Targeting Wnt/ß-catenin signaling using XAV939 nanoparticles in tumor microenvironment-conditioned macrophages promote immunogenicity.

The aberrant activation of Wnt/ß-catenin signaling in tumor cells and immune cells in the tumor microenvironment (TME) promotes malignant transformation, metastasis, immune evasion, and resistance to cancer treatments. The increased Wnt ligand expression in TME activates ß-catenin signaling in antigen (Ag)-presenting cells (APCs) and regulates anti-tumor immunity. Previously, we showed that activation of Wnt/ß-catenin signaling in dendritic cells (DCs) promotes induction of regulatory T cell responses over anti-tumor CD4+ and CD8+ effector T cell responses and promotes tumor progression. In addition to DCs, tumor-associated macrophages (TAMs) also serve as APCs and regulate anti-tumor immunity. However, the role of ß-catenin activation and its effect on TAM immunogenicity in TME is largely undefined. In this study, we investigated whether inhibiting ß-catenin in TME-conditioned macrophages promotes immunogenicity. Using nanoparticle formulation of XAV939 (XAV-Np), a tankyrase inhibitor that promotes ß-catenin degradation, we performed in vitro macrophage co-culture assays with melanoma cells (MC) or melanoma cell supernatants (MCS) to investigate the effect on macrophage immunogenicity. We show that XAV-Np-treatment of macrophages conditioned with MC or MCS significantly upregulates the cell surface expression of CD80 and CD86 and suppresses the expression of PD-L1 and CD206 compared to MC or MCS-conditioned macrophages treated with control nanoparticle (Con-Np). Further, XAV-Np-treated macrophages conditioned with MC or MCS significantly increased IL-6 and TNF-α production, with reduced IL-10 production compared to Con-Np-treated macrophages. Moreover, the co-culture of MC and XAV-Np-treated macrophages with T cells resulted in increased CD8+ T cell proliferation compared to Con-Np-treated macrophages. These data suggest that targeted ß-catenin inhibition in TAMs represents a promising therapeutic approach to promote anti-tumor immunity.

Role of IL-27 in HSV-1-Induced Herpetic Stromal Keratitis.

Herpetic stromal keratitis (HSK) is a painful and vision-impairing disease caused by recurrent HSV-1 infection of the cornea. The virus replication in the corneal epithelium and associated inflammation play a dominant role in HSK progression. Current HSK treatments targeting inflammation or virus replication are partially effective and promote HSV-1 latency, and long-term use can cause side effects. Thus, understanding molecular and cellular events that control HSV-1 replication and inflammation is crucial for developing novel HSK therapies. In this study, we report that ocular HSV-1 infection induces the expression of IL-27, a pleiotropic immunoregulatory cytokine. Our data indicate that HSV-1 infection stimulates IL-27 production by macrophages. Using a primary corneal HSV-1 infection mouse model and IL-27 receptor knockout mice, we show that IL-27 plays a critical role in controlling HSV-1 shedding from the cornea, the optimum induction of effector CD4+ T cell responses, and limiting HSK progression. Using in vitro bone marrow-derived macrophages, we show that IL-27 plays an antiviral role by regulating macrophage-mediated HSV-1 killing, IFN-ß production, and IFN-stimulated gene expression after HSV-1 infection. Furthermore, we report that IL-27 is critical for macrophage survival, Ag uptake, and the expression of costimulatory molecules involved in the optimum induction of effector T cell responses. Our results indicate that IL-27 promotes endogenous antiviral and anti-inflammatory responses and represents a promising target for suppressing HSK progression.

Targeting ß-catenin using XAV939 nanoparticle promotes immunogenic cell death and suppresses conjunctival melanoma progression.

Many tumors dysregulate Wnt/ß-catenin pathway to promote stem-cell-like phenotype, tumorigenesis, immunosuppression, and resistance to targeted cancer immunotherapies. Therefore, targeting this pathway is a promising therapeutic approach to suppress tumor progression and elicit robust anti-tumor immunity. In this study, using a nanoparticle formulation for XAV939 (XAV-Np), a tankyrase inhibitor that promotes ß-catenin degradation, we investigated the effect of ß-catenin inhibition on melanoma cell viability, migration, and tumor progression using a mouse model of conjunctival melanoma. XAV-Nps were uniform and displayed near-spherical morphology with size stability for upto 5 days. We show that XAV-Np treatment of mouse melanoma cells significantly suppresses cell viability, tumor cell migration, and tumor spheroid formation compared to control nanoparticle (Con-Np) or free XAV939-treated groups. Further, we demonstrate that XAV-Np promotes immunogenic cell death (ICD) of tumor cells with a significant extracellular release or expression of ICD molecules, including high mobility group box 1 protein (HMGB1), calreticulin (CRT), and adenosine triphosphate (ATP). Finally, we show that local intra-tumoral delivery of XAV-Nps during conjunctival melanoma progression significantly suppresses tumor size and conjunctival melanoma progression compared to Con-Nps-treated animals. Collectively, our data suggest that selective inhibition of ß-catenin in tumor cells using nanoparticle-based targeted delivery represents a novel approach to suppress tumor progression through increased tumor cell ICD.

Development of OX40 agonists for canine cancer immunotherapy.

Recent breakthroughs in cancer immunotherapy have provided unprecedented clinical benefits to human cancer patients. Cancer is also one of the most common causes of death in pet dogs. Thus, canine-specific immune therapies targeting similar signaling pathways can provide better treatment options for canine cancer patients. Here, we describe the development and characterization of two canine-specific anti-OX40 agonists to activate OX40 signaling. We show that canine OX40, like human OX40, is not expressed on resting T cells, and its expression is markedly increased on canine CD4 T cells and Tregs after stimulation with concanavalin A (Con-A). cOX40 is also expressed on tumor-infiltrating lymphocytes (TILs) in canine osteosarcoma patients. The canine-specific OX40 agonists strongly activates cPBMCs by increasing IFN-γ expression and do not require Fc receptor-mediated cross-linking for OX40 agonism. Together, these results suggest that cFcOX40L proteins are potent OX40 agonists and have the potential to enhance antitumor immunity in canine cancer patients.

Optical Genome Mapping and Single Nucleotide Polymorphism Microarray: An Integrated Approach for Investigating Products of Conception.

Conventional cytogenetic analysis of products of conception (POC) is of limited utility because of failed cultures, as well as microbial and maternal cell contamination (MCC). Optical genome mapping (OGM) is an emerging technology that has the potential to replace conventional cytogenetic methods. The use of OGM precludes the requirement for culturing (and related microbial contamination). However, a high percentage of MCC impedes a definitive diagnosis, which can be addressed by an additional pre-analytical quality control step that includes histological assessment of H&E stained slides from formalin-fixed paraffin embedded (FFPE) tissue with macro-dissection for chorionic villi to enrich fetal tissue component for single nucleotide polymorphism microarray (SNPM) analysis. To improve the diagnostic yield, an integrated workflow was devised that included MCC characterization of POC tissue, followed by OGM for MCC-negative cases or SNPM with histological assessment for MCC-positive cases. A result was obtained in 93% (29/31) of cases with a diagnostic yield of 45.1% (14/31) with the proposed workflow, compared to 9.6% (3/31) and 6.4% (2/31) with routine workflow, respectively. The integrated workflow with these technologies demonstrates the clinical utility and higher diagnostic yield in evaluating POC specimens.

Nanostring-Based Identification of the Gene Expression Profile in Trigger Finger Samples.

Trigger finger is a common yet vastly understudied fibroproliferative hand pathology, severely affecting patients' quality of life. Consistent trauma due to inadequate positioning within the afflicted finger's tendon/pulley system leads to cellular dysregulation and eventual fibrosis. While the genetic characteristics of the fibrotic tissue in the trigger finger have been studied, the pathways that govern the initiation and propagation of fibrosis are still unknown. The complete gene expression profile of the trigger finger has never been explored. Our study has used the Nanostring nCounter gene expression assay to investigate the molecular signaling involved in trigger finger pathogenesis. We collected samples from patients undergoing trigger finger (n = 4) release surgery and compared the gene expression to carpal tunnel tissue (n = 4). Nanostring nCounter analysis identified 165 genes that were differentially regulated; 145 of these genes were upregulated, whereas 20 genes were downregulated. We found that several collagen genes were significantly upregulated, and a regulatory matrix metalloproteinase (MMP), MMP-3, was downregulated. Bioinformatic analysis revealed that several known signaling pathways were dysregulated, such as the TGF-ß1 and Wnt signaling pathways. We also found several novel signaling pathways (e.g., PI3K, MAPK, JAK-STAT, and Notch) differentially regulated in trigger finger. The outcome of our study helps in understanding the molecular signaling pathway involved in the pathogenesis of the trigger finger.

IFN-λ Regulates Neutrophil Biology to Suppress Inflammation in Herpes Simplex Virus-1-Induced Corneal Immunopathology.

HSV-1 infection of the cornea causes a severe immunoinflammatory and vision-impairing condition called herpetic stromal keratitis (SK). The virus replication in corneal epithelium followed by neutrophil- and CD4+ T cell-mediated inflammation plays a dominant role in SK. Although previous studies demonstrate critical functions of type I IFNs (IFN-α/ß) in HSV-1 infection, the role of recently discovered IFN-λ (type III IFN), specifically at the corneal mucosa, is poorly defined. Our study using a mouse model of SK pathogenesis shows that HSV-1 infection induces a robust IFN-λ response compared with type I IFN production at the corneal mucosal surface. However, the normal progression of SK indicates that the endogenous IFN responses are insufficient to suppress HSV-1-induced corneal pathology. Therefore, we examined the therapeutic efficacy of exogenous rIFN-λ during SK progression. Our results show that rIFN-λ therapy suppressed inflammatory cell infiltration in the cornea and significantly reduced the SK pathologic condition. Early rIFN-λ treatment significantly reduced neutrophil and macrophage infiltration, and IL-6, IL-1ß, and CXCL-1 production in the cornea. Notably, the virucidal capacity of neutrophils and macrophages measured by reactive oxygen species generation was not affected. Similarly, ex vivo rIFN-λ treatment of HSV-1-stimulated bone marrow-derived neutrophils significantly promoted IFN-stimulated genes without affecting reactive oxygen species production. Collectively, our data demonstrate that exogenous topical rIFN-λ treatment during the development and progression of SK could represent a novel therapeutic approach to control HSV-1-induced inflammation and associated vision impairment.

Differentiation of bovine herpesvirus1 subtypes based on UL0.5 gene sequencing.

Infectious bovine rhinotracheitis/infectious pustular vulvovaginitis is one of the high economic importance diseases of cattle and caused by bovine herpesvirus1 (BoHV1). Based on the restriction endonuclease fingerprinting of viral DNA, the BoHV1 can be divided into three subtypes viz., BoHV1.1, 1.2a, and 1.2b. Since this method requires a pure viral DNA, it is time-consuming and labour intense. In the current study, the UL0.5 gene based PCR sequencing has been used for the subtyping of BoHV1. Out of five isolates, four had BoHV1-like signatures and one isolate had BoHV1.2-like signatures. Further, these viruses phylogenetically clustered under the respective subtypes. These results indicate that the UL 0.5 gene based PCR sequencing could be used as an alternate method of subtyping of BoHV1.

Modulation of miRNAs by Vitamin C in Human Bone Marrow Stromal Cells.

MicroRNAs (miRNAs) are small (18-25 nucleotides), noncoding RNAs that have been identified as potential regulators of bone marrow stromal cell (BMSC) proliferation, differentiation, and musculoskeletal development. Vitamin C is known to play a vital role in such types of biological processes through various different mechanisms by altering mRNA expression. We hypothesized that vitamin C mediates these biological processes partially through miRNA regulation. We performed global miRNA expression analysis on human BMSCs following vitamin C treatment using microarrays containing human precursor and mature miRNA probes. Bioinformatics analyses were performed on differentially expressed miRNAs to identify novel target genes and signaling pathways. Our bioinformatics analysis suggested that the miRNAs may regulate multiple stem cell-specific signaling pathways such as cell adhesion molecules (CAMs), fatty acid biosynthesis and hormone signaling pathways. Furthermore, our analysis predicted novel stem cell proliferation and differentiation gene targets. The findings of the present study demonstrate that vitamin C can have positive effects on BMSCs in part by regulating miRNA expression.

A novel immunohistochemical score to predict early mortality in acute myeloid leukemia patients based on indoleamine 2,3 dioxygenase expression.

Indoleamine 2,3 dioxygenase-1 (IDO-1) is an enzyme in the kynurenine pathway which augments tumor-induced immune tolerance. Previous studies in childhood acute myeloid leukemia (AML) have shown a negative correlation of IDO-1 mRNA expression with outcomes. The aim of our study was to develop a practical and objective immunohistochemical technique to quantify IDO-1 expression on diagnostic bone marrow biopsies of AML patients in order to facilitate its use in routine clinical practice. IDO-1 mRNA was extracted from diagnostic bone marrow specimens from 29 AML patients. IDO-1 protein expression was assessed in 40 cases via immunohistochemistry and quantified by a novel 'composite IDO-1 score'. In a univariate analysis, higher age (p = 0.0018), male gender (p = 0.019), high risk cytogenetics (p = 0.002), higher IDO-1 mRNA (p = 0.005), higher composite IDO-1 score (p < 0.0001) and not undergoing allogeneic stem cell transplant (SCT, p = 0.0005) predicted poor overall survival. In a multivariate model that included the aforementioned variables, higher composite IDO-1 score (p = 0.007) and not undergoing allogeneic SCT (p = 0.007) was found to significantly predict poor outcomes. Further, patients who failed induction had higher composite IDO-1 score (p = 0.01). In conclusion, 'composite IDO-1 score' is a prognostic tool that can help identify a certain subset of AML patients with 'early mortality'. This unique subset of patients can potentially benefit from specific IDO-1 inhibitor therapy, currently in clinical trials.

Gender-specific differential expression of exosomal miRNA in synovial fluid of patients with osteoarthritis.

The pathogenesis of osteoarthritis (OA) is poorly understood, and therapeutic approaches are limited to preventing progression of the disease. Recent studies have shown that exosomes play a vital role in cell-to-cell communication, and pathogenesis of many age-related diseases. Molecular profiling of synovial fluid derived exosomal miRNAs may increase our understanding of OA progression and may lead to the discovery of novel biomarkers and therapeutic targets. In this article we report the first characterization of exosomes miRNAs from human synovial fluid. The synovial fluid exosomes share similar characteristics (size, surface marker, miRNA content) with previously described exosomes in other body fluids. MiRNA microarray analysis showed OA specific exosomal miRNA of male and female OA. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified gender-specific target genes/signaling pathways. These pathway analyses showed that female OA specific miRNAs are estrogen responsive and target TLR (toll-like receptor) signaling pathways. Furthermore, articular chondrocytes treated with OA derived extracellular vesicles had decreased expression of anabolic genes and elevated expression of catabolic and inflammatory genes. In conclusion, synovial fluid exosomal miRNA content is altered in patients with OA and these changes are gender specific.