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Despite advances in therapy,glioblastoma remains an incurable disease with a dismal prognosis. Recent studies have implicated cancer stem cells within glioblastoma (glioma stem cells,GSCs) as mediators of therapeutic resistance and tumor progression. In this study,we investigated the role of the transforming growth factor-$\beta$ (TGF-$\beta$) superfamily,which has been found to play an integral role in the maintenance of stem cell homeostasis within multiple stem cell systems,as a mediator of stem-like cells in glioblastoma. We find that BMP and TGF-$\beta$ signaling define divergent molecular and functional identities in glioblastoma,and mark relatively quiescent and proliferative GSCs,respectively. Treatment of GSCs with BMP inhibits cell proliferation,but does not abrogate their stem-ness,as measured by self-renewal and tumorigencity. Further,BMP pathway activation confers relative resistance to radiation and temozolomide chemotherapy. Our findings define a quiescent cancer stem cell population in glioblastoma that may be a cellular reservoir for tumor recurrence following cytotoxic therapy. View Publication

Protein disulfide isomerase (PDI,PDIA1) is an emerging therapeutic target in oncology. PDI inhibitors have demonstrated a unique propensity to selectively induce apoptosis in cancer cells and overcome resistance to existing therapies,although drug candidates have not yet progressed to the stage of clinical development. We recently reported the discovery of lead indene compound E64FC26 as a potent pan-PDI inhibitor that enhances the cytotoxic effects of proteasome inhibitors in panels of Multiple Myeloma (MM) cells and MM mouse models. An extensive medicinal chemistry program has led to the generation of a diverse library of indene-containing molecules with varying degrees of proteasome inhibitor potentiating activity. These compounds were generated by a novel nucleophilic aromatic ring cyclization and dehydration reaction from the precursor ketones. The results provide detailed structure activity relationships (SAR) around this indene pharmacophore and show a high degree of correlation between potency of PDI inhibition and bortezomib (Btz) potentiation in MM cells. Inhibition of PDI leads to ER and oxidative stress characterized by the accumulation of misfolded poly-ubiquitinated proteins and the induction of UPR biomarkers ATF4,CHOP,and Nrf2. This work characterizes the synthesis and SAR of a new chemical class and further validates PDI as a therapeutic target in MM as a single agent and in combination with proteasome inhibitors. View Publication

Lambda interferons (IFN-$\lambda$s) are a major component of the innate immune defense to viruses,bacteria,and fungi. In human liver,IFN-$\lambda$ not only drives antiviral responses,but also promotes inflammation and fibrosis in viral and non-viral diseases. Here we demonstrate that macrophages are primary responders to IFN-$\lambda$,uniquely positioned to bridge the gap between IFN-$\lambda$ producing cells and lymphocyte populations that are not intrinsically responsive to IFN-$\lambda$. While CD14+ monocytes do not express the IFN-$\lambda$ receptor,IFNLR1,sensitivity is quickly gained upon differentiation to macrophages in vitro. IFN-$\lambda$ stimulates macrophage cytotoxicity and phagocytosis as well as the secretion of pro-inflammatory cytokines and interferon stimulated genes that mediate immune cell chemotaxis and effector functions. In particular,IFN-$\lambda$ induced CCR5 and CXCR3 chemokines,stimulating T and NK cell migration,as well as subsequent NK cell cytotoxicity. Using immunofluorescence and cell sorting techniques,we confirmed that human liver macrophages expressing CD14 and CD68 are highly responsive to IFN-$\lambda$ ex vivo. Together,these data highlight a novel role for macrophages in shaping IFN-$\lambda$ dependent immune responses both directly through pro-inflammatory activity and indirectly by recruiting and activating IFN-$\lambda$ unresponsive lymphocytes. View Publication

Hodgkin Lymphoma (HL) is a malignancy that frequently affects young adults. Although,there are effective treatments not every patient responds,necessitating the development of novel therapeutic approaches,especially for relapsed and refractory cases. The two TNF receptor family members CD30 and CD137 are expressed on Hodgkin and Reed Sternberg (HRS) cells,the malignant cells in HL. We found that this co-expression is specific for HRS cells. Based on this discovery we developed a bispecific antibody that binds preferentially to the CD30,CD137-double positive HRS cells. The CD30,CD137 bispecific antibody gets internalized into HRS cells opening up the possibility to use it as a carrier for a toxin. This antibody also induces antibody-dependent,cell-mediated cytotoxicity in CD30,CD137-double positive HRS cells. The enhances specificity of the CD30,CD137 bispecific antibody to HRS cells makes it a promising candidate for development as a novel HL treatment. View Publication

(1) Background: The cannabinoid 2 receptor (CB2R) is a promising anti-inflammatory drug target and development of selective CB2R ligands may be useful for treating sight-threatening ocular inflammation. (2) Methods: This study examined the pharmacology of three novel chemically-diverse selective CB2R ligands: CB2R agonists,RO6871304,and RO6871085,as well as a CB2R inverse agonist,RO6851228. In silico molecular modelling and in vitro cell-based receptor assays were used to verify CB2R interactions,binding,cell signaling ({\ss}-arrestin and cAMP) and early absorption,distribution,metabolism,excretion,and toxicology (ADMET) profiling of these receptor ligands. All ligands were evaluated for their efficacy to modulate leukocyte-neutrophil activity,in comparison to the reported CB2R ligand,HU910,using an in vivo mouse model of endotoxin-induced uveitis (EIU) in wild-type (WT) and CB2R-/- mice. The actions of RO6871304 on neutrophil migration and adhesion were examined in vitro using isolated neutrophils from WT and CB2R-/- mice,and in vivo in WT mice with EIU using adoptive transfer of WT and CB2R-/- neutrophils,respectively. (3) Results: Molecular docking studies indicated that RO6871304 and RO6871085 bind to the orthosteric site of CB2R. Binding studies and cell signaling assays for RO6871304 and RO6871085 confirmed high-affinity binding to CB2R and selectivity for CB2R {\textgreater} CB1R,with both ligands acting as full agonists in cAMP and {\ss}-arrestin assays (EC50s in low nM range). When tested in EIU,topical application of RO6871304 and RO6871085 decreased leukocyte-endothelial adhesion and this effect was antagonized by the inverse agonist,RO6851228. The CB2R agonist,RO6871304,decreased in vitro neutrophil migration of WT neutrophils but not neutrophils from CB2R-/-,and attenuated adhesion of adoptively-transferred leukocytes in EIU. (4) Conclusions: These unique ligands are potent and selective for CB2R and have good immunomodulating actions in the eye. RO6871304 and RO6871085,as well as HU910,decreased leukocyte adhesion in EIU through inhibition of resident ocular immune cells. The data generated with these three structurally-diverse and highly-selective CB2R agonists support selective targeting of CB2R for treating ocular inflammatory diseases. View Publication

The aim of this study was to vascularize brain organoids with a patient's own endothelial cells (ECs). Induced pluripotent stem cells (iPSCs) of one UC Davis patient were grown into whole-brain organoids. Simultaneously,iPSCs from the same patient were differentiated into ECs. On day 34,the organoid was re-embedded in Matrigel with 250 000 ECs. Vascularized organoids were grown in vitro for 3-5 weeks or transplanted into immunodeficient mice on day 54,and animals were perfused on day 68. Coating of brain organoids on day 34 with ECs led to robust vascularization of the organoid after 3-5 weeks in vitro and 2 weeks in vivo. Human CD31-positive blood vessels were found inside and in-between rosettes within the center of the organoid after transplantation. Vascularization of brain organoids with a patient's own iPSC-derived ECs is technically feasible. View Publication

Abdominal aortic aneurysm (AAA) is a prevalent life-threatening disease,where aortic wall degradation is mediated by accumulated immune cells. Although cytokines regulate inflammation within the aorta,their contribution to AAA via distant alterations,particularly in the control of hematopoietic stem cell (HSC) differentiation,remains poorly defined. Here we report a pathogenic role for the interleukin-27 receptor (IL-27R) in AAA,as genetic ablation of IL-27R protects mice from the disease development. Mitigation of AAA is associated with a blunted accumulation of myeloid cells in the aorta due to the attenuation of Angiotensin II (Ang II)-induced HSC expansion. IL-27R signaling is required to induce transcriptional programming to overcome HSC quiescence and increase differentiation and output of mature myeloid cells in response to stress stimuli to promote their accumulation in the diseased aorta. Overall,our studies illuminate how a prominent vascular disease can be distantly driven by a cytokine-dependent regulation of bone marrow precursors. View Publication

Microenvironmental factors such as oxygen concentration mediate key effects on the biology of mesenchymal stromal cells (MSCs). Herein,we performed an in-depth characterization of the metabolic behavior of MSCs derived from the placenta,umbilical cord,and adipose tissue (termed hPMSCs,UC-MSCs,and AD-MSCs,respectively) at physiological (hypoxic; 5{\%} oxygen [O2]) and standardized (normoxic; 21{\%} O2) O2 concentrations using chemical isotope labeling liquid chromatography-mass spectrometry. 12C- and 13C-isotope dansylation (Dns) labeling was used to analyze the amine/phenol submetabolome,and 2574 peak pairs or metabolites were detected and quantified,from which 52 metabolites were positively identified using a library of 275 Dns-metabolite standards; 2189 metabolites were putatively identified. Next,we identified six metabolites using the Dns library,as well as 14 hypoxic biomarkers from the human metabolome database out of 96 altered metabolites. Ultimately,metabolic pathway analyses were performed to evaluate the associated pathways. Based on pathways identified using the Kyoto Encyclopedia of Genes and Genomes,we identified significant changes in the metabolic profiles of MSCs in response to different O2 concentrations. These results collectively suggest that O2 concentration has the strongest influence on hPMSCs metabolic characteristics,and that 5{\%} O2 promotes arginine and proline metabolism in hPMSCs and UC-MSCs but decreases gluconeogenesis (alanine-glucose) rates in hPMSCs and AD-MSCs. These changes indicate that MSCs derived from different sources exhibit distinct metabolic profiles. View Publication

Products derived from bacterial members of the gut microbiota evoke immune signalling pathways of the host that promote immunity and barrier function in the intestine. How immune reactions to enteric viruses support intestinal homeostasis is unknown. We recently demonstrated that infection by murine norovirus (MNV) reverses intestinal abnormalities following depletion of bacteria,indicating that an intestinal animal virus can provide cues to the host that are typically attributed to the microbiota. Here,we elucidate mechanisms by which MNV evokes protective responses from the host. We identify an important role for the viral protein NS1/2 in establishing local replication and a type I interferon (IFN-I) response in the colon. We further show that IFN-I acts on intestinal epithelial cells to increase the proportion of CCR2-dependent macrophages and interleukin (IL)-22-producing innate lymphoid cells,which in turn promote pSTAT3 signalling in intestinal epithelial cells and protection from intestinal injury. In addition,we demonstrate that MNV provides a striking IL-22-dependent protection against early-life lethal infection by Citrobacter rodentium. These findings demonstrate novel ways in which a viral member of the microbiota fortifies the intestinal barrier during chemical injury and infectious challenges. View Publication

PURPOSE The radiosensitivity of the normal intestinal epithelium is the major limiting factor for definitive radiotherapy against abdominal malignancies. Radiosensitizers,which can be used without augmenting radiation toxicity to normal tissue,are still an unmet need. Inhibition of proteosomal degradation is being developed as a major therapeutic strategy for anticancer therapy as cancer cells are more susceptible to proteasomal inhibition-induced cytotoxicity compared with normal cells. Auranofin,a gold-containing antirheumatoid drug,blocks proteosomal degradation by inhibiting deubiquitinase inhibitors. In this study,we have examined whether auranofin selectively radiosensitizes colon tumors without promoting radiation toxicity in normal intestine. EXPERIMENTAL DESIGN The effect of auranofin (10 mg/kg i.p.) on the radiation response of subcutaneous CT26 colon tumors and the normal gastrointestinal epithelium was determined using a mouse model of abdominal radiation. The effect of auranofin was also examined in a paired human colonic organoid system using malignant and nonmalignant tissues from the same patient. RESULTS Both in the mouse model of intestinal injury and in the human nonmalignant colon organoid culture,auranofin pretreatment prevented radiation toxicity and improved survival with the activation of p53/p21-mediated reversible cell-cycle arrest. However,in a mouse model of abdominal tumor and in human malignant colonic organoids,auranofin inhibited malignant tissue growth with inhibition of proteosomal degradation,induction of endoplasmic reticulum stress/unfolded protein response,and apoptosis. CONCLUSIONS Our data suggest that auranofin is a potential candidate to be considered as a combination therapy with radiation to improve therapeutic efficacy against abdominal malignancies. View Publication

Experimental autoimmune uveoretinitis (EAU) is a mouse model of human autoimmune uveitis marked by ocular autoantigen-specific regulatory immunity in the spleen. The melanocortin 5 receptor (MC5r) and adenosine 2 A receptor (A2Ar) are required for induction of post-EAU regulatory T cells (Tregs) which provide resistance to EAU. We show that blocking the PD-1/PD-L1 pathway prevented suppression of EAU by post-EAU Tregs. A2Ar induction of PD-1+FoxP3+ Tregs in uveitis patients was similar compared to healthy controls,but was significantly reduced with melanocortin stimulation. Further,lower body mass index correlated with responsiveness to stimulation of this pathway. These observations indicate an importance of the PD-1/PD-L1 pathway to provide resistance to relapsing uveitis and shows a reduced capacity of uveitis patients to induce Tregs when stimulated through melanocortin receptors,but that it is possible to bypass this part of the pathway through direct stimulation of A2Ar. View Publication

Aim Reactive oxygen species (ROS) produced by enzymes of the NADPH oxidase family serve as second messengers for cellular signaling. Processes such as differentiation and proliferation are regulated by NADPH oxidases. In the intestine,due to the exceedingly fast and constant renewal of the epithelium both processes have to be highly controlled and balanced. Nox1 is the major NADPH oxidase expressed in the gut,and its function is regulated by cytosolic subunits such as NoxO1. We hypothesize that the NoxO1-controlled activity of Nox1 contributes to a proper epithelial homeostasis and renewal in the gut. Results NoxO1 is highly expressed in the colon. Knockout of NoxO1 reduces the production of superoxide in colon crypts and is not subsidized by an elevated expression of its homolog p47phox. Knockout of NoxO1 increases the proliferative capacity and prevents apoptosis of colon epithelial cells. In mouse models of dextran sulfate sodium (DSS)-induced colitis and azoxymethane/DSS induced colon cancer,NoxO1 has a protective role and may influence the population of natural killer cells. Conclusion NoxO1 affects colon epithelium homeostasis and prevents inflammation. View Publication