Are beta defensin 1 and beta defensin 2 key innate immune effector peptides against urinary tract infection in women?

PhD ThesisIntroduction & Hypothesis Recurrent urinary tract infection (rUTI) is a debilitating problem affecting 5% of women. Current treatment using intermittent or long-term antibiotics gives limited symptomatic benefit and encourages bacterial resistance. The aetiology of rUTI is unclear but may involve altered innate defence mechanisms in susceptible individuals. Colonisation of vaginal mucosa by uropathogenic Escherichia coli (UPEC) is the initiating event for UTI, with subsequent migration up the urethra and attachment to bladder epithelium. Protective innate immunological mechanisms include epithelial synthesis of cationic antimicrobial peptides (AMPs) such as the beta defensins. These may be expressed constitutively or induced via Toll-like-receptor (TLR) activation by pathogen associated molecular patterns (PAMPs). This study investigates the hypothesis that women suffering from rUTI have altered tolerance to infecting bacteria related to differences in expression of endogenous AMPs and that identification of such deficiency gives a potentially useful opportunity for novel preventive therapy. Methods and Patients A synergistic methodological approach of in vitro modelling with validation in clinical samples from the relevant patient group and controls was used. In vitro, cell culture was carried out using RT4 immortalised urothelial-cells, VK2 E6/E7 immortalised vaginal cells and finite primary culture of normal human urothelium. Cells were challenged with E. coli and PAMPs. Assays for Beta defensin AMP gene expression, secretion and antimicrobial activity were carried out. Clinically, 98 women (60 rUTI, 38 controls) were recruited with ethical approval. All subjects provided symptom and health state questionnaires; blood for single nucleotide polymorphism (SNP) analysis; vaginal and bladder biopsies for AMP gene expression analysis; plus vaginal washings and overnight urine samples for AMP peptide secretion assays. Results In vitro, cell culture experiments demonstrated that beta defensin 1 (BD1) was constitutively expressed and secreted in urothelial and vaginal cells. Beta defensin 2 (BD2) expression and secretion was induced by E. coli flagellin and is a potent antimicrobial against UPEC. In vaginal cells, BD2 expression and secretion was enhanced by estrogen. Clinically, women with rUTI were identified as having significantly lower basal levels of vaginal BD2 expression and secretion than controls but no difference in BD1 expression. Postmenopausal women had significantly lower BD2 levels than pre-menopausal women. 392Stop During active UTI, women with history of rUTI and the TLR5 SNP showed significantly lower BD2 expression and secretion in both the bladder and vagina than women with wild type TLR5 gene and rUTI. Discussion This study identifies flagellin induced BD2 expression as a novel and important urogenital innate immune response against invading E. coli, which is reduced in a significant proportion of women with rUTI particularly those with the TLR5 392Stop SNP. Observations in vitro on the BD2 inducing effect of estrogen, and clinically in pre- and post-menopausal women, raise the possibility that BD2 expression can be modulated by exogenous factors.The Wellcome Trus

Alternative to prophylactic antibiotics for the treatment of recurrent urinary tract infections in women: multicentre, open label, randomised, non-inferiority trial.

OBJECTIVE: To test and compare the efficacy of methenamine hippurate for prevention of recurrent urinary tract infections with the current standard prophylaxis of daily low dose antibiotics. DESIGN: Multicentre, open label, randomised, non-inferiority trial. SETTING: Eight centres in the UK, recruiting from June 2016 to June 2018. PARTICIPANTS: Women aged ≥18 years with recurrent urinary tract infections, requiring prophylactic treatment. INTERVENTIONS: Random assignment (1:1, using permuted blocks of variable length via a web based system) to receive antibiotic prophylaxis or methenamine hippurate for 12 months. Treatment allocation was not masked and crossover between arms was allowed. MAIN OUTCOME MEASURE: Absolute difference in incidence of symptomatic, antibiotic treated, urinary tract infections during treatment. A patient and public involvement group predefined the non-inferiority margin as one episode of urinary tract infection per person year. Analyses performed in a modified intention-to-treat population comprised all participants observed for at least six months. RESULTS: Participants were randomly assigned to antibiotic prophylaxis (n=120) or methenamine hippurate (n=120). The modified intention-to-treat analysis comprised 205 (85%) participants (antibiotics, n=102 (85%); methenamine hippurate, n=103 (86%)). Incidence of antibiotic treated urinary tract infections during the 12 month treatment period was 0.89 episodes per person year (95% confidence interval 0.65 to 1.12) in the antibiotics group and 1.38 (1.05 to 1.72) in the methenamine hippurate group, with an absolute difference of 0.49 (90% confidence interval 0.15 to 0.84) confirming non-inferiority. Adverse reactions were reported by 34/142 (24%) in the antibiotic group and 35/127 (28%) in the methenamine group and most reactions were mild. CONCLUSION: Non-antibiotic prophylactic treatment with methenamine hippurate might be appropriate for women with a history of recurrent episodes of urinary tract infections, informed by patient preferences and antibiotic stewardship initiatives, given the demonstration of non-inferiority to daily antibiotic prophylaxis seen in this trial. TRIAL REGISTRATION: ISRCTN70219762

Comparing an integrated amphiphilic surfactant to traditional hydrophilic coatings for the reduction of catheter-associated urethral microtrauma

Hydrophilic-coated intermittent catheters have improved the experience of intermittent urinary catheterization for patients compared to conventional gel-lubricated uncoated catheters. However, the incorporation of polyvinylpyrrolidone (PVP) within hydrophilic coatings can lead to significant issues with coating dry-out. Consequently, increased force on catheter withdrawal may cause complications, including urethral microtrauma and pain. Standard methods of evaluating catheter lubricity lack physiological relevance and an understanding of the surface interaction with the urethra. The tribological performance and urethral interaction of commercially available hydrophilic PVP-coated catheters and a coating-free integrated amphiphilic surfactant (IAS) catheter were evaluated by using a biomimetic urethral model designed from a modified coefficient of friction (CoF) assay. T24 human urothelial cells were cultured on customized silicone sheets as an alternate countersurface for CoF testing. Hydrophilic PVP-coated and coating-free IAS catheters were hydrated and the CoF obtained immediately following hydration, or after 2 min, mimicking in vivo indwell time for urine drainage. The model was observed for urethral epithelial cell damage postcatheterization. The majority of hydrophilic PVP-coated catheters caused significantly greater removal of cells from the monolayer after 2 min indwell time, compared to the IAS catheter. Hydrophilic PVP-coated catheters were shown to cause more cell damage than the coating-free IAS catheter. A biomimetic urethral model provides a more physiologically relevant model for understanding the factors that govern the frictional interface between a catheter surface and urethral tissue. From these findings, the use of coating-free IAS catheters instead of hydrophilic PVP-coated catheters may help reduce urethral microtrauma experienced during catheter withdrawal from the bladder, which may lead to a lower risk of infection

Datasets for “Comparing an integrated amphiphilic surfactant to traditional hydrophilic coatings for the reduction of catheter-associated urethral microtrauma”

Open access datasets to support the manuscript: “Comparing an integrated amphiphilic surfactant to traditional hydrophilic coatings for the reduction of catheter-associated urethral microtrauma” Abstract: Hydrophilic-coated intermittent catheters have improved the experience of intermittent urinary catheterization for patients compared to conventional gel lubricated uncoated catheters. However, the incorporation of polyvinylpyrrolidone (PVP) within hydrophilic coatings can lead to significant issues with coating dry-out. Consequently, increased force on catheter withdrawal may cause complications including urethral microtrauma and pain. Standard methods of evaluating catheter lubricity lack physiological relevance and an understanding of surface interaction with the urethra. The tribological performance and urethral interaction of commercially available hydrophilic PVP-coated catheters and a coating-free integrated amphiphilic surfactant (IAS) catheter were evaluated using a biomimetic urethral model designed from a modified coefficient of friction (CoF) assay. T24 human urothelial cells were cultured on customized silicone sheets as an alternate countersurface for CoF testing. Hydrophilic PVP-coated and coating-free IAS catheters were hydrated and the CoF obtained immediately following hydration, or after 2 minutes, mimicking in vivo indwell time for urine drainage. The model was observed for urethral epithelial cell damage post-catheterization. The majority of hydrophilic PVP-coated catheters caused significantly greater removal of cells from the monolayer after 2 minutes indwell time, compared to the IAS catheter. Hydrophilic PVP-coated catheters were shown to cause more cell damage than the coating-free IAS catheter. A biomimetic urethral model provides a more physiologically relevant model for understanding of the factors that govern the frictional interface between a catheter surface and urethral tissue. From these findings, the use of coating-free IAS catheters instead of hydrophilic PVP-coated catheters may help reduce urethral microtrauma experienced during catheter withdrawal from the bladder which may lead to a lower risk of infection. Dataset for public access at request of the publishing journal ACS Omega. Access to the dataset ensures transparency to the readers of the manuscript. The dataset was created and obtained between 1/11/21 and 31/12/2023 as part of two one year funded projects with Convatec Ltd. The dataset may help readers and researchers adapt the novel biomimetic model described within the manuscript for their own research. During the project, many images were obtained of urothelial cells for assessment of micro trauma and damage. Moreover, these images were assessed in a blinded grading survey. Access to the dataset shows images that were unable to be included in the manuscript due to shear volume of the data. The data in the dataset includes Coefficient of friction analysis, calculation of cell monolayer coverage, calculation of the percentage of intermittent catheter surfaces covered with cells after catheterisation in the biomimetic model, results form the blinded grading survey and a copy of the survey. Images in the dataset show cell images included in the blinded grading survey, images of cells before and after catheterisation in the biomimetic model, images of cells adhered to the catheter surface and images of the catheters before and after catheterisation showing visual change in the surface observed. Images of catheter delamination also shown. We hope the data and manuscript will help inform readers, researchers and clinicians of the issues associated with intermittent catheters and lead a new direction in intermittent catheter design. Ultimately, improving the life of patients who use intermittent catheters

Urethral bulking therapy for treating stress urinary incontinence in women

Objectives: This is a protocol for a Cochrane Review (intervention). The objectives are as follows:. To assess the effects of urethral bulking injections for treating stress urinary incontinence in women; and summarise the principal findings of relevant economic evaluations

Prostate specific antigen enhances the innate defence of prostatic epithelium against Escherichia coli infection

BACKGROUND: This study investigated whether the increase in serum prostate specific antigen (PSA) typically seen during male urinary tract infection (UTI) is incidental or reflects an innate defence mechanism of the prostate. The protective roles of the whey-acid-motif-4-disulphide core (WFDC) proteins, secretory leukoproteinase inhibitor (SLPI) and WFDC2, in the prostate were also examined. METHODS: UTI recurrence was assessed retrospectively in men following initial UTI by patient interview. PSA, SLPI, and WFDC2 gene expression were assessed using biopsy samples. LNCaP and DU145 in vitro prostate cell models were utilized to assess the effects of an Escherichia coli challenge on PSA and WFDC gene expression, and bacterial invasion of the prostate epithelium. The effects of PSA on WFDC antimicrobial properties were studied using recombinant peptides and time-kill assays. RESULTS: Men presenting with PSA >4 ng/ml at initial UTI were less likely to have recurrent (r) UTI than those with PSA <4 ng/ml [2/15 (13%) vs. 7/10 (70%), P < 0.01]. Genes encoding PSA, SLPI and WFDC2, were expressed in prostatic epithelium, and the PSA and SLPI proteins co-localized in vivo. Challenging LNCaP (PSA-positive) cells with E. coli increased PSA, SLPI, and WFDC2 gene expression (P < 0.05), and PSA synthesis (P < 0.05), and reduced bacterial invasion. Pre-incubation of DU145 (PSA-negative) cells with PSA also decreased bacterial invasion. In vitro incubation of recombinant SLPI and WFDC2 with PSA resulted in peptide proteolysis and increased E. coli killing. CONCLUSIONS: Increased PSA during UTI appears protective against rUTI and in vitro is linked to proteolysis of WFDC proteins supporting enhanced prostate innate defences