The Gut Microbiome and Inflammatory Bowel Diseases.

Inflammatory bowel diseases (IBD) arise from a convergence of genetic risk, environmental factors, and gut microbiota, where each is necessary but not sufficient to cause disease. Emerging evidence supports a bidirectional relationship between disease progression and changes in microbiota membership and function. Thus, the study of the gut microbiome and host-microbe interactions should provide critical insights into disease pathogenesis as well as leads for developing microbiome-based diagnostics and interventions for IBD. In this article, we review the most recent advances in understanding the relationship between the gut microbiota and IBD and highlight the importance of going beyond establishing description and association to gain mechanistic insights into causes and consequences of IBD. The review aims to contextualize recent findings to form conceptional frameworks for understanding the etiopathogenesis of IBD and for the future development of microbiome-based diagnostics and interventions.

Classification of IDH wild-type glioblastoma tumorspheres into low- and high-invasion groups based on their transcriptional program.

BACKGROUND: Glioblastoma (GBM), one of the most lethal tumors, exhibits a highly infiltrative phenotype. Here, we identified transcription factors (TFs) that collectively modulate invasion-related genes in GBM. METHODS: The invasiveness of tumorspheres (TSs) were quantified using collagen-based 3D invasion assays. TF activities were quantified by enrichment analysis using GBM transcriptome, and confirmed by cell-magnified analysis of proteome imaging. Invasion-associated TFs were knocked down using siRNA or shRNA, and TSs were orthotopically implanted into mice. RESULTS: After classifying 23 patient-derived GBM TSs into low- and high-invasion groups, we identified active TFs in each group-PCBP1 for low invasion, and STAT3 and SRF for high invasion. Knockdown of these TFs reversed the phenotype and invasion-associated-marker expression of GBM TSs. Notably, MRI revealed consistent patterns of invasiveness between TSs and the originating tumors, with an association between high invasiveness and poor prognosis. Compared to controls, mice implanted with STAT3- or SRF-downregulated GBM TSs showed reduced normal tissue infiltration and tumor growth, and prolonged survival, indicating a therapeutic response. CONCLUSIONS: Our integrative transcriptome analysis revealed three invasion-associated TFs in GBM. Based on the relationship among the transcriptional program, invasive phenotype, and prognosis, we suggest these TFs as potential targets for GBM therapy.

Inflammatory Bowel Diseases (IBD) and the Microbiome-Searching the Crime Scene for Clues.

Inflammatory bowel diseases (IBD) develop via convergence of environmental, microbial, immunological, and genetic factors. Alterations in the gut microbiota have been associated with development and progression of IBD, but it is not clear which populations of microbes are involved or how they might contribute to IBD. We review the genetic and environmental factors affecting the gut microbiota, the roles of gut microbes and their bioproducts in the development and clinical course of IBD, and strategies by which microbiome-based therapies can be used to prevent, manage, and eventually cure IBD. We discuss research findings that help bridge the gap between the basic sciences and clinical application.

Investigation of PRDM10 and PRDM13 Expression in Developing Mouse Embryos by an Optimized PACT-Based Embryo Clearing Method.

Recent developments in tissue clearing methods have significantly advanced the three-dimensional analysis of biological structures in whole, intact tissue, providing a greater understanding of spatial relationships and biological circuits. Nonetheless, studies have reported issues with maintaining structural integrity and preventing tissue disintegration, limiting the wide application of these techniques to fragile tissues such as developing embryos. Here, we present an optimized passive tissue clearing technique (PACT)-based embryo clearing method, initial embedding PACT (IMPACT)-Basic, that improves tissue rigidity without compromising optical transparency. We also present IMPACT-Advance, which is specifically optimized for thin slices of mouse embryos past E13.5. We demonstrate proof-of-concept by investigating the expression of two relatively understudied PR domain (PRDM) proteins, PRDM10 and PRDM13, in intact cleared mouse embryos at various stages of development. We observed strong PRDM10 and PRDM13 expression in the developing nervous system and skeletal cartilage, suggesting a functional role for these proteins in these tissues throughout embryogenesis.

Potassium intake, skeletal muscle mass, and effect modification by sex: data from the 2008-2011 KNHANES.

BACKGROUND: A loss of muscle mass may be influenced by multiple factors. Insulin sensitivity and metabolic acidosis are associated with muscle wasting and may be improved with potassium intake. This study evaluated the association between dietary potassium intake and skeletal muscle mass. METHODS: We performed a cross-sectional study with data obtained from the Korean National Health and Nutrition Examination Survey (KNHANES) (2008-2011). Participant's daily food intake was assessed using a 24-h recall method. Appendicular skeletal muscle mass (ASM) was calculated as the sum of muscle mass in both arms and legs, measured using dual energy X-ray absorptiometry. The skeletal muscle index (SMI) was calculated as ASM divided by height2 (kg/m2). Low muscle mass was defined as a SMI < 7.0 kg/m2 for men and < 5.4 kg/m2 for women. RESULTS: Data from 16,558 participants (age ≥ 19 years) were analyzed. Participants were categorized into quintiles according to their potassium intake. Sex-specific differences were found in the association between potassium intake and muscle mass (PInteraction < 0.001). In men, higher potassium intake was associated with lower odds for low muscle mass; the fully adjusted odds ratios (95% confidence intervals) were 0.78 (0.60-1.03), 0.71 (0.54-0.93), 0.68 (0.51-0.90), and 0.71 (0.51-0.98) for the top four quintiles (referenced against the lowest quintile), respectively. However, this association was attenuated in women after adjusting for total energy intake. Higher potassium intakes were also associated with a greater SMI. CONCLUSIONS: Higher dietary potassium intake decreased the odds of low muscle mass in men but not in women.

An in vivo cell-based assay for investigating the specific interaction between the SARS-CoV N-protein and its viral RNA packaging sequence.

The SARS-CoV nucleocapsid (N) protein serves multiple functions in viral replication, transcription, and assembly of the viral genome complex. Coronaviruses specifically package genomic RNA into assembled virions, and in SARS-CoV, it is reported that this process is driven by an interaction between the N-protein and a packaging signal encoded within the viral RNA. While recent studies have uncovered the sequence of this packaging signal, little is known about the specific interaction between the N-protein and the packaging signal sequence, and the mechanisms by which this interaction drives viral genome packaging. In this study, we developed a novel in vivo cell-based assay for examining this interaction between the N-protein and packaging signal RNA for SARS-CoV, as well as other viruses within the coronaviridae family. Our results demonstrate that the N-protein specifically recognizes the SARS-CoV packaging signal with greater affinity compared to signals from other coronaviruses or non-coronavirus species. We also use deletion mapping to identify a 151-nt region within the packaging signal sequence that is critical for N-protein-RNA binding, and conversely, we show that both the N-terminal and C-terminal domains of the N protein are necessary for recognizing the packaging RNA. These results describe, for the first time, in vivo evidence for an interaction between the SARS-CoV N-protein and its packaging signal RNA, and demonstrate the feasibility of using this cell-based assay to further probe viral RNA-protein interactions in future studies.

Clinical and Echocardiographic Characteristics of Acute Cardiac Dysfunction Associated With Acute Brain Hemorrhage　- Difference From Takotsubo Cardiomyopathy.

BACKGROUND: Cardiac dysfunction (CD) associated with brain hemorrhage is similar to that with takotsubo cardiomyopathy but still not well understood. We aimed to investigate the clinical and echocardiographic findings of acute CD (ACD) related to brain hemorrhage. METHODS AND RESULTS: Between 2013 and 2014, consecutive patients diagnosed with spontaneous and traumatic brain hemorrhage were prospectively enrolled. Electrocardiography, cardiac enzymes, and echocardiography were performed. Left ventricular (LV) systolic dysfunction on echocardiography was defined as ACD related to brain hemorrhage when all the following conditions were satisfied: abnormal ECG and cardiac troponin level, LV wall motion abnormality or decreased LV systolic function on echocardiography, and no previous history of cardiac disease. Otherwise, LV dysfunction was considered to be other CD unrelated to brain hemorrhage. In a total of 208 patients, 15 (7.2%) showed ACD. Of them, 8 patients were men and 8 showed apex-sparing LV hypokinesia and 9 died in hospital. Other cardiac abnormalities observed in the study patients were NT-proBNP elevation (n=123), QT interval prolongation (n=95), LV hypertrophy (n=89), and troponin I elevation (n=47). There were 36 in-hospital deaths (17.3%). Glasgow coma score and ACD were independently associated with in-hospital death. CONCLUSIONS: ACD was observed in patients with various brain hemorrhages. Unlike takotsubo cardiomyopathy, high proportions of male sex, apex-sparing LV dysfunction, and in-hospital death were observed for ACD associated with brain hemorrhage. (Circ J 2016; 80: 2026-2032).

RapGAP9 regulation of the morphogenesis and development in Dictyostelium.

Recent reports have demonstrated that the importance of Rap1-specific GTPase-activating proteins (GAPs) in the spatial and temporal regulation of Rap1 activity during cell migration and development in Dictyostelium. Here, we identified another putative Rap1 GAP-domain containing protein, showing high sequence homologies with those of human Rap1GAP and Dictyotelium RapGAP3, by bioinformatic search. Loss of RapGAP9 resulted in some defects in morphogenesis and development in Dicytostelium. rapGAP9 null cells were more flattened and spread, and highly multinucleated. Compared to wild-type cells, cells lacking RapGAP9 exhibited increased levels of F-actin and more filopodia. These results suggest that RapGAP9 is involved in the regulation of cytoskeleton reorganization and cytokinesis. rapGAP9 null cells showed a small increase of cell-substratum attachment and slightly lower moving speed and directionality compared to wild-type cells. In addition, the loss of RapGAP9 resulted in an altered morphology of fruiting body with a shorter length of stalk and spore. Identification and characterization of RapGAP9 in this study will provide further insights into the molecular mechanism by which Rap1 regulates cytoskeleton reorganization and morphogenesis in Dictyostelium.

Clinical features of right-sided infective endocarditis occurring in non-drug users.

Right-sided infective endocarditis (RIE) occurs predominantly in intravenous drug users in western countries, and it has a relatively good prognosis. Clinical features and prognosis of RIE occurring in non-drug users are not well known. We investigated the clinical findings of RIE in non-drug users. We retrospectively reviewed 345 cases diagnosed with IE. Cases with RIE or left-sided infective endocarditis (LIE) defined by the vegetation site were included and cases having no vegetation or both-side vegetation were excluded. Clinical findings and in-hospital outcome of RIE were compared to those of LIE. Among the 245 cases, 39 (16%) cases had RIE and 206 (84%) cases had LIE. RIE patients were younger (40 ± 19 yr vs 50 ± 18 yr, P=0.004), and had a higher incidence of congenital heart disease (CHD) (36% vs 13%, P<0.001) and central venous catheter (CVC) (21% vs 4%, P=0.001) compared to LIE patients. A large vegetation was more common in RIE (33% vs 9%, P<0.001). Staphylococcus aureus was the most common cause of RIE, while Streptococcus viridans were the most common cause of LIE. In-hospital mortality and cardiac surgery were not different between the two groups. CHD and use of CVC were common in non-drug users with RIE. The short-term clinical outcome of RIE is not different from that of LIE.

Customized Hydrogel Films for MicroRNA Super-Resolution Imaging in Liquid Biopsies.

Tissue biopsy for early diagnosis and monitoring comes with several challenges, such as its invasiveness, and issues related to tissue heterogeneity in sampling. To address these issues, researchers have proposed a noninvasive approach called liquid biopsy, which uses blood samples to detect specific noncoding RNA (microRNA, miRNA). However, the current process of isolating and amplifying miRNA can be time-consuming and yield nonspecific results. In this study, a new super-resolution imaging tool is introduced that utilizes a thin, hydrogel-based liquid view (LV) film. This film can undergo a ninefold expansion and allows the analysis of cells obtained from liquid biopsy. The potential of the LV film is validated as a tool for early diagnosis and prognosis by testing biofluids derived from a variety of diseases. This method is confirmed to accurately analyze a greater number of miRNAs with higher sensitivity in a shorter time compared to other analytical methods. These findings suggest that the LV film provides high specificity, and multiplexing in detecting small amounts of miRNAs within cells, making it suitable for 3D implementation. It is proposed that liquid biopsy with LV films can be a solution to limitations related to the invasiveness, cost, and time-consuming nature of molecular analysis.

Preprocedural transthoracic echocardiography for predicting outcomes of transcatheter edge-to-edge repair for chronic primary mitral regurgitation.

INTRODUCTION AND OBJECTIVES: Limited data exist on the prognostic usefulness of transthoracic echocardiography preceding MitraClip for chronic primary mitral regurgitation (MR). We evaluated the predictive ability of transthoracic echocardiography in this setting. METHODS: A total of 410 patients (median age, 83 years, 60.7% males) were included in the study. The primary outcome was the 1-year composite of all-cause mortality or heart failure hospitalization. Secondary endpoints encompassed individual elements of the primary outcome, the persistence of significant functional impairment or above-moderate MR at 1 year, and above-mild MR at 1-month. RESULTS: The only parameter associated with the risk of the primary outcome was a ventricular end systolic diameter index of ≥2.1 cm/m2, corresponding to the cohort's 4th quartile (HR, 2.44; 95%CI, 1.09-4.68; P=.022). Concurrently, higher left atrial volume index (LAVi) and a mid-diastolic medial-lateral mitral annular diameter (MAD) equal to or above the cohort's median of 32.2mm were linked to a higher probability of death and heart failure hospitalization, respectively. LAVi of ≥ 60mL/m2, above-mild mitral annular calcification, and above-moderate tricuspid regurgitation conferred higher odds of functional class III-IV or above-moderate MR persistence. All variables except LAVi and MAD, as well as indexed mid-diastolic medial-lateral MAD of ≥ 20.2mm/m2 and mitral effective regurgitant orifice area of ≥ 0.40 cm2, were associated with greater-than-mild MR at 1 month. CONCLUSIONS: Preprocedural increased indexed left heart dimensions, mainly left ventricular end-systolic diameter index, MAD, mitral annular calcification, mitral effective regurgitant orifice area, and tricuspid regurgitation mark a less favorable course post-MitraClip for chronic primary MR.

Implications of Mitral Annular Calcification on Outcomes Following Mitral Transcatheter Edge-to-Edge Repair.

BACKGROUND: Limited data exist regarding the impact of mitral annular calcification (MAC) on outcomes of transcatheter edge-to-edge repair for mitral regurgitation (MR). METHODS: We retrospectively analyzed 968 individuals (median age, 79 [interquartile range, 70-86] years; 60.0% males; 51.8% with functional MR) who underwent an isolated, first-time intervention. Stratified by MAC extent per baseline transthoracic echocardiogram, the cohort was assessed for residual MR, functional status, all-cause mortality, heart failure hospitalizations, and mitral reinterventions post-procedure. RESULTS: Patients with above-mild MAC (n=101; 10.4%) were older and more likely to be female, exhibited a greater burden of comorbidities, and presented more often with severe, primary MR. Procedural aspects and technical success rate were unaffected by MAC magnitude, as was the significant improvement from baseline in MR severity and functional status along the first postprocedural year. However, the persistence of above-moderate MR or functional classes III and IV at 1 year and the cumulative incidence of reinterventions at 2 years were overall more pronounced within the above-mild MAC group (significant MR or functional impairment, 44.7% versus 29.9%, P=0.060; reinterventions, 11.9% versus 6.2%, P=0.033; log-rank P=0.035). No link was demonstrated between MAC degree and the cumulative incidence or risk of mortality and mortality or heart failure hospitalizations. Differences in outcomes frequencies were mostly confined to the primary MR subgroup, in which patients with above-mild MAC also experienced earlier, more frequent 2-year heart failure hospitalizations (20.8% versus 9.6%; P=0.016; log-rank P=0.020). CONCLUSIONS: Mitral transcatheter edge-to-edge repair in patients with and without above-mild MAC is equally feasible and safe; however, its postprocedural course is less favorable among those with primary MR.

MicroRNA super-resolution imaging in blood for Alzheimer's disease.

We propose a novel blood biomarker detection method that uses miRNA super-resolution imaging to enable the early diagnosis of Alzheimer's disease (AD). Here, we report a singlemolecule detection method for visualizing disease-specific miRNA in tissue from an AD mice model, and peripheral blood mononuclear cells (PBMCs) from AD patients. Using optimized Magnified Analysis of Proteome (MAPs), we confirmed that five miRNAs contribute to neurodegenerative disease in the brain hippocampi of 5XFAD and wild-type mice. We also assessed PBMCs isolated from the whole blood of AD patients and a healthy control group, and subsequently analyzed those samples using miRNA super-resolution imaging. We detected more miR-200a-3p expression in the cornu ammonis 1 and dentate gyrus regions of 3 month-old 5XFAD mice than in wild-type mice. Additionally, miRNA super-resolution imaging of blood provides AD diagnosis platform for studying miRNA regulation inside cells at the single molecule level. Our results present a potential liquid biopsy method that could improve the diagnosis of early stage AD and other diseases. [BMB Reports 2023; 56(3): 190-195].

Repeat Mitral Transcatheter Edge-to-Edge Repair for Recurrent Significant Mitral Regurgitation.

Background There are limited data on repeat mitral transcatheter edge-to-edge repair for recurrent significant mitral regurgitation (MR). Methods and Results We conducted a single-center, retrospective analysis of consecutive patients referred to a second mitral transcatheter edge-to-edge repair after a technically successful first procedure. Clinical, laboratory, and echocardiographic measures were assessed up to 1 year after the intervention. The composite of all-cause death or heart failure (HF) hospitalizations constituted the primary outcome. A total of 52 patients (median age, 81 [interquartile range, 76-87] years, 29 [55.8%] men, 26 [50.0%] with functional MR) met the inclusion criteria. MR recurrences were mostly related to progression of the underlying cardiac pathology. All procedures were technically successful. At 1 year, most patients with available records (n=24; 96.0%) experienced improvement in MR severity or New York Heart Association functional class that was statistically significant but numerically modest. Fourteen (26.9%) patients died or were hospitalized due to HF. These were higher-risk cases with predominantly functional MR who mostly underwent an urgent procedure and exhibited more severe HF indices before the intervention, as well as an attenuated 1-month clinical and echocardiographic response. Overall, 1-year course was comparable to that experienced by patients who underwent only a first transcatheter edge-to-edge repair at our institution (n=902). Tricuspid regurgitation of greater than moderate grade was the only baseline parameter to independently predict the primary outcome. Conclusions Repeat mitral transcatheter edge-to-edge repair is feasible, safe, and clinically effective, especially in non-functional MR patients without concomitant significant tricuspid regurgitation.

Left ventricular morphologic progression in apical hypertrophic cardiomyopathy.

BACKGROUND: Left ventricular (LV) morphologic progression in apical hypertrophic cardiomyopathy (AHC) has not been well studied. We evaluated serial echocardiographic changes in LV morphology. METHODS: Serial echocardiograms in AHC patients were assessed. LV morphology was categorized according to the presence of an apical pouch or aneurysm, and LV hypertrophic severity and extent; relative, pure, and apical-mid type defined as mild (<15 mm thickness) apical hypertrophy, significant (≥15 mm) apical hypertrophy, and both apical and midventricular hypertrophy, respectively. Adverse clinical events and late gadolinium enhancement (LGE) extent on cardiac magnetic resonance were evaluated for each morphologic type. RESULTS: In 41 patients, 165 echocardiograms (maximal interval: 4.2 [IQR, 2.3-11.8] years) were evaluated. Morphologic changes were observed in 19 (46%) patients. Eleven (27%) patients displayed the progression of LV hypertrophy toward pure or apical-mid type. Five (12%) and 6 (15%) patients developed new pouches and aneurysms. Patients with progression tended to be younger (50 ± 15.6 vs 59 ± 14.4 years, P = 0.058) and had a longer period of follow-up (12 [5-14] vs 3 [2-4] years, P < 0.001). During a follow-up of 7.6 (IQR 3.0-12.1) years, 21 (51%) experienced clinical events. The relative, pure, and apical-mid types showed different LGE extents (2%, 6%, and 19%, P = 0.004). Patients with severe hypertrophic and apical involvement showed higher clinical event rates. CONCLUSIONS: About half of AHC patients had a progression of LV morphology to more hypertrophic involvement and/or an apical pouch or aneurysm formation. Advanced AHC morphologic types were associated with higher event rates and scar burdens.

Optimization of the optical transparency of bones by PACT-based passive tissue clearing.

Recent developments in tissue clearing methods such as the passive clearing technique (PACT) have allowed three-dimensional analysis of biological structures in whole, intact tissues, thereby providing a greater understanding of spatial relationships and biological circuits. Nonetheless, the issues that remain in maintaining structural integrity and preventing tissue expansion/shrinkage with rapid clearing still inhibit the wide application of these techniques in hard bone tissues, such as femurs and tibias. Here, we present an optimized PACT-based bone-clearing method, Bone-mPACT+, that protects biological structures. Bone-mPACT+ and four different decalcifying procedures were tested for their ability to improve bone tissue clearing efficiency without sacrificing optical transparency; they rendered nearly all types of bone tissues transparent. Both mouse and rat bones were nearly transparent after the clearing process. We also present a further modification, the Bone-mPACT+ Advance protocol, which is specifically optimized for processing the largest and hardest rat bones for easy clearing and imaging using established tissue clearing methods.

Correlates and prognostic implications of LVEF reduction after transcatheter edge-to-edge repair for primary mitral regurgitation.

AIMS: To explore the characteristics and outcomes of patients undergoing transcatheter edge-to-edge repair (TEER) for primary mitral regurgitation (MR) according to the presence of left ventricular ejection fraction (LVEF) reduction post-procedure. METHODS AND RESULTS: We retrospectively analysed 317 individuals [median age 83 (interquartile range, 75-88) years, 197 (62.1%) males] treated with an isolated, first-time TEER that was concluded by a successful clip deployment. Stratified by LVEF change at 1-month compared with baseline, the cohort was evaluated for residual MR and heart failure (HF) indices up to 1-year, as well as all-cause mortality and HF hospitalizations at 2-years. Overall, 212 (66.9%) patients displayed LVEF reduction, which was mainly driven by lowered total stroke volume and diffuse hypocontractility. While post-procedural MR, transmitral mean pressure gradient, and functional status were comparable in the two study groups, patients with LVEF reduction exhibited a greater decline in filling pressures intra-procedurally; left ventricular mass index, pulmonary arterial systolic pressure, and serum natriuretic peptide level at 1-month; and walking limitation at 1-year. Also, by 2 years, they were less likely to die (13.3% vs. 5.7%, P = 0.019), be readmitted for HF (17.1% vs. 9.0%, P = 0.033), and experience either of the two (23.8% vs. 12.7%, P = 0.012). Lastly, LVEF reduction was the only 1-month echocardiographic parameter to independently confer an attenuated risk for the composite of deaths or HF hospitalizations (HR 0.28, 95% CI 0.10-0.78, P = 0.016). CONCLUSION: LVEF reduction at 1-month post-TEER for primary MR is associated with better clinical outcomes, possibly reflecting a more pronounced unloading effect of the procedure.

Prognostic value of mitral valve haemodynamic parameters obtained by intraprocedural echocardiography in transcatheter edge-to-edge repair.

AIMS: To assess whether intraprocedural transesophageal echocardiographic (TEE)-derived haemodynamic parameters predict outcomes in patients undergoing transcatheter edge-to-edge repair (TEER) for mitral regurgitation (MR). METHODS AND RESULTS: This is a single-centre, retrospective analysis encompassing 458 (IQR, 104-1035) days of follow-up after 926 consecutive patients [481 (52%) with functional MR] referred to an isolated mitral TEER between 2013 and 2020. Cases without actual clip deployment, or in whom prior mitral procedures had taken place, were excluded. The primary outcome was the combined rate of all-cause mortality or heart failure (HF) hospitalizations. Secondary endpoints included single components of the primary outcome, as well as MR severity at one month and one year following the procedure. A multivariable analysis identified two intraprocedural echocardiographic observations made after clip deployment as independent predictors of the primary outcome: an above mild MR (HR for whole study period 1.49, 95% CI 1.05-2.13, P = 0.026) and a 100% or more increase from baseline in the transmitral mean pressure gradient (TMPG) (HR for whole study period 1.32, 95% CI 1.01-1.72, P = 0.039). Also, MR grade of above mild and the absence of a normal pulmonary venous flow pattern (PVFP) bilaterally were associated with an increased risk for HF hospitalizations and greater-than-mild 1-month MR. No prognostic role was demonstrated for the change in MR severity, the absolute TMPG, or the mere improvement in PVFP. CONCLUSION: Immediate post-TEER MR severity and the relative change in TMPG are predictive of clinical and echocardiographic outcomes following the procedure.

A case of ANCA-associated vasculitis presenting with calf claudication.

Wegener's granulomatosis (WG), Churg-Strauss syndrome, and microscopic polyangiitis (MPA) are closely related small vessel vasculitides characterized by anti-neutrophil cytoplasmic antibodies (ANCA). Although there were some reports of MPA presenting with claudication, there are very few reports on WG presenting with claudication of calf muscles. We report an unusual case of ANCA-associated vasculitis in a 75-year-old man who presented with bilateral calf claudication. Comprehensive evaluations, including electromyography, nerve conduction study, lower extremity magnetic resonance imaging, and Doppler scan, did not reveal any other cause of bilateral calf claudication. P-ANCA and anti-myeloperoxidase antibody was positive, but the anti-proteinase 3 antibody was negative. Chest computed tomography scan showed subpleural honeycombing and reticulation, predominantly in both basal lung areas. Biopsy of the calf muscle showed granulomatous vasculitis. Kidney biopsy was also performed which revealed focal necrotizing glomerulonephritis. Our patient does not exhibit typical clinical features for WG, but histopathologic findings of necrotizing granulomatous vasculitis in calf muscle biopsy is highly suggestive of WG.

Kidney Mesenchymal Stem Cell-derived Extracellular Vesicles Engineered to Express Erythropoietin Improve Renal Anemia in Mice with Chronic Kidney Disease.

Extracellular vesicles (EVs) shed from kidney mesenchymal stem cells (KMSCs) show protective effects against acute kidney injury and progressive kidney fibrosis via mRNA transfer. Previous studies report improvement of renal anemia following administration of genetically modified MSCs or peritoneal mesothelial cells that secrete erythropoietin (EPO). Here, we determined whether EPO-secreting KMSC-derived EVs (EPO(+)-EVs) can improve renal anemia in mouse models of chronic kidney disease (CKD). The mouse CKD and renal anemia model was induced by electrocoagulation of the right renal cortex and sequential left nephrectomy. At six weeks post-nephrectomy, we observed significantly lower hemoglobin (10.4 ± 0.2 vs. 13.2 ± 0.2 g/dL) and significantly higher blood urea nitrogen and serum creatinine levels in CKD mice relative to controls (60.5 ± 0.5 and 0.37 ± 0.09 mg/dL vs. 19.9 ± 0.5 and 0.12 ± 0.02 mg/dL, respectively). Genetically engineered EPO(+)-KMSCs secreted 71 IU/mL EPO/106 cells/24 h in vitro, and EPO(+)-EVs isolated by differential ultracentrifugation expressed EPO mRNA and horizontally transferred EPO mRNA into target cells in vitro and in vivo. Furthermore, at two weeks post-injection of EPO(+)-KMSCs or EPO(+)-EVs into CKD mice with renal anemia, we observed significant increases in hemoglobin levels (11.7 ± 0.2 and 11.5 ± 0.2 vs. 10.1 ± 0.2 g/dL, respectively) and significantly lower serum creatinine levels at eight weeks in comparison to mice receiving vehicle control (0.30 ± 0.00 and 0.23 ± 0.03 vs. 0.43 ± 0.06 mg/dL, respectively). These results demonstrate that intraperitoneal administration of EPO(+)-EVs significantly increased hemoglobin levels and renal function in CKD mice, suggesting the efficacy of these genetically engineered EVs as a promising novel strategy for the treatment of renal anemia.