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Background Chronic inflammatory pain significantly reduces the quality of life and lacks effective interventions. In recent years, human umbilical cord mesenchymal stem cells (huc-MSCs)-derived exosomes have been used to relieve neuropathic pain and other inflammatory diseases as a promising cell-free therapeutic strategy. However, the therapeutic value of huc-MSCs-derived exosomes in complete Freund's adjuvant (CFA)-induced inflammatory pain remains to be confirmed. In this study, we investigated the therapeutic effect and related mechanisms of huc-MSCs-derived exosomes in a chronic inflammatory pain model. Methods C57BL/6J male mice were used to establish a CFA-induced inflammatory pain model, and huc-MSCs-derived exosomes were intrathecally injected for 4 consecutive days. BV2 microglia cells were stimulated with lipopolysaccharide (LPS) plus adenosine triphosphate (ATP) to investigate the effect of huc-MSCs-derived exosomes on pyroptosis and autophagy. Bioinformatic analysis and rescue experiments were used to demonstrate the role of miR-146a-5p/ TRAF6 in regulating pyroptosis and autophagy. Western blotting, RT-qPCR, small interfering RNA and Yo-Pro-1 dye staining were performed to investigate the related mechanisms. Results Huc-MSCs-derived exosomes alleviated mechanical allodynia and thermal hyperalgesia in CFA-induced inflammatory pain. Furthermore, huc-MSCs-derived exosomes attenuated neuroinflammation by increasing the expression of autophagy-related proteins (LC3-II and beclin1) and inhibiting the activation of NLRP3 inflammasomes in the spinal cord dorsal horn. In vitro, NLRP3 inflammasome components (NLRP3, caspase1-p20, ASC) and gasdermin D (GSDMD-F, GSDMD-N) were inhibited in BV2 cells pretreated with huc-MSCs-derived exosomes. Western blot and Yo-Pro-1 dye staining demonstrated that 3-MA, an autophagy inhibitor, weakened the protective effect of huc-MSCs-derived exosomes on BV2 cell pyroptosis. Importantly, huc-MSCs-derived exosomes transfected with miR-146a-5p mimic promoted autophagy and inhibited BV2 cell pyroptosis. TRAF6, as a target gene of miR-146a-5p, was knocked down via small-interfering RNA, which increased pyroptosis and inhibited autophagy. Conclusion Huc-MSCs-derived exosomes attenuated inflammatory pain via miR-146a-5p/TRAF6, which increased the level of autophagy and inhibited pyroptosis. Graphical Abstract

Omega-3 polyunsaturated fatty acid (ω-3 PUFA) have been reported to have beneficial cardiovascular effects, but its mechanism of protection against acute myocardial infarction (AMI) who are under guideline-based therapy is not fully understood. Here, we used a metabolomic approach to systematically analyze the eicosanoid metabolites induced by ω-3 PUFA supplementation and investigated the underlying mechanisms.Participants with AMI after successful percutaneous coronary intervention were randomized to 3 months of 2 g daily ω-3 PUFA and guideline-adjusted therapy (n = 30, ω-3 therapy) or guideline-adjusted therapy alone (n = 30, Usual therapy). Functional PUFA-derived eicosanoids in plasma were profiled by metabolomics. Clinical and laboratory tests were obtained before and 3 months after baseline and after the study therapy.By intent-to-treat analysis, the content of 11-HDoHE, 20-HDoHE and 16,17-EDP and that of epoxyeicosatetraenoic acids (EEQs), derived from docosahexaenoic acid and eicosapentaenoic acid, respectively, were significantly higher with ω-3 group than Usual therapy, whereas that of prostaglandin J2 (PGJ2) and leukotriene B4, derived from arachidonic acid, was significantly decreased. As compared with Usual therapy, ω-3 PUFA therapy significantly reduced levels of triglycerides (-6.3%, P 0.05), apolipoprotein B (-4.9%, P 0.05) and lipoprotein(a) (-37.0%, P 0.05) and increased nitric oxide level (62.2%, P 0.05). In addition, the levels of these variables were positively correlated with change in 16,17-EDP and EEQs content but negatively with change in PGJ2 content.ω-3 PUFA supplementation may improve lipid metabolism and endothelial function possibly by affecting eicosanoid metabolic status at a systemic level during convalescent healing after AMI.URL: http://www.chictr.org.cn. Unique identifier: ChiCTR1900025859.

Purpose: Prediction models for acute myeloid leukemia (AML) are useful, but have considerable inaccuracy and imprecision. No current model includes covariates related to immune cells in the AML microenvironment. Here, an immune risk score was explored to predict the survival of patients with AML. Experimental Design: We evaluated the predictive accuracy of several in silico algorithms for immune composition in AML based on a reference of multi-parameter flow cytometry. CIBERSORTx was chosen to enumerate immune cells from public datasets and develop an immune risk score for survival in a training cohort using least absolute shrinkage and selection operator Cox regression model. Results: Six flow cytometry–validated immune cell features were informative. The model had high predictive accuracy in the training and four external validation cohorts. Subjects in the training cohort with low scores had prolonged survival compared with subjects with high scores, with 5-year survival rates of 46% versus 19% (P < 0.001). Parallel survival rates in validation cohorts-1, -2, -3, and -4 were 46% versus 6% (P < 0.001), 44% versus 18% (P = 0.041), 44% versus 24% (P = 0.004), and 62% versus 32% (P < 0.001). Gene set enrichment analysis indicated significant enrichment of immune relation pathways in the low-score cohort. In multivariable analyses, high-risk score independently predicted shorter survival with HRs of 1.45 (P = 0.005), 2.12 (P = 0.004), 2.02 (P = 0.034), 1.66 (P = 0.019), and 1.59 (P = 0.001) in the training and validation cohorts, respectively. Conclusions: Our immune risk score complements current AML prediction models.

Glycerophospholipids (GPs) and sphingolipids (SPs) are important lipid components in the body and play biological functions. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are important nutrients, and their supplements are commonly used for preventing some diseases. However, the effect of n-3 PUFAs on the human glycerophospholipidome and sphingolipidome is unclear. We used targeted lipidomics to study the GP and SP profile of healthy individuals after supplementation with n-3 PUFAs for 3, 7, 14 and 21 days. Fuzzy c-means clustering was used to cluster the lipid species into six classes reflecting different changed-content patterns after n-3 PUFA supplementation. Among the species with significantly changed content, lysophospholipids were the most sensitive; their content started to increase on day 3. The content of phosphatidylserines increased at a later stage. The content of most of the phosphatidylcholines and alkylphosphatidylcholines decreased on day 21. A correlation network analysis of lipid species suggested that some enzymes involved in the metabolism of lysophospholipids and phosphatidylserines were regulated by n-3 PUFAs. Levels of creatine kinase-MB (CK-MB), urea, glucose, triglycerides and total bilirubin were altered by n-3 PUFA at 21 days. Correlation analysis revealed that the level of CK-MB was negatively correlated with those of species in lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine and phosphatidylserine classes, which were increased by n-3 PUFA supplementation. With the analysis in this work, we demonstrated the regular pattern of n-3 PUFAs on GP and SP metabolism, which provides a pharmacological basis for n-3 PUFAs for clinical application.

Chronic pain, such as persistent inflammatory pain, remains a public health problem that has no effective treatment at present. Bromodomain-containing protein 4 (BRD4) inhibition, induced by JQ1 injection or BRD4 knockdown, has been used to attenuate inflammatory pain; However, it remains elusive whether BRD4 aggravates inflammatory pain by regulating inflammasome. Western blot and immunofluorescence staining showed that BRD4 expression increased after administration of complete Freund’s adjuvant (CFA) and reached its peak on day 3. Immunofluorescence staining showed that BRD4 was mainly colocalized with NeuN-positive neurons in the spinal cord, which was accompanied by upregulation of inflammasome component proteins, such as NLRP3, gasdermin D, and caspase-1. JQ1 was intrathecally injected into mice 1 h before CFA administration, and the mechanical and thermal hyperalgesia levels were measured on days 1, 3, and 7 after CFA administration. CFA-induced inflammatory pain, paw inflammation, and swelling were attenuated by pre-treatment with JQ1. To our knowledge, this study was the first to prove that NLRP3 inflammasome-induced neuronal pyroptosis participates in inflammatory pain. BRD4 inhibition decreased the expression of pyroptosis-related proteins by inhibiting the activation of NF-κB signaling pathway, both in vivo and in vitro. Taken together, BRD4 inhibition exerted analgesic and anti-inflammatory effects against inflammatory pain by inhibiting NF-κB and inflammasome activation, which protected neural cells from pyroptosis.

Terminthia paniculata (Sanyeqi) is widely used for treating inflammation and rheumatic arthritis in the folk areas of Yunnan province, China. Its total extract was first revealed with xanthine oxidase (XO) inhibitory activity in vitro and anti-hyperuricemic effect in vivo. Bioassay-guided separation on Fr. A5 yielded six chalcone-flavonone heterodimers, termipaniculatones A-F. Their structures were elucidated based on extensive spectroscopic analyses involving HRESIMS, 1D and 2D NMR, UV, IR and [α]D, and the absolute configuration of termipaniculatone F was verified by ECD calculation. Termipaniculatones A and E showed obvious XO inhibitory activity with IC50 values of 55.6 and 89.5 μM, respectively, which took effects via a mix-type mode. A molecular modeling study revealed that termipaniculatone A was well located into the active site of XO by interacting with Glu802, Arg880, Thr1010 and Val1011 residues. Termipaniculatone A showed anti-hyperuricemic effects by decreasing serum uric acid levels and inhibiting XO activity in both serum and liver on potassium oxonate (PO)-induced hyperuricemia mice, and anti-inflammatory activity through alleviating paw swelling on monosodium urate (MSU)-induced mice, at the concentration of 20 mg/kg. This is the first time to reveal the anti-hyperuricemic and anti-acute gouty arthritis potency of T. paniculata and the characteristic biflavonoids as active constituents, which provides valuable information for searching new XO inhibitors from natural sources.

Ethnopharmacological relevance Traditional Chinese medicines (TCMs) are fascinating sources for natural drug candidates. Uncaria rhynchophylla (Gouteng) is a famous TCM used for alleviating central nervous system (CNS) disorders, while its antidepressant constituents are still disputed. Aim of the study The present study was designed to assess the antidepressant property of U. rhynchophylla and characterize the active constituents targeting melatonin receptors which are closely related to CNS diseases. Materials and methods The total extract and each fraction of U. rhynchophylla were extensively assessed for their agonistic activity on melatonin receptors in vitro. The following bioassay-guided fractionation yielded the active constituents, whose activity was confirmed by dose-dependent bioassay and antagonistic experiment on HEK293 cells. Their antidepressant effects were evaluated on forced swimming test (FST), tail suspension test (TST) and open-field test (OFT) mice models in vivo. Their metabolic profiles in mice plasma were analyzed by LCMS-IT-TOF. Results The stems and hooks of U. rhynchophylla were revealed with agonistic activity on melatonin receptors (MT1 and MT2). Under the guidance of bioassay, two flavanols, catechin and epicatechin were obtained and showed obviously activity agitating MT1 (EC50 = 25.8 and 156.1 μM) and MT2 (EC50 = 47.3 and 208.8 μM) receptors. The agonistic activity of catechin on melatonin receptors can be antagonized by luzindole at the concentrations of 1.57–100 μM. Catechin could significantly reduce the immobility time in both FST and TST mice models at doses of 80 and 40 mg/kg, without obvious effect on locomotor activity in OFT mice model. Five phase II (M1-M5) and one phase I (M6) metabolites of catechin were detected in mice plasma after intragastric (i.g.) administration. Conclusion Catechin is a potent antidepressant candidate from U. rhynchophylla by targeting melatonin receptors. The main metabolic pathways of catechin in mice plasma are glucuronidation (M3) and methylated glucuronidation (M4 and M5). This study provides valuable information for understanding the antidepressant potency of Gouteng and its active constituents.

Electromyography (EMG) signals can be used for clinical diagnosis and biomedical applications. It is very important to reduce noise and to acquire accurate signals for the usage of the EMG signals in biomedical engineering. Since EMG signal noise has the time-varying and random characteristics, the present study proposes an adaptive Kalman filter (AKF) denoising method based on an autoregressive (AR) model. The AR model is built by applying the EMG signal, and the relevant parameters are integrated to find the state space model required to optimally estimate AKF, eliminate the noise in the EMG signal, and restore the damaged EMG signal. To be specific, AR autoregressive dynamic modeling and repair for distorted signals are affected by noise, and AKF adaptively can filter time-varying noise. The denoising method based on the self-learning mechanism of AKF exhibits certain capabilities to achieve signal tracking and adaptive filtering. It is capable of adaptively regulating the model parameters in the absence of any prior statistical knowledge regarding the signal and noise, which is aimed at achieving a stable denoising effect. By comparatively analyzing the denoising effects exerted by different methods, the EMG signal denoising method based on the AR-AKF model is demonstrated to exhibit obvious advantages.

Depression is associated with high mortality and morbidity rates worldwide. By our random screening, it was first revealed that 23 magnolol derivatives were synthesized followed by in vitro and in vivo evaluation of their antidepressive potential. Compound 7c was found to be the most promising compound, with EC50 values of 396.5 and 383.0 μM agitating on MT1 and MT2 receptors, respectively. Additionally, we carried out in vivo experiments to confirm the efficacy and safety of compound 7c; the compound was found to be orally bioavailable and highly effective, leading to a significant reduction of immobility time in a mouse model of depression (forced swimming test and tail suspension test); the acting mechanism was explored by determining its effect on the levels of monoamine neurotransmitters and their metabolites in different mice brain regions; the acute toxicity study showed that the 50% lethal dose (LD50) of 7c was higher than 2000 mg/kg, p. o. A total of 25 metabolites of 7c were identified, including 5 metabolites in phase I and 20 metabolites in phase II. Altogether, these results indicate that magnolol derivative 7c is a promising lead compound for the development of a new chemical class of antidepressant drugs.

Primary immune thrombocytopenia (ITP) is a bleeding disorder commonly encountered in clinical practice. The International Working Group (IWG) on ITP has published several landmark papers on terminology, definitions, outcome criteria, bleeding assessment, diagnosis, and management of ITP. The Chinese consensus reports for diagnosis and management of adult ITP have been updated to the 4th edition. Based on current consensus positions and new emerging clinical evidence, the thrombosis and hemostasis group of the Chinese Society of Hematology issued Chinese guidelines for management of adult ITP, which aim to provide evidence-based recommendations for clinical decision making.

Salidroside (Sal) is a natural antioxidant that elicits cardioprotective and neuroprotective effects in vivo and in vitro; however, its impact on hepatic ischemia/reperfusion (I/R) injury remains unclear. The purpose of this study was to investigate the hepatoprotective effects of salidroside against segmental (70%) warm hepatic I/R injury in rats. Animals were randomized into Sham, Sham+salidroside pretreatment (Sal), Sham+Sal+carboxyatractyloside (CATR), Sham+CATR, I/R, I/R+Sal, I/R+Sal+CATR and I/R+CATR groups. The hepatic artery, left portal vein and median liver lobes were occluded for 60min and then unclamped to allow reperfusion. Pretreatment with salidroside (20mg/kg/day for 7 days, intraperitoneally) significantly decreased serum alanine aminotransferase (sALT) and serum aspartate aminotransferase (sAST) levels after 6h and 24h of reperfusion and protected the liver against I/R-induced injury. However, this protective effect could be reversed by CATR, a mitochondrial permeability transition pore (MPTP) opener (5mg/kg 30min before I/R insult, intraperitoneally). Mechanistic studies have revealed that salidroside inhibits glycogen synthase kinase-3 beta (GSK-3β) activity and enhances the NF-E2-related factor (Nrf2)-dependent antioxidant response by activating the Akt signaling pathway, thereby reducing mitochondrial reactive oxygen species generation, increasing MPTP resistance and preventing apoptosis by suppressing cytochrome c release and caspase activation during reperfusion. Therefore, salidroside ameliorates hepatocyte death and apoptosis through activation of the GSK-3β/Nrf2-dependent antioxidant response and subsequent MPTP inhibition. These results provide experimental evidence supporting the clinical use of salidroside for hepatoprotection in surgical settings.

Background Adaptor proteins bridge the gap between cell surface receptors and their downstream signaling elements. The clinicopathological and prognostic values of adaptor proteins remain poorly understood. The purpose of the present study was to explore the expression and prognostic value of three adaptor proteins: GRB2-associated binding protein 2 (GAB2), CRK-like protein (CRKL) and fibroblast growth factor receptor substrate 2 (FRS2) in pancreatic ductal adenocarcinoma (PDAC). Methods The expression of GAB2, CRKL, and FRS2 in 77 formalin fixed paraffin embedded (FFPE) samples from 77 PDAC patients, along with three paired fresh PDAC and matched normal tissues from 3 PDAC patients was analyzed by immunohistochemistry and western blot, respectively. The association between the expression of the three proteins and the clinicopathological factors of PDAC was assessed by χ 2 test. The correlation between the expression levels of the three proteins was analyzed by Spearman rank correlation analyses; Kaplan-Meier survival analyses were also performed. Results IHC was successful in 75, 76, and 77 cases for GAB2, CRKL, and FRS2, respectively. Of which, the positive rate of GAB2, CRKL, and FRS2 protein expression was 40.00% (30/75), 53.95% (41/76) and 35.06% (27/77), respectively. The positive rate of GAB2, CRKL and FRS2 co-expression was 16.88% (13/77). Though there was no association between GAB2 expression, CRKL expression, FRS2 expression, GAB2/CRKL/FRS2 co-expression and the clinicopathological parameters of PDAC, positive correlations were observed between the expressions of the three proteins. Further, univariate survival analysis showed that positive expression of GAB2, CRKL and FRS2 and co-expression of GAB2/CRKL/FRS2 of PDAC predicted poor clinical outcomes, and multivariate survival analysis suggested that positive expression of GAB2 and positive co-expression of GAB2/CRKL/FRS2 were independent prognostic factors for disease-free survival (DFS) and overall survival (OS), respectively. Conclusion In conclusion, GAB2, CRKL, and FRS2 may be potential prognosticators and therapeutic targets for PDAC patients.

The immune system plays a pivotal role in defending against infection and cancer immunosurveillance during the onset and procession of malignant disease. Cancer patients are frequently immunocompromised and subject to refractory infection and relapse of leukemia, due to the cytotoxic agents and immunosuppressive glucocorticoids in the chemotherapy regimens. Bu Shen Hui Yang Fang (BSHY), a traditional Chinese compound, was widely used in China to enhance the immune system of leukemia patients combined with chemotherapy and effectively lowered their risk of infection, with specific mechanism unknown yet. Thus, we investigated the effects of BSHY on the immune system using immunosuppressive mouse models. By analyzing the immune system of immunosuppressed BALB/C mice induced by hydrocortisone, we found an increase of CD4+ and CD8+ lymphocytes in the spleens of mice after BSHY treatment. Furthermore, we found the enhanced immune system in BSHY treated group was due to increased proliferation and decreased apoptosis of lymphocytes. Cytokine array analysis revealed that interleukin 4 (IL-4) was reduced in the plasma of immunosuppressed mice but returned to a normal level after BSHY treatment. Moreover, we found IL-4 was an adverse prognostic factor in acute myeloid leukemia patients and part of them could be elevated by BSHY. Mechanistically, we found BSHY enhances the proliferation of lymphocytes in a Stat6-dependent manner. In summary, our current study demonstrates that BSHY enhances the proliferation of lymphocytes in the immunosuppressed mice via upregulating IL-4 signaling.

Background Programmed death ligand 1 (PD-L1) has shown potential as a therapeutic target in numerous solid tumors. Its prognostic significance has also been established in pancreatic ductal adenocarcinoma (PDAC). The present study aimed to explore PD-L1 expression in PDAC cases in a large Chinese cohort using an in vitro diagnostic (IVD) assay to provide further insight into the potential value of programmed cell death protein 1 (PD-1) as a therapeutic target. Methods Three hundred seventy-three PDAC patients were retrospectively recruited in this study. Tissue microarray (TMA) blocks were made from available formalin-fixed and paraffin-embedded (FFPE) tumor and matched adjacent tissue specimens. We evaluated PD-L1 protein expression via immunohistochemistry (IHC) using a U.S. Food and Drug Administration (FDA)-approved IVD assay. The relationships between PD-L1 positivity and both clinicopathological characteristics and patient prognosis were analyzed. PD-1 expression and clinicopathological significance were also evaluated. Results PD-L1 and PD-1 positivity were observed in 3.2% and 7.5% of cases, respectively. PD-L1 showed a predominantly membranous pattern in tumor cells, while no positive PD-L1 staining was observed in normal regions. Statistical analyses revealed that PD-L1 expression was associated with lymph node metastasis. PD-L1 positivity was a prognostic indicator of progression-free survival (PFS) and overall survival (OS) in univariate analyses, but only PFS remained statistically significant in multivariate analysis. PD-1 expression was detected in lymphocytes and was not associated with any clinicopathological feature except a history of pancreatitis. Conclusions The PD-L1 positivity rate is low in PDAC when evaluated using a companion diagnostic assay. It remains an independent prognostic factor for poor PFS. Electronic supplementary material The online version of this article (10.1186/s13000-017-0678-4) contains supplementary material, which is available to authorized users.

// Hui Zhang 1,* , Huanwen Wu 1,* , Jian Guan 2 , Li Wang 1 , Xinyu Ren 1 , Xiaohua Shi 1 , Zhiyong Liang 1 and Tonghua Liu 1 1 Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, PR China 2 Department of Pathology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences, Beijing, PR China * These authors contributed equally to this work Correspondence: Tonghua Liu, email: // Zhiyong Liang, email: // Keywords : Pancreatic cancer, Pancreatic stellate cells, Chemoresistance, SDF-1α, IL-6 Received : November 23, 2014 Accepted : December 25, 2014 Published : December 30, 2014 Abstract Pancreatic cancer exhibits the poorest prognosis among all tumors and is characterized by high resistance to the currently available chemotherapeutic agents. Our previous studies have suggested that stromal components could promote the chemoresistance of pancreatic cancer cells (PCCs). Here, we explored the roles of pancreatic stellate cells (PSCs) and the SDF-1α/CXCR4 axis in pancreatic cancer chemoresitance. Our results showed that primary PSCs typically expressed SDF-1α, whereas its receptor CXCR4 was highly expressed in PCCs. PSC-conditioned medium (PSC-CM) inhibited Gemcitabine (GEM)-induced cytotoxicity and apoptosis in the human PCC line Panc-1, which was antagonized by an SDF-1α neutralizing Ab. Recombinant human SDF-1α (rhSDF-1α) increased IL-6 expression and secretion in Panc-1 cells in a time and dose-dependent manner, and this effect was suppressed by the CXCR4 antagonist AMD3100. rhSDF-1α protected Panc-1 cells from GEM-induced apoptosis, and the protective effect was significantly reduced by blocking IL-6 using a neutralizing antibody. Moreover, rhSDF-1α increased FAK, ERK1/2, AKT and P38 phosphorylation in Panc-1 cells, and either FAK or ERK1/2 inhibition suppressed SDF-1α-upregulated IL-6 expression. SDF-1α-induced AKT activation was almost completely blocked by FAK inhibition. In conclusion, we demonstrate for the first time that PSCs promote the chemoresistance of PCCs to GEM, and this effect is mediated by paracrine SDF-1α/CXCR4 signaling-induced activation of the intracellular FAK-AKT and ERK1/2 signaling pathways and a subsequent IL-6 autocrine loop in PCCs. Our findings indicate that blocking the PSC-PCC interaction by inhibiting SDF-1α/CXCR4 signaling may be a promising therapeutic strategy for overcoming chemoresistance in pancreatic cancer.

5-Hydroxytryptamine 2C (5-HT 2C ) receptor is one of the major targets of anti-obesity agents, due to its role in regulation of appetite. In the present study, the 70% EtOH extract of the roots of Bupleurum chinense was revealed to have agonistic activity on 5-HT 2C receptor, and the subsequent bioassay-guided isolation led to identification of several saikosaponins as the active constituents with 5-HT 2C receptor agonistic activity in vitro and anti-obesity activity in vivo . The new compound, 22-oxosaikosaponin d (1), was determined by extensive spectroscopic analyses (HR-ESI-MS, IR, and 1D and 2D NMR). The primary structure-activity relationship study suggested that the intramolecular ether bond between C-13 and C-28 and the number of sugars at C-3 position were closely related to the 5-HT 2C receptor agonistic activity. Saikosaponin a (3), the main saponin in B. chinense , showed obviously agonistic activity on 5-HT 2C receptor with an EC 50 value of 21.08 ± 0.33 μmol·L −1 in vitro and could reduce food intake by 39.1% and 69.2%, and weight gain by 13.6% and 16.4%, respectively, at 3.0 and 6.0 mg·kg −1 in vivo . This investigation provided valuable information for the potential use of B. chinense as anti-obesity agent.

Cancer diagnosis depending on microarray technology has drawn more and more attention in the past few years. Accurate and fast diagnosis results make gene expression profiling produced from microarray widely used by a large range of researchers. Much research work highlights the importance of gene selection and gains good results. However, the minimum sets of genes derived from different methods are seldom overlapping and often inconsistent even for the same set of data, partially because of the complexity of cancer disease. In this paper, cancer classification was attempted in an alternative way of the whole gene expression profile for all samples instead of partial gene sets. Here, the three common sets of data were tested by NIPALS-KPLS method for acute leukemia, prostate cancer and lung cancer respectively. Compared to other conventional methods, the results showed wide improvement in classification accuracy. This paper indicates that sample profile of gene expression may be explored as a better indicator for cancer classification, which deserves further investigation.

Objective To observe the effects of Tangnaikang (TNK), a compound traditional Chinese herbal medicine, on glucose metabolism and insulin resistance in obese Zucker rats. Methods Twelve male obese Zucker rats, 6 weeks old, were randomly divided into control group and TNK group (3.24 g/kg) after being fed for 2 weeks. All rats received high-fat diet and 4-week treatment. Body weight and blood glucose were tested every week. Oral glucose tolerance test (OGTT) was performed and fasting insulin level was tested on days 0, 14 and 28. Triglyceride, cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and free fatty acids (FFA) were tested on day 28. Glucose infusion rate (GIR) was tested by hyperinsulinemic-euglycemic clamp from day 29. The protein expressions of protein kinase B (Akt), phospho-Akt (p-Akt) (Thr308) and glucose transporter protein 4 (GLUT4) in skeletal muscle and GLUT4 in adipose tissue were measured after hyperinsulinemic-euglycemic clamp test. Results Compared with the control group, the fed blood glucose level and glucose level of OGTT at 120 min had a significant decline in TNK group on day 28, and TNK caused no alteration of the fasting serum insulin, and the GIR increased significantly in hyperinsulinemic-euglycemic clamp study. Furthermore, TNK increased Akt and p-Akt (Thr308) protein expressions in skeletal muscle and decreased the protein expression of GLUT4 in white adipose tissue. Body weight, and triglyceride, cholesterol, LDL-C and FFA contents were slightly decreased in the TNK group, but there were no statistically significant effects. Conclusion TNK increases the protein expressions of Akt and p-Akt (Thr308) of the signal transduction pathway to influence the translocation of GLUT4 in skeletal muscle and improves glucose metabolism by reducing insulin resistance.

Context: The molecular pathogenesis of sporadic insulinomas is unknown. There is a lack of biomarker to distinguish benign and malignant form of insulinoma. Objective: Our objective was to confirm the occurrence of microsatellite instability (MSI) in insulinomas, to identify alterations of mismatch repair (MMR) genes in the tumors, and to evaluate the possibility to distinguish benign and malignant insulinoma or to predict the clinical outcome of patients with these alterations. Design and Patients: We detected MSI and inactivation of MLH1 gene in 55 sporadic insulinomas by PCR, immunohistochemical staining, allelic typing, analysis of promoter methylation, and exon mutations. Their correlations with clinicopathological characteristics were analyzed with univariate and multivariate statistic analysis. Results: A high rate of MSI (MSI-H) was found in 33% of sporadic insulinomas. Reduced expression of mutL homolog 1 (MLH1) protein was observed in 36% of insulinomas and correlated with MSI-H (P = 0.008). Promoter methylation and loss of heterozygosity of MLH1 gene was found in 31 and 49% of insulinomas, respectively. Reduced expression of MLH1 and MSI-H were significantly associated with both tumor malignancy (P = 0.033 and P = 4.8 × 10−6, respectively) and incurable disease (P = 0.006 and P = 0.001, respectively). Conclusion: High frequency of MSI occurred in sporadic insulinomas. The silencing of MLH1 gene may partially contribute to the MSI-H in the tumors. Assessing MSI-H and expressions of MLH1 could be used to distinguish benign and malignant insulinomas and to predict the outcome of patients. Detecting of a high rate of microsatellite instability can be used to distinguish malignancy from benign, and predict clinical outcome of the sporadic insulinomas.

OBJECTIVES To avoid unnecessary lymphadenectomy for renal cell cancer (RCC) in patients with retroperitoneal enlarged lymph nodes (ELNs). METHODS Frozen section examination (FSE) of ELNs was used to evaluate the lymphatic status. In the present study, 114 patients with RCC underwent FSE of ELNs and concurrent regional lymphadenectomy. The results of FSE were compared with the final histopathologic results of lymphadenectomy. Some clinical tumor characteristics were also considered to improve the evaluation effect of the FSE. Multiple regression analysis was applied to define the independent risk factors for lymphatic metastasis. RESULTS The final histopathologic results indicated that 36 patients (31.6%) had nodal metastases. In these 36 patients, the FSE of ELNs revealed positive findings in 32 patients and negative findings in 4 patients. The sensitivity, specificity, concordance, and false-negative rate of FSE was 88.9%, 100%, 96.5%, and 11.1%, respectively. Multivariate analysis revealed that distant metastasis and high T stage (T3-T4) were independent risk factors for lymphatic metastasis. When FSE indicated negative results, no nodal metastases were found (64 patients) without these 2 risk factors. CONCLUSIONS ELNs in patients with RCC do not necessarily indicate metastatic disease, and more than one half of ELNs were benign. FSE of ELNs can be used to evaluate the lymphatic status. Using the findings from FSE and the clinical characteristics of the primary tumor, we can avoid unnecessary lymphadenectomy in patients with retroperitoneal ELNs.

Objective This study aimed to determine a contrast medium injection rate that ensures both accuracy for data and safety for operation by comparing hemodynamic parameters of brain CT perfusion imaging with varied injection rates. Methods Twenty-four healthy volunteers were divided into three groups based on contrast medium injection rates (4.5, 6.0, and 7.5 ml/s). For all subjects, CT perfusion scanning was started at 4 s after antecubital venous bolus of contrast media injection. A perfusion-analyzing software package was used to produce a time–density curve in the anterior cerebral artery and the superior sagittal sinus and calculate the regional cerebral blood flow (rCBF) in the gray matter and the white matter. The hemodynamic indices were compared among the three groups, and statistical analysis was carried out using the F test. Results The time for the arterial rise to reach the peak value for the 7.5-ml/s group was only 0.2 s ahead of the initiation time for the rise of the superior sagittal sinus. The differences of rCBF in the gray matter and the white matter among the three groups were statistically significant. rCBF in the gray matter and the white matter for the 7.5-ml/s group was 52.8 ml · min −1 100 g −1 · (±3.1) and 21.9 ml · min −1 · 100 g −1 (±2.4), respectively. Conclusions The use of the 7.5-ml/s injection rate can meet the prerequisite of the maximum slope model, and the resulting rCBF can be very close to that measured by positron emission tomography. Therefore, 7.5 ml/s was an ideal contrast medium injection rate.

Background and Aim: To our knowledge there are few reports on the K-ras mutation pattern of pancreatic adenocarcinoma from Chinese mainland patients. We examined surgically resected formalin-fixed, paraffin-embedded primary pancreatic adenocarcinoma tissue blocks for the presence of activating point mutations at codon 12 and 13 of the K-ras gene. Methods: Mutations were detected through the use of microdissection, polymerase chain reaction (PCR) and direct sequencing. The results were confirmed by reverse sequencing. Results: The combination of microdissection, PCR and direct sequencing techniques resulted in a rapid and sensitive detection of K-ras mutations at codon 12 and 13. Twenty-five (83%) of the 30 pancreatic adenocarcinomas examined harbored K-ras mutation. Among the 25 pancreatic adenocarcinomas, 24 showed K-ras mutation at codon 12 (11 with GGT-GTT, seven with GGT-GAT, four with GGT-CGT, and two with GGT-TGT), and only one showed a GGC-TGC mutation at codon 13. In this study most of K-ras mutations at codon 12 were at the second base (72%, 18/25) with a transition/transversion ratio of 1 : 1.57 (7/11). Conclusions: The mutation profiles of K-ras at codon 12 in our pancreatic adenocarcinoma samples were significantly different from those of European and Japanese samples.

To explore the potential decrease of serum erythropoietin (EPO) level in patients with multiple sclerosis (MS) complicated with anemia.The serum EPO levels were detected in the patients with MS complicated with anemia (MS group, n=31), patients with iron deficiency anemia (IDA group, n=33), and healthy subjects (normal control group, n=80) by enzyme-linked immunosorbent assay (ELISA). Blood routine test, reticulocyte count, hemoglobin, and indexes of liver and kidney function were also detected.The serum EPO level in MS group was significantly lower than those in IDA group [(101.3±17.6)U/L vs.(166.1±8.7)U/L, P0.01]. Moreover, the serum EPO level decreased as the severity of anemia in the MS group increased: it was (95.7±9.6), (101.7±8.1), and (123.7±9.3) U/L in patients with mild, moderate, and severe anemia, respectively (P0.05). Other indicators including blood routine findings, reticulocyte count, hemoglobin, and liver and kidney function parameters showed no significant difference between the MS group and the IDA group (P0.05).The serum EPO level decreases in patients with multiple sclerosis complicated with anemia, and the decreasing levels are related with the severity of anemia. Thus EPO therapy may be beneficial for these patients.

To observe the immunohistochemical staining of IgG4 in nonspecific interstitial pneumonia (NSIP) and to study the clinicopathological features of IgG4-related NSIP.Retrospective analysis was carried out for 32 patients with NSIP who had been admitted into Peking Union Medical College Hospital from November 2002 to October 2010. The diagnosis of NSIP was established by surgical lung biopsy and all specimens were fixed in neutral formalin and embedded in paraffin. Sections were cut for HE and immunohistochemical stain. According to the diagnostic criteria for IgG4-related disease, 4 cases were confirmed to be IgG4-related NSIP. The clinicopathological features including clinical history, laboratory examination, and pathologic evaluation were studied.The 4 patients with IgG4-related NSIP included 1 man and 3 women, with a median age of 48 years (range, 44 - 56 years). The presenting symptoms were dry cough or shortness of breath. One patient (1/4, 25.0%) was found to have a positive autoantibody but no cases showed positive RF in serum. The histological finding of the 4 cases was characterized by inflammatory cell infiltration in interstitium with fibrosis, and 1 case showed obliterative arteritis. The numbers of IgG4-positive plasma cells in the 4 cases were 42/hpf, 22/hpf, 11/hpf, and 33/hpf respectively, while the percentages of IgG4-positive to IgG-positive plasma cells were 70%, 71%, 57%, 43% respectively.IgG4-related interstitial pulmonary disease can be characterized as the NSIP pattern. The pathological features of IgG4-related NSIP include infiltration of lympho-plasmacytes and eosinophils in interstitium with fibrosis, and lymphoid follicles are frequently identified in the area of lymphocyte aggregation, but obliterative arteritis is infrequently identified in the lesion. Immunohistochemical staining of IgG and IgG4 is very helpful for a definite diagnosis of IgG4-related disease.

To investigate the differences between primary mediastinal B-cell lymphoma (PMBCL) and non-mediastinal conventional diffuse large B-cell common lymphoma (DLBCL) in immunoglobulin gene rearrangement and EB virus infections.Twenty cases of PMBCL and 30 cases of non-mediastinal DLBCL were collected from September, 2000 to May, 2011. Pathological data were retrospectively analysed. Immunoglobulin heavy chain and light chain gene rearrangements and EBER in-situ hybridization were performed.Six of 20 cases of PMBCL showed monoclonal gene rearrangement, all of which were weakly detected. Twenty-seven of 30 cases of ordinary diffuse large B-cell lymphoma showed monoclonal gene rearrangement, which were strongly detected (90.0%). Only 1 of 20 cases PMBCL and 2 of 30 cases of DLBCL were positive for EBER in-situ hybridization.The detection rate of immunoglobulin gene rearrangement is significantly lower in PMBCL than that of non-mediastinal DLBCL. However, EB virus infection rates are very low in both types of lymphomas.

To observe the expressing model of galectin-1 in colorectal carcinoma (CRC) progress.The expression of galectin-1 in peritumor, adenoma and CRC tissues using immunohistochemistry, western-blotting and RT-PCR was detected. Serum galectin-1 concentrations were detected by ELISA.Galectin-1 intensities in peritumor, adenoma and CRC tissues increased serially. Galectin- 1 expressed in the extracellular matrix of peritumor and pathological colorectal tissues mainly, while almost none was detected in colorectal epithelia. Different colorectal tissues expressed galectin-1 with different intensities. Non-parametric tests showed increased galectin-1 intensities were in peritumor, adenoma and carcinoma tissues (p0.001). The over-expressing rates of galectin-1 in CRCs from Dukes A to D stage were increased significantly (p0.001). Differently expressing intensities of galectin-1 were in different invasive locations of CRCs. RT-PCR showed that no statistically significant differences of galectin-1 mRNA were observed among peritumor colorectal tissue, adenoma and CRC tissues (p0.05). Serum concentrations of galectin-1 decreased from healthy individuals to Dukes D CRCs. The differences between any of the groups were significant (p0.05).Stronger intensities of galectin-1 protein in the extracellular matrix of CRC tissues than those of peritumor and adenoma tissues, with no significant differences in the mRNA levels of galectin-1. Interestingly, serum galectin- 1 concentrations in patients with CRC decreased significantly. Above all, we presume that overexpression of galectin-1 protein in the CRCs is derived possibly from secreted galectin-1 from other normal tissues that cumulate in the stroma.

To study the immunophenotype and gene rearrangement pattern of pulmonary lymphomatoid granulomatosis.Nine cases of pulmonary lymphomatoid granulomatosis, included 5 cases of open lung biopsy, 3 cases of lobectomy specimen and 1 case of autopsy, were retrospectively analyzed by immunohistochemistry, in-situ hybridization for Epstein-Barr virus-encoded RNA, immunoglobulin and T-cell receptor gene rearrangement studies.The age of patients ranged from 3 to 59 years. The male-to-female ratio was 3: 6. Histologically, all cases showed lymphocytic infiltration surrounding the blood vessels and in the perivascular areas. Most of these lymphoid cells expressed T-cell marker CD3. There were also variable numbers of CD20-positive B cells. The staining for CD56 was negative. According to the WHO classification, there were 4 cases of grade I , 1 case of grade II and 4 cases of grade III lesions. Six cases had gene rearrangement studies performed and 3 of them demonstrated clonal immunoglobulin gene rearrangement (including 1 of the grade II and 2 of the grade III lesions). No T-cell receptor gene rearrangement was detected.Pulmonary lymphomatoid granulomatosis may represent a heterogeneous group of lymphoproliferative disorders. Some of the cases show B-cell immunophenotype and clonal immunoglobulin gene rearrangement, especially the grade II and grade lesions. They are likely of lymphomatous nature.

The effects of silybin and tetrandrine on the survival of spontaneously hypertensive rats subjected to acute coronary artery occlusion were investigated. The mortality after acute coronary occlusion in spontaneously hypertensive rats (66.7%) was higher than that of control Wistar-Kyoto rats (20%, P < 0.05). Oral administration of silybin (300 mg/kg daily) for 8-12 days reduced mortality in spontaneously hypertensive rats (0, P < 0.01 in comparison with untreated spontaneously hypertensive rats). Administration of tetrandrine 40 mg/kg daily for 8-12 days reduced the mortality to some extent (22.2%, P = 0.051, as compared with control rats). Silybin reduced blood pressure and the incidence of post-occlusion arrhythmias in spontaneously hypertensive rats to the same extent as tetrandrine. Both silybin and tetrandrine decreased the severity of ventricular hypertrophy. Although there were significant decreases in risk zone and infarct zone in silybin- and tetrandrine-treated rats, the ratio of infarct to risk zone was not changed. The results implies that silybin may be beneficial when used in hypertensive patients who develop acute myocardial infarction.

BackgroundThis study was designed to investigate EGFR protein expression, EGFR copy number and EGFR mutations in lung adenocarcinomas, to explore the relationship of the three markers.MethodsEGFR status was analyzed in surgically resected lung adenocarcinoma samples from 133 Chinese patients by three methods: protein expression (n = 133) by standardized immunohistochemistry (IHC), gene copy number (n = 133) by fluorescence in situ hybridization (FISH), and mutation analysis using the Scorpion amplification refractory mutation system (ARMS) (n = 133).ResultsThe results showed that 68.4% of the samples were positive by IHC, 42.1% were positive by FISH, and 63.9% contained activating kinase domain mutations. EGFR mutations were more frequent in non-smoking patients (p = 0.008), and EGFR mutations were associated with EGFR FISH positivity (p < 0.0001). When using 10% positivity and 2+ as cutoffs, EGFR protein expression was significantly correlated with EGFR FISH positivity (p = 0.012) and EGFR mutations (p = 0.008) after Bonferroni correction.ConclusionEGFR protein expression, EGFR copy number and EGFR mutations were closely related to each other. Standard methods and interpretation criteria need to be established.

Cardiac resynchronization therapy (CRT) is a major breakthrough in therapy for advanced heart failure patients; however, a number of key clinical research questions remain, perhaps most importantly the issue of why apparently suitable patients do not respond to CRT.Seven patients, six males and one female, aged (56.43 +/- 6.13) years, all diagnosed with dilated cardiomyopathy, were included in this study. They were all non-responders to CRT who underwent routine optimization postoperatively, and received optimal drug therapy. On the basis of biventricular pacing, titrating various atrioventricular (AV) intervals were performed to get the true fusional QRS complexes composed of biventricular pacing and AV intrinsic conduction. Then, the effects of AV intrinsic conduction during CRT were evaluated.On the setting of AV intrinsic conduction during CRT, the true fusional QRS complexes were the narrowest, and all patients showed alleviation of symptoms, improvement of exercise tolerance, life quality and hemodynamic parameters during more than 6 months of follow-up.Titrating AV intervals to get the true fusional QRS complexes composed of biventricular pacing and AV intrinsic conduction will be beneficial for non-responders to CRT. Maintaining AV intrinsic conduction during CRT may decrease the rates of non-responders to CRT.

To study the clinicopathologic characteristics and immunohistochemical profile of lung adenocarcinomas with a micropapillary pattern (MPP).Among 135 cases of lung adenocarcinomas, the clinical, histological and immunohistochemical features of 48 cases of lung adenocarcinomas with a micropapillary components (the micropapillary componentsor = 10%) were studied. The literature was reviewed.All the 135 cases were resected pulmonary adenocarcinomas. Among 48 cases of lung adenocarcinomas with a micropapillary components, the age of patients ranged from 43 to 85 years (mean = 60.7 years). The male-to-female ratio was 9:7. Histologically, 36 cases of lung adenocarcinomas with the MPP were characterized by small papillary tufts lacking a central fibrovascular core lying freely within alveolar spaces (IA type) or in the clefts of fibrous tissue just like those in MPP breast cancers (IB type). Another type of the micropapillary pattern consisted of 12 cases, the micropapillary tufts floating within cystic spaces lined by tumor cells (II type). In micropapillary pattern-positive cases, lymphatic invasion and lymph node metastasis were identified significantly more frequently than in micropapillary pattern-negative cases (P0.01). The percentages of cases positive for various markers were 97.9% (47/48) for E-cadherin, 89.5% (43/48) for beta-catenin, 91.7% (44/48) for Muc-1, 70.8% (34/48) for epidermal growth factor receptor, 35.4% (17/48) for p53, 93.8% (45/48) for Ki-67. The percentages of cases with high expression (including 3+ or 4+) for these markers were 72.3% (34/47) for E-cadherin, 90.7% (39/43) for beta-catenin, 88.6% (39/44) for Muc-1, 52.8% (19/36) for epidermal growth factor receptor, 58.8% (10/17) for p53, 46.7% (16/36) for Ki-67. Adequate clinical follow-up information was available for 36 patients. The mean follow-up time was 21.1 months. Among these, 16 of 36 patients (44.4%) were alive with no evidence of tumor, 12(33.3%) were died, and 8 (22.2%) were alive with tumor.Lung adenocarcinomas with the MPP correlates positively with lymphatic invasion and lymph node metastasis, and are likely to have a potential for high malignancy, suggesting a poor prognosis.

Matrix metalloproteinase (MMP)-26 has been implicated in genesis, progression, invasion and metastasis of several types of human cancers. This study was to investigate the expression of MMP-26 protein in invasive non-small cell lung cancer (NSCLC), pre-invasive lung cancer and normal lung tissues, thus to explore the role of MMP-26 in the progression and prognosis of NSCLC.SP immunohistochemistry was used to measure the expression of MMP-26 protein in 72 specimens of NSCLC, 14 specimens of atypical hyperplasia and 10 specimens of normal lung tissues.The high expression rate of MMP-26 was 0 (0/10) in normal lung tissues, 14.3% (2/14) in atypical hyperplasia and 59.7% (43/72) in NSCLC. The expression rate of MMP-26 protein was significantly higher in NSCLC than in atypical hyperplasia and normal lung tissues (p0.01); the expression was higher in atypical hyperplasia than in normal lung tissues, but the difference was not significant (p0.05). The high expression rate of MMP-26 protein was significantly correlated to stage (p0.05) and lymph node metastasis (p0.05), but not to age, gender, tumor size and differentiation (p0.05). Multivariate analysis showed that MMP-26 and stage were independent prognostic factors of NSCLC (p0.05). The disease-free survival and overall survival were shorter in NSCLC patients with high expression of MMP-26 than in those with low expression of MMP-26 (log-rank = 19.34 and 23.2, both p0.001).High expression of MMP-26 protein is correlated to carcinogenesis, lymph node metastasis, clinical stage and prognosis of NSCLC. Therefore, MMP-26 may be served as a tumor marker in monitoring progression and predicting prognosis of NSCLC.

To investigate EGFR mutations and gene copy number status in non-small cell lung carcinomas in the Chinese patients.Using formalin fixed and paraffin embedded tissue samples, EGFR mutations were investigated in 290 cases of non-small cell lung carcinomas by microdissection and scorpions amplification refractory mutation system. The status of EGFR gene copy number was investigated by FISH. Furthermore, the relationship between EGFR mutations and gene copy number, and the relationship between EGFR gene status and clinicopathological variables of non-small cell lung carcinoma were analyzed.The overall mutation rate of EGFR was 41.7% (121/290). The mutation rates in adenocarcinoma, large cell carcinoma and squamous carcinoma were 48.4%, 16.7% and 0, respectively. Ninety-two of 121 cases with mutations had exon 19 deletion and L858R mutation, and 6 tumors contained both types of the mutation. The overall FISH positive rate of EGFR was 51.2% (107/209). FISH positive rates in adenocarcinoma, large cell carcinoma and squamous carcinoma were 52.1%, 75.0% and 11.1%, respectively. Therefore, EGFR mutations mainly occurred in the adenocarcinoma, and was significantly correlated with EGFR high copy number.There are higher EGFR mutation rate and FISH positive rate in non-small cell lung carcinoma in Chinese patients. Combined analysis of EGFR mutation and gene copy number by FISH may provide a superior approach in selecting patients who may benefit anti-EGFR target therapy.

To investigate 18q21 LOH in human pancreatic ductal adenocarcinomas and chronic pancreatitis by fluorescence in-situ hybrydization (FISH) technique, and to analyze the relationship between 18q21 LOH and clinicopathologic characteristics.RP11-729G3 and RP11-850A17, the regions on 18q21, were selected as the target fragments, the region RP11-621L6, close to the centromere of chromosome 18, was selected as the reference fragment. The specific BAC clones were used to isolate and purify the corresponding genomic DNA, which were labeled with biotin or DIG by nick translation into dual color probes. 18q21 LOH was assessed by dual-color FISH in 30 cases of pancreatic ductal adenocarcinoma and 10 cases of chronic pancreatitis. All samples were 10% formalin fixed and paraffin embedded. The relationship between 18q21 LOH and clinicopathologic characteristics was analyzed.Among 30 cases of pancreatic ductal adenocarcinoma, 25 cases showed LOH at the region RP11-729G3 (83.3%), and 26 cases showed LOH at the region RP11-850A17 (86.6%). Among these, 25 cases with LOH at both regions, 1 case showed LOH only at the region of RP11-850A17. No LOH was found in 10 cases of chronic pancreatitis.18q21 LOH is a high-frequency event in human pancreatic ductal adenocarcinomas. LOH at the regions RP11-729G3 and RP11-850A17 demonstrates a high concordance. 18q21 may play an important role during pancreatic carcinogenesis and tumor progression. 18q21 LOH may be used as a diagnostic marker for pancreatic ductal adenocarcinoma.

Cancer is a complex disease involving multiple oncogenes with diverse actions. Inhibiting only one oncogene is unlikely to eliminate the malignancy of cancer cells. The goal of this study was to investigate whether synergistic effects can be achieved by combined silencing of two oncogenes, K-ras and Akt2, which are key players in the Ras/MAPK and PI3K/Akt signaling pathways. The pancreatic cancer cell line, Panc-1, was selected for these studies as it has elevated expression of K-ras and Akt2. Compared with inhibiting each oncogene alone, simultaneously silencing the two oncogenes with RNA interference (RNAi) more effectively inhibited Panc-1 cell proliferation and colony formation, induced a significantly higher percentage of apoptosis and resulted in greater inhibition of c-myc expression in vitro. Furthermore, when delivered by polyethyleneimine into Panc-1 tumors in nude mice, RNAi simultaneously targeting K-ras and Akt2 inhibited tumor growth more efficiently than RNAi targeting the individual oncogenes. Therefore, RNAi simultaneously silencing the oncogenes K-ras and Akt2 may offer potential opportunities for pancreatic cancer gene therapy.

To investigate the genetic status of 13q and its role in the oncogenesis and progress of soft tissue tumors.Forty-one soft tissue tumors, including 9 benign tumors, 9 tumors of malignant potential and 23 sarcomas, were studied by fluorescence in-situ hybridization (FISH) using dual color probes. The probes were generated from BAC clones RP11-685I15, RP11-352N7 and RP11-505F3, corresponding to Rb, RFP2, KCNRG and KLF5 genes respectively.Loss of heterozygosity (LOH) of RP11-685I15 were found in 8/41 cases, LOH of RP11-352N7 was seen in 4/41 cases and LOH of RP11-505F3 was present in 3/41 cases. LOH of all 3 loci were detected in 2 cases. LOH of RP11-61K9, an internal control locus, was detected in 2 cases. One case of malignant peripheral nerve sheath tumor showed amplification at all 3 loci. Amplification of RP11-505F3 was seen in another 2 cases.A significant percentage of soft tissue tumors exhibited chromosomal instability, reflected by an increase of LOH at tumor-suppressing gene loci. The incidence of 13q abnormality was different in various types of soft tissue tumors, indicating that alterations of Rb, RFP2, KCNRG and KLF5 tumor suppressing genes may play diverse roles in different types of soft tissue tumor.

To investigate the protein expression and gene copy number of EGFR and HER2, and the correlation between the two markers in colorectal carcinomas in Chinese.Total 42 samples of paraffin-embedded colorectal carcinomas in tissue microarray format were studied by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) for EGFR and HER2 protein expression and gene copy number status, respectively.Among 42 cases evaluated, EGFR scores were 0 in 18 cases, 1+ in 10 cases, 2+ in 5 cases and 3+ in 9 cases. HER2 expression was negative in 39 tumors, 1+ in 1 tumor, 2+ in 1 tumor and 3+ in 1 tumor. For FISH assessing EGFR, 18 (42.9%) cases showed no apparent copy number changes, including 14 (33.3%) cases of disomy and 4 (9.5%) cases of low trisomy, 24 (57.1%) cases showed increased gene copy numbers including high trisomy in 3/42 (7.1%), low polysomy in 9/42 (21.4%) and high polysomy in 12/42 (28.6%) cases. Gene amplification of EGFR is not detected. Four of 42 patients (9.5%) had increased HER2 gene copy number, including 3 patients with high polysomy and 1 patient with gene amplification. Significant association was not seen between EGFR protein expression and the gene copy number, nor between two markers and tumor differentiation. There was a highly significant concordance between the gene amplification and IHC 3+ for HER2 similar to that of breast cancer.Protein expression and/or increased gene copy number of EGFR is common in colorectal carcinomas but unrelated to pathological features in this cohort. HER2 protein overexpression and/or gene amplification are rare.

To investigate the changes of topoisomerase IIalpha (TOP2A) and HER2/neu genes in pancreatic ductal adenocarcinomas of Chinese patients, and to determine their roles during carcinogenesis and tumor progression.Expressions of TOP2A and HER2/neu proteins were detected by using immunohistochemistry, while gene amplifications of TOP2A and HER2/neu were assessed by using multi-color fluorescence in situ hybridization (FISH). All the samples were of paraffin embedded and 10% formalin fixed tissue, including 26 cases of pancreatic ductal adenocarcinomas with adjacent non-neoplastic pancreatic tissues, 10 cases of chronic panreatitis, and 10 cases of normal pancreas. The correlation between TOP2A and HER2/neu gene status was analyzed.By immunohistochemistry, the nuclear positive index of TOP2A in pancreatic ductal adenocarcinomas varied from 0.5% to 70%, and the positive rate of HER2/neu in pancreatic ductal adenocarcinomas was 46.2% (12/26). By FISH, 9/10 TOP2A amplified adenocarcinomas showed TOP2A and HER2/neu gene coamplification, while one case with HER2/neu gene amplification adenocarcinoma showed no TOP2A amplification. No expression of TOP2A, HER2/neu proteins and no amplification of TOP2A and HER2/neu gene were detected in adjacent non-neoplastic pancreatic tissues, chronic pancreatitis tissues and normal pancreas. No relationship was found between protein expression and gene amplification of TOP2A and HER2/neu (P0.05). TOP2A gene amplification was significantly correlated with HER2/neu gene amplification (P0.01).Protein expression of TOP2A and HER2/neu are not associated with the gene amplification. There is a significant correlation between TOP2A amplification and HER2/neu gene amplification. Co-amplification of TOP2A and HER2/neu may play an important role in the carcinogenesis and progression of pancreatic carcinoma. Evaluation of the status of TOP2A and HER2/neu may be helpful to achieve target therapy of pancreatic carcinoma.

To study the gain of chromosome 8 and c-myc gene and lipoprotein lipase gene status in prostatic adenocarcinoma of Chinese patients, and to analyze the relationship between chromosome 8 alterations and Gleason score of prostatic cancer.Formalin-fixed, paraffin-embedded prostatic biopsy tissues from 34 Chinese patients with untreated prostatic adenocarcinoma were studied by three-color fluorescence in situ hybridization (FISH) using ProVysion(TM) probe kit. The materials included 1 case with Gleason score 5, 10 cases with Gleason score 6, 14 cases with Gleason score 7, 4 cases with Gleason score 8 and 5 cases with Gleason score 9. The relationship between Gleason score and chromosome 8 aneusomy, c-myc and lipoprotein lipase gene copy number, including gene amplification or deletion, were analyzed.Seventeen (50%) of the 34 cases studied had gain of chromosome 8, while 21 cases (61.8%) had gain of c-myc gene copy number, 15 cases (44.1%) had lipoprotein lipase gene monosomy, 23 cases (67.6%) had c-myc gene amplification, 21 cases (61.8%) had deletion of lipoprotein lipase gene and 16 cases (47.1%) had lipoprotein lipase gene deletion coupled with c-myc gene amplification. In general, at least one type of chromosome 8 alteration was identified in 85.3% of cases (29/34). Gain of chromosome 8 was strong significant associated with Gleason score (P = 0.0006). A positive correlation between increased c-myc copy number and high Gleason score was also noted (P = 0.0035). On the other hand, loss of lipoprotein lipase gene was negatively correlated with high Gleason score (P = 0.0383). In addition, the association of c-myc gene amplification with high Gleason score was noted after age adjustment (P = 0.0462).Alterations in chromosome 8 are common in prostatic adenocarcinoma occurring in Chinese patients. There is a correlation between Gleason score and gain of chromosome 8, increased c-myc gene copy number, c-myc gene amplification and lipoprotein lipase gene deletion. C-myc gene amplification accompanied by lipoprotein lipase gene deletion is also a common occurrence in prostatic cancer. Our data suggest that chromosome 8 alterations may play some roles in the development and progression of prostatic adenocarcinoma.

To study the clinicopathologic features of osteomalacia or rickets-associated mesenchymal tumors.The clinical and pathologic findings of 10 cases of osteomalacia or rickets-associated mesenchymal tumors were evaluated. Hematoxylin and eosin stain, immunohistochemistry and histochemistry were performed on the archival paraffin sections.Amongst the 10 patients studied, 6 were males and 4 were females. Their age at the time of operation ranged from 28 to 69 years ( mean = 45.6 years). A history of long-standing bone pain, arthralgia, limitation in movement, hypophosphatemia and hyperphosphaturia was present in all cases. The duration of symptoms ranged from 2 to 27 years (mean = 9.6 years). The tumor size ranged from 1 to 7 cm (mean size = 3.52 cm). Microscopically, the tumors were composed of various mesenchymal cells, including spindled fibroblast-like cells, adipocytes, chondroid cells and mucinous cells. The background was rich in blood vessels. In 8 of the 10 cases, there was also dystrophic calcification in an unusual flocculent or "grungy" pattern. Peripheral woven bone shell formation was noted in 2 cases and non-urate crystal deposition in 2 cases. Mitotic figures were rare in 9 cases. In 1 of the 10 cases however, mitotic figures and bizarre cells were commonly encountered. On immunohistochemical study, the tumor cells were all positive for vimentin. There was focal positivity for smooth muscle actin and CD34 in 5 and 3 cases respectively. The staining for desmin, S-100 and AE1/AE3 was negative. Ki-67 proliferation index was less than 4% in 8 cases and 30% in 1 case. Alcian blue-positive mucinous matrix and mucinous degeneration around vessels were noted in 8 cases.Most of the osteomalacia or rickets-associated tumors are either benign or low-grade malignant mesenchymal tumors. They can be mistaken as other neoplasms due to the morphologic heterogeneity present. Thorough understanding of the associated clinical features and laboratory investigation results is helpful in arriving at the correct diagnosis.

To investigate the clinicopathologic characteristics, immunophenotype and TCR gene rearrangements of hepatosplenic T-cell lymphoma in eight Chinese patients.Eight Chinese patients with hepatosplenic gammadelta T-cell lymphomas were studied. Hematoxylin-eosin-stained slides and clinical histories were reviewed. We also carried out immunohistochemical staining for CD3, CD4, CD8, CD20, CD43, CD56, CD79a, UCHL-1, and TCR gammadelta. Rearrangements of TCR gamma and delta chain genes were also studied.The spleens were enlarged and the cut surfaces were homogeneous and red-purple in color without identifiable gross lesions or enlarged hilar lymph nodes. Histologically, lymphoma cells infiltrated the cords of Billroth and often packed the sinuses. Liver biopsy showed lymphoma cell infiltrations in the sinusoids, and three cases showed involvements of the portal tracts. Immunohistochemically lymphoma cells were positive for CD3, CD43, and CD56 in all cases. Four of eight cases were positive for CD8, and all cases were negative for CD4 (6/6). Monoclonal rearrangements of TCR gamma gene were demonstrated by PCR analysis in five out of the eight cases. TCR delta gene rearrangements were detected in six out of the eight cases, which demonstrated single bands on PAGE gel, and the amplification products in two cases were confirmed by sequencing.The clinicopathology of hepatosplenic gammadelta T-cell lymphoma in Chinese patients is similar to what was previously reported except that the splenomegaly is not so massive, and CD8 is positive.

The pathogenesis of sporadic insulinomas is not clear, and there are no reliable genetic determinants that are useful to distinguish malignant and benign forms of this tumor. It was reported that 1q LOH might contribute to pathogenesis in gastrinomas and was correlated with tumor progression. However, little data are available on 1q LOH in sporadic insulinomas. In our study, we determine whether 1q LOH occurs in sporadic insulinomas and is associated with tumor malignancy by performing 1q allelotyping with 17 markers in 40 tumors and pair normal DNA. Thirty-five (88%) insulinomas had 1q LOH. Of the 35 insulinomas with 1q LOH, 14 (40%) had 1q21.3-23.2 LOH over a 7.5 cM region (SRO-1), whereas LOH in 21 tumors (60%) occurred at 1q31.3 over an 11.4 cM area (SRO-2). Of 24 tumors without MEN1 LOH, 20 had either SRO-1 or SRO-2 LOH (83%), whereas in 16 tumors with MEN1 LOH, 9 were shown to have LOH at either SRO-1 or SRO-2 (56%) (p = 0.065). This result suggests that LOH at 2 SRO might be MEN1 gene independent and may contribute to the pathogenesis in a subset of insulinomas without MEN1 gene LOH. The presence of 1q21.3-23.2 LOH is significantly associated with malignancy of insulinomas (p = 0.014). The high frequency of LOH at 1q 21.3-23.2 and 1q31.3 suggests these 2 areas may harbor putative tumor suppressor genes that may play an important role in the tumorigenesis of a subset of insulinomas. LOH at 1q21.3-23.2, which was associated with tumor malignancy, could be one of the genetic markers for identifying malignancy in sporadic insulinomas.

To determine the expression status of survivin gene in pancreatic carcinoma.Expression of survivin gene was evaluated by immunohistochemistry, Western Blot and RT-PCR in 59 cases of pancreatic carcinoma along with their corresponding adjacent benign tissues, 11 cases of chronic pancreatitis, and 7 pancreatic carcinoma cell lines.The positive expression rate of survivin in pancreatic carcinoma was 72.8% (43/59). There was no relationship between the expression of survivin and tumor stage and differentiation. No expression of survivin was detected in benign tissue adjacent to the tumors as well as in samples of chronic pancreatitis. All 7 pancreatic carcinoma cell lines showed a positive expression of survivin mRNA and protein.The expression of survivin appears to be tumor specific to some extent in our pancreatic carcinoma samples. Survivin may be an ideal target for therapy against pancreatic carcinoma.

To study the clinical and pathological characteristics of pulmonary epithelioid hemangioendothelioma.Four cases of pulmonary epithelioid hemangioendothelioma were studied by histopathologic and immunohistochemical examination of lung biopsy specimens.There were 3 female and 1 male, age 28 to 40 years. Clinically the tumor presented as multiple bilateral small nodules in the lung. Histologically, crown-like clusters of epithelioid tumor cells were obtained which filled in the alveoli locating at the periphery of the tumor nodules, while the central part of the nodules contained myxoid to hyaline matrix. The overall architecture of the lung was still preserved. Additionally, intracytoplasmic vacuoles were seen in tumor cells within which red blood cells were sometimes identified. Tumor cells generally lacked pleomorphism, mitotic activity and necrosis. They were immunohistochemically positive for CD31 and CD34. AE1/AE3 staining was positive in some cases.Pulmonary epithelioid hemangioendothelioma often occurs in a middle-aged woman and represents a distinct clinical pathological entity.

To investigate the correlation between loss of Y chromosome and development of pancreatic cancer.The status of Y chromosome was analyzed by two color interphase fluorescence in situ hybridization. performed on paraffin sections of pancreatic cancer tissues from 15 Chinese males. The probes located on the heterochromatin region of chromosome Y (maps to Yq12) and on the alpha satellite of X chromosome (maps to centromeric region) were selected for testing and as control respectively.The cancer cells from 10 out of the 15 pancreatic cancer patients studied showed loss of chromosome Y. The Y chromosome in the cells of surrounding non-neoplastic pancreatic tissues was intact.Loss of chromosome Y occurs non-randomly in tumor cells of Chinese male patients with pancreatic cancer. This cytogenetic aberration, which happens in high frequency, may serve as one of the markers for pancreatic malignancy.

To elucidate the etiology, pathohistology, clinical characteristic and differential diagnosis, reduce missed diagnosis and improve the early detection and treatment of Penicillium Marneffei infection, by means of this case report and literature review.A patient hospitalized Penicillium Marneffei infection were presented here, together with 27 cases in the literature, among which 10 patients had complications of AIDS and 5 with other diseases.Penicillium Marneffei is a temperature-sensitive, two-phase fungus, which can infect healthy and immunocompromised subjects. The common symptoms are lymphadenopathy and infection of the lung. The infection may be local or diffuse, involving the intestinal tract, soft tissue, bone, liver, spleen and bone marrow etc. The lesion can be classified into the granuloma type, suppurative type and anergy/necrosis type histologically. The yeast-like fungus were mainly found in the cytoplasm of macrophages, which were demonstrated by PAS and Giemsa staining. The wine red color developed on the culture confirms the diagnosis.The diagnosis of Penicillium Marneffei infection should be considered when tuberculosis is suspected but not confirmed, and if the patient has a history of having lived or traveled in Southeast Asia, is anemic or resistant to anti-tuberculosis treatment. The major differential diagnosis is histoplasmosis. Early administration of anti-fungus drugs is essential for recovery.

To detecte whether loss of heterozygosity (LOH) at the MEN-1 locus as well as 22q occurs in sporadic insulinoma and if LOH can be used as a genetic marker to differentiate malignant and benign insulinomas.MEN-1 gene and 22q allelotyping were performed by PCR with microsatallite markers in DNA from microdissected normal and tumor tissues from archived or frozen insulinomas (8 malignant and 32 benign, from 38 patients). The significance was calculated using t test and Cochran-Mantel-Haenszel Statistics, P0.05 was considered significant.Sixteen of the 40 insulinomas (40.0%) had MEN-1 LOH and 22q LOH was shown in 30 of the 40 tumors (75.0%). Eleven of the 30 tumors (37.0%) with 22q LOH had 22q12 LOH over a 3 cm region, whereas LOH in 14 tumors (47.0%) occurred at 22q13.3. Eight tumors with D22S 280 locus (22q12) LOH were shown without MEN-1 LOH while 14 of the 30 tumors without D22S280 LOH had MEN-1 LOH (0% vs 47%, P = 0.016). Six of the 14 tumors (43.0%) with 22q13.3 LOH were malignant, whereas 2 of 26 tumors without 22q13.3 LOH (8.0%) are malignant (P = 0.0088).MEN-1 gene LOH may contribute to a proportion of insulinomas, and 22q LOH occurs frequently in insulinomas. D22S 280 (22q12) LOH may independently contribute to the tumorogenesis of sporadic insulinoams, whereas detecting 22q13.3 LOH in insulinomas can be a potential genetic marker to distinguish malignancy from benign tumors.

To investigate the expression of vascular endothelial growth factor (VEGF), CD44v6 in non-small cell lung carcinoma.The expression of VEGF and CD44v6 in 35 cases I approximately II stages, 27 cases III approximately IV stages and 50 cases with the metastasis of lymph nodes, 12 cases without metastasis of non-small cell lung carcinoma (NSCLC) tissues were studied by SP immunohistochemistry staining, and the 3 years survival rates were calculated in 42 cases patients.The positive rates of VEGF and CD44v6 in NSCLC were 73% and 69%, respectively. They were both higher than those of matched normal lung tissues, P0.01. The expression of VEGF and CD44v6 were related to clinical stages and metastasis of lymph nodes significantly. The expression rates of the two markers in NSCLC with the metastasis of lymph nodes were higher than those without metastasis, chi(2) = 7.146 and 5.376 respectively, and those of III approximately IV stages were higher than those of I approximately II Stages, chi(2) = 6.392 and 12.152 respectively. Survival rates of three years were lower to those patients with positive expression of the two makers than those with negative expression. In a multivariate analysis, besides TNM stages and metastasis of lymph nodes, the positive expression of CD44v6 and the positive expression of VEGF were also the independent prognostic factors to affect the Survival time.The VEGF and CD44v6 protein may act as one of important indexes to indicate the process of infiltration and metastasis in NSCLC.