Independent risk factors for true malignancy in atypical cytologic diagnostic category in EUS-FNA/FNB of the pancreas: A novel prediction model.

Background and Objects: An atypical cytologic diagnosis arises from inflammation or early neoplastic process. It is commonly found in EUS-guided fine-needle aspiration/biopsy (EUS-FNA/FNB) tissue sampling of pancreatic malignancies. The aims of this study were to evaluate the diagnostic performance of EUS-FNA/FNB in patients with cytologic diagnosis of atypical cells and to develop a prediction model for malignant tumors of the pancreas in the atypical cytologic diagnostic category. Methods: Two hundred and twenty-six patients in the atypical cytologic diagnostic category were analyzed. Multivariate logistic regression analyses were performed to determine predictive factors for pancreatic malignancies. The final diagnoses were confirmed by repeat biopsy; surgical pathology, or clinical follow-up for at least 6 months. Results: The atypical cytologic diagnosis using EUS-FNA/FNB was associated with an absolute risk of malignancy (82.3%). Multivariate logistic regression analyses revealed that older age, long axis of the mass, and increased carbohydrate antigen 19-9 (CA19-9) were independent risk factors for true malignant pancreatic tumors among patients in the atypical cytologic diagnostic category. The calibration curve had a slope of 0.96, and a regression coefficient (R2) of 0.91. The area under the receiver operating characteristic curve of the validation group was 0.803. Conclusions: Atypical lesions of EUS-FNA/FNB have a higher risk of malignancy. Older age, the long axis of the mass, and elevated serum CA19-9 level were identified as independent risk factors for true malignant pancreatic tumors among patients in the atypical cytologic diagnostic category.

Endogenous neurotrophin-3 promotes neuronal sprouting from dorsal root ganglia.

In the present study, we investigated the role of endogenous neurotrophin-3 in nerve terminal sprouting 2 months after spinal cord dorsal root rhizotomy. The left L1-5 and L7-S2 dorsal root ganglia in adult cats were exposed and removed, preserving the L6 dorsal root ganglia. Neurotrophin-3 was mainly expressed in large neurons in the dorsal root ganglia and in some neurons in spinal lamina II. Two months after rhizotomy, the number of neurotrophin-3-positive neurons in the spared dorsal root ganglia and the density of neurite sprouts emerging from these ganglia were increased. Intraperitoneal injection of an antibody against neurotrophin-3 decreased the density of neurite sprouts. These findings suggest that endogenous neurotrophin-3 is involved in spinal cord plasticity and regeneration, and that it promotes axonal sprouting from the dorsal root ganglia after spinal cord dorsal root rhizotomy.

Effect of glycyrrhizin on traumatic brain injury in rats and its mechanism.

OBJECTIVE: To investigate the neuroprotective effects of glycyrrhizin (Gly) as well as its effect on expression of high-mobility group box 1 (HMGB1) in rats after traumatic brain injury (TBI). METHODS: Male Sprague-Dawley rats were randomly divided into three groups: sham group, TBI group, and TBI+Gly group (n=36 per group). Rat TBI model was made by using the modified Feeney's method. In TBI+Gly group, Gly was administered intravenously at a dosage of 10 mg/kg 30 min after TBI. At 24 h after TBI, motor function and brain water content were evaluated. Meanwhile, HMGB1/HMGB1 receptors including toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE)/nuclear factor-κB(NF-κB) signaling pathway and inflammatory cytokines in the injured brain tissues were detected using quantitative real-time polymerase chain reaction, western blot, electrophoretic mobility shift assay and enzyme-linked immunosorbent assay. Furthermore, HMGB1, RAGE and TLR4 immunohistochemistry and apoptosis were analyzed. RESULTS: Beam walking performance impairment and brain edema were significantly reduced in TBI+Gly group compared with TBI group; meanwhile, the over-expressions of HMGB1/HMGB1 receptors (TLR4 and RAGE)/NF-κB DNA-binding activity and inflammatory cytokines were inhibited. The percentages of HMGB1, RAGE and TLR4-positive cells and apoptotic cells were respectively 58.37% ± 5.06%, 54.15% ± 4.65%, 65.50% ± 4.83%, 52.02% ± 4.63% in TBI group and 39.99% ± 4.99%, 34.87% ± 5.02%, 43.33% ± 4.54%, 37.84% ± 5.16% in TBI+Gly group (all P<0.01 compared with TBI group). CONCLUSION: Gly can reduce secondary brain injury and improve outcomes in rat following TBI by down-regulation of HMGB1/HMGB1 receptors (TLR4 and RAGE)/NF-κB-mediated inflammatory responses in the injured rat brain.

Anisomycin induces glioma cell death via down-regulation of PP2A catalytic subunit in vitro.

AIM: To examine the effects of anisomycin on glioma cells and the related mechanisms in vitro. METHODS: The U251 and U87 human glioblastoma cell lines were tested. The growth of the cells was analyzed using a CCK-8 cell viability assay. Apoptosis was detected using a flow cytometry assay. The expression of proteins and phosphorylated kinases was detected using Western blotting. RESULTS: Treatment of U251 and U87 cells with anisomycin (0.01-8 µmol/L) inhibited the cell growth in time- and concentration-dependent manners (the IC(50) values at 48 h were 0.233±0.021 and 0.192±0.018 µmol/L, respectively). Anisomycin (4 µmol/L) caused 21.5%±2.2% and 25.3%±3.1% of apoptosis proportion, respectively, in U251 and U87 cells. In the two cell lines, anisomycin (4 µmol/L) activated p38 MAPK and JNK, and inactivated ERK1/2. However, neither the p38 MAPK inhibitor SB203580 (10 µmol/L) nor the JNK inhibitor SP600125 (10 µmol/L) prevented anisomycin-induced cell death. On the other hand, anisomycin (4 µmol/L) reduced the level of PP2A/C subunit (catalytic subunit) in a time-dependent manner in the two cell lines. Treatment of the two cell lines with the PP2A inhibitor okadaic acid (100 nmol/L) caused marked cell death. CONCLUSION: Anisomycin induces glioma cell death via down-regulation of PP2A catalytic subunit. The regulation of PP2A/C exression by anisomycin provides a clue to further study on its role in glioma therapy.

Pharmacokinetics and the bystander effect in CD::UPRT/5-FC bi-gene therapy of glioma.

BACKGROUND: Cytosine deaminase (CD) converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) in CD/5-FC gene therapy, 5-FU will be mostly converted into nontoxic beta-alanine without uracil phosphoribosyltransferase (UPRT). UPRT catalyzes the conversion of 5-FU to 5-fluorouridine monophosphate, which directly kills CD::UPRT-expressing cells and surrounding cells via the bystander effect. But the pharmacokinetics and the bystander effect of CD::UPRT/5-FC has not been verified in vivo and in vitro. Before the CD::UPRT/5-FC bi-gene therapy system is used in clinical trial, it is essential to monitor the transgene expression and function in vivo. Thus, we developed a preclinical tumor model to investigate the feasibility of using (19)F-magnetic resonance spectroscopy ((19)F-MRS) and optical imaging to measure non-invasive CD and UPRT expression and its bystander effect. METHODS: C6 and C6-CD::UPRT cells were cultured with 5-FC. The medium, cells and their mixture were analyzed by (19)F-MRS. Rats with intracranial xenografted encephalic C6-CD::UPRT glioma were injected intraperitoneally with 5-FC and their (19)F-MRS spectra recorded. Then the pharmacokinetics of 5-FC was proved. Mixtures of C6 and C6-CD::UPRT cells at different ratios were cultured with 5-FC and the cytotoxic efficacy and survival rate of cells recorded. To determine the mechanism of the bystander effect, the culture media from cell comprising 25% and 75% C6-CD::UPRT cells were examined by (19)F-MRS. A comparative study of mean was performed using analysis of variance (ANOVA). RESULTS: (19)F-MRS on samples from C6-CD::UPRT cells cultured with 5-FC showed three broad resonance signals corresponding to 5-FC, 5-FU and fluorinated nucleotides (F-Nuctd). For the C6 mixture, only the 5-FC peak was detected. In vivo serial (19)F-MRS spectra showed a strong 5-FC peak and a weak 5-FU peak at 20 minutes after 5-FC injection. The 5-FU concentration reached a maximum at about 50 minutes. The F-Nuctd signal appeared after about 1 hour, reached a maximum at around 160 minutes, and was detectable for several hours. At a 10% ratio of C6-CD::UPRT cells, the survival rate was (79.55 +/- 0.88)% (P < 0.01). As the C6-CD::UPRT ratio increased, the survival rate of the cells decreased. (19)F-MRS showed that the signals for 5-FU and F-Nuctd in the culture medium increased as the ratio of C6-CD::UPRT in the mixture increased. CONCLUSIONS: (19)F-MRS studies indicated that C6-CD::UPRT cells could effectively express CD and UPRT enzymes. The CD::UPRT/5-FC system showed an obvious bystander effect. This study demonstrated that CD::UPRT/5-FC gene therapy is suitable for 5-FC to F-Nuctd metabolism; and (19)F-MRS can monitor transferred CD::UPRT gene expression and catalysis of substrates noninvasively, dynamically and quantitatively.