Citations

Abstract Introduction Asymptomatic and mildly symptomatic dominantly inherited Alzheimer's disease mutation carriers (DIAD‐MC) are ideal candidates for preventative treatment trials aimed at delaying or preventing dementia onset. Brain atrophy is an early feature of DIAD‐MC and could help predict risk for dementia during trial enrollment. Methods We created a dementia risk score by entering standardized gray‐matter volumes from 231 DIAD‐MC into a logistic regression to classify participants with and without dementia. The score's predictive utility was assessed using Cox models and receiver operating curves on a separate group of 65 DIAD‐MC followed longitudinally. Results Our risk score separated asymptomatic versus demented DIAD‐MC with 96.4% (standard error = 0.02) and predicted conversion to dementia at next visit (hazard ratio = 1.32, 95% confidence interval [CI: 1.15, 1.49]) and within 2 years (area under the curve = 90.3%, 95% CI [82.3%–98.2%]) and improved prediction beyond established methods based on familial age of onset. Discussion Individualized risk scores based on brain atrophy could be useful for establishing enrollment criteria and stratifying DIAD‐MC participants for prevention trials.

The present forward mutagenesis screen strategies in vertebrates are difficult to identify genes and mutants affecting a specific organ of interest. Here, we report a strategy to enrich cardiac mutants by leveraging the transparency of zebrafish embryos and the high efficiency of pGBT-RP2 (RP2), a gene-breaking transposon-based in vivo protein trap cassette. By selectively raising up founder fish with Red Fluorescence Protein (RFP) reporter expression in the heart, we successfully generated 15 cardiac mutant lines. All tagged loci exhibit cardiac expression and the overall knockdown efficiency at transcript level is greater than 95%. One of the cardiac lines, RP2_ #135, is a homozygous embryonic lethal line that is caused by a RP2 insertion in methionine adenosyltransferase 2a (mat2a) . Taking advantage of the LoxP sites inside the RP2 vector, we further demonstrated that the cardiac phenotypes in RP2_ #135 is due to a disrupted expression of mat2a in cardiomyocytes, as indicated by a conditional rescue assay using Tg(cmlc2:Cre-YFP) , a myocardium specific Cre transgenic line. Finally, we show that the reduced cardiomyocyte number, but not size, in the mat2a mutant is conveyed by the p53-mediated apoptosis pathway. Together, our data prove the feasibility of a facile approach to generate a vertebrate cardiac mutant collection with the following two unique features. First, the expression dynamics of each tagged gene is reported by RFP. Second, the causality between the tissue-specific gene expression and the resulted cardiac phenotypes can be attested genetically.

Background: While proper heart contraction requires an intact sarcomere structure, contraction reciprocally regulates sarcomere assembly and cardiomyocyte shape during heart morphogenesis and heart remodeling. However, how contraction modulates heart morphogenesis and the underlying molecular mechanism remains unclear. Here we address this question by manipulating cardiac contractility in a developing zebrafish embryo. Results: Cessation of heart contraction was achieved by treating zebrafish embryos with blebbistatin, a myosin II specific inhibitor, or Niferdipine, a Ca 2+ channel inhibitor. Loss of cardiac contractility reduces ventricular chamber size in 1 dpf embryo, but significantly increases ventricular chamber size in 2 dpf and 3 dpf embryos. In both circumstances, cessation of contraction consistently prevents or disrupts the lateral fusion of myofibril. Cessation of contraction-induced heart enlargement is reversible and also dependent on the time of drug treatment. This heart-remodeling event in 2dpf embryo, which manifests as increased cardiomyocyte surface area accompanied by reduced thickness, can be attenuated by reducing the transmural pressure towards the ventricle wall. This mechanotransductive process occurs in the myocardium without need of endocardium. At the molecular level, by screening several compounds, we found that PI3K signaling confers this contraction-regulated cardiomyocyte shape remodeling process without affecting the lateral fusion of sarcomere. Conclusion: Our data in an in vivo animal model distinguish two different and relatively independent functions of contraction in regulating lateral fusion of sarcomere and cardiomyocytes shape remodeling. The contraction-regulated heart shape remodeling is conveyed by PI3k signaling mediated mechanotransduction independent of endocardium.

The exact molecular mechanisms involved in hemodialysis arteriovenous fistula (AVF) failure caused by venous neointimal hyperplasia (VNH) are not clear. It has been observed that there is an accumulation of extracellular matrix and up regulation of pro-fibrotic genes accompanied with presence of fibroblasts, smooth muscle cells, and inflammatory cells in the stenotic veins. Previous studies have demonstrated that adventitial and medial fibroblasts have a pivotal role(s) in VNH formation. MicroRNA-21 (miR-21) contributes to fibroblast to myofibroblast differentiation and dysregulation of miR-21 plays a pathological role in failure of coronary artery bypass grafts. The aim of the present study was to determine the role of miR-21 in VNH associated with AVF. We assessed miR-21 expression using qRT-PCR in the outflow veins of AVFs compared to control (contralateral jugular veins) veins in the C57BL/6J mice with chronic kidney disease (CKD). MiR-21 expression was upregulated accompanied with down regulation of miR-21 target genes; PPAR-α, PTEN and TIMP-3. In addition, gene expression of fibroblast specific protein (FSP) -1, TGF (transforming growth factor) -β1, matrix metalloproteinases (MMP)-2, -9, collagen-I, and IV were significantly increased at day 7 after AVF creation. Immunohistochemistry revealed that there was a significant increase in proliferating cell index (Ki-67) and fibroblast index (FSP-1) in the outflow veins of AVFs. Hypoxia has been shown to increase fibroblast to myofibroblast differentiation and this is predicted to be an early step in VNH formation. Therefore we assessed miR-21 expression in hypoxic (1%O 2 ) mouse pulmonary vein fibroblasts compared to normoxic cells in vitro and it was found that miR-21and TGF-β1 significantly elevated with down regulation of miR-21 target genes PTEN and TIMP-3. Furthermore, miR-21 knockdown in hypoxic fibroblasts attenuated TGF-β1 expression with a significant upregulation of genes targeted by miR-21 compared to controls. Together these results indicate that upregulation of miR-21 expression may result in fibroblast to myofibroblast differentiation resulting in VNH formation.

Introduction: Venous neointimal hyperplasia (VNH) is a major cause of hemodialysis arteriovenous fistula (AVF) vascular access failure. CX3CR1 mediates macrophage infiltration into the vasculature. Mice were used which had been genetically engineered to knock in the human CX3CR1 gene. Hypothesis: The hypothesis to be tested is that increased CX3CR1 gene expression results in VNH formation associated with AVF. Methods: We used 50 CX3CR1 knock in mice which were divided into 2 groups: 1. CX3CR1 antibody (30 mg/kg administered intraperitoneally two times per week) or vehicle (equal amount of volume used for CX3CR1 antibody administered intraperitoneally). AVFs were created by connecting the carotid artery to ipsilateral jugular vein 28 days after nephrectomy was performed to induce chronic kidney disease. Histology and Immunohistochemistry analysis at the outflow vein of AVF were performed to assess the pathological changes. Results: There was a significant decrease in neointima area in the outflow veins of AVF in CX3CR1 antibody group compared to the vehicle group at day 28 (43085 ± 12678 vs. 58963.4 ± 9273 μm 2 , P<0.05). There was a significant decrease in the ratio of neointima/media+adventitia area in the outflow veins of AVF in CX3CR1 antibody group compared to the vehicle group at day 28 (0.34 ± 0.1 vs. 0.61 ± 0.1, P<0.05). Cell density in neointima area in CX3CR1 group outflow veins was significantly lower than the vehicle group at day 14 (9600 ± 1000 vs. 13000 ± 3000 /mm 2 , P<0.01). There was a significant decrease in the average CD68-positive cell density in the CX3CR1 group outflow veins compared to the vehicle group (0.96 ± 0.34 vs.15.9 ±10, P<0.001) at day 14. Conclusion: Decreasing CX3CR1 significantly reduces macrophages infiltration and results in a significant reduction in VNH. This study provides a rationale for using CX3CR1 antibody in reducing VNH formation.

AbstractContextInterleukin-2 (IL-2), a proinflammatory cytokine, has been used to treat malignancies. Increased cortisol and adrenocorticotropin (ACTH) were noted, but growth hormone (GH) secretion was not investigated in detail.ObjectiveWe quantified GH secretion after a single subcutaneous injection of IL-2 in 17 young and 18 older healthy men in relation to dose, age, and body composition.MethodsThis was a placebo-controlled, blinded, prospectively randomized, crossover study. At 20:00 hours IL-2 (3 or 6 million units/m2) or saline was injected subcutaneously. Lights were off between 23:00 and 07:00 hours. Blood was sampled at 10-minute intervals for 24 hours. Outcome measures included convolution analysis of GH secretion.ResultsGH profiles were pulsatile under both experimental conditions and lower in older than young volunteers. Since the effect of IL-2 might be time limited, GH analyses were performed on the complete 24-hour series and the 6 hours after IL-2 administration. Total and pulsatile 24-hour GH secretion decreased nonsignificantly. Pulsatile secretion fell over the first 6 hours after IL-2 (P = .03), with visceral fat as a covariate (P = .003), but not age (P = .10). Plots of cumulative 2-hour bins of GH pulse mass showed a distinction by treatment and age groups: A temporary GH decrease of 32% and 28% occurred in the first 2-hour bins after midnight (P = .02 and .04) in young participants, whereas in older individuals no differences were present at any time point.ConclusionThis study demonstrates that IL-2 temporarily diminishes GH secretion in young, but not older, men.

Abstract Background and Aims: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor superfamily, is predominantly expressed on immune cells. Although TRAIL biology has garnered considerable interest as a potential anti-cancer strategy, TR agonists have had very limited anti-cancer activity in humans. TRAIL signaling in T cells may also potentially provide an immune checkpoint function as it can inhibit T cell activation and proliferation by interfering with T cell receptor signaling. However, this potential immune checkpoint function of TRAIL has not been examined in cancer biology. Cholangiocarcinoma (CCA), a malignancy of the bile ducts, provides a model to examine the potential immune checkpoint function of TRAIL. Methods: Using a syngeneic, orthotopic murine model of CCA (PMID: 29464042), murine CCA cells (SB cells) that express both TRAIL and TRAIL receptor (TR) were implanted into livers of WT C57BL/6J and Tr−/− mice. Hence, in this model the host immune cells express TRAIL but not the receptor; they would be capable of inducing TRAIL-mediated apoptosis in CCA cells but would be resistant to potential TRAIL-mediated immunosuppression. After 4 weeks of tumor growth, mice were sacrificed and tumor and immune characterization (via flow cytometry) was conducted. Results: Implantation of SB cells into Tr−/− mice result in a significant reduction in tumor volumes compared to WT mice. Tumor-bearing Tr−/− mice had a significant infiltration of cytotoxic T lymphocytes (CTLs) (CD45+CD3+CD8+CD11a+) and enhanced CTL effector function. Moreover, Tr−/− mice tumors had a significant decrease in granulocytic and monocytic myeloid-derived suppressor cell (MDSCs) as well as tumor-associated macrophages (TAMs) (CD45+F4/80+CD11b+CD206+) compared to WT mice tumors. Conclusion: Using a unique syngeneic orthotopic implantation model of murine CCA, we demonstrate that CCA cells expressing TRAIL in a mouse genetically deficient for TR exhibit reduced tumor volumes enhanced CTL infiltration and function, and reduced MDSC and TAM infiltration into the tumors. These data suggest that TRAIL has a potential immune checkpoint function, and targeting TRAIL signaling with consequent augmentation of CTL function maybe a promising therapeutic approach in human cancers. Citation Format: Emilien Loeuillard, Jingchun Yang, Haidong Dong, Gregory J. Gores, Sumera Ilyas. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates tumor immune evasion in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2738.

Abstract Richter's transformation (RT) is a clinically challenging and aggressive transformation of CLL (Chronic lymphocytic leukemia) to lymphoma. Immune-checkpoint therapy has improved outcomes in selective lymphoma types. Nearly 40% of RT patients respond to anti-PD1, while the underlying CLL do not show any response, and 10% of CLL patients progress post-anti-PD1 treatment. Previous studies mostly focused on T-cell responses elicited by anti-PD1, however, intrinsic changes in CLL and RT B-cells induced by the treatment remains elusive. We hypothesized that pre-existing active transcriptional circuitry and distinct B-cell subsets in the CLL and RT B-cells may determine the response to anti-PD1 treatment. Peripheral blood mononuclear cells (PBMCs) from 3 patients who received pembrolizumab(anti-PD1) as a single agent. Samples were obtained at baseline and 3 months post-treatment initiation. Among these patients, 2 are progressors while 1 is a responder. We performed single-cell RNAseq (10X Genomics) on B-cells enriched from PBMCs derived from 3 samples (1 Responder: RS2, 2 progressors: CLL14 and CLL17) before and after treatment using chromium single cell 3' gene expression library. A total of ~10000 cells were captured with an average detection of 1000 genes/sample. To delineate the transcriptional changes upon the anti-PD1 treatment, we first integrated the single-cell data from 6 samples (before and after Pembro) and performed unsupervised clustering analysis using UMAP (Uniform Manifold Approximation and projection). We identified 8 different clusters based on highly expressed marker genes. Cluster 2(C2) was increased in the responder as compared to the progressors post-pem treatment. To determine the activated pathways in each cluster, we performed gene-set enrichment analysis (GSEA) using the Enrichr method. This analysis revealed upregulation of distinct pathways related to IFNG/MYC(C1), IFNG(C2), ER stress(C3), B-cell dependent T cell activation related pathway(C4), MYC targets/TNFα(C5,6,7) indicating intra-tumor transcriptional heterogeneity. To decipher the intratumor cell differentiation changes, we performed pseudotime trajectory analysis using Monocle 2 coupled with B cell developmental stage markers. We identified 7 different states, with distinct changes of state in responder sample RS2 (State 4,5, 6, and 7 were increased in RS2 post-treatment) while CLL samples(progressors) did not have distinct changes. Integration with B cell development revealed that aggressive B cells with CD38 expression were highly induced upon anti-PD1 in responder RS2 while regulatory (CD1d) and memory B cells (CD27) remained the same, suggesting PD1 induced intrinsic changes contributing to the disease response. We identified distinct transcriptional states in B cells within responders and progressors which may govern the intra-tumor and inter-tumor heterogeneity to the anti-PD1 response. Citation Format: Prajish Sundaram Iyer, Lu Yang, Yang Zhi Zhang, Hyo Jin Kim, Charla Secreto, Sutapa Sinha, Neil Kay, Stephen Ansell, Wei Ding, Lili Wang. Tumor intrinsic changes determine immune-checkpoint response in Richter's transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1612.

Abstract3D printing (additive manufacturing (AM)) has enormous potential for rapid tooling and mass production due to its design flexibility and significant reduction of the timeline from design to manufacturing. The current state‐of‐the‐art in 3D printing focuses on material manufacturability and engineering applications. However, there still exists the bottleneck of low printing resolution and processing rates, especially when nanomaterials need tailorable orders at different scales. An interesting phenomenon is the preferential alignment of nanoparticles that enhance material properties. Therefore, this review emphasizes the landscape of nanoparticle alignment in the context of 3D printing. Herein, a brief overview of 3D printing is provided, followed by a comprehensive summary of the 3D printing‐enabled nanoparticle alignment in well‐established and in‐house customized 3D printing mechanisms that can lead to selective deposition and preferential orientation of nanoparticles. Subsequently, it is listed that typical applications that utilized the properties of ordered nanoparticles (e.g., structural composites, heat conductors, chemo‐resistive sensors, engineered surfaces, tissue scaffolds, and actuators based on structural and functional property improvement). This review's emphasis is on the particle alignment methodology and the performance of composites incorporating aligned nanoparticles. In the end, significant limitations of current 3D printing techniques are identified together with future perspectives.

Abstract Androgen receptor (AR) is a major survival factor for prostate cancer. Inflammation is implicated in many cancer types, including prostate cancer. Activation of MAP3K7 (also termed TAK1) and downstream IκB kinase β (IKKβ) by proinflammatory cytokines such as TNFα stimulates NF-κB survival pathways. Paradoxically, MAP3K7 is often deleted in human prostate cancer. Here, we demonstrate that AR protein expression is lower in inflammatory tumor areas compared with non-inflammatory tissues in patients with prostate cancer. Map3k7 knockout increased AR protein levels and activity in the mouse prostate, and MAP3K7 and AR protein levels were inversely correlated in prostate cancer patient specimens. TNFα treatment increased AR protein ubiquitination and proteasomal degradation. Mechanistically, activation of IKKβ by TNFα induced phosphorylation and TRCP1/2 E3 ligase–mediated polyubiquitination and degradation of AR protein. TNFα suppressed prostate cancer proliferation, which could be rescued by blockade of AR degradation. These findings reveal a previously unrecognized tumor suppressive function of the inflammation-activated MAP3K7–IKKβ axis in degrading AR protein. Moreover, they suggest that aberrant elevation of AR protein could be a prognostic biomarker and therapeutic target for MAP3K7-deficient prostate cancer. Significance: This study identifies that MAP3K7–IKKβ signaling plays a tumor-suppressive role in prostate cancer by degrading AR, revealing potential prognostic and therapeutic strategies for MAP3K7-deficient tumors.