A prospective study of surgeons' workloads and associated factors in real-world practice.

New technologies such as laparoscopic and robotic surgery are spreading, and there is a demand for physicians to keep up with novel methods. In contrast to the recent focus on healthcare professional burnout, the mental and physical costs during surgery are not well-understood. We aimed to quantify surgeons' workloads in daily urological surgical practice and clarify potential background factors associated with such workloads. Urologists in Hokkaido, Japan, were invited to this study. Between December 2020 and December 2021, participants repeatedly reported workloads, which were assessed using the National Aeronautics and Space Administration-Task Load Index (NASA-TLX), after each surgery in conjunction with participants' names, patients' backgrounds, their roles (independent operator, operator under supervision, instructor, and 1st or 2nd assistant), and surgical outcomes, via SurveyMonkey®. Because of the heterogeneity among individuals, a linear mixed-effects model was utilized to analyze factors associated with NASA-TLX, calculating the parameter estimates (PE) of regression coefficients for each factor and their 95% confidence interval (CI). Sixty-five urologists (5 women) joined the study, and 2169 data were collected within 7 days after surgeries. A linear mixed-effects model revealed that female surgeons (PE + 15.56, 95% CI 2.36-28.77), urgent/emergency surgery (PE + 6.65, 95% CI 4.59-8.70), intraoperative complications (PE + 9.26, 95% CI 6.76-11.76), and near-miss incidents (PE + 3.81, 95% CI 2.27-5.36) were associated with higher workloads. Regarding the surgeons' role, operator under supervision (PE + 12.46, 95% CI 9.86-15.06) showed the highest workloads. Surgeons' workloads decreased as the number of previous cases of the same procedure increased. Surgeons' workloads were associated with various factors. Given that the highest workloads were for operators under supervision, instructors should be aware of trainees' high workloads and devise appropriate instructional interventions.

Cross-sectional and longitudinal analyses of urinary extracellular vesicle mRNA markers in urothelial bladder cancer patients.

We designed this multi-center prospective study with the following objectives: (1) the cross-sectional validation of extracellular vesicles (EV) mRNA markers to detect urothelial bladder cancer (UBC) before transurethral resection of bladder cancer (TURBT), and (2) the longitudinal validation of EV mRNA markers to monitor non-muscle invasive bladder cancer (NMIBC) recurrence after TURBT. EV mRNA markers evaluated in this study were KRT17, GPRC5A, and SLC2A1 in addition to two additional markers from literatures, MDK and CXCR2, and measured by quantitative RT-PCR with normalization by a reference gene (ALDOB). Diagnostic performances of EV mRNA markers were compared to conventional markers. Regarding the first objective, we confirmed that EV mRNA biomarkers in urine were higher in UBC patients, particularly those with higher stage/grade tumors, than in those without UBC (n = 278 in total) and the diagnostic performance of EV mRNA MDK and KRT17 outperformed conventional biomarkers with AUC 0.760 and 0.730, respectively. Concerning the second objective, we prospectively analyzed the time courses of EV mRNA markers while NMIBC patients (n = 189) (median follow-up 19 months). The expression of EV mRNA KRT17 was significantly high in patients with recurrence, while it gradually decreased over time in those without recurrence (p < 0.01).

The impact of second transurethral resection on survival outcomes in patients with non-muscle-invasive bladder cancer treated with bacillus Calmette-Guérin therapy.

OBJECTIVE: Several guidelines recommended that second transurethral resection should be performed in patients with diagnosis of high-risk non-muscle-invasive bladder cancer. However, therapeutic benefits of second transurethral resection before bacillus Calmette-Guérin intravesical instillation were conflicting amongst previous studies. We investigated the prognostic impact of second transurethral resection before bacillus Calmette-Guérin instillation in high-risk non-muscle-invasive bladder cancer patients. METHODS: This retrospective study included 3104 non-muscle-invasive bladder cancer patients who received bacillus Calmette-Guérin instillations between 2000 and 2019 at 31 collaborative institutions. Univariate and multivariate Cox proportional hazards models were used to assess the risk factors of intravesical recurrence, disease progression, cancer-specific mortality and overall mortality. RESULTS: In the entire population, patients undergoing second transurethral resection (33%, 1026/3104) had a lower risk of intravesical recurrence on univariate analysis (hazard ratio 0.85, 95% confidence interval 0.73-0.98, P = 0.027), although it did not remain significant on multivariate analysis (hazard ratio 0.90, 95% confidence interval 0.76-1.07, P = 0.24). Subgroup analysis revealed that, in pT1 patients (n = 1487), second transurethral resection was significantly correlated with a lower risk of intravesical recurrence on multivariate analysis (hazard ratio 0.80, 95% confidence interval 0.64-1.00, P = 0.048), but lower risks of disease progression (hazard ratio 0.75, 95% confidence interval 0.56-1.00, P = 0.049), cancer-specific mortality (hazard ratio 0.54, 95% confidence interval 0.35-0.85, P = 0.007) and overall mortality (hazard ratio 0.73, 95% confidence interval 0.55-0.97, P = 0.027) on univariate analysis. CONCLUSIONS: Second transurethral resection confers accurate pathological staging and could be used to safely select good candidates for intravesical bacillus Calmette-Guérin instillation. We further confirm that second transurethral resection could confer an oncological benefit in pT1 bladder cancer patients treated by bacillus Calmette-Guérin instillation, and so strongly recommend second transurethral resection in this patient population.

Impact of postoperative complications on long-term survival in bladder cancer patients.

OBJECTIVE: To determine the impact of postoperative complications on long-term survival outcomes in patients with bladder cancer undergoing radical cystectomy. METHODS: This retrospective multi-institutional study included 766 bladder cancer patients who underwent radical cystectomy between 2011 and 2017. Patient characteristics, perioperative outcomes, all complications within 90 days after surgery and survival outcomes were collected. Each complication was graded based on the Clavien-Dindo system, and grouped using a standardized grouping method. The Comprehensive Complication Index, which incorporates all complications into a single formula weighted by their severity, was utilized. Overall survival and recurrence-free survival (local, distant or urothelial recurrences) were stratified by Comprehensive Complication Index (high: ≥26.2; low: <26.2). A multivariate model was utilized to identify independent prognostic factors. RESULTS: The incidence of any and major complications (≥Clavien-Dindo grade III) was 70 and 24%, respectively. In terms of Comprehensive Complication Index, 34% (261/766) of the patients had ≥26.2. Patients with Comprehensive Complication Index ≥ 26.2 had shorter overall survival (4-year, 59.5 vs. 69.8%, respectively, log-rank test, P = 0.0037) and recurrence free survival (51.9 vs. 60.1%, respectively, P = 0.0234), than those with Comprehensive Complication Index < 26.2. The Cox multivariate model identified the age, performance status, pT-stage, pN-stage and higher CCI (overall survival: HR = 1.35, P = 0.0174, recurrence-free survival: HR = 1.26, P = 0.0443) as independent predictors of both overall survivial and recurrence-free survival. CONCLUSIONS: Postoperative complications assessed by Comprehensive Complication Index had adverse effects on long-term survival outcomes. Physicians should be aware that major postoperative complications can adversely affect long-term disease control.

Role of reactive oxygen species (ROS) in the regulation of adipogenic differentiation of human maxillary/mandibular bone marrow-derived mesenchymal stem cells.

BACKGROUND: Maxillary/mandibular bone marrow-derived mesenchymal stem cells (MBMSCs) exhibit a unique property of lower adipogenic potential than other bone marrow-derived MSCs. However, the molecular mechanisms regulating the adipogenesis of MBMSCs remain unclear. This study aimed to explore the roles of mitochondrial function and reactive oxygen species (ROS) in regulating the adipogenesis of MBMSCs. METHODS AND RESULTS: MBMSCs exhibited significantly lower lipid droplet formation than iliac BMSCs (IBMSCs). Moreover, the expression levels of CCAAT/enhancer-binding protein ß (C/EBPß), C/EBPδ, and early B cell factor 1 (Ebf-1), which are early adipogenic transcription factors, and those of peroxisome proliferator-activated receptor-γ (PPARγ) and C/EBPα, which are late adipogenic transcription factors, were downregulated in MBMSCs compared to those in IBMSCs. Adipogenic induction increased the mitochondrial membrane potential and mitochondrial biogenesis in MBMSCs and IBMSCs, with no significant difference between the two cell types; however, intracellular ROS production was significantly enhanced only in IBMSCs. Furthermore, NAD(P)H oxidase 4 (NOX4) expression was significantly lower in MBMSCs than in IBMSCs. Increased ROS production in MBMSCs by NOX4 overexpression or treatment with menadione promoted the expression of early adipogenic transcription factors but did not induce that of late adipogenic transcription factors or lipid droplet accumulation. CONCLUSIONS: These results suggest that ROS may be partially involved in the process of MBMSC adipogenic differentiation from undifferentiated cells to immature adipocytes. This study provides important insights into the tissue-specific properties of MBMSCs.

A Case of Horizontal Stent-assisted Coiling for an Aneurysm Arising from Fenestration of the Vertebral Artery: A Technical Case Report.

Horizontal stenting protects the aneurysm neck with stent deployment across the aneurysm neck via the circle of Willis. A saccular aneurysm associated with intracranial arterial fenestration is very rare. Herein, we describe the first case of an unruptured aneurysm related to intracranial arterial fenestration treated with horizontal stenting. A 23-year-old woman presented with a 7-mm broad-necked aneurysm at the fenestration of the right intracranial vertebral artery (VA), which was incidentally found on magnetic resonance imaging. The patient underwent endovascular treatment with horizontal stenting via the vertebrobasilar junction from the contralateral left VA, followed by coil embolization using a jailed microcatheter from the ipsilateral right VA. The procedure was finished with sufficient embolization, and no complications occurred. Horizontal stent delivery via the vertebrobasilar junction for coil embolization of a broad-necked aneurysm arising from the fenestration of the VA is a safe and effective therapeutic strategy.

Cisplatin resistance driver claspin is a target for immunotherapy in urothelial carcinoma.

Bladder cancer is a major and fatal urological disease. Cisplatin is a key drug for the treatment of bladder cancer, especially in muscle-invasive cases. In most cases of bladder cancer, cisplatin is effective; however, resistance to cisplatin has a significant negative impact on prognosis. Thus, a treatment strategy for cisplatin-resistant bladder cancer is essential to improve the prognosis. In this study, we established a cisplatin-resistant (CR) bladder cancer cell line using an urothelial carcinoma cell lines (UM-UC-3 and J82). We screened for potential targets in CR cells and found that claspin (CLSPN) was overexpressed. CLSPN mRNA knockdown revealed that CLSPN had a role in cisplatin resistance in CR cells. In our previous study, we identified human leukocyte antigen (HLA)-A*02:01-restricted CLSPN peptide by HLA ligandome analysis. Thus, we generated a CLSPN peptide-specific cytotoxic T lymphocyte clone that recognized CR cells at a higher level than wild-type UM-UC-3 cells. These findings indicate that CLSPN is a driver of cisplatin resistance and CLSPN peptide-specific immunotherapy may be effective for cisplatin-resistant cases.

Elucidation of adipogenic differentiation regulatory mechanism in human maxillary/mandibular bone marrow-derived stem cells.

OBJECTIVE: This study aims to investigate the underlying molecular mechanisms that regulate the adipogenic differentiation of maxillary/mandibular bone marrow-derived mesenchymal stem cells (MBMSCs). DESIGN: MBMSCs and iliac bone marrow-derived MSCs (IBMSCs) were compared for osteogenic, chondrogenic, and adipogenic differentiation. Cell surface antigen expression was examined using flow cytometry, and stem cell marker expression was assessed using real-time polymerase chain reaction (PCR). Various adipogenic regulatory factors' expression was evaluated using real-time PCR and western blotting. RESULTS: No significant differences in cell surface antigen profiles or stem cell marker expression in MBMSCs and IBMSCs were observed. MBMSCs and IBMSCs displayed similar osteogenic and chondrogenic potentials, whereas MBMSCs showed significantly lower adipogenic potentials than those shown by IBMSCs. Expression of CCAAT/enhancer binding protein ß (C/EBPß), C/EBPδ, early B-cell factor 1 (Ebf-1), and Krüppel-like factor 5 (KLF5), which are early adipogenic differentiation factors, was suppressed in MBMSCs compared to that in IBMSCs. Peroxisome proliferator-activated receptor-γ (PPARγ) and C/EBPα, which play important roles in the terminal differentiation of adipocytes, was lower in MBMSCs than that in IBMSCs. Furthermore, the level of zinc finger protein 423 (Zfp423), which is involved in the commitment of undifferentiated MSCs to the adipocyte lineage, was significantly lower in MBMSCs than that in IBMSCs. CONCLUSIONS: MBMSCs are negatively regulated in the commitment of undifferentiated MSCs to the adipocyte lineage (preadipocytes) as well as in the terminal differentiation of preadipocytes into mature adipocytes. These results may elucidate the site-specific characteristics of MBMSCs.

Thoracic spinal metastasis as recurrence of borderline Brenner tumor without local recurrence: A case report.

Background: Brenner tumor is a rare epithelial ovarian neoplasm that accounts for 2-3% of all ovarian neoplasms. Herein, we report the first case of thoracic spinal metastasis of recurrent Brenner tumor without local recurrence.Case Description.A 70-year-old female presented with a feeling of abdominal distension. Computed tomography revealed cystic lesions in her bilateral ovaries. Blood examination revealed high CA-125 [74.9 U/ml]. We excised bilateral ovaries, uterus, and omentum. Borderline Brenner tumor was diagnosed [Ki-67 labeling index: 10 %]. Follow-up abdominal echo and CA-125 examination revealed no local recurrence. 26 months later she developed paraplegia. Magnetic resonance imaging revealed tumor in the 5th-9th thoracic vertebra and compression of spinal cord at the 6th thoracic vertebra level. Her paraplegia was progressive. We performed semi-urgent partial resection of tumor and release of spinal cord compression. Spinal metastasis from Brenner tumor was diagnosed [Ki-67 labeling index: 50-60 %]. She received adjuvant radiation of 30 Gy in 10 fractions to the 4th-10th thoracic vertebra. After radiation and rehabilitation, she was discharged home on foot. She received adjuvant radiation and chemotherapy but died 11 months after spinal surgery. An autopsy has not been performed on her, and the cause of death is unknown. Conclusion: We report the first case of thoracic metastasis of recurrent Brenner tumor without local recurrence.

Piper retrofractum extract and its component piperine promote lymphangiogenesis via an AKT- and ERK-dependent mechanism.

Administration of Piper retrofractum extract (PRE) has been reported to alleviate edema, but the mechanism underlying this effect is unknown. Promotion of lymphangiogenesis is known to improve lymphedema, but the effect of PRE on lymphangiogenesis remains unclear. In the present study, we investigated whether PRE and specifically, piperine, the main component of PRE, can induce lymphangiogenesis. Treatments with PRE and piperine significantly promoted the proliferation, migration, and tube formation in human dermal lymphatic microvascular endothelial cells (HDLECs) but had no effect on the expression of lymphangiogenic factors. Furthermore, PRE and piperine significantly promoted the phosphorylation of the AKT and ERK proteins in HDLECs, and pretreatment with AKT and ERK inhibitors significantly attenuated the PRE- and piperine-induced lymphangiogenesis. These results indicate that PRE and piperine promote lymphangiogenesis via an AKT- and ERK-dependent mechanism. PRACTICAL APPLICATIONS: The lymphatic system plays various roles such as maintaining tissue fluid homeostasis, immune defense, and metabolism. Disruption of the lymphatic system results in insufficient fluid drainage, which causes edema. Currently, there are no effective treatments for lymphedema; therefore, the development of novel treatment strategies is desirable. In this study, we showed that PRE and its main component piperine promote lymphangiogenesis in lymphatic endothelial cells. Therefore, PRE has the potential to be used as a novel functional food for relieving lymphedema.

Purple sweet potato leaf extracts suppress adipogenic differentiation of human bone marrow-derived mesenchymal stem cells.

Purple sweet potato (Ipomoea batatas L.) leaf extract (PSPLE) is known to exhibit various biological effects. However, the anti-adipogenic effects of PSPLE on mesenchymal stem cells (MSCs) remain unknown. In the present study, we investigated the effect of PSPLE on the adipogenic differentiation of human bone marrow MSCs. PSPLE treatment significantly reduced lipid accumulation and triglyceride levels during adipogenic differentiation. PSPLE suppressed the expression of PPARγ and C/EBPα, which are the master transcription factors orchestrating adipogenesis; moreover, it inhibited the expression of adiponectin, adipocyte protein 2 (aP2), and lipoprotein lipase (LPL), which are downstream target genes involved in adipogenic differentiation. Furthermore, PSPLE treatment suppressed glucose transporter 4 expression and intracellular glucose uptake and significantly inhibited the adipogenic differentiation induced factor-stimulated Akt signaling activation. These results indicate that PSPLE suppresses the differentiation of undifferentiated MSCs into adipocyte lineages and inhibits the terminal differentiation from preadipocytes into mature adipocytes. PRACTICAL APPLICATION: The increase in the prevalence of obesity worldwide is a problem today. Obesity is induced by an excessive accumulation of adipocytes and causes obesity-related diseases, such as diabetes, hypertension, and hyperlipidemia. Natural compounds derived from plants and fruits have a variety of biological activities and are expected to exert therapeutic effects against various diseases. This study shows that purple sweet potato (Ipomoea batatas L.) leaf extract (PSPLE) suppresses adipogenesis of bone marrow-derived mesenchymal stem cells. Thus, PSPLE may be a novel functional food for controlling obesity.

Thoracic spinal metastasis as recurrence of borderline Brenner tumor without local recurrence: A case report.

Background: Brenner tumor is a rare epithelial ovarian neoplasm that accounts for 2-3% of all ovarian neoplasms. Herein, we report the first case of thoracic spinal metastasis of recurrent Brenner tumor without local recurrence.Case Description.A 70-year-old female presented with a feeling of abdominal distension. Computed tomography revealed cystic lesions in her bilateral ovaries. Blood examination revealed high CA-125 [74.9 U/ml]. We excised bilateral ovaries, uterus, and omentum. Borderline Brenner tumor was diagnosed [Ki-67 labeling index: 10 %]. Follow-up abdominal echo and CA-125 examination revealed no local recurrence. 26 months later she developed paraplegia. Magnetic resonance imaging revealed tumor in the 5th-9th thoracic vertebra and compression of spinal cord at the 6th thoracic vertebra level. Her paraplegia was progressive. We performed semi-urgent partial resection of tumor and release of spinal cord compression. Spinal metastasis from Brenner tumor was diagnosed [Ki-67 labeling index: 50-60 %]. She received adjuvant radiation of 30 Gy in 10 fractions to the 4th-10th thoracic vertebra. After radiation and rehabilitation, she was discharged home on foot. She received adjuvant radiation and chemotherapy but died 11 months after spinal surgery. An autopsy has not been performed on her, and the cause of death is unknown. Conclusion: We report the first case of thoracic metastasis of recurrent Brenner tumor without local recurrence.

Efficacy of nivolumab plus ipilimumab as first-line therapy for primary tumors in patients with renal cell carcinoma.

OBJECTIVES: With the emergence of several effective combination therapies, information on their effects at the primary site will be crucial for planning future cytoreductive nephrectomy (CN). The present study focused exclusively on changes in primary tumor sizes following treatment with nivolumab plus ipilimumab and investigated the clinical factors associated with a good response in primary tumors. METHODS AND MATERIALS: We retrospectively assessed 27 patients diagnosed with advanced renal cell carcinoma (RCC) who started treatment with nivolumab plus ipilimumab. Changes in tumor sizes at the primary site were described using waterfall and spider plots, respectively. We analyzed the correlation of tumor shrinkage between primary and metastatic site. The parameters analyzed between responders and non-responders according to primary tumor sizes were International Metastatic Renal Cell Carcinoma Database Consortium (IMDC) risk scores, peripheral blood markers, and CRP. RESULTS: The median age and follow-up period were 66 years and 9.3 months, respectively. The median IMDC risk score was 3 (range: 1-6). Nineteen patients were diagnosed with clear-cell RCC (ccRCC) and 8 patients with non-ccRCC. Among ccRCC patients, 9 (47.4%) achieved a significant response with a maximum reduction of 30% or more in the size of the primary tumor from baseline within 4 months, while 3 (37.5%) out of 8 patients with non-ccRCC achieved a significant response. Shrinkage of the primary tumor correlated with the metastatic tumors in both ccRCC and non-ccRCC cases. Of note, 6 patients underwent CN and no viable tumor cells were detected in the surgical specimens of 3 patients whose primary tumors shrank by approximately 50%-60% with a reduction to 4 cm or less. Among ccRCC patients, the neutrophil-to-lymphocyte ratio and monocyte-to-lymphocyte ratio were slightly lower in responders than in non-responders (P = 0.0944 and P = 0.0691). The platelet-to-lymphocyte ratio was significantly lower in responders than in non-responder (P = 0.0391). CONCLUSIONS: Significant responses in primary tumors to nivolumab plus ipilimumab were observed in 50% of ccRCC patients, while responses varied among non-ccRCC patients. Inflammation markers may be predictive factors of treatment responses in primary tumors. Although further studies are needed, the present results suggest the importance of considering CN from radiological and pathological viewpoints.

Comparative study of postoperative complications after radical cystectomy during the past two decades in Japan: Radical cystectomy remains associated with significant postoperative morbidities.

OBJECTIVES: During the past 2 decades, in order to improve perioperative and oncological outcomes, a minimally invasive approach, neoadjuvant chemotherapy (NAC), and an enhanced postoperative recovery program after surgery have been introduced into routine clinical practice of radical cystectomy (RC). Our aim was to examine the differences in clinical practice and postoperative complications after RC by comparing our previous and current cohorts. MATERIALS AND METHODS: A retrospective multi-institutional study. We collected all complications within 90 days after surgery between 2011 and 2017 (current cohort), and categorized them according to a standardized methodology. Then, we compared the outcomes with those in our previous study (previous cohort, 1997-2010). A multivariate logistic regression model was utilized to determine predictors of complications in the current cohort. RESULTS: A total of 838 patients were newly collected (current cohort), and 919 from the previous cohort were included in the subsequent analyses. In the current cohort, the rate of performing NAC was significantly higher (13% vs. 4%, respectively, P < 0.0001), and 26% (222/838) underwent laparoscopic RC (LRC, without robotic assistance: n = 210, with robotic assistance: n = 12). There was no significant difference in the overall complication [69% (580/838) vs. 68% (629/919), respectively, P = 0.7284] or major complication (Grades 3-5) [25% (211/838) vs. 22% (201/919), respectively, P = 0.1022] rates between the 2 cohorts. In both cohorts, the most frequent categories were infectious, gastrointestinal, wound-related, and genitourinary. In the current cohort, the performance status (odds ratio, OR = 2.11, P = 0.0013) and operative time (OR = 1.003, P = 0.0016) remained significant predictors of major complications. NAC was not associated with any or major complications. CONCLUSIONS: Surgical complications related to RC still remain significant problems, despite the recent improvements in surgical techniques and perioperative care. NAC did not increase the complications.

GRIK2 is a target for bladder cancer stem-like cell-targeting immunotherapy.

Recent studies have revealed that treatment-resistant cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) can be targeted by cytotoxic T lymphocytes (CTLs). CTLs recognize antigenic peptides derived from tumor-associated antigens; thus, the identification of tumor-associated antigens expressed by CSCs/CICs is essential. Human leucocyte antigen (HLA) ligandome analysis using mass spectrometry enables the analysis of naturally expressed antigenic peptides; however, HLA ligandome analysis requires a large number of cells and is challenging for CSCs/CICs. In this study, we established a novel bladder CSC/CIC model from a bladder cancer cell line (UM-UC-3 cells) using an ALDEFLUOR assay. CSCs/CICs were isolated as aldehyde dehydrogenase (ALDH)-high cells and several ALDHhigh clone cells were established. ALDHhigh clone cells were enriched with CSCs/CICs by sphere formation and tumorigenicity in immunodeficient mice. HLA ligandome analysis and cap analysis of gene expression using ALDHhigh clone cells revealed a distinctive antigenic peptide repertoire in bladder CSCs/CICs, and we found that a glutamate receptor, ionotropic, kainite 2 (GRIK2)-derived antigenic peptide (LMYDAVHVV) was specifically expressed by CSCs/CICs. A GRIK2 peptide-specific CTL clone recognized GRIK2-overexpressing UM-UC-3 cells and ALDHhigh clone cells, indicating that GRIK2 peptide can be a novel target for bladder CSC/CIC-targeting immunotherapy.

DNA/RNA heteroduplex oligonucleotide technology for regulating lymphocytes in vivo.

Manipulating lymphocyte functions with gene silencing approaches is promising for treating autoimmunity, inflammation, and cancer. Although oligonucleotide therapy has been proven to be successful in treating several conditions, efficient in vivo delivery of oligonucleotide to lymphocyte populations remains a challenge. Here, we demonstrate that intravenous injection of a heteroduplex oligonucleotide (HDO), comprised of an antisense oligonucleotide (ASO) and its complementary RNA conjugated to α-tocopherol, silences lymphocyte endogenous gene expression with higher potency, efficacy, and longer retention time than ASOs. Importantly, reduction of Itga4 by HDO ameliorates symptoms in both adoptive transfer and active experimental autoimmune encephalomyelitis models. Our findings reveal the advantages of HDO with enhanced gene knockdown effect and different delivery mechanisms compared with ASO. Thus, regulation of lymphocyte functions by HDO is a potential therapeutic option for immune-mediated diseases.

PON1 Q192R is associated with high platelet reactivity with clopidogrel in patients undergoing elective neurointervention: A prospective single-center cohort study.

BACKGROUND AND PURPOSE: The impact of the paraoxonase-1 (PON1) polymorphism, Q192R, on platelet inhibition in response to clopidogrel remains controversial. We aimed to investigate the association between carrier status of PON1 Q192R and high platelet reactivity (HPR) with clopidogrel in patients undergoing elective neurointervention. METHODS: Post-clopidogrel platelet reactivity was measured using a VerifyNow® P2Y12 assay in P2Y12 reaction units (PRU) for consecutive patients before the treatment. Genotype testing was performed for PON1 Q192R and CYP2C19*2 and *3 (no function alleles), and *17. PRU was corrected on the basis of hematocrit. We investigated associations between factors including carrying ≥1 PON1 192R allele and HPR defined as original and corrected PRU ≥208. RESULTS: Of 475 patients (232 men, median age, 68 years), HPR by original and corrected PRU was observed in 259 and 199 patients (54.5% and 41.9%), respectively. Carriers of ≥1 PON1 192R allele more frequently had HPR by original and corrected PRU compared with non-carriers (91.5% vs 85.2%, P = 0.031 and 92.5% vs 85.9%, P = 0.026, respectively). In multivariate analyses, carrying ≥1 PON1 192R allele was associated with HPR by original (odds ratio [OR] 1.96, 95% confidence interval [CI] 1.03-3.76) and corrected PRU (OR 2.34, 95% CI 1.21-4.74) after adjustment for age, sex, treatment with antihypertensive medications, hematocrit, platelet count, total cholesterol, and carrying ≥1 CYP2C19 no function allele. CONCLUSIONS: Carrying ≥1 PON1 192R allele is associated with HPR by original and corrected PRU with clopidogrel in patients undergoing elective neurointervention, although alternative results related to other genetic polymorphisms cannot be excluded.

Cholesterol-functionalized DNA/RNA heteroduplexes cross the blood-brain barrier and knock down genes in the rodent CNS.

Achieving regulation of endogenous gene expression in the central nervous system (CNS) with antisense oligonucleotides (ASOs) administered systemically would facilitate the development of ASO-based therapies for neurological diseases. We demonstrate that DNA/RNA heteroduplex oligonucleotides (HDOs) conjugated to cholesterol or α-tocopherol at the 5' end of the RNA strand reach the CNS after subcutaneous or intravenous administration in mice and rats. The HDOs distribute throughout the brain, spinal cord and peripheral tissues and suppress the expression of four target genes by up to 90% in the CNS, whereas single-stranded ASOs conjugated to cholesterol have limited activity. Gene knockdown was observed in major CNS cell types and was greatest in neurons and microglial cells. Side effects, such as thrombocytopenia and focal brain necrosis, were limited by using subcutaneous delivery or by dividing intravenous injections. By crossing the blood-brain barrier more effectively, cholesterol-conjugated HDOs may overcome the limited efficacy of ASOs targeting the CNS without requiring intrathecal administration.

Human tauopathy-derived tau strains determine the substrates recruited for templated amplification.

Tauopathies are a subset of neurodegenerative diseases characterized by abnormal tau inclusions. Specifically, three-repeat tau and four-repeat tau in Alzheimer's disease, three-repeat tau in Pick's disease (PiD) and four-repeat tau in progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) form amyloid-like fibrous structures that accumulate in neurons and/or glial cells. Amplification and cell-to-cell transmission of abnormal tau based on the prion hypothesis are believed to explain the onset and progression of tauopathies. Recent studies support not only the self-propagation of abnormal tau, but also the presence of conformationally distinct tau aggregates, namely tau strains. Cryogenic electron microscopy analyses of patient-derived tau filaments have revealed disease-specific ordered tau structures. However, it remains unclear whether the ultrastructural and biochemical properties of tau strains are inherited during the amplification of abnormal tau in the brain. In this study, we investigated template-dependent amplification of tau aggregates using a cellular model of seeded aggregation. Tau strains extracted from human tauopathies caused strain-dependent accumulation of insoluble filamentous tau in SH-SY5Y cells. The seeding activity towards full-length four-repeat tau substrate was highest in CBD-tau seeds, followed by PSP-tau and Alzheimer's disease (AD)-tau seeds, while AD-tau seeds showed higher seeding activity than PiD-tau seeds towards three-repeat tau substrate. Abnormal tau amplified in cells inherited the ultrastructural and biochemical properties of the original seeds. These results strongly suggest that the structural differences of patient-derived tau strains underlie the diversity of tauopathies, and that seeded aggregation and filament formation mimicking the pathogenesis of sporadic tauopathy can be reproduced in cultured cells. Our results indicate that the disease-specific conformation of tau aggregates determines the tau isoform substrate that is recruited for templated amplification, and also influences the prion-like seeding activity.

Hemodynamic Force as a Potential Regulator of Inflammation-Mediated Focal Growth of Saccular Aneurysms in a Rat Model.

Past studies have elucidated the crucial role of macrophage-mediated inflammation in the growth of intracranial aneurysms (IAs), but the contributions of hemodynamics are unclear. Considering the size of the arteries, we induced de novo aneurysms at the bifurcations created by end-to-side anastomoses with the bilateral common carotid arteries in rats. Sequential morphological data of induced aneurysms were acquired by magnetic resonance angiography. Computational fluid dynamics analyses and macrophage imaging by ferumoxytol were performed. Using this model, we found that de novo saccular aneurysms with a median size of 3.2 mm were induced in 20/45 (44%) of animals. These aneurysms mimicked human IAs both in morphology and pathology. We detected the focal growth of induced aneurysms between the 10th and 17th day after the anastomosis. The regional maps of hemodynamic parameters demonstrated the area exposed to low wall shear stress (WSS) and high oscillatory shear index (OSI) colocalized with the regions of growth. WSS values were significantly lower in the growing regions than in ones without growth. Macrophage imaging showed colocalization of macrophage infiltration with the growing regions. This experimental model demonstrates the potential contribution of low WSS and high OSI to the macrophage-mediated growth of saccular aneurysms.