KMT2E Haploinsufficiency Manifests Autism-Like Behaviors and Amygdala Neuronal Development Dysfunction in Mice.

Autism spectrum disorders (ASD) are highly heterogeneous neurodevelopmental disorders characterized by impaired social interaction skills. Whole exome sequencing has identified loss-of-function mutations in lysine methyltransferase 2E (KMT2E, also named MLL5) in ASD patients and it is listed as an ASD high-risk gene in humans. However, experimental evidence of KMT2E in association with ASD-like manifestations or neuronal function is still missing. Relying on KMT2E+/- mice, through animal behavior analyses, positron emission tomography (PET) imaging, and neuronal morphological analyses, we explored the role of KMT2E haploinsufficiency in ASD-like symptoms. Behavioral results revealed that KMT2E haploinsufficiency was sufficient to produce social deficit, accompanied by anxiety in mice. Whole-brain 18F-FDG-PET analysis identified that relative amygdala glycometabolism was selectively decreased in KMT2E+/- mice compared to wild-type mice. The numbers and soma sizes of amygdala neurons in KMT2E+/- mice were prominently increased. Additionally, KMT2E mRNA levels in human amygdala were significantly decreased after birth during brain development. Our findings support a causative role of KMT2E in ASD development and suggest that amygdala neuronal development abnormality is likely a major underlying mechanism.

Al18 F labeled sulfonamide-conjugated positron emission tomography tracer in vivo tumor-targeted imaging.

An ideal positron emission tomography (PET) tracer should be highly extractable by the tumor tissue or organ that contains low toxicity and can provide high-resolution images in vivo. In this work, the aim was to evaluate the application of Al18 F-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid containing sulfonamide group (18 F-Al-NOTA-SN) as a potential tumor-targeting signal-enhanced radioactive tracer in PET. SN as a tumor-targeting group was incorporated to NOTA to make a ligand. Subsequently, this ligand reacted with Na18 F and AlCl3 to produce a compound 18 F-Al-NOTA-SN. This compound was further characterized and its property in regard to cell cytotoxicity assay, microPET imaging, biodistribution, cell uptake assay, and tumor selectivity in vitro and in vivo, was also investigated. 18 F-Al-NOTA-SN possessed low cell cytotoxicity and uptake to COS-7 and 293T healthy cells and high cell cytotoxicity and uptake to MDA-MB-231, HepG2, and HeLa tumor cells in vitro. Moreover, 18 F-Al-NOTA-SN showed good tumor-targeting property and high PET signal enhancement of HeLa tumors, liver, and kidneys in mice, as well as the uptake ratios of tumor to blood and tumor to muscle, were 4.98 and 3.87, respectively. 18 F-Al-NOTA-SN can be accepted to be kidney and liver eliminated earlier and show a potential tumor-targeting signal-enhanced radioactive tracer in PET.

Whole Body Positron Emission Tomography/Computed Tomography (PET/CT) in the Evaluation of Ophthalmic Tumors.

The clinical value of whole body positron emission tomography/computed tomography (PET/CT) as an imaging tool in diagnosis of ophthalmic tumors was investigated. The retrospective observational case series were performed on the patients with suspected ophthalmic tumors who underwent whole body PET/CT. The golden standard of diagnosis was the final pathological diagnosis or the results of long-term follow-up for patients without surgery/biopsy. PET/CT findings were compared with the golden standard. The sensitivity, specificity, accuracy and positive likelihood ratio of PET/CT in the detection of ophthalmic tumors were calculated. The clinical application of PET/CT in different types of ophthalmic tumors was evaluated. The results showed that 30 patients (18 males and 12 females) with a mean age of 43.0 years (range 4-63 years) were collected. The mean sizes of orbital tumors and intraocular tumors were 26.8 mm×17.8 mm and 11.2 mm×6.1 mm, respectively. The overall sensitivity, specificity, accuracy and positive likelihood ratio of whole body PET/CT in ophthalmic tumors were 76.5%, 71.4%, 75.0% and 2.67, and were 62.5%, 100% and 70.0% in intraocular tumors, and those were 100%, 60.0% and 84.6% in orbital tumors, respectively. PET/CT findings were applied to help make appropriate treatment options in 27 out of 30 patients (90.0%), and 12 (40.0%) patients changed the treatment strategy. False negative results in 4 cases and false positive results in 2 cases were observed in this series. It was suggested that PET/CT was an effective imaging modality in detecting, diagnosing and developing therapeutic schedule for patients with ophthalmic tumors. It was more sensitive and accurate for detecting orbital tumors than for detecting intraocular tumors.

Sema3E/PlexinD1 inhibition is a therapeutic strategy for improving cerebral perfusion and restoring functional loss after stroke in aged rats.

Brain tissue survival and functional recovery after ischemic stroke greatly depend on cerebral vessel perfusion and functional collateral circulation in the ischemic area. Semaphorin 3E (Sema3E), one of the class 3 secreted semaphorins, has been demonstrated to be a critical regulator in embryonic and postnatal vascular formation via binding to its receptor PlexinD1. However, whether Sema3E/PlexinD1 signaling is involved in poststroke neovascularization remains unknown. To determine the contribution of Sema3E/PlexinD1 signaling to poststroke recovery, aged rats (18 months) were subjected to a transient middle cerebral artery occlusion. We found that depletion of Sema3E/PlexinD1 signaling with lentivirus-mediated PlexinD1-specific-shRNA improves tissue survival and functional outcome. Sema3E/PlexinD1 inhibition not only increases cortical perfusion but also ameliorates blood-brain barrier damage, as determined by positron emission tomography and magnetic resonance imaging. Mechanistically, we demonstrated that Sema3E suppresses endothelial cell proliferation and angiogenic capacity. More importantly, Sema3E/PlexinD1 signaling inhibits recruitment of pericytes by decreasing production of platelet derived growth factor-BB in endothelial cells. Overall, our study revealed that inhibition of Sema3E/PlexinD1 signaling in the ischemic penumbra, which increases both endothelial angiogenic capacity and recruitment of pericytes, contributed to functional neovascularization and blood-brain barrier integrity in the aged rats. Our findings imply that Sema3E/PlexinD1 signaling is a novel therapeutic target for improving brain tissue survival and functional recovery after ischemic stroke.

[Study of infective efficiency of two adenovirus vectors to rBMSC and its mechanism].

OBJECTIVE: To investigate the infective efficiency of Ad5/EGFP versus AdF35/EGFP to rat bone marrow mesenchymal stem cells (rBMSC) and understand its mechanism. METHODS: rBMSC were isolated from rat bone marrow by density gradient method. The expressions of CD90, CD29, CD44, CD34, CD11b and CD45 on the surface of rBMSC were measured by flow cytometry. The EGFP-carrying Ad5 and AdF35 were infected into rBMSC respectively. The effect of Ad5/EGFP and AdF35/EGFP with different MOIs to rBMSC was tested by MTT. Infected rBMSC were observed by fluorescence microscopy. The infective efficiency and mean fluorescence intensity were determined by flow cytometry after a cellular infection for 1 - 9 days. The expressions of CAR and CD46 on rBMSC were measured by real-time PCR. RESULTS: The expressions of CD90, CD29 and CD44 on rBMSC were positive while those of CD34, CD11b and CD45 were negative. When MOI

[Myocardial single photon emission tomography imaging of reporter gene expression in rabbits].

OBJECTIVE: To explore the feasibility of single photon emission computed tomography (SPECT) detection of heart reporter gene expression and observed the optimal transfecting titer and imaging time by using herpes simplex virus 1-thymidine kinase (HSV1-tk) as reporter gene and 131I-2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil (131I-FIAU) as reporter probe in rabbit myocardium. METHODS: The recombinant Ad-tk carrying HSV1-tk gene and adenovirus (Ad) as vector was constructed and intramyocardially injected to rabbits at various concentrations (1 x 10(9) pfu, 5 x 10(8) pfu, 1 x 10(8) pfu, 5 x 10(7) pfu, 1 x 10(7) pfu). Two days later, rabbits were injected with 600 microCi 131I-FIAU in ear-margin vein and then underwent SPECT myocardium imaging for detection of HSV1-tk expression at 6 h, 24 h, 48 h and 72 h after injection, rabbits with 1 x 10(9) pfu Ad-tk injection were imaged at 96 h and 120 h. Rabbits were sacrificed after imaging and the total myocardial 131I-FIAU accumulation was quantified in percent of injected dose per gram myocardium (% ID/g). The myocardial Ad-tk expression was determined with RT-PCR. RESULTS: Reporter gene was detected by SPECT imaging in the injection site while not detected in the control myocardium and site remote from injection. RT-PCR results also evidenced HSV1-tk express in the injection site. The SPECT target/nontarget ratio was correlated with ex vivo gamma-counting (r2 = 0.933, P<0.01) and expression of HSV1-tk (r2 = 0.877, P<0.01). Myocardial accumulation could be identified at viral titers as low as 1 x 10(7) pfu by SPECT imaging. CONCLUSION: The cardiac SPECT reporter gene imaging with HSV1-tk as reporter gene and 131I-FIAU as reporter probe is feasible.

The antiproliferative effects of somatostatin receptor subtype 2 in breast cancer cells.

AIM: Somatostatin receptor subtype 2 (SSTR2) is the principal mediator of somatostatin's (SST) antiproliferative effects on normal and cancer cells. Therefore, we investigated whether the enhanced expression of SSTR2 could inhibit the proliferation of tumor cells, and, if so, the mechanisms that might be involved. METHODS: SSTR2 expression levels were determined by qRT-PCR in several tumor cell lines. Then, a plasmid pIRES2-EGFP-SSTR2 (pSIG) was constructed and stably transfected into MCF-7 cells (MCF-7/pSIG). After SSTR2 overexpression was identified by qRT-PCR, immunofluorescence staining and a receptor binding assay, the MCF-7/pSIG cells were analyzed by PI staining for apoptosis and cell cycle arrest was tested by flow cytometry for epidermal growth factor receptor (EGFR) expression. The EGF-stimulated proliferation of MCF-7 cells was assayed by MTT. RESULTS: The human breast cancer cell line MCF-7 expresses a lower level of SSTR2, thereby partly accounting for the decreased response to SST. The overexpression of SSTR2 in MCF-7 cells resulted in apoptosis, cytostasis and G(1)/S cell cycle arrest. Furthermore, the expression of EGFR, together with EGF-stimulated proliferation, was markedly decreased in the MCF-7/pSIG cells. CONCLUSION: Enhanced SSTR2 expression played an antiproliferative role in MCF-7 cells through inducing apoptosis and G(1)/S cell cycle arrest, and also by decreasing EGFR expression, thereby counteracting the growth-stimulating effect of EGF. Our data seem to indicate that developing a new therapeutic agent capable of upregulating SSTR expression could potentially be a way to block tumor progression.Acta Pharmacologica Sinica (2009) 30: 1053-1059; doi: 10.1038/aps.2009.59.

[Autoradiography reporter gene (Ad5-tk carrying HSV1-tk gene) expression imaging in rat myocardium].

OBJECTIVE: Radionuclide imaging of reporter gene expression holds promise for noninvasive monitoring of gene therapy. Herpes simplex virus 1-thymidine kinase (HSV1-tk) has been successfully applied to the tumor tissue.We explored the feasibility of the expression imaging of HSV1-tk reporter gene in rat myocardium by using SPECT reporter probe (131)I-2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil ((131)I-FIAU) and autoradiography (ARG). METHODS: The recombinant Ad5-tk carrying HSV1-tk gene and adenovirus (Ad5-null) as vector were constructed and intramyocardially injected into SD rats. Experiment was grouped for different aims as follows: (1) Influence of time on the imaging after transfection reporter gene: rats were injected with (131)I-FIAU at day 1, 2, 3, 5 and 7 after transfection of 1 x 10(8)pfu Ad5-tk; (2) Influence of various titers on the imaging: rats underwent intramyocardial injection with various titers of Ad5-tk (5 x 10(8), 1 x 10(8), 5 x 10(7), 1 x 10(7)pfu). After 2 days, rats were injected with (131)I-FIAU in tail vein. Equal volume Ad-nulls was intramyocardially injected to control rats. Rats were killed 24 h after injection of (131)I-FIAU and the hearts were rapidly dissected for gamma counts measurement. The total myocardial (131)I-FIAU accumulation was quantified in percent of injected dose per gram myocardium (%ID/g). The myocardial reporter gene expression was semi-quantitatively determined by ARG and RT-PCR. RESULTS: ARG and RT-PCR showed that the local expression of reporter gene increased in proportion with increasing titer and decreased in proportion with time post injection. The semi-quantitative assay showed there were significant correlations among %ID/g, RT-PCR and ARG: r(2) = 0.963, P < 0.05 for RT-PCR and ARG; r(2) = 0.996, P < 0.01 for %ID/g and ARG in rats received various reporter gene titers at identical time point post injection; r(2) = 0.950, P < 0.05 for RT-PCR and ARG; r(2) = 0.980, P < 0.01 for %ID/g and ARG for rats received identical reporter gene titer on various time points post injection. CONCLUSIONS: The present study showed that cardiac reporter gene imaging with HSV1-tk as reporter gene and (131)I-FIAU as reporter probe was feasible in rats. The optimal Ad5-tk titer is 1 x 10(8) pfu and the optimal imaging time is 24 h to 48 h post gene transfer. HSV1-tk/FIAU may be used for the noninvasive monitoring of cardiac gene therapy.

[Feasibility of apoptosis-imaging agent 99mTc-HYNIC-annexin V in early assessment of chemotherapeutic effect on tumor models].

OBJECTIVE: To evaluate if 99mTc-HYNIC-annexin V may be used to detect the early chemotherapeutic effect and to determine the best timing for detecting apoptosis in vivo. METHODS: Annexin V was labeled with 99mTc using HYNIC as a bifunctional agent. Normal Kunming mice received inoculation of Ehrlich ascites cells into the right upper limb. After the tumor reached 1 cm in diameter, the mice were randomly divided into saline treatment group as control and cyclophosphamide (150 mg/kg injected intraperitoneally) treatment group. 99mTc-HYNIC-annexin V was injected intravenously at 1 h and 24 h after treatment. Region of interest technique (ROI) from the SPECT images taken at different time was used to get the ratio of tumor/limb in each group. TUNEL staining was used to detect apoptotic cells and the rates of positive stained cells were calculated. RESULTS: After treatment with saline, only little amount of the radiolabeled tracer could be seen in the tumor and showed weak image of the tumor. But after 24 h of treatment with cyclophosphamide, clear image on the tumor could be seen. 24 h after the treatment of cyclophosphamide, the ratio of tumor/limb was (6.27 +/- 0.24) which was much higher than that at 24 h after treatment with saline (2.36 +/- 0.18) and that at 1 h after cyclophosphamide treatment (4.00 +/- 0.38). At 24 h after cyclophosphamide treatment, TUNEL staining showed a significantly higher rate of apoptotic cells in the mice. CONCLUSION: 99mTc-HYNIC-annexin V can be used as an apoptosis-imaging agent to detect and evaluate the early curative effect after chemotherapy. The effective detection of apoptotic response in tumor with 99mTc-HYNIC-annexin V requires a 24 h interval after chemotherapy. SPECT images can be obtained at 60 min after injection of the imaging agent. It suggests that 99mTc-HYNIC-annexin V may become a promising agent for apoptosis-imaging in clinical application.

Uptake kinetics of 99mTc-MAG3-antisense oligonucleotide to PCNA and effect on gene expression in vascular smooth muscle cells.

UNLABELLED: To investigate the feasibility of in vivo imaging study of atherosclerotic plaque and restenosis using antisense probe, we evaluated the uptake kinetics of radiolabeled oligonucleotides to the messenger RNA (mRNA) of proliferating cell nucleus antigen (PCNA) in vascular smooth muscle cells (VSMCs) and the effect on gene expression. METHODS: The antisense oligonucleotide to PCNA was radiolabeled with (99m)Tc through bifunctional chelator mercaptoacetyltriglycine (MAG(3)). The labeling efficiency was assessed by Sephadex G25 chromatography. The radiochemical purity, in vitro stability, and ability of the labeled antisense oligonucleotide to hybridize to its complement were analyzed by Sep-Pak C18 column chromatography. The uptake kinetics of (99m)Tc-labeled antisense oligonucleotide and sense oligonucleotide were studied in VSMCs in log and plateau phases. To study whether the antisense probe can hybridize to a respective sequence on the whole PCNA mRNA strand after radiolabeling, we performed reverse-transcriptase polymerase chain reaction to assay the PCNA mRNA level after the VSMCs had been incubated with the (99m)Tc-labeled antisense oligonucleotide and sense oligonucleotide. RESULTS: The labeling efficiency of (99m)Tc-MAG(3)-antisense oligonucleotide was 60.1% (n = 5), the specific activity was 1,960 kBq/microg, and the radiochemical purity was more than 95% after purification. (99m)Tc-MAG(3)-antisense oligonucleotide was stable in vitro and retained the ability to hybridize with its complementary chain. Antisense oligonucleotide showed significantly higher accumulation than sense oligonucleotide in log phase, with peak values of 15.2% +/- 0.58% and 5.6% +/- 0.42%, respectively (P < 0.05). No significant difference was found between uptake of antisense oligonucleotide and uptake of sense oligonucleotide in plateau phase (P > 0.05), but higher accumulation of antisense oligonucleotide was found in log phase than in plateau phase (P < 0.05). The retention rate of antisense oligonucleotide in log phase was much higher than that of sense oligonucleotide (79.6% +/- 0.96% vs. 59.8% +/- 0.75%, P < 0.05) at 240 min. The 2 probes did not significantly differ in plateau phase (P > 0.05). The efflux of antisense oligonucleotide was obviously slower in log phase than in plateau phase (P < 0.05). Compared with (99m)Tc-MAG(3)-sense oligonucleotide, (99m)Tc-MAG(3)-antisense oligonucleotide could inhibit the expression of PCNA mRNA significantly. CONCLUSION: This in vitro study in VSMCs provided evidence that the (99m)Tc-labeled antisense oligonucleotide to PCNA can be used for in vivo imaging of atherosclerotic plaque and restenosis in further study.

An imaging study using laminin peptide 99mTc-YIGSR in mice bearing Ehrlich ascites tumour.

BACKGROUND: The YIGSR is a pentapeptide, from the laminin-1 of the beta1 chain, which can mediate cell adhesion and bind the 67 kD laminin receptor. The purpose is to evaluate the usefulness of (99m)Tc-YIGSR, a novel tumour radiotracer, in the receptor imaging of Ehrlich ascites tumour. METHODS: Using S-acetly-NH3-MAG3 as chelate, YIGSR, a pentapeptide from laminin, was tagged with (99m)Tc. (99m)Tc-YIGSR was detected in the tumour group bearing Ehrlich ascites tumour and blocked group. Tumour, normal, inflammatory and blocked groups were imaged. RESULTS: Through reverse phase Sep-Pak C18 chromatogram, it was revealed that YIGSR could conjugate with S-acetly-NH3-MAG3, and be radiolabelled at room temperature and neutral pH with a radiolabelling yield of 62%, and of 4% without chelate. (99m)Tc-YIGSR was rapidly cleared from kidney, then liver. The imaging findings showed tumour tissue accumulated initial radioactivity at fifteen minutes after injection in the tumour group, and the uptake increased to peak at three hours with a tumour/muscle ratio (T/M) of 11.36, then cleared slowly to a T/M of 7.50 at eight hours. The tumour uptake of radiotracer in blocked group was significantly lower with T/M of 4.61 at three hours and 0.89 at eight hours. The T/M was only 3.72 at three hours and 1.29 at eight hours after injection in inflammatory group. Compared with inflammatory group and control obstructive group, the ratio of T/M in tumour group was significantly different (P < 0.001). CONCLUSIONS: Using S-acetly-NH3-MAG3, we radiolabelled YIGSR with (99m)Tc. (99m)Tc-YIGSR possesses many merits of tumour imaging: rapid visualization, high sensitivity and specificity, and satisfactory target/nontarget ratio. Our data suggest (99m)Tc-YIGSR is a promising tumour radiotracer.

[The change of adrenergic receptor-adenyl cyclase system on myocardial ischemic preconditioning in rats].

AIM: To study the varies and effects of ischemic preconditioning of myocardium on every part of adrenergic receptor-adenyl cyclase system in rats in vivo. METHODS: SD rats were randomly divided into three groups: CON group (n = 6), IP group (n = 12) and I/R group (n = 12). Surgical procedure included intermittent left coronary artery occlusion and reperfusion. After the procedure, the hearts were extracted. We analyzed the infarct size by TTC staining, measured serum myocardial enzymes, studied the beta-AR Bmax and KD by radioligand binding assay of receptors (RAB), and checked the activity of AC and the content of cAMP by radioimmunoassay (RIA). RESULTS: Infarct area were much smaller in IP group than in I/R group (P < 0.05). CK, CK-MB, LDH were significantly higher in I/R group (P < 0.01). The Bmax of beta-AR in IP group were much higher than in I/R group (P < 0.01). No difference of KD could be seen between IP and I/R group. In IP group, the activity of AC and the content of cAMP were higher than I/R group (P < 0.05). CONCLUSION: Ischemic preconditioning can protect the heart from necrosis and reduce endo-enzyme leakage. Ischemic preconditioning can increase the density of beta-AR, the activity of AC and the content of cAMP, which shows that the system of adrenergic receptor-adenyl cyclase system probably takes part in the protection of IP.