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 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.

Vaginal langerhans cells nonproductively transporting HIV-1 mediate infection of T cells.

Although implied by other models, proof that Langerhans cells (LCs) in the human vagina participate in dissemination of infectious human immunodeficiency virus type 1 (HIV-1) has been lacking. Here, we show that LCs migrate from HIV-1-exposed vaginal epithelia and pass infectious virus to CD4+ T cells without being productively infected themselves, and we point to a pathway that might enable HIV-1 to avoid degradation in vaginal LCs. Transport by migratory LCs to local lymphatics in a nonproductive but infectious form may aid HIV-1 in evasion of topical microbicides that target its intracellular productive life cycle.

Fosfomycin Trometamol for the Prevention of Infectious Complications After Prostate Biopsy: A Consensus Statement by an International Multidisciplinary Group.

CONTEXT: Transrectal ultrasound-guided prostate biopsy (TRPB) has been a standard of care for diagnosing prostate cancer but is associated with a high incidence of infectious complications. OBJECTIVE: To achieve an expert consensus on whether fosfomycin trometamol provides adequate prophylaxis in TRPB and discuss its role as prophylaxis in transperineal prostate biopsy (TPPB). EVIDENCE ACQUISITION: An international multidisciplinary group of experts convened remotely to discuss how to best use fosfomycin in various clinical settings and patient situations. Six statements related to prostate biopsy and the role of fosfomycin were developed, based on literature searches and relevant clinical experience. EVIDENCE SYNTHESIS: Consensus was reached for all six statements. The group of experts was unanimous regarding fosfomycin as a preferred candidate for antimicrobial prophylaxis in TRPB. Fosfomycin potentially also meets the requirements for empiric prophylaxis in TPPB, although further clinical studies are needed to confirm or refute its utility in this setting. There is a risk of bias due to sponsorship by a pharmaceutical company. CONCLUSIONS: Antimicrobial prophylaxis is mandatory in TRPB, and fosfomycin trometamol is an appropriate candidate due to low rates of resistance, a good safety profile, sufficient prostate concentrations, and demonstrated efficacy in reducing the risk of infectious complications following TRPB. PATIENT SUMMARY: Patients undergoing transrectal ultrasound-guided prostate biopsy (TRPB) have a high risk of infectious complications, and antimicrobial prophylaxis is mandatory. However, increasing antimicrobial resistance, as well as safety concerns with fluoroquinolones, has restricted the number of antimicrobial options. Fosfomycin trometamol meets the requirements for a preferred antimicrobial in the prophylaxis of TRPB.

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.

Ex vivo comparison of microbicide efficacies for preventing HIV-1 genomic integration in intraepithelial vaginal cells.

Vaginally applied microbicides hold promise as a strategy to prevent sexual HIV transmission. Several nonspecific microbicides, including the polyanion cellulose sulfate, have been evaluated in large-scale clinical trials but have failed to show significant efficacy. These findings have prompted a renewed search for preclinical testing systems that can predict negative outcomes of microbicide trials. Moreover, the pipeline of potential topical microbicides has been expanded to include antiretroviral agents, such as reverse transcriptase, fusion, and integrase inhibitors. Using a novel ex vivo model of vaginal HIV-1 infection, we compared the prophylactic potentials of two forms of the fusion inhibitor T-20, the CCR5 antagonist TAK-778, the integrase inhibitor 118-D-24, and cellulose sulfate (Ushercell). The T-20 peptide with free N- and C-terminal amino acids was the most efficacious compound, causing significantly greater inhibition of viral genomic integration in intraepithelial vaginal leukocytes, measured by an optimized real-time PCR assay, than the more water-soluble N-acetylated T-20 peptide (Fuzeon) (50% inhibitory concentration [IC50], 0.153 microM versus 51.2 microM [0.687 ng/ml versus 230 ng/ml]; P<0.0001). In contrast, no significant difference in IC50s was noted in peripheral blood cells (IC50, 13.58 microM versus 7.57 microM [61 ng/ml versus 34 ng/ml]; P=0.0614). Cellulose sulfate was the least effective of all the compounds tested (IC50, 1.8 microg/ml). These results highlight the merit of our model for screening the mucosal efficacies of novel microbicides and their formulations and potentially rank ordering candidates for clinical evaluation.

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.

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.

A curriculum for Burch colposuspension and diagnostic cystoscopy evaluated by an objective structured assessment of technical skills.

OBJECTIVE: The purpose of this study was to develop an effective curriculum for teaching colposuspension and diagnostic cystoscopy. STUDY DESIGN: Fifty-five residents underwent an Objective Structured Assessment of Technical Skills that was composed of a task-specific checklist and validated global assessment. Thirty of the residents had been exposed to a training curriculum, and 25 residents served as untrained control subjects. RESULTS: For the colposuspension and cystoscopy checklists, the reliability coefficient was 0.85 and 0.72, and the interrater reliability was 0.92 and 0.68, respectively. Although residents who were provided the curriculum performed better on both task-specific checklists, the differences did not reach statistical significance. Senior residents performed consistently better than junior residents for both tasks. When a comparison was made of the junior residents separately to account for previous experience, trained residents performed significantly better on the cystoscopy checklist (P = .029). CONCLUSION: This curriculum is an effective way to teach diagnostic cystoscopy to junior residents. The checklist for this Objective Structured Assessment of Technical Skills has good reliability and construct validity.

A comprehensive women's health care center: are gynecologists offering primary care?

OBJECTIVE: The purpose of this study was to compare patient characteristics and diagnoses in a comprehensive Women's Health Care Clinic between gynecologists and internal medicine physicians. STUDY DESIGN: This retrospective cohort study evaluated International Classification of Diseases, 9th Revision, codes that were recorded between January 1, 2001, and January 4, 2004, at the Women's Health Care Clinic. We compared demographics and prevalence of diagnosis codes for patients who were seen by obstetricians/gynecologists and internal medicine physicians and compared these data with national survey statistics. A sampling of charts was reviewed for coding verification. RESULTS: We analyzed 13,462 visits at the Women's Health Care Clinic. Patients who were seen by internists were older, had greater racial diversity, and had more gender-nonspecific medical disorders (hypertension, depression). Gynecologists saw more specific women's health problems (P < .05). The diagnoses of menstrual disorders, menopause, pelvic pain, and abnormal cytologic findings within the Women's Health Care Clinic follow closely with the national ambulatory survey data. CONCLUSION: Within this multispecialty Women's Health Care Clinic, internal medicine physicians are practicing primary care and obstetricians/gynecologists are providing specialty care.

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.

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.

Most DC-SIGNR transcripts at mucosal HIV transmission sites are alternatively spliced isoforms.

The repeat region of DC-SIGNR (CD209L) is polymorphic on the genomic level, and, in a separate study, we observed a correlation between the DC-SIGNR genotype and HIV-1 susceptibility during sexual contact. However, previous investigations using immunohistochemistry failed to detect membrane-bound DC-SIGNR on cells in the genital and rectal mucosa. We therefore explored the presence of DC-SIGNR in these compartments with a more sensitive limiting dilution RT-PCR, which also allowed for quantification of alternatively spliced mRNA isoforms. DC-SIGN (CD209) and DC-SIGNR mRNA transcript isoforms were found in all 12 vaginal and two rectal biopsies obtained from 14 healthy individuals. For DC-SIGNR, we detected significantly more isoform than full-length transcripts (mean copy numbers/mug RNA: 602 vs 26; P=0.0009). Four mucosal samples lacked full-length DC-SIGNR transcripts entirely. Cloning and sequencing of DC-SIGNR mRNA in three additional individuals revealed a diverse repertoire of DC-SIGNR isoforms, many of which encoded for proteins predicted to be soluble and secreted. Indeed, in one vaginal sample, we detected only soluble isoforms. In conjunction with our prior observation that the DC-SIGNR genotype has an effect on HIV-1 transmission in vivo, these findings emphasize that DC-SIGNR, in addition to DC-SIGN, should be considered as a cofactor in sexual HIV-1 transmission. Soluble isoforms, in particular, may modulate the efficiency of viral transmission and dissemination.

A six-year study of surgical teaching and skills evaluation for obstetric/gynecologic residents in porcine and inanimate surgical models.

OBJECTIVE: This study was undertaken to evaluate an ongoing teaching and objective surgical skills testing program for obstetric/gynecologic residents in a laboratory setting, and assess the impact on residents of having 4 years of a surgical laboratory curriculum. STUDY DESIGN: From 1997 through 2002, we conducted surgical skills training sessions for all obstetric/gynecologic residents, using both inanimate and animal (porcine) models. Once a year we tested each resident on 12 structured surgical bench tasks. At the end of each year, we conducted formal objective structured assessment of technical skills (OSATS) with all residents attempting multiple surgical procedures. We compared residents who had 4 years of laboratory training with those who started residency earlier and had only 1 or 2 years of the new curriculum. We also compared residents' own performance from year to year and cohort performance by resident year. RESULTS: PGY3 and PGY4s who had 4 years of surgical laboratory training did significantly better on bench laboratory skills than PGY3 and PGY4s with fewer years of training sessions (total scores of 48.8 vs 30.3, P < .001). However, no significant improvement in surgical procedures as measured by global OSATS was found. When comparing residents' own performance between the beginning and the end of 1 year, global OSATS scores improved significantly on laparoscopic salpingotomy (P < .001) and open oophorectomy (P < .001). For the cohort of PGY4s completing 4 years of laboratory training, average global OSATS scores showed statistically significant improvement (PGY1, PGY2 < PGY3 < PGY4, P < .001). CONCLUSION: Residents who completed the 4-year curriculum showed significantly better technical skills on bench tasks but not on OSATS compared with those with less training. Resident surgical skills evaluated by OSATS significantly improve over time both individually and as a cohort by resident year.

Self-assessment of resident surgical skills: is it feasible?

OBJECTIVE: The purpose of this study was to examine obstetrics and gynecology residents' self-assessment of proficiency on a variety of surgical bench procedures and to compare their ratings with those ratings of trained faculty observers who used instruments that have been shown to be reliable and valid. STUDY DESIGN: As part of a 6-station Objective Structured Assessment of Technical Skills, 74 residents at 5 institutions estimated their overall open and laparoscopic skill level before the testing. After completing each station, residents evaluated their overall and global skills performance. RESULTS: Residents rated their proficiency higher on open skills than on laparoscopic skills. Task-specific, overall, and global assessments were correlated significantly with the faculty ratings (P < .001). Residents tended to rate themselves lower than did faculty on almost all measures; even those residents with poor skills indicated that they were aware of their deficiencies. Overall and global self-assessments increased with each resident level, which indicated good construct validity. CONCLUSION: Residents can rate their overall open and laparoscopic skills, task-specific performance, and global skills with good reliability and validity. Although they tended to score themselves lower than did faculty observers, the correlations are high (ie, residents who give themselves a higher score tended to receive a higher score from faculty, and vice versa). One of the concerns about self-assessment is that residents with poor skills might not be aware of their deficiencies. We did not find that to be the case. Therefore, when residents work on self-directed exercises, task-specific and global checklists can be used for both learning and self-assessment.

Musculoskeletal orofacial pain and other signs and symptoms of temporomandibular disorders during pregnancy: a prospective study.

AIMS: To describe the course of reported musculoskeletal pain in the temporomandibular region and other signs and symptoms of temporomandibular disorders (TMD) as well as psychological distress over the course of pregnancy and 1 year postpartum. METHODS: Women with musculoskeletal orofacial pain (n = 19) and pain-free comparison subjects (n = 16) in the first trimester of pregnancy were selected through records review from the population of a large health maintenance organization. Subjects completed a self-administered questionnaire assessing pain, depression, and somatic symptoms; provided a sample of whole unstimulated saliva; and underwent a standardized clinical examination during the third, sixth, and ninth months of pregnancy and 1 year postpartum. RESULTS: At baseline (third month of pregnancy), 16 of the 19 patients with musculoskeletal orofacial pain met criteria for an RDC/TMD diagnosis. Reported musculoskeletal orofacial pain diminished significantly during the second or third trimester of pregnancy and increased again postpartum. Measures of mandibular opening increased over pregnancy in both cases and comparison subjects and remained high postpartum. Depression and somatic symptoms changed little over the course of pregnancy but were substantially lowered at 1 year postpartum for both groups. As expected, subjects with pain had higher levels of palpation pain, diminished mandibular range of motion, and higher levels of psychological distress compared to subjects without orofacial pain. CONCLUSION: Musculoskeletal orofacial pain and related symptoms appear to improve over the course of pregnancy. This improvement occurs in the presence of increased joint laxity and is not paralleled by improvements in psychological distress. Thus, it was concluded that the improvement in pain is most likely associated with the dramatic hormonal changes occurring during pregnancy.

Resident experiences with and post-training plans for cystoscopy at the time of hysterectomy.

AIMS: To determine OB/GYN residents' experience with and opinions toward cystoscopy at the time of hysterectomy. STUDY DESIGN: An internet survey of OB/GYN residents assessed utilization of cystoscopy at the time of hysterectomy, familiarity with literature regarding universal versus selective cystoscopy, and plans post residency. RESULTS: Cystoscopy was performed universally in the minority of cases of vaginal hysterectomy (12%), laparoscopically assisted vaginal hysterectomy (14%), supracervical hysterectomy (0%), total abdominal hysterectomy (2%), laparoscopic supracervical hysterectomy (9%), total laparoscopic hysterectomy (27%), and hysterectomy with adnexa removal (5%). Residents planned universal cystoscopy post-training more frequently for all hysterectomy types. CONCLUSION: Cystoscopy at the time of hysterectomy was performed universally in the minority for all hysterectomy categories. For all hysterectomy types, residents planned post-graduation to utilize universal cystoscopy at the time of hysterectomy more often than occurred in training.

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.

Training surgical residents: the current Canadian perspective.

BACKGROUND: Numerous constraints may limit the practical experience of surgical residents. Current research supports a need for structured curricula, skills acquisition, and feedback outside the operating room (OR) and formal assessment of technical skills. We examined the current state of surgical skill teaching and evaluation for Canadian residents across surgical specialties. METHODS: One hundred twenty-three surveys were mailed to surgical residency program directors of all 17 Canadian medical schools. Nine surgical specialties were identified. Program demographics, details of surgical skill teaching methods, and several surgical skill assessment modalities used from admission to graduation were surveyed. Obstetrics and Gynecology (ObGyn) survey results were compared with other surgical specialties. RESULTS: Seventy surveys (57%) from 15 medical schools (88%) were returned. All specialties were represented in responses. ObGyn residents had fewer surgical training months per year than other residents (4.9 versus 8.5 months; p = 0.001). The most common teaching method was didactic lectures (86%; 95% confidence interval [CI], 75%-93%) for all programs. Inanimate and/or animate skills laboratories and didactic lectures were equally used by 90% (95% CI, 55%-100%) of ObGyn programs. Virtual reality simulators for procedure-specific surgical skills ranked low at 30% (95% CI, 7%-65%) use in ObGyn programs. Most programs had a dedicated space to teach and practice skills outside the OR. Assessment, feedback, and determination of surgical competence were most commonly performed via subjective evaluation by surgical mentors. Forty percent of ObGyn programs versus 76% of other programs used a local program-specific surgical training curriculum (p = 0.054). Most program directors who did not have access to a standard training curriculum wished to have one implemented. CONCLUSIONS: Lectures and subjective evaluations are the most common educational tools in Canadian surgical residencies. Despite the availability of validated surgical teaching and evaluation tools remote from the OR, standard training curricula are not the norm in Canadian ObGyn residency programs.

Targeting temporomandibular disorder pain treatment to hormonal fluctuations: a randomized clinical trial.

Mounting evidence supports the importance of hormonal fluctuations in temporomandibular disorder (TMD) pain among women. Stabilizing influential hormones or having a plan and skills for coping with hormonally related increases in TMD pain, therefore, may be beneficial for women with TMD pain. This randomized clinical trial evaluated the short- and long-term efficacy of 3 interventions for women with TMD pain: (1) dental hygienist-delivered pain self-management training (SMT; n=59); (2) the same dental hygienist-delivered pain self-management training, but with a focus on menstrual cycle-related changes in pain and other symptoms (targeted SMT, or TSMT; n=55); and (3) continuous oral contraceptive therapy (6-month trial) aimed at stabilizing hormones believed to be influential in TMD pain (COCT; n=57). Study participants completed outcome (pain, activity interference, depression) and process (pain beliefs, catastrophizing, coping effectiveness) measures before randomization, and 6 and 12months later. Intent-to-treat analyses supported the benefits of the SMT and TSMT interventions relative to COCT. Targeting the self-management treatment to menstrual cycle-related symptoms did not increase the treatment's efficacy. The benefits of the self-management interventions relative to COCT for pain and activity interference were statistically significant at 12 months, but not at 6 months, whereas the benefits for the process measures generally were apparent at both time points. COCT was associated with multiple adverse events (none serious). The study provides further support for long-term benefits of a safe, low-intensity (2 in-person sessions and 6 brief telephone contacts), dental hygienist-delivered self-management treatment for TMD pain.