[Clinicopathological features and prognosis of anorectal melanoma: A report of 68 cases].

OBJECTIVE: To investigate the clinicopathological characteristics of anorectal mucosal melanoma (ARMM), and to evaluate the prognostic factors. METHODS: A total of 68 primary ARMM surgical specimens from 2010 to 2018 were retrospectively studied. Slides were reviewed to evaluate pathological features. Slingluff staging method was used for staging. RESULTS: (1) Clinical features: The median age at diagnosis in this group was 61.5 years, with a male-to-female ratio 1 ∶1.62. The most common complaint was blooding (49 cases). For anatomic site, anorectum was the prevalent (66.2%), followed by rectum (20.6%). At the time of diagnosis, 28 cases were stage Ⅰ (localized stage, 41.2%), 25 cases were stage Ⅱ (regional lymph node metastasis, 36.8%), and 15 cases were stage Ⅲ (distant metastasis, 22.1%). Five patients underwent wide local excision, the rest abdominoperineal resection, and 48 patients received adjuvant therapy after surgery. (2) Pathological features: Grossly 88.2% of the tumors were exophytic polypoid masses, with the median tumor size 3.5 cm and the median tumor thickness 1.25 cm. Depth of invasion below lamina muscularis mucosae ranged from 0-5.00 cm (median 1.00 cm). The deepest site of tumor invasion reached muscular layer in 27 cases, and perirectal tissue in 16 cases. Melanin pigmentation was absent or not obvious in 67.6% of the cases. The predominant cytology was epithelioid (45 cases, 66.2%). The rate for ulceration, necrosis, lymphovascular invasion, and perineural invasion was 89.7%, 35.3%, 55.9%, and 30.9%, respectively. The median mitotic count was 18/mm2. The positive rate of S100, HMB-45 and Melan-A were 92.0%, 92.6% and 98.0%, respectively. The median of Ki-67 was 50%. The incidences of mutations within CKIT, BRAF and NRAS genes were 17.0% (9 cases), 3.8% (2 cases) and 9.4% (5 cases), respectively. (3) Prognosis: Survival data were available in 66 patients, with a median follow-up of 17 months and a median survival time of 17.4 months. The 1-year, 2-year and 5-year overall survival rate was 76.8%, 36.8% and 17.2%, respectively. The rate of lymphatic metastasis at diagnosis was 56.3%. Forty-nine patients (84.5%) suffered from distant metastasis, and the most frequent metastatic site was liver. Univariate analysis revealed that tumor size (>3.5 cm), depth of invasion below lamina muscularis mucosae (>1.0 cm), necrosis, lymphovascular invasion, BRAF gene mutation, lack of adjuvant therapy after surgery, deep site of tumor invasion, and high stage at diagnosis were all poor prognostic factors for overall survival. Multivariate model showed that lymphovascular invasion and BRAF gene mutation were independent risk factors for lower overall survival, and high stage at diagnosis showed borderline negative correlation with overall survival. CONCLUSION: The overall prognosis of ARMM is poor, and lymphovascular invasion and BRAF gene mutation are independent factors of poor prognosis. Slingluff staging suggests prognosis effectively, and detailed assessment of pathological features, clear staging and genetic testing should be carried out when possible. Depth of invasion below lamina muscularis mucosae of the tumor might be a better prognostic indicator than tumor thickness.

[Analysis of clinicopathological and molecular abnormalities of angioimmunoblastic T-cell lymphoma].

OBJECTIVE: To analyze the clinicopathological features, molecular changes and prognostic factors in angioimmunoblastic T-cell lymphoma (AITL). METHODS: Sixty-one cases AITL diagnosed by Department of Pathology of Peking University Cancer Hospital were collected with their clinical data. Morphologically, they were classified as typeⅠ[lymphoid tissue reactive hyperplasia (LRH) like]; typeⅡ[marginal zone lymphoma(MZL)like] and type Ⅲ [peripheral T-cell lymphoma, not specified (PTCL-NOS) like]. Immunohistochemical staining was used to evaluate the presence of follicular helper T-cell (TFH) phenotype, proliferation of extra germinal center (GC) follicular dendritic cells (FDCs), presence of Hodgkin and Reed-Sternberg (HRS)-like cells and large B transformation. The density of Epstein-Barr virus (EBV) + cells was counted with slides stained by Epstein-Barr virus encoded RNA (EBER) in situ hybridization on high power field (HPF). T-cell receptor / immunoglobulin gene (TCR/IG) clonality and targeted exome sequencing (TES) test were performed when necessary. SPSS 22.0 software was used for statistical analysis. RESULTS: Morphological subtype (%): 11.4% (7/61) cases were classified as type Ⅰ; 50.8% (31/61) as type Ⅱ; 37.8% (23/61) as type Ⅲ. 83.6% (51/61) cases showed classical TFH immunophenotype. With variable extra-GC FDC meshwork proliferation (median 20.0%); 23.0% (14/61) had HRS-like cells; 11.5% (7/61) with large B transformation. 42.6% (26/61) of cases with high counts of EBV. 57.9% (11/19) TCR+/IG-, 26.3% (5/19) TCR+/IG+, 10.5% (2/19) were TCR－/IG－, and 5.3% (1/19) TCR－/IG+. Mutation frequencies by TES were 66.7% (20/30) for RHOA, 23.3% (7/30) for IDH2 mutation, 80.0% (24/30) for TET2 mutation, and 33.3% (10/30) DNMT3A mutation. Integrated analysis divided into four groups: (1) IDH2 and RHOA co-mutation group (7 cases): 6 cases were type Ⅱ, 1 case was type Ⅲ; all with typical TFH phenotype; HRS-like cells and large B transformation were not found; (2) RHOA single mutation group (13 cases): 1 case was type Ⅰ, 6 cases were type Ⅱ, 6 cases were type Ⅲ; 5 cases without typical TFH phenotype; 6 cases had HRS-like cells, and 2 cases with large B transformation. Atypically, 1 case showed TCR－/IG－, 1 case with TCR－/IG+, and 1 case with TCR+/IG+; (3) TET2 and/or DNMT3A mutation alone group (7 cases): 3 cases were type Ⅱ, 4 cases were type Ⅲ, all cases were found with typical TFH phenotype; 2 cases had HRS-like cells, 2 cases with large B transformation, and atypically; (4) non-mutation group (3 cases), all were type Ⅱ, with typical TFH phenotype, with significant extra-GC FDC proliferation, without HRS-like cells and large B transformation. Atypically, 1 case was TCR－/IG－. Univariate analysis confirmed that higher density of EBV positive cell was independent adverse prognostic factors for both overall survival (OS) and progression free survival(PFS), (P=0.017 and P=0.046). CONCLUSION: Pathological diagnoses of ALTL cases with HRS-like cells, large B transformation or type Ⅰ are difficult. Although TCR/IG gene rearrangement test is helpful but still with limitation. TES involving RHOA, IDH2, TET2, DNMT3A can robustly assist in the differential diagnosis of those difficult cases. Higher density of EBV positive cells counts in tumor tissue might be an indicator for poor survival.

[Synchronization isolation method for multiple types of cells from mouse liver].

Objective: To explore a simple and feasible method for the isolation and purification of hepatocytes, hepatic stellate cells (HSC), and lymphocytes from mice. Methods: The cell suspension was obtained from male C57bl/6 mice by hepatic perfusion through the portal vein digestion method and then isolated and purified by discontinuous Percoll gradient centrifugation. Trypan blue exclusion was used to determine cell viability. Glycogen staining, cytokeratin 18, and transmission electron microscopy were used to identify hepatic cells. Immunofluorescence was used to detect α-smooth muscle actin combined with desmin in HSCs. Flow cytometry was used to analyze lymphocyte subsets in the liver. Results: After isolation and purification, about 2.7×10(7) hepatocytes, 5.7×10(5) HSCS, and 4.6×106 hepatic mononuclear cells were obtained from the liver of mice with a body weight of about 22g. The cell survival rate in each group was > 95%. Hepatocytes were apparent in glycogen deposited purple-red granules and cytokeratin 18. Electron microscopy showed that there were abundant organelles in hepatocytes and tight junctions between cells. HSC had expressed α-smooth muscle actin and desmin. Flow cytometry showed hepatic mononuclear cells, including lymphocyte subsets such as CD4, CD8, NKs, and NKTs. Conclusion: The hepatic perfusion through the portal vein digestion method can isolate multiple primary cells from the liver of mice at once and has the features of simplicity and efficiency.

[Difficulties and challenges in pathological diagnosis of gastrointestinal pancreatic neuroendocrine tumors].

In the 5th edition of WHO Classification of Digestive System Tumours, the classification and designation of gastroenteropancreatic neuroendocrine neoplasm (GEP-NEN) were updated, and G3 was redefined. Therefore, the differential diagnosis between well-differentiated neuroendocrine tumour G3(NETG3) and poorly differentiated neuroendocrine carcinomas (NEC) is both important and difficult, which has important clinical treatment and prognostic significance. Based on the latest WHO and 2020 Chinese consensus on pathological diagnosis of GEP-NEN, this paper proposed a comprehensive analysis on the differential diagnosis of gastrointestinal pancreatic neuroendocrine tumor from four aspects, including tumor morphological differentiation、tumor proliferative activity, gene change and disease progression. However, even with the above four diagnostic tools, there are still some difficulties and challenges in the work of pathological diagnosis. This paper briefly expounds and discusses them together, in order to improve the clinical application value.

Cytomegalovirus shedding in seropositive healthy women of reproductive age in Tianjin, China.

Cytomegalovirus (CMV) enters latency after primary infection and can reactivate periodically with virus excreted in body fluids which can be called shedding. CMV shedding during the early stage of pregnancy is associated with adverse pregnancy outcome. The shedding pattern in healthy seropositive women who plan to have babies has not been well characterised. Vaginal swabs, urine and blood were collected from 1262 CMV IgG-positive women who intended to have babies and tested for CMV DNA by fluorogenic quantitative PCR method. Serum IgM was also detected. The association between sociodemographic characteristics and CMV shedding prevalence was analysed. Among 1262 seropositive women, 12.8% (161/1262) were detected CMV DNA positive in at least one body fluid. CMV DNA was more frequently detected in vaginal secretion (10.5%) than in urine (3.2%) and blood (0.6%) also with higher viral loads (P < 0.00). CMV shedding was more likely detected in IgM-positive women than IgM-negative women (29.5% (13/44) vs. 12.2% (148/1218); OR 3.03, 95% CI 1.55-5.93; P = 0.001). CMV shedding in vaginal secretion was highly correlated with shedding in urine, the immune state of IgM, the adverse pregnant history and younger age. CMV shedding was more commonly detected in vaginal secretion than in urine or blood with higher viral loads among healthy seropositive women of reproductive age. Further studies are needed to figure out whether the shedding is occasional or continuous and whether it is associated with adverse pregnancy outcomes.

Knockdown of CERB expression inhibits proliferation and migration of glioma cells line U251.

BACKGROUND: Glioma is a type of tumor that occurs in the brain and accounts for almost 30 % of all brain and central nervous system tumors and 80 % of all malignant brain tumors. In this study, we investigate the role of cAMP response element-binding protein (CREB) in the progression of glioma. METHODS: Tissue samples from glioma patients were collected and examined for expression of CREB and its correlation with tumor grades. CREB was then knocked down via siRNA to see if reduced expression of CREB affects cell proliferation and migration. Factors involved in cell cycles, adhesion and apoptosis were examined as well. Moreover, CRESP/CAS9 mediated knockout of CREB was conducted and athymic Nude mice model was used to investigate CREB's role in vivo. RESULTS: The evaluated expression level of CREB in glioma patients was correlated with tumor grades. Knockdown of CREB via siRNA in glioma cell line U251 significantly inhibited the proliferation and migration of tumor cells. Moreover, CyclinD1 and Bcl-2 expression were reduced, as well as phosphorylation of IRK1/2 and AKT. Additionally, knockout of CREB via CRESP/CAS9 inhibited tumor formation of U251 cells in athymic Nude mice model. CONCLUSIONS: In conclusion, our data suggest that over expression of CREB may contribute to progression of glioma and knockdown of CREB expression may serve as a novel target for therapy (Tab. 1, Fig. 6, Ref. 25).

Genetic polymorphisms of loci D18S53, D18S59, and D18S488 in fetuses from a Chinese Tianjin Han population.

We investigated the genetic polymorphisms of three short tandem repeat (STR) loci, D18S53, D18S59, and D18S488, on chromosome 18 in fetuses from a Chinese Tianjin Han population. Sixty-four villus samples and 374 amniotic fluid samples were collected from fetuses. Quantitative fluorescence polymerase chain reaction was performed to amplify the STR loci, followed by scanned electrophoresis and quantitative analysis of the fluorescence signals. Hardy-Weinberg equilibrium (HWE) analysis was performed based on the genotype distributions of the STR loci to obtain the following population genetic data: genotype frequency, heterozygosity of observation (HO), polymorphism information content (PIC), probability of discrimination power (PD), and probability of exclusion (PE). We detected 15, 13, and 15 alleles of D18S53, D18S59, and D18S488, respectively. The genotype frequencies were found to be in line with HWE. The HO values of the three loci, D18S53, D18S59, and D18S488, were 0.797, 0.847, and 0.792; the PIC values were 0.81, 0.75, and 0.73; the PD values were 0.944, 0.901, and 0.881; and the PE values were 0.593, 0.689, and 0.585, respectively. D18S53, D18S59, and D18S488 loci are good genetic markers of chromosome 18, and show potential for use in the prenatal genetic diagnosis of Edwards' syndrome.

[Effect of double-J stent indwelling time in treatment of ureteric calculi complicated with infection].

OBJECTIVE: To evaluate the effect and double-J stent indwelling time in the treatment of acute upper urinary tract infection caused by ureteral calculi. METHODS: A prospective non-randomized controlled study was carried out. From January 2011 to August 2015, a total of 142 patients treated in Department of Urology of Shanghai First People's Hospital for ureteral calculi with systematic inflammatory response syndrome were divided into double-J stent indwelling 7 days group (n=63) and double-J stent indwelling > 7 days group (n=79). The preoperative routine blood test, urinalysis, and urine culture, the urinalysis and urine culture immediately after double-J indwelling, and the routine blood test, urinalysis, and urine culture 7 days after indwelling were compared between the two groups. Postoperative complications after lithotripsy were analyzed. RESULTS: There were no statistically significant differences in preoperative blood white blood cell (WBC) count, urine WBC count, and positive bacterial culture between the two groups (all P>0.05). Seven days after double-J stent indwelling, significant improvement was seen in both the 7 d group and the >7 d group in blood WBC count, urine WBC count, and positive urine culture [(8.1±1.7) vs (14.8±3.1) ×10(9)/L, (356±44) vs (3 077±643)/µl, 1.6% vs 52.4%; (7.9±1.1) vs (15.1±3.8) ×10(9)/L, (363±52) vs (3 122±805)/µl, 3.8% vs 48.1%], and infection was managed effectively, while no statistically significant inter-group differences were observed. There was no significant difference in stone clearance rate between the two groups (96.8% vs 96.2%, P>0.05). There was no significant difference in the incidence of postoperative complications, especially infectious complications (all P>0.05). CONCLUSIONS: Indwelling double-J stents for 7 days could effectively manage the infection in patients with acute upper urinary tract infection secondary to ureteral calculi. These patients can be treated with lithotripsy safely. The time of double-J stent indwelling could be shortened.

[Expression of epidermal growth factor receptor mutation specific antibodies in lung adenocarcinoma: evaluation of sensitivity, specificity and relationship to histologic subtypes].

OBJECTIVE: To evaluate the expression of epidermal growth factor receptor (EGFR) mutation specific antibodies in invasive lung adenocarcinomas, and their sensitivity, specificity, as well as relationship to histological subtypes. METHODS: Immunostaining with EGFR mutation-specific antibodies, del E746-A750 in exon 19 and L858R in exon 21, was performed in tissue microarrays of 884 cases of resection specimens to study the relationship between the immunophenotypes and morphologic subtypes. The sensitivity and specificity of the stains were compared with gene mutations detected by amplified refractory mutation system-polymerase chain reaction (ARMS-PCR). RESULTS: Of the 884 cases, the expression of del E746-A750 in exon 19 was 3+ , 2+ , 1+ and 0 in 7 cases (0.79%), 38 cases (4.30%), 129 cases (14.59%) and 710 cases (80.32%), respectively. For L858R in exon 21, 3+ , 2+ , 1+ and 0 staining were seen in 82 cases (9.28%), 93 cases (10.52%), 82 cases (9.28%) and 627 cases (70.93%), respectively. For both antibodies, positive expression (1+ or more) was mainly observed in lepidic, acinar and papillary predominant subtypes, and rarely seen in solid subtype or invasive mucinous adenocarcinoma (P=0.014 and 0.016). If 1+ to 3+ expression was set as positive, the specificity of exon 19/exon 21 reached 98.59%/92.98%, while the sensitivity was relatively lower (62.86%/88.89%). If 2+ to 3+ expression was read as positive, the specificity and sensitivity were 99.30%/97.37% and 25.71%/74.60% for exon 19/exon 21. If only 3+ expression was considered positive, the specificity was 100.0% for both antibodies, with a low sensitivity (8.57% for exon 19 and 34.92% for exon 21). Of the 18 cases with E746-A750 del in exon 19 based on molecular detection, the sensitivity of immunohistochemistry for exon 19 was 88.89% if a positive cutoff value ≥1+ was used; in contrast, of the 8 cases harboring other deletions in exon 19, only two cases were positive as 1+ . CONCLUSIONS: Both the EGFR mutation specific antibodies del E746-A750 in exon 19 and L858R in exon 21 demonstrate high specificity and relatively low sensitivity, and are mostly expressed in lepidic, acinar and papillary predominant subtypes, but rarely in solid subtype or invasive mucinous adenocarcinoma. For cases with 3+ expression, a mutational statue for EGFR is likely. For the 2+ positive cases, the accuracy to predict mutation almost reaches 90%, but molecular detection for confirmation is desirable. For the 1+ and negative cases, DNA-based test is essential to avoid false negativity.

[Effect of hydroquinone on the histone deacetylase in human bone marrow mononuclear cells].

OBJECTIVE: To observe the activity of histone deacetylase and the mRNA expression level of HDAC1 and HDAC2 in human bone marrow mononuclear cells, which induced by hydroquinone and exposed to hydroquinone plus Trichostatin as a histone deacetylase inhibitor for 10 hours respectively. METHODS: Collect the bone marrow mononuclear cells suspension,divided into control group,HQ group (3 h, 6 h, 12 h, 24 h) , HQ+TSA 10 h group and HQ 10 h group. Extract the nuclear proteins and RNA, test the activity of histone deacetylase with the colorimetric HDAC assay kit and detect the mRNA expression level of HDAC1 and HDAC2 by real-time Polymerase Chain Reaction (PCR). RESULTS: The HDAC activity of HQ3 h group, HQ6 h group and HQ12 h group were 1.31 times, 1.53 times and 1.148 times than that of control group respectively. And the difference was statistically significant (P<0.05). Except the HQ24 h group (P>0.05) , the HDAC1 mRNA expression of HQ3 h group, HQ6 h group and HQ12 h group were 1.173 times, 1.901 times and 2.348 times than that of control group respectively. And the difference was statistically significant (P<0.05). The HDAC2 mRNA expression of HQ6 h group and HQ12 h group were 1.426 times and 1.766 times than that of the control group respectively. And the difference was statistically significant (P<0.05). No significant difference was observed between HQ3 h group, HQ24 h group and control group (P>0.05). The cells were treated by hydroquinone plus TSA for 10 hours. The HDAC activity of HQ+TSA 10h group was reduced by 25.6% than that of HQ 10 h group (P<0.05) and rised 13.0% compared to the control group (P<0.05). And the difference was statistically significant between groups (P<0.05) .The cells were treated by hydroquinone plus TSA for 10 hours. The HDAC1 mRNA expression of the HQ+TSA 10h group is reduced by 26.9% than that of HQ10h group. The HDAC2 mRNA expression is reduced by 19.3% compared to the HQ 10h group.And the difference was statistically significant between groups (P<0.05). The HDAC1 and HDAC2 mRNA expression is obviously higher than the control group, the difference was statistically significant (P<0.05). CONCLUSION: Treatment of hydroquinone, the histone deacetylase activity and the mRNA expression of HDAC1 and HDAC2 were increased in a certain time range. The histone deacetylase inhibitor (TSA) can reduce the histone deacetylase activity and the mRNA expression level of HDAC1 and HDAC2 in the bone marrow mononuclear cell induce by hydroquinone.It can be confirmed that hematopoietic damage induced by the benzene metabolites is related to the histone acetylation modification level.

[Clinical outcomes of transoral endoscopic thyroidectomy vestibular approach assisted with submental mini-incision for early thyroid papillary carcinoma].

Objective: To investigate the efficacy of transoral endoscopic thyroidectomy vestibular approach (TOETVA) assisted with submental mini-incision in early thyroid papillary carcinoma. Methods: A total of 63 patients with early papillary thyroid carcinoma (cT1N0M0) were included who underwent TOETVA from December 2019 to May 2021 in Department of Thyroid Surgery of the Affiliated Hospital of Jining Medical University. There were 4 males and 59 females, aged from 17 to 46 years old. Of those 36 patients received traditional TOETVA as control and 27 patients accepted modified TOETVA assisted with submental mini-incision. The clinical outcomes of patients in two groups were compared. Chi-square test and t test were used in statistical analyses. Results: Compared to control group, modified TOETVA group had the less mean operation time [(146.63±38.62) minutes vs. (167.78±36.71) minutes, t=-2.21, P=0.031], the shorter time required for returning to normal diet after operation [(2.11±0.89) days vs. (2.72±1.16) days, t=-2.28, P=0.026], and the lower probability of mandibular numbness (0 vs. 16.67%, χ2=4.97, P=0.026). There was no significant difference between two groups in intraoperative blood loss, postoperative drainage volume, number of central lymph nodes dissection, and postoperative complications such as gas embolism, postoperative bleeding, postoperative infection, skin burns, subcutaneous effusion and so on(all P>0.05). After 6 months of operation, the thyroid ultrasound of the patients in two groups showed no recurrence, and the patients were satisfied with their surgical incision appearances. Conclusion: Both the modified and traditional TOETVA show similar efficacies for treatments of early thyroid papillary carcinoma, but the modified TOETVA can reduce the operation time and improve the quality of life.

[Investigation for pathological interpretation criteria and its prognostic value for P53 expression in Chinese diffuse large B-cell lymphoma].

Objective: To explore the feasibility of predicting TP53 mutation risk by immunohistochemical staining (IHC) pattern of P53 in Chinese diffuse large B-cell lymphoma (DLBCL) and its correlation with a prognostic difference. Methods: Between January 2021 and December 2021, 51 DLBCL cases at Beijing Boren Hospital were gathered. These cases had both IHC and next-generation sequencing (NGS) results. IHC classified the P53 protein expression pattern into a loss (<1% ) , diffuse (>80% ) , and heterogeneous (1% -80% ) . The sensitivity and specificity of the predicting TP53 mutation by IHC were assessed by comparing the results of the NGS, and the TP53 high mutation risk group included both loss and diffuse expression of P53. From June 2016 to September 2019, Peking University Cancer Hospital collected 131 DLBCL cases with thorough clinicopathological and follow-up data. From their tumor blocks, tissue microarray blocks were made for IHC evaluation of P53 expression pattern, and prognosis effect of P53 studies. Results: Among 51 cases with both IHC and NGS results, 23 cases were classified as TP53 high mutation risk (7 cases loss and 16 cases diffuse) , 22/23 cases were proved with mutated TP53 by NGS. Only 1 of the 28 cases classified as TP53 low mutation risk was proved with mutated TP53 by NGS. IHC had a sensitivity and specificity of 95.7% and 96.4% for predicting TP53 mutation. NGS identified a total of 26 TP53 mutations with a mutation frequency of 61.57% (13.41% -86.25% ) . In the diffuse group, 16 missense mutations and 2 splice mutations were detected; 6 truncating mutations and 1 splice mutation were detected in the loss group; 1 truncating mutation was detected in the heterogeneous group. Multivariate analysis demonstrated that TP53 cases with high mutation risk have impartial adverse significance for the 131 patients included in survival analysis (HR=2.612, 95% CI 1.145-5.956, P=0.022) . Conclusion: IHC of P53 exhibiting loss (<1% ) or diffuse (>80% ) pattern indicated TP53 high mutation risk, IHC can predict TP53 mutation with high specificity and sensitivity. TP53 high mutation risk is an independent predictor for adverse survival.

Zinc-alpha2-glycoprotein is involved in regulation of body weight through inhibition of lipogenic enzymes in adipose tissue.

CONTEXT: Zinc-alpha2-glycoprotein (ZAG) was found to influence lipolysis in adipose tissue and has recently been proposed as a candidate factor in the regulation of body weight. OBJECTIVE: To elucidate the association of serum ZAG level with body weight and percentage of body fat in normal, obese subjects and high-fat diet (HFD)-induced obese mice. DESIGN: The relationship between serum ZAG and obesity-related parameters was studied in 44 human subjects and 36 mice fed standard food and HFD. Furthermore, the effects of ZAG overexpression on adipose tissue of mice was also evaluated by using a liposome transfection method. RESULTS: Serum ZAG level was significantly lower in obese patients and obese mice in comparison to that in people and mice with normal weight. The further statistical analysis demonstrated that ZAG level was negatively correlated with body weight (r=-0.62, P<0.001), body mass index (r=-0.64, P<0.001), waist circumference(r=-0.68, P<0.001), hip circumference (r=-0.60, P<0.001), percentage of body fat (r=-0.52, P=0.03) and fat mass(r=-0.59, P=0.01) in human subjects after adjustment for age and sex. Furthermore, ZAG overexpression in mice reduced body weight and the percentage of epididymal fat. The decreased FAS, ACC1 and DGAT mRNA and the increased HSL mRNA were also observed in epididymal adipose tissue in ZAG overexpression mice. CONCLUSION: ZAG is closely linked to obesity. Serum ZAG level is inversely associated with body weight and percentage of body fat. The action of ZAG is associated with downregulated lipogenic enzymes and upregulated lipolytic enzyme expressions in adipose tissue of mice.

Downregulated lncRNA ADAMTS9-AS2 in breast cancer enhances tamoxifen resistance by activating microRNA-130a-5p.

OBJECTIVE: The aim of this study was to elucidate the regulatory effect of long non-coding RNA (lncRNA) ADAMTS9-AS2 on Tamoxifen (TAM) resistance in breast cancer (BC), and to explore its underlying mechanism. PATIENTS AND METHODS: TAM-resistant BC cell lines were first verified. Subsequently, cell proliferation and apoptosis were detected using cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) assay and flow cytometry, respectively. Protein levels of ABCB1, ABCC1, and ABCG2 in TAM-treated MCF-7 and MCF-7R cells were determined by Western blot. ADAMTS9-AS2 expression in BC tissues, para-cancerous tissues, as well as MCF-7 and MCF-7R cells, was accessed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The correlation between ADAMTS9-AS2 expression with pathological grade and tumor size of BC was explored. Chromatin fractionation was conducted to elucidate the subcellular distribution of ADAMTS9-AS2. The binding condition between ADAMTS9-AS2 and microRNA-130a-5p, as well as microRNA-130a-5p and PTEN, was verified by the dual-luciferase reporter gene assay. Furthermore, regulatory effects of ADAMTS9-AS2, microRNA-130a-5p, and PTEN on the proliferation and apoptosis of MCF-7R cells were determined. RESULTS: MCF-7R cells were identified with TAM resistance. ADAMTS9-AS2 was lowly expressed in BC tissues and MCF-7R cells. Particularly, ADAMTS9-AS2 expression in BC tissues with grade III-IV or tumor size ≥2 cm was significantly lower than that of controls. Dual-luciferase reporter gene assay confirmed the binding condition between ADAMTS9-AS2 and microRNA-130a-5p, showing a negative correlation indicated by Pearson correlation analysis. PTEN was positively correlated with ADAMTS9-AS2. Overexpression of ADAMTS9-AS2 reversed the increased viability and decreased apoptosis induced by microRNA-130a-5p mimics transfection. In addition, PTEN knockdown reversed the decreased viability and accelerated apoptosis caused by ADAMTS9-AS2 overexpression. CONCLUSIONS: We found that ADAMTS9-AS2 is lowly expressed in BC tissues and drug-resistant BC cells. Low expression of ADAMTS9-AS2 inhibits PTEN expression and enhances tamoxifen resistance through targeting microRNA-130a-5p.

Bacteriocins from lactic acid bacteria increases tumor necrosis factor-alpha expression in a rat kidney model of chronic rejection.

Chronic allograft dysfunction is the primary cause of graft loss after the first posttransplantation year. Bacteriocins are biologically active proteins exhibiting antimicrobial properties against other bacterial species, which are usually closely related to the producer organism. The objective of our study was to determine whether lactic acid bacterial bacteriocins were associated with tumor necrosis factor (TNF)-alpha observed in a rat kidney model of chronic rejection. Using a kidney model of chronic rejection in the rat, we administered cyclosporine (CsA) immunosuppression (5 mg/kg/d). One group of animals was treated with bacteriocins, and the other was left untreated. Grafts were harvested after transplantation for standard histological studies. The expression of TNF-alpha was demonstrated using immunohistochemistry of frozen sections of the grafts. We observed a greater increase in the expression of TNF-alpha among the group treated with bacteriocins compared with the untreated group. These results showed that lactic acid bacterial bacteriocins were associated with TNF-alpha in our kidney graft model.

TNFα-activated mesenchymal stromal cells promote breast cancer metastasis by recruiting CXCR2+ neutrophils.

Mesenchymal stromal cells (MSCs) tend to infiltrate into tumors and form a major component of the tumor microenvironment. Our previous work demonstrated that tumor necrosis factor α (TNFα)-activated MSCs significantly promoted tumor growth. However, the role of TNFα-treated MSCs in tumor metastasis remains elusive. Employing a lung metastasis model of murine breast cancer, we found that TNFα-activated MSCs strikingly enhanced tumor metastasis compared with normal MSCs. We analyzed the chemokine profiles and found that the expression of CCL5, CCR2 and CXCR2 ligands were enhanced in TNFα-activated MSCs. Using genetic or pharmacological strategies to inhibit CCL5 or CCR2, we demonstrated that CCL5 and CCR2 ligands were indispensable in supporting TNFα-activated MSCs to promote tumor metastasis. Analysis of immune cells revealed that CXCR2 ligands (CXCL1, CXCL 2 and CXCL5) expressed by TNFα-activated MSCs efficiently recruited CXCR2+ neutrophils into tumor. These neutrophils were responsible for the pro-metastatic effect of MSCs since inhibition of this chemotaxis abolished increased neutrophil recruitment and tumor metastasis. The interaction between neutrophils and tumor cells resulted in markedly elevated metastasis-related genes by tumor cells, including CXCR4, CXCR7, MMP12, MMP13, IL-6 and TGFß. Importantly, in IL8high human breast cancer samples, we also observed similar alterations of gene expression. Collectively, our findings demonstrate that TNFα-activated MSCs promote tumor metastasis via CXCR2+ neutrophil recruitment.

Downregulation of CXCL12 in mesenchymal stromal cells by TGFß promotes breast cancer metastasis.

Mesenchymal stromal cells (MSCs) are one of major components of the tumour microenvironment. Recent studies have shown that MSC tumour residence and their close interactions with inflammatory factors are important factors that affect tumour progression. Among tumour-associated inflammatory factors, transforming growth factor ß (TGFß) is regarded as a key determinant of malignancy. By employing a lung metastasis model of a murine breast cancer, we show here that the prometastatic effect of MSCs was dependent on their response to TGFß. Interestingly, we found that MSC-produced CXCL12, an important chemokine in tumour metastasis, was markedly inhibited by TGFß. Furthermore, silencing of CXCL12 in TGFß-unresponsive MSCs restored their ability to promote tumour metastasis. We found that 4T1 breast cancer cells expressed high levels of CXCR7, but not of CXCR4, both of which are CXCL12 receptors. In presence of CXCL12, CXCR7 expression on tumour cells was decreased. Indeed, when CXCR7 was silenced in breast cancer cells, their metastatic ability was inhibited. Therefore, our data demonstrated that sustained expression of CXCL12 by MSCs in the primary tumour site inhibits metastasis through reduction of CXCR7, while, in the presence of TGFß, this CXCL12 effect of MSCs on tumour cells is relieved. Importantly, elevated CXCR7 and depressed CXCL12 expression levels were prominent features of clinical breast cancer lesions and were related significantly with poor survival. Our findings reveal a novel mechanism of MSC effects on malignant cells through which crosstalk between MSCs and TGFß regulates tumour metastasis.

High rates of ras codon 61 mutation in thyroid tumors in an iodide-deficient area.

Using polymerase chain reaction and sequence-specific oligonucleotide hybridization, the frequency of three ras oncogene mutations (N-ras, Ha-ras, and K-ras) in thyroid tumors (25 adenomas, 16 follicular carcinomas, and 22 papillary carcinomas) was investigated in both iodide-deficient and iodide-sufficient areas. The ras oncogene mutation rate was significantly higher in the iodide-deficient area, being 85 versus 17% in the adenomas, and 50 versus 10% in the follicular carcinomas. No mutations were found in papillary carcinomas. The most common mutation site was Ha-ras codon 61 with Gln----Arg substitution. Two ras mutations at codon 61 (Gln----Lys in N-ras and Gln----Arg in Ha-ras) were found in a microfollicular adenoma specimen from Eastern Hungary. We conclude that dietary iodine may modulate ras oncogene mutations, and that in the iodide-deficient area, ras oncogene activation may play a more important role in the initiation and/or maintenance of follicular tumors. Additional factors are, however, necessary to initiate carcinogenesis.