Macrophage-depleted young mice are beneficial in vivo models to assess the translocation of Klebsiella pneumonia from the gastrointestinal tract to the liver in the elderly.

Pathobionts are commensal intestinal microbiota capable of causing systemic infections under specific conditions, such as environmental changes or aging. However, it is unclear how pathobionts are recognized by the intestinal mucosal immune system under physiological conditions. This study demonstrates that the gut pathobiont Klebsiella pneumoniae causes injury to the epithelium and translocates to the liver in specific pathogen-free mice treated with clodronate-liposomes that depleted macrophages. In the clodronate-liposome-treated mice, indigenous classical K. pneumoniae (cKp) with non-K1/K2 capsular serotypes were isolated from the liver, indicating that gut commensal cKp translocated from the gastrointestinal tract to the liver due to the depletion of intestinal macrophages. Oral inoculation of isolated cKp to clodronate-liposome-treated mice significantly reduced the survival rates compared to that of non-treated mice. Our findings demonstrate that intestinal mucosal macrophages play a pivotal role in sensing commensal cKp and suppressing their translocation to the liver. This study demonstrates that clodronate-liposome-treated mouse models are effective for screening and evaluating drugs that prevent the translocation of cKp to the liver, providing new insights into the development of preventive protocols against K. pneumoniae infection.

Profile of miRNAs in small extracellular vesicles released from glioblastoma cells treated by boron neutron capture therapy.

PURPOSE: Boron neutron capture therapy (BNCT) is a tumor cell-selective particle-radiation therapy. In BNCT, administered p-boronophenylalanine (BPA) is selectively taken up by tumor cells, and the tumor is irradiated with thermal neutrons. High-LET α-particles and recoil 7Li, which have a path length of 5-9 µm, are generated by the capture reaction between 10B and thermal neutrons and selectively kill tumor cells that have uptaken 10B. Although BNCT has prolonged the survival time of malignant glioma patients, recurrences are still to be resolved. miRNAs, that are encapsulated in small extracellular vesicles (sEVs) in body fluids and exist stably may serve critical role in recurrence. In this study, we comprehensively investigated microRNAs (miRNAs) in sEVs released from post-BNCT glioblastoma cells. METHOD: Glioblastoma U87 MG cells were treated with 25 ppm of BPA in the culture media and irradiated with thermal neutrons. After irradiation, they were plated into dishes and cultured for 3 days in the 5% CO2 incubator. Then, sEVs released into the medium were collected by column chromatography, and miRNAs in sEVs were comprehensively investigated using microarrays. RESULT: An increase in 20 individual miRNAs (ratio > 2) and a decrease in 2 individual miRNAs (ratio < 0.5) were detected in BNCT cells compared with non-irradiated cells. Among detected miRNAs, 20 miRNAs were associated with worse prognosis of glioma in Kaplan Meier Survival Analysis of overall survival in TCGA. CONCLUSION: These miRNA after BNCT may proceed tumors, modulate radiation resistance, or inhibit invasion and affect the prognosis of glioma.

Potential utility of pretreatment serum miRNAs for optimal treatment selection in advanced high-grade serous ovarian cancer.

OBJECTIVE: The primary treatment of patients with advanced ovarian cancer is selected from whether primary debulking surgery or neoadjuvant chemotherapy. We investigated whether pretreatment serum microRNA profiles are useful for selecting patients with advanced high-grade serous ovarian cancer who obtain better outcomes from undergoing primary debulking surgery or neoadjuvant chemotherapy. METHODS: Consecutive patients with clinical stage IIIB-IVB and serum microRNA data were selected. Patients who underwent primary debulking surgery or neoadjuvant chemotherapy were subjected to 1:1 propensity score matching before comparing their progression-free survival using Cox modelling. Progression-free probabilities for the selected microRNA profiles were calculated, and the estimated progression-free survival with the recommended primary treatment was determined and compared with the actual progression-free survival of the patients. RESULTS: Of the 108 patients with stage IIIB-IVB disease, the data of 24 who underwent primary debulking surgery or neoadjuvant chemotherapy were compared. Eleven and three microRNAs were independent predictors of progression-free survival in patients who underwent primary debulking surgery and neoadjuvant chemotherapy, respectively. Two microRNAs correlated significantly with complete resection of the tumours in primary debulking surgery. No differences were found between the actual and estimated progression-free survival in the primary debulking surgery and neoadjuvant chemotherapy groups (P > 0.05). The recommended and actual primary treatments were identical in 27 (56.3%) of the 48 patients. The median improved survival times between recommended and actual treatment were 11.7 and 32.6 months for patients with actual primary debulking surgery and neoadjuvant chemotherapy, respectively. CONCLUSIONS: Pretreatment microRNA profiles could be used to select subgroups of patients who benefited more from primary debulking surgery or neoadjuvant chemotherapy and might contribute to selecting the optimal primary treatment modality in advanced high-grade serous ovarian cancer patients.

A comparative study on dietary diversity and gut microbial diversity in children with autism spectrum disorder, attention-deficit hyperactivity disorder, their neurotypical siblings, and non-related neurotypical volunteers: a cross-sectional study.

BACKGROUND: Previous research has shown a significant link between gut microbiota in children with autism spectrum disorder (ASD) and attention-deficit hyperactivity disorder (ADHD). However, much remains unknown because of the heterogeneity of disorders and the potential confounders such as dietary patterns and control group variations. METHODS: Children aged 6-12 years who had been clinically diagnosed with ASD and/or ADHD, their unaffected neurotypical siblings, and non-related neurotypical volunteers were recruited cross-sectionally. The ASD diagnosis was confirmed using the Autism Diagnostic Observation Schedule-2 (ADOS-2) in all patients, including those with ADHD. Standardized DNA extraction and sequencing methods were used to compare gut microbial alpha-diversity among the groups. Dietary diversity was calculated from a standardized dietary questionnaire form. We compared the difference in gut microbiome between patients with ASD and/or ADHD with neurotypical siblings and non-related neurotypical controls. RESULTS: Ninety-eight subjects were included in the study (18 with ASD, 19 with ADHD, 20 with both ASD and ADHD, 13 neurotypical siblings, and 28 non-related neurotypical controls). The alpha-diversity indices, such as Chao 1 and Shannon index, showed a significant difference between the groups in a Linear mixed-effect model (F(4, 93) = 4.539, p = .02), (F(4, 93) = 3.185, p = .017), respectively. In a post-hoc pairwise comparison, patients with ASD had lower alpha-diversity compared with non-related controls after Bonferroni correction. Dietary diversity shown in Shannon index did not differ among the groups (F(4, 84) = 1.494, p = .211). CONCLUSIONS: Our study indicates disorder-specific microbiome differences in patients with ASD. In future research on gut microbiota in neurodevelopmental disorders, it is necessary to consider the impact of ASD and ADHD co-occurrence, and strictly control for background information such as diet, to elucidate the gut-microbiota interaction in ASD and ADHD for exploring the potential of therapeutic interventions.

Plasma extracellular vesicle microRNAs reflecting the therapeutic effect of the CBP/ß-catenin inhibitor PRI-724 in patients with liver cirrhosis.

There is an unmet need for antifibrotic therapies to prevent the progression of liver cirrhosis. Previously, we conducted an exploratory trial to assess the safety and antifibrotic efficacy of PRI-724, a selective CBP/ß-catenin inhibitor, in patients with liver cirrhosis. PRI-724 was well tolerated and exerted a potential antifibrotic effect. Here, we investigated whether the profiles of circulating microRNAs packaged in extracellular vesicles (EV-miRNAs) are associated with responses to liver fibrosis treatments. Eighteen patients who received PRI-724 for 12 weeks in a phase 1/2a study were classified as responders (n = 10) or non-responders (n = 8) based on changes in liver stiffness. Plasma samples were obtained before and after PRI-724 administration and the levels of EV-miRNAs were analyzed. Three miRNAs (miR-6510-5p, miR-6772-5p, and miR-4261) were identified as predictors of response or non-response to PRI-724, and the levels of three other miRNAs (miR-939-3p, miR-887-3p, and miR-7112-5p) correlated with the efficacy of treatment. Expression of miR-887-3p was detected in hepatocytes and was decreased significantly in liver tissue following PRI-724 treatment. In addition, transfection of a miR-887-3p mimic activated hepatic stellate cells. Thus, decreases in the miR-887-3p level in blood may reflect recovery from liver fibroses in patients with liver cirrhosis treated with PRI-724, although further validation studies are warranted to confirm this.

Intestinal metabolites predict treatment resistance of patients with depression and anxiety.

BACKGROUND: The impact of the gut microbiota on neuropsychiatric disorders has gained much attention in recent years; however, comprehensive data on the relationship between the gut microbiome and its metabolites and resistance to treatment for depression and anxiety is lacking. Here, we investigated intestinal metabolites in patients with depression and anxiety disorders, and their possible roles in treatment resistance. RESULTS: We analyzed fecal metabolites and microbiomes in 34 participants with depression and anxiety disorders. Fecal samples were obtained three times for each participant during the treatment. Propensity score matching led us to analyze data from nine treatment responders and nine non-responders, and the results were validated in the residual sample sets. Using elastic net regression analysis, we identified several metabolites, including N-ε-acetyllysine; baseline levels of the former were low in responders (AUC = 0.86; 95% confidence interval, 0.69-1). In addition, fecal levels of N-ε-acetyllysine were negatively associated with the abundance of Odoribacter. N-ε-acetyllysine levels increased as symptoms improved with treatment. CONCLUSION: Fecal N-ε-acetyllysine levels before treatment may be a predictive biomarker of treatment-refractory depression and anxiety. Odoribacter may play a role in the homeostasis of intestinal L-lysine levels. More attention should be paid to the importance of L-lysine metabolism in those with depression and anxiety.

Identifying high-grade serous ovarian carcinoma-specific extracellular vesicles by polyketone-coated nanowires.

Cancer cell-derived extracellular vesicles (EVs) have unique protein profiles, making them promising targets as disease biomarkers. High-grade serous ovarian carcinoma (HGSOC) is the deadly subtype of epithelial ovarian cancer, and we aimed to identify HGSOC-specific membrane proteins. Small EVs (sEVs) and medium/large EVs (m/lEVs) from cell lines or patient serum and ascites were analyzed by LC-MS/MS, revealing that both EV subtypes had unique proteomic characteristics. Multivalidation steps identified FRα, Claudin-3, and TACSTD2 as HGSOC-specific sEV proteins, but m/lEV-associated candidates were not identified. In addition, for using a simple-to-use microfluidic device for EV isolation, polyketone-coated nanowires (pNWs) were developed, which efficiently purify sEVs from biofluids. Multiplexed array assays of sEVs isolated by pNW showed specific detectability in cancer patients and predicted clinical status. In summary, the HGSOC-specific marker detection by pNW are a promising platform as clinical biomarkers, and these insights provide detailed proteomic aspects of diverse EVs in HGSOC patients.

Gas6 ameliorates intestinal mucosal immunosenescence to prevent the translocation of a gut pathobiont, Klebsiella pneumoniae, to the liver.

Immunosenescence refers to the development of weakened and/or dysfunctional immune responses associated with aging. Several commensal bacteria can be pathogenic in immunosuppressed individuals. Although Klebsiella pneumoniae is a commensal bacterium that colonizes human mucosal surfaces, the gastrointestinal tract, and the oropharynx, it can cause serious infectious diseases, such as pneumonia, urinary tract infections, and liver abscesses, primarily in elderly patients. However, the reason why K. pneumoniae is a more prevalent cause of infection in the elderly population remains unclear. This study aimed to determine how the host's intestinal immune response to K. pneumoniae varies with age. To this end, the study analyzed an in vivo K. pneumoniae infection model using aged mice, as well as an in vitro K. pneumoniae infection model using a Transwell insert co-culture system comprising epithelial cells and macrophages. In this study, we demonstrate that growth arrest-specific 6 (Gas6), released by intestinal macrophages that recognize K. pneumoniae, inhibits bacterial translocation from the gastrointestinal tract by enhancing tight-junction barriers in the intestinal epithelium. However, in aging mice, Gas6 was hardly secreted under K. pneumoniae infection due to decreasing intestinal mucosal macrophages; therefore, K. pneumoniae can easily invade the intestinal epithelium and subsequently translocate to the liver. Moreover, the administration of Gas6 recombinant protein to elderly mice prevented the translocation of K. pneumoniae from the gastrointestinal tract and significantly prolonged their survival. From these findings, we conclude that the age-related decrease in Gas6 secretion in the intestinal mucosa is the reason why K. pneumoniae can be pathogenic in the elderly, thereby indicating that Gas6 could be effective in protecting the elderly against infectious diseases caused by gut pathogens.

Evidence for in vitro extensive proliferation of adult hepatocytes and biliary epithelial cells.

Over the last several years, a method has emerged that endows adult hepatocytes with in vitro proliferative capacity, producing chemically induced liver progenitors (CLiPs). However, there is a growing controversy regarding the origin of these cells. Here, we provide lineage tracing-based evidence that adult hepatocytes acquire proliferative capacity in vitro using rat and mouse models. Unexpectedly, we also found that the CLiP method allows biliary epithelial cells to acquire extensive proliferative capacity. Interestingly, after long-term culture, hepatocyte-derived cells (hepCLiPs) and biliary epithelial cell-derived cells (bilCLiPs) become similar in their gene expression patterns, and they both exhibit differentiation capacity to form hepatocyte-like cells. Finally, we provide evidence that hepCLiPs can repopulate injured mouse livers, reinforcing our earlier argument that CLiPs can be a cell source for liver regenerative medicine. This study advances our understanding of the origin of CLiPs and motivates the application of this technique in liver regenerative medicine.

A validation study for the utility of serum microRNA as a diagnostic and prognostic marker in patients with osteosarcoma.

In our previous study, osteosarcoma advanced locally, and metastasis was promoted through the secretion of large number of small extracellular vesicles, followed by suppressing osteoclastogenesis via the upregulation of microRNA (miR)-146a-5p. An additional 12 miRNAs in small extracellular vesicles were also detected ≥6× as frequently in high-grade malignancy with the capacity to metastasize as in those with a low metastatic potential. However, the utility of these 13 miRNAs for determining the prognosis or diagnosis of osteosarcoma has not been validated in the clinical setting. In the present study, the utility of these miRNAs as prognostic and diagnostic markers was therefore assessed. In total, 30 patients with osteosarcoma were retrospectively reviewed, and the survival rate was compared according to the serum miRNA levels in 27 patients treated with chemotherapy and surgery. In addition, to confirm diagnostic competency for osteosarcoma, the serum miRNA levels were compared with those in patients with other bone tumors (n=112) and healthy controls (n=275). The patients with osteosarcoma with high serum levels of several miRNAs (miR-146a-5p, miR-1260a, miR-487b-3p, miR-1260b and miR-4758-3p) exhibited an improved survival rate compared with those with low levels. In particular, patients with high serum levels of miR-1260a exhibited a significantly improved overall survival rate, metastasis-free survival rate and disease-free survival rate compared with those with low levels. Thus, serum miR-1260a may potentially be a prognostic marker for patients with osteosarcoma. Moreover, patients with osteosarcoma had higher serum miR-1261 levels than those with benign or intermediate-grade bone tumors and thus may be a potential therapeutic target, in addition to being useful for differentiating whether or not a bone tumor is high-grade. A larger investigation is required to clarify the actual utility of these miRNAs in the clinical setting.

Evidence for in vitro extensive proliferation of adult hepatocytes and biliary epithelial cells.

Over the last several years, a method has emerged which endows adult hepatocytes with in vitro proliferative capacity, producing chemically-induced liver progenitors (CLiPs). However, a recent study questioned the origin of these cells, suggesting that resident liver progenitor cells, but not hepatocytes, proliferate. Here, we provide lineage tracing-based evidence that adult hepatocytes acquire proliferative capacity in vitro . Unexpectedly, we also found that the CLiP method allows biliary epithelial cells to acquire extensive proliferative capacity. Interestingly, after long-term culture, hepatocyte-derived cells (hepCLiPs) and biliary-derived cells (bilCLiPs) become similar in their gene expression patterns, and they both exhibit differentiation capacity to form hepatocyte-like cells. Finally, we provide evidence that hepCLiPs can repopulate chronically injured mouse livers, reinforcing our earlier argument that CLiPs can be a cell source for liver regenerative medicine. Moreover, this study offers bilCLiPs as a potential cell source for liver regenerative medicine.

Prediction of tissue-of-origin of early stage cancers using serum miRNomes.

BACKGROUND: Noninvasive detection of early stage cancers with accurate prediction of tumor tissue-of-origin could improve patient prognosis. Because miRNA profiles differ between organs, circulating miRNomics represent a promising method for early detection of cancers, but this has not been shown conclusively. METHODS: A serum miRNA profile (miRNomes)-based classifier was evaluated for its ability to discriminate cancer types using advanced machine learning. The training set comprised 7931 serum samples from patients with 13 types of solid cancers and 5013 noncancer samples. The validation set consisted of 1990 cancer and 1256 noncancer samples. The contribution of each miRNA to the cancer-type classification was evaluated, and those with a high contribution were identified. RESULTS: Cancer type was predicted with an accuracy of 0.88 (95% confidence interval [CI] = 0.87 to 0.90) in all stages and an accuracy of 0.90 (95% CI = 0.88 to 0.91) in resectable stages (stages 0-II). The F1 score for the discrimination of the 13 cancer types was 0.93. Optimal classification performance was achieved with at least 100 miRNAs that contributed the strongest to accurate prediction of cancer type. Assessment of tissue expression patterns of these miRNAs suggested that miRNAs secreted from the tumor environment could be used to establish cancer type-specific serum miRNomes. CONCLUSIONS: This study demonstrates that large-scale serum miRNomics in combination with machine learning could lead to the development of a blood-based cancer classification system. Further investigations of the regulating mechanisms of the miRNAs that contributed strongly to accurate prediction of cancer type could pave the way for the clinical use of circulating miRNA diagnostics.

Serum microRNAs as new criteria for referral to early palliative care services in treatment-naïve advanced cancer patients.

A major obstacle to the implementation of early palliative care (EPC) is the lack of objective criteria for referral to EPC. Circulating microRNAs (miRNAs) have been recognized as promising biomarkers. The present study investigated objective definitions for referral to EPC using microRNA. A total of 178 serum samples were obtained from patients with lung, gastrointestinal, colorectal, bile duct, pancreas and bladder cancers who were treatment-naïve and received chemotherapy between January 2011 and December 2013 at National Cancer Center Hospital East. We investigated expression levels of miRNAs using microarrays. The primary outcome was prediction of admission to a palliative care unit ≤6 months after first visit. Diagnostic models using clinical characteristics, miRNAs and combinations of both were constructed. The miRNA models were constructed using 6 miRNA levels. The best areas under the receiver operating characteristic curve (AUCs) of the clinical model was 0.741, while the average AUCs of miRNA-based models and combination models were 0.769 and 0.806, respectively. Combination models showed higher AUCs than the clinical model (p < 0.023). The present combination models might offer new objective definitions for referral to EPC and thus contribute to real-world implementation of EPC.

Circulating microRNA profiling for prediction of oncological outcomes in prostate cancer patients following radical prostatectomy.

BACKGROUND: Although radical prostatectomy is associated with good long-term oncological outcomes, approximately 30% of patients present biochemical recurrence, whereupon salvage treatments are required. Identification of novel molecular biomarkers to predict cancer behavior is clinically important. Here, we developed a novel microRNA (miRNA)-based prognostic model for patients who underwent radical prostatectomy. METHODS: We retrospectively investigated the clinical records of 295 patients who underwent radical prostatectomy between 2009 and 2017. We randomly assigned these cases into training or validation sets. The prognostic model was constructed using Fisher linear discriminant analysis in the training set, and we evaluated its performance in the validation set. RESULTS: Overall, 72 patients had biochemical recurrence. A prediction model was constructed using a combination of three miRNAs (miR-3147, miR-4513, and miR-4728-5p) and two pathological factors (pathological T stage and Gleason score). In the validation set, the predictive performance of the model was confirmed to be accurate (area under the receiver operating characteristic curve: 0.80; sensitivity: 0.78; specificity: 0.76). Additionally, Kaplan-Meier analysis revealed that the patients with a low prediction index had significantly longer recurrence-free survival than those with a high index (p < 0.001). CONCLUSIONS: Circulating miRNA profiles can provide information to predict recurrence after prostatectomy. Our model may be helpful for physicians to decide follow-up strategies for patients.

Circulating microRNAs: Challenges with their use as liquid biopsy biomarkers.

Circulating microRNA (miRNA) is a major focus in liquid biopsy studies. The circulating levels of certain miRNAs have been suggested to reflect specific physiological conditions, and several studies have reported their potential use as biomarkers for the detection and prognosis of cancer, as well as for predicting responses to chemotherapy or radiotherapy. Alongside these biomarker studies, research into the effects of specific background factors on circulating miRNA levels is progressing. Indeed, several studies have shown that a number of factors, including blood sample collection and processing methods, as well as subject-specific factors such as age, sex, and other physiological conditions, can affect the normal levels of circulating miRNAs. Unfortunately, the evidence supporting these effects is not yet strong enough to support a definite conclusion and further research is warranted. Here, we summarize the findings of several studies that have addressed these concerns and identify important topics that should be considered when analyzing circulating miRNA levels in liquid biopsy studies.

Independent verification of circulating miRNA as diagnostic biomarkers for urothelial carcinoma.

Urothelial carcinoma (UC) is an umbrella term for bladder cancers (BCa) and upper-tract urothelial carcinoma (UTUC), with BCa and UTUC sometimes detected concomitantly. The methods of detection for UC are often inaccurate or highly invasive, and, therefore, are thought to be unsatisfactory. Previously, we reported seven serum miRNAs as diagnostic markers for BCa. Here, we re-evaluated potential diagnostic miRNAs in different institutions. We prospectively analyzed serum samples obtained from 126 UC patients (BCa: 106 samples; UTUC: 14 samples; UTUC with BCa: six samples) and 50 noncancer controls by microarray analysis. We randomly assigned these samples into a training or a validation set. Biomarker candidate miRNAs were selected based on cross-validation scores in the training set of samples, with diagnostic power confirmed in the validation set. Among the diagnostic miRNAs identified in this way, miR-1343-5p and miR-6087 had been identified as potential diagnostic miRNAs in our previous study. In addition, we evaluated the association between the serum levels of identified miRNAs and the presence of UC risk conditions. The expression levels of several miRNAs correlate with the risk factors in participants without UC, which may be explained by the presence of a microscopic tumor or a precancerous lesion. In conclusion, we identified two robust miRNA diagnostic markers for UC detection. Further functional analysis is required to elucidate the mechanism by which alterations in the expression of these miRNAs occur.

Efficacy and safety of low-dose rifabutin-based 7-day triple therapy as a third- or later-line Helicobacter pylori eradication regimen.

BACKGROUND: Rifabutin-based regimens are used as rescue therapy for refractory Helicobacter pylori infection; however, the duration for which treatment is required and side effects are concerning. This study assessed the efficacy and safety of 7-day rifabutin, amoxicillin, and vonoprazan triple therapy as third- or later-line treatment for H. pylori infection. MATERIALS AND METHODS: Patients who did not respond to second-line therapy were enrolled. After H. pylori infection was confirmed with the culture method, the patients received rifabutin-containing triple therapy (20 mg vonoprazan b.i.d., 500 mg amoxicillin q.i.d., and 150 mg rifabutin q.d.) for 7 days. Twelve weeks after the eradication therapy, successful eradication was confirmed using a 13 C urea breath test or the H. pylori stool antigen test. The results obtained from our previous study that reported a 10-day or 14-day esomeprazole based rifabutin-containing triple therapy as a third- or fourth-line rescue therapy treated patients were used as historical control. We determined the minimum inhibitory concentrations of amoxicillin and rifabutin. We also evaluated whether the patients were positive for the mutation of the rpoB gene. RESULTS: Intention-to-treat and per-protocol analyses showed that our regimen resulted in a high eradication rate (91.2%, 95% CI: 84%-99% and 92.7%, 95% CI: 86%-100%, respectively). Adverse events occurred in 31.6% of the patients, and two patients discontinued the therapy. CONCLUSIONS: This is the first study to evaluate the efficacy and safety of a 7-day low-dose rifabutin-based triple therapy with vonoprazan and amoxicillin. Our results suggest that our regimen was effective and safe as a third- or later-line H. pylori eradication regimen. To clarify what component in this regimen are critical, subsequent studies using a factorial design (comparing vonoprazan-amoxicillin dual therapy vs. vonoprazan-rifabutin triple therapy) will be needed.

Identification of circulating microRNAs as potential biomarkers for hepatic necroinflammation in patients with autoimmune hepatitis.

OBJECTIVE: MicroRNAs (miRNAs) are implicated in the pathogenesis of autoimmune diseases and could be biomarkers of disease activity. This study aimed to identify highly expressed circulating miRNAs in patients with autoimmune hepatitis (AIH) and to evaluate their association with clinical characteristics. METHODS: Microarray analyses were performed, and miRNA expression profiling for AIH, primary biliary cholangitis (PBC) and overlap syndrome (OS) using the serum of patients and healthy individuals was done. Samples were divided into discovery and test sets to identify candidate miRNAs that could discriminate AIH from PBC; the former included 21 AIH and 23 PBC samples, while the latter included five AIH and eight PBC samples. RESULTS: Among 11 candidate miRNAs extracted in the discovery set, 4 (miR-3196, miR-6125, miR-4725-3 p and miR-4634) were specifically and highly expressed in patients with AIH in the test set. These four miRNAs discriminated AIH from PBC with high sensitivity (0.80-1.00) and specificity (0.88-1.00). In situ hybridisation analysis revealed that these miRNAs were expressed in the cytoplasm of hepatocytes in patients with AIH. Their expression levels were highest in untreated patients with AIH, followed by those in untreated patients with OS. They drastically or moderately decreased after prednisolone treatment. Histological analysis demonstrated that the expression levels of miR-3196, miR-6125 and miR-4634 in patients with AIH and OS were correlated with severe hepatic necroinflammatory activity. CONCLUSION: These circulating miRNAs are suggested to reflect hepatic necroinflammatory activity and serve as AIH-related and treatment-responsive biomarkers. These miRNAs could be beneficial in developing new therapeutic strategies for AIH.

Serum microRNA as liquid biopsy biomarker for the prediction of oncological outcomes in patients with bladder cancer.

OBJECTIVES: Radical cystectomy is the gold-standard treatment for muscle-invasive bladder cancer and aggressive non-muscle-invasive bladder cancer. To enhance clinical decision-making regarding patients with bladder cancer who underwent radical cystectomy, a recurrence prediction biomarker with high accuracy is urgently needed. In this study, we developed a model for the prediction of bladder cancer recurrence after radical cystectomy by combining serum microRNA and a pathological factor. METHODS: We retrospectively analyzed the clinical records of 81 patients with bladder cancer who underwent radical cystectomy between 2008 and 2016. The dataset was divided into two, and Fisher linear discriminant analysis was used to construct a prognostic model for future recurrence in the training set (n = 41). The performance of the model was evaluated in the validation set (n = 40). RESULTS: Thirty patients had recurrence after having undergone radical cystectomy. A prognostic model for recurrence was constructed by combining a pathological factor (i.e. positive pathological lymph node status) and three microRNAs (miR-23a-3p, miR-3679-3p, and miR-3195). The model showed a sensitivity of 0.87, a specificity of 0.80, and an area under the receiver operating characteristic curve of 0.88 (0.77-0.98) in the validation set. Furthermore, Kaplan-Meier analysis revealed that patients with a low prediction index have significantly longer overall survival than patients with a high prediction index (P = 0.041). CONCLUSION: A combination of serum microRNA profiles and lymph node statuses is useful for the prediction of oncological outcomes after radical cystectomy in patients with bladder cancer.