obtaining consistent flow cytometric analyses of isolated genital cells from four study sites in the US and Africa. These data represent an important step towards implementing mucosal cell sampling in international clinical trials of HIV prevention.