Background Extracellular vesicles (EVs) are released by most cell types and are involved in multiple basic biological processes. Medium/large EVs (m/lEVs), which are of a different size from exosomes, play an important role in the coagulation in blood, and are secreted from cancer cells, etc., suggesting functions related to malignant transformation. The m/lEVs levels in blood or urine may help unravel pathophysiological findings in many diseases. However, it remains unclear how many naturally-occurring m/lEV subtypes exist as well as how their characteristics and functions differ from one another. Methods We used the blood and urinal sample from each 10 healthy donors for analysis. Using a flow cytometer, we focus on characterization of EVs with large sizes (>200 nm) that are different from exosomes. We also searched for a membrane protein for characterization with a flow cytometer using shotgun proteomics. We then identified m/lEVs pelleted from plasma and urine samples by differential centrifugation and characterized by flow cytometry. Results Using proteomic profiling, we identified several proteins involved in m/lEV biogenesis including adhesion molecules, peptidases and exocytosis regulatory proteins. In healthy human plasma, we could distinguish m/lEVs derived from platelets, erythrocytes, monocytes/macrophages, T and B cells, and vascular endothelial cells with more than two positive surface antigens. The ratio of phosphatidylserine appearing on the membrane surface differed depending on the cell-derived m/lEVs. In urine, 50% of m/lEVs were Annexin V negative but contained various membrane peptidases derived from renal tubular villi. Urinary m/lEVs, but not plasma m/lEVs, showed peptidase activity. The knowledge of the new characteristics is considered to be useful as a diagnostic material and the newly developed method suggests the possibility of clinical application.

Background: MicroRNAs (miRNA) are expected as useful biomarkers for various diseases. We studied the pre-analytical factors causing variation in the analysis of miRNA.Material and Methods: Blood samples were collected from 25 healthy subjects. Plasma and serum were obtained from the same samples. The levels of miR-451, -16, -126, and -223 were analyzed using RT-qPCR. Cel-miR-39 was added as a spiked-in control in each sample.Results: With the exception of miR-451, the levels of the miRNAs in plasma were higher than in serum. After high-speed centrifugation, the levels of miRNAs were almost equal between plasma and serum except for miR-451. Membrane filtration with 0.45 µm pore size reduced the levels of plasma miRNAs. The coagulation accelerators for serum processing did not affect the analysis of miRNA. The use of fraction containing particles of > 0.45 µm in size showed the inhibitory effect on the analysis of plasma miR-451. The RNase inhibitor was effective for protecting against the degradation of miRNAs.Conclusion: Plasma contains factors modifying miRNA profiles. The immediate processing of plasma with membrane filtration and RNase inhibitor may be a relevant method for achieving the stable analysis of miRNA

Early detection and long‐term monitoring are important for urothelial carcinoma of the bladder (UCB). Urine cytology and existing markers have insufficient diagnostic performance. Here, we examined medium‐sized extracellular vesicles (EVs) in urine to identify specific markers for UCB and evaluated their usefulness as diagnostic material. To identify specific markers in urinary EVs derived from UCB, we undertook shotgun proteomics using urine from four UCB patients and four healthy subjects. Next, 29 healthy specimens, 18 noncancer specimens, and 33 UCB specimens, all from men, were analyzed for urinary EVs by flow cytometry to evaluate the diagnostic performance of UCB‐specific EVs. Nanoparticle‐tracking analysis indicated that the size of EVs extracted from urine was mostly <400 nm. By shotgun proteomics, we detected several proteins characteristic of UCB and found that carcinoembryonic antigen‐related adhesion molecule (CEACAM) proteins were increased in patients. Flow cytometric analysis revealed that the degree of expression of CEACAM1, CEACAM5, and CEACAM6 proteins on the surface of EVs varied among patients. Extracellular vesicles expressing CEACAM proteins also expressed mucin 1, suggesting that they were derived from tumorigenic uroepithelial cells. The number of EVs expressing CEACAM1, 5, and 6 proteins was significantly increased in UCB (mean ± SD, 8.6 ± 13%) compared to non‐UCB (0.69 ± 0.46) and healthy (0.46 ± 0.34) by flow cytometry. The results of receiver operating characteristic (ROC) analysis showed a good score of area under the ROC curve of 0.907. We identified EVs that specifically express CEACAM proteins in urine and have potential for diagnostic applications. These EVs are potential targets in a new liquid biopsy test for UCB patients.

Medium/large extracellular vesicles (m/lEVs) are released by most cell types and are involved in multiple basic biological processes. Analysis of m/lEV levels in blood or urine may help unravel pathophysiological findings in many diseases. However, it remains unclear how many naturally-occurring m/lEV subtypes exist as well as how their characteristics and functions differ from one another. Here, we identified m/lEVs pelleted from plasma and urine samples by differential centrifugation and showed by flow cytometry that they typically possessed diameters between 200 nm and 800 nm. Using proteomic profiling, we identified several proteins involved in m/lEV biogenesis including adhesion molecules, peptidases and exocytosis regulatory proteins. In healthy human plasma, we could distinguish m/lEVs derived from platelets, erythrocytes, monocytes/macrophages, T and B cells, and vascular endothelial cells using various surface antigens. m/lEVs derived from erythrocytes and monocytes were Annexin V positive. In urine, 50% of m/lEVs were Annexin V negative but contained various membrane peptidases derived from renal tubular villi. Urinary m/lEVs, but not plasma m/lEVs, showed peptidase activity. The method we have developed to characterize cellderived m/lEVs suggests the possibility of clinical applications.

Purpose: Early detection and long-term monitoring are important for urothelial carcinoma of the bladder (UCB). Urine cytology and existing markers have insufficient diagnostic performance. Here, we used medium-sized extracellular vesicles (EVs) in urine to find specific markers for UCB and evaluate their usefulness as diagnostic material. Experimental Design: To search for specific markers in urinary EVs derived from UCB, we performed a comparative analysis using shotgun proteomics with urine from four UCB patients and four healthy subjects. Next, we used male 24 healthy specimens, 18 non-cancer specimens, and 29 UCB specimens for analysis of urinary EVs using a flow cytometer to evaluate the diagnostic performance of UCB-specific EVs. Results. NTA analysis showed that the size of EVs extracted from urine by centrifugation was mostly less than 400 nm. By shotgun analysis of EVs, we detected several proteins characteristic of UCB and found that CEACAM proteins was increased in patients. Flow cytometric analysis revealed that the degree of expression of CEACAM1, 5, and 6 antigens on the surface of EVs varied among patients. EVs expressing CEACAM antigens also expressed MUC1, suggesting that they were derived from tumorigenic uroepithelial cells. The number of EVs expressing CEACAM1, 5, and 6 proteins (CEACAMs+EVs) was significantly increased in UCB (Mean9.5%±SD14%), non-bladder cancer (0.69±0.46), and healthy (0.37±0.31) by flow cytometry. The results of ROC analysis showed a good score of AUC=0.929. A provisional CEACAMs+EVs cutoff % was established with a sensitivity of 83% and specificity of 98%. Here, seven out of nine cases of negative urine cytology (class 2) were determined to be positive. Conclusions: The presence of CEA antigen in the urine of patients with bladder cancer is known, but it has not yet been practically used for diagnosis. We have discovered EVs that specifically express these antigens and have obtained useful diagnostic performance. EVs expressing CEACAM proteins in urine are expecting target as a new liquid biopsy for UCB patients. Citation Format: Ko Igami, Takeshi Uchiumi, Masaki Shiota, Saori Ueda, Masatoshi Eto, Dongchon Kang. Extracellular vesicles expressing CEACAM proteins in the urine of bladder cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5104.

The 3243A &gt; G in mtDNA is a representative mutation in mitochondrial diseases. Mitochondrial protein synthesis is impaired due to decoding disorder caused by severe reduction of 5-taurinomethyluridine (τm5U) modification of the mutant mt-tRNALeu(UUR) bearing 3243A &gt; G mutation. The 3243A &gt; G heteroplasmy in peripheral blood reportedly decreases exponentially with age. Here, we found three cases with mild respiratory symptoms despite bearing high rate of 3243A &gt; G mutation (&gt;90%) in blood mtDNA. These patients had the 3290T &gt; C haplotypic mutation in addition to 3243A &gt; G pathogenic mutation in mt-tRNALeu(UUR) gene. We generated cybrid cells of these cases to examine the effects of the 3290T &gt; C mutation on mitochondrial function and found that 3290T &gt; C mutation improved mitochondrial translation, formation of respiratory chain complex, and oxygen consumption rate of pathogenic cells associated with 3243A &gt; G mutation. We measured τm5U frequency of mt-tRNALeu(UUR) with 3243A &gt; G mutation in the cybrids by a primer extension method assisted with chemical derivatization of τm5U, showing that hypomodification of τm5U was significantly restored by the 3290T &gt; C haplotypic mutation. We concluded that the 3290T &gt; C is a haplotypic mutation that suppresses respiratory deficiency of mitochondrial disease by restoring hypomodified τm5U in mt-tRNALeu(UUR) with 3243A &gt; G mutation, implying a potential therapeutic measure for mitochondrial disease associated with pathogenic mutations in mt-tRNAs.