The STING in the tale of Teflon®: Delayed ureteric obstruction after subureteric transurethral injection with polytetrafluoroethylene paste for vesicoureteral reflux.

INTRODUCTION: "Subureteric Teflon INGection" (STING) of polytetrafluoroethylene (PTFE/polytef) paste to treat vesicoureteral reflux (VUR) in children was popularised in 1984. It was later abandoned as an implantation material because of the possibility of migration from the injection site. Giant-cell foreign-body granuloma to Polytef in the bladder is a rare cause of ureteric obstruction. Only a handful of cases have been reported in the literature. METHODS: We performed a prospective analysis of a series of 6 adult patients who had childhood STING and presented with foreign-body granuloma to Polytef in the bladder. We report their clinical presentation, findings and treatment. RESULTS: 1 male and 5 females with a history of STING procedure in childhood for VUR presented in later life with foreign-body granuloma to Polytef. The median age at first STING procedure and at presentation to the Urology Department was 3 and 34 years respectively. The most common clinical presentations were flank pain and urinary tract infection (UTI) and all patients had radiological findings of calcified lesions at the vesicoureteric junction(s). 4 patients had histological findings of giant-cell foreign-body granuloma. 4 patients required definitive ureteric reimplantation. CONCLUSION: Polytef granuloma causing distal ureteric obstruction may give rise to significant morbidity and renal damage. Due to the likelihood of progression of the granuloma, excision and ureteric reimplantation is considered the standard approach in the management of patients with viable kidneys. LEVEL OF EVIDENCE: Level 5.

End-to-end collaboration to transform biopharmaceutical development and manufacturing.

An ambitious 10-year collaborative program is described to invent, design, demonstrate, and support commercialization of integrated biopharmaceutical manufacturing technology intended to transform the industry. Our goal is to enable improved control, robustness, and security of supply, dramatically reduced capital and operating cost, flexibility to supply an extremely diverse and changing portfolio of products in the face of uncertainty and changing demand, and faster product development and supply chain velocity, with sustainable raw materials, components, and energy use. The program is organized into workstreams focused on end-to-end control strategy, equipment flexibility, next generation technology, sustainability, and a physical test bed to evaluate and demonstrate the technologies that are developed. The elements of the program are synergistic. For example, process intensification results in cost reduction as well as increased sustainability. Improved robustness leads to less inventory, which improves costs and supply chain velocity. Flexibility allows more products to be consolidated into fewer factories, reduces the need for new facilities, simplifies the acquisition of additional capacity if needed, and reduces changeover time, which improves cost and velocity. The program incorporates both drug substance and drug product manufacturing, but this paper will focus on the drug substance elements of the program.

Transcriptomic analysis of IgG4 Fc-fusion protein degradation in a panel of clonally-derived CHO cell lines using RNASeq.

In this study, we report an investigation of a panel of clonally-derived Chinese hamster ovary (CHO) cell lines exhibiting variability in the proportion of full-length IgG4 Fc-fusion protein produced. The recombinant protein was found to be degraded during cell culture into four shorter "clipped" species (three of the four cleavage sites occurred at arginine residues) and preliminary analyses suggested that a host cell enzyme was responsible for proteolysis. To identify the specific enzyme responsible, RNA sequencing was used to identify gene expression differences between the cell lines with a "high" and "low" clipping phenotype. From this analysis, six protease-encoding genes were found to be significantly upregulated in those cell lines yielding the lowest proportion of full-length IgG4 Fc-fusion protein. Four of these protease candidates were deprioritized after examination of their cleavage site specificity. The remaining enzymes, Adam19 and Furin, were found to be capable of cleavage at arginine residues, and inhibitors for both proteases were added to cell-free media to determine if the product degradation could be reduced. While the Adam19 inhibitor had no impact, Furin inhibitor I (specific for the proprotein convertase family of enzymes) was found to result in a 33-39% increase in complete IgG4 Fc-fusion protein when compared with untreated samples.

Evaluation of the Impact of Ureteroscope, Access Sheath, and Irrigation System Selection on Intrarenal Pressures in a Porcine Kidney Model.

Introduction: Elevated intrarenal pressure (IRP) during flexible ureterorenoscopy (FURS) is a predictor of postoperative complications. The aim of this study is to evaluate IRP during FURS in a porcine kidney model to determine the safest combination of irrigation device, ureteral access sheath (UAS), and ureteroscope. Methods: Urinary tracts were harvested from Landrace pigs slaughtered for the food chain. Two flexible ureteroscopes, 8.7F and 9.5F, were evaluated. Irrigation systems evaluated included the following: TraxerFlow™ (Rocamed, France), SAPS™ single action pumping system (Boston Scientific), Pathfinder Plus™ (Utah Medical), and a manual "bag squeeze." This experiment was conducted with no UAS, followed by an 11/13F UAS and then a 12/14F UAS. IRPs were measured in the prepared porcine kidney during all possible combinations of scope, UAS, and irrigation system. Results: Pressures were significantly reduced when using 12/14F UAS compared with 11/13F UAS (16.45 ± 5.3 cmH2O vs 32.73 ± 35.66 cmH2O, p = 0.006), and when using 11/13F UAS compared with no UAS (32.73 ± 35.66 cmH2O vs 49.5 ± 29.36 cmH2O, p = 0.02). Pressures were significantly reduced with the 8.7F scope compared with the 9.5F scope (24.1 ± 21.24 cmH2O vs 41.68 ± 34.5 cmH2O, p = 0.001). SAPS generates significantly greater IRP than TraxerFlow, Pathfinder Plus, and a "bag squeeze" (p < 0.05). The most dangerous combination was using the SAPS, no UAS, and larger ureteroscope leading to an IRP of 100.6 ± 16.1 cmH2O. The safest combination was using Pathfinder Plus with a 12/14F UAS and smaller ureteroscope giving an IRP of 11.6 ± 3.65 cmH2O. Conclusion: IRPs are reduced by selecting larger UAS and a small ureteroscope. The SAPS generates significantly higher IRPs than other irrigation systems. To maintain safe IRPs during FURS, urologists should use large UAS, narrow ureteroscopes, and be cautious in the selection of an irrigation device.

Evaluation of a new disposable flexible ureterorenoscope and comparison to an established disposable flexible ureterorenoscope: a prospective, observational study.

PURPOSE: To objectively and subjectively assess the performance and surgical outcomes of the new Innovex EU-scope™ single-use digital flexible ureteroscope (fURS). METHODS: A prospective cohort study was carried out (August 2019 to May 2020). The new single-use fURS (Innovex Medical Devices Co. Shanghai, China) was analysed with regard to visibility, manoeuvrability, laser interference and overall performance using a validated Likert scale. Outcomes are compared to the LithoVue™ (Boston Scientific, Marlborough, MA). RESULTS: One hundred patients were included in this study. 50 cases underwent retrograde fURS using the Innovex EU-scope™ and 50 with the LithoVue™. There were no differences in the patient demographics data, or operative data between the two groups. The Innovex EU-scope™ scored higher visibility scores compared to the LithoVue™, median 4, interquartile range (IQR) (4-4), vs. 3.5, IQR (3-5), p = 0.5086. Both scopes had similar manoeuvrability scores. The Innovex EU-scope™ scored significantly lower with regard to comfort compared to the LithoVue, median 4 IQR (3-4) vs. 4.5 IQR (4-5), p = 0.0445. Whereas, laser interference, affected the Innovex much less than the LithoVue™. Both scopes scored well for overall performance. The median overall performance score for the Innovex was 4 IQR (4-4) vs. 4 IQR (4-5). CONCLUSIONS: This Innovex EU-scope™ has good objective and subjective visibility and manoeuvrability profiles. This single-use flexible ureteroscope may achieve similar clinical outcomes to an established single use instrument.

Clonal variation in productivity and proteolytic clipping of an Fc-fusion protein in CHO cells: Proteomic analysis suggests a role for defective protein folding and the UPR.

Product degradation, such as clipping, is a common quality issue in the production of Fc-fusion proteins from Chinese hamster ovary (CHO) cells. Degradation of proteins is mainly due to the action of either intracellular or extracellular host cell proteases. This study was carried out to understand more fundamentally the intracellular events that may play a role in determining why cell lines from the same cell line development project can vary with regards to the extent of Fc-fusion protein clipping. The cell lines that displayed the highest levels of clipping also produced less product than the cell lines with a lower level of clipping. In this study we applied differential quantitative label-free LC-MS/MS proteomic analysis to group clonally-derived cell lines (CDCLs) based on the level of clipping of the Fc-fusion protein. The analysis was carried out over two times points in culture and clones were designated as either having 'high' or 'low' clipping phenotypes. We have identified 200 differentially expressed proteins using quantitative label-free LC-MS/MS analysis between the two experimental groups. Functional assessment of the resultant proteomic data using Gene Ontology analysis showed a significant enrichment of biological processes and molecular functions related to protein folding, response to unfolded protein and protein translation. The levels of several proteases were also increased. This study identified protein targets that could be modified using cell line engineering approaches to improve the quality of recombinant Fc-fusion protein production in the biopharmaceutical industry.

Helminth vaccines: from mining genomic information for vaccine targets to systems used for protein expression.

The control of helminth diseases of people and livestock continues to rely on the widespread use of anti-helminthic drugs. However, concerns with the appearance of drug resistant parasites and the presence of pesticide residues in food and the environment, has given further incentive to the goal of discovering molecular vaccines against these pathogens. The exponential rate at which gene and protein sequence information is accruing for many helminth parasites requires new methods for the assimilation and analysis of the data and for the identification of molecules capable of inducing immunological protection. Some promising vaccine candidates have been discovered, in particular cathepsin L proteases from Fasciola hepatica, aminopeptidases from Haemonchus contortus, and aspartic proteases from schistosomes and hookworms, all of which are secreted into the host tissues or into the parasite intestine where they play important roles in host-parasite interactions. Since secreted proteins, in general, are exposed to the immune system of the host they represent obvious candidates at which vaccines could be targeted. Therefore, in this article, we consider the potential values and uses of algorithms for characterising cDNAs amongst the collated helminth genomic information that encode secreted proteins, and methods for their selective isolation and cloning. We also review the variety of prokaryotic and eukaryotic cell expression systems that have been employed for the production and downstream purification of recombinant proteins in functionally active form, and provide an overview of the parameters that must be considered if these recombinant proteins are to be commercialised as vaccine therapeutics in humans and/or animals.

High-level production of a monoclonal antibody in murine myeloma cells by perfusion culture using a gravity settler.

A perfusion system is described for the production of a human monoclonal antibody in non-secreting murine myeloma (NS0) cells that was previously shown to be difficult to produce at high levels using fed-batch culture. The perfusion system was based on the use of a commercially available cell settler as the separation device to separate the cells from the culture. Separation efficiency of the cell settler was above 98%. Based on the growth and glucose consumption rates, fresh media was added to the culture and the turnover rate for the bioreactor was set at a maximum of 1.5 times the bioreactor volume per day. The perfusion process resulted in twice the maximum viable cell densities and up to three times the total protein production in a 53-day run period when compared to the fed-batch process. In addition, charge heterogeneity of the antibody as measured by ion exchange chromatography was lower for material purified from the perfusion runs compared to fed-batch. Perfusion mode of culture using a commercially available gravity settler is therefore a viable alternative to fed-batch mode for high-level production of this monoclonal antibody in NS0 cells.

Capsaicin receptor TRPV1 in urothelium of neurogenic human bladders and effect of intravesical resiniferatoxin.

OBJECTIVES: To study TRPV1 immunoreactivity in the urothelium of patients with neurogenic detrusor overactivity (NDO) before and after treatment with resiniferatoxin (RTX) and controls. Functional capsaicin TRPV1 receptors have been demonstrated in urothelial cells of rodent urinary bladder, and TRPV1-knockout mice exhibit diminished nitric oxide and stretch-evoked adenosine triphosphate release from urothelial cells. In patients with NDO, TRPV1 suburothelial nerve density is increased, which is reversed by successful treatment with intravesical RTX. However, the role of urothelial TRPV1 in human bladder disorders is unknown. METHODS: Flexible cystoscopic bladder biopsies were obtained from 14 patients with NDO before and after treatment with RTX and from 8 control patients. Using a specific antibody for immunostaining, TRPV1 immunoreactivity in the urothelium was quantified by image analysis. RESULTS: TRPV1 immunoreactivity was observed in basal and apical urothelial cells. Basal cell layer TRPV1 immunoreactivity was significantly increased in NDO compared with control bladders (P = 0.003). In 5 patients who responded clinically to RTX, basal cell layer and total urothelial TRPV1 immunoreactivity decreased significantly after treatment (P = 0.032 and P = 0.016, respectively). The decreases in the basal cell layer TRPV1 immunoreactivity after RTX were comparable to the decreases in suburothelial TRPV1 nerve fibers in the biopsies previously studied from the same patients. CONCLUSIONS: Increased urothelial TRPV1 in patients with NDO may play a role in the pathophysiology, in concert with increased TRPV1 nerve fibers. Although it is not known whether similar pathogenic mechanisms are involved in the increase of urothelial and neuronal TRPV1, both may be targeted by successful RTX therapy.

Maximum urethral closure pressure and sphincter volume in women with urinary retention.

PURPOSE: In 1988 a syndrome of isolated urinary retention in young women that is associated with electromyographic abnormality of the striated urethral sphincter was described. It was hypothesised that urinary retention resulted from a failure of sphincter relaxation. The electromyographic abnormality causes overactivity of the muscle and may induce changes of work hypertrophy. If the hypothesis that the electromyographic abnormality is the cause of urinary retention is correct, we would expect the urethral sphincter to be enlarged and the urethral pressure profile to be increased in these women. We evaluated the role of static urethral pressure profilometry and transvaginal ultrasound in women in urinary retention. MATERIALS AND METHODS: A total of 66 women in complete or partial urinary retention underwent electromyography of the striated urethral sphincter using a concentric needle electrode, followed by urethral pressure profile and/or urethral sphincter volume measurement by transvaginal ultrasound. RESULTS: Maximum urethral closure pressure plus or minus standard deviation was significantly increased in patients with versus without the electromyographic abnormality (103 +/- 26.4 versus 76.7 +/- 18.4 cm. water, p <0.001). Maximum urethral sphincter volume was also increased in women with versus without the abnormality (2.29 +/- 0.64 versus 1.62 +/- 0.32 cm.3, p <0.001). CONCLUSIONS: The results of this study are consistent with the hypothesis that a local sphincter abnormality is the cause of urinary retention in a subgroup of women. Urethral pressure profilometry and sphincter volume measurement are useful for assessing these cases, especially when sphincter electromyography is not readily available.

Recombinant PfEMP1 peptide inhibits and reverses cytoadherence of clinical Plasmodium falciparum isolates in vivo.

The parasite ligand Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) and host endothelial receptors represent potential targets for antiadhesive therapy for cytoadherence. In the present study, the major host receptor CD36 was targeted in vitro and in vivo with a recombinant peptide, PpMC-179, corresponding to the minimal CD36-binding domain from the cysteine-rich interdomain region 1 (CIDR1) within the MCvar1 PfEMP1. The in vitro inhibitory effect of PpMC-179 on human dermal microvascular endothelial cells (HDMECs) expressing multiple relevant adhesion molecules was investigated using a parallel-plate flow chamber. Pretreatment of endothelial monolayers with PpMC-179 (2 microM) inhibited the adhesion of infected erythrocytes (IRBCs) from all clinical isolates tested by 84.4% on resting and 62.8% on tumor necrosis factor alpha (TNF-alpha)-stimulated monolayers. Adhesion to stimulated cells was further inhibited (90.4%) when PpMC-179 was administered with an inhibitory anti-intercellular adhesion molecule 1 (ICAM-1) monoclonal antibody 84H10 (5 microg/mL). To determine the in vivo effectiveness of PpMC-179, we used a human/severe combined immunodeficiency (SCID) mouse chimeric model that allowed direct visualization of cytoadherence on intact human microvasculature. In unstimulated skin grafts, PpMC-179 inhibited adhesion by 86.3% and by 84.6% in TNF-alpha-stimulated skin grafts. More importantly, PpMC-179 administration resulted in the detachment of already adherent IRBCs by 80.7% and 83.3% on resting and stimulated skin grafts, respectively. The antiadhesive effect of PpMC-179 was rapid and sustained in vivo for at least 30 minutes. Our data indicate that targeting cytoadhesion in vivo is feasible and may offer a rapid antimalarial therapy.

P2X3-immunoreactive nerve fibres in neurogenic detrusor overactivity and the effect of intravesical resiniferatoxin.

OBJECTIVES: The ATP-gated purinergic receptor P2X3 is expressed by small diameter sensory neurons and has been identified in normal and neurogenic human bladder suburothelial fibres. Animal models have shown that ATP is released by the urothelium during bladder distension, suggesting a mechanosensory role for P2X3 receptors in normal bladder function. Successful treatment of spinal neurogenic detrusor overactivity (NDO) with intravesical resiniferatoxin (RTX), which partly acts on suburothelial C fibres, provides evidence for the emergence of a C fibre-mediated spinal reflex. The aim of this study was to investigate the possible role of P2X3-positive innervation in this pathological voiding reflex by comparing suburothelial P2X3 immunoreactivity of controls and in patients with NDO before and after intravesical RTX. METHODS: Bladder biopsies were obtained from 8 controls and 20 patients with refractory NDO enrolled in a trial of intravesical RTX. P2X3 nerve fibre density and intensity were studied in the specimens by immunohistochemistry. RESULTS: P2X3-IR nerve fibres were significantly increased in patients with NDO compared to controls (p=0.014). Thirteen patients had pre- and post-RTX biopsies available for immunohistochemistry; 5 of them responded clinically and 8 were non-responders. In the 5 patients who responded to RTX, there was a significant decrease in P2X3-positive fibres (p=0.032), whereas in non-responders, P2X3-IR nerve fibre density did not change significantly. CONCLUSIONS: In patients with NDO, the numbers of P2X3-IR nerve fibres were increased in the suburothelium. There was a significant decrease in P2X3 immunoreactivity in responders to RTX, indicating a potential pathophysiological role for the P2X3 expressing fibres.

The ultrastructure of bladder lamina propria nerves in healthy subjects and patients with detrusor hyperreflexia.

PURPOSE: Detrusor hyperreflexia is a common finding in patients with neurological disease involving the spinal cord. In animal models it has been attributed to an emergent reflex mediated mostly by unmyelinated C-fibers. We describe and measure ultrastructural features of these nerves in the lamina propria in healthy subjects and patients with detrusor hyperreflexia. MATERIALS AND METHODS: Flexible cystoscopic bladder biopsies were obtained from 51 patients (8 controls, 8 with tropical spastic paraparesis, 23 with multiple sclerosis and 12 with spinal cord disease). Electron micrographs were obtained of every nerve profile seen in the midpoint of the biopsy specimen, and in each nerve profile a number of variables were measured and recorded. RESULTS: The mean nerve profile diameter was greater in patients with tropical spastic paraparesis (mean 2.19 microm.) compared to controls (1.59 microm.) and patients with multiple sclerosis (1.55 microm.) (p <0.001). We observed a sparse urothelial innervation by naked axonal varicosities but similar bare varicosities were more frequent in the superficial layer of the lamina propria. In deeper layers close membrane contacts between axonal varicosities and cells with cytological characteristics of myofibroblasts were seen. CONCLUSIONS: We described and measured ultrastructural characteristics of human bladder lamina propria nerves. The mean profile diameter is larger in patients with tropical spastic paraparesis compared to controls and patients with multiple sclerosis. This study provides a baseline to which other bladder disorders can be compared and may allow the effect of intravesical treatments on these nerves to be assessed. Some possible functional aspects of observed structural interrelationships are presented.