Production of afucosylated antibodies in CHO cells by coexpression of an anti-FUT8 intrabody.

Some effector functions prompted by immunoglobulin G (IgG) antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC), strongly depend on the N-glycans linked to asparagine 297 of the Fc region of the protein. A single α-(1,6)-fucosyltransferase (FUT8) is responsible for catalyzing the addition of an α-1,6-linked fucose residue to the first GlcNAc residue of the N-linked glycans. Antibodies missing this core fucose show a significantly enhanced ADCC and increased antitumor activity, which could help reduce therapeutic dose requirement, potentially translating into reduced safety concerns and manufacturing costs. Several approaches have been developed to modify glycans and improve the biological functions of antibodies. Here, we demonstrate that expression of a membrane-associated anti-FUT8 intrabody engineered to reside in the endoplasmic reticulum and Golgi apparatus can efficiently reduce FUT8 activity and therefore the core-fucosylation of the Fc N-glycan of an antibody. IgG1-producing CHO cells expressing the intrabody secrete antibodies with reduced core fucosylation as demonstrated by lectin blot analysis and UPLC-HILIC glycan analysis. Cells engineered to inhibit directly and specifically alpha-(1,6)-fucosyltransferase activity allows for the production of g/L levels of IgGs with strongly enhanced ADCC effector function, for which the level of fucosylation can be selected. The quick and efficient method described here should have broad practical applicability for the development of next-generation therapeutic antibodies with enhanced effector functions.

Prenatal vesico-allantoic cyst outcome - a spectrum from patent urachus to bladder exstrophy.

OBJECTIVE: The purpose of this study is to assess the prognostic significance of a cystic vesico-allantoic communication discovered on prenatal ultrasound in terms of its effect on the developing urinary tract. METHODS: Multi-institution review of prenatal screening ultrasound was performed between 2004 and 2015 to identify cases of in utero communication between the fetal urinary bladder and a cystic dilatation of the umbilical cord. Gestational age at diagnosis, perinatal evolution, and eventual urinary tract outcome are described and compared with existing literature. RESULTS: Five cases of cystic vesico-allantoic communication were identified on second-trimester screening ultrasound. Serial ultrasounds showed an increase in the size of the umbilical cystic component with gestational age, followed by its eventual rupture prior to delivery. All neonates had urinary leakage through the inferior portion of the umbilicus with bladder prolapse ranging from patent urachus to partial bladder exstrophy. CONCLUSIONS: An isolated prenatal ultrasound finding of an hourglass communication between the fetal bladder and a cyst of the umbilical cord should be considered predictive of a spectrum from patent urachus to bladder exstrophy. Given the significant ramications on the developing urinary tract, the prenatal finding of vesico-allantoic cyst warrants referral to a high-risk obstetrical center with urologic consultation.

Bladder triplication associated with exstrophy variant.

A one-day old full-term female neonate presented with a duplicate bladder and exstrophy variant including a patch of exstrophic or ectopic mucosa, duplicate vagina, uterus, and two complete bladders. We report on the surgical management performed in this case and functional urinary results based on a synchronous urodynamic study of the duplicate bladders. To our knowledge, the occurrence of duplicate bladder exstrophy variant with complete urinary continence has not been previously reported.

Arsenal of nanobodies shows broad-spectrum neutralization against SARS-CoV-2 variants of concern in vitro and in vivo in hamster models.

Nanobodies offer several potential advantages over mAbs for the control of SARS-CoV-2. Their ability to access cryptic epitopes conserved across SARS-CoV-2 variants of concern (VoCs) and feasibility to engineer modular, multimeric designs, make these antibody fragments ideal candidates for developing broad-spectrum therapeutics against current and continually emerging SARS-CoV-2 VoCs. Here we describe a diverse collection of 37 anti-SARS-CoV-2 spike glycoprotein nanobodies extensively characterized as both monovalent and IgG Fc-fused bivalent modalities. The nanobodies were collectively shown to have high intrinsic affinity; high thermal, thermodynamic and aerosolization stability; broad subunit/domain specificity and cross-reactivity across existing VoCs; wide-ranging epitopic and mechanistic diversity and high and broad in vitro neutralization potencies. A select set of Fc-fused nanobodies showed high neutralization efficacies in hamster models of SARS-CoV-2 infection, reducing viral burden by up to six orders of magnitude to below detectable levels. In vivo protection was demonstrated with anti-RBD and previously unreported anti-NTD and anti-S2 nanobodies. This collection of nanobodies provides a potential therapeutic toolbox from which various cocktails or multi-paratopic formats could be built to combat multiple SARS-CoV-2 variants.

Burnout among caregivers in the era of the COVID-19 pandemic: Insights and challenges.

The arrival of COVID-19 modified the way we live, care, teach, and self-care tremendously. Indeed, our personal and professional lives were much affected, and despite the fact that Canadian healthcare workers (HCW) and systems had some time to prepare in comparison to other countries, we faced a scenario that resembled no other. Adding the negative effects of a never-before-seen pandemic to the already well-known risk of burnout among HCW could surely increase the likelihood of colleagues, as well as ourselves, being affected. We hereby reviewed the numerous factors that could increase the risk of burnout amid this ongoing pandemic.

Burnout in Canadian urology: Cohort analysis from the 2018 Canadian Urological Association census.

INTRODUCTION: Physician burnout is associated with medical error, patient dissatisfaction, and poorer physician health. Urologists have reported high levels of burnout and poor work-life integration compared with other physicians. Burnout rates among Canadian urologists has not been previously investigated. We aimed to establish the prevalence of Canadian urologist burnout and associated factors. METHODS: In the 2018 Canadian Urological Association census, the Maslach Burnout Inventory questions were assigned to all respondents. Responses from 105 practicing urologists were weighted by region and age group to represent 609 urologists in Canada. Burnout was defined as scoring high on the scales of emotional exhaustion or depersonalization. Demographic and practice variables were assessed to establish factors associated with burnout. Comparisons were made to the results of the 2016 American Urological Association census. RESULTS: Overall, 31.8% of respondents met the criteria for burnout. There was no effect of subspecialty practice or practice setting on burnout. On univariate analysis, rates of burnout were highest among urologists under financial strain (50.8%), female urologists (45.3%), and early-to-mid-career urologists (37.7-41.8%). Factors associated with demanding practices and poor work-life integration were predictive of burnout. A total of 12.2% of urologists reported seeking burnout resources and 54.0% wished there were better resources available. CONCLUSIONS: Urologist burnout in Canada is lower than reported in other countries, but contributing factors are similar. Urologists who report demanding clinical practices (particularly in early-to-mid career), poor work-life integration, financial strain, and female gender may benefit from directed intervention for prevention and management of burnout. Burnout resources for Canadian urologists require further development.

Survey of Canadian urology programs: Which aspects of the Canadian Residency Matching Service (CaRMS) application are the most important?

INTRODUCTION: For medical students, determining which aspects of the Canadian Residency Matching Service (CaRMS) application are the most important when applying to residency programs can be challenging. Due to the lack of current and reliable information on the selection criteria of Canadian urology residency programs, we surveyed each program about which criteria are the most important when selecting future residents in order to provide medical students with more transparency and programs with a better idea of how their criteria compare to those of others. METHODS: An electronic survey was sent to all 13 Canadian urology residency programs (both program directors and selection committee members). It asked respondents to rate each aspect of the application on a five-point Likert scale. Following a 100% response rate from program directors, the same survey was sent to selection committee members. A numeric mean score was calculated for each individual aspect surveyed to create an overall rank list of the components. Independent samples t-tests (two groups) were used to compare the scores of program directors vs. program committee members and of francophone programs vs. anglophone programs. RESULTS: Forty-three urologists involved in the application process answered. The three most important aspects were rotation performance at the respondent's institution (4.95±0.21), quality of reference letters from a urologist (4.60±0.62), and interview performance (4.49±0.63). There were no statistically significant differences between program directors and committee members for mean score of any aspect surveyed. Compared to anglophone programs, francophone programs gave statistically more significant importance to French proficiency (p<0.001) and pre-clinical academic performance (p=0.0272), while giving less importance to English proficiency (p<0.001). CONCLUSIONS: Canadian urology residency programs are similar in that they rank "clinical performance during a rotation at their school" as the most important selection criteria when choosing a future urology trainee. Graduate degrees, career plans, and reference letters from non-urologists have less impact when choosing future urology residents. Francophone schools and anglophone schools differ in the importance of language proficiency and preclinical grades as selection criteria for urology residency. This study will provide future urology applicants with more information and transparency when applying to urology programs in Canada and be of use to urology residency programs that must now publish their selection criteria.

Topical triamcinolone for persistent phimosis.

PURPOSE: Between 2% and 5% of uncircumcised boys have persistent or pathological phimosis. Traditional treatment is usually circumcision. Recently medical treatment with topical corticosteroids has become more popular. We evaluated the efficacy of the topical steroid triamcinolone compared to foreskin retraction with an emollient cream and verified the long-term success rate of these treatments. MATERIALS AND METHODS: We performed a double-blind, randomized, placebo controlled study to compare 2-month twice daily treatment with emollient cream (placebo group 1) vs 0.1% triamcinolone (experimental group 2). Boys between ages 3 and 12 years with persistent or pathological phimosis were included in analysis. Study EXCLUSION criteria were previous treatment with topical corticosteroid, untreated balanitis and any known medical condition with immune system impairment. Patients were seen 2, 4 and 12 months after treatment initiation. Success was defined as complete, easy foreskin retraction at 4 and 12 months. Statistical analysis was done using Fisher's exact test. RESULTS: We enrolled 63 patients, of whom 43 completed the study. Despite multiple attempts 20 patients had incomplete followup and were excluded from study. Placebo group 1 included 25 patients and triamcinolone group 2 included 21. In group 1 the success rate was significantly lower than in group 2 (9 patients or 39% vs 16 or 76%, p = 0.0086). At 2 months 5 and 16 nonresponders in groups 2 and 1, respectively, were treated in nonblinded fashion with topical triamcinolone. In this subgroup 1 of 3 group 2 patients and 6 of 13 in group 1 achieved complete, easy retraction. Two and 1 patients were lost to followup in groups 1 and 2, respectively. Circumcision was required in only 5 patients (11.6%), including 4 (17.4%) initially in group 1. No complications were noted in either group. CONCLUSIONS: Triamcinolone is a highly effective and safe short-term treatment for persistent physiological or pathological phimosis. However, at long-term followup recurrence is frequent and not rare with triamcinolone and it may require re-treatment or circumcision.

Paratesticular rhabdomyosarcoma presenting as thickening of the tunica vaginalis.

Pediatric paratesticular primary malignant tumors are rare, and most of them correspond to rhabdomyosarcomas (RMS). Paratesticular RMS has a better prognosis than RMS originating in other locations, because of its favorable histology and possible early diagnosis. The diagnosis relies upon the US findings of a paratesticular hypervascular mass in an adequate clinical setting. We report the unusual appearance of an infiltrating RMS tumor in a 3-year-old boy. The tumor presented as thickening of the tunica vaginalis. Atypical US appearance and discordance with clinical features caused suspicion of a tumoral process, allowing early diagnosis and treatment.

Burnout in Urology: Findings from the 2016 AUA Annual Census.

INTRODUCTION: Physician burnout is linked to decreased job performance, increased medical errors, interpersonal conflicts and depression. Two recent multispecialty studies showed that urologists had the highest rate of burnout. However, these reports were limited by a low sample size of urologists (119). We aimed to establish the prevalence of urologist burnout and associated factors. METHODS: In the 2016 American Urological Association Census, Maslach Burnout Inventory questions were randomly assigned to half of the respondents. Using matrix sampling, the 1,126 practicing urologists who received and answered the Maslach Burnout Inventory questions represented the entire 2,301 who completed the census. Burnout was defined as scoring high on the scales of emotional exhaustion or depersonalization. Demographic and practice variables were assessed to establish factors correlating to burnout. RESULTS: Overall 38.8% of urologists met the criteria for burnout, with 17.2% scoring high for emotional exhaustion and 37.1% scoring high for depersonalization. Multivariate analysis revealed that urologist burnout is associated with more patient visits per week, younger age, being in a subspecialty area other than pediatric or oncology, in solo or multispecialty practice, practice size greater than 2 and greater number of work hours per week. CONCLUSIONS: These results suggest that the burnout rate for urologists is lower than previously reported, and are consistent with rates reported in other medical and surgical specialties. However, burnout continues to be an important issue. Greater workload correlated with increased burnout while other practice patterns appeared to be protective. It is critical to keep urologists in the workforce to help lessen projected shortages.

Ossifying Renal Tumor of Infancy: Report of a Case With Positive WT1 Immunohistochemistry and High Mitotic Index and Review of the Literature.

Ossifying renal tumor of infancy (ORTI) is a rare, benign pediatric tumor of the kidney. Since first reported by Chatten in 1980, 23 cases have been published. Previous authors have argued that ORTI might originate from nephrogenic rests, thereby sharing a pathogenic relationship with Wilms' tumor (WT). ORTI is characterized histologically by a population of polygonal osteoblast-like cells around an osteoid core and densely cellular component of blastemal-like or spindle cells. While the immunohistochemical profile of the cellular components has been reported, to the best of our knowledge, the status of WT1 expression has only been reported once, where it showed negative marking. Mitoses have been described only sporadically in this neoplasm. We report on a case of ORTI with positive WT1 immunohistochemical marking and numerous mitoses. This case highlights a possible pitfall for misdiagnosing ORTI as a WT and provides additional insight into its pathogenesis.

Role of specific protein kinase C isoforms in modulation of beta1- and beta2-adrenergic receptors.

The function of beta-adrenergic receptor (betaAR) is modulated by the activity status of alpha1-adrenergic receptors (alpha1ARs) via molecular crosstalk, and this becomes evident when measuring cardiac contractile responses to adrenergic stimulation. The molecular mechanism underlying this crosstalk is unknown. We have previously demonstrated that overexpression of alpha1B-adrenergic receptor (alpha1BAR) in transgenic mice leads to a marked desensitization of betaAR-mediated adenylyl cyclase stimulation which is correlated with increased levels of activated protein kinase C (PKC) beta, delta and [J. Mol. Cell. Cardiol. 30 (1998) 1827]. Therefore, we wished to determine which PKC isoforms play a role in heterologous betaAR desensitization and also which isoforms of the betaAR were the molecular target(s) for PKC. In experiments using constitutively activated PKC expression constructs transfected into HEK 293 cells also expressing the beta2AR, constitutively active (CA)-PKC overexpression was first confirmed by immunoblots using specific anti-PKC antibodies. We then demonstrated that the different PKC subtypes lead to a decreased maximal cAMP accumulation following isoproterenol stimulation with a rank order of PKCalpha > or = PKCzeta>PKC>PKCbetaII. However, a much more dramatic desensitization of adenylyl cyclase stimulation was observed in cells co-transfected with different PKC isoforms and beta1AR. Further, the modulation of beta1AR by PKC isoforms had a different rank order than for the beta2AR: PKCbetaII>PKCalpha>PKC>PKCzeta. PKC-mediated desensitization was reduced by mutating consensus cAMP-dependent protein kinase (PKA)/PKC sites in the third intracellular loop and/or the carboxy-terminal tail of either receptor. Our results demonstrate therefore that the beta1AR is the most likely molecular target for PKC-mediated heterologous desensitization in the mammalian heart and that modulation of adrenergic receptor activity in any given cell type will depend on the complement of PKC isoforms present.

Long-term functional outcomes after penoscrotal hypospadias repair: A retrospective comparative study of proximal TIP, Onlay, and Duckett.

INTRODUCTION: A variety of techniques are available for proximal hypospadias repair. Onlay, proximal tubularized incised plate (TIP), and Duckett are among the popular choices because they can be performed as a one-step procedure. However, the decision to select a procedure often comes down to the surgeon's preference rather than that supported by evidence-based data. In particular, there is a paucity of literature on the long-term urinary outcomes after proximal hypospadias repair. OBJECTIVE: The aim of this study was to evaluate the evolution of long-term uroflowmetry parameters after proximal hypospadias surgery over a long-term follow-up including the adolescent period. STUDY DESIGN: Files from patients who underwent primary proximal hypospadias repair at our institution between 1997 and 2001 were reviewed. Only patients with documented serial postoperative uroflowmetry profiles at follow-up visits were included. Comparison between surgeries (pTIP vs. Onlay vs. Duckett) was performed according to the following postoperative time interval endpoints: 0-1 years, 1-2, >2-4, >4-6, >6-10, >10-12, and >12 years. Maximal urinary flow rate (Qmax) in relation to voiding volume (VV) adjusted for age or body surface area (BSA) were also evaluated in comparison to normal children using established Miskolc nomograms and compared between surgery techniques. RESULTS: Fifty-two patients met the inclusion criteria with a median follow-up of 10 years: 25 (59.6%) TIP, 18 (34/6%) Onlay, and nine (17.3%) Duckett. Overall, Qmax increased progressively according to time and age in particular during the period covering adolescence. At follow-up 12 years postoperatively, median Qmax values were 18.5 mL/second, 13.8 mL/second and 16.6 mL/second for TIP, Onlay, and Duckett, respectively, with no significant differenced detected between groups (p = 0.5) see figure). Compared with normal children when adjusted for voiding volume and BSA, the proportion of obstructive uroflowmetry patterns defined as Qmax<25th percentile of nomograms was more prevalent in patients aged 3-7 years old at 83.8% but decreased to less than 21.2% in patients aged >13 years for all procedures (see figure). DISCUSSION: These results are consistent with previous work showing frequently reduced Qmax after hypospadias surgery with great potential for improvement at puberty. CONCLUSION: These results suggest that the obstructive urinary flow pattern observed in patients early on is a frequent finding after proximal hypospadias surgery. However, because of the remarkable improvement observed at puberty a watchful waiting approach is proposed in order to avoid unnecessary intervention.

Characterization of the rat RALDH1 promoter. A functional CCAAT and octamer motif are critical for basal promoter activity.

Retinal dehydrogenase type 1 (RALDH1) catalyzes the oxidation of retinal to retinoic acid (RA), a metabolite of vitamin A important for embryogenesis and tissue differentiation. Rat RALDH1 is expressed to high levels in developing kidney, and in stomach, intestine epithelia. To understand the mechanisms of the transcriptional regulation of rat RALDH1, we cloned a 1360-base pair (bp) 5'-flanking region of RALDH1 gene. Using luciferase reporter constructs transfected into HEK 293 and LLCPK (kidney-derived) cells, basal promoter activity was associated with sequences between -80 and +43. In this minimal promoter region, TATA and CCAAT cis-acting elements as well as SP1, AP1 and octamer (Oct)-binding sites were present. The CCAAT box and Oct-binding site, located between positions -72 and -68 and -56 and -49, respectively, were shown by deletion analysis and site-directed mutation to be critical for promoter activity. Nuclear extracts from kidney cells contain proteins specifically binding the Oct and CCAAT sequences, resulting in the formation of six complexes, while different patterns of complexes were observed with non-kidney cell extracts. Gel shift assays using either single or double mutations of the Oct and CCAAT sequences as well as super shift assays demonstrated single and double occupancy of these two sites by Oct-1 and CBF-A. In addition, unidentified proteins also bound the Oct motif specifically in the absence of CBF-A binding. These results demonstrate specific involvement of Oct and CCAAT-binding proteins in the regulation of RALDH1 gene.

Long-term functional outcomes of distal hypospadias repair: a single center retrospective comparative study of TIPs, Mathieu and MAGPI.

INTRODUCTION: Tubularized Incised-Plate (TIP) urethroplasty is currently the preferred technique for distal hypospadias repair. Nevertheless, concerns have been raised on the long-term functionality of the reconstructed neourethra. OBJECTIVE: The aim of this study is to evaluate long-term uroflowmetry parameters' evolution after TIP surgery over a long-term follow-up including the adolescent period. TIP patients were compared to normal children using established Miskolc nomograms, as well as to patients who underwent Mathieu and Meatal Advancement and Glanuloplasty (MAGPI) surgery repairs for distal hypospadias. STUDY DESIGN: Files from patients who underwent primary distal hypospadias repair at our institution between January 1, 1997 and January 31, 2001 were reviewed. Only patients with documented serial postoperative uroflowmetry profiles at follow-up visits were included. Comparison between surgeries (TIP vs. Mathieu vs. MAGPI) was performed according to the following postoperative time interval endpoints: 0-6 months, 6-12 months, 12-24 months, 24-48 months, 4-6 years, 6-10 years and >10 years. Maximal urinary flow rate (Qmax) in relation to Voiding Volume (VV) adjusted for Age or Body Surface Area (BSA) were also evaluated in comparison to normal children using established Miskolc nomograms and compared between surgery techniques. RESULTS: 153 patients met the inclusion criteria: 70 (43%) TIP, 24 (17%) Mathieu and 59 (35%) MAGPI. Overall, Qmax increases progressively according to time and age and in particular during the period covering adolescence with a similar trend regardless of the type of surgery. Uroflowmetry profiles in terms of Qmax, VV and PVR were equivalent between surgeries at each examined time point. At 10 years of follow-up postopertively, mean Qmax were 17.2 ml/s, 18.8 ml/s and 21.6 ml/s respectively with no significant difference detected between groups (p = 0.344). Compared to normal children when adjusted for voiding volume and BSA, the proportion of obstructive uroflowmetry patterns defined as Qmax< 5th percentile of nomograms was more prevalent in patients aged 2-7 years old at 60% but decreased to less than 10%in patients aged >13 years for all procedures combined (see abstract figure) but without detected differences between surgery types (p = 0.276). DISCUSSION: After sub-optimal obstructive maximal urinary flows in the early postoperative period, hypospadias patients treated with TIP exhibit favourable long-term evolution with age and during adolescence in particular compared to normal children. In addition, a similar trend was found for patients treated with Mathieu and MAGPI with no significant differences detected between procedures. Nevertheless, because of the relatively small sample size we cannot exclude that a statistical difference between surgeries would have been detected if the study was adequately powered on every endpoint. Nevertheless and also as suggested by the values obtained, this potential difference may be quite small and not clinically relevant. CONCLUSION: These results suggest that the obstructive urinary flow pattern observed in patients early on is possibly an intrinsic feature associated to the malformation itself and may be less of a consequence of the surgical technique.

Renal transplantation in children with reconstructed bladders.

In this article, we review the outcome and complications of renal transplantation in patients with reconstructed bladders and address specific issues and controversies regarding the management of such cases. Twenty-five articles covering the subjects of renal transplantation, lower urinary tract anomalies, and bladder reconstruction have been selected. Although urologic complications are higher when kidneys are transplanted into reconstructed bladders or urinary diversions, the graft and patient survival rates in most series are comparable with those transplanted into nonreconstructed bladders. The reported series of renal transplantation into abnormal bladders are small, and controlled studies are lacking. Bladder reconstruction should be performed before transplantation when clinically indicated.

Laparoscopic orchidopexy: the easy way to go.

BACKGROUND/PURPOSE: Intraabdominal testes represent less than 10% of cryptorchid testicles, and yet, they are the most challenging to correct. In the last 15 years, the two-stage Fowler-Stephens orchidopexy has gained popularity. The traditional approach includes laparoscopic or open clipping of the testicular vessels (first stage) and open inguinal orchidopexy (second stage). We present our experience with 2-stage orchidopexy with both stages done through a laparoscopic approach. MATERIALS AND METHODS: Over a recent 5-year period, we reviewed patients operated for intraabdominal testis using a two-stage laparoscopic orchidopexy with a minimum of 1-year follow-up. In this study, success is defined as a nonatrophic, intrascrotal testis. Fifteen patients met the inclusion criteria, and none were lost to follow-up. RESULTS: In the 15 patients, 11 had a unilateral intraabdominal testis, and 4 had bilateral cryptorchidism, with one of the 2 testes intraabdominal. The first stage was done at a mean age of 32 months, and the average time between the two stages was 9.7 months. All procedures (31) were done on an outpatient basis. Only 2 complications occurred, one scrotal hematoma and one redo first stage because of unsuccessful clipping noted at the time of planned second stage. The success rate is 93.3% (14/15). All testicles are intrascrotal, and all but 1 have maintained preoperative volume. CONCLUSION: Two-stage laparoscopic orchidopexy is a fairly easy surgical procedure with minimum morbidity and high short term success rate. A larger cohort of patients with long-term follow-up is needed to substantiate these findings.