Cell Survival Function Research Articles

Abstract 3252: OrthoCARs: Engineered human IL-2/IL-2Rb orthogonal pairs selectively enhance CAR T cell anti-tumor efficacy and durability of response

Abstract CAR T cell therapy has demonstrated remarkable clinical efficacy against relapsed and refractory hematological malignancies, such as B cell non-Hodgkin lymphoma (NHL) and acute lymphoblastic leukemia (ALL). Despite these advances, prominent barriers including poor T cell effector function, lack of proliferation, and limited CAR T cell persistence prevent CAR T cell therapies from reaching their full curative potential. Interleukin-2 (IL-2) is a potent stimulator of CD4 and CD8 effector T cell proliferation, survival, and cytotoxic function, thereby making it an attractive molecule to support CAR T cell therapy. However, therapeutic use of IL-2 is limited by systemic toxicity due its promiscuous activation of undesired immune cell populations, including non-tumor reactive T cells and NK cells. To facilitate selective in vivo expansion of engineered T cells we have developed an orthogonal (ortho) ligand/receptor system consisting of a pegylated, IL-2 mutein (STK-009) and a mutated IL-2 Receptor Beta (orthoIL-2Rβ) that selectively bind each another, but do not interact with their wild type receptor and cytokine counterparts. This system allows for in-vivo IL-2 signaling in engineered adoptive cell therapies that express the orthoIL-2Rβ while avoiding signaling in non-tumor reactive T cells and NK cells. Here, we demonstrate the ability of STK-009/orthoIL-2Rβ pair to selectively potentiate human orthoIL-2Rβ expressing CD19 CAR T cells in vitro and in vivo. We incorporated orthoIL-2Rβ into a CD19 directed CAR lentiviral construct utilizing a T2A peptide linker, allowing the use of a single lentiviral plasmid to generate orthoCAR T cells. Transduction of donor T cells with the CAR+orthoIL-2Rβ lentivirus and the use of STK-009 during in vitro manufacturing enabled selective proliferation and enrichment of orthoCAR transduced T cells. orthoCAR T cells grown in STK-009 maintained a similar immunophenotype and cytotoxic function compared to T cells manufactured with wild type IL-2. Subcutaneous administration of STK-009 dramatically enhanced the expansion (>100-fold) and efficacy of orthoCAR T cells in an aggressive pre-clinical mouse model of B cell malignancy (Raji), demonstrating 100% durable complete responses, compared to 50% relapsed and refractory lymphoma in CD19 CAR alone. Immunoprofiling of in vivo orthoCAR T cells revealed that STK-009 treatment significantly increased both naïve and effector memory RA (TEMRA) populations. RNAseq and IHC analysis also showed that STK-009 treatment increases expression of cytotoxic molecules such as Granzyme B and perforin specifically in the CAR T cells, without inducing GranzymeB in other cells. These findings validate a potent platform that selectively harnesses the potent anti-tumoral T cell effector functions of IL-2 to improve the efficacy and durability CAR T cell therapy. Citation Format: Paul-Joseph Aspuria, Michele Bauer, Sandro Vivona, Steven E. Kauder, Scott McCauley, Romina Riener, Rene De Waal Malefyte, Navneet Ratti, Deepti Rokkam, Jan Emmerich, Patrick J. Lupardus, Rob A. Kastelein, Martin Oft. OrthoCARs: Engineered human IL-2/IL-2Rb orthogonal pairs selectively enhance CAR T cell anti-tumor efficacy and durability of response [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3252.

Abstract 1017: RICTOR/mTOR signaling regulates novel immune checkpoints in non-small cell lung cancer (NSCLC)

Abstract Background: RICTOR is a critical component of the mTOR2 complex. Through activation of AKT, SGK1, PKC and other downstream pathways, the RICTOR/mTOR signaling is essential in controlling cell survival, proliferation, metabolism, migration, protein degradation and cytoskeleton functions. Moreover, our recent work implicates that RICTOR amplification may define a unique subtype of NSCLC that can potentially be targeted by mTOR1/2 inhibitors. Additionally, previous evidence also suggest that the PI3K/AKT/mTOR pathway may be involved in immune regulation. Immunotherapy targeting PD-1/PD-L1 has significantly shifted the treatment landscape in NSCLC. PD-1/PD-L1 belongs to the B7 family of ligands and CD28 family of receptors, which play crucial roles in the regulation of T-cell responses. B7x, HHLA2 and B7-H3 are recently discovered B7 family ligands, which function as T-cell co-inhibitory molecules. These new immune checkpoints are widely expressed in NSCLC and in particular, these alternative immune checkpoints are commonly found in PD-L1 negative tumors. In the current study, we investigated the potential roles of the RICTOR/mTOR signaling on immune modulation, especially the regulation of immune checkpoints. Results: Using multiple RICTOR amplified NSCLC cells lines (H23, H1703 and H1734), we found that knockdown of RICTOR by siRNA or inducible shRNA resulted in decreased whole lysate and cell surface PD-L1 and HHLA-2 expression but not B7x or B7-H3. RICTOR knockdown can also downregulate IFN-gamma induced PD-L1 expression. Moreover, inducible RICTOR overexpression upregulates PD-L1 levels and also modestly HHLA2 expression. Our preliminary data also indicate that this regulation is likely occurring at a post-transcriptional level. In addition to potential modulation of immune checkpoint molecules, preliminary analysis of 14,698 NSCLC cases indicate that tumors with RICTOR amplification more often have higher levels of tumor mutation burden (TMB) than other lung cancers: the mean TMB was 14.9 mut/Mb in RICTOR-amplified vs 9.2 mut/Mb in non-amplified cases. TMB is a surrogate for increased number of expressed tumor neoantigens and is an important biomarker for response to immune checkpoint inhibitors. Conclusion: Our study provides new insight into the regulation of the tumor immune microenvironment by RICTOR/mTOR signaling. This may pave the way for designing or improving immunotherapeutic strategies in lung cancer by exploiting the novel immune escape mechanisms in these tumors. Citation Format: Mariam Alexander, Ni Fan, Xiaoxin Ren, Jeffrey Ross, Hao Wang, Feng Wang, Balazs Halmos, Roman Perez-Soler, Xingxing Zang, Haiying Cheng. RICTOR/mTOR signaling regulates novel immune checkpoints in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1017.

Loss of Clustered Protocadherin Diversity Alters the Spatial Distribution of Cortical Interneurons in Mice.

Cortical interneurons (cINs) are locally projecting inhibitory neurons that are distributed throughout the cortex. Due to their relatively limited range of influence, their arrangement in the cortex is critical to their function. cINs achieve this arrangement through a process of tangential and radial migration and apoptosis during development. In this study, we investigated the role of clustered protocadherins (cPcdhs) in establishing the spatial patterning of cINs through the use of genetic cPcdh knockout mice. cPcdhs are expressed in cINs and are known to play key functions in cell spacing and cell survival, but their role in cINs is poorly understood. Using spatial statistical analysis, we found that the 2 main subclasses of cINs, parvalbumin-expressing and somatostatin-expressing (SST) cINs, are nonrandomly spaced within subclass but randomly with respect to each other. We also found that the relative laminar distribution of each subclass was distinctly altered in whole α- or β-cluster mutants. Examination of perinatal time points revealed that the mutant phenotypes emerged relatively late, suggesting that cPcdhs may be acting during cIN morphological elaboration and synaptogenesis. We then analyzed an isoform-specific knockout for pcdh-αc2 and found that it recapitulated the α-cluster knockout but only in SST cells, suggesting that subtype-specific expression of cPcdh isoforms may help govern subtype-specific spatial distribution.

Rationalized inhibition of mixed lineage kinase 3 and CD70 enhances life span and antitumor efficacy of CD8+ T cells

BackgroundThe mitogen-activated protein kinases (MAPKs) are important for T cell survival and their effector function. Mixed lineage kinase 3 (MLK3) (MAP3K11) is an upstream regulator of MAP kinases and emerging...

Modulation of Host Lipid Pathways by Pathogenic Intracellular Bacteria.

Lipids are a broad group of molecules required for cell maintenance and homeostasis. Various intracellular pathogens have developed mechanisms of modulating and sequestering host lipid processes for a large array of functions for both bacterial and host cell survival. Among the host cell lipid functions that intracellular bacteria exploit for infection are the modulation of host plasma membrane microdomains (lipid rafts) required for efficient bacterial entry; the recruitment of specific lipids for membrane integrity of intracellular vacuoles; and the utilization of host lipid droplets for the regulation of immune responses and for energy production through fatty acid β-oxidation and oxidative phosphorylation. The majority of published studies on the utilization of these host lipid pathways during infection have focused on intracellular bacterial pathogens that reside within a vacuole during infection and, thus, have vastly different requirements for host lipid metabolites when compared to those intracellular pathogens that are released into the host cytosol upon infection. Here we summarize the mechanisms by which intracellular bacteria sequester host lipid species and compare the modulation of host lipid pathways and metabolites during host cell infection by intracellular pathogens residing in either a vacuole or within the cytosol of infected mammalian cells. This review will also highlight common and unique host pathways necessary for intracellular bacterial growth that could potentially be targeted for therapeutic intervention.

Shank-associated RH domain interactor signaling in tumorigenesis.

Shank-associated RH domain interactor (SHARPIN) is a component of the linear ubiquitin chain activation complex, which is essential for p53 signaling and inflammation. Previous studies have demonstrated that SHARPIN functions in tumor cell survival, growth, invasion and tumorigenesis. These functions include the regulation of p53 proteins via poly-ubiquitination, interaction with a type II protein arginine methyltransferase 5 in melanoma cells, modulating ras-associated protein-1 through p38 and c-Jun N-terminal kinases/c-Jun signaling, and mediating phosphoinositide 3-kinase/AKT signaling via phosphatase and tensin homologue deleted on chromosome 10. Hence, SHARPIN not only participates in the inflammatory response but also serves a critical role in tumor cells. The present review summarizes the biological functions of the absence or presence of SHARPIN with regard to activating the canonical NF-κB signaling pathway and the effects on p53 and other signaling pathways for the modulation of tumorigenesis. Therefore, this review provides insight into the underlying role and mechanisms of SHARPIN in tumorigenesis, as well as its potential application in cancer therapy.

CD137 costimulation enhances the antiviral activity of V\u03b39V\u03b42-T cells against influenza virus

Influenza epidemics and pandemics are constant threats to global public health. Although strategies including vaccines and antiviral drugs have achieved great advances in controlling influenza virus infection, the efficacy of these strategies is limited by the highly frequent mutations in the viral genome and the emergence of drug-resistant strains. Our previous study indicated that boosting the immunity of human Vγ9Vδ2-T cells with the phosphoantigen pamidronate could be a therapeutic strategy to treat seasonal and avian influenza virus infections. However, one notable drawback of γδ-T cell-based immunotherapy is the rapid exhaustion of proliferation and effector responses due to repeated treatments with phosphoantigens. Here, we found that the expression of CD137 was inducible in Vγ9Vδ2-T cells following antigenic stimulation. CD137+ Vγ9Vδ2-T cells displayed more potent antiviral activity against influenza virus than their CD137− counterparts in vitro and in Rag2-/- γc-/- mice. We further demonstrated that CD137 costimulation was essential for Vγ9Vδ2-T cell activation, proliferation, survival and effector functions. In humanized mice reconstituted with human peripheral blood mononuclear cells, CD137 costimulation with a recombinant human CD137L protein boosted the therapeutic effects of pamidronate against influenza virus. Our study provides a novel strategy of targeting CD137 to improve the efficacy of Vγ9Vδ2-T cell-based immunotherapy.

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment.

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

Astaxanthin improves osteopenia caused by aldehyde-stress resulting from Aldh2 mutation due to impaired osteoblastogenesis

Aldehyde dehydrogenase 2 (ALDH2) plays major roles in aldehyde detoxification and in the catalysis of amino acids. ALDH2∗2, a dominant-negative transgenic expressing aldehyde dehydrogenase 2 (ALDH2) protein, is produced by a single nucleotide polymorphism (rs671) and is involved in the development of osteoporosis and hip fracture with aging. In a previous study, transgenic mice expressing Aldh2∗2(Aldh2∗2 Tg) osteoblastic cells or acetaldehyde -treated MC3T3-E1 showed impaired osteoblastogenesis and caused osteoporosis [1].In this study, we demonstrated the effects of astaxanthin for differentiation to osteoblasts of MC3T3-E1 by the addition of acetaldehyde and Aldh2∗2 Tg mesenchymal stem cells in bone marrow. Astaxanthin restores the inhibited osteoblastogenesis by acetaldehyde in MC 3T3-E1 and in bone marrow mesenchymal stem cells of Aldh2∗2 Tg mice. Additionally, astaxanthin administration improved femur bone density in Aldh2∗2 Tg mice. Furthermore, astaxanthin improved cell survival and mitochondrial function in acetaldehyde-treated MC 3T3-E1 cells.Our results suggested that astaxanthin had restorative effects on osteoblast formation and provide new insight into the regulation of osteoporosis and suggest a novel strategy to promote bone formation in osteopenic diseases caused by impaired acetaldehyde metabolism.

Phosphorylated GSK‑3β protects stress‑induced apoptosis of myoblasts via the PI3K/Akt signaling pathway.

Facial jaw muscle is involved in the occurrence, development, treatment and maintenance of maxillofacial deformities. The structure and function of this tissue can be altered by changes in external stimuli, and orthodontists can regulate its reconstruction using orthopedic forces. The PI3K/Akt signaling pathway is most well-known for its biological functions in cell proliferation, survival and apoptosis. In the present study, the effects of the PI3K/Akt signaling pathway in cyclic stretch-induced myoblast apoptosis were investigated. For this purpose, L6 rat myoblasts were cultured under mechanical stimulation and treated with the PI3K kinase inhibitor, LY294002, to elucidate the role of the PI3K/Akt signaling pathway. Cells were stained with Hoechst 33258 to visualize morphological changes and apoptosis of myoblasts, and western blotting was performed to detect expression of Akt, phosphorylated (p)-Akt (Ser473), glycogen synthase kinase 3β (GSK-3β) and p-GSK-3β (Ser9). After addition of PI3K inhibitor, the expression of total Akt and GSK-3β did not significantly differ among groups; however, the levels of p-Akt and p-GSK-3β were lower in inhibitor-treated groups than in those treated with loading stress alone. In addition, the rate of apoptosis in myoblasts subjected to cyclic stretch increased in a time-dependent manner, peaking at 24 h. Collectively, it was also demonstrated that the PI3K/Akt/GSK-3β pathway plays an important role in stretch-induced myoblast apoptosis.

S1P-lyase deficiency uncouples ganglioside formation – Potential contribution to tumorigenic capacity

Sphingosine-1-phosphate (S1P) is not only a catabolic intermediate of all sphingolipids but also an evolutionary conserved bioactive lipid with critical functions in cell survival, differentiation, and migration as well as in immunity and angiogenesis. S1P-lyase (SGPL1) irreversibly cleaves S1P in the final step of sphingolipid catabolism. As sphingoid bases and their 1-phosphates are not only metabolic intermediates but also highly bioactive lipids that modulate a wide range of physiological processes, it would be predicted that their elevation might induce adjustments in other facets of sphingolipid metabolism and/or alter cell behavior. We actually found in a previous study that in terminally differentiated neurons SGPL1 deficiency increases sphingolipid formation via recycling at the expense of de novo synthesis. We now investigated whether and how SGPL1 deficiency affects the metabolism of (glyco)sphingolipids in mouse embryonic fibroblasts (MEFs). According to our previous experiments in neurons, we found a strong accumulation of S1P in SGPL1-deficient MEFs. Surprisingly, a completely different situation arose as we analyzed sphingolipid metabolism in this non-differentiated cell type. The production of biosynthetic precursors of complex glycosphingolipids including ceramide, glucosylceramide and also ganglioside GM3 via de novo synthesis and recycling pathway was substantially increased whereas the amount of more complex gangliosides dropped significantly.

Emerging Role of Angiotensin AT2 Receptor in Anti-Inflammation: An Update.

The hyperactive RAS and inflammation are closely associated. The angiotensin-II/AT1R axis of the RAS has been explored extensively for its role in inflammation and a plethora of pathological conditions. Understanding the role of AT2R in inflammation is an emerging area of research. The AT2R is expressed on a variety of immune and non-immune cells, which upon activation triggers the release of a host of cytokines and has multiple effects that coalesce to anti-inflammation and prevents maladaptive repair. The anti-inflammatory outcomes of AT2R activation are linked to its well-established signaling pathways involving formation of nitric oxide and activation of phosphatases. Collectively, these effects promote cell survival and tissue function. The consideration of AT2R as a therapeutic target requires further investigations.

Bioprinting 101: Design, Fabrication, and Evaluation of Cell-Laden 3D Bioprinted Scaffolds.

3D bioprinting is an additive manufacturing technique that recapitulates the native architecture of tissues. This is accomplished through the precise deposition of cell-containing bioinks. The spatiotemporal control over bioink deposition permits for improved communication between cells and the extracellular matrix, facilitates fabrication of anatomically and physiologically relevant structures. The physiochemical properties of bioinks, before and after crosslinking, are crucial for bioprinting complex tissue structures. Specifically, the rheological properties of bioinks determines printability, structural fidelity, and cell viability during the printing process, whereas postcrosslinking of bioinks are critical for their mechanical integrity, physiological stability, cell survival, and cell functions. In this review, we critically evaluate bioink design criteria, specifically for extrusion-based 3D bioprinting techniques, to fabricate complex constructs. The effects of various processing parameters on the biophysical and biochemical characteristics of bioinks are discussed. Furthermore, emerging trends and future directions in the area of bioinks and bioprinting are also highlighted. Graphical abstract [Figure: see text] Impact statement Extrusion-based 3D bioprinting is an emerging additive manufacturing approach for fabricating cell-laden tissue engineered constructs. This review critically evaluates bioink design criteria to fabricate complex tissue constructs. Specifically, pre- and post-printing evaluation approaches are described, as well as new research directions in the field of bioink development and functional bioprinting are highlighted.

Fatty acids - from energy substrates to key regulators of cell survival, proliferation and effector function.

Recent advances in immunology and cancer research show that fatty acids, their metabolism and their sensing have a crucial role in the biology of many different cell types. Indeed, they are able to affect cellular behaviour with great implications for pathophysiology. Both the catabolic and anabolic pathways of fatty acids present us with a number of enzymes, receptors and agonists/antagonists that are potential therapeutic targets, some of which have already been successfully pursued. Fatty acids can affect the differentiation of immune cells, particularly T cells, as well as their activation and function, with important consequences for the balance between anti- and pro-inflammatory signals in immune diseases, such as rheumatoid arthritis, psoriasis, diabetes, obesity and cardiovascular conditions. In the context of cancer biology, fatty acids mainly provide substrates for energy production, which is of crucial importance to meet the energy demands of these highly proliferating cells. Fatty acids can also be involved in a broader transcriptional programme as they trigger signals necessary for tumorigenesis and can confer to cancer cells the ability to migrate and generate distant metastasis. For these reasons, the study of fatty acids represents a new research direction that can generate detailed insight and provide novel tools for the understanding of immune and cancer cell biology, and, more importantly, support the development of novel, efficient and fine-tuned clinical interventions. Here, we review the recent literature focusing on the involvement of fatty acids in the biology of immune cells, with emphasis on T cells, and cancer cells, from sensing and binding, to metabolism and downstream effects in cell signalling.

Nudix Hydrolase NUDT16 Regulates 53BP1 Protein by Reversing 53BP1 ADP-Ribosylation.

53BP1 controls two downstream subpathways, one mediated by PTIP and Artemis and the other by RIF1 and MAD2L2/Shieldin, to coordinate DNA repair pathway choices. However, the upstream regulator(s) of 53BP1 function in DNA repair remain unknown. We and others recently reported that TIRR associates with 53BP1 to stabilize it and prevents 53BP1 localization to DNA damage sites by blocking 53BP1 Tudor domain binding to H4K20me2 sites. Here, we report that the Nudix hydrolase NUDT16, a TIRR homolog, regulates 53BP1 stability. We identified a novel posttranslational modification of 53BP1 by ADP-ribosylation that is targeted by a PAR-binding E3 ubiquitin ligase, RNF146, leading to 53BP1 polyubiquitination and degradation. In response to DNA damage, ADP-ribosylated 53BP1 increased significantly, resulting in its ubiquitination and degradation. These data suggest that NUDT16 plays a major role in controlling 53BP1 levels under both normal growth conditions and during DNA damage. Notably, overexpression of a NUDT16 catalytically inactive mutant blocked 53BP1 localization to double-strand breaks because (i) the mutant binding to TIRR increased after IR; (ii) the mutant enhanced 53BP1 Tudor domain binding to TIRR, and (iii) the mutant impaired the interaction of 53BP1 Tudor domain with H4K20me2. Moreover, NUDT16's catalytic hydrolase activity was required for 53BP1 de-ADP-ribosylation, 53BP1 protein stability, and its function in cell survival. In summary, we demonstrate that NUDT16 regulates 53BP1 stability and 53BP1 recruitment at double-strand breaks, providing yet another mechanism of 53BP1 regulation.Significance: This study provides a novel mechanism of 53BP1 regulation by demonstrating that NUDT16 has hydrolase activities that remove ADP-ribosylation of 53BP1 to regulate 53BP1 stability and 53BP1 localization at DSBs.

The role of coffee derivatives in the regulation of pancreatic beta cell function in Type 2 Diabetes

AbstractCoffee contains several components other than caffeine such as chlorogenic acids (CGAs), which are a family of polyphenols. Non-caffeinated coffee has been shown to reduce the risk of Type 2 diabetes, but it is unclear whether this effect is primarily due to a beneficial action on glucose regulation in peripheral tissues or whether it is partly mediated via a direct, functional modulation of insulin-secreting beta cells from the pancreas. This study aims to explore the specific role of coffee compounds derived from the polyphenolic family of CGAs (caffeic acid (CA) and ferulic acid (FA)) and their metabolites (dihydroferulic acid (diFA) and ferulic acid 4-O-sulphate (FA-4-OS)) in the regulation of beta cell survival and secretory function. To investigate this role, the cells were initially exposed to conditions of glucotoxicity (30mmol/l glucose), lipotoxicity (0.5mmol/l palmitate), glucolipotoxicity (30mmol/l glucose + 0.5mmol/l palmitate) and cytokine-induced cell toxicity (25U/ml IL1b + 500U/ml TNFα) for 20 and 48 h. INS1 beta cells were subsequently treated with or without 100nmol/l of the CGA compounds for 48 h followed by 20 h exposure to glucolipotoxicity to measure cellular ATP content and 3/7 caspase activity, respectively, as an indication of cell viability and apoptosis. Additionally, insulin release was assessed by radioimmunoassay following 1 h static incubations with or without CA, FA and metabolites. Data were analysed by One-Way ANOVA using GraphPad prism software (version 8). Glucotoxicity, lipotoxicity and glucotoxicity or glucolipotoxicity combined with cytokines significantly reduced INS1 cell viability compared to control at 20 and 48 h (p < 0.001 vs 11mmol/l glucose, p < 0.05; Glucotoxicity vs 11mmol/l glucose at 20 h, n = 6), whereas cytokines alone did not significantly affect cellular ATP content. Moreover, pre-treatment for 48 h with CGAs alone or in combination did not affect INS1 beta-cell viability under basal conditions (n = 3, p > 0.2). Additional exposure to glucolipotoxicity for 20 h significantly decreased beta cell viability and survival and was not alleviated by pre- or co-treatment with CGAs (n = 3, p > 0.05). However, insulin release in response to 1 h incubation with 20 mmol/l glucose + 10μmol/l forskolin (FSK) + 100μmol/l 3-isobutyl-1-methylxanthine (IBMX) was significantly higher from cells pre-treated with CA and FA combined compared to controls (7.64 + 2.89pg insulin/5,000 cells/h vs 2.17 + 0.35pg insulin/5,000 cells/h; p < 0.01 vs. 20mmol/l glucose + 10μmol/l FSK + 100μmol/l IBMX only, n = 5). The results suggest that CGA derivatives from coffee do not directly modulate INS1 beta cell viability and apoptosis whereas the compounds may play a role in the regulation of beta cell secretory function.

3D bioprinting of liver spheroids derived from human induced pluripotent stem cells sustain liver function and viability in vitro

The liver is responsible for many metabolic, endocrine and exocrine functions. Approximately 2 million deaths per year are associated with liver failure. Modern 3D bioprinting technologies allied with autologous induced pluripotent stem cells (iPS)-derived grafts could represent a relevant tissue engineering approach to treat end stage liver disease patients. However, protocols that accurately recapitulates liver’s epithelial parenchyma through bioprinting are still underdeveloped. Here we evaluated the impacts of using single cell dispersion (i.e. obtained from conventional bidimensional differentiation) of iPS-derived parenchymal (i.e. hepatocyte-like cells) versus using iPS-derived hepatocyte-like cells spheroids (i.e. three-dimensional cell culture), both in combination with non-parenchymal cells (e.g. mesenchymal and endothelial cells), into final liver tissue functionality. Single cell constructs showed reduced cell survival and hepatic function and unbalanced protein/amino acid metabolism when compared to spheroid printed constructs after 18 days in culture. In addition, single cell printed constructs revealed epithelial-mesenchymal transition, resulting in rapid loss of hepatocyte phenotype. These results indicates the advantage of using spheroid-based bioprinting, contributing to improve current liver bioprinting technology towards future regenerative medicine applications and liver physiology and disease modeling.

Drug Screen Identifies Leflunomide for Treatment of Inflammatory Bowel Disease Caused by TTC7A Deficiency

Mutations in the tetratricopeptide repeat domain 7A gene (TTC7A) cause intestinal epithelial and immune defects. Patients can become immune deficient anddevelop apoptotic enterocolitis, multiple intestinal atresia, and recurrent intestinal stenosis. The intestinal disease in patients with TTC7A deficiency is severe and untreatable, and it recurs despite resection or allogeneic hematopoietic stem cell transplant. We screened drugs for those that prevent apoptosis of in cells with TTC7A deficiency and tested their effects in an animal model of the disease. We developed a high-throughput screen to identify compounds approved by the US Food and Drug Administration that reduce activity of caspases 3 and 7 in TTC7A-knockout (TTC7A-KO) HAP1 (human haploid) cells and reduce the susceptibility to apoptosis. We validated the effects of identified agents in HeLa cells that stably express TTC7A with point mutations found in patients. Signaling pathways in cells were analyzed by immunoblots. We tested the effects of identified agents in zebrafish with disruption of ttc7a, which develop intestinal defects, and colonoids derived from biopsy samples of patients with and without mutations in TTC7A. We performed real-time imaging of intestinal peristalsis in zebrafish and histologic analyses of intestinal tissues from patients and zebrafish. Colonoids were analyzed by immunofluorescence and for ion transport. TTC7A-KO HAP1cells have abnormal morphology and undergo apoptosis, due to increased levels of active caspases 3 and 7. We identified drugs that increased cell viability; leflunomide (used to treat patients with inflammatory conditions) reduced caspase 3 and 7 activity in cells by 96%. TTC7A-KO cells contained cleaved caspase 3 and had reduced levels of phosphorylated AKT and X-linked inhibitor of apoptosis (XIAP); incubation of these cells with leflunomide increased levels of phosphorylated AKT and XIAP and reduced levels of cleaved caspase 3. Administration of leflunomide to ttc7a-/- zebrafish increased gut motility, reduced intestinal tract narrowing, increased intestinal cell survival, increased sizes of intestinal luminal spaces, and restored villi and goblet cell morphology. Exposure of patient-derived colonoids to leflunomide increased cell survival, polarity, and transport function. In a drug screen, we identified leflunomide as an agent that reduces apoptosis and activates AKT signaling in TTC7A-KO cells. In zebrafish with disruption of ttc7a, leflunomide restores gut motility, reduces intestinal tract narrowing, and increases intestinal cell survival. This drug might be repurposed for treatment of TTC7A deficiency.

Mitochondrial dysfunction plays a key role in the development of neurodegenerative diseases in diabetes.

Mitochondria have an essential function in cell survival due to their role in bioenergetics, reactive oxygen species generation, calcium buffering, and other metabolic activities. Mitochondrial dysfunctions are commonly found in neurodegenerative diseases (NDs), and diabetes is a risk factor for NDs. However, the role of mitochondria in diabetic neurodegeneration is still unclear. In the present study, we review the latest evidence on the role of mitochondrial dysfunctions in the development of diabetes-related NDs and the underlying molecular mechanisms. Hypoglycemic agents, especially metformin, have been proven to have neuroprotective effects in the treatment of diabetes, in which mitochondria could act as one of the underlying mechanisms. Other hypoglycemic agents, including thiazolidinediones (TZDs), dipeptidyl peptidase 4 (DPP-4) inhibitors, and glucagon-like peptide 1 (GLP-1) receptor agonists, have gained more attention because of their beneficial effects on NDs, presumably by improving mitochondrial function. Our review highlights the notion that mitochondria could be a promising therapeutic target in the treatment of NDs in patients with diabetes.

Rhes, a striatal-enriched protein, promotes mitophagy via Nix

Elimination of dysfunctional mitochondria via mitophagy is essential for cell survival and neuronal functions. But, how impaired mitophagy participates in tissue-specific vulnerability in the brain remains unclear. Here, we find that striatal-enriched protein, Rhes, is a critical regulator of mitophagy and striatal vulnerability in brain. In vivo interactome and density fractionation reveal that Rhes coimmunoprecipitates and cosediments with mitochondrial and lysosomal proteins. Live-cell imaging of cultured striatal neuronal cell line shows Rhes surrounds globular mitochondria, recruits lysosomes, and ultimately degrades mitochondria. In the presence of 3-nitropropionic acid (3-NP), an inhibitor of succinate dehydrogenase, Rhes disrupts mitochondrial membrane potential (ΔΨ m ) and promotes excessive mitophagy and cell death. Ultrastructural analysis reveals that systemic injection of 3-NP in mice promotes globular mitochondria, accumulation of mitophagosomes, and striatal lesion only in the wild-type (WT), but not in the Rhes knockout (KO), striatum, suggesting that Rhes is critical for mitophagy and neuronal death in vivo. Mechanistically, Rhes requires Nix (BNIP3L), a known receptor of mitophagy, to disrupt ΔΨ m and promote mitophagy and cell death. Rhes interacts with Nix via SUMO E3-ligase domain, and Nix depletion totally abrogates Rhes-mediated mitophagy and cell death in the cultured striatal neuronal cell line. Finally, we find that Rhes, which travels from cell to cell via tunneling nanotube (TNT)-like cellular protrusions, interacts with dysfunctional mitochondria in the neighboring cell in a Nix-dependent manner. Collectively, Rhes is a major regulator of mitophagy via Nix, which may determine striatal vulnerability in the brain.