Figure legends for Supplementary Figures 1-7.

Figure S1. Aspirin and Aspirin-PC does not block the release of TGF from platelets following co-culture with colon cancer cells. Figure S2. Aspirin and Aspirin-PC block platelet induced upregulation of the EMT marker SNAIL in MC-26 cells. Figure S3. Aspirin and Aspirin-PC decrease collagen-induced activation of platelets obtained from the AOM/DSS cancer model. Figure S4. Time course of aspirin (ASA) test drugs on body weight in AOM/DSS mouse colon cancer model. Figure S5. Aspirin and Aspirin-PC do not alter changes in body weight induced by AOM/DSS. Figure S6. Effect of aspirin (ASA) test drugs on fecal hemoglobin in AOM/DSS mouse cancer model. Figure S7. Effect of aspirin (ASA) test drugs on hematocrit in AOM/DSS mouse cancer model.

Objective: We have previously shown changes in protein, immune cell, nitric oxide a neurotrophic factor content and distribution in the olfactory bulb of an endotoxin-treated rat model, as all these factors have been shown to be involved in neurodegeneration. We expanded our studies by fluorescently imaging smooth muscle actin and endothelial nitric oxide synthase (eNOS), both markers of blood vessels, to investigate loss of vasculature content, as well as imaging locations and quantities of immune cells. The work was performed to shed further light on associations between vessel integrity and immune cell initiated endothelial disruption. Our goal was to demonstrate that cytokine production, NOS induction and immune cell increases, are likely part of the process that leads to a loss of olfaction and dopaminergic signaling and includes vascular perturbations. Methods: Rats were sacrificed following lipopolysaccharide (LPS) treatment. Olfactory bulbs were harvested, sectioned from top to bottom to include the tract and sensory neurons, and probed for markers of inflammation. Inducible nitric oxide synthase (iNOS), neuronal nitric oxide synthase (nNOS), eNOS, interleukin-1 beta (IL-1β), TNF-α, interleukin-6, glial cell derived neurotrophic factor (GDNF) and circulating nitric oxide (NO) were imaged together with tagged macrophages, T-cells, B-cells and neutrophils. Results: Serum NO levels indicated that an inflammatory episode had occurred, being significantly higher in treated animals, with tissue levels of NOS elevated for an extended period of time. Immune cell clusters were seen in a number of areas and the localization of NOS isomers suggests that they have divergent roles in neurodegeneration. For instance, eNOS was associated with blood vessels, iNOS with glial and matrix cells and nNOS with glial cells and neurons. T and B-cell numbers showed a sustained increase; neutrophil numbers rapidly increased then returned to baseline levels; macrophage numbers increased and remained high; LAMP positive cell numbers (NK-cells) increased and remained high; GDNF content increased; IL-6, TNF-α and IL-1β levels all rapidly increased, before dropping to untreated levels, while circulating, NO levels increased dramatically. Of interest, the images of vascular content, immune cell content, eNOS and smooth muscle actin, allowed us to show detrimental interactions between cells, factors and vessels. Our data show that the majority of the vessels were intact, though sections of interest were ‘extracted’ to reveal possible leaky areas. Specific sites of IL-6 positive lymphocyte clustering were noted around vessels, suggesting that interactions are occurring that lead to disruptions of blood vessel tunicae, allowing the internalization of circulating cells and subsequent cytokine-initiated endothelial cell death. Conclusion: Our findings suggest that protective GDNF and eNOS, which maintains vascular tone, are possibly synthesized too late to combat cytokine initiated neuron damage, glial activation and chronic loss of vascular integrity.

BackgroundThere is a need for drug treatment for 2nd degree burn injury that can be applied quickly to both relieve pain and promote healing. Currently used drugs for chronic pain have substantial side‐effects which limit their usefulness. These include opioids and their addictive potential, gabapentin and its sedation, and coxibs with their risk of cardiovascular failure. Nonsteroidal anti‐inflammatory drugs (NSAIDs) are potent analgesics and provide relief from burn pain, but their use is limited due to side effects related to gastrointestinal (GI) bleeding insusceptible patients.Objective/AimThe goal of this project is to develop rodent model systems to evaluate the analgesic efficacy and side‐effects of a novel treatment for 2nd degree burn injury to alleviate pain and promote healing using a new class of NSAIDs that are complexed with the soy phosphatidylcholine (PC) that can be administered as a sterile topical alone, parenterally or in combination.MethodsEmploying two established methods, for the induction of 2nd degree burn injury in rats to either the dorsal skin or the hindlimb we investigated the effects of the PC‐NSAIDs, Ibuprofen‐PC and Indomethacin‐PC vs the unmodified NSAIDs administered (either subcutaneously (sc) or topically to the affected skin) daily over a 10‐day study at a dose of 20 mg/kg and 2mg/kg respectively. We then assessed pain sensation behaviorally, the depth of skin injury by light and fluorescent microscopy and tissue inflammation biochemically and by non‐invasive IVIS imaging.Results & ConclusionBoth Indomethacin‐PC and Ibuprofen‐PC when sc administered provided moderate hyperalgesia at 3‐ and 5‐days post‐burn injury, which was similar to the unmodified NSAIDs and in some cases more efficacious as analgesic/anti‐inflammatory agents. Topical administration of our test PC‐NSAIDs provided more variable responses using the two rodent model systems, with the most consistent analgesic/anti‐inflammatory effect being recorded with Indomethacin‐PC. Morphological analysis revealed that the depth of skin injury was reduced in rats treated with the PC‐NSAIDs with no evidence of GI bleeding. Supported by a USARMC grant, W81XWH‐14‐2‐0016.Support or Funding InformationSupported by a USARMC grant, W81XWH‐14‐2‐0016.

Abstract The mechanism by which aspirin consumption is linked to significant reductions in the incidence of multiple forms of cancer and metastatic spread to distant tissues, resulting in increased cancer patient survival is not well understood. In this study, using colon cancer as an example, we provide both in vitro (cell culture) and in vivo (chemically induced mouse model of colon cancer) evidence that this profound antineoplastic action may be associated with aspirin's ability to irreversibly inhibit COX-1–mediated platelet activation, thereby blocking platelet–cancer cell interactions, which promote cancer cell number and invasive potential. This process may be driven by platelet-induced epithelial–mesenchymal transition (EMT), as assessed using confocal microscopy, based upon changes in cell morphology, growth characteristics and fibronectin expression, and biochemical/molecular analysis by measuring changes in the expression of the EMT markers; vimentin, β-catenin, and SNAIL. We also provide evidence that a novel, gastrointestinal-safe phosphatidylcholine (PC)-associated aspirin, PL2200 Aspirin, possesses the same or more pronounced actions versus unmodified aspirin with regard to antiplatelet effects (in vitro: reducing platelet activation as determined by measuring the release of thromboxane and VEGF in culture medium; in vivo: inhibiting platelet number/activation and extravasation into tumor tissue) and chemoprevention (in vitro: inhibiting colonic cell growth and invasive activity; in vivo: inhibiting colonic dysplasia, inflammation, and tumor mass). These results suggest that aspirin's chemopreventive effects may be due, in part, to the drug blocking the proneoplastic action of platelets, and the potential use of Aspirin-PC/PL2200 as an effective and safer chemopreventive agent for colorectal cancer and possibly other cancers. Cancer Prev Res; 10(2); 142–52. ©2016 AACR.

Background:Specific factors in Parkinson’s disease have become targets as to their protective and degenerative effects. We have demonstrated that cytokines and PD-CSF detrimentally affect microglia and astrocyte growth. While glial cell-derived neurotrophic factor (GDNF) has been recognized as a possible neuron-rescue agent, nitric oxide synthase (NOS) has been implicated in neurodegenerative processes.Objective:To demonstrate that glial cell activation, cytokine production, and NOS induction, play an intimate role in the loss of dopaminergic signaling,viamechanisms that are a result of inflammation and inflammatory stimuli.Methods:Study animals were sacrificed following endotoxin treatment and tissue sections were harvested and probed for GDNF and NOS isomers by fluorescence deconvolution microscopy. Fluorescence was mapped and quantified for each probeResults:An immune cell influx into ‘vulnerable’ areas of the brain was seen, and three NOS isomers, inducible (iNOS), neuronal (nNOS) and endothelial (eNOS), were synthesized in the brains, a finding which suggests that each isomer has a role in neurodegeneration. eNOS was found associated with blood vessels, while iNOS was associated with glial and matrix cells and nNOS was located with both glia and neurons. Following endotoxin treatment, serum levels of nitric oxide were higher at 6-8 hours, while tissue levels of NOS were elevated for much longer. Thus, induction of NOS occurred earlier than the induction of GDNF.Conclusion:Our findings suggest that the protective abilities of GDNF to combat neural destruction are not available rapidly enough, and do not remain at sufficiently high levels long enough to assert its protective effects. (250).

To document the prevalence of risk factors for cardiovascular disease in an unstudied community, a survey of 130 participants from different areas of Sierra Leone was performed.The focus was on cardiovascular disease because its prevalence had not been studied and World Health Organization (WHO) reports on Non-Communicable Diseases (NCD) prevalence were extrapolations and not from actual data.Resting blood pressure, fasting blood glucose, fasting lipid levels and carotid intima media thickness (CIMT) were measured.Mean blood pressure in men was slightly lower than in women (132/87 mmHg and 139/90 mmHg respectively), mean fasting blood glucose levels were <100 mg/dL in both groups and mean fasting total cholesterol was higher in women than in men (206 mg versus 193.5 mg/dL).Mean fasting low density lipoprotein levels were similar in both groups, 129 mg/dL in men and 133 mg/dL in women.Mean fasting high density cholesterol was 47.2 mg/dL in men and 55.5 mg/dL in women and CIMT values were similar in both groups, with 60% above the 75 th percentile.This study demonstrated a prevalence of cardiovascular risk factors in this population with mean BP placing both men and women in pre-hypertension or stage 1 hypertension ranges and elevated mean low density lipoprotein levels.Fasting total cholesterol, blood glucose and high density lipoprotein were within normal ranges.Mean CIMT values for men and women placed them at risk for subclinical atherosclerosis.

A suitable therapy for combating Parkinson’s disease (PD) neurodegeneration has not been forthcoming and although treatments are available to slow progression, there is no cure and no medical repair procedures. Of great interest to neurologists and neurology researchers have been the new findings with neurotrophic factors such as glial-cell derived neurotrophic factor and brain-derived neurotrophic factor, the neurotrophins being shown to have the ability to slow neurodegeneration and, in certain instances, rescue neurons. Unfortunately, outcomes of treatments using these proteins have been disappointing or inconclusive at best, and this includes direct infusion into areas of the brain. There is a consensus that once this disease starts there is little chance of halting its progress. One of the first indications that PD is a future possibility in a patient is a loss of olfaction. We have taken this as a starting point, maybe the earliest indication that neurotrophin treatment should be initiated with the possibility that if this isn’t a cure, it is a potent retardant of inevitable neuron loss and attenuating at least some of the many associated problems. Our work showed in cell cultures and an endotoxin animal model that synthesis of GDNF lagged behind inflammatory cell invasion, release of cytokines, protein folding problems and microglial activation. Our hypothesis is, that early interventions with neurotrophins might delay devastating neuronal loss and overcome some of the inflammatory effects, and that this treatment could begin at the time of loss of olfaction, together with prescribing anti-inflammatories. Couple this with blocking nitric oxide synthase increases and a reasonably early starting point for treatment might well be found. Journal of Nature and Science (JNSCI), 2(11):e249, 2016

Inflammation plays a significant role in dopaminergic neuron damage associated with the development and progression of Parkinson’s disease (PD). Microglia within the central nervous system play are known to be activated during the inflammatory process and activated microglia can induce pleiotropic downstream sequellae. Activated microglia can serve a neuronal protective role, including their synthesis of glial cell line‐derived neurotrophic factor (GDNF). However, chronic or uncontrolled activation of microglia can lead to the production of pro‐inflammatory cytokines and other neurotoxic factors. A rat model of inflammation involving exposure to lipopolysaccharide (LPS) demonstrates many of the patho‐physiologic markers of PD. Furthermore our data has shown that there is a close temporal relationship between GDNF appearance and freely diffusible nitric oxide (NO) production. In the cardiovascular system an inflammatory response leads to a multiphasic production of NO and expression of different isoforms of nitric oxide synthase (NOS) which in turn activate protective pathways. We hypothesized that in the CNS, NO production can also regulate the activation of protective pathways. Our studies employed LPS exposure in both cell cultures and animal models. Cells and tissues were probed for the presence of GDNF and inducible, neuronal and endothelial nitric oxide synthase (iNOS, nNOS, eNOS), by fluorescence deconvolution microscopy in a time dependent manner after exposure to LPS. Both microglial cells and astrocytes in culture synthesized GDNF and nitric oxide synthase isoforms following LPS treatment. A similar time dependent production of these markers was observed in our animal model. Furthermore, we observed these differential changes were not generalized within the brain sections but occurred in specific areas.

This study examined inflammatory cell and cytokine production in brain tissue from a lipopolysaccharide (LPS) treated rat model that mimics many of the neuropathologic changes associated with neurodegenerative diseases. We monitored the appearance of glial cell line‐derived neurotrophic factor (GDNF) and circulating nitric oxide (NO) levels, as well as immune system associated cells in a selected area of the brain, the olfactory lobe. Our hypothesis was that LPS treatment stimulates temporal changes within the brain and these responses included immune cell recruitment, increased tissue levels of immune modulating cytokines and NO, as well as greater glial cell activation resulting in increased production of GDNF. Our animal model of systemic LPS treatment lead to an increase in the concentrations of circulating cytokines including TNF‐α, IL‐Iβ and IL‐6, with a maximum response 6 hours post LPS. Concomitant with cytokine elevations, circulating NO levels were elevated for several hours post LPS. Brain content of GDNF was also elevated over a similar time frame. Lymphocytes, neutrophils, macrophages, plasma cells and cytokines were all seen in various areas of LPS treated brains. Our results suggest an involvement of both the peripheral and the central nervous system immune components in response to inflammation and inflammatory episodes.

Purpose . To provide an ovine model of ventricular remodeling and reverse remodeling by creating congestive heart failure (CHF) and then treating it by implanting a left ventricular assist device (LVAD). Methods . We induced volume-overload heart failure in 2 sheep; 20 weeks later, we implanted an LVAD and assessed recovery 11 weeks thereafter. We examined changes in histologic and hemodynamic data and levels of cellular markers of CHF. Results . After CHF induction, we found increases in LV end-diastolic pressure, LV systolic and diastolic dimensions, wall thickness, left atrial diameter, and atrial natriuretic protein (ANP) and endothelin-1 (ET-1) levels; β-adrenergic receptor (BAR) and dystrophin expression decreased markedly. Biopsies confirmed LV remodeling. After LVAD support, LV systolic and diastolic dimensions, wall thickness, and mass, and ANP and ET-1 levels decreased. Histopathologic and hemodynamic markers improved, and BAR and dystrophin expression normalized. Conclusions . We describe a successful sheep model for ventricular and reverse remodeling.

OBJECTIVE:To determine alterations in quantities and distributions of natural antimicrobials following ischemia-reperfusion injury. We hypothesized that these compounds would be upregulated in areas of small intestine where changes in permeability and cellular disruption were likely and where protective mechanisms would be initiated.METHODS:Rats with ischemia-reperfusion underwent superior mesenteric artery clamping and reperfusion. Shams were subjected to laparotomy but no clamping. Ileum and jejunum were harvested and sectioned, and subjected to fluorescence deconvolution microscopy for determinations of content and localization of rat beta defensins, 1, 2, 3; rat neutrophil protein-1; and cathelicidin LL-37. Modeling was performed to determine cellular location of antimicrobials.RESULTS:Ischemia-reperfusion increased neutrophil defensin alpha (RNP-1) in jejunum; rat beta defensin 1 was increased 2-fold in ileal mucosa and slightly reduced in jejunal mucosa; rat beta defensin 2 was reduced by ischemia-reperfusion in ileum, but slightly increased in jejunum; rat beta defensin 3 was concentrated in the muscularis externa and myenteric plexus of the jejunum; ischemia-reperfusion did not alter cathelicidin LL-37 content in the small intestine, although a greater concentration was seen in jejunum compared with ileum.CONCLUSION:Ischemia-reperfusion injury caused changes in antimicrobial content in defined areas, and these different regulations might reflect the specific roles of jejunum versus ileum.

Background Excessive and abnormal accumulation of alpha-synuclein (α-synuclein) is a factor contributing to pathogenic cell death in Parkinson's disease. The purpose of this study, based on earlier observations of Parkinson's disease cerebrospinal fluid (PD-CSF) initiated cell death, was to determine the effects of CSF from PD patients on the functionally different microglia and astrocyte glial cell lines. Microglia cells from human glioblastoma and astrocytes from fetal brain tissue were cultured, grown to confluence, treated with fixed concentrations of PD-CSF, non-PD disease control CSF, or control no-CSF medium, then photographed and fluorescently probed for α-synuclein content by deconvolution fluorescence microscopy. Outcome measures included manually counted cell growth patterns from day 1-8; α-synuclein density and distribution by antibody tagged 3D model stacked deconvoluted fluorescent imaging. Results After PD-CSF treatment, microglia growth was reduced extensively, and a non-confluent pattern with morphological changes developed, that was not evident in disease control CSF and no-CSF treated cultures. Astrocyte growth rates were similarly reduced by exposure to PD-CSF, but morphological changes were not consistently noted. PD-CSF treated microglia showed a significant increase in α-synuclein content by day 4 compared to other treatments (p ≤ 0.02). In microglia only, α-synuclein aggregated and redistributed to peri-nuclear locations. Conclusions Cultured microglia and astrocytes are differentially affected by PD-CSF exposure compared to non-PD-CSF controls. PD-CSF dramatically impacts microglia cell growth, morphology, and α-synuclein deposition compared to astrocytes, supporting the hypothesis of cell specific susceptibility to PD-CSF toxicity.

We have recently shown that ventricular unloading with an implantable left ventricular assist device (LVAD) leads to improved calcium handling and membrane integrity and redistribution of alpha adrenoreceptors (AARs) and betaadrenoreceptors (BARs).Here, we used fluorescence deconvolution microscopy to examine the effect of LVAD type (pulsatile vs non-pulsatile) on upregulation and redistribution of adrenoreceptors in core biopsy samples of the myocardium before and after the removal LVAD.We noted no major differences between the pulsatile and non-pulsatile groups; however, an individual patient's 'recovery' in adrenoreceptor numbers depended on the pre-LVAD number of receptors.These findings suggest that ventricular unloading is beneficial regardless of LVAD type; however, the degree of repair and recovery may correlate with the patient's level of ventricular dysfunction at implant and the pre-LVAD number of adrenoreceptors (130).

Objective: The purpose of this study was to quantify volumes of specific subcortical gray matter nuclei implicated in Parkinson's disease (PD) as a preliminary step for identifying a non-invasive clinical biomarker for PD. We hypothesized that REM sleep behavior disorder (RBD) patients, at risk for developing PD, will demonstrate a pattern of neuronal degeneration reflected in reduced striatal volumes on T1-weighted MRI.Methods: We compared measures of RBD patients confirmed by polysomnography (PSG) with groups of age/gender-matched Control subjects and early PD (EPD) patients (Hoehn & Yahr < 2). Clinical measurements included the Unified Parkinson's disease Rating Scales (UPDRS), timed gait and finger tapping tasks, the Parkinson's Disease Questionnaire (PDQ-39), and a time-synchronized video recorded single-night PSG. Volumetric measurements were derived from high-resolution T1-weighted 3 T MRI images.Results: The matched Control and EPD groups were statistically similar to the RBD group in age, gender, handedness, and total brain volumes. The RBD group had smaller bilateral putamen volumes (both raw and normalized by brain tissue volume), in addition to some clinical impairment on the UPDRS and PDQ-39.Conclusions: Reduced putamen volumes may be a structural marker for RBD and reflect a pattern of neurodegeneration that predicts the development of PD. (C) 2010 Elsevier Ltd. All rights reserved,