细粒棘球蚴抗原EPC1基因的克隆、表达及其免疫诊断的研究

目的克隆、表达细粒棘球蚴EPC1(EgEPC1)基因,鉴定重组抗原的反应原性,并用棘球蚴病患者血清评价其诊断价值。方法从绵羊肝脏棘球蚴囊中分离原头节,提取其总RNA,采用RT-PCR扩增EgEPC1基因,将其克隆至pGEM-T载体,再将其与原核表达载体PET28a(+)连接构建重组质粒PET28a-EgEPC1,转化大肠埃希菌(E.coli)BL21(DE3),经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达重组蛋白,用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹(Western blotting)对纯化的重组抗原进行鉴定分析。以该重组蛋白作为包被抗原建立ELISA方法,检测细粒棘球蚴病患者血清(60份)特异性IgG抗体,同时以多房棘球蚴病(37份)、囊尾蚴病(16份)、华支睾吸虫病(7份)、日本血吸虫病(4份)患者和健康人血清(33份)作为对照,评价重组抗原EgEPC1的免疫诊断效果。结果双酶切鉴定和测序结果均显示重组质粒PET28a-EgEPC1构建成功。SDS-PAGE和Western blotting分析显示,重组质粒PET28a-EgEPC1在E.coli BL21(DE3)...     

Magnetically directed self-assembly of electrospun superparamagnetic fibrous bundles to form three-dimensional tissues with a highly ordered architecture

Engineering three-dimensional (3D) cell-dense tissues with a well-organized structure remains a challenge in tissue engineering. In this study, highly oriented fibrous bundles, consisted of composite fibers of poly(L-lactide-co-glycolide)/superparamagnetic iron oxide nanoparticles, were fabricated using an electrospinning technique. The magnetic properties of the fabricated fibrous bundles were examined by a vibrating sample magnetometer and a superconducting quantum interference device; the results demonstrate that the fabricated fibrous bundles revealed superparamagnetic behavior without magnetic hysteresis. After seeding C2C12 myoblasts on the fibrous bundles, cells were grown along the direction of the underlying fibers (cell rods), an aligned pattern similar to those in native skeletal muscle tissues. When treated with the differentiation medium, myoblasts were fused together and formed multinucleated myotubes. As soon as applying an external magnetic field, the cell rods can spontaneously response to the magnetic control and self-assemble into 3D tissues with a highly ordered architecture. These findings demonstrate that the magnetically susceptible fibrous bundles not only can serve as a functional unit providing the topographic cue for cell orientation, but also can be magnetically manipulated for the creation of 3D cell-dense constructs. This technique may be applied to various cell types and scaffold configurations, thus advancing the design of engineered tissues that more closely replicate native tissues.     

Enhanced systemic T-cell activation after in situ gene therapy with radiotherapy in prostate cancer patients

PURPOSE: In situ cytotoxic gene therapy can potentially trigger a systemic immune response, which could impact occult metastatic disease. We are currently conducting three clinical trials using in situ adenoviral vector mediated herpes simplex virus-thymidine kinase (HSV-tk) gene delivery followed by the HSV-tk prodrug ganciclovir (GCV) or valacyclovir (VCV). This study evaluates the systemic T-cell response after gene therapy in each trial. METHODS AND MATERIALS: The study protocol included three separate clinical trials in the Baylor Prostate Cancer SPORE Program: Trial A gene therapy in prostate cancer patients failing radiotherapy (36 patients), Trial B neoadjuvant gene therapy in pre-radical prostatectomy patients (22 patients), and Trial C gene therapy in combination with radiotherapy for prostate cancer (27 patients). Heparinized blood was collected at the time of vector injection and at selected intervals afterward. A complete blood count was performed, and peripheral blood lymphocytes were analyzed by fluorescent antibody cell sorting after labeling with dual color-labeled antibody pairs. RESULTS: The pretreatment mean percentage of activated CD8+ T cells (DR+CD8+ T cells) was 12.23%, 16.72%, and 14.09% (Trials A, B, and C, respectively). Two weeks posttreatment, this increased to 22.87%, 26.15%, and 39.04% (Trials A, B, and C, respectively), and these increases were statistically significant (p = 0.0188, p = 0.0010, p < 0.0001, respectively). The increase of DR+CD8+ T cells was significantly larger in Trial C than in Trial A (p = 0.0044) or Trial B (p = 0.0288). Total CD8+ T cells significantly increased at 2 weeks posttreatment in Trial B and C (p = 0.0013, p = 0.0004, respectively). Interestingly, only in Trial C were significant increases in activated CD4+ T cells seen at 2 weeks (p = 0.0035). CONCLUSIONS: This is the first report of systemic T-cell responses after HSV-tk+GCV/VCV gene therapy under three clinical trial conditions. There was an increase in activated CD8+ T cells in the peripheral blood after vector injection, suggesting the potential for activation of components of cell-mediated immune response in all trial conditions. The addition of radiotherapy to in situ gene therapy seems to further increase the total CD8+ T cells and activated CD4+ T cells.     

Psychometric validation and normative data of a second Chinese version of the Hooper Visual Organization Test in children

The Hooper Visual Organization Test (HVOT) is a measure of visuosynthetic ability. Previously, the psychometric properties of the HVOT have been evaluated for Chinese-speaking children aged 5-11 years. This study reports development and further evidence of reliability and validity for a second version involving an extended age range of healthy children and children with developmental disabilities (DD) from 5 to 14 years of age. Rasch analysis revealed that after deletion of 6 items, a 24-item version conformed to a unidimensional scale. The test showed satisfactory internal consistency; 3-week test-retest coefficients all exceeded .85 for three DD subsamples. The second version was able to successfully differentiate between the three DD subgroups (attention-deficit hyperactivity disorder, autism spectrum disorders, and mental retardation) and the healthy control group, with correct classification rates ranging from 86.6% to 94.1%. Its construct validity was supported by expected correlations. Accordingly, age-based normative data were established as a basis for interpretation of performance. In sum, the second Chinese version of the HVOT has good psychometric properties and norms that are suited for use in clinical practice.     

Clinical management of cervical intraepithelial neoplasia in pregnant and postpartum women

Objective

To evaluate the clinical management of cervical intraepithelial neoplasia (CIN) and cervical microinvasive squamous cell carcinoma in pregnant and postpartum women.

Methods

This prospective study enrolled 27,230 pregnant women undergoing routine gestational examinations between August 1, 2007 and July 31, 2010 in the Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Colposcopy and cervical biopsy were performed for patients with abnormal Thin Prep^® Papanicolaou test (TCT) results. Periodic colposcopy was performed every 8–12 weeks and cervical biopsy was performed if progression was suspected. Cervical cold knife conization was recommended to patients diagnosed with CINIII or microinvasive cervical carcinoma 6–12 weeks after delivery.

Results

A total of 2,260 patients had abnormal TCT results (8.12 %). Colposcopy and cervical biopsy were performed for 369 patients. Fifteen patients had microinvasive squamous cell carcinoma, 116 patients had cervicitis, and the number of CIN patients with histological grades I, II, and III were 124, 49, and 65, respectively. Tumor progression during pregnancy was found in 253 patients (CINI or above). Prognosis varied depending on the highest grade of pathological diagnosis results during pregnancy or initial pathological diagnosis results performed 6–12 weeks after delivery by cervical biopsy under colposcopy. Treatment and follow-up were carried out according to diagnoses, state of progression, and reversion (if any).

Conclusion

These findings underline a need for cervical lesion screening for all women during pregnancy, and colposcopy should be performed for pregnant women who have abnormal TCT results. Appropriate treatment and follow-up were recommended according to different diagnosis of CIN.     

Cold-induced exodus of postsynaptic proteins from dendritic spines

Dendritic spines are small protrusions on neuronal dendrites and the major target of the excitatory inputs in mammalian brains. Cultured neurons and brain slices are important tools in studying the biochemical and cellular properties of dendritic spines. During the processes of immunocytochemical studies of neurons and the preparation of brain slices, neurons were often kept at temperatures lower than 37 degrees C for varied lengths of time. This study sought to investigate whether and how cold treatment would affect the protein composition of dendritic spines. The results indicated that upon cold treatment four postsynaptic proteins, namely, alpha,beta-tubulins, calcium, calmodulin-dependent protein kinase IIalpha, and cytoplasmic dynein heavy chain and microtubule-associated protein 2, but not PSD-95 or AMPA receptors, exited from the majority of dendritic spines of cultured rat hippocampal neurons in a Gd(3+)-sensitive manner. The cold-induced exit of tubulins from dendritic spines was further found to be an energy-dependent process involving the activation of Gd(3+)-sensitive calcium channels and ryanodine receptors. The results thus indicate that changes in temperature, calcium concentration, and energy supply of the medium surrounding neurons would affect the protein composition of the dendritic spines and conceivably the protein composition of the subcellular organizations, such as the postsynaptic density, in the cytoplasm of dendritic spines.     

Associations of polymorphisms of <Emphasis Type="Italic">CYP2D6</Emphasis> and <Emphasis Type="Italic">CYP2C9</Emphasis> with early onset severe pre-eclampsia and response to labetalol therapy

Background and aims

Natural orifice translumenal endoscopic surgery (NOTES) procedure describes a surgical approach using natural orifices. We describe a prospective non-comparative clinical study on transgastric salpingo-oophorectomy in humans.

Methods

Patients with indication for salpingo-oophorectomy were offered the transgastric approach. This paper presents the data of the first 6 patients, in whom the procedure was performed. After gastroscopic incision in the anterior corpus wall we advanced the flexible gastroscope into the abdominal cavity. With the help of a transvaginally introduced 10 mm trocar and, if the uterus was present, an intrauterine manipulator, the salpingo-oophorectomy was performed. The colpotomy was dilated and the specimens were extracted in a bag. The gastrotomy was closed with an over-the-scope-clip and the colpotomy with a running suture.

Results

The planned salpingo-oophorectomy was performed successfully in all patients. All patients recovered quickly and were discharged between the third and fifth day. In one patient a gastric bleeding was seen on the first postoperative day. The bleeding was clipped gastroscopically, all other follow ups were uneventful.

Conclusion

Our study demonstrates the feasibility of the transgastric access to the pelvis. The gastroscope provided excellent optical control and good tissue preparation. Therefore, we conclude that pure NOTES procedures using the transgastric access to the adnexa are feasible. An increasing role of transgastric procedures for diseases in the pelvic region can be expected in particular if new endoscopic platforms with better means of instrumentation and tissue management become available.