[Expression and significance of PD-1 and PD-L1 in the specimens of epithelial ovarian cancer].

Objective: To examine the expression of programmed cell death 1 (PD-1) and its ligand (PD-L1) in epithelial ovarian cancer (EOC) tissues, and investigate the correlation among their expression, clinicopathological features and prognosis. Methods: The specimens of 180 patients with EOC treated in the First Affiliated Hospital of Dalian Medical University from October 2002 to December 2013 were confirmed by pathological examination. The pathological tissue specimens of subtypes ，included 120 cases of serous carcinoma, 30 cases of mucinous carcinoma, 20 cases of endometrioid carcinoma, and 20 cases of clear cell carcinoma. The normal paracancerous tissues of 50 cases randomly selected from the 180 patients as control group. Immunohistochemical SP method was used to detect the expressions of both PD-1 and PD-L1 in epithelial ovarian cancer tissues, and the relationships among their expressions,the clinicopathological parameters and prognosis were respectively analyzed. Results: （1） PD-1 was expressed in lymphocytes infiltrated in EOC tissues, and PD-L1 was expressed in the cell membranes of cancer tissues. In all EOC cases, 33 cases (18.3%, 33/180) of both PD-1 and PD-L1 were highly expressed, and only 1 (2.0%, 1/50) of control group showed high expression. There was statistically significant difference between two groups (P<0.01). (2) Among the four subtypes tissue specimens of EOC, the high expression rate of PD-1 was 25.0% (30/120) for serous carcinoma, 3/15 for endometrioid carcinoma, 0 (0/30) for mucinous carcinoma, and 0 (0/15) for clear cell carcinoma. The high expression rate of PD-L1 was 23.3% (28/120) for serous carcinoma, 3.3% (1/30) for mucinous carcinoma, 2/15 for endometrioid carcinoma, and 2/15 for clear cell carcinoma. Both PD-1 and PD-L1 expressions in the four sub-types of tissue specimens were significantly different (P<0.05). The high expression rate of both PD-1 and PD-L1 was 9.2% (8/87) in the early stage and 26.9% (25/93) in the late stage. There was a statistically significant difference between the two groups (P<0.01). Similarly, the expression of both PD-1 and PD-L1 were significantly higher in the cases of high-grade EOC （type Ⅱ） than those of low-grade （type Ⅰ） and in the cases of EOC distributed bilaterally than that distributed unilaterally, and there were statistically significant differences (P<0.05). (3) The Kaplan-Meier survival analysis showed that the survival time were respectively 35 and 36 months in the cases with high expressions of both PD-1 and PD-L1, and the survival time were the same as 61 months in the cases with low expression of both PD-1 and PD-L1, and the comparison was statistically significant (P<0.05). Conclusions: The expression levels of PD-1 and PD-L1 in EOC tissues are higher than those in adjacent tissues, especially in serous carcinomas. The expression of both PD-1 and PD-L1 is higher in specimens of the patients with advanced stages. The results showed that the high expression of both PD-1 and PD-L1 is an indicator of poor prognosis of patients suffering from EOC.

[Expression and significance of MAPK/ERK in the specimens and cells of epithelial ovarian cancer].

Objective: To detect phosphorylated-extracellular signal-regulated kinase (p-ERK1/2) protein expression in epithelial ovarian cancer and cell lines, and to examine the effects of mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor AZD6244 on cell proliferation, apoptosis as well as cell cycle of ovarian cancer cells. To explore the function and significance of MAPK/extracellular signal-regulated kinase (ERK) signaling pathway in the development of ovarian cancer. Methods: (1) A total of 104 cases of patients with ovarian cancer who accepted the treatment of gynecological surgery and being confirmed by pathological examination in First Affiliated Hospital, Dalian Medical University from January 2004 to December 2013 were selected. The expressions of p-ERK1/2 protein were detected by immunohistochemistry in ovarian cancer specimens, and the relationship between the expressions of p-ERK1/2 and the clinical features of patients was analyzed. (2) p-ERK1/2 and other related proteins were determined by western blot in various ovarian cancer cells, including SKOV3, OV2008, C13, A2780S, A2780CP, OVCAR4, OVCAR5, OVCAR8 and CAOV3 treated with or without MEK inhibitor. The cellular proliferation, apoptosis and cell cycle of ovarian cancer cells after treatment with MEK inhibitor were analyzed by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry, respectively. Results: (1) The immunohistochemical method showed that p-ERK1/2 between low grade serous carcinoma and clear cell carcinoma were not significantly higher expressed (P>0.05) . However, a lower level of the p-ERK1/2 expression were observed among high grade serous carcinoma, mucinous carcinoma and endometrioid carcinoma (all P<0.05) . There was no significant correlation between the protein expression of p-ERK1/2 and patients' age, pathological stage of surgery, and preoperative serum CA(125) level (P>0.05). (2) Western blot showed that the protein p-ERK1/2 was widely expressed in various ovarian cancer cell lines such as SKOV3, OV2008, C13, A2780S, A2780CP, OVCAR4, OVCAR5, OVCAR8 and CAOV3. After treatment with AZD6244 (5, 10 µmol/L), the level of p-ERK1/2 in OVCAR5 and OVCAR8 decreased significantly in dose-dependent manner. Additionally, we found a reduction of the expression level of cyclin D1, caspase-3 and appeared cleaved poly adenosine diphosphate ribose polymerase (PARP) in OVCAR5 and OVCAR8, compared with control groups. MTT assays showed that OVCAR5, OVCAR8 and A2780S were differently inhibited in the dose-dependent manner after being treated with different concentrations of AZD6244 (0, 2.5, 5, 10, 25, 50 and 100 µmol/L, all P<0.05). Further tested by flow cytometry, the results showed that AZD6244 (5, 10 µmol/L) was able to induce the apoptosis of OVCAR5, OVCAR8 and A2780S, as well as G(0)/G(1) phase arrest, both in a dose-dependent manner (P<0.05). Conclusions: As the main active and functional unit of MAPK/ERK signaling pathway, p-ERK1/2 protein is expressed in both the tissues and various ovarian cancer cell lines. AZD6244 could down-regulated the expression of p-ERK1/2 in ovarian cancer cells, accompanied by the decreased proliferation and increased cell apoptosis of ovarian cancer cells. In conclusion, MAPK/ERK signaling pathway might play a role in the development and progression of ovarian cancer, and may be provide a novel option for molecular targeted therapies of the disease.

[Expression and significance of IKKε in the specimens and cells of epithelial ovarian cancer].

Objective: To examine the expressions of IKKε protein in the specimens and cells of epithelial ovarian cancer and investigate the effect of IKKε inhibitor on cell proliferation and apoptosis. Methods: (1) A total of 118 cases of patients with the median age of 59 who have accepted surgical treatment due to ovarian cancer in the First Affiliated Hospital of Dalian Medical University from January 2006 to April 2013 were selected. Twenty cases of patients with the median age of 55 who have accepted hysterectomy and salpingo-oophorectomy due to uterine leiomyoma during the same period were selected as the control. The expressions of IKKε protein were detected by immunohistochemistry in normal ovarian tissues and epithelial ovarian cancer specimens, and the relationship between the expressions of IKKε and the clinical features of patients was analyzed. IKKε protein was determined by western blot in various ovarian cancer cells, including SKOV3, OV2008, C13, A2780S, A2780CP, OV4, OV5, OV8, and CAOV3 treated with or without IKKε inhibitor. The cellular proliferation and apoptosis of ovarian cancer cells after 48 hours treatment of IKKε inhibitor were analyzed by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry, respectively. Results: (1) The immunohistochemical results showed that IKKε was highly expressed in epithelial ovarian cancer specimens with the expression rate 66.1% (78/118), compared with normal ovarian tissue with the expression rate 35.0% (7/20), which exhibited statistically significant difference (χ(2)=6.993, P=0.008). The expression of IKKε protein was correlated with International Federation of Gynecology and Obstetrics (FIGO) stage, histological grade, the level of CA(125) in preoperative serum and distribution of the tumor (P<0.05), but no correlation with age, histological type, the incidence pattern, and tumor size (all P>0.05). (2) IKKε was widely overexpressed in different levels in SKOV3, OV2008, C13, A2780S, A2780CP, OV4, OV5, OV8, and CAOV3 cells, and the expression of IKKε decreased as the increase of the concentration of IKKε inhibitor (0.1 and 0.5 µmol/L) in OV2008, C13, A2780S, and A2780CP cells after 48 hours treatment. Different concentrations of IKKε inhibitor (0.05, 0.1, 0.5, 1, 5, 10, and 25 µmol/L) significantly inhibited the proliferation of OV2008, C13, A2780S, A2780CP, and SKOV3 cells in a concentration-dependent manner (P<0.05), and the half maximal inhibitory concentration (IC(50)) was 0.43, 0.86, 0.10, 0.19, and 0.24 µmol/L, respectively. The cell apoptotic rate of OV2008, C13, A2780S, A2780CP, and SKOV3 cells was significantly increased after 48 hours treatment of IKKε inhibitor with the concentration of 0.1 and 0.5 µmol/L (P<0.05). Conclusions: The IKKε protein in epithelial ovarian cancer specimens and cells is overexpressed. IKKε inhibitor could inhibit cellular proliferation and induce apoptosis in a concentration-dependent manner. Together, the result indicated that IKKε may be a candidate target for the treatment of ovarian cancer in future.

[Intestinal barrier dysfunction and its related factors in patients with sepsis].

Objective: To investigate the relationship between related factors of intestinal barrier dysfunction in patients with sepsis or septic shock and severity of the condition. Methods: A prospective observational study was conducted in 31 sepsis patients, 28 septic shock patients, and 21 postoperative patients without sepsis (control group) who were admitted to intensive care unit (ICU) of General Hospital of Ningxia Medical University between November 2015 and June 2016. Blood samples were collected from the patients within 24 hours following admission to ICU. D-lactic acid and endotoxin levels were measured by enzymatic method, serum high-sensitivity C-reactive protein (hsCRP) level by immune scatter turbidimetry. An arterial blood gas (ABG) measurement was carried out every 8 hours within the first 24 hours after admission to ICU, and average arterial blood lactate levels were calculated. Acute Physiology and Chronic Health Evaluation Ⅱ (APACHE Ⅱ) score, Sequential Organ Failure Assessment (SOFA) score of the patients within 24 hours following ICU admission were recorded. The patients with sepsis or septic shock were followed up for 28 days after admission to ICU, and divided into survival group (n=44) and death group (n=15). The patients with sepsis or septic shock were divided into two groups according to the site of infection, i. e. intra-abdominal infection group (n=37) and extra-abdominal infection group (n=22). Results: (1) In the control, sepsis, and septic shock groups, D-lactic acid [mg/L, M(P25, P75) ] were 11.68(7.49, 14.92), 19.78 (12.25, 34.85), and 32.45 (16.03, 46.95), respectively; endotoxin levels [U/L, M(P25, P75)] were 10.60(7.59, 13.39), 16.12(10.09, 20.23), and 17.31(14.09, 23.77), respectively. The levels of serum D-lactic acid and endotoxin in the patients with sepsis or septic shock were significantly higher than those in the control group (all P<0.01); while no statistically significant differences existed in these two indices between the sepsis and septic shock groups (both P>0.05). There were no statistically significant differences in serum D-lactic acid and endotoxin levels between the intra-abdominal infection group and the extra-abdominal infection group [20.07(14.70, 38.97)vs 21.65 (14.53, 56.56)mg/L; 17.23(13.38, 20.85)vs 17.17(9.93, 20.81)U/L; both P>0.05]. There were no statistically significant differences in levels of serum D-lactic acid and endotoxin between the survival group and the death group [21.65(15.11, 39.00) vs 19.78(14.41, 80.93)mg/L; 17.09(12.62, 20.42) vs 19.26(13.22, 26.27)U/L, both P>0.05]. (2) In the sepsis and septic shock patients, serum D-lactate level was significantly related to mean arterial blood lactate concentration, APACHE Ⅱ score, and SOFA score in the first 24 hours after admission to ICU (r=0.499, 0.447, 0.469, all P<0.01); serum endotoxin level was correlated with hsCRP, APACHE Ⅱ score, and SOFA score (r=0.224, 0.388, 0.393, all P<0.05). (3) Multivariate linear regression analysis showed that D-lactic acid level was independently associated with average arterial blood lactate concentration as well as with SOFA score(R2=0.34, F=19.91, P<0.01), and endotoxin was independently associated with only SOFA score(R2=0.14, F=12.68, P<0.01). Conclusions: Regardless of the site of infection, patients with sepsis or septic shock often have intestinal barrier injury, which is correlated with the severity of disease, but does not independently affect patient outcome. Tissue hypoperfusion in the early stage of sepsis may be one of the causes of intestinal barrier injury.

Early third trimester maternal response to glucose challenge and pregnancy outcome in Chinese women-relationship between upper distribution level and recommended diagnostic criteria.

BACKGROUND/OBJECTIVES: The objective of this study was to examine the relationship between upper distribution levels of glucose values in the 75-g oral glucose tolerance test (OGTT) and recommended diagnostic criteria for gestational diabetes mellitus (GDM) and adverse pregnancy outcomes. SUBJECTS/METHODS: The distribution of the OGTT 2-h values of 13,501 pregnant women, which were below the World Health Organization (WHO) threshold for overt diabetes mellitus (DM), and managed in one teaching hospital in China, was reviewed and related to maternal characteristics and pregnancy outcomes. RESULTS: For the entire group, the 90th and 95th percentile values of the OGTT 2-h glucose level, respectively, were close to the diagnostic cutoff values of the WHO and International Association of Diabetes and Pregnancy Study Groups (IADPSG) criteria. For adverse maternal outcomes, glucose level above the 90th percentile value was associated with increased hypertensive disorders, whereas no difference was seen with cutoff using the 95th percentile value. For perinatal outcomes, the 90th percentile was associated with increased neonatal intensive care unit admission and hypoglycemia, whereas the 95th percentile showed in addition association with phototherapy for jaundice and 5th-minute Apgar score <7. Although no differences in the incidence of adverse pregnancy outcomes were found using the different cutoffs, the >95th percentile cutoff value would have missed out 33.3-56.7% of the cases of adverse outcomes that would otherwise have been attributed to GDM. CONCLUSIONS: Further studies are warranted to clarify which diagnostic criterion is most appropriate universally to identify adverse pregnancy outcomes attributed to GDM, and which could be mitigated with treatment specific for GDM.

The influence of aquaporin-1 and microvessel density on ovarian carcinogenesis and ascites formation.

This study aimed at investigating aquaporin-1 (AQP1) distribution and expression in primary ovarian epithelial tumors, correlating with clinicopathologic variables and intratumoral microvessel density (IMD). The AQP1 expression and IMD in 105 cases with primary epithelial ovarian tumors were measured by semiquantitative immunohistochemical technique. AQP1 was located mainly in microvessels and small vessels but seldom in tumor cells. Expression of AQP1 and IMD in ovarian malignant tumors was significantly higher than that in borderline tumors (P= 0.000, P= 0.001, respectively), and that in borderline tumors was higher than in benign tumors (P= 0.008, P= 0.028, respectively). The expression of AQP1 in FIGO stage III-IV was more than that in stage I-II (P= 0.001), and cases with ascites volume greater than 1000 mL were more than cases with ascites volume less than 1000 mL (P= 0.000). There is a positively correlated relationship between expression of AQP1 and IMD (correlation coefficient 0.60, P= 0.000) and between expression of AQP1 and ascites volume (correlation coefficient 0.57, P= 0.000). These data implicate that high AQP1 expression may play an important role in the ovarian carcinogenesis, progression, and ascites formation. Further studies into the mechanism of AQP1 regulation and the relationship between AQP1 expression and tumor angiogenesis may lead to novel therapies for ovarian carcinoma.