Inflammatory markers for improved recurrent UTI diagnosis in postmenopausal women.

Recurrent urinary tract infection (rUTI) severely impacts postmenopausal women. The lack of rapid and accurate diagnostic tools is a major obstacle in rUTI management as current gold standard methods have >24-h diagnostic windows. Work in animal models and limited human cohorts have identified robust inflammatory responses activated during UTI. Consequently, urinary inflammatory cytokines secreted during UTI may function as diagnostic biomarkers. This study aimed to identify urinary cytokines that could accurately diagnose UTI in a controlled cohort of postmenopausal women. Women passing study exclusion criteria were classified into no UTI and active rUTI groups, and urinary cytokine levels were measured by immunoassay. Pro-inflammatory cytokines IL-8, IL-18, IL-1ß, and monocyte chemoattractant protein-1 were significantly elevated in the active rUTI group, and anti-inflammatory cytokines IL-13 and IL-4 were elevated in women without UTI. We evaluated cytokine diagnostic performance and found that an IL-8, prostaglandin E2, and IL-13 multivariable model had the lowest misclassification rate and highest sensitivity. Our data identify urinary IL-8, prostaglandin E2, and IL-13 as candidate biomarkers that may be useful in the development of immunoassay-based UTI diagnostics.

Self-assembly of a fluorescent virus-like particle for imaging in tissues with high autofluorescence.

Virus-like particles (VLPs) are engineered nanoparticles that mimic the properties of viruses-like high tolerance to heat and proteases-but lack a viral genome, making them non-infectious. They are easily modified chemically and genetically, making them useful in drug delivery, enhancing vaccine efficacy, gene delivery, and cancer immunotherapy. One such VLP is Qß, which has an affinity towards an RNA hairpin structure found in its viral RNA that drives the self-assembly of the capsid. It is possible to usurp the native way infectious Qß self-assembles to encapsidate its RNA to place enzymes inside the VLP's lumen as a protease-resistant cage. Further, using RNA templates that mimic the natural self-assembly of the native capsid, fluorescent proteins (FPs) have been placed inside VLPs in a "one pot" expression system. Autofluorescence in tissues can lead to misinterpretation of results and unreliable science, so we created a single-pot expression system that uses the fluorescent protein smURFP, which avoids autofluorescence and has spectral properties compatible with standard commercial filter sets on confocal microscopes. In this work, we were able to simplify the existing "one-pot" expression system while creating high-yielding fluorescent VLP nanoparticles that could easily be imaged inside lung epithelial tissue.

Reduced urothelial expression of uroplakin-IIIa in cystitis areas in bladders of postmenopausal women with recurrent urinary tract infections: pilot study.

PURPOSE: To study human bladder biopsies to investigate urothelial response to UTI, expression of uroplakin, and urothelial response after healing from cystoscopy with electrofulguration (CEF) treatment for antibiotic-recalcitrant RUTI. METHODS: Following IRB approval, cold cup bladder biopsies from "no cystitis" and "cystitis" regions were obtained from women with antibiotic-recalcitrant rUTI undergoing CEF under anesthesia. "No cystitis" and "cystitis" biopsies from 14 patients (5 had prior CEF, 9 naïve) were analyzed by immunofluorescence (IF) confocal microscopy using antibodies against uroplakin-IIIa. For an additional 6 patients (2 prior CEF, 4 naïve), only "cystitis" area biopsies were taken and analyzed. Cytokeratin 5 (marker for squamous metaplasia) staining was performed on 14 patients. RESULTS: In healthy tissue, uroplakin-IIIa staining was observed as a contiguous line on the luminal surface of umbrella cells. In "cystitis" areas for 19/20 patients, there was either no uroplakin-IIIa staining observed or spotty (+) staining. The "cystitis" regions of all patients had less intense uroplakin-IIIa staining compared to the matched "no cystitis" area in the same patient. In patients post-CEF but requiring repeat EF for persistent RUTI lesions, healed areas served as control and in 3 of 7 patients no uroplakin-IIIa staining was observed. Squamous metaplasia was observed in 10 patients. CONCLUSION: In bladders of postmenopausal women with antibiotic-recalcitrant RUTI, areas with visible cystitis expressed less uroplakin-IIIa, supporting the model of urothelial exfoliation in response to UTI.

PhotothermalPhage: A Virus-Based Photothermal Therapeutic Agent.

Virus-like particles (VLPs) are multifunctional nanocarriers that mimic the architecture of viruses. They can serve as a safe platform for specific functionalization and immunization, which provides benefits in a wide range of biomedical applications. In this work, a new generation immunophotothermal agent is developed that adjuvants photothermal ablation using a chemically modified VLP called bacteriophage Qß. The design is based on the conjugation of near-infrared absorbing croconium dyes to lysine residues located on the surface of Qß, which turns it to a powerful NIR-absorber called PhotothermalPhage. This system can generate more heat upon 808 nm NIR laser radiation than free dye and possesses a photothermal efficiency comparable to gold nanostructures, yet it is biodegradable and acts as an immunoadjuvant combined with the heat it produces. The synergistic combination of thermal ablation with the mild immunogenicity of the VLP leads to effective suppression of primary tumors, reduced lung metastasis, and increased survival time.

Metal-Organic Framework Encapsulated Whole-Cell Vaccines Enhance Humoral Immunity against Bacterial Infection.

The increasing rate of resistance of bacterial infection against antibiotics requires next generation approaches to fight potential pandemic spread. The development of vaccines against pathogenic bacteria has been difficult owing, in part, to the genetic diversity of bacteria. Hence, there are many potential target antigens and little a priori knowledge of which antigen/s will elicit protective immunity. The painstaking process of selecting appropriate antigens could be avoided with whole-cell bacteria; however, whole-cell formulations typically fail to produce long-term and durable immune responses. These complications are one reason why no vaccine against any type of pathogenic E. coli has been successfully clinically translated. As a proof of principle, we demonstrate a method to enhance the immunogenicity of a model pathogenic E. coli strain by forming a slow releasing depot. The E. coli strain CFT073 was biomimetically mineralized within a metal-organic framework (MOF). This process encapsulates the bacteria within 30 min in water and at ambient temperatures. Vaccination with this formulation substantially enhances antibody production and results in significantly enhanced survival in a mouse model of bacteremia compared to standard inactivated formulations.

Urinary prostaglandin E2 as a biomarker for recurrent UTI in postmenopausal women.

Urinary tract infection (UTI) is one of the most common adult bacterial infections and exhibits high recurrence rates, especially in postmenopausal women. Studies in mouse models suggest that cyclooxygenase-2 (COX-2)-mediated inflammation sensitizes the bladder to recurrent UTI (rUTI). However, COX-2-mediated inflammation has not been robustly studied in human rUTI. We used human cohorts to assess urothelial COX-2 production and evaluate its product, PGE2, as a biomarker for rUTI in postmenopausal women. We found that the percentage of COX-2-positive cells was elevated in inflamed versus uninflamed bladder regions. We analyzed the performance of urinary PGE2 as a biomarker for rUTI in a controlled cohort of 92 postmenopausal women and PGE2 consistently outperformed all other tested clinical variables as a predictor of rUTI status. Furthermore, time-to-relapse analysis indicated that the risk of rUTI relapse was 3.6 times higher in women with above median urinary PGE2 levels than with below median levels. Taken together, these data suggest that urinary PGE2 may be a clinically useful diagnostic and prognostic biomarker for rUTI in postmenopausal women.

Stromal Hedgehog pathway activation by IHH suppresses lung adenocarcinoma growth and metastasis by limiting reactive oxygen species.

Activation of the Hedgehog (Hh) signaling pathway by mutations within its components drives the growth of several cancers. However, the role of Hh pathway activation in lung cancers has been controversial. Here, we demonstrate that the canonical Hh signaling pathway is activated in lung stroma by Hh ligands secreted from transformed lung epithelia. Genetic deletion of Shh, the primary Hh ligand expressed in the lung, in KrasG12D/+;Trp53fl/fl autochthonous murine lung adenocarcinoma had no effect on survival. Early abrogation of the pathway by an anti-SHH/IHH antibody 5E1 led to significantly worse survival with increased tumor and metastatic burden. Loss of IHH, another Hh ligand, by in vivo CRISPR led to more aggressive tumor growth suggesting that IHH, rather than SHH, activates the pathway in stroma to drive its tumor suppressive effects-a novel role for IHH in the lung. Tumors from mice treated with 5E1 had decreased blood vessel density and increased DNA damage suggestive of reactive oxygen species (ROS) activity. Treatment of KrasG12D/+;Trp53fl/fl mice with 5E1 and N-acetylcysteine, as a ROS scavenger, decreased tumor DNA damage, inhibited tumor growth and prolonged mouse survival. Thus, IHH induces stromal activation of the canonical Hh signaling pathway to suppress tumor growth and metastases, in part, by limiting ROS activity.

Detection of Tissue-resident Bacteria in Bladder Biopsies by 16S rRNA Fluorescence In Situ Hybridization.

Visualization of the interaction of bacteria with host mucosal surfaces and tissues can provide valuable insight into mechanisms of pathogenesis. While visualization of bacterial pathogens in animal models of infection can rely on bacterial strains engineered to express fluorescent proteins such as GFP, visualization of bacteria within the mucosa of biopsies or tissue obtained from human patients requires an unbiased method. Here, we describe an efficient method for the detection of tissue-associated bacteria in human biopsy sections. This method utilizes fluorescent in situ hybridization (FISH) with a fluorescently labeled universal oligonucleotide probe for 16S rRNA to label tissue-associated bacteria within bladder biopsy sections acquired from patients suffering from recurrent urinary tract infection. Through use of a universal 16S rRNA probe, bacteria can be detected without prior knowledge of species, genera, or biochemical characteristics, such as lipopolysaccharide (LPS), that would be required for detection by immunofluorescence experiments. We describe a complete protocol for 16S rRNA FISH from biopsy fixation to imaging by confocal microscopy. This protocol can be adapted for use in almost any type of tissue and represents a powerful tool for the unbiased visualization of clinically-relevant bacterial-host interactions in patient tissue. Furthermore, using species or genera-specific probes, this protocol can be adapted for the detection of specific bacterial pathogens within patient tissue.

p63 and SOX2 Dictate Glucose Reliance and Metabolic Vulnerabilities in Squamous Cell Carcinomas.

Squamous cell carcinoma (SCC), a malignancy arising across multiple anatomical sites, is responsible for significant cancer mortality due to insufficient therapeutic options. Here, we identify exceptional glucose reliance among SCCs dictated by hyperactive GLUT1-mediated glucose influx. Mechanistically, squamous lineage transcription factors p63 and SOX2 transactivate the intronic enhancer cluster of SLC2A1. Elevated glucose influx fuels generation of NADPH and GSH, thereby heightening the anti-oxidative capacity in SCC tumors. Systemic glucose restriction by ketogenic diet and inhibiting renal glucose reabsorption with SGLT2 inhibitor precipitate intratumoral oxidative stress and tumor growth inhibition. Furthermore, reduction of blood glucose lowers blood insulin levels, which suppresses PI3K/AKT signaling in SCC cells. Clinically, we demonstrate a robust correlation between blood glucose concentration and worse survival among SCC patients. Collectively, this study identifies the exceptional glucose reliance of SCC and suggests its candidacy as a highly vulnerable cancer type to be targeted by systemic glucose restriction.