[Preclinical study of T cell receptor specifically reactive with KRAS G12V mutation in the treatment of malignant tumors].

OBJECTIVE: KRAS gene is one of the most common mutations of proto-oncogenes in human tumors, G12V is one of the most common mutation types for KRAS. It's challenging to chemically acquire the targeted drug for this mutation. Recent studies reported that this mutation peptides can form a neoepitope for T cell recognition. Our study aims to clone the T cell receptor (TCR) which specifically recognizes the neoepitope for KRAS G12V mutation and constructs TCR engineered T cells (TCR-T), and to investigate if TCR-Ts have strong antitumor response ability. METHODS: In this study, tumor infiltrating lymphocytes were obtained from one colorectal cancer patient carrying KRAS G12V mutation. Tumor-reactive TCR was obtained by single-cell RT-5' rapid-amplification of cDNA ends PCR analysis and introduced into peripheral blood lymphocytes to generate TCR-Ts. RESULTS: We obtained a high-affinity TCR sequence that specifically recognized the HLA-A*11:01-restricted KRAS G12V8-16 epitope: KVA11-01. KVA11-01 TCR-T could significantly kill various tumor cells such as PANC-1, SW480 and HeLa (overexpressing HLA-A*11:01 and KRAS G12V), and secreting high levels of interferon-γ (IFN-γ). Non-specific killing experiments suggested KVA11-01 specifically recognized tumor cells expressing both mutant KRAS G12V and HLA-A*11:01. In vivo assay, tumor inhibition experiments demonstrated that infusion of approximately 1E7 KVA11-01 TCR-T could significantly inhibit the growth of subcuta-neously transplanted tumors of PANC-1 and HeLa (overexpressing HLA-A*11:01 and KRAS G12V) cells in nude mice. No destruction of the morphologies of the liver, spleen and brain were observed. We also found that KVA11-01 TCR-T could significantly infiltrate into tumor tissue and had a better homing ability. CONCLUSION: KVA11-01 TCR-T cells can effectively target a variety of malignant tumor cells carrying KRAS G12V mutation through in vitro and in vivo assay. KVA11-01 TCR-T cells have excellent biological activity, high specificity of target antigen and homing ability into solid tumor tissue. KVA11-01 TCR-T is expected to be an effective treatment for patients with KRAS G12V mutant solid malignancies.

[Effects of tumor protein translation control antisense RNA1 on radiosensitivity, proliferation, migration and invasion of hepatocellular carcinoma cells by targeting miR-30c-5p].

Objective: To investigate the effects of tumor protein translation control antisense RNA1 (TPT1-AS1) on the radiosensitivity, cell proliferation, migration and invasion of hepatocellular carcinoma cells by targeting microRNA-30c-5p (miR-30c-5p). Methods: Thirty-four cases of liver cancer tissues and adjacent normal tissues were derived from liver cancer patients who were admitted to Shanxi Provincial People's Hospital from March 2016 to March 2018. Liver cancer HepG2 cell was transfected with negative control siRNA (si-NC group), si-TPT1-AS1 (si-TPT1-AS1 group), pcDNA3.1 (pcDNA3.1 group), pcDNA3.1-TPT1-AS1 (pcDNA3.1-TPT1-AS1 group), si-TPT1-AS1 and anti-miR-NC (si-TPT1-AS1+ anti-miR-NC group), si-TPT1-AS1 and anti-miR-30c-5p (si-TPT1-AS1+ anti-miR-30c-5p group), respectively. Real-time quantitative reverse transcription polymerase chain reaction (qPCR) was used to detect the transcription levels of TPT1-AS1 and miR-30c-5p in normal tissues adjacent to cancer and liver cancer tissues, the clone formation test was used to test the radiosensitivity of HepG2 cells, and the Methyl Thiazolyl Tetrazolium (MTT) test was used to test the proliferation of HepG2 cells. Cell cycle distribution was detected by flow cytometry, Transwell array was used to detect the migration and invasion ability of HepG2 cells, dual luciferase reporter array was used to verify the targeting relationship of TPT1-AS1 and miR-30c-5p, western blot was used to detect the expressions of proliferation, migration and invasion-related proteins. Results: The expression levels of TPT1-AS1 and miR-30c-5p in liver cancer tissues were 0.84±0.08 and 0.13±0.01, statistically different from 0.31±0.03 and 0.50±0.05 in normal tissues adjacent to cancer (P<0.05). When the cells were treated with 2, 4, 6, 8 Gy irradiation, the cell survival scores of the si-TPT1-AS1 group were 0.280±0.040, 0.069±0.011, 0.020±0.003 and 0.005±0.001, respectively, lower than 0.648±0.070, 0.348±0.080, 0.130±0.020 and 0.060±0.009 of the si-NC group (P<0.05), the radiosensitization ratio of the si-TPT1-AS1 group was 1.672. The number of cell migration and invasion in the si-TPT1-AS1 group were (50.00±4.36) and (44.00±4.03), respectively, which were lower than (109.00±8.68) and (94.00±7.49) in the si-NC group (P<0.05), the cell absorbance (A) values at 24, 48 and 72 hours were 0.28±0.03, 0.43±0.04 and 0.68±0.07, respectively, lower than 0.46±0.04, 0.87±0.08 and 1.35±0.13 of the si-NC group (P<0.05), the protein expression levels of Cyclin D1, p21, E-cadherin and MMP-2 were 0.25±0.02, 0.65±0.06, 0.68±0.07 and 0.27±0.03, respectively, statistically different from 0.88±0.08, 0.17±0.02, 0.14±0.01 and 0.89±0.09 of si-NC group (P<0.05), the proportions of S phase and G(2) phase in the si-TPT1-AS1 group were (17.82±1.03)% and (34.15±2.29)%, respectively, significantly different from (35.14±2.61)% and (16.84±1.21)% in the si-NC group (P<0.05). The luciferase activity of cells in the WT-TPT1-AS1+ miR-30c-5p group was 0.26±0.02, lower than 0.92±0.09 in the WT-TPT1-AS1+ miR-NC group (P<0.05). The cell survival scores in the si-TPT1-AS1+ anti-miR-30c-5p group were 0.450±0.081, 0.200+ 0.045, 0.070±0.010, 0.026±0.004 after treatment with 2, 4, 6, 8 Gy irradiation, higher than 0.285±0.043, 0.075±0.014, 0.028±0.004, 0.006±0.001 of si-TPT1-AS1+ anti-miR-NC group (P<0.05). The radiosensitization ratio of the si-TPT1-AS1+ anti-miR-30c-5p group was 0.694. The number of migration and invasion in the si-TPT1-AS1+ anti-miR-30c-5p group were 79.00±6.65 and 68.00±6.33, higher than (52.00±4.41) and (46.00±4.06) of si-TPT1-AS1+ anti-miR-NC Group (P<0.05), the A values at 24, 48 and 72 hours were 0.37±0.03, 0.64±0.06 and 0.96±0.09, respectively, higher than 0.26±0.03, 0.41±0.04, and 0.65±0.06 of si-TPT1-AS1+ anti-miR-NC group (P<0.05), the expression levels of Cyclin D1, p21, E-cadherin and MMP-2 protein were 0.57±0.06, 0.43±0.04, 0.43±0.04 and 0.64±0.06, statistically different from 0.24±0.02, 0.66±0.06, 0.65±0.06 and 0.28±0.03 of the si-TPT1-AS1+ anti-miR-NC group (P<0.05). Conclusions: The expression of TPT1-AS1 up-regulates in the liver cancer tissues. TPT1-AS1 may down-regulate miR-30c-5p expression, reduce the radiosensitivity of liver cancer cells, and promote the proliferation, migration and invasion of liver cancer cells.

[Clinical characteristics and prognostic analysis of double primary breast cancer and thyroid cancer].

Objective: To explore and describe clinicopathological characteristics and prognosis of patients with double primary breast cancer (BC) and thyroid cancer (TC). Methods: Medical records of 98 patients diagnosed with double primary breast and thyroid cancer in National Cancer Center (NCC)/Cancer Hospital between January 1, 2001 and December 31, 2020 were retrospectively collected. All of the patients were followed up until January 1, 2021 to acquire survival data. Univariate survival analysis was conducted by Kaplan-Meier method, and multivariate survival analysis was carried out using the Cox proportional hazard model. Results: All of 98 patients in the group were women. The age at diagnosis of the first tumor ranged from 26-72 years old, and the median age was 47 years old. The BC recurring TC (breast methyl) group included 18 cases, TC recurring BC (methyl breast) group included 60 cases, BC and TC simultaneously occurred group (the two are diagnosed within 3 months) included 20 cases. There were statistically significant differences in breast cancer pathological grading, breast cancer postoperative radiotherapy, and combined with other tumors in breast methyl group, methyl breast group and the simultaneous group (P<0.05). Among the 98 patients, 14 had recurrence and metastasis, and 7 died. The patients who died from tumors were all those with TC recurrence of BC. There were no statistically significant differences in the death, recurrence and metastasis of patients in the breast methyl group, methyl breast group and the simultaneous group (P>0.05). Univariate analysis showed that BC stage and estrogen receptor (ER) were related to overall survival (P<0.05), while the family history of BC, BC stage, and ER were not related with the recurrence and metastasis (P<0.05). Multivariate analysis showed that BC family history, ER positive, and the order of tumor diagnosis (TC recurring BC) were independent influencing factors for the recurrence and metastasis (P<0.05). Conclusion: ER negative is a poor prognostic factor for the double primary breast and thyroid cancer.

[Exploration of the high-risk factors of catheter-related thrombosis in breast cancer].

Objective: To explore the high risk factors of catheter-related thrombosis (CRT) in breast cancer patients, and provide the basis for the development of appropriate prevention and treatment strategies. Methods: A total of 1 432 breast cancer patients scheduled to receive central venous catheterization in National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College from January 1, 2015 to August 31, 2019 were selected. Baseline information and catheterization information of patients were collected. The occurrence of CRT was confirmed by vascular ultrasound examination, and the influencing factors of CRT were analyzed. Results: The total number of catheter days were 121, 980 days in 1 432 patients with breast cancer, and the average number of catheter days in each patient was 85.2 days. The incidence of CRT was 6.8% (97/1 432), which was 0.79 cases/1 000 catheter days. Among 815 patients with centrally inserted central venous catheters (CICC), 43 (5.3%) had CRT, which was 0.70 cases/1 000 catheter days. Among 617 patients with peripherally inserted central venous catheters (PICC), 54 (8.8%) developed CRT, which was 0.90 cases/1 000 catheter days. CRT was most common in subclavian vein (63.9%). Multivariate regression analysis showed that age ≥ 60 years old (OR=1.712, 95% CI: 1.056-2.775, P=0.029), PICC (OR=1.732, 95% CI: 1.130-2.656, P=0.012), the catheter position except subclavian vein (OR=10.420, 95% CI: 1.207-89.991), secondary adjustment of catheter position (OR=3.985, 95% CI: 1.510-10.521, P=0.005) and high D-Dimer level (OR=1.129, 95% CI: 1.026-1.241, P=0.012)were independent risk factors for CRT. Conclusions: The CRT problem can't be ignored in the clinical treatment of breast cancer patients with central venous catheterization. Screening the appropriate age of patients and the type of central venous catheters, reducing the secondary adjustment of catheter position, and timely monitoring the level of D-dimer are helpful to the prevention and treatment of CRT.

Rationale for the advancement of PI3K pathway inhibitors for personalized chordoma therapy.

PURPOSE: Chordomas are rare and serious tumors with few effective treatments outside of aggressive surgery and radiation. Targeted therapies may present a more effective option for a subset of patients with lesions possessing certain genetic biomarkers. METHODS: A small molecule inhibitor library was tested in patient-derived UM-Chor1 cells to identify targeted therapies with potential efficacy. Targeted exome sequencing of UM-Chor1 and UM-Chor2 cells was performed to investigate genetic aberrations in relevant pathways. Chordoma cell lines were treated with inhibitors of the phosphotidylinositol 3-kinase (PI3K), epidermal growth factor receptor (EGFR), and cyclin dependent kinase (CDK) pathways, and responses were determined using resazurin cell viability assays, Annexin V apoptosis assays, and western blotting. Pan-PI3K inhibitor BKM120 was also tested in five chordoma xenograft models. RESULTS: Unbiased small molecule profiling nominated PI3K-AKT-mTOR pathway inhibitors as a promising therapy in chordoma, and genetic analyses of UM-Chor1 and UM-Chor2 cell lines revealed aberrations in PTEN, EGFR, and CDKN2A. Treatment of UM-Chor1 and UM-Chor2 with targeted PI3K, EGFR, and CDK inhibitors inhibited growth and proliferation and induced apoptosis more robustly than imatinib, a currently used chordoma therapy. Furthermore, BKM120 significantly inhibited tumor growth in a subset of the xenograft models tested. CONCLUSION: Targeted therapies, especially those inhibiting PI3K, display promising effects in multiple chordoma cell line and xenograft models. Nevertheless, the limited effects of PI3K, EGFR, and CDK targeting agents in other models reveal the presence of resistance mechanisms, which motivates future research to both identify biomarkers of response and develop combination therapies.

LncRNA CDKN2B-AS1 promotes the progression of ovarian cancer by miR-143-3p/SMAD3 axis and predicts a poor prognosis.

Long noncoding RNAs (LncRNAs) show great potential as the therapeutic targets attributing to their implication in the progression of various human cancers, including ovarian cancer (OC). Here, we aimed to explore the biological function of lncRNA cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) in OC and its mechanism of action. The abundances of CDKN2B-AS1, miR-143-3p, and SMAD3 mRNA were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Cell Counting Kit-8 (CCK8) was performed to analyze cell proliferation. Cell apoptosis was assessed by flow cytometry and western blot analyses. Transwell assay was utilized to analyze cell migration and invasion abilities. Tumor xenograft was performed to confirm the role of CDKN2B-AS1 in ovarian tumor growth in vivo. The protein level of SMAD3 was examined by western blot assay. The interaction between CDKN2B-AS1 and miR-143-3p, or miR-143-3p and SMAD3 was demonstrated by bioinformatic, luciferase reporter, qRT-PCR and western blot analyses. CDKN2B-AS1 was upregulated in OC and correlated with clinicopathologic features. The knockdown of CDKN2B-AS1 hampered the development of OC, as reflected by the suppression of cell proliferation, migration, and invasion, and the enhancement of cell apoptosis, whereas the effects could be rescued by the overexpression of SMAD3. The absence of CDKN2B-AS1 blocked tumor growth in vivo. CDKN2B-AS1 served as a molecular sponge for miR-143-3p, leading to the derepression of miR-143-3p target SMAD3, which eventually triggered the progression of OC. In conclusion, CDKN2B-AS1 promoted tumor growth, invasion, and migration of OC by regulation of miR-143-3p/SMAD3 axis, hinting that CDKN2B-AS1 might be a potential biomarker for OC diagnosis and treatment.

[Combined transperineal and transpubic urethroplasty for patients with complex male pelvic fracture urethral distraction defect].

OBJECTIVE: To evaluate the clinical effects and characteristics of combined transperineal and transpubic urethroplasty for patients with complex pelvic fracture urethral distraction defect (PFUDD). METHODS: We retrospectively reviewed the clinical data of 17 male patients with complex posterior PFUDD from January 2010 to December 2019. The complications included urethrorectal fistulas in 2 patients (11.8%), urethroperineal fistula in 1 patient (5.9%). Ten patients had undergone previous treatments: dilatation in 3 patients (17.6%), internal urethrotomy in 1 patient, failed urethroplasty in 6 patients (35.3%), of whom 2 patients had two times of failed urethroplasties. All the patients were performed with urethroplasty by combined transperineal and transpubic approach with removing the entire pubic bone followed by the anastomosis. RESULTS: The mean age of the patients included in this study was 35.5 (range: 21-62) years. The mean length of stricture was 5.5 (range: 4.5-7.0) cm, the mean follow-up was 27 (range: 7-110) months, the mean time of operation was 190 (range: 150-260) min, the mean evaluated blood loss was 460 (range: 200-1 200) mL. There were 5 patients who needed blood transfusion intraoperatively or postoperatively. Wound infection was seen in 4 out of 17 patients and thrombosis of lower extremities in 1 out of 17 patients. The last follow-up showed that the mean postoperative maximum urinary flow rate was 22.7 (range: 15.5-40.7) mL/s. After removing the catheter, one patient presented with decreased urinary flow and symptoms of urinary infection. Cystoscopy showed the recurrent anastomotic stricture, which was cured by internal urethrotomy. In our series, the success rate of the combined transperineal and transpubic urethroplasty was 94.1% (16/17). CONCLUSION: Combined transperineal and transpubic urtheroplasty can achieve a tension free anastomosis after removing the entire wedge of pubis in some patients with complex PFUDD. However, this procedure should be completed in a regional referral hospital due to the complexity of the operation and the high percentage of complications.

[Effect of lncRNA HOTAIR on the radiosensitivity of HCCLM3 cells].

Objective: To investigate the effect of down-regulating long non-coding RNA (lncRNA) and HOX transcript antisense RNA (HOTAIR) targeting miR-761 on the radiosensitivity of HCCLM3. Methods: The expression of HOTAIR in liver cancer cells HuH-7, SNU-449, HCCLM3 and normal liver cells L-02 were measured by real-time quantitative PCR. HCCLM3 cells were divided into control, Sh-NC (transfected shRNA negative control), Sh-HOTAIR (transfected HOTAIR shRNA), RAD+Sh-NC (transfected shRNA negative control and irradiated with 8Gy dose), and RAD+Sh-HOTAIR (HOTAIR shRNA was transfected and irradiated with 8Gy dose) group. Apoptosis was detected by flow cytometry, Bcl-2 Associated X Protein (Bx-2), cleaved cysteine-containing Cleaved cysteinyl aspartate specific proteinase 3 (C-Caspase-3) protein expression. Sh-NC, Sh-HOTAIR cells were irradiated with 0, 2, 4, 6, 8 Gy, and plate-clone experiments were used to determine radiosensitivity. Bioinformatics software predicted that miR-761 might be a target gene of HOTAIR, and the luciferase reporter system identified the targeting relationship. The miR-761 inhibitor, HOTAIR shRNA and inhibitor negative control, and HOTAIR shRNA were co-transfected into HCCLM3 cells, respectively. Cell apoptosis and Bax and C-cysteine-containing aspartate proteins were also measured using the above method, as well as the hydrolase-3 protein expression and cell survival fraction. Results: The expression levels of HOTAIR in liver cancer cells HuH-7, SNU-449, and HCCLM3 were higher than those in normal liver cells L-02 (1.85±0.12, 2.27±0.23, 2.68±0.15 vs 1.00±0.09, P<0.05). Compared with Sh-NC, the apoptosis rate of Sh-HOTAIR, RAD+Sh-NC cells and Bax, C-Caspase-3 protein levels are higher [Apoptotic rate: (13.47±1.32)%, (12.84±1.19)% vs (2.98±0.27)%; Bax protein: 0.74±0.08, 0.72±0.06 vs 0.42±0.06; C-Caspase-3 protein: 0.56±0.06, 0.54±0.08 vs 0.25±0.04, all P<0.05]. Compared with Sh-HOTAIR and RAD+Sh-NC, RAD+Sh-HOTAIR cell apoptosis rate and Bax, C-Caspase-3 protein levels are higher [apoptosis rate:(22.57±2.36)% vs (13.47±1.32)%, (12.84±1.19)%, Bax protein: 0.99±0.11 vs 0.74±0.08, 0.72±0.06, C-Caspase-3 protein: 1.03±0.12 vs 0.56±0.06, 0.54±0.08,all P<0.05]. Compared with Sh-NC, Sh-HOTAIR cells had lower survival scores and higher radiosensitivity (P<0.05). HOTAIR targets negative regulation of miR-761 expression. Compared with cells co-transfected with inhibitor negative control and HOTAIR shRNA, cells co-transfected with miR-761 inhibitor and HOTAIR shRNA had lower apoptosis rate after radiation treatment [(10.24±1.32)% vs (21.84±2.01))%], Bax (0.50±0.06 vs 1.01±0.10) and C-Caspase-3 protein (0.56±0.07 vs 1.05±0.14) had lower expression and higher cell survival scores (all P<0.05). Conclusion: Down-regulating lncRNA HOTAIR targets miR-761 to increase the radiosensitivity of HCCLM3.

[Clinical characteristics and prognosis of renal cell carcinoma patients with bone metastases].

Objective: To determine the status of bone metastasis (BM) and prognosis factors of patients with renal cell carcinoma (RCC) in our center. Methods: The clinical and medical records of RCC patients with BM, who were admitted to the Department of Urology, Bone Oncology and Spine Surgery, Beijing Jishuitan Hospital from August 2009 to August 2017 were collected. The gender, age, time of BM, location of BM, numbers of BM, presence or absence of visceral metastasis, pathological types of BM were investigated. The patients were followed up regularly, and the survival curves were analyzed by Kaplan-Meier method. Cox proportional hazard regression model was used to estimate the prognostic factors. Results: A total of 51 RCC patients with bone metastasis were collected. The age of patients ranged from 38 to 76 (58.6±8.2) years old, including 39 males (76.5%) and 12 females (23.5%). The ratio of male to female was 3.25∶1. The patients were followed up for 8 to 109 months, with a median follow-up time of 30 months. The follow-up rate was 90.2%. Thirty-one (60.8%) patients died at the last follow-up, with a median overall survival (OS) time of 25 months. The median OS was 38 months and 20 months in the solitary BM group (26 cases, 51.0%) and BM ≥ 2 group (25 cases, 49.0%), respectively. The difference between the two groups was statistically significant (P=0.021). The median OS was 30 months, 69 months and 17 months in the axis BM group (22 cases, 43.1%), appendicular BM group (19 cases, 37.3%) and both the axis and appendicular BM group (10 cases, 19.6%), respectively. The difference between the groups was statistically significant (P=0.012). The median OS was 22 months and 38 months in the patients with (15 cases, 29.4%) and without (36 cases, 70.6%) visceral metastases groups, respectively. The difference between the two groups was statistically significant (P=0.007). Univariate and multivariate Cox regression analysis showed that the numbers of BM (HR=3.130, 95%CI: 1.502-6.520, P=0.035) and visceral metastasis (HR=4.699, 95%CI: 1.810-9.545, P=0.001) were independent prognostic factors for RCC with BM. Conclusions: Solitary BM, no visceral metastasis are good prognostic factors for RCC with BM. For these patients, radical resection of BM is feasible to improve survival rate.

[The value of conventional magnetic resonance imaging based radiomic model in predicting the texture of pituitary macroadenoma].

Objective: To investigate the value of conventional magnetic resonance imaging (MRI) based radiomic model in predicting the texture of pituitary macroadenoma. Methods: The complete data of 101 patients with pituitary macroadenoma confirmed by surgery and pathology in Yijishan Hospital of Wannan Medical College from December 2014 to December 2019 were retrospectively analyzed. According to the texture of the intraoperative pituitary tumor, patients were divided into soft group (n=58) and hard group (n=43). They were randomly divided into training group (n=72) and validation group (n=29) at a ratio of 7∶3. All patients underwent conventional MRI scan of the pituitary gland. Itk-snap software was used to manually outline the T(1)-weighted image (T(1)WI), T(2)-weighted image (T(2)WI) and enhanced T(1)WI image section by section on tumor area of interest (ROI) and perform three-dimensional fusion. Then AK software was imported to extract texture features. The regression analysis methods of minimum redundancy maximum relevance (mRMR) and least absolute shrinkage and selection operator (LASSO) were used for feature selection and radiomic signature establishment. The reliability of the model was verified by 100 leave-group-out cross validation (LGOCV), and the predictive ability of the model was evaluated by drawing the receiver operating characteristic (ROC) curve. The decision curve analysis (DCA) was used to evaluate the clinical application value of the model. Results: The AUC (Area Under the ROC Curve) (95%CI) values of T1WI, T2WI, enhanced T1WI, and the combined sequence model to predict the texture of pituitary macroadenomas in the training and validation groups were 0.91 (0.84-0.98) and 0.90 (0.78-1.00), 0.86 (0.78-0.95) and 0.83 (0.64-1.00), 0.90 (0.83-0.97) and 0.89 (0.77-1.00),0.92 (0.85-0.98) and 0.91 (0.79-1.00), respectively. DCA demonstrated that T(1)WI, T(2)WI, enhanced T(1)WI, and combined sequence model all had good net benefits in clinical practice. Conclusions: T(1)WI, T(2)WI, enhanced T(1)WI, and combined sequence model of conventional MRI all had high efficacy in predicting the texture of pituitary macroadenoma, which provided a new quantitative method for predicting the texture of pituitary macroadenoma.

[Protective effect and mechanism of mild hypothermia on liver injury after cardiopulmonary resuscitation in pigs].

Objective: To investigate the effect of mild hypothermia therapy on liver after cardiopulmonary resuscitation. Methods: Thirty-three inbred Chinese Wuzhishan (WZS) minipigs, weighted (28±2) kg, were used to establish a ventricular fibrillation model. And 30 animals survived after cardiopulmonary resuscitation reached basic life support. The surviving animals were randomly divided into two groups: mild hypothermia group (group M, n=15) and conventional treatment group (group C, n=15). All the animals were observed for 24 hours. Blood samples were extracted at baseline, 0.5, 1, 2, 4, 6, 12 and 24 h after successful resuscitation. The concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected at the time points. The enzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α). The data were compared between the two groups, LSD test was used when the variance was homogeneous, and Tamhane T2 test was used when the variance was uneven. Results: Eleven pigs (73.3%) in the group M and 8(53.3%) in the group C survived at 24 h after successful resuscitation, with no statistically significant difference between the two groups (χ(2)=1.229, P=0.225). After successful resuscitation, the AST, ALT increased in both group but less in M group (all P<0.05). After successful resuscitation, the concentrations of TFN-α and IL-6 in the blood increased in both groups, reached the peak at 4h, and then decreased gradually. The concentrations of TFN-α in group M were lower than those in group C at 0.5, 2, 4 and 6 h after successful resuscitation (t=0.01, 0.01, 0.87, 0.86, all P<0.05). The concentrations of IL-6 in the group M were lower than those in group C at 0.5, 1, 2 and 4 h after successful resuscitation (t=0.23, 0.78, 0.11, 0.80, all P<0.05). Conclusions: After successful resuscitation, the release of inflammatory mediators, such as TNF-α and IL-6, and cell apoptosis may involve in liver ischemia reperfusion injury. After successful resuscitation, the liver undergoes ischemia-reperfusion injury, which may be related to the release of inflammatory mediators such as TNF-α and IL-6. Mild hypothermia therapy can prevent the release of TNF-α, IL-6 to reduce the degree of liver damage after resuscitation.

[Single-stage repair of penile urethral stricture using combined dorsal onlay oral mucosa grafting with ventral onlay penile skin flap].

OBJECTIVE: To evaluate the clinical effect of single-stage repair of penile urethral stricture using combined dorsal onlay oral mucosa grafting with ventral onlay penile skin flap. METHODS: We retrospectively reviewed the clinical database of 22 male patients with penile urethral stricture who received single-stage repair using combined dorsal onlay oral mucosa grafting with ventral onlay penile skin flap from November 2015 to October 2018. All the cases had no complications, such as skin fistula. The causes of stricture included iatrogenic (14/22, 63.6%), inflammation (2/22, 9.1%) and idiopathic (6/22, 27.3%). A ventral urethrotomy was made in the segment of stricture and extended proximally and distally until the normal calibre urethra was encountered. The oral mucosa graft was secured to the corpus spongiosum in dorsal onlay fashion or underlying corpora cavernosum after resection of the severe scarred urethra. Then the prepared Orandi fasciocutaneous penile skin flap was secured to edges of corpus spongiosum or oral mucosa graft. A 16 F or 14 F Foley catheter was left in situ for a minimum of 3 weeks, at which time a urethrogram was performed to look for extravasation, and the urethroscopy was performed if necessary. Success was defined as an open urethra with Qmax≥15 mL/s and no need for further surgical intervention. RESULTS: all the 22 patients with a mean age of 52.6 (18-73) years underwent the combined tissue-transfer technique. The mean length of the penile urethral stricture was 5.3 (2.5-10.0) cm and the mean preoperative Qmax was 6.7 mL/s. the mean length of oral mucosa grafts and fasciocutaneous skin flaps were 5.5 (3.2-10.5) cm and 6.0 (3.5-11.0) cm, respectively. The mean operation time was 225 (150-420) minutes and the mean evaluated blood loss was 53 (20.0-110.0) mL. The grafts included buccal mucosa (19/22, 86.4%) and lingual mucosa (3/22, 13.6%). The mean postoperative Q max was 21.2 (15-32) mL/s. A case of skin fistula and 2 cases of recurrent stricture were found, so the technique success rate was 81.8% (18/22) at a mean follow-up of 20.5 (5-51) months. The perioperative complications included 2 cases of infection and skin necrosis, which healed well after conservative treatment. CONCLUSION: Single-stage repair of penile urethral stricture using combined dorsal onlay oral mucosa grafting with ventral onlay penile skin flap appears to be an excellent option to repair penile urethral stricture with unsalvageable urethral plate and the penile skin is available. The present clinical series showed a successful rate of 81.8% (18/22).

Association between air pollution and upper respiratory tract infection in hospital outpatients aged 0-14 years in Hefei, China: a time series study.

OBJECTIVE: To investigate the association between air pollution and upper respiratory tract infection (URTI) in children aged 0-14 years in Hefei, China in 2014-2015. STUDY DESIGN: An ecological method (i.e. generalised additive model [GAM]) was used to explore the effects of air pollutants on paediatric hospital outpatients with URTI. METHODS: GAM was used to evaluate the lag effects (including lag0 to lag6, lag01 and lag06) between daily changes in particulate matter (PM10), fine particulate matter (PM2.5), sulphur dioxide (SO2), nitrogen dioxide (NO2), ozone (O3) and carbon monoxide (CO) and the number of hospital outpatients with URTI in 2014-2015, after controlling for the confounding effects of long-term trends, seasonality, day of the week, public holidays and meteorological factors. RESULTS: PM10, PM2.5, SO2, NO2 and CO in the single-pollutant models had significant positive effects on the number of paediatric hospital outpatients with URTI. It was found that per 10 µg/m3 increasing in concentrations of PM10 at lag3, PM2.5, SO2, NO2 and CO at lag06 were associated with an increase of Excess risk (ER) with 0.15% (95% CI: 0.07%∼0.23%), 0.38% (95% CI: 0.17%∼0.60%), 2.92% (95% CI: 1.88%∼3.97%), 4.47% (95% CI: 3.69%∼5.25%) and 0.05% (95% CI: 0.02%∼0.08%), respectively. Only NO2 remained significantly positively associated with the number of hospital outpatients with URTI in the full-pollutant models, and ERs were 4.72% (95% CI = 3.76%-5.69%) and 4.70% (95% CI = 3.76%-5.65%) per 10 µg/m3 increase in NO2 in Model 1 (including PM10, SO2, NO2, O3 and CO) and Model 2 (including PM2.5, SO2, NO2, O3 and CO), respectively. CONCLUSION: This study showed that short-term exposure to air pollution was associated with increased risk of URTI among paediatric hospital outpatients aged 0-14 years in Hefei. NO2 was the major air pollutant affecting the daily number of paediatric hospital outpatients with URTI.

[Progressive pseudorheumatoid dysplasia misdiagnosed as ankylosing spondylitis: a case report].

In this study, we reported a case of progressive pseudorheumatoid dysplasia in Peking University Third Hospital. A 56-year-old male patient presented with hip joint pain for more than 40 years and multiple joints pain with limitation of movements of these joints for 28 years. This patient suffered from joint pain and impaired range of motion of the hip, knee, elbow and shoulder gradually, associated with difficulty in walking and inability to take care of himself. He was diagnosed with "femoral head necrosis" or "ankylosing spondylitis" in local hospitals, but the treatment of nonsteroidal antiinflammatory drugs (NSAIDs) and sulfasalazine was not effective. Up to the age of 14, the patient displayed normal physical development, with the highest height was about 158 cm, according to the patient recall. However, his height was 153 cm at present. There was no history of similar illness in any family member. Physical examinations descried limitation of movement of almost all joints. Enlargement and flexion deformity of the proximal interphalangeal (PIP) joints of the hands resulted in the claw hand appearance. Limited abduction and internal and external rotation of the shoulder and hip could be find. He had normal laboratory findings for blood routine test, biochemical indexes and acute phase reactants such as C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). Furthermore, HLA-B27 and autoimmune antibodies such as rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP) antibody and antinuclear antibody (ANA) were all negative. X-ray of the hip showed loss of the joint space and irregularities of the femoral head, both femoral head were flattened, it could be see hyperplasia, osteophytes, bilateral femoral neck thicken, neck dry angle turned smaller. The radiological findings of the spinal vertebra indicated kyphosis deformity, narrowing of the intervertebral discs, vertebral syndesmophytes and flattening of the vertebra. However, there was no clues of bone marrow edema in the lumbar MRI. At last, genetic testing for the Wnt1-inducible signaling pathway protein 3 (WISP3) gene was done and indicated compound heterozygous mutations: 756C>G and c.866dupA. These two mutations were derived from the patient's mother and father (the patient's parents each had a heterozygous mutation). Two exons of the WISP3 gene had nucleotide changes leading to amino acid mutations. According to the patient's history, symptoms, physical examinations, radiological findings and genetic testing, the final definitive diagnosis was progressive pseudorheumatic dysplasia.

[A meta-analysis of prognosis after bipolar umbilical cord coagulation or radiofrequency ablation to reduce complicated monochorionic multiple pregnancies].

Objective: To evaluate the prognosis and complications after reduction of monochorionic multifetal pregnancies using bipolar umbilical cord coagulation (BCC) or radiofrequency ablation (RFA). Methods: A literature search were performed by using online databases including PubMed, Embase, and COCHRANE.The publications that described prognosis and complications after selective reduction of monochorionic twin pregnancies using either BCC or RFA for studies with clear outcome data were identified. Results: We identified five retrospective cohort studies for the meta-analysis and 231 cases of BCC and 174 cases of RFA.There was no statistical difference in overall survival after reduction between BCC group (79.2%) and RFA groups (76.4%) (RR=0.96; 95%CI: 0.86~1.08; P=0.48). Neonatal mortality was similar in both groups (8.2% vs 11.1%, respectively; RR=1.34; 95%CI: 0.60~2.99; P=0.48). However, intrauterine fetal death (IUFD) in the RFA group was 13.6%, and it significantly higher than that in the BCC group 7.7% (RR=2.15; 95%CI: 1.10~4.21; P=0.03). In contrast, after reduction, those in the RFA group had less preterm premature rupture of membranes (PPROM) compared with the BCC group (17.1% vs 27.5%, RR=0.58; 95%CI: 0.39~0.86; P=0.007). Conclusions: RFA and BCC groups have similar overall survival for complicated monochromic multiple pregnancies, but RFA was more prone to IUFD, and BCC tended to have more PPROM.Thus, procedures should be chosen according to specific clinical situations and pregnancy conditions.

[Analysis of the entrance and suture method of umbilical incision in gynecological laparoscopy].

Objective: To explore the entrance and suture method of umbilical incision in gynecological laparoscopy. Methods: A total of 204 cases of gynecologic laparoscopy in our hospital from 2013 to 2016 were reviewed respectively. All the cases used two kinds of approach of umbilical incision: intra-umbilical incision and peri-umbilical incision (longitudinal/transverse oblique/arc incision according to the bellybutton natural skin folds) and two methods of suture: the suture of "U" and the suture of the whole subcutaneous tissue. Two groups were randomly assigned based on the entrance and suture method with each group 102 cases. The peri-operative outcomes were compared, including intra-operative and postoperative bleeding, postoperative incision fat liquefaction and infection, incision pain, incision appearance satisfaction and incision healing satisfaction. Results: The difference was statistically significant in the intra-operative and postoperative bleeding between two groups of intra-umbilical incision and peri-umbilical incision (P<0.05). There was statistically significant difference in postoperative incision fat liquefaction, infection and incision pain between two groups of the suture of "U" and the suture of the whole subcutaneous tissue (P<0.05). The incision appearance satisfaction showed no difference (P>0.05), but the difference was statistically significant (P<0.05) in the incision healing satisfaction between two groups. Conclusion: The peri-umbilical incision (longitudinal/transverse oblique/arc incision according to the bellybutton natural skin folds) and suture of the whole subcutaneous tissue can be the feasible modified methods with high practicability and security, good cosmetic result. It should be promoted in gynecologic laparoscopy.

[The expressions and the roles of SDF-1/CXCR-4 and SDF-1/CXCR-4 in human endometriosis].

Objective: To investigate the expressions and significances of SDF-1 and its receptor CXCR-4 in endometriosis. Methods: 50 hysteromyoma patients treated at Beijing Tongren Hospital, Capital Medical University between 1(st) January 2016 and 31(st) December 2017 were divided into control group, that is, non-endometriosis group, while another 50 endometriosis patients were divided into experimental group.The endometrial tissues, endometriosis lesions, and peritoneal fluid samples of hysteromyoma patients were collected by operation.RT-PCR, ELISA and immunohistochemistry were adopted to detect the expressions of SDF-1 and CXCR-4 in the two groups.Cell count was used to analyze the roles of SDF-1 and CXCR4 in the mitosis and proliferation of endometrial stromal cells. Results: The mean value of SDF-1 and CXCR-4 expressions in ascites or peritoneal fluid of endometriosis patients were (2.56±0.33) mg/L and (4.47±0.32) mg/L, respectively. The mean concentrations in ascites or peritoneal fluid in hysteromyoma patients were (1.39±0.36) mg/L and (3.16±0.32) mg/L, respectively.The expressions of SDF-1 and CXCR-4 in ascites or peritoneal fluid of endometriosis patients were both significantly higher than those of patients in non-endometriosis group (P<0.05). SDF-1 and CXCR-4 were expressed in both endometriosis lesions and the glandular epithelial cells and mesenchymal cells of normal endometrial tissue.Positive staining sites were located in the cytoplasm.A value was used to calculate and analyze the expression of immune staining.The mean A value of SDF-1 and CXCR-4 in endometriosis group were 0.21±0.13 and 0.21±0.13, respectively, and the mean A value in normal endometrial tissues were 0.15±0.13 and 0.14±0.13, respectively.The expressions of these two in endometrial tissues were significantly higher than that in normal endometrial tissues, and the differences were statistically significant (P<0.05). The expression of CXCR-4 mRNA was abundant in the mesenchymal cells of endometriosis cultured in vitro.SDF-1 promoted the mitosis and proliferation of endometrial stromal cells cultured in vitro in a dose-dependent manner.The neutralizing antibody against CXCR-4 was obviously inhibited. Conclusion: The high expressions of SDF-1 and CXCR-4 in endometriosis as well as SDF-1 through its specific receptor CXCR-4 promoted the mitosis and proliferation of endometrial stromal cells, suggesting that SDF-1 and CXCR-4 played important roles in the pathogenesis of endometriosis.

Determination of Oleandrin in Blood and Liver Samples by LC-MS/MS.

OBJECTIVES: To develop a liquid chromatography-tandem mass spectrometry （LC-MS/MS） analytical method for the determination of oleandrin in blood and liver tissues, which could be applied to the cases of death caused by oleander poisoning. METHODS: Blood or liver tissues underwent a liquid-liquid extraction （LLE） using ethyl acetate, and the extract was separated on an Agilent ZORBAX SB-C18 column and eluted with a gradient of acetonitrile and 20 mmol/L ammonium acetate （containing 0.1% formic acid）. Oleandrin was detected using electrospray positive ionization （ESI+） with multiple-reaction monitoring （MRM） mode. RESULTS: Oleandrin showed excellent linearity in both blood and liver samples in the corresponding linear range （r>0.995 0）, with detection limits 1 ng/mL and 2 ng/g, respectively, extraction recovery rates greater than 70.50%, both intra- and inter-day precisions less than 10.71%, accuracies 98.42%-111.63%, and matrix effects 91.52%-106.39%. The method was successfully applied to a case of suspected oleander poisoning. Oleandrin was detected in the blood, urine, liver tissues, bile, stomach wall tissues and stomach contents of the cadaver, with the content ranging from 65.5 to 29 600.0 ng/mL （ng/g）. CONCLUSIONS: The method developed in this study is simple and convenient to operate with good selectivity, and is suitable for the analysis of oleandrin in biological samples such as blood and liver tissues, which can provide technical support for forensic identification and clinical diagnosis and treatment of oleander poisoning.

[Cause analysis of spinal surgery in ankylosing spondylitis].

OBJECTIVE: To investigate the common cause of spinal surgery in ankylosing spondylitis (AS) and to develop reasonable and effective treatment programs for rhematologists. METHODS: In this study, 79 AS patients (72 males, 7 females) hospitalized in the Department of Orthopedic and received spinal surgery in Peking University Third Hospital from January 2007 to July 2013 were investigated retrospectively. The causes of the spinal surgery were analyzed. RESULTS: The majority of the surgery were of cervical vertebra surgery for 59 cases (74.7%), followed by the thoracolumbar vertebra, thoracic vertebra, cervicothoracic vertebra, and lumbar vertebra. The common causes of AS spinal surgery were instability (53.2%) including fracture and dislocation caused by trauma mostly, followed by compression symptoms (34.1%). However, the orthopaedic surgery (12.7%) was not the predominant surgery causes. The most common cause of cervical surgery was cervical spondylosis (25 cases), followed by cervical fracture (22 cases) and cervical dislocation (10 cases). For cervical fracture, the most common site was C7 (8 cases). Atlanto-axial vertebral lesions (13 cases) induced by dislocation, subluxation, instability and fracture were common in cervical vertebra surgery. The age and disease duration of atlantoaxial surgical patient's were less than other parts of the cervical surgery. Thoracolumbar vertebra surgery included thoracolumbar kyphosis orthopaedic surgery (10 cases), and fractures (6 cases), which mainly occurred in T11-12 vertebrae. According to the time of admission for surgery, the cases of AS that received spinal surgery were 34 from 2007 to 2008. The number of the cases that accepted the surgery decreased gradually year by year. CONCLUSION: The majority surgery section was cervical vertebra, and atlanto-axial vertebral lesions were not rare as we thought. The main etiology of spinal surgery for AS patients was instability, e.g. fracture and compression symptoms. Of course, trauma accounted for the relative percentage of this phetonomenon. Although, the annual number of patients who receives spinal surgery is decreasing, to go to make early diagnosis and treatment, and to standardize patient education for AS patients are still important.

Structure-function analysis of SAP97, a modular scaffolding protein that drives dendrite growth.

Activation of AMPA receptors assembled with the GluA1 subunit can promote dendrite growth in a manner that depends on its direct binding partner, SAP97. SAP97 is a modular scaffolding protein that has at least seven recognizable protein-protein interaction domains. Several complementary approaches were employed to show that the dendrite branching promoting action of full length SAP97 depends on ligand(s) that bind to the PDZ3 domain. Ligand(s) to PDZ1, PDZ2 and I3 domains also contribute to dendrite growth. The ability of PDZ3 ligand(s) to promote dendrite growth depends on localization at the plasma membrane along with GluA1 and SAP97. These results suggest that the assembly of a multi-protein complex at or near synapses is vital for the translation of AMPA-R activity into dendrite growth.