Network pharmacological and molecular docking verification of the mechanism of Osteoking in preventing deep vein thrombosis of lower limb.

OBJECTIVE: The aim of this study was to predict the mechanism of Osteoking in preventing deep vein thrombosis (DVT) of the lower limb by network pharmacology and molecular docking. MATERIALS AND METHODS: The relevant active components and targets of Osteoking were collected through the TCMSP database, and the relevant disease targets of DVT were collected through the GeneCards, OMIM, and DisGeNET databases. The intersecting gene targets of Osteoking and DVT were obtained using Venny 2.1.0 software. PPI network construction and core target selection using Cytoscape 3.9.0 software. The Metascape database was used for GO and KEGG enrichment analysis of relevant targets. Finally, the molecular docking of the main active components and key targets was carried out. RESULTS: There are 361 potential targets and 71 core targets of Osteoking in preventing deep vein thrombosis of the lower limb. Signal pathways are involved in various diseases such as cancer, diabetic complications, atherosclerosis, and more. Some of the most common pathways include AGE-RAGE signaling pathway and Calcium signaling pathway. Molecular docking results showed that the main active components of Osteoking had relatively stable binding activities with the key targets. CONCLUSIONS: Osteoking can play a role through multiple targets and multiple signal pathways to prevent the formation of deep venous thrombosis of the lower limb after fracture.

[Lymphoplasmacytic lymphoma: a clinicopathological and prognostic analysis of 27 cases].

Objective: To study the clinical manifestations, pathologic features, diagnosis and differential diagnosis, treatment and prognosis of lymphoplasmacytic lymphoma/Waldenström's macroglobulinemia (LPL/WM). Methods: Twenty-seven cases of LPL from January 2016 to December 2020 at Guangdong Provincial People's Hospital were collected. The clinical data, histomorphology, immunophenotype, MYD88 L265P mutation, treatment and prognosis were analyzed retrospectively. Results: There were 19 males and 8 female patients, with median age of 63 years. The most common initial symptoms were fatigue related to anemia. Bone marrow was involved in all cases, lymphadenopathy was seen in 11 cases and splenomegaly in 10 cases. Monoclonal IgM type protein was detected in 25 cases, meeting the diagnostic criteria of WM. Microscopically, bone marrow and lymph nodes were infiltrated by small lymphocytes, plasmacytoid lymphocytes or plasma cells. The cells expressed pan B-cell markers and showed immunoglobulin light chain restriction. There was no expression of CD5, and low expression of CD23 and CD10; Ki-67 index was usually low. The positive rate of MYD88 L265P mutation was 73.9% (17/23). Most of the patients were treated with rituximab combined with alkylating agents, nucleoside analogues or immunomodulators, and the few patients with relapse or progression were treated with Ibutinib. During the 3-168 months' follow-up period, recurrence or progression were seen in nine cases. Thrombocytopenia, elevated ß2-microglobulin and high-risk group were associated with recurrence or progression of the disease (P<0.05). The overall survival (OS) and progression-free survival (PFS) of the high-risk patients were significantly lower than those of the low-medium risk patients (P<0.05). Conclusions: LPL/WM is an exclusive diagnosis; the detection of MYD88 L265P mutation has high diagnostic value, but it is not specific. These cases should be assessed comprehensively for their clinical manifestation, serum IgM protein level and immunophenotype. The overall prognosis of LPL/WM is good, but there are still a small number of high-risk patients with rapid progress, and so the symptomatic patients should be diagnosed accurately and treated in a timely manner.

[Effects of immunohistochemical conditions on the results of PD-L1 (22C3) staining].

Objective: To investigate the optimal experimental conditions (including antigen retrieval time, antibody titers and antibody incubation time) for reliable detection of programmed death-ligand 1 (PD-L1) expression using PD-L1 (22C3) antibody concentrate, and to establish a laboratory developed test for PD-L1 detection. Methods: Using Dako PD-L1 IHC 22C3 pharmDX staining procedure and scoring guidelines as the standard reference (group A), the PD-L1 expression in 25 tissue specimens (including 15 lung cancer tissues, 5 tonsil tissues and 5 placenta tissues) was detected with Flex+/HRP detection kit (EnVision) under 8 different experimental conditions (groups B1 to B8). The staining results were then compared to those in group A. Results: In group B1, 3 tissue samples showed the percentages of PD-L1 positive tumor cells were similar to those in group A, while the percentages of PD-L1 positive tumor cells were lower than those in group A in the other samples. In group B7, two case showed a positive rate higher than that in group A that was also above the positive cut-off value, and the rest of the samples had a percentage of PD-L1 positive tumor cells slightly higher than that in group A, but still below the positive cut-off value. The staining results of group B8 were the closest to those of group A compared with the other groups. Although the percentages of PD-L1 positive tumor cells in the B2 to B6 groups were decreased in various degrees as compared with group A, they were still concordant with group A's classification (positive vs. negative) and would not change the choice of clinical treatments. Conclusions: The experimental conditions are associated with detection rate of PD-L1 expression using 22C3 antibody. In the present study, the most-suitable alterative conditions in the PD-L1 detection using 22C3 antibody concentrate are those applied in the group B8 (including antigen retrieval in Dako PT Link tank at 97 ℃, pH 6.0 for 40 min and incubation with 22C3 antibodies (1∶100 dilution) at room temperature for 60 min, incubation with EnVision Flex+Linker at room temperature for 30 min, incubation with EnVision/HRP at room temperature for 30 min and DAB staining for 5 min), which could provide reliable results at minimum costs.

[Clinicopathological features of fibrin-associated diffuse large B cell lymphoma arising within concurrent atrial myxoma: report of six cases].

Objective: To investigate the clinicopathological features, treatment and prognosis of fibrin-associated diffuse large B cell lymphoma (DLBCL) arising within concurrent atrial myxoma. Methods: Six cases of fibrin-associated DLBCL arising within concurrent atrial myxoma diagnosed at the Department of Pathology, Guangdong General Hospital, from 2006 to 2019 were included. The histology, immunophenotype, treatment and prognoses were analyzed. Results: The patients' age ranged from 46 to 78 years (mean 59 years). There were 3 males and 3 females. The tumors were all discovered incidentally on histological examination of surgical pathology specimens excised for atrial myxoma. All patients appeared to have morphological features of DLBCL, B lineage immunophenotype, high proliferative index and latency type III of Epstein-Barr viral infection. They had complete tumor resections without adjuvant chemotherapy and were healthy at 5- to 120-month follow-ups. Conclusions: Fibrin-associated DLBCL arising within concurrent atrial myxoma is an unusual form of DLBCL associated with chronic inflammation, and its clinical outcome is indolent. The findings suggest that this type of lymphoma does not warrant excessive or unnecessary treatments after complete resection.

[Clinical features of patients with hypertrophic obstructive cardiomyopathy combining obstructive sleep apnea].

Objective: To investigate the clinical features of patients with hypertrophic obstructive cardiomyopathy (HOCM) combined with obstructive sleep apnea (OSA). Methods: From 2010 to 2018, a total of 299 patients who were diagnosed with hypertrophic cardiomyopathy and underwent sleep monitoring at Fuwai Hospital were retrospectively analyzed. General clinical features, data of echocardiography, and sleep breathing parameters were recorded. OSA was diagnosed by apnea-hypopnea index ≥ 5 events/hour. Clinical characteristics were compared between patients with and without OSA. Results: A total of 156 (52.2%) HOCM patients were diagnosed with OSA. Compared with patients without OSA, patients with OSA were older((54±10) years vs (45±14) years, P<0.001), had a higher body mass index ((27±3) kg/m(2) vs (25±3) kg/m(2), P<0.001), a higher prevalence of hypertension (54.4% (85/156) vs 21.0% (30/143), P<0.001), hyperlipidemia (37.2% (58/156) vs 13.3% (19/143), P<0.001) and smoking history (48.1% (75/156) vs 35.0% (50/143), P=0.022). Patients with OSA also had a higher incidence of New York Heart Association functional class Ⅱ or Ⅲ (P=0.017), atrial fibrillation (P=0.005), and higher levels of systolic and diastolic blood pressure, fast glucose and high-sensitive c-reactive protein (all P<0.001). Left ventricular end-diastolic diameter as well as ascending aorta diameter in patients with OSA were also greater than those without OSA (both P<0.001). Apnea-hypopnea index (AHI) value positively correlated with left ventricular end-diastolic diameter (r=0.346), ascending aorta diameter (r=0.357) and high-sensitive c-reactive protein (r=0.230) (all P<0.001). Conclusions: A high prevalence of OSA occurs in patients with HOCM. Severity of OSA correlates with cardiac remodeling and serum inflammatory factor level. As for HOCM patients, clinicians should actively monitor the sleep breathing parameters in order to recognize and treat potential OSA, thereby improving the prognosis of patients with HOCM.

[Analysis of follow-up results of chrono-chemotherapy or conventional chemotherapy combined with intensity modulated radiotherapy in locally advanced nasopharyngeal carcinoma].

Objective: To evaluate the long-term effect and safety of chrono-chemotherapy combined with intensity modulated radiotherapy (IMRT) in locally advanced nasopharyngeal carcinoma (NPC). Methods: 160 patients with locally advanced NPC were randomly divided into a chrono group and conventional group according to random number table. In the first stage, all patients underwent two cycles of induced chemotherapy, consisting of docetaxel, cisplatin and 5-Fu every 21 days. Notably, patients received chrono-moduated chemotherapy according to circadian rhythm in the chrono group, and conventional chemotherapy in the conventional group. Then, 21 days after the completion of first stage, three cycles of concurrent cisplatin chemotherapy every 21 days were given to all patients during IMRT. The median follow-up after the completion of radiotherapy was 31 months. Long-term side effects and the survival of patients were observed. Results: Patients in the chrono group had significantly lower rates of hearing loss (22.72%), dysphagia (0) and neck fibrosis (4.54%) compared with those in the conventional group (39.13%、8.69%, 15.94%, respectively, all P<0.05). Meanwhile, the 1- year overall survival rates (97.0% vs 92.8%), 3-year overall survival rates (80.3% vs 81.2%), 1-year progression free survival rates (95.5% vs 87.0%), 3-year progression free survival rates (71.2% vs 73.9%), 1-year locoregional relapse-free survival rates (97.0% vs 95.7%), 1-year locoregional relapse-free survival rates (92.4% vs 92.8%), 1-year distant metastasis-free survival rates (97.0% vs 98.6%) and 3-year distant metastasis-free survival rates (90.9% vs 91.3%) between the chrono group and the conventional group were not statistically significant (all P>0.05). Conclusions: Compared with conventional chemotherapy, chrono-chemotherapy combined with IMRT didn't affect long-term survival, but reducing the incidence of adverse events in patients with locally advanced NPC.

[Clinicopathological features of primary cardiac CD5-positive and bcl-2 and C-MYC double expression diffuse large B-cell lymphoma].

Objectives: To investigate the clinicopathological features, therapy and prognosis of primary cardiac CD5-positive diffuse large B-cell lymphoma with C-MYC and bcl-2 double expression. Methods: Two cases diagnosed at Guangdong Provincial People's Hospital were included, the clinical data were collected; the tumor morphology, immunophenotypic profiles, therapy and prognosis were analyzed. Results: Case 1 was a 55-year-old man and case 2 was a 61-year-old women. Intraoperatively, both cases showed large masses in the right atrium or ventricle, involving adjacent tissue. Pathologically, the tumors were composed of diffusely infiltrating large lymphoid cells with high mitotic activity and apoptosis. The tumor cells were positive for CD20, CD5, bcl-6, MUM1, C-MYC and bcl-2, and the Ki-67 index was equal or greater than 90%. Case 1 had bcl-6, but not bcl-2 or MYC gene rearrangements. No MYC, bcl-2 or bcl-6 gene rearrangements were detected in case 2. Case 1 defaulted chemotherapy after operation and died 1 month after diagnosis. Case 2 was treated with 4 cycles of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) therapy after surgery and attained partial remission, and was then treated with apatinib and ibrutinib, and remained stable 18 months after initial diagnosis. Conclusion: Primary cardiac CD5-positive diffuse large B-cell lymphoma with C-MYC and bcl-2 double expression usually shows large infiltrative mass in the right atrium or ventricle, non-germinal center like immunophenotype and high proliferation index, and this may contribute to the aggressiveness of primary cardiac lymphoma.

MiR-27a alleviates osteoarthritis in rabbits via inhibiting inflammation.

OBJECTIVE: To investigate the regulatory effect of micro ribonucleic acid-27a (miR-27a) on the nuclear factor-kappa B (NF-κB) pathway and to explore its effect on rabbits with osteoarthritis (OA). MATERIALS AND METHODS: Anterior cruciate ligament (ACL) cross-section method was adopted to establish OA rabbit models. Cartilage specimens were collected to detect expression levels of miR-27a in OA cartilage and normal cartilage tissues. Meanwhile, chondrocytes were isolated and cultured, and transfected with miR-27a mimics and miR-27a inhibitor. Blank control group was set up. Next, the changes in chondrocyte proliferation were detected using 5-ethynyl-2'-deoxyuridine (EdU) staining and cell counting kit-8 (CCK-8). Quantitative Real Time Polymerase Chain Reaction (PCR) was applied to detect the messenger RNA (mRNA) expression of inflammatory factors interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) in chondrocytes. Also, Western blot was adopted to detect the differential expression of NF-κB pathway-related proteins NF-κB and matrix metalloproteinase 13 (MMP-13). RESULTS: Compared with that in normal cartilage tissues, miR-27a in OA cartilage tissues was decreased evidently (p<0.05). The expression level of miR-27a was higher in miR-27a mimics group than in control group, while it significantly declined in miR-27a inhibitor group (p<0.05). EdU staining and CCK-8 method results showed that miR-27a mimics could promote the proliferation of chondrocytes, while miR-27a inhibitor inhibited the proliferation of chondrocytes. Compared with those in control group, the expression levels of inflammatory factors TNF-α and IL-6 in chondrocytes in miR-27a inhibitor group were increased significantly (p<0.05). MiR-27a mimics could evidently reduce the expression of inflammatory factor IL-6 (p<0.05), but did not significantly reduce the expression of TNF-α. Besides, the results of Western blot suggested that the expression levels of MMP-13 and NF-κB proteins were decreased significantly in miR-27a mimics group (p<0.05) and increased significantly in miR-27a inhibitor group (p<0.05). CONCLUSIONS: MiR-27a in OA cartilage tissues is evidently lower than in normal cartilage tissues. Transfection of miR-27a mimics can promote proliferation of chondrocytes, lower the expression of inflammatory factors, and reduce the expression of MMP-13 and NF-κB proteins. Therefore, the up-regulation of miR-27a can benefit the treatment of bone joints through the NF-κB pathway.

[Diagnostic and prognostic implications of MAML2 gene translocation in primary pulmonary mucoepidermoid carcinoma].

Objective: To investigate MAML2 gene-translocation in primary pulmonary mucoepidermoid carcinoma (PMEC) and pulmanary adenosquamous carcinoma, and the optimal diagnostic immunohistiochemical (IHC) panel in distinguishing PMEC from adenosqumous carcinoma. Methods: Twenty-four cases of PMEC and 44 adenosqumous carcinoma diagnosed in the Guangdong General Hospital were tested for MAML2 translocation by fluorescent in-situ hybridization (FISH) using tissue array. An IHC panel including TTF1, Napsin A, CK5/6, p63, p40 and Ki-67 was performed on the cohort. The clinical data for all cases were collected and all PMEC patients had follow-up information. Results: The patients' age ranged form 6 to 73 years, with a median age of 32 years. The male to female ratio was 1.4∶1.0. MAML2 translocation was found in 16/24 (66.7%) cases of PMEC whereas all 44 cases adenosqumous carcinoma were negative for translocation. All the cases of the PMEC were negative for TTF1 and Napsin A but positive for CK5/6, p63 and p40 in the intermediate cells and epidermal-like cells. In most PMEC cases, the Ki-67 expression index was lower than 10%. In contrast, most cases of adenosqumous carcinomas expressed TTF1 and Napsin A in the adenomatous component and CK5/6, p63 and p40 in the squamous component, which expression pattern was different from that of PMEC. Based on IHC staining, 2 cases of highly invasive ALK-positive adenocarcinoma mimicing PMEC were also found in the study. Conclusions: MAML2 gene translocation can be detected in about two-third of PMEC. Translocation of MAML2 gene and lower morphology grading are associated with good prognosis. The combined use of IHC antibodies panel is helpful to distinguish PMEC from the adenosqumous carcinoma and adenocarcinoma mimicing PMEC.

[Pathological features of duodenal-type follicular lymphoma].

Objective: To investigate the clinicopathological features, treatment and prognosis of duodenal-type follicular lymphoma. Methods: Four cases of duodenal-type follicular lymphoma diagnosed at Guangdong General Hospital from 2014 to 2015 with detailed clinical data were included. The histomorphology, immunophenotype, treatment and prognoses were analyzed. Results: The patients' age ranged from 51 to 57 years (mean 54 years), and there were 2 males and 2 females. The involved sites were gastric fundus in one case, second portion of the duodenum in two cases and terminal ileum in one case. All patients presented with multiple mucosal granules or nodules at endoscopy. Microscopically, there were multiple mucosal neoplastic follicles, constituting grade 1-2 disease based on nodal follicular lymphoma grading system. The tumor cells were positive for CD20, CD10, bcl-6 and bcl-2. CD21 highlighted the follicular dendritic meshwork mainly at the periphery of the follicles. Proliferation index was low. Three patients received rituximab monotherapy for 4 cycles, leading to complete remission. One patient refused therapy and the disease progressed to systemic lymphoma 15 months after the initial diagnosis. Conclusions: Duodenal-type follicular lymphoma is a special variant of follicular lymphoma with indolent clinical course. The tumor exhibits morphology of low grade follicular lymphoma with characteristic dendritic meshwork at the periphery of the follicles and a low proliferation index. Prognosis is excellent. Rituximab monotherapy is treatment of choice, but a small minority of patients may progress to systemic disease.

[Detection and application of bcl-2/IgH gene translocation and immunoglobulin gene rearrangement in follicular lymphoma].

Objective: To evaluate the application of FISH testing of bcl-2/IgH gene translocation and IgH/L gene rearrangement in different stages of follicular lymphoma. Methods: In 32 follicular lymphoma cases, which were collected at Guangdong General Hospital from September 2014 to December 2016, the bcl-2/IgH gene ectopic state was detected by FISH while the IgH/L gene rearrangement was tested using PCR-GeneScan to analyze the relationship between bcl-2/IgH gene translocation, different stages of follicular lymphoma and clonal immunoglobulin (IgH/L) gene rearrangements. Results: From the paraffin sections of all 32 follicular lymphomas, 17 cases showed bcl-2/IgH gene translocation, and the percentages of FL1, FL2 and FL3 translocation were 12/13, 3/5 and 2/14, respectively. Among the 24 cases of IgH/L gene arrangements identified from the total sample, the occurrence rates of FL1, FL2 and FL3 gene arrangement were 7/13, 4/5 and 13/14, respectively. Spearman's rank correlation analysis and χ(2) analysis showed that bcl-2/IgH gene translocation was negatively correlated with follicular lymphoma stage and the association was statistically significant. In more advanced stages of follicular lymphoma, the occurrence of bcl-2/IgH gene translocation tended to decrease with distinct FL1, FL2 and Fl3 gene expression (P<0.05). As IgH/L gene rearrangement in FL3 was higher than that in FL1 and FL2, its detection may be complimentary to FISH test for bcl-2/IgH gene translocation in diagnosing follicular lymphoma. Conclusions: The combined use of FISH and PCR-GeneScan increases the positive rate of follicular lymphoma diagnosis, and this combination is more sensitive than FISH or clonal analysis only to detect the chromosomal abnormality or the gene rearrangement.

[Expression of ßF1 and T cell receptor γ in T lymphoblastic lymphoma/leukemia].

Objective: To evaluate the expression of ßF1 and T cell receptor (TCR)γ in T lymphoblastic lymphoma/leukemia(T-LBL/ALL), and investigate the clinicopathological features. Methods: Fifty-one cases of T-LBL/ALL were collected at Guangdong General Hospital from 2010 to 2016, the expression of ßF1 and TCRγ was assessed by immunohistochemistry. Results: There were 13 cases of children and adolescents, and 38 cases of adults. The expression rates of ßF1 and TCRγ were 27.5%(14/51) and 15.7%(8/51) respectively. The proportion of adults in αß T-LBL/ALL, TCR-silent T-LBL/ALL and γδ T-LBL/ALL was 7/14, 79.3%(23/29)and 8/8 respectively, and the difference was significant (P=0.023). There was no statistical difference in sex, LDH, bone marrow involvement and Ann arbor stage among these three groups(P>0.05). γδ T-LBL/ALLs included 6 cases of CD4(-)/CD8(-) phenotype, whereas αß T-LBL/ALL included 7 cases of CD4(+) /CD8(+) phenotype. There was significant difference in CD4/CD8 expression among these three groups(P<0.01). Conclusions: γδ T-LBL/ALL occurred only in adults, with predominantly CD4(-)/CD8(-) phenotype. αß T-LBL/ALL occurred more common in children and adolescents, with predominantly CD4(+) /CD8(+) phenotype.

Environment-stimulated nanocarriers enabling multi-active sites for high drug encapsulation as an "on demand" drug release system.

Limited active sites in polyesters hinder fabrication of multifunctional biodegradable nanocarriers for successful clinical applications. Herein, poly(malic acid) (PMA)-based biodegradable polyesters bearing large carboxyl groups in their side chains were grafted with intracellular reductive-sensitive polyethylene glycol and imidazole to construct bioreducible nanocarriers (PLM-g-ss-EGA). The uniform spherical shape and high stability of the PLM-g-ss-EGA nanocarriers were demonstrated by dynamic light scattering (DLS) and dissipative particle dynamics (DPD) simulations. Enhanced interaction between the monomers in this novel nanocarrier doubled its drug loading efficiency (15%) as compared to that of traditional polyester nanocarriers (5-7%). Moreover, stimulus-responsive assessment and in vitro drug release studies showed that these bioreducible nanocarriers can balance extracellular stability in blood circulation and intracellular "on demand" release. In vitro and in vivo assays have demonstrated that these bioreducible nanocarriers not only can substantially enhance antitumor efficacy as compared to insensitive micelles and even comparably to free DOX·HCl, but can also greatly reduce unwanted side effects in other organs. The encouraging anticancer efficiency of these poly(malic acid)-based nanocarriers opens a new avenue to design multifunctional biodegradable polyester drug-delivery systems.

Prognostic significance of downregulated BMAL1 and upregulated Ki-67 proteins in nasopharyngeal carcinoma.

This study assessed the prognostic value of BMAL1 and Ki-67 expression in patients with nasopharyngeal carcinoma. Level of BMAL1 mRNA was assessed in tissue specimens from 36 nasopharyngeal carcinomas and 20 nasopharyngeal chronic inflammations using quantitative reverse transcriptase-polymerase chain reaction. Expression of BMAL1 and Ki-67 proteins was analyzed immunohistochemically in 90 paired nasopharyngeal carcinoma and distant normal tissues. The Kaplan-Meier curves and the Log-rank test were used to calculate prognostic significance stratified by BMAL1 and Ki67 protein expression and the COX regression model was to analyze the multivariate prognosis. BMAL1 mRNA was significantly reduced in nasopharyngeal carcinoma (4.67 ± 0.27 versus 6.64 ± 0.51 in chronic inflammation tissues, p = 0.002). Level of BMAL1 mRNA was associated with tumor distant metastasis (3.37 ± 0.66 versus 5.04 ± 0.27 compared with non-metastasis, p = 0.011). Level of BMAL1 protein was also reduced in tumor tissues and BMAL1 expression was associated with better 1-, 3- and 5-year overall survival (OS) of cancer patients (92.6%, 69.2% and 62.3% versus 59.1%, 40.9% and 0% in patients with low BMAL1 expressed tumors; p = 0.000). BMAL1 expression and age were independent prognostic factors for OS (p = 0.032). Furthermore, Ki-67 expression was high in tumor versus normal tissues and associated with poor OS of cancer patients (p = 0.035). The Pearson correlation analysis showed that there was an inverse association between BMAL1 and Ki-67 protein expression (p = 0.021). This study demonstrated that lost BMAL1 and Ki-67 overexpression were associated with poor OS of nasopharyngeal carcinoma patients.

[Experimental study on the immune response of fusion tumor vaccine of HepG2 and dendritic cells in vitro].

Objective: To estimate the immune response of HepG2/dendritic cell (DC) fusion cells vaccines against HepG2 cells in vitro. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by Ficoll-Hypaque density-gradient centrifugation.Then DC were obtain from PBMCs by culturing in medium containing granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 5 days.DC and HepG2 fusion cells were induced by polythyleneglycol (PEG). The fusion cells were examined under fluorescence microscope by labeling DCs and HepG2 with green and red fluorescein, respectively, and then the fusion rates were analyzed by flow cytometry.The capacity of fusion cells to secrete interleukin (IL)-12 and stimulate the proliferation of T lymphocyte was assessed by ELISA and Flow cytometry, respectively.ELISPOT was used to assess the interferon gamma (IFN-γ) produced by cytotoxicity T lymphocyte (CTL), and the specific killing ability of fusion cells induce-CTL targeting HepG2 was estimated. Results: The fusion rate of HepG2/DC was 54.5%, and the fusion cells expressed a higher levels of DC mature marker CD80 and costimulatory molecules CD83, CD86 and MHC-Ⅰ, MHC-Ⅱ molecules HLA-ABC and HLA-DR than those in immature DCs (P<0.01). HepG2/DC showed a greater capacity to secrete high level of IL-12 (P<0.05) and activate proliferation of lymphocytes in vitro, as compared with DCs alone and DCs mix HepG2 (P<0.01). The HepG2/DC -activated CTL generated higher IFN-γ level and had a specific killing ability against HepG2 cells at the effecter/target ratio 30∶1 (31.4%±2.4%) and 100∶1 (57.6%±7.3%) (P<0.01). Conclusions: HepG2/DC fusion cells could efficiently stimulate T lymphocytes to generate specific CTL targeting HepG2 cells.It might be a promising strategy of immunotherapy for HCC.

MicroRNA miR-590-5p inhibits breast cancer cell stemness and metastasis by targeting SOX2.

OBJECTIVE: SOX2 (Sry-related high-mobility box SOX-2) is a transcription factor, which is essential for maintaining the cancer cell stemness. However, the role of microRNAs targeting  SOX2 in cancer cell stemness remains unclear. We examined the effect of miR-590-5p, which targeted  SOX2, on the breast cancer cell stemness and metastasis. MATERIALS AND METHODS: We predicted and screened microRNA targeting SOX2, and further investigated the regulatory role of miR-590-5p on the level of SOX2 with Western blot, luciferase reporting assay and qRT-PCR analysis. Flow cytometry was performed to detect the effect of miR-590-5p on the breast cancer stem cell population with ALDEFLUOR Assay. We inoculated the breast cancer cells transfected with or without miR-590-5p to NOD/SCID mice to detect the tumorigenicity in vivo. Finally, forty-nine pairs of breast cancer samples and adjacent noncancerous tissues were obtained, and immunohistochemistry (IHC) with SOX2 antibody and qRT-PCR assay were used to quantify the expression of miR-590-5p in breast cancer samples. RESULTS: miR-590-5p significantly downregulated the SOX2 protein expression, and inhibition of miR-590-5p increased SOX2 expression. The luciferase reporter assay indicated that miR-590-5p decreased the SOX2 3'UTR (3' untranslated region) reporter activity but not the luciferase activity of the mutant reporter, in which the binding sites for miR-590-5p were mutated. ALDEFLUOR Assay showed that miR-590-5p significantly decreased breast cancer stem cells population. NOD/SCID nude mice experiments indicated that miR-590-5p significantly inhibited tumorigenicity of breast cancer cells. IHC assay and qRT-PCR suggested that miR-590-5p expression was downregulated in breast cancer patients, and negatively correlated with SOX2. CONCLUSIONS: miR-590-5p inhibited breast cancer cell stemness through targeting SOX2. Our study indicated that miR-590-5p might be a useful strategy for breast cancer treatment.

The role of Schwann cell differentiation in perineural invasion of adenoid cystic and mucoepidermoid carcinoma of the salivary glands.

Twenty cases of adenoid cystic carcinoma (ACC) and 18 cases of mucoepidermoid carcinoma (MEC), were examined for expression of the Schwann cell markers S100 protein and glial fibrillary acidic protein (GFAP) by immunohistochemical staining. The relationship between expression of S100 and GFAP and the occurrence of perineural invasion was assessed. Ultrastructural localization of S100 and GFAP was examined by immunoelectron microscopy, and the co-expression of S100 and muscle actin by double fluorescence immunostain. Perineural invasion was found in 11 ACCs (55%) and 0 MECs (0%). S100 and GFAP were expressed in most of the ACCs but none of the MECs; the difference in the rate of perineural invasion and expression of S100 and GFAP was significant between ACC and MEC (P<0.001). There was a correlation between the expression of S100 and GFAP and perineural invasion in salivary malignancy (P<0.001). The ultrastructures of S100- and GFAP-positive cells were consistent with the characteristics of myoepithelial cells. Double fluorescence immunostain also showed that S100 and muscle actin were expressed in the same type of ACC cells. These results indicate that Schwann cell differentiation correlates with perineural invasion in salivary malignancy, and occurs in modified myoepithelial cells of ACC.