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Background We performed a multicenter, open, randomized, clinical study of autologous cultured osteoblast injection for long-bone fracture, to evaluate the fracture healing acceleration effect and the safety of autologous cultured osteoblasts. Methods Sixty-four patients with long-bone fractures were randomly divided into two groups, i.e. those who received autologous cultured osteoblast injection and those who received no treatment. The sum of the difference in the callus formation scores after four and eight weeks, was used as the first efficacy variable. Results The autologous cultured osteoblast injection group showed fracture healing acceleration of statistical significance, and there were no specific patient complications when using this treatment. Conclusion Autologous cultured osteoblast injection should therefore be considered as a successful treatment option for treating long-bone fracture. Trial registration Current Controlled Trials ISRCTN10637905

BackgroundCo-stimulatory signals regulate the expansion, persistence, and function of chimeric antigen receptor (CAR) T cells. Most studies have focused on the co-stimulatory domains CD28 or 4-1BB. CAR T cell persistence is enhanced by 4-1BB co-stimulation leading to nuclear factor kappa B (NF-κB) signaling, while resistance to exhaustion is enhanced by mutations of the CD28 co-stimulatory domain.MethodsWe hypothesized that a third-generation CAR containing 4-1BB and CD28 with only PYAP signaling motif (mut06) would provide beneficial aspects of both. We designed CD19-specific CAR T cells with either 4-1BB or mut06 together with the combination of both and evaluated their immune-phenotype, cytokine secretion, real-time cytotoxic ability and polyfunctionality against CD19-expressing cells. We analyzed lymphocyte-specific protein tyrosine kinase (LCK) recruitment by the different constructs by immunoblotting. We further determined their ability to control growth of Raji cells in NOD scid gamma (NSG) mice. We also engineered bi-specific CARs against CD20/CD19 combining 4-1BB and mut06 and performed repeated in vitro antigenic stimulation experiments to evaluate their expansion, memory phenotype and phenotypic (PD1+CD39+) and functional exhaustion. Bi-specific CAR T cells were transferred into Raji or Nalm6-bearing mice to study their ability to eradicate CD20/CD19-expressing tumors.ResultsCo-stimulatory domains combining 4-1BB and mut06 confers CAR T cells with an increased central memory phenotype, expansion, and LCK recruitment to the CAR. This enhanced function was dependent on the positioning of the two co-stimulatory domains. A bi-specific CAR targeting CD20/CD19, incorporating 4-1BB and mut06 co-stimulation, showed enhanced antigen-dependent in vitro expansion with lower exhaustion-associated markers. Bi-specific CAR T cells exhibited improved in vivo antitumor activity with increased persistence and decreased exhaustion.ConclusionThese results demonstrate that co-stimulation combining 4-1BB with an optimized form of CD28 is a valid approach to optimize CAR T cell function. Cells with both mono-specific and bi-specific versions of this design showed enhanced in vitro and in vivo features such as expansion, persistence and resistance to exhaustion. Our observations validate the approach and justify clinical studies to test the efficacy and safety of this CAR in patients.

Chronic kidney disease (CKD) is one of the most common causes of mortality in wild non-domestic felidae. The molecular mechanism regulating renal fibrosis in nephropathy is not fully understood especially in the felidae. This study aimed to elucidate senescence marker protein 30 (SMP30) expression patterns and its relationship with epithelial-mesenchymal transition (EMT) by immunostaining in two necropsied Siberian tigers (Panthera tigris altaica) with CKD. Two kidney samples from male Siberian tigers were fixed and tissue sections were stained for histopathological assay. In CKD, renal tubular epithelial cells lost their tubular structures surrounded by severe interstitial fibrosis and were detached from the basement membrane. These damaged cells resembled the morphology of mesenchymal cells and showed much lower SMP30 expression compared with intact tubular epithelial cells. These cells also expressed vimentin, which is specifically expressed by mesenchymal cells, and through double staining, it was observed that vimentin was expressed in the tubular epithelial cells where SMP30 was not expressed. In addition, double-positive expression of pan-cytokeratin (pan-CK) and vimentin was found in damaged epithelial cells with mesenchymal features. We demonstrated possible evidence to understand the role of SMP30 as a new pivotal factor and the possibility of decreased SMP30 as a potential indicator of EMT at the end stage of CKD.

Stormwater pollution is the untreated contaminated water that drains into natural waterways from land uses within an urban catchment. Several studies have demonstrated the deterioration of water quality in receiving bodies of water caused by stormwater runoff. The data have reported that urban runoff play primary roles in degrading water quality in adjacent aquatic systems. The accurate estimation of non-pollutant loads from urban runoff and the prediction of water quality in receiving waters are important. The objective of this paper is to assess the applicability of the watershed scale hydrologic and water quality simulation models SWMM and HSPF to simulate the hydrology of a small watershed in the Han River Basin. Monitoring was performed in small scale watersheds, which is homogeneous land use. The applicability of SWMM and HSPF model was examined for small watersheds using hourly monitoring data. The results of SWMM were reasonably reflected with observed data in small scale urban area. HSPF model was effective at specifying parameters related to runoff and water quality when using hourly monitoring data. The watershed models used in this study adequately simulated watershed characteristics and are recommended to support watershed management.

BackgroundCo-stimulatory signals regulate the expansion, persistence, and function of chimeric antigen receptor (CAR) T cells. Most studies have focused on the co-stimulatory domains CD28 or 4-1BB. CAR T cell persistence is enhanced by 4-1BB co-stimulation leading to NF-κB signaling, while resistance to exhaustion is enhanced by mutations of the CD28 co-stimulatory domain.MethodsWe hypothesized that a third-generation CAR containing 4-1BB and CD28 with only PYAP signaling motif (mut06) would provide beneficial aspects of both. We designed CD19-specific CAR T cells with 4-1BB or mut06 together with the combination of both (BB06). We evaluated their immune-phenotype, cytokine secretion, real-time cytotoxic ability and polyfunctionality against CD19-expressing cells. We analyzed LCK recruitment by the different constructs by immunoblotting. We further determined their ability to control growth of Raji cells in NSG mice. Additionally, we engineered bi-specific CARs against CD20/CD19 combining 4-1BB and mut06 and performed repeated in vitro antigenic stimulation experiments to evaluate their expansion, memory phenotype and phenotypic (PD1+CD39+) and functional exhaustion. Bi-specific CAR T cells were transferred into Raji or Nalm6-bearing mice to study their ability to eradicate CD20/CD19-expressing tumors.ResultsCo-stimulatory domains combining 4-1BB and mut06 confers CAR T cells with an increased polyfunctionality and LCK recruitment to the CAR (figure 1A), after repeated-antigen stimulation these cells expanded significantly better than second-generation CAR T cells (figure 1B). A bi-specific CAR targeting CD20/CD19, incorporating 4-1BB and mut06 co-stimulation, showed enhanced antigen-dependent in vitro expansion with lower exhaustion-associated markers (figure 1C). Bi-specific CAR T cells exhibited improved in vivo anti-tumor activity with increased persistence and decreased exhaustion (figure 1D).Abstract 105 Figure 1A. pLCK is increased in h19BB06z CAR T cells and associated with the CAR. CAR T cells were stimulated with irradiated 3T3-hCD19 cells at a 10:1 E:T ratio for 24hr. Cells were lysed and CAR bound and unbound fractions were western blotted. A single-cell measure of polyfunctional strength index (PSI) of CAR T cells. B. h19BB06z CAR T cells have the highest proliferation after repeated antigen stimulations. 5x105 CAR T cells were stimulated with 1x105 irradiated 3T3-hCD19 cells. After 1 week, half of the cells were enumerated by flow cytometry and the other half was re-stimulated with 1x105 fresh irradiated 3T3-hCD19 cells. This was repeated for a total of 4 weeks. C. 5x105 CAR T cells were co-cultured with 5x105 target cells (Raji-CD19High). After 1 week half the cells were harvested enumerated and stained by flow cytometry while the other half was re-stimulated with 5x105 fresh target cells (indicated by arrows). This was repeated for a total of 4 weeks. Frequency of PD1+CD39+ cells within CD8+ CAR T cells. D. Raji-FFLuc-bearing NSG mice were treated with 1x106 CAR T cells 5 days after initial tumor cell injection. Tumor burden (average luminescence) of mice treated with bi-specific or monospecific CAR T cells, UT and tumor control. Each line represents an individual mouse. (n = 7 mice per group).ConclusionsThese results demonstrate that co-stimulation combining 4-1BB with an optimized form of CD28 is a valid approach to optimize CAR T cell function. Cells with both mono- and bi-specific versions of this design showed enhanced in vitro and in vivo features such as expansion, persistence and resistance to exhaustion. Our observations validate the approach and justify clinical studies to test the efficacy and safety of this CAR in patients.

This article describes the pilot study on the water reuse for agricultural irrigation in Korea. The project is a part of the application of wastewater reuse system for Agriculture project, a 21st Century Frontier R&D Program sponsored by the Ministry of Education, Science, and Technology and associated with the Sustainable Water Resources Research Program. The goal of the project was to develop infra-technologies necessary to reclaim wastewater for irrigation in agriculture. The project involved two phases: laboratory and field research. Reclamation techniques for irrigation and feasible reuse were developed as a first step in proposing appropriate water quality standards. Reclaimed wastewater of various qualities was used to irrigate cereal crops and vegetables, and possible adverse effects on crops, humans, and the environment were investigated. The optimal reclamation methods required to satisfy water quality standards were explored and the operational characteristics investigated. Moreover, an inventory of farmlands that could reuse reclaimed wastewater was established. Feasible delivery systems for irrigation were developed, and pilot project sites were identified. Finally, operational field data from pilot units were collected and analyzed. This research and development may help solve water shortage problems in Korea, which left unaddressed will have an adverse effect on future generations.

Chimeric antigen receptor T cell (CAR-T) therapy has led to significant improvements in patient survival. However, a subset of patients experience high-grade toxicities, including cytokine release syndrome (CRS) and immune cell-associated hematological toxicity (ICAHT). We utilized IL-2Ra knockout mice to model toxicities with elevated levels of IL-6, IFN-γ, and TNF-α and increased M1-like macrophages. Onset of CRS was accompanied by a reduction in peripheral blood neutrophils due to disruption of bone marrow neutrophil homeostasis characterized by an increase in apoptotic neutrophils and a decrease in proliferative and mature neutrophils. Both nontumor-bearing and Em-ALL tumor-bearing mice recapitulated the cooccurrence of CRS and neutropenia. IFN-γ-blockade alleviated CRS and neutropenia without affecting CAR-T efficacy. Mechanistically, a Th1-Th17 imbalance was observed to drive cooccurrence of CRS and neutropenia in an IFN-γ-dependent manner leading to decreased IL-17A and G-CSF, neutrophil production, and neutrophil survival. In patients, we observed an increase in the IFN-γ-to-IL-17A ratio in the peripheral blood during high-grade CRS and neutropenia. We have uncovered a biological basis for ICAHT and provide support for the use of IFN-γ blockade to reduce both CRS and neutropenia.

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of patients with relapsed/ refractory large B cell lymphoma (LBCL). Despite its promising efficacy, a subset of patients experiences primary resistance or relapse. In Chapter 2, we explore the tumor microenvironment (TME)-induced resistance mechanisms in patients with LBCL. Analysis of the intratumoral immune infiltrates in pre-infusion tumor biopsies revealed that the levels of immunoregulatory macrophages were elevated in patients who responded poorly to axicabtagene ciloleucel (axi-cel). Using preclinical mouse B cell tumor models, we found that the CAR T-cell-produced interferon-gamma (IFN-γ) enhanced the expression of inducible nitric oxide synthase (iNOS, NOS2) in tumor-associated macrophages (TAMs), impairing CAR T-cell effector function. Furthermore, metabolomics and stable isotope tracing showed that CAR T-cell metabolism was compromised by iNOS-dependent depletion of glycolytic intermediates and alterations in the TCA cycle. Inhibition of iNOS significantly improved CAR T-cell therapy outcomes in B-cell tumor-bearing mice, underscoring the importance of targeting iNOS in TAMs.In Chapter 3, we explore how serum inflammatory proteins correlate with severe immune-mediated toxicities and adverse clinical outcomes in patients with diffuse large B-cell lymphoma (DLBCL) undergoing treatment with axi-cel. We developed a straightforward stratification model using pre-lymphodepletion levels of C-reactive protein (CRP) and ferritin to categorize patients into low, intermediate, and high-risk groups. This model effectively predicted clinical outcomes, indicating that patients at high risk were more likely to experience grade ≥ 3 toxicities and had worse overall and progression-free survival rates. Validation through two independent international cohorts verified that patients classified as low-risk achieved outstanding efficacy and safety profiles. These results demonstrate the effectiveness of our risk stratification model, which relies on easily accessible lab tests, in guiding the selection of patients for CAR T-cell therapy.

The metabolic properties of tumor microenvironment (TME) are dynamically dysregulated to achieve immune escape and promote cancer cell survival. However, in vivo properties of glucose metabolism in cancer and immune cells are poorly understood and their clinical application to development of a biomarker reflecting immune functionality is still lacking. Methods: We analyzed RNA-seq and fluorodeoxyglucose (FDG) positron emission tomography profiles of 63 lung squamous cell carcinoma (LUSC) specimens to correlate FDG uptake, expression of glucose transporters (GLUT) by RNA-seq and immune cell enrichment score (ImmuneScore). Single cell RNA-seq analysis in five lung cancer specimens was performed. We tested the GLUT3/GLUT1 ratio, the GLUT-ratio, as a surrogate representing immune metabolic functionality by investigating the association with immunotherapy response in two melanoma cohorts. Results: ImmuneScore showed a negative correlation with GLUT1 (r = -0.70, p < 0.01) and a positive correlation with GLUT3 (r = 0.39, p < 0.01) in LUSC. Single-cell RNA-seq showed GLUT1 and GLUT3 were mostly expressed in cancer and immune cells, respectively. In immune-poor LUSC, FDG uptake was positively correlated with GLUT1 (r = 0.27, p = 0.04) and negatively correlated with ImmuneScore (r = -0.28, p = 0.04). In immune-rich LUSC, FDG uptake was positively correlated with both GLUT3 (r = 0.78, p = 0.01) and ImmuneScore (r = 0.58, p = 0.10). The GLUT-ratio was higher in anti-PD1 responders than nonresponders (p = 0.08 for baseline; p = 0.02 for on-treatment) and associated with a progression-free survival in melanoma patients who treated with anti-CTLA4 (p = 0.04). Conclusions: Competitive uptake of glucose by cancer and immune cells in TME could be mediated by differential GLUT expression in these cells.

Background Chimeric antigen receptor T- Cell (CAR-T) immunotherapy has been a breakthrough treatment for various hematological malignancies. However, cardiotoxicities such as new-onset heart failure, arrhythmia, acute coronary syndrome and cardiovascular death occur in 10–15% of patients treated with CAR-T. This study aims to investigate the changes in cardiac and inflammatory biomarkers in CAR-T therapy to determine the role of pro-inflammatory cytokines. Methods In this observational study, ninety consecutive patients treated with CAR-T underwent baseline cardiac investigation with electrocardiogram (ECG), transthoracic echocardiogram (TTE), troponin-I, and B-type natriuretic peptide (BNP). Follow-up ECG, troponin-I and BNP were obtained five days post- CAR-T. In a subset of patients (N = 53), serum inflammatory cytokines interleukin (IL)-2, IL-6, IL-15, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), and angiopoietin 1 & 2 were tested serially, including baseline and daily during hospitalization. Adverse cardiac events were defined as new-onset cardiomyopathy/heart failure, acute coronary syndrome, arrhythmia and cardiovascular death. Results Eleven patients (12%) had adverse cardiac events (one with new-onset cardiomyopathy and ten with new-onset atrial fibrillation). Adverse cardiac events appear to have occurred among patients with advanced age (77 vs. 66 years; p = 0.002), higher baseline creatinine (0.9 vs. 0.7 mg/dL; 0.007) and higher left atrial volume index (23.9 vs. 16.9mL/m 2 ; p = 0.042). Day 5 BNP levels (125 vs. 63pg/mL; p = 0.019), but not troponin-I, were higher in patients with adverse cardiac events, compared to those without. The maximum levels of IL-6 (3855.0 vs. 254.0 pg/mL; p = 0.021), IFN-γ (474.0 vs. 48.8pg/mL; p = 0.006) and IL-15 (70.2 vs. 39.2pg/mL; p = 0.026) were also higher in the adverse cardiac events group. However, cardiac and inflammatory biomarker levels were not associated with cardiac events. Patients who developed cardiac events did not exhibit worse survival compared to patients without cardiac events (Log-rank p = 0.200). Conclusion Adverse cardiac events, predominantly atrial fibrillation, occur commonly after CAR-T (12%). The changes in serial inflammatory cytokine after CAR-T in the setting of adverse cardiac events suggests pro-inflammation as a pathophysiology and require further investigation for their role in adverse cardiac events. Tweet brief handle CAR-T related Cardiotoxicity has elevated cardiac and inflammatory biomarkers. #CARTCell #CardioOnc #CardioImmunology.