To investigate the morphogenetic process of human adenovirus type 41 (HAdV-41), 293TE cells were infected with purified wild-type HAdV-41, and ultrathin sections of infected cells were prepared and observed under a transmission electron microscope. Results showed that HAdV-41 entered host cells mainly through three ways: non-clathrin-coated pit, clathrin-coated pit, and direct penetration of plasma membrane. In addition, cell microvilli might help HAdV-41 enter cells. After entering into cells, HAdV-41 virus particles could be found in vacuoles or lysosomes or be in a free state in cytoplasm. Only free virus particles could be found near nuclear pores (NP), suggesting that the virus needed to escape from lysosomes for effective infection and viral nucleoprotein entered the nucleus through NP. Progeny viruses were as-sembled in the nucleus. Three types of inclusion bodies, which were termed as fibrillous inclusion body, condense inclusion body, and stripped condense inclusion body, were involved in HAdV-41 morphogenesis. In the late phase of viral replication, the membrane integrity of the infected cells was lost and viral particles were released extracellularly. This study reveals the partial process of HAdV-41 morphogenesis and provides more biological information on HAdV-41.
Adenovirus Infections, Human ; virology ; Adenoviruses, Human ; genetics ; growth & development ; physiology ; ultrastructure ; Cell Membrane ; virology ; Cell Nucleus ; virology ; Humans ; Virus Release ; Virus Replication

Microscopy, Electron, Transmission ; methods ; Negative Staining ; methods ; Viruses ; ultrastructure

Humans ; Parvoviridae Infections ; virology ; Parvovirus B19, Human ; genetics ; metabolism ; physiology

SUMMARY To explore the clinical pathological characteristics and improve the recognition in the diag-nosis and treatment of basal cell carcinoma (BCC)of prostate.Three cases of BCC of prostate were re-ported and the relevant literature was reviewed to investigate the diagnosis and treatment of this disease. We analyzed three cases of prostatic BCC.Their ages were within a range of 57 to 83 years.One of them complained of hematuria and two complained of dysuria.All of them presented with prostatic hyperplasia. Two of them presented with high prostate specific antigen (PSA)and one with normal PSA.Case 1 had prostate cancer invasion of bladder,rectal fascia,with lymph node metastasis,bone metastasis and lung metastases.The patient received bladder resection +bilateral ureteral cutaneous ureterostomy +lymph node dissection on November 2,2014 .Postoperative pathological diagnosis showed BCC.Reexamination of pelvic enhanced MRI in January 8,2015 suggested pelvic recurrence.Abdominal enhanced CT showed multiple liver metastases and pancreatic metastasis on July 11,2015.Prostate cancer specific death occurred in October 2015.Case 2 was diagnosed as BCC in prostate biopsy on March 27,2015. Positron emission tomography and computed tomography (PET-CT)showed pulmonary metastasis and bone metastasis.Then the patient received chemotherapy,endocrine therapy and local radiation therapy. Reexamination of PET-CT on January 11,2016 showed that the lung metastase tumors and bone metas-tase tumors were larger than before.Up to January 10,2016,the patient was still alive.Postoperative pathological changes of transurethral resection of prostate (TURP)in case 3 showed BCC might be con-sidered.The PET-CT suggested residual prostate cancer,which might be associated with bilateral pelvic lymph node metastasis.In April 20,2016,the review of PET-CT showed pelvic huge irregular hybrid density shadow,about 14.5 cm ×10.0 cm ×12.9 cm in size,and tumor recurrence was considered. Then the patient received local radiation therapy.The patient survived in the followed upon January 10, 2016.BCC of prostate is a rare subtype.Due to the local infiltrative and distant metastatic potentiality, active management is preferred and a life-long follow-up is necessary.

Objective To build the experimental basement for the clinical use of cytokines induced killer(CIK)cells from the cord blood mononuclear cells(CBMNC)in tumor adoptive cellular immunotherapy, an effective protocol for their proliferation in vitro and cytotoxicity of CIK cells was established.Methods The lymphocytes from umbilical cord blood were isolated by density gradient centrifugation and suspended in medium with CD_3 mAb,rIL-2,rIL-1 and IFN-? as inducing agents to prepare CIK cells.At the same time, the lymphokine activated killer(LAK)and CBMNC were set as controls,which were only added IL-2 and not any cytokines during the whole culture.The changes of CIK cells before and after induction were observed with microscope and the phenotypes of the cells were analyzed by using flow cytometry.The proliferation of CIK cells were determined by trypan blue exclusion assay and the cytotoxic activity to lung cancer cell were tested with MTF method.Results According to the experiment,combining use of four types of cytokines could generate a great deal of CIK cells possessing highly cytotoxicity.From day 5 CIK cells became to prolif- erate and reached the peak at day 14.During the whole period,the relative percentage of CD_3~+ CD_(56)~+ cells in- creased significantly.Compared with LAK cells,which reached the proliferation peak at day 7 and then showed no evident proliferation.The control cells(CBMNC)showed no evident change of phenotypes and proliferation.CIK cells showed a higher antitumor activity on the tumor cells than LAK cells and CBMNC in vitro.Conclusion Umbilical cord blood can generate a great deal of CIK cells combining used with cy- tokines.Compared with classic LAK cells,umbilical cord blood CIK cells have the advantages of rapid prolif- eration speed and powerful cytotoxicity.CIK cells will be promising as a new strategy for the adoptive cellular immunotherapy of tumor.

Objective We performed experiments using Neuregulin-1β (NRG-1β) treatment to determine a mechanism for the protective role derived from its beneficial effects by remodeling gap junctions (GJs) during heart failure (HF). Methods Rat models of HF were established by aortocaval fistula. Forty-eight rats were divided randomly into the HF (HF, n = 16), NRG-1β treatment (NRG, n = 16), and sham operation (S, n = 16) group. The rats in the NRG group were administered NRG-1β (10 μg/kg per day) for 7 days via the tail vein, whereas the other groups were injected with the same doses of saline. Twelve weeks after operation, Connexin 43 (Cx43) expression in single myocytes obtained from the left ventricle was determined by immunocytochemistry. Total protein was extracted from frozen left ventricular tissues for immunoblotting assay, and the ultrastructure of myocytes was observed by transmission electron microscopy. Results Compared with the HF group, the cardiac function of rats in the NRG group was markedly improved, irregular distribution and deceased Cx43 expression were relieved. The ultrastructure of myocytes was seriously damaged in HF rats, and NRG-1β reduced these pathological damages. Conclusions Short-term NRG-1β treatment can rescue pump failure in experimental models of volume overload-induced HF, which is related to the recovery of GJs structure and the improvement of Cx43 expression.

OBJECTIVETo construct full-length hepatitis B core particles presenting preS1 aa 21-47 epitope and truncated core particles presenting preS1 aa 37-45 epitope on their surface and compare their antigenicity.

METHODSPreS1 aa21-47 epitope and aa 37-45 epitope were inserted respectively into full-length hepatitis B core (aa 1-183) and truncated HBcAg (aa 1-144), between the 78th (Asp) and 79th (Pro). The genes synthesized after the codon optimization were ligated to the pET43. 1a vector with the same cohesive terminal (NdeI and XhoI) and expressed in the E. coli expression system. The morphology of the proteins of interest were observed by electron microscope and characterized by ELISA and Western Blotting.

RESULTSThe morphology of the virus-like particles were confirmed by electron microscope. H2 were solid particles with a diameter of (31.61 +/- 1.27) nm, while H3 were hollow particles with a diameter of (28.46 +/- 1.16) nm. Statistical analysis showed that H2 is larger than H3 in the diameter (P < 0.01). The antigenicity of the inserted epitopes and carrier protein were identified by ELISA and Western Blotting.

CONCLUSIONChimeric hepatitis B core particles presenting the preS1 neutralizing epitopes on their surface have been expressed, purified and identified, which lays the foundation for its application in vaccine research.

Epitopes ; chemistry ; genetics ; immunology ; Hepatitis B ; immunology ; virology ; Hepatitis B Core Antigens ; chemistry ; genetics ; immunology ; Hepatitis B Surface Antigens ; chemistry ; genetics ; immunology ; Hepatitis B virus ; chemistry ; genetics ; immunology ; Humans ; Neutralization Tests ; Protein Precursors ; chemistry ; genetics ; immunology ; Recombinant Fusion Proteins ; chemistry ; genetics ; immunology

OBJECTIVETo compare the number of harvested perisplenic hilar lymph nodes by laparoscopy-assisted total gastrectomy (LATG) and conventional open total gastrectomy (OTG) for advanced upper and middle gastric cancer.

METHODSThree hundred twelve patients with advanced gastric cancer treated in a single institution between Sept 2008 and Jan 2011 were included in this study. They were divided into two groups: the LATG group and OTG (D2) group. All the surgical operations were performed by one surgeon or under his supervision. The lymph node clearance outcomes of the patients treated by those two surgical procedures were analyzed.

RESULTSThe harvested lymph node numbers of the two groups were (29.57 ± 9.62) and (29.38 ± 11.22) respectively, statistically with no significant difference (P = 0.875). The numbers of lymph node dissected around the splenic area in the LATG group and OTG group (Section 10, 11 group) were (2.01 ± 1.34) and (1.33 ± 1.11), respectively, indicating a significant difference (P = 0.000). The numbers of lymph nodes dissected around the celiac region (Section 7, 8, 9, 11p and 12a(2) group) were (7.90 ± 3.41) and (7.22 ± 2.65), respectively, with a non-significant difference (P = 0.050). There were also no significant differences while comparing with the numbers of lymph nodes dissected in the cardiac area (group 1, 2), pyloric region (5, 6 group) and the greater and lesser omentum area (group 3 and 4) between the two groups (P = 0.605, P = 0.248, P = 0.262).

CONCLUSIONShort-term results of this study indicate that laparoscopy-assisted total gastrectomy (D2) is better than conventional open surgery in perisplenic hilar lymph node dissection.

Adult ; Aged ; Female ; Gastrectomy ; methods ; Humans ; Laparoscopy ; Lymph Node Excision ; methods ; Lymph Nodes ; pathology ; surgery ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Staging ; Spleen ; Stomach ; Stomach Neoplasms ; pathology ; surgery

OBJECTIVETo establish a TaqMan-based Real-time PCR assay with internal control for the detection of parvovirus B19 DNA in human serum.

METHODSTwo DNA fragments in length of 113 bp each were artificially synthesized, cloned into T vector and used as standard DNA or internal control, respectively. One pair of primers and two probes were included in the Real-time PCR. The probes were labeled with different fluoresceins and could bind B19 DNA or internal control, respectively. Precision and specificity of the method were evaluated. Specimens of human serum were examined by this assay to find B19 DNA.

RESULTSThe standard curve was constructed using the quantified standard B19 DNA. The Real-time PCR method was established. It was stable according to precision evaluation by the intra- and inter-assay and specific without any evident cross-reaction with human hepatitis B virus (HBV). Among 160 samples of human serum, B19 DNA was detected in 2 with a concentration of 2.1 x 10(5) Geq/ml and 3.6 x 10(3) Geq/ml, respectively.

CONCLUSIONThe Real-time PCR for B19 DNA detection was developed successfully, in which the internal control was helpful to exclude false-negative results.

DNA, Viral ; blood ; Humans ; Parvovirus B19, Human ; genetics ; Polymerase Chain Reaction ; methods

Adenovirus type 40 and 41 (Ad40, Ad41), which belong to human adenovirus subgroup F, are called fastidious adenoviruses due to their property of poor growth in cultured cell lines in vitro The effect of expression of exogenous E1B55K in Hep2 on Ad41 replication in this cell line was investigated. E1B55K gene was amplified by PCR with DNA extracted from Ad41-positive feces supernatant as template. Eukaryotic expression plasmid (pcDNA3) carrying E1B55K was constructed, purified, and transferred into Hep2 cell. Expression of E1B55K in G418-resistant clones was assayed by RT-PCR, and one clone named as Hep2-E1B4#4 could produce more Ad41 progenies when compared with other clones by the method of inducing complete cytopathic effect (CPE) in 293 cells. Infection of equivalent Ad41 caused more significant cytopathic effect (CPE) in Hep2-E1B#4 than that in the control cells of Hep2 or Hep2-DNA3, also suggesting enhanced viral replication in Hep2-E1B#4. The titer of Ad41 was further determined by method of immunocytochemical staining, and semi-quantity PCR was employed to compare the copy number of Ad41 genome DNA. The results showed that the yield of Ad41 in Hep2-E1B#4 was more than 9 times of that in control cells when equal amount of seed viruses were incubated, and the copy number of Ad41 genome increased 4 times in the raw extract from the infected Hep2-E1B#4 when compared with that from control cells. In conclusion, E1B55K gene transfer improved the ability of Hep2 in packaging Ad41, and the Hep2-E1B#4 cell line, which expressed E1B55K constitutively, would be helpful in isolation, cultivation and amplification of Ad41.
Adenovirus E1B Proteins ; genetics ; metabolism ; Adenoviruses, Human ; genetics ; Cell Line, Tumor ; Gene Expression ; Humans ; Immunohistochemistry ; Reverse Transcriptase Polymerase Chain Reaction ; Virus Replication ; genetics