Survival Time Of Animals Research Articles

A model of acute antibody mediated rejection in mouse renal transplantation

Objective To establish a model of acute antibody mediated rejection (AMR) in mouse kidney transplantation. Methods 6-8 weeks old male C3H (H2k) and BALB/c (H2d) mice were used as skin or kidney graft donors and recipients, respectively. All animals were provided by Beijing vital river experimental animal technical limited company. Recipient animals were divided into 2 groups. AMR group, recipient animals were presensitized by transplanting skin grafts before one week of kidney transplantation (n=10). Cell mediated rejection (CMR) group, recipient animals received kidney grafts without being presensitized by skin transplantation (n=10). Normal balb/c mice acted as control (n=3). Animal survival time was recorded and renal allograft function of creatinine (Cr) and blood urea nitrogen (BUN) was monitored. On the fifth day after kidney transplantation, recipient animals were sacrificed, analysis of renal graft histology based on Banff 2013 for rejection diagnosis, immunohistochemistry for mouse IgG, complement fragment C3d deposition detection intragraft, and flow cytometry for circulating donor specific antibody (DSA)-IgG, IgM detection were performed. Results The mean survival time of AMR group was (4.4±1.1) d and longer than 60 days for CMR goup. Graft function of serum Cr [ (415.6±25.6) μmol/L] and BUN [ (2 394.0±94.0) pmol/L] were remarkably increased on postoperative 2 d in AMR group. CMR control group showed normal serum levels of Cr [ (77.8±7.2) μmol/L] and BUN [ (1 343.0±233.9) pmol/L] on 60 days after kidney transplantation. On 5 d after kidney transplantation, the macroscopic morphology of renal grafts in AMR group was significantly swelling while grafts in CMR group were normal. The circulating IgG DSA level in AMR group was remarkably higher than CMR group with mean fluorescence intensity (MFI) values 223.0±29.0 vs. 134.5±39.6. The serum IgM DSA level did not change significantly with MFI values 9.2±0.1 vs. 10.0±0.7. Grafts in AMR group demonstrated dilation of peritubular capillaries (PTC), peritubular capillaritis, glomerulitis, glomerular/tubular necrosis and interstitial inflammation, hemorrhage, and edema. Extensively deposition of IgG and C3d intragraft were also observed in AMR group, while mild changes presented in CMR group which showed no significant difference from the normal control BALB/c mice. Conclusion We successfully established a model of acute AMR in C3H→BALB/c mouse kidney transplantation by presensitizing recipient mice using donor-strain derived skin transplantation at 1 week prior to kidney transplantation, which showed significantly different from non-presensitized recipient mice. Key words: Kidney transplantation; Antibody mediated rejection; Model, animal; Mice

Abstract 5058: Lysine specific demethylase-1 (LSD-1) Inhibitor SYC-836 in combination with radiation prolongs animal survival in patient-derived posterior fossa ependymoma xenograft mouse models

Abstract Background: Ependymoma (EPN) is the third most common malignant pediatric brain tumor. Current standard therapy include maximally safe surgical resection followed by radiation and lead to a 5-year overall survival of 50-71%. Recent molecular subgrouping of EPN has identified one group, posterior fossa A (PFA), which accounts for 45% of all EPN cases, to have one of the worst prognosis and it is driven by epigenetic changes, suggesting targeting epigenetic changes in PFA EPN can potentially be effective. In this study, we examined the therapeutic efficacy of SYC-836, a novel LSD-1 inhibitor compound developed at Baylor College of Medicine, both in vitro and in vivo in PDOX models of posterior fossa EPN. Methods: To examine in vitro anti-tumor activities, paired primary cultured cells (both as attached cells and neurospheres) from an established PDOX model of posterior fossa EPN (ICb-4423EPN) were subjected to SYC-836 at various concentrations (0-25uM). Cell viability and proliferation were measured using Cell Counting Kit-8 assay at 5 different time points over 14 days. To validate the drug’s in vivo efficacy, two established posterior fossa EPN PDOX models, ICb-4423EPN and ICb-2002EPN, were utilized. 40 eight weeks old SCID mice per model were implanted with tumor cells. They were divided into 4 treatment groups (10 mice/group) each: 1) control (DPBS, 10uL/kg IP daily x 28 days), 2) radiation/standard therapy (2 Gy focal XRT daily x 5 days), 3) SYC-836 only (15mg/kg IP daily x 28 days), and 4) combination (radiation + SYC-836 per regimen above). Animal survival times were analyzed using log rank analysis. Changes of histone lysine methylation were examined through western hybridization. Results: SYC-836 demonstrated effective cell killing in vitro against both attached and neurosphere cultured cells in both time- and dose-dependent manner. IC50 was ~7.5uM. In vivo experiment was completed in 1 of the 2 EPN PDOX models (ICb-2002EPN) with the second model ongoing. Median survival times for each group is as followed: control 136 days, radiation 148 days, SYC-836 only 136 days, combination 180 days. There were no survival benefit with either XRT only (P=0.205) or SYC-836 only (P=0.186) when compared to the control group; however, when used in combination, the treatment strategy lead to significant improvement in animal survival (P=0.004). SYC-836 was well tolerated in mice. Conclusion: Our data showed that combining SYC-836 with current standard therapy of radiation synergistically prolongs animal survival significantly, although as a single agent SYC-836 was not effective against posterior fossa ependymoma. Our data suggest that SYC-836 may have a role in the clinical setting by either reducing radiation dosages, or be a potential adjuvant agent to other chemotherapy drugs in our treatment approach for ependymoma. Citation Format: Sibo Zhao, Huiyuan Zhang, Lin Qi, Holly Lindsay, Yuchen Du, Mari Kogiso, Frank Braun, Sarah Injac, Laszlo Perlaky, Donald W. Parsons, Murali Chintagumpala, Adekunle Adesina, Yongcheng Song, Xiao-Nan Li. Lysine specific demethylase-1 (LSD-1) Inhibitor SYC-836 in combination with radiation prolongs animal survival in patient-derived posterior fossa ependymoma xenograft mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5058. doi:10.1158/1538-7445.AM2017-5058

Abstract 4214: Identification of personalized active agents in pediatric gliomas through high-throughput drug screening in matching pairs of patient derived orthotopic xenograft (PDOX) neurosphere and monolayer cells

Abstract Objective: Glioblastoma (GBM) is one of the deadliest brain tumors both in adults and children. While next-generation sequencing rapidly identifies whole exome/genome gene mutations, the efficacy of targeting mutated genes/pathways has not been systematically analyzed. Additionally, it remains unknown if neurospheres (enriched with cancer stem cells) and non-stem monolayer tumor cells will respond equally to the same target therapies. Methods: Matching pairs of neurosphere and monolayer cell cultures from 4 molecularly characterized PDOX mouse models established from distinct clinical stages of pediatric glioma (treatment-naïve, early recurrent, terminal, and radiation-induced) were treated with 7,913 drugs (mostly in clinical use or trials) at 4 doses (0-10 uM) for 3 and 7 days. Dose-response curves and active area under the curve (AAUC) were constructed to identify active drugs and correlate with model-specific gene mutations. Results: Relatively long-term treatment (7 day screening) was achieved for high-throughput drug screening and revealed time- and dose-dependent effects in most of the 4,629 compounds that were active in at least one culture at one time point. When gene mutations were druggable, not all inhibitors of the same family were active. In IC-4687GBM (a treatment-naïve GBM), high NF1 mutation (>76-99% allele) frequency was found in patient tumor, xenografts, and cultured cells, but only 3/17 MEK inhibitors were active in neurospheres and 2/17 in monolayer cells (GDC0980 and PI-103 were active in both) on day 7. In IC-R0315GBM (from an autopsied terminal GBM) that carried PI3KCA mutation (allele frequency 22-27%), 5/33 PI3K inhibitors were active in neurospheres and 8/33 in monolayer cells (only Tremetinib was active in both) after 7 days. In IC-3752GBM (recurrent GBM) and ICb-1127AA (radiation-induced anaplastic astrocytoma), no druggable mutations were detected. The number of active drugs on day 7 was 366 in IC-4768GBM, 406 in IC-3752GBM, 284 in IC-R0315GBM, and 305 in ICb-1277AA. When the 4 matching pairs of neurospheres and monolayers were compared, the agents active in both cultures ranged from 36% to 60%, active only in neurosphere from 10.3% to 25%, and active only in monolayer cells from 14.8% to 53%. Subsequent in vivo validation using MLN8327 in IC-4687GBM and IC-R0315GBM showed that effective targeting of both neurosphere and monolayer was required for significantly-improved animal survival times. Conclusion: We showed that long-term treatment is feasible for high-throughput drug screening. Targeting druggable mutations can be achieved but only by a fraction of specific agents. Neurospheres and monolayer cells do not always respond equally toward the same drugs, and effective targeting of both subpopulations is needed to generate prolonged animal survival times. Citation Format: Lin Qi, Yuchen Du, Mari Kogiso, Goeun Bae, Frank Braun, Holly Lindsay, Huiyuan Zhang, Sibo Zhao, Sarah Injac, Patricia Baxter, Jack Su, Michael Mancini, Oliver Hampton, William Parsons, Murali Chintagumpala, Clifford Stephan, Peter Davies, Xiao-Nan Li, Xiao-Nan Li. Identification of personalized active agents in pediatric gliomas through high-throughput drug screening in matching pairs of patient derived orthotopic xenograft (PDOX) neurosphere and monolayer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4214. doi:10.1158/1538-7445.AM2017-4214

Abstract 4031: Mutant isocitrate dehydrogenase 1 (IDH1) inhibitor synergistically prolongs animal survival with standard therapies in patient-derived IDH1 mutant glioma xenograft mouse models

Abstract Background: Mutation in isocitrate dehydrogenase 1 (IDH1) occurs in > 70% of WHO grades II and III astrocytomas and oligodendrogliomas and secondary glioblastoma (GBM). The mutant enzyme catalyze the reduction of α-ketoglutaric acid to D-2-hydroxyglutaric acid, leading to cancer initiation. In this study, we examined the therapeutic efficacy of SYC-435 (1-hydroxypyridin-2-one), a newly developed mutant IDH1inhibitor, both in vitro and in vivo in IDH1 mutant gliomas as compared with IDH1 wild type GBMs. Methods: An established neurosphere line (BT142) of anaplastic oligoastrocytoma (AOA), and a patient-derived orthotopic xenograft (PDOX) model of recurrent AOA (IC-V0914AOA), and 2 PDOX models of GBM (IC-4687GBM and IC-3752GBM) were included. IDH1 mutations (R132H and R132C) were analyzed by pyrosequencing. To determine the in vitro antitumor activities, tumor cells were exposed to SYC-435 (0.02 to 20 µM) and examining for changes of cell proliferation every 3-4 days till day 13 by Cell Counting Kit-8 assay. For in vivo effects, orthotopic xenograft mouse models of IC-BT142AOA and IC-V0914AOA were treated with vehicle (as control), SYC-435 (i.p., 15 mg/kg/day x 28 days), temozolomide (TMZ, oral, 50 mg/kg/day x 5 days) + fractionated radiation (XRT, 2 Gy/day x 5 days) (as standard therapy), and combination of SYC-435 with standard therapy starting 2 weeks after intracranial tumor implantation. Animal survival times were analyzed by log rank analysis. Results: IDH1 R132H mutation (homozygous) was detected in BT142AOA neurosphere line and R132C mutation (mutant allele frequency 39-42%) in IC-V0914AOA xenograft cells, while the two GBM models (IC-4687GBM and IC-3752GBM) carried wild-type IDH1. Suppression of cell growth was observed in time- and dose-dependent manner by SYC-435, particularly at the IDH1 mutant models. At 0.5 µM, SYC-435 inhibited cell growth by 90% in BT142 and 60% in IC-V0914AOA cells, whereas in IDH wild-type GBMs only by 17% in IC-4687GBM and 19% in IC-3752GBM cells at day 13, indicating the high selectivity of SYC-435 of mutant over wild type IDH1. Systematic in vivo treatment with SYC-435 alone did not alter survival times in neither IC-BT142AOA nor IC-V0914AOA models when compared with the control group. Although standard therapy significantly prolonged animal survival times in both models (P<0.0005), combining SYC-435 with standard therapies further extended the median survival times from 106 days (in the standard therapy group) to 124 days (P<0.05) in IC-V0914AOA and exhibited similar trend in IC-BT142AOA. Conclusion: SYC-435 possesses antitumor effects that are highly selective in IDH1 mutant gliomas, and generated strong synergistic activities with standard therapies in vivo. Our data support the clinical testing of SYC-435 in patients with IDH1 mutant glioma. Citation Format: Mari Kogiso, Lin Qi, Huiyuan Zhang, Frank K. Braun, Yuchen Du, Yulun Huang, Holly Lindsay, Sibo Zhao, Sarah G. G. Injac, Zhen Liu, Patricia A. Baxter, Jack M. Su, Laszlo Perlaky, D. Williams Parsons, Murali Chintagumpala, Adekunle Adesina, Jialiang Wang, Yongcheng Song, Xiao-Nan Li. Mutant isocitrate dehydrogenase 1 (IDH1) inhibitor synergistically prolongs animal survival with standard therapies in patient-derived IDH1 mutant glioma xenograft mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4031. doi:10.1158/1538-7445.AM2017-4031

Abstract LB-063: Anti-tumor activity of the novel dual p70S6KAKT inhibitor M2698 in glioma stem cell mouse models

Abstract The efficacy of the dual p70S6K/AKT inhibitor M2698 was evaluated in a panel of patient-derived glioma stem cell (GSC) lines. The IC50 was determined from a 7-dose cell viability assay using Celltiter Glo. GSCs were classified as sensitive (IC50 ≤ 1µM) or resistant (IC50 >1µM). Two sensitive cell lines (GSC17 and GSC231) and two resistant cell lines (GSC272 and GSC20) were selected for in vivo experiments. To determine the efficacy of M2698 in orthotopic models of glioblastoma, GSCs were implanted into the basal ganglia of nude mice. After four days, mice were treated with vehicle (control) or M2698 at 20 mg/kg daily M-F. In the cell line GSC231 (sensitive in vitro), the median animal survival time was 47 days in the control-treated mice and 62 days in the M2698-treated mice (p=0.0288). The tumor volumes at 6, 7.5, 10 weeks were 4.98 ± 6.17 mm3, 48.48 ± 10.57 mm3, and 61.38 ± 28.00 mm3, respectively, in the control-treated mice and 0.33 ± 0.45 mm3, 9.96 ± 6.89 mm3, and 18.00 ± 5.59 mm3, respectively, in the M2698 -treated mice (p=0.322, p<0.001, and p=0.05, respectively). To determine the pharmacodynamic effect of the drug in mouse xenografts, the phospho-S6 (P-S6) expression level was evaluated over time (7.5 and 10 weeks). At 7.5 weeks, the P-S6 staining was 11.65 ± 1.17% per field in the control group and 2.61 ± 1.21% per field in the M2698 alone treated group (p<0.004). At 10 weeks , the P-S6 staining was 13.93 ± 3.46% per field in the control group and 3.10 ± 1.23% per field in the M2698 alone treated group (p=0.020). In the GSC272 (resistant line) xenograft mouse model, the median survival time was 72 days in the control-treated mice and 76 days in the M2698-treated mice. Although this difference was statistically significant (p=0.0022), the 4 day improvement in survival was minimal compared to the sensitive line GSC231. The tumor volumes at 6, 7.5, 10 weeks were 7.48 ± 4.10 mm3, 126.37 ± 41.98 mm3, and 158.79 ± 39.53 mm3, respectively in the control mice and 0.54 ± 0.47 mm3, 8.62 ± 3.88 mm3, and 64.3 ± 5.55 mm3, respectively in the M2698-treated mice (p=0.014, p<0.001, and p=0.008, respectively). The P-S6 expression level at 6 weeks, 7.5 weeks, and 10 weeks was 4.27 ± 3.27% per field, 13.83 ± 4.13% per field, and 22.3 ± 7.67% per field, respectively, in the control- treated mice and 1.40 ± 1.06% per field, 4.30 ± 2.26% per field, and 5.13 ± 1.02% per field, respectively, in the M2698-treated mice (p=0.302, p=0.005, and p=0.036, respectively). GSC17 and GSC20 lines that were resistant to M2698 were further evaluated to elucidate potential mechanisms of resistance to p70S6K/AKT inhibitor treatment. Reverse Phase Protein Array (RPPA) assay was performed 24 hours after treatment with M2698 in GSC17 and GSC20 in vitro. M2698 successfully prevented S6 phosphorylation, but also resulted in an increase in P-ERK1/2 and P-MAPK expression suggesting compensatory activation of the Ras/MEK/ERK signaling pathway. To validate these results in vivo, we performed immunohistochemistry staining of P-S6 and P-ERK1/2 in tumor tissue from the GSC17 xenograft model at the latest time points. The data revealed that tumors from M2698-treated mice had lower levels of P-S6 expression and high level of P-ERK1/2 expression compared with the untreated control group. These results suggest that tumors not responsive to p70S6K/AKT inhibitor monotherapy might benefit from combination treatment with a MEK inhibitor. Citation Format: yuji piao, Craig Thomas, Verlene Henry, Ningyi Tiao, Soon Young park, Martinez-ledesma Juan, Jianwen Dong, John frank de groot. Anti-tumor activity of the novel dual p70S6KAKT inhibitor M2698 in glioma stem cell mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-063. doi:10.1158/1538-7445.AM2017-LB-063

Quercetin suppresses the metastatic ability of lung cancer through inhibiting Snail-dependent Akt activation and Snail-independent ADAM9 expression pathways

Metastasis is the major cause of death from lung cancer. Quercetin, a widely distributed bioflavonoid, is well known to induce growth inhibition in a variety of human cancer cells, but how it affects lung cancer cell invasion and metastasis is unclear. Herein, we found that quercetin inhibited the migration/invasion of non-small cell lung cancer (NSCLC) cell lines and bone metastasis in an orthotopic A549 xenograft model by suppressing the Snail-mediated epithelial-to-mesenchymal transition (EMT). Moreover, survival times of animals were also prolonged after quercetin treatment. Mechanistic investigations found that quercetin suppressed Snail-dependent Akt activation by upregulating maspin and Snail-independent a disintegrin and metalloproteinase (ADAM) 9 expression pathways to modulate the invasive ability of NSCLC cells. In clinical samples, we observed that patients with Snailhigh/p-Akthigh tumors had the shortest survival times. In addition, a lower survival rate was also found in ADAM9high patients than in ADAM9low patients. Overall, our results provide new insights into the role of quercetin-induced molecular regulation in suppressing NSCLC metastasis and suggest that quercetin has potential therapeutic applications for metastatic NSCLC.

Multilayer photodynamic therapy for highly effective and safe cancer treatment.

Photodynamic therapy (PDT) is increasingly used as a minimally invasive, controllable and effective therapeutic procedure for cancer treatment. However, complete eradication of tumor cells by PDT alone remains challenging. In this study, we investigate the potential of multilayer PDT in cancer treatment with high efficacy and low side effects. Through PDT targeting two cancer biomarkers located at distinct subcellular localizations, remarkable synergistic effects in cancer cell killing and tumor inhibition were observed in both in vitro and in vivo experiments. This strategy may be widely applied to treat various cancer types by using strategically designed PDT photosensitizers that target corresponding upregulated receptors at tactical subcellular localization.

Cardiac prosthesis as an advanced surgical therapy for end-stage cardiac patients: current status and future perspectives.

This paper reviews the current status and future perspectives of the artificial heart research that was started in 1957 by Akutsu and Kolff. During the 1960's, although not much progress was made in increasing animal survival time with artificial hearts, clinical applications were already made for both a ventricular assist device in 1962 and total artificial heart (TAH) in 1969 followed by a second TAH application in 1981. Both TAH applications were done as bridges to heart transplantation. Meanwhile, the animal survival time improved during the 1970's because of the availability of better biomaterials, better understanding of the circulatory system, and improvement in surgical techniques. Continuous flow pumps were also investigated during the 1970's, which demonstrated feasibility for chronically supporting circulation in healthy animals. Four permanent cases of TAH application were done early 1980's for patients who could not be the candidates for heart transplantation. Although the patients were tethered to the external drive-console, one of them survived for nearly two years. Complications due to thromboembolism and infection were the major causes of death in these patients. The patients' quality of life was questionable and the permanent application of the TAH was then stopped to make improvements in the system in terms of implantability and biocompatibility. During the 1980's, efforts were then switcthed to development of totally implantable VAD and TAH systems, which led to the first discharge of a VAD patient from the hospital in 1992. In the early 1990's, implantable electric VADs, Novacor and ThermoCardio System (TCI), became available to support the circulation of end-stage cardiac patients until a donor heart could be found. The transplantation rate of the VAD patients ranged around 70% with the average waiting time of 80 to 100 days. The number of patients transplanted with VADs are more than 5000 and those with the pneumatic TAH exceed 200. Because of the larger size, requirement of heart valves, and complicated control mechanism of the pulsatile VADs, continuous flow pumps have been gaining popularity among clinicians. The clinical applications of the continuous flow devices have just begun, demonstrating remarkable performance in bridging to heart transplantation. In, the 21st century, we will see prevalent clinical applications of various circulatory support devices from pulsatile VAD, pulsatile TAH to continuous flow VAD. These devices will be combined with genetic treatment to re-generate the myocardium and recover the failing heart. Complete recovery of the myocardium may become possible through therapy combining circulatory assist devices and biotechnology.

TIGAR knockdown radiosensitizes TrxR1-overexpressing glioma in vitro and in vivo via inhibiting Trx1 nuclear transport

The up-regulation of thioredoxin reductase-1 (TrxR1) is detected in more than half of gliomas, which is significantly associated with increased malignancy grade and recurrence rate. The biological functions of NADPH-dependent TrxR1 are mainly associated with reduced thioredoxin-1 (Trx1) which plays critical roles in cellular redox signaling and tumour radio-resistance. Our previous work has proved that TP53 induced glycolysis and apoptosis regulator (TIGAR) knockdown could notably radiosensitize glioma cells. However, whether TrxR1-overexpressing glioma cells could be re-radiosensitized by TIGAR silence is still far from clear. In the present study, TrxR1 was stably over-expressed in U-87MG and T98G glioma cells. Both in vitro and in vivo data demonstrated that the radiosensitivity of glioma cells was considerably diminished by TrxR1 overexpression. TIGAR abrogation was able to radiosensitize TrxR1-overexpressing gliomas by inhibiting IR-induced Trx1 nuclear transport. Post-radiotherapy, TIGAR low-expression predicted significant longer survival time for animals suffering from TrxR1-overexpessing xenografts, which suggested that TIGAR abrogation might be a promising strategy for radiosensitizing TrxR1-overexpressing glial tumours.

Repurposing Mebendazole as a Replacement for Vincristine for the Treatment of Brain Tumors

The microtubule inhibitor vincristine is currently used to treat a variety of brain tumors, including low-grade glioma and anaplastic oligodendroglioma. Vincristine, however, does not penetrate well into brain tumor tissue, and moreover, it displays dose-limiting toxicities, including peripheral neuropathy. Mebendazole, a Food and Drug Administration-approved anthelmintic drug with a favorable safety profile, has recently been shown to display strong therapeutic efficacy in animal models of both glioma and medulloblastoma. Importantly, appropriate formulations of mebendazole yield therapeutically effective concentrations in the brain. Mebendazole has been shown to inhibit microtubule formation, but it is not known whether its potency against tumor cells is mediated by this inhibitory effect. To investigate this, we examined the effects of mebendazole on GL261 glioblastoma cell viability, microtubule polymerization and metaphase arrest, and found that the effective concentrations to inhibit these functions are very similar. In addition, using mebendazole as a seed for the National Cancer Institute (NCI) COMPARE program revealed that the top-scoring drugs were highly enriched in microtubule-targeting drugs. Taken together, these results indicate that the cell toxicity of mebendazole is indeed caused by inhibiting microtubule formation. We also compared the therapeutic efficacy of mebendazole and vincristine against GL261 orthotopic tumors. We found that mebendazole showed a significant increase in animal survival time, whereas vincristine, even at a dose close to its maximum tolerated dose, failed to show any efficacy. In conclusion, our results strongly support the clinical use of mebendazole as a replacement for vincristine for the treatment of brain tumors.

Pathophysiological features of rat hemorrhagic shock under high temperature conditions

Objective To investigate the pathophysiological features of rat hemorrhagic shock under high temperature conditions. Methods A total of 128 SD rats were assigned to high temperature group and normal temperature group according to the random number table, with 64 rats per group. Rats in high temperature group were pretreated at 34℃ for 12 h, and in normal temperature group were kept at 25℃. Hemorrhagic shock models in rats were produced by withdrawing 40% of the total blood volume via the femoral artery. Parameters of the two groups were measured including blood electrolytes (Na+ , K+ ), plasma osmotic pressure, liver function [aspartate aminotransferase (AST), alanine aminotransferase (ALT)], kidney function [creatinine (CREA)], arterial blood gases (pH, PO2 and PCO2) and cardiac function [heart rate (HR), cardiac output (CO), cardiac index (CI) and DO2]. Animal survival time and survival rate were detected. Results Before shock, high temperature group versus normal temperature group showed higher detections of Na+ concentration [(142.3±2.2)mmol/L ∶(139.1±1.5)mmol/L], plasma osmotic pressure [(304.8±4.7)mmol/L ∶(300.0±1.9)mmol/L], HR [(462±30)times/min ∶(402±44)times/min], CO [(0.892±0.190)L/min ∶(0.713±0.090)L/min] and CI [(0.0030±0.0006)L·min-1·cm-2 ∶(0.0023±0.0002)L·min-1·cm-2] (P<0.05). After shock, normal temperature group showed further increased concentrations of Na+ and K+ and significantly enhanced AST, ALT and CREA compared to normal temperature group (P<0.05). After shock, CO, CI and DO2 in high temperature group were decreased by 63%, 63% and 69% respectively compared to those before shock, but less decrease was observed in normal temperature group. Rat survival rate and survival time were significantly reduced in high temperature group compared to normal temperature group (P<0.05). Conclusion Pathophysiological changes of hemorrhagic shock in rats under high temperature are mainly manifested as impaired cardiac function, significantly increased concentrations of Na+ and K+ and significantly shortened survival time. Key words: Hyperthermia, induced; Shock, hemorrhagic; Homeostasis

Anterograde and retrograde tracing with high molecular weight biotinylated dextran amine through thalamocortical and corticothalamic pathways.

Biotinylated dextran amine (BDA) has been used for neural pathway tracing in the central nervous system for many decades, in which high molecular weight BDA appeared to be transported predominantly in the anterograde direction and less in the retrograde direction. In the current study, we reexamined the properties of neural labeling with high molecular weight BDA through a reciprocal neural pathway between thalamus and somatosensory cortex. After injection of BDA into the ventral posteromedial nucleus of thalamus (VPM) in the rat, the BDA labeling was sequentially examined on somatosensory cortex at 3, 5, 7, 10, and 14 survival days. Both of anterogradely labeled axonal terminals and retrogradely labeled neuronal cell bodies were observed simultaneously on the somatosensory cortex. With the increasing of survival times after injection, morphological changes occurred on the labeled axonal arbors and neuronal dendrites, in which the high quality of BDA labeling appeared on the tenth survival day. These results indicate that high molecular weight BDA is not only a sensitive anterograde tracer but also an excellent retrograde marker to be used for tracing through thalamocortical and corticothalamic pathways. And the detailed structure of neural labeling with BDA similar to Golgi-like resolution can be obtained at optimal survival times of animals after the injection of high molecular weight BDA.

Establishment of a new acute-on-chronic liver failure model

To establish an animal model of acute-on-chronic liver failure (ACLF) that would replicate the pathological process of ACLF in humans, rats were administered porcine serum (PS) for 11 weeks. Liver fibrosis was determined by pathological and biochemical assessments. The animals then were injected with d-galactosamine (d-gal) and lipopolysaccharide (LPS). The survival times of animals with cirrhosis and ACLF were determined over 48h. Other animals were killed at 0, 4, 8 and 12h after administration of d-gal/LPS. Liver injury was assessed by histopathological analysis and biochemical indices, and apoptosis was detected by Western blot and TUNEL analysis. After PS administration for 11 weeks the serum levels of hyaluronic acid and N-procollagen type III peptide increased significantly, and serious fibrosis and cirrhosis was observed at weeks 10 and 11. Cirrhotic rats were injected with d-gal/LPS to induced ACLF; the rate of mortality over 48h was 80%. ALT and AST levels increased markedly at 4h, but decreased significantly at 8 and 12h post-treatment. The total bilirubin, direct bilirubin, and total bile acids levels increased markedly at 8 and 12h. Clotting times, TNF-α and IL-6 levels increased significantly, except for 12h post-treatment. Apoptosis, inflammation and necrosis were elevated as determined by hematoxylin-eosin staining and TUNEL assays. BCL-2 levels decreased significantly, While BAX levels increased significantly. Cytochrome c expression peaked at 8h post-d-gal/LPS treatment. In conclusion, an ACLF model induced by PS and d-gal/LPS was established and the underlying mechanisms of ACLF development were explored.

Extraction of biofilm produced by Escherichia coli that are isolated from animals infected with diarrhea and study its protective role

This study was conducted to detect the ability of E.coli isolated from diarrhea to produce biofilm and protection against diseases cause by these bacteria compared with whole cell sonicated antigen. One hundred two fecal samples (52 fecal samples from cows, calves and 50 fecal samples from sheep,goat) were collected from College of veterinary medicine-university of Baghdad, College of Agriculture-university of Baghdad, Dora zone and Abu-Ghraib zone. Samples were cultured on MacConkey and Eosin Methylene Blue agar and after purification of cultured bacteria, biochemical tests, API 20 E System and RapIDTM ONE System kit were done. Results showed that 91 out of 102 fecal samples have the characteristics belong to E.coli ,the ability of these isolates to produce biofilm were detected and the results showed that 38 out of 49 E.coli isolates from fecal samples of cow produce biofilm (77.55%)and 39 out of 42 E.coli isolates from fecal samples of sheep produce biofilm (92.85%) with different thickness ranged between (0.2-2)mm, while 11 isolates from 49 fecal samples of cow and calves and 39 isolates from 42 fecal samples of sheep had not produce biofilm. The results showed that protein concentration of biofilm was 92 mg/ml for one isolate and 70 mg/ ml for the whole sonicated antigen of the bacteria that produce biofilm. Three types of bacterial antigens were prepared as follow; Whole cell sonicated antigen (WCA) of biofilm producer E.coli, biofilm extract antigen (biA) with protein concentration 3.5 mg/ml and biofilm extract antigen (biA) with protein concentration 14 mg/ml, then these antigens were injected in 50 healthy White BALB mice. Results showed that survival time of animals in the immunized group with biofilm antigen of high protein concentration (14 mg/ml) was longer (652.8 hrs) than animals immunized with whole sonicated antigen of protein concentration 3.5 mg/ml antigen (378 hrs ) and than the group with biofilm antigen of protein concentration 3.5 mg/ml (513.6 hrs) , heavy bacterial isolation were recorded in the internal organs of the immunized infected animals at (12-48) hours post infection, while moderate bacterial isolation at day 30 post infection. Histopathological examination showed large abscess which caused adhesion of liver with stomach and spleen with stomach and pancreas surround by dense cellular fibrous tissue, the result showed that all animals of control positive group,5 animals of group injected with(WCA) produce biofilm,3 animals of group injected with (biA) 3.5 mg/ml and 1 animal of group injected with (biA) 14 mg/ml were died during 12-48 hours post infection, acute suppurative reaction were seen in internal organs of animals died at 12-24 hours post infection, granulomatous lesions were seen in most internal organs of animals in group 2 , 3 and 4 at day 30 post challenge, while mild inflammatory reaction was recorded in most internal organs of group 4 at day 30 post challenge.

Effects of Different Crystalloid Solutions on Hemodynamics, Peripheral Perfusion, and the Microcirculation in Experimental Abdominal Sepsis.

Crystalloid solutions are used to restore intravascular volume in septic patients, but each solution has limitations. The authors compared the effects of three crystalloid solutions on hemodynamics, organ function, microcirculation, and survival in a sepsis model. Peritonitis was induced by injection of autologous feces in 21 anesthetized, mechanically ventilated adult sheep. After baseline measurements, animals were randomized to lactated Ringer's (LR), normal saline (NS), or PlasmaLyte as resuscitation fluid. The sublingual microcirculation was assessed using sidestream dark field videomicroscopy and muscle tissue oxygen saturation with near-infrared spectroscopy. NS administration was associated with hyperchloremic acidosis. NS-treated animals had lower cardiac index and left ventricular stroke work index than LR-treated animals from 8 h and lower mean arterial pressure than LR-treated animals from 12 h. NS-treated animals had a lower proportion of perfused vessels than LR-treated animals after 12 h (median, 82 [71 to 83] vs. 85 [82 to 89], P = 0.04) and greater heterogeneity of proportion of perfused vessels than PlasmaLyte or LR groups at 18 h. Muscle tissue oxygen saturation was lower at 16 h in the NS group than in the other groups. The survival time of NS-treated animals was shorter than that of the LR group (17 [14 to 20] vs. 26 [23 to 29] h, P < 0.01) but similar to that of the PlasmaLyte group (20 [12 to 28] h, P = 0.74). In this abdominal sepsis model, resuscitation with NS was associated with hyperchloremic acidosis, greater hemodynamic instability, a more altered microcirculation, and more severe organ dysfunction than with balanced fluids. Survival time was shorter than in the LR group.

Effect of the autonomic nervous system on cancer progression depends on the type of tumor: solid are more affected then ascitic tumors.

A number of recently published studies have shown that the sympathetic nervous system may influence cancer progression. There are, however, some ambiguities about the role of the parasympathetic nerves in the modulation of growth of different tumor types. Moreover, tumor models used for investigation of the autonomic neurotransmission role in the processes related to the cancer growth and progression are mainly of the solid nature. The knowledge about the nervous system involvement in the modulation of the development and progression of malignant ascites is only fragmental. Therefore, the aim of the present article was to summarize the results of our experimental studies focused on the elucidation of the role of the autonomic nervous system in the modulation of tumor growth in animals. We are summarizing data from studies, in which not only different experimental approaches in order to influence the autonomic neurotransmission, but also different tumor models have been used. Three different types of tumor models, namely solid rat intra-abdominal fibrosarcoma, solid murine subcutaneous melanoma, and rat ascites hepatoma, and three types of interventions have been used in order to modulate the autonomic neurotransmission, specifically chemical sympathectomy, subdiaphragmatic vagotomy, or the electric stimulation of the vagus nerve. We have proved a strong stimulatory effect of the sympathetic nerves on the development and growth in both solid tumors, rat fibrosarcoma as well as murine melanoma, and significant inhibitory impact on the survival time of tumor-bearing animals. The progression of ascites hepatoma in rats was not influenced by chemical sympathectomy. Modulation of parasympathetic signalization by vagotomy or vagal nerve stimulation does not affect fibrosarcoma and ascites hepatoma growth and survival of the tumor-bearing rats. Based on the obtained data, it seems that the solid types of tumors are suitable substrate for the direct action of neurotransmitters released especially from the sympathetic nerves. In contrast, it appears that the malignant ascites are not under the direct autonomic nerves control; however, an indirect action via the immune functions modulation cannot be excluded.

Combination of p53 and Rb in the local nano-therapy for rabbit VX2 model of hepatic metastasis carcinoma: the curative effect and safety evaluation

Objective Combination of nanoparticle with p53 and Rb gene therapy by gene targeting was applied to investigate its curative effect and safety evaluation on colorectal rabbit hepatic VX2 metastasis for tumor eradication and survival enhancement. Methods Recombinant expressing plasmids harboring wild type p53 and Rb were cotransferred or transferred separately to the rabbit hepatic VX2 metastasis by the emulsion of PLL-nHAP nanoplex and lipodiol through the hepatic artery in a tumor target manner. Subsequent co-expressions of p53 and Rb protein within the treated tumors were detected by Western blot and in situ analysis of confocal laser scanning microscope. The therapeutic effect was evaluated by the tumor growth velocity and the survival time of animals. Eventually, investigations of liver function were applied to evaluate the safety of the process. Results With safe procedure for the rabbits liver function, both p53 and Rb local nano-therapy showed favorable anti-tumor effects and increased animal survival time. p53+ Rb local nano-therapy could significantly inhibit hepatic VX2 metastasis and enhance the animal survival time compared with p53 local nano-therapy or Rb local nano-therapy. Local nano-therapy showed no significant influence to animal liver function. Conclusions Rb can work synergistically with p53 in the combined therapy mediated by PLL-nHAP nanoplex to augment the anti-tumor effect. The local nano-therapy with p53 and Rb is likely to be an effective and safe anti-tumor therapy for hepatic colorectal metastasis. Key words: Nanoparticles; Transfection; Neoplasm hepatic metastasis; Rabbits

Novel trypanocide from an extract of Pleurotus sajor-caju against Trypanosoma congolense

Context: Control of African trypanosomiasis relies on chemotherapy, but the development of resistance and the problem of drug residues require research for alternatives. Triterpenes and phenolics, the major constituents of Pleurotus sajor-caju (Fr.) Singer (Pleurotaceae), are reported to be effective against trypanosomiasis.Objective: Trypanocidal effect of whole Pleurotus sajor-caju aqueous extract was investigated in vivo against Trypanosoma congolense.Materials and methods: Mice (25–32 g) were divided into seven groups of six animals. Mice in groups A–F received 2.5 × 104 trypanosomes, while group G was uninfected. Extracts (100–250 mg/kg) were administered intraperitoneally for 5 days to groups A–D while diminazine aceturate (group E) and normal saline (group F) served as positive and negative controls, respectively. Parasitemia, survival time, body weight and haematological parameters were monitored for 60 days post-treatment.Results: Parasitemia decreased significantly (p < 0.01) post-treatment with 200 and 250 mg/kg of the extract and became undetectable by day 16 and 12 post-infection, respectively; the ED50 was 221.5 mg/kg. The packed cell volume (PCV) and the weight of mice treated with 250 mg/kg extract were 46.20 ± 2.6% and 32.05 ± 3.63 g, respectively, which is higher than the group treated with diminazine aceturate. The mean survival time of animals in groups D and E was >60 days, while that of group F was <4 days. Differential leucocyte count on day 68 post-infection in groups C, D and E were not significantly different.Conclusion: Pleurotus sajor-caju therefore could be a potential source of new trypanocidal drugs.

The Vitamin B12 Analog Cobinamide Is an Effective Antidote for Oral Cyanide Poisoning.

Cyanide is a major chemical threat, and cyanide ingestion carries a higher risk for a supra-lethal dose exposure compared to inhalation but provides an opportunity for effective treatment due to a longer treatment window and a gastrointestinal cyanide reservoir that could be neutralized prior to systemic absorption. We hypothesized that orally administered cobinamide may function as a high-binding affinity scavenger and that gastric alkalinization would reduce cyanide absorption and concurrently increase cobinamide binding, further enhancing antidote effectiveness. Thirty New Zealand white rabbits were divided into five groups and were given a lethal dose of oral cyanide poisoning (50mg). The survival time of animals was monitored with oral cyanide alone, oral cyanide with gastric alkalinization with oral sodium bicarbonate buffer (500mg), and in combination with either aquohydroxocobinamide or dinitrocobinamide (250mM). Red blood cell cyanide concentration, plasma cobinamide, and thiocyanate concentrations were measured from blood samples. In cyanide ingested animals, oral sodium bicarbonate alone significantly prolonged survival time to 20.3±8.6min compared to 10.5±4.3min in saline-treated controls, but did not lead to overall survival. Aquohydroxocobinamide and dinitrocobinamide increased survival time to 64±41 (p<0.05) and 75±16.4min (p<0.001), respectively. Compared to aquohydroxocobinamide, dinitrocobinamide showed greater systemic absorption and reduced blood pressure. Dinitrocobinamide also markedly increased the red blood cell cyanide concentration. Under all conditions, the plasma thiocyanate concentration gradually increased with time. This study demonstrates a promising new approach to treat high-dose cyanide ingestion, with gastric alkalinization alone and in combination with oral cobinamide for treating a supra-lethal dose of orally administered cyanide in rabbits.

Abstract 2668: The exosomes derived from BM-hMSCs home to glioma and deliver synthetic microRNA mimics after systemic administration

Abstract Glioblastoma is the most malignant type of glioma (World Health Organization grade 4) and has a median survival time of only 14.6 months even after the standard treatment of surgical resection followed by radiotherapy with concomitant and adjuvant temozolomide administration. Although some novel and promising anti-glioma agents have been developed, therapeutic advancement has remained disappointing due to the lack of efficient delivery tools. Here we focused on exosomes derived from mesenchymal stem cells (MSCs) as carriers to deliver biological therapeutic agents for the treatment of gliomas. These exosomes are expected to cross the blood-brain barrier and selectively home to the glioma tissue, similar to the parental cells (MSCs). Additionally, MSCs could produce sufficient amounts of exosomes on a clinically applicable scale. Thus, MSC exosomes present a potentially ideal cell-free delivery vehicle for glioma therapy. We hypothesized that exosomes derived from human bone marrow-derived MSCs (BM-hMSCs) can target gliomas and can be used as delivery vehicles following systemic administration. In this study, we employed microRNA (miR-128) as the therapeutic cargo, and exosomes were isolated from a cultured media of BM-hMSCs using ultracentrifugation. At first, exosomes labeled with fluorescent dye were incubated with glioma cells, and exosomes taken up by glioma cells were evaluated by confocal fluorescence microscopy and flow cytometry. Labeled exosomes were also systemically injected into a glioma xenograft model and the ability to home to glioma tissue was evaluated by an in vivo imaging system and histological study. The results showed that MSC exosomes can be taken up by glioma cells and can home to glioma tissues. Next, we cultured miR-128-enriched MSC exosomes with glioma cells. To check the function of miR-128 delivered to glioma cells by MSC exosomes, the down-regulation of the BMI-1 target gene of miR128 was evaluated using real-time polymerase chain reaction and western blot analyses. We also systemically injected these exosomes into glioma xenograft models and evaluated the function of miR-128 by analyzing the downregulation of BMI-1 at the tumor site and the animal survival time in vivo. We confirmed that miR-128 delivered by MSC exosomes could function at the target site both in vitro and in vivo. These results indicate that MSC exosomes may be an ideal delivery vehicle for the treatment of gliomas, especially for microRNA replacement therapy. Citation Format: Shinji Yamashita, Tal Shahar, Anwar Hossain, Brittany Parker Kerrigan, Joy Gumin, Shoudong Li, Feng Gao, Kouji Yamasaki, Yuzaburo Shimizu, Frederick F. Lang. The exosomes derived from BM-hMSCs home to glioma and deliver synthetic microRNA mimics after systemic administration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2668.