Positive correlation between latent Epstein-Barr virus infection and severity of illness in inflammatory bowel disease patients.

BACKGROUND: Emerging studies indicate the critical involvement of microorganisms, such as Epstein-Barr virus (EBV), in the pathogenesis of inflammatory bowel disease (IBD). Immunosuppressive therapies for IBD can reactivate latent EBV, complicating the clinical course of IBD. Moreover, the clinical significance of EBV expression in B lymphocytes derived from IBD patients' intestinal tissues has not been explored in detail. AIM: To explore the clinical significance of latent EBV infection in IBD patients. METHODS: Latent EBV infection was determined by double staining for EBV encoded RNA and CD20 in colon specimens of 43 IBD patients who underwent bowel resection. Based on the staining results, the patients were divided into two groups, according to their latent EBV infection states - negative (n = 33) and positive (n = 10). Illness severity of IBD were assigned according to Crohn's disease activity index (ulcerative colitis) and Mayo staging system (Crohn's disease). The clinic-pathological data were analyzed between the two different latent EBV groups and also between the mild-to-moderate and severe disease groups. RESULTS: Systolic pressure (P = 0.005), variety of disease (P = 0.005), the severity of illness (P = 0.002), and pre-op corticosteroids (P = 0.025) were significantly different between the EBV-negative and EBV-positive groups. Systolic pressure (P = 0.001), variety of disease (P = 0.000), pre-op corticosteroids (P = 0.011) and EBV infection (P = 0.003) were significantly different between the mild-to-moderate and severe disease groups. CONCLUSION: IBD patients with latent EBV infection may manifest more severe illnesses. It is suggested that the role of EBV in IBD development should be further investigated, latent EBV infection in patients with serious IBD should be closely monitored, and therapeutic course should be optimized.

RNF43 Mutations in IPMN Cases: A Potential Prognostic Factor.

An intraductal papillary mucinous neoplasm (IPMN) is a common pancreatic precursor lesion, and it often harbors mutations in KRAS, GNAS, and RNF43. To clarify the molecular profiles of IPMNs, we conducted mutation analysis of KRAS, GNAS, and RNF43 in 61 IPMN formalin-fixed, paraffin-embedded (FFPE) specimens. The mutation rates of codons 12, 13, and 61 in KRAS and codon 201 in GNAS were detected by Sanger sequencing. Next-generation sequencing was performed on RNF43, and the results were further verified by Sanger sequencing. We identified KRAS and GNAS mutations in 35 (57%) and 40 (66%) IPMN cases, respectively. GNAS mutations were significantly correlated with the morphologic subtype (P < 0.001) and were more prevalent in the intestinal subtype (93%) than in the gastric (55%) and pancreatobiliary subtypes (44%) but were absent in the oncocytic subtype. RNF43 mutations were found in 5 cases (8%), all of which occurred in high-grade dysplasia and invasive lesions (2/5 and 3/5). All 5 cases harboring RNF43 mutations also exhibited GNAS mutations. RNF43 mutations were associated with a worse prognosis in invasive IPMN patients (P = 0.002), while KRAS and GNAS mutations did not affect the prognosis of patients.

HAp/Ti2Ni coatings of high bonding strength on Ti-6Al-4V prepared by the eutectic melting bonding method.

Eutectic melting bonding (EMB) method is a useful technique for fabricating bioactive coatings with relatively high crystallinity and bonding strength with substrate on titanium substrates. Using the EMB method, hydroxyapatite/Ti2Ni coatings were prepared on the surface of Ti-6Al-4V at a relatively low temperature (1,050 °C) in a vacuum furnace. The coatings were then characterized in terms of phase components, microstructure, bonding strength and cytotoxicity. The results showed that the coatings were mainly composed of HAp and Ti2Ni, and the thickness of the coatings was approximately 300 µm. X-ray diffraction analysis showed that the coatings exhibited relatively high crystallinity. The tensile bonding strength between the coatings and the substrates was 69.68±5.15 MPa. The coatings had a porous and rough surface which is suitable for cell attachment and filopodia growth. The cell culture study showed that the number of MG-63 cells increased, and the cell morphology changed with the incubation time. This study showed that the EMB method can be utilized as a potentially powerful method to obtain high quality hydroxyapatite coatings with desired mechanical and biocompatibility properties on Ti-alloy substrates.

Up-regulation of urotensin II and its receptor contributes to human hepatocellular carcinoma growth via activation of the PKC, ERK1/2, and p38 MAPK signaling pathways.

Urotensin II (UII) and its receptor (UTR) have mitogenic effects on tumor growth. Our previous study demonstrated that the UII/UTR system is up-regulated in dithyinitrosamine-induced precancerous rat liver lesions. However, its role in human hepatocellular carcinoma remains unknown. In this study, the mRNA and protein expression of UII and its receptor (UTR) in human hepatocellular carcinoma samples and in the BEL-7402 human hepatoma cell line were evaluated. In addition, the effect of exogenous UII on the pathways that regulate proliferation in BEL-7402 cells in vitro were determined. Liver sections were subjected to immunohistochemical staining. mRNA expression was detected by real-time polymerase chain reaction analysis, and protein levels were evaluated by western blotting. Proliferating cells were detected by BrdU incorporation. The expression of UII/UT mRNA and protein significantly increased in human hepatocellular carcinoma samples, and in BEL-7402 cells. Administration with UII increased the phosphorylation of protein kinase C (PKC), extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinases (p38 MAPK). Furthermore, GF109203x, PD184352, and SB203580 partially abolished UII-induced proliferation of BEL-7402 cells. These results provide the first evidence that up-regulation of the UII/UT system may enhance proliferation of the human hepatoma cell line at least in part via PKC, ERK1/2, and p38 MAPK signaling pathways, and may provide novel therapeutic targets for inhibiting human hepatocellular carcinoma.

Oleanolic acid and ursolic acid inhibit proliferation in transformed rat hepatic oval cells.

AIM: To investigate H2O2-induced promotion proliferation and malignant transformation in WB-F344 cells and anti-tumor effects of ursolic acid (UA) and oleanolic acid (OA). METHODS: WB-F344 cells were continuously exposed to 7 x 10(-7) mol/L H2O2 for 21 d. Observations of cell morphology, colony formation rates, flow cytometric analysis of cell cycle changes and aneuploidy formation indicated that H2O2 was able to induce malignant transformation of WB-F344 cells. We treated malignantly transformed WB-F344 cells with 4 µmol/L OA or 8 µmol/L UA for 72 h and analyzed the cell cycle distribution by flow cytometry. RESULTS: MTT assay showed that 7 x 10(-7) mol/L H2O2 decreased G1 phase subpopulation from 73.8% to 49.6% compared with the control group, and increased S phase subpopulation from 14.5% to 31.8% (P < 0.05 vs control group). Cell morphology showed that nucleus to cytoplasm ratio increased, many mitotic cells, prokaryotes and even tumor giant cells were shown in H2O2-induced WB-F344 cells. Fluorescence activated cell sorting analysis showed that WB-F344 cell aneuploidy increased to 12% following H2O2 treatment. Flow cytometric analysis of the transformed WB-F344 cells following treatment with OA (4 µmol/L) and UA (8 µmol/L) showed that OA increased G1 subpopulation to 68.6%, compared to 49.7% in unexposed cells. UA increased G1 subpopulation to 67.4% compared to 49.7% in unexposed cells (P < 0.05 vs H2O2 model group). CONCLUSION: H2O2 causes the malignant transformation of WB-F344 cells. OA and UA exert anti-tumor effects by inhibiting the proliferation in malignantly transformed WB-F344 cells.

Giant panda (Ailuropoda melanoleuca) spermatozoon decondensation in vitro is not compromised by cryopreservation.

Natural breeding of giant pandas in captivity is compromised, making artificial insemination and spermatozoa cryopreservation essential for genetic management. This study examined the influence of freeze-thawing on traditional parameters such as motility and spermatozoon functionality, specifically decondensation in vitro. Giant panda spermatozoa were assessed before and after rapid cryopreservation (4 degrees C to -130 degrees C over 2 min) in liquid nitrogen vapour. Spermatozoa pre-incubated in medium for 6 h were co-incubated with cat zonae (2 zonae microL(-1)) for 30 min to effect capacitation and an acrosome reaction. Spermatozoa were then mixed with mature cat oocyte cytoplasm (2 cytoplasm microL(-1)) for 4 h and evaluated for decondensation. Frozen spermatozoa were less motile (P < 0.05) than fresh counterparts immediately post-thawing, but not after 6 h incubation. There were more (P < 0.05) spermatozoa with completely diffused chromatin post-thaw (10.4 +/- 1.3%; mean +/- s.e.m.) compared to fresh counterparts (5.1 +/- 1.0%). However, there was no overall difference (P > 0.05) in the incidence of decondensation between fresh (4 h, 69.8 +/- 5.9%) and thawed (4 h, 71.5 +/- 4.9%) spermatozoa after exposure to cat oocyte cytoplasm. It is concluded that the 'rapid' method now used to cryopreserve giant panda spermatozoa has little impact on spermatozoon decondensation.

Flow cytometric cell-cycle analysis of cultured fibroblasts from the giant panda, Ailuropoda melanoleuca L.

In animal cloning, it is generally believed that the inactive diploid G(0)or G(1)stage of the cell cycle is beneficial to initiate cell-cycle coordination and reprogramming following transfer of the donor nucleus. Previous experiments have demonstrated that serum starvation results in quiescent cell stage. Some experiments show that the majority of cells in a fully confluent cell culture are also in an inactive G(1)stage. In order to provide more G(0)/G(1)stage cells for giant panda cloning, we carried out a flow cytometric analysis of the cell cycle of fibroblasts from the abdominal muscle of a giant panda at different passage numbers under different growth conditions, and after different periods of serum starvation. The percentage of G(0)+G(1)stage cells differed significantly under different growth conditions. Serum starvation effectively increased the percentage of G(0)+G(1)stage cells, and the cell cycle characteristics following serum starvation for varying periods of time differed with this and the initial confluency of the cultures. The data should help in choosing the optimal stage for preparing donor cells as well as increasing the potential cloning efficiency in our study of giant panda cloning.

Interspecies implantation and mitochondria fate of panda-rabbit cloned embryos.

Somatic cell nuclei of giant pandas can dedifferentiate in enucleated rabbit ooplasm, and the reconstructed eggs can develop to blastocysts. In order to observe whether these interspecies cloned embryos can implant in the uterus of an animal other than the panda, we transferred approximately 2300 panda-rabbit cloned embryos into 100 synchronized rabbit recipients, and none became pregnant. In another approach, we cotransferred both panda-rabbit and cat-rabbit interspecies cloned embryos into the oviducts of 21 cat recipients. Fourteen recipients exhibited estrus within 35 days; five recipients exhibited estrus 43-48 days after embryo transfer; and the other two recipients died of pneumonia, one of which was found to be pregnant with six early fetuses when an autopsy was performed. Microsatellite DNA analysis of these early fetuses confirmed that two were from giant panda-rabbit cloned embryos. The results demonstrated that panda-rabbit cloned embryos can implant in the uterus of a third species, the domestic cat. By using mitochondrial-specific probes of panda and rabbit, we found that mitochondria from both panda somatic cells and rabbit ooplasm coexisted in early blastocysts, but mitochondria from rabbit ooplasm decreased, and those from panda donor cells dominated in early fetuses after implantation. Our results reveal that mitochondria from donor cells may substitute those from recipient oocytes in postimplanted, interspecies cloned embryos.

[Interspecies nuclear transfer using nonquiescent somatic cell].

It is the point at issue in intraspecies nuclear transfer whether quiescence is necessary for development of nuclear transfer reconstructed embryos. In the interspecies nuclear transfer, some reports have proved that quiescent cell is able to support preimplantation development of the interspecies reconstructed embryos. Are non-quiescent cells able to support preimplantation development of the interspecies reconstructed embryos? We used non-quiescent somatic cells from C57BL/6 mice and giant pandas as donors to transfer into enucleated rabbit oocytes. After electrofusion (the electrofusion rates were 62.2% and 71.6%, respectively) and electrical activation, 5.1% of those mouse-rabbit reconstructed embryos developed to blastocyst in vitro, and 4.2% of panda-rabbit reconstructed embryos developed to blastocyst after transferring into ligated rabbit oviduct. These results indicate that non-quiescent cell from C57BL/6 mouse and giant panda could be dedifferentiated in enucleated rabbit oocytes and support early embryo development.