To generate recombinant avian adeno-associated virus (rAAAV) for gene transfer studies in avian cells, the recombinant plasmid containing the whole genome of AAAV was digested with restriction enzymes to remove the Rep and Cap genes, resulting in AAAV transfer vector pAITR. GFP-expressing cassette was amplified by PCR and inserted into the AAAV transfer vector. The Rep-Cap gene of AAAV amplified by high fidelity PCR was subcloned into eukaryotic expression vector pcDNA3, resulting in an AAAV helper vector pcDNA-ARC. The Rep and Cap genes amplified by high fidelity PCR were subcloned separately into the co-expression vector pVITRO2-mcs, resulting in another AAAV helper vector pVITRO2-ARC. Using calcium phosphate precipitation method, rAAAV-GFP was generated by co-transfecting AAV-293 cells with a cocktail of pAITR-GFP, pcDNA-ARC or pVITRO2-ARC, and adenovirus helper vector pHelper. The three structural proteins VP1, VP2 and VP3 of correct molecular masses were detected by SDS-PAGE and the GFP reporter gene was detected by PCR in purified rAAAV-GFP virions. Chicken embryonic fibroblast (CEF) cells and CEL cell line were transduced with the recombinant virus, the GFP-positive cells were easily observed under fluorescent microscope, expression of which lasted for at least two weeks. These data demonstrate that an efficient helper virus-free packaging system has been established for generating recombinant AAAV particles for gene transfer studies in avian cells and for development of recombinant vaccines against avian diseases.
Animals ; Cell Line ; Cells, Cultured ; Chickens ; Dependovirus ; genetics ; Electrophoresis, Polyacrylamide Gel ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Microscopy, Fluorescence ; Models, Genetic ; Plasmids ; genetics ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo evaluate the feasibility of generating animal mammary gland bioreactors expressing human lysozyme (hLYZ).

METHODSThe recombinant vector p205C3-hLYZ, as a result of connecting the hLYZ cDNA with the mammry gland expression vector p205C3, was used to generate transfer genic mice by microinjection.

RESULTSA total of 136 F0 mice were obtained, of which 7 (2 females and 5 males) and 4 (1 females and 3 males) were found to contain the transfer-gene by PCR and Southern blotting respectively. The results of Western blotting indicated that the expressed protein had the same molecular weight as that of normal hLYZ. From the F1 generation on, the mice mated only with their brothers or sisters and a colony of F7 transgenic mice was obtained. Among the offspring, the female transgenic mice maintained and expressed the transfer-gene stably with an expression level as high as 750 mg/L. The expressed protein had strong tissue specificity, and in addition to the mammary glands, some degree of ectropic expression in the spleens and intestines of the transgenic mice was confirmed by dot blotting assay.

CONCLUSIONThese data indicate that the mice mammary gland bioreactors expressing hLYZ have been successfully generated.

Animals ; Blotting, Southern ; Blotting, Western ; Female ; Humans ; Male ; Mammary Glands, Animal ; metabolism ; Mice ; Mice, Transgenic ; Muramidase ; genetics ; metabolism ; Polymerase Chain Reaction

OBJECTIVETo examine granulocyte colony stimulating factor (G-CSF) expression in human non-small cell lung cancer (NSCLC) as well as discuss its clinicopathological significance.

METHODSSpecimens were obtained from 114 cases (53 cases with granulocyte infiltration) diagnosed pathologically as NSCLC in General Hospital of PLA. Paraffin-embedded tissues from these 114 cases of NSCLC were examined for expression of G-CSF by immunohistochemical staining. Correlation between G-CSF expression and pathological features, clinical manifestation, prognosis of patients with NSCLC was analyzed statistically. All the patients were retrospectively followed-up.

RESULTSFifty-five of the 114 NSCLC specimens expressed G-CSF, and among these 41 (41/54, 75.9%) were large cell carcinoma, nine (9/30, 30.0%) were adenocarcinoma and five (5/30, 16.7%) were squamous cell carcinoma. The expression was significantly correlated with infiltration of tumor mass by neutrophilic granulocytes, histological type, necrosis, differentiation, lymph node metastases, distant metastases, recurrence and survival period (P < 0.01). There was no significant correlation with primary tumor size (P > 0.05). Logistic multi-factor analysis revealed that necrosis, lymph nodes metastases and distant metastases RR (risk ratio) in G-CSF positive group was 5.57, 6.28 and 5.24 times higher than those of G-CSF negative group (P < 0.05). There were remarkable difference of 5-year survival rates (0 and 12.1% respectively) and survival period (42 and 62 months respectively) between positive and negative groups (P < 0.01).

CONCLUSIONSNSCLC with G-CSF excretion are mainly large cell lung cancer. The pathologic characteristics of these cases with G-CSF expression included poor differentiation, remarkable atypia, prominent necrosis and infiltration of tumor mass by neutrophils or emperipolesis. These tumors are usually more aggressive in biological behavior and have worse prognosis than those without G-CSF expression.

Adenocarcinoma ; metabolism ; pathology ; surgery ; Carcinoma, Large Cell ; metabolism ; pathology ; surgery ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; surgery ; Carcinoma, Squamous Cell ; metabolism ; pathology ; surgery ; Follow-Up Studies ; Granulocyte Colony-Stimulating Factor ; metabolism ; Humans ; Lung Neoplasms ; metabolism ; pathology ; surgery ; Lymphatic Metastasis ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Neoplasm Recurrence, Local ; Retrospective Studies ; Survival Rate

To examine whether or not the regulatory sequence of chicken ovalbumin gene can drive transgene expression specifically in hen oviduct, the authors constructed an oviduct-specific expression vector (pOV), containing 3.0 kilobases (kb) of the 5'-flanking sequence and 3.0 kb of the 3'-flanking sequence of the chicken ovalbumin gene. Jellyfish green fluorescence protein (EGFP) reporter gene and bacterial LacZ reporter gene were respectively inserted into the downstream of the 5'-regulatory region. The recombinants were named as pOVEGFP and pOVLacZ. Two transfer systems, in vitro and in vivo, were used to verify the function of the vector. In vitro, the plasmid DNA pOVEGFP and pEGFP-N1 were transfected respectively by the polyethyleneimine procedure into the primary chicken oviduct epithelium (PCOE) and fibroblasts cells isolated from laying hens. In vivo, the recombinant vector pOVLacZ was injected into egg-laying hens via wing vein and the tissues were collected for RT-PCR analysis. The results showed that expression of pEGFP-N1 was achieved at low level in oviduct epithelial cells and at high level in fibroblasts, but that the recombinant vector was not expressed in both cells. RT-PCR analysis showed that the LacZ gene was transcribed in the oviduct, but not in the heart, liver, kidney and spleen of the injected hens. Accordingly, the beta-galactosidase activity was only detected in the oviduct magnum (116.7 mU/ml) and eggs (16.47 mU/ml). These results indicated that the cloned regulation regions of chicken ovalbumin gene could drive exogenous gene expression specifically in the oviducts of hens. In vivo gene injection via wing vein may serve as a rapid production system of recombinant proteins in chicken eggs. In addition, the cultured primary oviduct cells from laying hens were not efficient temporary expression systems for analyzing the function of regulating elements of ovalbumin gene.
Animals ; Cells, Cultured ; Chickens ; Cloning, Molecular ; methods ; Fallopian Tubes ; metabolism ; Female ; Organ Specificity ; Ovalbumin ; biosynthesis ; genetics ; Protein Engineering ; methods ; Recombinant Proteins ; biosynthesis ; Transfection ; methods ; Women

To develop a gene therapy strategy for treating bovine mastitis, a new mammary-specific vector containing human lysozyme (hLYZ) cDNA and kanamycin resistance gene was constructed for intramammary expression and clinical studies. After one time acupuncture or intracisternal infusion of healthy cows with 400 microg of the p215C3LYZ vector, over 2.0 microg/ml of rhLYZ could be detected by enzymatic assay for about 3 weeks in the milk samples. Western blotting showed that rhLYZ secreted into milk samples from the vector-injected cows had molecular weight similar to that of the natural hLYZ in human colostrums. Twenty days after the primary injection, the quarters were re-injected with the same vector by quarter acupuncture and even higher concentrations of rhLYZ could be detected. Indirect competitive ELISA of milk samples showed that the vector injection did not induce detectable humoral immune response against hLYZ. Clinical studies showed that twice acupuncture of quarters with the p215C3LYZ vector had overt therapeutic effect on clinical and subclinical mastitis previously treated with antibiotics, including disappearance of clinical symptoms and relatively high microbiological cure rates. These data provide a solid rationale for using the vector to develop gene therapy for treating bovine mastitis.
Acupuncture ; Animals ; Cattle ; Female ; Genetic Therapy ; methods ; veterinary ; Genetic Vectors ; genetics ; Mastitis, Bovine ; genetics ; therapy ; Milk ; chemistry ; Muramidase ; biosynthesis ; genetics ; metabolism

OBJECTIVETo identify antigenic proteins secreted by Streptococcus suis (S. suis) type 2 strain SC84.

METHODSTwo-dimensional electrophoresis (2-DE), western-blot assay and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis were performed to search and identify antigenic proteins secreted by S. suis strain SC84, which triggered an outbreak of the disease in Sichuan province,China, in 2005.

RESULTSA total number of 14 western blot spots were found on PVDF membrane. 11 spots which could be found the existence of matching protein on coomassie G-250-stained 2-DE gel were identified by MALDI-TOF MS. The 11 proteins, all located at extra-cellular or cell wall, were classified into 8 kinds of proteins. Among of them, muramidase-released protein (MRP), suilysin (Sly) and extra-cellular factor (EF) were the known antigenic proteins, but several proteins such as putative 5'-nucleotidase, ribo-nucleases G and E, and predicted metal-loendo-peptidase were newly found antigenic proteins. All the identified protein were found to have had the coding gene in genomic of S. suis strain 05ZYH33, isolated from patients in Sichuan province, China in 2005.

CONCLUSIONThe newly found proteins could be used as voluntary antigens for detection and vaccination of S. suis.

Bacterial Proteins ; analysis ; immunology ; Humans ; Proteomics ; Streptococcal Infections ; Streptococcus suis ; immunology ; isolation & purification ; metabolism

Objective To study the integration site and arrangement of SfII and SfX prophages in Shigella flexneri serotype 2b strains. Methods A series of primers were designed based on potential integration site of SfII and SfX prophages in Shigella flexneri serotype 2b strains, and PCR were performed for 50 serotype 2b strains to amplify special genes located in host and prophages. PCR products were sequenced to identify integration sites and arrangement of SfII and SfX. Results In all the serotype 2b strains, prophage SfII and SfX were adjacent to each other, and integrated into the thrW tRNA gene of the host, which were located between genes proA and yaiC of host. Prophage SfX was located immediately upstream of prophage SfII in all the detected 50 serotype 2b strains exception for strain 51251. Conclusion This was the first report on the integration site and arrangement of serotype-converting prophages SfII and SfX in Shigella flexneri 2b strains.

BACKGROUNDOccult invasive cervical cancer discovered after simple hysterectomy is not common, radical parametrectomy (RP) is a preferred option for young women. However, the morbidity of RP was high. The aim of our study is to assess the incidence of parametrial involvement in patients who underwent radical parametrectomy for occult cervical cancer or radical hysterectomy for early-stage cervical cancer and to suggest an algorithm for the triage of patients with occult cervical cancer to avoid RP.

METHODSA total of 13 patients with occult cervical cancer who had undergone RP with an upper vaginectomy and pelvic lymphadenectomy were included in this retrospective study. Data on the clinicopathologic characteristics of the cases were collected. The published literature was also reviewed, and low risk factors for parametrial involvement in early-stage cervical cancer were analyzed.

RESULTSOf the 13 patients, 9 had a stage IB1 lesion, and 4 had a stage IA2 lesion. There were four patients with grade 1 disease, seven with grade 2 disease, and two with grade 3 disease. The median age of the entire patients was 41 years. The most common indication for extrafascial hysterectomy was cervical intraepithelial neoplasia 3. Three patients had visible lesions measuring 10-30 mm, in diameter and ten patients had cervical stromal invasions with depths ranging from 4 to 9 mm; only one patient had more than 50% stromal invasion, and four patients had lymph-vascular space invasion (LVSI). Perioperative complications included intraoperative bowel injury, blood transfusion, vesico-vaginal fistula, and ileus (1 case for each). Postoperative pathologic examination results did not show residual disease or parametrial involvement. One patient with positive lymph nodes received concurrent radiation therapy. Only one patient experienced recurrence.

CONCLUSIONSPerioperative complications following RP were common, whereas the incidence of parametrial involvement was very low among selected early-stage cervical cancer patients. Based on these results, we thought that patients with very low-risk parametrial involvement(tumor size ≤ 2 cm, no LVSI, less than 50% stromal invasion, negative lymph nodes) may benefit from omitting RP. Further prospective data are warranted.

Adult ; Cervical Intraepithelial Neoplasia ; Combined Modality Therapy ; Female ; Humans ; Hysterectomy ; Lymph Node Excision ; Middle Aged ; Neoplasm Recurrence, Local ; Neoplasms, Unknown Primary ; Postoperative Complications ; Retrospective Studies ; Risk Factors ; Uterine Cervical Neoplasms

It is of vital significance to conduct active post-marketing surveillance of Chinese medicine, as an active response to laws, rules and guidelines issued by the China food and drug administration. The standards for technological specifications based on expert consensus have been drafted. These will provide technological support in evaluating adverse drug reactions (ADRs) or adverse drug events (ADEs). The technological specifications for post-marketing surveillance focus on two surveillance designs; one is a large sample registry study to explore general population ADR/ADE characteristics, the other is a nested case-control study to explore the characteristic and mechanisms of ADRs.
China ; epidemiology ; Drug Monitoring ; adverse effects ; standards ; Drug-Related Side Effects and Adverse Reactions ; epidemiology ; etiology ; Drugs, Chinese Herbal ; adverse effects ; standards ; Hospital Information Systems ; Humans ; Product Surveillance, Postmarketing ; methods ; standards