OBJECTIVEThe present study was undertaken to design antisense oligodeoxynucleotides (AS-ODNs) of glial glutamate transporter-la (GLT-1a) and to evaluate the effectiveness of the designed AS-ODNs on the expression of GLT-1a.

METHODSFive sequences of GLT-1a AS-ODNs were designed according to the C terminus specific sequences of GLT-1a mRNA using antisense design software of IDT Com- pany. Western blot analysis was used to evaluate the inhibition effects of the five GLT-1a AS-ODNs on the expression of GLT-la.

RESULTSThe sequence of GLT-1a AS-ODNs with sequence of 5'-GGTTCTTCCTCAACACTGCA-3' could specifically inhibit the expression of GLT-1a in the hippocampal CA1 subfield of rats, while it had no effect on the expression of GLT-1b. This sequence showed similar inhibition on the expression of GLT-la in sham and ceftriaxone (Cef)-treated rats. It could also significantly inhibit the cerebral ischemic preconditioning (CIP)-induced up-regulation in the expression of GLT-1a. The magnitude of the inhibition in sham, Cef- or CIP-treated rats was similar by more than 60%.

CONCLUSIONFrom the designed five sequences of GLT-1a AS-ODNs, we obtained an effective sequence which can specifically inhibit the expression of GLT-1a.

Animals ; CA1 Region, Hippocampal ; metabolism ; Excitatory Amino Acid Transporter 2 ; antagonists & inhibitors ; metabolism ; Ischemic Preconditioning ; Oligonucleotides, Antisense ; genetics ; RNA, Messenger ; Rats ; Up-Regulation

Objective To explore the potential mechanisms of mesenchymal stem cell (MSC) therapy in ischemia/reperfusion injury (IRI)-induced acute kidney injury (AKI). Methods Forty-five C57/BL6 male mice were randomly divided into three groups: sham group, IRI group, and IRI+MSCs group, with 15 mice in each group. The IRI-induced AKI model in mice was reproduced by clamping both renal pedicles for 35 minutes. In the sham group, both kidneys were exposed, but their pedicles were not clamped. Six hours after reperfusion, mice in IRI+MSCs group received 100 μL of MSCs (1×104 /μL) isolated from the bone marrow from C57/BL6 mice via tail vein, while the mice in the IRI group received same amount of normal saline. Blood samples were harvested at 48 hours after reperfusion, and levels of serum creatinine (SCr) and blood urea nitrogen (BUN) were determined. The changes in renal pathology were observed by microscopy with PAS staining, and the tubular injury and acute tubular necrosis (ATN) scores were calculated. The number of leukocytes (CD45+) infiltrated in kidney at 24 hours and 72 hours after reperfusion was measured with flow cytometry. The number of neutrophils (Ly-6G+) and macrophages (F4/80+) infiltrated in kidneys at 24 hours and 72 hours after reperfusion was determined by immunofluorescence. Results There was significant increase in the related parameters in IRI group compared with those of sham group. The levels of SCr (μmol/L) and BUN (mmol/L) were 180.3±8.8 vs. 9.7±3.5, and 1 121.1±8.3 vs. 9.4±2.3, both P < 0.01. The score of tubular injury was 4.80±0.55 vs. 0 at 48 hours after reperfusion. The quantity of leukocyte (CD45+) infiltration in kidney at 24 hours and 72 hours after reperfusion was increased (×105 cells/g: 60.50±2.56 vs. 19.46±4.83, 42.00±1.87 vs. 14.70±3.74, both P < 0.01), and the number of neutrophils (Ly-6G+) and macrophages (F4/80+) infiltrated in kidney at 24 hours and 72 hours after reperfusion was also increase although the number of leukocytes infiltrated in kidney was significantly lower at 72 hours after reperfusion than that at 24 hours. There was significant lowering of the levels of SCr and BUN [SCr (μmol/L): 99.0±8.0 vs. 180.3±8.8, BUN (mmol/L): 84.5±7.6 vs. 112.1±8.3, both P < 0.01] in IRI+MSCs group, compared to IRI group. For the degree of tubular necrosis in two groups, the tubular injury scores were 2.60±0.55 vs. 4.80±0.55 (P < 0.05). The number of leukocytes infiltrated in kidney at 24 hours and 72 hours after reperfusion (×105 cells/g) were 24.20±4.53 vs. 60.50±2.56, 31.70±3.15 vs. 42.00±1.87 (both P < 0.01). The number of neutrophils was lowered despite (the number of macrophages was increased). However, the number of infiltrated leukocytes was significantly more in IRI+MSCs group at 72 hours than that at 24 hours (×105 cells/g: 31.70±3.15 vs. 24.20±4.53, P < 0.05). Conclusion MSCs could protect against IRI induced AKI by reducing the total number of leuckocytes, especially that of the neutrophils infiltrating into ischemic kidney and by recruiting macrophages into ischemic kidney.

OBJECTIVETo summarize the clinical effect of avulsed skin replantation of hand and foot via vacuum sealing drainage (VSD) combing low temperature technique.

METHODSFrom March 2012 to October 2013,13 cases with avulsed skin replantation of hand foot using combined technique included 8 males and 5 females with an average age of 32 years old ranging from 18 to 62 years. The time from injury to hospital was 1 to 4 hours (2.4 hour in average). The reasons of injury included machine injury in 7 cases and rolling over by cars in 6 cases. The parts of injuried involved finger in 2 cases,back of the hand in 5 cases and dorsum of foot in 6 cases. The area of avulsed skin was 5 cm x 6 cm to 12 cm x 16 cm,tendon and bone exposure was found in 4 cases. VSD was operated in all patients and the avulsed skin was refrigerated in the temperature of -4 °C or -80 °C. After 4 days, the skin stored in the -4 °C was replanted to the wounded place in 5 cases and in 3 cases the skin was planted to the donor site of flap. The skin stored in the -80 °C was replanted in 4 cases after 7 or 8 days, 1 case after 45 days.

RESULTSOf the 13 cases, 1 case of degloved injury from lower leg to dorsal foot,the replanted skin was necrosis completely; 1 case of degloving injury with fourth finger,the skin which replanted after 45 days survived approximately 30%,cured after skin-graft many times. In the other cases, the survival area of replanted skin was more than 85%, all cured after dressing. According to the standard of skin survival area evaluation by Jia et al, 11 cases showed excellent, 1 showed medium and 1 showed inferior. There were no complication about grafted skin rupture after the skin survived in 11 patients,after 4 to 22 months follow-up, the resiliency of grafted skin showed good. Sensation recovery was measured by BMRC standard: 3 cases of S3, 5 cases of S3, 3 cases of S2.

CONCLUSIONVSD combining lower temperature technique in skin replantation provides time and space for wound preparation and treatment plan for the patients who need second surgery, especially for the large area skin degloving,this method could utilize the degloved skin efficiently, decrease the donor site area, alleviate the pain and financial burden,reduce the scar formation of donor site and impediment.

Adolescent ; Adult ; Cryopreservation ; methods ; Drainage ; instrumentation ; methods ; Female ; Foot Injuries ; surgery ; Hand Injuries ; surgery ; Humans ; Male ; Middle Aged ; Replantation ; Skin ; injuries ; Skin Transplantation ; Young Adult

Although gene therapy was regarded as a promising approach for glioma treatment, its therapeutic efficacy was often disappointing because of the lack of efficient drug delivery systems. Mesenchymal stem cells(MSCs) have been reported to have a tropism for brain tumors and thus could be used as delivery vehicles for glioma therapy. Therefore, in this study, we attempted to treat glioma by using MSCs as a vehicle for delivering replication-competent adenovirus. We firstly compared the infectivity of type 3, type 5, and type 35 fiber-modified adenoviruses in MSCs. We also determined suitable adenovirus titer in vitro and then used this titer to analyze the ability of MSCs to deliver replication-competent adenovirus into glioma in vivo. Our results indicated that type 35 fiber-modified adenovirus showed higher infectivity than did naked type 3 or type 5 fiber-modified adenovirus. MSCs carrying replication-competent adenovirus significantly inhibited tumor growth in vivo compared with other control groups. In conclusion, MSCs are an effective vehicle that can successfully transport replication-competent adenovirus into glioma, making it a potential therapeutic strategy for treating malignant glioma.
Adenoviridae ; Animals ; Brain Neoplasms ; pathology ; therapy ; Cell Line, Tumor ; Genetic Vectors ; Glioma ; pathology ; therapy ; Humans ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Oncolytic Virotherapy ; Random Allocation ; Virus Replication ; Xenograft Model Antitumor Assays

ObjectiveTo evaluate the neurobiological characteristics of human histio-amniotic mesenchymal (hAMCs) and effect of hAMCs transplantation into the brain to treat Parkinson's disease(PD) modle mice.MethodsThe expressions of mesenchymal stem cells, neural stem cells, dopaminergic neurons and markers related to neurogenesis such as Vimentin, STRO-1, nestin, CD133, β-tubulin, TH, DAT, Ngn2 and mash-1 in hAMCs were evaluated through immunocytochemical stain; and the mRNA transcriptions of neural stem cell markers, Vimentin and nestin in hAMCs were detected by RT-PCR. The PD model was induced by MPTP(i.p.) in C57BL/6 mice transplanted with hAMCs into the right striatum. The therapeutical effect of hAMCs on PD mice was evaluated by spontaneous movement, rotating bar test and the immunohistochemistry of anti-human chondrosome and TH antibodies in striatum.ResultshAMCs induced by nerve cells culture medium, expressed mesenchymal stem cells, neural stem cells, dopaminergic neurons and other specific markers related to neurogenesis mentioned above. The frequency of spontaneous movement in PD mice was significantly increased(P<0-05), and the time of rotating bar was obviously prolonged(P<0-05) after transplantation with hAMCs.ConclusionhAMCs possess the characteristics of nerve cells after cultured in vitro and can significantly recover the damage of motor function induced by MPTP after transplantation into striatum in PD model mice.

OBJECTIVETo explore the molecular mechanism and prevention of retinoic acid syndrome (RAS).

METHODSSDF-1 alpha mRNA from healthy adult lung tissue was measured by RT-PCR, CXCR4 protein expression on the cell membrane of APL cells induced by ATRA (APL-ATRA) was tested by FCM, and the rotary cell culture system (RCCS) was used to build a modal for in vitro stimulation of APL-ATRA infiltrating human lung tissue. The ability of APL-ATRA in adhesion, migration and infiltration was observed by interference from DEX, Ara-C and DNR.

RESULTSThe APL-ATRA cells could evidently infiltrate into normal lung tissue. Mean fluorescence intensity (MFI) of CXCR4 on the cell membrane of APL-ATRA cells was 30.6 +/- 1.8, which was much higher than that on unspecialized APL cells (9.8 +/- 4.2). SDF-1 alpha mRNA expression was detected positive in all 6 lung tissue. Contrary to the control groups, DEX could dramatically restrain the ability of APL-ATRA cells in adhesion and migration [(27.2 +/- 2.6)% vs. (46.0 +/- 3.0)%, (28.1 +/- 4.0)% vs. (48.2 +/- 3.0)%], while Ara-C and DNR could distinctly depress the ability in adhesion, migration and infiltration [(28.1 +/- 3.0)%, (30.2 +/- 3.2)% vs. (46.0 +/- 3.0)%; (29.0 +/- 4.0)%, (23.0 +/- 5.2)% vs. (48.2 +/- 3.0)%; (16.8 +/- 7.6)%, (17.1 +/- 6.0)% vs. (43.6 +/- 5.0)%].

CONCLUSIONIn vitro APL-ATRA cells can infiltrate into the human lung tissue. High expression of CXCR4 on APL-ATRA and SDF-1 alpha in the lung tissue may be one of the molecular mechanisms of the lung infiltration and RAS. DEX, Ara-C and DNR can dramatically restrain the ability of APL-ATRA cells in adhesion, migration and infiltration.

Adolescent ; Adult ; Cell Adhesion ; Cell Culture Techniques ; Cell Movement ; Chemokine CXCL12 ; genetics ; metabolism ; Child ; Female ; Humans ; Leukemia, Promyelocytic, Acute ; metabolism ; pathology ; Male ; Middle Aged ; Neoplasm Invasiveness ; Receptors, CXCR4 ; genetics ; metabolism ; Tretinoin ; adverse effects ; Tumor Cells, Cultured

The activities of enzymes responsible for lignification in pepper, pre-inoculation with arbuscular mycorrhizal (AM) fungus of Glomus intraradices and/or infection with pathogenic strain of Phytophthora capsici, and the biological control effect of G. intraradices on Phytophthora blight in pepper were investigated. The experiment was carried out with four treatments: (1) plants pre-inoculated with G. intraradices (Gi), (2) plants pre-inoculated with G. intraradices and then infected with P. capsici (Gi+Pc), (3) plants infected with P. capsici (Pc), and (4) plants without any of the two microorganisms (C). Mycorrhizal colonization rate was reduced by about 10% in pathogen challenged plants. Root mortality caused by infection of P. capsici was completely eliminated by pre-inoculation with antagonistic G. intraradices. On the ninth day after pathogen infection, Peroxidase (POD) activity increased by 116.9% in Pc-treated roots but by only 21.2% in Gi+Pc-treated roots, compared with the control, respectively. Polyphenol oxidase (PPO) and Phenylalanine ammonia-lyase (PAL) activities gradually increased during the first 3 d and dramatically decreased in Pc-treated roots but slightly decreased in Gi+Pc-treated roots, respectively. On the ninth day after pathogen infection, PPO and PAL decreased by 62.8% and 73.9% in Pc-treated roots but by only 19.8% and 19.5% in Gi+Pc-treated roots, compared with the control, respectively. Three major POD isozymes (45,000, 53,000 and 114,000) were present in Pc-treated roots, while two major bands (53,000 and 114,000) and one minor band (45,000) were present in spectra of Gi+Pc-treated roots, the 45,000 POD isozyme was significantly suppressed by G. intraradices, suggesting that the 45,000 POD isozyme was induced by the pathogen infection but not induced by the antagonistic G. intraradices. A 60,000 PPO isozyme was induced in Pc-treated roots but not induced in Gi+Pc-treated roots. All these results showed the inoculation of antagonistic G. intraradices alleviates root mortality, activates changes of lignification-related enzymes and induces some of the isozymes in pepper plants infected by P. capsici. The results suggested that G. intraradices is a potentially effective protection agent against P. capsici.
Capsicum ; cytology ; enzymology ; microbiology ; Lignin ; metabolism ; Pest Control, Biological ; methods ; Phyllachorales ; cytology ; physiology ; Phytophthora ; cytology ; physiology ; Plant Proteins ; metabolism

BACKGROUNDAutoimmune encephalitis associated with antibodies against γ-aminobutyric acid B receptor (GABA B R) in patients with limbic encephalitis (LE) was first described in 2010. We present a series of Han Chinese patients for further clinical refinement.

METHODSSerum and cerebrospinal fluid (CSF) samples from patients referred to the program of encephalitis and paraneoplastic syndrome of Peking Union Medical College Hospital were tested with indirect immunofluorescence. Clinical information of patients with anti-GABA B R antibody positivity was retrospectively reviewed, and descriptive statistical analysis was performed.

RESULTSAll eighteen anti-GABA B R antibody-positive cases had limbic syndromes, and electroencephalogram (EEG) or neuroimaging evidence fulfilled the diagnostic criteria of LE. Four patients had additional antibodies against Hu in serum and one had anti-N-methyl-d-aspartate receptor antibody in both sera and CSF. Seventeen (17/18) patients presented with new-onset refractory seizure or status epileptics. Twelve (12/18) patients had memory deficits, 11 (11/18) patients had personality change, 7 (7/18) patients had disturbance of consciousness, and 3 (3/18) patients showed cerebellar dysfunction. One patient with LE had progressive motor and sensory polyneuropathy. Lung cancer was detected in 6 (6/18) patients. Ten (10/18) patients showed abnormality in bilateral or unilateral mediotemporal region on magnetic resonance imaging. Ten (10/18) patients had temporal lobe epileptic activity with or without general slowing on EEG. Seventeen patients received immunotherapy and 15 of them showed neurological improvement. Four patients with lung cancer died within 1-12 months due to neoplastic complications.

CONCLUSIONSOur study demonstrates that most Han Chinese patients with anti-GABA B R antibody-associated LE have prominent refractory epilepsy and show neurological improvement on immunotherapy. Patients with underlying lung tumor have a relatively poor prognosis. Testing for anti-GABA B R antibodies is necessary for patients with possible LE or new-onset epilepsy with unknown etiology.

Adult ; Autoantibodies ; immunology ; China ; Electroencephalography ; Epilepsy ; immunology ; pathology ; Female ; Humans ; Limbic Encephalitis ; immunology ; pathology ; Male ; Middle Aged ; Receptors, N-Methyl-D-Aspartate ; immunology ; Retrospective Studies ; gamma-Aminobutyric Acid ; metabolism

The residual protein mixture (the content is 4%, approximately), called Salvia miltiorrhiza antigen, was extracted from the Salvia miltiorrhiza cruel materials by mimicking the alcohol-deposit extracts process. Both rabbits and guinea pigs sensitized by Salvia miltiorrhiza could produce specified antibodies. Large molecular antigenic impurities were extracted from the Danshen injection and Xiangdan injection using the centrifugal filtering method. The test results of active systemic anaphylaxis (ASA) and passive cutaneous anaphylaxis (PCA) in guinea pigs confirmed that the extracted antigenic impurities could induce the anaphylaxis reaction in the animals which were sensitized by the Salvia miltiorrhiza antigen. Using the specified antibody produced from rabbits which were sensitiyed by Salvia miltiorrhiza, ELISA test method was developed to test the residual Salvia miltiorrhiza antigenic materials contained in the parenteral Chinese traditional medicines. Calculated as residual protein, the linear range was 0.08-5.12 microg x mL(-1) (r2 = 0.9906), the detection limit and quantization limit are 0.08 microg x mL(-1) and 0.4 microg x mL(-1), respectively. 308 batches of parenteral Chinese traditional medicines containing water-soluable components of Salvia miltiorrhiza were tested, and the Salvia miltiorrhiza antigenic impurities were spotted in 35 (11.4%) batches of samples. The test results show that the extracting process currently used can not remove the Salvia miltiorrhiza antigenic impurities completely, and this may be one of the reasons for anaphylactic reaction in clinics. The proposed ELISA method can be used for improving the manufacture process and for routine quality control of drug products.
Anaphylaxis ; chemically induced ; Animals ; Antigens, Plant ; analysis ; toxicity ; Drug Combinations ; Drugs, Chinese Herbal ; chemistry ; Enzyme-Linked Immunosorbent Assay ; methods ; Female ; Guinea Pigs ; Male ; Passive Cutaneous Anaphylaxis ; drug effects ; Phenanthrolines ; chemistry ; Quality Control ; Rabbits ; Salvia miltiorrhiza ; chemistry

OBJECTIVETo investigate the safety and efficacy of intracoronary transplantation of G-CSF mobilized autologous peripheral blood stem cells in patients with acute myocardial infarction (AMI).

METHODSPatients with AMI were randomly assigned to receive intracoronary PBSCs transplantation following bone marrow cells mobilization by granulocyte colony-stimulating factor (300-600 microg/day subcutaneously for 5 days) in addition to standard therapy (standard drug therapy and PCI, PBSCs transplantation group, n = 35) or standard therapy (standard drug therapy and PCI, n = 35). One day after G-CSF treatment was finished the patient's mononuclear cells were harvested by Baxter CS 3000 blood cell separator in a volume of 57 ml and then transferred into the infarct related artery by occluding the over the wire balloon and infusing artery through balloon center lumen. Complications during intervention and left ventricular function at baseline and 6 months thereafter were monitored.

RESULTSNo severe side effects of G-CSF treatment could be observed. Malignant arrhythmias were not observed either. Left ventricular function was significantly improved 6 months after G-CSF mobilized autologous peripheral blood stem cell transplantation compared to baseline (global left ventricular function ejection fraction: 57.1 +/- 7.8 vs. 50.0 +/- 8.2%, P < 0.0001; WMSI: 1.101 +/- 0.118 vs. 1.219 +/- 0.190, P < 0.0001; left end-systolic volume: 52.6 +/- 20.3 vs. 63.8 +/- 23.9 ml, P = 0.01 and left end-diastolic volume: 119.2 +/- 30.3 vs. 134.2 +/- 36.7 ml, P = 0.07) while these parameters remained unchanged in the control group.

CONCLUSIONThe present study demonstrates that G-CSF mobilized autologous intracoronary PBSCs transplantation is a safe and feasible treatment for patients with AMI and global left ventricular function is improved and left ventricular remodeling attenuated at six-month follow-up.

Aged ; Female ; Granulocyte Colony-Stimulating Factor ; therapeutic use ; Hematopoietic Stem Cell Mobilization ; methods ; Humans ; Male ; Middle Aged ; Myocardial Infarction ; surgery ; therapy ; Peripheral Blood Stem Cell Transplantation ; Transplantation, Autologous ; Treatment Outcome