Automatic and real-time tissue sensing for autonomous intestinal anastomosis using hybrid MLP-DC-CNN classifier-based optical coherence tomography.

Anastomosis is a common and critical part of reconstructive procedures within gastrointestinal, urologic, and gynecologic surgery. The use of autonomous surgical robots such as the smart tissue autonomous robot (STAR) system demonstrates an improved efficiency and consistency of the laparoscopic small bowel anastomosis over the current da Vinci surgical system. However, the STAR workflow requires auxiliary manual monitoring during the suturing procedure to avoid missed or wrong stitches. To eliminate this monitoring task from the operators, we integrated an optical coherence tomography (OCT) fiber sensor with the suture tool and developed an automatic tissue classification algorithm for detecting missed or wrong stitches in real time. The classification results were updated and sent to the control loop of STAR robot in real time. The suture tool was guided to approach the object by a dual-camera system. If the tissue inside the tool jaw was inconsistent with the desired suture pattern, a warning message would be generated. The proposed hybrid multilayer perceptron dual-channel convolutional neural network (MLP-DC-CNN) classification platform can automatically classify eight different abdominal tissue types that require different suture strategies for anastomosis. In MLP, numerous handcrafted features (∼1955) were utilized including optical properties and morphological features of one-dimensional (1D) OCT A-line signals. In DC-CNN, intensity-based features and depth-resolved tissues' attenuation coefficients were fully exploited. A decision fusion technique was applied to leverage the information collected from both classifiers to further increase the accuracy. The algorithm was evaluated on 69,773 testing A-line data. The results showed that our model can classify the 1D OCT signals of small bowels in real time with an accuracy of 90.06%, a precision of 88.34%, and a sensitivity of 87.29%, respectively. The refresh rate of the displayed A-line signals was set as 300 Hz, the maximum sensing depth of the fiber was 3.6 mm, and the running time of the image processing algorithm was ∼1.56 s for 1,024 A-lines. The proposed fully automated tissue sensing model outperformed the single classifier of CNN, MLP, or SVM with optimized architectures, showing the complementarity of different feature sets and network architectures in classifying intestinal OCT A-line signals. It can potentially reduce the manual involvement of robotic laparoscopic surgery, which is a crucial step towards a fully autonomous STAR system.

Generation of two isogenic sickle cell disease induced pluripotent stem cell lines from testicular fibroblasts.

Curative bone marrow transplantation (BMT) therapies for sickle cell disease (SCD) can cause infertility. The Fertility Preservation Program (FPP) in Pittsburgh cryopreserves testicular tissues for SCD patients prior to BMT in anticipation that those tissues can be thawed in the future and matured to produce sperm. Here, we generated and validated two isogenic patient-derived induced pluripotent stem cell (iPSC) lines from testicular biopsy fibroblasts of a 12-year-old SCD patient.

Fertility after Curative Therapy for Sickle Cell Disease: A Comprehensive Review to Guide Care.

Curative therapy for sickle cell disease (SCD) currently requires gonadotoxic conditioning that can impair future fertility. Fertility outcomes after curative therapy are likely affected by pre-transplant ovarian reserve or semen analysis parameters that may already be abnormal from SCD-related damage or hydroxyurea treatment. Outcomes are also likely affected by the conditioning regimen. Conditioning with myeloablative busulfan and cyclophosphamide causes serious gonadotoxicity particularly among post-pubertal females. Reduced-intensity and non-myeloablative conditioning may be acutely less gonadotoxic, but more short and long-term fertility outcome data after these approaches is needed. Fertility preservation including oocyte/embryo, ovarian tissue, sperm, and experimental testicular tissue cryopreservation should be offered to patients with SCD pursing curative therapy. Regardless of HSCT outcome, longitudinal post-HSCT fertility care is required.

Deep point cloud landmark localization for fringe projection profilometry.

Point clouds have been widely used due to their information being richer than images. Fringe projection profilometry (FPP) is one of the camera-based point cloud acquisition techniques that is being developed as a vision system for robotic surgery. For semi-autonomous robotic suturing, fluorescent fiducials were previously used on a target tissue as suture landmarks. This not only increases system complexity but also imposes safety concerns. To address these problems, we propose a numerical landmark localization algorithm based on a convolutional neural network (CNN) and a conditional random field (CRF). A CNN is applied to regress landmark heatmaps from the four-channel image data generated by the FPP. A CRF leveraging both local and global shape constraints is developed to better tune the landmark coordinates, reject extra landmarks, and recover missing landmarks. The robustness of the proposed method is demonstrated through ex vivo porcine intestine landmark localization experiments.

A Novel Propidium Monoazide-Based PCR Assay Can Measure Viable Uropathogenic E. coli In Vitro and In Vivo.

BACKGROUND: Polymerase chain reaction (PCR) is an important means by which to study the urine microbiome and is emerging as possible alternative to urine cultures to identify pathogens that cause urinary tract infection (UTI). However, PCR is limited by its inability to differentiate DNA originating from viable, metabolically active versus non-viable, inactive bacteria. This drawback has led to concerns that urobiome studies and PCR-based diagnosis of UTI are confounded by the presence of relic DNA from non-viable bacteria in urine. Propidium monoazide (PMA) dye can penetrate cells with compromised cell membranes and covalently bind to DNA, rendering it inaccessible to amplification by PCR. Although PMA has been shown to differentiate between non-viable and viable bacteria in various settings, its effectiveness in urine has not been previously studied. We sought to investigate the ability of PMA to differentiate between viable and non-viable bacteria in urine. METHODS: Varying amounts of viable or non-viable uropathogenic E. coli (UTI89) or buffer control were titrated with mouse urine. The samples were centrifuged to collect urine sediment or not centrifuged. Urine samples were incubated with PMA and DNA cross-linked using blue LED light. DNA was isolated and uidA gene-specific PCR was performed. For in vivo studies, mice were inoculated with UTI89, followed by ciprofloxacin treatment or no treatment. After the completion of ciprofloxacin treatment, an aliquot of urine was plated on non-selective LB agar and another aliquot was treated with PMA and subjected to uidA-specific PCR. RESULTS: PMA's efficiency in excluding DNA signal from non-viable bacteria was significantly higher in bacterial samples in phosphate-buffered saline (PBS, dCT=13.69) versus bacterial samples in unspun urine (dCT=1.58). This discrepancy was diminished by spinning down urine-based bacterial samples to collect sediment and resuspending it in PBS prior to PMA treatment. In 3 of 5 replicate groups of UTI89-infected mice, no bacteria grew in culture; however, there was PCR amplification of E. coli after PMA treatment in 2 of those 3 groups. CONCLUSION: We have successfully developed PMA-based PCR methods for amplifying DNA from live bacteria in urine. Our results suggest that non-PMA bound DNA from live bacteria can be present in urine, even after antibiotic treatment. This indicates that viable but non-culturable E. coli can be present following treatment of UTI, and may explain why some patients have persistent symptoms but negative urine cultures following UTI treatment.

Host tissue proteomics reveal insights into the molecular basis of Schistosoma haematobium-induced bladder pathology.

Urogenital schistosomiasis remains a major public health concern worldwide. In response to egg deposition, the host bladder undergoes gross and molecular morphological changes relevant for disease manifestation. However, limited mechanistic studies to date imply that the molecular mechanisms underlying pathology are not well-defined. We leveraged a mouse model of urogenital schistosomiasis to perform for the first time, proteome profiling of the early molecular events that occur in the bladder after exposure to S. haematobium eggs, and to elucidate the protein pathways involved in urogenital schistosomiasis-induced pathology. Purified S. haematobium eggs or control vehicle were microinjected into the bladder walls of mice. Mice were sacrificed seven days post-injection and bladder proteins isolated and processed for proteome profiling using mass spectrometry. We demonstrate that biological processes including carcinogenesis, immune and inflammatory responses, increased protein translation or turnover, oxidative stress responses, reduced cell adhesion and epithelial barrier integrity, and increased glucose metabolism were significantly enriched in S. haematobium infection. S. haematobium egg deposition in the bladder results in significant changes in proteins and pathways that play a role in pathology. Our findings highlight the potential bladder protein indicators for host-parasite interplay and provide new insights into the complex dynamics of pathology and characteristic bladder tissue changes in urogenital schistosomiasis. The findings will be relevant for development of improved interventions for disease control.

A Confidence-Based Supervised-Autonomous Control Strategy for Robotic Vaginal Cuff Closure.

Autonomous robotic suturing has the potential to improve surgery outcomes by leveraging accuracy, repeatability, and consistency compared to manual operations. However, achieving full autonomy in complex surgical environments is not practical and human supervision is required to guarantee safety. In this paper, we develop a confidence-based supervised autonomous suturing method to perform robotic suturing tasks via both Smart Tissue Autonomous Robot (STAR) and surgeon collaboratively with the highest possible degree of autonomy. Via the proposed method, STAR performs autonomous suturing when highly confident and otherwise asks the operator for possible assistance in suture positioning adjustments. We evaluate the accuracy of our proposed control method via robotic suturing tests on synthetic vaginal cuff tissues and compare them to the results of vaginal cuff closures performed by an experienced surgeon. Our test results indicate that by using the proposed confidence-based method, STAR can predict the success of pure autonomous suture placement with an accuracy of 94.74%. Moreover, via an additional 25% human intervention, STAR can achieve a 98.1% suture placement accuracy compared to an 85.4% accuracy of completely autonomous robotic suturing. Finally, our experiment results indicate that STAR using the proposed method achieves 1.6 times better consistency in suture spacing and 1.8 times better consistency in suture bite sizes than the manual results.

Varenicline limits ischemia reperfusion injury following testicular torsion in mice.

BACKGROUND: Torsion of the spermatic cord and the resulting testicular ischemia leads to the production of inflammatory cytokines and cell death due to impaired aerobic metabolism. Following reperfusion of the testis, a robust innate inflammatory response furthers tissue injury due to the production of reactive oxygen species and disruption of normal capillary function. Blunting the innate immune response with antioxidants, anti-inflammatory medications and targeted genetic interventions reduces long term testicular injury in animal models of torsion, however these approaches have limited clinical applicability. Mediated via α7 nACh receptors, the cholinergic anti-inflammatory pathway limits NFKB signaling and prevents renal fibrosis following warm renal ischemia. We identified varenicline as an FDA approved α7 nAChR agonist and hypothesized that varenicline administration would decrease long-term testicular atrophy and fibrosis in a murine model of testicular torsion. METHODS: Using an established model, unilateral testicular torsion was induced in mature male CD1 mice by rotating the right testicle 720° for 2 h. In the treatment group, 4 doses of varenicline (1mg/grm) were administered via intraperitoneal injection every 12 h, with the first dose given 1 h after the creation of testicular torsion. The acute inflammatory response was evaluated 48 h following reperfusion of the testis. Long term outcomes were evaluated 30 days following testicular perfusion. RESULTS: 48 h following reperfusion, the testis of animals treated with varenicline demonstrated a significant reduction in the inflammatory response as measured by the acute immune cell infiltrate, myeloperoxidase activity, concentration of reduced glutathione and expression of downstream NF-KB targets. 30 days following reperfusion, animals treated with varenicline, demonstrated decreased testicular atrophy (Summary Figure), fibrosis and expression of pro-fibrotic genes. CONCLUSION: Activation of a central immunosuppressive cascade with varenicline after the onset of testicular torsion reduces ischemia reperfusion injury and prevents long term testicular atrophy and fibrosis. Further studies are needed to define the optimum dose and varenicline administration regimen. Our results suggest that varenicline offers a novel, FDA approved, adjunct to the current management of testicular torsion.

Macroscopic and microscopic imaging modalities for diagnosis and monitoring of urogenital schistosomiasis.

Urogenital schistosomiasis remains a major global challenge. Optimal management of this infection depends upon imaging-based assessment of sequelae. Although established imaging modalities such as ultrasonography, plain radiography, magnetic resonance imaging (MRI), narrow band imaging, and computerized tomography (CT) have been used to determine tissue involvement by urogenital schistosomiasis, newer refinements in associated technologies may lead to improvements in patient care. Moreover, application of investigational imaging methods such as confocal laser endomicroscopy and two-photon microscopy in animal models of urogenital schistosomiasis are likely to contribute to our understanding of this infection's pathogenesis. This review discusses prior use of imaging in patients with urogenital schistosomiasis and experimentally infected animals, the advantages and limitations of these modalities, the latest radiologic developments relevant to this infection, and a proposed future diagnostic standard of care for management of afflicted patients.

A View from the past into our collective future: the oncofertility consortium vision statement.

PURPOSE: Today, male and female adult and pediatric cancer patients, individuals transitioning between gender identities, and other individuals facing health extending but fertility limiting treatments can look forward to a fertile future. This is, in part, due to the work of members associated with the Oncofertility Consortium. METHODS: The Oncofertility Consortium is an international, interdisciplinary initiative originally designed to explore the urgent unmet need associated with the reproductive future of cancer survivors. As the strategies for fertility management were invented, developed or applied, the individuals for who the program offered hope, similarly expanded. As a community of practice, Consortium participants share information in an open and rapid manner to addresses the complex health care and quality-of-life issues of cancer, transgender and other patients. To ensure that the organization remains contemporary to the needs of the community, the field designed a fully inclusive mechanism for strategic planning and here present the findings of this process. RESULTS: This interprofessional network of medical specialists, scientists, and scholars in the law, medical ethics, religious studies and other disciplines associated with human interventions, explore the relationships between health, disease, survivorship, treatment, gender and reproductive longevity. CONCLUSION: The goals are to continually integrate the best science in the service of the needs of patients and build a community of care that is ready for the challenges of the field in the future.

A single intravesical instillation of Lactobacillus rhamnosus GG is safe in children and adults with neuropathic bladder: A phase Ia clinical trial.

Context/objective: Manipulation of the microbiome is an emerging approach to promote health. We conducted a Phase Ia safety study of a single bladder instillation of probiotics in asymptomatic patients with neuropathic bladder to determine the tolerability and safety of a single Lactobacillus instillation.Design: Phase Ia safety study.Setting: Outpatient rehabilitation clinic at a rehabilitation hospital (adults) and urology clinic at a free-standing children's hospital (children).Participants: Ten patients with neuropathic bladder were included: five children with spina bifida and five adults with spinal cord injury.Interventions: A single Lactobacillus rhamnosus GG (Culturelle, 20 billion live organisms) instillation.Outcome measures: After the instillation, participants self-monitored symptoms using the Urinary Symptoms Questionnaire for People with Neuropathic Bladder using Intermittent Catheterization daily for one week. Repeat urinalysis, urine culture, and 16S bacterial rRNA-based microbiome analyses were performed 7-10 days after instillation.Results: Probiotic instillation was well-tolerated. One child had upper respiratory tract symptoms during the trial, and two had transient cloudy urine. No adults reported any symptoms following instillation. Lactobacillus did not grow on culture post-instillation. There were differences in beta diversity of the urine microbiome in children vs. adults with neuropathic bladder (P < 0.0156). Lactobacillus was present in the pre-instillation urinary microbiomes all of the adults and 4 out of 5 of the pediatric subjects, and identified in 4 out of 5 of both the adult and pediatric subjects' post-instillation urinary microbiomes.Conclusion: Intravesical instillation of Culturelle probiotic may be safe and well-tolerated in patients with neuropathic bladder.

Perspectives from the Society for Pediatric Research: Probiotic use in urinary tract infections, atopic dermatitis, and antibiotic-associated diarrhea: an overview.

Probiotics have received significant attention within both the scientific and lay communities for their potential health-promoting properties, including the treatment or prevention of various conditions in children. In this article, we review the published data on use of specific probiotic strains for three common pediatric conditions: the prevention of urinary tract infections and antibiotic-associated diarrhea and the treatment of atopic dermatitis. Research into the utility of specific probiotic strains is of varying quality, and data are often derived from small studies and case series. We discuss the scientific merit of these studies, their overall findings regarding the utility of probiotics for these indications, issues in reporting of methods, and results from these clinical trials, as well as future areas of investigation.

IPSE, a parasite-derived, host immunomodulatory infiltrin protein, alleviates resiniferatoxin-induced bladder pain.

The transient receptor potential cation channel subfamily V member 1 (TRPV1) receptor is an important mediator of nociception and its expression is enriched in nociceptive neurons. TRPV1 signaling has been implicated in bladder pain and is a potential analgesic target. Resiniferatoxin is the most potent known agonist of TRPV1. Acute exposure of the rat bladder to resiniferatoxin has been demonstrated to result in pain-related freezing and licking behaviors that are alleviated by virally encoded IL-4. The interleukin-4-inducing principle of Schistosoma mansoni eggs (IPSE) is a powerful inducer of IL-4 secretion, and is also known to alter host cell transcription through a nuclear localization sequence-based mechanism. We previously reported that IPSE ameliorates ifosfamide-induced bladder pain in an IL-4- and nuclear localization sequence-dependent manner. We hypothesized that pre-administration of IPSE to resiniferatoxin-challenged mice would dampen pain-related behaviors. IPSE indeed lessened resiniferatoxin-triggered freezing behaviors in mice. This was a nuclear localization sequence-dependent phenomenon, since administration of a nuclear localization sequence mutant version of IPSE abrogated IPSE's analgesic effect. In contrast, IPSE's analgesic effect did not seem IL-4-dependent, since use of anti-IL-4 antibody in mice given both IPSE and resiniferatoxin did not significantly affect freezing behaviors. RNA-Seq analysis of resiniferatoxin- and IPSE-exposed bladders revealed differential expression of TNF/NF-κb-related signaling pathway genes. In vitro testing of IPSE uptake by urothelial cells and TRPV1-expressing neuronal cells showed uptake by both cell types. Thus, IPSE's nuclear localization sequence-dependent therapeutic effects on TRPV1-mediated bladder pain may act on TRPV1-expressing neurons and/or may rely upon urothelial mechanisms.

IPSE, a urogenital parasite-derived immunomodulatory molecule, suppresses bladder pathogenesis and anti-microbial peptide gene expression in bacterial urinary tract infection.

BACKGROUND: Parasitic infections can increase susceptibility to bacterial co-infections. This may be true for urogenital schistosomiasis and bacterial urinary tract co-infections (UTI). We previously reported that this co-infection is facilitated by S. haematobium eggs triggering interleukin-4 (IL-4) production and sought to dissect the underlying mechanisms. The interleukin-4-inducing principle from Schistosoma mansoni eggs (IPSE) is one of the most abundant schistosome egg-secreted proteins and binds to IgE on the surface of basophils and mast cells to trigger IL-4 release. IPSE can also translocate into host nuclei using a nuclear localization sequence (NLS) to modulate host transcription. We hypothesized that IPSE is the factor responsible for the ability of S. haematobium eggs to worsen UTI pathogenesis. METHODS: Mice were intravenously administered a single 25 µg dose of recombinant S. haematobium-derived IPSE, an NLS mutant of IPSE or PBS. Following IPSE exposure, mice were serially weighed and organs analyzed by histology to assess for toxicity. Twenty-four hours after IPSE administration, mice were challenged with the uropathogenic E. coli strain UTI89 by urethral catheterization. Bacterial CFU were measured using urine. Bladders were examined histologically for UTI-triggered pathogenesis and by PCR for antimicrobial peptide and pattern recognition receptor expression. RESULTS: Unexpectedly, IPSE administration did not result in significant differences in urine bacterial CFU. However, IPSE administration did lead to a significant reduction in UTI-induced bladder pathogenesis and the expression of anti-microbial peptides in the bladder. Despite the profound effect of IPSE on UTI-triggered bladder pathogenesis and anti-microbial peptide production, mice did not demonstrate systemic ill effects from IPSE exposure. CONCLUSIONS: Our data show that IPSE may play a major role in S. haematobium-associated urinary tract co-infection, albeit in an unexpected fashion. These findings also indicate that IPSE either works in concert with other IL-4-inducing factors to increase susceptibility of S. haematobium-infected hosts to bacterial co-infection or does not contribute to enhancing vulnerability to this co-infection.

IPSE, an abundant egg-secreted protein of the carcinogenic helminth Schistosoma haematobium, promotes proliferation of bladder cancer cells and angiogenesis.

BACKGROUND: Schistosoma haematobium, the helminth causing urogenital schistosomiasis, is a known bladder carcinogen. Despite the causal link between S. haematobium and bladder cancer, the underlying mechanisms are poorly understood. S. haematobium oviposition in the bladder is associated with angiogenesis and urothelial hyperplasia. These changes may be pre-carcinogenic events in the bladder. We hypothesized that the Interleukin-4-inducing principle of Schistosoma mansoni eggs (IPSE), an S. haematobium egg-secreted "infiltrin" protein that enters host cell nuclei to alter cellular activity, is sufficient to induce angiogenesis and urothelial hyperplasia. Methods: Mouse bladders injected with S. haematobium eggs were analyzed via microscopy for angiogenesis and urothelial hyperplasia. Endothelial and urothelial cell lines were incubated with recombinant IPSE protein or an IPSE mutant protein that lacks the native nuclear localization sequence (NLS-) and proliferation measured using CFSE staining and real-time monitoring of cell growth. IPSE's effects on urothelial cell cycle status was assayed through propidium iodide staining. Endothelial and urothelial cell uptake of fluorophore-labeled IPSE was measured. Findings: Injection of S. haematobium eggs into the bladder triggers angiogenesis, enhances leakiness of bladder blood vessels, and drives urothelial hyperplasia. Wild type IPSE, but not NLS-, increases proliferation of endothelial and urothelial cells and skews urothelial cells towards S phase. Finally, IPSE is internalized by both endothelial and urothelial cells. Interpretation: IPSE drives endothelial and urothelial proliferation, which may depend on internalization of the molecule. The urothelial effects of IPSE depend upon its NLS. Thus, IPSE is a candidate pro-carcinogenic molecule of S. haematobium. SUMMARY: Schistosoma haematobium acts as a bladder carcinogen through unclear mechanisms. The S. haematobium homolog of IPSE, a secreted schistosome egg immunomodulatory molecule, enhances angiogenesis and urothelial proliferation, hallmarks of pre-carcinogenesis, suggesting IPSE is a key pro-oncogenic molecule of S. haematobium.

A comparative proteomics analysis of the egg secretions of three major schistosome species.

Morbidity associated with hepatic and urogenital schistosomiasis stems primarily from the host immune response directed against schistosome eggs. When eggs become entrapped in host tissues, the development of fibrotic plaques drives downstream pathology. These events occur due to the antigenic nature of egg excretory/secretory products (ESPs). Both Schistosoma mansoni and S. japonicum ESPs have been shown to interact with several cell populations in the host liver including hepatocytes, macrophages, and hepatic stellate cells, with both immunomodulatory and pathological consequences. Several protein components of the ESPs of S. mansoni and S. japonicum eggs have been characterised; however, studies into the collective contents of schistosome egg ESPs are lacking. Utilising shotgun mass spectrometry and an array of in silico analyses, we identified 266, 90 and 50 proteins within the S. mansoni, S. japonicum and S. haematobium egg secretomes respectively. We identified numerous proteins with already established immunomodulatory activities, vaccine candidates and vesicle markers. Relatively few common orthologues within the ESPs were identified by BLAST, indicating that the three egg secretomes differ in content significantly. Having a clearer understanding of these components may lead to the identification of new proteins with uncharacterised immunomodulatory potential or pathological relevance. This will enhance our understanding of host-parasite interactions, particularly those occurring during chronic schistosomiasis, and pave the way towards novel therapeutics and vaccines.

Novel catheter design enables transurethral catheterization of male mice.

The male mouse is underrepresented in research of the urinary tract due to the difficulty of transurethral catheterization. As a result, there is a lack of analysis of sex differences in urinary tract research. Here, we present a novel catheter design and technique that enables urethral catheterization of male mice for bladder inoculation. Our catheterization technique uses the resistance met at the level of the external urinary sphincter and prostate to guide the retraction, positioning, and advancement of the catheter into the urinary bladder. We have shown that this method can be used to reproducibly catheterize 12 male mice with minimal urogenital trauma but cannot be used as a cystometric technique. This method will facilitate the expansion of research into sex differences in various genitourinary conditions that require transurethral catheterization of mice.

A cross-sectional analysis of the urine microbiome of children with neuropathic bladders.

BACKGROUND: Distinguishing a urinary tract infection (UTI) from asymptomatic bacteriuria (ASB) in children with neuropathic bladders is difficult. Currently used markers of infection, such as the routine urinalysis, lack specificity for UTI in this population. The urinary microbiome may help differentiate these states. OBJECTIVE: The objective of this work was to describe the baseline microbiome in children with neuropathic bladders, and to determine if differences exist among the urine microbiomes of children with neuropathic bladders who have negative urine cultures, ASB, or UTI. STUDY DESIGN: This is a cross-sectional study of children with neuropathic bladders who use clean intermittent catheterization for bladder management who had a urine culture sent as part of clinical management. Residual urine, initially collected via catheter for urine culture, was obtained for use in this work. Microbial DNA was isolated, and the V4 region of the 16SrRNA gene sequenced. The relative abundance of each bacteria was measured in each group. Alpha diversity, measured by Chao1 and the Shannon Diversity Index, was also measured in each group. PERMANOVA was used to compare the microbiota between groups. RESULTS: 36 children with neuropathic bladders were included in this study (UTI = 11, ASB = 19, negative cultures = 4). The most abundant bacteria were unspecified Enterobacteriaceae, Klebsiella, Staphylococcus, Streptococcus, and Enterococcus. Children who catheterize their urethra have a higher proportion of Staphylococcus, while the urine microbiome of those who catheterize through a Mitrofanoff consists predominantly of members of the family Enterobacteriaceae. Given the low numbers of patients with Mitrofanoffs and augmented bladders, we did not statistically compare the urine microbiomes between these patients. There was no difference in either alpha diversity or the overall microbiota between children with neuropathic bladders with UTI, ASB, and negative cultures. DISCUSSION: In this pilot cohort of children with neuropathic bladders, bacteria that are members of the family Enterobacteriaceae are the most predominant bacteria in the urine microbiomes. There was no difference in the urine microbiome between those with UTI, ASB, and negative cultures. Route of catheterization may affect the composition of the urine microbiome, although due to limited sample size, this was not confirmed statistically. CONCLUSION: There was no difference in the urine microbiome between patients with negative urine cultures, ASB, and UTI. Further work is needed to determine if the urine microbiome varies based on either the route of catheterization or the presence of augmented bladder.

Interleukin-4 Signaling Plays a Major Role in Urogenital Schistosomiasis-Associated Bladder Pathogenesis.

Interleukin-4 (IL-4) is crucial in many helminth infections, but its role in urogenital schistosomiasis, infection with Schistosoma haematobium worms, remains poorly understood due to a historical lack of animal models. The bladder pathology of urogenital schistosomiasis is caused by immune responses to eggs deposited in the bladder wall. A range of pathology occurs, including urothelial hyperplasia and cancer, but associated mechanisms and links to IL-4 are largely unknown. We modeled urogenital schistosomiasis by injecting the bladder walls of IL-4 receptor-alpha knockout (Il4ra-/- ) and wild-type mice with S. haematobium eggs. Readouts included bladder histology and ex vivo assessments of urothelial proliferation, cell cycle, and ploidy status. We also quantified the effects of exogenous IL-4 on urothelial cell proliferation in vitro, including cell cycle status and phosphorylation patterns of major downstream regulators in the IL-4 signaling pathway. There was a significant decrease in the intensity of granulomatous responses to bladder-wall-injected S. haematobium eggs in Il4ra-/- versus wild-type mice. S. haematobium egg injection triggered significant urothelial proliferation, including evidence of urothelial hyper-diploidy and cell cycle skewing in wild-type but not Il4ra-/- mice. Urothelial exposure to IL-4 in vitro led to cell cycle polarization and increased phosphorylation of AKT. Our results show that IL-4 signaling is required for key pathogenic features of urogenital schistosomiasis and that particular aspects of this signaling pathway may exert these effects directly on the urothelium. These findings point to potential mechanisms by which urogenital schistosomiasis promotes bladder carcinogenesis.