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Uncarboxylated osteocalcin (ucOC) is a hormone secreted by osteoblasts that strengthens bone during mineralization and is a biomarker for ongoing bone formation. It also regulates glucose homeostasis by stimulating insulin secretion from pancreatic ß-cells. However, its effect on ß-cells under hyperglycemic diabetic conditions is unclear. The objective of this study was to investigate ucOC's effect on insulin secretion in ß-cells maintained under high glucose conditions. We hypothesized that hyperglycemia potentiates insulin secretion in response to ucOC stimulation. Using INS-1 cells, we performed insulin secretion experiments, intracellular calcium recordings, and RT-qPCR to determine ucOC's effect on glucose-stimulated insulin secretion (GSIS)-related genes. The results reveal that ucOC significantly increased insulin secretion under hyperglycemic conditions compared to lower glucose levels. High glucose conditions also potentiated the effect of ucOC on calcium signals, which enhanced insulin secretion. The increase in intracellular calcium was due to an influx from the extracellular space via voltage-dependent calcium channels (VDCCs). Interestingly, the treatment of cells with NPS-2143, a GPRC6A blocker, failed to abolish the calcium signals. Uncarboxylated osteocalcin upregulated the expression of GSIS-related genes under high glucose conditions (450 mg/dL) compared to cells under standard culture conditions (200 mg/dL). In conclusion, hyperglycemia potentiates ucOC-induced insulin secretion in ß-cells by opening VDCCs and upregulating GSIS genes. These findings provide a better understanding of ucOC's mechanism in the diabetic state and could lead to alternative treatments to stimulate insulin secretion.

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Background: Efficiently delivering nucleic acid into mammalian cells is essential to overexpress genes for assessing gene functions. Human bone marrow stem cells (hBMSCs) are the most studied tissue-derived stem cells. Adeno-associated viruses (AAVs) have been used to deliver DNA into hBMSCs for various purposes. Current literature reported that transduction efficiencies of up to 65% could be achieved by AAV gene delivery into hBMSCs. Further improvement of efficiency is needed and possible. This study tested a selection of AAV serotypes for high-efficient DNA delivery into hBMSCs. Methods: hBMSCs from different donors were infected with different serotypes of AAVs containing the enhanced green fluorescence protein (eGFP) reporter gene driven by the CMV promoter. Green fluorescence was monitored in the infected cells at five-day intervals. Cells were collected at designated time points after the infection for reverse-transcription polymerase chain reaction (RT-PCR) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to assess eGFP mRNA transcription. Results: The results indicated that the order of transduction efficiency of the AAV serotypes was AAV2 > AAV2.7m8 > AAV6 > AAV6.2 > AAV1 > AAV-DJ. AAV2 could achieve almost 100% transduction at the multiplicity of infection (MOI) greater than 100K. Over 90% of cells could be transduced at 20K to 50K MOI. About 80% transduction was seen at MOIs of 10K and 15K. RT-PCR analysis showed that eGFP mRNA could be detected from day 5 to day 30 post-AAV infection. The differences in the observed transduction efficiencies of the hBMSCs from different patients indicate donor-to-donor variability, and increased eGFP mRNA was generally seen after day 15 post-AAV2 infection. Maximal eGFP transcription was detected on day 30 post-infection. Conclusions: We conclude that AAV2 and AAV2.7m8 at an MOI of 100K or greater can efficiently deliver transgene into hBMSCs with up to near 100% transduction efficiency for sustained expression over one month. However, donor-to-donor variation exists in transduction efficiency and transgene expression, especially at MOIs less than 100K.

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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.

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In this trial, nasopharyngeal 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 April and September 2014, we enrolled 90 patients who met the enrolment criteria, and assigned them to three groups (n=30). Throughout the trial, injection site reaction was the most common reaction (81%), and fever was least common (31%); however, there was no difference among the three groups. When the immune responses pre- and post-vaccination were compared, we found that the CSC-specific and -nonspecific response in the medium- and high-dose groups were both significantly enhanced. This study is the first clinical trial of a nasopharyngeal CSC vaccine and preliminarily proves its safety and efficacy.

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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.

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OBJECTIVE: The aim of this study was to retrospectively assess the effect of comprehensive cryosurgery (ablation of intrapancreatic and extrapancreatic tumors) plus immunotherapy in metastatic pancreatic cancer. METHODS: We divided 106 patients (57 men, 49 women; median age, 65 years) into the cryoimmunotherapy (31 patients), cryotherapy (36 patients), immunotherapy (17 patients), and chemotherapy groups (22 patients). Pretreatment immune function was tested in patients who underwent immunotherapy. Overall survival (OS) after diagnosis of metastatic pancreatic cancer was assessed after a 4-year follow-up. RESULTS: Median OS was higher in the cryoimmunotherapy (13 months) and cryotherapy groups (7 months) than in the chemotherapy group (3.5 months; both P < 0.001) and was higher in the cryoimmunotherapy group than in the cryotherapy (P < 0.05) and immunotherapy groups (5 months; P < 0.001). In both the cryoimmunotherapy and cryotherapy groups, median OS was higher after multiple cryoablations than after a single cryoablation (P = 0.0048 and 0.041, respectively). In both groups, the median OS was higher in patients with normal immunologic function than in those with immune dysfunction (P < 0.0001 and P = 0.0004, respectively). CONCLUSIONS: Cryoimmunotherapy significantly increased OS in metastatic pancreatic cancer. Multiple cryoablations and normal pretreatment immunologic function were associated with better prognosis.

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BACKGROUND: P. vivax infection is characterised by relapsing fever, indicating reinfection by previously hidden parasites in the host. Relapsed infection can lead to the activation of the memory T cell pool, which may lead to protective immunity. This study aims to characterise immune responses in acute P. vivax-infected patients living in an area of central China characterised by only P. vivax infection. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a cross-sectional immune-phenotypic analysis of adults using the following inclusion criteria: acute P. vivax infection (N=37), a history of P. vivax infection (N=17), and no known history of P. vivax infection (N=21). We also conducted a 2-week longitudinal analysis following acute P. vivax infection, in which PBMC proliferation was measured in response to P. vivax and P. falciparum blood stage lysates. Using flow cytometry, we showed elevated memory T cells in the blood during acute P. vivax infection. The levels of γδ T cells were two-fold higher than those measured in naive controls. This result suggested that in the two populations, memory and γδ T cells promptly responded to P. vivax parasites. Interestingly, P. falciparum antigens stimulated T cells obtained from P. vivax-infected patients during a day 14-convalescence, whereas lymphocytes from the naïve control group responded to a lower degree of convalescence. CONCLUSIONS/SIGNIFICANCE: Cell-mediated immunity during the convalescent period of the P. vivax-infected hosts was comprised of T cells that were specifically able to recognise P. falciparum antigens. Although the magnitude of the response was only half that measured after stimulation with P. vivax antigens, the matter of cross-antigenic stimulation is of great interest.

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Malignant mesothelioma (MM) is an aggressive neoplasm usually arising from the mesothelial surfaces of the pleural or peritoneal cavity. Currently, no standard therapy is available. The most commonly used therapy is cytoreductive surgery combined with systematic chemotherapy, but the median overall survival (OS) is less than 12 months; moreover, treatments are lacking for patients in whom chemotherapy has failed and/or who cannot withstand surgery. We investigated multiple minimally invasive therapies (cryosurgery, photodynamic therapy and intracavity chemotherapy) for the treatment of MM patients in whom systemic chemotherapy had failed. Twenty-seven patients were divided into comprehensive (combination of the three therapies) and palliative (intracavity chemotherapy only) treatment groups. The OS of patients who received comprehensive treatment was significantly longer than that of those who received palliative treatment (median OS: 64 vs. 9 months, P<0.001). This interesting result was not associated with treatment timing, but was closely associated with repeated treatments.

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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.

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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.

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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%.

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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.

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