Microbiomes of Urine and the Prostate Are Linked to Human Prostate Cancer Risk Groups.

BACKGROUND: Bacteria play a suspected role in the development of several cancer types, and associations between the presence of particular bacteria and prostate cancer have been reported. OBJECTIVE: To provide improved characterisation of the prostate and urine microbiome and to investigate the prognostic potential of the bacteria present. DESIGN, SETTING, AND PARTICIPANTS: Microbiome profiles were interrogated in sample collections of patient urine (sediment microscopy: n = 318, 16S ribosomal amplicon sequencing: n = 46; and extracellular vesicle RNA-seq: n = 40) and cancer tissue (n = 204). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Microbiomes were assessed using anaerobic culture, population-level 16S analysis, RNA-seq, and whole genome DNA sequencing. RESULTS AND LIMITATIONS: We demonstrate an association between the presence of bacteria in urine sediments and higher D'Amico risk prostate cancer (discovery, n = 215 patients, p < 0.001; validation, n = 103, p < 0.001, χ2 test for trend). Characterisation of the bacterial community led to the (1) identification of four novel bacteria (Porphyromonas sp. nov., Varibaculum sp. nov., Peptoniphilus sp. nov., and Fenollaria sp. nov.) that were frequently found in patient urine, and (2) definition of a patient subgroup associated with metastasis development (p = 0.015, log-rank test). The presence of five specific anaerobic genera, which includes three of the novel isolates, was associated with cancer risk group, in urine sediment (p = 0.045, log-rank test), urine extracellular vesicles (p = 0.039), and cancer tissue (p = 0.035), with a meta-analysis hazard ratio for disease progression of 2.60 (95% confidence interval: 1.39-4.85; p = 0.003; Cox regression). A limitation is that functional links to cancer development are not yet established. CONCLUSIONS: This study characterises prostate and urine microbiomes, and indicates that specific anaerobic bacteria genera have prognostic potential. PATIENT SUMMARY: In this study, we investigated the presence of bacteria in patient urine and the prostate. We identified four novel bacteria and suggest a potential prognostic utility for the microbiome in prostate cancer.

Ciprofloxacin: single versus multiple doses in transrectal ultrasound guided prostate biopsy.

INTRODUCTION: There is rising concern regarding overuse of fluoroquinolones due to severe musculoskeletal and neurological side effects, and development of resistant microorganisms. In June 2019, the European Commission recommended fluoroquinolones should not be used routinely for prophylaxis in urological surgical procedures. Methods to reduce unnecessary exposure to fluroquinolones should be investigated.The aim of this article was to determine differences in hospital admission secondary to sepsis following transrectal ultrasound (TRUS) guided prostate biopsies between patients who received single vs. multiple doses of fluoroquinolones. MATERIAL AND METHODS: A retrospective analysis (June 2017-September 2018) of 200 consecutive TRUS biopsies at a single centre was undertaken. Group 1 (n = 100) received 750 mg ciprofloxacin 1-hr before their procedure followed by 3 days of ciprofloxacin 250 mg BD. Group 2 (n = 100) received a single dose of 750 mg ciprofloxacin 1-hr before the procedure. Midstream urine (MSU) culture results were examined pre-biopsy and 7 days post-biopsy. Data was also gathered on readmission rates to hospital as a result of urosepsis. RESULTS: A total of 1% of patients in each group required hospital admission secondary to Escherichia coli sepsis. A further 4% (n = 4) in Group 1 developed a urinary tract infection requiring antibiotic treatment post biopsy compared with 1% (n = 1) in Group 2. There was no statistically significant difference in development of infectious complications post-biopsy between the two groups (p >0.05). CONCLUSIONS: A single prophylactic dose of 750 mg of ciprofloxacin 1-hour pre-biopsy is as effective as multiple doses for TRUS guided prostate biopsy. Avoiding an unnecessary and prolonged course of fluoroquinolones has advantages in reducing potential side effects and development of resistant pathogens.

Methodology for the at-home collection of urine samples for prostate cancer detection.

Urine from patients with prostate cancer (PCa) contains gene transcripts that have been used for PCa diagnosis and prognosis. Historically, patient urine samples have been collected after a digital rectal examination of the prostate, which was thought necessary to boost the levels of prostatic secretions in the urine. We herein describe methodology that allows urine to be collected by patients at home and then posted to a laboratory for analysis. RNA yields and quality were comparable to those for post digital rectal examination urine, and there was improved sensitivity for the detection of TMPRSS2:ERG transcripts by RT-PCR. The At-Home collection protocol has opened up the potential to perform large-scale PCa studies without the inconvenience, cost, discomfort and expense of patients having to visit the clinic.

Vigorous intensity aerobic interval exercise in bladder cancer patients prior to radical cystectomy: a feasibility randomised controlled trial.

PURPOSE: Strategies to improve pre-operative cardiopulmonary fitness could positively impact recovery after surgery. This study investigated the feasibility of vigorous intensity aerobic interval exercise in bladder cancer patients prior to radical cystectomy (RC). METHODS: A total of 60 patients were randomised (1:1) to exercise or control following a cardiopulmonary exercise test (CPET). The exercise group was offered twice-weekly pre-operative supervised vigorous intensity aerobic interval exercise in addition to standard treatment. The controls received standard treatment only. A repeat CPET was undertaken before surgery and post-operative recovery outcomes were recorded. RESULTS: Over half of the 112 eligible patients approached in the clinic were recruited to the study (53.5%), with recruited patients attending a median of 8 (range 1-10) exercise sessions over a pre-operative period of 3-6 weeks. Improvements in peak values of oxygen pulse (P = 0.001), minute ventilation (P = 0.002) and power output (P < 0.001) were observed at the follow-up CPET in the exercise group versus controls and there were no adverse events. Although this feasibility study was not powered to detect changes in post-operative recovery outcomes, there were marginal (non-significant) differences in favour of the exercise group in post-operative Clavien-Dindo score and need for high dependency unit inotropic support. CONCLUSIONS: Bladder cancer patients respond well to pre-surgical aerobic interval exercise, and the improvements in cardiopulmonary fitness variables could have important implications for post-operative recuperation after RC. These findings provide a strong foundation for an adequately powered randomised controlled trial.

A renal colic fast track pathway to improve waiting times and outcomes for patients presenting to the emergency department.

INTRODUCTION: Renal colic is commonly encountered in the emergency department (ED). We validated a fast track renal colic (FTRC) initiative to decrease patient waiting times and streamline patient flow. METHOD: The FTRC pathway was devised according to the National Institute for Health and Care Excellence clinical summary criteria for the management of patients with suspected renal colic. ED triage nurses use the pathway to identify patients with likely renal colic suitable for fast track to analgesia, investigation and management. Investigations, diagnosis and patient demographics were recorded for 1157 consecutive patients coded as renal colic at a single-center ED over 12 months. RESULTS: Three hundred and two patients were suitable for the FTRC pathway (26.1%), while 855 were seen by the ED clinicians prior to onward referral. Also, 83.9% of patients underwent computed tomography scan. In the FTRC group, 57.3% of patients had radiologically confirmed calculi versus 53.8% in the non-FTRC group (p=0.31). Alternative diagnoses among FTRC patients (2.6%) included ovarian pathology (n=1), diverticulitis (n=2) and incidental renal cell carcinoma (n=2), while 26.1% had no identifiable pathology. No immediately life-threatening diagnoses were identified on imaging. Computed tomography scans performed in the non-FTRC group identified two ruptured abdominal aortic aneurysms and alternative diagnoses (2.57%) including ovarian pathology (n=7), cholecystitis (n=2), incidental renal cell carcinoma (n=3) and inflammatory bowel disease (n=1); 31.2% identified no pathology. Time in ED and time to radiologist-reported imaging were lower for the FTRC group versus non-FTRC group (p<0.0001). CONCLUSION: The FTRC pathway is a safe and efficacious method of reducing diagnostic delay and improving patient flow in the ED.

Is There a Cosmetic Advantage to Single-Incision Laparoscopic Surgical Techniques Over Standard Laparoscopic Surgery? A Systematic Review and Meta-analysis.

Single-incision laparoscopic surgery represents an evolution of minimally invasive techniques, but has been a controversial development. A cosmetic advantage is stated by many authors, but has not been found to be universally present or even of considerable importance by patients. This systematic review and meta-analysis demonstrates that there is a cosmetic advantage of the technique regardless of the operation type. The treatment effect in terms of cosmetic improvement is of the order of 0.63.

Free deep inferior epigastric perforator flap used for management of post-pneumonectomy space empyema.

Various solutions exist for management of post-pneumonectomy space empyema. We describe the use of a free deep inferior epigastric perforator (DIEP) flap to fill the space and close a pleural window. Previously, flaps involving abdominal muscle or omentum have been used for this purpose. Abdominal surgery to harvest such flaps can impair ventilatory mechanics. The DIEP flap--harvested from the abdomen, and composed primarily of skin and muscle avoids this problem, thus is a desirable technique in patients with impaired lung function. We believe this is the first report of the DIEP flap to close a postpneumonectomy empyema space.

Benign emptying of postpneumonectomy space due to severe dehydration.

Causes of benign emptying of the postpneumonectomy space include small bronchopleural fistulas with spontaneous healing and escape of fluid into the chest wall or diaphragm. We present an additional cause: severe dehydration. As postpneumonectomy empyema usually involves drainage of the pleural space, it is important to be aware of this uncommon cause so as to avoid unnecessary instrumentation and contamination of the postpneumonectomy space.

Blood patch for persistent air leak: a review of the current literature.

PURPOSE OF REVIEW: Persistent air leak (PAL) poses a significant challenge to the thoracic surgeon. Of the numerous methods employed to manage this problem, autologous blood 'patch' pleurodesis (ABPP) remains one of the most controversial, seemingly due to a lack of robust data and consensus of opinion regarding its efficacy, technique of application and its role in clinical practice. Despite a lack of randomized control trials, the evidence to-date has shown ABPP to be an efficacious, cheap, simple, well tolerated and readily available treatment, with minimal side effects and broad range of applications, allowing for earlier chest drain removal, decreased complications and decreased hospital stay. A review is therefore required to assess the role for ABPP in contemporary clinical practice. RECENT FINDINGS: Recent studies have demonstrated that ABPP is an effective management for PAL in specific patient groups and there is an argument that it has the potential to be the gold-standard or first-line treatment in certain clinical scenarios such as for patients with interstitial lung disease or acute respiratory distress syndrome. SUMMARY: This review aims to discuss the relevance of recent findings and to suggest a firm role for ABPP in current practice. In addition, the evidence for the efficacy of ABPP will be assessed and compared with other established methods of pleurodesis. Finally, the review will include a summary of relevant research to-date in order to suggest an evidence-based standardized protocol for the application of ABPP.

Human cytomegalovirus escapes a naturally occurring neutralizing antibody by incorporating it into assembling virions.

Human cytomegalovirus (CMV) is a common but difficult to treat infection of immunocompromised patients. MSL-109 is a human monoclonal IgG isolated from a CMV seropositive individual that recognizes the viral glycoprotein H (gH) surface antigen complexes that mediate entry. Although MSL-109 blocks CMV infection in vitro, it lacked sufficient efficacy in human trials, and CMV isolated from treated patients suggested the evolution of MSL-109 resistance. To understand how CMV escapes MSL-109, we characterized a MSL-109-resistant CMV strain. Our results elucidate a nongenetic escape mechanism in which the antibody is selectively taken up by infected cells and incorporated into assembling virions in a dose-dependent manner. The resistant virus then utilizes the Fc domain of the incorporated antibody to infect naive nonimmune cells. This resistance mechanism may explain the clinical failure of MSL-109, illustrate a general mechanism of viral antibody escape, and inform antiviral vaccine and therapeutic development.

The glycoprotein B disintegrin-like domain binds beta 1 integrin to mediate cytomegalovirus entry.

Cellular integrins were identified as human cytomegalovirus (HCMV) entry receptors and signaling mediators in both fibroblasts and endothelial cells. The goal of these studies was to determine the mechanism by which HCMV binds to cellular integrins to mediate virus entry. HCMV envelope glycoprotein B (gB) has sequence similarity to the integrin-binding disintegrin-like domain found in the ADAM (a disintegrin and metalloprotease) family of proteins. To test the ability of this region to bind to cellular integrins, we generated a recombinant soluble version of the gB disintegrin-like domain (gB-DLD). The gB-DLD protein bound to human fibroblasts in a specific, dose-dependent and saturable manner that required the expression of an intact beta1 integrin ectodomain. Furthermore, a physical association between gB-DLD and beta1 integrin was demonstrated through in vitro pull-down assays. The function of this interaction was shown by the ability of cell-bound gB-DLD to efficiently block HCMV entry and the infectivity of multiple in vivo target cells. Additionally, rabbit polyclonal antibodies raised against gB-DLD neutralized HCMV infection. Mimicry of the ADAM family disintegrin-like domain by HCMV gB represents a novel mechanism for integrin engagement by a virus and reveals a unique therapeutic target for HCMV neutralization. The strong conservation of the DLD across beta- and gammaherpesviruses suggests that integrin recognition and utilization may be a more broadly conserved feature throughout the Herpesviridae.

Transcriptional regulation of BK virus by nuclear factor of activated T cells.

The human polyomavirus BK virus (BKV) is a common virus for which 80 to 90% of the adult population is seropositive. BKV reactivation in immunosuppressed patients or renal transplant patients is the primary cause of polyomavirus-associated nephropathy (PVN). Using the Dunlop strain of BKV, we found that nuclear factor of activated T cells (NFAT) plays an important regulatory role in BKV infection. Luciferase reporter assays and chromatin immunoprecipitation assays demonstrated that NFAT4 bound to the viral promoter and regulated viral transcription and infection. The mutational analysis of the NFAT binding sites demonstrated complex functional interactions between NFAT, c-fos, c-jun, and the p65 subunit of NF-kappaB that together influence promoter activity and viral growth. These data indicate that NFAT is required for BKV infection and is involved in a complex regulatory network that both positively and negatively influences promoter activity and viral infection.

Human alpha-defensins inhibit BK virus infection by aggregating virions and blocking binding to host cells.

BK virus (BKV) is a polyomavirus that establishes a lifelong persistence in most humans and is a major impediment to success of kidney grafts. The function of the innate immune system in BKV infection and pathology has not been investigated. Here we examine the role of antimicrobial defensins in BKV infection of Vero cells. Our data show that alpha-defensin human neutrophil protein 1 (HNP1) and human alpha-defensin 5 (HD5) inhibit BKV infection by targeting an early event in the viral lifecycle. HD5 treatment of BKV reduced viral attachment to cells, whereas cellular treatment with HD5 did not. Colocalization studies indicated that HD5 interacts directly with BKV. Ultrastructural analysis revealed HD5-induced aggregation of virions. HD5 also inhibited infection of cells by other related polyomaviruses. This is the first study to demonstrate polyomavirus sensitivity to defensins. We also show a novel mechanism whereby HD5 binds to BKV leading to aggregation of virion particles preventing normal virus binding to the cell surface and uptake into cells.

Nuclear factor of activated T-cells (NFAT) plays a role in SV40 infection.

Recent evidence highlighted a role for the transcription factor, nuclear factor of activated T-cells (NFAT), in the transcription of the human polyomavirus JCV. Here we show that NFAT is also important in the transcriptional control of the related polyomavirus, Simian Virus 40 (SV40). Inhibition of NFAT activity reduced SV40 infection of Vero, 293A, and HeLa cells, and this block occurred at the stage of viral transcription. Both NFAT3 and NFAT4 bound to the SV40 promoter through kappaB sites located within the 72 bp repeated enhancer region. In Vero cells, NFAT was involved in late transcription, but in HeLa and 293A cells both early and late viral transcription required NFAT activity. SV40 large T-Ag was found to increase NFAT activity and provided a positive feedback loop to transactivate the SV40 promoter.

Identification of amino acid residues in BK virus VP1 that are critical for viability and growth.

BK virus (BKV) is a ubiquitous pathogen that establishes a persistent infection in the urinary tract of 80% of the human population. Like other polyomaviruses, the major capsid protein of BKV, virion protein 1 (VP1), is critical for host cell receptor recognition and for proper virion assembly. BKV uses a carbohydrate complex containing alpha(2,3)-linked sialic acid attached to glycoprotein and glycolipid motifs as a cellular receptor. To determine the amino acids important for BKV binding to the sialic acid portion of the complex, we generated a series of 17 point mutations in VP1 and scored them for viral growth. The first set of mutants behaved identically to wild-type virus, suggesting that these amino acids were not critical for virus propagation. Another group of VP1 mutants rendered the virus nonviable. These mutations failed to protect viral DNA from DNase I digestion, indicating a role for these domains in capsid assembly and/or packaging of DNA. A third group of VP1 mutations packaged DNA similarly to the wild type but failed to propagate. The initial burst size of these mutations was similar to that of the wild type, indicating that there is no defect in the lytic release of the mutated virions. Binding experiments revealed that a subset of the BKV mutants were unable to attach to their host cells. These motifs are likely important for sialic acid recognition. We next mapped these mutations onto a model of BKV VP1 to provide atomic insight into the role of these sites in the binding of sialic acid to VP1.

Microarray analysis of glial cells resistant to JCV infection suggests a correlation between viral infection and inflammatory cytokine gene expression.

The human polyomavirus, JCV, has a highly restricted tropism and primarily infects glial cells. The mechanisms restricting infection of cells by JCV are poorly understood. Previously we developed and described a glial cell line that was resistant to JCV infection with the aim of using these cells to identify factors that determine JCV tropism. Gene expression profiling of susceptible and resistant glial cells revealed a direct correlation between the expression of inflammatory cytokines and susceptibility to JCV infection. This correlation manifested at the level of viral gene transcription. Previous studies have suggested a link between an increase in cytokine gene expression in HIV patients and the development of PML and these data supports this hypothesis.

NFAT4 is required for JC virus infection of glial cells.

The human polyomavirus JC virus (JCV) infects 70% of the population worldwide. In immunosuppressed patients, JCV infection can lead to progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system (CNS). The majority of PML cases occur in the setting of human immunodeficiency virus (HIV) infection, and it has been suggested that the link between HIV and the development of PML is in part related to the production of numerous cytokines in the CNS during HIV infection. To examine the link between the expression of inflammatory cytokines and JCV infection, we tested an anti-inflammatory compound, cyclosporine A (CsA), for its ability to block JCV infection of glial cells. We found that CsA inhibited JCV infection by preventing the activation of the transcription factor nuclear factor of activated T cells 4 (NFAT4). Luciferase reporter assays and chromatin immunoprecipitation assays revealed that NFAT4 directly bound the JCV promoter during infection and was important for the activation of both early and late transcription. In addition, the expression of the JCV early viral gene products increased NFAT activity to further aid viral transcription. The necessity of NFAT for JCV infection suggests that calcium signaling and the activation of NFAT in glial cells are required for JCV infection of the CNS.

The human polyomavirus, JCV, uses serotonin receptors to infect cells.

The human polyomavirus, JCV, causes the fatal demyelinating disease progressive multifocal leukoencephalopathy in immunocompromised patients. We found that the serotonergic receptor 5HT2AR could act as the cellular receptor for JCV on human glial cells. The 5HT2A receptor antagonists inhibited JCV infection, and monoclonal antibodies directed at 5HT2A receptors blocked infection of glial cells by JCV, but not by SV40. Transfection of 5HT2A receptor-negative HeLa cells with a 5HT2A receptor rescued virus infection, and this infection was blocked by antibody to the 5HT2A receptor. A tagged 5HT2A receptor colocalized with labeled JCV in an endosomal compartment following internalization. Serotonin receptor antagonists may thus be useful in the treatment of progressive multifocal leukoencephalopathy.

Derivation of a JC virus-resistant human glial cell line: implications for the identification of host cell factors that determine viral tropism.

JC virus (JCV) is a common human polyomavirus that infects 70-80% of the population worldwide. In immunosuppressed individuals, JCV infects oligodendrocytes and causes a fatal demyelinating disease known as progressive multifocal leukoencephalopathy (PML). The tropism of JCV is restricted to oligodendrocytes, astrocytes, and B lymphocytes. Several mechanisms may contribute to the restricted tropism of JCV, including the presence or absence of cell-type-specific transcription and replication factors and the presence or absence of cell-type-specific receptors. We have established a system to investigate cellular factors that influence viral tropism by selecting JCV-resistant cells from a susceptible glial cell line (SVG-A). SVG-A cells were subjected to several rounds of viral infection using JC virus (M1/SVE Delta). A population of resistant cells emerged (SVGR2) that were refractory to infection with the Mad-4 strain of JCV, the hybrid virus M1/SVE Delta, as well as to the related polyomavirus SV40. SVGR2 cells were as susceptible as the SVG-A cells to infection with an unrelated amphotropic retrovirus. The stage at which these cells are resistant to infection was investigated and the block appears to be at early viral gene transcription. This system should ultimately allow us to identify glial specific factors that influence the tropism of JCV.