Orthotopic model of SHG-44 in the enhanced green fluorescent protein nude mouse.

Naturally fluorescent proteins have been widely used in biological research. In this study, we found that the simple and effective way to obtain enhanced green fluorescent protein (EGFP) nude mice is to cross transgenic EGFP C57BL/6J mice with nude (nu/nu) mice. EGFP expression is identified by tail genotyping. Establishment of the orthotopic EGFP nude mouse model used surgical orthotopic implantation. The morphology and human glioma cell markers, such as glial fibrillary acidic protein (GFAP) and S-100, remain unchanged in this mouse model. The tumor blood vessels obtained from the orthotopic model show brilliant EGFP fluorescence as observed by fluorescence microscopy. These findings suggested that this is an ideal mouse model with which to study interaction among host, tumor, and tumor microenvironment; the findings also suggested that the host (EGFP nude mouse) was involved in tumor angiogenesis.

Rapamycin induces autophagy and exacerbates metabolism associated complications in a mouse model of type 1 diabetes.

Type 1 diabetes mellitus (T1DM) is characterized by lack of insulin production as a consequence of massive beta cells destruction. The contributions of autophagy to loss of beta cell mass were not clearly elucidated. Rapamycin is a specific and potent inhibitor of mammalian target of rapamycin (mTOR) and is used as the central immunosuppressant in T1DM patients especially for those who received islet transplantation. In the present study, effects of rapamycin on autophagy of T1DM were investigated in a mouse model treated with multiple low doses of streptozotocin. Rapamycin treatment led to hyperglycemia, weight loss, increased intake of food and drinking water, and islet inflammation in T1DM mice. Pathological changes including autophagy and apoptosis in pancreas, kidney, spleen and thymus, accompanied with an accumulation of LC3-II, Beclin1 and Caspase-3 protein were observed. The results indicate that rapamycin may exacerbate metabolism associated complications by activating autophagy and apoptosis in T1DM.

Pharmacokinetics and metabolism of lithospermic acid by LC/MS/MS in rats.

The pharmacokinetics and metabolism of lithospermic acid (LA), a component isolated from Salvia miltiorrhiza, and its two O-methylated metabolites (3'-monomethyl- and 3',3''-dimethyl-lithospermic acid), were analyzed by a rapid and specific isocratic liquid chromatography-tandem mass spectrometry (LC/MS/MS) method. Rat serum samples collected after intravenous and oral administration were analyzed for obtaining pharmacokinetic data of LA. Two O-methylated metabolites, namely one 3'-monomethyl- and one 3',3''-dimethyl-lithospermic acid were detected in rat serum and bile samples after intravenous and oral administration of LA, respectively. An oral bioavailability of 1.15% was found, with the AUC(0-t) values of 301.89 and 3.46mgh/L for intravenous and oral administration, respectively. The total recovery from bile was 75.36% (0.46% for LA, 17.23% for M1, and 57.67% for M2) after intravenous administration, and 4.26% (0.00% for LA, 0.10% for M1, and 4.16% for M2) after oral administration. These results indicate that methylation is the main metabolic pathway of LA, and that LA is excreted into rat bile and finally into feces.

[Transplantation of human glioma stem cells in nude mice with green fluorescent protein expression].

OBJECTIVES: To investigate the possibility of transplantation of human glioma stem cells (HGSCs) in nude mice stably expressing green fluorescent protein (GFP) so as to clearly identify the incubated HGSCs from the host tissues. METHODS: Transgenic C57BL/6J mice expressing GFP was crossed with nude mice of the line NC, then hairless male nude mice expressing GFP were crossed with hairy female pubescent mice to obtain nude mice with GFP expression the expression of GFP in the skin and organs of these nude mice were evaluated by naked eyes, and immunohistochemical and immunofluorescence assays. HGSCs were transplanted orthotopically into the caudate nuclei of nude mice expressing GFP. Immunohistochemistry was used to observe the transplanted tumor. RESULTS: The structures rich in adipose tissue of the 8th generation nude mice were dark green and the other organs were light green. However, green fluorescence was emitted from all tissues under fluorescence microscopy. Confocal fluorescence microscopy showed that the tumor cells were stained red, distinguished from the host cells distinctly in the brains bearing tumor transplanted orthotopically. CONCLUSION: Nude mice expressing GFP can be obtained by crossing the transgenic mice bearing naive immunity with nude mice. Orthotopic transplantation of HGSCs may be used in the investigation of tumor tissue reconstitution because of the easy identification between the transplantation tumor and host tissue.

[Immune response and immunopathology in inducible costimulatory molecule (ICOS) transgenic mice infected with Schistosoma japonicum].

OBJECTIVE: To establish the ICOS transgenic mice schistosomiasis japonica model and to observe the immune response and immunopathology of the model. METHODS: The transgenic mice were infected with Schistosoma japonicum. Spleen cells and sera of mice were harvested at week 4, 6, and 8 after infection. The cytokines IFN-gamma and IL-4 were measured in culture supernatans by ELISA. The serum IgG, IgGI and IgG2a were measured by ELISA at different period of infection. Liver tissue sections were prepared with HE staining. Liver granuloma formation was observed under microscope. RESULTS: The expression level of IFN-y showed no significant difference between ICOS transgenic mice and control, while that of IL-4 in ICOS transgenic mice was significantly up-regulated to 20.81+/-1.95 and 25.31+/-3.37 pg/ml at week 6 and 8 respectively (P<0.01). The serum IgG and IgGl in ICOS transgenic mice were also significantly higher than those in control. Th2 differentiation index and lgC1/IgG2a were used to evaluate the immune regulation balance of Thl/Th2, and results showed that Th2 response in ICOS transgenic mice was significantly stronger than that of the control. The egg granuloma response in ICOS transgenic mice was also significantly stronger than that in control (P<0.01). The rate of egg granuloma enlargement was 24.48% and 26.37% at week 6 and 8 respectively. CONCLUSION: The findings suggest that there is stronger Th2 type response in ICOS transgenic mice infected with Schistosoma japonicum and ICOS may play an important role in the egg granuloma formation of Schistosoma japonicum.

Vitamin D induces autophagy of pancreatic ß-cells and enhances insulin secretion.

Epidemiological evidence indicates that vitamin D is involved in defense against diabetes; however, the precise underlying mechanism remains to be elucidated. In the present study, the effect of vitamin D on the pathogenesis of diabetes was investigated, with an emphasis on its direct effect on pancreatic ß­cells. A streptozotocin (STZ)­induced type 1 diabetes mellitus (T1DM) mouse model and MIN6 mouse insulinoma ß­cells were subjected to vitamin D treatment. Histopathological analysis of pancreatic islets was performed to investigate insulitis, and reverse transcription-quantitative polymerase chain reaction and western blotting were used to determine the mRNA and protein expression levels of markers of autophagy [microtubule-associated protein 1A/1B­light chain 3 (LC3) and Beclin 1] and regulation of apoptosis [B-cell lymphoma 2 (Bcl-2)]. Apoptosis of MIN6 cells was examined by flow cytometry following annexin V/propidium iodide labeling. The secretion of insulin was measured by ELISA. The results revealed that vitamin D reduced the incidence of T1DM, enhanced insulin secretion and relieved pancreatic inflammation in STZ­treated mice. Furthermore, vitamin D increased the mRNA expression levels of LC3 and Beclin 1, and increased Bcl­2 protein expression levels in STZ­treated MIN6 cells, while decreasing the apoptosis rate. The results of the present study demonstrated, for the first time to the best of our knowledge, that vitamin D induces autophagy and suppresses apoptosis of pancreatic ß­cells, as well as preventing insulitis. These findings regarding vitamin D provide insights into its involvement in diabetes, and suggest a potential novel strategy for the treatment of diabetes via agents enhancing autophagy in pancreatic ß-cells.

Adeno-associated virus-mediated survivin mutant Thr34Ala cooperates with oxaliplatin to inhibit tumor growth and angiogenesis in colon cancer.

Colon cancer is one of the most common cancers. Survivin is overexpressed in human colon cancer and correlate with chemoresistance, angiogenesis and poor prognosis. Oxaliplatin, a platinum derivative cancer drug, has been used for treating human colorectal cancers. In the present study, we investigated the effect of the adeno-associated virus (AAV)-mediated survivin mutant Thr34Ala [rAAV-Sur-Mut(T34A)] on colon cancer growth. Infection with rAAV-Sur-Mut(T34A) inhibited cell proliferation, induced apoptosis and mitotic catastrophe, and sensitized colon cancer cells to chemotherapeutic drugs in vitro. Treatment with rAAV-Sur-Mut(T34A) significantly induced apoptosis, reduced angiogenesis and inhibited colon cancer growth in vivo. More importantly, rAAV-Sur-Mut(T34A) treatment strongly enhanced the anti-tumor activity of oxaliplatin and prolonged animal survival. Thus, the use of rAAV-Sur-Mut(T34A) in combination with chemotherapy may be a promising strategy for colon cancer therapy.

Anti-human FLT3 monoclonal antibody that inhibits proliferation of monocytic leukemia cell line SHI-1.

FLT3, a transmembrane molecule, was found on hematopoietic stem/progenitor cells and leukemia cells and determined to be a promising target in leukemia diagnosis and therapy. In this study a functional anti-human FLT3, monoclonal antibody (MAb) 10G6, was obtained and the specificity of this MAb was verified by flow cytometry. This MAb effectively recognized the FLT3 molecule expressed on a series of malignant cell lines. Furthermore, we demonstrated that MAb 10G6 inhibited the proliferation and migration ability and induced the apoptosis of SHI-1 cells that derived from a human monocytic leukemia. This functional anti-human FLT3 MAb provides a valuable tool for further study targeting the FLT3 on leukemia cells.

Anti-human CD133 monoclonal antibody that could inhibit the proliferation of colorectal cancer cells.

CD133, a five-transmembrane molecule, has been found on many types of cancers and determined to be a cancer stem cell biomarker. In this study a functional anti-human CD133 MAb 6B6 was obtained, and the specificity of this MAb was verified by flow cytometry. This MAb effectively recognized the CD133 molecule expressed on a series of malignant cell lines. Immunohistochemistry staining showed the CD133 was expressed on colorectal tumor tissue. Furthermore, we demonstrated that MAb 6B6 could inhibit the proliferation of Caco-2 cells that were derived from a human colorectal carcinoma. This functional anti-human CD133 MAb provides a valuable tool for further study of biological functions of cancer stem cell that expressed CD133.

Defibrillation and resuscitation in a piglet model of pediatric ventricular fibrillation following AHA 2005 guidelines.

OBJECTIVE: To evaluate the efficacy and safety of defibrillation on children according to AHA 2005 recommendations METHODS: Pig resembles human in the chest configuration, anatomy and physiology of the cardiovascular and pulmonary systems. Piglets weighing 7.0 Kg ± 1.4kg, 14.0kg ± 2.8kg, 25.0kg ± 5.0kg respectively, which represented children 1 to 8 yr old were induced ventricular fibrillation (VF). An adult biphasic AED was used in conjunction with pediatric attenuating electrodes which could deliver 50-J shock for 2 min and two min of cardiopulmonary resuscitation (CPR) immediately followed it. If VF did not reverse, 70-J shock combined with CPR was used, and the protocol was repeated five times. If an organized cardiac rhythm with mean aortic pressure more than 60 mmHg persisted for an interval of 5 minutes, the animal was regarded as successfully resuscitated. If the AED recognized a "non-shockable" rhythm, CPR was also performed immediately for 2 min. The same resuscitation program was exercised on piglets of manual defibrillator group. Neurologic alertness score, hemodynamic and myocardial functions were evaluated, autopsy was routinely performed to document possible injuries. RESULTS: In the AED group, 14 out of 15 animals, were successfully resuscitated, among them 11 piglets were resuscitated by 50-J defibrillation combined with cardiopulmonary resuscitation, and other three recovered to normal by 1 or 2 times of 70-J shocks and CPR. All animals in manual defibrillator group were successfully resuscitated by 50-J shocks and CPR. Left ventricular ejection fraction and fractional area change were reduced significantly during 3-4 hr post-resuscitation (P<0.05) and returned to baseline ranges at the end of 72 hr. There was no evidence of myocardial and pulmonary damage during autopsy, and neurologic recovery was also normal. Data of blood gas analysis, blood electrolytes and myocardial enzymes does not show any statistically significant difference (P> 0.05) in the groups. 50 J biphasic dose defibrillation combined with effective CPR, successfully terminated VF without adverse effects on myocardial function and survival in a piglet defibrillation model for young children 1 to 8 yr of age. CONCLUSIONS: The new guidelines recommendation that one shock immediately followed by CPR is reasonable. Adults AED combined with pediatric electrodes is feasible to the diagnosis and treatment of pediatric VF model. But the user should not rely too much on AED's "automatic" function, but should accumulate and integrate his experience with AED technology.