Fashion In Mice Research Articles

Ascorbic acid antagonizes the sedative effect of diazepam possibly through inhibition of GABA(Aρ₁) and GABA(B1) receptors

Gamma-aminobutyric acid (GABA) receptors belong to a ligand-gated ion channels family and are markedly expressed at the axon terminals of retinal bipolar cells. Ascorbic acid (AA), a known and vital antioxidant in the brain can modulate GABA receptors. We postulate that AA would antagonize benzodiazepines' effect via GABA receptor(s) interacting pathway. Here, we evaluated the modulatory sedative effect of AA on diazepam (DZP)'s anxiolytic effects in Swiss albino mice. The anxiolytic study was accomplished by using open-field, hole-board, and by swing and light-dark tests taking DZP as a standard anxiolytic drug. To understand the possible modulatory effects of AA, animals were co-administered with AA and DZP and/or its antagonist flumazenil (FLU). Additionally, an in-silico study was undertaken against GABA(A1), GABA(B1), and GABA(Aρ₁) receptors. Data suggest that AA at 25 mg/kg (i.p.) increased (p<0.05) the number of field cross, rearing, number of hole cross, and swing and residence, while decreased grooming and dark residence parameters as compared to the control and DZP groups. In addition, AA and/or FLU combined with DZP (2 mg/kg, i.p.) reversed DZP-mediated sedative effects in mice. Results from in silico study suggest that AA has good interactions with GABA(Aρ₁) and GABA(B1) receptors. In conclusion, DZP is a GABA receptor agonist and AA may reverse DZP-mediated sedative effects in a non-competitive binding fashion in mice through inhibition of GABA(Aρ₁) and GABA(B1) receptors.

Matrix Metalloproteinase-9 Mediates the Deleterious Effects of α2-Antiplasmin on Blood–Brain Barrier Breakdown and Ischemic Brain Injury in Experimental Stroke

During acute brain ischemia, α2-antiplasmin markedly enhances brain injury, blood–brain barrier breakdown and matrix metalloproteinase-9 (MMP-9) expression. Although α2-antiplasmin inhibits fibrin thrombus-degradation, and MMP-9 is a collagen-degrading enzyme altering blood–brain barrier, both have similar deleterious effects on the ischemic brain. We examined the hypothesis that MMP-9 is an essential downstream mediator of α2-antiplasmin’s deleterious effects during brain ischemia. Middle cerebral artery thromboembolic stroke was induced in a randomized, blinded fashion in mice with increased blood levels of α2-antiplasmin. There was a robust increase in MMP-9 expression (immunofluorescence) in the ischemic vs. the non-ischemic hemisphere of MMP-9+/+ but not MMP-9−/− mice, 24 h after stroke. Brain swelling and hemorrhage were significantly increased in the ischemic vs. the non-ischemic hemisphere of MMP-9+/+ mice. By comparison to MMP-9+/+ mice, the ischemic hemispheres of MMP-9−/− mice showed a ∼6-fold reduction in brain swelling (p < 0.001) and a ∼9-fold reduction in brain hemorrhage. Brain infarction (p < 0.0001) and TUNEL-positive cell death (p < 0.001) were significantly diminished in the ischemic hemisphere of MMP-9−/− mice vs. MMP-9+/+ mice. Ischemic breakdown of the blood–brain barrier and fibrin deposition were also significantly reduced in MMP-9−/− mice vs. MMP-9+/+ mice (p < 0.05), as measured by quantitative immunofluorescence. We conclude that MMP-9 deficiency ablates many of the deleterious effects of high α2-antiplasmin levels, significantly reducing blood–brain barrier breakdown, TUNEL-positive cell death, brain hemorrhage, swelling and infarction. This suggests that the two molecules may be in a shared pathway in which MMP-9 is essential downstream for the deleterious effects of α2-antiplasmin in ischemic stroke.

Ultrasonic neuromodulation of the mouse cortex increases cardiorespiratory activity

Focused ultrasound (FUS) has demonstrated its ability to modulate neuronal activity in both cortical and subcortical brain regions in a noninvasive and safe fashion in mice, non-human primates, and humans. Rodent studies have shown that ultrasonic neuromodulation (UNMOD) can elicit motor responses in limbs and trigger pupil dilation. Little is known about the effect of ultrasonic neuromodulation of the CNS on the autonomic activity in mice, primarily due to the cardiorespiratory depression caused by the anesthesia used. However, urethane has limited effects on autonomic activity and brain hemodynamics. In this paper, we demonstrate that UNMOD of the cortex increases cardiorespiratory activity in mice. A 1.9-MHz single-element focused ultrasound transducer was used to apply pulsed ultrasound to the mouse cortex. Each animal was injected intraperitoneally with urethane (1500 mg/kg), allowing for a stable plane of anesthesia and experimental efficacy window often exceeding 4 hours with minimal effects on autonomic activity. Sonications were performed in a grid spanning the cortex centered at the Bregma skull suture and heart and breathing rates were acquired using a pulse oximeter. FUS resulted in significant increases in both breathing and heart rates immediately following sonication. This study demonstrated that FUS can have an autonomous excitatory effect invivo.

Dezocine Antagonizes Morphine Analgesia upon Simultaneous Administration in Rodent Models of Acute Nociception

Background: Dezocine is a powerful analgesic that can be less addictive than morphine, yet how the two drugs interact in vivo is poorly understood. Here we administered dezocine alone or in combination with morphine to different acute nociception paradigms to explore the interactions of the 2 drugs upon co-administration. Objective: To evaluate how dezocine interacts with morphine in different acute nociception paradigms. Study Design: Laboratory animal study. Setting: Zhejiang University School of Medicine, Hangzhou, China. Methods: Healthy mice were treated with saline, dezocine (0.625 – 2.5 µg), or a combination of dezocine with morphine (2.5 µg). Tail withdrawal latency (TWL) was analyzed prior to and 30 minutes after drug administration. Rats were treated with saline, morphine (3 mg/kg), dezocine (3 mg/kg), or a combination of both drugs. The animals were then left uninjured, subjected to plantar incision, or underwent formaldehyde-induced acute inflammation. Nociception was then analyzed in terms of mechanical threshold (MT) to von Frey stimulation and paw withdrawal latency (PWL) to thermal stimulation. Formaldehyde-induced pain score was calculated based on the duration of biting and elevating of the animal’s legs. Phosphorylation of extracellular signal-regulated kinase (pERK) was also measured after plantar incision as a molecular index of nociception. Results: Dezocine enhanced TWL but inhibited morphine analgesia in a dose-dependent fashion in mice. Usage of morphine or dezocine alone in uninjured rats increased MT, but co-administering both drugs did not further increase MT. Usage of one drug alone, and both drugs together increased MT and PWL relative to saline at 30 minutes after incision. Usage of one drug alone, but not both drugs together, increased MT and PWL at 120 minutes after incision. Dezocine reduced formaldehyde-induced nociception but co-administering both drugs did not further reduce pain behavior. Limitations: The results were obtained from animal study; clinical investigations will be needed to clarify their interaction. Conclusion: Dezocine antagonizes morphine analgesia on acute nociception upon simultaneous administration. Key words: Dezocine, morphine, acute nociception, analgesia

Neto2 Assembles with Kainate Receptors in DRG Neurons during Development and Modulates Neurite Outgrowth in Adult Sensory Neurons.

Peripheral sensory neurons in the dorsal root ganglia (DRG) are the initial transducers of sensory stimuli, including painful stimuli, from the periphery to central sensory and pain-processing centers. Small- to medium-diameter non-peptidergic neurons in the neonatal DRG express functional kainate receptors (KARs), one of three subfamilies of ionotropic glutamate receptors, as well as the putative KAR auxiliary subunit Neuropilin- and tolloid-like 2 (Neto2). Neto2 alters recombinant KAR function markedly but has yet to be confirmed as an auxiliary subunit that assembles with and alters the function of endogenous KARs. KARs in neonatal DRG require the GluK1 subunit as a necessary constituent, but it is unclear to what extent other KAR subunits contribute to the function and proposed roles of KARs in sensory ganglia, which include promotion of neurite outgrowth and modulation of glutamate release at the DRG-dorsal horn synapse. In addition, KARs containing the GluK1 subunit are implicated in modes of persistent but not acute pain signaling. We show here that the Neto2 protein is highly expressed in neonatal DRG and modifies KAR gating in DRG neurons in a developmentally regulated fashion in mice. Although normally at very low levels in adult DRG neurons, Neto2 protein expression can be upregulated via MEK/ERK signaling and after sciatic nerve crush and Neto2-/- neurons from adult mice have stunted neurite outgrowth. These data confirm that Neto2 is a bona fide KAR auxiliary subunit that is an important constituent of KARs early in sensory neuron development and suggest that Neto2 assembly is critical to KAR modulation of DRG neuron process outgrowth.SIGNIFICANCE STATEMENT Pain-transducing peripheral sensory neurons of the dorsal root ganglia (DRG) express kainate receptors (KARs), a subfamily of glutamate receptors that modulate neurite outgrowth and regulate glutamate release at the DRG-dorsal horn synapse. The putative KAR auxiliary subunit Neuropilin- and tolloid-like 2 (Neto2) is also expressed in DRG. We show here that it is a developmentally downregulated but dynamic component of KARs in these neurons, that it contributes to regulated neurite regrowth in adult neurons, and that it is increased in adult mice after nerve injury. Our data confirm Neto2 as a KAR auxiliary subunit and expand our knowledge of the molecular composition of KARs in nociceptive neurons, a key piece in understanding the mechanistic contribution of KAR signaling to pain-processing circuits.

L-3,4-Dihydroxyphenylalanine induces ptosis through a GPR143-independent mechanism in mice

Through its conversion to dopamine by aromatic l-amino acid decarboxylase (AADC), l-3,4-dihydroxyphenylalanine (l-DOPA) replenishes depleted brain dopamine in Parkinson's disease patients. We recently identified GPR143 as a candidate receptor for l-DOPA. In this study, we investigated the behavioral actions of l-DOPA in wild type (wt) and Gpr143-deficient mice. l-DOPA dose-dependently (10–100 mg/kg, i.p.) induced ptosis under treatment with 3-hydroxybenzylhydrazine, a centrally acting AADC inhibitor. This effect was not mimicked by 3-O-methyldopa. l-DOPA-induced ptosis in Gpr143-deficient mice to a similar extent as in wt mice. These results suggest that l-DOPA induces ptosis in a GPR143-independent fashion in mice.

Sulfate-reducing bacteria slow intestinal transit in a bismuth-reversible fashion in mice.

Hydrogen sulfide (H2 S) serves as a mammalian cell-derived gaseous neurotransmitter. The intestines are exposed to a second source of this gas by sulfate-reducing bacteria (SRB). Bismuth subsalicylate binds H2 S rendering it insoluble. The aim of this study was to test the hypothesis that SRB may slow intestinal transit in a bismuth-reversible fashion. Eighty mice were randomized to five groups consisting of Live SRB, Killed SRB, SRB+Bismuth, Bismuth, and Saline. Desulfovibrio vulgaris, a common strain of SRB, was administered by gavage at the dose of 1.0×109 cells along with rhodamine, a fluorescent dye. Intestinal transit was measured 50minutes after gavage by euthanizing the animals, removing the small intestine between the pyloric sphincter and the ileocecal valve and visualizing the distribution of rhodamine across the intestine using an imaging system (IVIS, Perkin-Elmer). Intestinal transit (n=50) was compared using geometric center (1=minimal movement, 100=maximal movement). H2 S concentration (n=30) was also measured when small intestinal luminal content was allowed to generate this gas. The Live SRB group had slower intestinal transit as represented by a geometric center score of 40.2±5.7 when compared to Saline: 73.6±5.7, Killed SRB: 77.9±6.9, SRB+Bismuth: 81.0±2.0, and Bismuth: 73.3±4.2 (P<.0001). Correspondingly, the Live SRB group had the highest luminal H2 S concentration of 4181.0±968.0ppb compared to 0±0ppb for the SRB+Bismuth group (P<.0001). Live SRB slow intestinal transit in a bismuth-reversible fashion in mice. Our results demonstrate that intestinal transit is slowed by SRB and this effect could be abolished by H2 S-binding bismuth.

Tranylcypromine, a lysine-specific demethylase 1 (LSD1) inhibitor, suppresses lesion growth and improves generalized hyperalgesia in mouse with induced endometriosis.

BackgroundGrowing evidence indicates that endometriosis is an epigenetic disease. Encouragingly, histone deacetylases (HDACs) and DNA methyltransferases have been shown to be promising targets by numerous in vitro studies. However, only a few studies have shown promising effects of HDAC inhibition in preclinical studies in endometriosis. While lysine-specific demethylase 1 (LSD1) is recently found to be aberrantly expressed in endometriosis, and that the treatment of endometriotic stromal cells with tranylcypromine (TC), an LSD1 inhibitor, significantly reduced cellular proliferation, cell cycle progression, and invasiveness, the in vivo effect of TC treatment is currently lacking. This study sought to evaluate the effect of TC in a mouse model of endometriosis.MethodsForty-seven female C57BL/6 mice were used in this experimentation. All mice, except those randomly selected to form Sham surgery (M) and specificity control (S) groups, received an endometriosis-inducing surgery. Group S was set up mainly to ensure that the reduced generalized hyperalgesia in mice treated with TC is not due to any possible analgesic effect of TC, but rather resulting from the treatment effect specific to endometriosis. Two weeks after the surgery, mice that received surgery were further divided randomly into 3 groups: 1) untreated group (U); 2) low-dose TC group (L); 3) high-dose TC group (H). Group S received the same treatment as in group H. Two weeks after treatment, all mice were sacrificed and their ectopic endometrial tissues were harvested and analyzed by immunohistochemistry analysis. Hotplate test was administrated to all mice before the induction, treatment and sacrifice. Lesion size, hotplate latency, immunoreactivity against markers of proliferation, angiogenesis, H3K4 methylation, and of epithelial-mesenchymal transition (EMT).ResultsTC treatment significantly and substantially reduced the lesion size and improved generalized hyperalgesia in a dose-dependent fashion in mice with induced endometriosis. In addition, TC treatment resulted in reduced immunoreactivity to biomarkers of proliferation, angiogenesis, and H3K4 methylation, leading to arrested EMT and lesion growth.ConclusionIn light of our previously reported reduced cellular proliferation, cell cycle progression and invasiveness resulting from the LSD1 inhibition in in vitro studies, our data strongly suggest that LSD1 is a promising therapeutic target for endometriosis.Trial registrationNot applicable.

Abstract 10941: Hepatic Overexpression of Endothelial Lipase Induces a Markedly Low HDL-Cholesterol but Maintains Macrophage Reverse Cholesterol Transport in Mice in an ABCA1-dependent Fashion

We previously showed that hepatic overexpression of endothelial lipase (EL) by adenoviral vectors markedly reduced plasma HDL levels but maintained macrophage reverse cholesterol transport (RCT) in an SR-BI-dependent fashion in mice. In the present study, we further investigated roles of ABCA1 using probucol, an ABCA1 inhibitor, under EL overexpression. In vivo RCT assay demonstrated that hepatic EL overexpression reduced macrophages-derived 3H-cholesterol in plasma, especially HDL fractions but maintained fecal 3H-cholesterol excretion as compared with the control under normal chow. Probucol diet reduced both 3H- and non-radiolabeled-cholesterol levels in HDL fraction, which were further exacerbated by EL overexpression. Interestingly, probucol diet combined with EL overexpression clearly promoted fecal excretion of macrophages-derived 3H-cholesterol, indicating that EL overexpression and ABCA1 inhibition synergistically enhanced macrophage RCT despite of extremely low HDL levels.

416 Live but Not Killed Sulfate-Reducing Bacteria Slow Intestinal Transit in a Bismuth-Reversible Fashion in Mice

416 Live but Not Killed Sulfate-Reducing Bacteria Slow Intestinal Transit in a Bismuth-Reversible Fashion in Mice

Chronic Intermittent Hypoxia Induces Thioredoxin System Changes in a Gender-Specific Fashion in Mice

Obstructive sleep apnea (OSA) is an independent risk factor of multisystem injury including liver and cardiovascular system. Chronic intermittent hypoxia (CIH) associated with recurrent apneas in patients with OSA is one of the most important causes of the increased various systems injury and oxidative stress induced by CIH is an important pathogenic mechanism. Reports indicated that females are less susceptible to oxidative stress injury. The goal of this study was to explore if there exists gender deference of thioredoxin system (Trx/ Txnip) alterations by CIH and to clarify a clue for studying gender disparity of OSA-related multisystem injury. C57BL/6J mice of each gender were exposed to CIH with a fractional inspired O2 (FiO2) nadir of 5%. The oxidative and antioxidant biomarkers were evaluated, including serum OxLDL level and Trx/Txnip expression of liver tissue. Male mice exposed to CIH exhibited significant increases in serum OxLDL level than that of the male control (73.24 ± 22.43 μg/dL, 45.20 ± 28.53 μg/dL, P 5 0.032) but no significant difference in the females. Male mice exposed to CIH also exhibited decreased expression of Trx than the female (0.4460 ± 0.1023 versus 1.0454 ± 0.1777, P = 0.013) and increased expression of Txnip than the female (0.0123 ± 0.0476 versus 0.0065 ± 0.0058, P 5 0.022). These data suggest that CIH induces thioredoxin system change in a gender-specific fashion in mice.

Annexin A5 prevents post-interventional accelerated atherosclerosis development in a dose-dependent fashion in mice

BackgroundActivated cells in atherosclerotic lesions expose phosphatidylserine (PS) on their surface. Annexin A5 (AnxA5) binds to PS and is used for imaging atherosclerotic lesions. Recently, AnxA5 was shown to inhibit vascular inflammatory processes after vein grafting. Here, we report a therapeutic role for AnxA5 in post-interventional vascular remodeling in a mouse model mimicking percutaneous coronary intervention (PCI). Methods and resultsAssociations between the rs4833229 (OR=1.29 (CI 95%), pallelic=0.011) and rs6830321 (OR=1.35 (CI 95%), pallelic=0.003) SNPs in the AnxA5 gene and increased restenosis-risk in patients undergoing PCI were found in the GENDER study. To evaluate AnxA5 effects on post-interventional vascular remodeling and accelerated atherosclerosis development in vivo, hypercholesterolemic ApoE−/− mice underwent femoral arterial cuff placement to induce intimal thickening. Dose-dependent effects were investigated after 3days (effects on inflammation and leukocyte recruitment) or 14days (effects on remodeling) after cuff placement. Systemically administered AnxA5 in doses of 0.1, 0.3 and 1.0mg/kg compared to vehicle reduced early leukocyte and macrophage adherence up to 48.3% (p=0.001) and diminished atherosclerosis development by 71.2% (p=0.012) with a reduction in macrophage/foam cell presence. Moreover, it reduced the expression of the endoplasmic reticulum stress marker GRP78/BiP, indicating lower inflammatory activity of the cells present. ConclusionsAnxA5 SNPs could serve as markers for restenosis after PCI and AnxA5 therapeutically prevents vascular remodeling in a dose-dependent fashion, together indicating clinical potential for AnxA5 against post-interventional remodeling.

The ageing systemic milieu negatively regulates neurogenesis and cognitive function

SummaryIn the central nervous system (CNS), aging results in a precipitous decline in adult neural stem/progenitor cells (NPCs) and neurogenesis, with concomitant impairments in cognitive functions1. Interestingly, such impairments can be ameliorated through systemic perturbations such as exercise1. Here, using heterochronic parabiosis we show that blood-borne factors present in the systemic milieu can inhibit or promote adult neurogenesis in an age dependent fashion in mice. Accordingly, exposing a young animal to an old systemic environment, or to plasma from old mice, decreased synaptic plasticity and impaired contextual fear conditioning and spatial learning and memory. We identify chemokines - including CCL11/Eotaxin – whose plasma levels correlate with reduced neurogenesis in heterochronic parabionts and aged mice, and whose levels are increased in plasma and cerebral spinal fluid of healthy aging humans. Finally, increasing peripheral CCL11 chemokine levels in vivo in young mice decreased adult neurogenesis and impaired learning and memory. Together our data indicate that the decline in neurogenesis, and cognitive impairments, observed during aging can be in part attributed to changes in blood-borne factors.

Amyloid-beta oligomers impair fear conditioned memory in a calcineurin-dependent fashion in mice.

Soluble oligomeric aggregates of the amyloid-beta (A beta) peptide are believed to be the most neurotoxic A beta species affecting the brain in Alzheimer disease (AD), a terminal neurodegenerative disorder involving severe cognitive decline underscored by initial synaptic dysfunction and later extensive neuronal death in the CNS. Recent evidence indicates that A beta oligomers are recruited at the synapse, oppose expression of long-term potentiation (LTP), perturb intracellular calcium balance, disrupt dendritic spines, and induce memory deficits. However, the molecular mechanisms behind these outcomes are only partially understood; achieving such insight is necessary for the comprehension of A beta-mediated neuronal dysfunction. We have investigated the role of the phosphatase calcineurin (CaN) in these pathological processes of AD. CaN is especially abundant in the CNS, where it is involved in synaptic activity, LTP, and memory function. Here, we describe how oligomeric A beta treatment causes memory deficits and depresses LTP expression in a CaN-dependent fashion. Mice given a single intracerebroventricular injection of A beta oligomers exhibited increased CaN activity and decreased pCREB, a transcription factor involved in proper synaptic function, accompanied by decreased memory in a fear conditioning task. These effects were reversed by treatment with the CaN inhibitor FK506. We further found that expression of hippocampal LTP in acutely cultured rodent brain slices was opposed by A beta oligomers and that this effect was also reversed by FK506. Collectively, these results indicate that CaN activation may play a central role in mediating synaptic and memory disruption induced by acute oligomeric A beta treatment in mice.

Cross-species comparison of in vivo PK/PD relationships for second-generation antisense oligonucleotides targeting apolipoprotein B-100

The in vivo pharmacokinetics/pharmacodynamics of 2′- O-(2-methoxyethyl) (2′-MOE) modified antisense oligonucleotides (ASOs), targeting apolipoprotein B-100 (apoB-100), were characterized in multiple species. The species-specific apoB antisense inhibitors demonstrated target apoB mRNA reduction in a drug concentration and time-dependent fashion in mice, monkeys, and humans. Consistent with the concentration-dependent decreases in liver apoB mRNA, reductions in serum apoB, and LDL-C, and total cholesterol were concurrently observed in animal models and humans. Additionally, the long duration of effect after cessation of dosing correlated well with the elimination half-life of 2′-MOE modified apoB ASOs studied in mice ( t 1/2 ≅ 20 days) and humans ( t 1/2 ≅ 30 days) following parental administrations. The plasma concentrations of ISIS 301012, observed in the terminal elimination phase of both mice and monkeys were in equilibrium with liver. The partition ratios between liver and plasma were similar, approximately 6000:1, across species, and thus provide a surrogate for tissue exposure in humans. Using an inhibitory E max model, the ASO liver EC 50s were 101 ± 32, 119 ± 15, and 300 ± 191 μg/g of ASO in high-fat-fed (HF) mice, transgenic mice containing the human apoB transgene, and monkeys, respectively. The estimated liver EC 50 in man, extrapolated from trough plasma exposure, was 81 ± 122 μg/g. Therefore, extraordinary consistency of the exposure–response relationship for the apoB antisense inhibitor was observed across species, including human. The cross-species PK/PD relationships provide confidence in the use of pharmacology animal models to predict human dosing for second-generation ASOs targeting the liver.

The Tumor Suppressive Reagent Taurolidine Inhibits Growth of Malignant Melanoma—a Mouse Model

The tumor suppressive agent taurolidine (TRD) inhibits tumor growth of more than 30 cell lines in vitro and reduces tumor load in early and advanced stages of neoplastic disease in animals. TRD has been shown to induce apoptosis of melanoma cells in vitro. Therefore, the effects of TRD on disseminated melanoma were evaluated in a mice model. After general anesthesia, a midline laparotomy was performed and 1.5 million malignant melanoma cells (B78-D14) were applied in the spleen and 1 million cells at the back (C57BL/6). Animals were randomized and either treated intraperitoneally (i.p., n = 40, 7 days, 12 hourly) or intravenously (i.v., n = 40, 2 days, 12 hourly) with 1%, 2%, or 3% TRD or with Ringer's solution (control group). On day 28, all animals were sacrificed and the total tumor weight and the number of metastatic lesions were determined by two investigators blinded for randomization. The i.p. therapy caused a dose-dependent inhibition of total tumor growth (P = 0.003) and i.p. tumor growth (P = < 0.001), whereas subcutaneous (s.c.) tumor growth was not affected (P = 0.132) compared with the i.p. control group. The i.v. therapy reduced the total tumor growth (P = 0.013) and the s.c. tumor growth (P = 0.016), whereas the i.p. tumor load was not reduced (P = 0.122) compared with the control group. Both i.p. and i.v. therapy with 3% TRD significantly decreased the total number of metastatic lesions. The animal weight was not affected. The i.p. and i.v. therapies reduce total tumor weight and number of metastatic lesions of disseminated malignant melanoma in a dose-dependent fashion in mice. Our encouraging findings should be further confirmed in clinical studies examining the influence of TRD in patients with disseminated malignant melanoma for whom prognosis still remains dismal.

Probing inner retinal circuits in the rod pathway: A comparison ofc-fosactivation in mutant mice

We have used wild-type mice and mice possessing defects in specific retinal circuits in order to more clearly define functional circuits of the inner retina. The retina of the nob mouse lacks communication between photoreceptors and depolarizing bipolar cells (DBCs). Thus, all light driven activity in the nob mouse is mediated via remaining hyperpolarizing bipolar cell (HBC) circuits. Transducin null (Tr alpha-/-) mice lack rod photoreceptor activity and thus remaining retinal circuits are solely generated via cone photoreceptor activity. Activation in inner retinal circuits in each of these mice was identified by monitoring light-induced expression of an immediate early gene, c-fos. The number of cells expressing c-fos in the inner retina was dependent upon stimulus intensity and was altered in a systematic fashion in mice with known retinal mutations. To determine whether c-fos is activated via circuits other than photoreceptors in the outer retina, we examined c-fos expression in tulp1-/- mice that lack photoreceptors in the outer retina; these mice showed virtually no c-fos activity following light exposure. Double-labeling immunohistochemical studies were carried out to more clearly define the population of c-fos expressing amacrine cells. Our results indicate that c-fos may be used to map functional circuits in the retina.

963. Development and Testing of Anti-Cancer Sleeping Beauty Transposons for Localized Tumors and Use in Metastasis Suppression

Top of pageAbstract We have developed a series of anti-cancer Sleeping Beauty (SB) transposons designed for transfer of anti-angiogenic genes or Herpes Simplex Virus Thymidine Kinase (HSVTK) fusion genes into tumor cells, tumor-associated stroma, or organs undergoing metastatic attack. All genes were placed in SB T2 transposon vectors driven by the chicken beta-actin/CMV immediate early promoter/rabbit globin intron (CAGGS) promoter construct. Glioblastoma multiforme (GBM) and at least 6 other cancers overexpress IL-13 receptors. In order to take advantage of this, the human IL-13 ligand was fused to the N-terminus of HSVTK to make a secreted, tumor-targeted, suicide gene. Cell culture media collected from cells transfected with IL13-HSVTK is able to specifically sensitize IL-13 receptor positive GBM-derived cells to ganciclovir. Furthermore, we have delivered the IL13-HSVTK transposon intratumorally as plasmid DNA vector in polyethylenimine (PEI) to abolish glioma xenografts in immunodeficient nu/nu mice. We have also used angiostatin-endostatin fusion and dominant-negative VEGF transposons, delivered intratumorally as DNA/PEI complexes, to reduce glioma xenograft size and halt tumor growth. GBM generally causes death quickly as a primary tumor, however metastatic disease such as occurs in late-stage melanoma also remains difficult to treat. Therefore, we have investigated delivering anti-angiogenic transposons into the mouse lung as DNA/PEI complexes to prevent metastatic engraftment of syngenic B16F10 melanoma cells. Both the quantity and volume of lung metastases was reduced in a transposase-dependent fashion in mice treated 2 months prior to melanoma challenge. SB anti-cancer transposons represent a novel, non-viral vector system that could be used to augment conventional therapy for localized and metastatic cancers.

Studies on the anti-inflammatory properties of Entada abyssinica

The defatted methanolic extract of Entada abyssinica was evaluated for anti-inflammatory activity in acute and chronic models of inflammation. The extract (50–200 mg/kg, p.o.) exhibited dose-dependent and significant inhibition of both the carrageenan-induced rat paw oedema and granuloma tissue formation in rats. The extract (50–200 mg/kg, p.o.) was also found to inhibit the acetic acid-induced vascular permeability in a dose-dependent fashion in mice. This study demonstrated the anti-inflammatory activity of Entada abyssinica.

Inhibition of intrathymic T cell development by expression of a transgenic antagonist peptide.

The mature T cell receptor (TCR) repertoire is shaped by positive- and negative-selection events taking place during T cell development. These events are regulated by interactions between the TCR and major histocompatibility complex molecules presenting self-peptides. It has been shown that many antagonist peptides are efficient at mediating positive selection. In this study we analyzed the effects of a transgene encoding an antagonist peptide (influenza NP34) that is presented by H-2Db in a Tap-1-independent fashion in mice expressing the influenza NP68-specific TCR F5. We find that the transgenic peptide does not mediate positive or negative selection in F5(+)Tap-1(-/-) mice, but inhibits maturation of CD8(+) single positive thymocytes in F5(+)Tap-1(+) mice without inducing signs of negative selection. We conclude that antagonism of antigen recognition occurs not only at the level of mature T cells but also in T cell development.