Michael Fialkow

Initial Events in Establishing Vaginal Entry and Infection by Human Immunodeficiency Virus Type-1

Summary Understanding the initial events in the establishment of vaginal human immunodeficiency virus type-1 (HIV-1) entry and infection has been hampered by the lack of appropriate experimental models. Here, we show in an ex vivo human organ culture system that upon contact in situ, HIV-1 rapidly penetrated both intraepithelial vaginal Langerhans and CD4+ T cells. HIV-1 entered CD4+ T cells almost exclusively by CD4 and CCR5 receptor-mediated direct fusion, without requiring passage from Langerhans cells, and overt productive infection ensued. By contrast, HIV-1 entered CD1a+ Langerhans cells primarily by endocytosis, by means of multiple receptors, and virions persisted intact within the cytoplasm for several days. Our findings shed light on the very earliest steps of mucosal HIV infection in vivo and may guide the design of effective strategies to block local transmission and prevent HIV-1 spread.

Lifetime risk of surgical management for pelvic organ prolapse or urinary incontinence.

The objective of our study was to estimate the age-specific incidence and lifetime risk of surgically managed pelvic organ prolapse (POP) and urinary incontinence (UI). Women aged 20 and older who underwent primary surgical management of POP or UI in 1993 were identified from the database of a health maintenance organization using ICD-9 codes and confirmed through chart abstraction. From a population of 147,719 women, 135 were identified who underwent prolapse surgery only, 82 incontinence only, and 34 surgery for both conditions. From the age-specific incidence, we estimated the lifetime risk of undergoing an operation by age 80 to be 11.8%. Our findings agree with a previous estimate that ∼11% of women will undergo surgery for POP or UI by age 80. POP and UI appear to be common problems, undoubtedly affecting an even larger proportion of the women than suggested by this high cumulative incidence of surgery.

Vaginal Langerhans Cells Nonproductively Transporting HIV-1 Mediate Infection of T Cells

ABSTRACT 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.

Reoperation for urinary incontinence

OBJECTIVE(S) The objective of the study was to describe the rate and associated factors of reoperation for urinary incontinence. STUDY DESIGN A cohort study using Washington state hospitalization records from 1987 to 2005 of inpatient urinary incontinence surgeries. The cumulative reoperation rate was estimated for the entire cohort and by procedure. Cox regression was used to estimate the hazard of reoperation. RESULTS A total of 41,705 women underwent either a sling or retropubic colposuspension (Burch); 1895 underwent reoperation for urinary incontinence (8.6%; 95% confidence interval, 7.8-9.5%), a rate of 5.5 per 1000 woman-years. Women undergoing Burch had a lower reoperation rate than those undergoing slings (4.2 vs 6.7 per 1000 woman-years; P < .001). Concomitant hysterectomy was associated with a lower reoperation rate for Burch and sling repairs (5.4-2.9 and 7.7-4.2 per 1000 woman-years). CONCLUSION(S) Reoperation for urinary incontinence occurs commonly in the general population. The variable reoperation rate observed should be further investigated, given current trends toward increased Sling use.

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/μg 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.

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 α4β7 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.

Ex Vivo Comparison of Microbicide Efficacies for Preventing HIV-1 Genomic Integration in Intraepithelial Vaginal Cells

ABSTRACT 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 μM versus 51.2 μM [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 μM versus 7.57 μM [61 ng/ml versus 34 ng/ml]; P = 0.0614). Cellulose sulfate was the least effective of all the compounds tested (IC50, 1.8 μg/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.

Training the Next Generation of Minimally Invasive Surgeons

A paradigm shift is occurring in the educational approach to surgical procedures. A variety of pressures are forcing the initial education of surgeons of all disciplines out of the operating department and into simulation. Fortunately, increasing evidence suggests that surgeons can learn many fundamental skills and specific procedures with simulators. Evidence also supports the expectation that surgeons trained in simulation laboratories initially perform better in the operating department than those who are not. Minimally invasive procedures lend themselves to simulation particularly well. Currently, many different models are available for training and improvement in skills are seen with both low- and high-fidelity models. Developing an effective curriculum principally requires a commitment to the concept, and the time and space, for residents to learn and practice. Although many questions remain about how to optimally apply and evaluate the educational tools being developed, it appears certain that surgical simulation, in some form, is the educational paradigm of the present and future.

Reoperation After Sacral Neuromodulation Therapy: A Single-institution Experience

Objectives Sacral neuromodulation has become an accepted treatment for various types of lower urinary tract dysfunction. However, despite technologic advances in device implantation and a trial stimulation period, sacral neuromodulation still has a significant reoperation rate. We report our single-institution experience of reoperation rates. Methods We performed a retrospective review of our patients who had undergone the implantation of the InterStim device from April 1999 to December 2011 for lower urinary tract dysfunction. Results A total of 155 InterStim devices were implanted by 2 surgeons. Of the 142 patients with complete follow-up, 55 (38.2%) patients required reoperation, for either revision or explantation of the device. Revisions were performed in 30 (21.1%) patients, most commonly for mechanical failure of device, battery end-of-service, and pain, either at the site of the implanted pulse generator or with stimulation. Of the 30 patients who underwent revision, 14 had successful results, 6 had persistent symptoms, and 10 progressed to eventual explantation. The overall explantation rate was 24.6% (35 of 142 patients), and the average time to removal was 44 months. Most of the explantations were performed for poor symptom control and failure to maintain response (74.3%). Reoperation was not associated with age, sex, obesity, diabetes, chronic pain, use of the tined lead, or type of lower urinary tract dysfunction. Conclusions Sacral neuromodulation has a substantial revision and explantation rate, without any clear predictors for these complications. Patients should be counseled to these complications before surgery.

An in situ standardized patient-based simulation to train postpartum hemorrhage and team skills on a labor and delivery unit.

Postpartum hemorrhage (PPH) is the leading cause of maternal mortality worldwide, contributing to approximately 140,000 deaths per year. Because of readily available medical and surgical management, the number of deaths in the United States is low; nonetheless, PPH remains a leading cause of pregnancy-related morbidity and mortality. Early recognition of known risk factors for PPH and the use of third-stage intravenous oxytocin have been shown to significantly lower the incidence of PPH. Unfortunately, diagnosing clinically significant PPH is notoriously difficult because health care providers typically underestimate blood loss and, in pregnancy, signs of hypovolemia are also delayed. Appropriate management of PPH therefore typically requires an urgent interdisciplinary coordinated effort to safely administer a variety of medications and perform any necessary procedures to stop the patient’s blood loss. In situ simulation of PPH is a particularly valuable process. First and foremost, it provides a platform for interdisciplinary team practice while maintaining the environmental and system factors present during actual patient care events. Second, it allows the entire interdisciplinary team to practice management of the PPH patient, including diagnosis, medical (nonoperative) management, and resuscitation of the acutely decompensating patient. Finally, the debriefing process allows for the (1) identification of gaps in knowledge and teamwork behaviors and (2) detection of environmental and system deficiencies before an actual adverse event. This article describes the development, content validation, and in situ implementation of a standardized patient-based, interdisciplinary PPH scenario. The training received expedited approval from the University of Washington Institutional Review Board.