Prospective study of the safety and efficacy of a pancreatic cancer stem cell vaccine.

INTRODUCTION: In this trial, we isolated and cultured pancreatic cancer stem cells (CSCs) to produce a vaccine and prospectively evaluated its safety and efficacy in low-, medium-, and high-dose groups. MATERIAL AND METHODS: Between February and October 2014, we enrolled 90 patients who met the enrollment criteria and assigned them to three groups (n = 30). CSC-specific and CSC-non-specific immunity pre- and post-vaccination were compared by Dunnett's multiple comparison test (one-way ANOVA). The data are presented as the mean±standard deviation. Local and systemic adverse events were recorded in the nursing records and compared using the Chi-square test. All statistical analyses were conducted using GraphPad software (GraphPad, San Diego, CA, USA). RESULTS: Throughout the trial, an injection site reaction was the most common reaction (54 %), and fever was least common (9 %). The incidence of these side effects did not vary among the three groups. When the pre- and post-vaccination immunity was compared, we found that both CSC-nonspecific and CSC-specific responses were significantly increased in the high-dose group. CONCLUSION: This study is the first clinical trial of a pancreatic CSC vaccine and preliminarily proves its safety and efficacy.

Safety and efficacy study of lung cancer stem cell vaccine.

In this trial, lung cancer stem cells (CSCs) were separated and cultured to produce a vaccine; its safety and efficacy were prospectively evaluated in low-, medium-, and high-dose groups. Between February and September 2014, we enrolled 90 patients who met the enrollment criteria and assigned them to three groups (n = 30). Throughout the trial, injection site reaction was the most common reaction (63 %), and fever was least common (16 %); however, there was no difference among the three groups. When the immune responses pre- and post-vaccination were compared, we found that the CSC-nonspecific and CSC-specific responses were both significantly enhanced in the medium- and high-dose groups. This study is the first clinical trial of a lung CSC vaccine and preliminarily proves its safety and efficacy.

[Eukaryotic expression of recombinant porcine single-chain IL-12 and its biologic activity].

AIM: To express the recombinant porcine single-chain interleukin-12 (pscIL-12) gene in CHO-K1 cells, and identify biological activity of pscIL-12 fusion protein. METHODS: The recombinant pcDNA3.1(+)-pscIL-12 plasmid was transfected into the CHO-K1 cells using Sofast(TM); reagent. The levels of pscIL-12 fusion protein and IFN-γ produced by human peripheral blood mononuclear cells (PBMCs) stimulated with the supernatant of CHO-K1 cells were detected by ELISA. NK cell cytotoxicity was tested by MTT assay. The lymphocyte proliferation was examined by flow cytometry. RESULTS: ELISA showed that the transfected CHO-K1 cells had a relatively higher expression of pscIL-12 fusion protein. And the pscIL-12 fusion protein possessed strong bioactivities in enhancing the NK cell cytotoxicity, increasing the IFN-γ production, and stimulating the lymphocyte proliferation. CONCLUSION: CHO-K1 cells transfected with pcDNA3.1(+)-pscIL-12 recombinant plasmid can successfully express pscIL-12 fusion protein, which has the expected biological activities.

[Construction and eukaryotic expression of recombinant porcine single-chain interleukin-12].

OBJECTIVE: To clone the p40 and p35 subunit cDNA of porcine IL-12(pIL-12) and construct the fusion gene of recombinant porcine single-chain interleukin-12 (pscIL-12). METHODS: The total RNAs were extracted from porcine peripheral blood mononuclear cells (PBMCs) and porcine splenic lymphocytes for cloning pIL-12 p35 and p40 cDNA by RT-PCR. A hydrophobic polypeptide linker (Gly4Ser)3 was used for splicing two different gene fragments (pIL-12) p40+linker+p35 (pscIL-12) by recombinant PCR to construct pscIL-12 fusion gene. The pscIL-12 fusion gene was then inserted into pcDNA3.1(+) eukaryotic expression plasmid, and the resulted pcDNA3.1(+)-pscIL-12 was transfected into CHO-K1 cells via lipofectin. The expression of pscIL-12 mRNA in the transfected cells was identified by RT-PCR. RESULTS: The sequence of the cloned porcine IL-12 p40 and p35 cDNA and the constructed pscIL-12 fusion gene were verified by PCR, restriction enzyme digestion and sequencing. The mRNA of pscIL-12 fusion gene was detected in the transfected CHO-K1 cells by RT-PCR. CONCLUSION: The constructed pcDNA3.1(+)-pscIL-12 eukaryotic expression plasmid allows expression of pscIL-12 in CHO-K1 cells, thus facilitating further study of the biological activity and adjuvant effect of pscIL-12 fusion protein.

[Construction of full-length cDNA library of erythrocytic stage Plasmodium vivax].

Blood samples were collected from vivax malaria patients without antimalarial drug therapy. After filtration through Plasmodipur filter to remove white blood cells, Plasmodium vivax-infected RBCs were enriched by Percoll. Total P. vivax in red blood cells was isolated. A full-length cDNA library of erythrocytic stage P. vivax was constructed by the SMART cDNA library construction kit. The volume and recombinant rate of the library were evaluated. The inserted fragments were identified by PCR amplification. The titer of cDNA library was 1.14 x 10(6). The length of inserted fragment ranged from 900 to 2 500 bp, and the recombination efficiency accounted for 97.2%.

Oncogenic B-RAF negatively regulates the tumor suppressor LKB1 to promote melanoma cell proliferation.

The LKB1-AMPK signaling pathway serves as a critical cellular sensor coupling energy homeostasis to cell growth, proliferation, and survival. However, how tumor cells suppress this signaling pathway to gain growth advantage under conditions of energy stress is largely unknown. Here, we show that AMPK activation is suppressed in melanoma cells with the B-RAF V600E mutation and that downregulation of B-RAF signaling activates AMPK. We find that in these cells LKB1 is phosphorylated by ERK and Rsk, two kinases downstream of B-RAF, and that this phosphorylation compromises the ability of LKB1 to bind and activate AMPK. Furthermore, expression of a phosphorylation-deficient mutant of LKB1 allows activation of AMPK and inhibits melanoma cell proliferation and anchorage-independent cell growth. Our findings provide a molecular linkage between the LKB1-AMPK and the RAF-MEK-ERK pathways and suggest that suppression of LKB1 function by B-RAF V600E plays an important role in B-RAF V600E-driven tumorigenesis.