Development and evaluation of a droplet digital PCR assay to detect Brucella in human whole blood.

BACKGROUND: With the development of domestic animal husbandry, the spread of brucellosis has accelerated, and the scope of the epidemic has expanded. The timely and accurate diagnosis of human brucellosis continues to challenge clinicians in endemic areas. Droplet digital PCR (ddPCR) technology can quickly and accurately determine DNA load in samples, providing laboratory evidence for diagnosis, prognosis and management of brucellosis patients. In this study, a ddPCR method was established to accurately quantify Brucella DNA load in whole blood samples, and its diagnostic, prognostic, and therapeutic value for human brucellosis was evaluated. METHODS: Annealing temperature, primers, and probe targeting the Brucella bcsp31 gene were optimised, and the sensitivity, specificity and repeatability of the ddPCR assay were assessed using 94 whole blood samples from 61 confirmed and 33 suspected cases. Results were compared with those of quantitative PCR (qPCR). Nine follow-up brucellosis patients were also analysed by the two methods after 2 and 6 months of treatment. RESULTS: Optimal primer and probe concentrations were 800 nmol/L and 400 nmol/L, respectively, and the optimal annealing temperature was 55.3 °C. The ddPCR results showed that the limit of detection was 1.87 copies per reaction, with high repeatability. The positive rates for ddPCR and qPCR were 88.5% and 75.4% among 61 serum agglutination test (SAT) positive patients. In addition, 57.6% (19/33) of suspected sero-negative samples were positive by ddPCR, but only 36.3% (12/33) were positive by qPCR. Analysis of nine post-therapy follow-up brucellosis patients revealed that the Brucella DNA load in the whole blood samples decreased after 2 and 6 months of treatment, and was slightly increased following relapse and continuous exposure. CONCLUSION: The ddPCR assay showed good accuracy for whole blood samples, and could be a potential diagnostic and prognostic tool for detecting Brucella.

NFKB1 -94 insertion/deletion polymorphism and cancer risk: a meta-analysis.

Previous studies on the associations of the NFKB1 -94 insertion/deletion polymorphism with cancer risk have produced conflicting results. The purpose of this meta-analysis is to define the effect of the NFKB1 -94 insertion/deletion polymorphism on cancer risk. A search of the literature by PubMed was performed to identify studies based on the predetermined inclusion criteria. Twenty-three studies consisting of 6,494 cases and 9,884 controls were identified and analyzed. Overall, significant association was observed between the polymorphism and cancer risk under all genetic models. Subgroup analysis according to ethnicity and cancer type also detected significant association. The NFKB1 -94 insertion/deletion polymorphism was associated with cancer risk in Asian population (dominant model: OR=1.52, 95 % CI=1.17-1.98; recessive model: OR=1.50, 95 % CI=1.26-1.79; II vs. DD: OR=1.90, 95 % CI=1.37-2.65; ID vs. DD: OR=1.32, 95 % CI=1.05-1.66; I vs. D: OR=1.37, 95 % CI=1.17-1.60), but not in Caucasian population. In addition, significant associations in OC, HCC, and OSCC were observed, but significant associations were not found in BC and LC. The current meta-analysis suggested that NFKB1 -94 insertion/deletion polymorphism may influence cancer risk in Asian population.

Non-association of IL-16 rs4778889 T/C polymorphism with cancer risk in Asians: a meta-analysis.

The IL-16 rs4778889 T/C polymorphism is associated with cancer risk. However, the results are conflicting. We performed this meta-analysis to derive a more precise estimation of the relationship. A comprehensive literature search was performed using PubMed, Embase and Web of Science databases. Odds ratio (OR) and 95% confidence interval (CI) were used to assess the strength of association. A total of 6 studies including 1,603 cases and 2,342 controls were identified. With all studies involved, results showed no statistically significant association between IL-16 rs4778889 T/C polymorphism and cancer risk (CC vs. CT+TT: OR=0.74, 95%CI: 0.55-1.02, Ph=0.15; CC+CT vs. TT: OR=0.89, 95%CI: 0.72-1.10, Ph =0.03; CC vs. TT: OR=0.73, 95%CI: 0.53- 1.00, Ph =0.08; CT vs. TT: OR=0.91, 95%CI: 0.79-1.05, Ph =0.08; C vs. T: OR=0.89, 95%CI: 0.74-1.07, Ph =0.02). In addition, the results were not changed when studies were stratified by cancer type. However, to verify our findings, it is essential to perform more well-designed studies with larger sample sizes in the future.

Low-dose paclitaxel enhances the anti-tumor efficacy of GM-CSF surface-modified whole-tumor-cell vaccine in mouse model of prostate cancer.

Chemotherapy combined with a tumor vaccine is an attractive approach in cancer therapy. This study was designed to investigate the optimal schedule and mechanisms of action of a novel GM-CSF (granulocyte-macrophage colony-stimulating factor) surface-modified tumor-cell vaccine in combination with paclitaxel in the treatment of mouse RM-1 prostate cancer. First, the anti-tumor efficiencies of various dosage of paclitaxel (4, 20, 40 mg/kg) in combination with the vaccine in different administration sequences were examined in the mouse RM-1 prostate cancer model. Then, the in vivo and in vitro effects of various dosage of paclitaxel on RM-1 cells, T cells, and DCs (dendritic cells) were evaluated. The results showed that: (a) the GM-CSF-surface-modified tumor-cell vaccine was more potent at inducing the uptake of tumor antigens by DCs than irradiated tumor cells plus free GM-CSF; (b) 4 mg/kg paclitaxel combined with the GM-CSF-surface-modified tumor-cell vaccine was the most effective at enhancing tumor regression in RM-1 prostate cancer mice when the vaccine was administrated 2 days after paclitaxel; and (c) administration of 4 mg/kg paclitaxel followed by the vaccine induced the highest degree of CD8(+) T-cell infiltration in tumor tissue, suggesting that the induction of tumor-specific immune response had occurred. These findings suggested that the GM-CSF-surface-modified tumor-cell vaccine may have potential clinical benefit for patients with prostate cancer when it is combined with paclitaxel. Furthermore, the effect of immunochemotherapy depends on careful selection of paclitaxel dosage and the sequence of paclitaxel/vaccine administration.

[Preparation and bioactivity evaluation of SA-mIL15 fusing protein].

AIM: To prepare and characterize streptavidin-tagged murine interleukin-15 fusion proteins. METHODS: pET24a-SA-L-mIL15 and pET21a-mIL15-L-SA plasmids were constructed and expressed in Rosetta (DE3) host bacteria to generate SA/mIL15 fusion proteins. SA-mIL15 fusion protein was purified through the Ni-NTA affinity chromatography, and mIL15-SA fusion protein through anion exchange chromatography, followed by refolding. The efficiency of surface modification of the fusion proteins on the biotinylated RM-1 tumor cells was evaluated by a flow cytometer. MTT method was used to evaluate the proliferating effect of SA/mIL15 fusion proteins on mouse spleen lymphocytes stimulated by ConA. RESULTS: Both SA-mIL15 and mIL15-SA fusion proteins were highly expressed in Rosetta (DE3) at about 20% of total bacterial proteins. They exhibited the bi-functionality: proliferation-promoting activity of mIL15 on mouse spleen lymphocytes with the specific activity of 1x10(6); IU/mg for SA-mIL15 or 2 x 10(5); IU/mg for mIL15-SA, and SA-mediated high-affinity binding to the biotinylated surfaces of RM-1 tumor cells with about 95% surface modification efficiency. CONCLUSION: SA/mIL15 bi-functional fusion proteins were generated, which made feasible the development of mIL15-surface-modified cancer cell vaccine.

A novel type of N-formylation and related reactions of amines via cyanides and esters as formylating agents.

A novel N-formylation and related reactions proceed from cyanides promoted by esters.