To address the shortcomings of urine cytology and cystoscopy for probing and grading urinary bladder cancer (BC), we applied (1)H nuclear magnetic resonance (NMR) spectroscopy as a surrogate method for the identification of BC. This study includes 99 serum samples comprising low-grade (LG; n = 36) and high-grade (HG; n = 31) BC as well as healthy controls (HC; n = 32). (1)H NMR-derived serum data were analyzed using orthogonal partial least-squares discriminant analysis (OPLS-DA). OPLS-DA-derived model validity was confirmed using an internal and external cross-validation. Internal validation was performed using the initial samples (n = 99) data set. External validation was performed on a new batch of suspected BC patients (n = 106) through a double-blind study. Receiver operating characteristic (ROC) curve analysis was also performed. OPLS-DA-derived serum metabolomics (six biomarkers, ROC; 0.99) were able to discriminate 95% of BC cases with 96% sensitivity and 94% specificity when compared to HC. Likewise (three biomarkers, ROC; 0.99), 98% of cases of LG were able to differentiate from HG with 97% sensitivity and 99% specificity. External validation reveals comparable results to the internal validation. (1)H NMR-based serum metabolic screening appears to be a promising and less invasive approach for probing and grading BC in contrast to the highly invasive and painful cystoscopic approach for BC detection.

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Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene lead to cystic fibrosis (CF). The most common mutation F508del inhibits folding and processing of CFTR protein. FDA-approved correctors rescue the biosynthetic processing of F508del-CFTR protein, while potentiators improve the rescued CFTR channel function. Transforming growth factor (TGF-β1), overexpressed in many CF patients, blocks corrector/potentiator rescue by inhibiting CFTR mRNA in vitro. Increased TGF-β1 signaling and acquired CFTR dysfunction are present in other lung diseases. To study the mechanism of TGF-β1 repression of CFTR, we used molecular, biochemical, and functional approaches in primary human bronchial epithelial cells from over 50 donors. TGF-β1 destabilized CFTR mRNA in cells from lungs with chronic disease, including CF, and impaired F508del-CFTR rescue by new-generation correctors. TGF-β1 increased the active pool of selected micro(mi)RNAs validated as CFTR inhibitors, recruiting them to the RNA-induced silencing complex (RISC). Expression of F508del-CFTR globally modulated TGF-β1-induced changes in the miRNA landscape, creating a permissive environment required for degradation of F508del-CFTR mRNA. In conclusion, TGF-β1 may impede the full benefit of corrector/potentiator therapy in CF patients. Studying miRNA recruitment to RISC under disease-specific conditions may help to better characterize the miRNAs utilized by TGF-β1 to destabilize CFTR mRNA.

Rationale: Tissue-resident memory T cells (T RM ) play a critical role in the defense against inhaled pathogens. The isolation and study of human lung tissue-resident memory T cells and lung-resident macrophages (M LR ) are limited by experimental constraints.Objectives: To characterize the spatial and functional relationship between M LR and human lung tissue-resident memory T cells using ex vivo lung perfusion (EVLP).Methods: T RM and M LR were isolated using EVLP and intraperfusate-labeled CD45 antibody. Cells isolated after 6 hours of EVLP were analyzed using spectral flow cytometry. Spatial relationships between CD3 1 and CD68 1 cells were explored with multiplexed immunohistochemistry. Functional relationships were determined by using coculture and T-cell-receptor complex signal transduction.Measurements and Main Results: Lungs from 8 researchconsenting organ donors underwent EVLP for 6 hours. We show that human lung T RM and M LR colocalize within the human lung, preferentially around the airways. Furthermore, we found that human lung CD8 1 T RM are composed of two functionally distinct populations on the basis of PD1 (programed cell death receptor 1) and ZNF683 (HOBIT) protein expression. We show that M LR provide costimulatory signaling to PD1 hi CD4 1 and CD8 1 lung T RM, , augmenting the effector cytokine production and degranulation of T RM .Conclusions: EVLP provides an innovative technique to study resident immune populations in humans. Human M LR colocalize with and provide costimulation signaling to T RM , augmenting their effector function.