Joshua T. Piotrowski

WASH Knockout T Cells Demonstrate Defective Receptor Trafficking, Proliferation, and Effector Function

ABSTRACT WASH is an Arp2/3 activator of the Wiskott-Aldrich syndrome protein superfamily that functions during endosomal trafficking processes in collaboration with the retromer and sorting nexins, but its in vivo function has not been examined. To elucidate the physiological role of WASH in T cells, we generated a WASH conditional knockout (WASHout) mouse model. Using CD4Cre deletion, we found that thymocyte development and naive T cell activation are unaltered in the absence of WASH. Surprisingly, despite normal T cell receptor (TCR) signaling and interleukin-2 production, WASHout T cells demonstrate significantly reduced proliferative potential and fail to effectively induce experimental autoimmune encephalomyelitis. Interestingly, after activation, WASHout T cells fail to maintain surface levels of TCR, CD28, and LFA-1. Moreover, the levels of the glucose transporter, GLUT1, are also reduced compared to wild-type T cells. We further demonstrate that the loss of surface expression of these receptors in WASHout cells results from aberrant accumulation within the collapsed endosomal compartment, ultimately leading to degradation within the lysosome. Subsequently, activated WASHout T cells experience reduced glucose uptake and metabolic output. Thus, we found that WASH is a newly recognized regulator of TCR, CD28, LFA-1, and GLUT1 endosome-to-membrane recycling. Aberrant trafficking of these key T cell proteins may potentially lead to attenuated proliferation and effector function.

Perioperative Complications following Artificial Urinary Sphincter Placement.

PURPOSE We evaluated perioperative complications in patients undergoing primary artificial urinary sphincter placement and the potential impact of these complications on device outcomes. MATERIALS AND METHODS During the 2-year period from 2012 to 2014 we retrospectively evaluated the outcomes of 197 consecutive artificial urinary sphincter implantation procedures performed at our institution for post-prostatectomy incontinence. Of these cases 100 that were primary implantations comprise the study cohort. Perioperative complications, defined as those occurring within 6 weeks postoperatively, were classified by the Clavien-Dindo classification. After office evaluation at 6 weeks patients were followed for symptoms. Patient followup was obtained through office examination and telephone correspondence. RESULTS Patients undergoing primary artificial urinary sphincter implantation had a median age of 71.5 years (IQR 66, 76). The overall rate of any complication (Clavien I-V) within 6 weeks of surgery was 35%, including urinary retention in 31% of cases, cellulitis in 1%, device infection in 2% and urethral erosion in 2%. No significant differences in pertinent clinical comorbidities such as age (p = 0.69), hypertension (p = 0.95), coronary artery disease (p = 0.57), diabetes mellitus (p = 0.17), body mass index (p = 0.47), prior pelvic radiation therapy (p = 0.45), prior urethral sling placement (p = 0.91) or transcorporeal urethral cuff placement (p = 0.22) were found between patients with and without complications. Median followup was similar between those with and without postoperative urinary retention (p = 0.14). Postoperative urinary retention was associated with adverse 6-month device survival (76% vs 89%, p = 0.04). CONCLUSIONS The most common complication of artificial urinary sphincter placement is urinary retention. Serious adverse events following artificial urinary sphincter placement are rare. Postoperative urinary retention is associated with adverse short-term device survival rates.

Neonatal Dacryostenosis as a Risk Factor for Anisometropia

OBJECTIVE To determine whether there is a relationship between congenital nasolacrimal duct obstruction (CNLDO) and subsequent refractive error disorders in children. METHODS The medical records of children 5 years and younger diagnosed as having CNLDO between January 1, 2000, and December 31, 2007, were retrospectively reviewed. RESULTS Three hundred five consecutive children were diagnosed as having CNLDO at a median age of 12.3 months (range, 0.8 months to 4.8 years). Thirty children (9.8%) were diagnosed as having anisometropia with (n = 16) or without (n = 14) amblyopia at a median age of 19.2 months (range, 3.6 months to 7.4 years). Twenty-six of the 30 patients had hyperopic anisometropia; more severe hyperopia occurred in the eye with CNLDO in 23 patients (88.5%), 2 patients had more severe hyperopia in the fellow eye, and 1 patient had bilateral CNLDO. The median initial (P = .005) and final (P < .001) refractive error was significantly more hyperopic in those with both CNLDO and anisometropia compared with those with CNLDO alone. CONCLUSIONS The development of anisometropia with or without amblyopia seems to be more frequent in children examined by an ophthalmologist for CNLDO compared with that reported for the general public. The laterality of more severe hyperopia and amblyopia is generally on the side of the previous dacryostenosis.

SNX17 Affects T Cell Activation by Regulating TCR and Integrin Recycling

A key component in T cell activation is the endosomal recycling of receptors to the cell surface, thereby allowing continual integration of signaling and Ag recognition. One protein potentially involved in TCR transport is sorting nexin 17 (SNX17). SNX proteins have been found to bind proteins involved in T cell activation, but specifically the role of SNX17 in receptor recycling and T cell activation is unknown. Using immunofluorescence, we find that SNX17 colocalizes with TCR and localizes to the immune synapse in T– conjugates. Significantly, knockdown of the SNX17 resulted in fewer T–APC conjugates, lower CD69, TCR, and LFA-1 surface expression, as well as lower overall TCR recycling compared with control T cells. Lastly, we identified the 4.1/ezrin/radixin/moesin domain of SNX17 as being responsible in the binding and trafficking of TCR and LFA-1 to the cell surface. These data suggest that SNX17 plays a role in the maintenance of normal surface levels of activating receptors and integrins to permit optimum T cell activation at the immune synapse.

Dendritic Cells Utilize the Evolutionarily Conserved WASH and Retromer Complexes to Promote MHCII Recycling and Helper T Cell Priming

Immature dendritic cells (DCs) maintain a highly dynamic pool of recycling MHCII that promotes sampling of environmental antigens for presentation to T helper cells. However, the molecular basis of MHCII recycling and the cellular machinery that orchestrates MHCII trafficking are incompletely understood. Using a mouse model we show that WASH, an actin regulatory protein that facilitates retromer function, is essential for MHCII recycling and efficient priming of T helper cells. We further demonstrate that WASH deficiency results in impaired MHCII surface levels, recycling, and an accumulation of polyubiquitinated MHCII complexes, which are subsequently slated for premature lysosomal degradation. Consequently, conditional deletion of the Wash gene in DCs impairs priming of both conventional and autoimmune T helper cells in vivo and attenuates disease progression in a model of experimental autoimmune encephalitis (EAE). Thus, we identify a novel mechanism in which DCs employ the evolutionarily conserved WASH and retromer complex for MHCII recycling in order to regulate T helper cell priming.

An American Association for the Surgery of Trauma (AAST) prospective multi-center research protocol: outcomes of urethral realignment versus suprapubic cystostomy after pelvic fracture urethral injury

Background Pelvic fracture urethral injuries (PFUI) occur in up to 10% of pelvic fractures. It remains controversial whether initial primary urethral realignment (PR) after PFUI decreases the incidence of urethral obstruction and the need for subsequent urethral procedures. We present methodology for a prospective cohort study analyzing the outcomes of PR versus suprapubic cystostomy tube (SPT) after PFUI. Methods A prospective cohort trial was designed to compare outcomes between PR (group 1) and SPT placement (group 2). Centers are assigned to a group upon entry into the study. All patients will undergo retrograde attempted catheter placement; if this fails a cystoscopy exam is done to confirm a complete urethral disruption and attempt at gentle retrograde catheter placement. If catheter placement fails, group 1 will undergo urethral realignment and group 2 will undergo SPT. The primary outcome measure will be the rate of urethral obstruction preventing atraumatic passage of a flexible cystoscope. Secondary outcome measures include: subsequent urethral interventions, post-injury complications, urethroplasty complexity, erectile dysfunction (ED) and urinary incontinence rates. Results Prior studies demonstrate PR is associated with a 15% to 50% reduction in urethral obstruction. Ninety-six men (48 per treatment group) are required to detect a 15% treatment effect (80% power, 0.05 significance level, 20% loss to follow up/death rate). Busy trauma centers treat complete PFUI approximately 1–6 times per year, thus our goal is to recruit 25 trauma centers and enroll patients for 3 years with a goal of 100 or more total patients with complete urethral disruption. Conclusions The proposed prospective multi-institutional cohort study should determine the utility of acute urethral realignment after PFUI.

MP88-16 A PROSPECTIVE EVALUATION OF COMPLICATIONS AFTER ARTIFICIAL URINARY SPHINCTER PLACEMENT AND THEIR IMPACT ON DEVICE SURVIVAL

within the space of Retzius (SOR) may be challenging and subject to troublesome complications. We report our longitudinal experience utilizing a novel high submuscular (HSM) PRB placement technique and compare functional outcomes to traditional SOR placement of the PRB. METHODS: We reviewed a prospectively maintained database of AUS patients between July 2007 and January 2014. Only 61-70 cm H2O PRBs were placed through a trans-scrotal approach via an HSM tunnel (2011-2014) or within the SOR (2007-2010). Our HSM technique consisted of uniform placement of the PRB beneath the rectus abdominis muscle while SOR placement involved perforation of the transversalis fascia at the pubic tubercle, beneath the external inguinal ring. Demographics, patient data, cuff durability, and functional outcomes were compared between groups. RESULTS: 232 consecutive patients underwent AUS placement with amean follow up of 38months. SOR placement was performed in 139 (60%) patients while HSM placement was performed in 93 (40%). Functional outcomes including continence (defined as0-1 pads/day) rates (88% vs. 81%, p1⁄40.15), erosion rates (9% vs. 5%, p1⁄40.32), and explantation rates (12% vs. 10%, p1⁄40.83) were similar between groups. Fewer AUS revisions for persistent incontinence were required in patients undergoing HSM PRB placement (6.5% vs. 18%, p1⁄40.01). Although mean follow-up was longer for patients undergoing SOR placement (51 vs. 20 months, p<0.001), Kaplan-Meier analysis revealed no difference between groups with regards to rates of explantation (p1⁄40.71) or revision (p1⁄40.36). CONCLUSIONS: High submuscular placement of the PRB at the time of AUS surgery offers a safe and effective alternative with equivalent functional outcomes to traditional SOR.