Hr Postinjection Research Articles

Maternal immune activation affects precursor cell populations and developmental processes in the rodent spinal cord in a temporally and spatially dependent manner

Studies suggest that maternal immune activation (MIA) during pregnancy may be implicated in development of neurological disorders with timing of immune challenge and spatial differences playing an important role. However, further research is necessary in order to elucidate fully the CNS regions, cell populations and timing of gestational vulnerability. As the development of the spinal cord (SC) is well characterised, here we investigate how MIA affects cell populations and developmental processes in this CNS region.Time‐mated dams received an intraperitoneal injection of 100μg/kg lipopolysaccharide (LPS) or saline on embryonic day (E)12 or 16. Embryos were harvested 5hr post‐injection, or delivered and sacrificed at postnatal day (P)14. Offspring were fixed, frozen whole or following SC dissection and cryosectioned. In order to determine how MIA affects oligodendrocyte precursors, microglia, and reelin expression during development, sections were immunostained with Olig2, Iba‐1 and reelin. In addition total RNA was extracted from fresh frozen SCs and prepared for microarray, RNAseq and small RNAseq.At E12, numbers of Olig2+ and Iba‐1+ cells were unchanged 5hrs post‐LPS injection but Olig2+ cells were decreased in the ventral grey matter of the rostral SC at P14. The number of Iba‐1+ cells were unchanged at P14. At E16, Olig2+ and Iba‐1+ cells were decreased in grey and white matter 5 hrs post‐LPS, however by P14 there was no difference between the LPS and saline treated groups. Reelin expression following MIA at E12 appeared unchanged at 5hrs and P14 however, at E16, reelin expression was decreased 5hrs after MIA. Total cell numbers at E16, at both the 5hr and P14 survival times, were unchanged following LPS injection.These findings suggest MIA affects development in the SC in a temporally and spatially precise manner. At E12 MIA does not appear to affect olig2+ oligodendrocyte precursors acutely but may affect their survival to P14. In contrast, at E16, MIA affects Olig2+, Iba‐1+ and reelin expression acutely. Ongoing work is characterising postnatal reelin expression following LPS at E16, and the phenotype of Iba1+ cells to understand more fully the detailed cellular perturbations resulting from MIA.Support or Funding InformationAnatomical Society, Graduate Entry Medical School, Health Research Institute, University of LimerickThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Carbohydrate tolerance in the fruit-eating fish Piaractus mesopotamicus (Holmberg, 1887)

Some fish species have a limited ability to metabolize dietary carbohydrates. An important tool for understanding carbohydrate metabolism is the application of the glucose tolerance test, which can be performed orally or intraperitoneally. To evaluate carbohydrate tolerance in the fruit-eating fish pacu, two experiments were performed, one with oral administration by gavage of three carbohydrate types (glucose, fructose and starch, 2.0 g/kg body weight (BW)) and the other with intraperitoneal injection (IP) of glucose (500 mg/kg BW). Oral glucose resulted in an increase in plasma glucose 2 hr later with the peak at 4 hr (8.30 mmol/L), and return to baseline between 6 and 12 hr; starch administration promoted a peak after 4 hr (7.70 mmol/L), returning to the baseline at 6 hr. The administration of fructose promoted a moderate peak after 2 hr (5.71 mmol/L), and return to baseline for the time points that followed. Elevated serum cholesterol levels were observed 2 and 24 hr after administration of glucose and starch. Hepatic glycogen levels increased within 24 hr, regardless of the type of carbohydrate administered. IP glucose load resulted in a peak of plasma glucose 3 hr post injection (6.91 mmol/L), returning to baseline 6 hr later. There was a reduction in the concentration of triglycerides at 24 hr. The results demonstrate that pacu metabolize both oral (glucose or starch) and intraperitoneal (glucose) carbohydrate loads after 6 hr, suggesting good ability to deal with dietary carbohydrates.

The effect of a single injection of irinotecan on the development of enamel in the Wistar rats

Cancer is the second most frequent cause of death in children. Because the prognosis for childhood malignancies has improved, attention has now focused on long‐term consequences of cancer treatment. The immediate effects of chemotherapy on soft tissues have been well described; however, there is less information about long‐term effects of chemotherapy on the development of dental tissues. To test the association between the effect of chemotherapy on enamel development, we examined two groups of rats: one that had received an intraperitoneal dose of 200 mg/kg of irinotecan, whereas the other (control) group had received vehicle only. Rats were killed at 6, 48 and 96 hr post‐injection; the mandibles dissected out, fixed for histological evaluation and scanned for mineralization defects by Micro‐CT. Our results showed structural changes in the ameloblast layer along with a significant reduction in mineralization and thickness of enamel at 96 hr after chemotherapy. These data demonstrate that irinotecan induces structural changes in forming enamel that become apparent after anticancer chemotherapy treatment.

Local and systemic immune responses to different types of phytohemagglutinin in the green anole: Lessons for field ecoimmunologists

The phytohemagglutinin (PHA) skin test is commonly used by ecologists to assess cell-mediated immune function of wild animals. It can be performed quickly and easily in the field, involving injection of PHA and measurement of the resultant swelling. There are multiple formulations of PHA used in ecological studies, with potentially differing outcomes that could produce inconsistent results. We tested two common types of PHA in the green anole (Anolis carolinensis) to identify local and systemic immune responses underlying the resultant swelling at 6, 18, 24, and 48hr post injection. There were differences in both local (injection site) and systemic (blood) leukocyte responses to PHA-L versus PHA-P. PHA-P injection produced a greater overall increase in local heterophil count at the injection site compared with PHA-L, and this response was greatest at 6 and 24hr post injection. Systemically, heterophil percentage was higher in the blood of PHA-P- versus PHA-L-injected anoles at 24hr post injection; the time point at which heterophil percentage peaked in PHA-P-injected anoles. These results indicate that although both PHA types are effective tests of immune function in green anoles, the PHA-P swelling response invokes a much stronger heterophilic response. PHA-L is a more specific test of lymphocyte function, particularly at 24hr post injection, making it preferable for ecoimmunology studies.

Differential Change Patterns of Main Antimicrobial Peptide Genes During Infection of Entomopathogenic Nematodes and Their Symbiotic Bacteria.

The expression of antimicrobial peptides (AMPs) as the main humoral defense reactions of insects during infection by entomopathogenic nematodes (EPNs) and their symbiont is addressed herein. Three AMPs, attacin, cecropin, and spodoptericin, were evaluated in the fifth instar larvae of Spodoptera exigua Hübner (beet armyworm) when challenged with Steinernema carpocapsae or Heterorhabditis bacteriophora. The results indicated that attacin was expressed to a greater extent than either cecropin or spodoptericin. While spodoptericin was expressed to a much lesser extent, this AMP was induced against Gram-positive bacteria, and thus not expressed after penetration of Xenorhabdus nematophila and Photorhabdus luminescens. Attacin and cecropin in the larvae treated with S. carpocapsae at 8 hr post-injection (PI) attained the maximum expression levels and were 138.42-fold and 65.84-fold greater than those of larvae infected with H. bacteriophora, respectively. Generally, the ability of H. bacteriophora to suppress attacin, cecropin, and spodoptericin was greater than that of S. carpocapsae. According to the results, the expression of AMPs by Sp. exigua larvae against S. carpocapsae was determined in the 4 statuses of monoxenic nematode, axenic nematode, live symbiotic bacterium, and dead symbiotic bacterium. The expression of attacin in larvae treated with a monoxenic nematode and live bacterium at 8 and 2 hr PI, respectively, were increased to the maximum amount. Live X. nematophila was the strongest agent for the suppression of attacin. The expression of cecropin against monoxenic nematodes and live symbiotic bacteria at 8 and 4 hr PI, respectively, reached the maximum amount while the expression levels of attacin and cecropin for axenic nematodes were lesser and stable. The results highlighted that the ability of P. luminescens in AMPs suppression was much more than X. nematophila. The results also showed that the effect of symbiotic bacterium in suppressing attacin and cecropin expression was greater than that of a monoxenic nematode; this result provided deep insight into the expression pattern parallels and fluctuations of the main AMPs during nematode infection.

Structural and functional changes to lymph nodes in ageing mice.

SummaryLymph nodes (LN) are secondary lymphoid organs spread throughout the lymphatic system. They function to filter pathogenic material from the lymphatic fluid to maintain the health of the organism. Subcapsular sinus macrophages (SCSM) are among the first‐responders within the LN due to their strategic location within the subcapsular sinus region. These macrophages aid the delivery of immune complexes to B cells and follicular dendritic cells (FDC) within the LN. Here we show an increase in SCSM and other macrophage populations within aged LN. However, immune complex uptake by macrophages within LN was not altered with age, nor was immune complex uptake by B cells. LN stromal cell populations, important in immune responses and the localization and survival of leucocytes, were altered in their representation and distribution in aged LN. In particular, FDC regions were decreased in size and had decreased chemokine CXCL13 expression. Furthermore, the retention of immune complexes by FDC was decreased in aged LN at 24 hr post‐injection. As FDC are important in the maintenance of germinal centre responses, the decreased retention of immune complex in aged LN may contribute to the reduced germinal centre responses observed in aged mice.

Neurotransmitter-Regulated Regeneration in the Zebrafish Retina.

SummaryCurrent efforts to repair damaged or diseased mammalian retinas are inefficient and largely incapable of fully restoring vision. Conversely, the zebrafish retina is capable of spontaneous regeneration upon damage using Müller glia (MG)-derived progenitors. Understanding how zebrafish MG initiate regeneration may help develop new treatments that prompt mammalian retinas to regenerate. We show that inhibition of γ-aminobutyric acid (GABA) signaling facilitates initiation of MG proliferation. GABA levels decrease following damage, and MG are positioned to detect decreased ambient levels and undergo dedifferentiation. Using pharmacological and genetic approaches, we demonstrate that GABAA receptor inhibition stimulates regeneration in undamaged retinas while activation inhibits regeneration in damaged retinas.

Amelioration of Immune Thrombocytopenia in Human CD44 Transgenic Mice By Anti-Human CD44 Antibodies

Selected monoclonal antibodies to murine CD44 are able to successfully ameliorate murine immune thrombocytopenia (ITP) and other inflammatory diseases at a 3 log fold lower dose than IVIg. Since not all murine CD44 antibodies were successful and, like IVIg, the anti-inflammatory mechanism involved is speculative, progress toward human antibodies has been stymied. To potentially develop a therapeutic antibody for patients, we generated transgenic mice expressing the extracellular and transmembrane sequences of human CD44 linked to the murine intracellular CD44 sequence (huCD44) with knockout of murine CD44. Flow cytometric analysis revealed that these mice express huCD44 on leukocytes, but not on platelets or red blood cells.HuCD44 mice injected with the anti-platelet antibody MWReg30 develop significant thrombocytopenia comparable to normal mice. Utilizing the huCD44 mouse model, we tested 5 monoclonal antibodies reactive with human CD44 of different isotypes; mouse IgG1, mouse IgG2a, mouse IgG2b, rat IgG2a, and rat IgG2b. We report here that pretreatment of mice with all of these antibodies, with the exception of the mouse IgG2b clone all ameliorated thrombocytopenia to the same extent as IVIg, but given at a 3 log-fold lower dosage than IVIg. Interestingly, the mouse IgG2b antibody that was not successful bound to white blood cells as well as the successful antibodies. When mice were re-injected with anti-platelet antibody 24 hr post anti-huCD44 antibody injection, they were still protected from thrombocytopenia at 48 hr, suggesting that these antibodies exhibit durable protection.The clinical effectiveness of these antibodies does not appear to be related to their extent of huCD44 binding or IgG subtype. In addition, the effectiveness of these antibodies may not be restricted or related to specific activating IgG Fc receptors (FcγR) as the successful mouse IgG1 subtype is only known to interact with FcγRIII, while the IgG2b subtype can bind activating FcγRI, III and IV with only one-of-two of this subtype having therapeutic activity.In conclusion we have developed, to our knowledge, the first huCD44 transgenic mouse and demonstrate herein that selected antibodies to huCD44 can ameliorate immune thrombocytopenia. These data suggest that some anti-CD44 antibodies may offer a new therapeutic modality for patients with ITP. DisclosuresNo relevant conflicts of interest to declare.

The role of necrosis, acute hypoxia and chronic hypoxia in 18F-FMISO PET image contrast: a computational modelling study

Positron emission tomography (PET) using 18F-fluoromisonidazole (FMISO) is a promising technique for imaging tumour hypoxia, and a potential target for radiotherapy dose-painting. However, the relationship between FMISO uptake and oxygen partial pressure () is yet to be quantified fully. Tissue oxygenation varies over distances much smaller than clinical PET resolution (<100 μm versus ∼4 mm), and cyclic variations in tumour perfusion have been observed on timescales shorter than typical FMISO PET studies (∼20 min versus a few hours). Furthermore, tracer uptake may be decreased in voxels containing some degree of necrosis.This work develops a computational model of FMISO uptake in millimetre-scale tumour regions. Coupled partial differential equations govern the evolution of oxygen and FMISO distributions, and a dynamic vascular source map represents temporal variations in perfusion. Local FMISO binding capacity is modulated by the necrotic fraction. Outputs include spatiotemporal maps of and tracer accumulation, enabling calculation of tissue-to-blood ratios (TBRs) and time-activity curves (TACs) as a function of mean tissue oxygenation.The model is characterised using experimental data, finding half-maximal FMISO binding at local of 1.4 mmHg (95% CI: 0.3–2.6 mmHg) and half-maximal necrosis at 1.2 mmHg (0.1–4.9 mmHg). Simulations predict a non-linear non-monotonic relationship between FMISO activity (4 hr post-injection) and mean tissue : tracer uptake rises sharply from negligible levels in avascular tissue, peaking at ∼5 mmHg and declining towards blood activity in well-oxygenated conditions. Greater temporal variation in perfusion increases peak TBRs (range 2.20–5.27) as a result of smaller predicted necrotic fraction, rather than fundamental differences in FMISO accumulation under acute hypoxia. Identical late FMISO uptake can occur in regions with differing and necrotic fraction, but simulated TACs indicate that additional early-phase information may allow discrimination of hypoxic and necrotic signals.We conclude that a robust approach to FMISO interpretation (and dose-painting prescription) is likely to be based on dynamic PET analysis.

Synthesis and preliminary evaluation of a 18 F-labeled ethisterone derivative [18 F]EAEF for progesterone receptor targeting.

To develop a novel progesterone receptor-targeting probe for positron emission tomography imaging, an ethisterone derivative [18 F]EAEF was designed and prepared in high decay-corrected radiochemical yield (30-35%) with good radiochemical purity (>98%). [18 F]EAEF is a lipophilic tracer (logP=0.53±0.06) with very good stability in saline and serum. In the biodistribution study, high radioactivity accumulation of [18 F]EAEF were found in uterus (5.73±1.83% ID/g) and ovary (4.05±0.73% ID/g) at 2hr postinjection (p.i.), which have high progesterone receptor expression after treated with estradiol, while the muscle background has very low uptake (0.50±0.17% ID/g). For positron emission tomography imaging, [18 F]EAEF showed high uptake in progesterone receptor-positive MCF-7 tumor (3.15±0.07% ID/g at 2hr p.i.) with good tumor to muscle ratio (2.90), and obvious lower tumor uptakes were observed in MCF-7 with EAEF blocking (1.84±0.05% ID/g at 2hr p.i.) or in progesterone receptor-negative MDA-MB-231 tumor (1.80±0.03% ID/g at 2hr p.i.). Based on the good stability and specificity of [18 F]EAEF, it may be a good candidate for imaging progesterone receptor and worth further investigation.

Development of 153Sm-DTPA-SPION as a theranostic dual contrast agents in SPECT/MRI.

This study describes the preparation, biodistribution of 153Sm-DTPA-SPION after intravenous injection in rats. The chelator DTPA dianhydride was conjugated to SPION using a small modification of the well-known cyclic anhydride method. Conjugation was done at a 1: 4 (SPION:ccDTPA) molar ratio. Conjugation reaction was purified with magnetic assorting column (MACs) using high gradient magnetic field following incubation, the radio labeled conjugate was checked using RTLC method for labeling and purity checked. The RTLC showed that labeling yield was above 99% after purification and the compound have good in vitro stabilities until 48 hr post injection in the presence of human serum. The biodistribution of 153Sm-DTPA-SPION in rats showed dramatic uptake in the reticuloendothelial system (RES) and their clearance is so fast in other organs especially in the blood. Biodistribution results show that after 30 min post injection more than 84% of injected activities were taken up by the liver and spleen (about 64% and 20%, respectively). Due to magnificent uptakes of this radiotracer in the liver and spleen and their fast clearance from other tissues, especially in blood, it is suggested that this radiotracer would be a potential candidate for RES theranostic purposes.

Hepatoprotective and antioxidant effects of dietary Glycyrrhiza polysaccharide against TCDD-induced hepatic injury and RT-PCR quantification of AHR2, ARNT2, CYP1A mRNA in Jian Carp (Cyprinus carpio var. Jian)

To evaluate the protective effects of Glycyrrhiza polysaccharide (GPS) against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced hepatotoxicity in Jian carp, the fish were fed diets containing GPS at doses of 0.1, 0.5 and 1.0g/kg for 60days before an intraperitoneal injection of 0.6μg/kg TCDD at a volume of 0.05mL/10g body weight. At 72hr post-injection, blood and liver samples were taken for biochemical analysis and the fish liver samples were used for the preparation of pathological slices. The results showed that increases in alanine aminotransferase (GPT), aspartate aminotransferase (GOT), lactate dehydrogenase (LDH), and alkaline phosphatase (AKP) in serum induced by TCDD were significantly inhibited by pre-treatment with 1.0g/kg GPS. Following the 1.0g/kg GPS pre-treatment, total protein (TP), albumin (Alb), catalase (CAT), glutathione peroxidase (GPx), total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activities in liver tissue increased significantly, malondialdehyde (MDA) formation (P<0.05 or P<0.01) was significantly inhibited, and the expression of cytochrome P4501A (CYP1A), aryl hydrocarbon receptor 2 (AHR2) and aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) mRNA (P<0.05) was significantly enhanced. Histological observations on fish liver were obtained by preparing paraffin tissue sections via HE staining, and the results showed that histological changes were obviously reduced by 0.5 and 1.0g/kg GPS. GPS significantly reduced liver tissue damage caused by TCDD. Overall, these results proved the hepatoprotective effect of GPS in protecting against fish liver injury induced by TCDD, and supported the use of GPS (1.0g/kg) as a hepatoprotective and antioxidant agent in fish.

Comparative analysis of metallic nanoparticles as exogenous soft tissue contrast for live in vivo micro-computed tomography imaging of avian embryonic morphogenesis.

Gestationally survivable congenital malformations arise during mid-late stages of development that are inaccessible in vivo with traditional optical imaging for assessing long-term abnormal patterning. MicroCT is an attractive technology to rapidly and inexpensively generate quantitative three-dimensional (3D) datasets but requires exogenous contrast media. Here we establish dose-dependent toxicity, persistence, and biodistribution of three different metallic nanoparticles in day 4 chick embryos. We determined that 110-nm alkaline earth metal particles were nontoxic and persisted in the chick embryo for up to 24 hr postinjection with contrast enhancement levels at high as 1,600 Hounsfield units (HU). The 15-nm gold nanoparticles persisted with x-ray attenuation higher than that of the surrounding yolk and albumen for up to 8 hr postinjection, while 1.9-nm particles resulted in lethality by 8 hr. We identified spatial and temporally heterogeneous contrast enhancement ranging from 250 to 1,600 HU. With the most optimal 110-nm alkaline earth metal particles, we quantified an exponential increase in the tissue perfusion vs. distance from the dorsal aorta into the flank over 8 hr with a peak perfusion rate of 0.7 μm(2) /s measured at a distance of 0.3 mm. These results demonstrate the safety, efficacy, and opportunity of nanoparticle based contrast media in live embryos for quantitative analysis of embryogenesis. Developmental Dynamics 245:1001-1010, 2016. © 2016 Wiley Periodicals, Inc.

Alternation of apoptotic and implanting genes expression of mouse embryos after re-vitrification

Nowadays, oocytes and embryos vitrification has become a routine technique. Based on clinical judgment, re-vitrification maybe required. But little is known about re-vitrification impact on genes expression. The impact of re-vitrification on apoptotic and implanting genes, Bax, Bcl-2 and ErbB4, at compaction stage embryos were evaluated in this study. In this experimental study, 8 cell embryos (n=240) were collected from female mature mice, 60-62 hr post HCG injection. The embryos were divided randomly to 3 groups included: fresh (n=80), vitrified at 8 cell stage (n=80), vitrified at 8 cell stage thawed and re-vitrified at compaction stage (n=80). Embryos were vitrified by using cryolock, (open system) described by Kuwayama. Q-PCR was used to examine the expression of Bax, Bcl2 ErbB4 genes in derived blastocysts. Our result showed that expanded blastocyst rate was similar between vitrified and re-vitrified groups, while re-vitrified embryos showed significant decrease in expanded blastocyst rate comparing with fresh embryos (p=0.03). In addition, significant difference was observed on apoptotic gene expression when comparing re-vitrified and fresh embryos (p=0.004), however expression of Bax and Bcl-2 (apoptotic) genes didn't demonstrate a significant difference between re-vitrified and vitrified groups. The expression rate of ErbB4, an implantation gene was decreased in re-vitrified embryos comparing with fresh embryos (p=0.003), but it was similar between re-vitrified and vitrified embryos. Re-vitrification can alter the expression of Bax, Bcl-2 and ErbB4 genes and developmental rate of mouse embryos in compaction stage.

MOAP-1 Mediates Fas-Induced Apoptosis in Liver by Facilitating tBid Recruitment to Mitochondria.

Fas apoptotic signaling regulates diverse physiological processes. Acute activation of Fas signaling triggers massive apoptosis in liver. Upon Fas receptor stimulation, the BH3-only protein Bid is cleaved into the active form, tBid. Subsequent tBid recruitment to mitochondria, which is facilitated by its receptor MTCH2 at the outer mitochondrial membrane (OMM), is a critical step for commitment to apoptosis via the effector proteins Bax or Bak. MOAP-1 is a Bax-binding protein enriched at the OMM. Here, we show that MOAP-1-deficient mice are resistant to Fas-induced hepatocellular apoptosis and lethality. In the absence of MOAP-1, mitochondrial accumulation of tBid is markedly impaired. MOAP-1 binds to MTCH2, and this interaction appears necessary for MTCH2 to engage tBid. These findings reveal a role for MOAP-1 in Fas signaling in the liver by promoting MTCH2-mediated tBid recruitment to mitochondria.

Humoral Immune Response And Midgut Histopatholical Changes In Grasshopper Schistocerca Gregaria Fed On Four Different Plants And Infected With Bacillus Thuringiensis

The present study investigated the effect of dietary changes on differential and total haemocyte counts (THCs) and midgut histopathologiocal changes of S. gregaria injected with Bacillus thuringiensis israelensis (Bti). THCs of Bti-injected adults significantly decreased in all insects fed on all experimental plants. In Bti-injected locusts, prohaemocytes (PRs) percent decreased in insects fed on grass and quietly disappeared in other treatments. plasmatocytes (PLs) percent increased, while the granulocytes (GRs) decreased in all tested insects fed on sorghum, sesban, grass and clover than those of the control. The percentages of oenocytoids (OEs) increased in all treatments except in insect fed on sesaban. The results indicated that insects fed on clover had the highest number of haemocytes, while those fed on grass had the lowest number. Certain pathological symptoms in the midgut of Bti treated adults were observed after 48 hr post injection, such as (1) disorganisation, disintegration and vacuolization of epithelial cells, (2) lysis of the basement membrane and surrounding muscles and (3) detachment of microvilli from epithelial cells. The most deformed group to Bti was grass fed locust. Subsequently, the host plant could be a major factor that affect on the response of herbivorous insects to pathogens.

The metabolic cost of mounting an immune response in male brown anoles (Anolis sagrei).

The tradeoff between reproduction and survival is central to life-history theory and is thought to reflect underlying energetic tradeoffs between reproduction and self-maintenance. Immune responses to parasites and pathogens are important components of self-maintenance in many species, but whether these defenses impose significant energetic costs has only been tested in a handful of organisms. We tested for a metabolic cost of mounting an immune response in the male brown anole (Anolis sagrei), a lizard in which we have previously shown that reproduction causes a marked reduction in immune response to the novel antigen phytohaemagglutinin (PHA). We treated captive male anoles with a subcutaneous injection of either PHA, which induces an immune response that manifests as localized swelling, or saline vehicle as a control. Prior to injection and at 24, 48, and 72 hr post-injection, we measured swelling at the site of injection and whole-animal resting metabolic rate (RMR) using stop-flow respirometry. Although we detected a robust swelling response to PHA at 24, 48, and 72 hr post-injection, mean RMR did not differ between treatments at any of these time points. However, within the PHA treatment group, RMR increased with the extent of swelling, suggesting a variable metabolic cost that scales with the magnitude of the induced immune response. Although individual anoles varied considerably in the extent to which they responded to PHA challenge, our results suggest that an immune response can impose a substantial metabolic cost (potentially as much as 63% above baseline RMR) for individuals that do respond maximally. J. Exp. Zool. 323A: 689-695, 2015. © 2015 Wiley Periodicals, Inc.

Abstract 5516: Biodistribution of liposomal docetaxel prodrug MNK-010 in mice bearing A549 human NSCLC xenografts

Abstract The anti-mitotic taxanes belong to one of the most important families of anticancer drugs that have broad anti-tumor activity and are widely used for a variety of human cancer types. However, the taxanes are limited by a number of serious pharmacological and toxicological effects. We are developing a unique delivery system for docetaxel, comprised of a PEGylated liposomal nanoparticle containing a prodrug of docetaxel to improve solubility, tolerability and increase efficacy through improved pharmacokinetics and biodistribution. In this study we evaluated the biodistribution of MNK-010, a liposomal formulation of a docetaxel prodrug in immunodeficient mice bearing A549 Human Non-Small Cell Lung Carcinoma (NSCLC) xenografts. This study tested the hypothesis that MNK-010 can produce greater plasma and tumor accumulation of docetaxel than with delivery of standard of care free docetaxel in tumor-bearing mice. Female nude mice bearing A549 human NSCLC xenografts were given a single intravenous (IV) dose of MNK-010 (40 mg/kg docetaxel molar equivalent) or free docetaxel (50 mg/kg). For each treatment group, three animals were sacrificed at various time points between 1 hr and 504 hr post injection. After euthanasia, blood, tumor, liver, kidney, spleen, lung and skeletal muscle specimens were collected. Tissue and blood samples were assayed to determine docetaxel concentrations. Administration of MNK-010 IV provided a reservoir for the continual slow release of docetaxel in the circulation and in tumors. MNK-010 increased systemic docetaxel exposure (AUC) ten-fold greater than free docetaxel injected at similar doses. MNK-010 produced sustained docetaxel levels in tumor through the 21 day observation period, resulting in four-fold greater docetaxel tumor exposure (AUC) compared to free docetaxel. The increase in MNK-010 plasma exposure as well as the enhanced permeability effect observed with liposomes likely contributed to the increased tumor exposure. In addition to improved accumulating in tumor tissue, docetaxel derived from MNK-010 also accumulated in liver, spleen and kidney. These tissues contain phagocytic cells (macrophages or mesangial) and serve as organs of elimination for chemotherapeutics. Docetaxel derived from MNK-010 was not detectable in skeletal muscle and was only found in lung over the first 24 hours post administration. In contrast, IV injection of free docetaxel produced high initial concentrations of docetaxel in all tissues evaluated, before falling quickly over time. Tumor levels decreased below levels of quantitation after nine days. This study demonstrated that Mallinckrodt Pharmaceuticals’ PEGylated liposomal docetaxel prodrug MNK-010 provided a reservoir for the continual release of docetaxel into the systemic circulation and produced an enhanced accumulation of docetaxel in A549 human NSCLC xenografts in mice compared to the standard of care docetaxel. Citation Format: Richard M. Fitch, James H. Wible, James A. Blackledge, William D. McGhee. Biodistribution of liposomal docetaxel prodrug MNK-010 in mice bearing A549 human NSCLC xenografts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5516. doi:10.1158/1538-7445.AM2015-5516

Abstract CT222: Ferumoxytol enhanced MRI for lymph node staging in genitourinary cancers

Abstract Background: Conventional imaging has limited accuracy in genitourinary (GU) cancer staging. This study examines the utility of ferumoxytol enhanced MRI in lymph node (LN) staging of GU cancers. Methods: This ongoing IRB-approved phase II clinical trial enrolls patients with prostate cancer, renal cell carcinoma, or bladder cancer at high risk for LN metastases. Patients undergo baseline T2 and T2* weighted MRI scans followed by injection of 7.5mg/Kg ferumoxytol. Repeat scans are acquired at 24hr and 48hr post-injection. The criterion for positive LNs was preservation of hyper-intense signal indicating failure to take up ferumoxytol. Validation was by histopathology when available or on clinical grounds, for which LNs that changed size on routine imaging were considered true positives. Results: To date, 13 patients have completed the study. Of 11 prostate cancer patients, one was studied pre-operatively while 10 had suspected therapy failure. Median age and PSA were 65yrs (36-75) and 5.6ng/mL (0.3-201). The other 2 patients had renal cell carcinoma and bladder cancer. Overall, 20 LNs were identified with mean size 1.9cm (0.7-3.8) long axis by 1.3cm (0.6-2.6) short axis. There were 14 true positive LNs, 1 false positive, 1 false negative, and 4 nodes pending validation. Validation was by histopathology for 7 LNs, with 2 nodes pending biopsy, and clinical grounds for 13 LNs, with 2 inconclusive nodes awaiting further validation. Ferumoxytol correctly identified LN status in 9 of 10 patients with validated nodes (Table 1). Conclusions: Ferumoxytol enhanced MRI shows promise in detecting malignant LNs &amp;gt;6mm in GU cancer patients. Since the method involves a conventional MRI unit with off-label use of an FDA-approved agent, it could be widely available. However, further validation is necessary before routine use. Table 1: Preliminary results for LN staging in GU cancer patients using ferumoxytol enhanced MRI SubjectStudy ArmGenderAge (yr)PSA at study initiation (ng/mL)LN numberLN locationsize/long axis (cm)size/short axis (cm)Ferumoxytol positive? 1 = yes, 0 = noResult1prostate cancerM6325.061R ext iliac3.02.61TP2L ext iliac1.30.81TP2prostate cancerM6573.961L RP1.61.41TP2L int iliac3.81.91TP3prostate cancerM6410.491R ext iliac3.00.91pending2L ext iliac1.91.10pending4prostate cancerM742.061L RP1.71.51TP5prostate cancerM642.891R ext iliac1.61.11TP6prostate cancerM650.281L int iliac1.50.81TP7prostate cancerM752.871R int iliac0.70.71TP8prostate cancerM6427.911R common iliac1.71.71inconclusive9prostate cancerM72201.21L RP2.31.70FN10prostate cancerM736.771L int iliac1.51.01TP11prostate cancerM361.351R femoral0.80.81FP2L ext iliac1.51.01inconclusive3R perirectal0.80.61TP12renal cell carcinomaF41N/A1aortocaval2.82.21TP2R RP3.62.31TP3R int iliac2.01.51TP13bladder cancerM59N/A1aortocaval1.51.01TPRP = retroperitoneal, TP = true positive, FN = false negative, FP = false positive, ext = external, int = internal Citation Format: Anna M. Brown, Sandeep Sankineni, Marcelino Bernardo, Dagane Daar, Juanita Weaver, Yolanda McKinney, Anna Couvillon, James L. Gulley, Bradford J. Wood, Peter A. Pinto, William L. Dahut, Ravi Amrit Madan, Peter L. Choyke, Baris Turkbey. Ferumoxytol enhanced MRI for lymph node staging in genitourinary cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT222. doi:10.1158/1538-7445.AM2015-CT222

Preclinical evaluation of a novel CEA-targeting near-infrared fluorescent tracer delineating colorectal and pancreatic tumors.

Surgery is the cornerstone of oncologic therapy with curative intent. However, identification of tumor cells in the resection margins is difficult, resulting in nonradical resections, increased cancer recurrence and subsequent decreased patient survival. Novel imaging techniques that aid in demarcating tumor margins during surgery are needed. Overexpression of carcinoembryonic antigen (CEA) is found in the majority of gastrointestinal carcinomas, including colorectal and pancreas. We developed ssSM3E/800CW, a novel CEA-targeted near-infrared fluorescent (NIRF) tracer, based on a disulfide-stabilized single-chain antibody fragment (ssScFv), to visualize colorectal and pancreatic tumors in a clinically translatable setting. The applicability of the tracer was tested for cell and tissue binding characteristics and dosing using immunohistochemistry, flow cytometry, cell-based plate assays and orthotopic colorectal (HT-29, well differentiated) and pancreatic (BXPC-3, poorly differentiated) xenogeneic human-mouse models. NIRF signals were visualized using the clinically compatible FLARE™ imaging system. Calculated clinically relevant doses of ssSM3E/800CW selectively accumulated in colorectal and pancreatic tumors/cells, with highest tumor-to-background ratios of 5.1 ± 0.6 at 72 hr postinjection, which proved suitable for intraoperative detection and delineation of tumor boarders and small (residual) tumor nodules in mice, between 8 and 96 hr postinjection. Ex vivo fluorescence imaging and pathologic examination confirmed tumor specificity and the distribution of the tracer. Our results indicate that ssSM3E/800CW shows promise as a diagnostic tool to recognize colorectal and pancreatic cancers for fluorescent-guided surgery applications. If successfully translated clinically, this tracer could help improve the completeness of surgery and thus survival.