Tumour­derived exosomes and their emerging roles in leukaemia (Review).

Exosomes are small vesicles with a diameter of ~40-100 nm that are secreted by the majority of endogenous cells under normal and pathological conditions. They contain abundant proteins, lipids, microRNAs, and biomolecules such as signal transduction molecules, adhesion factors and cytoskeletal proteins, and play an important role in exchanging materials and transmitting information between cells. Recent studies have shown that exosomes are involved in the pathophysiology of leukaemia by affecting the bone marrow microenvironment, apoptosis, tumour angiogenesis, immune escape and chemotherapy resistance. Furthermore, exosomes are potential biomarkers and drug carriers for leukaemia, impacting the diagnosis and treatment of leukaemia. The present study describes the biogenesis and general characteristics of exosomes, and then highlight the emerging roles of exosomes in different types of leukaemia. Finally, the value of clinical application of exosomes as biomarkers and drug carriers is discussed with the aim to provide novel strategies for the treatment of leukaemia.

Current progress in CAR-T cell therapy for tumor treatment.

Chimeric antigen receptor T (CAR-T) cells are a type of tumor immunotherapy that is a breakthrough technology in the clinical treatment of tumors. The basic principle of this method is to extract the patient's T cells and equip them with targeting recognition receptors of tumor cells and return them to the patient's body to recognize and kill tumor cells specifically. Most CAR-T cell therapies treat hematological diseases such as leukemia or lymphoma and achieved encouraging results. The safety and effectiveness of CAR-T cell technology in solid tumor treatment require to be improved, although it has demonstrated promising efficacy in treating hematological malignancies. It is worth noting that certain patients may experience fatal adverse reactions after receiving CAR-T cell therapy. At present, the difficulty of this therapy mainly lies in how to reduce adverse reactions and target escape effects during the course of treatment. The improvement of CAR-T cell therapy mainly focuses on improving CAR-T structure, finding suitable tumor targets and combining them with immune checkpoint inhibitors to the enhance efficacy and safety of treatment. The problems in the rapid development of CAR-T cell therapy provide both obstacles and opportunities. The present review elaborates on the clinical application of CAR-T cell technology to provide a reference for clinical practice and research on tumor treatment.

Chimeric specific antigen epitope-carrying dendritic cells induce interleukin-17(+) regulatory T cells to suppress food allergy.

BACKGROUND: The on-purpose-modulated dendritic cells (DCs) have shown charming effects on restoring immune regulatory functions in subjects with immune diseases. OBJECTIVE: This study aims to construct DCs carrying chimerical antigen (Ag) peptides (CAP-DCs) to induce interleukin (IL)-17+ inducible Tregs (iTregs) to alleviate food allergy (FA) in a murine model. METHODS: In this study, we constructed CAP-DCs. The CAP is a fusion protein, consisting of a segment of recombinant scFv of anti-DEC205 antibody and an ovalbumin (OVA) epitope (IC). A murine OVA-FA model was developed to test the effects of CAP-DCs on suppressing the allergic response in the intestine. RESULTS: The CAP-DCs are characterized as that a complex of scFv-IC is presented on the surface of the cells, moderately express CD80 and CD86 as well as IL-6, IL-23, transforming growth factor (TGF)-ß and CCR9. After being passively transferred with CAP-DCs or injection of scFv-IC, Ag-specific IL-17+ Foxp3+ iTregs were induced in the intestinal lamina propria of FA mice. The iTregs showed immune suppressive effects on Ag-specific Th2 response. FA mice were adoptively transferred with the CAP-DCs or scFv-IC injection, which resulted in a significant decrease in the number of Ag-specific Th2 cells and suppression of FA response in an Ag-specific manner. CONCLUSIONS AND CLINICAL RELEVANCE: CAP-DCs can ameliorate FA response by inducing Ag-specific IL-17+ Foxp3+ iTregs and suppressing Ag-specific Th2 response. To generate CAP-DCs has the translational potential in the treatment of FA.

Association of BCSC-1 and MMP-14 with human breast cancer.

Breast cancer suppressor candidate-1 (BCSC-1) is a candidate tumor suppressor gene that was identified recently. Decreased levels of BCSC-1 have been detected in a variety of cancer types in previous studies. Matrix metalloproteinase (MMP)-14 is a membrane-type MMP that plays an important role in tumor progression and prognosis. Previous research has indicated that MMP-14 is highly expressed in different cancer types and promotes tumor invasion or metastasis by remodeling the extracellular matrix. However, there have been few reports on BCSC-1 and MMP-14 in human breast cancer in recent years. In the present study, the association of BCSC-1 and MMP-14 with human breast cancer was investigated. The immunohistochemical analysis results revealed reduced expression of BCSC-1 and overexpression of MMP-14 in breast cancer tissues compared with adjacent normal breast tissues. Quantitative polymerase chain reaction and western blot analyses also showed that BCSC-1 was expressed at significantly lower levels, and that MMP-14 was expressed at significantly higher levels in breast cancer tissues compared with healthy breast tissue. Furthermore, decreased expression of BCSC-1 and overexpression of MMP-14 were associated with tumor cellular differentiation, lymph node metastasis and distant metastasis. A correlational analysis between BCSC-1 and MMP-14 was also conducted, and the results indicated a negative correlation between the two. In conclusion, the current findings indicate that BCSC-1 is downregulated, while MMP-14 is overexpressed in human breast cancer. These two genes may play important roles during the process of human breast cancer development.

BCSC-1 suppresses human breast cancer metastasis by inhibiting NF-κB signaling.

Breast cancer suppressor candidate-1 (BCSC-1; also termed von Willebrand factor A domain containing 5A and LOH11CR2A) is a newly identified candidate tumor suppressor gene that has been implicated in several types of cancer in previous studies. However, there have been few reports about the association between BCSC-1 and human breast cancer in recent years. In the present study, the expression of BCSC-1 in breast cancer was determined by immunohistochemistry (IHC) staining of tissue microarrays and clinical tissue specimens. Subsequently, BCSC-1 gene expression was evaluated in different breast cancer cell lines by quantitative polymerase chain reaction and the MDA-MB-231 cell line was selected for further use in subsequent experiments, due to its low BCSC-1 expression. An MDA-MB-231 cell line with stable overexpression of BCSC-1 was established through transfection with plasmid containing the BCSC-1 gene, and then screening for G418 resistance. Wound-healing, migration and invasion assays were conducted to detect the effect of BCSC-1 on MDA-MB-231 cells. Furthermore, changes in matrix metalloproteinases (MMPs), osteopontin (OPN) and the nuclear factor-κB (NF-κB) pathway were detected in the current study. Additionally, stable silencing of BCSC-1 expression in MCF-7 cells was performed using a lentivirus. The results of IHC indicated that BCSC-1 is expressed at low levels in breast cancer tissues compared with in normal breast tissue. Results of the wound healing, migration and invasion assays demonstrated that BCSC-1 overexpression reduced the metastasis ability of MDA-MB-231 cells in vitro. Further research confirmed that the BCSC-1 overexpression reduced the expression levels of MMP7, MMP9 and OPN, and the phosphorylation of NF-κB p65. Furthermore, inhibition of BCSC-1 via lentivirus-mediated RNA interference revealed that the downregulation of BCSC-1 increased the invasive ability of MCF-7 cells. In summary, the results demonstrated that BCSC-1 is expressed at low levels in breast cancer tissues, and that it can suppress human breast cancer cell migration and invasion, potentially altering the expression of MMP7, MMP9, OPN, and the activity of the NF-κB pathway. Therefore, BCSC-1 may be useful as a biomarker for the treatment of breast cancer in the future.

Adenovirus-mediated interleukin 21 gene transfer enhances antitumor immunity and reduces tumorigenicity of Hepa1-6 in mice.

In the present study, adenovirus-mediated interleukin 21 (Ad5-IL-21-EGFP) gene expression was induced in Hepa1-6 cells to investigate whether IL-21 was capable of enhancing antitumor immunity and reducing tumorigenicity of Hepa1-6 in a mouse model. Mice were inoculated intradermally into the right flank with Hepa1-6 cells or Hepa1-6 cells infected with Ad5 or Ad5-IL-21. Four weeks later, the mice were sacrificed humanely, and the tumor volume, tumor weight and mouse spleen index were measured. The levels of IL-21, IL-4 and interferon (IFN)-γ levels in mouse serum and tumor tissues were detected by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. Cell counting kit-8 (CCK-8) assay was used to detect the killing ability of spleen T cells and natural killer (NK) cells, and the proliferation ability of T cells. The expression of IL-21 was confirmed by reverse transcription-polymerase chain reaction, western blot analysis and ELISA assay in Ad5-IL-21-EGFP-infected Hepa1-6 cells. The overexpression of IL-21 significantly reduced the tumorigenicity of Hepa1-6 cells. The tumor volumes and tumor weights in Ad5-IL-21-Hepa1-6 mice were much smaller than those in the Ad5-Hepa1-6 group and Hepa1-6 wild-type group. The immunohistochemistry and ELISA assay demonstrated that IL-21 and IFN-γ levels were much higher while the IL-4 level was much lower in the Ad5-IL-21-Hepa1-6 group than in the other two groups. CCK-8 assay revealed that the killing ability of NK cells and T cells, and the proliferation ability of T cells in Ad5-IL-21-Hepa1-6 mice were higher than in the other two groups; the spleen index of Ad5-IL-21-Hepa1-6 mice was also higher than in the other groups. The data had a significant difference (P<0.01). In conclusion, IL-21 reduces tumorigenicity of Hepa1-6 by a mechanism involving enhanced activation of cell-mediated immunity in tumor-bearing mice.

Downregulation of human intercellular adhesion molecule-1 attenuates the metastatic ability in human breast cancer cell lines.

Intercellular adhesion molecule-1 (ICAM-1) is a cell surface glycoprotein that belongs to immunoglobulin superfamily and plays an important role in tumor cell expansion or metastasis. However, the detailed mechanisms of ICAM-1 in breast cancer remain unclear. In this study, we evaluated the expression level of ICAM-1 in breast cancer using tissue microarray and clinical tissue specimens by immunohistochemical method, and the results revealed that ICAM-1 is highly expressed in the breast cancer tissues. To investigate whether ICAM-1 can affect the metastasis ability in breast cancer, we knocked down ICAM-1 expression in breast cancer cell line MCF-7 by using lentivirus-mediated RNA interference (RNAi). As a result, we stably silenced ICAM-1 expression in MCF-7 cells by infection with lentivirus expressing green fluorescent protein (GFP), the change of metastatic ability of MCF-7 cells was assessed by wound-healing assay, Transwell assay or clone formation assay. Our results showed that silencing of ICAM-1 can inhibit the metastatic ability of MCF-7 cell lines in vitro significantly, and the decreased migration and invasion was accompanied by a reduction of MMP-14. These results implying that ICAM-1 might be involved in the progression of breast cancer metastasis and lentivirus-mediated silencing of ICAM-1 might be a potential therapeutic approach for the treatment of breast cancer.

[Down-regulation of human intercellular adhesion molecule-1 expression in MCF-7 cells infected by lentiviral short hairpin RNA interference vectors].

OBJECTIVE: To construct lentiviral interference vectors of human intercellular adhesion molecule-1 (ICAM-1), then infect human breast cancer MCF-7 cells and identify the interference effects. METHODS: Three short hairpin RNA (shRNA) interference sequences targeting human ICAM-1 gene (ICAM-1 shRNA1, ICAM-1 shRNA2 and ICAM-1 shRNA3) and a negative control sequence (NS) were designed, synthesized and cloned into the pLKO.1-SP6-PGK-GFP vector. After DNA sequencing, three plasmid-based lentiviral packaging system (vector plasmid-psPAX2-pMD2.G) was used to transfect HEK293T cells to package lentiviruses. The supernatants containing viruses were harvested to detect the viral titer. Human MCF-7 breast cancer cells were infected with the lentiviruses and the interference efficiency was detected by real-time quantitative PCR (qRT-PCR) and Western blotting. RESULTS: PCR showed that the designed sequences were successfully inserted into the pLKO.1-SP6-PGK-GFP vector and DNA sequencing results were correct. The qRT-PCR and Western blotting showed that the mRNA and protein expression levels of ICAM-1 in the infected MCF-7 cells decreased significantly in the ICAM-1 shRNA3 group. CONCLUSION: Lentiviral interference vectors of human ICAM-1 were constructed successfully and the expression of ICAM-1 in MCF-7 cells was down-regulated by ICAM-1 shRNA.

Validation of the Andon KD-5851 upper arm blood pressure monitor, for self-measurement according to the European Society of Hypertension International Protocol revision 2010.

OBJECTIVE: This study aimed to validate the Andon KD-5851 upper arm blood pressure (BP) monitor according to the European Society of Hypertension International Protocol (ESH-IP) revision 2010. METHODS: A total of 33 eligible participants were included in the study. Sequential measurements of BPs were performed using a mercury sphygmomanometer and the device, and the data analysis was carried out following precisely the ESH-IP revision 2010. RESULTS: The device had 82, 98, and 99 measurements within 5, 10, and 15 mmHg for systolic blood pressure and 85, 95, and 99 measurements for diastolic blood pressure, respectively. The average device-observer difference was -0.53±4.00 mmHg for systolic blood pressure and -1.15±4.06 mmHg for diastolic blood pressure. The device passed all the criteria according to the ESH-IP revision 2010. CONCLUSION: According to the validation results on the basis of the ESH-IP revision 2010, the Andon KD-5851 upper arm BP monitor can be recommended for self/home measurement in adults.

[The expression of human IL-1ß gene containing human erythropoietin signal peptide in HepG2 cells].

OBJECTIVE: To construct two lentiviruses secreting human IL-1ß through either classical or nonclassical pathway and analyze their expressions in HepG2 cells after packaging lentiviruses and infecting hepatoma carcinoma HepG2 cells. METHODS: Human full-length IL-1ß gene and chimeric gene containing human erythropoietin(EPO) signal peptide and mature IL-1ß protein coding sequence were respectively amplified from pIRES2-EGFP-proIL-1ß and pIRES2-EGFP-epoIL-1ß using PCR. The sequences were subsequently cloned into lentiviral expression vector pLenti6/V5 to construct pLenti6/V5-proIL-1ß, which expressed IL-1ß through nonclassical pathway, and pLenti6/V5-epoIL-1ß, which expressed IL-1ß through signal-peptide mediated classical pathway. Lentiviruses expressing human IL-1ß through either classical or nonclassical pathway were packaged in HEK293T cells using a three-plasmid packaging system, and then these viruses were used to infect HepG2 cells. The level of IL-1ß in both cytoplasm and culture supernatant were detected by sandwich ELISA and Western blotting. RESULTS: pLenti6/V5-proIL-1ß expressing human full-length IL-1ß gene and pLenti6/V5-epoIL-1ß expressing human EPO signal peptide and mature IL-1ß gene were successfully constructed and confirmed through enzymatic assay and DNA sequencing. The lentiviruses expressing IL-1ß through different pathways were prepared using a three-plasmid packaging system in HEK293T cells. Compared with the cells infected with control virus, levels of supernatant and cytoplasmic IL-1ß in the cells infected with two lentiviruses expressing IL-1ß through different pathways were markedly elevated (P<0.01). However, level of mature IL-1ß in supernatant of HepG2/epoIL-1ß cells was much higher than that of HepG2/proIL-1ß cells, while total IL-1ß level in cytoplasm of HepG2/proIL-1ß cells was significantly higher than that in HepG2/epoIL-1ß cells. CONCLUSION: Both classical and nonclassical pathway secretion vectors could express human IL-1ß in HepG2 cells, but EPO signal-peptide mediated classical pathway secreted much higher mature IL-1ß than that of nonclassical pathway.

[Preparation and characterization of monoclonal antibodies for human BCSC-1 protein].

AIM: To obtain monoclonal antibodies against human BCSC-1 protein for further study of the structure and function of human BCSC-1 protein. METHODS: pET-30a-BCSC-1 plasmid was constructed and transformed into E.coli BL21 (DE3) to express recombinant protein. BALB/c mice were immunized with recombinant human BCSC-1 protein, hybridoma cell lines secreting monoclonal antibodies against human BCSC-1protein were screened by regular cell fusion and subcloning approach. The specificity of these monoclonal antibodies were determined by ELISA and Western blotting. Expression of BCSC-1 protein in pcDNA3.1/v5-HisB-BCSC-1transfected MCF-7 cells was identified by Immunohistochemistry. RESULTS: Successfully constructed a prokaryotic expression vector pET30a-BCSC-1.BCSC-1 protein was expressed in E.coli BL21 (DE3). One hybridoma cell line 1B3 stable in secreting specific monoclonal antibodies wa successfully obtained. It could bind specifically to BCSC-1 protein proved by Western blotting and Immunohistochemistry assay. CONCLUSION: Monoclonal antibodies of high specificity against human BCSC-1 protein has been successfully prepared, which laid the foundation for the further study of human BCSC-1 protein.

[Establishment of the transfected cell line expressing the mouse OX40 gene and its potency to induce differentiation of B cells].

AIM: To establish HUVECs line expressing mouse OX40(CD134) and to study its promotive effect on proliferation of B cells. METHODS: The cDNA fragment encoding mouse OX40 was obtained from the total RNA of ConA-activated lymphocytes of thymus by using RT-PCR and cloned into pUCm-T vector. The cDNA was then inserted into the eukaryotic expression vector pIRES2-EGFP. The recombinant vector was transfected into HUVECs with lipofectin reagent, and the positive cellular clones were selected by G418. Expression of mouse OX40 in the transfected cells was analyzed by FCM. The differentiation of B cells in vitro induced by OX40 signal was studied by means of (3)H-TdR method. RESULTS: The cDNA fragment in the recombinant plasmid was consistent with the reported mouse OX40 cDNA in GenBank, which was confirmed by DNA sequencing, PCR and enzyme digestion. The HUVECs stably expressing the mouse OX40 were successfully cloned. The transfected cells promoted the differentiation of B cells in vitro and there existed a synergic effect between OX40/OX40L and CD40/CD40L signals. CONCLUSION: Transfected cell line expressing the mouse OX40 gene is established successfully. OX40 enhances the proliferation of B cells.