A novel redox/pH dual-responsive and hyaluronic acid-decorated multifunctional magnetic complex micelle for targeted gambogic acid delivery for the treatment of triple negative breast cancer.

Gambogic acid (GA) is a naturally derived potent anticancer agent with extremely poor biocompatibility. In the present study, a novel of redox/pH dual-responsive multifunctional magnetic complex micelle (sPEG/HA/CSO-SS-Hex/Fe3O4/GA), which consisted of a reducible hexadecanol-modified chitosan oligosaccharide polymer micelle (CSO-SS-Hex) coated with hyaluronic acid (HA) and DCA grafted sheddable PEG-PLL (sPEG) copolymers and loaded with gambogic acid (GA) and Fe3O4 nanoparticles were developed for parenteral delivery for the treatment of triple negative breast cancer (TNBC). The ex vivo study showed that the sPEG shielded cationic HA/CSO-SS-Hex/Fe3O4/GA core at physiological pH but quickly shed off to re-expose the core due to its charge reversible property. The sPEG/HA/CSO-SS-Hex/Fe3O4/GA micelles effectively facilitated tumor-targeted GA delivery by HA, which is a targeting ligand for CD44 receptor of TNBC cells, meanwhile increase GA uptake at the acidic condition but diminished the drug uptake at neutral pH. The in vitro cellular uptake study and in vivo biodistribution and antitumor activity of the formulations were determined, all results showed that the complex micelle enhanced TNBC tumor cellular uptake and fast drug release due to the combined effect of magnet targeting, CD44 receptor-mediated internalization and redox/pH dual-responsive drug release. Hence, tumor-targeted delivery of GA with redox/pH dual-responsive multifunctional magnetic complex micelle sPEG/HA/CSO-SS-Hex/Fe3O4/GA might have potential implications for the chemotherapy of TNBC.

Clinical Significance of CD163+ and CD68+ Tumor-associated Macrophages in High-risk HPV-related Cervical Cancer.

Objective. To explore the influence of M2-polarized tumor-associated macrophages (TAMs) on high-risk human papillomavirus (hr-HPV)-related cervical carcinogenesis and metastasis. Methods. CD68+ and CD163+ macrophages were examined immunohistochemically in a series of 130 samples, including 26 cases of normal cervical tissues, 59 cases of cervical intraepithelial neoplasia (CIN), and 45 cases of squamous cell carcinoma (SCC), and the results were statistically analyzed. The macrophage count was corrected for the epithelial and stromal compartments respectively. Clinical data were also obtained. Results. High counts of CD68+ and CD163+ macrophages were associated with hr-HPV infection (both p < 0.05) and positively correlated with cervical carcinogenesis (Spearman's rho = 0.478, p = 0.000; Spearman's rho = 0.676, p =0.000, respectively). The immunostaining pattern of CD163 exhibited clearer background than that of CD68. CD163+ macrophages showed a more obviously increasing migration into the epithelium along with the progression of CIN to invasive cancer. Notably, a high index of CD163+ macrophages was significantly associated with higher FIGO stages (p = 0.009) and lymph node metastasis (p = 0.012), but a similar finding was not found for CD68+ macrophages (p = 0.067, p = 0.079, respectively). Conclusions. Our study supported a critical role of TAMs as a prospective predictor for hr-HPV-related cervical carcinogenesis. CD163, as a promising TAMs marker, is superior to CD68 for predicting the malignant transformation and metastatic potential of cervical cancer.

Orthotopic Xenograft Mouse Model of Cervical Cancer for Studying the Role of MicroRNA-21 in Promoting Lymph Node Metastasis.

Cervical cancer is the most frequent cause of gynecologic cancer-associated death worldwide. Animal models that demonstrate metastatic patterns consistent with the clinical course of cervical cancer are urgently needed to conduct studies focused on understanding the mechanisms of the disease and identifying optimal treatments. To address this, we established an orthotopic xenograft model of cervical cancer in female NOD-SCID mice using SiHa and ME180 cell lines stably expressing green fluorescent protein to evaluate the role of microRNA-21 (miR-21) in spontaneous lymph node metastasis in vivo. In this case, SiHa and ME180 cells were transduced by lentivirus to stably express green fluorescent protein and miR-21. Overexpression of miR-21 promoted proliferation, migration, and invasion of SiHa and ME180 cells in vitro. Finally, an orthotopic xenograft model of human cervical cancer was successfully established in NOD-SCID mice. Using this model, we confirmed that overexpression of miR-21 resulted in an increase in the size of primary tumors and in the frequency of spontaneous lymph node metastasis at the time of excision. Therefore, the use of the orthotopic xenograft model should allow for the investigation of novel factors that affect metastasis of cervical cancer and presents an opportunity to evaluate potential therapeutic agents that may inhibit the spread of the disease.

HPV16 integration probably contributes to cervical oncogenesis through interrupting tumor suppressor genes and inducing chromosome instability.

BACKGROUND: The integration of human papilloma virus (HPV) into host genome is one of the critical steps that lead to the progression of precancerous lesion into cancer. However, the mechanisms and consequences of such integration events are poorly understood. This study aims to explore those questions by studying high risk HPV16 integration in women with cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (SCC). METHODS: Specifically, HPV integration status of 13 HPV16-infected patients were investigated by ligation-mediated PCR (DIPS-PCR) followed by DNA sequencing. RESULTS: In total, 8 HPV16 integration sites were identified inside or around genes associated with cancer development. In particular, the well-studied tumor suppressor genes SCAI was found to be integrated by HPV16, which would likely disrupt its expression and therefore facilitate the migration of tumor. On top of that, we observed several cases of chromosome translocation events coincide with HPV integration, which suggests the existence of chromosome instability. Additionally, short overlapping sequences were observed between viral derived and host derived fragments in viral-cellular junctions, indicating that integration was mediated by micro homology-mediated DNA repair pathway. CONCLUSIONS: Overall, our study suggests a model in which HPV16 might contribute to oncogenesis not only by disrupting tumor suppressor genes, but also by inducing chromosome instability.

Accuracy of microRNAs for the diagnosis of hepatocellular carcinoma: A systematic review and meta-analysis.

Due to the high morbidity, mortality and late detection of hepatocellular carcinoma (HCC), it becomes a major public health challenge. MicroRNAs (miRNAs) have been reported to be aberrantly expressed in patients with HCC and thus may serve as potential diagnostic biomarkers. The aim of this study was to systematically assess the diagnostic accuracy of miRNAs as biomarkers for diagnosing HCC through a meta-analysis. Our results indicated that serum miRNAs had a relatively high diagnostic value for HCC diagnosis; the combination of serum α-fetoprotein (AFP) and miRNAs displayed a better diagnostic accuracy than using AFP or miRNAs alone; the combination of multiple miRNAs assay in serum had the highest diagnostic accuracy in HCC diagnosis based on the published data. In conclusion, our meta-analysis suggests that miRNAs, especially serum miRNAs, had a relatively high diagnostic value for HCC diagnosis, which can discriminate HCC from healthy subjects and those with chronic hepatitis and cirrhosis easily, and may serve as a novel, less-invasive screening tool.

Serological and molecular study of hepatitis E virus among illegal blood donors.

AIM: To investigate the seroprevalence and molecular characteristics of hepatitis E virus (HEV) in the illegal blood donors (IBDs) of central China in the early 1990s. METHODS: A total of 546 blood samples were collected from the IBDs in Maanshan city, a questionnaire was completed by each subject, detailing the age, sex, and periods of blood or plasma donation. Anhui Province and tested for the anti-HEV antibodies. The seropositive samples were subjected to nested reverse transcription-polymerase chain reaction and sequencing to analyze HEV partial genome. RESULTS: The prevalence of IgG and IgM HEV antibody in IBDs was 22.7% and 1.8%, and genotype 4 was the dominant circulating HEV type in IBDs. The prevalence of anti-HEV IgG was significantly related to sex (OR = 4.905, P = 0.004) and increased with age (OR = 2.78, P = 0.022), which ranged from 13.0% in those < 40 years old to 30.6% among older persons aged > 60 years. Moreover, frequency of blood donation was significantly associated with HEV seropositivity (OR = 2.06, P = 0.006). HEV partial sequences of ORF2 and obtained 3 sequences in serum samples of 10 IBDs which developed HEV specific IgM. CONCLUSION: This study helps define one of the possible routes of transmission of sporadic HEV infection and provides guidance to screen HEV in the blood donors so as to guarantee safe blood banks in China.

[Therapeutic effects of B and T lymphocyte attenuator extracellular domain and heat shock protein 70 antigen peptide on cervical cancer in mouse model].

OBJECTIVE: To investigate the synergistic therapy effects of B and T lymphocyte attenuator (BTLA) extracellular domain in combination with heat shock protein 70 (HSP70)-TC-1 antigen peptide complex on the mouse model of cervical cancer and the related immunological mechanisms. METHODS: (1) Detecting the BTLA and herpesvirus entry mediator (HVEM) gene expression in the tumor microenvironment after C57BL/6 mice were inoculated with TC-1 tumor cells by realtime PCR; BTLA, HVEM expression on tumor infiltrating lymphocytes cell surface were detected by flow cytometry (fluorescence intensity). (2) According to different treatments, tumor-bearing mice were divided into 5 groups, which was injected with pcDNA3.1 (empty vector plasmid as control), psBTLA (vector plasmid which expresses BTLA extracellular domain), HSP70 (HSP70-TC-1 cell peptide complex), HSP70 + pcDNA3.1 or HSP70 + psBTLA, respectively. The weight of tumor was recorded. The expression of immunoregulatory genes in tumor microenvironment were detected. The change of lymphocyte amount and cytotoxicity were detected too; lymphocyte proliferation activity was measured by tritium thymidine incorporation assay; the concentration of interleukin (IL)2 and interferon-γ (IFN-γ) in supernatants of spleen lymphocyte were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: (1) BTLA gene expression was gradually increased after tumor cells inoculation. The highest expression level was 2.83 ± 0.35 at 14th day, which had statistical significance difference with the 7th day expression of 1.66 ± 0.25 (P < 0.05). While HVEM mRNA expression did not change significantly (P > 0.05). The 7th and 14th day after TC-1 cells inoculation, the average fluorescence intensity of BTLA expression on the surface of tumor infiltrating lymphocytes was 33.5 and 51.8, respectively, in which there was statistically significant difference (P < 0.05); while the difference of HVEM expression was not statistically significant (57.2 vs 49.3, P > 0.05). (2) The 28th day after inoculation, tumor inhibition rate of HSP70 + psBTLA group was 88%, which was significantly higher than other treatment groups (P < 0.05). The 28th day after TC-1 cells inoculation, combination therapy not only promoted IFN-γ and IL-2 gene (3.12 ± 0.71, 3.20 ± 0.62) expression but also reduced transforming growth factor-ß (TGF-ß), Foxp3 and IL-10 expression (0.25 ± 0.03, 0.19 ± 0.03, 0.31 ± 0.04; P < 0.05). It also promoted CD8(+) T lymphocyte infiltration (52 ± 6)/high power field, cytotoxicity (65.5 ± 2.4)%, proliferation (15.0 × 10³ cpm) and cytokine IL-2, IFN-γ secretion (824 ± 51), (1096 ± 112) pg/ml, which were all significantly higher than other groups (P < 0.05). CONCLUSION: The effect of immunotherapy on tumor can be augmented by the combination of psBTLA which expresses extracellular domain of BTLA and HSP70-TC-1 tumor antigen peptide complex, which could improve the expression of the related immunoregulatory genes to establish a much better microenvironment in favor of anti-tumor immune response against the mice model of the cervix carcinoma.

[Effect of HPV16 peptide vaccine in combination with paclitaxel-cisplatin chemotherapy on cervical cancer in vitro and in vivo].

OBJECTIVE: to the explore the effect of Human papillomavirus (HPV) 16 peptide vaccine in combination with paclitaxel-cisplatin (TP) chemotherapy on cervical cancer in vitro and in vivo. METHODS: (1) the major histocompatibility complex (MHC) class I restricted T cell epitopes were studied by bioinformatics for transporter associated with antigen processing (TAP). Their effects were compared and E7Pa had the most dramatic effect. (2) In vivo, the C57BL/6 mice were divided equally into 6 groups randomly after loading with TC-1 cells (HPV 16 positive tumor cells from C57BL/6 mouse), named as E7Pa + CpG + TP, E7Pa + CpG, CpG + TP, TP and CpG group as experiment groups and control (blank injection with physiological saline). The tumor volumes were measured regularly by tumor growth curve to compare the therapeutic effects in different groups; the related cell factors in murine peripheral blood were evaluated by enzyme-linked immunosorbent assay (ELISA); the TUNEL test kit was used to explore cellular apoptosis in tumor tissue; the survival curve was drawn from the TC-1 cell loading to natural death; safety was tested by pathological test and leucocyte count. RESULTS: at day 60 of tumor growth, the tumor volume of immunotherapy plus TP chemotherapy group was (0.013 ± 0.010) cm(3) versus the control (1.900 ± 0.075) cm(3) with a great significant deviation (P < 0.01). Meanwhile, the volumes were E7Pa + CpG group (0.340 ± 0.038) cm(3), TP + CpG group (0.650 ± 0.029) cm(3), TP group (1.100 ± 0.052) cm(3) and that of CpG group was (0.890 ± 0.047) cm(3) separately. And these groups had significant difference with the controls (P < 0.05). The average survival time of different groups were E7Pa + CpG + TP group (108.50 ± 8.97) d, E7Pa + CpG group (100.02 ± 2.27) d, CpG + TP group (79.63 ± 4.05) d, TP group (73.24 ± 3.11) d, CpG group (68.63 ± 1.38) d and controls (52.37 ± 2.47) d. And the difference between the E7Pa + CpG + TP and E7Pa + CpG groups had great significance with the controls (P < 0.01). Furthermore, the immune system was effectively stimulated for suppressing tumor growth in the immunotherapy group while cell apoptosis was significantly induced in the chemotherapy group. The combination of immunotherapy and chemotherapy was significantly efficacious than either of them alone. And it could thoroughly stimulate the immune effects and enhance the anti-tumor function of chemotherapeutical drugs. In safety test, there was no significant difference among all groups. CONCLUSION: the HPV16 peptide vaccine in combination with TP chemotherapy can treat the HPV16 E7 positive tumor effectively in experiment.

[Preparation of human papillomavirus 16 E7 peptide vaccine and its effectiveness in vitro and in vivo.].

OBJECTIVE: To prepare the human papillomavirus (HPV) 16 peptide vaccine and explore the effect in vitro and in vivo. METHODS: (1) Prediction of the major histocompatibility complex (MHC) class I restricted T cell epitopes by bioinformatics target at transporter associated with antigen processing (TAP) and named by E7Pa, E7Pb, E7Pc separately. (2) In vivo, the C57BL/6 mice were divided into five groups with same amounts randomly after loading with TC-1 cells (HPV 16 positive tumor cells from C57BL/6 mouse), named as E7Pa + CpG, E7Pb + CpG, E7Pc + CpG (as experiment groups, and added 50 microg/ml E7Pa, E7Pb, E7Pc, respectively), CpG (as positive control group and added Con A with 12 mg/L final concentration) and blank control group (without any treatment). The T cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay at different time points; the lactate dehydrogenase (LDH) delivery method was used to test the cytolytic T lymphocyte (CTL) activity of mouse splenic lymphocyte in different ratio of effector cells and target cells (E:T); the related cytokines in tumor tissue and mouse peripheral blood were evaluated by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The tumor volumes were measured to contrast the therapeutic effect in different groups. RESULTS: (1) Three peptide named E7Pa, E7Pb, E7Pc were successfully preparated which had high affinity and specificity. (2) After vaccination of 24, 48, 72, 96 hours, MTT results shown that the proliferation rate in E7Pa + CpG group were (131 +/- 32)%, (302 +/- 15)%, (552 +/- 28)%, (731 +/- 24)% individually, which were much higher than those in blank control [(72 +/- 15)%, (120 +/- 57)%, (176 +/- 41)%, (288 +/- 29)%; P < 0.01], and the other groups i.e. E7Pb + CpG, E7Pc + CpG and CpG groups all proliferated much higher than those in blank control group with statistic signification (P < 0.05), but there was no significant difference between groups (P > 0.05); the LDH delivery assay showed that when the ratio of E:T was 100:1, the activity of CTL in the E7Pa + CpG group was most powerful than the other groups with statistic signification (P < 0.01).Meanwhile, the ratio of E:T was concentration-dependent. Compared E7Pb + CpG, E7Pc + CpG or CpG groups with blank control group, there were significantly difference (P < 0.05), while there was no significant difference between groups (P > 0.05). The mRNA levels of interferon gamma (IFN-gamma), interleukin-2 (IL-2) in tumor tissue and peripheral blood in E7Pa + CpG group were significantly higher than those in blank control group (P < 0.01), which was the similar results when compared E7Pb + CpG, E7Pc + CpG or CpG groups with control group (P < 0.05), and without significant difference between groups (P > 0.05). The tumor volumes were suppressed obviously in all the experiment groups, especially at the 60th days, the volumes in E7Pa + CpG group were much smaller than that in blank control group with statistic signification (P < 0.01), which was the similar results that E7Pb + CpG, E7Pc + CpG or CpG groups had difference than blank control group with statistic signification (P < 0.05), and without significant difference between groups (P > 0.05). CONCLUSION: The HPV16 E7 peptide target at TAP combination with CpG as a vaccine could treat effectively the HPV16 E7 positive tumor in experiment.

Soluble PD-1 facilitates 4-1BBL-triggered antitumor immunity against murine H22 hepatocarcinoma in vivo.

PURPOSE: The use of costimulatory molecules targeting distinct T-cell signaling pathways has provided a means for triggering and enhancing antitumor immunity; however, it is still not fully understood what types of costimulatory molecules are suitable for the combination in tumor therapy. Our purpose in this study is to establish an effective antitumor immune approach by using costimulatory molecule 4-1BBL in combination with soluble PD-1. EXPERIMENTAL DESIGN: The murine H22 hepatocarcinoma served as an ectopic tumor model. Local gene transfer was done by injection with naked plasmid p4-1BBL and/or psPD-1. The synergistic mechanism of dual-gene therapy was elucidated by detecting the change of gene expression of immunoregulatory factors in tumor microenvironment. The effects of immunotherapy were evaluated by testing the function of tumor-specific T cells, measuring tumor weight or volume, survival of mice, and H&E staining of tissues. RESULTS: 4-1BBL expressed by normal nonimmune cells effectively enhanced antitumor immune response but up-regulated PD-L1 and did not reduce IL-10 and transforming growth factor-beta (TGF-beta). sPD-1 synergized with 4-1BBL to establish efficient antitumor immune environment, including down-regulation of IL-10 and TGF-beta, further up-regulation of interleukin (IL)-2 and IFN-gamma, and higher CD8(+) T-cell infiltration. The combined treatment by 4-1BBL/sPD-1 eradicated tumors from mice with small amounts of preexistent tumor cells or tumors from approximately 60% of individuals with larger amounts of preexistent tumor cells. CONCLUSIONS: Our findings in this report imply a great potential of 4-1BBL in combination with sPD-1 in tumor therapeutics with the in vivo existent tumor cells as antigens.

Soluble form of T cell Ig mucin 3 is an inhibitory molecule in T cell-mediated immune response.

T cell Ig mucin 3 (Tim-3) has been found to play an important role in Th1-mediated auto- and alloimmune responses, but the function of soluble form of Tim-3 (sTim-3) remains to be elucidated. In this study, we report the inhibitory effect of sTim-3 on T cell-mediated immune response. In this study, sTim-3 mRNA was found, among different tissues and organs, only in splenic cells, and the activation of splenocytes resulted in up-regulated production of both sTim-3 mRNA and protein. We constructed a eukaryotic expression plasmid, psTim-3, which expresses functional murine sTim-3. In C57BL/6 mice inoculated with B16F1 melanoma cells, the growth of tumor was facilitated by the expression of this plasmid in vivo. Furthermore, sTim-3 inhibited the responses of T cells to Ag-specific stimulation or anti-CD3 mAb plus anti-CD28 mAb costimulation and the production of cytokines IL-2 and IFN-gamma in vitro. In tumor rejection model, sTim-3 significantly impaired T cell antitumor immunity, evidenced by decreased antitumor CTL activity and reduced amount of tumor-infiltrating lymphocytes in tumor. Real-time PCR analysis of gene expression in tumor microenvironment revealed the decreased expression of Th1 cytokine genes and the unchanged profile of the genes related to T regulatory cell function, suggesting that the inhibitory effect of sTim-3 on the generation of Ag-specific T cells in vivo is dominated by T effector cells rather than T regulatory cells. Our studies thus define sTim-3 as an immunoregulatory molecule that may be involved in the negative regulation of T cell-mediated immune response.