Highly sensitive detection of cancer-related genes based on complete fluorescence restoration of a molecular beacon with a functional overhang.

The accurate detection of cancer-related genes is of great significance for early diagnosis and targeted therapy of cancer. In this contribution, an automatically cycling operation of a functional overhang-containing molecular beacon (OMB)-based sensing system was proposed to perform amplification detection of the p53 gene. Contrary to the common molecular beacon (MB), a target DNA is designated to hybridize with a label-free recognition probe (RP) with a hairpin structure rather than OMB. In the presence of a target DNA of interest, the locked primer in RP opens and triggers the subsequent amplification procedures. The newly-developed OMB is not only capable of accomplishing cyclical nucleic acid strand-displacement polymerization (CNDP) with the help of polymerase and nicking endonuclease, but is also cleaved by restriction endonucleases, removing the quencher away from the fluorophore. Thus, the target DNA at an extremely low concentration is expected to generate a considerable amount of double-stranded and cleaved OMBs, and the quenched fluorescence is completely restored, leading to a dramatic increase in fluorescence intensity. Utilizing this sensing platform, the target gene can be detected down to 8.2 pM in a homogeneous way, and a linear response range of 0.01 to 150 nM could be obtained. More strikingly, the mutant genes can be easily distinguished from the wild-type ones. The proof-of-concept demonstrations reported herein are expected to promote the development of DNA biosensing systems, showing great potential in basic research and clinical diagnosis.

[Effect of epitope-based peptide-DNA dual vaccines against Schistosoma japonicum in mice].

A C-T-B PDDV mixture of the three constructed epitope-based peptide-DNA dual vaccines (PDDV) containing the CTL (C), Th (T) and B-cell (B) epitopes from Sj22.6 tegument (C-PDDV, T-PDDV and B-PDDV) with a 1:1:1 ratio was prepared. Thirty-six mice were randomly divided into six groups averagely named as 18K group, PBS group, C-PDDV group, T-PDDV group, B-PDDV group, and C-T-B PDDV group. All the mice received three immunizations at 2-week intervals with the same dose of antigen (10 microg DNA+28 microg peptide). One week after the last immunization, the mice were sacrificed, the spleens were removed and splenocytes were collected. Splenocyte proliferation was assayed by[3H] TdR incorporation after stimulation with soluble worm antigen (SWA). Levels of IFN-gamma and IL-4 in the splenocyte culture supernatants were determined by ELISA. The results showed that IFN-gamma content in T-PDDV group [(76.0 +/- 11.2) pg/ml] was higher than that of PBS [(13.0 +/- 2.1) pg/ml] and 18K control groups [(14.0 +/- 3.2) pg/ml] (P<0.01). IL-4 level in T-PDDV [(152.0 +/- 21.1) pg/ml] and C-T-B mixture groups [(86.0 +/- 12.2) pg/ml] was higher than others (P<0.01 and P<0.05). The splenocytes from T-PDDV group showed a significant increase in proliferation compared with PBS and 18K control groups after stimulation by SWA (P<0.01). However, there was no significant difference in splenocyte proliferation among C-T-B, PBS and 18K control groups (P>0.05). These findings indicate that T-PDDV and C-T-B PDDV mixture induces stronger immune response than that of C-PDDV or B-PDDV.

Diagnosis of intestinal acariasis with avidin-biotin system enzyme-linked immunosorbent assay.

AIM: To explore the value of avidin-biotin system enzyme-linked immunosorbent assay (ABC-ELISA) in diagnosis of intestinal acariasis. METHODS: Mite-specific IgG levels in serum of 48 patients with intestinal acariasis were measured with ABC-ELISA. The sensitivity of this method was compared with that of staphylococcal protein A enzyme-linked immunosorbent assay (SPA-ELISA). RESULTS: The positive rate of mite-specific IgG detected with ABC-ELISA and SPA-ELISA was 89.58% (43/48) and 56.25% (27/48), respectively. The positive rate with ABC-ELISA was statistically higher than that with SPA-ELISA (chi2=13.50, P<0.01). CONCLUSION: ABC-ELISA is an effective method for the diagnosis of intestinal acariasis.

Clinical and epidemiological features of patients with clonorchiasis.

AIM: To study the clinical and epidemiological features of patients with clonorchiasis so as to provide scientific evidences for the diagnosis and prevention of clonorchiasis. METHODS: Stools from 282 subjects suspected of having clonorchiasis were examined for helminth eggs with modified Kato's thick smear and sedimentation methods, and their sera were tested for HAV-DNA, HBV-DNA, HCV-RNA, HDV-RNA and HEV-RNA with polymerase chain reaction (PCR). Clinical symptoms of patients with clonorchiasis only were analyzed, and their blood samples were tested for circulating antigen (CAg) with Dot-ELISA, eosinophilic granulocyte count, and alanine aminotransferase (ALT). Meanwhile, they were asked to provide data of occupation, eating habit, hygienic habit and knowledge of clonorchiasis. In addition, the ecosystem of the environment in epidemic areas was surveyed. RESULTS: Among the 282 patients, 61 (21.43%) were infected with clonorchis sinensis only, 97 (34.64%) were co-infected with clonorchis sinensis and other pathogens, 92 (32.86%) were infected with hepatitis virus only and 31 (11.07%) neither with clonorchis sinensis nor hepatitis virus. Among the 61 patients with clonorchiasis only, there were 14 (22.95%) subjects with discomfort over hepatic region or epigasfrium, 12 (19.67%) with general malaise or discomfort and inertia in total body, 6 (9.84%) with anorexia, indigestion and nausea, 4 (6.56%) with fever, dizziness and headache (6.56%), and 25 (40.98%) without any symptoms; sixty one (100%) with CAg (+), 98.33% (59/60) with eosinophilic granulocytes increased and 65.00% (39/60) with ALT increased. B-mode ultrasonography revealed 61 cases with dilated and thickened walls of intrahepatic bile duct, and blurred patchy echo acoustic image in liver. Twenty-six cases had stones in the bile duct, 39 cases had slightly enlarged liver with diffuse coarse spots in liver parenchyma. Twenty cases had enlarged gallbladder with thickened coarse wall and image of floating plagues, 9 cases had slightly enlarged spleen. By analysis of epidemiological data, we found that the ecologic environment was favorable for the epidemiology of clonorchiasis. Most patients with clonorchiasis were lack of knowledge about the disease. Their living environment, hygienic habits, eating habits and their occupations were the related factors that caused the prevalence of the disease. CONCLUSION: The clinical symptoms of clonorchiasis are non-specific, and the main evidences for diagnosis of clonorchiasis should be provided by etiologic examination, B-mode ultrasonography and clinical history. The infection of clonorchis sinensis is related to occupations, bad eating habits and lack of knowledge about prevention of the disease.

[Immune regulation of T-bet adjuvant on Ag85B DNA vaccine against Mycobacterium tuberculosis].

AIM: To construct a novel Ag85B DNA vaccine based on the genetic adjuvant of T-bet known as Th1 transcription factor and to research the immunoregulation function of the DNA complex vaccine. METHODS: Ag85B gene and T-bet gene were amplified by RT-PCR, and cloned into pcDNA3.1 plasmid to construct recombinant plasmids pcDNA3-Ag85B and pcDNA3-T-bet, respectively. The recombinant plasmids were transfected into RAW264.7 cells using Lipofectamine(TM); 2000 reagent to detect the expressions of Ag85B and T-bet proteins by Western blotting. BALB/c mice were immunized by three intramuscular inoculations with pcDNA3.1-FLAG-T-bet in combination with pcDNA3.1-FLAG-Ag85B. Two weeks after the last immunization, the anti-Ag85B antibody titres in sera were tested by ELISA. Meanwhile, spleen lymphocyte suspension was cultured in the context of Ag85B, and then the secretion of cytokines in the culture fluid was tested by ELISA. RESULTS: Plasmid proteins were successfully expressed in a dose-dependent manner. Additionally, T-bet/Ag85B complex not only induced obviously higher IgG2a titre with the lower IgG1, but also stimulated the increased secretion of IFN-γ and IL-2 with the concomitant repression of IL-4 and IL-10. CONCLUSION: T-bet can enhance Ag85B-specific IgG2a antibody response and convert T cell subsets to a Th1-predominant immune response.

Th1 immunity is not required for the effect of lipopolysaccharide exposure on modifying asthmatic responses of mice before sensitization.

BACKGROUND: Disequilibrium of Th1/Th2 is known as an important cause of allergic asthma with a biased Th2 type response. It has been shown that lipopolysaccharide (LPS) administration during post-sensitization modified the inflammation of asthma via upregulating the Th1 response that decrease the Th2 immunity. We would like to know if, during pre-sensitization, the elevated Th1 response is necessary for LPS exposure to modify the asthmatic response. METHODS: During pre- or post-sensitization, 40 microg LPS were intraperitoneal injected (i.p.) to asthmatic mice sensitized and challenged by Dermatophagoides farinae (D. farinea). Inflammation was assessed by examining bronchoalveolar lavage fluid (BALF) for the number and identity of cells and by cytokine titers measured by ELISA. Semi-quantified RT-PCR was used to evaluate the level of Toll-like receptor 4 (TLR4) mRNA in dendritic cells (DCs) from bone marrow (BMDCs). RESULTS: These investigations demonstrated that LPS exposure during pre-sensitization inhibited the Th2 cytokine and inflammatory infiltration, the same as with LPS exposure during post-sensitization in allergic asthma mice. Contrary to post-sensitization LPS exposure, the Th1 cytokines were not upregulated by pre-sensitization with LPS. Finally, the study failed to show any significant difference between TLR4 mRNA expressed in BMDCs with the two times of LPS exposure. CONCLUSIONS: Our data suggest that elevated Th1 immunity is not required for the modification of the Th2 response induced by LPS exposure during pre-sensitization in asthmatic mice and that pre-sensitization differs from post-sensitization. Immune modulation with treatment is independent of TLR4 expression in BMDCs. This study implicates a potential way to protect from allergic disease and an inflammatory response.

[The effect of rural exposure on allergic asthma in Anhui province].

OBJECTIVE: To establish the evidence of exposure to rural areas would reduce the risk of atopic asthma and sensitization. METHODS: A cross-sectional survey was carried out in 2986 school-age children and their parents completed standardized questionnaires on atopic asthma and sensitization, wheezing. A radioallergosorbent technique-fluorescence enzyme immunoassay (RAST-FEIA) was used to measure the level of specific IgE in serum. RESULTS: The risks of atopic and non-atopic asthma (OR = 0.45, 95% CI:0.13-0.96 and OR=0.41, 95% CI:0.15-0.95), atopic sensitization. and wheezing (OR= 0.32, 95% CI:0.11-0.62; OR =0.44, 95% CI:0.13-0.91) were lower in subjects living in village area compared with those living in towns. The risks of atopic asthma and sensitization were lower in subjects exposed to stables in first year (OR=0.23, 95% CI:0.04-0.91 and OR =0.32, 95% CI:0.17-0.78) and were lowest in those exposed continually until the age of 6 (OR = 0.21, 95% CI:0.03-0.87 and OR = 0.31, 95% CI:0.15-0.78) compared with those non-exposed in the first 6 years. CONCLUSION: Exposure to rural environment might have a protective effect on children against atopic asthma and sensitization while continual exposure could strengthen the effects.

[Study on the relations between concentration of endotoxin in dwelling and atopic asthma in school-age children].

OBJECTIVE: To explore the effect of endotoxin concentration in dwellings on the prevalence of atopic asthma in children. METHODS: Standardized questionnaires of asthma were distributed to the parents of 2986 school children aged between 8 and 12 years and endotoxin content in children's mattress was measured by a kinetic limulus assay. A radioallergosorbent technique--fluorescence enzyme immunoassay (RAST-FEIA) was used to measure the level of specific IgE in serum. RESULTS: Complete data was available for 904 children with males more than females. There were both negative associations seen between endotoxin levels and both atopic asthma (OR = 0.48, 95% CI: 0.32-0.72, P < 0.05) and atopic sensitization (OR = 0.65, 95% CI: 0.49-0.94, P < 0.05) but not with non-atopic asthma and wheeze. Comparing with normal people, patients with atopic sensitization, atopic wheeze and atopic asthma had a higher levels of endotoxin (M-W U: 15 138.0, P < 0.01, M-W U: 4858.0, P < 0.01, M-W U: 4041.0, P < 0.01). CONCLUSION: Exposure to endotoxin in early lives of children might have a protective effect on atopic asthma and sensitization.