2009年 · 第122卷第11期

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults, which accounts for approximately 50% of all gliomas. Its prognosis is particularly disappointing with a median life expectancy less than a year even when the patients are treated with the most aggressive regimens.1 Over the past 10 years, a number of trials have tried to establish whether adjuvant chemotherapy, as well as molecularly targeted therapy, provides GBM patients with clinically meaningful benefits。

Abstract：Background Glioblastoma multiforme (GBM) is the most malignant kind of astrocytic tumors and is associated with a poor prognosis. In this retrospective study, we assessed the clinical, radiological, genetic molecular and treatment factors that influence clinical outcomes of patients with GBM.Methods A total of 116 patients with GBM who received surgery and radiation between January 2006 and December 2007 were included in this study. Kaplan-Meier survival analysis and Cox regression analysis were used to find the factors independently influencing patients' progression free survival (PFS) time and overall survival (OS) time.Results Age, preoperative Kamofsky Performance Scale (KPS) score, KPS score change at 2 weeks after operation, neurological deficit symptoms, tumor resection extent, maximal tumor diameter, involvement of eloquent cortex or deep structure, involvement of brain lobe, Ki-67 expression level and adjuvant chemotherapy were statistically significant factors (P <0.05) for both PFS and OS in the univariate analysis. Cox proportional hazards modeling revealed that age ≤50 years, preoperative KPS score ≥80, KPS score change after operation ≥0, involvement of single frontal lobe,non-eloquent area or deep structure involvement, low Ki-67 expression and adjuvant chemotherapy were independent favorable factors (P <0.05) for patients' clinical outcomes.Conclusions Age at diagnosis, preoperative KPS score, KPS score change at 2 weeks postoperation, involvement of brain lobe, involvement of eloquent cortex or deep structure, Ki-67 expression level and adjuvant chemotherapy correlate significantly with the prognosis of patients with GBM。

Abstract：Background Bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor, has shown promising activity in recurrent malignant gliomas. We reported the treatment response for the combination of bevacizumab and chemotherapy in a series of six patients with recurrent malignant glioma and investigated the molecular alterations in cancer pathways using the surgical biopsies from these patients.Methods Standard therapy with primary resection followed by adjuvant chemoradiotherapy had failed in all patients.Bevacizumab was administered at a dose of 10 mg/kg every 2 weeks. Concomitantly, four patients received temozolomide (50 mg-m-2-d-1), one patient irinotecan (125 mg/m2 every 2 weeks) and one patient topotecan (1.2 mg.m-2.d-1). Response to therapy was mainly determined by magnetic resonance imaging. The expression of Ras,phosphorylated mitogen activated protein kinase (p-MAPK), phosphorylated AKT (p-AKT), phosphorylated mammalian target of rapamycin (p-mTOR) and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) were semiquantitatively assessed by immunohistochemistry using surgical biopsies before the initial treatment.Results Five of the six patients had a radiographic response. Three were complete response, and two were partial response. Only one patient had progressive disease. The 6-month progession-free survival (PFS) was 33% and the median PFS was 15 weeks, with a range of 6 to more than 60 weeks. Of the three core pathways analyzed in this study,the Ras/MAPK and phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR pathways were more likely to be associated with the treatment response to bevacizumab. In two younger patients (ages <50) with complete response, simultaneous overexpression of p-MAPK, p.AKT and p-mTOR might be the crucial feature.Conclusions Bevacizumab in combination with chemotherapeutic agents may be an effective strategy for patients with recurrent malignant glioma. Activated MAPK and AKT might be possible biomarkers for selecting suitable patients for this targeted therapy。

Abstract：Background Recent studies have demonstrated the existence of a small fraction of cells with features of primitive neural progenitor cells and tumor-initiating function in brain tumors. These cells might represent primary therapeutic target for complete eradication of the tumors. This study aimed to determine the resistant phenotype of glioblastoma stem cells (GSCs) to temozolomide (TMZ) and to explore the possible molecular mechanisms underlying TMZ resistance.Methods Freshly resected glioblastoma specimen was collected and magnetic isolation of GSCs was carded out using the Miltenyi Biotec CD133 Celt isolation kit. The cytotoxic effect of TMZ on CD133+ and CD133- glioblastoma cells was determined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Autophagy-related proteins (Beclin-1, LC3 and Atg5) and cleaved caspase-3 (p17) were analyzed by Westem blotting. Immunofluorescent staining was used to detect Atg5, glial fibrillary acidic protein (GFAP) and CD133 expression in glioblastoma cells. Statistical analysis was carried out using SPSS 10.0 software. For all tests, the level of statistical significance was set at P <0.05.Results CD133+ glioblastoma cells exhibited neurosphere-like growth in vitro and high expression of CD133 stem cell marker. The growth-inhibiting rate in CD133- glioblastoma cells treated with 5 or 50 pmol/L TMZ was significantly higher than that in CD133+ glioblastoma cells ((14.36±3.75)% vs (2.54±1.36)% or (25.95±5.25)% vs (2.72±1.84)%, respectively, P <0.05). Atg5, LC3-ll and Beclin-1 levels were significantly lower in CD133+ glioblastoma cells than those in autologous CD133- cells after TMZ treatment (P <0.05). Caspase-3 was mildly activated only in CD133- glioblastoma cells after exposure to TMZ (P <0.05). Immunofluorescent staining revealed elevated expression of Atg5 in GFAP* cells following TMZ treatment.Conclusions The GSCs display strong capability of tumor's resistance to TMZ. This resistance is probably contributed by the CD133+ cells with down-regulation of autophagy-related proteins. Future treatment should target this small population of cancer stem cells in tumors to improve survival of patients。

Abstract：Background Glioma is the most common primary brain tumor with poor prognosis. Temozolomide has been used with thalidomide to treat gliomas. We investigated the synergistic mechanism of these two drugs in vitro.Methods Human malignant glioma cells U251-MG were cultured and assigned to four groups with different treatments for 3 days: temozolomide group (100 pmol/L), thalidomide group (100 pg/L), temozolomide (100 IJmol/L) plus thalidomide group (100 pg/L) and control group. MTT assay was applied to evaluate the cell viability. Cell cycle was analyzed by flow cytometry. The ultra-structural features of autophagosomes were observed with electron microscope. Acridine orange and monodansylcadavedne were adopted to label autophagosomes and flow cytometry was applied for quantification of autophagosomes. The expression of autophagy-associated protein was detected by Western blotting.Results Proliferation of tumor cell was obviously suppressed by temozolomide with thalidomide treatment than by either drug used alone (P=-0.000 for each day). The combination treatment induced cell cycle arrest at G0/G1 phase.Typical autophagic ultra-structural character was found after the combined treatment. Thalidomide promoted the autophagy induced by temozolomide. The autophagy-associated proteins - microtubule associated protein 1 light chain 3 (MAPILC3) and Beclinl were more significantly up-regulated by the combined treatment than temozolomide used alone (MAP1LC3, P=-0.000; Beclinl, P=-0.004). The expression level of phosphatase and tensin homolog deleted on chromosome ten (PTEN), which promoted autophagy by suppressing PI3K/Akt/mTOR signaling pathway, was elevated by thalidomide (thalidomide group: P=-0.000; combined group: P=0.002).Conclusions Thalidomide enhances the cytotoxicity of temozolomide by promoting the autophagy induced by temozolomide. Contributing to the up-regulation of PTEN by thalidomide, the expression of autophagy associated protein-MAP1LC3 and Beclinl was enhanced, which leads to a reinforced autophagy in the combined treatment of temozolomide and thalidomide in vitro。

Abstract：Background Cytosine deaminase (CD) converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) in CD/5-FC gene therapy, 5-FU will be mostly converted into nontoxic β-alanine without uracil phosphoribosyltransferase (UPRT). UPRT catalyzes the conversion of 5-FU to 5-fluorouridine monophosphate, which directly kills CD::UPRT-expressing ceils and surrounding cells via the bystander effect. But the pharmacokinetics and the bystander effect of CD::UPRT/5-FC has not been verified in vivo and in vitro. Before the CD::UPRT/5-FC bi-gene therapy system is used in clinical trial, it is essential to monitor the transgene expression and function in vivo. Thus, we developed a preclinical tumor model to investigate the feasibility of using 19F-magnetic resonance spectroscopy (19F-MRS) and optical imaging to measure non-invasive CD and UPRT expression and its bystander effect.Methods C6 and C6-CD::UPRT cells were cultured with 5-FC. The medium, cells and their mixture were analyzed by 19F-MRS. Rats with intracranial xenografted encephalic C6-CD::UPRT glioma were injected intraperitoneally with 5-FC and their 19F-MRS spectra recorded. Then the pharmacokinetics of 5-FC was proved. Mixtures of C6 and C6-CD::UPRT cells at different ratios were cultured with 5-FC and the cytotoxic efficacy and survival rate of cells recorded. To determine the mechanism of the bystander effect, the culture media from cell comprising 25% and 75% C6-CD::UPRT cells were examined by19F-MRS. A comparative study of mean was performed using analysis of variance (ANOVA). Results 19F-MRS on samples from C6-CD::UPRT cells cultured with 5-FC showed three broad resonance signals corresponding to 5-FC, 5-FU and fluorinated nucleotides (F-Nuctd). For the C6 mixture, only the 5-FC peak was detected. In vivo serial 19F-MRS spectra showed a strong 5-FC peak and a weak 5-FU peak at 20 minutes after 5-FC injection. The 5-FU concentration reached a maximum at about 50 minutes. The F-Nuctd signal appeared after about I hour, reached a maximum at around 160 minutes, and was detectable for several hours. At a 10% ratio of C6-CD::UPRT cells, the survival rate was (79.55±0.88)% (P <0.01). As the C6-CD::UPRT ratio increased, the survival rate of the cells decreased.19F-MRS showed that the signals for 5-FU and F-Nuctd in the culture medium increased as the ratio of C6-CD::UPRT in the mixture increased.Conclusions 19F-MRS studies indicated that C6-CD::UPRT cells could effectively express CD and UPRT enzymes.The CD::UPRT/5-FC system showed an obvious bystander effect. This study demonstrated that CD::UPRT/5-FC gene therapy is suitable for 5-FC to F-Nuctd metabolism; and 19F-MRS can monitor transferred CD::UPRT gene expression and catalysis of substrates noninvasively, dynamically and quantitatively.

Abstract：Background Apelin is an adipokine that contributes to the pathogenesis of type 2 diabetes. The plasma levels of apelin increased in obese patients and diabetic subjects. This study aimed to investigate the effects of apelin genetic variants on type 2 diabetes and related quantitative traits.Methods We selected three single nucleotide polymorphisms (SNPs) that could capture all common variants in APLN gene region and genotyped them in 1892 type 2 diabetic patients and 1808 normal glucose regulation controls. The clinical features related to glucose metabolism were measured in the controls. The comparison of allele and genotype distribution in the cases and controls were performed by using X2 tests. The association between SNPs and quantitative traits were analyzed using Wilcoxon's rank-sum test.Results None of the SNPs or haplotypes showed evidence of association to type 2 diabetes. However, rs2235306 was nominally associated with fasting plasma glucose levels in the male subjects with normal glucose regulation ((4.93±0.03)vs (5.01±0.03) mmol/L, P=0.04). No significant difference was observed between all three SNPs and other variables. Conclusions APLN SNP rs2235306 was associated with fasting plasma glucose levels in males. It suggests that APLN genetic variants may contribute to clinical features related to glucose metabolism in Chinese population。

Abstract：Background Previous cytogenetic studies revealed aberrations varied among the throe subtypes of rhabdomyosarcoma. We profiled chromosomal imbalances in the different subtypes and investigated the relationships between clinical parameters and genomic aberrations.Methods Comparative genomic hybridization was used to investigate genomic imbalances in 25 cases of primary rhabdomyosarcomas and two rhabdomyosarcoma cell lines. Specimens were reviewed to determine histological type, pathological grading and clinical staging.Results Changes involving one or more regions of the genome were seen in all rhabdomyosarcomal patients. For rhabdomyosarcoma, DNA sequence gains were most frequently (>30%) seen in chromosomes 2p, 12q, 6p, 9q, 10q, 1p,2q, 6q, 8q, 15q and 18q; losses from 3p, 11p and 6p. In aggressive alveolar rhabdomyosarcoma, frequent gains were seen on chromosomes 12q, 2p, 6p, 2q, 4q, 10q and 15q; losses from 3p, 6p, 1q and 5q. For embryonic rhabdomyosarcoma, frequent gains were on 7p, 9q, 2p, 18q, 1p and 8q; losses only from 11p. Frequently gained chromosome arms of translocation associated with rhabdomyosarcoma were 12q, 2, 6, 10q, 4q and 15q; losses from 3p,6p and 5q. The frequently gained chromosome arms of nontranslocation associated with rhabdomyosarcoma were 2p,9q and 18q, while 11p and 14q were the frequently lost chromosome arms. Gains on chromosome 12q were significantly correlated with translocation type. Gains on chromosome 9q were significantly correlated with clinical staging. Conclusions Gains on chromosomes 2p, 12q, 6p, 9q, 10q, 1p, 2q, 6q, 8q, 15q and 18q and losses on chromosomes 3p, 11p and 6p may be related to rhabdomyosarcomal carcinogenesis. Furthermore, gains on chromosome 12q may be correlated with translocation and gains on chromosome 9q with the early stages of rhabdomyosarcoma.

Abstract：Background Human bocavirus (HBoV) is a newly identified human parvovirus that was originally detected in the respiratory secretions of children with respiratory infections. This study aimed to learn about the importance of HBoV infections by revealing the prevalence of serum antibodies against HBoV in Beijing population.Methods Two batches of serum specimens collected in different periods were tested by Western blotting for specific IgG against HBoV using recombinant VP2 as antigen.Results Out of 677 serum specimens collected during April 1996 to March 1997, 400 (59.1%) were positive and antibody positive rate for another batch of 141 serum specimens collected in August, 2005 from adults aged from 20 years to over 60 years was 78.7% (111/141). Comparison of the sero-prevalence profiles for serum specimens collected during 1996-1997 to those collected in 2005 indicated that the antibody positive rate for specimens collected in 2005 was higher than that of the corresponding age groups collected during 1996-1997.Conclusions The data suggest that HBoV has been circulating in Beijing population for at least over 10 years, and most of children had been exposed to HBoV by age of 7 years. Higher HBoV antibody positive rate shown in the serum specimens collected in 2005 suggested that infections by HBoV have been increased in Beijing population in recent years。

Abstract：Background The usefulness of in-phase/opposed-phase imaging and diffusion weighted imaging (DWI) in differentiating benign and neoplastic vertebral fractures has been described. In this study, we aimed to evaluate the influence of the severity of vertebral damage on the diagnostic performance of these two technologies.Methods Totally 59 patients with 68 acute benign vertebral fractures and 43 patients with 79 vertebral metastases were included in this study. The MR protocol included DWls and sagittal in-phase/opposed-phase gradient recalled sequence.The severity of vertebral damage was expressed by lesion ratio (LR, the ratio of lesion area to vertebral area on the slices of largest abnormal signal area in the T1-weighted sequence). Quantitative (signal intensity ratio (SIR) defined as signal intensity (SI) on opposed-phase gradient recalled echo (GRE) images divided by SI on in-phase; apparent diffusion coefficient (ADC) value derived from DWI analysis was performed, the relationships between LR and the measurements of these two technologies were analyzed using linear regression. The covariate-specific receiver operating characteristic (ROC) curves were also fitted to evaluate the influence of LR on the diagnostic performance of ADC and SIR. Results The difference in both SIR and ADC for vertebral metastasis and acute benign vertebral fractures was significant (P <0.001). A positive correlation between the LR and the SIR was found in benign fractures (P <0.05). The severity of vertebral damage had a significant influence on the AUC (area under ROC curve) for SIR (P<0.05) but ADC (P >0.05). More severe cases were associated with increased AUC for SIR.Conclusions LR is capable of affecting the diagnostic performances of chemical shift imaging. Thus, when applying these tests to make diagnoses on vertebral fractures, the severity of the vertebral damage should be taken into account.The covariate-specific ROC model is recommended because it substantially improves the ability to avoid bias when evaluating tests。

Abstract：Background Hyaluronidase (Hyase) is an enzyme which hydrolyses hyaluronan (HA), a large nonsulfated glycosaminoglycan. Several genes have been identified to code for hyaluronidases in humans. Its role has only recently been underlined in the invasion of prostate cancer, colonic cancer, and breast cancer. Moreover, the findings were in agreement with some experimental results which showed that HA-derived oligosaccharides had angiogenesis-promoting activity. All these findings prompted us to investigate factors that had been characterized as putative invasive factors in different human breast cancer-derived cell lines.Methods We selected two series of human breast cancer-derived cell lines whose expression of estrogen receptors (ER) was previously published. Hyaluronidase secretion in culture medium and expression of matrix metallo-proteinase (MMP)-9, cathepsin-D (cath-D) and vascular endothelial growth factor (VEGF) by cells were determined. We also investigated cell invasiveness in the Matrigel invasion assay, and studied the capability of cancer cells to promote in vitro formation of tubules by endothelial cells.Results ER(-) cells secreted significantly more hyaluronidase (P <0.001) and expressed significantly more VEGF (P <0.01), MMP-9 (P <0.05) and cath-D (P <0.0001) than ER(+) cells. Invasion through Matdgel by ER(-) Hyase(+) cells was significantly higher than that by ER(+) Hyase(-) cells (P<0.05). In both cases, invasion was decreased by heparin (P <0.05). When ECV-304 endothelial cells were co-cultivated in millicell chambers with cancer cells, ECV-304 cells were induced to form tubules. Tubule formation was demonstrated to be more prominent with ER(-) Hyase(+) cells than with ER(+) Hyase(-) cells (P <0.05).Conclusion Invasive features of ER(-) breast cancer cells can be characterized in vitro by an invasive Matrigel assay,as the induction of tubule formation by ECV-304 endothelial cells, higher secretion of hyaluronidase, and higher expression of proteinases MMP-9, cath-D, and the angiogenesis promoting factor VEGF。

Abstract：Background Nosocomial infection caused by methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) could lead to increased morbidity and mortality. In 2006, VRE nosocomial spread became a reality in our hospital since the first VRE nosocomial infection in 2003. Little is known about the prevalence of coexistence with VRE and MRSA in the patients. The primary objective of the study was to identify the molecular characteristics of epidemic MRSA clones in our hospital and the prevalence of the coexistence with MRSA and VRE in same patients during the 2-year period, 2006-2007.Methods The clinical features, laboratory test results, and therapeutic outcomes of 129 cases who isolated MRSAcollected from January 2006 to December 2007 were retrospectively analyzed. Polymerase chain reaction (PCR) was used to determine mecA-femB type and staphylococcal cassette chromosome mec (SCCmec) type. All the participants were screened for clinical and microbiological data to identify the coexistence of VRE strains with MRSA.Results One hundred and twenty-nine MRSA isolates were included in the study: 71 (55%) from the intensive care unit,35 (27.2%) from the surgical wards and 23 (17.8%) from the medical wards. The most frequent source of isolation of MRSA was sputum (76.7%). From seven patients we isolated MRSA and VRE (E. faecium) simultaneously during their inpatient stay. One hundred and twenty-seven (127/129, 98.4%) MRSA isolates harboured SCCmec type Ⅲ, only 2 MRSA strains contained SCCmec type Ⅱ. All of the 129 MRSA isolates remained sensitive to vancomycin, teicoplanin and linezolid. Higher sensitivity rates were noted for chloramphenicol 99.2% (128/129). Only 20.2% (26/129) of the MRSA isolates were sensitive to rifampin. All isolates presented resistance to multiple antimicrobial agents with high minimum inhibitory concentrations (MICs), including: β-lactams (penicillin, oxacillin, cefoxitin, and cefazolin), tetracycline,erythromycin, gentamicin, and quinolones (ciprofloxacin, levofloxacin, and moxifloxacin).Conclusions The predominant MRSA clone at Beijing Chaoyang Hospital from 2006 to 2007 had the type Ⅲ SCCmec element. All of the MRSA isolates were multiresistant to antimicrobial agents. Emergence of coexistence of MRSA and VRE in the same patient was not rare. Physicians should pay more attention to infections resulting from MRSA and VRE. Aggressive infection control measures should be taken to prevent the transmission of the multidrug resistance organism。

During the last decade, much progress has been made in exploring the mechanisms of alterations elicited by foreign compounds in xeno- and endobiotic metabolism regulated by the human nuclear pregnane X receptor (PXR). PXR, identified as a human nuclear receptor in 1998 and generally regarded as a sensor activated by exogenous and endogenous chemicals,regulates a large number of enzymes and transporters involved in the response of mammals to their chemical environment) PXR activation is ligand dependent.Following ligand binding, PXR forms a heterodimer with the retinoid X receptor (RXR) that binds to PXR response dements, resulting in their transcriptional activation. PXR is mainly associated with the cellular response to xenobiotics, including induction of enzymes involved in drug oxidation and conjugation, as well as induction of xenobiotic and endobiotic transporters. Unlike other nuclear receptors such as the steroid receptors that interact selectively with their physiological ligands, PXR ligands are structurally diverse and include prescription drugs,herbal medicines, dietary supplements, environmental pollutants, and endobiotics. PXR exhibits the ability to bind multiple ligands, and each receptor's ligand specificity is species-dependent. The detailed information of PXR was summarized (Table 1). The purpose of this review is to highlight the promiscuous ligand-binding properties of PXR, its positive effect in regulation of the body's garbage-disposal system and negative effect in harmful drug-drug interactions and endocrine disruption。

O6-Methylguanine-DNA methyltransferase (MGMT) is a cellular DNA repair protein that rapidly reverses alkylation (e.g. methylation) at the O6 position of guanine,thereby neutralizing the cytotoxic effects of alkylating agent therapy such as temozolomide (TMZ) and carmustine.1It has been shown that epigenetic silencing of the MGMT gene by promoter methylation shuts down gene transcription2 and reflects a common alteration in primary human tumors leading to MGMT deficiency)Epigenetic silencing of the MGMT gene has been shown to correlate with improved survival in several studies of glioma" patients treated with the alkylating agent. 56therapy4 and has been substantiated in two clinical trials.5.6

The global commitment to providing antiretroviral therapy (ART) to people living with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) in low-income countries has raised hope that the increasing momentum in the fight against the worldwide HIV/AIDS pandemic will be sufficient to control it. However, improved availability of subsidized antiretroviral (ARV) treatments in low-income countries raises complex ethical issues.1,2 In many resource-constrained countries the number of individuals infected with HIV in need of treatment far exceeds the supply of ARV medication. Resource allocation decisions can be made on the basis of many epidemiological,ethical, or preferential treatment priority criteria,Healthcare systems and funding in low-income countries are limited, requiring a step-by-step aipproach to scalingup programs to reach their stated aims。

China's rapidly evolving HIV/AIDS epidemic calls for a dramatic expansion of both prevention and treatment services.1,2 Official state media recently reported that for the first time, in 2008, HIV/AIDS became China's leading cause of death among infectious diseases.3 Estimates from the Ministry of Health indicate that around 700 000 people were living with HIV and 85 000 people had AIDS in 2007.4 Initially, HIV-1 infection was confined primarily to certain high-risk populations such as injection drug users (IDU) along drug-trafficking routes, and former plasma donors (FPD) in rural communities in east-central China.1,5-7 Now, however,HIV prevalence is increasing among female sex workers (FSW) and men who have sex with men (MSM).4,8 It is estimated that in 2008, approximately 45% of new HIV cases in China were attributed to heterosexual transmission and 12% to MSM; the proportion of women infected has also doubled in the past decade。

Transradial coronary stenting using 5-French (5F) guiding catheters has been associated with a higher procedural success rate, a lower frequency of vascular access complications and is well tolerated, particularly in the subgroup of patients with small radial artery diameters.1

With the development of laparoscopic techniques,laparoscopic hepatectomy is feasible for hepatocellular carcinoma as reported in recent years.Although several reports have been published on laparoscopic surgery for metastatic liver cancer,1,2 few of them deals with second laparoscopic resection of recurrent hepatocellular carcinoma. We report a case of second laparoscopic resection for recurrent hepatocellular carcinoma after initial laparoscopic hepatectomy。