A carcinoembryonic antigen-specific cell therapy selectively targets tumor cells with HLA loss of heterozygosity in vitro and in vivo.

The CEACAM5 gene product [carcinoembryonic antigen (CEA)] is an attractive target for colorectal cancer because of its high expression in virtually all colorectal tumors and limited expression in most healthy adult tissues. However, highly active CEA-directed investigational therapeutics have been reported to be toxic, causing severe colitis because CEA is expressed on normal gut epithelial cells. Here, we developed a strategy to address this toxicity problem: the Tmod dual-signal integrator. CEA Tmod cells use two receptors: a chimeric antigen receptor (CAR) activated by CEA and a leukocyte Ig-like receptor 1 (LIR-1)-based inhibitory receptor triggered by human leukocyte antigen (HLA)-A*02. CEA Tmod cells exploit instances of HLA heterozygous gene loss in tumors to protect the patient from on-target, off-tumor toxicity. CEA Tmod cells potently killed CEA-expressing tumor cells in vitro and in vivo. But in contrast to a traditional CEA-specific T cell receptor transgenic T cell, Tmod cells were highly selective for tumor cells even when mixed with HLA-A*02-expressing cells. These data support further development of the CEA Tmod construct as a therapeutic candidate for colorectal cancer.

Chimeric Antigen Receptors Directed at Mutant KRAS Exhibit an Inverse Relationship Between Functional Potency and Neoantigen Selectivity.

Neoantigens are among the most intriguing potential immuno-oncology targets because, unlike many cancer targets that are expressed on normal tissues, they are by definition restricted to cancer cells. Medicines directed at common neoantigens such as mutant KRAS are especially interesting because they may offer the convenience and cost of an off-the-shelf therapy. However, all common KRAS mutations produce proteins that differ from the wild type at a single amino acid, creating challenges for molecular discrimination. We have undertaken an effort to optimize single-chain variable fragments (scFv) against peptide/major histocompatibility antigen complexes composed of HLA-A*11 and either G12V- or G12D-mutant KRAS peptides. These scFvs could in principle be used in chimeric antigen receptor (CAR) T-cell therapies for selected patients whose tumors bear either of these mutations. Here we show that optimization of such CARs involves a trade-off between potency and selectivity. We further show that targeting this family without high selectivity engenders risks of cross-reactivity against other members of the G-protein family to which KRAS belongs. Significance: We report an effort to generate high potency, selective CARs directed at mutant KRAS peptides. Although the heavily optimized CARs maintain high selectivity against wild-type KRAS, they lose selectivity against other KRAS-related peptides derived from human proteins. To our knowledge, this work is the first to examine the trade-off between potency and selectivity with regard to KRAS pMHC-directed CARs, illustrating the challenge to achieve both sufficient potency and high selectivity.

Potent, Selective CARs as Potential T-Cell Therapeutics for HPV-positive Cancers.

Next-generation T-cell therapies will likely continue to utilize T-cell receptors (TCRs) and chimeric antigen receptors (CARs) because each receptor type has advantages. TCRs often possess exceptional properties even when tested unmodified from patients' T cells. CARs are generally less sensitive, possibly because their ligand-binding domains are grafted from antibodies selected for binding affinity or avidity and not broadly optimized for a functional response. Because of the disconnect between binding and function among these receptor types, the ultimate potential of CARs optimized for sensitivity and selectivity is not clear. Here, we focus on a thoroughly studied immuno-oncology target, the HLA-A*02/HPV-E629-38 complex, and show that CARs can be optimized by a combination of high-throughput binding screens and low-throughput functional assays to have comparable activity to clinical TCRs in acute assays in vitro. These results provide a case study for the challenges and opportunities of optimizing high-performing CARs, especially in the context of targets utilized naturally by TCRs.

Re-examination of MAGE-A3 as a T-cell Therapeutic Target.

In 2013, an innovative MAGE-A3-directed cancer therapeutic of great potential value was terminated in the clinic because of neurotoxicity. The safety problems were hypothesized to originate from off-target T-cell receptor activity against a closely related MAGE-A12 peptide. A combination of published and new data led us to test this hypothesis with current technology. Our results call into question MAGE-A12 as the source of the neurotoxicity. Rather, the data imply that an alternative related peptide from EPS8L2 may be responsible. Given the qualities of MAGE-A3 as an onco-testis antigen widely expressed in tumors and largely absent from normal adult tissues, these findings suggest that MAGE-A3 may deserve further consideration as a cancer target. As a step in this direction, the authors isolated 2 MAGE-A3 peptide-major histocompatibility complex-directed chimeric antigen receptors, 1 targeting the same peptide as the clinical T-cell receptor. Both chimeric antigen receptors have improved selectivity over the EPS8L2 peptide that represents a significant risk for MAGE-A3-targeted therapeutics, showing that there may be other options for MAGE-A3 cell therapy.

Structure-function relationships of chimeric antigen receptors in acute T cell responses to antigen.

Chimeric antigen receptors (CARs) and their parent signaling molecule, the T cell receptor (TCR), are fascinating proteins of increasing relevance to disease therapy. Here we use a collection of 1221 pMHC-directed CAR constructs representing 10 pMHC targets to study aspects of CAR structure-activity relationships (SAR), with particular focus on the extracellular and transmembrane structural components. These experiments that involve pMHC targets whose number/cell can be manipulated by peptide dosing in vitro enable systematic analysis of the SAR of CARs in carefully controlled experimental situations (Harris and Kranz, 2016). We find that CARs tolerate a wide range of structural variation, with the ligand-binding domains (LBDs) dominating the SAR of CAR antigen sensitivity. Notwithstanding the critical role of the LBD, CAR antigen-binding on the cell surface, measured by pMHC tetramer staining, is not an effective predictor of functional sensitivity. These results have important implications for the design and testing of CARs aimed toward the clinic.

Short PlGF-derived peptides bind VEGFR-1 and VEGFR-2 in vitro and on the surface of endothelial cells.

The placental growth factor (PlGF), a member of VEGF family, plays a crucial role in pathological angiogenesis, especially ischemia, inflammation, and cancer. This activity is mediated by its selective binding to VEGF receptor 1 (VEGFR-1), which occurs predominantly through receptor domains 2 and 3. The PlGF ß-hairpin region spanning residues Q87 to V100 is one of the key binding elements on the protein side. We have undertaken a study on the design, preparation, and functional characterization of the peptide reproducing this region and of a set of analogues where glycine 94, occurring at the corner of the hairpin in the native protein, is replaced by charged as well as hydrophobic residues. Also, some peptides with arginine 96 replaced by other residues have been studied. We find that the parent peptide weakly binds VEGFR-1, but replacement of G94 with residues bearing H-bond donating residues significantly improves the affinity. Replacement of R96 instead blocks the interaction between the peptide and the domain. The strongest affinity is observed with the G94H (peptide PlGF-2) and G94W (peptide PlGF-10) mutants, while the peptide PlGF-8, bearing the R96G mutation, is totally inactive. The PlGF-1 and PlGF-2 peptides also bind the VEGFR-2 receptors, though with a reduced affinity, and are able to interfere with the VEGF-induced receptor signaling on endothelial cells. The peptides also bind VEGFR-2 on the surface of cells, while PlGF-8 is inactive. Data suggest that these peptides have potential applications as PlGF/VEGF mimic in various experimental settings.

Immunodominant West Nile Virus T Cell Epitopes Are Fewer in Number and Fashionably Late.

Class I HLA molecules mark infected cells for immune targeting by presenting pathogen-encoded peptides on the cell surface. Characterization of viral peptides unique to infected cells is important for understanding CD8(+) T cell responses and for the development of T cell-based immunotherapies. Having previously reported a series of West Nile virus (WNV) epitopes that are naturally presented by HLA-A*02:01, in this study we generated TCR mimic (TCRm) mAbs to three of these peptide/HLA complexes-the immunodominant SVG9 (E protein), the subdominant SLF9 (NS4B protein), and the immunorecessive YTM9 (NS3 protein)-and used these TCRm mAbs to stain WNV-infected cell lines and primary APCs. TCRm staining of WNV-infected cells demonstrated that the immunorecessive YTM9 appeared several hours earlier and at 5- to 10-fold greater density than the more immunogenic SLF9 and SVG9 ligands, respectively. Moreover, staining following inhibition of the TAP demonstrated that all three viral ligands were presented in a TAP-dependent manner despite originating from different cellular compartments. To our knowledge, this study represents the first use of TCRm mAbs to define the kinetics and magnitude of HLA presentation for a series of epitopes encoded by one virus, and the results depict a pattern whereby individual epitopes differ considerably in abundance and availability. The observations that immunodominant ligands can be found at lower levels and at later time points after infection suggest that a reevaluation of the factors that combine to shape T cell reactivity may be warranted.

The "flowerpot" sign: inference of poor renal function in high grade vesicoureteral reflux by calyceal orientation.

INTRODUCTION/OBJECTIVE: Modern radiographic advances have allowed for detailed and accurate imaging of not only urologic anatomy but also urologic function. The art of observational inference of subtle anatomic features and function from a static radiograph is being traded for new, more precise, and more expensive modalities. While the superiority of these methods cannot be denied, the total information provided in simpler tests should not be ignored. The relationship between high grade vesicoureteral reflux with the dilated calyces arranged cephalad to a dilated funnel-shaped renal pelvis on VCUG and reduced differential renal function has not been previously described, but has been anecdotally designated a "flowerpot" sign by our clinicians. We hypothesize that the appearance of a "flowerpot" kidney as described herein is an indicator of poor renal function in the setting of high grade VUR. STUDY DESIGN: IRB approval was obtained and 315 patients were identified from system-wide VCUG reports from 2004-2012 with diagnosed "high grade" or "severe" vesicoureteral reflux. Inclusion into the study required grade IV or V VUR on initial VCUG and an initial radionuclide study for determination of differential function. Patients with a solitary kidney, posterior urethral valve, multicystic dysplastic kidney, renal ectopia, or duplex collecting systems were excluded. Grade of reflux, angle of the inferior-superior calyceal axis relative to the lumbar spine, and differential uptake were recorded along with presence of the new "flowerpot" sign. Variables were analyzed using the Mann-Whitney U test to determine statistical significance. RESULTS: Fifty seven patients met inclusion criteria with 11 being designated as "flowerpot" kidneys. These "flowerpot" kidneys could be objectively differentiated from other kidneys with grade IV and/or grade V VUR both by inferior-superior calyceal axis (median angle, 52° [37-66] vs. 13° [2-37], respectively p < 0.001) and by differential renal uptake (median, 23% [5-49] vs. 45% [15-81], respectively p < 0.001). Likewise, there was no difference between either calyceal axis (median angle, 13° [3-20] vs. 13° [2-37]) or differential function (median, 48% [24-81] vs. 40% [15-66], p = 0.129) when comparing kidneys with grade IV and grade V VUR, respectively, that did not demonstrate the "flowerpot" sign. DISCUSSION/CONCLUSION: Grading of VUR is used to provide a common language for scientific discussion and determine prognosis for children with similar attributes. The dysmorphic calyceal system in the "flowerpot" kidneys supports the theory of abnormal renal blastema induction associated with abnormal differentiation of the ureteral bud. Even in the absence of urinary tract infections and/or pyelonephritis, renal abnormalities and decreased differential function can be observed on renal scintigraphy. This study also confirms the male predominance and functional similarities between grade 4 and 5 refluxing renal units. Recognizing this is a limited observational study based on imaging alone, the "flowerpot" sign is an indicator of the most severe form of grade 5 VUR but is only one factor in predicting long term overall renal prognosis. However, 14% (8/57) of our cohort had a relative uptake of less than 20% with 5 of these exhibiting the "flowerpot" sign. The "flowerpot" sign on VCUG can be used as indirect evidence of poor differential renal function and, therefore, useful in guiding parental expectations prior to formal functional imaging.

The prevalence of bell clapper anomaly in the solitary testis in cases of prior perinatal torsion.

PURPOSE: Bell clapper anomaly is associated with an increased risk of intravaginal testicular torsion. However, perinatal torsion is thought to be secondary to an extravaginal process. We quantified the contralateral prevalence of bell clapper anomaly in children found to have atrophic testicular nubbins secondary to presumed torsion during gestation to better define the subsequent risk of metachronous testicular torsion. MATERIALS AND METHODS: Inspection results for the presence of contralateral bell clapper anomaly was recorded by a single surgeon in 50 consecutive cases in which exploration for nonpalpable testes revealed a testicular nubbin. For comparison data were collected in 27 consecutive cases of acute testicular torsion. Anatomy of the normal contralateral testis was compared between the 2 groups. RESULTS: Average age at surgery in the perinatal torsion group was 15 months vs 12.7 years in the acute torsion group. One case of partial contralateral bell clapper anomaly was discovered in the perinatal torsion group but no complete anomaly was found. In contrast, in older boys with acute testicular torsion complete bell clapper anomaly was found in 21 of the 27 contralateral testes (78%). CONCLUSIONS: In older boys with acute testicular torsion contralateral bell clapper anomaly is highly prevalent, supporting the standard practice of contralateral testicular fixation in this clinical situation. However, the prevalence of contralateral bell clapper anomaly is exceedingly small in cases of monorchism after perinatal torsion, substantiating an insufficient risk of subsequent torsion to justify routine fixation of the solitary testis.

Most infants with dilating vesicoureteral reflux can be treated nonoperatively.

PURPOSE: Dilating vesicoureteral reflux provokes concern for physicians and parents that often leads to corrective surgery in young children. Since there are limited data describing the natural history of dilating vesicoureteral reflux in infants, we identified factors predictive of resolution/improvement in infants initially treated nonoperatively. MATERIALS AND METHODS: We reviewed the medical records of 90 infants 6 months old or younger from 2004 to 2010 who were referred for prenatal hydronephrosis or initial febrile urinary tract infection and found to have dilating vesicoureteral reflux (grade 3 or greater). Variables of interest included presentation, dimercapto-succinic acid results, sex, breakthrough febrile urinary tract infections, reflux grade and bilateral reflux. Cox regression analysis was performed to determine predictors of spontaneous resolution and/or improvement to reflux grade less than 3 as well as predictors of surgical intervention. RESULTS: Included in final analysis were 80 infants (113 renal units). Of the patients 51 (64%) experienced spontaneous resolution/improvement with a mean followup of 29 months before resolution, discharge home and/or end of followup. Only 20 patients (25%) underwent surgery. Cox regression analysis revealed that a normal initial dimercapto-succinic acid scan, initial reflux grade less than 5 and absent breakthrough febrile urinary tract infections were predictive of reflux resolution/improvement (p <0.05). Dimercapto-succinic acid scan abnormalities, prenatal hydronephrosis and breakthrough febrile urinary tract infections were significant predictors of surgery (p <0.05). CONCLUSIONS: Dilating vesicoureteral reflux in infancy often resolves/improves spontaneously. Therefore, surgery should be directed toward patients unlikely to experience resolution, ie those with an abnormal initial dimercapto-succinic acid scan, grade 5 vesicoureteral reflux and breakthrough febrile urinary tract infections.

Stereoscopic augmented reality for laparoscopic surgery.

BACKGROUND: Conventional laparoscopes provide a flat representation of the three-dimensional (3D) operating field and are incapable of visualizing internal structures located beneath visible organ surfaces. Computed tomography (CT) and magnetic resonance (MR) images are difficult to fuse in real time with laparoscopic views due to the deformable nature of soft-tissue organs. Utilizing emerging camera technology, we have developed a real-time stereoscopic augmented-reality (AR) system for laparoscopic surgery by merging live laparoscopic ultrasound (LUS) with stereoscopic video. The system creates two new visual cues: (1) perception of true depth with improved understanding of 3D spatial relationships among anatomical structures, and (2) visualization of critical internal structures along with a more comprehensive visualization of the operating field. METHODS: The stereoscopic AR system has been designed for near-term clinical translation with seamless integration into the existing surgical workflow. It is composed of a stereoscopic vision system, a LUS system, and an optical tracker. Specialized software processes streams of imaging data from the tracked devices and registers those in real time. The resulting two ultrasound-augmented video streams (one for the left and one for the right eye) give a live stereoscopic AR view of the operating field. The team conducted a series of stereoscopic AR interrogations of the liver, gallbladder, biliary tree, and kidneys in two swine. RESULTS: The preclinical studies demonstrated the feasibility of the stereoscopic AR system during in vivo procedures. Major internal structures could be easily identified. The system exhibited unobservable latency with acceptable image-to-video registration accuracy. CONCLUSIONS: We presented the first in vivo use of a complete system with stereoscopic AR visualization capability. This new capability introduces new visual cues and enhances visualization of the surgical anatomy. The system shows promise to improve the precision and expand the capacity of minimally invasive laparoscopic surgeries.

Do Not Judge a Kidney by Its Pelvis: A Rare Case of an Unobstructed Dilated Renal Pelvis.

We present a rare case of an unobstructed dilated renal pelvis in a newborn female. Prenatal imaging documented a large abdominal cyst in a stable fetus. Postnatal imaging confirmed persistence of this large cyst but with an unclear etiology. The child was taken to surgery by the pediatric surgeons with concern for a possible harmful nonurologic diagnosis. Intraoperative findings were that of a severely dilated renal pelvis; however, in the absence of an expected ureteropelvic junction obstruction. Reduction pyeloplasty without interference of the ureteropelvic junction proved successful.

Cost analysis of open radical cystectomy versus robot-assisted radical cystectomy.

Bladder cancer is the fourth and ninth most common malignancy in males and females, respectively, in the U.S. and one of the most costly cancers to manage. With the current economic condition, physicians will need to become more aware of cost-effective therapies for the treatment of various malignancies. Robot-assisted radical cystectomy (RARC) is the latest minimally invasive surgical option for muscle-invasive bladder cancer. Current reports have shown less blood loss, a shorter hospital stay, and a lower morbidity with RARC, as compared with the traditional open radical cystectomy (ORC), although long-term oncologic results of RARC are still maturing. There are few studies that have assessed the cost outcomes of RARC as compared with ORC. Currently, ORC appears to offer a direct cost advantage due to the high purchase and maintenance cost of the robotic platform, although when the indirect costs of complications and extended hospital stay with ORC are considered, RARC may be less expensive than the traditional open procedure. In order to accurately evaluate the cost effectiveness of RARC versus ORC, prospective randomized trials between the two surgical techniques with long-term oncologic efficacy are needed.

Laparoscopic bilateral native nephrectomies with simultaneous kidney transplantation.

UNLABELLED: Study Type--Therapy (case series) Level of Evidence 4. What's known on the subject? and What does the study add? Extirpation of polycystic kidneys for various medical reasons has been performed using many different approaches in attempts to limit morbidity from such a large operation. In indicated patients, it has usually been offered in a staged approach with renal transplantation to avoid graft complications. We published the first case of simultaneous laparoscopic bilateral native nephrectomy with kidney transplant in 2008. The present study shows our continued experience with offering this minimally invasive, single surgery alternative. The results are comparable to a staged laparoscopic approach with significantly shorter total hospital stay and one recovery for the patient and his/her family. OBJECTIVE: • To analyse the perioperative outcomes of native bilateral laparoscopic nephrectomy (BLN) with simultaneous kidney transplantation. PATIENTS AND METHODS: • From November 2000 to April 2011, 37 patients were seen for renal failure secondary to autosomal-dominant polycystic kidney disease (ADPKD) and underwent renal transplant with native nephrectomies at a single tertiary academic centre. • In all, 15 patients underwent BLN for ADPKD followed by simultaneous kidney transplantation. • The other 22 patients underwent BLN for ADPKD with kidney transplant performed at a separate setting. • Demographic data, perioperative outcomes, complications regardless of need for intervention, and graft function were analysed in both groups. RESULTS: • The combined surgery was completed without intraoperative complication in all cases. • The median total operative duration was 372 min, estimated blood loss was 300 mL with two patients requiring transfusion, and the median (range) hospital stay was 5 (3-7) days. • All patients had immediate graft function with additional relief of compressive symptoms. • In comparison to our staged cohort, the simultaneous group had a significantly shorter total hospital stay. • All other outcomes and complication rates were comparable. CONCLUSION: • In ADPKD, a less invasive laparoscopic approach for native nephrectomies with simultaneous renal transplant offers comparable morbidity without graft compromise and the convenience of one operation and one recovery for the patient.

Incontinence after radical prostatectomy: a patient centered analysis and implications for preoperative counseling.

PURPOSE: Incontinence after radical prostatectomy is common yet poorly defined in the current literature. We aimed to accurately characterize incontinence after robot-assisted radical prostatectomy to achieve improved preoperative patient counseling. MATERIALS AND METHODS: After receiving institutional review board approval we performed a cross-sectional survey of the first 600 patients with prostate cancer who underwent robot-assisted radical prostatectomy at our institution. The International Consultation on Incontinence Modular Questionnaire-Lower Urinary Tract Symptoms Quality of Life and Urinary Incontinence Short Form were used to evaluate incontinence and quality of life after robot-assisted radical prostatectomy. Surveys were mailed by a third party. Data were analyzed on the prevalence of incontinence after robot-assisted radical prostatectomy. More specifically we characterized in detail the nature of incontinence and its effect on quality of life. RESULTS: The response rate was 68% (408 of 600 participants). Response time since surgery was 2.5 months to 4 years. Overall incontinence bother scores and ratings of life interference were quite low. Patients reported that most incontinence occurred during physical activity but 35% reported interference with sleep. Of the patients 31% experienced some anxiety due to urinary difficulties and 51% had to occasionally change clothes due to leakage. Patients did not report much interference with traveling, visiting friends or family and family life. The most bothersome aspects of incontinence were its effects on partner relationship, sexual life and energy levels. CONCLUSIONS: Despite patient concerns of incontinence after prostatectomy they report little interference with quality of life.

Robot-assisted radical cystectomy versus open radical cystectomy: a complete cost analysis.

OBJECTIVES: To perform a complete cost analysis comparing robot assisted radical cystectomy (RARC) versus open radical cystectomy (ORC). MATERIAL AND METHODS: After institutional review board approval for data collection, we prospectively recorded perioperative outcomes and costs, such as hospital stay, transfusion rate, readmission rate, and medications for consecutive patients undergoing RARC or ORC. Using actual cost data, we developed a cost decision tree model to determine typical perioperative costs for both RARC and ORC. Multivariate sensitivity analysis was performed to elucidate which variables had the greatest impact on overall cost. Breakeven points with ORC were calculated using our model to better evaluate variable influence. In addition to the above modeled analysis, actual patient costs, including complications 30 days from surgery, were also compared for each procedure. RESULTS: Our model analysis showed that operative time and length of stay had the greatest impact on perioperative costs. Robotic cystectomy became more expensive than open cystectomy at the following break-even points: operating room (OR) time greater than 361 minutes, length of stay greater than 6.6 days, or robotic OR supply cost exceeding $5853. RARC was 16% more expensive when only comparing direct operative costs. Interestingly, actual total patient costs revealed a 38% cost advantage favoring RARC due to increased hospitalization costs for ORC in our cohort. CONCLUSIONS: RARC can provide a cost-effective alternative to ORC with operative time and length of stay being the most critical cost determinants. Higher complication rates with ORC make total actual costs much higher than RARC.

Bleeding after holmium laser enucleation of the prostate: lessons learned the hard way.

OBJECTIVE: To examine specific causes of postoperative bleeding requiring transfusion after holmium laser enucleation of the prostate (HoLEP) in order to enhance preoperative screening and counseling. MATERIALS AND METHODS: After Institutional Review Board (IRB) approval, a retrospective review of a single surgeon's experience of 130 consecutive HoLEPs was performed to specifically examine patients requiring perioperative blood transfusions. All patients from August 2007 to April 2009 who underwent a HoLEP at our institution since its inception were included. These patients' charts were reviewed to gain insight into their bleeding diathesis. A case series report was compiled and compared with the relevant published literature. RESULTS: Of the 130 patients, eight (6.7%) were found to require transfusion postoperatively. Four of these patients required a second operation for completion. These patients had a variety of causes for increased bleeding and subsequent transfusion including: chronic anticoagulation (n = 1), significant cardiac disease requiring maintenance of hemoglobin (n = 4), sepsis with secondary disseminated intravascular coagulation (n = 1), large prostate size (>150 g) (n = 4), underlying prostate cancer (n = 1) and inadequate anesthesia during the procedure leading to patient movement (n = 1). All patients made a full recovery with satisfactory urinary symptom improvement except for one patient with residual incontinence at last follow-up. CONCLUSIONS: Despite the many benefits of holmium laser enucleation, all patients should be counseled regarding the real potential for postoperative blood transfusion. When feasible, any known bleeding risk should be minimized by the surgeon as long it is done safely for the benefit of the patient considering their co-morbidities.

Reactive nitrogen and oxygen species, and iron sequestration contribute to macrophage-mediated control of Encephalitozoon cuniculi (Phylum Microsporidia) infection in vitro and in vivo.

Encephalitozoon cuniculi (Phylum Microsporidia) infects a wide range of mammals, and replicates within resting macrophages. Activated macrophages, conversely, inhibit replication and destroy intracellular organisms. These studies were performed to assess mechanisms of innate immune responses expressed by macrophages to control E. cuniculi infection. Addition of reactive oxygen and nitrogen species inhibitors to activated murine peritoneal macrophages statistically significantly, rescued E. cuniculi infection ex vivo. Mice deficient in reactive oxygen species, reactive nitrogen species, or both survived ip inoculation of E. cuniculi, but carried significantly higher peritoneal parasite burdens than wild-type mice at 1 and 2 weeks post inoculation. Infected peritoneal macrophages could still be identified 4 weeks post inoculation in mice deficient in reactive nitrogen species. L-tryptophan supplementation of activated murine peritoneal macrophage cultures ex vivo failed to rescue microsporidia infection. Addition of ferric citrate to supplement iron, however, did significantly rescue E. cuniculi infection in activated macrophages and further increased parasite replication in non-activated macrophages over non-treated resting control macrophages. These results demonstrate the contribution of reactive oxygen and nitrogen species, as well as iron sequestration, to innate immune responses expressed by macrophages to control E. cuniculi infection.

Robot-assisted radical cystectomy: intermediate survival results at a mean follow-up of 25 months.

OBJECTIVE: To assess the overall and disease-specific survival rates of patients undergoing robot-assisted radical cystectomy (RARC) compared with historical open cystectomy. PATIENTS AND METHODS: Survival, pathological and demographic data were collected on all patients undergoing RARC for bladder cancer from both Tulane University Medical Center and Mayo Clinic Arizona. Of a total of 80 RARCs we only included those with a follow-up of > or =6 months from surgery. Survival curves were compared with those from historical series of open cystectomy. RESULTS: Of the 80 patients 59 were identified as having a follow-up of > or =6 months from the date of surgery. The mean (range) follow-up was 25 (6-49) months. Overall survival rates at 12 and 36 months were 82% and 69%, respectively, and disease-specific survival rates were 82% and 72% at 12 and 36 months, respectively. These results are comparable to survival rates from open cystectomy. As expected, patients with lymph node-positive disease fared worse than those with lymph node-negative disease. Patients with extravesical lymph node-negative disease (pT3, pT4) fared worse than patients with organ-confined lymph node-negative disease. Also, patients with lymph node-positive disease fared worse than those with extravesical lymph node-negative disease, which is consistent with historical results of open cystectomy. CONCLUSIONS: RARC has a comparable survival rate to open cystectomy in the intermediate follow-up. Further study with a longer follow-up and more patients is necessary to determine any long-term survival benefits.

Outpatient prostatectomy: too much too soon or just what the patient ordered.

OBJECTIVES: To evaluate the feasibility of performing a robot-assisted radical prostatectomy (RARP) as an outpatient procedure while maintaining patient satisfaction and safety. Herein we report our experience, selection criteria, and discharge criteria for outpatient RARP. METHODS: We performed a prospective study with 11 patients undergoing extraperitoneal RARP. These patients were counseled before the procedure that they would go home the same evening of the procedure. The patients were then surveyed by a third party shortly after they returned home, using the Patient Judgement System-24, a previously validated instrument for patient satisfaction. Sociodemographic data, comorbidities, and outcomes were collected for analysis. RESULTS: All patients were successfully discharged the same day of surgery. Mean patient age was 62.2 years with a mean body mass index of 26 kg/m(2). Mean operative time was 117.6 minutes, console time was 76.7 minutes, and estimated blood loss was 168.2 mL. Mean indwelling catheter time was 7.5 days. No complications occurred in this series of patients. Satisfaction was unanimously high in all patients surveyed, with most scores over 90% on the Patient Judgement System-24. No patient reported any ill effects from the shortened stay or felt rushed to leave the hospital. CONCLUSIONS: The early experience with extraperitoneal RARP as a same day surgery is promising. Preoperative patient counseling and selection is paramount. Patient satisfaction is not adversely affected by the shortened stay. Surgeon experience, assessment of intraoperative findings, and adequate postoperative assessment are essential.