Extraskeletal Ewing's Sarcoma with CD7 Positivity and T-cell Receptor/Immunoglobulin Rearrangement Masquerading as T-lymphoblastic Lymphoma.

Background: Extraskeletal Ewing's Sarcoma (EES) may harbor more than one tumor-specific genetic abnormality, leading to diagnostic difficulties. Case report: We report a nine-year-old boy with recurrent mass of his right thigh. Tumor cells were round, with scant cytoplasm, finely dispersed chromatin, and inapparent, small nucleoli. The initial misdiagnosis was T-lymphoblastic lymphoma due to CD7 and TCR/Ig monoclonal rearrangement. As it expressed NKX2.2 and harbored an EWSR1-FLI1 fusion transcript, the diagnosis was changed to EES. The child underwent EES therapy with good initial response, but had a subcutaneous relapse at 22 months. Conclusion: In addition to typical genetic alterations, Ewing sarcoma can also express CD7 and TCR/Ig rearrangement, which are not limited to lymphoma.

Key factors affecting photoactivated fungicidal activity of sodium pheophorbide a against Pestalotiopsis neglecta.

Recently, photodynamic therapy (PDT) and photoactivated pesticides have attracted considerable research attention. In the present study, we aimed to investigate the photodynamic activity of a chlorophyllous derivative, sodium pheophorbide a (SPA), and to evaluate its potential as a photoactivated fungicide. The singlet oxygen quantum yield, the photoreaction process, the anti-photobleaching ability in sterile water (H2O), the effect of light conditions on its antifungal activity, and its stability were all investigated. SPA showed significant fungicidal activity and photostability, during which Type I and Type II photodynamic reactions occurred simultaneously on Pestalotiopsis neglecta, and the influence of Type I was slightly larger than that of Type II. In addition, light promoted the antifungal activity of SPA. In particular, the antifungal activity was enhanced with increasing light intensity, and was strongest under 8000 lx conditions. Under monochromatic light sources, antifungal activity was strongest under green light s; however, the effect of monochromatic light was not as good as that of white light. From 0 to 24 h, the antifungal effect of the SPA solution was enhanced; however, the activity of the solution began to weaken after 24 h. Furthermore, our study confirmed that the antifungal activity of SPA was stable under different temperatures, pH values, and UV irradiation durations.

Plasma SCF/c-kit Levels in Patients with Dipper and Non-Dipper Hypertension.

Objective The aim of this study was to investigate the relationship between peripheral plasma stem cell factor (SCF)/c-kit levels and the types of dipper and non-dipper hypertension in hypertensive patients. Methods This cross-sectional study included newly diagnosed hypertensive patients who underwent 24-hour ambulatory blood pressure monitor (ABPM) between January 2009 and 2012 in Jiangning city. Patients were divided into the dipper group and the non-dipper group according to ABPM measurements. The levels of SCF and its receptor c-kit, tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) in peripheral blood were measured via enzyme-linked immunosorbent assays. The serum levels of glucose and lipid were examined as well. The levels of SCF/c-kit were compared between the dippers and the non-dippers; and their correlation with 24-hour mean systolic blood pressure (MSBP), 24-hour mean diastolic blood pressure (MDBP), TNF-α and IL-6 were investigated using linear regression analyses statistically. Results A total of 247 patients with newly diagnosed hypertension were recruited into the study, including 116 non-dippers and 131 dippers. The levels of peripheral plasma SCF were higher in non-dipper group (907.1±52.7 ng/L vs. 778.7±44.6 ng/L; t=2.837, P<0.01), and the levels of c-kit were higher in non-dipper group too (13.2±1.7 µg/L vs 9.57±1.4 µg/L; t=2.831, P<0.01). Linear regression analysis revealed that SCF/c-kit levels were significantly positively correlated with MSBP, MDBP, plasma TNF-α, and IL-6 levels (all P<0.01). Conclusions Peripheral plasma SCF/c-kit levels are higher in patients with non-dipper hypertension than those with dipper one, and significantly correlate with 24-hour MSBP, 24-hour MDBP, serum TNF-α and IL-6 levels.

[Effect of Chinese Drugs for Breaking Blood Expelling Stasis on Acute Cerebral Hemorrhage: a Prospective Randomized Double-blind Controlled Study].

OBJECTIVE: To study the safety of using Chinese drugs for breaking blood expelling stasis (CDBBES) in hypertension patients with intracerebral hemorrhage within 6 h, and to observe whether they would result in hematoma enlargement. METHODS: A prospective randomized double-blind controlled clinical study was employed. Totally 128 cerebral hemorrhage patients within 6 h were recruited from 8 research centers from October 2013 to March 2015, and finally 76 of them were included. These patients were assigned to 3 groups by simple random sampling, group A, B, and C. Patients in group A (26 cases) took whole CDBBES recipe (containing leeches and equivalent insects). Those in group B (25 cases) took CDBBES recipe (removing leech and gradfly). Those in group C (25 cases) took placebos. Medication lasted for 10 successive days. The hematoma enlargement rate within 24 h, the occurrence of adverse reactions and adverse events were observed. To guarantee the safety of this trial, an interim analysis of first level unblinding was used. RESULTS: The hematoma enlargement rate was 11. 5% (3/26) in group A, 16. 0% (4/25) in group B, and 20. 0% (5/25) in group C. There was no statistical difference in the hematoma enlargement rate among the 3 groups (X² =0. 823, P =0. 682). Adverse reactions and adverse events occurred in 7 cases, 1 patient with acute myocardial infarction, 1 with chest op- pression and palpitation, 2 with diarrhea in group A. No patient had adverse reaction or adverse event in group B. And diarrhea occurred in 3 patients of group C. CONCLUSION: The interim analysis of first level unblinding showed that hematoma enlargement within 6 h was not resulted from using CDBBES.

[Research and Implementation of Wood Species Recognition System with Wood Near Infrared Spectral Reflection Features].

This paper proposes a novel wood species recognition scheme based on the spectral reflection features of wood surface, aiming to address the following three issues in terms of the noise filtering, feature selection and radian's optimal design . First, noises occur in some bands of wood spectral reflection curve so that these noisy bands should be deleted. Second, the wood spectral band is 350~2 500 nm, which is a 2 150D vector with a spectral sampling interval of 1 nm. Therefore, both noise filtering and feature selection should be performed to wood spectral data. In this paper, to simultaneously and efficiently solve the two problems of feature selection and noise filtering, both a feature selection procedure and a noise filtering procedure are performed by solving the eigenvalues of dispersion matrix. This scheme is novel and produces a good outcome. Third, to make the spectral reflection curves picked up by the spectral instrument have the best pattern recognition information; an optimal design is performed for the indoor radian's mounting height. The genetic algorithm is used to solve the optimal radian's height so that the spectral reflection curves have the best classification information for wood species. Therefore, the optimal design scheme for the radian's mounting height can improve the pattern classification accuracy of the wood species to some extents, which is novel with excellent executive feasibility. Many experiments made with our developed software system on the five ordinary wood species in northeast region of China (i.e., including Betula platyphylla, Populus davidiana, Pinus Sylvestris, Picea jezoensis, Larix gmelinii) are performed for approximately 105 times. It indicates that the overall recognition rate reaches to a good recognition accuracy of 95% for five wood species with an ideal recognition velocity. The selected feature wavelengths by using of our feature selection algorithm based on dispersion matrix are mainly in the near infrared band.

Congenital cystic adenomatoid malformation with associated mucinous bronchioloalveolar carcinoma in a neonate.

Congenital cystic adenomatoid malformation (CCAM) of lung is a rare hamartomatous disorder characterized by abnormal branching morphogenesis of the lung. We report an unusual case of a 2-day-old male newborn with a pulmonary cystic lesion and lobectomy revealed a CCAM of the lung that has overlapping features of type 1 and type 2, complicating with multifocal mucinous bronchioloalveolar carcinoma (BAC). The case indicates that malignant transformation can occur in very early stage of the infancy in the patients with CCAM of lung.

Deficits in LTP induction by 5-HT2A receptor antagonist in a mouse model for fragile X syndrome.

Fragile X syndrome is a common inherited form of mental retardation caused by the lack of fragile X mental retardation protein (FMRP) because of Fmr1 gene silencing. Serotonin (5-HT) is significantly increased in the null mutants of Drosophila Fmr1, and elevated 5-HT brain levels result in cognitive and behavioral deficits in human patients. The serotonin type 2A receptor (5-HT2AR) is highly expressed in the cerebral cortex; it acts on pyramidal cells and GABAergic interneurons to modulate cortical functions. 5-HT2AR and FMRP both regulate synaptic plasticity. Therefore, the lack of FMRP may affect serotoninergic activity. In this study, we determined the involvement of FMRP in the 5-HT modulation of synaptic potentiation with the use of primary cortical neuron culture and brain slice recording. Pharmacological inhibition of 5-HT2AR by R-96544 or ketanserin facilitated long-term potentiation (LTP) in the anterior cingulate cortex (ACC) of WT mice. The prefrontal LTP induction was dependent on the activation of NMDARs and elevation of postsynaptic Ca(2+) concentrations. By contrast, inhibition of 5-HT2AR could not restore the induction of LTP in the ACC of Fmr1 knock-out mice. Furthermore, 5-HT2AR inhibition induced AMPA receptor GluR1 subtype surface insertion in the cultured ACC neurons of Fmr1 WT mice, however, GluR1 surface insertion by inhibition of 5-HT2AR was impaired in the neurons of Fmr1KO mice. These findings suggested that FMRP was involved in serotonin receptor signaling and contributed in GluR1 surface expression induced by 5-HT2AR inactivation.

Transepithelial photorefractive keratectomy mode using SCHWIND-ESIRIS excimer laser: initial clinical results.

AIM: To evaluate postoperative pain, uncorrected visual acuity (UCVA), and cornea haze value after transepithelial photorefractive keratectomy (T-PRK) performed with aspherical ablation profile using SCHWIND ESIRIS excimer laser. METHODS: Retrospective case series. Fifty-nice eyes (32 patients) with myopia associated with or without astigmatism underwent phototherapeutic keratectomy (PTK) followed by photorefractive keratectomy (PRK) which performed by Optimized Refractive Keratecomy (ORK)-CAM software based on aspherical ablation profile using SCHWIND ESIRIS excimer laser. Postoperative pain scale was measured on a questionnaire through five levels. Haze was graded by five grades, and UCVA, manifest refraction spherical equivalent (MRSE) were analyzed. RESULTS: Mean pain level was (1.37±0.613) (range: 1 to 3), the mean time picking out the soft contact lens was (6.22±1.73) days, at 3 months, UCVA was 1.0 for 40 eyes (67.8%), 0.5 for all eyes (100.0%). The UCVA was significantly less than the preoperative best spectacle corrected visual acuity (BSCVA) (t=-2.84, P=0.006), haze value was (0.27±0.25), no patients had a haze grade up to 2. Mean MRSE was (0.76±0.96) diopter(D) by 3 months. CONCLUSION: The outcomes from this study show that using the SCHWIND ESIRIS aspherical ablation profile for transepithelial PRK has a good visual result. The primary advantage is related to a spherical ablation profile, automatically considers the ablation volume of the stroma and the accurate and smooth removal of the epithelium with PTK. Additional studies are needed to determine long-term outcomes.

Axin induces cell death and reduces cell proliferation in astrocytoma by activating the p53 pathway.

Astrocytic tumors are the most common brain tumors with various genetic defects. As a tumor suppressor gene, Axin could control cell death and growth. Axin possesses a separate domain that directly interacts with p53 and regulates the activity of p53 pathway. Our aims were to elucidate the effects of Axin on the progression of astrocytoma. We examined the expression of Axin in 96 cases of astrocytoma using immunohistochemistry. The apoptotic index, proliferactive acitivity and the expression levels of p53 and its downstream genes, p21 and Cyclin D1, were evaluated in the C6 astrocytoma cells with overexpression or silencing of Axin. The results showed the levels of Axin correlated significantly inversely with the grades of astrocytoma (R=-0.286, P<0.05) and correlated negatively with Ki-67 labeling index (R=-0.227, P<0.05). Overexpression of Axin in C6 cells induces cell death, and reduces the cell proliferation, up-regulates the expression of p53. Silencing of Axin reduces p53 expression. The p53 inhibitor, pifithrin-alpha, was able to counteract the effect of Axin in C6 cells. Our data demonstrate that the expression of Axin is associated negatively with the progression of astrocytoma. In conclusion, Axin induces cell death and reduces cell proliferation, partially by activating the p53 pathway in astrocytoma cells. This knowledge is helpful in understanding the role of Axin in the progression of astrocytoma.

Expression of CD147 as a significantly unfavorable prognostic factor in hepatocellular carcinoma.

The aim of this study is to investigate whether the expression of CD147 could be a prognostic factor for hepatocellular carcinoma. Tissue samples from 111 hepatocellular carcinoma patients were immunohistochemically stained with anti-CD147, anti-matrix metalloproteinases-2 and anti-vascular endothelial growth factor antibodies. Tumor microvessel density was evaluated using CD34. The survival curves were estimated by Kaplan-Meier analysis and the prognostic significance of the marker was analyzed using the log-rank test. In addition, the identification of relevant prognostic factors was performed by multivariate Cox regression analysis. CD147 was mainly expressed in cancerous lesions and its expression was positively correlated with metalloproteinases-2 (P<0.0001), vascular endothelial growth factor (P<0.0001) and microvessel density CD34 (P<0.0001). Furthermore, CD147 was significantly associated with the presence of venous invasion (P=0.0013), tumor size (P<0.0001) and pTNM tumor stages (P=0.0001), as well as serum alpha-fetoprotein level (P<0.0001). Patients with positive expression of CD147 had poorer tumor recurrence-free survival than those with negative expression of CD147 (P<0.0001). Analyzed by a proportional hazard model, strong expression of CD147 had the highest risk ratio of recurrence among these markers (P<0.0001). The findings suggest that CD147 may be a significant independent predictor of poor survival in patients with hepatocellular carcinoma, and may be involved in tumor growth, invasion and angiogenesis in hepatocellular carcinoma.

[Construction of the prokaryotic expression vector and expression of human CIDE-3 gene].

AIM: To construct a prokaryotic expression vector for human CIDE-3 gene, and to express the gene in E.coli BL21(DE3). METHODS: The total RNA was extracted from human hepatocellular carcinoma cell line HepG2. CIDE-3 gene fragment was amplified by RT-PCR and was cloned into the pET28a(+) vector. The recombinant vector was identified by restriction endonuclease digestion analysis and DNA sequencing, and then it was transformed into E.coli BL21(DE3) through IPTG induction to express the target protein bearing His tag. RESULTS: A 516 bp of CIDE-3 gene fragment was obtained. After E.coli BL21(DE3) was transformed with recombinant vector pET28a(+)-CIDE-3 and through IPTG induction, the recombinant protein with relative molecular masse about 23,000 was obtained. SDS-PAGE analysis showed that the expressed product was mainly inclusion bodies, accounting for 32% of the total bacterial proteins. CONCLUSION: Recombinant expression vector pET28a(+)-CIDE-3 is constructed successfully. The expressed CIDE-3 protein will be helpful to our further research.

[Effect of exogenous RGS16 gene transfection on growth of glioma cell line C6].

BACKGROUND & OBJECTIVE: Wild type p53 could induce RGS16 mRNA expression, and RGS16 protein is related with carcinogenesis of glioma. This study was designed to investigate the effects of exogenous RGS16 gene transfection on the growth of rat glioma cell line C6. METHODS: A eukaryotic expression plasmid pIRES2-EGFP-RGS16 was constructed, and transfected into C6 cells. The cells were selected by G418. The expression of enhanced green fluorescent protein (EGFP) was observed under fluorescent microscope, and the expression of RGS16 mRNA and protein was detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. The effects of RGS16 gene on cell cycle progression, cell growth, and proliferation of C6 cells were measured by flow cytometry, cell growth curve, and clonogenic assay in plate. RESULTS: C6-RGS16 and C6-GFP cells, which separately expressed RGS16 and pIRES2-EGFP vector, were constructed successfully. S phase proportion was significantly higher in C6-RGS16 cells than in C6-GFP and C6 cells (28.5% vs. 18.9% and 14.3%, P<0.05); C6-RGS16 cells grew faster than C6-GFP and C6 cells. The clone formation rate was significantly lower in C6-RGS16 cells than in C6-GFP and C6 cells (12% vs. 25% and 25%, P<0.05). CONCLUSION: Exogenous RGS16 gene could promote cell cycle progression of glioma C6 cells from G1 phase to S phase, and accelerate cell growth, but can't increase the clone formation.

[Construction of the eukaryotic expression vector pIRES2-EGFP-Axin and its expression in glioma cells].

AIM: To construct the eukaryotic expression vector pIRES2-EGFP-Axin, and to express Axin in C6 glioma cells. METHODS: The Axin gene was amplified by PCR using pCMV5-HA-Axin as a template, and confirmed by DNA sequencing. The eukaryotic expression vector pIRES2-EGFP-Axin was constructed by introducing Axin DNA fragment into the sites of Nhe I and Sal I of pIRES2-EGFP vector. The plasmid was transfected into the C6 cells using lipofectamine. The expressed EGFP was observed under fluorescent microscope and the Axin protein expression was detected by immunostaining using anti-Axin antibody. RESULTS: The eukaryotic expression vector pIRES2-EGFP-Axin was constructed and transfected successfully into C6 glioma cells. The green fluorescence of EGFP was observed in the plasma and nuclei of transfected cells, and Axin protein was only found in the plasma. CONCLUSION: The recombinant expression vector pIRES2-EGFP-Axin was constructed, and the EGFP and Axin gene could be co-expressed in the C6 cells. This study laid a foundation for the further research of the function of Axin in cell differentiation, growth and tumorigenesis.

Heat shock protein 70/MAGE-3 fusion protein vaccine can enhance cellular and humoral immune responses to MAGE-3 in vivo.

MAGE-3, a member of melanoma antigen (MAGE) gene family, is recognized as an ideal candidate for tumor vaccine because it is expressed in a significant proportion of tumors of various histological types and can induce antigen-specific immune response in vivo. There is now substantial evidence that heat shock proteins HSPs isolated from cancer cells and virus-infected cells can be used as vaccines to produce cancer-specific or virus-specific immunity. In this research, we investigated whether M. tuberculosis HSP70 can be used as vehicle to elicit immune response to its accompanying MAGE-3 protein. A recombinant protein expression vector was constructed that permitted the production of fusion protein linking amino acids 195-314 of MAGE-3 to the C terminus of HSP70. We found that HSP70-MAGE-3 fusion protein can elicit stronger cellular and humoral immune responses against MAGE-3 expressing murine tumor than those elicited by MAGE-3 protein in vivo, which resulted in potent antitumor immunity against MAGE-3-expressing tumors. Covalent linkage of HSP70 to MAGE-3 was necessary to elicit immune response to MAGE-3. These results indicate that linkage of HSP70 to MAGE-3 enhanced immune responses to MAGE-3 in vivo and HSP70 can be exploited to enhance the cellular and humoral immune responses against any attached tumor-specific antigens.

[Telomerase SiRNA inhibits KB cell growth in human oral squamous cell carcinoma].

OBJECTIVE: To test the telomerase SiRNA on telomerase mRNA and on KB cell growth of oral squamous cell carcinoma. METHODS: We synthesized 21-nucleotide SiRNA duplexes with symmetric 2-nucleotide 3' overhangs corresponding to the target sequence (2 657 approximately 2 675 nucleotide downstream of the start codon) of telomerase mRNA. Telomerase activity, cell proliferation, cell cycle and apoptosis were measured after transfection. RESULTS: Twenty one-nucleotide small interfering RNA (SiRNA) duplexes specifically suppressed expression of endogenous telomerase mRNA in human oral squamous carcinoma KB cells. This inhibitory effect lasted only for about 48 h after transfection. Telomerase activity reduction corresponded to the mRNA suppression. Cell proliferation decreased by 30% at 48 h after transfection and lasted for 120 h after treatment. This inhibitory effect resulted from the block of G(1) to S transition. Apoptosis was not involved in this process. CONCLUSIONS: SiRNA is a powerful tool for studying gene function and can be used as gene-specific therapeutics.

Identification of HLA-A2-restricted CTL epitope encoded by the MAGE-n gene of human hepatocellular carcinoma.

BACKGROUND: Identification of the cytotoxic T lymphocytes (CTL) restricted epitopes of tumor antigens opens up possibilities of developing a new cancer vaccine. For the MAGE-n has been demonstrated closely associated with hepatocellular carcinoma (HCC) and HLA-A2.1 is found in over 50% of HCC patients in China, we aim at identifying MAGE-n-encoded peptide presented by HLA-A2.1. MATERIALS: A HLA-A2.1-restricted CTL epitope was identified by using an improved "reverse immunology" strategy: (a) computer-based epitope prediction from the amino acid sequence of MAGE-n antigen; (b) peptide-binding assay to determine the affinity of the predicted peptide with HLA-A2.1 molecule; (c) stimulation of primary T-cell response against the predicted peptides in vitro; and (d) testing of the induced CTLs toward HCC cells expressing MAGE-n antigen and HLA-A2.1. RESULTS: Of the five tested peptides, effectors induced by a peptide of MAGE-n at residue position 159-167(QLVFGIEVV) lysed HCC cells expressing both MAGE-n and HLA-A2.1. Our results indicated that peptide QLVFGIEVV was a new HLA-A2.1-restricted CTL epitope capable of inducing MAGE-n specific CTLs in vitro. CONCLUSIONS: Identification of the MAGE-n /HLA-A2.1 peptide QLVFGIEVV may facilitate peptide-based specific immunotherapy for HCC. The combination of epitope prediction, epitope reconstruction method and immunological methods can improve the efficiency and accuracy of CTL epitope studies.

[Establishment of mouse melanoma B16 cell lines co-expressing MAGE-1 and EGFP].

AIM: To construct the melanoma antigen-1(MAGE-1) eukaryotic expression plasmid and express MAGE-1 in mouse melanoma B16 cells. METHODS: The MAGE-1 gene was amplified by PCR and cloned into the eukaryotic expression vector pIRES2-EGFP to construct the pIRES2-EGFP-MAGE-1 plasmid. The plasmid was transfected into the B16 cells. The EGFP expression was detected under fluoroscent microscope and the MAGE-1 expression was detected by immunohistochemistry staining. RESULTS: The eukaryotic expression vector pIRES2-EGFP-MAGE-1 was constructed and transfected successfully into B16 cells, and the EGFP and MAGE-1 genes were co-expressed in the B16 cells. CONCLUSION: A mouse melanoma cell line B16 co-expressing MAGE-1 and EGFP genes has been established successfully, which lays the foundation for the research on application of MAGE-1 in the tumor immunotherapy.

Heat shock protein 70 / MAGE-1 tumor vaccine can enhance the potency of MAGE-1-specific cellular immune responses in vivo.

The cancer-testis antigen encoded by the MAGE-1 gene is an attractive antigen in tumor immunotherapy because it can be processed as a foreign antigen by the immune system and generate tumor-specific cellular immune response in vivo. However, increase of the potency of MAGE-1 DNA vaccines is still needed. The high degree of sequence homology and intrinsic immunogenicity of heat shock protein 70 (HSP70) have prompted the suggestion that HSP70 might have immunotherapeutic potential, as HSP70 purified from malignant and virally infected cells can transfer and deliver antigenic peptides to antigen-presenting cells to elicit peptide-specific immunity. In this research, we evaluated the enhancement of linkage of Mycobacterium tuberculosis HSP70 to MAGE-1 gene of the potency of antigen-specific immunity elicited by naked DNA vaccines. We found that vaccines containing MAGE-1-HSP70 fusion genes enhanced the frequency of MAGE-1-specific cytotoxic T cells in contract to vaccines containing the MAGE-1 gene alone. More importantly, the fusion converted a less effective DNA vaccine into one with significant potency against established MAGE-1-expressing tumors. These results indicate that linkage of HSP70 to MAGE-1 gene may greatly enhance the potency of DNA vaccines, and generate specific antitumor immunity against MAGE-1-expressing tumors.