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Radiation induced intestinal damage histology and collagen deposition information.

The effect of ascorbate and catalase on clonogenic cell survival in PDAC.

RNAi targeting sequences, Supplemental Figure Legends, & Supplemental Figures S1-S11

Existing anticancer strategies focused on disrupting integrin functions in tumor cells or tumor-involved endothelial cells have met limited success. An alternative strategy is to augment integrin-mediated pathways that suppress tumor progression, but how integrins can signal to restrain malignant behavior remains unclear. To address this issue, we generated an in vivo model of prostate cancer metastasis via depletion of α3β1 integrin, a correlation observed in a significant proportion of prostate cancers. Our data describe a mechanism whereby α3β1 signals through Abl family kinases to restrain Rho GTPase activity, support Hippo pathway suppressor functions, and restrain prostate cancer migration, invasion, and anchorage-independent growth. This α3β1-Abl kinase-Hippo suppressor pathway identified α3 integrin–deficient prostate cancers as potential candidates for Hippo-targeted therapies currently under development, suggesting new strategies for targeting metastatic prostate cancer based on integrin expression. Our data also revealed paradoxical tumor suppressor functions for Abl kinases in prostate cancer that may help to explain the failure of Abl kinase inhibitor imatinib in prostate cancer clinical trials. Cancer Res; 76(22); 6577–87. ©2016 AACR.

Chemoradiation therapy is the mainstay for treatment of locally advanced, borderline resectable pancreatic cancer. Pharmacologic ascorbate (P-AscH−, i.e., intravenous infusions of ascorbic acid, vitamin C), but not oral ascorbate, produces high plasma concentrations capable of selective cytotoxicity to tumor cells. In doses achievable in humans, P-AscH− decreases the viability and proliferative capacity of pancreatic cancer via a hydrogen peroxide (H2O2)-mediated mechanism. In this study, we demonstrate that P-AscH− radiosensitizes pancreatic cancer cells but inhibits radiation-induced damage to normal cells. Specifically, radiation-induced decreases in clonogenic survival and double-stranded DNA breaks in tumor cells, but not in normal cells, were enhanced by P-AscH−, while radiation-induced intestinal damage, collagen deposition, and oxidative stress were also reduced with P-AscH− in normal tissue. We also report on our first-in-human phase I trial that infused P-AscH− during the radiotherapy “beam on.” Specifically, treatment with P-AscH− increased median overall survival compared with our institutional average (21.7 vs. 12.7 months, P = 0.08) and the E4201 trial (21.7 vs. 11.1 months). Progression-free survival in P-AscH−–treated subjects was also greater than our institutional average (13.7 vs. 4.6 months, P < 0.05) and the E4201 trial (6.0 months). Results indicated that P-AscH− in combination with gemcitabine and radiotherapy for locally advanced pancreatic adenocarcinoma is safe and well tolerated with suggestions of efficacy. Because of the potential effect size and minimal toxicity, our findings suggest that investigation of P-AscH− efficacy is warranted in a phase II clinical trial.Significance:These findings demonstrate that pharmacologic ascorbate enhances pancreatic tumor cell radiation cytotoxicity in addition to offering potential protection from radiation damage in normal surrounding tissue, making it an optimal agent for improving treatment of locally advanced pancreatic adenocarcinoma.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system, and among all the genes associated with MS, HLA class II genes have the highest relative risk. Although IL-17A is thought to be a major cytokine in the pathogenesis of MS, the exact role of IL-17A in the MS is still poorly understood. Specifically, it is unclear whether IL-17A is required for disease initiation or disease severity or in both. Therefore, in the present study, utilizing HLA-DR3 transgenic mice lacking IL-17A (HLA-DR3.IL-17A−/−), we investigated the importance of IL-17A in disease development and progression in experimental autoimmune encephalomnyelitis (EAE) an animal model of MS. We observed that HLA-DR3.IL-17A−/− mice had a higher frequency of CD4+CD25+FoxP3+ regulatory T cells (Treg)s and developed milder EAE compared to IL-17 sufficient HLA-DR3 mice. Further characterization of mechanism identified a novel role of IL-17A in regulating the disease progression through modulation of immunoregulatory responses. We also identified an important role of gut microbiota in IL-17A dependent disease regulation specifically depletion of gut bacteria with ability to promote Treg population in IL-17A sufficient HLA-DR3 mice. Fecal transplantation of HLA.DR3 mice with HLA-DR3. IL17A−/− showed milder EAE and an increase in Treg cells in HLA-DR3 mice confirming a role of gut microbiota in shaping Treg repertoire and function. Additionally, we observed higher frequency of metabolic pathway involved with Treg promoting short chain fatty acid (SCFA) metabolism in HLA-DR3.IL17A−/− mice. Thus our study shows a novel role of IL-17A in immune homeostasis and inflammation by regulating levels of Tregs through modulation of gut microbiota.

Interleukin-1 alpha (IL-1α) is a pleiotropic cytokine involved in inflammation and immune response and is upregulated in many solid tumors including head and neck squamous cell carcinomas (HNSCCs). Although IL-1α expression is generally associated with poor prognosis, the implications of the subcellular localization of IL-1α expression in patient outcomes are poorly understood. This study is aimed at investigating the clinical relevance of immunohistochemical IL-1α expression in HNSCCs. Tissue microarrays (TMAs) containing human papillomavirus (HPV)-positive and HPV-negative HNSCCs were analyzed for IL-1α and epidermal growth factor receptor (EGFR) expression by immunohistochemistry. Nuclear and cytoplasmic IL-1α and EGFR expression scores were correlated with clinicopathological parameters and patient outcomes. IL-1α expression was observed in the nuclear and/or cytoplasm compartments in 98% of evaluable tumors and 78% of tumors expressed IL-1α in both compartments. A higher percentage of tumors with combined high nuclear and moderate cytoplasmic IL-1α expression were observed in HPV-negative tumors compared to HPV-positive tumors. In HPV-negative tumors a combined EGFR-negative and high nuclear IL-1α expression profile was associated with a low risk of tumor recurrence and favorable overall survival compared to all other EGFR/IL-1α expression profiles. Lastly, a higher frequency of tumors with combined high nuclear and moderate cytoplasmic IL-1α expression was observed in cetuximab monotherapy responders compared to non-responders. Altogether, IL-1α in combination with EGFR expression may be a strong indicator of risk of recurrence in HPV-negative HNSCCs and warrants further study as a tissue biomarker for predicting HNSCC patient outcomes. Citation Format: Andrean L. Simons, Anand Rajan, Katherine Gibson-Corley, Georgina Ofori-Amanfo, Patrick Ten Eyck, Madelyn Espinosa-Cotton, Sandra Schmitz, Joseph Coppock, Steven Sperry. Impact of interleukin-1 alpha and EGFR expression on recurrence and survival outcomes in head and neck squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3150.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system. We and others have shown that gut microbiota play an important role in disease pathogenesis of MS as there is an enrichment or depletion of specific gut bacteria compared to healthy controls (HC). Thus the specific human gut bacteria found depleted or showing lower abundance in MS patients can be used as potential drug to treat MS. Further, we and others have shown that patients with MS have either lower abundance of Prevotella compared to HC or an increased level of Prevotella in patients receiving disease modifying therapies such as Copaxone and/or Interferon beta (IFN-β). We have previously identified a specific strain Prevotella histicola within Prevotella genus which can suppress disease in experimental autoimmune encephalomyelitis (EAE) animal model of MS. In this present study we compared disease suppressing ability of P. histicola to Copaxone or IFN-β in HLA-DR3.DQ8 transgenic mouse model of MS. We observed that P. histicola suppresses EAE as efficiently as Copaxone and IFN-β. P. histicola treated mice showed higher level of CD4+FoxP3+ regulatory T cell frequencies and decreased levels of pro-inflammatory cytokines IFNγ and IL17 suggesting that P. histicola suppresses disease through immunomodulation. Fecal microbiota analysis of different groups showed that group receiving Prevotella histicola had a distinct gut microbiota than the control group suggesting that P. histicola as might suppresses disease through modulation of gut microbiota. In conclusion, our study suggests that human gut commensal Prevotella histicola can provide an alternate treatment option for MS patients.

Our recent multiple sclerosis (MS) microbiome study indicates an important role of gut microbiota in pathogenesis of MS, an autoimmune disease of central nervous system. Gut microbiota help in maintaining immune-homeostasis by regulating balance between CD4+CD25+FoxP3+ regulatory T cells (Tregs) and pro-inflammatory Th1/Th17 cells. A shift of balance towards Th1/Th17 cells leading to inflammation and demyelination in central nervous system (CNS), is suggested to be responsible of disease initiation and/or relapses in MS. Therefore, gut commensals capable of restoring the microbiome to a healthy state could provide novel therapeutic options for treating autoimmune diseases including MS. Here, we report identification of human gut-derived commensal bacteria, Prevotella histicola (P. histicola), which can suppress an autoimmune disease in HLA class-II transgenic model of experimental autoimmune encephalomyelitis (EAE), an experimental model of MS. P. histicola suppresses disease through modulation of systemic immune response. P. histicola challenge led to a decrease in pro-inflammatory Th1 (IFNγ) and Th17 (IL17) cytokines, and increase in the frequencies of CD4+FoxP3+ Tregs, tolerogenic dendritic cells and suppressive macrophages. We also observed that mice with EAE had a altered gut microbiota compared to naïve mice and treatment with P. histicola restored gut flora to a normal state which further support an important role of gut microbiota in EAE/MS. Our study provides compelling evidence that administration of gut commensals may regulate a systemic immune response and have a possible role in the treatment of autoimmune diseases.

Recently we demonstrated that the late-pregnancy cardiovascular disorder, preeclampsia, is characterized by robust, early and sustained vasopressin (AVP) hypersecretion in all three trimesters of pregnancy in humans. We hypothesize a causative role for elevated AVP in the pathogenesis of preeclampsia. This concept was tested by chronically infusing AVP (0.24 - 240 ng/hr, s.c., or saline vehicle) in wildtype C57BL/6J female mice throughout pregnancy. In pregnant mice, AVP infusion caused pregnancy-specific increases in systolic blood pressure by tail-cuff plethysmography with significant increases in the pregnant cohort (saline n=16: 110±3, vs 24 ng/hr AVP n=11: 120±3 mmHg on GD15/16, P0.05). In addition, the AVP infused mice exhibited increased proteinuria (0.24 ng/hr n=2: 66±73, 2.4 ng/hr n=5: 348±56, 24 ng/hr n=2: 799±297 mg/d, Ppregnancy-specific physiological model of preeclampsia in mice. These data support our hypothesis that AVP hypersecretion during pregnancy may be causative for the development of preeclampsia. Ongoing experiments are aimed at identifying the causes of AVP hypersecretion in human preeclampsia, the target tissues and receptors involved, and the utility of targeting AVP signaling as a novel therapeutic for preeclampsia.

Lipophosphoglycans (LPG) of Leishmania spp. are known to alter innate immune responses and be critical for parasite binding to the vector gut wall. LPG displays various oligosaccharides capping the terminal and branch apexes. However, it is unknown how these capping oligosaccharides serve as immunomodulators in the absence of a conjugated protein. Several oligosaccharides including dimannose and trimannose, were synthesized and covalently linked to latex beads to study their effect in a Leishmania infection model. In vitro incubation of activated J774 or bone marrow derived macrophages with di- and tri-mannose resulted in differential modulation of IL-12p40, a key cytokine to control intracellular pathogens by driving a Th1 response. Co-inoculation of trimannose and L. major into the footpad of C57BL/6 mice increased IL-12p40 and IFN-γ production 48 hours post-infection, a significantly Th1 skewed response. Noticeably at 14 days post-infection, L. major infected, trimannose treated mice had significantly decreased lesion size and decreased parasite load compared to infected untreated, controls. Cytokines from draining lymph node supernatants measured via Luminex at 14 days post infection showed decreased IL-12p40 and IL-10 secretion and increased IFN-γ in the co-inoculated mice. In vitro, the effects of trimannose were mediated by macrophage TLR and mannose receptors as well as T cell proliferation. Leishmania-derived trimannose represents a novel immunomodulator that provides early Th1 skewed cytokine production to control parasite load and alter the course of infection.

The drawbacks of iliac crest autograft as graft material for spine fusion are well reported. Despite continued modifications to improve bone healing capacity, the efficacy of synthetic graft materials as stand-alone replacements remains uncertain. The rabbit posterolateral fusion model is an established environment for testing of fusion concepts. It offers the opportunity to obtain radiographic, biomechanical and histological data on novel fusion materials. The objective of this study was to compare the spine fusion capability of two synthetic bone graft products in an established rabbit posterolateral spine fusion (PLF) model: Signafuse® Bioactive Bone Graft Putty and Actifuse® ABX.Bilateral intertransverse spine fusion was performed at the L5-L6 transverse processes (TPs) of New Zealand White rabbits using either Signafuse or Actifuse ABX as the bone graft material. Bone remodeling and spine fusion were assessed at 6 and 12 weeks using radiographic, biomechanical and histological endpoints.Fusion rate by manual palpation at 6 weeks was greater for Signafuse (33%) compared to Actifuse ABX (0%), and equivalent in both groups at 12 weeks (50%). Biomechanical fusion rate based on flexion-extension data was 80% in Signafuse group and 44% for Actifuse ABX. Histology revealed a normal healing response in both groups. MicroCT and histomorphometric data at 6 weeks showed greater new bone formation in the Signafuse group compared to Actifuse ABX (p0.05), with no differences detected at 12 weeks. Histological fusion scores were greater in the Signafuse group at 6 and 12 weeks, indicated by higher degree structural remodeling and tendency towards complete bridging of the fusion bed compared to the Actifuse ABX group.Confirmed by several metrics, Signafuse outperformed Actifuse ABX as a standalone synthetic bone graft in an established PLF model, demonstrating greater rates of bone remodeling and spine fusion. The combination of 45S5 bioactive glass and biphasic HA/βTCP granules of Signafuse appear to provide greater bone healing capability in comparison to the 0.8% silicate-substituted hydroxyapatite material of Actifuse ABX.