The cis and trans isomers separation of 2-butene-1,4-diol and lafutidine were studied by HPLC on two kinds of chiral columns: (S,S)-Whelk-O 1 and ChiraSpher. The isomers of 2-butene-1,4-diol can be separated on both chiral columns while the isomers of lafutidine can only be resolved on ChiraSpher column. The influence of different type and amount of mobile phase modifier on the isomers separation was extensively studied. The resolution of cis and trans isomers of 2-butene-1,4-diol was 2.61 on (S,S)-Whelk-O 1 column with hexane-ethanol (97:3, v/v) as the mobile phase. The resolution of lafutidine was 1.89 on ChiraSpher column with hexane-ethanol-THF-diethylamine (92:3:5:0.1, v/v/v/v) as the mobile phase. LC-MS methods were developed to identify the isomer peaks.
Acetamides ; analysis ; chemistry ; Butylene Glycols ; analysis ; chemistry ; Chemical Fractionation ; methods ; Chromatography, High Pressure Liquid ; methods ; Isomerism ; Mass Spectrometry ; methods ; Piperidines ; analysis ; chemistry ; Pyridines ; analysis ; chemistry

The specimens of ductal carcinoma in situ (DCIS) with early invasion, and specimens collected by core needle biopsy (CNB) tend to contain limited amount of invasive component, so it is imperative to explore a new technique which can assess HER2 gene status accurately for the limited invasive cancer component in these specimens. Dual staining technique of combining immunohistochemistry (IHC) for myoepithelial cells and single or dual probe chromogenic in situ hybridization (CISH) for HER2 gene was performed on routinely processed paraffin sections from 20 cases diagnosed as having DCIS with invasive cancer. Among them, 10 had fluorescence in situ hybridization (FISH)-confirmed amplification of HER2 and 10 had FISH-confirmed non-amplification of HER2. We successfully detected HER2 genetic signals and myoepithelial IHC markers (SMM-HC or CK5/6) simultaneously on a single section in all 20 specimens. Myoepithelial markers and HER2 signals detected by dual staining assay were consistent with those by individual technique performed alone. HER2 gene amplification results determined by dual staining assay were 100% consistent with those of FISH. Dual staining technique which allows simultaneous detection of myoepithelial marker protein and cancerous HER2 gene is feasible, and it has potential to be used in clinical practice for effective determination of HER2 amplification in limited invasive component.
Biomarkers, Tumor ; metabolism ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Chromogenic Compounds ; Female ; Gene Expression Profiling ; methods ; Humans ; Immunohistochemistry ; methods ; In Situ Hybridization, Fluorescence ; methods ; Neoplasm Invasiveness ; pathology ; physiopathology ; Receptor, ErbB-2 ; genetics ; metabolism ; Reproducibility of Results ; Sensitivity and Specificity

BACKGROUNDThrombin is a multifunctional serine protease that plays a crucial role in hemostasis following tissue injury. In addition to its procoagulation effect, thrombin is also a potent mesenchymal cell mitogen, therefore it plays important roles in the local proliferation of mesenchymal cells in the tissue repair process. Reactive oxygen species (ROS) can induce some human cells to proliferate at lower rates while at higher concentrations they promote cells to undergo apoptosis or necrosis. Accumulative evidence suggests that thrombin can induce some cells to produce ROS. Based on these observations, we provide a hypothesis that thrombin can stimulate human lung fibroblasts to produce ROS, which play an important role in human lung fibroblast proliferation.

METHODSROS were detected in fibroblasts at 30 minutes and 60 minutes following thrombin (20 U/ml) exposure using flow cytometry. The ratio of reduced glutathione/oxidized glutathione (GSH/GSSG) was assayed in lung fibroblasts using a commercial kit following treatment with thrombin at different concentrations. NADPH oxidase and the extracellular regulated kinase1/2 (ERK1/2) signaling pathway were detected by Western blotting after thrombin stimulation to lung fibroblasts.

RESULTSThrombin, at 20 U/ml, stimulated human lung fibroblasts (HLF) to generate ROS in a time dependent manner. The ratio of GSH/GSSG in fibroblasts treated with thrombin showed a significant decrease. NADPH oxidase was activated and the ERK1/2 signal pathway was involved in the proliferation process of fibroblasts treated with thrombin.

CONCLUSIONThe activation of NADPH oxidase by thrombin leads to the production of ROS, which promotes fibroblasts proliferation via activation of the ERK1/2 signaling pathway.

Cell Proliferation ; drug effects ; Cells, Cultured ; Extracellular Signal-Regulated MAP Kinases ; analysis ; physiology ; Fibroblasts ; drug effects ; physiology ; Flow Cytometry ; Glutathione ; metabolism ; Humans ; Lung ; cytology ; NADPH Oxidases ; analysis ; physiology ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; physiology ; Thrombin ; pharmacology

OBJECTIVETo study the effect of delayed sequential bone marrow transplantation on acute graft-versus-host disease (aGVHD) in major H-2 incompatible mouse transplantation.

METHODSC57BL/6 (H-2b) mice were used as donors and BALB/c (H-2d) mice as recipients. BALB/c mice were given 8.0 Gys total body irradiation (TBI) on day 0 and infused with a blend of bone marrow cells and spleen cells in different time. Transplantation was carried out as follows: group I TBI on day 0 and transplantation at 4 h after TBI; groups of II TBI on day 0 and transplantation at 4 h, d1, d2, d3 after TBI; groups III TBI on day 0 and transplantation at day 4 after TBI; groups IV TBI on day 0 and transplantation at day 4 through day 7 after TBI. Recipient's spleen H-2b cells were detected by flow cytometry and the level of serum cytokines (IL-2, IL4, IL-6, IL-10 and IFN-gamma) by ELISA. The survival, aGVHD and hematopoietic recovery were observed.

RESULTSaGVHD occurred in group I and the mice all died within 3 weeks after transplantation. The 60 day survival rates of groups of II and III were 30% and 50% respectively. The degree of aGVHD in group III was modest and the survival rate was higher than that in other groups (P <0.05). The peak time of IL-2, IFN-gamma, IL-4 and IL-10 in groups III and IV were later than that in group I. The levels of IL-4 and IL-10 in groups III and IV were higher than that in group I and for the levels of IL-2 and IFN-gamma were on the contrary (P < 0.05). The level of IL-6 in all groups peaked on day 5 to day 10 after TBI and was higher in group I than in others (P <0.05). In group IV the mean value of donor H-2b cells was (98.1 +/- 1.1)% on day 60 and WBC counts recovered normal on day 20.

CONCLUSIONSDelayed sequential transplantation can reduce the morbidity of aGVHD ,improve the survival rate and not affect the engraftment and reconstitution of hematopoiesis in mouse allo-BMT. The mechanism of aGVHD prevention may be related to the reducing of type 1 cytokines of T lymphocyte and the increasing of type 2 cytokines.

Animals ; Bone Marrow Transplantation ; immunology ; methods ; Disease Models, Animal ; Female ; Graft vs Host Disease ; immunology ; prevention & control ; Interleukin-2 ; blood ; Interleukin-4 ; blood ; Interleukin-6 ; blood ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL

OBJECTIVETo observe the effect of Wnt3a-transduced mouse bone marrow mesenchymal stem cells (MSC) on the proliferation of T lymphocytes.

METHODSMSC were isolated from C57BL/6 mouse bone marrow and expanded in vitro, then identified by flow cytometry and their differentiation capacity into osteocytes and adipocytes were determined. Recombinant plasmids containing Wnt3a gene, were transfected with lipofectamine into HEK293 cells by the AdEasy system. Viral particles were collected to infect MSC and adenovirus vector expressing GFP (Ad-GFP) was used as control. The expression of GFP in MSC was observed using fluorescence microscopy and the protein levels of Wnt3a and β-catenin were determined by Western blot. Wnt3a-transduced and Ad-GFP transduced MSC were separately cocultured with spleen lymphocytes stimulated by ConA, at the ratio of 1:100, 1:50 or 1:10 respectively. The proliferation rate of T lymphocytes was estimated by Cell Cout Kit-8 (CCK-8) and the level of cytokine by ELISA.

RESULTSFCM analysis showed that the MSC were highly positive for CD90.2, CD44 and negative for CD34, CD45, they could differentiate into osteoblasts and adipocytes after induction; The titer of recombinant adenoviruses was up to 1 × 10(10) pfu/ml. After infected with the adenoviruses, MSC had the strongest GFP expression at 72 h and the efficiency of infection was 50%-60%. The expressions of Wnt3a and β-catenin protein in the Wnt3a-transduced MSC were significantly increased. MSC could suppress the proliferation of T lymphocytes in a dose-dependent manner. When MSC cocultured with spleen lymphocytes at 1:10 ratio, T lymphocyte proliferation rate and the level of IFN-γ were (55.41 ± 1.75)% and (326.70 ± 14.41) pg/ml respectively in Ad-GFP transduced MSC group, while in Wnt3a-transduced MSC group, they were (37.27 ± 2.66)% and (218.80 ± 12.93) pg/ml respectively. There was no effect on the production of IL-2.

CONCLUSIONCompared to Ad-GFP transduced MSC, Wnt3a-transduced MSC exhibit a more potent inhibitory effect on the proliferation of T lymphocytes.

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; Cell Proliferation ; Female ; Lymphocyte Activation ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; T-Lymphocytes ; cytology ; Transduction, Genetic ; methods ; Wnt3A Protein ; genetics ; metabolism

OBJECTIVETo study the number of CECs in patients with acute myocardial infarction (AMI) and unstable angina (UA), and to investigate its relationship with inflammatory related cytokines.

METHODS37 patients with AMI, 12 patients with UA, and 42 health controls were studied. CECs were isolated from peripheral blood by using of immunomagnetic beads coated with antibodies against CD146. Their endothelial origin was confirmed by the positive labelling of von Willebrand Factor (vWF), CD31 and electron microscope. Annexin V-FITC/PI kit was used to measure the apoptosis of CECs. Inflammatory related cytokines were analyzed turbidimetrically or ELISA using of commercially available testing kit.

RESULTSCECs number was significantly higher in AMI and UA patients [medians (interquartile range) were 52 (28 approximately 81.5) cells/ml and 29 (18 approximately 61) cells/ml respectively] compared with health control [10.5 (6-16.5)cells/ml, P < 0.001]. After excluding diabetes patients, the number of CECs and CRP in AMI and UA group (n = 26) were still significantly higher than controls. The necrotic rate of CECs in AMI and UA was significantly higher than controls (P < 0.01). Correlation analysis revealed a significant positive correlation between CECs and CRP, or IL-6 (r = 0.677, 0.316, P = 0.000, 0.002). The multivariate linear regression analysis showed that CRP and Diabetes increased the number of CECs significantly (OR = 0.620, 0.164, 95% CI 3.985-6.751, 0.301-21.877, P = 0.000, 0.044).

CONCLUSIONThe mechanism responsible for the increase of CECs in acute coronary disease may be due to the vessel injury caused by inflammation.

Acute Coronary Syndrome ; blood ; Aged ; Angina, Unstable ; blood ; C-Reactive Protein ; metabolism ; Case-Control Studies ; Endothelial Cells ; cytology ; Endothelium, Vascular ; chemistry ; cytology ; Female ; Humans ; Inflammation ; Interleukin-6 ; metabolism ; Linear Models ; Male ; Middle Aged ; Multivariate Analysis ; Myocardial Infarction ; blood ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo explore the difference on the prevalence and the risk factors of type 2 diabetes(T2DM) between Yi farmer and immigrants in Liangshan, Sichuan province.

METHODSA representative sample of 2878 Yi people (including 1549 farmers and 1329 immigrants) aged from 16 to 76 was selected by the method of multistage and cluster sampling in Liangshan, Sichuan province, China, during 2007 - 2008. The samples were divided into 5 groups by the factor of age (16-, 25-, 35-, 45- and 55-76). The standardized prevalence of T2DM was calculated by the national census data in 2000. Logistic regression analysis was used to study the related risk factors of T2DM.

RESULTSThe prevalence of Yi farmer was 4.33% (67/1549) (male: 6.15% (42/683), female: 2.89% (25/866)), and that of Yi migrants was 9.03% (120/1329) (male: 11.31% (88/778), female: 5.81% (32/551)). The standardized prevalence (SP) was calculated by the data of national census 2000. The SP of Yi farmer was 5.97%in male, and that of the female was 2.40%. The SP of Yi migrant was 10.25% in male, and that of the female was 6.29%. For Yi people, sex (male versus female, OR = 1.69, 95%CI: 1.02 - 2.81), age (versus the group aged 16- and 25-, group aged 35 to 54: OR = 5.04, 95%CI: 2.93 - 8.69; group aged above 54: OR = 6.19, 95%CI: 3.23 - 11.86), hypertension (versus normal group, borderline hypertension value: OR = 1.61, 95%CI: 1.08 - 2.38; hypertension group: OR = 2.40, 95%CI: 1.37 - 4.22), smoking (OR = 1.55, 95%CI: 1.01 - 2.37), triglyceride (TG) level (OR = 1.65, 95%CI: 1.10 - 2.46) and high-density lipoprotein cholesterol (HDL-C) level (OR = 1.64, 95%CI: 1.13 - 2.37) were the positive factors correlated with T2DM, and drinking (the alcohol volume from 22.67 to 52.50 g/d ) was negative factor (OR = 0.53, 95%CI: 0.30 - 0.95) correlated with T2DM.

CONCLUSIONThe prevalence of T2DM in Yi immigrants was higher than that in Yi farmers;sex, age, blood pressure, smoking, TG, HDL-C, drinking were influencing factors of T2DM.

Adolescent ; Adult ; Aged ; China ; epidemiology ; Diabetes Mellitus, Type 2 ; epidemiology ; Ethnic Groups ; Female ; Humans ; Male ; Middle Aged ; Prevalence ; Risk Factors ; Rural Population ; Young Adult

BACKGROUND AND OBJECTIVERadiotherapy is effective in treating nasopharyngeal carcinoma (NPC). This study evaluated the treatment efficacy, toxicity, and prognostic factors of intensity-modulated radiotherapy (IMRT) in the treatment NPC.

METHODSBetween September 2003 and September 2006, 305 patients with NPC were treated with IMRT in Fujian Provincial Cancer Hospital. IMRT was delivered as follows: gross tumor volume (GTV) received 66.0-69.8 Gy in 30-33 fractions, high-risk clinical target volume (CTV-1) received 60.0-66.65 Gy, low-risk clinical target volume (CTV-2) and clinical target volume of cervical lymph node regions (CTV-N) received 54.0-55.8 Gy. Patients with stages III or IV disease also received cisplatin-based chemotherapy. All patients were assessed for local-regional control, survival, and toxicity.

RESULTSWith a median follow-up of 35 months (range, 5-61 months), there were 16, 8, and 39 patients who had developed local, regional, and distant recurrence, respectively. The 3-year rates of local control, regional control, metastasis-free survival, disease-free survival, and overall survival were 94.3%, 97.7%, 86.1%, 80.3%, and 89.1%, respectively. Multivariate analyses revealed that T-classification had no predictive value for local control and survival, whereas N-classification was a significant prognostic factor for overall survival (P < 0.001), metastasis-free survival (P < 0.001), and disease-free survival (P = 0.003). For stages III-IV disease, concurrent and adjuvant chemotherapy did not influence prognosis. The most severe acute toxicities included Grade III mucositis in 14 patients (4.6%), Grade III skin desquamation in 90 (29.5%), and Grades III-IV leucocytopenia in 20 (6.5%). There were 7% patients with Grade II xerostomia after 2 years of IMRT, no Grades 3 or 4 xerostomia was detected.

CONCLUSIONSIMRT provided favorable locoregional control and survival rates for patients with NPC, even in those with locally advanced disease. The acute and late toxicities were acceptable. N-classification was the main factor of prognosis. Further study is needed on chemotherapy for patients with NPC.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Child ; Cisplatin ; administration & dosage ; Combined Modality Therapy ; Disease-Free Survival ; Female ; Follow-Up Studies ; Humans ; Leukopenia ; etiology ; Lymphatic Metastasis ; Male ; Middle Aged ; Mucositis ; etiology ; Nasopharyngeal Neoplasms ; drug therapy ; pathology ; radiotherapy ; Neoplasm Recurrence, Local ; Neoplasm Staging ; Radiotherapy Dosage ; Radiotherapy, Intensity-Modulated ; adverse effects ; methods ; Retrospective Studies ; Survival Rate ; Xerostomia ; etiology ; Young Adult

This study is to explore the effect of ginsenoside Rb1 on the process of beta-amyloid peptide(25-35) (Abeta(25-35)) -induced hyperphosphorylation of tau protein, and on the level of cyclin-dependent kinase 5 activator, p25/p35. Western blotting and/or immunocytochemical staining were used to detect the levels of phosphorylation of tau protein at the sites of Thr205, Ser396, Ser404 in hippocampal neurons, cdk5 and p25/p35. After exposure to Abeta(25-35) (20 micromol x L(-1)) for 12 h, the levels of tau protein phosphorylation at the sites of Thr205, Ser396, Ser404 were enhanced, the level of p25 was increased, but the level of protein cdk5 was not changed markedly. Pretreatment with ginsenoside Rb1 reduced Abeta(25-35) -induced hyperphosphorylation of tau protein and decreased the lever of p25, but had no effect on cdk5. Ginsenoside Rb1 can attenuate Abeta(25-35) -induced hyperphosphorylation of tau protein through CDK5 signal pathway.
Amyloid beta-Peptides ; antagonists & inhibitors ; Animals ; Cyclin-Dependent Kinase 5 ; metabolism ; Fetus ; Ginsenosides ; isolation & purification ; pharmacology ; Hippocampus ; cytology ; Nerve Tissue Proteins ; metabolism ; Neurons ; metabolism ; Panax ; chemistry ; Phosphorylation ; drug effects ; Plants, Medicinal ; chemistry ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; tau Proteins ; metabolism

BACKGROUNDOssification of the ligamentum flavum (OLF) is being increasingly recognized as a cause of thoracic myelopathy. This study was to describe a rare clinical entity of spinal cord kinking (SK) in thoracic myelopathy secondary to OLF.

METHODSThe data of 95 patients with thoracic myelopathy secondary to OLF were analyzed retrospectively. The incidence and location of SK were determined using preoperative magnetic resonance imaging (MRI). The clinical presentation and radiological characteristics in patients with SK were analyzed. Posterior en bloc laminectomy with OLF was performed, and the surgical results were evaluated.

RESULTSSK was found in seven patients (7.4%) based on preoperative MRI. The patients included one male and six females with an average age of 55.6 years (range, 48-64 years). Five patients presented with radiculomyelopathy and two presented with typical thoracic myelopathy of spastic paraparesis. In all cases, the kinking was located just above the end of the spinal cord where the conus medullaris (CM) was compressed by the OLF. The degree of SK varied from mild to severe. The tip of the CM was located between the upper third of T11 to the lower third of L1, above the lower edge of L1. With an average follow-up of 30.4 months, the modified Japanese Orthopedic Association score significantly improved from 5.7 ± 1.8 preoperatively to 8.9 ± 1.4 postoperatively (t = 12.05; P < 0.0001) with an improvement rate of 63.1 ± 12.3%.

CONCLUSIONSSK is a rare radiological phenomenon. It is typically located at the thoracolumbar junction, where the CM is compressed by the OLF. Our findings indicate that these patients may benefit from a posterior decompressive procedure.

Female ; Humans ; Ligamentum Flavum ; pathology ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Ossification, Heterotopic ; complications ; Radiography ; Spinal Cord Compression ; diagnosis ; diagnostic imaging ; surgery ; Spinal Cord Diseases ; diagnosis ; diagnostic imaging ; etiology ; surgery