Hr Postinjection Research Articles

Glymphatic clearance of simulated silicon dispersion in mouse brain analyzed by laser induced breakdown spectroscopy

Silicon-based devices, such as neural probes, are increasingly used as electrodes for receiving electrical signals from neural tissue. Neural probes used chronically have been known to induce inflammation and elicit an immune response. The current study detects and evaluates silicon dispersion from a concentrated source in the mouse brain using laser induced breakdown spectroscopy. Element lines for Si (I) were found at the injection site at approximately 288 nm at 3hr post-implantation, even with tissue perfusion, indicating possible infusion into neural tissue. At 24hr and 1-week post-implantation, no silicon lines were found, indicating clearance. An isolated immune response was found by CD68 macrophage response at 24hr post injection. Future studies should measure chronic silicon exposure to determine if the inflammatory response is proportional to silicon administration. The present type of protocol, coupling laser induced breakdown spectroscopy, neuroimaging, histology, immunohistochemistry, and determination of clearance could be used to investigate the glymphatic system and different tissue states such as in disease (e.g. Alzheimer's).

Longitudinal In Vivo Imaging of Atherosclerotic Disease Development in The apoE Deficient Zucker Rat

One obstacle hindering the development of therapies to treat diabetic vasculopathies, is access to an affordable and appropriate small animal model that recapitulates human pathology. There are currently no well‐established small animal models to study diabetes and atherosclerosis combined. Longitudinal in vivo imaging of inflammation in atherosclerosis is a promising tool to evaluate progression of cardiovascular complications. However, vascular size of current mouse models of atherosclerosis does not allow for detailed molecular imaging. Radiolabeled DANBIRT has been used as an in vivo molecular imaging tool on environmental exposure, aging, and atherosclerosis. Our group has modified the lipid metabolism in Lean Zucker (LZ: Lepr+/?) rats to promote dyslipidemia and development of atherosclerosis by knocking out the apolipoprotein E (apoE) gene using CRISPR/Cas9. We hypothesize that apoE knockout, LZ rats (apoE−/−, Lepr+/?) will develop atherosclerosis with dietary exposure to high fat, high cholesterol (HFHC) diet. [111In]‐DANBIRT will be able to localize inflammation in vivo and allow longitudinal monitoring of vascular atherosclerotic disease using SPECT/CT imaging.MethodsapoE−/−;LZ adult rats were imaged for 1hr post injection of ~900 μCi of [111In]‐DANBIRT for SPECT/CT imaging using a NanoSPECT/CT®. Rats were placed on either HFHC or standard rodent chow for 10 weeks starting at 12 weeks of age (baseline). apoE+/+;LZ under normal and HFHC diet were used as controls. Regions of Interests (ROI) are identified and determined for muscle (background tissue), carotid arteries, aortic arch, and descending aorta. Activity is decay corrected and compared between and among experimental groups. Serum chemistry and histologic analysis of en face aortas and aortic root were performed. Data is represented as mean ± SEM. Two‐way ANOVA with Tukey’s correction was performed.ResultsAll apoE−/− rats developed severe hypercholesterolemia particularly under a HFHC diet. After 10 weeks of HFHC diet apoE−/−, LZ rats have higher [111In]‐DANBIRT uptake in the descending aorta uniquely compared to baseline (p<0.05) and to normal diet fed rats (ns). Histologic analysis showed apoE−/−, LZ have increased significant atherosclerosis on the aortas and the aortic sinus, which correlates to our imaging results. apoE+/+, LZ rats did not develop atherosclerosis disease as expected.ConclusionTo our knowledge, we have the first polygenic rat model able to develop atherosclerosis translationally under HFHC. [111In]‐DANBIRT longitudinally evidenced atherosclerotic disease in the abdominal aorta in vivo correlated to ex vivo histologic analysis.Support or Funding InformationSupported by the NORC PPF (P30 DK056350) to EMB and the CRTG (Burroughs Wellcome Fund) to RIM.

Population pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin in clinically diseased or injured Eastern box turtles (Terrapene carolina carolina), yellow-bellied sliders (Trachemys scripta scripta), and river cooters (Pseudemys concinna).

Enrofloxacin is frequently administered to turtles in wildlife clinics during rehabilitation due to its wide spectrum of antibacterial activity and availability of injectable formulations. However, sufficient pharmacokinetic data to guide dosing are lacking. The objective of this study was to determine pharmacokinetic parameters of enrofloxacin and its active metabolite, ciprofloxacin, in chelonians presenting injured to a wildlife clinic. Thirty-six Eastern box turtles (EBT, Terrapene carolina carolina), 23 yellow-bellied sliders (YBS, Trachemys scripta scripta), and 13 river cooters (RC, Pseudemys concinna) received a single subcutaneous injection of enrofloxacin at 10mg/kg. Blood samples were collected between 0 and 240hr postinjection. Pharmacokinetic parameters were determined using nonlinear mixed-effects modeling (NMLE). Overall elimination half-life (T½) was over 75hr, and varied among species. T½ was 63hr in EBT and 79hr in YBS, which islonger than in previous reports. The volume of distribution (steady-state) was 1.4L/kg across turtle species, but highly variable-ranging from 0.4L/kg in RC to 1.9L/kg in YBS. Antibiotic concentrations were above a minimum inhibitory concentration value of 0.5µg/ml for over 200hr. These results indicate variable pharmacokinetic parameters for enrofloxacin among turtle species, which will help guide appropriate dosing protocols in injured turtles.

Effects of human chorionic gonadotropin and gonadotropin releasing hormone analogue on plasma steroid hormones and spawning performances in golden rabbitfish Siganus guttatus

In this experiment, golden rabbitfish (Siganus guttatus) were allocated between three treatment groups. The fish were injected with saline, human chorionic gonadotropin (hCG) and D-Ala6, Pro9-Net-mGnRH. After injection, in 6 hr intervals, blood plasma samples were collected for steroid hormone (testosterone [T] in males and estradiol-17β [E2] in females) using enzyme immune assay (EIA). In male fish, T levels significantly increased and reached 170 and 650 pg/ml for hCG and D-Ala6, Pro9-Net-mGnRH treatments, respectively. Then T levels slightly decreased until 24 hr post injection. There were no significant changes of T levels in saline treatment. In female fish, we found significant changes in E2 levels at 2,567 and 524 pg/ml at 12 hr post injection in hCG and D-Ala6, Pro9-Net-mGnRH treatments, respectively. No significant differences of E2 levels were observed in saline group. In the second experiment, we injected 100 golden rabbitfish with both hCG and D-Ala6, Pro9-Net-mGnRH. Fish spawned successfully when hCG and D-Ala6, Pro9-Net-mGnRH were given individually and in combination. Latency periods were between 46–64 hr with an average fertilization rate of 70%–90% and hatching rate of 56%–74%. The embryonic duration was 16–20 hr. The saline-injected group produced no spawning. Our findings contribute to further understanding of exogenous hormones impact on golden rabbitfish reproductive endocrinology, refining breeding protocol and implications for fish propagation.

Regulation of food intake by astrocytes in the brainstem dorsal vagal complex.

A role for glial cells in brain circuits controlling feeding has begun to be identified with hypothalamic astrocyte signaling implicated in regulating energy homeostasis. The nucleus of the solitary tract (NTS), within the brainstem dorsal vagal complex (DVC), integrates vagal afferent information from the viscera and plays a role in regulating food intake. We hypothesized that astrocytes in this nucleus respond to, and influence, food intake. Mice fed high‐fat chow for 12 hr during the dark phase showed NTS astrocyte activation, reflected in an increase in the number (65%) and morphological complexity of glial‐fibrillary acidic protein (GFAP)‐immunoreactive cells adjacent to the area postrema (AP), compared to control chow fed mice. To measure the impact of astrocyte activation on food intake, we delivered designer receptors exclusively activated by designer drugs (DREADDs) to DVC astrocytes (encompassing NTS, AP, and dorsal motor nucleus of the vagus) using an adeno‐associated viral (AAV) vector (AAV‐GFAP‐hM3Dq_mCherry). Chemogenetic activation with clozapine‐N‐oxide (0.3 mg/kg) produced in greater morphological complexity in astrocytes and reduced dark‐phase feeding by 84% at 4 hr postinjection compared with vehicle treatment. hM3Dq‐activation of DVC astrocytes also reduced refeeding after an overnight fast (71% lower, 4 hr postinjection) when compared to AAV‐GFAP‐mCherry expressing control mice. DREADD‐mediated astrocyte activation did not impact locomotion. hM3Dq activation of DVC astrocytes induced c‐FOS in neighboring neuronal feeding circuits (including in the parabrachial nucleus). This indicates that NTS astrocytes respond to acute nutritional excess, are involved in the integration of peripheral satiety signals, and can reduce food intake when activated.

Effects of acute hyperglycaemia stress on indices of glucose metabolism and glucose transporter genes expression in cobia ( Rachycentron canadum )

The objective of the present study is to preliminarily clarify the mechanism of carbohydrates metabolism in cobia (Rachycentron canadum) (85 ± 3 g) receiving injection of glucose solution. We examined plasma glucose (GLU), total protein (TP), triglyceride (TG), cholesterol (CHOL), insulin, liver glycogen and muscle glycogen, activities of hepatic hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK), as well as relative expressions of glucose transporter 1 (GLUT1), GLUT2, GLUT3, GLUT4, GLUT5 and GLUT9 in hemocyte, liver and muscle of R. canadum when fish were injected with 200 μl of glucose solution (255 mg/ml) after 0, 1, 2, 4, 8, 12, 24 and 48 hr. Fish received injection of 0.68% saline served as control. Results indicated that the plasma GLU, TG and CHOL increased and reached peak at 1, 8 and 48 hr postinjection (hpi) respectively. The hepatic glycogen increased from 1 hpi, and reached peak at 8 hpi, plasma insulin increased at 1 hpi, and reached peak at 2 hpi, and activity of hepatic PK peaked at 8 hpi. Furthermore, the relative expressions of GLUT1, GLUT2, GLUT3, GLUT4 and GLUT5 in hemocytes reached peak at 1,4, 8, 4 and 8 hpi, respectively, relative expressions of GLUT2, GLUT3, GLUT5 and GLUT9 in liver reached peak at 24, 24, 12 and 24 hpi, respectively, and relative expressions of GLUT1 and GLUT3 in muscle were significantly higher at 2 and 2–4 hpi, respectively compared with those in controls. In conclusion, low ability of utilizing glucose in R. canadum may be attributed to insufficient insulin secretion, low activities of key glycolytic enzymes (HK, PFK and PK) regulated by glucose injection and slow increase of GLUTs.

Effect of dietary tryptophan supplementation on growth performance, immune response and anti-oxidant status of broiler chickens from 7 to 21 days.

BackgroundThis study was conducted to investigate the optimum dietary level of tryptophan (Trp) supplementation at which broiler chickens have better growth with efficient immune system and anti‐oxidant status.MethodOne hundred and twenty (n = 120) 1‐day‐old broiler chicks were fed a common commercial diet from days 1 to 7. On day 7, the chicks were randomly divided in three treatment groups, that is, Trp 0.2 [national research council (NRC) recommended level of tryptophan], Trp 0.3 (tryptophan supplemented at 0.3%) and Trp 0.5 (tryptophan supplemented at 0.5%). All the experimental diets were iso‐caloric (ME; 3,000 kcal/kg) and iso‐proteic (CP; 18.5%). Weekly data on feed intake and body weight gain (BWG) were recorded to calculate feed conversion ratio (FCR). On day 19, avian tuberculin was injected to note the cellular immunity. On day 21, two birds per replicate were killed to determine carcass and visceral organ weights. Blood serum samples were collected for analysis of humoral immune response against sheep red blood cells, total oxidant and anti‐oxidants by spectrophotometric method.ResultsFeed intake, carcass and visceral organ weights remained unaffected by dietary treatments while BWG and FCR tended to improve (p < .05) in broiler chicks fed the Trp 0.3 and the Trp 0.5 diets. Total oxidant status was also improved (p < .05) in broiler chicks fed the Trp 0.5 diet. Likewise, broiler chicks fed the Trp 0.3 and the Trp 0.5 diets tended to have better (p < .05) total anti‐oxidant status, catalase, glutathione peroxidase, glutathione reductase and arylesterase (ARE). The overall antibodies response and IgG improved (p < .05) by the Trp 0.3 and Trp 0.5 diets compared to control. However, IgM level remained similar across the treatment. The cellular immunity against avian tuberculin improved at 24 hr post‐injection but its effect disappeared at 48 hr.ConclusionThe results of present study revealed that Trp above the NRC recommended level may give better growth, immune response and anti‐oxidant status in broiler chickens.

The impact of sialylation linkage-type on the pharmacokinetics of recombinant butyrylcholinesterases.

Chinese hamster ovary (CHO) cells typically produce glycoproteins with N-glycans terminating in α-2,3 sialylation. Human cells produce glycoproteins that include α-2,3 and α-2,6 sialic acids. To examine the impact of altering protein sialylation on pharmacokinetic properties, recombinant human butyrylcholinesterase (BChE) was produced in CHO cells by knocking out the α-2,3 sialyltransferase genes followed by overexpression of the α-2,6 sialyltransferase (26BChE) enzyme. The N-glycan composition of 26BChE was compared to BChE with α-2,3 sialylation (23BChE) derived from wild-type CHO cells. Both 23BChE and 26BChE exhibited comparable antennarity distributions with bi-antennary di-sialylated glycans representing the most abundant glycoform. CD-1 mice were intravenously injected with the 23BChE or 26BChE, and residual BChE activities from blood collected at various time points for pharmacokinetic analyses. Although 23BChE contained a slightly lower initial sialylation level compared to 26BChE, the molecule exhibited higher residual activity between 5 and 24 hr postinjection. Pharmacokinetic analyses indicated that 23BChE exhibited an increase in area under the curve and a lower volume of distribution at steady state than that of 26BChE. These findings suggest that the type of sialylation linkage may play a significant role in the pharmacokinetic behavior of a biotherapeutic when tested in in vivo animal models.

EVALUATION OF THE EFFECTS OF MIDAZOLAM AND FLUMAZENIL IN THE BALL PYTHON (PYTHON REGIUS).

The study objective was to evaluate the sedative, muscle relaxant, and cardiorespiratory effects of midazolam and flumazenil in the ball python (Python regius). Ten healthy adult female ball pythons were used in a randomized and blinded crossover trial evaluating the effects of two dosages (1 and 2 mg/kg intramuscular [i.m.] in the cranial third of the body). In a subsequent open trial, nine ball pythons received 1 mg/kg i.m. of midazolam followed by 0.08 mg/kg i.m. of flumazenil 60 min later. Heart rate, respiratory rate, temperature, and the level of sedation and muscle relaxation (using a semiobjective scoring system) were evaluated. There were no significant differences between midazolam dosages for any of the parameters evaluated. Sedation scores were significantly increased compared with baseline from 15 min (1 mg/kg) and 10 min (2 mg/kg) postinjection up until 56 hr (1 mg/kg) and 72 hr (2 mg/kg) postinjection. Peak effect was reached 60 min postinjection, with 60% of snakes (6/10) being unable to right themselves. One snake developed paradoxical excitation with the 2 mg/kg dosage. Heart rates were significantly lower than baseline from 30 min to 128 hr postinjection with both midazolam dosages. Respiratory rates were significantly lower than baseline at four time points, with the highest dosage only: 15, 45, 60 min, and 8 hr postinjection. Flumazenil resulted in reversal of sedation and muscle relaxation in all snakes within 10 min of administration. However, resedation was evident in all snakes 3 hr after reversal. Midazolam administered at 1 and 2 mg/kg i.m. provides a moderate to profound, although prolonged, sedation and muscle relaxation in ball pythons. Flumazenil reverses the effects of midazolam in ball pythons, but its duration of action at the evaluated dosage is much shorter than midazolam, leading to resedation.

Pharmacokinetics of midazolam and its major metabolite 1-hydroxymidazolam in the ball python (Python regius) after intracardiac and intramuscular administrations.

Midazolam is a benzodiazepine with sedative, muscle relaxant, anxiolytic, and anticonvulsant effects. Twelve ball pythons (Python regius) were used in a parallel study evaluating the pharmacokinetics of 1mg/kg midazolam following a single intracardiac (IC) or intramuscular (IM) administration. Blood was collected from a central venous catheter placed 7days prior, or by cardiocentesis, at 15 time points starting just prior to and up to 72hr after drug administration. Plasma concentrations of midazolam and 1-hydroxymidazolam were determined by the use of high-performance liquid chromatography tandem-mass spectrometry and pharmacokinetic parameters were estimated using noncompartmental analysis. The mean±SD terminal half-lives of IC and IM midazolam were 12.04±3.25hr and 16.54±7.10hr, respectively. The area under the concentration-time curve extrapolated to infinity, clearance, and apparent volume of distribution in steady-state of IC midazolam were 19,112.3±3,095.9ng*hr/ml, 0.053±0.008 Lhr-1 kg-1 , and 0.865±0.289L/kg, respectively. The bioavailability of IM midazolam was estimated at 89%. Maximum plasma concentrations following an IM administration were reached 2.33±0.98hr and 24.00±14.12hr postinjection for midazolam and 1-hydroxymidazolam, respectively, and 22.33±20.26hr postinjection for 1-hydroxymidazolam following IC administration.

Nerve growth factor-induced muscle hyperalgesia facilitates ischaemic contraction-evoked pain.

Intramuscular injection of Nerve Growth Factor (NGF) may influence the responsiveness of active chemo-sensitive channels affecting muscle pain sensitivity. This double-blinded crossover study in healthy humans assessed contraction-evoked pain responses and pain sensitivity during acute ischaemia in the tibialis anterior (TA) muscle before and 24hr after five distributed NGF injections (1µg, 4cm interval) compared with control injections (isotonic-saline). Twenty-one subjects participated in two experimental phases, each including five sessions over 7days, with a gap of 4weeks in-between. Muscle pain intensity evoked with daily functional tasks (Likert scale score) was collected using a paper diary. Pain intensity evoked by ischaemic and non-ischaemic contractions numerical rating scale (NRS) was collected at Day0 and Day1. Pressure pain thresholds (PPTs) on the TA were recorded before (Day0), 3hr, 1, 3, and 7days post-injection, and after the ischaemic-contractions and post-cuff deflation at Day0 and Day1. Increased Likert scores of pain were present for 7days after NGF compared to control injections (p<.05). Higher NRS pain scores of ischaemic-contractions were seen when contracting the muscle injected with NGF compared to baseline (p=.003) and control (p=.012). Pain during non-ischaemic contractions was not significantly affected by NGF injections. Decreased PPTs were found at 3hr, Day1 and Day3 post-injection (p<.05) in both conditions. Compared with pre-contractions, PPTs were increased following ischaemic contractions at Day0 (p<.05) and Day1 (p<.05) in both conditions. This study showed that ischaemic contraction-evoked pain was facilitated in an NGF-sensitized muscle. Acidification of the muscle environment may affect muscle nociceptors and pain by different mechanisms, including activation of ASIC3 and TRPV1. In this study, pain evoked following ischaemic contractions was increased in the Nerve Growth Factor (NGF)-sensitized muscle compared with non-ischaemic contractions and in the non-sensitized muscle. These findings illustrate that responses of peripheral afferents under ischaemic conditions are altered by a pre-sensitized muscle. This highlights the role of growth factors, including NGF, in peripheral muscle sensitization with clinical implications for ischaemic myalgia.

Changes in immune responses of Helicoverpa armigera Hübner followed by feeding on Knotgrass, Polygonum persicaria agglutinin.

There is no study implying the effect of plant lectins on insect immune elements in both challenged and non-challenged conditions with entomopathogenic agents. Lectins may bind to immune receptors on the surface of insect hemocytes, thus inducing or even disabling common immune functions including hemocyte counts, nodulation/encapsulation, phenoloxidase activity, and synthesis of antimicrobial peptides. In the present study, effect of Polygonum persicaria L. agglutinin (PPA) on immune responses of Helicoverpa armigera Hübner was investigated by feeding artificial diet treated to the larvae. Subsequently hemocyte count and expression of some immune-related genes were considered for analyses. The two groups of larvae including control and PPA-treated (1%) were divided into four subgroups of intact, Tween-80 injected, latex-bead injected and Beauveria bassiana-injected. Except for intact larvae, the highest numbers of total and differential hemocyte counts were recorded 12 hr postinjection, however, the PPA-fed larvae showed a significantly lower hemocyte counts compared to control. The number of nodules in PPA-fed larvae was significantly lower than control, but the injected larvae of both control and PPA showed the highest nodulation 24 hr postinjection. Although the highest activity of phenoloxidase was observed 12 and 24 hr postinjection but its activity significantly decreased in PPA-fed larvae compared to control. Gene expression of antimicrobial peptides including attacin, cecropin, and peptidoglycan receptor proteins were significantly decreased in artificial diet-fed larvae containing PPA and then injected by B. bassiana spores and latex bead compared to control. These results clearly indicate adverse effects of PPA on immune responses in H. armigera.

Development and preclinical validation of an Integrin αvβ6-targeting imaging agent for near-infrared fluorescent-guided surgery in pancreatic cancer

Background: Margin positive resections or early recurrence due to occult metastases after surgery of pancreatic ductal adenocarcinoma (PDAC) are common. Intraoperative near-infrared fluorescent (NIRF) imaging allows cancer visualization to improve surgical accuracy, however targeted agents are lacking. Integrin αvβ6 is a target specific for PDAC, able to detect positive lymph nodes and differentiate between pancreatitis and PDAC. We developed R01-MG-IRDye800, a clinically translatable αvβ6-targeting NIRF agent, based on a cysteine knot peptide, to visualize PDAC. Methods: The applicability of the tracer was tested for cell and tissue binding using cell-based plate assays, and subcutaneous (BxPC-3, αvβ6 positive), orthotopic pancreatic (AsPC-1, αvβ6 positive), and spontaneous transgenic PDAC-mouse models. The agent was compared to a scrambled control peptide. NIRF signals were visualized using PEARL imaging system. Results: R01-MG-IRDye800 accumulated selectively in PDAC with highest tumor-to-background ratios of 7.6, which proved suitable for intraoperative detection and delineation of PDAC and small tumor nodules in mice, between 6 and 24 hr postinjection. The agent outperformed the control peptide. Ex vivo fluorescence and pathologic examination confirmed tumor specificity and distribution. Conclusion: Our Results indicate that R01-MG-IRDye800 shows promise to recognize PDAC for fluorescent-guided surgery. This tracer can help improve the stratification of patients for potentially curative, margin-negative resection.

Nanocontainer designed from an infectious hypodermal and hematopoietic necrosis virus (IHHNV) has excellent physical stability and ability to deliver shrimp tissues.

BackgroundA virus-like particle (VLP) is an excellent tool for a compound delivery system due to its simple composition, symmetrical structure and self-assembly. Its surface modification both chemically and genetically is established, leading to the target-specific delivery and improved encapsulation efficiency. However, its physical stabilities against many harsh conditions that guarantee long term storage and oral administration have been much less studied.MethodsIHHNV-VLPs were reconstructed from recombinant IHHNV capsid protein in E. coli. Their physical properties against three strong physical conditions including long term storage (0–30 days) in 4 °C, physical stabilities against broad ranged pH (4–9) and against three types of digestive enzymes were tested. Disassembly and reassembly of VLPs for encapsidating an enhanced green fluorescent protein tagged plasmid DNA (EGFP-VLPs) were controlled by the use of reducing agent (DTT) and calcium specific chelating agent (EGTA). Lastly, delivering ability of EGFP-VLPs was performed in vivo by intramuscular injection and traced the expression of GFP in the shrimp tissues 24 hr post-injection.ResultsUpon its purification, IHHNV-VLPs were able to be kept at 4 °C up to 30 days with only slight degradation. They were very stable in basic condition (pH 8–9) and to a lesser extent in acidic condition (pH 4–6) while they could stand digestions of trypsin and chymotrypsin better than pepsin. As similar with many other non-enveloped viruses, the assembly of IHHNV-VLPs was dependent on both disulfide bridging and calcium ions which allowed us to control disassembly and reassembly of these VLPs to pack EGFP plasmid DNA. IHHNV-VLPs could deliver EGFP plasmids into shrimp muscles and gills as evident by RT-PCR and confocal microscopy demonstrating the expression of GFP in the targeted tissues.DiscussionThere are extensive data in which capsid proteins of the non-enveloped viruses in the form of VLPs are constructed and used as nano-containers for therapeutic compound delivery. However, the bottleneck of its application as an excellent delivery container for oral administration would rely solely on physical stability and interacting ability of VLPs to the host cells. These properties are retained for IHHNV-VLPs reported herein. Thus, IHHNV-VLPs would stand as a good applicable nanocontainer to carry therapeutic agents towards the targeting tissues against ionic and digestive conditions via oral administration in aquaculture field.

Preliminary application of micro-SPECT/CT imaging by 99m Tc-tricine-EDDA-HYNIC-c-Met for non-small-cell lung cancer.

A novel 99m Tc-tricine-EDDA-Hynic-c-Met molecular probe was synthetized, and nude mice models of human non-small-cell lung cancer (NSCLC) were established in order to preliminarily investigate that whether the molecular probe can be used to screen c-Met inhibitor for targeted drug therapy in NSCLC. With Hynic as the chelating agent, 99m TcO4 -labeled c-Met receptor was used to synthetize 99m Tc-tricine-EDDA-HYNIC-c-Met. Two nude mice successfully transplanted with H1993 tumor and two nude mice transplanted with H292 tumor were injected with 99m Tc-tricine-EDDA-HYNIC-c-Met through the tail vein. After 2 and 4hr, micro-SPECT/CT imaging was performed. micro-SPECT/CT imaging of nude mice transplanted with H1993 and H292 tumors using 99m Tc-tricine-EDDA-HYNIC-c-Met showed that uptake of 99m Tc-tricine-EDDA-HYNIC-c-Met was high in H1993 tumor, while it was mild in H292 tumor both at 2 and 4hr postinjection. Semiquantitative analysis of the ratio of radioactivity intensity at tumor site to radioactivity intensity of adjacent muscles (T/N value) showed that the average T/N value of H1993 and H292 tumors at 2hr was 3.90 and 2.85, while it was 4.88 and 2.36 at 4hr. micro-SPECT/CT imaging through 99m Tc-tricine-EDDA-HYNIC-c-Met can be used to screen c-Met indicators of NSCLC in H1993 nude mice models for targeted drug therapy.

Technetium-99m radiolabeling and biological study of epirubicin for in vivo imaging of multi-drug-resistant Staphylococcus aureus infections via single photon emission computed tomography.

The development of functional imaging is a promising strategy for diagnosis and treatment of infectious and cancerous diseases. In this study, epirubicin was developed as a [99m Tc]-labeled radiopharmaceutical for the imaging of multi-drug-resistant Staphylococcus aureus infections. The labeling was carried out using sodium pertechnetate (Na99m TcO4 ; ~370MBq). The other parameters such as amount of ligand, reducing agent (SnCl2 .2H2 O), and pH were optimized. The highest labeling yield ≥96.98% was achieved when 0.3mg epirubicin, 13μg SnCl2 .2H2 O, and ~370MBq Na99m TcO4 were incubated at pH 7 for 15min in the presence of ascorbic acid at room temperature. Radiochemical purity, stability, charge, and glomerular filtration rate were studied to evaluate the biological compatibility for in vivo administration. Biodistribution investigations showed radiotracer uptake (13.89±1.56%ID/gm organ) by liver and 7.79±0.38%ID/gm organ by kidneys at 30min post-injection which promisingly wash out at 24hr post-injection. Scintigraphy study showed selective uptake in S.aureus-infected tissues in contrast to turpentine oil-induced inflamed tissues. Target-to-non-target ratio (6.7±0.05) was calculated at 1hr post-injection using SPECT gamma camera. The results of this study reveal that the [99m Tc]-epirubicin can be a choice of imaging and monitoring the treatment process of multi-drug resistant S.aureus bacterial infections.

Abstract 3220: Preliminary evaluation of 212Pb BB2r targeted radiotherapy in prostate cancer

Abstract Introduction: Treating castration resistant prostate cancer (CRPC) continues to be a challenge for clinicians. We have recently shown that gamma emitting 203Pb-RM2 (DOTA-4-amino-1-carboxymethyl-piperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) efficiently targets the BB2r expressed on CRPC cells in vivo. We are now exploring the potential of using the alpha-emitting radioisotope 212Pb to generate the radiotherapeutic 212Pb-RM2. Using 212Pb-RM2, we have performed in vitro cell studies to examine treatment effects in terms of DNA damage, clonogenic potential, and cell viability and preliminary in vivo xenograft targeting proof of principle studies. Methods: 212Pb-RM2 was prepared using an automated radiosynthesizer using 212Pb eluted from a 212Ra/212Pb generator. DNA damage, colony formation, and cell viability were evaluated in PC-3 cells following treatment with 212Pb-RM2 at 0.51, 1, 2, and 4uCi/160uL. DNA damage was assessed using 53BP1 foci following a 4hr incubation with 212Pb-RM2 (T0) and at 24hrs and 48hrs. Colony formation assays were performed on cells treated with TRT for 4hrs, and analyzed at 12-14 days. The MTT assay was conducted up to 144hrs. All cell experiments were performed in triplicate on different occurrences. PC-3 xenografted SCID mice were administered 212Pb-RM2 and organs/tissues were harvested and counted at 15 min, 1hr, 4hr, and 24hrs post injection. Results: DNA damage was found to be sustained over time with 53BP1 foci/cell at 48hrs of 14.8+1.6, 33.8+2. 6, 29.6+1.1, and 40.6+1.6 (avg+SEM) at 0.51, 1, 2, and 4uCi of 212Pb-RM2/160uL, respectively. Colony formation followed a dose dependent response with surviving fraction decreasing as TRT increased with 17.7+3.2, 4.4+2.9, 0.5+0.5, and 0.0+0.0 (avg+SEM) at 0.51, 1, 2, and 4uCi of 212Pb-RM2/160uL, respectively. 212Pb-RM2 tumor uptake in PC-3 xenografts was found to be 5.2+0.7, 5.0+0.9, 3.9+0.7, and 2.6+0.3 %ID/g at 15min, 1hr, 4hr, and 24hrs respectively. In vivo BB2r blocking studies confirmed 212Pb-RM2 targeting of the BB2r with &amp;gt; 72% blocking of normal pancreas uptake measured at 4hr post injection. Conclusions: Treatment with alpha emitting 212Pb-RM2 TRT showed sustained levels of DNA damage which was confirmed by impaired colony formation at all dose levels of 212Pb-RM2 TRT studied. Preliminary in vivo tumor targeting and retention of 212Pb-RM2 was observed out to 24hrs post injection warranting further evaluation of 212Pb-RM2 in multiple CRPC cell line models assessing potential therapeutic efficacy. Acknowledgements: US Veterans Administration (VA) BX001699 and USVA Research Career Scientist (TJH) Citation Format: Tammy L. Rold, Nkemakonam C. Okoye, Hoffman Timothy. Preliminary evaluation of 212Pb BB2r targeted radiotherapy in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3220.

Effects of dietary phytoncides extracted from Korean pine (Pinus koraiensis) cone on performance, egg quality, gut microflora, and immune response in laying hens.

This study was conducted to evaluate the effects of dietary phytoncides extracted from discarded Korean pine cones (Pinus koraiensis) on the performance, egg quality, immune response and gut microflora in laying hens. A total of 400 Hy-Line brown laying hens (50-week old) were allotted into four dietary treatments including a control diet or a diet supplemented with phytoncides at 0.002%, 0.004% and 0.008%. During the 6weeks of experimental feeding, 0.008% of dietary phytoncides improved egg production, feed conversion ratio (p<0.05), but not feed intake, egg weight or feed efficiency. Although dietary phytoncides had no effect on egg quality, decreases in Haugh units depending on storage periods were improved by 0.008% of dietary phytoncides (p<0.05). To investigate the roles of dietary phytoncides on the alteration of the immune response during inflammation, lipopolysaccharide (LPS) or saline was intraperitoneally injected into 10 hens per diet group on the end date of the experimental feeding period. Serum immunoglobulins and splenic cytokine expression at mRNA levels were then measured at 4hr postinjection. Although the levels of IgA were decreased by LPS injection in all dietary groups, dietary phytoncides at 0.008% showed a higher level of IgA by LPS (p<0.05). Interestingly, although LPS injection resulted in an enhanced expression of proinflammatory cytokines such as IL-1β and IL-6, dietary phytoncides at 0.008% showed less increased levels of them (p<0.05). Gut microflora was examined from 10 hens per diet group at the end of the experimental period. While the number of Lactobacillus spp. was increased (p<0.05), Escherichia coli counts in the cecal contents were decreased by 0.008% of dietary phytoncides. Taken together, these results demonstrate that dietary supplementation of 0.008% phytoncides improved the egg production, immune responses during inflammation and gut microflora in laying hens.

CXCL12/CXCR4 Signaling Enhances Human PSC-Derived Hematopoietic ProgenitorFunction and Overcomes Early InVivo Transplantation Failure.

SummaryHuman pluripotent stem cells (hPSCs) generate hematopoietic progenitor cells (HPCs) but fail to engraft xenograft models used to detect adult/somatic hematopoietic stem cells (HSCs) from donors. Recent progress to derive hPSC-derived HSCs has relied on cell-autonomous forced expression of transcription factors; however, the relationship of bone marrow to transplanted cells remains unknown. Here, we quantified a failure of hPSC-HPCs to survive even 24 hr post transplantation. Across several hPSC-HPC differentiation methodologies, we identified the lack of CXCR4 expression and function. Ectopic CXCR4 conferred CXCL12 ligand-dependent signaling of hPSC-HPCs in biochemical assays and increased migration/chemotaxis, hematopoietic progenitor capacity, and survival and proliferation following in vivo transplantation. This was accompanied by a transcriptional shift of hPSC-HPCs toward somatic/adult sources, but this approach failed to produce long-term HSC xenograft reconstitution. Our results reveal that networks involving CXCR4 should be targeted to generate putative HSCs with in vivo function from hPSCs.

Post mortem Survival of Gallibacterium anatis in a Laying Hen Experimental Infection Model.

To assess the survival of Gallibacterium anatis in dead laying hens, 21-wk-old laying hens were injected intraperitoneally with 0.5 ml brain hearth infusion broth containing 108 colony-forming units (CFU) of G. anatis 12656-12 liver ( n = 16), Escherichia coli ST141 ( n = 16), or a mix of G. anatis 12656-12 liver and E. coli ST141 ( n = 16), respectively. Birds were euthanatized 24 hr post injection. From each group eight dead birds were kept at 4 C and eight at room temperature. Swab samples were taken at different time points post euthanatization and streaked on blood agar plates. From the birds kept at 4 C, G. anatis was reisolated from the G. anatis and the G. anatis- E. coli co-injected groups at least 12 days post euthanization. From birds kept at room temperature, G. anatis was reisolated up to 2 days post euthanatization. When using the gyrB-based G. anatis-specific quantitative PCR (qPCR), G. anatis was detected within at least 5 days, and up to 5 days post euthanatization, from birds kept at room temperature and 4 C, respectively. Escherichia coli was reisolated from all the time points independent of how the birds were kept. No difference was observed between the reisolation rates for G. anatis or E. coli when comparing similar detection methods. For birds kept at 4 C, bacterial cultivation was a more sensitive method for detecting G. anatis ( P < 0.05), whereas for birds kept at room temperature, the G. anatis-specific qPCR outperformed bacterial culture ( P < 0.05). In conclusion, we demonstrated that G. anatis has a poorer survival rate than does E. coli in dead chickens kept at room temperature. That finding may affect the overall diagnostic sensitivity and lead to underdiagnosis of G. anatis in a normal production setting.